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Virol JVirology Journal1743-422XBioMed Central 1743-422X-7-262012889710.1186/1743-422X-7-26ResearchInhibition of Tomato Yellow Leaf Curl Virus (TYLCV) using whey proteins Abdelbacki Ashraf M [email protected] Soad H [email protected] Mahmoud Z [email protected] Abdelgawad I Abou [email protected] Mahmoud M Abd-El [email protected] Adel A [email protected] Plant Pathology Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt2 Dairy Science Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt3 Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt4 Virus and Mycoplasma Department, Agriculture Research Center, Giza 12619, Egypt2010 3 2 2010 7 26 26 25 8 2009 3 2 2010 Copyright ©2010 Abdelbacki et al; licensee BioMed Central Ltd.2010Abdelbacki et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The antiviral activity of native and esterified whey proteins fractions (α-lactalbumin, β-lactoglobulin, and lactoferrin) was studied to inhibit tomato yellow leaf curl virus (TYLCV) on infected tomato plants. Whey proteins fractions and their esterified derivatives were sprayed into TYLCV-infected plants. Samples were collected from infected leaves before treatment, 7 and 15 days after treatment for DNA and molecular hybridization analysis. The most evident inhibition of virus replication was observed after 7 and 15 days using α-lactoferrin and α-lactalbumin, respectively. Native and esterified lactoferrin showed complete inhibition after 7 days. On the other hand, native β-lactoglobulin showed inhibition after 7 and 15 days whereas esterified β-lactoglobulin was comparatively more effective after 7 days. The relative amount of viral DNA was less affected by the esterified α-lactalbumin whereas native α-lactalbumin inhibited virus replication completely after 15 days. These results indicate that native or modified whey proteins fractions can be used for controlling the TYLCV-infected plants.
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Introduction
Tomato yellow leaf curl virus (TYLCV) is one of the major and serious diseases of tomato which causes considerable amount of yield loss in Egypt [1-3]. One hundred twenty five million tons of tomatoes were produced in the world in 2007. China, the largest producer, accounted for about one quarter of the global output, followed by the United States, Turkey, India and Egypt. http://www.fas.usda.gov/htp/2009%20Tomato%20Article.pdf. Losses from plant diseases can have a significant economic impact, causing a reduction in income for crop producers, distributors, and higher prices for consumers.
In order to control TYLC-disease, it was found that frequent spray (at 7 days interval) of insecticide, like Cypermethrin (0.01%) or Dimethoate (0.1%) is effective to minimize the disease by controlling its vector whitefly (Bemisia tabaci) [4,5].
Researches focused on the use of alternative method to avoid the undesirable effects of the insecticides. In 1940s several investigators suggested the use of milk as spraying or dipping of seedlings for reducing the incidence of virus infections. Recent studies demonstrated the effectiveness of milk in reducing infection of tobacco mosaic virus (TMV) in pepper, tomato, and tobacco [6,7].
Whey represents a rich and heterogeneous mixture of secreted proteins with wide ranging nutritional, biological and food functional attributes. The main constituents of whey are α-lactalbumin (ALA), β-lactoglobulin (BLG) and two small globular proteins that account for approximately 70-80% of total whey protein. Historically, whey has been considered a waste product and disposed of in the most cost-effective manner, or processed into relatively low value commodities such as whey powder and various grades of whey protein concentrate/isolate (WPC, WPI). Nowadays, whey proteins and their derivatives are widely used in the food industry due to the excellent functional and nutritive properties adding to the commercial value of the processed foods [8]. The biological components of whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, lactoperoxidase, immunoglobulins and glycomacropeptides, demonstrate a wide range of immune enhancing properties, and act as antioxidant, antihypertensive, antitumer, antiviral, antimicrobial and chelating agent. They also improve muscle strength and body composition and prevent cardiovascular, cancer diseases and osteoporosis [9].
In spite of their high biological properties, native whey proteins are not hydrolyzed easily by means of digestion enzymes as pepsin and trypsin, due to disulfide bonds in the protein molecules. The poor digestibility of whey proteins is considered to be the reason for their allergenicity [10]. Therefore, modification of whey proteins to enhance or alter their biological and functional properties may increase its applications. Whey protein modification can be accomplished by chemical, enzymatic, or physical techniques [11,12]. Acetylation, succinylation, esterification, amidation, phosphorylation, and thiolation are chemical modifications that induce significant alterations of the structure and functional behavior of whey proteins.
Relatively small alterations of structure, brought about through chemical derivatization, often can be reflected in significant changes of physical and biological properties [13,14].
Many studies concerned with the antiviral activity of native and modified whey proteins in human [15,16]. Other studies focused on the use of milk or milk components to control plant viruses [17]. Therefore the objective of this work was to find and study possible antiviral compounds that would provide effective disease control under practical conditions, while also minimizing environmental impacts using native and modified whey proteins fractions (α-lactalbumin, β-lactoglobuline and lactoferrine) to control TYLCV.
Materials and methods
Materials
Healthy tomato, Lycopersicon esculentum Mill (Castlerock) seedlings and the severe strain of TYLCV-Is Tomato yellow leaf curl virus-Israel (TYLCV-Is [Sever]) [18] were obtained from Virus and Mycoplasma Department, Plant Pathology Research Institute, Agriculture Research Center, Giza - Egypt [19-21]. α-lactalbumin (97.46% protein), β-lactoglobulin (97.8% protein) were kindly obtained from Davisco food international (USA) and lactoferrine (95% protein) were kindly obtained from Armor Proteins (France). All other chemicals used in this study were of analytical grade.
Methods
1-Protein Esterification
The procedure of [12] was used for esterification of whey proteins fractions using >99.5% methyl alcohol, at 4°C for 10 h. as follows:
Native whey proteins fractions were dispersed (5%, w/v) in methyl alcohol 99.5%. Amounts of hydrochloric acid equivalent to 50 molar ratio of acidity (MR, mole acid/mole carboxyl group) were added drop-wise at the start of the reaction time. All the reaction mixtures were kept at 4°C under continuous stirring. At the end of the reaction (6 h), the samples were centrifuged at 10000 g for 10 min. The resulting supernatant was discarded and the residue was dispersed in a volume of alcohol (99.7% ethanol) equal to that of the discarded supernatant, and well mixed before re-centrifuging at the same conditions. This washing step was repeated three times. The final precipitate was dissolved in an appropriate amount of distilled water then submitted to freeze-drying. The lyophilized samples were kept at -20°C until analysis. The color reaction using hydroxylamine hydrochloride was used according to [22] to quantify the extent of esterification of proteins.
2-Experimental
Agro-Infiltration with TYLCV-IS infectious clone
Tomato plants previously transformed using optimized Agrobacerium-Mediated protocol [19] were agro-infiltrated with the infectious clone of TYLCV-IS using the syringe spotted technique (SST) [19-21].
Treatments
Tomato plants were planted under green house conditions taking into consideration all the environmental requirements conditions of irrigation, fertilization....etc. Plants were then transferred to large coercive after 20 days from planting (5 plants for each treatment). They were submitted to virus infection after 7-10 days from transferring using Agro-Infiltration with TYLCV-IS infectious clone. After 10 - 20 days from infection, each plant was sprayed using 20 ml of the native and chemically modified whey proteins fraction at concentration of 1 mg/ml. Leaves were collected from new growth produced after inoculation before treatment, 7 and 15 days after treatment in which total nucleic acids and molecular hybridization analysis were carried out.
3-Analytical
Detection and quantification of viral DNA
Viral DNA was extracted from tomato tissues using the modified Dellaporta extraction method [23,24].
Antiviral activity of modified whey proteins fractions
Antiviral activity was assessed on TYLCV particles replicated in plant tissue, using DNA non-radioactive hybridization [see Additional file 1 for data] to detect the presence and the absence of TYLCV in the treated plants using DNA sequence according to [19-21,24].
The dried DNA pellets were resuspended in 50 μl of TE-RNase buffer (Tris EDTA-RNase buffer) and 5 μl of each sample were dot onto the positively charged nylon membrane. The hybridization experiments were curried out using Gene Images AlkPhos and Chemiluminescent Detection System signal generation and detection with CDP-Star (Amersham, Biosciences, UK Limited) as described by [25-27].
Results
Extent of esterification
The whey proteins fractions α-lactalbumin, β-lactoglobulin and lactoferrine were modified at the extent of 68%, 100% and 100% respectively which indicate less esterification susceptibility of α-lactalbumin as compared to both of β-lactoglobulin and lactoferrin. The observed extents of such esterification are in accordance with [12].
Evaluation of TYLCV infection
Results obtained from PCR carried out on samples taken 10-15 days after infection using two TYLCV specific Primers, TYv 2337, (5'-ACG TAG GTC TTG ACA TCT GTT GAG CTC-'3) and TYc138 (5'-AAG TGG GTC CCA CAT ATT GCA AGA C-'3) [20] indicated that the plants were completely infected by the virus. Infected plants are stunted or dwarfed since only new growth produced after infection is reduced in size. Leaflets are rolled upwards and inwards and leaves are often bent downwards and are stiff, thicker than normal have a leathery texture, show interveinal chlorosis and are wrinkled. Young leaves are slightly chlorotic (yellowish).
Antiviral activity of whey proteins fractions against TYLCV
1-Antiviral activity of α-Lactalbumin (ALA)
Data presented in Fig. 1(A) &1(B) shows that the virus replication was completely inhibited after 15 days using native ALA (Fig. 1A). In contrast, modified form of ALA (68% methylation extent) gave the same antiviral action such as the native protein after 7 days of application (Fig. 1B).
Figure 1 Antiviral activity of α-Lactalbumin (ALA). Antiviral activity of α-lactalbumin (ALA) on infected tomato plants treated with: A) native α-Lactalbumin, B) modified α-Lactalbumin (5 plants for each treatment). 1) Before treatment (zero time), 2) 7 days after treatment, 3) 15 days treatment. 4) Positive control (without treatment), 5) negative control (Healthy plants), 6) infected plants sprayed with water.
2-Antiviral activity of β-lactoglobulin (BLG)
As shown in Fig. 2(A) &2(B), the native and modified forms of BLG had a little antiviral activity.
Figure 2 Antiviral activity of β-lactoglobulin (BLG). Antiviral activity of β-lactoglobulin ((BLG) on infected tomato plants treated with: A) native β-lactoglobulin, B) modified β-lactoglobulin (5 plants for each treatment). 1) Before treatment (zero time), 2) 7 days after treatment, 3) 15 days treatment. 4) Positive control (without treatment), 5) negative control (Healthy plants), 6) infected plants sprayed with water.
3-Antiviral activity of Lactoferrin
Fig. 3(A) &3(B) shows that lactoferrin inhibits the virus replication completely in infected plants either the native or the modified form even after 7 days from spraying.
Figure 3 Antiviral activity of Lactoferrin. Antiviral activity of lactoferrin on infected tomato plants treated with: A) native lactoferrin, B) modified lactoferrin (5 plants for each treatment). 1) Before treatment (zero time), 2) 7 days after treatment, 3) 15 days treatment. 4) Positive control (without treatment), 5) negative control (Healthy plants), 6) infected plants sprayed with water.
Discussion
Esterification is an important and easy tool of protein modification. Esterification blocks free carboxyl groups raising thus the net positive charge and rendering more basic the modified protein. It has been recently reported that increased basicity of dairy proteins after their esterification endow them with DNA-binding properties [12,14,28].
Early studies led to several hypotheses about milk's mode of action. The first one was in the 1930s suggested that milk inhibited infection by somehow reducing the plant's susceptibility to the virus [7]. The second one in the 1940s suggested that the milk "inactivated" the virus by forming a loose "molecular union" which, if broken, results in re-activation of the virus. That is, the inhibiting effects were reversible and the effect was on the virus and not the plant. The studies of an Australian scientist in the 1950s supported the earlier hypothesis that milk contains a substance that inhibits virus infection due to its effect on the plant, by supposedly inducing some type of resistance. It was also found that the inhibitory effects were restricted only in the treated part of the plant. Furthermore, investigations suggested that the active substance in the milk was a protein. The conclusion that the active substance is a protein component or number of such components is supported by recent work carried out by USDA scientists. But the answer to how exactly milk inhibits or reduces virus infection is still unknown [6,7].
Milk is rather heterogeneous suspension of oil (butter-fat), protein (cassein), sugar (lactose) and a multitude of possibly bioactive trace ingredients, including minerals, enzymes and vitamins. Possible modes of action of milk-based sprays were provided by [29]. These include an increase in the pH of the leaf surface [30], the establishment of a protective barrier, the establishment of possibly antagonistic organisms [31,32] the direct induction of systemic resistance [33] and/or the production of biocidal compounds [34]. All of the above processes will probably be highly dependent on the environmental conditions and the timing of the epidemic with respect to the phenology of the crop.
Milk contains several salts and amino-acids. These substances have been shown to be effective in controlling powdery mildew and other diseases [31-33,35-37].
The obtained results reveal that the antiviral activity of lactoferrin (either native or purified form) is greater than α-lactalbumin or β-lactoglobulin.
Milk whey proteins acquire net positive charges after esterification with methanol or ethanol enabling them to interact with negatively charged macromolecules such as nucleic acids or some proteins [12]. Consequently, these basic proteins may interact with viral DNA or RNA. Esterification not only increases the gross positive charge of the protein but also its hydrophobicity by grafting hydrophobic methyl or ethyl groups on the carboxyl groups of aspartyl and glutamyl residues. Enhanced hydrophobicity may also promote hydrophobic interactions with the hydrophobic binding sites formed by viral capsid proteins. Some antiviral inhibitory effects were already explained by the entry of hydrophobic inhibitory molecules in the hydrophobic binding cavities on the viral surface [38-40].
The interaction of antiviral proteins such as LF with receptors on cell surface and/or with viral envelope proteins is critical to blocking viral entry to target cells. The charge on the antiviral protein plays an indispensable role in this interaction. Chemical modifications lead to changes in the charges on milk proteins which can enhance their antiviral properties [41,42].
The results indicate that the inhibition of TYLCV may be related to the degree of cationisation of esterified whey proteins as well as to the size of the backbone protein which could be due to:1) Saturating binding to viral DNA by purely coulombic interactions, inhibiting its replication and transcription; 2) Hydrophobic interactions with viral capsid proteins; 3) Perturbation of viral DNA-protein interactions, hence inhibition of the translation of viral proteins; 4) Interference with/saturation of viral entry sites on the cellular membranes.
Many researchers recommend the use of milk to reduce the spread of virus particles between plants. Techniques using milk are frequently used in nurseries to stop the spread of virus between susceptible hosts when people touch the plant, during pruning. They reported milk proteins inactivated the capsid protein of the virus. Milk is not a potential environmental or food contaminant; consequently it can be used in organic agriculture.
Also, the data of [43-45] indicated that whey was effectively used to control powdery mildew in cucumber and zucchini and they recommended further studies to optimize the concentration and timing of whey applications for mildew management in commercial crops.
The antiviral effect of the used whey protein fractions can be arranged in descending order as follows: lactoferrin (native or modified form) > native α-lactalbumine > modified β-lactoglobulin > modified α-lactalbumin = native β-lactoglobulin. More studies are needed to improve the antiviral activity of both of α-lactalbumin and β-lactoglobulin.
In future experiments, we will examine combined regimen of alternating milk-based and chemical sprays and also using different concentration of whey, whey protein fractions and skim milk. These strategies may provide adequate protection against this disease, while reducing the chemical load on the environment and forestalling the development of resistant strains.
Finally the use of alternative "green" methods would have its advantage in the market, as many consumers are ready to pay more for pesticide-free products. This point could be of enough interest to justify the present work.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AMA conceived the research, performed the experiments, and wrote the manuscript; SHT developed the conceptual aspects of the work and edited the manuscript; MIS conceived of the study, and participated in its design and coordination; AZA participated in the design of the study; MMA conceived the research, performed the experiments, and edited the manuscript; AAR carried out the molecular genetic studies. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Antiviral activity of modified whey proteins fractions. The data provided represent the DNA sequence used in DNA non-radioactive hybridization.
Click here for file
Acknowledgements
We thank Dr. Ali Mamoun for helpful discussions. We also thank Davisco food international (USA) and Armor Proteins (France) for their kindly provided offers.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2023190209-PONE-RA-14985R110.1371/journal.pone.0009616Research ArticleBiochemistry/Cell Signaling and Trafficking StructuresCell Biology/Cell SignalingCell Biology/Cellular Death and Stress ResponsesRegulation of Proapoptotic Mammalian ste20–Like Kinase MST2 by the IGF1-Akt Pathway IGF1/Akt Regulates MST2Kim Donghwa Shu Shaokun Coppola Marc D. Kaneko Satoshi
¤a
Yuan Zeng-qiang
¤b
Cheng Jin Q.
*
Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America
Bergmann Andreas EditorUniversity of Texas MD Anderson Cancer Center, United States of America* E-mail: [email protected] and designed the experiments: JQC. Performed the experiments: DK SS MDC SK Z-qY. Analyzed the data: DK JQC. Contributed reagents/materials/analysis tools: SK. Wrote the paper: JQC.
¤a Current address: University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
¤b Current address: Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
2010 9 3 2010 5 3 e961617 12 2009 18 2 2010 Kim et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.
Methodology/Principal Findings
Using immunoblotting, in vitro kinase and in vivo labeling assays, we show that IGF1 inhibits MST2 cleavage and activation induced by DNA damage through the phosphatidylinosotol 3-kinase (PI3K)/Akt pathway. Akt phosphorylates a highly conserved threonine-117 residue of MST2 in vitro and in vivo, which leads to inhibition of MST2 cleavage, nuclear translocation, autophosphorylation-Thr180 and kinase activity. As a result, MST2 proapoptotic and growth arrest function was significantly reduced. Further, inverse correlation between pMST2-T117/pAkt and pMST2-T180 was observed in human breast tumors.
Conclusions/Significance
Our findings demonstrate for the first time that extracellular cell survival signal IGF1 regulates MST2 and that Akt is a key upstream regulator of MST2.
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Introduction
MST2 and its close homologue MST1 are members of the germinal center kinase group II (GCK II) family of mitogen-activated protein kinase (MAPK)–related kinases that includes the more distantly related kinases MST3, MST4, LOK, SOK, and SLK. Unlike other members, MST1 and MST2 contain a Ste20-related kinase catalytic domain in the N-terminal region followed by a noncatalytic tail that contains an autoinhibitory domain, a dimerization domain, and two nuclear export sequences at the COOH terminus [1], [2], [3]. It has been shown that the noncatalytic tail is cleaved by caspase upon various apoptotic stimuli [4], [5], [6]. Ectopic expression of MST1/2 induces striking morphological changes characteristic of apoptosis in both nucleus and cytoplasm. During the execution phase of apoptosis in mammalian cells induced by proapoptotic stimuli, MST1 and MST2 are activated by caspase cleavage and subsequently translocated to the nucleus. In the case of MST1, this leads to a constitutive phosphorylation of H2B, resulting in nuclear DNA fragmentation [7]. It has been shown that the protective function of the Raf1 (including kinase-dead Raf-1) against apoptosis involves the inhibition of MST2 activity by direct sequestration and inhibition of MST2 activation [8], [9]. In addition, RASSF (Ras association domain family) proteins RASSF1A and RASSF5 have been demonstrated to bind MST1 [10], [11], [12]. RASSF1A also releases MST2 from the inhibitory effect of Raf-1[13]. RASSF1A also releases MST2 from the inhibitory effect of Raf-1 [13]. Ultimately, RASSF1A and RASSF5 activate NDR1, NDR2, and LATS1 to induce apoptosis [13], [14], [15], [16]. These findings suggest that RASSF1A and RASSF5 stimulate MST signaling. A recent study shows that RASSF6 interacts with MST2 and inhibits MST2 activity. However, RASSF6 caused apoptosis when released from activated MST2 in a manner dependent on WW45 [17]. These findings suggest that activation of MST2 causes apoptosis through the canonical pathway, as well as through a RASSF6-mediated pathway [17].
In Drosophila, Hippo, a homolog of mammalian MST2, restricts cell growth and cell proliferation and promotes cell death by interaction with the tumor suppressors Salvador (Sav)/WW45 and Warts (Wts)/Lats1/Lats2, which result in inhibition of transcription and/or degradation of cyclin E and DIAPs [18], [19], through phosphorylation of Yorkie, which is the Drosophila ortholog of the mammalian transcription co-activator yes-associated protein (YAP) [20]. YAP and Yorkie have recently been shown to be negatively regulated by the Hippo/MST pathway and play an important role in mediating cell contact inhibition, organ size and tumorigenesis [21], [22].
Accumulated evidence shows that Akt and its downstream targets constitute a major cell survival pathway. Akt inhibits the programmed cell death in a number of cell types induced by a variety of stimuli through regulation of down stream molecules [23], [24]. Akt phosphorylates BAD on serine 136, which promotes the association of BAD a pro-apoptotic protein in Bcl-2 family, with 14-3-3 proteins in the cytosol, thus inactivating its pro-apoptotic function [25]. In addition, Akt reduces the transcription of a subset of pro-apoptotic genes by phosphorylation of Forkhead transcription factors, which causes their nuclear exclusion and inactivation [26], including FOXO1, FOXO3a, and FOXO4 and the phosphorylation by Akt negatively regulates FOXO activity by relocalizing FOXO from nucleus to the cytoplasm, where it is sequestered away from target genes through interacting with 14-3-3 [27]. In addition, several pro-apoptotic and anti-apoptotic proteins are also phosphorylated by Akt, including ASK1 [28], XAIP [24], Par-4 [29], BAX [30], [31], HtrA2 [32], which leads to direct activation of cell survival pathway.
A previous study showed that EGF stimulation caused a drop of MST1 kinase activity [33]. However, the regulation of MST2/Hippo by cell survival signaling remains largely unknown. In this report, we demonstrate that MST2 is inhibited by IGF1 through the PI3K/Akt pathway. Akt phosphorylates MST2 at Thr117 in vitro and in vivo, which leads to inhibition of MST2 cleavage and kinase activity as well as nuclear translocation. Furthermore, Akt activation is inversely correlated with autophosphorylation of MST2-T180 but paralleled with MST2-T117 phosphorylation in breast tumors. Collectively, our findings suggest that MST2 is a bona fide substrate of Akt and that Akt could play a critical role in regulation of the Hippo/MST2 pathway.
Materials and Methods
Reagents, Cell Culture and Breast Tumor Specimens
Stauroporine, LY294002 were obtained from Sigma (St. Louis, MO). DMEM and fetal bovine serum were purchased from Invitrogen Co. (Grand Island, NY). Anti-MST2 (#3952), -pMST2-Thr180 (#3681), -Akt (#9272), -pAkt-Ser473 (#9271), -actin (#4967) and -cleaved PARP (#9541 and #9544) antibodies were from the Cell Signaling Technology (Beverly, MA). Anti-GFP antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). COS7 and human embryonic kidney (HEK) 293 cells were purchased from The American Type Culture Collection (ATCC; Manassas, VA) and cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum. Eighty primary human breast cancer specimens were obtained from patients who underwent surgery at H. Lee Moffitt Cancer Center and approved by Institutional Review Board. Each sample contains at least 80% tumor cells, confirmed by microscopic examination.
Expression Constructs
Flag-tagged MST2 was created by PCR amplification of human Fetal Marathon-Ready cDNA (Clontech). The PCR products were cloned to p3XFLAG-CMV-10 vector (Sigma) at EcoRI-BamHI sites. MST2 specific primers are: ccggaattcatggagcagccgccggcg (5′primer), and cgcggatccaaagttttgctgccttct (3′primer). Flag-MST2-T117A and Flag-MST2-T117D were produced with mutagenesis kit (Stratagene) using wild-type MST2 as template. Wild type MST2 and mutant MST2 were also cloned to pEGFP-C1 (Clontech) at EcoRI-BamHI sites. GST-MST2 and GST-MST2-T117A were created by PCR amplification of 150 nucleotide fragment (a.a. 100–150) which include T117 and T117A sites using Flag-MST2-T117A and Flag-MST2-T117D as templates. The fragments were cloned to pGEX-4.1 vector at BamHI-EcoRI sites. The MST2 plasmids were confirmed by DNA sequencing. Akt expression constructs were previously described [32].
Immunoprecipitation, Immunoblotting and In Vitro Kinase Assay
Immunoprecipitation and immunoblotting were performed as previously described [24]. The immunoprecipitates were subjected to Western blotting analysis or in vitro kinase assay. Protein kinase assays were performed as previously described [34]. Briefly, reactions were carried out in the presence of 10 µCi of [γ-32P] ATP and 3 µM cold ATP in 30 µl buffer containing 20 mM Hepes (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, and 1 mM dithiothreitol using myelin basic protein (MBP) as substrate. After incubation at room temperature for 30 min the reaction was stopped by adding protein loading buffer and proteins were separated on SDS-PAGE gels. Each experiment was repeated three times and the relative amounts of incorporated radioactivity were determined by autoradiography and quantified with a Phosphoimager (Molecular Dynamics).
In Vivo [32P] Pi Labeling
COS7 cells were co-transfected with kinase-dead Flag-MST2 and constitutively active Akt. Following 48 h incubation, cells were labeled with [32P] Pi (0.5 mCi/ml) in phosphate- and serum-free DMEM medium for 4 h and then lysed. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were separated by 7.5% SDS-PAGE and transferred to membranes. The phosphorylated MST2 band was visualized by autoradiography.
Cell Death Assay, TUNEL Assay, Caspase3/7 Assay
Cells were seeded into 60-mm dishes and grown in DMEM supplemented with 10% FBS for 24h, treated with STS (30 µM). Apoptosis was determined by Tunel assay using an in situ cell death detection kit (Boehringer Mannheim, Indianapolis, IN) and caspase 3/7 assay kit (Promega). These experiments were performed three times in triplicate.
Results
IGF1 Inhibits MST2 through the PI3K/Akt Pathway and Akt Regulates MST2 Activation
Since MST2 is cleaved and activated upon apoptotic stimuli and is required for DNA damage-induced apoptosis in different types of cells, we initially examined if extracellular cell survival signal regulates MST2 cleavage and activation. COS7 cells were treated with staurosporine (STS; 1 µM) together with and without IGF1 for 2 h. Consistent with previous studies, STS alone induces MST2 cleavage and autophosphorylation of Thr180, an indicator of MST2 activation, as well as apoptosis. However, addition of IGF1 largely reduced STS effect towards MST2. PI3K inhibitor LY294002 inhibited the IGF1 action (Figure 1a). Further, LY294002 and Akt inhibitor API-2 [35] were able to induce MST2 activation and cleavage, which is inhibited by pan-caspase inhibitor Z-VAD (Figure 1b). These results imply that STS-induced MST2 cleavage and activation are inhibited by IGF1 through the PI3K/Akt pathway.
10.1371/journal.pone.0009616.g001Figure 1 IGF1/PI3K/Akt inhibits MST2 cleavage, activation and MST2-induced cell death.
(a) IGF1 inhibits MST2 through the PI3K/Akt pathway. Top panel is domain structure of MST2. DELD is a caspase cleavage motif and NES stands for nuclear export signal. Middle panels are immunoblots of COS7 cells, which were treated with IGF1 (50 µM) or/and LY294002 (20 µM) for 30 min prior to exposure to STS (1 µM) for 1 h, with indicated antibodies. Bottom panel shows the apoptosis measured with Tunel assay in three experiments in triplicate. (b) Inhibitors of PI3K (LY294002) and Akt (API-2) activate MST2. After treatment with indicated compounds for 3 h, COS7 cells were subjected to immunoblotting with indicated antibodies (upper panels) and apoptosis analysis (bottom). (c) Reconstitution of Akt in Akt1-null MEFs reduces MST2 activation induced by STS. Akt1-knockout MEFs were infected with adenovirus expressing Akt and then treated with 0.2 µM STS for 1h. Immunoblotting (upper) and apoptosis (bottom) analyses were performed as described above. (d) Knockdown of Akt enhances doxorubicin-activated MST2. MDA-MB-468 cells were transfected/treated with Akt/shRNA together with or without doxorubicin, and then subjected to immunoblotting (upper) and Tunel (bottom) analyses. (e) and (f) Constitutively active (Ac) Akt inhibits but dominant-negative (DN) Akt induces MST2 activation. COS7 cells were transfected with indicated plasmids. Following 48 h of incubation, cells were assayed for autophospho-T180 (top panel of e) and in vitro MST2 kinase using MBP as substrate (top panel of f). Middle and bottom panels show expression of the transfected plasmids.
We next examined the effect of Akt on MST2 activation. Akt-knockout MEFs were infected with adenovirus expressing Akt and adenovirus vector alone as control. STS treatment at low concentration (0.2 µM) considerably induces MST2 activation and cleavage as well as apoptosis in Akt-null MEF but not Akt-reconstituted cells (Figure 1c). Moreover, knockdown of Akt1 induces MST2 activation and enhances doxorubicin-activated MST2 and apoptosis in PTEN-mutated MDA-MB-468 cells (Figure 1d). Further, ectopic expression of dominant-negative Akt induces whereas constitutively active Akt represses MST2-Thr180 autophosphorylation in COS7 cells (Figure 1e). Constitutively active Akt also inhibited the apoptosis induced by ectopic expression of MST2 in MCF10A cells (Figure S1). In vitro MST2 kinase activity was also inhibited by ectopic expression of wild-type and constitutively active Akt (Figure 1f).
Akt Interacts with and Phosphorylates MST2
We next investigated whether Akt forms a complex with MST2. COS7 cells were co-transfected with HA-Akt and Flag-MST2. Co-immunoprecipitation experiments revealed that Flag-MST2 was readily detected in HA-Akt immunoprecipitates and vice versa (Figure 2a). Having demonstrated constitutively active Akt inhibition of MST2 but dominant-negative Akt activation of MST2 (Figure 1), we reasoned Akt regulation of MST2 through a kinase-dependent mechanism. Sequence analysis shows a well-conserved Akt phosphorylation consensus motif (112
RLRNKT
117) in a kinase domain of MST2 (Figure 2b). In vivo [32P]orthophosphate labeling and immunoblotting analysis with Akt substrate antibody revealed that constitutively active Akt induced MST2 phosphorylation (Figures 2c and 2d). Further, we created wild-type and T117A GST-MST2 (a.a. 100–150) fusion proteins which were used as substrate for in vitro Akt kinase assay. Figure 2e shows that Akt phosphorylates wild-type but not T117A MST2. These data suggest that MST2 is an Akt substrate.
10.1371/journal.pone.0009616.g002Figure 2 Akt interacts with and phosphorylates MST2.
(a) MST2 binds to Akt. COS7 cells were co-transfected with Flag-MST2 and HA-Akt. After 48 h of transfection, cells were lysed, immunoprecipitated with anti-Flag and detected with HA antibody (top) and vice versa (bottom). (b) Sequence alignment shows a highly conserved Akt phosphorylation consensus site of MST2 among different species. (c) and (d) Akt phosphorylates MST2 in vivo. COS7 cells were transfected with kinase-dead Flag-MST2 together with or without constitutively active (Ac) dominant-negative (DN) Akt. Following 36 h of transfection, cells were either labeled by [32P]-orthophosphate (c) or immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Akt-substrate antibody (top panel of d). For in vivo labeled cells, immunoprecipitation was performed with anti-Flag antibody and exposed to a film (top panel of c). Middle and bottom panels show expression of the transfected plasmids. (e) Akt phosphorylates MST2-T117 in vitro. In vitro Akt kinase assay was carried out by incubation of recombinant active Akt and GST-WT-MST2 and -MST2-T117A fused proteins (top). Bottom panel is coomassie blue staining of GST-MST2 proteins.
Akt Phosphorylates MST2-T117 In Vivo and the pMST2-T117 Inhibits Thr180 Autophosphorylation
To demonstrate in vivo phosphorylation of MST2-T117 by Akt, we generated specific phospho-MST2-T117 antibody by immunization of a rabbit with phospho-peptides (Ac-IRLRNK(pT)LIEDEIA-amide). We first characterized the specificity of this antibody. HEK293 cells were co-transfected with constitutively active HA-Akt and full-length wild-type and T117A mutant Flag-MST2. After immunoprecipitation with anti-Flag antibody, immunoblotting analysis with anti-pMST2-T117 antibody revealed that Akt phosphorylates wild-type but not T117A mutant MST2 (Figure 3a). We further showed that ectopic expression of wild-type or constitutively active Akt induced MST2-T117 phosphorylation whereas dominant negative Akt decreased the phosphorylation compared to that of the cells transfected with vector alone (Figure 3b).
10.1371/journal.pone.0009616.g003Figure 3 Akt phosphorylates Thr117 of MST2 in vivo and the pMST2-T117 regulates autophospho-MST2-T180.
(a) Characterization of anti-pMST-T117 antibody. HEK293 cells were transfected with constitutively active Akt and Flag-MST2 or Flag-MST2-T117A and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-pMST2-T117 antibody (top). Middle and bottom panels show expression of the transfected plasmids. (b) Akt phosphorylates endogenous MST2-T117. Following transfection of HEK293 cells with indicated different Akt, cells were lysed and immunoblotted with anti-pMST2-T117 (top), -MST2 (panel 2), -HA (panel 3) and -actin (bottom) antibodies. (c) IGF1 induces pMST2-T117 and represses pMST2-T180. After serum starvation for 12 h, HEK293 cells were stimulated with IGF1 for 1 h and then immunoblotted with indicated antibodies. (d) Re-expression of Akt in Akt1-/- MEF increases pMST2-T117 and reduces pMST2-T180 level. Akt1-null MEFs were infected with adenovirus expressing Akt or control vector and immunoblotted with indicated antibodies. (e) Knockdown of Akt decreases pMST2-T117 and increases pMST2-T180. MDA-MB-468 cells were transfected with AKT1/shRNA and control shRNA. After 72 h incubation, cells were lysed and immunoblotted with indicated antibodies. (f) Phospho-Thr117 regulates MST2 autophosphorylation of Thr180. HEK293 cells were transfected with indicated plasmids and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-pMST2-T180 (top) and -Flag (bottom) antibodies.
In addition, serum starvation reduces whereas IGF1 induces pMST2-T117 levels (Figure 3c). Introduction of Akt into Akt-null MEFs also increases pMST2-T117 (Figure 3d). Notably, pMST2-T117 is inversely correlated with autophosphorylation of Thr180 (Figures 3c and 3d). We also observed that knockdown of Akt in MDA-MB-468 cells reduced pMST2-T117 but increased pMST2-T180 (Figure 3e). Based on these findings, we hypothesized that Akt phosphorylation of Thr117 is required for inhibition of autophosphorylation of Thr180 of MST2. To test this, we introduced Akt phosphomimetic Flag-MST2-T117D and nonphosphorylatable Flag-MST2-T117A as well as wild-type Flag-MST2 into HEK293 cells. After immunoprecipitation with Flag antibody, immunoblotting analysis revealed that antophosphorylation of MST2-T180 was detected in the cells transfected with MST2-T117A and MST2 but not with MST2-T117D (Figure 3f). Collectively, we conclude that MST2 is bona fide substrate of Akt and the phosphorylation of Thr117 negatively regulates pMST2-T180.
Akt Inhibits MST2 Nuclear Translocation, Cleavage and Kinase Activity via a pThr117-Dependent Mechanism
Because the MST2 cleavage and nuclear translocation are critical steps for MST2 function and because Akt phosphorylates MST2-T117, we reasoned that Akt could inhibit MST2 nuclear translocation and that this action could depend on phosphorylation of Thr117. To this end, we created GFP-tagged wild-type MST2, Akt phosphomimetic MST2-T117D and nonphosphorylatable MST2-T117A. After transfection of COS7 cells with the various forms of GFP-MST2 together with and without constitutively active Akt, subcellular localization of GFP-MST2 was examined under a fluorescence microscope. Figure 4a shows that STS treatment induces WT-MST2 nuclear translocation which was inhibited by constitutively active Akt. Under non-DNA damage (e.g., STS) condition, constitutively active Akt also reduced approximately half of ectopically expressed wild-type MST2 nuclear translocation (Figure 4b). MST2-T117A exhibited nuclear localization whereas MST2-T117D located at cytoplasm. Expression of constitutively active Akt had no effect on subcellular localization of MST2-T117A and MST2-T117D (Figure 4b).
10.1371/journal.pone.0009616.g004Figure 4 Akt phosphorylation of Thr117 reduces MST2 nuclear translocation and activation.
(a) and (b), Akt inhibits wild-type MST2 nuclear translocation but has no effects on MST2-T117A and MST2-T117D. Panel (a) shows that STS-induced MST2 nuclear translocation was inhibited by Akt. COS7 cells were transfected with GFP-MST2 together with and without constitutively active Akt. After STS treatment for 2 h, cellular distribution of MST2 was examined (top) and quantified (bottom) under fluorescent microscopy. In panel b, COS7 cells were transfected with different forms of GFP-MST2 together with and without constitutively active Akt. After 36 h incubation, cells were examined and quantified for the nuclear MST2. The experiments were repeated for three times and 200 cells/transfection were examined. (c) Phospho-Thr117 reduces MST2 cleavage and kinase activity. COS7 cells were transfected with indicated plasmids. Following treatment with and without STS, cells were subjected to immunoblotting (top) and immunoprecipitation with anti-Flag antibody. The immunoprecipitates were subjected to in vitro MST2 kinase assay using MBP as substrate (bottom). (d) Akt failed to inhibit STS-induced MST2-T117A cleavage and kinase activity. COS7 cells were transfected/treated with indicated plasmids and reagent and then subjected to Western blot (top) and in vitro MST2 kinase assay (bottom) as described above.
Having demonstrated Akt inhibition of MST2 nuclear translocation through phosphorylation of Thr117, we next examined if Akt inhibition of MST2 cleavage and kinase activity depends on phosphorylation of Thr117. After transfection with Flag-tagged wild-type and mutant MST2 together with and without constitutively active Akt, COS7 cells were treated with STS or vehicle. Immunoblotting analysis shows that STS treatment induced the cleavage of MST2-T117A and wild type MST2, but had no significant effect on the cleavage of MST2-T117D (Figure 4c). Moreover, ectopic expression of constitutively active Akt reduced STS-stimulated wild-type MST2 but not MST2-T117A cleavage (Figure 4d). We have also examined whether the Akt phosphorylation of thr-117 affects MST2 kinase activity. In vitro kinase assays revealed that basal kinase activity was considerably reduced in phosphomimetic MST2-T117D whereas nonphosphorylatable MST2-T117A exhibited much higher kinase activity compared to wild type MST2 in the absence or presence of STS (Figure 4c). In combination of the findings in Figure 2b, these data indicate that full-length MST2-T117A is able to translocate to the nucleus and exhibits high level of kinase activity whereas MST2-T117D remains in cytoplasm and loses its kinase activity. Further, Akt had no effect on MST2-T117A kinase activity (Figure 4d). Taken collectively, these results indicate that phosphorylation of Thr117 is required for Akt inhibition of MST2 cleavage, nuclear translocation and kinase activity.
Phosphorylation of Thr117 Reduces MST2-Induced Apoptosis and Growth Arrest and Is Associated with pAkt in Breast Cancer
We next examined the effects of phosphorylation Thr117 on MST2-regulated cell survival and growth. Figure 5a shows that ectopic expression of MST2 and MST2-T117A in HEK293 cells induced PARP cleavage whereas MST2-T117D had no effect on PARP cleavage compared to the cell transfected with vector alone. Caspase-3/7 activity was also increased by expression of MST2 and MST2-T117A but not MST2-T117D. Moreover, constitutively active Akt reduced the effect of wild-type MST2 but had no influence on MST2-T117A and MST2-T117D (Figure 5b). Previous studies have also shown that MST2 activation inhibits cell proliferation [36]. Thus, we have performed cell proliferation assay and observed that cell growth was significantly decreased in MST2-T117A cells whereas increased in MTS2-T117D cells when compared to wild-type-MST2 and vector-transfected cells (Figure 5c). Because Akt phosphorylation of MST2-T117 inhibits autophospho-MST2-T180 (Figures 2 and 3), we also investigated whether this regulation also existed in tumor tissues. Western blot analysis was performed in 80 human primary breast carcinomas. Elevated levels of phospho-Akt were detected in 40 specimens (Figure 5d and data not shown), 33 of which exhibited high levels of phospho-MST2-T117 whereas 35 of which express low or undetectable level of phospho-MST2-T180. Notably, all 33 tumors with high levels of phospho-Akt/phospho-MST2-T117 had low phospho-MST2-T180. Statistic analysis revealed that phospho-Akt significantly links to phospho-MST2-T117 (p<0.0001) which inversely correlates with phospho-MST2-T180 (p<0.0001; Figure 5e). In addition, overall survival of the patients with elevated pMST2-T117/low pMST2-T180 is significantly lower than those with low pMST2-T117/high p-MST1-T180 (Figure 5f). Taken together, we conclude that MST2 is a bona fide substrate of Akt and that pMST2-T117 could be a prognostic marker in human breast cancer.
10.1371/journal.pone.0009616.g005Figure 5 Akt inhibition of MST2 cellular function depends on the phosphorylation of Thr117.
(a) MST2-T117A increases PARP cleavage. HEK293 cells were transfected with indicated plasmids and then immunoblotted with indicated antibodies. (b) Akt inhibits WT-MST2 but not MST2-T117A-induced apoptosis. HEK293 cells were transfected with indicated plasmids. After 36 h of transfection, caspase3/7 activity was measured. The experiment was repeated three times in triplicate. (c) Cell growth curve. Indicated plasmids were introduced into HEK293 cells. Cell number was accounted daily for three days. MST2-T117A significantly inhibited whereas MST2-T117D increased cell growth compared to cells transfected with MST2 or vector alone. (d) Representative breast cancer specimens were lysed and immunoblotted with indicated antibodies. (e) Inverse expression of pMST2-117/Akt-S473 and pMST2-T180 was detected in majority of human breast tumors. (f) Overall survival (OS) in patients with high pMST2-T117/low pMST2-T180 (n = 32) versus low pMST2-T117/high pMST2-T180 patients (n = 29) was plotted by the Kaplan-Meier method. Statistical comparison of survival between groups with the log-rank statistic analysis suggests that patients whose tumors express pMST2-T117 (+)/pMST2-T180 (−) had poor survival compared to those with pMST2-T117 (−)/pMST2-T180 (+) (P = 0.01).
Discussion
MST1 and MST2 are human homologes of Hippo, however, protein sequence similarity between MST2 and Hippo (63.5%) is higher than that of MST1 versus Hippo (50%). Previous studies have shown that Hippo/MST is autophosphorylated and cleaved by caspases in response to apoptotic stimuli. The cleaved N-terminal kinase domain (e.g., activated MST2) subsequently translocates into the nucleus where it interacts and phosphorylates tumor suppressors Salvador (WW45) and Warts (Lats1/2), which result in inhibition of transcription and/or degradation of DIAPs and cyclin E leading to apoptosis and cell growth arrest [18], [19], [37]. In this study, we demonstrate that IGF1 inhibits MST2 cleavage and activation through the PI3K/Akt pathway. Small molecule inhibitors of PI3K/Akt or depletion of Akt activate MST2. Further, Akt interacts with MST2 and phosphorylates MST2-T117 in vitro and in vivo leading to inhibition of MST2 cleavage, autophosphorylation and kinase activity. The phosphorylation of Thr117 abrogates MST2 proapoptotic and cell growth-inhibitory function. These results indicate Akt as a key upstream regulator of MST2 and provide a mechanism by which Akt promotes cell survival through direct phosphorylation of MST2 at Thr117 (Figure 6).
10.1371/journal.pone.0009616.g006Figure 6 A diagram represents the regulation of MST2 by IGF1/Akt signal.
Under stress conditions, MST2 is activated through caspase-3 cleavage and autophosphorylation-Thr180. The activated/cleaved MST2 translocates to the nucleus and promotes apoptosis. IGF1/Akt inhibits these processes through phosphorylation of MST2-T117.
A previous study has demonstrated that Raf-1 kinase binds to MST2 and prevents its dimerization and autophosphorylation of Thr180, which results in inhibition of MST2 activation and proapoptotic activity [8]. Intriguingly, this regulation is independently of Raf-1 protein kinase activity because kinase-negative Raf-1 also could inhibit MST2 activation and apoptosis [8]. While Akt interacts with MST2, the regulation of MST2 by Akt depends on its kinase activity since constitutively active Akt inhibits whereas dominant-negative Akt induces MST2 activation (Figures 1 and 4). In addition, Akt mediates IGF1 signal towards MST2 and directly phosphorylates Thr117. The phosphorylation of Thr117 is required for Akt inhibition of MST2. Thus, our findings provide the evidence that IGF1/Akt survival signal regulates MST2 through a phosphorylation of Thr117-dependent mechanism.
Our study shows that MST2 possesses an Akt phosphorylation site (112RLRNKT
117) within its N-terminal kinase domain (Figure 1a), which is highly conserved among yeast, Drosophila, Xenopus, mouse, and human. Moreover, this motif also exists in MST1 (Figure 2b). The Hippo pathway was initially identified in the fly to control organ size. Its core components are evolutionally conserved in mammals. Hippo, Sav, Wts and Mats in the fly are homologous to mammalian MST1/2, WW45, LATS1/2, and Mob1, respectively [38]. Previous studies also showed that the Drosophila insulin receptor transduces signals that positively regulate cell and organ growth through its downstream molecule Chico/Dp110/Dakt1 [39], [40]. Overexpression of the Dakt1 dramatically increases clonal size in wing imaginal disc through an enlargement of the cells [39]. These studies suggest that the crosstalk between Akt and Hippo/MST regulates cell growth and survival and that Akt phosphorylation of MTS2-T117 represents a major regulatory mechanism of the MST2/Hippo pathway among different species.
A previous study showed that Akt phosphorylates Thr387 at C-terminal region of MST1 to abrogate MST1 function [41]. However, Thr387 is not conserved in MST2 and Hippo. As described above, Thr117 of MST2 is conserved in MST1 (Figure 2b). Thus, Akt could regulate both MST1 and MST2 through phosphorylation of the same (e.g., Thr117/Thr120) and different (e.g., Thr387) sites. In addition, it has been shown that full-length and cleaved forms of MST1 and MST2 bind to C-terminal hydrophobic region of Akt and inhibit Akt activation (42), suggesting a negative feedback regulation loop between Akt and MST1/MST2 (Fig. 6). Further investigations are required to define the physiological importance of these Akt phosphorylation sites and feedback regulation between of Akt-MST in knock-in mouse model.
Supporting Information
Figure S1 MST2-induced apoptosis is inhibited by constitutively active Akt. MCF10A cells were transfected with Flag-MST2 and constitutively active HA-myr-Akt. After 36 h of incubation, cells were subjected to Tunel assay (A) and immunoblotting with indicated antibodies (B).
(2.13 MB TIF)
Click here for additional data file.
We are grateful for Tissue Procurement and DNA Sequence and Core Facilities at H. Lee Moffitt Cancer Center. We also thank M.J. Birnbaum for Akt-null MEF.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by grants from National Institutes of Health CA107078 and CA137041, Department of Defense W81XWH-08-2-0101 and Bankhead-Coley Grant ID 09BB-05. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J Minim Access SurgJMASJournal of Minimal Access Surgery0972-99411998-3921Medknow Publications India 20407567JMAS-05-9310.4103/0972-9941.59306Original ArticlePediatric cholelithiasis and laparoscopic management: A review of twenty two cases Deepak J Agarwal Prakash Bagdi R K Balagopal S Madhu R Balamourougane P Department of Pediatric Surgery, Sri Ramachandra Medical College, Porur, Chennai - 600 116, IndiaAddress for correspondence: Dr. J Deepak, Department of Paediatric Surgery, Sri Ramachandra Medical College, Porur, Chennai - 600 116, India. E-mail: [email protected] 2009 5 4 93 96 23 8 2009 07 11 2009 © Journal of Minimal Access Surgery2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AIM:
To evaluate the role of laparoscopic cholecystectomy (LC) in the management of cholelithiasis in children.
MATERIALS AND METHODS:
A retrospective review of our experience with LC for cholelithiasis at our institution, between April 2006 and March 2009 was done. Data points reviewed included patient demographics, clinical history, haematological investigations, imaging studies, operative techniques, postoperative complications, postoperative recovery and final histopathological diagnosis.
RESULTS:
During the study period of 36 months, 22 children (10 males and 12 females) with cholelithiasis were treated by LC. The mean age was 9.4 years (range 3 to 18 years). Twenty-one children had symptoms of biliary tract disease and one child was incidentally detected with cholelithiasis during an ultrasonogram of the abdomen for an unrelated cause. Only five (22.7%) children had definitive etiological risk factors for cholelithiasis and the remaining 13(77.3%) cases were idiopathic. Twenty cases had pigmented gallstones and two had cholesterol gallstones. All the 22 patients underwent LC, 21 elective and one emergency LC. The mean operative duration was 74.2 minutes (range 50-180 minutes). Postoperative complications occurred in two (9.1%) patients. The average duration of hospital stay was 4.1 days (range 3-6 days).
CONCLUSION:
Laparoscopic chloecystectomy is confirmed to be a safe and efficacious treatment for pediatric cholelithiasis. The cause for an increased incidence of pediatric gallstones and their natural history need to be further evaluated.
Childrencholelithiasislaparoscopic cholecystectomy
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INTRODUCTION
Cholelithiasis, although increasing in frequency in children, is still far less common than in the adult population.[1] In a population-based study prevalence of gallstones in children was 1.9%.[2] The nature of the disease process is different in children as compared to adults, with a higher proportion of pigment stones and less cholesterol-based stone disease in the pediatric population, especially in those younger than 10 years.[3] Laparoscopic cholecystectomy (LC) is considered to be the ‘gold standard’ surgical procedure for cholelithiasis in adults, with a vast amount of published data supporting this. However, there is a paucity of reports in the literature pertaining to the clinicopathological characteristics and laparoscopic management of gallstones in children.
MATERIALS AND METHODS
A retrospective review of all the children who underwent LC for cholelithiasis in our institution between April 2006 and March 2009 was done. The patient medical records were examined and the data pertaining to demographic information, clinical history, diagnosis, operative findings and operative technique, postoperative complications and recovery and the final histopathological diagnosis were obtained. LC was performed by different surgeons using the standard four ports technique [Figure 1]. A 10 mm umbilical camera port was inserted using the open technique in all the cases, which was also used for retrieval of the gall bladder specimen. Two 5 mm working ports for the surgeon were placed in the epigastrium and right midclavicular line in the hypochondrium or the lumbar region. Another 5 mm port was inserted in the right anterior axillary line, to help the assistant surgeon grasp the fundus of the gall bladder for retraction. The positions of the ports were adjusted, (by placing them away from the site of the surgery) according to the size of the child. Intraoperative cholangiography was not deemed to be necessary in any of our patients. In a pediatric population the dissection around the Calot's triangle is easier and faster compared to adults, as the fat deposit is very minimal and the peritoneal covering layer is thin, allowing clear visualization of the anatomy [Figure 2]. The patients were discharged when they were able to tolerate a regular diet and were ambulatory. They were followed up in the outpatient clinic at least once after the discharge.
Figure 1 Diagrammatic representation of port placement for laparoscopic cholecystectomy in children
Figure 2 Laparoscopic intraoperative view of Calot's triangle showing clear anatomy
RESULTS
During the study period, 22 patients underwent LC for cholelithiasis. The mean age was 9.4 years (range 3 to 18 years). Two children were less than five years, 14 were aged between five and 12 years and six were adolescents. Fourteen (63.6%) children had typical symptoms of biliary tract disease (right upper quadrant or epigastric pain, nausea, vomiting and food intolerance), seven (31.9%) had fever in addition to the above-mentioned symptoms (calculous cholecystitis), and one child had asymptomatic gallstones, which were diagnosed incidentally on ultrasound examination of the abdomen, done for an unrelated cause.
Duration of symptoms at diagnosis varied from one month to 12 months (mean-2.9 months). Risk factors for development of gallstones were present in five (22.7%) children only. Two had a family history of gallstones, two were obese (BMI > 30) and one child had undergone previous abdominal surgery and had received an injection of Ceftriaxone for 14 days. After a complete workup, it was found that none of our patients were detected with having haemolytic disorders such as sickle cell disease, thalassemia or hereditary spherocytosis. A complete haemogram, peripheral blood smear and liver function tests were within normal limits in all the patients. All the children underwent an abdominal ultrasound and were detected to have single or multiple gallstones. In addition, ten (45.4%) children had inflammatory features around the gall bladder.
Twenty-one children underwent elective LC and one child was taken up for emergency LC after treating acute cholecystitis with intravenous antibiotics for two days. The mean operative duration was 74.2 minutes (range 50-80 minutes). Operative findings included omental or small bowel adhesions around the gall bladder (with or without edematous gallbladder) in 13 (59.1%) patients. The child who underwent emergency LC had empyema along with the above-mentioned inflammatory features. Twenty children had pigmented stones [Figure 3] and two had cholesterol stones. Among the 16 children with pigmented gallstones, two had multiple gravel-like (< 1 mm) stones. Tube drains were placed in three (13.6%) cases, wherein intraoperative bile spillage or gallbladder fossa ooze was present.
Figure 3 Gall bladder specimen with multiple pigment gallstones
Two drains were removed within 24 hours; the remaining one was kept for 96 hours, as significant serous fluid discharge was present during the first two days postoperatively. The average duration of hospital stay was 4.1 days (range 3-6 days). Postoperative complications occurred in two (9.1%) patients. One child had significant prolonged serous discharge from the tube drain as mentioned earlier, which resolved spontaneously. The other child who underwent emergency LC had postoperative fever for three days, which resolved with intravenous antibiotics.
Histopathological analysis of the cholecystectomy specimen revealed chronic cholecystitis in 18 cases, chronic cholecystitis with focal ulceration in two cases and one each of acute cholecystitis and acute chronic cholecystitis. Follow-up duration ranged from four months to 35 months (average 17 months) and there were no cases of retained common bile duct stones in our study.
DISCUSSION
Cholelithiasis is considered as an uncommon condition in children, however, recent studies have documented increasing incidents of this disorder.[24] This may be explained by the increased availability and use of the abdominal ultrasonogram in children. Pediatric cholelithiasis was viewed as a disease of prematurity, usually related to total parenteral nutrition. Various risk factors for cholelithiasis in children include haemolytic disorders, obesity, family history of gallstones, abdominal surgery, IgA deficiency, cystic fibrosis, therapy with ceftriaxone and Gilbert's disease.
In our series, only 22.7% of the patients had the above-mentioned risk factors, the remaining 77.3% had idiopathic cholelithiasis. The incidence of idiopathic cholelithiasis in other reported series varies from 23 to 52.5%.[56] The trend of the increasing incidence of non-haemolytic cholelithiasis is also reflected in our series, with all of them belonging to the non-haemolytic cholelithiasis category. The mechanism of gallstone formation in these children is probably due to a combination of interacting processes, including, dehydration, transient hepatic dysfunction, dietary, inflammatory, hereditary and endocrine influences, which affect the composition of bile.[7]
Approximately 80% of the adults with gallstone are asymptomatic.[89] However, in children, asymptomatic gallstones are less frequent, with a reported incidence of 10 and 33% in two different studies.[67] In our study 4.5% children had asymptomatic gallstones. The incidence of gallstones among boys and girls is almost equal, with a slightly high incidence among boys.[51011] The sex ratio in our study was slightly in favour of females. LC in children differs from that in adults in various aspects. First and foremost it is the constraint of space. The importance of positioning the epigastric cannula in the left upper quadrant in small children cannot be overemphasized. Similarly, the working and retracting ports on the right side should be placed in the lumbar or iliac region in younger children. Second, as mentioned earlier, the dissection at the Calot's triangle is relatively easier and faster in children, as the fat deposit is less and the peritoneal covering layer is thin in children.
Routine intraoperative assessment for common duct stones was not done in our series. Shawn et al., have reported that the incidence of subclinical common bile duct stones is low in children.[1] This finding has also been described in a small prospective pediatric study.[12] In the present series, as there was no evidence of common bile duct stones or altered liver function tests preoperatively in any of the patients, an intraoperative cholangiogram was not done. None of the patients had any evidence of residual ductal stones during the follow up period. Hence, a routine intraoperative cholangiogram was not recommended for the children.
The natural history of cholelithiasis in children is not known,[5] hence, the treatment remains controversial. The clinical presentation, findings on ultrasound imaging, intraoperative findings and the final histopathological diagnosis of the gall bladder specimen did not correlate completely in our study. While only seven (31.9%) patients had fever suggestive of clinical inflammation, 10 (45.4%) patients had ultrasound findings suggestive of inflammation and 13 (59.1%) had intraoperative evidence of inflammation. However, on histological analysis, all the 22 resected gallbladder specimens showed either acute or chronic inflammation. As the natural history of gallstones in the children was not known and histological evidence of inflammation was present in all the cases of cholelithiasis in our series, we suggested an LC for all children with cholelithiasis. A recently conducted multicenter study also reported structural alterations in the majority of gallbladders removed for cholelithiasis.[5] These authors also suggest that because of the expectancy of long life for children, expectant management of cholelithiasis may not be safe. However, in adults where the natural history is well-documented, only 1 to 4% per year develop symptoms or complications of gallstone disease, only 10% develop symptoms in the first five years after diagnosis and approximately 20% by 20 years.[13–15]
The mean operative duration for LC was 74.2 minutes in our study. This duration was between 70 and 80 minutes in other reported series.[111] The comparison of various parameters between LC and open cholecystectomy in one study reported significantly less duration of hospital stay and decreased overall cost in patients undergoing LC.[11] The other advantages of LC, such as, decreased pain, avoidance of upper abdominal muscle cutting incision, faster return to activity and cosmetically better scar, are well-documented.[16–18]
CONCLUSIONS
Laparoscopic cholecystectomy (LC) is confirmed to be a safe and efficacious treatment modality for pediatric cholelithiasis. The cause for increased incidence of pediatric cholelithiasis and their natural history need to be further evaluated. LC is much simpler in children compared to adult population, when it is performed by an experienced surgeon.
Source of Support: Nil
Conflict of Interest: None declared.
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J Int AIDS SocJ Int AIDS SocJournal of the International AIDS Society1758-2652The International AIDS Society 1758-2652-13-72019685610.1186/1758-2652-13-7ResearchAre Nepali students at risk of HIV? A cross-sectional study of condom use at first sexual intercourse among college students in Kathmandu Adhikari Ramesh [email protected] Geography and Population Department, Mahendra Ratna Campus, Tribhuvan University, Kathmandu, Nepal2 Institute for Population and Social Research, Mahidol University, Salaya, Thailand2010 2 3 2010 13 7 7 28 10 2009 2 3 2010 Copyright ©2010 Adhikari; licensee BioMed Central Ltd.2010Adhikari; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Condoms offer the best protection against unintended pregnancies and sexually transmitted infections. Little research has been conducted to determine the prevalence and investigate the influencing factors of condom use at first sexual intercourse among college students.
Methods
A self-administered questionnaire was completed by 1137 college students (573 male and 564 female) in the Kathmandu Valley. Analyses were confined to 428 students who reported that they have ever had sexual intercourse. The association between condom use at first sexual intercourse and the explanatory variables was assessed in bivariate analysis using Chi-square tests. The associations were further explored using multivariate logistic analysis in order to identify the significant predictors after controlling for other variables.
Results
Among the sexually active students, less than half (48%) had used condoms during first sexual intercourse. The results from the logistic regression analysis revealed that age, caste and/or ethnicity, age at first sexual intercourse, types of first sex partner, alcohol consumption and mass media exposure are significant predictors for condom use at first sexual intercourse among the college students. Students in the older age groups who had first sex were about four times (16 to 19 years old) (OR = 3.5) more likely and nine times (20 or older) (OR = 8.9) more likely than the students who had sex before 16 years of age to use condoms at first sexual intercourse.
Moreover, those students who had first sex with commercial sex worker were five times (OR = 4.9) more likely than those who had first sex with their spouse to use condoms at first sex. Furthermore, students who had higher exposure to both print and electronic media were about twice (OR = 1.75) as likely as those who had lower media exposure to use condoms. On the other hand, students who frequently consumed alcohol were 54% (OR = 0.46) less likely to use condoms at first sexual intercourse than those who never or rarely consumed alcohol.
Conclusions
The rate of condom use at first sexual intercourse is low among the students. It indicates students are exposed to health hazards through their sexual behaviour. If low use of condom at first sex continues, vulnerable sexual networks will grow among them that allow quicker spreading of sexually transmitted diseases and HIV. Findings from this study point to areas that policy and programmes can address to provide youth with access to the kinds of information and services they need to achieve healthy sexual and reproductive lives.
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Background
The Federal Democratic Republic of Nepal is a landlocked country in south Asia. It lies between the two of the most populous countries of the world: China in the north and India in the south, east and west. These countries are ranked as the first and the second largest countries of the world, respectively, in size of population. The total population of Nepal was 27.5 million in 2009 [1]. About one in five of Nepal's people are in the age group of 15 to 24 years [2].
HIV/AIDS has become a global problem and has spread all over the world. The latest statistics estimate that approximately 33.4 million people worldwide were living with HIV/AIDS by the end of 2008. Of these, 4.7 million people were in Asia. India, Nepal's neighbour, accounts for roughly half of Asia's HIV prevalence. With the exception of Thailand, every country in Asia has an adult HIV prevalence of less than 1%. However, owing to the region's large population, Asia's comparatively low HIV prevalence translates into a substantial portion of the global HIV burden in terms of numbers [3].
Like other countries in Asia, Nepal is susceptible to HIV. The country is indeed facing critical challenges posed by the rapid spread of HIV/AIDS. By October 2009, about 15,000 cases of HIV infection and about 2600 cases of AIDS had been officially reported. Among these HIV cases, more than two in five cases (41%) were in the college/university-going (15-29 years) age group [4]. However, given the limitations of Nepal's public health surveillance system, the actual number of infections is thought to be much higher. The Joint United Nations Programme on HIV/AIDS (UNAIDS) estimated that 75,000 people were living with HIV at the end of 2007 [5]. It has been found that one in every 200 young males (0.5%) and one in every 300 females (0.3%) aged 15 to 24 are infected by HIV in Nepal [1].
Studies show that college students engage in a variety of behaviours that put them at risk for serious health problems [6,7]. College students are at risk of sexually transmitted infections, including HIV, due to their propensity to take risks, often with multiple partners, accompanied by an inconsistent use of condoms [8,9]. Ample research has also examined the prevalence of excessive alcohol and other substance use, risky sexual behaviour, and other harmful health behaviours that are not uncommon among college students [10-13].
Similarly, a study found that young adults represent one of the groups at highest risk for HIV infection [14]. A study conducted in colleges in Nepal showed that about two in five male college students (39%) had premarital sexual experiences. Among these, more than half reported that they had multiple sex partners. Furthermore, more than one in five (23%) had sexual experience with commercial sex workers, and less than half of them (49%) had used condoms in every act of sexual intercourse with a sex worker [15]. Such risky sexual behaviour increases the risk of contracting an STI or HIV which jeopardizes academic achievement and performance of the students [13,16,17].
Differences in levels of condom use in various population groups and settings have been identified in the literature. In particular, groups with the lowest levels of education have consistently been the least likely to use condoms, both with non-marital and marital partners [18-22]. Some studies also revealed an increase in condom use in the younger age groups [18,23,24] compared to the older groups. Some studies found that later sexual activity has also been associated with an increased use of condoms [25-27]. Furthermore, alcohol use is highly prevalent among college students, and contributes to rising rates of sexual risk taking [28-31]. Empirical evidence also suggests that the mass media has an important role in shaping individual reproductive attitudes and behaviours [32-34]. In fact, the mass media variable is a reliable predictor of condom use among individuals [35,36]. One study showed that men who heard about AIDS through either electronic or print media were 30% to 50% more likely to have used a condom than men not exposed to these media [37].
Many researchers and public health policy makers are particularly interested in the subject of first sexual contact. A person's first sexual intercourse often occurs before the age of 20. This is linked to two factors: the association between behaviours in the first sexual relationship and the establishment of behavioural patterns throughout life [38-40]; and the recognition that sexual initiation at a very young age is a risk factor for pregnancies before the age of 20 and acquiring sexually transmitted diseases, including HIV [26,41].
Condom use at first sex is easily remembered. Some studies have found a strong link between first condom use and lifetime use: a 20-fold increase in lifetime use and a 10-fold increase in current use if a condom was used at first sexual contact [42]. There is a limited body of literature that points to condom use at first sexual contact among young people, especially students, and none of the studies has analyzed the data on condom use at first sex in Nepal.
It is useful to know about condom use during first sexual intercourse in Nepal so that priorities and approaches for interventions to prevent risky sexual behaviour can be better designed. This article aims to determine the prevalence of and investigate the factors associated with condom use at first sexual intercourse among college students in Nepal.
Specifically, it is assumed that students who have sex at an earlier age, who consume alcohol frequently, who have sex with irregular partners, and who have lower exposure to mass media use condoms less at first sexual intercourse. The findings of this study address the gap in knowledge by providing the information on condom use at first sexual intercourse that could assist programme managers of government agencies and non-governmental organizations and the Government of Nepal in designing appropriate and timely education-based interventions in institutions of secondary and higher education.
Methods
Data and sampling framework
The data used in this paper comes from a cross-sectional survey on attitude and behaviour towards premarital sex among college students of Kathmandu, the capital of Nepal, carried out in 2006. The survey involved a total of 1137 students (573 male and 564 female) studying in 12 colleges affiliated to Tribhuvan University in Kathmandu. Analyses were confined to 428 students who reported that they have ever had sexual intercourse. The scientific committee, which included the Ethical Review Board of the University Grant Commission in Nepal, approved the proposal and provided funding for this study.
A two-stage random sampling technique was applied. The first stage included a random selection of 12 colleges in Kathmandu. In order to select these colleges, a list of all the private and public colleges affiliated with Tribhuvan University and located in the Kathmandu Valley (which includes three districts: Bhaktapur, Lalitpur and Kathmandu) was obtained from the office of the Vice Chancellor in Kathmandu. This list included colleges that provide intermediate (commonly known as Grades 11 and 12), undergraduate and graduate degrees. In the second stage, two classes were randomly selected from each sampled college. The number of students in a class ranged from 40 to 60. All the colleges are co-educational, and all male and female students present on the day of the interview in the sampled classes were requested to participate in the study.
Research instrument and data collection
Due to the sensitive nature of the study and the educational background of the respondents, a self-administrated, structured questionnaire in the Nepali language was used to obtain information. The questionnaires were first developed in English and then translated into Nepali. Almost all sections pertaining to the behavioural aspect were based on Behavioral Surveillance Survey questionnaire developed by Family Health International/Impact [43]. However, necessary modifications were made to suit the sample population. The questionnaires were pre-tested among students in a college that had not been selected as part of the study, and later refined as required. The pre-test was conducted to determine whether the questionnaires were in sequential order and the wording in Nepali was understandable or not. Most of the questions were close ended; a few open-ended questions were also included.
Female and male students filled in the questionnaire separately in different classrooms. Each student was allocated a separate bench, as in an exam setting, before the questionnaire was distributed to them. A male researcher supervised the male students' class while a female researcher supervised the female students' class. Students were then requested to place the filled-in questionnaire on a table in the corner of each class.
Ethical considerations
Before starting the study in a sampled college, approval from the campus administrative authority was obtained. All the participants involved in the study were fully informed about the nature of the study, the research objectives, and the confidentiality of the data. After this, verbal consent was obtained from the participants before they were enrolled in the study. The consent form was also written in the local language, stating the study's objectives, nature of the participant's involvement, risk and benefits, and confidentiality of the data. Students were requested to read the consent form carefully. They were given clear options on voluntary participation. It was also made clear that they could refuse to answer any of the questions and terminate the interview if and when they desired.
All of the approached students agreed to participate in the study. Confidentiality of information was ensured by removing personal identifiers from the completed questionnaires. The names of sampled colleges were not made public and thus, it is not possible for anyone outside the research team to trace reported incidents of sexual behaviour to respondents. Respondents were thus protected from any possible adverse repercussions of participating in the study.
Variables
The measurable outcome of the study is condom use at first sexual intercourse, a dichotomous variable indicating whether or not the respondent had used a condom during the first sexual encounter.
The independent variables used in the study were: sex of the respondent; age; caste and/or ethnicity; level of education; age at first sexual intercourse; marital status; types of first sexual partner; permanent place of residence; alcohol consumption; mass media exposure; and living arrangement. All these variables were organized into two or three categories, based on those used in other literature, as well as on the frequency distribution of the variables.
The indicators of exposure to mass media include an exposure to radio, television and newspapers. The majority of the students rent rooms in the Kathmandu Valley so it is assumed that not all of them have TVs in their rooms. Thus, radio and TV were combined and treated as electronic media, and newspaper as print media. Almost all students were exposed to at least one type of media (either electronic or print). Because of this, the mass media variable was organized into two categories. If a student was exposed to only one type of media, it was considered as low exposure; if a student was exposed to both print and electronic media, it was considered as high exposure.
Similarly, some ethnic groups in Nepal offer alcohol to the gods in religious ceremonies, and people have to consume alcohol in these ceremonies. Therefore, this variable was categorized into two: (1) never/rarely consume alcohol and (2) frequently consume alcohol (two or three times a week).
The variable, "living arrangement", is also organized into two categories: those students who live with their family members were considered as "with biological family"; those who live away from family members were considered as "without biological family". The other independent variables were categorized in the same way.
Methods of analysis
All completed survey questionnaires were entered into a database after manual coding and validation. Data entry and validity checks were performed for all the questionnaires by using the computer software programme, dBase IV. The cleaned and validated data was transferred into the SPSS software programme for further processing and analysis.
Both bivariate and multivariate techniques were applied in the analysis. The Chi-square test was used to test the association between the variables. Those variables that were significant in the bivariate analysis were further reexamined in the multivariate analysis (binary logistic regression) in order to identify the significant predictors after controlling for other variables. Before the multivariate analysis, multicollinearity among variables was assessed, and the least important variable, which was highly correlated to other variables, was removed from the logistic model.
Results
Characteristics of the respondents
Among the students, about two in five (39%) (47% boys; 28% girls) had sexual intercourse irrespective of their marital status. Among these sexually active students (n = 428), around 27% were aged 15 to 19. Eleven percent reported that they had had sex before the age of 16. A large majority of the sexually active students were from outside of the Kathmandu Valley. More than a third (34%) of the students consumed alcohol frequently (two or three times a week). Almost half of the students resided with their biological families. Almost all students were exposed to at least one type of mass media (either electronic or print). Furthermore, both male and female college students were generally aware of HIV/AIDS and knew of at least one mode of transmission of HIV/AIDS (data not shown).
Socio-demographics correlate with condom use
Among the sexually active students, just less than half (48%) had used condoms during their first sexual contacts. Table 1 shows the clear association between condom use at first sexual intercourse and different socio-demographic characteristics. Of those who used condoms at their first sex, a significantly higher proportion than their comparison group: were males; were aged 15 to 19; were from the Brahmin and Chhetri communities; had first sex at age 16 or older; were unmarried; had sex with a boyfriend/girlfriend; were from outside the Kathmandu Valley; had high exposure to the mass media; and lived with their biological families (Table 1).
Table 1 Condom use at first sexual intercourse by background characteristics (n = 428)
Condom use
Yes No Percent Number
Sex of the respondents*
Female 40.3 59.7 100.0 159
Male 52.0 48.0 100.0 269
Age group**
15-19 58.6 41.4 100.0 116
20 and above 43.6 56.4 100.0 312
Caste/ethnicity**
Brahmin/Chhetri 53.1 46.9 100.0 243
Other 40.5 59.5 100.0 185
Level of education
Intermediate 52.3 47.7 100.0 88
Undergraduate 47.2 52.8 100.0 233
Graduate degree 44.9 55.1 100.0 107
Age at first sexual intercourse**
Up to 15 years 23.4 76.6 100.0 47
16-19 years 50.3 49.7 100.0 195
20 or more years 51.1 48.9 100.0 186
Marital status***
Married 34.5 65.5 100.0 165
Unmarried 55.9 44.1 100.0 263
Types of first sex partner***
Spouse 28.9 71.1 100.0 121
Boyfriend/girlfriend 57.2 42.8 100.0 180
Commercial sex worker 52.0 48.0 100.0 127
Permanent place of residence*
Outside Kathmandu Valley 49.5 50.5 100.0 378
Kathmandu Valley 34.0 66.0 100.0 50
Alcohol consumption*
Never/rarely consumed 52.7 47.3 100.0 283
Frequently consumed 37.9 62.1 100.0 145
Mass media exposure*
Low exposure 44.5 55.5 100.0 290
High exposure (both print and electronic media) 54.3 45.7 100.0 138
Living arrangement
With biological family 50.9 49.1 100.0 224
Without biological family (friends/alone) 44.1 55.9 100.0 204
Total 47.7 52.3 100.0 428
Note: *** = p < 0.001 ** = p < 0.01 * = p < 0.5
Binary logistic regression analysis was used to measure the strength of the association between various independent variables and the probabilities of using condoms at first sex. Only those variables that had significant association in bivariate analysis were reassessed in the logistic model. Before the multivariate analysis, multicollinearity among the variables was assessed. It was found that the variables, "marital status" and "types of first sex partner", were highly correlated (r = 0.7). Therefore, the variable, "marital status", was not included in the logistic model.
Analysis from logistic regression showed that age group, caste and/or ethnicity, age at first sexual intercourse, types of first sex partner, alcohol consumption, and mass media exposure were significant predictors for condom use at first sexual intercourse. Students aged 20 or older were less likely to use condoms (OR = 0.40) than students aged 15 to 19. Similarly, students from castes other than Brahmin and Chhetri were 40% (OR = 0.60) less likely to use condoms during their first sexual intercourses than those from other castes and ethnic groups. Furthermore, students who had sexual intercourse for the first time in the age groups of 16 to 19 and 20 or older were almost four times (OR = 3.5) and nine times (OR = 8.9) more likely, respectively, than students who had first sex before 16 years to use condoms at first sexual intercourse (Table 2).
Table 2 Adjusted odds ratio (OR) and 95% confidence interval (CI) for using condoms at first sexual intercourse by selected predictors
Selected predictors OR 95% CI
Sex of the respondents
Female (ref.) 1.00
Male 1.34 0.81-2.21
Age group
15-19 (ref.) 1.00
20 and above 0.40** 0.23-0.71
Caste/ethnicity
Brahmin/Chhetri (ref.) 1.00
Other 0.60* 0.39-0.94
Age at first sexual intercourse
Up to 15 years (ref.) 1.00
16-19 years 3.53** 1.58-7.90
20 or more years 8.96*** 3.75-21.42
Types of first sex partner
Spouse (ref.) 1.00
Boyfriend/Girlfriend 4.52*** 2.48-8.22
Commercial sex worker 4.98*** 2.54-9.74
Permanent place of residence
Outside Kathmandu valley (ref.) 1.00
Kathmandu valley 0.64 0.32-1.30
Alcohol consumption*
Never/rarely consumed (ref.) 1.00
Frequently consumed 0.46** 0.28-0.75
Mass media exposure
Low exposure (ref.) 1.00
High exposure (both print and electronic media) 1.75* 1.08-2.83
2 Log likelihood 504.68
Cox & Snell R Square 0.185
Note: *** = p < 0.001 ** = p < 0.01 * = p < 0.5
Those students who had first sex with a commercial sex worker were about five times more likely than those who had first sex with their spouse to use condoms during first sexual intercourse. An inverse relationship was observed between alcohol consumption and condom use. Those students who had frequently consumed alcohol were 54% (OR = 0.46) less likely to use condoms at first sexual intercourse than those who did not consume alcohol. On the other hand, students who had higher exposure to both print and electronic media were about twice (OR = 1.75) as likely as who had lower media exposure to use condoms during their first sexual intercourses (Table 2).
Discussion
This study shows that condom use at first sexual intercourse is low among college students in Kathmandu, Nepal. If condom use at first sex remains low, vulnerable sexual networks will grow among the students, allowing faster spreading of sexually transmitted diseases and HIV. Our study found that condom use at first sexual intercourse varied according to different criteria. Age group, caste and/or ethnicity, age at first sexual intercourse, types of first sex partner, alcohol consumption and mass media exposure were significant predictors for using condoms at first sexual intercourse.
The study also found that students aged 15 to 19 were more likely to use condoms during first sexual intercourse compared with students aged 20 or older. Results from this study are consistent with those of many other studies [18,23,24]. The finding of this study was also consistent with that of other literature: that later sexual intercourse has also been associated with increased use of condoms [25-27].
Many other studies have found significant differences in condom use according to the type of relationship with sex partners, defined as casual or fixed/steady [44-46]. Our study also supported the finding of other studies that the level of condom use is higher in those who had first sex with casual or non-steady partners than in those who had first sex with spouses or steady partners. Regarding alcohol consumption, results from this study are similar to most other studies: alcohol use contributes to a lower use of condoms and elevated rates of sexual risk [28-31].
Mass media exposure is another important predictor for condom use at first sexual intercourse. Those students who were exposed to both electronic and print media were more likely to use condoms than those who had low exposure to these media. This finding is similar to that of other studies [35,36]. Diffusion theorists postulate that the mass media effect contraceptive use by stimulating partners to discuss contraceptive use [47]. Through the sharing of information and mutual feedback, people give meaning to information, understand each other's views and influence each other [48]. Thus, discussion of contraceptive use leads to the development of a better understanding between partners of their reproductive health goals.
There are some limitations to this study. First, because of the cross-sectional design of the study and the nature of the items used in the logistic regression analysis, the analysis can only provide evidence of statistical association between those items and condom use at first sex and can not show cause-effect relationships. Second, all measures were self-reported. Thus, responses may have been biased by recall errors or intentional misreporting of behaviour. However, the privacy conditions around the study and the use of self-administered questionnaires are likely to have minimized purposeful misreporting.
Conclusions
The rate of condom use at first sexual intercourse is low among the students. It indicates students are exposed to health hazards through their sexual behaviour. If low use of condoms at first sex continues, vulnerable sexual networks will increase among the students, allowing more growth in the spread of sexually transmitted diseases and HIV.
Our study showed a positive effect of the mass media on condom use at first sex; information about condom use and sexual risk behaviour, including unsafe sex, should be provided through the mass media. Similarly, students who had sex at a later age were more likely to use condoms at first sex. Therefore, awareness programmes that encourage postponement of sexual debuts could benefit the students. Findings from this study point to areas that policy and programmes can address to provide youth with access to the kinds of information and services they need to achieve healthy sexual and reproductive lives.
Competing interests
The author declares that they have no competing interests.
Authors' contributions
RA conceived the study and its design, undertook the analysis, and wrote the manuscript.
Acknowledgements
The University Grant Commission, Nepal, provided funding for this study. The author wishes to thank the administrators of all the sampled college in the Kathmandu Valley for their support. He also thanks the students for their participation in the study.
==== Refs
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Asian Spine JASJAsian Spine Journal1976-19021976-7846Korean Society of Spine Surgery 10.4184/asj.2009.3.2.45Basic StudiesMechanical Properties of Blood-Mixed Polymethylmetacrylate in Percutaneous Vertebroplasty Ahn Dong Ki *Lee Song *Choi Dea Jung *Park Soon Yeol *Woo Dae Gon †Kim Chi Hoon †Kim Han Sung †* Department of Orthopedic Surgery, Seoul Sacred Heart General Hospital, Seoul, Korea.† Biomedical Engineering Department, Yonsei University, Wonju, Korea.Corresponding author: Dae Jung Choi, MD. Department of Orthopedic Surgery, Seoul Sacred Heart General Hospital, 40-12 Chungryangri-dong, Dongdaemoon-gu, Seoul 130-011, Korea. Tel: +82-2-968-2394, Fax: +82-2-966-1616, [email protected] 2009 31 12 2009 3 2 45 52 27 7 2009 06 10 2009 06 10 2009 Copyright © 2009 by Korean Society of Spine Surgery2009This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Study Design
Mechanical study of polymethylmetacrylate (PMMA) mixed with blood as a filler.
Purpose
An attempt was made to modify the properties of PMMA to make it more suitable for percutaneous vertebroplasty (PVP).
Overview of Literature
The expected mechanical changes by adding a filler into PMMA included decreasing the Young's modulus, polymerization temperature and setting time. These changes in PMMA were considered to be more suitable and adaptable conditions in PVP for an osteoporotic vertebral compression fracture.
Methods
Porous PMMA were produced by mixing 2 ml (B2), 4 ml (B4) and 6 ml (B6) of blood as a filler with 20 g of regular PMMA. The mechanical properties were examined and compared with regular PMMA(R) in view of the Young's modulus, polymerization temperature, setting time and optimal passing-time within an injectable viscosity (20-50 N-needed) through a 2.8 mm-diameter cement-filler tube. The porosity was examined using microcomputed tomography.
Results
The Young's modulus decreased from 919.5 MPa (R) to 701.0 MPa (B2), 693.5 Mpa (B4), and 545.6 MPa (B6). The polymerization temperature decreased from 74.2℃ (R) to 59.8℃ (B2), 54.2℃ (B4) and 47.5℃ (B6). The setting time decreased from 1,065 seconds (R) to 624 seconds (B2), 678 seconds (B4), and 606 seconds (B6), and the optimal passing-time decreased from 75.6 seconds (R) to 46.6 seconds (B2), 65.0 seconds (B4), and 79.0 seconds (B6). The porosity increased from 4.2% (R) to 27.6% (B2), 27.5% (B4) and 29.5% (B6). A homogenous microstructure with very fine pores was observed in all blood-mixed PMMAs.
Conclusions
Blood is an excellent filler for PMMA. Group B6 showed more suitable mechanical properties, including a lower elastic modulus due to the higher porosity, less heating and retarded optimal passing-time by the serum barrier, which reduced the level of friction between PMMA and a cement-filler tube.
FillerBone cementVertebroplastyOsteoporotic verterbral compression fracture
==== Body
Introduction
Vertebroplasty is a popular treatment for osteoporotic vertebral compression fractures, and various preventive strategies have been introduced to reduce the number and severity of complications. Bone cement leakage and embolism can occur when a lower viscosity cement is infused through a cement filling tube with a relatively small diameter1,2. The cement that leaks into the spinal canal or neural canal can cause stenosis, and a high polymerization temperature can cause severe thermal injury to the nerve tissue3,4. In addition, the vertebral body strengthened with the cement itself might induce an adjacent vertebral compression fracture5-7. To reduce these complications from vertebroplasty, there have been several reports on the ideal mechanical properties of the cement for vertebroplasty, including methods for infusing the cement with high viscosity and the use of cement with less strength and a lower polymerization temperature8-10. The porosity generated by filler materials may allow the release of antibiotics loaded in cement more easily but their Young's modulus should be decreased. On the other hand, the cement must be beneficial in vertebroplasty and have other modified properties, including a reduced setting time due to the faster polymerization time and the lower polymerization temperature10-12. This study examined whether mixing blood into cement can make the mechanical properties more suitable to vertebroplasty to reduce the number of complications.
Materials and Methods
1. Materials and mixing methods
Exolent Spine (Elmdown Ltd., London, UK) contained 20 g (20 ml) of polymer and 9.2 g (8 ml) of monomer per one pack. The specimens were mixed in a operating room with a room temperature of 18℃ and 21% humidity with the cement preserved at room temperature for more than 24 hours. Six specimens were made in each group including the regular cement group (R) and blood-mixed groups (B2, B4, B6), which contained separately 2 ml, 4 ml, and 6 ml of blood in a single pack of cement, respectively. One researcher gave blood, which had a hemoglobin and hematocrit level of 16.7 mg/dl and 34.8%, respectively. It was sampled immediately before being mixed with the cement and should be mixed within a short time (about 1 minute after sampling) prior to hematoma formation. Cement was mixed evenly at a 2 Hz speed for approximately 45 seconds and more 15 seconds after mixing the blood. The mixed cement was transferred into a 20 ml regular syringe and filled into a stainless mold with a 10 mm inner diameter and a 30 mm height to make one block for the Young's modulus, and into three filler tubes with a 2.8 mm inner diameter and 215 mm height to measure the optimal passing-time. The cement remaining in the syringe was transferred into a bowel to measure the polymerization temperature and setting time. The cement block was easily separated by light impact with a rod after 24 hours when the cement specimen had hardened completely. The height and diameter was manipulated accurately with sandpaper and inspected with Venier Calipers (Mitutoyo, Kawasaki, Japan) with 0.05 mm accuracy. The same procedures were repeated six times to make six samples and collect the data in each group.
2. Study methods
The Young's modulus was measured using an Instron Micro-test system (Instron, Norwood, MA, USA) under a compressive speed and load of 5 mm/minutes and 2 kN, respectively. The Young's modulus was defined as the slope of the stress-strain curve. The displacement by load was measured at 20 Hz and the data collected was analyzed using Bluehill 2 software (Instron). The polymerization temperature reached a maximum after mixing the polymer and monomer, and was measured using an Infrared Thermometer (AR852B; ARCO, Guangzhou, China) under the condition of 0.9 emissivity and within 10 mm from the specimen for the optimal optic-distance ratio. The setting time was defined as the interval time from a mixing-start point to a point reaching polymerization temperature12. The optimal passing time was defined as the possible interval time for the cement with an optimal viscosity to pass through a filler tube with a inner diameter of 2.8 mm using a 20-50 N hand pushing pressure. The cement viscosity that could be passed by less than 20 N hand pressure showed more running fluid character, and the cement viscosity that could be passed with 20-50 N showed sticky, tenacious and malleable properties. The hardened cement that required more than 60 N hand pressure could not be passed through the cement filler tube and fixed to the pusher within the tube. Considering these properties as cement hardened, the optimal passing-time was determined to be the interval showing a certain viscosity equal to the 20-50 N hand pressure. The pressure passing viscous fluid through a tube was determined to be the viscosity coefficient and diameter of the tube8. A larger diameter tube has been already used for higher viscosity cement in order to allow easy passing clinically. Therefore, this study used a tube with a 2.8 mm inner diameter and 215 mm length, which is generally used for kyphoplasty. A zig was required to connect the filler tube and a digital pressure gauge, FGP-5 (NIDEC-SHIMPO Corporation, Kyoto, Japan) tightly and avoid unnecessary resistant pressure evoked by a sagged axis. The minimal pressure required to pass the cement through the tube was measured (Fig. 1). The porosity of the cement specimens was measured using SkyScan 1076 Micro-CT System (Skyscan, Aartselaar, Begium). The data was analyzed using the SPSS 15.0 (SPSS Inc., Chicago, IL, USA). The Young's modulus, polymerization temperature, setting time, optimal passing-time and porosity were compared statistically using a one-way ANOVA test to test the null hypothesis, and a Tukey's-b test with a Post Hoc multiple comparison test was used to identify differences between the groups.
Results
The Young's modulus of group R, B2, B4 and B6 was 919.5±148.2 MPa, 701.0±76.1 MPa, 693.5±104.1 MPa, and 545.6±93.1 MPa, respectively. The Young's moduli of the Blood-mixed polymethylmetacrylate (PMMA) groups were significantly lower than that of group R. The more blood-mixed PMMA showed a lower modulus but the difference between the groups was not significant according to the Post Hoc multiple comparison test (Table 1). The polymerization temperature of group R, B2, B4 and B6 was 74.2±2.6℃, 59.8±2.2℃, 54.2±1.6℃, and 47.5±1.0℃, respectively. The blood-mixed PMMA groups showed a significantly lower polymerization temperature than that of group R. In addition, the polymerization temperature and setting time decreased with increasing amount of blood.
The setting time of the group R, B2, B4 and B6 was 1065±15 seconds, 624±8 seconds, 678±3 seconds, and 606±8 seconds, respectively. The optimal passing-time of group R, B2, B4 and B6 was 75.6±2.6 seconds, 46.6±2.3 seconds, 65.0±6.1 seconds, and 79.0±4.2 seconds, respectively. The optimal passing-time increased with increasing amount of mixed blood but the setting time decreased. The optimal passing-time of B2 was almost the same as that of group R in the Post Hoc test.
The porosity of group R, B2, B4 and B6 in micro-CT was found to be 4.2±0.6%, 27.6±1.7%, 27.5±1.4% and 29.5±1.6%, respectively. The blood-mixed PMMA groups showed similar porosity regardless of the amount of mixed blood according to the Post Hoc test. Micro-CT revealed micropores distributed evenly in the blood-mixed PMMA groups (Fig. 2).
Discussion
Vertebroplasty with PMMA for osteoporotic vertebral compression fracture is considered to be very successful in that stabilization of fractured osteoporotic vertebral body and dramatic pain relief. However, with the increasing use of the procedure, many complications have been reported. One of them was adjacent vertebral compression fractures, which might be caused by outward factors including stress transferred from a treated vertebra with PMMA, poor sagittal balance and compensation of the upper body shifting after pain relief, intradiscal cement leakage, cement configuration and an infused volume in the vertebral body as well as an inward resistant factor, i.e. bone density6,7,13-17. The strength of PMMA was eight to forty times higher than that of the osteoporotic vertebra. Therefore, harder PMMA in the verterbral body could cause more stress transfer to the surrounding cancellous bone, causing continuous refracturing of a treated body or into the adjacent vertebral endplate, resulting in an additional vertebral fracture5,18. Lower modulus PMMA or PMMA substitutes are recommended to reduce the newly increasing stress transfer after vertebroplasty19,20. The optimal modulus of PMMA for vertebroplasty cannot be determined due to the combined mechanical effects of the variety of causative factors on the adjacent vertebral fractures. However, considering the strength of PMMA only, a sufficient volume of PMMA supporting both the upper and lower endplates in the vertebral body was reported to increase the level of stress transfer by 670% and 200% using 3,000 MPa and 100 MPa PMMA, respectively21. Therefore, the use of commercially available PMMA with a modulus of 1,000 MPa for verterboplasty can cause approximately 200-400% stress transfer. In addition, the modulus of osteoporotic vertebral body was considered in stress resistance. The elastic modulus of the osteoporotic vertebral body was reported to be 34 MPa, 804 MPa, and 670 MPa in cancellous bone, cortical bone and endplate, respectively22, and the early recollapse rate was reported to be 16% in vertebroplasty with lower modulus substitutes, such as calcium phosphate cement23. The optimal modulus of PMMA was the modulus near the vertebral body to prevent a treated veterbral body from refracture and reduce the increase of stress transfer. The polymer to monomer ratio was changed to reduce the modulus of PMMA by 19.5% from 2,210 MPa to 1,780 MPa, but could not reach the clinical requirements11. Fifty percent mixing with a radiopaque material could decrease the modulus by 95.7%, from 2,800 MPa to 120 MPa, but the result was also ineffective clinically because PMMA with such a low modulus could not recover the strength of a treated vertebra to the preinjured level24. These results showed that 4 ml and 6 ml blood mixed PMMA showed a modulus of 701 MPa and 545.6 MPa, respectively, which is similar to the modulus of cortical and endplate in an osteoporotic vertebral body.
The moduli and porosities of the blood-mixed PMMAs were unaffected by the volume of blood. The lack on an increase in porosity with increasing blood volume was due to the previous mixing of the polymer and monomer for 45 seconds, which might already produce a certain level of polymerization, and the mixing methods with the hands in which there could be a limitation of flourishing porosity over a certain point. The early mixing of blood and PMMA might allow considerable porosity to the blood-mixed PMMA, but more porous PMMA was not required. Overplus blood, on the other hand, which did not be mixed with PMMA, was shifted to the surface of PMMA and produced a larger plasma barrier on the PMMA specimen.
Porosity caused by the mixing of a filler can reduce the modulus of PMMA. A filler which is available in verterbroplasty to endow porosity, should have high viscosity, easy resorption and release from PMMA, water solubility, biocompatibility and biodegradability10. Boger et al.10 reported that when a pack of PMMA was mixed with sodium hyaluronate at a 35% (13 ml) volume ratio, it resulted in a 56% porosity, a decrease in modulus and polymerization temperature from 1,837 MPa to 477 MPa and from 68℃ to 41℃, respectively, which could prevent thermal injury to the surrounding soft tissues. A very small amount of sodium hyaluronate is already used in a bone substitute product. However, such a large volume of sodium hyaluronate might cause an embolism in the blood. Hence, the safety cannot be guaranteed except for topical use in those regions from which sodium hyaluronate could not be resorbed directly into the blood25.
A filler and additive mixed with PMMA can reduce the polymerization temperature. A filler might produce pores in PMMA, which can allow the polymerization heat to dissipate quickly, and an additive may induce a new polymerization reaction with a lower heat of reaction. One of the theories of pain relief by verterbroplasty was the thermal necrosis of the peripheral endings of the nerve fibers in the vertebral body. However, calcium phosphate cement with no exothermic effect can show similar pain relief. On the other hand, a microscopic study showed that micronecrosis around PMMA after verterbroplasty was due to a resorption process rather than to a foreign body reaction caused by PMMA reaction heating or radiopaque barium26,27. The central temperature of PMMA in polymerization reached 49-112℃, and the duration in which the temperature exceeded 50℃ lasted for almost 8 minutes, which might be enough to injure the nerve tissue. If PMMA with the emission of significant reaction heat leaked into the spinal canal, heat transfer could be mostly intercepted by the continuous flow of the cerebrospinal fluid (CSF), but the mass effect could provoke stenosing neurologic symptoms. However, it would cause thermal injury to the nerve tissue if it leaked into the neural foramen and bordered on a root ganglion without the flow of CSF. Therefore, the reaction heat in the polymerization of PMMA should be lower3,4,28,29. In the case of blood-mixed PMMA, the polymerization temperature was as high as 47.5-54.2℃, which is not believed to be enough to prevent thermal injury but the temperature may be decreased easily to a safe level on surrounding tissues in a shorter time than that of regular PMMA.
According to the Hagen-Poiseuille law, the pressure required in passing liquid PMMA through a filler tube is dependent on the viscosity of the PMMA in direct proportion8.
The setting time decreased in the blood-mixed PMMA, which is similar to the same results of various filler-mixed PMMAs. A decrease in setting times means that the viscosity is elevated faster and PMMA hardens earlier. Viscous PMMA should be passed through a filler tube of a smaller diameter during verterbroplasty rather than kyphoplasty. If the viscosity is elevated over a certain level and an optimal passing-point is missed, sufficient volume of PMMA could not be infused into the verterbral body and become stuck in the tube. However, the optimal passing-time of blood-mixed PMMA was lengthened in proportion to the volume of blood regardless of the shortened setting time, because of the plasma barrier formed on the surface of the PMMA specimen, which worked effectively as a lubricant between the PMMA and the tube. The optimal passing-time of group B6 was similar to that of group R but the setting time of group B6 was reduced by 43% compared to group R. Hence, the viscosity of group B6 was much higher than that of group R at the same point of infusion, which can clinically reduce the risk of extravasation from a vertebral body or an embolism of PMMA.
Micro-CT revealed micropores which were distributed evenly but not interconnected, so the PMMA and cancellous bone could not be suspected to be conglutinated in the processing of bone union.
In some point of view, bone substitutes were suspected to be replaced for PMMA even in treatment for osteoporotic vertebral compression fractures. Bone substitutes undergo a process of crystallization without an exothermic effect in the body temperature rather than polymerization. In addition, they can be resorbed by osteoclasts followed by the remodeling of host bone, and do not release toxic substances like the monomer in PMMA20,30,31. However, calcium carbonate cement was resorbed very early within two to four months after infusion, making unable to support a collapsed body for sufficient time32, and calcium phosphate cement has a risk of recollapse of the fractured vertebral body due to the very low strength23,33. Calcium sulphate cement can restore the treated vertebral strength to a similar strength through regular PMMA but it has also a risk of refracture of the treated verterbra due to the rapid resorption34,35. It is believed that the advantage of resorption and replacement of calcium phosphate cement by the normal host bone does not exactly mean the recovery of bone density and strength to those of the young and healthy vertebra that would be sufficient to prevent refracture of an osteoporotic vertebral body. In addition, the use of these substitutes carries a risk of embolism, and the disintegration tendency can deteriorate the pulmonary and arterial oxygen tension in the aqueous environment of continuous blood flow in a vertebral body in an experimental study36. Hydroxyapatite is believed to overcome the disadvantages of regular PMMA and calcium phosphate cement. However, its high viscosity can make handling and infusion more difficult and the resorption property can be obscure. Its use is not yet prevalent because of its cost effectiveness37,38.
There were several limitations in this study due to the aims of the study, which was focused on identifying the clinical problems and solutions during performing vertebroplasty, and the measurement criteria and methods were thought to be possibly affected by the subjective opinion by researchers. The optic-distance ratio must be considered when using an infrared thermometer as a noncontact measurement. The recorded temperature would have been lower than the real temperature if the temperature was measured far from the acceptable distance according to optic-distance ratio when the larger measured area was detected by a non-contact measurement method. An infrared thermometer calculates the average temperature over a certain area. The emissivity of PMMA was believed to be 0.8-0.9, which is similar to plaster or brick. Steinless showed lower emissivity. Therefore, the measured temperature would have been much lower than the real polymerization temperature if the polymerization temperature was measured on the surface of a steinless mold containing PMMA. Accordingly, more studies using similar instruments and objective methods would be done to overcome these limitations and more advisable and objective methods could be commented.
Conclusions
Blood was used as a biocompatible filler to modify the properties of bone cement to make it more suitable to vertebroplasty by reducing the Young's modulus to that of the osteoporotic vertebral body and lowering the polymerization temperature. The blood-mixed cement is believed to have clinical benefits in vertebroplasty, in that a lower modulus can reduce the level of stress to the adjacent vertebrae. In addition, the lubricant effect of the plasma membrane can allow a high viscosity cement to pass smoothly under the same pressure for passing regular cement to reduce a risk of cement leakage and embolism. Furthermore, a lower polymerization temperature might also reduce a risk of thermal injury to the nerve tissue.
Fig. 1 Methods for measuring the pressure-related manipulation time. A cement filler with a 2.8 mm diameter was connected to FGP-5. A jig can hold a cement filler tightly along the parallel axis to FGP-5 to prevent unexpected resistant pressure during cement-pushing.
Fig. 2 Micro-configuration on micro-CT. Blood-mixed polymethylmetacrylate's (2B, 4B and 6B) showed homogenous structures with fine regular pores.
Table 1 Homogenous subsets by Tukey B test
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2039638209-PONE-RA-10669R210.1371/journal.pone.0010085Research ArticleCardiovascular Disorders/MyopathiesDiabetes and Endocrinology/ObesityGeriatrics/Geriatric CardiologyInteraction between Age and Obesity on Cardiomyocyte Contractile
Function: Role of Leptin and Stress Signaling Obesity, Age, Cardiac FunctionRen Jun
1
2
3
*
Dong Feng
2
Cai Guo-Jun
3
Zhao Peng
2
Nunn Jennifer M.
2
Wold Loren E.
4
Pei Jianming
1
*
1
Department of Physiology, Fourth Military Medical University,
Xi'an, China
2
University of Wyoming College of Health Sciences, Laramie, Wyoming,
United States of America
3
School of Pharmacy, Second Military Medical University, Shanghai,
China
4
Center for Cardiovascular and Pulmonary Research, The Research Institute
at Nationwide Children's Hospital and the Department of Pediatrics, The
Ohio State University, Columbus, Ohio, United States of America
Calbet Jose A. L. EditorUniversity of Las Palmas de Gran Canaria, Spain* E-mail: [email protected] (JR); [email protected] (JP)Conceived and designed the experiments: JR FD GJC JP. Performed the
experiments: JR FD PZ JMN. Analyzed the data: JR FD GJC PZ JMN. Wrote the
paper: JR LEW. Conducted the statistical analysis: JR FD.
2010 9 4 2010 5 4 e1008528 5 2009 17 3 2010 Ren et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Objectives
This study was designed to evaluate the interaction between aging and obesity
on cardiac contractile and intracellular Ca2+
properties.
Methods
Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the
leptin deficient ob/ob obese mice were treated with leptin
(0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet
(45% calorie from fat) and the leptin receptor mutant
db/db obesity models at young and older age were used
for comparison. Cardiomyocyte contractile and intracellular
Ca2+ properties were evaluated including peak
shortening (PS), maximal velocity of shortening/relengthening (±
dL/dt), time-to-PS (TPS), time-to-90% relengthening
(TR90), intracellular Ca2+ levels and
decay. O2
− levels were measured by
dihydroethidium fluorescence.
Results
Our results revealed reduced survival in ob/ob mice. Aging
and obesity reduced PS, ± dL/dt, intracellular
Ca2+ rise, prolonged TR90 and
intracellular Ca2+ decay, enhanced
O2
− production and
p
47phox expression
without an additive effect of the two, with the exception of intracellular
Ca2+ rise. Western blot analysis exhibited reduced
Ob-R expression and STAT-3 phosphorylation in both young and aging
ob/ob mice, which was restored by leptin. Aging and
obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK
and pIκB. Low levels of leptin reconciled contractile, intracellular
Ca2+ and cell signaling defects as well as
O2
− production and
p
47phox upregulation in
young but not aging ob/ob mice. High level of leptin (50
nM) compromised contractile and intracellular Ca2+
response as well as O2
− production and
stress signaling in all groups. High fat diet-induced and
db/db obesity displayed somewhat comparable
aging-induced mechanical but not leptin response.
Conclusions
Taken together, our data suggest that aging and obesity compromise cardiac
contractile function possibly via phosphorylation of Akt, eNOS and stress
signaling-associated O2
− release.
==== Body
Introduction
Obesity is a devastating health problem afflicting all ages, races and socioeconomic
classes in both genders. Over the past decade, only modest success has been achieved
in combating the escalating prevalence of obesity and metabolic syndrome [1], [2]. The
current obesity epidemic may be attributed to many factors including environmental
(e.g., caloric and nutrient intake), genetic and even evolutionary (e.g.,
interaction between human biology and human culture over the long period of human
evolution) [1], [3]. With
today's prolonged human lifespan, aging has also been considered as an
obesogenic factor given the increased visceral fat associated with aging [4].
Paradoxically, visceral fat accumulation may in turn influence longevity, thus
prompting the speculation that obesity could be a condition of premature aging [4]. Although
effective physiological adjustments are present to counterbalance the potentially
detrimental health outcome of obesity such as altered respiratory
mechanical/muscular function peculiar to the aging condition [5], a number of
obesity-associated comorbidities such as cancer, endocrine, cardiovascular and
immune disorders may ultimately contribute to premature aging and the shortened
lifespan. Therefore, the concept of health promotion, especially on nutrition and
life style, has become an important part of health care in older adults [6].
Among a wide array of comorbidities associated with obesity including type 2
diabetes, hypertension, cancer and sleep apnea [1], heart disease, which is
mainly manifested by cardiac hypertrophy and compromised ventricular function, may
lead to heart failure or premature death [7]–[9]. The
pathophysiological alterations associated with establishing and perpetuating
obesity-induced heart disease are complex but are becoming more clear, including the
interaction of sympathetic overactivation and endothelial dysfunction [10]. In an
effort to better understand the pathophysiology of human obesity, several rodent
models of obesity have been developed and implemented including high fat diet
feeding and spontaneous mutants of the 16 KD obesity gene product leptin or its
receptor such as ob/ob and
db/db mice. A common feature of these obese
animal models is the overtly compromised cardiac contractile function associated
with a marked increase in visceral fat and hyperinsulinemia [11]–[14], similar
to human obesity. Accumulating evidence has also implicated a role of the obese gene
product leptin, which regulates food intake and energy expenditure, in the
regulation of cardiac function, while the disruption of which contributes to
obesity-associated cardiac contractile and morphometric defects [15], [16]. Human
circulating leptin levels are elevated in obesity, vascular and coronary heart
diseases, favoring a contemporary perception of hyperleptinemia being an independent
risk factor for cardiovascular diseases [16], [17]. This notion is further
supported by the experimental evidence that leptin may contribute to cardiac
hypertrophy, atherosclerosis and thrombosis possibly through accumulation of
reactive oxygen species [16], [18], [19]. Elevated leptin level or
hyperleptinemia is correlated with hyperphagia, insulin resistance, hyperlipidemia
and hypertension, independent of total adiposity [16]. Data from our lab
revealed that leptin directly suppresses cardiomyocyte contraction and intracellular
Ca2+ handling through mechanism(s) related to endothelial
nitric oxide synthase (eNOS), superoxide (O2
−)
production, activation of Janus kinase (JAK)/signal transducer and activator of
transcription (STAT) and stress signaling pathways including Jun N-terminal kinase
(JNK) and p38 mitogen-activated protein (MAP) kinase [20]–[22]. Further
evidence from our lab as well as others also indicated that leptin deficiency
paradoxically triggers cardiac hypertrophy and contractile dysfunction in
ob/ob obese mice with a mutant leptin gene, the effect of which
is reconciled by leptin supplementation [12], [15]. Both hyperleptinemia
and leptin-deficiency have been shown to be associated with increased apoptosis, DNA
damage and mortality, suggesting a potential association between leptin signaling
and aging-related DNA damage and premature death [23]. Nevertheless, the
interaction between obesity and aging on cardiac function, with a focus on leptin
signaling, has not been elaborated. Given the prevalence of metabolic syndrome in
older adults and the detrimental impact of metabolic syndrome especially obesity on
life expectancy and comorbidity in the elderly [24], the present study was
designed to evaluate the influence of aging on basal and leptin-elicited cardiac
contractile response in the leptin-deficient ob/ob mice. Expression
and activation of the leptin receptor Ob-R and post-receptor signaling STAT-3,
O2
− producing enzyme NADPH oxidase
(p
47phox subunit) [25], Akt,
eNOS, AMP-activated kinase (AMPK) and the stress signaling molecules p38 MAP kinase,
JNK, extracellular signaling regulated kinase (ERK) and NFκB were also
examined in young and aging lean C57 and ob/ob leptin deficient
obese mice. Twelve- and eighteen-month-old mice were chosen for the aging group
largely due to the reduced lifespan and high mortality seen after one year of age in
ob/ob mice [26]. Cardiomyocyte contractile function was also
examined in the high fat diet-induced and the leptin receptor mutant hyperleptinemic
db/db obesity models for comparison.
Materials and Methods
Experimental animals and high fat diet feeding
All animal procedures were conducted in accordance with humane animal care
standards outlined in NIH Guide for the Care and Use of Experimental and were
approved by the University of Wyoming and University of North Dakota Animal Care
and Use Committees. In brief, young (4-month-old) and aging (12- or
18-month-old) male homozygous B6.V-lep<ob>/J leptin deficient
ob/ob and B6.Cg-m +/+ Leprdb/J leptin
receptor mutant db/db obese mice were housed in our
institutional animal facilities. Age- and gender-matched wild-type C57BL/6J mice
were used as lean controls. All animals were allowed free access to standard lab
chow and tap water. For high fat diet-induced obesity model, 4- and 12-month-old
male C57BL/6J mice (4 per group) were randomly assigned to a low fat
(10% of total calorie) or a high fat (45% of total
calorie) diet (Research Diets Inc., New Brunswick, NJ, USA) for 16 weeks [13].
Blood glucose was monitored with a glucometer (Accu-ChekII, model 792,
Boehringer Mannheim Diagnostics, Indianapolis, IN, USA). All mice used for
lifespan analysis (the Kaplan-Meier survival curve and log-rank test) were
assigned to a longevity cohort at birth and were not used for any biochemical,
immunoblotting or mechanical function tests. Only male mice were used for this
study.
Body fat composition measurement
Body composition was measured using Dual Energy X-ray Absorptiometry (DEXA),
which is a clinical measure of lean tissue mass, adipose tissue mass, and bone
mineral mass and density. A low level pencil-beam x-ray moved transversely from
the head to the tail across the sedated mouse. Difference in absorbance of the
X-ray was detected according to tissue density. Percent fat was calculated using
fat and body mass [27].
Cardiomyocyte isolation and in vitro leptin
treatment
Mouse hearts were removed under anesthesia (ketamine/xylazine at 3∶1,
1.32 mg/kg) and were perfused with oxygenated (5%
CO2–95% O2) Krebs-Henseleit
bicarbonate (KHB) buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 1.25
CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25
NaHCO3, 10 HEPES, and 11.1 glucose. Hearts were perfused with a
Ca2+-free KHB containing Liberase Blendzyme 4
(Hoffmann-La Roche Inc., Indianapolis, IN, USA) for 20 min. After perfusion,
left ventricles were removed and minced to disperse cardiomyocytes in
Ca2+-free KHB buffer. Extracellular
Ca2+ was added incrementally back up to 1.25 mM [12].
Myocyte yield was ∼75% which was not affected by obesity or
age. Cohorts of cardiomyocytes were incubated with leptin (0.5, 1.0 and 50 nM)
for 4 hrs in a serum-free defined medium consisting of Medium 199 (Sigma) with
Earle's salts. The concentrations of leptin were chosen to cover
physiological (0.5 and 1.0 nM) as well as pharmacological levels [16], [22],
[28]. Cardiomyocytes with obvious sarcolemmal
blebs or spontaneous contractions were not used for mechanical recording.
Cell shortening/relengthening
Mechanical properties of cardiomyocytes were assessed using a SoftEdge
MyoCam® system (IonOptix Corporation, Milton, MA, USA) [12]. In
brief, cardiomyocytes were placed in a chamber mounted on the stage of an
inverted microscope (Olympus Incorporation, Model IX-70, Tokyo, Japan) and
superfused at 25°C with a buffer containing (in mM): 131 NaCl, 4 KCl, 1
CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The
cells were field stimulated with suprathreshold voltage (150% of the
threshold voltage of cell contraction) at a frequency of 0.5 Hz, 3 msec
duration, using a pair of platinum wires placed on opposite sides of the chamber
connected to a FHC stimulator (Brunswick, NE, USA). The myocyte being studied
was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix
SoftEdge software was used to capture changes in cell length during shortening
and relengthening. Cell shortening and relengthening were assessed using the
following indices: peak shortening (PS), the amplitude myocytes shortened upon
electrical stimulation, an indicative of peak ventricular contractility;
time-to-PS (TPS), the duration of myocyte shortening, an indicative of systolic
duration; time-to-90% relengthening (TR90), the duration
to reach 90% relengthening, an indicative of diastolic duration
(90% rather 100% relengthening was used to avoid noisy
signal at baseline level); and maximal velocities of shortening/relengthening,
maximal slope (derivative) of shortening and relengthening phases, indicatives
of maximal velocities of ventricular pressure increase/decrease.
Intracellular Ca2+ transient measurement
Intracellular Ca2+ was measured using a dual-excitation,
single-emission photomultiplier system (IonOptix) in myocytes loaded with
Fura-2-AM (0.5 µM). Myocytes were placed on an inverted microscope and
imaged through an Olympus (IX-70) Fluor ×40 oil objective. Myocytes
were exposed to light emitted by a 75-W halogen lamp through either a 360- or
380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence
emissions were detected between 480 and 520 nm by a photomultiplier tube after
initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of
the recording protocol. The 360-nm excitation reading was repeated at the end of
the protocol. Qualitative evaluation of intracellular Ca2+
was inferred from fluorescence intensity changes. Myocyte shortening was also
evaluated in a cohort of the fura-2-loaded ventricular myocytes simultaneously
to compare their temporal relationship with the fluorescence signal. However,
their mechanical properties were not used for data summary due to the apparent
Ca2+ buffering effect of fura-2 [12].
Western blot analysis
Following leptin treatment, cardiomyocytes from young and aging C57 and
ob/ob mice were collected and sonicated in a lysis buffer
containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton, 0.1% SDS and protease inhibitor cocktail. The protein
concentration of the supernatant was evaluated using the protein assay reagent
(Bio-Rad, Hercules, CA, USA). Equal-amounts (30 µg) of protein and
prestained molecular weight marker (GIBCO, Gaithersburg, MD, USA) were loaded
onto 7%∼10% SDS-polyacrylamide gels in a minigel
apparatus (Mini-PROTEAN II, Bio-Rad), separated, and transferred to
nitrocellulose membranes (0.2 µm pore size, Bio-Rad). Membranes were
incubated for 1 hr in a blocking solution containing 5% nonfat milk
in TBS-T before being washed in TBS-T. Membranes were then incubated overnight
at 4°C with anti-p47phox (1∶1000, kindly provided by Dr. Mark T. Quinn from Montana
State University, Bozeman, MT), anti-p38 MAP kinase and anti-phospho-p38 MAP
kinase (pp38, 1∶1,000), anti-ERK (1∶1,000;),
anti-phospho-ERK (pERK; 1∶1,000), anti-SAPK/JNK (1∶1,000),
anti-phospho-SAPK/JNK (pJNK, 1∶1,000), anti-IκB
(1∶1,000), anti-phospho-IκB (pIκB, 1∶1,000),
anti-Akt (1∶1,000), anti-phospho-Akt (pAkt, 1∶1,000),
anti-eNOS, anti-phospho-eNOS (peNOS, 1∶1000), anti-STAT3
(1∶1,000), anti-phospho-STAT3 (pSTAT3, 1∶1,000), anti-Ob-R
(long form isoform Ob-Rb, 1∶1000), anti-AMPK (1∶1000) and
anti-phospho-AMPK (pAMPK,1∶1000) antibodies. GAPDH was used as the
internal loading control (1∶5,000). All antibodies were obtained from
Cell Signaling Technology (Beverly, MA, USA) or Santa Cruz Biotechnology (Santa
Cruz, CA, USA) unless otherwise specified. After incubation with the primary
antibodies, blots were incubated with either anti-mouse or anti-rabbit IgG
horseradish peroxidase-linked antibodies at a dilution of 1∶5,000 for
60 min at room temperature. Immunoreactive bands were detected using the Super
Signal West Dura Extended Duration Substrate (Pierce, Milwaukee, WI). Intensity
of the bands was measured with a scanning densitometer (model GS-800; Bio-Rad)
coupled with Bio-Rad personal computer analysis software.
Intracellular fluorescence measurement of superoxide
(O2
−)
Intracellular superoxide were monitored by changes in fluorescence intensity
resulting from intracellular probe oxidation according to a previously described
method [22]. Following leptin (0.5, 1.0 and 50 nM)
treatment, cardiomyocytes from young and aging C57 lean and
ob/ob mice were loaded with 5 µM dihydroethidium
(DHE) (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C and washed
with PBS buffer. Cells were sampled randomly using an Olympus BX-51 microscope
equipped with an Olympus MagnaFire™ SP digital camera and ImagePro
image analysis software (Media Cybernetics, Silver Spring, MD). Fluorescence was
calibrated with InSpeck microspheres (Molecular Probes). More than 150 cells per
group were evaluated using the grid crossing method for cell selection in more
than 15 visual fields per experiment.
Statistical analysis
Data are presented as mean ± SEM. Statistical significance
(p<0.05) was determined by a one- or two-way analysis of variance (ANOVA)
followed by a Tukey's post hoc analysis.
Results
General features of experimental animals
As expected, young ob/ob mice displayed significantly greater
body, heart, liver and kidney weights compared with age-matched young C57 mice.
The organ size (when normalized to tibial length) was also significantly greater
in young ob/ob mice compared with the young C57 group. Body and
organ (except kidney in aging ob/ob mice) weights were
significantly heavier in aging (12-month-old) C57 or ob/ob mice
compared with respective young groups, as expected. Consistently, the organ size
(normalized to tibial length) was significantly greater in the 12-month-old
aging C57 mice compared with young C57 mice. Nonetheless, liver size but not
that of heart and kidney was significantly enhanced with aging in
ob/ob mice. Body fat composition was significantly elevated
with aging and obesity, with an additive effect between the two. There were no
significant differences in tibial length and fasting glucose levels among the
four mouse groups, excluding growth-related factor and the presence of
full-blown diabetes mellitus (Table 1). The 18-month-old C57 and ob/ob mice
displayed a comparable biometric profile somewhat similar to their 12-month-old
counterparts (data not shown). The Kaplan-Meier survival curve comparison
depicts that ob/ob mice display significantly reduced survival
rates when compared to C57 lean mice. The median lifespan was 27.0 and 18.0
months for C57 and ob/ob mice, respectively
(p = 0.0007). Survival curves of the two mouse
groups began to separate after ∼6 months of age with
ob/ob mice exhibiting a greater mortality rate (Fig. 1).
10.1371/journal.pone.0010085.g001Figure 1 Cumulative survival curve (Kaplan-Meier survival plot) of male C57
lean and ob/ob obese mice.
The cumulative survival rate was plotted against age in months. The Log
rank test was performed to compare the two mouse groups
(p = 0.0007).
n = 26 and 16 mice for C57 and
ob/ob mice, respectively.
10.1371/journal.pone.0010085.t001Table 1 General features of young (4-month-old) or aging (12-month-old) lean
C57 and ob/ob mice.
C57-young C57-aging
ob/ob-young
ob/ob-aging
Body Weight (g) 24.0±0.5 29.0±0.5* 54.3±0.9** 63.6±2.9*,**
Heart Weight (mg) 144±3 178±3* 316±5** 350±8*,**
Tibial Length (mm) 16.6±0.2 17.0±0.2 16.5±0.2 17.1±0.3
HW/TL (mg/mm) 8.66±0.19 10.4±0.2* 19.1±0.3** 20.5±0.5**
Liver Weight (g) 1.28±0.08 1.58±0.07* 3.12±0.13** 4.58±0.17*,**
LW/TL (mg/mm) 77.2±4.8 92.5±4.2* 189.3±7.6** 267.8±9.9*,**
Kidney Weight (g) 0.27±0.02 0.37±0.02* 0.46±0.02** 0.49±0.02**
KW/TL (mg/mm) 16.1±0.6 21.8±1.1* 28.1±1.0** 28.9±0.9**
Body Fat Composition (%) 18.2±1.2 26.5±1.2* 69.1±2.1** 78.6±1.7*,**
Blood Glucose (mM) 5.50±0.12 5.60±0.21 5.79±0.13 5.79±0.20
HW = heart weight; LW
= liver weight; KW
= kidney weight; TL
= tibial length; Mean
± SEM, * p<0.05 vs.
corresponding young group, ** p<0.05
vs. corresponding C57 group,
n = 13 and 14 mice for C57 and
ob/ob groups, respectively.
Mechanical and intracellular Ca2+ properties of
cardiomyocytes in ob/ob obesity
Neither obesity nor aging significantly affected the myocyte yield or overall
appearance (Fig. 2). The
resting cell length was significantly greater in young ob/ob
and aging (both 12 and 18 months of age) C57 mice compared with young C57 mice.
Obesity further augmented aging-elicited elongation in cardiomyocyte resting
cell length at both 12 and 18 months of age. Short-term leptin treatment did not
affect resting cell length in either young or aging C57 and
ob/ob mice (Fig. 3A). Both obesity and aging (12 and 18 months) significantly
reduced peak shortening (PS) amplitude and maximal velocity of
shortening/relengthening (± dL/dt), prolonged time-to-90%
relengthening (TR90) without affecting time-to-peak shortening (TPS).
There was little difference in the aging-induced change in mechanical parameters
between 12 and 18 months of age. In addition, there was no discernible
synergistic effect between obesity and age on these mechanical indices. Leptin
supplementation at physiological levels (0.5 and 1.0 nM) effectively nullified
obesity-induced mechanical deficiencies (PS, ± dL/dt and
TR90) in young but not aging (12-month) mouse groups. However,
leptin treatment (0.5 and 1.0 nM) did not alter aging-induced mechanical changes
in PS, ± dL/dt and TR90 (Fig. 3B-3F). Consistent with our previous
report [22], pharmacological level of leptin (50 nM)
overtly impaired cardiomyocyte mechanical function including depressed PS and
± dL/dt as well as prolonged TPS and TR90 in both young
and aging C57 or ob/ob mouse groups (Fig. 3A–3F). To explore the
possible role of intracellular Ca2+ homeostasis in obesity
and/or aging-induced mechanical responses, we evaluated intracellular
Ca2+ transients using the Fura-2 fluorescence
measurement. Our results indicated that both obesity and aging enhanced resting
intracellular Ca2+ levels without any additive effects. The
rise of intracellular Ca2+ in response to electrical stimuli
was significantly reduced by either obesity or aging (12- and 18-month) with an
overt additive effective between the two. Both obesity and aging reduced the
intracellular Ca2+ clearing rate (single and bi-exponential
decay) with no additive effect. Consistent with its effect on cardiomyocyte
shortening, there was little difference in the aging-induced change in
intracellular Ca2+ property between 12 and 18 months of age.
Furthermore, short-term leptin supplementation at physiological levels (0.5 and
1.0 nM) significantly attenuated or ablated intracellular
Ca2+ abnormalities in young but not aging
ob/ob mice. Consistent with its response in cardiomyocyte
shortening, short-term leptin treatment at physiological levels (0.5 and 1.0 nM)
failed to affect aging-induced changes in intracellular Ca2+
handling although pharmacological level of leptin (50 nM) drastically
interrupted cardiomyocyte intracellular Ca2+ homeostasis
including elevated resting intracellular Ca2+ levels,
depressed intracellular Ca2+ rise in response to electrical
stimuli and prolonged intracellular Ca2+ decay in both young
and aging C57 or ob/ob mouse groups (Fig. 4). Given that 12 and 18 months of age
produced reminiscent mechanical changes in C57 lean and ob/ob
mice, 12 months of age was chosen as the only aging group the remaining of
ob/ob study.
10.1371/journal.pone.0010085.g002Figure 2 Light microscopic images of cardiomyocytes freshly isolated from
young (4-month-old) and aging (12- or 18-month-old) lean (C57) and
ob/ob mice.
200x, scale bar = 100 µm.
10.1371/journal.pone.0010085.g003Figure 3 Contractile properties of cardiomyocytes freshly isolated from young
(4-month-old) and aging (12- or 18-month-old) lean (C57) and
ob/ob mice treated with or without leptin (0.5, 1.0
and 50 nM) for 4 hrs.
A: Resting cell length; B: Peak shortening (PS, normalized to cell
length); C: Maximal velocity of shortening (+ dL/dt); D:
Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening
(TPS); F: Time-to-90% relengthening (TR90); Mean
± SEM, n = 50–53
cells from 3 mice per group, * p<0.05
vs. respective C57 group, **
p<0.05 vs. young C57 group, # p<0.05
vs. respective ob/ob group.
10.1371/journal.pone.0010085.g004Figure 4 Intracellular Ca2+ transient properties of
cardiomyocytes freshly isolated from young (4-month-old) and aging (12-
or 18-month-old) lean (C57) and ob/ob mice treated with
or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.
A: Resting intracellular Ca2+ fluorescence intensity;
B: Rise in intracellular Ca2+ fluorescence intensity
in response to electrical stimuli; C: Single-exponential
Ca2+ transient decay rate and D: Bi-exponential
Ca2+ transient decay rate. Mean ±
SEM, n = 36–38 cells from 3
mice per group, * p<0.05 vs. respective
C57 group, ** p<0.05 vs. young
C57 group, # p<0.05 vs. respective
ob/ob group.
Influence of age and ob/ob obesity on
O2
− production and NADPH oxidase
(p47phox subunit) expression
Depending on the level of exposure, leptin is known to elicit a paradoxical
effect on cardiomyocyte contractile function through either inhibition or
stimulation of O2
− production [22,22].
To determine whether O2
− production plays a role
in the disparate leptin effects between young and aging ob/ob
mice, we evaluated O2
− production and expression
of the rate-limiting enzyme for O2
− production
NADPH oxidase (p
47phox subunit)
[25] using DHE fluorescence and Western blot
analysis, respectively. Our data suggested that obesity and aging (12-month)
significantly enhanced O2
− production and
upregulated expression of p
47phox
NADPH oxidase without an additive effect of the two. Leptin supplementation at
physiological levels (0.5 and 1.0 nM) ablated obesity-induced
O2
− production and
p
47phox NADPH oxidase
expression in young but not aging ob/ob mice. Nonetheless,
leptin treatment at 0.5 and 1.0 nM failed to reconcile aging-induced effects on
O2
− production and
p
47phox NADPH oxidase
expression. Consistent with the mechanical and intracellular
Ca2+ response, pharmacological level of leptin (50 nM)
directly enhanced O2
− production and upregulated
expression of p
47phox NADPH oxidase
in both young and aging C57 or ob/ob mouse groups (Fig. 5).
10.1371/journal.pone.0010085.g005Figure 5 O2
− production (Panel A) and
p
47
phox
NADPH oxidase subunit expression (Panel B) measured by DHE
fluorescence and immunoblotting, respectively, in cardiomyocytes
freshly isolated from young (4-month-old) or aging (12-month-old)
lean (C57) and
ob/ob
mice treated with or without leptin (0.5, 1.0 and 50 nM)
for 4 hrs.
Insets: Representative gel blots of
p
47phox NADPH oxidase
subunit using specific
anti-p
47phox
antibody. GAPDH was used as the loading control. Mean ± SEM,
n = 12–14 (Panel A) and
9–11 (Panel B) per group, * p<0.05
vs. respective C57 group, **
p<0.05 vs. young C57 group, # p<0.05
vs. respective ob/ob group.
Influence of age and ob/ob obesity on leptin receptor (Ob-R)
and STAT3 activation
To determine the potential involvement of leptin signaling in obesity and/or
age-induced effects on cardiomyocyte contractile function, intracellular
Ca2+ homeostasis and O2
−
production, we evaluated the leptin receptor Ob-R, the post-leptin receptor
signaling molecule STAT-3 and STAT-3 phosphorylation. Our results shown in Fig. 6 revealed that obesity,
but not aging (12-month), significantly reduced Ob-R protein expression and its
post-receptor signaling STAT-3 phosphorylation without affecting the total
STAT-3 expression. Interestingly, short-term leptin supplementation at both 1.0
nM and 50 nM significantly upregulated Ob-R expression in young but not aging
ob/ob mice and stimulated STAT-3 phosphorylation in both
young and aging ob/ob groups. Leptin treatment at 1.0 and 50 nM
did not alter the expression of Ob-R and STAT-3 in lean mice although the high
but not the low concentration of leptin directly stimulated STAT-3
phosphorylation in lean mice.
10.1371/journal.pone.0010085.g006Figure 6 The leptin receptor Ob-R expression (Panel A) and phosphorylation of
the leptin receptor downstream signaling molecule STAT-3 (pSTAT3, Panel
B) in cardiomyocytes freshly isolated from young (4-month-old) or aging
(12-month-old) lean (C57) and ob/ob mice treated with
or without leptin (1.0 and 50 nM) for 4 hrs.
Protein expression of Ob-R and pSTAT-3 was normalized to the loading
control GAPDH or total STAT-3, respectively. Insets: Representative gel
blots of Ob-R, pSTAT-3 and STAT-3 proteins using specific antibodies.
Mean ± SEM, n = 3
– 6 isolations, * p<0.05 vs.
respective C57 group, # p<0.05 vs. respective
ob/ob group.
Influence of age and ob/ob obesity on Akt, eNOS and
AMPK
A number of signaling molecules have been shown to participate in obesity and
aging-induced biological responses and regulation of cardiac function, including
Akt, the Akt downstream signal eNOS and the cellular fuel AMPK [29], [30]. We examined the expression of Akt, eNOS,
AMPK and their phosphorylation in young and aging (12-month) C57 lean and
ob/ob mouse cardiomyocytes. Our results revealed that
either obesity or age independently dampened the phosphorylation of Akt and its
downstream signaling molecule eNOS without affecting expression of Akt and eNOS.
There was no interaction between obesity and age on the phosphorylation of Akt
and eNOS. Short-term leptin treatment at physiological level (1.0 nM) reconciled
the reduced phosphorylation of Akt and eNOS in young ob/ob, but
not the aging mice. While obesity and age alone failed to affect AMPK and its
phosphorylation, the combination of the two significantly attenuated AMPK
phosphorylation but not total AMPK expression. Short-term leptin treatment at
physiological level (1.0 nM) reduced AMPK phosphorylation in young
ob/ob mice but not other groups. Short-term leptin
treatment at physiological level did not affect the expression of
non-phosphorylated Akt, eNOS and AMPK. Interestingly, short-term treatment of
leptin at pharmacological level (50 nM) significantly suppressed the
phosphorylation of Akt, eNOS and AMPK in all mouse groups (with the exception of
AMPK phosphorylation in aging ob/ob group) without affecting the expression of
non-phosphorylated Akt, eNOS and AMPK (Fig. 7).
10.1371/journal.pone.0010085.g007Figure 7 Panel A: Representative gel blots of total and phosphorylated Akt,
eNOS and AMPK in cardiomyocytes freshly isolated from young
(4-month-old) or aging (12-month-old) lean (C57) and
ob/ob mice treated with or without leptin (1.0 and
50 nM) for 4 hrs using specific antibodies; Panel B: Phosphorylation of
Akt expressed as pAkt-to-Akt ratio; Panel C: Phosphorylation of eNOS
expressed as peNOS-to-eNOS ratio; and Panel D: Phosphorylation of AMPK
expressed as pAMPK-to-AMPK ratio.
Mean ± SEM, n = 4 –
6 isolations, * p<0.05 vs. respective
C57 group, ** p<0.05 vs. young
C57 group, # p<0.05 vs. respective
ob/ob group.
Influence of age and ob/ob obesity on p38 MAP kinase, JNK,
ERK and IκB
To further examine the possible role of stress signaling pathways in obesity,
aging and leptin-induced cardiac responses, expression of p38 MAP kinase, JNK,
ERK and the NFκB inhibitor IκB as well as their phosphorylation
were examined in young and aging C57 lean and ob/ob mouse
cardiomyocytes. Our results revealed that both obesity and age significantly
inhibited and stimulated phosphorylation of p38 MAP kinase and JNK,
respectively, without affecting expression of total p38 MAP kinase or JNK. There
was no additive effect between obesity and age on the phosphorylation of p38 MAP
kinase and JNK. Short-term leptin supplementation at physiological level (1.0
nM) restored obesity-induced changes in the phosphorylation of p38 MAP kinase
and JNK in young mice without affecting that in aging mice. Neither obesity nor
age affected expression of total and phosphorylated ERK, although the
combination of the two significantly reduced ERK phosphorylation. Leptin at 1.0
nM reconciled the reduced ERK phosphorylation in aging ob/ob
mice without affecting any other mouse groups. Expression of non-phosphorylated
ERK was unaffected by short-term leptin treatment at 1.0 nM. Our data further
revealed that either obesity or aging significantly enhanced phosphorylation of
IκB with no additive effect between the two. IκB is an inhibitor
of NFκB where enhanced IκB phosphorylation removes its
inhibition on NFκB). Similar to its effect on other stress signaling
molecules, short-term leptin treatment at physiological level (1.0 nM) removed
obesity-induced phosphorylation of IκB in young but not aging mice. Last
but not the least, short-term treatment of leptin at pharmacological level (50
nM) significantly activated the stress signaling molecules p38, JNK, ERK and
NFκB (via enhanced phosphorylation of IκB) in all mouse groups
without affecting the expression of non-phosphorylated proteins (Fig. 8).
10.1371/journal.pone.0010085.g008Figure 8 Total and phosphorylated protein expression of p38 MAP kinase, JNK,
ERK and IκB in cardiomyocytes isolated from young (4-month-old)
or aging (12-month-old) lean (C57) and ob/ob mice
treated with or without leptin (1.0 and 50 nM) for 4 hrs.
Panel A: Phosphorylation of p38 expressed as pp38-to-p38 ratio; Panel B:
Phosphorylation of JNK expressed as pJNK-to-JNK ratio; Panel C:
Phosphorylation of ERK expressed as pERK-to-ERK ratio; and Panel D:
Phosphorylation of IκB expressed as p IκB-to-IκB
ratio. Insets: Representative gel blots of total and phosphorylated p38,
JNK, ERK and IκB proteins using specific antibodies. Mean
± SEM, n = 4–8
isolations, * p<0.05 vs. respective C57
group, ** p<0.05 vs. young C57
group, # p<0.05 vs. respective
ob/ob group.
Influence of age and obesity on cardiomyocyte function in high fat-induced
and db/db obesity
To further elucidate the interaction between aging and obesity on cardiac
contractile function, we went on to examine the high fat diet-induced and the
leptin receptor mutant db/db obesity models. A 16-week high fat
diet feeding regimen was applied to young (4-month-old) and aging (12-month-old)
C57 mice. Both young and aging mice were euglycemic (data not shown) and
displayed a comparable separation in body weight in response to low and high fat
diet feeding (Young: Low fat: 26.6±0.6 g vs. High
fat: 30.6±0.6 g; Aging: Low fat: 28.8±0.4 g
vs. High fat: 32.7±0.4 g,
n = 4 mice per group). The resting cell length
was significantly greater in high fat diet and aging groups without any additive
effect between the two. Short-term leptin treatment (1.0 nM) failed to affect
resting cell length in either low or high fat diet groups at both ages. Both
high fat diet feeding and aging independently and significantly reduced PS and
± dL/dt, prolonged TR90 without affecting TPS. There was
no additive or synergistic effect between the two on the mechanical responses.
Leptin supplementation (1.0 nM) failed to reconcile high fat diet or
aging-induced mechanical alteration in PS, ± dL/dt and
TR90. Furthermore, leptin treatment (1.0 nM) did not alter any of
the mechanical indices tested (Fig.
9). Our further study using the db/db obese model
revealed a somewhat comparable euglycemic (data not shown) body weight gain
between C57 (4-month: 24.0±0.8 g; 12-month: 28.0±0.9 g;
18-month: 30.2±0.8 g) and db/db (4-month:
48.2±2.0; 12-month: 56.7±2.3; 18-month:
59.9±2.9%, n = 4 mice per
group, p<0.05 vs. corresponding C57 group) mice. The
resting cell length was significantly greater in db/db and
aging groups without any additive effect between the two. Both
db/db obesity and aging significantly reduced PS and
± dL/dt, prolonged TR90 without affecting TPS.
Interestingly, aging and db/db obesity exerted an additive
inhibitory effect on PS and ± dL/dt without affecting TR90
at 18 but not 12 months of age (Fig. 10).
10.1371/journal.pone.0010085.g009Figure 9 Contractile properties of cardiomyocytes isolated from young
(4-month-old) and aging (12-month-old) male C57 mice fed a low
(10%) or high (45%) fat diet for 16 weeks.
Cohorts of cardiomyocytes were treated with or without leptin (1.0 nM)
for 4 hrs prior to mechanical study. A: Resting cell length; B: Peak
shortening (PS, normalized to cell length); C: Maximal velocity of
shortening (+ dL/dt); D: Maximal velocity of relengthening (-
dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90%
relengthening (TR90); Mean ± SEM,
n = 50–51 cells from 3 mice
per group, * p<0.05 vs. respective low
fat group, ** p<0.05 vs. young
low fat group.
10.1371/journal.pone.0010085.g010Figure 10 Contractile properties of cardiomyocytes isolated from young
(4-month-old) and aging (12- or 18-month-old) male C57 lean and the
leptin receptor-deficient db/db obese mice.
A: Resting cell length; B: Peak shortening (PS, normalized to cell
length); C: Maximal velocity of shortening (+ dL/dt); D:
Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening
(TPS); F: Time-to-90% relengthening (TR90); Mean
± SEM, n = 102–103
cells from 3 mice per group, * p<0.05
vs. respective C57 group, **
p<0.05 vs. young C57 (4-month) group, #
p<0.05 vs. young db/db
(4-month) group.
Discussion
The major findings of our current study revealed that increased age mimicked
leptin-deficient ob/ob obesity-induced changes in cardiomyocyte
contractile function, intracellular Ca2+ homeostasis, NADPH
oxidase expression, O2
− accumulation, Akt/eNOS and
stress signaling (p38, JNK and NFκB). Little additive or synergistic actions
were noted between aging and ob/ob obesity on the above-mentioned
parameters, with the exception of a rise in intracellular Ca2+.
Short-term treatment of leptin at physiological levels (0.5 and 1.0 nM) elicited a
beneficial effect on cardiomyocyte contractile and intracellular
Ca2+ responses in young but not aging ob/ob
mice whereas pharmacological level of leptin (50 nM) compromised cardiomyocyte
contractile function, intracellular Ca2+ handling, NADPH oxidase
expression, O2
− accumulation, Akt/eNOS and stress
signaling. The disparity between young and aging mice in physiological leptin
level-induced mechanical responses was closely mirrored by an accumulation of
O2
− and expression of NADPH oxidase
(p
47phox), the enzyme responsible
for O2
− production. Further scrutiny depicted that
aging and obesity independently decreased the phosphorylation of Akt and its
downstream signaling molecule eNOS, stimulated JNK and IκB phosphorylation
as well as inhibited p38 phosphorylation without overt interactions between the two.
Consistent with its responsiveness to mechanical function,
O2
− production and
p
47phox expression,
physiological levels of leptin effectively restored leptin deficiency-induced
changes in the phosphorylation of Akt, eNOS, JNK, IκB and p38 in young but
not aging ob/ob mice. These data favor a role of post-insulin
receptor signaling and stress signaling in obesity-associated cardiac mechanical
defects and O2
− accumulation. Our data did not favor
a major role for the leptin receptor (Ob-R), its post-receptor signal STAT-3, ERK or
AMPK in leptin-elicited beneficial effects in ob/ob obese mice.
Given that leptin (at physiological levels) failed to reconcile aging-induced
detrimental effects in cardiomyocytes, it appears that aging may produce cardiac
contractile and intracellular Ca2+ defects associated with
O2
− accumulation reminiscent of leptin-deficient
obesity through a mechanism(s) independent of interrupted leptin signaling.
Development of obesity and its associated complications may be attributed to multiple
factors including genetic, dietary, environmental and evolutionary components,
although pinpointing each specific influence has been rather difficult [1], [3].
Although human obesity is usually accompanied by hyperleptinemia [16], both
hypo- and hyper-leptinemia have been shown to induce obesity due to interrupted
leptin signaling and energy expenditure [31]. Sustained obesity
(diet-induced or genetically predisposed) impairs cardiac contractile function in a
manner reminiscent of pre-diabetic insulin resistance and full-blown diabetes [32]–[35], indicating a role for
insulin resistance in obesity-induced cardiac contractile dysfunction. This is
supported by our current observation of dephosphorylated Akt and eNOS in young and
aging ob/ob mice. In our study, the leptin deficient
ob/ob mice were euglycemic at both ages, thus excluding
possible contribution from full-blown diabetes to the cardiac anomalies of the
ob/ob mice. Our data revealed a somewhat similar tibial length
among young and aging C57 or ob/ob mice, excluding the possible
contribution of disparate growth and development in these mice. These data are in
line with the notion that tibial length reached plateau when body growth slowed down
after postnatal day 70 in mice [36]. Nonetheless, these ob/ob mice
were hyperinsulinemic, hypertriglyceridemic and glucose intolerant based on our
earlier studies [12], [14], indicating the presence of insulin resistance.
More importantly, our DEXA study depicted an age-dependent increase in body fat
composition in both C57 lean and ob/ob obese mice, favoring aging
itself as an independent obesogenic factor [4]. Data from our study
indicated that aging itself produced a cascade of cardiomyocyte mechanical defects
reminiscent of young ob/ob or db/db as well as
high fat diet-induced obese mice. In all three murine obesity models used in our
study, both aging and obesity independently triggered an elongation in resting cell
length, depression in peak shortening (PS) amplitude and maximal velocity of
shortening/relengthening amplitude (± dL/dt), as well as prolongation in
relengthening duration (TR90) but not duration of shortening (TPS). These
data are consistent with our previous observations from aged or obese mice [14], [37], [38].
Interestingly, there was little interaction between aging and obesity on
cardiomyocyte contractile parameters with the exception of further depressed PS and
± dL/dt in 18-month-old db/db mice. These data seem to
favor the notion that aging and obesity may share somewhat similar cellular
mechanisms en route to cardiomyocyte mechanical dysfunction. The
apparent disparity between ob/ob and db/db mice on
the synergistic effect of aging (18 months) and obesity depicts presence of an overt
age-related difference between the two leptin mutant murine obesity models. Thus
caution should be taken to derive experimental conclusions using various rodent
obesity models. Given our further observation that physiological leptin treatment
failed to reconcile high fat diet- or age-induced detrimental effects in
cardiomyocytes, the convergence between aging and obesity in cardiac contractile and
intracellular Ca2+ defects as well as
O2
− accumulation likely occurs at a point
downstream of or independent of leptin signaling.
The Kaplan-Meier survival curve (Fig.
1) revealed a greatly elevated mortality in ob/ob mice,
supporting the hypothesis that obesity may be considered a status of premature aging
[4]. It
is worth mentioning that the 12 or 18 months of age selected for our
“aging” mice was not as old as other studies have used. However
the mortality rate of ob/ob mice after one year of life is much
higher than other mouse types [26], [38]. Although limited information is available for
the precise cause of death for these ob/ob obese mice, it may be
speculated that obesity-associated tumorigenesis (e.g., colon and skin cancer),
chronic inflammation, immune deficiency and cardiovascular complications are among
the leading causes of death in these mice [39]. In our study,
the young and aging ob/ob mice exhibited significantly greater fat
composition, heavier body and heart weights compared with the age-matched lean
control group. Moreover, the aging ob/ob mice displayed an
additional increase in body weight compared with the young ob/ob
mice. Considering the comparable cardiomyocyte functional profiles between young and
aging ob/ob mice, it appears that the extra body weight gain and
body fat composition in aging ob/ob mice had little effect on
cardiac dysfunction associated with obesity. Nonetheless, the additional increase in
body and fat mass was mirrored by a drop in the rise of intracellular
Ca2+ seen with aging. The greater cardiomyocyte cell length
in ob/ob mice at both ages was not affected by short-term
physiological leptin treatment, likely due to the fact that cardiac hypertrophy
resulting from interrupted leptin signaling in ob/ob mice is a
chronic process [23], [40]. During the chronic
cardiac remodeling process with interrupted leptin signaling, the heart transforms
from compensated to decompensated states accompanied by deteriorated cardiac
function.
Our study revealed that both aging and obesity impaired intracellular
Ca2+ handling shown as delayed intracellular
Ca2+ clearance and reduced intracellular
Ca2+ rise, consistent with our previous studies [14], [37], [38]. Unlike
the observation from cardiomyocyte mechanical assessment, the obesity-induced
decline in intracellular Ca2+ release was further accentuated
with aging, indicating a possible change in myofilament Ca2+
sensitivity in the aging ob/ob murine cardiomyocytes. These
observations favor the idea that dysregulated intracellular Ca2+
regulation may contribute to cardiomyocyte contractile dysfunction (prolonged
TR90, reduced PS and ± dL/dt) under aging, obesity or
both. Our data further revealed that physiological leptin reconciled intracellular
Ca2+ mishandling in young but not aging
ob/ob mice, indicating that intracellular
Ca2+ handling may contribute to the beneficial mechanical
response of leptin in young ob/ob mice. Our observation of elevated
O2
- production and upregulated
p
47phox subunit of NADPH
oxidase in both aging and obese groups (without interaction between the two)
suggests a likely role of NADPH oxidase-dependent
O2
− release in aging and/or obesity-elicited
cardiomyocyte intracellular Ca2+ handling and contractile
dysfunction. The NADPH oxidase-dependent O2
−
production and other reactive oxygen species are known to cause cardiomyocyte
mechanical dysfunction [22], [25]. The fact that
physiological leptin alleviated obesity-elicited increases in
O2
− production and
p
47phox expression in young but
not aging ob/ob mice favors a role for NADPH oxidase-dependent
O2
− production in the disparate cardiac response
of short-term leptin treatment. Our further observation in Ob-R expression and
STAT-3 phosphorylation depicted reduced Ob-R expression and STAT-3 phosphorylation
in both ob/ob age groups. To our surprise, unlike its effect on
cardiomyocyte contractile function, intracellular Ca2+
homeostasis and O2
− production, leptin treatment
restored downregulated Ob-R expression and STAT-3 activation in both
ob/ob age groups without any effect in lean groups. These data
indicate that the likely mechanism responsible for the age-dependent disparity of
cardiac leptin responses may not reside at the levels of the Ob-R or STAT-3. This
notion received further support from our observation that the pharmacological
concentration of leptin (50 nM) promoted Ob-R/STAT-3 signaling while compromising
cardiomyocyte contractile function, intracellular Ca2+ handling,
NADPH oxidase expression, O2
− accumulation, Akt/eNOS
and stress signaling. Data from our previous study revealed that pharmacological
levels of leptin (50 and 100 nM) compromised cardiac contractile function and
intracellular Ca2+ homeostasis through an ET-1 receptor-/NADPH
oxidase-dependent accumulation of reactive oxygen species [22]. Our current data
revealed unchanged Ob-R (long form) expression and reduced STAT-3 activation in
murine hearts at 12 months of age. Limited information is available with regards to
aging-induced changes in Ob-R expression and STAT-3 activation. The leptin-induced
STAT-3 phosphorylation was found to be higher along with an upregulated hypothalamic
expression of the Ob-R at 14 - 18 months of mouse age compared with 2 months of age
[41],
indicating increased leptin sensitivity with aging in the mouse brain. However,
little information is available on the heart with regards to the impact of aging on
leptin sensitivity.
Our results showed that aging and obesity independently depressed the phosphorylation
of Akt and eNOS, stimulated JNK and IκB phosphorylation as well as inhibited
p38 phosphorylation without overt interaction between the two. Meanwhile, leptin
supplementation at physiological levels rescued the dampened Akt/eNOS/p38
phosphorylation in young ob/ob mice, the effect of which was
obliterated by aging. These data are consistent with the basal and leptin-elicited
responses on cardiomyocyte contractile function, intracellular
Ca2+ handling, O2
−
production and NADPH oxidase expression. Under-activation of the key cardiac
survival factor Akt and its downstream signaling molecule eNOS has been demonstrated
in various models of cardiac dysfunction and heart failure [14], [34], suggesting a crucial
role of Akt/eNOS in the maintenance of cardiac function. It is noteworthy that the
dampened Akt/eNOS phosphorylation observed in our ob/ob mice may
contribute to enhanced cardiac oxidative stress and compromised cardiac function
since the Akt-eNOS cascade is known for its role in cardiac survival, glucose uptake
and maintenance of cardiac contractile function [30]. In our hands, both
obesity and aging independently decreased the phosphorylation of Akt and eNOS
without any additive effects between the two, consistent with our data on
mechanical, intracellular Ca2+ and
O2
− production. These observations favor a key
role for Akt/eNOS signaling in leptin-deficient obesity and age-induced cardiac
dysfunction. Our data revealed reduced p38 MAP kinase phosphorylation in aged and
obese mice, while leptin effectively restored p38 phosphorylation in young
ob/ob but not aging mice. These results favor a beneficial role
of p38 phosphorylation in the maintenance of cardiomyocyte function, which is
supported by the previous finding that inhibition of p38 MAP kinase reduces insulin
sensitivity and glucose uptake in human myotubes [42]. This is also in line
with the finding that leptin directly stimulates p38 MAP kinase phosphorylation
[43],
[44]. JNK
and NFκB may be turned on by pro-inflammatory cytokines and free fatty acids
in aging and obesity, resulting in interrupted insulin signaling and development of
cardiac dysfunction [45]. Our observation of enhanced phosphorylation of
JNK and IκB (which removes its inhibition on NFκB) in aging and
obesity are consistent with the reduced phosphorylation of Akt and eNOS. In
addition, our results revealed that neither obesity nor age significantly affected
the total expression and phosphorylation of ERK and AMPK. Interestingly, combining
obesity and age significantly attenuated phosphorylation of ERK and AMPK, the
effects of which were ablated and unaffected, respectively, by leptin treatment.
Although we are unable to offer any precise explanation for the combined effect
between age and obesity on ERK and AMPK at this time, our data do not favor a
significant role for ERK and AMPK in the reminiscent cardiac defects between aging
and obesity as well as the disparity in the leptin cardiac responsiveness. Further
study is warranted to better address the interplay among various cell signaling
pathways such as sirtuin, a key signaling molecule in longevity and lifespan [46], or
RAGE, which plays a key role in aging-associated cardiomyocyte dysfunction via
NFκB activation [47], in the aging- and obesity-associated cardiac
contractile dysfunction.
Experimental limitations: Although our study provided a likely causal relationship
among cardiac mechanical function, intracellular Ca2+
homeostasis, NADPH oxidase, O2
− accumulation,
Akt/eNOS and stress signaling activation between aging and obesity, caution should
be taken for the interpretation of the precise interaction between aging and obesity
in cardiac dysfunction in the human setting. First and foremost, the short-term
in vitro leptin incubation used in our study may not best
represent the in vivo longer term effect of leptin on phenotypic
changes in obesity. Oxidative modification of intracellular Ca2+
handling proteins is known to contribute to altered cardiomyocyte mechanics such as
prolonged relaxation in obesity [37]. It may be speculated that short-term incubation
of physiological levels of leptin may interrupt the oxidative processes (i.e.
scavenging reactive oxygen species) thus shifting the redox balance towards reducing
processes and promoting reactions to temporarily reverse oxidative modification of
Ca2+ handling proteins. Nonetheless, this may not truly
reflect the physiological setting in vivo. In our study, only male
mice were used which ignored the important gender disparity in obesity and aging
[2]. In
our cell isolation procedure, butanedione monoxime was used to uncouple
cardiomyocyte contractile elements and maintain cell viability for a prolonged
period of time, which may unevenly alter the true in vivo
cardiomyocyte mechanics and thus bias cardiomyocyte function from lean and
ob/ob groups. Measurement of contractile performance in
isolated cardiomyocytes has been established to provide a fundamental assessment of
cardiac contractile function in pathological states. However, as in any study of
this nature, caution needs to be taken when correlating our present cellular
findings to whole heart function, as the latter is composed of heterogeneous cell
types, including nerve terminals and fibroblasts, as well as the connective tissue
alluded to above. Furthermore, cardiomyocytes beat at a high frequency in
vivo as opposed to the non-physiological slow pace (0.5 Hz) used in our
study despite the fact that a low frequency contraction is deemed as a
“slow motion” to maximally reveal the cell mechanical defect.
Last but not least, the long–form Ob-Rb receptor monoclonal antibody used
in our study may cross-act with the short-form leptin receptors although the latter
cannot turn on the full JAK-STAT leptin signaling due to the absence of the
essential box-2 motif.
In summary, data from our present study suggested that aging and the leptin deficient
ob/ob obesity compromise cardiac contractile function and
intracellular Ca2+ homeostasis via comparable mechanisms
involving NADPH oxidase-dependent O2
− production,
phosphorylation of Akt, eNOS as well as the stress signaling molecules p38, JNK and
NFκB. Our study further revealed an age-associated disparity in
physiological leptin level-elicited responsiveness in cardiomyocyte contraction,
intracellular Ca2+ handling and
O2
− production. Collectively, these data favor a
role for NADPH oxidase, O2
− generation, Akt, eNOS
and the stress signaling molecules p38, JNK and NFκB, rather than Ob-R and
STAT-3, in the basal and leptin-elicited cardiac response during aging and obesity.
Our data further revealed both similarity and disparity in aging-associated
cardiomyocyte mechanical response between ob/ob obesity and high
fat diet-induced or the hyperleptinemic db/db obesity. Given the
lack of knowledge of aging-induced changes in adiposity and leptin signaling, the
precise interplay between aging and obesity, and contribution of leptin signaling
and downstream stress signaling activation, if any, to the cardiac contractile
dysfunction in the state of concurrent aging and obesity warrant further
research.
The authors wish to express our appreciation to Dr. Derek Smith and Mr. Christopher
Dorozynski from University of Wyoming College of Health Sciences for their
assistance in Dual Energy X-ray Absorptiometry.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported in part by grants from the American Heart Association
Pacific Mountain Affiliate (#0355521Z), American Diabetes Association
(7-08-RA-130) and National Institutes of Health/National Center for Research
Resources (NIH/NCRR) North Rockies Regional INBRE (P20 RR 016474) to J.R. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2041912809-PONE-RA-13203R410.1371/journal.pone.0010105Research ArticleOncologyCell Biology/Cellular Death and Stress ResponsesCell Biology/CytoskeletonOncology/SarcomasVimentin Is a Novel Anti-Cancer Therapeutic Target; Insights from In Vitro and In Vivo Mice Xenograft Studies WFA Induces Vimentin CleavageLahat Guy
1
2
Zhu Quan-Sheng
1
2
Huang Kai-Lieh
2
3
Wang Suizhao
1
2
Bolshakov Svetlana
1
2
Liu Jeffery
1
2
Torres Keila
1
2
Langley Robert R.
3
Lazar Alexander J.
2
4
Hung Mien Chie
5
Lev Dina
2
3
*
1
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
2
Sarcoma Research Center, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
3
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
4
Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
5
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States of America
Bauer Joseph Alan EditorBauer Research Foundation, United States of America* E-mail: [email protected] and designed the experiments: GL QSZ KLH MCH DL. Performed the experiments: GL QSZ KLH SW SB JL KT AJFL DL. Analyzed the data: GL QSZ RL AJFL MCH DL. Contributed reagents/materials/analysis tools: RL MCH DL. Wrote the paper: GL DL. Assisted in revision and in generating data to address review: KLH.
2010 16 4 2010 5 4 e1010526 9 2009 3 3 2010 Lahat et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting “epithelial to mesenchymal transition” phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses.
Methods and Findings
Withaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens.
Conclusions
In light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and “epithelial to mesenchymal transition” clinical contexts is warranted.
==== Body
Introduction
Comprising more than 50 histological subtypes, soft tissue sarcoma (STS) can be classified into two major groups: those with specific genetic alterations (translocations or point mutations) and relatively simple karyotypes, and those with complex, unbalanced aneuploid karyotypes [1], [2], [3], [4], [5], [6]. Enhanced understanding of the molecular aberrations driving the inception and progression of several STS subtypes belonging to the first group (e.g., c-Kit mutations in gastrointestinal stromal tumors [GIST] or the 17;22 translocation leading to PDGF-B over-expression in dermatofibrosarcoma protuberans; [DFSP]), has resulted in clinical applications of effective targeted therapies (e.g. Imatinib mesylate) with significantly improved outcome [6]. Such therapeutic advancements are very encouraging and highlight the need to identify other novel molecular targets in additional STS subtypes.
The majority of STS belong to the second group harboring aneuploid karyotypes; this group mainly consists of malignant fibrous histiocytoma (MFH, also termed unclassified pleomorphic sarcoma [UPS]), leiomyosarcomas, malignant peripheral nerve sheath tumors (MPNST), and dedifferentiated or pleomorphic liposarcomas. While clinical presentation and disease manifestations vary depending on the specific histological subtype, as a whole, these complex karyotype STS have a dismal prognosis, with a 5-year survival rate of less than 50%. Despite initial local control (achieved by surgery with or without radiation), local recurrence and systemic spread commonly occur and the lack of effective therapeutic options in these clinical scenarios is the major unresolved problem in STS. In contrast to the genetic simplicity of STS in the first group, the biological and molecular diversity of complex karyotype STS markedly limits the identification of single and specific “oncogenic addiction” aberrations. While challenging, elucidating novel therapies that might have utility for a broad range of complex karyotype STS is crucial.
Since the early 1960s, plants and microbes have yielded several useful, naturally derived, small organic molecules possessing anti-cancer properties [7], [8], [9], [10]. Withania somnifera (ashwagandha) is a medicinal plant commonly used in Indian traditional medicine to treat a wide spectrum of disorders [11], [12], [13], [14], [15]. Withaferin-A (WFA), a highly oxygenated C-28 ergostane-type steroidal lactone, is a bioactive compound isolated from Withania somnifera. WFA exhibits diverse pharmacologic activities, including anti-inflammatory, immunomodulatory and antiangiogenic effects [15], [16], [17], [18]. Several lines of evidence suggest that WFA has anti-cancer properties, manifested by directly targeting tumor cells and indirectly impeding [tumor-associated neovasculature [19], [20]. Recent studies have shown that WFA suppresses human breast and prostate cancer cells' growth in vitro and in vivo by inducing marked apoptosis [19], [21], [22]. WFA-induced cytoskeletal architecture alteration [23], [24], [25], reactive oxygen species generation [26], [27], mitochondrial dysfunction [26], and proteosomal inhibition [21] have also been suggested. Normal, nontumorigenic cells were found to be more resistant than tumor cells to WFA-induced apoptosis [22]; selectivity for malignant cells while sparing normal cells is a highly desirable feature of potential anti-cancer therapeutic agents.
The exact mechanisms of WFA action have not been conclusively defined. Several molecular targets have been proposed, including the transcription factor NF-κB [28], [29]; the signaling molecule AKT [27]; the proapoptotic molecules PAR-4, FOXO-3, and Bim [22]; and the proteosomal chymotrypsin subunit β5 [21]. A recent study utilized a chemical genetic and proteomic investigational approach [30], in which a biotinylated WFA analog was used as a probe to pull down WFA binding partners in endothelial cells. This strategy resulted in the identification of vimentin, a type III intermediate filament, as a novel WFA substrate. Furthermore, WFA-modified vimentin was found to elicit significant proapoptotic and antiangiogenic effects, whereas vimentin knockdown resulted in decreased WFA sensitivity. These experiments offer insight into WFA activity and highlight the potential utility of vimentin as a novel anti-cancer therapeutic target.
STS are mesenchymal and therefore all express vimentin, regardless of their histological subtype. Consequently, it is plausible that anti-vimentin therapeutic strategies might elicit anti-STS effects in a broad range of STS. This hypothesis prompted us to determine the impact of WFA on complex karyotype STS in vitro and in vivo. We used human cell lines representing leiomyosarcoma, MPNST, fibrosarcoma, and UPS/pleomorphic liposarcoma to assess the impact of vimentin on WFA sensitivity. Our results suggest that STS are highly sensitive to WFA, an effect that is much more pronounced than in vimentin-negative epithelial cancers. WFA induces a caspase-dependent degradation of vimentin, resulting in marked anoikis-independent apoptosis and STS-associated antiangiogenic effects. The results strongly support the evaluation of WFA or its analogs as a novel clinical strategy for patients harboring these devastating malignancies.
Results
Sarcoma cells are highly sensitive to WFA
To evaluate the effect of WFA on complex karyotype STS we selected a panel of human STS cell lines representing fibrosarcoma (HT1080), leiomyosarcoma (SKLMS1), MPNST (STS26T), and high grade pleomorphic sarcoma/liposarcoma (PLS-1). This latter cell line has recently been established in our laboratory (see Data S1 and Figure S1 for further details). Treatment of the above cells with WFA resulted in a significant decrease in the number of attached cells and marked morphological changes, including cell-rounding and nuclear condensation (Figure 1A). The effects of WFA occurred as early as 2 h after treatment initiation and were dose- and time-dependent (Figure S2). Cell growth assays demonstrated a WFA-induced, dose-dependent decrease in STS cell growth (Figure 1B). Mean±SD WFA IC50 values (after 24 h of treatment) were recorded as 0.4 µM±0.07, 0.41 µM±0.03, 0.37 µM±0.12, and 0.53 µM±0.1, for HT1080, SKLMS1, STS26T, and PLS-1, respectively. Similarly, low doses of WFA (0.5 µM) markedly inhibited STS cell colony formation capacity (Figure 1C). Lastly, the effect of WFA on STS anchorage independent growth was investigated. All STS cells evaluated demonstrated a capacity to grow in soft agar; this growth was abrogated after 24 h of WFA (0.5 µM) treatment (Figure 1D). Taken together, these data suggest that human STS cells are highly sensitive to the growth inhibitory effects of WFA.
10.1371/journal.pone.0010105.g001Figure 1 WFA inhibits STS cell growth.
A) WFA treatment (1 µM/24 h) results in marked morphological changes, including cell-rounding and nuclear condensation, in STS cells; B) MTS assays demonstrate a WFA-induced, dose-dependent decrease in STS cell growth; C) WFA (0.5 µM/24 h) markedly inhibits STS cell colony formation capacity measured after ten days; D) WFA (0.5 µM/24 h) abrogates STS cell anchorage independent growth measured after three weeks. Graphs represent the average of three repeated experiments ±SD.
WFA induces marked apoptosis in STS cells but less apoptosis in normal human fibroblasts and myogenic cells
To evaluate the effect of WFA on STS cell survival, we conducted Annexin V/FACS analyses. STS cells were treated with increasing concentrations of WFA (0–5 µM) for 4 h and 24 h; a significant induction of apoptosis was apparent even within the short time frame especially when high doses were used (>2.5 µM, p<0.05; Figure 2A). A dose- and time-dependent increase in tumor cell apoptosis was observed in all cells tested. Apoptosis induction was also reflected in the observed increase in activated caspase 3 and PARP cleavage (Western blot analysis [WB]; Figure 2B).
10.1371/journal.pone.0010105.g002Figure 2 WFA induces marked apoptosis in STS cells.
A) Annexin-V/FACS analyses demonstrating marked WFA-induced apoptosis in STS cells (black bars represent 4 h of WFA treatment and gray bars represent 24 h); B) WFA (1 µM/24 hr) induces caspase-3 (Casp-3) cleavage and PARP activation in STS cells (WB analysis); C) STS cells are resistant to anoikis as compared to normal human dermal fibroblasts (NHDF). WFA (1 µM/24 hr) induces apoptosis in both attached and floating STS cells; D) Transmission electron microscopy (TEM) photographs depicting STS cell apoptosis (large arrow–nuclear condensation, small arrow–cytoplasmic blebbing) in response to WFA. Necrosis is demonstrated in floating STS cells; E) NHDF are more resistant to the effects of WFA (IC50: 3.7 µM±0.15) as compared to STS cells. Graphs represent the average of three repeated experiments ±SD.
Next, we evaluated whether WFA-induced apoptosis could be attributed to tumor cell loss of adhesion and detachment from the culture plate, resulting in anoikis. Normal human dermal fibroblasts (NHDF) underwent significant apoptosis when cultured in suspension, while STS cells were resistant to anoikis and no significant increase in apoptosis could be observed (Figure 2C). WFA enhanced apoptosis of both attached and floating STS cells.
WFA-induced apoptosis was further confirmed using transmission electron microscopy (TEM). Signs of apoptosis, including chromatin condensation and cytoplasm shrinkage, were evident within 4 h. After 24 h, marked apoptosis was observed, along with nuclear membrane loss and cytoplasmic blebbing; these effects were noticed in both attached and floating STS cells. Floating necrotic STS cells were also observed (∼20–30% of total floating cells).
Last, we evaluated the effect of WFA on normal mesenchymal cells (Figure 2E and S3). Primary cultures of NHDF and human intestinal smooth muscle cells (HISMC) were treated with increasing doses of WFA for 24 h. Contrary to our observations in STS cells, decreases in cell number and morphological changes were evident by microscopy only after high doses of WFA (>2.5 µM). Mean±SD WFA IC50 values were 3.7 µM±0.15 and 3.2 µM±0.21 for NHDF and HISMC, respectively. These values were more than 9 times higher than those observed in STS cells. Similarly, WFA induced a significantly lower rate of apoptosis in these normal cells (P<0.05). Taken together, these data suggest that WFA is a potent proapoptotic compound. STS cells are highly sensitive to WFA, while normal mesenchymal cells are more resistant to its effects.
WFA abrogates STS cell migration and invasion
We next evaluated the effect of WFA on STS cell migration and invasion. STS cells were pretreated with low doses of WFA (0.5 µM) for 4 h, at which point WFA was washed off carefully and a scratch wound healing assay was conducted. We observed a marked decrease in migration (Figure S4A). In addition, modified Boyden chambers were used to quantitate the effect of WFA on migration and invasion. STS cells were pretreated with a low dose of WFA (0.5 µM) for 4 h. After discontinuation of WFA, cells were washed and counted; only viable cells were further utilized. WFA significantly inhibited migration and invasion in all STS cells examined (P<0.05; Figure S4B).
One potential caveat to these experiments is that the impact demonstrated might reflect the antiapoptotic or growth-inhibitory effects of WFA rather than its direct effects on motility and/or invasion. To address this possibility, in conjunction with the above experiments, we pretreated STS cells with a low dose of WFA (0.5 µM for 4 h) or DMSO (control); at discontinuation of WFA, cells were washed and counted, and viable cells were re-plated. Cell counting at the end of the experiment revealed only a small decrease (<10%) in the number of viable WFA-treated cells relative to the number of control-treated cells (data not shown). Together, these results suggest that WFA abrogates STS cell motility and invasion.
WFA induces vimentin degradation and vimentin knockdown decreases cells' sensitivity to WFA
A recent study identified vimentin as the possible WFA molecular target [30]. Therefore, we evaluated the effect of WFA on vimentin expression and degradation. WFA treatment resulted in decreased full-length vimentin levels and increased expression of vimentin degradation products in all STS cells tested (Figure 3A). WFA effect on vimentin is dose and time dependent; high WFA concentrations result in vimentin cleavage after only 4 h of treatment, while vimentin degradation is noticed after 24 h with lower doses.
10.1371/journal.pone.0010105.g003Figure 3 WFA induces vimentin degradation and vimentin knockdown decreases cells' sensitivity to WFA.
A) WFA treatment (5 µM/4 h) results in decreased full-length (FL) vimentin levels and increased expression of vimentin degradation products (VDP) in all STS cells tested. A WFA dose dependent effect in SKLMS1 cells treated for 4 h is also shown. Vimentin cleavage is noticed secondary to low WFA concentrations after 24 h of treatment; B) Anti-vimentin SMARTpool siRNA (20 nM) elicits a marked decrease in vimentin expression in SKLMS1 cells (WB). Vimentin knockdown substantially blocks WFA-induced (1 µM/24 hr) apoptosis; C) Endogenous vimentin was first knocked down in SKLMS1 cells using anti-vimentin antisense phosphorodiamidate morpholino oligomers. After knockdown, vimentin was forcefully re-expressed in the cells (WB). Similar to the results of siRNA knockdown, anti-vimentin morpholino oligomers significantly blocks WFA-induced (1 µM/24 hr) apoptosis. Re-expression of vimentin restores SKLMS1 sensitivity to WFA; D) STS cells (SKLMS1 and PLS1) express significantly higher levels of soluble vimentin as compared to normal mesenchymal cells (smooth muscle cells: HA-SMC and HC-SMC and fibroblasts: NHDF). (NT siRNA = non targeting siRNA, Vim siRNA = anti-vimentin siRNA smartpool; NT morpholino = non targeting morpholino; Vim KD = vimentin knockdown)
To determine whether the effect of WFA on STS cells is at least partially mediated through vimentin, we elected to use a vimentin knockdown approach. Anti-vimentin SMARTpool siRNA elicited a substantial decrease in vimentin expression in SKLMS1 cells (WB; Figure 3B). Vimentin knockdown per se did not induce significant apoptosis in STS cells, as compared to mock or non-targeting siRNA transfection. As expected per the experiments above, WFA treatment resulted in significant apoptosis in mock and non-targeting siRNA transfected cells. However, vimentin knockdown substantially blocked WFA-induced apoptosis. Together, these data suggest that WFA-induced vimentin degradation is necessary for enhanced therapeutic effect.
To confirm the potential role of vimentin in WFA-induced apoptosis, we used a rescue experimental approach. Endogenous vimentin was first knocked down in SKLMS1 cells using anti-vimentin antisense phosphorodiamidate morpholino oligomers. These morpholinos target vimentin pre-mRNA, thereby enabling the forced re-expression of vimentin by transfecting a vimentin construct resistant to the continuous presence of the morpholino oligomers. After knockdown, vimentin was forcefully re-expressed in the cells (WB, Figure 3C); cells were treated with WFA or DMSO as control and subjected to Annexin V/FACS analysis. Similar to the results of siRNA knockdown, anti-vimentin morpholino oligomers treatment significantly blocked WFA-induced apoptosis. Re-expression of vimentin restored SKLMS1 sensitivity to WFA (Figure 3C).
Both STS cells and normal mesenchymal cells express vimentin. However, as shown in Figure 2, WFA induces more significant pro-apoptotic effects in STS cells as compared to normal mesenchymal cells (i.e. fibroblasts and muscle cells). The previous published data described above (30) identified WFA to bind to tetrameric, soluble vimentin. Thus, we sought to evaluate the levels of this vimentin fraction in normal vs. STS cells. As shown in Figure 3D, tumor cells express significantly higher levels of soluble, free vimentin as compared to normal mesenchymal cells. This finding offers a possible explanation for our observed differential WFA effects.
WFA-induced vimentin degradation is caspase-dependent
Vimentin degradation commonly occurs through the activation of the caspase pathway. To evaluate whether WFA-induced vimentin degradation is a result of caspase cleavage, we pretreated STS with Z-VAD (Promega, Madison, WI), a pan-caspase inhibitor, before WFA therapy (for 24 h). Z-VAD pretreatment resulted in decreased vimentin degradation in conjunction with lower levels of both cleaved caspase-3 and activated PARP (Figure 4A). Furthermore, Z-VAD pretreatment significantly abrogated WFA-induced apoptosis (after 24 h of WFA treatment) in STS cells (Figure 4B). Next, after vimentin knockdown using morpholino oligos, SKLMS1 cells were transfected to express either wild-type vimentin or vimentin mutated at caspase cleavage sites (D85N and D259N). WFA (1 µM) induced marked apoptosis in wild-type vimentin transfected cells in which vimentin degradation and caspase-3 activation were observed (Figure 4C). However, we observed a significant decrease in WFA-induced apoptosis in cells expressing the mutated vimentin, and we noticed a dramatic decrease in both vimentin degradation and caspase-3 activation (Figure 4C). Taken together, these data suggest a possible WFA-induced vicious cycle, wherein WFA binding to vimentin elicits its degradation by caspases and said degradation results in additional caspase activation and apoptosis.
10.1371/journal.pone.0010105.g004Figure 4 WFA-induced vimentin degradation is caspase-dependent.
A) Pretreatement (4 h) of SKLMS1 cells with Z-VAD (a pan-caspase inhibitor) results in decreased WFA-induced (24 h) vimentin degradation in conjunction with lower levels of both cleaved caspase-3 and activated PARP; B) Z-VAD (4 h) pretreatment significantly abrogates WFA-induced (24 h) apoptosis in SKLMS1 cells; C) Vimentin knocked down SKLMS1 cells were transfected to express either wild-type vimentin or vimentin mutated at caspase cleavage sites (D85N and D259N). WFA (1 µM/24 hr) induces marked apoptosis in wild-type vimentin transfected cells in which vimentin degradation and caspase-3 activation can be detected (WB). However, a significant decrease in WFA-induced apoptosis in cells expressing the mutated vimentin, is noticed as well as a decrease in both vimentin degradation and caspase-3 activation. (WT Vim = wild type vimentin; Mut Vim = mutated vimentin; Vim FL = full length vimentin; VDP = vimentin degradation products)
WFA-induced molecular deregulations are, at least partly, mediated by vimentin
Several molecular mechanisms have previously been proposed to underlie WFA anti-tumorigenic effects including inhibition of AKT phosphorylation [27], abrogation of NF-κB function [20], [31], and direct proteosomal inhibition [21]. We first sought to evaluate whether these molecular deregulations occur in STS cells in response to WFA treatment. We observed a WFA dose- and time-dependent decrease in pAKT levels, but not total AKT levels, in STS cells (Figure 5A). Similarly, a dose-dependent decrease in NF-κB (p65) was also demonstrated, although this decrease occurred only after 24 h of treatment. NF-κB activity, on the other hand, was shown to be inhibited early after treatment, suggesting mechanisms other than decreased NF-κB protein expression affecting NF-κB activity. In addition, we found a marked dose- and time-dependent accumulation of ubiquitinated proteins, supporting WFA-induced proteosomal inhibition in these cells.
10.1371/journal.pone.0010105.g005Figure 5 WFA-induced molecular deregulations are, at least partly, mediated by vimentin.
A) WFA treatment induces a dose- and time- dependent decrease in pAKT without effect on total AKT. Similarly, a dose-dependent decrease in NF-κB (p65) protein expression is also seen, although this decrease occurs only after 24 h of treatment. NF-κB activity in PLS1 cells, as depicted in graphs representing luciferase reporter assay results, is shown to be inhibited early after treatment (4 h). A marked dose- and time-dependent accumulation of ubiquitinated proteins is also shown; B) Vimentin knockdown in SKLMS1 cells abrogates WFA (2.5 µM/24 h)-induced pAKT inhibition, NF-κB protein decrease, and increased levels of protein ubiquitination. Similarly, vimentin knockdown blocks WFA (1 µM/4 h)-induced decrease in NF-κB activity. Graphs represent the average of three repeated experiments ±SD.
Our results suggest a pivotal role for vimentin in cells' response to WFA. Thus, we sought to evaluate whether the effects of WFA on these diverse pathways can, at least partly, be mediated through vimentin. SKLMS1 cells were transiently transfected with anti-vimentin siRNA or non-targeting siRNA and then treated with WFA. Vimentin knockdown abrogated WFA-induced pAKT inhibition, NF-κB protein decrease and activity blockade, and increased levels of protein ubiquitination (Figure 5B). These experiments provide further evidence that the effects of WFA are mediated through vimentin and highlight novel potential vimentin functions.
WFA induces apoptosis in endothelial cells cultured in STS-conditioned media
Analogous to solid malignancies, STS consist of both tumor and tumor-associated normal cells; STS growth, migration, and dissemination depend on cross-talk between these two compartments. STS are generally highly vascular and angiogenic, resulting in increased metastatic potential. Previous data suggest that WFA might harbor antiangiogenic properties [17], [30]. To further expand these initial observations and evaluate the potential antiangiogenic effects of WFA in the context of the STS microenvironment, we evaluated the effect of WFA on endothelial cell grown in regular control medium (lacking angiogenic factors, mimicking quiescent endothelial cells) and on endothelial cells cultured in STS-conditioned medium (CM; mimicking proliferating angiogenic endothelial cells). We used both human endothelial (HDMEC) and murine endothelial cells (LEC) and observed a significantly higher WFA-induced growth inhibition in endothelial cells cultured in STS-CM than in control medium (Mean±SD WFA IC50: 0.42 µM±0.16 vs. 1.4 µM±0.04 in HDMEC and 0.38 µM±0.09 vs. 2.3 µM±0.1 in LEC; P<0.05, Figure 6A). Similarly, WFA induced higher levels of apoptosis in endothelial cells grown in STS-CM than in control medium (Figure 6B).
10.1371/journal.pone.0010105.g006Figure 6 WFA induces apoptosis in endothelial cells cultured in STS-conditioned media.
A) A significantly higher rate of WFA-induced (24 h) growth inhibition is seen in endothelial cells (human dermal microvessel endothelial cells–HDMEC and murine lung endothelial cells–LEC) cultured in STS conditioned medium (CM) than in control regular medium (RM); B) WFA(1 µM/24 hr) induces higher levels of apoptosis in endothelial cells grown in STS-CM than in control medium; C) WFA (1 µM/24 hr) induces significantly higher rates of vimentin degradation and caspase-3 activation in endothelial cells grown in STS-CM than in quiescent endothelial cells; D) WFA (1 µM) abrogates migration and invasion of endothelial cells cultured in STS-CM; E) WFA (2 mg/kg) results in a significant decrease in the mean number of CD31-positive (red) blood vessels compared to control DMSO treatment in an in vivo gelfoam assay. CD-31(red)/TUNEL(green) double staining reveals endothelial cell apoptosis in WFA-treated mice. Graphs represent the average of three repeated experiments ±SD. (Vim FL = full length vimentin; VDP = vimentin degradation products)
Endothelial cells are mesenchymal in origin and thus express vimentin, so we evaluated the effect of WFA on vimentin expression and cleavage in both regular media and STS-CM. Interestingly, WFA induced significantly higher rates of vimentin degradation and caspase-3 activation in endothelial cells grown in STS-CM than in quiescent endothelial cells (Figure 6C). Next, we evaluated the effect of WFA on endothelial cell migration and invasion. Similar to the previously described experimental approach utilized with STS cells, we used viable endothelial cells pretreated with short-term WFA (4 h) in modified Boyden chamber assays. As anticipated, STS-CM enhanced endothelial cell migration and invasion. More important, WFA abrogated migration and invasion of endothelial cells cultured in STS-CM, but not that of those grown in regular medium (Figure 6D).
Last, to confirm that the observed WFA antiangiogenic effect occurs in vivo as well, we performed a Gelfoam angiogenesis assay Gelfoam sponges were incubated in STS-CM and implanted subcutaneously into the flanks of SCID mice. Two days after implantation, mice were treated with i.p. WFA (2 mg/kg; n = 4) or DMSO (n = 4) for 10 consecutive days; at the termination of the study, the Gelfoams were excised and subjected to IHC analysis (Figure 6E). WFA treatment resulted in a significant decrease in the mean number of CD31-positive blood vessels compared to control DMSO treatment (41±7.2 vs. 6±5.3, respectively; P<0.05). CD-31/TUNEL double staining revealed endothelial cell apoptosis in WFA-treated mice. These results suggest that WFA potentially abrogates cell growth, inhibits migration and invasion, and induces apoptosis in proliferating endothelial cells within the STS microenvironment.
Epithelial origin cancers' sensitivity to WFA is enhanced in cells exhibiting epithelial to mesenchymal transition (EMT)
Our central hypothesis is that cells expressing vimentin are expected to demonstrate a higher WFA sensitivity. Unlike STS, the more common epithelial origin cancers do not naturally express vimentin. However, substantial data indicate that both invasion and metastasis may critically depend on the acquisition of a “mesenchymal” phenotype by the incipient cancer cell [32], [33], [34]. One of the hallmarks of this epithelial to mesenchymal transition, is induced vimentin expression. Taking this into account, we analyzed vimentin expression in a panel of carcinoma cell lines and evaluated their response to WFA. Three of the cells (MCF7, HT29, and TMK1) exhibited no vimentin mRNA and protein expression, and the other two (MDA231 and A549) expressed vimentin (Figure 7A). Cells expressing vimentin were significantly more sensitive to WFA than those not expressing vimentin (Mean±SD WFA IC50 values in MDA231 and A549 cells were 1.3 µM±0.42 and 1.8 µM±0.37, respectively, and 2.9 µM±0.31, 7.8 µM±2.34, 3.1 µM±0.18 in MCF-7, HT29, and TMK1 cells, respectively; P<0.05). Similarly, WFA (1 µM for 24 h) elicited a significantly higher apoptotic rate in vimentin-expressing carcinoma cells (P<0.05; Figure 7B). WB analysis further revealed vimentin degradation products in MDA231 cells in conjunction with enhanced cleaved caspase-3 and activated PARP expression levels (Figure 7C). In contrast, only minimal or no expression of cleaved caspase-3 and activated PARP was demonstrated in MCF7 and HT29 cells, respectively. The data presented here further support the role of vimentin as a WFA molecular target, suggesting the possible therapeutic efficacy of WFA on epithelial cancers exhibiting EMT phenotypes.
10.1371/journal.pone.0010105.g007Figure 7 Epithelial origin cancers' sensitivity to WFA is enhanced in cells exhibiting epithelial to mesenchymal transition (EMT).
A) WFA-induced growth inhibition corresponds to vimentin expression level in epithelial origin cancer cells; B) WFA (1 µM for 24 h) elicits a significantly higher apoptotic rate in vimentin-expressing carcinoma cells; C) WB analysis demonstrating vimentin degradation MDA231 cells in conjunction with enhanced cleaved caspase-3 and activated PARP expression levels. In contrast, only minimal or no expression of cleaved caspase-3 and activated PARP are seen in the vimentin-negative MCF7 and HT29 cells. Graphs represent the average of three repeated experiments ±SD. (Vim FL = full length vimentin; VDP = vimentin degradation products)
WFA abrogates STS growth, angiogenesis, recurrence, and metastasis in vivo
To test the potential in vivo significance of the above observations, we investigated the effect of WFA on STS growth, local recurrence, and metastasis using several human STS xenograft mouse models representing different complex karyotype STS histological subtypes.
First, we investigated the effect of WFA on leiomyosarcoma (SKLMS1) and fibrosarcoma (HT-1080) growth in SCID mice. In a 2-arm study (n = 10/group), we compared the effects of WFA (2 mg/kg) to that of the carrier (DMSO) alone. Previous studies demonstrated in vivo efficacy and no significant side effects in breast and prostate cancer xenografts when a WFA dose of 4 mg/kg was utilized [19]; estimating enhanced sensitivity in STS xenografts, we selected a lower WFA dose. Therapy was initiated after tumor establishment, as defined in Methods. The treatment regimen was highly tolerated; we observed no significant weight loss. WFA caused significant growth retardation in both tumor types (Figure 8A); at the termination of the study, mean±SD SKLMS1 tumor volumes were 501.6 mm3±114.2 in the WFA group vs. 1365.7 mm3±155.5 in the control group (P = 0.032), and HT1080 tumor volumes were 210 mm3±41.4 vs. 1050.1 mm3±51.9, respectively (P = 0.001). Similarly, WFA treatment significantly reduced tumor weight. Mean ± SD tumor weights at the termination of the study were 779.5 mg±175.4 and 770.6 mg±218.4 in control mice and 230.6 mg±64.4 and 211.8 mg±94.1 in WFA-treated mice bearing SKLMS1 and HT1080 xenografts, respectively (P = 0.017 and P = 0.036; Figure 8A).
10.1371/journal.pone.0010105.g008Figure 8 WFA abrogates STS growth, angiogenesis, recurrence, and metastasis in vivo.
A) WFA (2 mg/kg) significantly inhibits STS local growth and tumor weight; B) WFA treatment decreases tumor cell proliferation (PCNA), enhances apoptosis (TUNEL), and inhibits STS associated angiogenesis (CD31). CD31 (red)/TUNEL (green) double immunofluorescence analysis demonstrating apoptotic endothelial cells in WFA-treated tumors; C) WB analysis of tissue samples demonstrating vimentin degradation and caspase-3 activation in WFA-treated tumors; D) Kaplan Meier curves demonstrating a statistically significant (P = 0.02) delay in SKLMS1 local recurrence in WFA treated mice (black curve) compared to control (gray curve); E) Control mice (DMSO treated) exhibit numerous large MPNST lung metastases almost completely replacing the lung parenchyma. In contrast, WFA-treated mice exhibit markedly fewer microscopic small lung nodules (circle). WFA treatment significantly decreases average lung weights as compared to controls (box plots). (Vim FL = full length vimentin; VDP = vimentin degradation products).
Tumor sections containing viable cells from each treatment group were selected for IHC studies (Figure 8B). PCNA staining revealed a significant decrease in tumor cell proliferation (the mean±SD PCNA scores were 88%±10.2% and 73%±14.7% in control tumors and 25%±10.2% and 15%±6.5% in WFA-treated tumors of SKLMS1 and HT1080 xenografts, respectively; P = 0.025 and P = 0.04). TUNEL staining demonstrated significant apoptosis in WFA-treated cells (mean±SD TUNEL scores were 2±1 and 5±2 in control tumors and 77±13 and 63±10 in WFA-treated tumors bearing SKLMS1 and HT1080 xenografts, respectively; P = 0.001 and P = 0.017). Furthermore, WFA induced a significant decrease in tumor-associated blood vessels, and small collapsed CD31-positive vessels were detected in the treated tumors (mean±SD microvascular density [MVD] was 38.2±17.1 and 64.3±21.6 in control tumors and 11.4±7.8 and 6.1±5.8 in WFA-treated tumors of SKLMS1 and HT1080 xenografts, respectively; P = 0.021 and P<0.0001). CD31/TUNEL double immunofluorescence analysis identified apoptotic endothelial cells in WFA-treated tumors. WB analysis of tissue samples revealed vimentin degradation and caspase-3 activation in WFA-treated tumors, confirming the effects of WFA on vimentin in vivo (Figure 8C).
One of the major clinical challenges in STS management is successfully interdicting their enhanced propensity for local recurrence. To evaluate whether WFA can inhibit STS local failure, we established SKLMS1 tumor xenografts and surgically enucleated (macroscopically resected) them when the tumor size reached an average of 1.5 cm in diameter. Two days after tumor resection, mice were allocated to 1 of 2 groups as previously described and observed for tumor recurrence (Figure 8D). Six of 7 mice (86%) in the control group developed tumor recurrence within approximately 3 weeks of surgery, whereas only 2 of 8 (25%) WFA-treated mice experienced local failure within this same time frame (P = 0.02). These results suggest that adjuvant WFA treatment might be potentially useful in abrogating or delaying STS local recurrence.
To evaluate the effect of WFA on metastatic growth, we utilized an experimental MPNST lung metastasis model. STS26T cells were injected into the SCID mice tail veins. Treatment was initiated 10 days after injection, a time point at which 95–100% of mice usually harbor established lung metastasis. The mice were treated with WFA or DMSO as control (n = 10/group) for two weeks. At the termination of the study, the lungs were harvested, weighed, and evaluated for metastases. All control mice exhibited numerous large lung metastases that almost completely replaced the lung parenchyma. In contrast, WFA-treated mice exhibited markedly fewer small lung nodules (Figure 8E). Macroscopic findings were confirmed by H&E staining, demonstrating large lung tumor deposits in control mice and smaller, microscopic lesions in WFA-treated mice. The average±SD lung weight of control mice (635.6 mg±119.1) was significantly higher than that of WFA-treated mice (329.1 mg±114.4; P = 0.011).
These data demonstrate the pronounced and broad anti-STS effects of WFA in vivo. The models utilized represent clinically relevant STS-related scenarios and incorporate a diverse panel of STS histological subtypes. These findings recapitulate the results obtained in vitro, suggesting that WFA treatment results in vimentin degradation and marked apoptosis, targeting both tumor cells and sarcoma-associated endothelial cells. Taken together, our findings strongly support further evaluation of WFA and/or its analogs in STS clinical contexts.
Discussion
Vimentin is one of the most widely expressed mammalian intermediate filament proteins. Its expression commences at E8.5 of mouse development in the primary mesenchymal cells forming the primitive streak [35]. In adults, vimentin is present in all mesenchymal cells and tissues [36] and is frequently used as a marker of differentiation. Like other intermediate filaments, the vimentin network, spreading from the nucleus to the plasma membrane, is believed to act as a scaffold, providing cellular mechano-structural support and thereby maintaining cell and tissue integrity [37].
Studies in genetically engineered mice have previously demonstrated that the vimentin −/− phenotype is rather mild and that the structural role of vimentin is possibly redundant and compensated via other cellular components [38]. This observation is of potential importance if vimentin is to be considered an antitumor molecular therapeutic target as suggested by the results presented here. Moreover, an increasing body of evidence suggests that under cellular stress, functions of vimentin extend well beyond its mechanical and structural properties [39] to include demonstrable roles in adhesion, migration, survival, and cell signaling [40], [41], [42], [43], [44], [45], [46], [47], [48]. For example, vimentin plays a key role in endothelial cell adhesion by regulating integrin functions [49], and it has also been identified as a major contributor to leukocyte transmigration [50], [51].
While the exact mechanisms of vimentin function are not yet fully elucidated, the unique properties described above have been attributed to the dynamic disassembly/assembly and spatial reorganization of vimentin in response several stimuli [35], [52], [53], [54], [55], [56], [57], [58], [59], [60]. Post-translation modifications of vimentin, specifically phosphorylation, are thought to regulate the dynamic states of vimentin [55], [61], [62], [63], [64], [65], [66], [67]. Vimentin contains a highly complex phosphorylation pattern involving a multitude of kinase-specific sites and has been recognized as a substrate for several kinases, including Rho kinase, protein kinase C (PKC), cGMP kinase, Yes kinase, Raf-1 kinase, PAK kinase, and Aurora B kinase [68], [69], [70], [71], [72], [73]. Phosphorylation enhances the disassembly of vimentin into nonfilamentous (monomeric, dimeric, and tetrameric) particles, shifting the equilibrium between polymeric and depolymerized vimentin [74]. For example, in cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation by PAK, resulting in partial disassembly and spatial reorientation of vimentin [75], [76]. In addition, external stress results in vimentin depolymerization in fibroblasts secondary to Rho kinase phosphorylation [70], [77]. The resulting pool of free vimentin may mediate its effects on motility, adhesion, cell signaling, and cell survival. For example, after axonal injury, a soluble form of vimentin has been shown to facilitate activated MAPK transport to the nucleus [78]. Similarly, phosphorylated, disassembled vimentin has been demonstrated to enhance the recycling of integrins subjected to endocytosis to the plasma membrane during cell migration [67].
Taken together, these and other studies raise the possibility that under quiescent conditions, polymeric vimentin maintains cellular integrity, a physiological, “traditional” role that, without vimentin, might be compensated (or performed) by other intermediate filaments. However, under stress and stimulatory conditions, vimentin phosphorylation impairs the steady state of vimentin in favor of increased free, depolymerized vimentin; this “activated” vimentin pool in turn mediates the “nontraditional” vimentin functions.
“Hijacking” of normal physiological processes is a hallmark of cancer. The plethora of described functions of vimentin might contribute to the protumorigenic, prometastatic properties of vimentin-expressing cancer cells, i.e., STS and epithelial cancers featuring EMT [32], [33], [34]. Several studies have demonstrated that vimentin expression levels correlate with poor outcome in epithelial-origin cancers [79], [80], [81], [82], [83]. Recently vimentin knockdown was reported to inhibit vimentin-expressing epithelial cell migration and adhesion [84]. While not previously reported, it is logical that in the context of cancer cells, which are known to express a multitude of activated kinases, the steady state of vimentin will be shifted toward the presence of a large pool of free, depolymerized “activated” vimentin. Our results support this hypothesis, demonstrating a significantly higher level of soluble vimentin in tumor vs. normal cells. In that case, “activated” vimentin might be a novel, unique anti-cancer therapeutic target, as supported by our findings described here.
WFA was previously shown to bind to tetrameric vimentin at a unique pocket between the pair of head-to-tail α-helical dimers [30]. It is possible that free, soluble vimentin—rather than polymerized, filamentous vimentin—is the target of WFA. This notion might offer an explanation for our observation that WFA induces an effect that is selective to vimentin-expressing cancer cells and proliferating stimulated tumor-associated endothelial cells but only minimally affects normal mesenchymal cells under quiescent conditions. Our findings further support previous data demonstrating WFA selectivity to cancer cells rather than normal cells [19], [85], a property of significant potential clinical relevance.
The potential utility of vimentin as an anti-cancer molecular therapeutic target is highlighted by recent findings suggesting marked proapoptotic effects induced by vimentin cleavage [86]. Vimentin undergoes rapid caspase-induced proteolysis (at caspase cleavage sites: Asp85, Asp259, and Asp429; [86] upon diverse proapoptotic stimuli, including ionizing radiation, Fas, TRAIL, TNFα, and tamoxifen administration [87], [88], [89]. This cleavage results in an irreversible disruption of vimentin filaments that precedes the dramatic reorganization of the cytoskeleton that typifies apoptotic cell death. It is possible that vimentin degradation and collapse contribute to many of the morphological manifestations of apoptosis, including cellular rounding, nuclear condensation, and packaging of the debris of dying cells into apoptotic bodies. Furthermore, vimentin degradation releases potential proapoptotic proteolytic fragments that can markedly enhance apoptosis [86]. For example, the generation of a short N-terminal cleavage product (amino acids 1–85) has been shown to play an active proapoptotic role; overexpression of this peptide in MCF-7 cells resulted in significant caspase-dependent apoptosis [86]. A positive feedback loop is suggested, whereby activated caspases induce the cleavage of vimentin and these cleavage products in turn activate caspases to amplify apoptosis.
Our study further demonstrates the role of vimentin in apoptosis. WFA was previously shown to induce marked apoptosis in cancer cells [18], [19], [22], [26], [85], [90]. Data presented here validated these findings and demonstrated that WFA-induced apoptosis is significantly more pronounced in vimentin-expressing cells. Furthermore, vimentin knockdown, as well as inhibition of vimentin degradation (using either caspase inhibition or overexpression of caspase-resistant vimentin), abrogated WFA-induced apoptosis. Taken together, these findings suggest that vimentin may function as a potential double-edged sword for STS and other vimentin-expressing cancers. On one hand, vimentin's inherent properties elicit protumorigenic, prometastatic effects; on the other hand, its cleavage strongly incites proapoptotic signals. Therefore, further evaluation of vimentin as a molecular therapeutic target is warranted.
Whether vimentin is the exclusive target of WFA cannot be ascertained based on the current study, and several additional WFA-induced molecular mechanisms have been previously demonstrated [17], [18], [20], [22]. However, the role of vimentin in WFA-induced toxicity is strongly supported and is reflected in the marked sensitivity of vimentin-expressing cells to the compound when compared to other cancer cells (as evidenced by IC50 values in the nanomolar vs. micromolar range, respectively). Furthermore, it is also possible that several previously identified WFA-induced molecular effects, such as decreased AKT phosphorylation [18], reduced NF-κB activity [31], and proteosomal inhibition [21], are at least in part mediated by vimentin degradation, as reflected in our study. The mechanisms of vimentin effect on these pathways are unknown and merit further consideration.
The relative rarity of STS, compounded by its clinical and molecular diversity, has significantly hampered progress in developing improved therapeutic approaches for this cohort of devastating malignant neoplasms. To our knowledge, the studies presented here are the first to demonstrate the potent anti-STS effects of WFA in vitro and in vivo. We observed these effects in a panel of diverse human complex karyotype STS histological subtypes, suggesting the potential broad applicability of WFA in STS. Furthermore, these effects were independent of p53 mutational status; three of the cell lines tested (SKLMS1, STS26T, and PLS1) harbor mutated p53, whereas HT1080 exhibits wild type p53. This finding is important because p53 mutations are very common in STS, and p53-mutated STS are more therapeutically resistant [91].
Lastly, considering the marked angiogenic nature of STS, our data further supports the antiangiogenic effects of WFA, thereby buttressing previously published studies [17], [30], [92] while suggesting the potential for significant clinical relevance. The likelihood of greater genetic stability in co-opted endothelial cells than in STS tumor cells per se would hopefully lead to their being less likely to acquire chemoresistance during the toxic stress selection of such therapies, raising the possibility that therapies targeting both sarcoma cells and their tumor-associated endothelial cells may lead to improved STS treatment.
Methods
Cell culture and reagents
Human STS cell lines SKLMS1 (leiomyosarcoma) and HT1080 and were purchased from the American Type Culture Collection (ATCC); ST26T cell line (malignant peripheral nerve sheath tumor) was a kind gift from Dr. Steven Porcelli (Albert Einstein College of Medicine, NY, NY; [93]). PLS1 cell line was established in our laboratory (see Data S1 for further information); This study was conducted with institutional review board (IRB) approval from the University of Texas M.D.Anderson Cancer Center and with patient written informed consent. The epithelial cancer cell lines MDA-231 and MCF7 (breast cancer); A549 (lung cancer), HT29 (colon cancer), and TMK1 (gastric cancer) were purchased from ATCC. LEC (murine lung endothelial cells) have been described previously [94]; HDMEC (human dermal microvascular endothelial cells) and NHDF (Normal human dermal fibroblasts) primary cultures were purchased from PromoCell (Heidelberg, Germany. HA-SMC and HC-SMC (human normal smooth muscle cells) primary cultures were purchased from ScienCell Research Laboratories (Carlsbad, CA). All cells were maintained and cultured as per suppliers' recommendations.
Withaferin-A (WFA) was purchased from Chromadex (Irvine, CA). For in vitro studies the drug was dissolved in DMSO and stored in −20°C; for in vivo experiments stock solutions were freshly prepared. The Caspase inhibitor Z-VAD was purchased from Promega (Madison, WI); the inhibitor was dissolved in DMSO and stored in −20°c. Commercially available antibodies were used for immunoblot or immunohistochemical detection of: caspase-3, cleaved caspase-3, cleaved PARP, AKT, phospho-AKT, NF-κB (p65), ubiquitin (Cell Signaling, Beverly, MA); Vimentin (V9), β actin (Santa Cruz Biotechnology, Santa Cruz, CA); PCNA (DAKO A/S, Copenhagen, Denmark); CD31-PECAM-1 (PharMingen, San Diego, CA); peroxidase-conjugated goat anti-rat IgG (Jackson Research Laboratories, West Grove, CA); peroxidase-conjugated rat anti-mouse IgG2a (Serotec, Indianapolis, IN). Stable 3,3′-diaminobenzidine (Research Genetics, Huntsville, AL) and Gill's hematoxylin (Sigma, St. Louis, MO) were used for visualization of IHC reaction and counterstaining, respectively. TUNEL was performed using a commercial apoptosis detection kit (Promega Corp., Madison, WI). Additional reagents are described below.
Cell growth assays
MTS assays: these were conducted using CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega Corp, Madison, WI), per manufacturer's instructions. Absorbance was measured at a wavelength of 490 nm, and the absorbance values of treated cells are presented as a percentage of the absorbance of untreated cells. Drug concentrations required to inhibit cell growth by 50% (IC50) were determined by interpolation of dose-response curves. Colony formation assay: Soft tissue sarcoma cells were treated in culture dishes for 24 h with DMSO (control) and WFA (0.5 µM). One hundred viable cells per well were re-plated and allowed to grow in normal medium for 10 days and then stained for 30 min at room temperature with a 6% glutaraldehyde, 0.5% crystal violet solution. Pictures were captured digitally and colonies were counted. Anchorage independent growth: STS cells were treated with DMSO (control) or WFA (0.5 µM) for 24 h in a 6-well plate. 1×103 viable cells were plated in a 24-well plate in culture medium containing 0.35% agarose overlying a 0.7% agarose layer. Cells were incubated for 3 weeks at 37°C. Cells were stained with p-iodonitrotetrazolium violet (1 mg/ml) for 24 h at 37°. Number of colonies per well were counted. All experiments were repeated 4 times for each cell line.
Apoptosis assays
Apoptosis was measured using the Apoptosis Detection kit I (BD Biosciences) per manufacturers' recommendations. As a standard, 1×106/mL of cells per treatment condition were fixed and stained with 5 µL Annexin V–FITC (BD PharMingen, San Diego, CA) and 5 µL propidium iodide (Sigma, St Louis, MO). Flow cytometric analysis was performed for 1×104 cells and analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ) using a single laser emitting excitation light at 488 nm. Data were analyzed by CellQuest software (Becton Dickson, Franklin Lakes, NJ). To determine apoptosis in cells grown in suspension (Anoikis), 5×105 cells/ml SKLMS1 and NHDF cells were placed in 15 ml conical tubes with filter caps in a total volume of 8 ml complete DMEM/F12 media, and tubes were placed in a continuous rotator in a cell culture incubator with and without WFA (1 µM). After 24 h, 1×106/mL of cells per treatment condition were fixed and subjected to Annexin V/PI FACS as per above.
Western blot analysis
Western blot analysis was performed by standard methods. Briefly, 25 to 50 µg of total proteins extracted from cultured cells were separated by SDS-PAGE and transferred onto nitrocellulose membranes. In case of vimentin, extracted proteins represent both the soluble and polymeric fractions. To further determine expression levels of soluble and insoluble, polymerized vimentin, cells were washed with cold PBS before lysis in a buffer containing 1% Triton X-100 (150 mM NaCl, 50 mM Tris, pH 7.4, 10% glycerol, 1 mM EGTA, 10 mM NaF, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail). Lysate was transferred into centrifuge conical tubes (Beckman) after incubation on ice for 1 hour. Cells were then centrifuged at 200,000 g for 30 min. Supernatants (soluble vimentin) were transferred into new tubes, mixed with 5X sample buffer (312.5 mM Tris, pH 6.8, 50% glycerol, 10% SDS, 25% 2-mercaptoethanol and 0.25% bromophenol blue). The remaining pellets (insoluble vimentin) were denatured in Laemmli sample buffer (62.5 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 5% 2-mercaptoethanol and 0.05% bromophenol blue). All samples were boiled for 10 min before separation on a 10% SDS PAGE. Membranes were blocked and blotted with relevant antibodies. HRPconjugated secondary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences, Pittsburgh, PA). IRdye680-conjugated and IRdye800-conjugated secondary antibodies (Molecular Probes) were detected using Odyssey Imaging (LICOR Biosciences, Lincoln, NE).
Reverse transcription-PCR
RT-PCR was done as previously described [95]. Briefly, total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA) as per manufacturer instructions. Total RNA was reverse-transcribed using superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), and 2 uL of the product were used as templates for multiplex PCR containing both target vimentin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers for normalization. PCR primers were designed using primer 3 software: vimentin, 5′-TCCAGCAGCTTCCTGTAGGT-3′ and 5′-CCCTCACCTGTGAAGTGGAT- 3′; GAPDH, 5′-GAGCCACATCGCTCAGAC-3′and 5′-CTTCTCATGGTTCACACCC-3′. PCR consisted of denaturation for 3 min at 94°C, 28 cycles of denaturation for 30 s at 94°C, annealing for 40 s at 56°C, and an extension for 50 s at 72°C. PCR cycles were terminated by an extension at 72°C for 7 min and products were resolved on a 2% agarose gel.
Transmission electron microscopy
SKLMS1 and STS26T cells were grown on glass coverslips and treated with WFA or DMSO alone for 4 h (5 µM) or 24 h (1 µM) and then fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in cacodylate buffer (pH 7.3) for 1 h. After fixation, the samples were washed and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained en-bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in Spurr's low viscosity medium. The samples were polymerized in a 70°C oven for 2 days. Ultra-thin sections were cut in a Leica Ultracut microtome (Leica, Deerlake, IL), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM-1010 transmission electron microscope (JEOL USA Inc., Peabody, MA) at an accelerating voltage of 80 kV. Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques, Danvers, MA).
Migration and invasion assays
Migration and invasion assays were conducted as described previously [96]. In brief, 2×105 Cells were plated in a 6-well plate and treated for 4 h with WFA (0.5 µM) or DMSO. The drug was washed with PBS and the cell monolayers were carefully wounded with a 200 uL pipette tip. The cells were photographed after 6 h utilizing a light microscope. BioCoat cell culture inserts and polycarbonate filters with 8-µm pores (Becton Dickinson Labware, Franklin Lakes, NJ) in 24-well tissue culture plates were used for modified Boyden chamber migration assays. Lower chamber compartments contained DMEM supplemented by 1% bovine serum albumin or 1% fetal bovine serum as chemoattractants. Cells (5×104) after 4 hr treatment with WFA (0.5 µM) were seeded in the upper compartment and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Invasion assays were conducted similarly using 24-well BioCoat Matrigel invasion chambers with 8-µm pore size polycarbonate filters coated with Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ). After incubation, filters were fixed with 4% formaldehyde and stained with 0.2% crystal violet (Baxter Healthcare, Houston TX). Cells on the upper surface of the filters were removed by wiping with a cotton swab, and migratory and invasive activities were determined by counting the number of cells per high-power field (×200) that had migrated to the lower side of the filter.
Constructs and transfection procedures
Wild-type human vimentin cDNA (pcDNA3-VIM) was a gift from Dr. Vincent Cryns (Northwestern University, Chicago, IL). Site-directed mutagenesis was performed according to the manufacturer's protocol (Stratagene, Cedar Creek, TX) to replace aspartic acid-85 and aspartic acid-259 (vimentin caspase cleavage sites) with aspargine (VIMD85N/D259N). All constructs were confirmed by DNA sequencing. On-TARGET plus SMARTpool siRNA constructs targeting vimentin (cat. no. L-003551-00), as well as non-targeting siRNA, were purchased from Dharmacon (Lafayette, CO). Anti-vimentin antisense phosphorodiamidate Morpholino oligomers targeting the splicing junction between the first exon and second intron of human vimentin pre-mRNA (sequence, TTGCATGGGCGCAGCCTTACTTCTC) were purchased from Gene Tools (Philomath, OR).
Plasmid DNA and siRNA were introduced into cells using Lipofectamine 2000 (Invitrogen) per manufacturer instructions. Briefly, 2×105 cells were plated in each well of a six-well plate and incubated overnight. Cells were then incubated with a mixture of plasmid DNA (4 µg) or siRNA (20–80 nM) and Lipofectamine 2000 (10 µl) diluted in Dulbecco's modified Eagle medium (DMEM) for 24 h, followed by incubation in regular medium. Cells were harvested at indicated time points for specific experiments. Anti-vimentin morpholino oligos and standard non-targeting control morpholino oligos were delivered into cells by Endo-Porter delivery reagent per manufacturer's protocol (Gene Tools). Briefly, 3×105 cells plated in each well of a six-well plate were incubated overnight with regular culture medium. The medium was then replaced with fresh medium containing morpholino oligos (10 µM). After thorough mixing, Endo-Porter reagent (12 µl) was added. Cells were harvested for further studies at indicated time points.
NF-κB reporter assay
The NF-κB dual-luciferase reporter plasmid was purchased from SABioscience (Fredrick, MD) and the procedure was conducted based on manufacture's instructions. STS cells (parental or 24 hr after anti-vimentin siRNA or non-targeting siRNA transfection) were first transfected with the reporter plasmid using sureFECT reagent (SABioscience). After 24–48 hr cells were further treated with WFA or DMSO prior to lysis with passive lysis buffer (Promega, Madison, WI). Lysates were analyzed using the Dual-Glo Luciferase reporter assay system kit (Promega, Madison, WI). Luminescence was measured by a programmed DTX multimode detector (Beckman Coulter). Promoter activity values were measured as arbitrary units using a renilla reporter for internal normalization and were reported as relative luciferase activity as compared to control untreated samples. All experiments were performed in triplicates and the standard deviation was calculated.
Gelfoam angiogenesis assay
These experiments were approved by the MD Anderson Cancer Center Institutional Animal Care and Usage Committee. Gel-foam sponges (Pharmacia & Upjohn, Peapack, NJ) were cut into approximately 0.5×0.5 cm square fragments and saturated overnight in PBS at 4°C. The next day, the sponges were placed on sterile filter paper to allow excess PBS to be drawn out. Sponges were incubated with conditioned media from SKLMS1 cells. The sponges were allowed to sit at room temperature for approximately 1 hour and then implanted subcutaneously into the flank of SCID mice (n = 8), as previously described [97]. Mice were assigned to two treatment groups: WFA (2 mg/kg/once daily) vs. DMSO (control group). After ten consecutive treatment days the gel-foam sponges were harvested and frozen in OCT (Sakura Fineter, Torrance, CA). The frozen samples were later sectioned and probed for CD31 and TUNEL.
In vivo animal models
All animal procedures and care were approved by the MD Anderson Cancer Center Institutional Animal Care and Usage Committee. Animals received humane care as per the Animal Welfare Act and the NIH “Guide for the Care and Use of Laboratory Animals.” Animal models were utilized as previously described [98]. Trypan blue staining confirmed viable STS cells (SKLMS1 and HT1080 1×106/0.1 mL HBSS/mouse) were injected subcutaneously into the flank of six week old female SCID mice (n = 20/experiment), growth was measured twice weekly; after establishment of palpable lesions mice were assigned to two treatment groups (10 mice per group): control (vehicle only) and WFA, i.p. (2 mg/kg/day). Mice were followed for tumor size, well being, and body weight and sacrificed when control group tumors reached an average of 1.5 cm in their largest dimension. Tumors were resected, weighed, and frozen or fixed in formalin and paraffin-embedded for immunohistochemical studies.
To evaluate the effect of WFA on STS local recurrence, SKLMS1 cells (1×106/0.1 mL HBSS/mouse) were injected subcutaneously into the flank of female SCID mice (n = 15). Tumors were left to grow until they reached an average of 1.5 cm at their largest dimension at which time they were resected within their pseudo-capsule, thus mimicking a complete macroscopic resection with positive microscopic margins. Two days after surgical procedure mice were allocated to two treatment groups as per above and were monitored for tumor recurrence.
An experimental lung metastasis STS model was used to evaluate metastases growth. STS26T cells (1×106/0.1 mL HBSS/mouse) were injected into the tail vein of female SCID mice (n = 20). Ten days after injection (a time-point by which 95–100% of mice develop established lung metastases) mice were allocated to two treatment groups as per above. Mice were followed for body weight and well being and sacrificed after two weeks of treatment. Lungs were resected, evaluated macroscopically for tumor load, weighed, and fixed in formalin and paraffin-embedded for immunohistochemical studies.
Immunohistochemistry and TUNEL assays
Immunohistochemistry, immunoflorescence, double immunoflorescence, and TUNEL assays were performed as previously described [99]. Staining scoring was conducted by 2 independent reviewers (GL and AJL). PCNA scoring was determined as the average of the percent of positive immunoreactive cells evaluated by counting tumor cells in 5 high-power fields (x400). For TUNEL scoring, the average number of positive nuclei was calculated in 5 high-power microscopic fields (x400) selected from a central region in viable tumor areas, avoiding areas containing necrosis. Five of the most vascularized areas within a tumor (“hot spots”) identified based on CD31 positivity were chosen at low magnification and vessels were counted in a representative high-power (x400) field in each of these areas. Blood vessel density was calculated as the summation of all counts divided by 5.
Statistics
Cell culture-based assays were repeated at least 3 times and mean ± SD was calculated. Cell lines were examined separately. For outcomes that were measured at a single time point, two-sample t-tests were used to assess the differences. Differences in xenograft growth (tumor/metastases) in vivo were assessed using a two-tailed Student's t-test. Significance was set at P≤0.05.
Supporting Information
Data S1 Supplemetal Materials and Methods
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Figure S1 Isolation and characterization of the human pleomorphic liposarcoma cell line PLS1. A) H+E staining of original human tumor; B) PLS-1 cells morphology in culture; C) PLS1 cells exhibit anchorage independent growth; D) PLS-1harbor an aneuploid karyotype; E) G-banding reveals complex karyotype; F) PLS1 cells grow as xenografts in SCID mice, histomorphology is similar to that of the original human tumor.
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Figure S2 WFA induced morphological changes in human STS and vimentin- expressing epithelial cancer cell lines.
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Figure S3 Human intestinal smooth muscle cells (HC-SMC) are more resistant to the effects of WFA as compared to STS cells. Graphs represent the average of three repeated experiments ±SD.
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Figure S4 WFA abrogates STS cell migration and invasion. A) Wound healing scratch assays demonstrating the effect of WFA on STS cell migration; B) modified Boyden chamber assays depicting the inhibitory effects of WFA on STS cell migration and invasion. Graphs represent the average of three repeated experiments ±SD.
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We thank Dr. Vincent Cryns (Division of Endocrinology, Northwestern University, Chicago, IL) for providing the wild-type human vimentin cDNA, and Dr. Steven Porcelli (Albert Einstein College of Medicine, NY, NY) for the STS26T Cell line. Dr Raphael Pollock is thanked for his critical review, Kenneth Dunner Jr. for assistance with electron microscopy. Kim Vu is thanked for aid in figure preparation and Markeda Wade and Kathryn Carnes for assistance with scientific editing.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was partially supported by NCI/NIH R01 grant CA138345 (to DL) and an Amschwand Sarcoma Cancer Foundation seed grant (to SW). The MD Anderson Cancer Center cell line characterization and cytogenetic Core Facilities are both supported by an NCI Cancer Center Support Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central 1471-2474-11-642037466210.1186/1471-2474-11-64Research articleThe effect of a sports chiropractic manual therapy intervention on the prevention of back pain, hamstring and lower limb injuries in semi-elite Australian Rules footballers: a randomized controlled trial Hoskins Wayne [email protected] Henry [email protected] Macquarie Injury Management Group, Department of Chiropractic, Faculty of Science, Macquarie University, NSW 2109, Australia2010 8 4 2010 11 64 64 27 10 2009 8 4 2010 Copyright ©2010 Hoskins and Pollard; licensee BioMed Central Ltd.2010Hoskins and Pollard; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Hamstring injuries are the most common injury in Australian Rules football. It was the aims to investigate whether a sports chiropractic manual therapy intervention protocol provided in addition to the current best practice management could prevent the occurrence of and weeks missed due to hamstring and other lower-limb injuries at the semi-elite level of Australian football.
Methods
Sixty male subjects were assessed for eligibility with 59 meeting entry requirements and randomly allocated to an intervention (n = 29) or control group (n = 30), being matched for age and hamstring injury history. Twenty-eight intervention and 29 control group participants completed the trial. Both groups received the current best practice medical and sports science management, which acted as the control. Additionally, the intervention group received a sports chiropractic intervention. Treatment for the intervention group was individually determined and could involve manipulation/mobilization and/or soft tissue therapies to the spine and extremity. Minimum scheduling was: 1 treatment per week for 6 weeks, 1 treatment per fortnight for 3 months, 1 treatment per month for the remainder of the season (3 months). The main outcome measure was an injury surveillance with a missed match injury definition.
Results
After 24 matches there was no statistical significant difference between the groups for the incidence of hamstring injury (OR:0.116, 95% CI:0.013-1.019, p = 0.051) and primary non-contact knee injury (OR:0.116, 95% CI:0.013-1.019, p = 0.051). The difference for primary lower-limb muscle strains was significant (OR:0.097, 95%CI:0.011-0.839, p = 0.025). There was no significant difference for weeks missed due to hamstring injury (4 v14, χ2:1.12, p = 0.29) and lower-limb muscle strains (4 v 21, χ2:2.66, p = 0.10). A significant difference in weeks missed due to non-contact knee injury was noted (1 v 24, χ2:6.70, p = 0.01).
Conclusions
This study demonstrated a trend towards lower limb injury prevention with a significant reduction in primary lower limb muscle strains and weeks missed due to non-contact knee injuries through the addition of a sports chiropractic intervention to the current best practice management.
Trial registration
The study was registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12608000533392).
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Background
Australian Rules football is a unique body contact sport. It is played on a natural grass, oval shaped field, with the size varying between 135 - 185 meters in length and 110 - 155 meters in width. Teams consist of 18 players per side plus four on an unlimited interchange bench. Each game is played over four 20 minute quarters plus stoppage time. Physical requirements of players include: repeated rapid acceleration and deceleration efforts often involving change of direction, agility, jumping, bending to pick up the oval shaped ball, tackling and other collisions [1]. There is a continuous nature of play requiring high aerobic capacity, although the speed of the game has increased and now involves a greater number of shorter high intensity play periods and longer stop periods [2]. The most important means of ball progression is by punt kicking. Australian Rules football has the highest rates of non-contact soft tissue injuries when compared with other body contact football codes such as rugby league and rugby union [3], with the incidence of lower limb muscle strains at the elite national competition, the Australian Football League (AFL), being 35% per season [4].
Hamstring injuries are the most prevalent injury in Australian Rules football at the AFL [4] and feature prominently at other levels of play [5]. Per season in the AFL hamstring injuries afflict 16% of players, cause 3.4 missed matches per injury, account for the most time missed due to injury and have the highest rates of injury recurrence, with one in three injuries recurring on return to play [4]. On return to play, player performance is significantly lower [6]. Hamstring injuries are also the most common muscle injury in running based sports [7]. Knowledge surrounding optimal preventative measures is therefore critical.
The prevention of hamstring injuries has long been recognized as a priority effort. By contrast, Bahr and Holme [8] have opined that well designed prospective hamstring injury prevention studies are lacking. Recent literature reviews have been universal in their depiction of the lack of evidence for the prevention of hamstring injuries and the requisite for evidence based approaches to be determined through randomized controlled trials (RCTs) [7,9,10]. Prevention of injury becomes more crucial as the most established predictors for hamstring injury in Australian Rules football are immutable in nature, namely a current or recent history of a hamstring injury and age [11].
Conventional injury prevention has focused on local hamstring factors. Orchard [11] has said that sports medicine dogma advises that poor flexibility, fatigue, lack of warm up and weakness are risk factors for injury. The evidence to support this tenet for hamstring injury is lacking [7]. However, a growing body of literature, largely of an indirect nature, suggests that several non-local hamstring factors may have an association with injury [12-16], whilst a Cochrane systematic review of the literature has stated that consideration should be given to the lumbar spine, sacroiliac and pelvic alignment and postural control mechanisms when managing hamstring injuries [17]. Despite the knowledge that non-local factors may exist, the literature appears almost devoid of research investigating their possible identification and documenting the effects, if any, of addressing non-local factors in hamstring injury management [12,16,18]. A recent review of the literature stated that newer approaches that incorporate manipulation in multi-modal management approaches for hamstring injury prevention should be further investigated [9]. Thus it was the objective of this RCT to investigate whether a sports chiropractic intervention consisting of pragmatically and individually determined high-velocity low-amplitude (HVLA) manipulation, mobilization and/or supporting soft tissue therapies to the spine, pelvis and extremity could reduce local and non-local hamstring injury risk factors to prevent the occurrence of hamstring and other non-contact lower limb injuries and decrease low back pain (LBP) and alter health outcomes in semi-elite Australian Rules footballers.
Methods
Protocol
Four of the thirteen clubs competing in the semi-elite state based Victorian Football League (VFL) were approached and agreed to provide players for this study during the 2005 season. However, a change in club staff resulted in two clubs withdrawing support prior to subject recruitment. VFL players train and play in the same competition as elite AFL players not selected for first grade competition and receive financial remuneration without being full time in their playing and training commitments. Players were eligible to participate if they were listed players on their respective VFL squad and excluded on the basis of: "red flag" conditions including: fractures, infections, inflammatory diseases, tumours and other causes of destructive lesions of the spine; "yellow flag" conditions including: insurance claims, litigation; history of malignant disease; clinical signs suggesting inguinal or femoral hernia; vascular disease; history of motor vehicle accident, or other serious fall or accident in the last three months; neurological signs and symptoms (muscle wasting, nerve root signs, bowel, bladder or sexual dysfunction); organic kidney, urinary tract or reproductive disease; previous recent spinal surgery (less than 2 years); club doctor or medical staff excludes the players participation; severe history of chronic hamstring problems; serious injury or surgery preventing play for the remainder of the season. Before the start of the study the subjects, coaches and medical personal were informed about the purpose and design of the study. Club staff gave permission to participate in the study.
Assignment
Players completed a self-reported questionnaire at their training location prior to randomization. The questionnaire consisted of the validated and reliable McGill Pain Questionnaire (short form) (MPQ-SF) for LBP, the 39 item Health Status Questionnaire (SF-39) as well as self reported questions on knee and hamstring injury history (incidence during the previous month, 6 months, year, 2 years, greater than 2 years or not at all). At each of the two clubs after completion of the baseline questionnaire, players were randomly allocated into one of two groups such that allocation was concealed. Eligible players were stratified by age and hamstring injury history and allocated using a computer generated randomization list for each club within these strata, as these are the most recognized predictors for injury [11]. Randomization was completed within each club to prevent an element of randomness in a clubs injury profile each season impacting on the results of the study. After all subjects had been allocated the two groups at each club were then randomly allocated to either the intervention or control with a coin toss.
Intervention
All of the players in both the intervention and control group continued to receive what is considered the current best practice medical, paramedical and sports science management including medication, manipulative physiotherapy, massage, strength and conditioning and rehabilitation as directed by club staff, which acted as the control. All treatment from club staff was independently administered without restriction or interference from the study authors. All staff were employed by the club and had no limitation in the number or type of treatment they could render. In addition to this, the intervention group received a sports chiropractic approach administered by a single practitioner. Treatment was pragmatically and individually determined by the therapist and could involve HVLA manipulation (either manual or mechanically assisted techniques), mobilization and/or supporting soft tissue therapies: various stretching and soft tissue massage techniques to the spine, pelvis and extremity. According to Mierau et al. [19] manual manipulation involves a brief, shallow, sudden carefully administered thrust (high velocity in nature). Mechanically assisted manipulation is performed through the assistance of devices (for example drop pieces) or impulse type instruments, being non-cavitational and high velocity in intent. Mobilization occurs when a joint is passively moved within its normal range of motion (usually a slow oscillatory movement). Treatment scheduling was also pragmatically determined. The minimum scheduling adhered to was: 1 treatment per week for 6 weeks (phase 1) followed by 1 treatment per fortnight for 3 months and 1 treatment per month for the remainder of the season (3 months) (phase 2).
Outcome Measures
The study was divided into two phases. Phase 1 (6 weeks) involved the late pre-season period where pre-season matches and the intervention commenced but no injury surveillance was conducted. Phase 2 (24 weeks) occurred where regular season (home and away) and finals matches were conducted weekly and an injury surveillance was conducted. The injury surveillance commenced after a period of more intense treatment scheduling such that the treatment effects, if any, would be observed in a changed injury pattern. At the mid point of the season (12 home and away season matches, 18 weeks of intervention) players completed the MPQ-SF and the SF-39 as secondary outcome measures at their training location.
The injury definition and injury surveillance conducted was a reproduction of the AFL's injury surveillance and used as a primary outcome measure for the prevention of hamstring injuries, lower limb muscle strains and non-contact knee injuries [4]. The definition of an injury was: "any physical or medical condition that prevents a player from participating in a regular season (home and away) or finals match". The missed match injury definition is currently considered the most reliable injury surveillance method in team sports [20]. The number of games missed due to injury was also determined. Injury diagnoses were determined by club medical staff who were blinded to group allocation using either clinical features of injury, advanced imaging or both at their discretion with blinded club recorders completing the injury surveillance. Clinical parameters of injury were also recorded including mechanism of injury (contact or non-contact). In this way separation of injuries could be made retrospectively and allocated into groups for statistical analysis. To attain this, the player was interviewed at the first available opportunity following injury. The club medical and coaching staff independently determined selection in matches. There was no interference from the study authors.
In addition a secondary injury surveillance for adverse outcomes resulting from the intervention was established for the duration of the study with an injury definition of: "any undue pain, discomfort or disability arising during, immediately after or subsequent to chiropractic therapy that resulted in missed participation in a match or training session, required additional medical consultation or treatment or was acknowledged by a player as not reasonably being associated or expected with the normal course of treatment". If an injury occurred, further details on the type of injury, timing of symptom onset, duration of symptoms and severity were to be determined.
Statistical Analysis
All data collected were manually entered using Microsoft Excel and analyzed using SPSS for Windows (version 12.0) or for weeks missed due to injury, SAS version 9.1.3 and PROC GENMOD. Pearson's "exact" Chi-squared test based on Monte Carlo simulation was used to assess the efficacy of the intervention with respect to the number of injuries. Odds ratios and 95% confidence intervals were also included. As such this calculation is just an approximation and is included as it is believed that confidence intervals should always be stated [21]. Negative binomial models were used to calculate significance for weeks missed due to injury. Two independent sample t-tests were used to compare group age, hamstring and knee injury history, MPQ-SF and SF-39 at baseline, or if distributions were mixed, Fisher's exact test was used. Repeated measures and regression models were used to determine change for the MPQ-SF and SF-39. If data were extremely skewed in distribution, transformation of scores was required. Between group differences were obtained from two independent sample t-tests. For global statistical tests, a p value < 0.05 was considered significant.
Statistical power calculation
Based on historical AFL data [4] the assumed hamstring incidence level for the null hypothesis is 15%. For a 5% significance level and 80% power, a total sample size of 117 is required to detect a 50% reduction in the incidence of hamstring injuries.
Ethical considerations
All players gave their written informed consent to participate and ethical approval was obtained from the Macquarie University Human Ethics Committee (Ethics Approval Number: HE27AUG2004-RO3066).
Results
Participants
Sixty male Australian Rules football players were recruited as subjects. Figure 1 shows a flow chart describing progress of subjects through the trial for the primary and secondary outcome measures. Players were randomly allocated to the intervention (n = 29) or control group (n = 30) with no baseline differences for age (mean/SD/range intervention 20.2/1.8/18-27, control 20.2/1.8/18-25), self-reported hamstring and knee injury history, MPQ-SF and SF-39 (all p > 0.05).
Figure 1 CONSORT flow chart indicating progress of subjects through the trial.
Injury Surveillance
Table 1 presents the results for the difference in injury incidence between the groups at the completion of the season (24 matches, 30 weeks of intervention). There was no statistical difference in the prevention of hamstring injuries (p = 0.051) or weeks missed due to hamstring injury (χ2 1.12, p = 0.29). For primary hamstring injuries, the incidence was 3.6% for the intervention group and 17.2% for the control group, with the recurrence rate being 40.0%. The intervention group missed 4 matches due to hamstring injury and the control 14 matches. The intervention group was at a statistically significant reduced risk of suffering a primary lower limb muscle strain injury (p = 0.025), equating to 3.6% of the intervention group and 27.6% of the control group. The intervention group missed 4 matches with a lower limb muscle strain and the control group 21 matches (χ2 2.66, p = 0.10). The difference in primary non-contact knee injury incidence was not statistically significant (p = 0.051), with the incidence being 3.6% for the intervention group and 24.1% for the control group. The intervention group missed 1 match with a primary non-contact knee injury and the control group 24 matches, the difference being statistically significant (χ2 6.70, p = 0.01). No players reported an adverse reaction to the intervention.
Table 1 Difference between the intervention and control group for injury incidence at the completion of the season (24 matches, 30 weeks of intervention)
Injury Intervention incidence
(n = 28) Control incidence
(n = 29) P value Odds ratio 95% CI
Hamstring injury 1 7 0.051 0.116 0.013-1.019
1° Hamstring 1 5 0.191 0.178 0.019-1.631
2° Hamstring 0 2 - - -
1° Lower limb muscle strain 1 8 0.025* 0.097 0.011-0.839
1° Non-contact knee 1 7 0.051 0.116 0.013-1.019
* Bold type face indicates significant difference
Low Back Pain
Table 2 presents the results for the change in baseline MPQ-SF at the mid point of the season. A positive and statistical significant change for the intervention group was achieved for overall (p = 0.006) and current LBP (p = 0.026). No significant change was noted for the other components of the MPQ-SF (p > 0.05).
Table 2 Lower Back Pain (as measured by the MPQ-SF): estimated marginal means for baseline and eighteen weeks by group and estimated change within and between groups
Variable Baseline 18 weeks Δ within groups Δ between groups
intervention control intervention control intervention control mean p-value
Current mean 33.26 32.71 21.44 34.36 -11.81 1.64 -13.46 .026
95% CI 24.00, 42.52 23.62, 41.81 12.77, 30.12 25.84, 42.88 -22.90, -0.73 -8.8, 12.14 -28.35, 1.44
Overall mean 26.67 22.86 17.04 27.86 -9.63 5.00 -14.63 .006
95% CI 20.54, 32.80 16.84, 28.88 10.67, 23.41 21.60, 34.11 -16.35, -2.91 -3.07, 13.07 -24.93, -4.33 (.034)1
Sensory mean 12.57 15.26 13.02 18.20 0.45 2.94 -2.49 .461
95% CI 8.56, 16.59 11.32, 19.20 8.02, 18.03 13.29, 23.11 -3.67, 4.57 -2.54, 8.43 -9.24, 4.25
Affective mean 4.95 4.76 8.33 10.72 3.38 5.96 -2.58 .411
95% CI 1.78, 8.12 1.65, 7.87 3.96, 12.70 6.43, 15.01 -0.73, 7.48 1.07, 10.85 -8.84, 3.6
Total mean 10.53 12.56 11.81 16.19 1.28 3.63 -2.35 .436
95% CI 7.0, 14.06 9.10, 16.02 7.24, 16.38 11.70, 20.67 -2.57, 5.12 -1.14, 8.40 -8.36, 3.66
* Bold type face indicates significant difference between groups at 18 weeks.
1 Regression analysis showed a significant difference between groups: p-value calculated using regression analysis.
Health Status
Table 3 presents the results for the change in baseline SF-39 at the mid point of the season. A positive statistical change for the intervention group was achieved for role limitations due to physical health (p = 0.004), bodily pain (p = 0.034), general health (p = 0.027), and physical summary score (p = 0.013). No other statistically significant change was noted for other health status components (p > 0.05).
Table 3 Health status (as measure by the SF-39): estimated marginal means for baseline and eighteen weeks by group and estimated change within and between groups
Variable Baseline 18 weeks Δ within groups Δ between groups
intervention control intervention control intervention control mean p-value
Physical functioning % at 100 65.50 60.00 55.60 33.30 -2.412 -4.262 1.852 .569
95%CI 48.20, 82.80 42.47, 77.53 36.86, 74.34 15.52, 51.08 -5.72, 0.90 -11.59, 3.07 -6.08, 9.79
Role limitation-physical % at 100 72.40 80.00 85.20 40.70 9.262 -18.522 27.782 .004
95%CI 56.13, 88.67 65.69, 94.31 71.81, 98.59 22.17, 59.23 -2.43, 20.94 -31.84, -5.20 10.48, 45.08
Bodily pain Mean 69.67 72.56 74.22 65.26 4.56 -7.30 11.85 .034
95%CI 63.68, 75.66 66.57, 78.55 67.43, 81.01 58.47, 72.05 -4.09, 13.21 -14.36, -0.24 0.95, 22.75
General health Mean 81.30 79.00 83.96 74.52 2.67 -4.48 7.15 .027
95%CI 76.51, 86.08 74.22, 83.78 78.51, 89.42 69.06, 79.97 -2.53, 7.86 -10.27, 1.31 -0.45, 14.74
Vitality Mean 60.37 61.11 67.04 59.44 6.67 -1.67 8.33 .050
95%CI 53.90, 66.84 54.64, 67.59 61.67, 72.40 54.08, 64.81 -0.57, 13.90 -9.15, 5.81 -1.83, 18.49
Social functioning % at 100 51.70 53.30 58.10 44.40 0.462 -5.092 5.562 .770
95%CI 33.51, 69.89 35.45, 71.15 39.49, 76.71 25.66, 63.14 -4.76, 5.68 -11.56, 1.38 -2.56, 13.67
Role limitation-emotional % at 100 75.90 83.30 92.60 66.70 8.642 -6.172 14.812 .142
95%CI 60.33, 91.47 69.95, 96.65 82.73, 102.47 48.92, 84.48 -0.75, 18.03 -16.54, 4.20 1.16, 28.47
Mental health Mean 77.63 77.93 76.59 71.11 -1.04 -6.81 5.78 .151
95%CI 72.50, 82.76 72.80, 83.06 71.61, 81.57 66.13, 76.09 -6.60, 4.52 -12.78, -0.85 -2.18, 13.74
Physical summary score Mean 52.66 52.03 53.80 49.06 1.15 -2.97 4.12 .013
95%CI 50.63, 54.68 50.00, 54.06 51.76, 55.85 47.01, 51.10 -1.14, 3.43 -5.36, -0.58 0.89, 7.35
Mental summary score Mean 50.04 50.55 51.41 48.48 1.37 -2.07 3.45 .103
95%CI 47.21, 52.87 47.72, 53.38 49.00, 53.83 46.06, 50.89 -1.38, 4.12 -5.34, 1.19 -0.72, 7.61
Depression % at 100 58.60 73.30 85.20 59.30 9.892 -3.702 13.592 .050
95%CI 40.67, 76.53 57.47, 89.13 71.81, 98.59 40.77, 77.83 1.87, 17.90 -12.15, 4.74 2.23, 24.96
* Bold type face indicates significant difference between groups at 18 weeks.
Intervention
Table 4 provides a description of the treatment rendered to the intervention group for the course of the study.
Table 4 Description of the treatment rendered to the intervention group
Intervention group (n = 29)
Number of treatments 487 (mean per player 17)
Amount of manipulation and/or mobilization to joint regions 2000 (47% total treatment, mean 4 per treatment)
Location of manipulation and/or mobilization Thoracic spine 21%, knee 18%, hip 18%, lumbar spine 15%, sacroiliac joint 12%
Manipulation and mobilization breakdown HVLA manipulation only 56%, HVLA manipulation and mobilization 36%, Mobilization only 8%
Amount of soft tissue techniques to soft tissue regions 2258 (53% total treatment, mean 4 per treatment)
Location of soft tissue techniques Gluteal region 22%, lumbar spine 12%, hip flexors 10%, knee 9%, posterior thigh 6%
* Soft tissue structures are defined as surrounding the involved joint (muscle, tendon, ligament, fascia etc.)
Discussion
This RCT demonstrated that a sports chiropractic manual therapy intervention provided at the semi-elite level of Australian Rules football in addition to the current best practice multi-disciplinary medical, paramedical and sports science management resulted in the prevention of primary lower limb muscle strain injuries, although no statistical significance was noted for hamstring injury and primary non-contact knee injury. The addition of the intervention was associated with a reduced number of matches missed due primary non-contact knee injury, although no statistical significance was noted for hamstring injury and primary lower limb muscle strains. In addition, reduction in LBP was observed along with improvements in some aspects of the physical components of health status as measured by the SF-39. Treatment was predominantly directed at non-local to hamstring areas, which supports the view that several non-local factors may potentially contribute to hamstring and lower limb injury occurrence [7,9], which may be addressed through multimodal and multidisciplinary management [16]. These findings are important due to their potential for injury reduction, performance benefit and cost saving practices for a relatively low cost intervention.
There are limitations in the presented study. Because the required subject numbers as determined by the power analysis was not achieved, care is needed in the interpretation of the results. The late withdrawal of two clubs reduced the subject numbers recruited and meant that the required target of subject numbers would not be reached. Due to the late withdrawal it was decided to continue with the study. However, the number of subjects determined by the power analysis is based on an arbitrary determined effect size, and the numbers required would have been different if another effect size had been chosen. Moreover, the results that are presented report statistical significance and it is difficult to determine what difference in the raw figures would be clinically significant. As the level of significance for prevention of hamstring injuries and primary non-contact knee injuries was p = 0.051, given that the study was short of the number of subjects required by the power analysis, there is a strong likelihood of a type 2 error, especially considering how close each of these results were to p < 0.05. With regards to the fact that lower limb muscle strain injury incidence was significantly lower while the missed weeks was not, this implies that many minor grade strain injuries may have been prevented, but the one injury causing 4 missed matches skewed the results and meant the comparison would not be statistically significant. This is important in a small sample study such as the prevention of one serious injury (or not) can significantly alter the weeks lost profile of a particular treatment approach. Only studies with much larger sample sizes can really effectively confirm this important research observation.
Furthermore, a question could be raised regarding control group selection. It was felt that using the club based best practice medical, paramedical and sports science management as the control was valid because no change in hamstring injury rates using this same approach have been documented in the AFL's long running injury surveillance [4]. Corroborating this viewpoint, the hamstring injury incidence reported for the control group (17%) was very similar to that reported in AFL players (16%) using the same methodology. Difficulty arises in attempting to perform research on high-level professional or semi-professional athletes due to clubs not being overly enthusiastic for researchers to perform interventions on their contracted and paid players, particularly if the intervention to be performed is purely for control purposes. To counter this dilemma a pragmatic approach to research design was taken which created a further limitation in that subjects were not blinded to group allocation, meaning it cannot be ruled out that the intervention effect was due purely to placebo or Hawthorne effects, particularly as there was no blinding of the therapist. However, more modern research design often requires new interventions to be compared with the existing best practice approach [22], which was done in this study.
The injury surveillance for adverse reactions to treatment may be limited due to the subjectivity of aspects of the injury definition. Players may not have self-reported injury. If injury was delayed or transient, players may not have attributed injury to the intervention, instead attributing it to training/competition activities or other medical, paramedical or sports science management. Such a problem exists in any multi-modal management scenario. Conversely, given the reliability of the missed match injury definition [20], it is highly unlikely that a more severe injury resulting in loss of competition match play was missed.
The diagnosis of hamstring strains is usually made on clinical grounds [23]. Hamstring strains are commonly diagnosed through history (acute onset, non-contact mechanism) and examination (local tenderness, reproducible pain on straight leg raise testing and/or resisted knee flexion) [24]. In professional sport, MRI assessment is often used to support the clinical diagnosis and provide further assessment of the extent and severity of the injury. However, costs and availability preclude the use of this modality for routine assessment outside of professional sport. Additionally, both clinical examination and MRI findings are strongly correlated with the time required to return to competition, suggesting MRI is not required for estimating the duration of rehabilitation of an acute minor or moderate hamstring injury [25]. MRI imaging to confirm diagnosis of hamstring strains was not routinely performed in this study. There are limitations in relying on both clinical methods of diagnosis and MRI as hamstring injuries can appear clinically but not on MRI and they also may appear on MRI but not clinically [25]. As MRI was not routinely used, there is a possibility that some of the hamstring injuries in this study may have been MRI negative which are often considered "back related". There is some controversy regarding "back related" hamstring injuries as to whether a muscle strain is the cause, particularly for minor strains where causes for the pain may include referred pain from neuromeningeal or myofascial structures such as the lumbar spine and sciatic nerve or from nearby muscles such as the gluteal and pirifomis [23]. However, "back related" hamstring injury is an undefined term generally signifying both local hamstring signs and positive lumbar signs [23]. It should be noted that none of the hamstring injuries in the study had positive lumbar signs present at the time of diagnosis, but the lack of MRI diagnosis remains as a limitation of the study.
The intervention applied in this study was based largely on indirect evidence and speculative reasoning that local and non-local factors could potentially contribute to hamstring injury, which have been suggested to act as a guide to a complete prevention program [9]. Similar hypotheses could be made regarding other lower limb muscle strain injuries and non-contact knee injuries. As a uni-modal approach was not adopted to address a single risk factor, it is unclear as to what the specific mechanism of improvement was or what component of the protocol resulted in injury prevention. The multi-modal intervention was decided upon on the basis that it more accurately represents sports chiropractic clinical practice [26-28], and because sports injuries, including hamstring injuries, result from a complex interaction of multiple risk factors and events, of which only a fraction have been identified [8]. For the reversible risk factors that exist for hamstring injury, no definitive evidence exists to support them [7]. It has been suggested that waiting for a substantial body of evidence to exist to support a risk factor in its role in injury before conducting a RCT may be considered unethical [8].
Whilst a multi-modal approach was adopted, we speculate that the most significant difference between the control and the intervention groups was the inclusion of a significant amount of HVLA manipulation, as soft tissue therapies were habitually administered to the athletes in this cohort. Although data were not recorded in this study, manipulation if used by manipulative physiotherapists (as in the control group) has a tendency to be slow velocity or mobilization in nature and if HVLA techniques are rendered they are characteristically done so sparingly [29]. In the paper by Flynn et al. [29] they state that in the previously reported low back pain literature high velocity spinal manipulation utilization rates for low back pain to be between 2.8% and 8.9%, with rates in a heavily evidence based education system to be 36.2%. Alternatively, in the cited studies low velocity mobilization is used between 27.2% and 72.0% of the time. Despite these figures being the most up to date yet published, these figures represent United States, Ireland and United Kingdom physiotherapists and the figures may not be representative of current practice in those geographical locations or in Australian physiotherapists in particular. In contrast, the sports chiropractic intervention provided to the intervention group had a greater emphasis on performing HVLA manipulative techniques to both spinal and extremity joints, with 92% of total joint based treatment involving some form of HVLA manipulation technique. Future research would benefit from recording the nature of the control interventions in order to clarify the differences between interventions or to specifically address the role of HVLA based manipulative techniques. Future studies could specifically document the scope of the manual treatment delivered by all treating practitioners in both groups, which would assist in comparing outcomes. A criticism of manual therapy interventions is that its effects are short term in nature. Because of this, it was decided that an ongoing treatment approach with adequate spacing of treatments during the season would be applied. This would also best manage ongoing injury and sub-clinical micro-trauma or gradual onset injury that could occur to players over the course of the season. The decision on the minimum scheduling of treatment decided upon for the intervention group was made such that there would be a likely treatment effect. Treatment scheduling in this pragmatic arrangement was then based upon current and previous player medical history, examination findings, practicality, player preference and practitioner experience. As the intervention was provided by a single practitioner, this removed issues associated with inter-practitioner reliability. As mentioned in the results there was an average of 17 treatment consultations administered per player in the intervention group, but due to the pragmatic nature of the design, not all players received the same amount of treatment.
Sherry and Best [18] have suggested that neuromuscular control of the lumbopelvic region is needed to create optimal function of the hamstrings. They further suggest that changes in neuromuscular control could lead to changes in length tension relationships or force-velocity relationships of the hamstrings, predisposing injury. This hypothesis could extend to other muscle groups including quadriceps and groin muscles. Other authors have also hypothesized that dysfunction of the axial skeleton may predispose abnormal hamstring functioning that may relate to a greater incidence of injuries [7,9,16], which is supported by evidence documenting lumbopelvic factors as risk factors for hamstring injury [12-15]. Supporting this mechanism of injury is the large body of literature showing that LBP is associated with changes in lumbopelvic muscle activation and recruitment [30,31], including early activation of biceps femoris and alteration in neuromuscular control strategies [32], all of which could contribute to injury. In athletes, changes in lumbopelvic stabilization exist following clinical recovery of LBP [33]. Noteworthy is the high prevalence, frequency and severity of LBP occurring in the subjects recruited for this study [34].
Although the neurophysiological mechanisms underlying HVLA manipulation are not fully known or understood [35], evidence exists showing it is capable of stimulating muscle spindles, pacinian corpuscles and golgi tendon organs greater than that achieved by slow velocity mobilization [36]. Panjabi [37] has hypothesized that injured spinal mechanoreceptors may alter afferent input, effecting motor unit recruitment. Alterations in the recruitment of motor units of the deep lumbopelvic muscles may result in altered lumbopelvic stabilization strategies and insufficient force generated by the hamstrings and other muscles attached to the pelvis, or may result in excessive force production, causing subsequent injury. Alterations in hamstring motor units may also occur. Stimulation of mechanoreceptors by HVLA manipulation may improve afferent feedback required to update and modify motor functions. This may improve neuromuscular control of the lumbopelvic region and/or the coordination of hamstring and pelvic muscle function, preventing injury. In support of such a view, Solomonow et al. [38] have demonstrated that discharge of spinal proprioception can produce change in multifidus activation. Additionally, HVLA spinal manipulation has been shown to produce significant improvements in feed forward activation times of deep abdominal musculature [39], whilst case reports have shown it may improve the ability to perform transversus abdominus [40] and multifidus contraction [41]. Collectively these deficiencies have been found to be associated with LBP, with transverses abdominus and multifidus being key stabilizers in lumbopelvic stabilization [30,42]. Studies have also indicated that HVLA manipulation may improve muscle function through either facilitation or disinhibition of neural pathways [35]. These effects, combined with spinal manipulation improving hamstring strength [12], and increased joint mobility through mechanical stretching and neurophysiological mechanisms [35], may have lead to improvements in hamstring and other lower limb muscle functioning and subsequent injury prevention noted in this study. Due to the complex multi-factorial etiology underlying hamstring and lower limb muscle injury, it is probable that more than one possibly interacting mechanism occurred to prevent injury. Additionally, the targeted inclusion of soft tissue therapies and extremity joint mobilization and manipulation stretching soft tissues and improving joint mobility may have potentially contributed to injury prevention.
The trend towards reduction in primary non-contact knee injuries and significant improvements in weeks missed due to these injuries may appear surprising. However, recent literature has documented the more precise details of the biceps femoris anatomy, which have not previously been appreciated [43]. The authors hypothesized that there may be a synergistic effect between biceps femoris and popliteus, signifying bicep femoris' important role in knee joint stabilization [43]. This may highlight the important bidirectional inter-play between hamstring and knee function. Thus, soft tissue treatments delivered to the popliteus and knee region and HVLA manipulation to the knee may have assisted with knee function and therefore led to prevention of knee injury. Lastly, research has shown that HVLA spinal manipulation can reduce knee extensor inhibition associated with anterior knee pain [44], which could lead to improvements in knee function and injury prevention. Of interest, LBP has also been associated with inhibition of the knee extensor muscles [45], which could imply a link between lumbopelvic and knee function. The potential role of the knee in hamstring injury has been discussed elsewhere in further detail [7].
Due to constraints in manuscript size, we are unable to describe the entire treatment provided in this study or to speculate on all proposed mechanisms of improvement, which will be the subject of a subsequent publication. It should be noted that treatment provided was patient specific and addressed both kinetic and kinematic chain variables. The treatment was representative of the 'modern multimodal' (MMM) chiropractic approach that has been described in the sports chiropractic literature [26,27], and recommended in selecting a chiropractor for the management of athletic injuries [28].
Conclusions
Based on the limitations of this study which includes a low sample size, this RCT demonstrated that a sports chiropractic intervention comprising significant amounts of HVLA manipulation and soft tissue therapies provided in addition to the current best practice medical, paramedical and sports science management appears to be beneficial for the prevention of lower limb muscle strain injuries, weeks missed due to primary non-contact knee injuries and reduction of LBP and improvement in physical components of health status. In addition, although not statistically significant, there was a trend towards prevention of hamstring and primary non-contact knee injuries and there were no reported adverse outcomes from the intervention. The interesting trend in results but non-statistically significance should be replicated using a larger sample size to remove the short comings of this study. Based on the findings of this study due consideration should be given for the inclusion of sports chiropractic in the management options of elite athletes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WH and HP conceived the idea of the study. WH performed recruitment of subjects, data entry and the intervention. WH and HP contributed to writing draft documents and the final manuscript. Both authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2474/11/64/prepub
Acknowledgements
We would like to acknowledge the statistical assistance provided by Ann Eyland and Don McNeil and the participation and assistance of the staff and players at the Northern Bullants and the Coburg Tigers football clubs.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2043667110-PONE-RA-16346R110.1371/journal.pone.0010350Research ArticleCell Biology/Cell SignalingCell Biology/Neuronal Signaling MechanismsDevelopmental Biology/Stem CellsOncology/Neuro-OncologyUpregulation of PTEN in Glioma Cells by Cord Blood Mesenchymal Stem Cells Inhibits Migration via Downregulation of the PI3K/Akt Pathway Stem Cells Upregulate PTENDasari Venkata Ramesh
1
Kaur Kiranpreet
1
Velpula Kiran Kumar
1
Gujrati Meena
2
Fassett Daniel
3
Klopfenstein Jeffrey D.
3
Dinh Dzung H.
3
Rao Jasti S.
1
3
*
1
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
2
Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
3
Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
Lesniak Maciej EditorThe University of Chicago, United States of America* E-mail: [email protected] and designed the experiments: VRD JR. Performed the experiments: VRD KK KKV. Analyzed the data: VRD MG DF JDK DHD JR. Contributed reagents/materials/analysis tools: JR. Wrote the paper: VRD.
2010 26 4 2010 5 4 e1035016 2 2010 1 4 2010 Dasari et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumor suppressor gene implicated in a wide variety of human cancers, including glioblastoma. PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Most human gliomas show high levels of activated Akt, whereas less than half of these tumors carry PTEN mutations or homozygous deletions. The unique ability of mesenchymal stem cells to track down tumor cells makes them as potential therapeutic agents. Based on this capability, new therapeutic approaches have been developed using mesenchymal stem cells to cure glioblastoma. However, molecular mechanisms of interactions between glioma cells and stem cells are still unknown.
Methodology/Principal Findings
In order to study the mechanisms by which migration of glioma cells can be inhibited by the upregulation of the PTEN gene, we studied two glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310) alone and in co-culture with human umbilical cord blood-derived mesenchymal stem cells (hUCBSC). Co-cultures of glioma cells showed increased expression of PTEN as evaluated by immunofluorescence and immunoblotting assays. Upregulation of PTEN gene is correlated with the downregulation of many genes including Akt, JUN, MAPK14, PDK2, PI3K, PTK2, RAS and RAF1 as revealed by cDNA microarray analysis. These results have been confirmed by reverse-transcription based PCR analysis of PTEN and Akt genes. Upregulation of PTEN resulted in the inhibition of migration capability of glioma cells under in vitro conditions. Also, wound healing capability of glioma cells was significantly inhibited in co-culture with hUCBSC. Under in vivo conditions, intracranial tumor growth was inhibited by hUCBSC in nude mice. Further, hUCBSC upregulated PTEN and decreased the levels of XIAP and Akt, which are responsible for the inhibition of tumor growth in the mouse brain.
Conclusions/Significance
Our studies indicated that upregulation of PTEN by hUCBSC in glioma cells and in the nude mice tumors downregulated Akt and PI3K signaling pathway molecules. This resulted in the inhibition of migration as well as wound healing property of the glioma cells. Taken together, our results suggest hUCBSC as a therapeutic agent in treating malignant gliomas.
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Introduction
Despite many advances in the treatment of malignant glioblastoma via surgery, radiotherapy and chemotherapy, patients afflicted with this disease continue to have a very poor prognosis [1]–[3]. Malignant glioblastoma is characterized by rapid cell proliferation, high invasion and genetic alterations [4]–[6]. A number of genetic alterations are involved in oncogenesis, including deactivation of tumor suppressor genes such as PTEN (phosphatase and tensin homologue deleted on chromosome ten) [7]. PTEN is a tumor suppressor gene implicated in a wide variety of human cancers and is a major negative regulator of the PI3K/Akt signaling pathway. Most human glioblastomas show high levels of activated Akt, whereas less than half carry PTEN mutations or homozygous deletions. There are several lines of evidence implicating PTEN in the regulation of cellular migration and invasion. It has also been suggested that PTEN may regulate cell migration by directly dephosphorylating FAK in the DBTRG-05MG glioblastoma cell line [8]. PTEN plays a significant role in inducing G1 cell cycle arrest and apoptosis, along with regulating cell adhesion, migration and differentiation [9], [10]. Dey et al. [11] studied glioma cell migration on vitronectin, which binds αvβ3 integrin, and showed that PTEN's protein phosphatase activity negatively regulated RAC1 indirectly by regulating the activity of the SRC-family kinase, FYN. Recent insights into the biology of gliomas include the finding that tyrosine kinase receptors and signal transduction pathways play a role in tumor initiation and maintenance [12]. PTEN is a tumor suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Davidson's recent data provides a novel tool to address the significance of PTEN's separable lipid and protein phosphatase activities and suggests that both activities suppress proliferation and both activities are required in concert to achieve efficient inhibition of invasion [13]. However, it is not clear whether PTEN genuinely regulates cell migration, tumor invasiveness and metastasis in vivo using the mechanisms and pathways defined by in vitro systems [14].
Recent studies have indicated that mesenchymal stem cells (MSCs) have the capacity to target therapeutic genes to malignant glioma [15]–[17]. Human umbilical cord blood is a rich source of both hematopoietic stem cells and MSCs [18], [19]. Stem cells derived from umbilical cord show higher proliferation and expansion potential than adult bone marrow stem cells [20], [21]. Human umbilical cord-derived mesenchymal stem cells (hUCBSC) have been regarded as an alternative cell source for cell transplantation and cell therapy because of their hematopoietic and non-hematopoietic (mesenchymal) potential [19], [22], [23].
To study the mechanisms by which migration of glioma cells can be inhibited by the upregulation of PTEN gene, we used two glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310) alone and in co-culture with hUCBSC. We evaluated whether hUCBSC are capable of inhibiting the migration capability of glioma cells both in vitro and in vivo, and whether this effect is mediated by downregulation of the PI3K-Akt pathway.
Results
Co-culture of glioma cells with hUCBSC upregulates PTEN
For all the experiments of this study, we used hUCBSC which are positive for CD29 and CD81, as confirmed by immunocytochemistryand FACS analyses (data not shown). To evaluate the efficiency of hUCBSC, we tested the effect of hUCBSC on glioma cells in co-cultures. All of the four glioma cell lines of the present study were co-cultured with hUCBSC for 3 days and the total RNA was extracted and reverse-transcribed to cDNA. We ran cDNA microarrays for PI3K-Akt pathway as described in Materials and Methods. We found that PTEN is upregulated many times over in the four tested cell lines. Many genes related to PI3K-Akt pathway were downregulated in SNB19, U251 and 4910 cells, whereas most of the genes in 5310 cells were brought down to normal levels. The upregulation of PTEN gene was correlated with the downregulation of numerous genes including Akt, JUN, MAPK14, PDK2, PI3K, PTK2, RAS and RAF1 as revealed by cDNA microarrays (Table 1). To check for PTEN expression levels, we carried out immunofluorescence assays with the PTEN antibody and found that with hUCBSC treatment, significant upregulation of PTEN took place in all of the glioma cells in the present study (Fig. 1A). This indicates that hUCBSC upregulated PTEN in glioma cells. To confirm these results, we checked the expression of PTEN, Akt and PI3K at both the transcriptional and translational levels. In both cases, PTEN was upregulated in hUCBSC-treated cancer cells whereas Akt, phospho-Akt and PI3K were downregulated as compared to control cells (Figs. 1B and 1C). To evaluate whether hUCBSC undergo any changes after co-culturing with glioma cells, hUCBSC were grown in conditioned media of glioma cells and observed for changes in PTEN expression at both transcriptional and translational levels. We did not observe any significant changes in the levels of PTEN in hUCBSC grown in glioma conditioned media (Figs. 1D and 1E). These results confirm that hUCBSC upregulates PTEN in glioma cells and shows a negative effect on PI3K and Akt levels as well as the phosphorylation status of the AktSer473 molecules.
10.1371/journal.pone.0010350.g001Figure 1 Upregulation of PTEN in hUCBSC-treated co-cultures of glioma cells.
(A) Fluorescent microscopic images demonstrate PTEN expression (red fluorescence). SNB19, U251, 4910 and 5310 cells were co-cultured with hUCBSC for 3 days and processed for immunofluorescence. Immunostaining was performed with Alexa flour-594 conjugated PTEN antibodies. All sections were stained with DAPI to show nuclear localization. Insets show DAPI. n ≥ 3. Scale bar = 100 µm. (B) Equal amounts of protein (40 µg) from single cultures and co-cultures were loaded onto 12% SDS gels and transferred onto nitrocellulose membranes, which were then probed with respective antibodies. GAPDH was used as a positive loading control. (C) Reverse transcription-based PCR analysis of PTEN, Akt and PI3K in co-cultures. β-actin was used as a positive loading control. Each blot and gel is representative of experiments performed in duplicate with each sample (n = 3). hUCBSC were grown in conditioned media from hUCBSC (control), SNB19, U251, 4910 and 5310 conditioned media for 3 days and the lysates (80 µg for each lane) were subjected to (D) Western analysis for PTEN or (E) Reverse-transcription based PCR analysis. CM = conditioned medium.
10.1371/journal.pone.0010350.t001Table 1 Effect of hUCBSC on glioma cells after co-culture (cDNA microarray results of PI3K-AKT pathway).
Gene Description
Fold up or down regulation SNB19 U251 5310 4910
AKT1/PKB V-akt murine thymoma viral oncogene homolog 1
−4.53
−2.35
−1.12
−4.00
FOXO1 Forkhead box O1
−3.43
−3.81
1.03
−6.96
JUN Jun oncogene
−18.13
−3.10
−2.01
−13.93
MAPK14 Mitogen-activated protein kinase 14
−1.72
−3.32
−1.69
−10.56
P110 (PIK3CA) Phosphoinositide-3-kinase, catalytic, alpha polypeptide
−7.89
1.77
1.14
−6.06
P27, KIP I (CDKN1B) Cyclin-dependent kinase inhibitor 1B (p27, Kip1)
−4.23
−1.30
1.45
−10.56
PAK1 P21 protein (Cdc42/Rac)-activated kinase 1
−38.85
−3.43
1.06
−13.00
PDGFRA Platelet-derived growth factor receptor, alpha polypeptide
1.09
−5.39
−1.08
1.52
PDK2 Pyruvate dehydrogenase kinase, isozyme 2
−41.64
−10.06
−1.47
−48.50
PI3K (PIK3CG) Phosphoinositide-3-kinase, catalytic, gamma polypeptide
−6.87
−1.26
1.61
−4.29
PIK3R2 Phosphoinositide-3-kinase, regulatory subunit 2 (beta)
−13.74
−4.08
1.67
−22.63
PTEN Phosphatase and tensin homolog
7.06
2.17
2.13
3.73
PTK2 PTK2 protein tyrosine kinase 2
−11.16
−2.35
−1.01
−16.00
RAS (RASA1) RAS p21 protein activator (GTPase activating protein) 1
−19.43
−1.66
1.36
−5.66
RAF1 V-raf-1 murine leukemia viral oncogene homolog 1
−15.78
−5.98
−2.08
−4.00
Human PI3K-Akt PCR arrays (SA Biosciences) were run using cDNA from single and co-cultures of glioma cells with hUCBSC. Real time PCR was carried out and changes in gene expression were illustrated as a fold increase/decrease according to manufacturer's instructions. The cut-off induction determining expression was 2.0 or −2.0 fold changes. Genes that met these criteria were considered to be upregulated or downregulated.
Conditioned media from co-cultured glioma and hUCBSC inhibits spheroid migration
To determine the effects of PTEN upregulation in glioma cells, we performed the spheroid migration assay using conditioned media from co-cultured glioma and hUCBSC cells. The spheroid model is a three-dimensional cell culture system that more closely resembles the in vivo situation inside a tumor [24]. Spheroid growth reflects the proliferation of tumor cells, while the migration assay measures the ability of the cells organized in a three-dimensional structure to migrate and proliferate [25]. The cell migration away from the spheroid was monitored over a period of 24 h to 48 h by photographing the mid plane of the spheroids at intervals of 24 h with an inverted Olympus phase contrast microscope. In conditioned media of untreated glioma cells, the cells from spheroids started migrating as early as 24 h, whereas in conditioned media of co-cultures, spheroid migration was delayed significantly, even after 48 h (Fig. 2A). We observed that spheroid migration was significantly inhibited in 5310 cells (51.19%) followed by 4910 (47.86%), U251 (41.95%) and SNB19 (41.4%) cells (Fig. 2B). In order to prove that PTEN is responsible for the inhibition of spheroid migration, glioma cells were grown in conditioned media from single and co-cultures. Similar to co-culture cell lysates, glioma cells grown in conditioned media also showed upregulated PTEN (Fig. 2C). In another experiment, spheroids were transfected with siRNA to PTEN (siPTEN) and then grown in conditioned media from co-cultures. We did not observe any significant change in the inhibition of spheroid migration (Figs. S1A and S1B). These results prove that upregulation of PTEN in glioma cells by conditioned media from co-cultured glioma and hUCBSC cells inhibit spheroid migration.
10.1371/journal.pone.0010350.g002Figure 2 Conditioned medium from co-culture of glioma cells with hUCBSC inhibits spheroid migration.
(A) SNB19, U251, 4910 and 5310 cells were cultured in 96-well low attachment plates at a concentration of 5×104 cells, and spheroids were allowed to grow for 24 h at 37°C with shaking at 40–60 rpm. The spheroids were then transferred to 48-well plates and maintained for another 24–48 h in conditioned media from single cultures and co-cultures. Spheroid migration was analyzed using a phase-contrast microscope. Scale bar = 1000 µm. (B) Quantitative analysis of spheroid migration from (A). Error bars indicate SEM. *p <0.05. **p <0.01. n = 3. (C) Immunoblot analysis of PTEN from glioma cells grown in their respective control conditioned media and co-culture conditioned media.
Inhibition of the wound healing capacity of glioma cells by hUCBSC
Glioma cells, in general, have very good wound healing capacity. In order to evaluate the effect of hUCBSC on wound healing, we checked the wound healing capacity of glioma cells in single cultures and co-cultures with hUCBSC. A wound was made in a sub-confluent cell monolayer and cells were allowed to migrate into the cell-free area. The distance moved by the cells in control and co-cultured plates, respectively, was compared. The mobility of glioma cells was inhibited in co-cultures compared to single cultures. We observed that SNB19 and U251 cells repair their wounds in 8 h; 4910 cells heal in 9 h and 5310 cells heal in 7 h. In co-cultures, hUCBSC inhibited this wound healing capacity (Fig. 3A). The wound healing capacity is significantly inhibited in 4910 cells (63.75%) as compared to U251 (53.74%), 5310 (50.99%) and SNB19 (49.99%) cells (Fig. 3B). In order to confirm, whether inhibition of wound healing is by soluble factors present in the conditioned media of co-cultures or due to cell-to-cell contact between glioma cells and hUCBSC, we performed another experiment with conditioned media from single cultures and co-cultures. Compared to complete media, glioma cells took long time to repair the wounds in conditioned media. For example, in SNB19 cells wound healing was observed after 23 h in conditioned medium compared to 8 h in complete medium. Similarly, wound healing was complete after 24 h in U251, after 21 h in 4910 and after 22 h in 5310 cells. However, glioma cells grown in hUCBSC conditioned medium and co-cultured conditioned medium did not show any inhibition of wound healing (Fig. S2A and S2B). Overall, these results indicate that cell-to-cell contact between hUCBSC and glioma cells is necessary which significantly decreased wound healing capacity of glioma cells.
10.1371/journal.pone.0010350.g003Figure 3 Monolayer wound-induced migration assay.
A line was scratched with a 200-µm plastic pipette tip in SNB19, U251, 5310 and 4910 cultures and co-cultures with hUCBSC. They were allowed to grow at 37°C in 5% CO2 atmosphere. Every three hours, cells that had migrated to the wounded areas were photographed under a microscope for quantification of cell migration. Images are representative of three separate experiments. Scale bar = 500 µm. (B) Quantitative analysis of wound-induced migration assay from (A). The results are presented as mean ± SEM of three experiments done in duplicate. *p <0.05. **p <0.001.
Upregulation of PTEN by hUCBSC treatment has an anti-tumor effect in U251 and 5310 glioma nude mice models
Our in vitro experiments have proved that co-culture with hUCBSC can efficiently inhibit glioma cell migration and wound healing capacity. Therefore, we further investigated the anti-tumor effect of these stem cells in vivo using U251 and 5310 cells in nude mice. After the mice were implanted with U251, 5310 and hUCBSC as described in Materials and Methods, the mice were observed for 21 days. At that point, tumor samples were taken, and paraffin-embedded sections were prepared for immunohistopathological examination. Hematoxylin and Eosin (H&E) staining of the in vivo sections clearly showed that the tumors in hUCBSC-treated mice were inhibited significantly and were one-third of the size of the tumors in control mice brains (Fig. 4A). U251 tumors were much bigger in size and more invasive than 5310 tumors. Next, we checked for the presence of hUCBSC in tumor areas of the brains of both control and hUCBSC-treated mice brain sections by immunofluorescence. In hUCBSC-treated mice brain tissue, the presence of hUCBSC as confirmed by mesenchymal stem cells markers CD29 and CD81, clearly establishes the fact that hUCBSC are responsible for tumor size reduction observed in the hUCBSC-treated brains (Figs. 4B and 4C). These CD29 and CD81 positive cells were observed in tumor regions only. We could not detect them in normal areas of the brains. Further, we evaluated PTEN expression in nude mice brain sections by colocalization studies using PTEN and CD81 antibodies. Nude mice brains treated with hUCBSC clearly show that PTEN was highly upregulated in hUCBSC-treated mice (Fig. 4C). CD81 was observed in hUCBSC-treated mice sections only and they were absent in control tumor sections. Colocalization of PTEN and CD81 in hUCBSC-treated mice confirms that the upregulation of PTEN was the result of presence of hUCBSC. It is plausible that hUCBSC not only upregulated PTEN in glioma cells which are in contact with them but also in surrounding glioma cells. We also evaluated whether upregulation of PTEN in mice brains had any effect on X-linked inhibitor of apoptosis protein (XIAP) levels in tumor brains. For this, we did DAB immunohistochemistry on both control tumor and stem cell-treated brains. Tumor brains show high levels of XIAP expression, whereas hUCBSC-treated brains show reduced levels of XIAP expression (Fig. 4D). This confirms that upregulation of PTEN in tumors significantly decreased the levels of XIAP as to induce the cellular death of the glioma cells.
10.1371/journal.pone.0010350.g004Figure 4 Inhibition of intracranial tumors by hUCBSC in vivo.
(A) Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with hUCBSC by intracranial injection (2.5×105). Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with Hematoxylin and Eosin (n≥3). Inset pictures show higher magnification at scale bar = 100 µm. (B) Characterization of hUCBSC in tumor areas in nude mice brain sections: Fourteen days after hUCBSC administration, the brains were harvested, sectioned and immunoprobed with mesenchymal stem cell markers CD29 and CD81 using Alexa flour-594 secondary antibody. (n ≥ 3). Scale bar = 100 µm. (C) Upregulation of PTEN in nude mice: Mice brain sections were immunoprobed with PTEN and CD81 using appropriate fluorescence-conjugated secondary antibodies. Secondary antibodies used for PTEN and CD81 were: goat anti-mouse Alexa flour-594 for PTEN and donkey anti-goat Alexa Fluor 488 for CD81, respectively. Scale bar = 100 µm. (D) Downregulation of XIAP in mice: Mice brain sections were probed with XIAP antibody by DAB immunohistochemistry and counterstained with DAPI. Scale bar = 100 µm. Inset pictures show DAPI. (n = >3).
Further, to understand the molecular mechanisms of PTEN-induced tumor regression, we evaluated the tissue lysates of both control tumor brains and hUCBSC-treated tumor brains by immunoblotting. Similar to the in vitro results, the expression levels of PTEN is upregulated, whereas Akt, p-Akt, FAK and XIAP in tumor specimens from hUCBSC-treated mice were prominently downregulated (Fig. 5A). Next, we assessed the mRNA expression of PTEN, XIAP, FAK and PDGFR. All of these genes were highly downregulated after the hUCBSC treatment with the upregulation of PTEN (Figs. 5C and 5D).
10.1371/journal.pone.0010350.g005Figure 5
In vivo expression of PTEN and other signaling proteins.
Equal amounts of protein (40 µg) from tissue lysates of untreated and treated mice brains were loaded onto 10–14% SDS gels and transferred onto nitrocellulose membranes, which were then probed with respective antibodies. GAPDH was used as a positive loading control. (A) PTEN, Akt, pAkt, FAK and XIAP proteins with respect to GAPDH. (B) Western blot analysis of PI3K pathway related proteins. (C) Reverse transcription-based PCR analysis of PTEN, XIAP, FAK and PDGFR in brain tissue lysates. Each blot or gel is representative of experiments performed in duplicate with each sample (n = 3). (D) Real-Time PCR analysis of FAK, PTEN, XIAP and PDGFR genes of in vivo samples. n = 3. Error bars indicate SEM. *p <0.05.
Signaling through the PI3Ks is frequently activated in many human cancers, including glioblastoma, because of loss of PTEN. Deletion or loss of PTEN function leads to failure to convert PIP3 back to PIP2, resulting in the deregulation of PI3K in the absence of upstream signals from receptor tyrosine kinases. Hence, we determined to check the expression of proteins related to the PI3K/Akt pathway (e.g., PI3K, RhoA, RAC1, CDC42 and PDGFR). We found that all were downregulated in mice brains treated with hUCBSC (Fig. 5B), showing that PTEN upregulation is inhibiting the PI3K/Akt pathway, thereby regulating the growth of tumor cells. These results confirm the anti-tumor effect of hUCBSC in vivo and that tumor cell migration in vivo is efficiently regulated by hUCBSC.
Discussion
Malignant glioblastoma is a highly invasive tumor of the central nervous system. Currently available therapies offer only limited benefit for patients with glioblastoma. As such, there is an immediate necessity to develop new therapeutic approaches and to better understand the molecular pathogenesis of glioblastoma. PTEN mediates many of its effects on proliferation, growth, survival and migration through its PtdIns(3,4,5)P3 lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways [26]. Re-expression of PTEN in mammalian cells lacking the enzyme has been found to inhibit the motility of several lineages of such cells, including mouse embryo fibroblasts and tumor-derived cells of glial and prostate origin [8], [27], [28], although most of these studies have not addressed the mechanism of action of PTEN. In this study, we investigated the effects of hUCBSC on the effects of migration of glioma cells by upregulation of PTEN. We observed that the stem cells are able to upregulate PTEN simultaneously downregulating the PI3K/Akt pathway and inhibiting the growth and migration of the cancer cells.
The Akt signaling pathway is very important in glioblastoma multiforme (GBM) progression, and this pathway is activated in the majority of primary GBM samples [29], [30] as well as in xenografts derived from GBM tumor samples [31]. Akt represents a nodal point in cell signaling and can be activated by several upstream events, including epidermal growth factor receptor amplification or mutation, loss of PTEN, and PIK3CA mutation. As such, targeting this pathway may be able to block glioblastoma multiforme proliferation secondary to a variety of upstream etiologies [32]. We observed a similar rate of Akt pathway activation in all four GBM cell lines used in this study (Fig. 1). When these glioma cells are co-cultured with hUCBSC, upregulation of PTEN has been observed with concomitant downregulation of Akt and phosphorylated form of Akt.
Phosphatidyl-inositol-3-kinase (PI3K) can phosphorylate and activate Akt while PI3K is negatively regulated by the tumor suppressor gene PTEN, which has been shown to be non-functional in 20 to 40% of GBM [33]–[36]. Akt is over-activated in many glioblastomas due to the loss of PTEN function [37], [38]. Akt regulates the function of numerous downstream signaling proteins involved in cell cycle, proliferation, apoptosis and invasion, which are all important to tumorigenesis [39]. Activated Akt deregulates cell growth by stabilization of cyclin D and promotion of the nuclear entry of MDM2, leading to the degradation of p53 [40]. Akt might also inhibit p21 expression through its phosphorylation and activation of MDM2 and subsequent downregulation of p53-mediated transcription of p21 [41], [42]. On the other hand, activated Akt exerts anti-apoptotic activity by phosphorylating and inactivating pro-apoptotic signaling proteins, such as BAD and caspase 9 [39], [43]. The involvement of Akt in diverse tumorigenic activities suggests that Akt activation alone might be sufficient to induce cancer [44]. Moreover, Akt activation may contribute to tumor invasion/metastasis by stimulating secretion of matrix metalloproteinases [45]. Therefore, dysregulation of the PI3K/Akt signaling pathway may play an important role in tumor development and progression.
In the present study, hUCBSC are able to upregulate PTEN under both in vitro and in vivo conditions. This upregulation of PTEN decreased the expression of Akt and its phosphorylated form pAkt. Also, hUCBSC treatment resulted in the downregulation of XIAP, which is highly upregulated in glioma cells, both in vitro and in vivo. This in turn resulted in the dysregulation of PI3K/Akt signaling pathway and ultimately inhibited the survival of glioma cells. In addition, hUCBSC treatment downregulated PDGFR and Akt genes at the transcriptional and translational levels, this resulted in the inhibition of glioma migration. In order to understand the anti-cancer effect of hUCBSC, we examined their effect on migration and wound healing capacity of glioma cells. Through our experiments, we confirmed that hUCBSC effect on glioma cells decreased the levels of phosphorylated Akt, which alters both cell migration and wound healing capacity. Tumor invasion requires both tumor cell migration and the degradation of the extracellular matrix [46]. Cell motility is one of the crucial points of metastasis which is necessary for the tumor cell to move through the matrix and enter the circulation so that it can travel to a distant site [47]. In our study, we demonstrated that hUCBSC significantly reduced the migration of glioma cells from the spheroids (Fig. 2). Wound-healing assays showed a slower wound-closure following co-culture with hUCBSC treatment, indicating decreased glioma cell motility (Fig. 3).
In our studies, we observed that PTEN upregulation accompanied XIAP downregulation. Previous studies support a role for XIAP in negatively regulating PTEN content in vitro and in vivo
[48]. Decrease of XIAP protein levels in colon cancer cells in response to the apoptosis-inducing agent mesalazine is accompanied, among others, by an increase of PTEN content [49]. Exposure of mice to the apoptosis-inducing agent rosiglitazone modestly upregulates PTEN protein levels in HCT116-XIAP+/+ cell-derived tumors but markedly increases PTEN content in HCT116-XIAP−/− cell-derived tumors [50]. Recently, it was proposed that XIAP acts as an E3 ubiquitin ligase for PTEN and promotes Akt activity by regulating PTEN content and compartmentalization [48]. Akt is one of the anti-apoptotic factors that must be activated through phosphorylation. The phosphorylation of Akt has previously been shown to be promoted by XIAP, another anti-apoptotic protein dictating the fate of normal and cancer cells [51], [52]. Our results are in accordance with the previous reports that downregulation of XIAP did result in the downregulation of Akt and inhibition of glioma cell growth.
Activation of PI3K occurs commonly in cancers including glioblastoma, the most common primary brain tumor. Activation of PI3K is associated with increased metabolism, suggesting potential dependence of cancer cells on PI3K signaling, and raising the possibility that blockade of PI3K signaling in glioma should effectively kill these cells [53]. PTEN encodes a phosphatase that dephosphorylates phosphatidylinositol-3,4,5 triphosphate to convert it to phosphatidylinositol-4, 5 bisphosphate. Therefore, inactivation of PTEN leads to increased levels of phosphatidylinositol-3,4,5 triphosphate and increased Akt activation [33]. Conversely, restoration of PTEN leads to inhibition of Akt. Recently, it was reported that survival times were significantly reduced in patients whose tumors showed PI3K pathway activation [54]. In our study with hUCBSC, we observed that PTEN upregulation decreases levels of PI3K and its associated signaling molecules, along with increased Akt inactivation. This ultimately resulted in the regression of tumor growth in nude mice brains.
There have been no reports regarding the possible mechanisms by which hUCBSC are capable of upregulating PTEN in glioma cells. In our in vivo studies we observed that CD81, a mesenchymal stem cell marker, is co-localized with PTEN in glioma regions of the brain. This suggests that hUCBSC are in contact with the glioma cells and are able to upregulate PTEN in glioma cells in their vicinity. These results support previous reports from our laboratory, which show that cell-to-cell contact between hUCBSC and glioma cells is necessary to induce apoptosis in glioma cells by hUCBSC (58). Taken together, our results demonstrate that PTEN is an important component of the PI3K/Akt signaling pathway. The growth retardation of glioblastoma cells treated with hUCBSC is not only due to the reduction in migration and wound healing capability, but also due to the downregulation of Akt and PI3K pathway-related genes. Targeting PTEN using hUCBSC may be an effective new strategy for the molecular therapy of human cancers.
Materials and Methods
Ethics Statement
After obtaining informed consent, human umbilical cord blood was collected from healthy volunteers according to a protocol approved by the Peoria Institutional Review Board, Peoria, IL, USA. The consent was written and approved. The approved protocol number is 06–014, dated December 10, 2009. The Institutional Animal Care and Use Committee of the University Of Illinois College Of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. The consent was written and approved. The approved protocol number is 851, dated November 20, 2009.
Culture of glioma cell lines
Two high-grade human glioma cell lines (SNB19 and U251) and two xenograft cell lines (4910 and 5310) were used for this study. SNB19 and U251 cells lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). Two xenograft cell lines (4910 and 5310) were kindly provided by Dr. David James at University of California, San Francisco. SNB19 and U251 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. Xenograft cell lines (4910 and 5310) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. For all the cells, medium was replaced every 2 days. In experiments with conditioned medium, medium was replaced every day.
Isolation and culture of hUCBSC
After obtaining informed consent, human umbilical cord blood was collected from healthy volunteers according to a protocol approved by the Peoria Institutional Review Board, Peoria, IL, USA. Human umbilical cord blood was enriched by sequential Ficoll density gradient purification. Next, we selected cells using CD29+ and CD81+ markers as described previously [55]. Briefly, the nucleated cells were suspended at a concentration of 1×106/ µL in Mesencult medium (Stem cell Technologies, Vancouver, Canada) supplemented with 20% fetal bovine serum (FBS) (Hyclone, Logan, UT), 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) and plated in 100-mm culture dishes. The cells were incubated for three days and the non-adherent cells were removed with medium replacement. After the cultures reached confluency, the cells were lifted by incubation with 0.25% trypsin and 1 mM ethylene diamine tetraacetic acid (EDTA) at 37°C for 3 to 4 min. Cells were diluted at a ratio of 1∶3 and replated and cultured at 37°C in an incubator with a 5% CO2 atmosphere. For co-culture experiments, hUCBSC and glioma cells were cultured at a ratio of 1∶4. Co-cultures were grown in the medium in which single glioma cells were grown. For all the cells, medium was replaced every 2 days. In experiments with conditioned medium, medium was replaced every day.
Spheroid migration assay
Spheroid migration was assayed as described previously [56] with some modifications. Spheroids of SNB19, U251, 5310 and 4910 cells were prepared by seeding a suspension of 5×104 cells in their respective media on ultra low attachment 96-well plates and cultured until spheroid aggregates formed. Single glioma spheroids were placed in the center of each well of a 0.5% agarose-coated 48-well microplate and 200 µL of conditioned media of single cultures and co-cultures with hUCBSC was added to each well. Spheroids were incubated at 37°C for 48 h, after which the spheroids were fixed and stained with Hema-3 (Fisher Scientific, Pittsburgh, PA) and photographed. The migration of cells from spheroids to monolayers was quantified using a microscope calibrated with a stage and ocular micrometer and represented graphically.
Transfection of siPTEN
The glioma cells were cultured as mentioned previously. siRNA to PTEN was obtained from Cell Signaling Technology (Danvers, MA). Cells at 60–70% confluency in 100 mm tissue culture plates were transfected with 100 nM of siPTEN and control siRNA using Fugene HD as per manufacturer's instructions (Roche, Indianapolis, IN). Following transfection, after 60–72 h depending on the cell line, cell lysates were assessed for expression levels of PTEN using western blot analysis as per standard protocols.
Wound healing assay
These experiments were done in either single and co-cultures in complete media or in conditioned media of single and co-cultures. Glioma cells (1×106) were seeded in a 100-mm culture plate and then cultured to at least 95% confluence. In a similar fashion, glioma cells were co-cultured with hUCBSC. Monolayer cells were washed with their respective media and then scraped with a plastic 200 µL pipette tip and then placed back in a 37°C incubator. The “wounded” areas were photographed by phase contrast microscopy at various time points (0, 3, 6, 8, 9, 10, 12, 21, 22, 23 and 24 h after scraping) depending on the cell line. The relative migration distance was calculated by the following formula: the relative migration distance (%) = 100 (A–B)/A, where A is the width of cell wounds before incubation, and B is the width of cell wounds after incubation. Results are expressed as the mean ± SEM.
Immunocytochemistry
Cultured hUCBSC were checked for mesenchymal markers by immunocytochemistry. Cultured cells plated in 2-well chamber slides were rinsed twice with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde. After additional PBS rinses, cells were blocked with 0.1 M PBS with 1% bovine serum albumin (BSA) for 1 h. Primary antibodies (1∶100 dilutions) specific for mesenchymal markers: mouse anti-CD29 (Millipore, Danvers, MA) and goat anti-CD81 (Santa Cruz Biotechnology, Santa Cruz, CA) and primary antibody specific for PTEN were diluted in goat serum and applied overnight at 4°C. Texas-Red conjugated anti-mouse or anti-goat secondary antibodies were diluted (1∶200) in goat serum and applied individually for 1 to 2 h at room temperature. Before mounting, the cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI). The cells were observed using a fluorescence microscope (Olympus IX71, Olympus, Melville, NY) and/or a confocal microscope (Olympus Fluoview, Olympus, Melville, NY) and photographed.
RNA extraction and quantitative real time PCR
All primer sequences were determined using established human GenBank sequences. Primer sequences were designed using Primer3 software (v.0.4.0). For real time polymerase chain reaction (RT-PCR) analysis and RT-PCR-based microarray analysis (RT2 Profiler PCR Array, SuperArray, Frederick, MD), total RNA was isolated from control and hUCBSC-treated cancer cells. Total cellular RNA was extracted using RNeasy kit (Qiagen, Valencia, CA), and RNA quality was determined by running a sample with RNA loading dye on a 1% agarose gel and checking for distinct 18S and 28S rRNA bands, indicating lack of degradation. Quantity of RNA was determined by A260 measurement. We used RNA whose A260∶A280 ratio is greater than 2.0. Samples were either used immediately or frozen at −80°C until use in RT-PCR. Total RNA was reverse transcribed into first strand cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Each cDNA was tested by running PCR using GAPDH and β-actin primers as a control for assessing PCR efficiency and for subsequent analysis by 2% agarose gel electrophoresis. PCR amplification was performed using the primer sets, amplified by 35 cycles (94°C, 1 min; 60°C, 1 min; 72°C, 1 min) of PCR using 20 pM of specific primers. Further quantitative analysis of genes was done by SYBR green based real-time PCR using Bio-Rad iCycler iQ Real-Time PCR Detection System. Each sample was measured in triplicate and normalized to the reference GAPDH or β-actin gene expression. The value of each well was determined and the average of the three wells of each sample was calculated. For samples that showed no expression of the test gene, the value of minimum expression was used for statistical analysis. Delta CT (ΔCT) and ΔΔCT values were calculated and the fold change in the test gene expression was finally calculated. A statistical evaluation of real-time PCR results was performed using one-way analysis of variance (ANOVA) to compare test gene expression between cancer cells and their co-cultures with hUCBSC.
Primers used for PCR
FAK Sense 5′ggtgcaatggagcgagtatt3′
Antisense 5′gccagtgaacctcctctga3′
PTEN Sense 5′ccaggaccagaggaaacct3′
Antisense 5′gctagcctctggatttga3′
Akt Sense 5′catcacaccacctgaccaa3′
Antisense 5′ctcaaatgcacccgagaaat3′
PI3K Sense 5′cccctccatcaacttcttca3′
Antisense 5′cggttgcctactggttcaat3′
XIAP Sense 5′ggccagactatgcccattta3′
Antisense 5′cgaagaagcagttgggaaa3′
PDGFR Sense 5′ctctgacggccatgagtaca3′
Antisense 5′catgatcttcagctccgaca3′
β-Actin Sense 5′gtcgtaccactggcattgt3′
Antisense 5′cagctgtggtggtgaagct3′
cDNA microarray analysis
We used PI3K-Akt pathway finder RT2 Profiler PCR Array (SuperArray Biosciences, Frederick, MD) because of its advantage of real-time PCR performance combined with the ability of microarrays to detect the expression of many genes simultaneously. Each array contains a panel of 96 primer sets of 84 relevant, pathway-focused genes, plus five housekeeping genes and three RNA and PCR quality controls. Real-time PCR was carried out under the following conditions: one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. Data were exported to Excel files and analyzed using SuperArray RT2 Profiler PCR Array Data Analysis Template (v3.0). Relative gene expression levels were calculated based on the ratio of the mean of housekeeping signals of all experiments. The formula used to calculate the relative gene expression level (2 ∧ (-Δ Ct)) in the “Results” worksheet is: Δ Ct = Ct (GOI) – avg. (Ct (HKG)), where GOI is each gene of interest, and HKG are the housekeeping genes chosen for the “Sample-Control Gene” worksheet. Scatter plots were made from normalized signals. Changes in gene expression were illustrated as a fold increase/decrease. The cut-off induction determining expression was 2.0 or −2.0 fold changes. Genes, which met these criteria, were considered to be upregulated or downregulated. We performed these experiments in duplicate.
Immunoblot analysis of proteins
Single and co-cultures of glioma cells or nude mice brain tissues were harvested and homogenized in four volumes of homogenization buffer (pH 7.4; 250 mM sucrose, 10 mM HEPES, 10 mM Tris-HCl, 10 mM KCl, 1% NP-40, 1 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF plus protease inhibitors: 1 µg/mL pepstatin, 10 µg/mL leupeptin and 10 µg/mL aprotinin) using a Teflon-fitted glass homogenizer. The homogenate was centrifuged at 20,000 g for 15 min at 4°C, and the protein levels in the supernatant were determined using the BCA assay (Pierce, Rockford, IL). Samples (40–50 µg of total protein/well) were subjected to 10–14% SDS-PAGE and transferred onto nitrocellulose membranes. The following antibodies were used for Western blot analysis: mouse anti-PTEN (1∶200; Santa Cruz Biotechnology Inc, Santa Cruz, CA), mouse anti-Fak (1∶500; Santa Cruz Biotechnology Inc), goat PI3K (1∶500; Santa Cruz Biotechnology Inc.), mouse anti-XIAP (1∶5000; BD Biosciences, Franklin Lakes, New Jersey), rabbit anti-AKT (1∶1000; Cell Signaling Technology), mouse anti-phospho-AKT (Ser473) (1∶1000; Cell Signaling Technology) mouse anti-Rho-A (1∶200; Santa Cruz Biotechnology Inc), rabbit anti-CDC42 [Phospho-Rac1/cdc42 (Ser71) antibody (1∶1000; Cell Signaling Technology)], mouse anti-PDGFR (1∶1000; Cell Signaling Technology), and mouse anti-RAC1(1∶1000; BD Biosciences). The membranes were blocked with 5% nonfat skim milk in PBS for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. The membranes were then processed with horse-radish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive bands were visualized using chemiluminescence ECL Western blotting detection reagents (Amersham, Piscataway, NJ). Immunoblots were stripped and re-developed with GAPDH antibody [mouse anti-GAPDH (1∶1000; Novus Biologicals, Littleton, CO)] to ensure equal loading levels. Experiments were performed in triplicates. Values for treated and untreated samples were compared using one-way ANOVA. A p value of <0.05 was considered significant.
Intracranial tumor growth
The Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. U251 (1×106 cells) and 5310 (8×105 cells) tumor cells were intracerebrally injected into the right side of the brains of nude mice, as described previously [57]. Seven days after tumor implantation, the mice were injected with hUCBSC near the left side of the brain. The ratio of the hUCBSC to cancer cells was maintained at 1∶4. Three weeks after tumor inoculation, six mice from each group were sacrificed by cardiac perfusion with 4% formaldehyde in PBS, their brains were removed, and paraffin sections were prepared. Sections were stained with H&E to visualize tumor cells and to examine tumor volume. The sections were blind reviewed by a neuropathologist and scored semiquantitatively for tumor size. Whole-mount images of brains were also taken to determine infiltrative tumor morphology. The average tumor area per section integrated to the number of sections where the tumor was visible was used to calculate tumor volume; tumor volumes were compared between controls and treated groups. RT-PCR was done on fresh brain tissue for FAK, PTEN, XIAP, PDGFR and β-actin.
Immunohistochemical analysis
Brains of control and hUCBSC-treated mice brains were fixed in formaldehyde and embedded in paraffin as per standard protocols. Sections were deparaffinized as per standard protocol. Sections were blocked in 1% BSA in PBS for 1 h, and the sections were subsequently transferred to primary antibody diluted in 1% BSA in PBS (1∶100). Sections were allowed to incubate in the primary antibody solution overnight at 4°C in a humidified chamber. Sections were then washed in 1% BSA in PBS, incubated with the appropriate secondary antibody for 1 h and visualized using a confocal microscope. Transmitted light images were obtained after H&E staining as per standard protocol to visualize the morphology of the sections. For immunofluorescence, sections were treated with primary antibodies overnight at 4°C and then treated with appropriate Alexa flour secondary antibodies at room temperature for 1 h. Negative controls were maintained either without primary antibody or using IgG.
Statistical analysis
Quantitative data from cell counts, Western blot analysis, and other assays were evaluated for statistical significance using one-way analysis of variance (ANOVA). Data for each treatment group were represented as mean ± SEM and compared with other groups for significance by one-way ANOVA followed by Bonferroni's post hoc test (multiple comparison tests) using Graph Pad Prism version 3.02, a statistical software package. Results were considered statistically significant at a p value less than 0.05.
Supporting Information
Figure S1 Spheroid migration in siPTEN transfected spheroids. (A) SNB19, U251, 4910 and 5310 cells were cultured in 96-well low attachment plates at a concentration of 5×104 cells, and spheroids were allowed to grow for 24 h at 37°C with shaking at 40–60 rpm. The spheroids were then transferred to 48-well plates and were transfected with siPTEN for 60 h and then grown in conditioned medium from co-cultures and maintained for another 24–48 h. Spheroid migration was analyzed using a phase-contrast microscope. Scale bar = 1000 µm. (B) Quantitative analysis of spheroid migration from (A). Error bars indicate SEM. n = 3. Control = without any treatment and grown in glioma conditioned media; siPTEN = transfected with siPTEN and grown in glioma conditioned media; siPTEN + (Glioma cells +hUCBSC) = transfected with siPTEN and grown in conditioned media from glioma cells + hUCBSC.
(7.50 MB TIF)
Click here for additional data file.
Figure S2 Monolayer wound-induced migration assay in conditioned media. A line was scratched with a 200-µm plastic pipette tip in SNB19, U251, 4910 and 5310 cultures. They were allowed to grow at 37°C in 5% CO2 atmosphere in conditioned media of glioma cells, hUCBSC and co-cultures. Every three hours, cells that had migrated to the wounded areas were photographed under a microscope for quantification of cell migration. Images are representative of three separate experiments. Scale bar = 500 µm. (B) Quantitative analysis of wound-induced migration assay from (A). The results are presented as mean ± SEM of three experiments done in duplicate. CM = conditioned medium.
(7.93 MB TIF)
Click here for additional data file.
We thank Peggy Mankin and Noorjehan Ali for their technical assistance. We also thank Shellee Abraham for manuscript preparation and Diana Meister and Sushma Jasti for manuscript review.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The project was supported by Award Number NS057529 (J.S.R.) from the National Institute of Neurological Disorders and Stroke (NINDS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2045445709-PONE-RA-13455R210.1371/journal.pone.0010338Research ArticleBiochemistryCell BiologyMolecular BiologyHOXB13, a Target of DNMT3B, Is Methylated at an Upstream CpG Island, and Functions as a Tumor Suppressor in Primary Colorectal Tumors DNMT3B Targets in Colon CancerGhoshal Kalpana
1
2
*
Motiwala Tasneem
1
Claus Rainer
3
Yan Pearlly
2
4
Kutay Huban
1
Datta Jharna
1
Majumder Sarmila
1
2
Bai Shoumei
1
Majumder Arnab
1
Huang Tim
2
4
Plass Christoph
3
Jacob Samson T.
1
2
*
1
Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, Ohio, United States of America
2
Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, United States of America
3
Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany
4
Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio, United States of America
Hotchin Neil A. EditorUniversity of Birmingham, United Kingdom* E-mail: [email protected] (STJ); [email protected] (KG)Conceived and designed the experiments: kg SJ. Performed the experiments: TM RC PY HK JD SM SB AM. Analyzed the data: KG TM RC PY SM. Contributed reagents/materials/analysis tools: SB THH CP. Wrote the paper: KG TM SM SJ.
2010 29 4 2010 5 4 e103389 10 2009 16 3 2010 Ghoshal et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
A hallmark of cancer cells is hypermethylation of CpG islands (CGIs), which probably arises from upregulation of one or more DNA methyltransferases. The purpose of this study was to identify the targets of DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer.
Methodology/Principal Findings
Chromatin immunoprecipitation with DNMT3B specific antibody followed by CGI microarray identified genes with or without CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI in their promoters, were methylated in multiple colon cancer cell lines but not in normal colon epithelial cells. Further, these genes were reactivated in RKO cells after treatment with 5-aza-2′-deoxycytidine, a DNA hypomethylating agent. COBRA showed that the CGIs encompassing the promoter and/or coding region of DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located ∼4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated in DNMT1−/− HCT cells but was almost methylation free in cells lacking both DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 abolished tumor growth in nude mice.
Conclusions/Significance
This is the first report that identifies several important tumor suppressors and transcription factors as direct DNMT3B targets in colon cancer and as potential biomarkers for this cancer. Further, this study shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer.
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Introduction
Symmetrical methylation of DNA at position 5 of cytosine within a CpG dinucleotide is a major epigenetic modification (∼5% of the total cytosine in the mammalian genome) although a small amount of 5-hydroxymethylcytosine (5hmC) generated from 5-meC by a methylcytosine dioxygenase has recently been detected in certain cell types [1]–[3]. Very recently it has been shown that cytosine methylation at nonCpG sites, although rare, is involved in gene silencing in mammals [4]. DNA methylation is essential for mammalian development. DNA hypermethylation suppresses spurious promoters located within the repeat elements and proviruses in mammalian genome whereas hypomethylation induces genomic instability [5], [6]. DNA methylation is also involved in the regulation of genomic imprinting, inactivation of the silent X chromosome in females and expression of certain tissue specific genes [1], [6]. In humans, alterations in genomic methylation patterns are linked to imprinting disorders and other human diseases including cancer [7]–[9].
Although CpG is usually underrepresented in much of the genome, short (500–2000 bp long) CpG regions, designated CpG islands (CGI), are predominantly located in the proximal promoter regions of almost 50% of the mammalian genes. These regions are usually methylation free in normal cells with the exception of imprinted alleles and genes on the inactive X chromosome. Recent high throughput genome wide DNA methylation analysis identified many more CGIs located distal to promoters that are tissue-specifically methylated [5]. Furthermore, methylation also occurs in the coding regions of active genes and reversible DNA methylation can regulate gene expression in response to stimuli such as estrogen treatment and membrane depolarization [6].
DNA methylation in mammalian cells is established and maintained by DNA (cytosine-5) methyltransferases (DNMTs). Methylation is initiated by highly homologous DNMT3A and DNMT3B that prefer unmethylated DNA as the substrate [1], [10]. DNA methylation is heritably propagated by DNMT1 that prefers hemimethylated DNA as substrate. All three DNMTs are essential for development in mammals [11], [12]. Among these three enzymes, DNMT3B is directly linked to different diseases. For example, mutation of the DNMT3B gene causes immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a rare human disorder due to alteration in the methylation of minor satellite repeats [13] and genes regulating immune function and neuronal development [14]. Thus, DNMT3B deficiency in these patients cannot be compensated by other DNMTs. Studies in mutant mice have shown that DNMT3A and DNMT3B methylate distinct as well as overlapping regions of the genome [12]. For example, DNMT3A2 catalyzes methylation of imprinted genes in germ cells whereas tandem repeat elements are methylated by both DNMT3A and DNMT3B [2]. DNMT3B has also been linked to type 2 diabetes by regulating mitochondrial DNA copy numbers through fatty acid-induced non-CpG methylation of PGC-1α [4]. Emerging studies have shown that a variety of cofactors specifically target DNMTs to distinct chromosomal regions in vivo [15], as these enzymes demonstrate specificity only towards CpG base pairs in vitro
[2]. Gene silencing by DNMTs occurs predominantly by recruitment of repressors that include methyl CpG binding proteins (MBDs) and co-repressors such as histone deacetylases (HDAC) and histone methyltransferases (HMT), resulting in distortion of local chromatin structure [3], [9], [16].
Hypermethylation of CpG islands (CGIs) is a common epigenetic event in almost all malignancies [7], [9]. Upregulation of DNMT3B is also a characteristic of many cancer cells [17]. For example, in sporadic breast carcinoma, 30% of the patients showed increased expression of DNMT3B compared to minimal increase (3–5%) in DNMT1 and DNMT3A [18]. Significantly higher expression of DNMT3B was observed in acute myeloid leukemia compared to normal myeloid cells [19]. DNMT3B overexpression was associated with high tumor grade and CIMP (CpG island methylator phenotype) in colon cancer [17]. Furthermore, depletion of DNMT3B, but not DNMT3A, induced apoptosis specifically in human cancer cells [20]. It has also been reported that upregulation of DNMT3B is more dramatic and more frequent than DNMT1 and DNMT3A in cancers including bladder and colon [21]. Studies in a mouse model have shown that the overexpression of Dnmt3b but not Dnmt3a promoted colon tumorigenesis in ApcMin/+ mice [22]. These observations suggest that DNMT3B may play a causal role in tumorigenesis.
Different groups have identified methylation targets using different techniques [14], [22], in the present study we have identified direct DNMT3B target genes in colon cancer cells by performing chromatin immunoprecipitation followed by CpG island microarray analysis (ChIP-on-chip). Many DNMT3B targets are embedded in CpG islands and some are known tumor suppressors. We also report the methylation status of some of these genes with potential growth suppressor properties in primary colorectal tumors and colon cancer cell lines. Further, we examined tumor suppressive characteristics of two important transcription factors, HOXB13 and TBX18, in colon cancer cells.
Methods
Mice
Nude mice were purchased from Jackson laboratory. All mice were housed, handled, and euthanized in accordance with federal and institutional guidelines under the supervision of the Ohio State University Institutional Animal Care and Use Committee. All animals used in this study were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies.
Cell culture, treatment with 5-aza-2′-deoxy-cytidine (decitabine) and isolation of DNA
Human colon cancer cell lines (RKO, HCT116, SW480, SW837, Colo205, CaCo2 and DLD1b) and normal colon cell line (CCD841) were obtained from ATCC and grown in media as suggested by ATCC. RKO and HCT116 cells were treated with decitabine (0.1 to 5 µM) for 24–120 hours. The wild type, DNMT1−/−, DNMT3B−/− and DKO (DNMT1−/− DNMT3B−/−) cells (a generous gift from Bert Vogelstein) were cultured as described [23].
Western blot analysis
Affinity purified antibodies against DNMT3A and DNMT3B were used for western blot analysis as described [24], [25]. Anti-Flag antibody was from Sigma.
Primary Human Tumors
The tumor samples were obtained from patients at James Cancer Hospital (The Ohio State University). Complete pathologic classification is available for all tumor samples studied. All tissues used for this study were part of an institutional review board-approved protocol at the Ohio State University College of Medicine.
ChIP on Chip assay
Chromatin immunoprecipitation (ChIP) assay was performed as described [26] with some modifications. ChIP was performed on formaldehyde cross-linked chromatin (DNA fragmented to ∼600 bp to 3000 bp by sonication) from 108 RKO cells with antibody against DNMT3B [26], [27]. The anti-DNMT3B antibodies raised in our laboratory do not cross react with each other or with DNMT1 [28]. We used affinity purified DNMT3B antibodies to pull down DNA from formaldehyde cross-linked chromatin prepared from RKO cells. The chromatin was cleared with pre-immune IgG and protein A beads. The precipitated DNA was dissolved in RIPA buffer and subjected to a second round of immunoprecipitation with the same antibody to minimize pull down of false positive targets. This DNA was then separated on an agarose gel and DNA from 0.5 to 3 kb in size was purified by using Gel Extraction kit (Qiagen), labeled with Cy5-labeled dNTP and hybridized to a CpG island library coated on glass slides [29], [30]. The same amount of input DNA and DNA precipitated with preimmune-IgG) was used as control. We selected only those genes for sequence analysis where the signal in ChIP DNA was ≥2 fold compared to the control rabbit IgG signal. MIAME complaint data has been submitted to Geo database (accession number GSE18929).
Sequence analysis of clones
The construction of CpG island library has been described earlier [29]. The clones pulled own by DNMT3B antibodies were picked up from the CpG island library and sequenced in an automated sequencer.
RT-PCR and real-time RT-PCR analysis
RNA was isolated using guanidinium thiocyanate-acid phenol method, treated with DNase 1 to remove residual DNA, if any, and reverse transcribed using random hexamers following standard protocol. Real time RT-PCR was done using SYBR Green technology following published protocol [27], [31]. RT-PCR primers will be available upon request.
ChIP-CHOP analysis was performed as described [32]
Primer sequences are provided in the Table S1.
COBRA (Combined bisulfite-restriction analysis)
COBRA of genomic DNA was performed as described [33], [34]. CGIs different genes were amplified with primers specific for bisulfite converted DNA where unmethylated cytosines are converted to uracils. Primers were designed using Methprimer software (http://www.urogene.org/methprimer/index1.html). Primer sequences are provided in the Table S1.
Quantitative DNA methylation analysis of HOXB13 CGI by MassARRAY
DNA methylation analyses were carried out using the EpiTYPER application (Sequenom, San Diego) as described [35]. Briefly, genomic DNA was isolated, subjected to integrity control and subjected to bisulfite treatment. Regions of interest were amplified using primers for bisulfite treated DNA (primer sequences available upon request), amplified DNA was transcribed in vitro and cleaved using RNAse A. The molecular weight of the resulting fragments indicative of the DNA methylation state was analyzed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA methylation standards (0, 20, 40, 60, 80 and 100%) were used to control for PCR amplification bias. Equation fitting algorithms based on the R statistical computing environment were used for data correction. Display of methylation results as heat maps and unsupervised clustering were performed using the Multiple Experimental Viewer software (http://www.tm4.org/mev.html).
Cloning of HOXB13 and TBX18 cDNA and generation of RKO and HCT116 cell lines overexpressing these proteins
cDNA derived from total RNA from normal human colon (Clontech) was amplified with primers specific for HOXB13 and TBX18 coding region and cloned into pcDNA-3XFlag (Sigma). The authenticity of the cDNAs was confirmed by sequencing. The Flag-tagged cDNAs were then cloned into pBabe-puro and infectious retroviruses were generated in phoenix cells. Stable cell lines (RKO, HCT116 or DLD1b) overexpressing HOXB13 or TBX18 were generated by infecting these cells with the recombinant retroviruses and selected with puromycin.
Clonogenic survival was performed as described [36]
Cell growth was measured by MTT assay as described[37]
Tumor growth in nude mice was performed as described [38]
HOXB13 promoter activity assay
HOXB13 promoter regions spanning −1.2 kb to +0.2 kb and −5.2 kb to +0.2 kb with respect to transcription start site (TSS) were amplified from lymphocyte DNA using Accuprime polymerase, confirmed by sequencing, and cloned into pGL3 basic (firefly luciferase vector). These promoter reporter plasmids along with pRLTK (renilla luciferase vector) were transfected into RKO cells and luciferase activity was measured using Dual luciferase assay kit (Promega).
Results
ChIP on Chip analysis identified DNMT3B target genes in RKO, a colon cancer cell line
Aberrant DNA methylation is prevalent in colorectal carcinogenesis [39]–[41]. To identify hypermethylated genes in colon cancer (RKO) cells we performed ChIP with anti-DNMT3B followed by CpG island (CGI) microarray. We selected DNMT3B because its expression is significantly higher than DNMT3A and DNMT1 in RKO cells (
Figure 1A
) and it appears to play a causal role in colon tumorigenesis [22]. The specificity of the affinity purified DNMT3B antibody was confirmed by using extracts from DNMT3B null HCT cells. A major (∼98/96 kDa) polypeptide was detected by this antibody in RKO cells, in the wild type and DNMT1−/− HCT cells. That these polypeptides are different variants of DNMT3B was confirmed by the inability of the antibody to detect any protein in DNMT3B−/− and DKO (DNMT1−/− DNMT3B−/) cells. A few very minor polypeptides detected in cells expressing DNMT3B are probably its isoforms because it is known that DNMT3B exhibits different spliced variants [42]. To reduce nonspecific pull down, ChIP was performed twice with the same antibody and the precipitated DNA was resolved on an agarose gel to elute DNA of smaller sizes (0.5 to 3 kb) as probe for CpG island microarray (see Methods for detail) (
Figure 1B
).
10.1371/journal.pone.0010338.g001Figure 1 Expression of DNMT3B is higher in RKO cells.
A. Western blot analysis of whole cell extracts from RKO and HCT (wild type and mutant) cells with antibodies specific for DNMTs. Affinity purified antibodies against DNMT3B were used for immunoblotting. B. Real-time RT-PCR analysis of DNMTs in RKO cells was performed using SYBR Green method. Data are mean of triplicate assays. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively. C. Ethidium bromide staining of DNA immunoprecipitated by DNMT3B antibody and input DNA that were used for CpG island microarray analysis.
Next, we sequenced only those pulled-down genes that were at least 2 fold enriched in ChIP DNA compared to that pulled down by control rabbit IgG. The microarray data has been submitted to Geo database and it is MIAME complaint (accession number GSE18929). We classified these genes into four groups, i) genes with CpG island (CGI) but without repeat elements, ii) genes without CGI and repeat elements, iii) repeat elements, and iv) genomic contigs associated with repeat elements (
Table 1
and Table S2). Among the genes with CGIs, there are some known tumor suppressors, such as P16/MTS1, DDC, PGRMC1, CAVEOLIN1 and some disease susceptibility genes such as DISC1 (disrupted in Schizophrenia 1) [43], TBX5 (Congenital heart failure or Holt-Oram syndrome) [44] (
Table 1
). We also identified a few novel genes such as TBX18, DGKI, SLIT1 and GNA11 as DNMT3B targets.
10.1371/journal.pone.0010338.t001Table 1 Methylted DNMT3B Target Genes in RKO cells identified by ChIP on Chip.
Accession # Gene name Gene symbol
GROUP I: Genes harboring CpG island without repeat elements
NM_018662 disrupted in schizophrenia 1 DISC1
NM_003522 histone cluster 1, H2bf HIST1H2BF
NM_001080508 T-box 18 TBX18
NM_001753 Caveolin-1 CAVEOLIN1
NM_004717 diacylglycerol kinase iota DGKI
U26727 P16INK4/MTS1 CDKN2A
NM_021951 doublesex and mab-3 related transcription factor 1 DMRT1
NM_003692 transmembrane protein with EGF-like and two follistatin-like domains 1 TMEFF1
NM_003061 Slit-1 SLIT1
NM_018362 lin-7 homolog C (C. elegans) LIN7C
AF532858 Nogo-66 receptor homolog-1 NGRH1/RTN4RL2
BC031869 M.m. C.elegans ceh-10 homeo domain containing homolog Chx10
NM_181486 T-box 5 TBX5
NM_006361 homeodomain protein HOXB13 HOXB13
NM_005215 colorectal tumor suppressor DCC
NM_002067 guanine nucleotide-binding regulatory protein, alpha-11 GNA11
NM_006667 progesterone receptor membrane component 1 PGRMC1
NM_024784.3 zinc finger and BTB domain containing 3 ZBTB3
GROUP II: Genes with no CGIs or repeat elements
AY210419 CUB and sushi multiple domains 3 CSMD3
Association of DNMT3B to the target genes and their methylation status was determined by ChIP-CHOP assay. Formaldehyde-cross-linked chromatin from RKO cells was immunoprecipitated with anti-DNMT3B or pre-immune IgG. DNA pulled down was divided into three aliquots, which were either undigested or digested with Hpa II, Msp I and subjected to PCR with primers specific for CGIs of different genes that harbor Hpa II/Msp I sites. PCR products were resolved by agarose gel electrophoresis. Generation of PCR product in Hpa II digested DNA indicates methylation.
To confirm the association of DNMT3B with some of its putative target genes, we amplified their promoter regions from a different batch of chromatin immunoprecipitated DNA. We performed ChIP-CHOP analysis to determine methylation status of the target DNA. For this assay, the immunoprecipated DNA was divided into three identical aliquots for mock-digestion, digestion with Hpa II (methylation sensitive enzyme) or Msp I (methylation insensitive enzyme) (see
Figure 2A
for a schematic diagram). The digested DNA was then used to amplify the promoter of interest using primers that encompass one or more Hpa II/Msp I sites. Amplification of the promoter region from the mock-digested DNA indicates that the gene of interest is a target of DNMT3B whereas generation of Hpa II resistant PCR product demonstrates that the gene is methylated. PCR products are not obtained from Msp I digested DNA since Msp I digestion is not sensitive to methylation. Thus, ChIP-CHOP assay not only reveals association of a gene with a certain protein but also identifies the methylation status of the associated promoter at Hpa II sites. The data presented in
Figure 2B
demonstrated that some of the DNMT3B target genes such as P16, TBX5, TBX18, GNA11, DMRT1, HOXB13, CAVEOLIN1, PGRMC1, DCC, DGKi, CDH26, LIN7C, ZBTB3, DISC1 and the histone H2B variant (H2Bf) are methylated in RKO cells. Notably, TBX-18, HOXB13, DMRT1, DISC1, H2Bf and LIN7C CGIs are partially methylated in RKO cells at least at the Hpa II sites (as demonstrated by increased PCR product in mock-digested DNA than in Hpa II cleaved DNA) and DNMT3B is associated with these CGIs irrespective of their methylation status. In contrast, DNMT3B is predominantly associated with methylated CGIs of P16, TBX5, DCC and PGRMC1. None of these target genes could be amplified from chromatin immunoprecipitated with control rabbit IgG (data shown for only TBX5) (Fig. 2B) demonstrating that these are specific DNMT3B targets. Lack of amplification of GAPDH promoter, a house keeping gene, from RKO genomic DNA indicates that GAPDH CGI is methylation free and Hpa II digestion was complete. GAPDH was not pulled down by DNMT3B (data not shown).
10.1371/journal.pone.0010338.g002Figure 2 ChIP-CHOP assay to demonstrate association of DNMT3A and DNMT3B with the selected target genes.
A. Schematic diagram of ChIP-CHOP assay. B. Formaldehyde-cross-linked chromatin from RKO cells was immunoprecipitated with anti-DNMT3B or pre-immune IgG. DNAs pulled down were divided into three aliquots, which were either undigested or digested with Hpa II, Msp I and subjected to PCR with primers specific for CGIs of different genes that harbor Hpa II/Msp I sites. PCR products were resolved by agarose gel electrophoresis. Generation of PCR product in Hpa II digested DNA indicates methylation. U, H and M indicate PCR products obtained in mock-, Hpa II- digested and Msp I-digested DNA, respectively. Lack of PCR product with Msp I digested DNA confirmed complete digestion. C. Activation of several DNMT3B target genes in RKO cells after treatment with decitabine. RKO cells were treated with the drug for the indicate time periods. RNA from these cells was subjected to RT-PCR with gene-specific primers. Three hundred ng of cDNA for the test gene and 1 ng of cDNA were used for 18S rRNA.
Next, we tested methylation status of a few genes in other colon cancer cell lines (DLD1b, HCT116, SW480, CACo2, COLO205, SW837) by digestion of their genomic DNA with Hpa II/Msp I followed by PCR with gene specific primers. While TBX5 and DCC are methylated in all cell lines, other genes (TBX18, DGKI, PGRMC1, LIN7C and HOXB13) are differentially methylated (Table S3). It is noteworthy that none of these genes are methylated in normal colon epithelial cells CCD841.
Treatment of RKO cells with decitabine resulted in activation of the methylated genes
To determine whether methylation of some of the DNMT3B target genes indeed suppressed their expression in RKO cells, we treated these cells with the commonly used DNA hypomethylating agent, 5-aza-2′-deoxycytidine. RNA isolated from untreated and the inhibitor (1 and 2.5 µM) treated RKO cells harvested at 24, 48 or 72 hours was subjected to RT-PCR analysis with gene specific primers. TBX18, DCC and CAVEOLIN 1 were re-expressed in RKO cells treated with 1 and 2.5 µM decitabine as early as 24 hr and their expression persisted up to 72 hr (
Figure 2B
). In contrast, SLIT1 was induced at a very low level under these conditions whereas HOXB13 was reactivated only after exposure to the drug for 72 hr. On the contrary, GNA11 was expressed at high level in RKO cells indicating that the methylation of CGI located in intron 1 of this gene did not affect its expression.
CGIs of DNMT3B target genes are methylated in human primary colorectal tumors
Next, we extended our study to analyze the methylation status of a selected group of genes (DCC, TBX18, TBX5, SLIT1 and GNA11) in several primary human colorectal tumors and matching normal colon tissues by COBRA. The methylation status of these genes in a few pairs is presented in
Figure 3
. The CGI spanning promoter/exon 1 of DCC was methylated at Taq I site in 8 out of 10 tumors as demonstrated by almost complete digestion with Taq I whereas it was methylated in matching normal colon tissues only in sample #8 and #10 (
Figure 3
.i). Similarly, CGI of TBX18 was methylated at the BstU I site in 6 out of 7 tumors without significant methylation in matching normals (
Figure 3
.ii). TBX5 encodes 4 transcript variants that are generated by alternate transcription initiation sites and alternative splicing. We analyzed methylation status of CGIs spanning the promoter/exon 1 of variants 1 and 3 (TBX5L) and variant 4 (TBX5S) (
Figure 3
.iii). Interestingly, CGI of TBX5L was methylated at Taq I sites both in normal colon tissues and tumors but methylation was more pronounced in 3 out of 5 tumors (#1, 4 & 6) than in the matching normals. In contrast, CGI of TBX5S was specifically methylated in tumors in 5 out of 6 samples at the BstU I site. Thus, two CGIs located in close proximity demonstrated differential methylation status in the same sample at least with respect to Taq I and BstU I sites. CGI spanning promoter/exon 1 of SLIT1 was tumor-specifically methylated at the Taq I site in 5 out of 10 samples analyzed (
Figure 3
.iv). Notably CGI located in the intron of GNA11 was completely methylated both in normals and tumors as demonstrated by complete digestion of the PCR product with BstU I (
Figure 3
.v). Methylation at this intronic CGI did not silence GNA11 as demonstrated by its robust expression in RKO cells (
Figure 2c
). Complete bisulfite conversion was demonstrated by digestion of the amplicons with methylation insensitive Mse I or Tsp509 I (data not shown). These COBRA data showed that some of DNMT3B targets were hypermethylated in primary colorectal tumors albeit at different levels. However, we did not observe methylation of CGI located in the promoter as well as exon 1 region of the HOXB13 gene in primary colon cancer by COBRA (data not shown).
10.1371/journal.pone.0010338.g003Figure 3 Analysis of methylation status of CGI of selected genes by COBRA in primary human colorectal tumors.
Location of the CGI of each gene as appeared in UCSC genome browser is depicted in the accompanied schematic diagrams. Bisulfite converted genomic DNA was amplified with gene specific primers followed by digestion with a methylation sensitive enzyme Taq I or BstU I. T and N denote tumor and matching normal, respectively. Sample numbers shown in red identify tumors with gene-specific methylation.
CGI located 4.5 kb upstream of HOXB13 Transcription start site (TSS) is hypermethylated in colorectal tumors
Although COBRA did not reveal methylation of CGI located in the promoter and exon 1 region, HOXB13 gene was reactivated after treatment with decitabine (
Figure 2C
). This observation suggested that methylation of a CGI located at a different region of the gene or specific CpGs within the promoter region might regulate its expression in colon cancer cells or tissues. BLAT analysis identified two CGIs in human HOXB13 gene, one in the promoter/exon1 and the other ∼4.5 kb upstream of transcription start site (TSS) (
Figure 4A
). We, therefore, analyzed methylation status of CpGs spanning the promoter and upstream CGI (
Figure 4B
, and Tables S4 and S5) in primary colorectal tumors and in cell lines by MassARRAY because in this mass spectrometry based assay methylation status of CpGs can be estimated quantitatively. It is evident from the HEAT map that methylation at the upstream CGI is higher in tumors than in normals (
Figure 4C
.i). Quantification of the results showed that overall methylation density at the upstream CGIs was significantly higher (P = 0.02) in tumors compared to the normals (
Figure 4C
.ii). In contrast, CGI in the promoter region was essentially methylation free (
Figure 4C
.i), which correlated with the COBRA data (data not shown). MassARRAY analysis of colon cancer cell lines revealed hypermethylation at the upstream CGI in HCT, CaCo2 and RKO cells whereas relatively low level methylation was detected in RWPE and SW837 cells. Notably, CCD841, a normal colon epithelial cell line, was essentially unmethylated. Selective methylation of the upstream CGI but not the one located in the immediate upstream promoter region occurs in colon cancer cells probably due to distinct chromatin structure that is accessible to DNMTs.
10.1371/journal.pone.0010338.g004Figure 4 Methylation of the CpGs spanning upstream and promoter regions of HOXB13 gene in primary colorectal tumors and colon cancer cell lines by MassARRAY.
A and B. Potential methylatable CpGs in these regions are shown after bisulfite conversion. CpGs sequenced by MassARRAY are boxed and numbered. It is notable that this technique provides average methylation status of CpGs that are in close proximity (numbered together as 1, 2 etc). Gray bars indicate samples that could not be sequenced. C.i. HEAT map of methylation profile of CpGs located within upstream CGI and promoter (TSS) region. The same amplicon of methylation density ranging from 0 to 100% was used to generate standard curve. C.ii. Box plot of quantitative analysis of methylation density in the upstream CGI and promoter regions in primary colorectal tumors and normals. Significance was assessed by Welch test (adaptation of t test, parametric, unequal variance, one-tailed). D. Upstream region (−5.4 kb) activates
HOXB13
promoter activity in colon cancer cells.
HOXB13 promoter regions (−1.2 kb and −5.4 kb) cloned into pGL3 basic vector (RLU1) were transfected into HCT116 cells along with internal control pRLTK (RLU2), followed by treatment with 10 nM estradiol (E) in phenol red free medium containing 5% charcoal stripped serum for different time periods. E. Only the upstream CGI is methylated in HCT cells, which undergoes site-specific and global demethylation by DNMT1 alone and both DNMT1, DNMT3B, respectively. i) MassARRAY, ii) real-time RT-PCR analysis of HOXB13, and iii) RT-PCR analysis of ERα and GAPDH.
To investigate the role of the upstream CGI in HOXB13 expression we generated luciferase reporter constructs harboring −5.4 kb to +0.2 kb and −1.2 kb to +0.2 kb regions, respectively into pGL3basic vector and compared their ability to modulate firefly luciferase activity at 36 and 48 hr post transfection in HCT116 cells, because these cells can be transfected with high efficiency. The luciferase activity driven by −5.4 kb promoter was at least 2 fold higher than that contributed by −1.2 kb region (
Figure 4D
). Promoterless pGL3basic showed minimal activity (data not shown). Since HOXB13 is an estrogen responsive gene [45], we also measured activity of these two promoter regions by treating cells with estrogen 24 hr post-transfection with estradiol that increased the activity of both promoters at 12 and 24 hrs (
Figure 4D
). Taken together, these results demonstrated that the upstream promoter region of HOXB13 gene stimulated promoter activity but did not contribute to estrogen responsiveness.
To identify the DNA methyltransferase (DNMT) that catalyzes methylation of HOXB13, we measured its methylation status in the wild type and mutant HCT cell lines lacking DNMT1, DNMT3B or both. Massarray analysis showed that the upstream CGI is heavily methylated in the wild type and DNMT3B−/− cells but is significantly hypomethylated at certain CpGs in DNMT1−/− cells and almost completely in double knock out (DKO) cells (
Figure 4E
and Tables S4 and S5). These results indicate that although DNMT1 alone can methylate certain CpGs, its cooperation with DNMT3B is required for efficient methylation of the upstream CGI. Notably, promoter CGI is not methylated in any of these 4 cell lines. Surprisingly, HOXB13 expression is upregulated only in DNMT1−/− cells compared to the wild type cells (
Figure 4E
). Significant downregulation of ERα (
Figure 4E
), an activator of HOXB13 [45] probably accounts for HOXB13 suppression in DNMT3B−/− and DKO cells. Increase in HOXB13 expression in DNMT1−/− cells suggests that methylation at certain CpGs in the upstream CGI suppresses HOXB13 expression. Thus, the upstream CGI probably functions as an enhancer and its methylation partially suppresses but does not silence HOXB13 expression.
Ectopic expression of TBX18 and HOXB13 inhibits growth, clonogenic survival and anchorage independent growth of colon cancer cells
We next explored the anti-tumorigenic properties of TBX18 and HOXB13 in colon cancer cells. TBX18, a member of T-box family of transcription factor, is expressed in the segmented somites and in the limb bud [46]. TBX18 knock out mice die immediately after birth due to severe defects in organs deriving from the lateral sclerotome [47]. In contrast, ubiquitously expressed HOXB13 is a member of homeobox super family involved in establishing cell fate during embryonic development and maintaining differentiation state in adults [48], [49]. HOXB13 is upregulated in many solid tumors including cancers of the endometrium, cervix, ovary and prostate whereas it is down regulated in renal cell carcinoma, melanoma and colon cancer [45]. To study the potential role of HOXB13 and TBX18 in modulating tumorigenic property of colon cancer cells we expressed these proteins using retroviral vector (pBabe) in the nonexpressing colon cancer cell lines RKO and DLD1b. Ectopic expression of the proteins was measured in puromycin-selected cells by Western blot analysis with anti-Flag antibody (
Figure 5A
). The growth rates of TBX18 and HOXB13 expressing versus nonexpressing cells were assessed by MTT assay. Overexpression of these proteins resulted in a significant decrease in the growth rate of the cells compared to vector starting from day 1 in both cell lines (
Figure 5B
), which correlated with dramatic reduction in replication potential in RKO cells expressing TBX18 or HOXB13 compared to vector transfected cells (
Figure 5C
). Similarly, clonogenic survival of RKO cells expressing these transcription factors was significantly reduced (
Figure 5D
). Ectopic expression of HOXB13 and TBX18 in another nonexpressing cell line, DLD1b inhibited these properties (data not shown). Together, these results demonstrated that TBX18 and HOXB13 severely compromised tumorigenic potential of colon cancer cells in vitro.
10.1371/journal.pone.0010338.g005Figure 5 HOXB13 and TBX18 demonstrate anti-tumorignic property in colon cancer cells in vitro.
A. Western blot analysis of cell extracts prepared from RKO cells expressing Flag-tagged HOXB13 or TBX18. Analysis of cell growth by MTT assay (B), replication potential by 3H1-thymidine incorporation (C), clonogenic survival (D) of RKO cells (pool and a clone selected at random). Each assay was performed in triplicate. Single and double asterisks denote p values ≤0.05 and ≤0.01, respectively.
HOXB13 but not TBX18 inhibits growth of colon cancer cells in nude mice
We next investigated whether HOXB13 and TBX18 expression could inhibit ex vivo growth of colon cancer cells. For this purpose, RKO cells expressing either HOXB13 or TBX18 and vector transfected cells were injected into the flanks of nude mice. Mice were monitored for tumor growth every week and tumor size was measured. Tumor growth was visible as early as one week in mice injected with the control and TBX18 expressing cells. In contrast, the tumor was not detectable in most of the animals injected with HOXB13 expressing cells (
Figure 6A
). At the end of the experiment, the mice were sacrificed, tumors were removed and their weights and volumes were determined. Notably, HOXB13 expressing cells could not form tumors in majority of animals (
Figure 6B, C
). These cells formed only two visible tumors in mice. Surprisingly, no significant change in tumor growth in RKO cells expressing TBX18 was observed. Western blot analysis with anti-Flag antibody to detect ectopic TBX18 showed that the tumors developed nude mice expressed TBX18 (
Figure 6D
) suggesting that lack of inhibition of tumor growth in nude mice was not due to loss of TBX18 expression. It is likely that some host factor(s) in the tumor microenvironment antagonizes the tumor suppressor function of TBX18 in nude mice. Similar results were observed in HCT116 and DLD1b cells (data no shown). Thus, HOXB13 functions as a tumor suppressor in colon cancer cells both in vitro and ex vivo.
10.1371/journal.pone.0010338.g006Figure 6 HOXB13 but not TBX18 inhibited ability of RKO cells to form tumor in nude mice.
One million cells in PBS (100 µl) were injected into the flanks of nude mice and tumor size was monitored every week using a caliper. After 25 days tumors were excised and their weights were documented. A. Time course of the progression of tumor growth. B. Photograph of the tumors developed by vector-transfected, HoxB13 or TBX18 expressing cells. C. Tumor weight at the time of sacrifice. D. Western blot analysis of ectopic TBX-18 in the tumors T1 to T4 (shown in A) with anti-Flag antibody.
Discussion
It is now well established that hypermethylation is a common mechanism for silencing tumor suppressor genes in cancer cells. Because re-expression of these genes upon demethylation was perceived to be an alternate strategy for cancer therapy, considerable effort has been expended to identify novel tumor suppressor genes in specific cancer types that are silenced by methylation. Clinical trials of Vidaza and Dacogen against different cancers underscore the significance of epigenetic therapy in cancer [50], [51]. Further, differentially methylated genes could be potential biomarkers for colorectal cancer. Indeed, recent studies have shown that some of the hypermethylated genes could be detected in the stool of colon cancer patients [41], [52].
DNA methyltransferases, expressed at relatively low levels in somatic cells, are frequently upregulated in cancer cells. Gain of function studies have shown that Dnmt3b but not Dnmt3a promotes colon tumorigenesis in APCMin/+ mice by inducing de novo methylation of multiple genes harboring CpG islands [22]. It, therefore, becomes important to identify the targets of DNMT3B in colon cancer cells to understand its function in tumorigenesis. A recent study has used expression profiling to identify its targets in colon cancer cell lines [53]. To our knowledge, the present study is the first report on the identification of direct DNMT3B targets in colon cancer cells using ChIP-on-chip with antibodies that are specific for DNMT3B. The targets identified include not only well known tumor suppressors such as P16/INK4A, DCC, CAVEOLIN1, PGRMC1 but also novel genes like TBX18, TBX5, SLIT1, DGKI. Activation of some of these genes after treatment with demethylating agents confirmed that methylation indeed silenced their expression in colon cancer cells. DNMT1 and DNMT3B function co-operatively to methylate and silence many tumor suppressor genes in colon cancer cells [23]. It is, therefore, conceivable that both enzymes could act in concert to alter methylation status of the target genes, as observed in HOXB13 upstream CGI (Figure 4E).
An important observation is that a validated set of genes (TBX5, DCC, DGKI, CDH26, HOXB13, CAVEOLIN1, PGRMC1, GNA11, TBX18, ZBTB3 and DMRT1) are indeed associated with DNMT3B and that they are methylated in more than one colon cancer cell line relative to normal colon epithelial cells (CCD841). Among these, only DCC
[54] and CAVEOLIN1
[55] have recently been reported to be methylated in colorectal carcinoma. DMRT1 is methylated in gastric cancer [56] whereas HOXB13 is methylated in melanoma [57], renal cancer [58] and breast cancer [45]. Further, analysis of a subset of these genes (DCC, TBX18, TBX5, SLIT1) in primary colon cancer revealed tumor-specific methylation. Recently several investigators have identified genes methylated in colorectal cancer some of which were also detected in the stool of colorectal cancer patients [41]. Different etiology, genetic background and the techniques used probably account for the identification of distinct methylated genes [59]. Cluster analysis demonstrated that tumors with dense methylation at the upstream CGI of HOXB13 gene clustered together (Figure S1). It would be of interest to analyze a large cohort of colorectal tumors to determine whether methylation of HOXB13 occurs in specific type of tumors and this epigenetic modification can be used as a diagnostic or prognostic marker for colorectal cancer.
HOXB13 belongs to the homeobox family of transcription factors. It is a unique developmentally regulated protein that is up- or down-regulated depending upon the cellular context. While it is upregulated in ovarian [60] and endometrial cancers [61] where it functions as a tumor promoter, its expression is suppressed in malignant melanoma [57], renal [58], prostate [62], colorectal [63] and breast [45] cancer. HOXB13 is methylated in malignant melanoma, renal and breast cancer in the CGIs spanning the immediate upstream promoter and exon 1. Surprisingly, this region is essentially methylation free in normal colon and colorectal tumors (Figure 4). Methylation at an upstream CGI located ∼4.5 kb upstream of the HOXB13 transcription start site in primary colorectal tumors and colon cancer cell lines suggests that chromatin structure of this region acquires a unique conformation accessible to DNMTs. C/EBPα is another transcription factor that is tumor-specifically methylated at an upsteam CGI in lung [64] and head and neck cancer [65]. Recent high throughput analysis has identified many more genes that are methylated at far upstream CGIs and even in coding regions [6]. The upstream CGI of HOXB13 appears to contribute to its promoter activity in colon cancer cells and harbors several conserved cis-regulatory elements some of which encompass CpG dinucleotides. It is, therefore, likely that methylation of this region is involved in modulating expression of HOXB13 and that this mechanism is unique to colon cancer cells. It would be of interest to examine whether HOXB13 knockout mice are susceptible to colon tumorigenesis spontaneously, after crossing with Apc/Min+, Mlh1−/− mice or upon exposure to carcinogens. Similarly, identification of HOXB13 target genes in colon epithelial cells is likely to elucidate the mechanism of its tumor suppressor function. Generation of immunoprecipitation grade antibody for HOXB13 will help us to answer this question. Studies along these lines are in progress.
Supporting Information
Table S1 List of primers (ChIP-CHOP, COBRA, RT-PCR, cDNA cloning and promoter regions) used in the present study. COBRA primers were designed using Methprimer database (http://www.urogene.org/methprimer/index1.html).
(0.11 MB DOC)
Click here for additional data file.
Table S2 Chromatin from RKO cells were immunoprecipitated with affinity purified Dnmt3B antibodies or mock-immunoprecipitated. Precipitated DNA ranging in size from 0.6 to 3 kb were subjected to CpG island microarrary. The chromatin was cleared with pre-immune IgG and protein A beads. The precipitated DNA was dissolved in RIPA buffer and subjected to a second round of immunoprecipitation with the same antibody to minimize pull down of false positive targets. This DNA was then separated on an agarose gel and DNA from 0.5 to 3 kb in size was purified by using Gel Extraction kit (Qiagen), labeled with Cy5-labeled dNTP and hybridized to a CpG island library coated on glass slides. The same amount of input DNA and mock-immunoprecipitated DNA (with rabbit preimmune-IgG) was used as control. We selected only those genes for further analysis where the signal in ChIP DNA was greater than 2 fold compared to the control rabbit IgG signal.
(0.16 MB DOC)
Click here for additional data file.
Table S3 Genomic DNA from different colon cancer and normal colon epithelial (CCD841) cells were digested with Hpa II, Msp I or mock-digested and an aliquot (100 ng) of DNA from each was subjected to PCR with primers specific for CGI of each gene followed by separation of the PCR products on an agarose gel. The gene was codiered to be methylated if PCR product was generated in the Hpa II digested DNA but not in Msp I digested DNA.
(0.08 MB DOC)
Click here for additional data file.
Table S4 MassARRAY data of upstream CGI of HOXB13 gene in primary colon cancer and matching colon tissues and colon cell lines (normal and cancer). Methylation at each CpGs was determined based on a standard curve generated using methylation density ranging from 0% to 100% of the amplicon.
(0.02 MB XLS)
Click here for additional data file.
Table S5 MassARRAY data of promoter CGI of HOXB13 gene in primary colon cancer and matching colon tissues and colon cell lines (normal and cancer). Methylation at each CpGs was determined based on a standard curve generated using methylation density ranging from 0% to 100% of the amplicon.
(0.03 MB XLS)
Click here for additional data file.
Figure S1 Unsupervised clustering of human primary colorectal tumors (T) and matching normal colon tissues (N) methylation based on methylation density at upstream CGI of HOXB13 as determined by MassARRAY.
(9.75 MB TIF)
Click here for additional data file.
We thank Dr. Bert Vogelstein for providing wild type and mutant HCT cell lines, Spencer Smith for technical assistance, Sandya Liyanarachchi for assistance with the submission to Geo database, Bo Wang for critically reading the manuscript and Sarah Wilkins for editorial assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was supported, in part, by grants CA086978 and CA101956 from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2049872310-PONE-RA-15697R110.1371/journal.pone.0010581Research ArticleGenetics and Genomics/EpigeneticsGenetics and Genomics/Gene ExpressionMolecular Biology/Chromatin StructureMolecular Biology/DNA MethylationMolecular Biology/Histone ModificationSETDB1 Is Involved in Postembryonic DNA Methylation and Gene Silencing in Drosophila
SETDB1 Starts DNA MethylationGou Dawei
1
¤a
Rubalcava Monica
1
Sauer Silvia
1
Mora-Bermúdez Felipe
2
¤b
Erdjument-Bromage Hediye
3
Tempst Paul
3
Kremmer Elisabeth
4
Sauer Frank
1
2
*
1
Department of Biochemistry, University of California Riverside, Riverside, California, United States of America
2
Zentrum für Molekulare Biologie der Universität Heidelberg, Universität Heidelberg, Heidelberg, Germany
3
Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America
4
Institute of Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany
Veenstra Gert Jan C. EditorRadboud University Nijmegen, Netherlands* E-mail: [email protected] and designed the experiments: DG HEB PT FS. Performed the experiments: DG MR SS FMB HEB PT. Analyzed the data: DG MR SS FMB HEB PT FS. Contributed reagents/materials/analysis tools: EK. Wrote the paper: DG HEB FS.
¤a Current address: Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America
¤b Current address: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
2010 17 5 2010 5 5 e1058121 1 2010 29 3 2010 Gou et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.DNA methylation is fundamental for the stability and activity of genomes. Drosophila melanogaster and vertebrates establish a global DNA methylation pattern of their genome during early embryogenesis. Large-scale analyses of DNA methylation patterns have uncovered revealed that DNA methylation patterns are dynamic rather than static and change in a gene-specific fashion during development and in diseased cells. However, the factors and mechanisms involved in dynamic, postembryonic DNA methylation remain unclear. Methylation of lysine 9 in histone H3 (H3-K9) by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers embryonic DNA methylation in Arthropods and Chordates. Here, we demonstrate that Drosophila SETDB1 (dSETDB1) can mediate DNA methylation and silencing of genes and retrotransposons. We found that dSETDB1 tri-methylates H3-K9 and binds methylated CpA motifs. Tri-methylation of H3-K9 by dSETDB1 mediates recruitment of DNA methyltransferase 2 (Dnmt2) and Su(var)205, the Drosophila ortholog of mammalian “Heterochromatin Protein 1”, to target genes for dSETDB1. By enlisting Dnmt2 and Su(var)205, dSETDB1 triggers DNA methylation and silencing of genes and retrotransposons in Drosophila cells. DSETDB1 is involved in postembryonic DNA methylation and silencing of Rt1b{} retrotransposons and the tumor suppressor gene retinoblastoma family protein 1 (Rb) in imaginal discs. Collectively, our findings implicate dSETDB1 in postembryonic DNA methylation, provide a model for silencing of the tumor suppressor Rb, and uncover a role for cell type-specific DNA methylation in Drosophila development.
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Introduction
The enzymatic methylation of position C5 of cytosine in genomic DNA is phylogenetically conserved between prokaryotes and eukaryotes [1]–[2]. In eukaryotes, DNA methylation plays a fundamental role in regulating the structure and activity of DNA and chromatin [3]–[4]. Numerous factors, including DNA methyltransferases (Dnmts), methyl cytosine binding domain (MBD) proteins, chromatin remodeling factors, and enzymes, which support posttranslational modifications of histones (H1, H2A, H2B, H3, and H4), are involved in establishing, maintaining and interpreting DNA methylation [3], [4], [5]. Multiple, intricate DNA methylation machineries mediate and maintain DNA methylation in plants and vertebrates, whereas DNA methylation in Drosophila melanogaster (Drosophila) appears to involve only an elementary set of factors [5], [6], [7]. One putative Dnmt (Dnmt2) and 5 members of the MBD superfamily of proteins, among them Drosophila SETDB1 (dSETDB1) have been identified in Drosophila
[7]. Dnmt2 mediates DNA and tRNA methylation; silencing of genes, telomers, and transposable elements; as well as methylation of lysine 20 in histone H4 [8], [9], [10]. DSETDB1 is a member of the phylogenetically conserved family of SET/MBD proteins and contains a bifurcated SET domain and an MBD-like domain (MBDL). The SET domain of dSETDB1 methylates lysine 9 in histone H3 [11], [12], [13], [14].
Contrary to the predominant methylation of CpG motifs in vertebrate genomes [15]–[16], The DNA methylation machinery of Drosophila preferentially methylates CpA and CpT motifs [17], [18], [19]. The level of DNA methylation is highest in early Drosophila embryos, remains detectable in all developmental stages, and decreases in adult flies [17], [18], [19]. In plants, fungi and vertebrates, methylation of H3-K9 by members of the Su(var)3–9 family of histone methyltransferases (HMTs) triggers DNA methylation [20], [21], [22], [23]. In Drosophila, Su(var)3–9 mediates DNA methylation during early embryogenesis [9].
The ability of cells to mitotically and meiotically maintain DNA methylation patterns and the low frequency of postembryonic DNA methylation in many organisms supported the model of DNA methylation patterns being static rather than dynamic [3], [24]–[25]. However, the large-scale analyses of DNA methylation patterns in normal and diseased cells and tissues have revealed that DNA methylation is highly dynamic and differentially regulated in response to normal and aberrant intra- and extra-cellular signals [26], [27], [28], [29], [30], [31]. Whether tissue-specific DNA methylation involves DNA de-methylation, as has been suggested for the inactive vertebrate X chromosome [32], or DNA methylation, or both, remains unknown. In addition, how organisms such as Drosophila and vertebrates, which establish DNA methylation patterns during the earliest steps of embryogenesis, differentially methylate genes during later stages of development remains unclear [4].
Here, we provide evidence that dSETDB1 is involved in postembryonic DNA methylation and gene silencing in Drosophila Schneider 2 (S2) cells and developing imaginal discs. We found that the MBDL of dSETDB1 binds methylated CpA motifs. Methylation of H3-K9 by ectopically expressed dSETDB1 nucleates DNA methylation by Dnmt2 and gene silencing in Drosophila cells. In cells and imaginal discs, dSETDB1 cooperates with Dnmt2 and Su(var)205 in DNA methylation and silencing of transposable elements and euchromatic genes such as the tumor suppressor gene retinoblastoma family protein 1 (Rb). Ectopically expressed dSETDB1 propagates the spreading of methylated H3-K9 and DNA methylation on the Rb locus in S2 cells, which culminates in the formation of heterochromatin and silencing of Rb. Our results implicate dSETDB1 in DNA methylation and gene silencing and uncover a role for DNA methylation in postembryonic development of Drosophila.
Results
Trimethylation of H3-K9 by dSETDB1 mediates gene silencing
The presence of an MBDL and the described H3-K9-specific HMT activity supports the hypothesis of dSETDB1 being functionally linked to the Drosophila DNA methylation machinery [11], [12], [13], [14]. To test this hypothesis, we dissected the role and interplay of the SET domain and MBDL of dSETDB1 in DNA methylation and gene expression. First, we revisited the substrate specificity of the HMT activity of dSETDB1. DSETDB1 can mono-, di- and/or tri-methylate H3-K9 in vitro
[12], [13], [14], [33] and differentially methylates H3-K9 in a gene-specific fashion in vivo. In HMT assays, dSETDB1 methylated H3-K9 in endogenous, purified mononucleosomes and polynucleosomes but did not significantly methylate non-nucleosomal H3-K9 (Figure 1A,B, and Figure S1). The mutation of histone-residue 775 in dSETDB1 (dSETDB1H775L), which is invariant among SET-domain proteins and part of the adenosine-methionine binding pocket [12], attenuated the HMT activity of dSETDB1 (Figure 1A,B). In vitro HMT assays coupled to Edman microsequencing (Figure 1C), mass spectrometry (Figures 1D; Figures S2 and S3), and western blot analysis (Figure 1E; Figure S4) revealed that recombinant dSETDB1 tri-methylates H3-K9 in both mononucleosomes and polynucleosomes. Collectively, our data indicates that dSETDB1 preferentially tri-methylates H3-K9 in nucleosomal H3.
10.1371/journal.pone.0010581.g001Figure 1 DSETDB1 preferentially tri-methylates lysine 9 in nucleosomal H3.
(A) Schematic representation of dSETDB1 and dSETDB1(H775L), which contains a single amino acid exchange mutation of histidine (H) to leucine (L) at amino acid position 775. Rectangles mark the positions of the methyl cytosine binding (MBD)-like domain (MBDL), Pre-SET domain (Pre), SET-domain (SET), and Post-SET domain (Post). The dark box in the SET domain indicates the peptide insertion present in bifurcated SET domain of dSETDB1. (B) Coomassie-blue stained SDS-PAGE gel (left) and corresponding fluorogram (right) of histone methyltransferase (HMT) assays containing polynucleosomes, [3H]-S-adenosyl-methionine (SAM), and recombinant, immunopurified Flag-epitope–tagged dSETDB1 (lanes 1,3) or Flag-epitope–tagged dSETDB1(H775L) (lanes 2, 4). Asterisks mark the position of anti-Flag antibody light and heavy chain. (C) Microsequencing of radiolabeled nucleosomal H3. Polynucleosomes were incubated with recombinant dSETDB1 in the presence of [3H]-SAM. H3 was subjected to Edman degradation, and resulting amino acid fractions were analyzed by scintillation counting. The × axis shows amino acids 1–29 of H3. The y axis shows [3H]-labeling of amino acids in decays per minutes (D.p.m.). (D) Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) spectra of a high-performance liquid chromatography (HPLC) fraction containing the peptide 9K(me0–3)STGGKAPR of H3. Polynucleosomes were incubated with Flag-dSETDB1 and SAM. H3 was purified and proteolytically digested with trypsin. The resulting peptides were separated by HPLC. Fraction 4 containing 9K(me0–3)STGGKAPR was subjected to MALDI-TOF mass spectrometry. The × axis indicates the mass:charge ratio. The y axis indicates the abundance of peptides. The positions and m:z ratios of KSTGGKAPR peptides containing non-methylated lysine 9 in H3 (H3-K9), mono-methylated H3-K9 [(me1)H3-K9], di-methylated H3-K9 [(me2)H3-K9], and tri-methylated H3-K9 [(me3)H3-K9] are indicated. (E) Western blot analysis of HMT reactions programmed with recombinant Flag-dSETDB1, polynucleosomes, and SAM. Reaction products were separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and probed with antibodies to (me1)H3-K9, (me2)H3-K9, (me3)H3-K9, and H3.
Tri-methylated H3-K9 is one hallmark component of heterochromatin [34]; however, dSETDB1-mediated methylation of H3-K9 has been associated with activation and repression of transcription [13]–[14], [33]. To assess the role of dSETDB1-mediated tri-methylation of H3-K9 in gene expression, we performed transient transfection assays in S2 cells. Because dSETDB1 lacks a “classical” DNA binding domain, we used fusion proteins consisting of dSETDB1 and the DNA binding domain of the bacterial tetracycline repressor (TetR) [35]. TetRdSETDB1 fusion proteins repressed the expression of a chromosomal integrated TetR-dependent reporter gene, whereas the HMT-inactive TetRdSETDB1(H775L) and TetR did not (Figure 2A,B; Figure S5). Chromatin immunoprecipitation (ChIP) assays revealed that recruitment of TetRdSETDB1 but not TetRdSETDB1(H775L) mediated tri-methylation of H3-K9 at the reporter gene, which indicates that dSETDB1-mediated tri-methylation of H3-K9, mediates gene silencing (Figure 2C; Figure S6; Table S1).
10.1371/journal.pone.0010581.g002Figure 2 Methylation of H3-K9 by dSETDB1 mediates transcriptional silencing.
(A) Western blot analysis detecting dSETDB1 (black triangle) and tetracycline repressor (TetR)–SETDB1 (TetRSETDB1) fusion proteins (open triangle) in S2 cells transiently expressing dSETDB1, TetR, or fusion proteins of TetR with dSETDB1 or dSETDB1(H775L). Forty-eight hours after transfection, total cell extracts were prepared, separated by SDS-PAGE, and electrophoretically transferred onto nitrocellulose membrane for probing with antibody to dSETDB1. (B) Schematic representation of luciferase assays with whole-cell extracts prepared from cells described in (A). The tetO-tk-luc reporter gene contains tet operator (tetO) sequences, which are fused to the human thymidine kinase (tk) promoter driving the expression of luciferase (luc). The diagram shows the mean values calculated from data from 5 different experiments. Error bars indicate the standard error of the mean (SEM). (C) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays monitoring the presence of the promoter region of the tetO-tk-luc reporter gene in DNA pools obtained by chromatin immunoprecipitation (ChIP). Chromatin was isolated from (tetO-tk-luc)-S2 cells expressing TetR, TetRdSETDB1, or TetRdSETDB1(H775L) and immunoprecipitated with antibodies to dSETDB1, Su(var)205, Dnmt2, (me3)H3-K9, and methylated cytosine (5mC), or protein-A agarose (control-A) and protein-G agarose (control-G). Input indicates the amount of target DNA present in 1% of the input chromatin.
The MBDL of dSETDB1 binds methylated CpA motifs
Differences in the primary sequences of the MBD and MBDL have led to the speculation that MBDL proteins do not bind methylated DNA (mDNA) [36]. However, the MBDL of dSETDB1 (dSETDB1-MBDL) contains key amino acid residues (such as arginine 436) that are invariant in MBD proteins and essential for the interaction with mDNA [36] (Figure 3A). To test whether dSETDB1-MBDL binds mDNA, we performed in vitro protein–DNA binding assays. In our assay system, the MBD of mouse MeCP2, which preferentially binds methylated CpG motifs, interacted with methylated CpG but not CpA and CpT motifs [37]. This result reveals that our assay system can recapitulate the interaction of MBD proteins with mDNA (Figure S7). In contrast the dSETDB1-MBDL (Figure 3B) preferentially bound DNA containing one (Figure 3C; Table S2) or multiple (Figure 3D; Table S2) methylated CpA motifs (5mCpA) and bound methylated CpA motifs in DNA containing one methylated CpA, CpT and CpG motif (Figure S8). DSETDB1-MBDL[R436C], which contains a single amino acid exchange mutation of arginine (R) 436 to cysteine (C) (R436C) (Figure 3A,B), did not bind 5mCpA motifs (Figure 3E). Our results reveal that dSETDB1-MBDL binds 5mCpA motifs in vitro and imply that dSETDB1 interacts with mDNA in Drosophila cells.
10.1371/journal.pone.0010581.g003Figure 3 The MBDL of dSETDB1 binds methylated CpA motifs.
(A) Schematic representation of dSETDB1 and the mutant dSETDB1(R436C), which contains the single amino acid exchange mutation arginine (R) to cysteine (C) at position 436. Rectangles mark the positions of the MBDL, Pre-SET domain (Pre), SET domain (SET), and Post-SET domain (Post). (B) Coomassie blue-stained SDS-PAGE gel detecting affinity-purified GST, the fusion protein consisting of GST, the MBDL of dSETDB1 (MBDL), and the GST-MBDL(R436C) [MBDL(R436C)] fusion protein. Recombinant proteins were expressed in Escherichia coli, purified with glutathione-agarose and subjected to SDS-PAGE. The positions and relative molecular weights of protein standards are indicated. The arrow marks the position of GST-MBDL and GST-MBDL(R436C), the asterisk the position of GST. The protein samples and amounts shown were used for the binding assays shown in (C-E). (C) Autoradiogram of in vitro protein–DNA interactions detecting the association of [32P]-radiolabeled, non-methylated (-) and methylated (+) DNA oligonucleotides containing 1 symmetrically methylated CpG-, CpA- or CpT motif. After proteins were incubated with DNA, retained DNA was purified, separated on native PAGE, and detected by autoradiography. (D) Autoradiogram of in vitro protein–DNA interaction assays as described in (C) except that methylated DNA oligonucleotides contained 3 symmetrically methylated CpA-, CpT-, or CpG motifs. (E) Autoradiogram of in vitro protein–DNA interaction assays as described in (C) except that GST, MBDL, and MBDL(R436C) and a DNA oligonucleotide containing 3 symmetrically methylated CpA motifs [(5mCpA)3] and the corresponding non-methylated DNA oligonucleotide [(CpA)3] were used. (C-E) The arrowhead marks the position of retained, radiolabeled DNA.
DSETDB1-mediated tri-methylation of H3-K9 promotes DNA methylation
Next, we asked whether dSETDB1 is involved in silencing and DNA methylation in Drosophila. We used in vitro DNA–protein interaction assays designed to identify mDNA target sequences for dSETDB1. Genomic DNA was isolated from 0–12 h old Drosophila embryos, sheared and incubated with dSETDB1-MBDL or dSETDB1-MBDL[R436C]. We obtained several DNA sequences that specifically associated with dSETDB1-MBDL but not dSETDB1-MBDL[R436C] (Figure S9). Among the identified DNA sequences are enhancer regions of the genes Rb, Antennapedia (Antp), and CG2136, and 2 copies of the retrotransposon Rt1b: Rt1b{}779 and Rt1b{}999. The 5155-bp Rt1b retrotransposon encodes for a nucleocapsid protein Gag and reverse transcriptase (RvT) and is present in 60 copies in the genome [38]. Rb is the Drosophila ortholog of the mammalian Rb gene [39]. Rb proteins regulate the activity of E2F transcription factors and are key regulators of cell proliferation by controlling the G1 and S phases of the cell cycle [40]. During eye development, silencing of Rb in response to oncogenic Notch signaling involves DNA methylation [41]. The homeotic gene Antp is a member of the HOX gene family, which is conserved between Arthropods and Chordates, and involved in cell fate determination during various stages of development [42]. CG2316 encodes for a putative ABC transporter membrane protein [43].
Because S2 cells apparently do not express dSETDB1 (Figure S10) and the genome of S2 cells is apparently not methylated [44], we transiently expressed wild type and mutant dSETDB1 proteins in S2 cells to assess whether dSETDB1 is involved in the transcription of identified target genes. Rb, Antp, and CG2316 are transcribed in S2 cells (Figure 4A; Figures S11-S12). We detected weak transcription of Rt1b retrotransposons in S2 cells, which suggests that Rt1b{} retrotransposons are transcriptionally active in S2 cells (Figure 4A; Figures S11-S12). However, because of the large copy number of Rt1b retrotransposons in the fly genome, we were unable to discern whether the detected transcripts originate from Rt1b{}779 and Rt1b{}999.
10.1371/journal.pone.0010581.g004Figure 4 DSETDB1-mediated tri-methylation of H3-K9 initiates DNA methylation and silencing.
(A) Digital images of ethidium bromide-stained agarose gels showing the reaction products of RT-PCR assays monitoring the transcription of Rb, Antp, CG2316, Rt1b/gag, dSETDB1, Dnmt2, Su(var)205 and actin5C in total RNA pools isolated from S2 cells, S2 cells transiently expressing GFP (control), and S2 cells co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). (B) Digital images of ethidium bromide-stained agarose gels showing the products of PCR assays detecting the dSETDB1 target DNA sequence in Antp, CG2316, and Rt1b{}799 (see Figure S9) in DNA pools obtained by ChIP. Chromatin was isolated from cells described in (A) and immunoprecipitated with antibodies and controls as described in Figure 2C. 1% of the chromatin used for ChIP.
Transient, ectopic expression of SETDB1 repressed the transcription of Rb, Antp, Rt1b and CG2316 transcription in S2 cells, whereas dSETDB1(H775L) and dSETDB1(R436C) did not, which suggests that the SET domain and MBDL are involved in dSETDB1-mediated silencing (Figure 4A; Figures S11-S12; Table S1). Ectopic dSETDB1 expression did not significantly affect Dnmt2 and Su(var)205 transcription, which suggests that dSETDB1 is not involved in regulating Dnmt2 and Su(var)205 transcription (Figure S12).
Next, we used ChIP assays to investigate whether silencing of target genes coincides with recruitment of dSETDB1 and methylation of H3-K9 and DNA. Chromatin was isolated from S2 cells expressing dSETDB1 [dSETDB1 cells] or dSETDB1(H775L) [dSETDB1(H775L) cells] and precipitated with antibodies to dSETDB1 (Figure S12); mono-, di-, and tri-methylated H3-K9 [(me3)H3-K9] (Figure S4); methylated cytosine (5mC) (Figure S13); and Dnmt2 (Figure S14). In contrast to a recent study, we detected the expression of Dnmt2 in S2 cells (Figure S14) [44]. Immunoprecipitated DNA was purified and analyzed with PCR assays that detected the presence of target DNA in immunoprecipitated DNA pools (Table S1).
DSETDB1, (me3)H3-K9, 5mC, and Dnmt2 were not detected at the transcriptionally active Rb Antp, CG2316 and Rt1b{}779 loci (Figure 4A,B; Figures S15-S17) and Rb (Figure 5A) in S2 cells, but were present at the silent gene loci in dSETDB1 cells (Figures 4A,B and 5A; Figures S15-S18). Tri- but not mono- and di-methylated H3-K9 was detected at silent target genes (Figure S19). In contrast, we detected dSETDB1 but not (me3)H3-K9, Dnmt2, and 5mC at the target gene loci in dSETDB1(H775L) cells (Figures 4B,5B; Figures S15-S18). Similarly, recruitment of Dnmt2, DNA methylation, and silencing of the tetO-tk-luc reporter gene involved dSETDB1-mediated tri-methylation of H3-K9 (Figure 2C; Figures S6 and S19). These results suggest that tri-methylation of H3-K9 by dSETDB1 can instigate DNA methylation and silencing.
10.1371/journal.pone.0010581.g005Figure 5 DSETDB1-mediated tri-methylation of H3-K9 propagates spreading of DNA methylation and silencing of Rb.
(A) Digital images of ethidium bromide-stained agarose gels showing reaction products for the PDE and Exon-I of Rb indicated in (A) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP (control) and S2 cells co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. PCR assays detected the PDE, PPE and Exon-I in immunoprecipitated DNA pools. (A,C) Input represents the amount of target DNA present in 1% of the chromatin used for ChIP. (B) Digital images of ethidium bromide-stained agarose gels detecting the target DNA sequences for dSETDB1 in Antp, CG2136 and Rt1b{}779 (see Figure S9) in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells transiently expressing GFP and dSETDB1(R436C). Chromatin was immunoprecipitated with antibodies and agarose beads described in Figure 2C. (C) Schematic representation of the Rb locus. Boxes mark the position of exons I (Exon-I), II, and VIII. The positions of the promoter distal enhancer element (PDE), promoter proximal enhancer element (PPE), and Exon-I fragments detected in ChIP assays are indicated. (D) Digital images of ethidium bromide stained agarose gels showing the reaction products of methylation-sensitive restriction analyses of genomic DNA isolated from cells described in (B). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the PDE, Exon-I, and the promoter region of Peepsqueak (Psq) in treated DNA pools.
Silencing of Rb involves initiation and spreading of DNA methylation and heterochromatin
DSETDB1-MBDL is involved in silencing of target genes (Fig. 4A). We performed ChIP assays to assess the role of the dSETDB1-MBDL in silencing and DNA methylation. Chromatin was isolated from S2 cells expressing dSETDB1(R436C) [dSETDB1(R436C) cells]. DSETDB1, Dnmt2 and methylated H3-K9 and DNA were present at the transcriptionally silent dSETDB1 target genes in dSETDB1(R436C) cells (Figures 5A,B; Figures S18 and S20), which implies that the MBDL is not involved in DNA methylation. How does the MBDL of dSETDB1 silence target gene transcription? In S2 cells, silencing of Rb coincides with methylation of DNA and H3-K9 at a promoter distal promoter element (PDE) (Figure 5A-C; Figure S9). In developing eye imaginal discs, silencing of Rb coincides with DNA methylation at the PDE, a promoter-proximal enhancer fragment (PPE) and first exon (Exon-I) (Figure 5C) [41], thus raising the possibility that dSETDB1 facilitates the spreading of DNA and H3-K9 methylation from the PDE to the coding region. To test this hypothesis, we used ChIP assays to detect dSETDB1-mediated H3-K9 and DNA methylation patterns on the Rb locus. Chromatin was isolated from dSETDB1, dSETDB1(H775L), and dSETDB1(R436C) cells and precipitated with antibodies to dSETDB1, (me3)H3-K9, 5mC, and Dnmt2. Immunoprecipitated DNA was purified and subjected to PCR assays to detect the presence of the PDE, PPE and promoter in immunoprecipitated DNA pools.
We detected dSETDB1, (me3)H3-K9, 5mC, and Dnmt2 at the PDE, PPE and Exon-I of Rb in dSETDB1 cells (Figure 5A; Fig. S18). In dSETDB1(H775L) cells, only dSETDB1 was detected at the PDE (Figure 5A; Fig. S18). In dSETDB1(R436C) cells, SETDB1, (me3)H3-K9, 5mC, and Dnmt2 occupied the PDE but not the PPE or Exon-I, which indicates that MBDL mediates the spreading of (me3)H3-K9 and mDNA from the PDE to the coding region (Figure 5A; Fig. S18). This model is supported by the results of bisulfite DNA sequencing (Figures S16 and S21) and methylation-sensitive restriction analyses (Figure 5D), which reveal an involvement of the SET domain and MBDL in initiation and spreading, respectively, of DNA methylation at the Rb locus.
DSETDB1 cooperates with Dnmt2 and Su(var)205 in Rb silencing
The association of HP1 with H3-K9 is a hallmark of silencing in heterochromatin and euchromatin [34]. The interaction of HP1 and Dnmts in vertebrates raised the possibility that Su(var)205 contributes to Rb silencing by recruiting Dnmt2 [45]. In ChIP assays, Su(var)205 was detected at the transcriptionally silent Antp, CG2316, and Rt1b{}779 (Figure 4B), the tetO-tk-luc reporter (Figure 2C) and Rb (Figure 5A). In contrast, Su(var)205 and Dnmt2 were not detected at dSETDB1 target genes in the absence of dSETDB1-mediated methylation of H3-K9, which suggests that the association of Su(var)205 with (me3)H3-K9 mediates recruitment of Dnmt2 to Rb (Figures 2C, 4B, and 5C). Su(var)205 interacted with Dnmt2 but not dSETDB1 in vitro and in vivo, which suggests that Su(var)205 is involved in recruiting Dnmt2 to target genes for dSETDB1 (Figure S22). To test this, we asked whether destruction of Dnmt2 and Su(var)205 through RNA interference (RNAi) affects dSETDB1-mediated silencing (Figure S23). Knockdown of Dnmt2 or Su(var)205 attenuated dSETDB1-mediated silencing of Rb (Figure 6A,B; Figure S24), Antp, CG2316, and Rt1b{} retrotransposons (Figure 7A; Figure S25). Knockdown of Dnmt2 attenuated DNA methylation of Rb (Figure 6C,D; Figures S26-S28), Antp, CG2316, and Rtb{}799 (Figure 7B,D; Figures S28-S30), whereas knockdown of Su(var)205 prevented recruitment of Dnmt2 and DNA methylation at the Rb locus (Figure 6C, right panel, Figure 6D; Figure S26-28), and the Antp, CG2316, and Rtb{}799 loci (Figure 7C; Figure S31). Collectively, these results reveal that dSETDB1 cooperates with Su(var)205 and Dnmt2 in DNA methylation and silencing of genes and Rt1b{} retrotransposons.
10.1371/journal.pone.0010581.g006Figure 6 Dnmt2 and Su(var)205 cooperate with dSETDB1 in DNA methylation.
(A) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Rb transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small-interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control RNA targeting human GAPDH (mock-RNAi). (B) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (C) Digital images of ethidium bromide-stained agarose gels showing the presence of the PDE of Rb (Figure 5A) in DNA pools generated by ChIP with chromatin isolated from cells described in (a; left panel) and (b; right panel). Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C. (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D). PCR assays detected the presence of PDE (Figure 5B) in treated DNA pools.
10.1371/journal.pone.0010581.g007Figure 7 Dnmt2 and Su(var)205 mediate dSETDB1-dependent DNA methylation and silencing of Antp, CG2316 and Rt1b{}799.
(A) (Left panel) Digital images of ethidium bromide-stained agarose gels showing reaction products of PCR assays detecting Antp, CG2316, and Rt1b{} transcription in total RNA isolated from S2 cells, S2 cells expressing dSETDB1, and S2 cells transiently expressing dSETDB1 in the presence of small interfering RNA (siRNA) targeting Dnmt2 (Dnmt2-RNAi) or control siRNA targeting human GAPDH (mock-RNAi). (Right panel) PCR assays as in (A) except that S2 cells were treated with siRNA targeting Su(var)205 [Su(var)205 RNAi]. (B) Digital images of ethidium bromide-stained agarose gels detecting the presence of Antp, CG2316, and Rt1b{} in DNA pools generated by ChIP with chromatin isolated from cells described in (A; left panel) Chromatin was immunoprecipitated with the antibodies and controls described in Figure 2C (C) ChIP assays as described in (C) except that chromatin was isolated from cells described in (A; right panel). (D) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from cells described in (A,B). Assays were performed as described (Figure 5D) except that PCR assays detected the presence of Antp, CG2316, and Rt1b{'} in treated DNA pools.
DSETDB1-mediated DNA methylation facilitates silencing of Rb in the developing eye
In the developing eye imaginal disc, Rb is expressed in two stripes flanking the morphogenetic furrow (MF), which progresses in a posterior to anterior direction across the eye imaginal disc and induces photoreceptor cell differentiation [46]. Undifferentiated cells exiting the MF undergo a second round of cell proliferation, which includes a single round of synchronized mitosis (second mitotic wave). Rb controls the rate of cell proliferation and differentiation in developing eyes by repressing E2F target gene expression [e.g., proliferating-cell nuclear antigen (PCNA)] and mitosis during the second mitotic wave [47].
To assess whether silencing of Rb involves dSETDB1-mediated DNA methylation in Drosophila, we attenuated dSETDB1 expression in developing eye imaginal discs by RNAi using the binary Gal4/UAS system [11]. Eye imaginal discs were isolated from lzGal4;UAS-dSETDB1.IR third-instar larvae. The Gal4-dependent reporter gene UAS-dEset1.IR (for simplicity, termed UAS-dSETDB1.IR) [11] transcribes an interfering double-stranded RNA targeting the dSETDB1 mRNA. The lzGal4 driver expresses Gal4 in all cells posterior to the MF. Western blot and immunostaining assays indicated that dSETDB1 expression is significantly reduced in lzGal4;UAS-dSETDB1.IR eye discs (Figure S32).
Knockdown of dSETDB1 resulted in ectopic transcription of Rb in cells posterior to the MF (Figure 8A). As observed in imaginal discs expressing constitutively active Rb
[47], ectopic Rb expression (Figure S35) suppressed PCNA transcription (Figure 8B; Figure S33) and mitosis during the second mitotic wave (Figure 8C) and resulted in defective eye development, as evidenced by the presence of misshaped and fused ommatidia and lack of bristles (Figure 9A,B). To assess the role of Dnmt2 in Rb transcription, we monitored Rb and PCNA expression in eye imaginal discs lacking Dnmt2 through RNAi. Knockdown of Dnmt2 resulted in ectopic expression of Rb in cells posterior to the MF (Figure 8A), repression of PCNA (Figure 8B), and attenuation of mitosis (Figure 8C). Collectively, these results indicate that dSETDB1 and Dnmt2 are involved in Rb expression in developing eye imaginal discs
10.1371/journal.pone.0010581.g008Figure 8 dSETDB1 represses Rb expression in the developing eye.
(A, B) In situ hybridization assays detecting the transcription of (A) Rb and (B) proliferating-cell nuclear antigen (PCNA) in eye imaginal discs prepared from control 3rd-instar larvae containing the lzGal4 driver, the reporter UAS-dSETDB1.IR or UAS-Dnmt2, and lzGal4 with UAS-dSETDB1.IR (lzGal4;UAS-dSETDB1.IR) or lzGal4 with UAS-Dnmt2 (lzGal4;UAS-Dnmt2). (C) Immunostaining assays detecting the mitotic marker phosphorylated H3 (serine 10) in eye imaginal discs described in (A,B). The mitotic index is38+3 for lzGal4 eye discs, 37+4 for UAS-dSETDB1.IR discs, 36+2 for UAS-Dnmt2, 9+3 for lzGal4;UAS-dSETDB1.IR and 11+2 for lzGal4;UAS-Dnmt2 discs. (A-C) The white-filled arrowheads mark the position of the morphogenetic furrow (MF). (A) Blue arrowheads indicate areas of ectopic Rb transcription in the posterior region (rectangle) of eye imaginal discs lacking dSETDB1 or Dnmt2, as compared to controls (see area marked by rectangle in lzGal4 discs). (B) The rectangle marks the position of the posterior PCNA transcription domain. The transcription of the posterior PCNA domain (rectangle) is reduced in dSETDB1 or Dnmt2 deficient eye imaginal discs as compared to controls. (C) The dark arrowhead marks the position of mitotic cells in the second mitotic wave posterior to the morphogenetic furrow. Note that the number of mitotic cells in regions posterior to the morphogenetic furrow (rectangle) is significantly reduced in eye discs lacking dSETDB1 or Dnmt2 through RNAi.
Next we performed ChIP assays to assess whether dSETDB1-mediated repression of Rb involves DNA methylation in imaginal discs. We isolated cell stripes from cross-linked eye imaginal discs with the genotype lzGal4, and UAS-dSETDB1.IR and lzGal4;UAS-dSETDB1.IR. Two cell stripes were isolated: one corresponding to the posterior Rb transcription domain (PRbD); the second corresponding to the posterior cells flanking the posterior Rb transcription domain [posterior cell stripe (PCS)] (Figure 9C). Rb is transcribed in the PRbD of control, lzGal4, UAS-dSETDB1.IR, and lzGal4;UAS-dSETDB1.IR (Figure 8A). Rb is not transcribed in the PCS isolated from lzGal4 and UAS-dSETDB1.IR discs (Figure 8A) but is transcribed in the PCS of lzGal4;UAS-dSETDB1.IR discs (Figure 8A). Chromatin was isolated from 20 cell stripes, sheared, and immunoprecipitated with antibodies to dSETDB1, 5mC, and Dnmt2.
10.1371/journal.pone.0010581.g009Figure 9 DSETDB1-mediated DNA methylation mediates silencing of Rb in the developing eye.
(A) Scanning electron micrographs showing the adult eye phenotype of lzGAl4, UAS-dSETDB1.IR and lzGAL4;UAS-dSETDB1.IR flies (50-fold magnification). (B) Magnification of the areas marked by white rectangles in (A) (1,000-fold total magnification). The arrowhead marks the position of fused ommatidia and the arrow the position of misshaped ommatidia. (C) (Top) Digital photograph showing Rb transcription in eye imaginal disc. The position of the posterior Rb transcription domain (PRbD) and posterior cell stripe (PCS) are indicated. (Bottom) Digital images of ethidium bromide-stained agarose gels detecting the PDE of Rb in DNA pools obtained by ChIP. Chromatin was isolated from cell stripes representing the PRbD and the PCS of imaginal discs isolated from third-instar lzGAl4, UAS-dSETDB1.IR, and lzGAL4;UAS-dSETDB1.IR larvae. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, 5mC or rabbit serum (mock). Input represents the amount of target DNA present in 4% of the chromatin used for ChIP.
DSETDB1, 5mC and Dnmt2 were not detected at the transcriptionally active Rb promoter in the PRbD from lzGal4, UAS-dSETDB1.IR, and lzGal4;UAS-dSETDB1.IR discs (Figure 9C; Figure S34). In contrast, dSETDB1, 5mC and Dnmt2 were present at the transcriptionally silent Rb locus in the PCS from lzGal4 and UAS-dSETDB1.IR discs (Figure 9C; Figure S36). We did not detect dSETDB1, 5mC or Dnmt2 in the PCS from lzGal;UAS-dSETDB1.IR, which lacks dSETDB1 and transcribes Rb (Figure 9C; Figure S36). Collectively, the results reveal that dSETDB1-mediated DNA methylation is involved in silencing of Rb in the developing eye imaginal disc of Drosophila.
DSETDB1 is involved in DNA methylation and silencing of retrotransposons
Because dSETDB1 mediates silencing of Rt1b{} retrotransposons in S2 cells (Figures 4 and 7). To test whether dSETDB1 is involved in silencing of Rt1b[] retrotransposons in Drosophila, we monitored the transcriptional activity and DNA methylation status of Rt1b{} and HeT-A retrotransposons in developing wing imaginal discs, which lack dSETDB1 and/or Dnmt2 expression by RNAi [11]. HeT-A retrotransposons are integral components of Drosophila telomers and silencing of HeT-A retrotransposons involves dSETDB1-dependent DNA methylation [10]. Wing imaginal discs were isolated from Gal4(71B);UAS-dSETDB1.IR;UAS-Dnmt2 and control third-instar larvae. The Gal4-dependent reporter gene UAS-Dnmt2 transcribes an interfering double-stranded RNA targeting the Dnmt2 mRNA. The Gal4(71B) driver expresses Gal4 ubiquitously in imaginal discs. Knockdown of dSETDB1, Dnmt2, or both, resulted in ectopic transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs (Figure 10A-C; Figure S35).
10.1371/journal.pone.0010581.g010Figure 10 DSETDB1-mediated DNA methylation mediates silencing of Rt1b{} and HeT-A retrotransposons in the developing wing.
(A) In situ hybridization assays detecting Rt1b and HeT-A transcription in wing (W), haltere (H), and third-instar leg (L) imaginal discs isolated from third-instar larvae, which ubiquitously express Gal4 [Gal4(71B)], contain the reporter construct USA-Dnmt2 and UAS-dSETDB1.IR or lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR], Dnmt2 [Gal4(71B);USA-Dnmt2], or both [Gal4(71B);UAS-dSETDB1.IRGal4(71B);USA-Dnmt2] by RNAi. (B,C) RvT-PCR assays monitoring the transcription of Rt1b{} and HeT-A retrotransposons in wing imaginal discs described in (A). RNA was isolated from 50 wing discs and reverse transcribed. PCR detected the presence of (A) Rt1b{} and (D) HeT-A transcription. (D,E) Digital images of ethidium bromide-stained agarose gels showing the reaction products of PCR assays detecting the presence of (B) Rt1b{} and (E) HeT-A in DNA pools generated by ChIP. Chromatin was isolated from in vivo cross-linked wing imaginal discs isolated from larvae of the genotype described in (A,D). Chromatin was immunoprecipitated with antibodies to dSETDB1, 5mC, Dnmt2, Su(var)205, and rabbit serum (control). Input represents the amount of retrotransposons detectable in 2.5% of the input material. (F,G) Digital images of ethidium bromide-stained agarose gels showing the reaction products of methylation-sensitive restriction analyses with genomic DNA isolated from wing imaginal discs described in (A). Genomic DNA was isolated, incubated with bovine serum albumin (BSA) (mock), the methylation-sensitive restriction endonuclease HpaII, or the methylation-insensitive restriction enzyme MspI. PCR assays monitored the presence of the (C) Rt1b{} and (F) HeT-A in treated DNA pools.
Next, we performed methylation-sensitive restriction analyses and ChIP assays to assess whether dSETDB1-mediated repression of Rb involves DNA methylation. Chromatin was isolated from Gal4(71B);UAS-dSETDB1.IR;UAS-Dnmt2, Gal4(71B);UAS-dSETDB1.IR, Gal4(71B);UAS-Dnmt2 and control discs. We detected dSETDB1, 5mC, Su(var)205 and Dnmt2 at silent Rt1b[] and HeT-A retrotransposons. Knockdown of dSETDB1 and/or Dnmt2 resulted in loss of DNA methylation at Rt1b{} (Figures 10D,F; 1) and HeT-A (Figure 10E,G; Figures S36, and S37; Table S1) retrotransposons, which indicates that dSETDB1-mediated DNA methylation is involved in silencing of RT1b{} and HeT-A retrotransposons.
Discussion
Collectively, our results implicate dSETDB1 in postembryonic DNA methylation and silencing of genes and transposons. Despite significant progress, the functional importance of DNA methylation in Drosophila remains controversial, and important mechanistic aspects of the DNA methylation process remain unknown [4], [7]–[8]. DSETDB1 is involved in oogenesis, maintenance of heterochromatin, silences gene expression in pericentric heterochromatin, and activates and represses transcription in the peculiar chromatin of the fourth chromosome, respectively [11], [12], [13], [14], [33]. Our results reveal that dSETDB1 is an integral component of the Drosophila DNA methylation machinery. The functional dissection of the SET domain and MBDL reveals that dSETDB1 is an epigenetic repressor. The activity of the SET-domain initiates silencing and DNA methylation, whereas the MBDL can facilitate propagation of DNA methylation. DSETDB1 conveys epigenetic silencing by directly and indirectly supporting the placement of 2 epigenetic marks at target genes: (me3)H3-K9 and mDNA.
The dSETDB1-MBDL preferentially associates with methylated CpA motifs in vitro, which represents one of the two predominantly methylated DNA motifs in the Drosophila genome. In Arabidopsis thaliana, MBD proteins bind methylated CpG- and CpNG motifs and can associate with 5mC in any DNA sequence context [48]. The interaction of dSETDB1-MBDL with methylated CpA motifs suggests that Drosophila has evolved specialized factors to translate CpA methylation into biological function. Although the genomes of many Arthropods and vertebrates contain methylated CpN motifs and often express multiple MBDL proteins, the biological importance of CpN methylation in development and disease remains mysterious [15], [36], [49]. The association of dSETDB1-MBDL with methylated CpA motifs supports a model for MBDL proteins interpreting complex patterns of CpN methylation into distinct biological activities.
Recent studies revealed that dSETDB1 mono-, di-, and/or tri-methylates H3-K9 [11]–[12]. However, the mechanisms underlying the gene-specific, differential methylation of H3-K9 by dSETDB1 remain unknown. It appears possible that intra- and extra-organismal stimuli modulate the specificity of the catalytic activity of dSETDB1, which results in gene-specific mono-, di- or tri-methylation of H3-K9. Our studies uncover a role for dSETDB1-mediated tri-methylation of H3-K9 in DNA methylation, but do not exclude the possibility that mono- and/or di-methylation of H3-K9 by dSETDB1 might trigger DNA methylation in the context of other genes.
In Drosophila, plants, vertebrates, and Neurospora crassa, members of the Su(var)3–9 family play pivotal roles in DNA methylation [9], [20], [21], [22], [23]. Su(var)3–9 facilitates DNA methylation during Drosophila embryogenesis and is apparently not involved in postembryonic DNA methylation [9]. Because dSETDB1 is not expressed during the early stages of embryogenesis, when global DNA methylation occurs [11], it appears likely that dSETDB1 does not play a major role in embryonic DNA methylation. The involvement of dSETDB1 in DNA methylation and silencing of Rb and Rt1b{} retrotransposons in imaginal discs suggests that the differential activities of at least 2 distinct HMT pathways mediate embryonic and postembryonic DNA methylation in Drosophila: the Su(var)3–9 pathway acting during embryogenesis and dSETDB1 pathway during postembryonic stages. This hypothesis is supported by a recent study by Elgin and colleagues, which revealed that Su(var3–9) activity is restricted to early development, whereas dSETDB1 acts preferentially during later stages of development [33].
The role and function of Dnmt2 in DNA methylation remains controversial [8], [10]. In a recent study, Reuther and colleagues have linked Dnmt2 with DNA methylation at transposable elements and telomers [10]. The observed loss of DNA methylation and silencing of genes and retrotransposons in cells and imaginal discs, which lack Dnmt2 through RNAi supports the role for Dnmt2 in DNA methylation, differential gene expression, and silencing of transposon activity during Drosophila development. However, our results do not exclude the possibility that the observed Dnmt2-dependent DNA methylation does not involve the catalytic activity of Dnmt2 but rather other unknown Dnmts, which are recruited to dSETDB1 target genes in a Dnmt2-dependent fashion.
Silencing of retrotransposons is fundamental for the structural integrity of eukaryotic genomes. Our results reveal that dSETDB1 contributes to genome stability by silencing Rt1b{} and HeT-A retrotransposons. Our results reveal that Drosophila uses epigenetic regulators and mechanisms involved in heterochromatin formation to silence the activity of retrotransposons and genes such as the essential cell cycle regulator Rb and during development.
The results of transient expression experiments in S2 cells support a model for dSETDB1-mediated silencing of Rb: recruitment of dSETDB1 and subsequent methylation of H3-K9 resulted in recruitment of Su(var)205 and Dnmt2 to the PDE and DNA methylation. Because the MBDL can interact with methylated CpA motifs, methylation of CpA motifs may result in de novo recruitment of dSETDB1 proteins or allow PDE-associated dSETDB1 to bind mDNA downstream of the PDE. In both cases, recruitment of dSETDB1 triggers a self-perpetuating, self-renewing H3-K9 and DNA methylation cascade, which culminates in the silencing of Rb. Similarly, silencing of retrotransposons in Drosophila involves propagation of DNA methylation [10].
Why does silencing of Rb involve heterochromatin formation at the enhancer, promoter, and coding region? In vertebrates, methylation of promoter-proximal CpG islands has been linked to gene silencing [15]. DNA methylation can silence gene expression by preventing the interaction of transcription factors and the RNA polymerase II transcription machinery with target genes [5], [50]–[51]. However, CpG methylation and CpG islands are rare in Drosophila
[17], [18], [19]. Thus, in the absence of CpG islands, DNA methylation and subsequent heterochromatin formation at the enhancer, promoter and coding region of Rb may be necessary to prevent the association of transcriptional regulators with Rb and, consequently, the initiation and elongation steps of transcription.
Rb proteins play important roles in development, and the precise temporal and spatial expression of Rb is fundamental for cell proliferation and differentiation [40]. Our results suggest that epigenetic silencing of Rb during eye development in Drosophila involves dSETDB1-mediated DNA methylation and heterochromatin formation. Most cells in metazoan organisms are quiescent [52]. Epigenetic silencing of cell cycle regulators upon completion of development may maintain the quiescent state after completion of cell differentiation and proliferation. DNA methylation and silencing of tumor suppressor genes has been correlated with various human diseases such as cancer [53], [54], [55]. Our results shed new light on the mechanisms involved in DNA methylation and silencing of tumor suppressor genes and provide a foundation for the dissection of the role of SET/MBDL proteins in dynamic DNA methylation during cell proliferation in development and disease.
Materials and Methods
Plasmids
Detailed information about the recombinant DNA used in this study can be found in Text S1.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation was performed as described [56]. Cross-linked chromatin was isolated from FACS-sorted Drosophila melanogaster. S2 cells expressing wild type or mutant dSETDB1 proteins or TetRdSETDB1 derivatives, and wing imaginal discs and sections of eye imaginal discs, which were isolated from wild type and mutant larvae. Chromatin was immunoprecipitated with antibodies to anti-5-methyl cytosine (Megabase, #CP51000), anti-(me3)H3-K9 (Abcam, ab8898), anti-Dnmt2 (this study), anti-dSETDB1 (this study), and anti- Su(var)205 (this study). A detailed ChIP protocol is available in Text S1.
Mono- and polyclonal antibodies
The rat monoclonal antibody to dSETDB1 was developed against the peptide K-796-2 NNSTIYVDDENRC (amino acids 351–362). Immunization, fusion, and cloning were performed as described [59]. Polyclonal antibodies against Dnmt2 and Su(var)205 were generated in rabbits with use of the peptides Dnmt2-7A53-2 (amino acids 64–84) and Su(var)205 3C78-2 (amino acids 72–91). Polyclonal antibodies were produced by Biosynthesis (Lewisville, Texas, USA).
Protein-DNA binding assays
Protein-DNA binding assays were performed as described [37]. GST, GST-MBDL and GST-MBDL(R436C) were expressed in and purified from Escherichia coli XL1-Blue (Stratagene). The GST proteins were immobilized on glutathione-sepharose beads (Invitrogen). To reduce unspecific interactions, 100 µl of protein-loaded glutathione-sepharose beads were preincubated with 0.5 µg/µl yeast DNA in 500 µl binding buffer (BB) (25 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 5% [v/v] glycerol) at 4°C for 3 h. An amount of 10 µl of pre-incubated glutathione-sepharose beads loaded with 250–500 ng GST or GST-dSETDB1 derivatives were incubated with [32P]-labeled radiolabeled oligonucleotides (150,000 c.p.m./reaction) in BB at 4°C for 4 h. The DNA oligonucleotides were unmethylated or contained 1 or 3 mDNA motifs (Table S2). After incubation, beads were precipitated and washed twice with BB, 4 times with BB containing 500 mM NaCl, and twice with TE. Retained DNA was purified by phenol/chloroform extraction, separated on native polyacrylamide electrophoresis, and detected by autoradiography.
RNAi
Small interfering RNAs (siRNAs) targeting Su(var)205 and Dnmt2 were designed using the “siRNA at Whitehead” [57] generated by in vitro transcription using the silencer siRNA construction kit (Ambion). S2 cells, 5×106, were transfected with 1–4 µg siRNA by use of oligofectamine (Invitrogen) and harvested after 3–5 days. Western blot analyses confirmed destruction of the target protein. A detailed description of the siRNAs used for RNAi is available in Text S1.
Fly strains
The following fly strains were used: OregonR (Bloomington stock center: #5), 12196–48 [11], lzGal4
[58] (Bloomington stock center: #6313), gal4(71B) [59] and actin5CGal4 (Bloomington stock center: #3954). Fly strains were maintained and crossed using standard media and procedures [60].
Supporting Information
Text S1 Supporting Information: Materials and Methods.
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Figure S1 DSETDB1 methylates H3-K9 in nucleosomal H3. Coomassie blue-stained SDS-PAGE gel (left) and corresponding fluorogram (right) of HMT-assays containing a mixture of recombinant histones H2A, H2B, H3, and H4 (H-mix); purified, endogenous mononucleosomes (mono nucl); or purified, endogenous polynucleosomes (poly nucl). Histones were incubated with [3H]-SAM) (-), [3H]-SAM and “anti-Flag antibodies coupled to agarose beads” (“Flag-beads”), which had been incubated with Sf9 extract (Flag-beads), or [3H]-SAM and Flag-beads, which had been loaded with Flag-tagged, recombinant dSETDB1 (Flag-beads-dSETDB1). Reaction products were separated by SDS-PAGE and detected by fluorography. Asterisks indicate the positions of the anti-Flag antibody light and heavy chains.
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Figure S2 DSETDB1 methylates H3-K9. (A) MALDI-TOF spectrum of the HPLC fraction containing the peptide 9K(me0–3)STGGKAPR. Shown is the complete spectrum corresponding to the MALDI-TOF spectrum shown in Fig. 1D. In addition to 9K(me0–3)STGGKAPR peptides, the HPLC fraction contains two peptides (peptide A, measured mass 573.362; peptide B, measured mass 630.412). The inset represents a magnified area of the spectrum shown in Fig. 1D. The x-axis indicates the mass/charge ratio. The y-axis indicates the abundance of peptides. (B) Table indicating the measured and calculated masses of detected peptides 9K(me0–3)STGGKAPR. ΔM/M represents the relative errors between measured and calculated masses.
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Figure S3 DSETDB1 preferentially tri-methylates H3-K9 (A) Nano-ESI MS/MS spectrum of the spectrum of the precursor ion at m/z 472.28 of peptide 9K(me3)STGGKAPR, which was obtained from the HPLC fraction shown in Fig. S2. The y-axis indicates abundance of peptides (ion counts), the x-axis represents the mass/charge ratio (m/z). (B) Nano-ESI MS/MS spectrum as in (A) except that the precursor ion at m/z 465.28 of peptide 9K(me2)STGGKAPR was analyzed. (C) Nano-ESI MS/MS spectrum as in (A) except that the precursor ion at m/z 458.27 of peptide 9K(me1)STGGKAPR was analyzed. (A-C) The inset shows the fragmentation schematic for the b and y series of the corresponding precursor ion.
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Figure S4 Western blot assays testing the specificity of antibodies recognizing mono-, di-, and tri-methylated H3-K9. 2 µg H3 peptides (amino acids 1-40) containing mono-, di- or tri-methylated H3-K9 were separated by SDS-PAGE and electrophoretically transferred onto PVDF. Blots were developed with antibodies to mono-methylated H3-K9 [anti-(me1)H3-K9; top], di-methylated H3-K9 [anti-(me2)H3-K9; middle] and tri-methylated H3-K9 [anti-(me3)H3-K9; bottom].
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Figure S5 Basal transcription level of the luciferase (luc) gene in the stable cell line (tetO-tk-luc)-S2. RvT-PCR assays detecting the mRNA of (top) luc and (bottom) actin5C in total RNA pools isolated from S2 cells and the stable cell line (tetO-tk-luc)-S2 cells. Arrowheads indicate the position of the PCR products.
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Figure S6 DSETDB1-mediated tri-methylation of H3-K9 mediates repression and DNA methylation. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 2C. RT-PCR assays were performed using the same DNA pools used for conventional PCR. The degree of association of an antigen with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S7 The MBD of MeCP2 binds methylated CpG-motifs. Autoradiogram of DNA-protein interaction assays programmed with DNA oligonucleotides containing one symmetrically methylated CpG-, CpA-, or CpT-motif and recombinant GST or fusion proteins consisting of GST and the MBD of MeCP2. Retained DNA was purified, separated by native PAGE, and detected by autoradiography. The arrowhead marks the position of retained DNA oligonucleotides.
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Figure S8 The MBDL of dSETDB1 preferentially binds methylated CpA motifs. (Top) Schematic representation of the 105 bp target DNA fragment C(ATG)-1. The positions of EcoRI restriction sites, the length of the DNA fragments (a,b,c) resulting from EcoRI digest, and the methylated CpN motifs present in fragments a, b, and c are indicated. (Bottom) Autoradiogram of in vitro DNA-protein binding assays. The 105 bp target DNA was incubated with glutathione beads loaded with GST, GST-MBDL, or GST-MBDL(R436C) (see Figure 3). Bound DNA was digested and digested with EcoRI. Retained, radiolabeled DNA was eluted. Retained DNA was separated by native PAGE and detected by autoradiography. The positions of radiolabeled DNA fragments (a, b, and c) are indicated.
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Figure S9 Identification of target genes for dSETDB1. (A) Digital image of ethidium bromide-stained agarose gel showing the affinity-purified genomic DNA. DNA was isolated from 0-12 h old Drosophila embryos and sonicated (input, lane 1). Genomic DNA was sequentially incubated with the MBDL of dSETDB1 (lane 2), the mutant MBDL(R436C) (lane 3), and the MBDL of dSETDB1 (lane 4). Purified DNA was amplified by PCR (lanes 2-3) cloned and sequenced. Input represents 0.1% of the input DNA used for the affinity-purification assay. PCR products, which contained 0.0001% of the affinity-purified DNA obtained after each purification step, are shown in lanes 2-4. 15% of the PCR reaction products and 0.1% of the DNA input material were separated by agarose gel-electrophoresis. (B) Table describing the putative dSETDB1 target genes. Listed are genes and transposable elements, which associate with the MBDL of dSETDB1 in vitro. The table lists genes and transposable elements, the region of the genes and transposable elements, which were found to associate with dSETDB1 in vitro, and the corresponding reference sequences.
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Figure S10 Functional characterization of the monoclonal antibody to dSETDB1 in Western Blot and immunoprecipitation (IP) assays. (A) Digital image of IP assays detecting dSETDB1 in total cell extracts prepared from S2 cells and nuclear extract prepared from 0-8 h old Drosophila embryos. Extracts were incubated with rat monoclonal antibody to dSETDB1. Protein-antibody complexes were precipitated using protein-G agarose, separated by SDS-PAGE, and electrophoretically transferred onto PVDF membrane. Western blots were developed using anti-dSETDB1 monoclonal rat antibody. Asterisks indicate the positions of the light and heavy chains of the anti-Flag antibody. (B) Digital image of Western blot analysis detecting dSETDB1 in total cell extracts prepared from Sf9 cells or Sf9 cells infected with recombinant baculovirus expressing Flag-epitope tagged dSETDB1 (Flag-dSETDB1). Cell extracts were separated by SDS-PAGE and electrophoretically transferred onto PVDF membrane. Western blots were developed using monoclonal anti-rat antibody to dSETDB1. (C) Digital image of Western blot analysis of IP assays detecting Flag-dSETDB1 using rat monoclonal antibody to dSETDB1. Flag-dSETDB1 was immunoprecipitated from total Sf9 extracts containing Flag-dSETDB1 using 1 µg dSETDB1 antibody and protein-G agarose beads (left) or Flag-beads containing 5–10 µg anti-Flag antibodies (right). Protein-antibody complexes were precipitated, separated by SDS-PAGE, and electrophoretically transferred onto PVDF membrane. Western blots were developed using rat monoclonal antibody to dSETDB1. (D) Digital image of Coomassie-Blue stained SDS-polyacrylamide gel detecting the presence of dSETDB1 in protein pools, which had been immunoprecipitated from nuclear extracts prepared from S2 cells and 0–8 h old embryos with antibody to dSETDB1. Immunoprecipitated proteins were separated by SDS-PAGE and detected by Coomassie Blue staining. Mass-spectrometry confirmed the presence of dSETDB1 in the protein bands marked with arrowheads.
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Figure S11 DSETDB1 mediates repression of genes and retrotransposons. Schematic representation of Real-Time (RT) PCR assays corresponding to the Rvt-PCR assays shown in Figure 4A. RT-PCR assays were performed with the same cDNA pools used for conventional RvT-PCR. The level of transcription is presented in percent (%). The level of target gene transcription in S2 cells was set as 100%.
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Figure S12 DSETDB1 does not control Dnmt2 and Su(var)205 transcription. Schematic representation of Real-Time (RT) PCR assays corresponding to the RvT-PCR assays shown in Figure 4A. RT-PCR assays were performed with the same cDNA pools used for conventional RvT-PCR. The level of transcription is presented in percent (%). The level of target gene transcription in S2 cells was set as 100%.
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Figure S13 Functional characterization of the polyclonal antibody to 5mC. Autoradiogram of immunoprecipitation assays using rabbit serum or polyclonal antibody to 5mC. Antibodies were incubated with 0.1 µg [32P]-radiolabeled DNA oligonucleotides, which are not methylated (left) or contain three symmetrically methylated CpA-motifs (right). DNA-antibody complexes were precipitated using protein-A agarose. Retained DNA was purified, separated by native PAGE, and detected by autoradiography. The arrowhead marks the position of DNA oligonucleotides.
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Figure S14 The anti-Dnmt2 polyclonal antibody detects Dnmt2 in Western Blot and immunoprecipitation (IP) assays. (A) Digital image of Western blot assays detecting Dnmt2 in total cell extracts prepared from Sf9 cells or Sf9 cells infected with recombinant baculovirus expressing Flag-epitope tagged Dnmt2. (B) Western blot analysis detecting Dnmt2 in total cell, extract prepared from S2 cells and nuclear extract prepared from 0–8 h old Drosophila embryos. (C) Western blot analyses of IP assays. 0.5 mg total S2 cell extract was incubated with rabbit serum or rabbit polyclonal antibody to Dnmt2. Protein-antibody complexes were precipitated with protein-A agarose. Cell extracts (A,B) and immunoprecipitated proteins (C) were separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and developed using rabbit polyclonal antibody to Dnmt2 (A-C). Asterisks indicate the positions of the anti-Flag antibody light and heavy chains.
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Figure S15 DSETDB1-mediated tri-methylation of H3-K9 mediates silencing and DNA methylation. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 4B. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S16 The activities of the SET-domain and MBDL of dSETDB1 mediate initiation and spreading of DNA methylation at the Rb locus. Schematic representation of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells transiently expressing GFP (mock) and S2 cells transiently co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Genomic DNA was treated twice with bisulfite. The indicated regions of the PDE and Exon-I of Rb were amplified by PCR and cloned into pCR2.1-TOPO. 10 PCR products were sequenced for each bisulfite reaction. The y-axis shows the CpN methylation rate in percent (%) for regions within the PDE (left) and the Exon-I (right) of Rb. The CpN methylation rate was calculated by dividing the number of methylation events at CpN-motifs by the total number of CpN-motifs present in tested DNA fragments. The shown data represents the mean value of the CpN methylation rates obtained form a total of 20 clones generated in two different “bisulfite-treated DNA sequencing” assays. Error bars represent the standard error of the mean (SEM).
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Figure S17 DSETDB1 mediates DNA methylation at the Antp and CG2316 and Rt1b{}799 loci. Schematic representation of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells transiently expressing GFP (mock) and S2 cells transiently co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Bisulfite-assays were performed and analyzed as described in Figure S16 except that DNA methylation was monitored at DNA fragments corresponding to the enhancer region of Antp and CG2316 and Rt1b{}799.
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Figure S18 DSETDB1 controls initiation and spreading of DNA methylation on the Rb locus. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 5A. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S19 DSETDB1 preferentially tri-methylates H3-K9 at target genes. Digital images of ethidium bromide-stained agarose gel showing the PCR products for the PDE of Rb (see Figure 5) and the promoter of the tetO-tk-luc reporter gene in DNA pools obtained by ChIP. Chromatin was isolated from S2 cells (top) and tetO-tk-luc S2 cells (bottom) transiently expressing dSETDB1. Chromatin was immunoprecipitated with antibodies to mono-methylated H3-K9, di-methylated H3-K9, and tri-methylated H3-K9 or rabbit serum (mock). PCR detected the presence of the PPE in immunoprecipitated DNA pools. Input represents the amount of target DNA present in 1% of the chromatin used for ChIP.
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Figure S20 The MBDL of DSETDB1 is involved in silencing. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 5B. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S21 DSETDB1 mediates methylation at the Rb locus. Schematic representation of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells transiently expressing GFP (mock), and S2 cells transiently co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Genomic DNA was treated twice with bisulfite. The indicated regions of the PDE and Exon-I of Rb were amplified by PCR and cloned into pCR2.1-TOPO. 10 PCR products were sequenced for each bisulfite reaction. The grey boxes indicate the number of detected methylation events at individual CpN-motifs present in a highly methylated region within the PDE (left) and Exon-I (right) of Rb. DSETDB1 mediates methylation at the Rb locus. Schematic representation of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells transiently expressing GFP (mock), and S2 cells transiently co-expressing GFP and dSETDB1, dSETDB1(H775L), or dSETDB1(R436C). Genomic DNA was treated twice with bisulfite. The indicated regions of the PDE and Exon-I of Rb were amplified by PCR and cloned into pCR2.1-TOPO. 10 PCR products were sequenced for each bisulfite reaction. The grey boxes indicate the number of detected methylation events at individual CpN-motifs present in a highly methylated region within the PDE (left) and Exon-I (right) of Rb.
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Figure S22 Dnmt2 and Su(var)205 associate in vitro and in Drosophila. (A,B) Fluorograms of in vitro protein-protein interaction assays programmed with Flag-beads loaded with Sf9 cell extract (control) or recombinant, Flag-epitope tagged dSETDB1, Dnmt2, and Su(var)205. Protein-loaded Flag-beads were incubated with in vitro translated, [35S]-methionine labeled (A) Dnmt2 or (B) Su(var)205. “Input” represents 5% of the input material used in binding assays (C,D) Western blot analysis of immunoprecipitation assays detecting the association of Dnmt2 and Su(var)205 in total cell extracts prepared from 0-8 h old Drosophila embryos. Extracts were incubated with antibodies to dSETDB1, Dnmt2, and Su(var)205. Precipitated proteins were separated by SDS-PAGE, electrophoretically transferred onto PVDF-membrane, and developed using antibodies to (C) Dnmt2 and (D) Su(var)205.
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Figure S23 Knockdown of Dnmt2 and Su(var)205 through RNAi. (A,B) Digital images of Western blot assays using total cell extract isolated from (A-C) S2 cells treated with control siRNA, which targets human GAPDH (mock-RNAi); (A) S2 cells treated with siRNA Dnmt2(1) [Dnmt2(1)-RNAi] or siRNA Dnmt2(2) [Dnmt2(2)-siRNA], which target the Dnmt2 mRNA. Extracts were separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and developed with rabbit monoclonal antibody to Dnmt2. (B,C) S2 cells were treated with siRNA Su(var)205(1) [Su(var)205(1)-RNAi] or siRNA Su(var)205(2) [Su(var)205(2)-siRNA], which target the Su(var)205 mRNA. Extracts were prepared using denaturing buffer containing 8M urea (B) or PBS (C), separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and developed with rabbit monoclonal antibody to Su(var)205. Note that Su(var)205 migrates as a 40 KD protein band in the absence of urea (C).
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Figure S24 Dnmt2 and Su(var)205 regulate Rb transcription. Schematic representation of Real-Time (RT) PCR assays corresponding to the Rvt-PCR assays shown in Figure 6A,B. RT-PCR assays were performed with the same cDNA pools used for conventional RvT-PCR. The level of transcription is presented in percent (%). The level of target gene transcription in S2 cells was set as 100%.
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Figure S25 Dnmt2 and Su(var)205 regulate the transcription of genes and retrotransposons. Schematic representation of Real-Time (RT) PCR assays corresponding to the RvT-PCR assays shown in Figure 7A. RT-PCR assays were performed with the same cDNA pools used for conventional RvT-PCR. The level of transcription is presented in percent (%). The level of target gene transcription in S2 cells was set as 100%.
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Figure S26 Su(var)205 and Dnmt2 mediate DNA methylation of Rb. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 6C. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S27 Dnmt2 and Su(var)205 mediate spreading of DNA methylation on the Rb locus. Digital images of ethidium bromide-stained agarose gels showing the PCR product for the Exon-I fragment of Rb (Fig. 5C) in DNA pools obtained by ChIP. The DNA pools used for the PCR assays are the same DNA pools, which were used to detect the PDE of Rb (Fig. 5A). Chromatin was isolated from S2 cells which did (+) or did not (-) transiently express dSETDB1, and were treated with control siRNA, which targets human GAPDH (mock-RNAi); (left) S2 cells treated with siRNA Dnmt2(1) (Dnmt2-RNAi), which targets the Dnmt2 mRNA; and (right) S2 cells treated with siRNA (Su(var)205-RNAi), which targets the Su(var)205 mRNA. Chromatin was immunoprecipitated with antibodies to dSETDB1, Dnmt2, Su(var)205, or 5mC. Input represents the amount of PCR product for Exon-I of Rb detectable in 3% of the chromatin sample used in immunoprecipitation assays.
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Figure S28 Dnmt2-mediated DNA methylation at the Rb and Antp loci. Schematic representations of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells incubated with siRNA targeting human GAPDH (mock) or siRNA targeting Dnmt2 mRNA. Bisulfite-assays were performed and analyzed as described in Figure S21 except that DNA methylation was monitored at DNA fragments corresponding to the PDE of Rb and the enhancer region of Antp.
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Figure S29 Dnmt2 mediates DNA methylation at genes and Rt1b retrotransposons. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 7B. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S30 Dnmt2-mediated DNA methylation at the CG2316 and Rt1b{}799 loci. Schematic representations of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from S2 cells incubated with siRNA targeting human GAPDH (mock) or siRNA targeting Dnmt2 mRNA. Bisulfite-assays were performed and analyzed as described in Figure S21 except that DNA methylation was monitored at DNA fragments corresponding to CG2316 and Rt1b{}799.
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Figure S31 Su(var)205 regulates DNA methylation at genes and retrotransposons. Schematic representation of RT-PCR assays corresponding to the conventional PCR assays shown in Figure 7C. The same immunoprecipitated DNA pools used for conventional PCR and RT-PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S32 Knockdown of dSETDB1 expression through RNA interference (RNAi). (A) Digital image of Western blot assays of immunoprecipitation assays using whole protein extract prepared from 0.2 g 0–8 h old embryos and 1,000 eye imaginal discs containing the indicated Gal4 driver and Gal4-dependent reporter genes. The actin5CGal4 driver strain (Act5CGal4) expresses Gal4 ubiquitously in Drosophila embryos. The lozenge (lz) Gal4 driver (lzGal4) expresses Gal4 in cells posterior and “to a lesser extent” anterior to the morphogenetic furrow in developing eye imaginal discs. The Gal4-dependent reporter UAS-dSETDB1.IR, which transcribes a dsRNA targeting the dSETDB1 mRNA. Total protein extracts were incubated with rat monoclonal antibody to dSETDB1. Protein-antibody complexes were precipitated with protein-G agarose, separated by SDS-PAGE, electrophoretically transferred onto PVDF membrane, and analyzed by Western blot using antibody to dSETDB1. The asterisk indicates the position of the heavy chain of the dSETDB1 antibody. The positions and relative molecular weights (rMW) of protein standards are indicated to the left. (B) Digital images of immunostaining assays detecting dSETDB1 and histone H3 phosphorylated at serine 10 [phospho-H3(Ser10)], which is a marker of mitosis, in eye imaginal discs isolated from third instar larvae containing the Gal4 driver and reporter constructs described in (A). Eye imaginal discs were isolated from third instar larvae and incubated with rat monoclonal antibody to dSETDB1 and rabbit polyclonal antibody to phospho-H3(Ser10). DSETDB1 (purple/brown) was detected using anti-rat secondary antibody coupled to alkaline phosphatase and the “Red Alkaline Phosphatase Substrate kit” (Vector Laboratories). Phospho-H3(Ser10) (dark brown) was detected using an anti-rabbit secondary antibody coupled to horseradish peroxidase and diaminobenzidine and peroxidae as substrates. The arrowhead marks the position of the morphogenetic furrow (MF). The enhanced staining on the right site of the eye imaginal discs results from folding of the eye discs.
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Figure S33 DSETDB1 represses PCNA transcription. Schematic representation of Real-Time (RT) PCR assays monitoring Rb and PCNA transcription in posterior and anterior halves of eye imaginal discs. RNA pools were isolated from posterior and anterior halves of eye imaginal discs, which were isolated from 3rd instar larvae of the genotypes described in Figure 8. Discs were separated at the morphogenetic furrow. RNA was reverse transcribed and the resulting cDNA pools served as a template for RT-PCR assays monitoring Rb (A) and PCNA (B) transcription. The level of transcription is presented in percent (%). The level of target gene transcription in eye imaginal discs isolated from lzGal4, larvae was set to 100%.
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Figure S34 DSETDB1 mediates methylation and silencing of Rb in the developing eye. Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 9C. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S35 DSETDB1 silences the transcription of Rt1b{} and HeT-A retrotransposons. Schematic representation of Real-Time (RT) PCR assays corresponding to the RvT-PCR assays shown in Figure 10B,C. RT-PCR assays were performed with the same cDNA pools used for conventional RvT-PCR. The level of transcription is presented in percent (%). The level of target gene transcription in imaginal discs isolated from Gal4(71) larvae was set as 100%.
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Figure S36 DSETDB1 mediates methylation and silencing of Rt1b{} and HeT-A retrotransposons Schematic representation of Real-Time PCR assays corresponding to the conventional PCR assays shown in Figure 10D,E. RT-PCR assays were performed using the same immunoprecipitated DNA pools used for conventional PCR. The degree of association of the antigens with the target DNA was calculated as fold enrichment by comparing the number of target DNA molecules in DNA pools obtained in ChIP assays using control antibodies with reactions containing antibodies to specific antigens.
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Figure S37 Dnmt2 mediates DNA methylation of Rt1b{} and HeT-A retrotransposons in the developing wing. Schematic representations of “bisulfite-treated DNA sequencing” assays. Genomic DNA was isolated from 50 wing imaginal discs of 3rd instar larvae, which lack dSETDB1 [Gal4(71B);UAS-dSETDB1.IR] or Dnmt2 [Gal4(71B);UAS-Dnmt2] through RNAi or control larvae (UAS-dSETDB1.IR and UAS-Dnmt2). Genomic DNA was treated twice with bisulfite. DNA fragments containing the depicted DNA sequences were amplified by PCR and cloned into pCR2.1-TOPO. 10 PCR products were sequenced for each bisulfite reaction. The grey boxes indicate the number of detected methylation events at individual CpN-motifs present in a highly methylated region within the Rt1b{} (left) and HeT-A (right) retrotransposons.
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Table S1 DNA oligonucleotides used for PCR and RvT-PCR.
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Table S2 DNA oligonucleotides used for DNA-protein interaction assays.
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We thank A. Lambertsson for 12196–48 flies, M. Dominguez for primer sequences, A.P. Bird for MeCP2 cDNA, A.F. Stewart for pins-tetO-tk-luc plasmid, D. Carter for assistance with scanning electron microscopy, S. Pon for mass spectrometry, and B. Walter for FACS. We also acknowledge S. Bertani and D. Parker for criticism of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This research was supported in part by a grant (GM073776) from the National Institute of General Medical Sciences (www.nigms.nih.gov) to FS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central 1471-2105-11-S1-S132012218410.1186/1471-2105-11-S1-S13ResearchIn silico screening of herbal and nanoparticle lead compounds for effectivity against H5N1, H1N1 neuraminidase and telomerase Ganguli Sayak [email protected] Manjita [email protected] Protip [email protected] Paushali [email protected] Sayani [email protected] Abhijit [email protected] DBT-Centre for Bioinformatics, Presidency College, Kolkata, India2010 18 1 2010 11 Suppl 1 Selected articles from the Eighth Asia-Pacific Bioinformatics Conference (APBC 2010)Laxmi Parida and Gene MyersS13 S13 Copyright ©2010 Ganguli et al; licensee BioMed Central Ltd.2010Ganguli et al; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This article has been withdrawn from the public domain because of significant plagiarism. In the light of this situation, BioMed Central regrets that this article is no longer available. The authors apologise to all affected parties for the inconvenience.
18–21 January 2010 The Eighth Asia Pacific Bioinformatics Conference (APBC 2010) Bangalore, India | 20122184 | PMC2872820 | CC BY | 2021-01-04 19:09:20 | yes | BMC Bioinformatics. 2010 Jan 18; 11(Suppl 1):S13 |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central 1477-7819-8-352043372110.1186/1477-7819-8-35ResearchColon and rectal surgery for cancer without mechanical bowel preparation: One-center randomized prospective trial Scabini Stefano [email protected] Edoardo [email protected] Emanuele [email protected] Renato [email protected] Giampiero [email protected] Davide [email protected] Valter [email protected] Unit of Surgical Oncology, Department of Emato-Oncology, San Martino Hospital, Genoa, Italy2010 30 4 2010 8 35 35 7 2 2010 30 4 2010 Copyright ©2010 Scabini et al; licensee BioMed Central Ltd.2010Scabini et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Mechanical bowel preparation is routinely done before colon and rectal surgery, aimed at reducing the risk of postoperative infectious complications. The aim of the study was to assess whether elective colon and rectal surgery can be safely performed without preoperative mechanical bowel preparation.
Methods
Patients undergoing elective colon and rectal resections with primary anastomosis were prospectively randomized into two groups. Group A had mechanical bowel preparation with polyethylene glycol before surgery, and group B had their surgery without preoperative mechanical bowel preparation. Patients were followed up for 30 days for wound, anastomotic, and intra-abdominal infectious complications.
Results
Two hundred forty four patients were included in the study, 120 in group A and 124 in group B. Demographic characteristics, type of surgical procedure and type of anastomosis did not significantly differ between the two groups. There was no difference in the rate of surgical infectious complications between the two groups but the overall infectious complications rate was 20.0% in group A and 11.3% in group B (p .05). Wound infection (p = 0.18), anastomotic leak (p = 0.52), and intra-abdominal abscess (p = 0.36) occurred in 9.2%, 5.8%, and 5.0% versus 4.8%, 4.0%, and 2.4%, respectively. No mechanical bowel preparation seems to be safe also in rectal surgery.
Conclusions
These results suggest that elective colon and rectal surgery may be safely performed without mechanical preparation.
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Background
In the first half of the 20th century, mortality from colon and rectal surgery often exceeded 20%, [1] mainly attributed to sepsis. Modern surgical techniques and improved perioperative care have significantly lowered the mortality rate. Infectious complications, however, still are a major cause of morbidity in colorectal surgery, leading to increased cost, prolonged hospital stay, and occasional mortality [2].
Mechanical bowel preparation is aimed at cleaning the large bowel of fecal content, there by reducing the rate of infectious complications following surgery. Traditionally, bowel cleansing was achieved using enemas in combination with oral laxatives [3]. More recently, oral cathartic agents to induce diarrhea and cleanse the bowel from solid feces were developed. These new bowel preparation agents, such as polyethylene glycol and sodium phosphate, provide superior cleansing compared to the more traditional methods [4-6] and are used by most surgeons in preparation for colorectal surgery [7-9]. The practice of bowel cleansing before colorectal surgery has became a surgical dogma, and primary colonic anastomosis is considered unsafe in the face of an unprepared bowel. There is, however, a paucity of data showing that mechanical bowel preparation by itself, separately from other operative and perioperative measures, actually reduces the rate of infectious complications.
In urgent colon surgery for penetrating trauma, many studies have shown that primary colonic anastomosis is safe even though mechanical bowel preparation is not performed before surgery [10,11]. These data therefore may bring into question the utility of mechanical bowel preparation in elective colon and rectal surgery.
Recently two studies [12,13] show no benefit of mechanical bowel preparation in elective colorectal resection and Bretagnol [14] says that avoid bowel preparation may be associated with reduced postoperative morbidity in elective rectal cancer surgery.
Finally a Cochrane review [15] concluded that there is no statistically significant evidence that patients benefit from bowel preparation, but this study requires further research on patients submitted for elective colorectal surgery in whom bowel continuity is restored, with stratification for colonic and rectal surgery.
The aim of this study was to assess whether elective colon and rectal surgery may be safely performed without preoperative mechanical bowel preparation also considering stratification in patients underwent at colon or rectal surgery.
Methods
Patients undergoing elective colon and rectal surgery with primary anastomosis in our Oncologic Unit Surgery between july 2005 and september 2009 were prospectively randomized by individual computer-generated randomization into two groups. Patients in Group A (the "prep" group) received mechanical bowel preparation with four administration of polyethylene glycol 12 to 16 hours before surgery, and Group B (the "non-prep" group) had no preoperative mechanical bowel preparation. All patients were allowed to have a regular diet until midnight the evening before surgery (patients in the prep group usually took their mechanical preparation after the last solid meal). All of the patients received perioperative broad-spectrum intravenous antibiotics (cephalosporine 2 g and metronidazole 500 mg b.i.d.), which were continued for at least 24 hours postoperatively. Surgeons were allowed to continue the prophylactic intravenous antibiotics for more then 1 day if necessary. 5 surgeons were enrolled in the study, all with high specialisation in colorectal resections (more than 20 procedure/year).
Patients undergoing rectal surgery were given one enema on the day before surgery to avoid extrusion of stool when using a transanally inserted stapling device.
All patients gave their informed consent before randomization in the study.
Data relative to patients' demographic and clinical characteristics, operative procedures and findings, and 30-day postoperative follow-up were prospectively entered in a Microsoft Excel database. The main outcome was the rate of postoperative infectious complications, such as wound infection, anastomotic leak, and intra-abdominal abscess. Wound infection was defined as a wound requiring partial or complete opening for drainage of purulent collection, or erythema requiring initiation of antibiotic treatment. Anastomotic leak was identified if demonstrated by imaging or documented in surgery, or if fecal drainage was evident through a perianastomotic drain. Abdominal abscess was defined as fluid collection demonstrated by computed tomography scan, in conjunction with elevated temperature or white blood cell count.
Statistical analysis was performed using the Fisher exact test or unpaired t test and probability values of less than 0.05 were considered significant (XLStat software).
Results
Three hundred six patients were entered into the study between July 2005 and September 2009. Sixty-two patients were excluded after randomization due to the exclusion criteria (abdominoperineal resection, transanal resection for T1, TME with sphincter saving procedure after neo-adiuvant therapy for middle or low rectal cancer, R2-resection, randomisation in other studies, urgency or emergency procedures, patients who required a diverting stoma proximal to the anastomosis and those who were found to have an abdominal abscess at the time of surgery). One hundred twenty patients had their surgery with preoperative mechanical bowel preparation, while one hundred twenty-four did not have mechanical preparation. Demographic characteristics, type of surgery and type of anastomosis did not significantly differ between the two groups (Table 1).
Table 1 Demographics and clinical characteristics
Prep (n:120) Non-Prep (n:124)
Mean Age (SD) 71,3 (10.8) 69.8 (10.9)
Gender 120 124
Male 65 60
Female 55 64
Surgical procedure 120 124
Right colectomy 40 50
Transverse colectomy 9 4
Left colectomy 13 26
Sigmoidectomy 25 15
Anterior resection 33 29
Localisation 120 124
Colon 87 95
Rectum (upper) 23 29
Staging 120 124
Stage I 9 25
Stage II 52 34
Stage III 59 65
Anastomosis 120 124
Manual 70 88
Mechanical 50 36
The median length of postoperative antibiotic treatment was 2.7 (SD 0.8) days in the prep group and 2.7 (SD 0.7) days in the nonprep group (P = NS).
When assessing the main outcomes of this study, there was no significant difference in the rate of postoperative wound infections, clinical anastomotic leaks, or intra-abdominal abscesses between the prep and the non-prep group (Table 2). We found no difference in anastomotic leak with stratification for colonic and rectal surgery. The surgical infectious complications rate was 20.0% in the prep group and 11.3% in the non-prep group (p .05).
Table 2 Results: infectious complications
Prep (n:120) Non-Prep (n:124) p-Value
Wound infection 11 (9.2%) 6 (4.8%) 0.18
Anastomotic leakage 7 (5.8) 5 (4.0) 0.52
Colon 2 2 0.97
Rectum 5 3 0.44
Abdominal abscess 6 (5%) 3 (2.4) 0.36
Total 24 (20%) 14 (11.3) 0.05
There was no significant difference in the average days to the first bowel movement and the length of hospital stay between the prep group and the non-prep group (4.9 days vs. 4.1 days, and 11.9 days vs. 11.0 days, respectively).
Mortality occurred in four patients in group A and two patients in group B (3.3% in the prep group, and 1.6% in the non-prep group). One patient in each group died due to sepsis from an anastomotic leak. Although none of these patients underwent an autopsy, none of the other four deaths was attributed to surgical infectious complications (2 cardiac, 1 respiratory, 1 neurologic disease).
Discussion
Preparation for elective colon and rectal surgery with mechanical cleansing and antibiotic prophylaxis, in conjunction with improved surgical techniques and advances in perioperative care, served to reduce the rate of infectious complications in colorectal surgery. Although mechanical bowel preparation before elective colorectal surgery has become a surgical dogma, there is a paucity of scientific evidence demonstrating the efficacy of this practice in reducing the rate of infectious complications.
Whereas some animal studies have shown that mechanical preparation improved anastomotic bursting strength [16,17] and decreased septic complications, others failed to find a difference between groups of animals with or without bowel preparation [18]. Further evidence questioning the utility of mechanical bowel preparation in colorectal surgery comes from the literature regarding the management of urgent cases, such as patients with penetrating colonic trauma or acute colonic obstruction. In cases of penetrating trauma, prospective randomized studies have shown that primary colonic anastomosis is safe [19,20] even though the colon is not prepared, the mechanism of injury is not as controlled as in elective cases, and there is often a delay between the injury and the repair. These studies have led to a change in the standard of care of penetrating colonic trauma toward primary colonic repair é [14,15].
In cases of acute colonic obstruction, resection with primary anastomosis in one stage is not the common practice, as the colon is not prepared. Advanced techniques, such as on-table bowel lavage [21,22] or colonic metallic stents [23,24], have been used in an effort to allow mechanical bowel cleansing before primary anastomosis. Few authors, however, have challenged the dogma that colon resection with primary anastomosis is unsafe in patients with obstructing colon lesions. Few series suggested that anastomosis between the small bowel and the colon, as performed in right or subtotal colectomy, may be safe without mechanical preparation [25,26], since this type of anastomosis avoids the stool column proximal to the anastomosis. In a multicentric trial, [27] 97 patients with malignant left colonic obstruction were randomized to have either a segmental colon resection with on-table bowel lavage or a subtotal colectomy. The rates of intra-abdominal sepsis and anastomotic leaks did not significantly differ between the two groups. Other authors have suggested that colo-colonic anastomosis may also be safe in an unprepared bowel in the face of an obstructed colon é [25,28,29]. Recently, Naraynsingh et al. [30] reported a prospective series of 58 unselected patients with left colonic obstruction. All underwent segmental colon resection with primary colo-colonic anastomosis, without a proximal diverting stoma. There was one case of anastomotic leak and one mortality unrelated to infection.
Other published studies [31-34] have prospectively randomized patients undergoing elective colon and rectal surgery to having mechanical bowel preparation or no mechanical preparation. Although all of the prior studies are smaller in numbers then the current study, they also failed to show a benefit to mechanical bowel preparation in reducing the rate of infectious complications and anastomotic leaks.
Although the new agents used for mechanical bowel preparation such as polyethylene glycol and sodium phosphate are strong cathartic agents, the colon is frequently not completely clean and dry at the time of surgery. In our experience fluid or semifluid stool was often found in the patients of the prep group. When preparation is done for colonoscopy, liquid stool can be easily aspirated to provide adequate cleansing for a safe and effective colonoscopy. In contrast, when used as a preparation for surgery, it is more difficult to control liquid than solid stool, which may lead to the significantly higher rate of intraoperative spillage of contaminated bowel content. When mechanical bowel preparation is used, the use of a clear liquid diet before surgery, in conjunction with the cathartic agent, may potentially improve the quality of the preparation and reduce the rate of liquid colonic content.
Recently two studies [12,14] show no benefit of mechanical bowel preparation in elective colorectal resection and suggested that bowel preparation could be omitted before this type of surgery. And Bretagnol [13] says that avoid bowel preparation may be associated with reduced postoperative morbidity in elective rectal cancer surgery.
Finally a Cochrane review [15] that included a total of 13 RCTs (with 4777 participants: 2390 allocated to bowel preparation and 2387 to no preparation before elective colorectal surgery) concluded that there is no statistically significant evidence that patients benefit from bowel preparation.
Mechanical bowel preparation is not harmless. It almost invariably causes significant discomfort to the patient, including nausea, abdominal bloating, and diarrhea [4,6]. Mechanical bowel preparation is also associated with electrolyte imbalance and dehydration, [4,5] which may complicate the induction of anesthesia and perioperative care. Thus, in our view, mechanical bowel preparation should be treated as a medication and used only when indicated.
The results of this study strongly suggest that elective colon and rectal surgery may be safely performed without the use of routine mechanical bowel preparation. Bowel cleansing should therefore be used selectively for instance, in cases where intraoperative colonoscopy is likely to be required. The recent Cochrane review requires further research on patients submitted for elective colorectal surgery in whom bowel continuity is restored, with stratification for colonic and rectal surgery. In our experience, we not found differences in anastomotic leakage between groups in patients underwent at colon or rectal surgery, but further and larger studies are needed, also considering surgery of mid or low rectal cancer after neoadiuvant therapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SS, surgeon and principal investigator, participated in surgical procedures, design and coordination of the study. ER, ER, RS, GD, DP, surgeons, partecipated in surgical procedures VF, chief of Surgical Oncologt Unit, partecipated in surgical procedures. All authors read and approved the final manuscript.
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Youxin Jin
5
Jinquan Tan
1
2
*
1
Department of Immunology, Wuhan University School of Medicine, Wuhan, People's Republic of China
2
Laboratory of Allergy and Clinical Immunology, Institute of Allergy and Immune-related Diseases, Centre for Medical Research, Wuhan University School of Medicine, Wuhan, People's Republic of China
3
Section of Geriatrics, Departments of Internal Medicine, The Renmin University Hospital, Wuhan University, Wuhan, People's Republic of China
4
Wuhan University School of Public Health, Wuhan, People's Republic of China
5
The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, People's Republic of China
Brutkiewicz Randy R. EditorIndiana University School of Medicine, United States of America* E-mail: [email protected] (HY); [email protected] (TJ)Conceived and designed the experiments: HY TJ. Performed the experiments: HY XR JX LL CL XJ XW WY ZL ZR TX BY JYP JY TJ. Analyzed the data: HY XR JX LL CL XJ XW WY ZL ZR TX BY JYP JY TJ. Wrote the paper: HY TJ.
5 2010 20 5 2010 6 5 e100091510 7 2009 20 4 2010 Yuling et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The reports on the origin of human CD8+ Vα24+ T-cell receptor (TCR) natural killer T (NKT) cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV)-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8+ NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8+ NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8+ NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells) and CD8+ NKT cells (∼25% of NKT cells) is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8+ NKT cells undetectable, respectively). The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8+ NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8+ NKT cells display an activated memory phenotype (CD69+CD45ROhiCD161+CD62Llo). After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8+ NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or reaggregated thymic organ cultures. Thymic antigen-presenting EBV-infected dendritic cells are required for this process. IL-7, produced mainly by thymic dendritic cells, is a major and essential factor for CD8+ NKT cell differentiation in EBV-challenged human-thymus/liver-SCID chimeras and fetal thymic organ cultures. Additionally, these EBV-induced CD8+ NKT cells produce remarkably more perforin than that in counterpart CD4+ NKT cells, and predominately express CD8αα homodimer in their co-receptor. Thus, upon interaction with certain viruses, CD8 lineage-specific NKT cells are developed, differentiated and matured intrathymically, a finding with potential therapeutic importance against viral infections and tumors.
Author Summary
We show that the average frequency of total and CD8+ NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 patients with acute lytic EBV infection, 16 EBV-associated HL patients, and 16 EBV-negative normal subjects. The frequency of total and CD8+ NKT cells is remarkably increased in the lytic EBV-infected patients at year 1 post-onset. EBV-challenge promotes total and CD8+ NKT cell development in the thymus and liver of human-thymus/liver-SCID chimeras, compared to those in the unchallenged chimeras. After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8+ NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or reaggregated thymic organ cultures. Thymic EBV-infected dendritic cells are required for this process. IL-7 is an essential factor for CD8+ NKT cell differentiation. EBV-induced CD8+ NKT cells produce remarkably more perforin, and predominately express CD8αα homodimer. CD8 lineage-specific NKT cells are developed and differentiated intrathymically upon EBV-exposure, a finding with potential therapeutic importance against viral infections and tumors.
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Introduction
NKT cells are unconventional T cells that bridge the innate and adaptive immune systems [1]–[4]. Unlike conventional T cells, which recognize MHC-molecule-presented peptide antigens via their αβTCR, NKT cells recognize CD1d-presented glycolipids. Two subsets of functionally distinct CD1d-dependent NKT cells have been identified based on whether the cells express the semi-invariant Vα24-Jα18 TCR (Vα14-Jα18 in mice) [1], [2], [5]–[12] and whether they recognize the exogenous NKT cell ligand α-GalCer. Other NKT-like cells have been reported based on their CD1d-independence and CD161 (NK1.1 in mouse) or CD56 expression [12]–[16], or other semi-invariant Vα7.2-Jα33/Vβ2,13 TCR expression (Vα19/Vβ6,8 in mouse) [12].
In mice, conventional αβT cell development in the thymus proceeds through three major stages, i.e. CD4−CD8− (DN), CD4+CD8+ (DP), and CD4+CD8− or CD4−CD8+ (SP) [17]. The developing αβT cells undergo positive and negative selection based on TCR affinity of MHC expressed on antigen presenting cells. By contrast, the semi-invariant αβTCR DP NKT precursors interact with the CD1d-ligand complex either on cortical thymocytes to undergo positive selection [1]–[2], or on thymic dendritic cells (DCs) to undergo negative selection [18]. Positively selected DP NKT cell precursors mature by down-regulating CD8 to reach a CD4+CD44lo stage [1]–[2]. Unlike conventional T cells, which emigrate from the thymus as naïve cells, CD44lo NKT cells remain in the postnatal thymus and undergo a linear differentiation program including the expression of the terminal differentiation marker NK1.1 [19], [20]. However, a proportion of the immature NKT cells remains NK1.1− and leaves the thymus [19], [20]. The final NKT-differentiation step takes place in both thymus and periphery [21], [22]. Peripheral NKT cells reside preferentially in the liver [23], [24], but are also present in the spleen, lymph nodes, bone marrow, lung, and gut [1], [2]. Human NKT cells have not been detected in engrafted fetal thymus tissue in a hu-thy/liv-SCID model, leading to a presumption that the development of peripheral NKT cells is thymus independent [25]. In later studies, it was proposed that the human thymus has little or no role in generating peripheral NKT cells after birth. This hypothesis is based on the inverse correlation between NKT cell frequency in fetal thymus and gestational age, and on the lack of a clear NKT cell population in postnatal thymus but their definite presence in adult blood [26]–[28]. However, reports on the origin of human CD8+ Vα24+TCR NKT cells are still controversial.
In mice, it is believed that there are essentially no CD8+ NKT cells [1], [2], [29]. However, recent report shows that IL-15 expands CD8ααNK1.1+ cells [30]. In humans, the existence of CD8+ NKT cells in thymus and periphery is an area of controversy. CD8+ αβTCR NKT cells expressing CD8αα homodimer are reported in human PBMC [31]. While there are several reports questioning the existence of these cells [26], [32], it is widely believed that CD8 is expressed on a minor proportion of human NKT cells, and that the CD8 marker is usually acquired after egress from the thymus [27], [28], [33]–[37]. The finding of a limited correlation between human thymic CD4+ NKT cells and peripheral CD8+ NKT cells has raised the question of what is the origin of CD8+ NKT cells [27], [28]. As accumulation of findings on NKT cell development, the underlying mechanisms that control CD4-CD8 differentiation of human NKT cells are becoming better characterized.
Results
EBV-induced CD8+ NKT cells in various EBV-infected individuals
We studied 177 eligible patients and subjects including 128 healthy latent EBV-infected subjects [EBV+(La)], 17 newly-onset acute infectious mononucleosis patients [EBV+(IMa)], 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients [EBV+(HL)], and 16 EBV-negative normal control subjects (NS) (Table S1). None of the individuals had received treatment with anti-virals, antibiotics, or corticosteroids before entry into this study. The race of all individuals was Han as determined and registered by the physicians in this study. None of the individuals had other complicating clinical infectious symptoms when the study samples were taken. The average frequency of total NKT cells in PBMCs from the 128 EBV+(La) subjects (1.5±0.5%) was significantly higher than that from 16 EBV-negative NS subjects (0.18±0.2%), 17 new-onset EBV+(IMa) patients (0.15±0.1%) and 16 newly-diagnosed EBV+(HL) patients (0.1±0.1%) (Figure 1B). The frequency of total NKT cells in the EBV+(IMa) patients dramatically increased at year 1 post-onset [EBV+(IMy), 1.6±0.6%] (Figure 1B). The frequency of the CD8+ subset of NKT cells in PBMCs from the EBV+(La) subjects (17±4%) was remarkably higher than from EBV-negative NS subjects (2.1±0.3%), new-onset EBV+(IMa) patients (1.9±0.4%) and EBV+(HL) patients (1.1±0.2%) (Figure 1B). The frequency of CD8+ NKT cells in the EBV+(IMa) patients was significantly increased at year 1 post-onset [EBV+(IMy), 20±5%] (Figure 1B). However, the average frequencies of total T cells and the ratios of CD4+ versus CD8+ T cells in PBMCs among the EBV+(La), EBV+(HL), EBV+(IMy) and NS subjects were not significantly different (Figure 1C), except for a slight and temporary increase in the frequency of total T cells in the EBV+(IMa) patients (Figure 1C, some data not shown). These observations clearly indicate that the EBV status affects the frequency of NKT cells, particularly, the appearance of CD8+ NKT cells in PBMC.
10.1371/journal.ppat.1000915.g001Figure 1 Human NKT and T cells in the various EBV-infected and non-infected subjects.
(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in PBMC was illustrated. The data to establish negative staining gates with fluorochrome conjugated empty CD1d tetramers (eCD1d tetramer) and αβTCR isotype mAb (αβTCRIso) control was shown in rightmost panel of this subfigure. (B) and (C) Frequencies of total (left panel) and co-receptor-expressing (right panel) NKT (B) and conventional αβT cells (C) in PBMCs from healthy latent EBV-infected subjects [EBV+(La)], newly-onset acute infectious mononucleosis patients [EBV+(IMa)], IM patients at year 1 post-onset [EBV+(IMy)], EBV-associated HL patients [EBV+(HL)] and EBV-negative normal control subjects (NS) assessed by flow cytometry. The absolute numbers per ml of NKT cell subsets of various patient and subject were shown in bottom panel as means ± s.d. (the s.d. was not shown for simplicity of the figure) (B). Data were mean ± s.d. (For patient numbers see Table S1). *, p<0.001.
EBV induces intrathymic CD8+ NKT cell development
To investigate the mechanism of total and CD8-lineage differentiation of human NKT cells in the context of EBV, we established hu-thy/liv-SCID chimeras. The chimeras were challenged i.t. with EBV, a dsDNA virus, or with human T-cell leukaemia virus type 1 (HTLV-1), a retrovirus. The EBV-challenge efficiently promoted the generation of total NKT cells, whereas HTLV-1-challenge had no effect, but instead promoted a significant increase in the frequency of αβTCR thymocytes and spleen T cells (Figure 2A). The frequency of total NKT cells reached more than 3% of thymic cells and more than 2% of hepatic cells by week 5 post-challenge with EBV. By contrast, the total thymic and hepatic NKT cells were less than 0.01% within 5 weeks post-challenge with HTLV-1, comparable to the frequencies in unchallenged chimeras (Figure 2A). The frequencies of total thymic or hepatic T cells at week 5 were slightly but significantly increased following HTLV-1 infection. There were approximately 30,000–35,000 total NKT cells per million thymic cells, and 20,000–23,000 total NKT cells per million hepatic cells at week 5 in EBV-challenged chimeras (Table S2). The EBV-challenge did not significantly alter the generation of total mainstream αβT cells, whereas HTLV-1-challenge did promote the generation of the T cells, compared with those in unchallenged chimeras (Figure 2B). The frequency of total T cells reached ∼28% of thymic cells and ∼32% of spleen cells at week 5 in the chimeras challenged with EBV or HTLV-1, as well as in the unchallenged chimeras (Figure 2B). Cell phenotyping based on CD4 and CD8 expression (Figure 2C) revealed that EBV-challenge significantly promoted the generation of thymic CD8+ NKT cells and the appearance of hepatic CD8+ NKT cells in the chimeras transplanted i.t. with total thymocytes (NKT cell-depleted) plus thymic stromal cells, a population that includes DC (Figure 2E), compared to unchallenged chimeras (Figure 2D). By contrast, HTLV-1-challenge had no effect on the frequency of CD8+ NKT cells (data not shown). The frequency of CD8+ cells in the chimeras reached more than 25% of thymic NKT cells and more than 23% of hepatic NKT cells at week 5 post-EBV challenge (Figure 2E). The CD8+ cells were essentially undetectable among thymic and hepatic NKT cells at 5 weeks in the unchallenged chimeras (Figure 2D). The different thymic or hepatic NKT cell populations (DN, CD4+, and CD8+) in the HTLV-1-challenged chimeras were comparable to those in the unchallenged chimeras (data not shown). The frequencies of total and CD4/CD8 co-receptor-expressing NKT cells in peripheral blood correlated well with those in thymus and livers in both unchallenged and EBV-challenged hu-thy/liv-SCID chimeras (data not shown). An important role for DCs in the generation of thymic and hepatic CD8+ NKT cells is suggested by the finding that the frequency of these cells was rather low in both unchallenged and EBV-challenged chimeras transplanted i.t. with total fetal thymocytes plus DC-depleted thymic stromal cells (data not shown), an issue explored further below. The different thymic and spleen co-receptor-expressing mainstream T cells (DN, CD4+CD8lo, CD4+, CD8+) in the EBV- or HTLV-1-challenged chimeras were comparable to those in the unchallenged chimeras (Figure 2D, 2E, and some data not shown). The absolute numbers of NKT cells and αβT cells (thymocytes) in the organs from various hu-thy/liv-SCID chimers were shown in the Table S2.
10.1371/journal.ppat.1000915.g002Figure 2 EBV promotes CD8+ NKT cell development in vivo in hu-thy/liv-SCID chimeric mice.
(A) and (B) Development of total NKT (A) and T cells (B). The frequencies of NKT cells and T cells in thymus, liver, and spleen from hu-thy/liv-SCID chimeras challenged i.t. with EBV (EBV+) or HTLV-1 (HTLV-1+), were assessed by flow cytometry (middle and right panels) at the indicated timepoints. Unchallenged hu-thy/liv-SCID chimeras (Nil) were used as controls. Empty CD1d tetramer and isotype matched control Abs, which were used as staining controls, were not illustrated in this figure. The experimental and analysis scheme was shown in the leftmost panel. Data were mean ± s.d. (n = 8). *, p<0.001. EBV-challenged chimeras vs. non-challenged or HTLV-1-challenged chimeras. (C) The experimental and analysis scheme for detecting the co-receptor-expressing NKT cells and T cells in various organs from different hu-thy/liv-SCID chimeras was illustrated. The α-GalCer-loaded CD1d tetramer, αβTCR mAb and other relevant mAbs were used to identify the different subsets of NKT cells and T cells as illustrated. The appropriate isotype Abs and empty fluorochrome-conjugated CD1d tetramer were used as controls. (D) and (E) Data showed the frequencies of co-receptor-expressing NKT cells and T cells in the unchallenged (D, Nil) or EBV challenged (E, EBV) hu-thy/liv-SCID chimeras. The protocols for the establishment of the chimeras were described in the leftmost panels. The chimeras were sacrificed at the indicated time intervals following post-immune-reconstitution and viral challenge and the various organs and tissues were collected. +DC, thymic DCs were included. VL, very low level (below detectable levels). Data were mean ± s.d. (n = 7). *, p<0.001, EBV-challenged chimeras vs. non-challenged or HTLV-1-challenged chimeras.
Thymic CD8+ NKT cells from EBV-challenged hu-thy/liv-SCID chimeras displayed an activated memory phenotype (CD69+CD45ROhi), compared with thymic CD4+ NKT cells in same chimeras (Figure S1A). Hepatic CD8+ NKT cells expressed higher amounts of CD62L than thymic CD8+ NKT cells in the EBV-challenged chimeras, probably attributable to their egress from the thymus toward secondary lymphoid organs, whereas these hepatic CD8+ NKT cells expressed similar amounts of CD69 and CD45RO as thymic CD8+ NKT cells (data not shown). CD161, a maturation marker for human NKT cells, was uniformly highly expressed on thymic and hepatic CD8+ NKT cells in EBV-challenged chimeras; the frequency of CD8+ NKT cells in the unchallenged chimeras was too low to evaluate CD161 expression. On CD4+ NKT cells, CD161 expression was independent of EBV challenge and revealed two populations, CD161hi and CD161lo, although the former population expressed much higher levels of CD161 in the EBV treated mice (Figure S1A and some data not shown). In parallel, we also examined the expression of CD69, CD62L and CD45RO on thymic and spleenic CD4+, CD4+CD8lo, CD4+CD8+, and CD8+ T cells in EBV-challenged or unchallenged chimeras. Both CD4+ and CD8+ T cells in thymus and spleen from EBV-challenged hu-thy/liv-SCID chimeras displayed an activated memory phenotype (CD69+CD45ROhi), compared with T cells from unchallenged chimeras (Figure S2B and some data not shown). Spleen CD4+ and CD8+ T cells in EBV-challenged chimeras expressed higher amounts of CD62L (data not shown) than the thymic CD4+ and CD8+ T cells (Figure S1B), which might be attributable to their egress from the thymus to secondary lymph organs.
EBV-induced CD8+ NKT cell development depends on thymic DCs
We established different hu-thy/liv-SCID chimeras by i.t. transplantation with purified human fetal DP thymocytes plus thymic stromal cells (either DC-containing or DC-depleted). In chimeras transplanted DN thymocytes only, the frequency of total NKT cells was very low (<0.01% of thymic or hepatic cells) at week 5 in both unchallenged and EBV-challenged mice (Figure 3B, 3C). The great majority of cells were CD4-expressing in both chimeras. Less than 0.5% of thymic and hepatic CD8+ NKT cells were detected in EBV-challenged chimeras (Figure 3C). Both the frequency of total αβTCR-expressing T cells and the ratio of CD4+ to CD8+ T cells in thymus and spleen from EBV-challenged hu-thy/liv-SCID chimera transplanted with DP thymocytes only (Figure 3B, 3C) were comparable to those in EBV-challenged or unchallenged chimeras transplanted with total thymocytes (Figure 2). We further examined the ontogeny and distribution of NKT cells and T cells in hu-thy/liv-SCID chimeras transplanted i.t. with fetal DP thymocytes plus DC-containing thymic stromal cells. The frequency of total NKT cells was still rather low (∼0.02% of thymic or hepatic cells) at week 5 post-establishment in the unchallenged chimeras (Figure 3D), but was substantially increased (∼3% of thymic or hepatic cells) at week 5 post-establishment in EBV-challenged chimeras (Figure 3E). Up to 25% of thymic or hepatic NKT cells expressed CD8 in EBV-challenged chimeras, whereas the frequencies of thymic or hepatic CD4+ NKT cells were correspondingly lower than those in the unchallenged chimeras (Figure 3D, 3E). The frequency of total mature αβTCR-expressing T cells and the ratio of CD4+ to CD8+ T cells in thymus and spleen in EBV-challenged hu-thy/liv-SCID chimeras were comparable to those in unchallenged chimeras (Figure 3D, 3E). The development of thymic or hepatic CD8+ NKT cells was severely impaired by the DC-deletion. The frequency of thymic and hepatic CD8+ NKT cells was essentially below the level of detection in both unchallenged and EBV-challenged chimera transplanted i.t. with DP thymocytes plus DC-depleted thymic stromal cells (Figure 3F, 3G). We also examined the ontogeny and distribution of NKT cells and T cells in the hu-thy/liv-SCID chimeras transplanted i.t. with DP thymocytes plus purified thymic DC. The frequency of total thymic and hepatic NKT cells and the ratio of CD4+ to CD8+ NKT cells in EBV-challenged or unchallenged chimeras were comparable to those in the counterpart chimeras transplanted with DP thymocytes plus DC-included thymic stromal cells (data not shown). We further established hu-thy/liv-SCID chimeras by transplantation i.t. with fetal DP thymocytes plus i.v. syngeneic fetal BM-derived DCs. The frequency of total NKT cells was substantially increased to ∼2% of thymic or hepatic cells at week 5 post-establishment in the EBV-challenged chimeras (Figure 3I), compared to the unchallenged chimeras (∼0.01% of thymic or hepatic cells) (Figure 3H). Up to 23% of thymic and hepatic NKT cells expressed CD8 in the EBV-challenged chimeras, whereas the levels of thymic and hepatic CD4+ NKT cells were correspondingly lower than those in the unchallenged chimeras (Figure 3H, 3I). The frequency of total mature αβTCR-expressing T cells and the ratio of CD4+ to CD8+ T cells in thymus and spleen from EBV-challenged hu-thy/liv-SCID chimeras were comparable with those in the unchallenged chimeras (Figure 3H, 3I). The absolute numbers of NKT cells and αβT cells (thymocytes) in the organs from various hu-thy/liv-SCID chimers were shown in the Table S2.
10.1371/journal.ppat.1000915.g003Figure 3 EBV-induced CD8+ NKT cell development occurs at the DP thymocyte stage and depends upon thymic dendritic cells.
(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various organs from different hu-thy/liv-SCID chimeras is illustrated after collecting 2.0×106 total cell events. (B–I) Development of co-receptor-expressing NKT cells (middle panels) and T cells (right panels). Data illustrate the frequency of co-receptor-expressing NKT cells and T cells in thymus, liver and spleen from EBV-challenged hu-thy/liv-SCID chimeras (EBV). Unchallenged hu-thy/liv-SCID chimeras (Nil) were used as controls. The protocols for the establishment of the various hu-thy/liv-SCID chimeras were described in the leftmost panels. (B–G) Instead of total thymocytes, hu-thy/liv-SCID chimeras were established by intrathymic transplantation with DP thymocytes. The chimeras were sacrificed at the indicated time points following post-immune-reconstitution and viral challenge. Staining was performed as in Figure 2. +DC, thymic DCs were included. -DC, thymic DCs were depleted. In (H) and (I) the hu-thy/liv-SCID chimeras were established by intrathymic transplantation with DP thymocytes. Instead of transplantation with thymic stromal cells, BM-derived dendritic cells (1×106 cells) were injected i.v. into the hu-thy/liv-SCID chimeras at t = 0. VL, very low level (below detectable level). ND, no determination. Data (B–I) were mean ± s.d. (n = 8). *, p<0.001, EBV-challenged chimeras vs. non-challenged chimeras.
We next performed various RTOC of human fetal DP thymocytes reaggregated with syngeneic fetal thymic stromal cells (thymic DC-included), purified thymic DCs, or BM-derived DCs. Various stimuli (EBV-epitopes, infectious EBV or α-GalCer) were applied during the culture. The frequency of total NKT cells was low (<0.01% of RTOC cells) in the EBV- or α-GalCer-challenged RTOC established with only DP thymocytes (Figure 4B). In RTOC of DP thymocytes reaggregated with syngeneic fetal thymic stromal cells (thymic DC-included), purified thymic DCs, or BM-derived DCs, the EBV-challenge significantly promoted the generation of total NKT cells. After 14-day-culture, the frequency of total NKT cells was up to 2.5–2.8% of RTOC cells, whereas treatment with α-GalCer had no such effect (Figure 4B). Addition of HLA-matched or unmatched EBV-epitopes (BMLF1+EBNA1) had no significant effect on the frequency of total NKT cells (<0.01% of RTOC cells) compared to the un-stimulated RTOC (Figure 4B and data not shown). In RTOCs where thymic or BM-derived DCs were present, EBV-challenge substantially promoted the development of CD8+ NKT cells. Up to 25% of NKT cells expressed CD8 in EBV-challenged RTOCs (Figure 4C). In this case, HLA-matched EBV-epitopes moderately and significantly increased the development of CD8+ NKT cells (2.5% of NKT cells), compared with those in un-stimulated RTOCs (Figure 4C). In RTOC of DP thymocytes reaggregated with DC-depleted thymic stromal cells, the EBV-induced increase in CD8+ NKT was almost completely abolished (data not shown). Both the frequency of total mature αβTCR-expressing T cells and the ratio of CD4+ to CD8+ T cells in the different RTOC conditions were comparable (Figure 4B, 4D).
10.1371/journal.ppat.1000915.g004Figure 4 EBV-induced CD8+ NKT cell differentiation depends upon thymic dendritic cells.
(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various reaggregated fetal thymic organ cultures (RTOCs). After 14-days of culture, the various cell types were identified by flow cytometry as in Figure 2. (B–D) Frequency of total NKT cells and total T cells (B), co-receptor-expressing NKT cells (C) and T cells (D), in the different RTOCs. The protocols for the establishment of the RTOCs were described in the leftmost panels in each sub-figure: DP thymocytes were reaggregated with either total thymic stromal cells (DC-included), purified thymic dendritic cells, or BM-derived dendritic cells. The stimuli were added as indicated. Nil, no stimulus; EBV-epitopes, HLA-A2-restricted, derived from the lytic cycle protein BMLF1 and HLA-DRB1-restricted, derived from nuclear antigen EBNA1 (10 µg/ml each); EBV, infectious EBV (107 pfu); Solvent, 0.005% polysorbate 20; α-GalCer (0.1 µg/ml). The RTOCs were harvested, and assessed by flow cytometry. Only the data for CD4+ and CD8+ NKT cells, and CD4 SP and CD8 SP T cells were shown. VL, very low level (below detectable level). Data were mean ± s.d. (n = 10). *, p<0.001, EBV-challenged RTOCs vs. non-challenged RTOCs.
To further confirm that EBV mediated intrathymic CD8-lineage differentiation of human NKT cells, we focused our attention on detecting the actual EBV- or HTLV-1-infection of human progenitor thymocytes, thymic NKT cells and thymic DCs in the virus exposed hu-thy/liv-SCID chimeras. For detection of EBV-infection, five transformation-associated EBV-genes, LMP1, EBNA1, BZLFl, BALF2, and RAZ were examined. By Southern blot and Q-PCR, a high level of viral genes and mRNA transcripts were detected in EBV-exposed human DCs in the chimeras. Since mice are not the natural EBV host and their DCs are well-known to be unsusceptible to EBV, these results indicated that EBV infected only the transplanted human DCs in EBV-exposed hu-thy/liv-SCID chimeras during NKT cell development and differentiation. There was no evidence of EBV viral genes in EBV-exposed human chimeric DP thymocytes or mature CD4+ and CD8+ NKT cells (Figure S2A). For detection of HTLV-1-infection, 2 highly conserved viral X-region DNA sequences, SK43 and SK44, were examined. By Q-PCR assay, high levels of SK43 and SK44 were detected in human chimeric HTLV-1-exposed DP αβTCR-expressing T cells (Figure S2B) as well as in chimeric hepatic T cells (data not shown). There was no detectable HTLV-1 in thymic CD4+ and CD8+ NKT cells or in DCs in the HTLV-1-exposed hu-thy/liv-SCID chimeras, indicating that HTLV-1 virus does not correlate with NKT cell differentiation in EBV-exposed hu-thy/liv-SCID chimeras.
EBV-induced CD8+ NKT cell development is IL-7-dependent
IL-7 and IL-15 were known survival factors for T cells, and enhancers of NKT cell homeostatic proliferation [21], [26], [38], [39]. Both cytokines were used in attempts to differentiate and activate NKT cells from human peripheral and cord blood [35], [40], [41]. DP thymocytes expressed an increased level of IL-7Rα in EBV-challenged hu-thy/liv-SCID chimeras compared to unchallenged chimeras. The thymic DCs produced a considerable amount of IL-7 mRNA in unchallenged chimeras, and the levels increased substantially with EBV-challenge (Figure S3A). The thymic CD8+ NKT cells expressed a very high level of IL-7Rα mRNA in EBV-challenged hu-thy/liv-SCID chimeras compared with other types of thymic NKT cells and αβTCR-expressing T cells in both unchallenged and EBV-challenged chimeras (Figure S3A). The thymic DCs produced a considerable amount of IL-15 mRNA in both unchallenged and EBV-challenged chimeras. The thymic CD4+ and DN NKT cells expressed higher levels of IL-15Rα mRNA in both unchallenged and EBV-challenged hu-thy/liv-SCID chimeras compared with other types of thymic NKT cells and αβTCR-expressing T cells in both chimeras (Figure S3A). In a time course study, the freshly isolated fetal thymic DCs were found to express a low level of IL-7 mRNA. Thymic DCs in unchallenged chimeras expressed comparable levels of IL-7 mRNA at the different time intervals examined, 1, 3, and 5 weeks. By contrast, the thymic DCs rapidly increased the expression of IL-7 mRNA by week 1 post-EBV challenge, and maintained high levels throughout the course of the analysis (Figure S3B). The IL-15 mRNA was uniformly expressed in the thymic DCs of both unchallenged and EBV-challenged chimeras (Figure S3B). Thymic stromal cells (DC-depleted) expressed a uniformly low level of IL-7 and IL-15 mRNA in both unchallenged and EBV-challenged chimeras (data not shown). These observations on cytokine and cytokine receptor mRNA expression were confirmed at the protein level by intracellular flow cytometry (for IL-7 and IL-15) and conventional flow cytometry (for IL-7Rα and IL-15Rα) (data not shown). Thus, the thymic DCs are a major source of IL-7 during the thymus-dependent development of NKT cells.
The frequency of total NKT cells were low (<0.01% of FTOC cells) after 14-days of culture without adding any stimuli in FTOC (Figure 5B). Nearly all of the NKT cells were CD4-positive and CD8+ cells were undetectable in these conditions (Figure 5C). By contrast, in FTOC with added EBV, the frequency of total NKT cells was increased (1.5% of FTOC cells), and of these, 15% of cells expressed CD8, whereas, in FTOC with added HLA-matched EBV-epitopes, neither total nor CD8+ NKT cells were changed (<0.01% of FTOC cells, of which <1% were CD8+) (Figure 5C). Exogenous IL-7 or IL-15 alone slightly but significantly increased the total, but not CD8+ NKT cell differentiation in the FTOCs (<0.01% of FTOC cells) (Figure 5C). HLA-mismatched EBV-epitopes were non-functional in FTOCs (data not shown). In EBV-challenged FTOCs, exogenous IL-7, but not IL-15, could significantly further enhance the total and CD8+ NKT cell differentiation (∼2.5% of FTOC cells, of which 20% were CD8+), compared to the EBV-challenged but non-IL-7-stimulated FTOC and un-stimulated FTOC (Figure 5B, 5C). A mAb against IL-7 completely abolished the effect of IL-7 on the differentiation of CD8+ NKT cells in the EBV-challenged FTOCs, indicating an essential role of the cytokine in the differentiation of CD8+ NKT cells. In FTOCs containing added α-GalCer, IL-7 slightly enhanced total, but not CD8+ NKT cell differentiation (∼1% of FTOC cells, of which <0.8% were CD8+) (Figure 5B, 5C). The mAb against IL-7 completely inhibited the effect of IL-7 on the differentiation of total NKT cells in the FTOCs stimulated with α-GalCer. IL-15 had no such effect on the development of total NKT cells in the FTOCs stimulated with α-GalCer. The frequency of total mature αβTCR-expressing T cells, but not ratios of CD4+ to CD8+ T cells, was enhanced by IL-7 or/and IL-15 in the various FTOCs (Figure 5B, 5C).
10.1371/journal.ppat.1000915.g005Figure 5 IL-7 is required for EBV-induced CD8+ NKT cell differentiation in vitro.
(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various human fetal thymic organ cultures. After 14-day-culture, the various cell types were identified by flow cytometry as in Figure 2. (B–D) Frequency of total NKT cells and total T cells (B), co-receptor-expressing NKT cells (C) and T cells (D) in the different FTOCs. The protocols for the establishment of different FTOCs were described in the leftmost panels in each sub-figure. The various stimuli and blockers were added as indicated. Nil, no stimulus; EBV-epitopes, HLA-A2-restricted, derived from the lytic cycle protein BMLF1 and HLA-DRB1-restricted, derived from nuclear antigen EBNA1 (1 µg/ml each); EBV, infectious EBV (107 pfu); Solvent, 0.005% polysorbate 20; α-GalCer (0.1 µg/ml). IL-7 (10 ng/ml); IL-15 (10 ng/ml), Abs, either mouse anti-human IL-7 monoclonal Ab plus mouse anti-human IL-7Rα monoclonal Ab, or mouse anti-human IL-15 monoclonal Ab plus mouse anti-human IL-15Rα monoclonal Ab, respectively (5 µg/ml each); The FTOCs were harvested and assessed by flow cytometry. Only the data for CD4+ and CD8+ NKT cells and CD4 SP and CD8 SP T cells were shown. VL, very low level (below detectable level). Data were mean ± s.d. (n = 10). ** p<0.05. *, p<0.001, EBV-challenged FTOCs vs. non-challenged FTOCs; IL-7-challenged FTOCs vs. non-challenged FTOCs.
Consistent with the above in vitro findings in the FTOCs, administration of exogenous IL-7 further enhanced development of thymic and hepatic total and CD8+ NKT cells in the in vivo EBV-challenged hu-thy/liv-SCID chimeras (Figure 6B), compared to chimeras given exogenous IL-7 but not EBV (data not shown) and to EBV-challenged chimeras not treated with exogenous IL-7 (Figure 2E). The frequency of total NKT cells was significantly increased (∼3.8% of thymic and ∼3.2% hepatic cells) (Figure 6B). About 28% of thymic and hepatic NKT cells expressed CD8 (Figure 6B and Table S2). The administration of mAb against IL-7 (plus exogenous IL-7) completely blocked the function of IL-7 in vivo (Figure 6C and Table S2), indicating an essential role of the cytokine in the EBV-induced development of CD8+ NKT cells. The administration of exogenous IL-7 plus isotype-matched control Ab had no the blocking effect (Figure 6D and Table S2). Thus, EBV-induced increase in CD8+ NKT cell development is IL7-dependent.
10.1371/journal.ppat.1000915.g006Figure 6 IL-7 is required for EBV-induced CD8+ NKT cell development in vivo.
(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various organs from hu-thy/liv-SCID chimeras treated with IL-7. The chimeras were established by intrathymic transplantation with DP thymocytes. The chimeras were treated i.v. with IL-7 (0.1 µg/kg/d), or with IL-7 plus mAb against IL-7 (1 µg/kg/d) (IL-7+Ab), or IL-7 plus isotype Ab (1 µg/kg/d) (IL-7+Iso) as indicated. The chimeras were sacrificed at the indicated time points following post-immune-reconstitution and viral challenge. Staining was performed as in Figure 2. (B–D) Development of co-receptor-expressing NKT cells. Data show the frequency of co-receptor-expressing NKT cells in thymus or livers from hu-thy/liv-SCID chimera challenged i.t. with EBV (EBV), as assessed by flow cytometry. The protocols for the establishment of the chimeras were described in the leftmost panel. +DC, thymic DCs were included. VL, very low level (below detectable level). Data (B–D) were mean ± s.d. (n = 7 or 8). *, p<0.001, EBV- and IL-7-challenged chimeras vs. a-IL-7 Ab-treated chimeras.
To track NKT cells more accurately, we applied 6B11 mAb, which recognized TCR Vα24JαQ junction CDR3-loop, combined with mAb against Vα24TCR for gate of NKT cells by flow cytometry. The outcome of frequencies of total and co-receptor-expressing NKT cells gated by either CD1d tetramers vs. anti-αβTCR mAb or by anti-Vα24 mAb vs. 6B11 mAb were comparable in different human normal subjects or EBV-infected patients (Figure S4A). Comparable sets of data on frequencies of total and co-receptor-expressing NKT cells were also obtained in thymus and liver from EBV-challenged hu-thy/liv-SCID chimeras (Figure S4B), as well as in EBV-exposed RTOCs and FTOCs (data not shown), gated by either CD1d tetramers vs. anti-αβTCR mAb or anti-Vα24 mAb vs. 6B11 mAb by flow cytometry. These data confirmed the observations on EBV-induced development of CD8+ NKT cells, and ruled out possible contamination of activated conventional CD8+ T cells during the flow cytometry analysis.
EBV-induced CD8+ NKT cells produce abundant perforin
In our recent study [42], frequencies of CD8+ NKT cells in patients with EBV-associated malignancies were found significantly lower than those in healthy EBV carriers. CD8+ NKT cells in tumor patients were functionally impaired in terms of cytokine production and cytotoxicity. In hu-thy/liv-SCID chimeras, EBV-exposure efficiently augmented the generation of IFN-γ-biased CD8+ NKT cells, which were strongly cytotoxic to EBV-associated tumor-cells. IL-4-biased CD4+ NKT cells were predominately generated in unchallenged chimeras, which were non-cytotoxic. In tumor-transplanted hu-thy/liv-SCID chimeras, adoptive transfer with EBV-induced CD8+ NKT cells remarkably suppressed tumorigenesis by EBV-associated malignancies. CD4+ NKT cells were synergetic with CD8+ NKT cells, leading to a more pronounced T-cell anti-tumor response in the chimeras co-transferred with CD4+ and CD8+ NKT cells. In the present study, we further investigated the perforin expression in NKT cells (Figure 7). CD8+ NKT cells from healthy EBV+ humans, EBV-challenged hu-thy/liv-SCID chimeras, or EBV-exposed RTOCs and FTOCs produced much higher amount of perforin (Figure 7B) than that in counterpart CD4+ NKT cells (Figure 7A), indicating that high production of perforin in EBV-induced CD8+ NKT cells was an additional reason for their high cytotoxicity to EBV-associated tumor cells, besides their biased IFN-γ-production [42]. In the analysis, we applied flow cytometry using the gating of either CD1d tetramers vs. anti-αβTCR mAb and anti-Vα24 mAb vs. 6B11 mAb. Two groups of results were comparable (Figure 7).
10.1371/journal.ppat.1000915.g007Figure 7 Perforin expression in human and chimeric CD4+ and CD8+ NKT cells.
PBMC were from healthy latent EBV-infected subjects [EBV+(La)], IM patients at year 1 post-onset [EBV+(IMy)], EBV-associated HL patients [EBV+(HL)] and EBV-negative normal control subjects (NS). Thymic cell suspension were from EBV-exposed (EBV+), un-exposed (EBV−) or HTLV-1-exposed (HTLV-1+) hu-thy/liv-SCID chimeras, or from EBV-exposed (EBV+) or un-exposed (EBV−) RTOC and FTOC. For detection of intracellular expression of perforin, cells were stimulated with α-GalCer (1 µg/ml) for 24 hrs, intracellular stained, and assessed by flow cytometry using the experimental strategy shown in the upper panel of each subfigure. The NKT cells were gated by either CD1d tetramers vs. anti-αβTCR mAb (middel panels) or anti-Vα24 mAb vs. 6B11 mAb (bottom panels). Perforin positive cells (%) in gated CD4+ (A) and CD8+ (B) NKT cells were shon. Solvent for α-GalCer was 0.005% polysorbate 20 (not shown). Nil, negative stimulation control. ND, no determination. Data were mean ± s.d. (n = 8). **, p<0.05; *, p<0.001. CD8+ in (B) vs. counterpart CD4+ NKT cells in (A).
Moreover, we further analyzed the CD8α and CD8β expression on NKT cells. Data revealed that CD8αα homodimer was predominately expressed on CD8+ NKT cells in PBMCs from healthy latent EBV-infected subjects and IM patients at year 1 post-onset, as well as from normal control subjects (Figure 8B and 8C), which were consistent with the previous reports [31]. CD8αα homodimer was also expressed in the majority of CD8+ NKT cells in thymus and liver from hu-thy/liv-SCID chimeras challenged i.t. with EBV (Figure 8D and 8E) and in EBV-exposed RTOCs and FTOCs (data not shown). In the analysis, we applied flow cytometry using the gating of either CD1d tetramers vs. anti-αβTCR mAb and anti-Vα24 mAb vs. 6B11 mAb. Two groups of results were comparable (Figure 8).
10.1371/journal.ppat.1000915.g008Figure 8 NKT cells differentially express the CD8α and CD8β chain.
(A) The experimental and analysis scheme for detecting total and co-receptor CD8α- and CD8β-expressing NKT cells. (B) and (C) NKT cells in PBMCs from healthy latent EBV-infected subjects [EBV+(La)], IM patients at year 1 post-onset [EBV+(IMy)], EBV-associated HL patients [EBV+(HL)] and EBV-negative normal control subjects (NS) were assessed by flow cytometry using the gate of either CD1d tetramers vs. anti-αβTCR mAb (B) or anti-Vα24 mAb vs. 6B11 mAb (C). Further dot plot analysis of CD8α vs. CD8β in gated NKT cells was shown. (D) and (E), NKT cells in thymus (Thy) and liver (Liv) from hu-thy/liv-SCID chimeras challenged i.t. with EBV (EBV+) or unchallenged (EBV−) were assessed by flow cytometry using the gate of either CD1d tetramers vs. anti-αβTCR mAb (D) or anti-Vα24 mAb vs. 6B11 mAb (E). Further dot plot analysis of CD8α vs. CD8β in gated NKT cells was shown. Data were representatives of 5 similar experiments in each group.
Discussion
Taken advantage of the hu-thy/liv-SCID chimeric mouse model [43], [44], we have found that a sizable CD8+ fraction (up to 25%) of total human thymic NKT cells is generated in vivo after EBV-challenge. The development of CD8+ NKT cells is promoted in the thymus at the DP precursor stage, and requires participation of thymic DCs. CD4 versus CD8 lineage commitment is controlled by the EBV-challenge. The findings provide a crucial access point for unraveling the mechanism for NKT cell development and differentiation. This study led to two important insights. First, we provide direct evidence that certain pathogens, EBV in this case, are important contributors to CD8-lineage commitment of NKT cells. Second, we demonstrate that differential CD4 versus CD8 lineage commitment can be controlled not only by some known classical endogenous elements [1], [2], but also by exogenous pathogenic element(s) such as EBV.
The impact of different viral pathogens on NKT cell frequencies has been investigated. In humans, infection by HIV and HTLV-1 results in a decrease in NKT cells [45]–[52]. In mice, LCMV induces long-term loss of NKT cells since induction of apoptosis [53], [54]. The patients with severe immunodeficiency (XLP), lacking of NKT cells, is characterized by an extreme sensitivity to EBV infection [55], [56]. Our previous [42] and current works show that EBV-infection promotes generation of IFN-γ- and perforin-biased CD8+ NKT cells, and IL-4-biased CD4+ NKT cells. A protective role of CD8+ NKT cells synergized with their counterpart CD4+ NKT cells against EBV-associated malignancies has been verified. The beneficial role against the persistence of EBV-infection could be speculated. The observation on induction of dominate populations of CD8αα+ NKT cells by EBV-infection is in agreement with previous report on that CD8αα+ NKT cells control expansion of total and EBV-specific T cells in humans [31], and is supporting the observation in mice [30].
There remain several controversial issues concerning CD8+ NKT cell development. For example, why does the frequency of human CD8+ NKT cells show such a limited correlation between different sites (thymus and blood) and among different ages (fetus, neonate and adults), and why are CD8+ NKT cells the most numerically variable NKT cell subset in humans, particularly under pathophysiological circumstances [1], [2], [26]–[28], [33]–[37]. In the present study, we were able to monitor the intrathymic and extrathymic development of human NKT cells in different organs in hu-thy/liv-SCID chimeric mice. Since all mature NKT cells were depleted from the thymocytes prior to cell transplantation (Figure S6), any co-receptor-expressing human NKT cells detected in the mice should have developed and differentiated post-cell-transplantation. Since all fetal samples have been from non-EBV-infected mothers, the EBV-challenge in this animal model accurately reflects the viral effects on the differentiation of CD8+ NKT cells. Nevertheless, more evidences are needed to rule out the possibility that EBV is capable of influencing NKT cell expansion/differentiation in the periphery.
Given the functional distinction between CD4+ and CD8+ NKT cells [26]–[28], [33]–[37] and their potential therapeutic importance such as in cancer treatment [42], it is crucial to identify the factors that induce the development and differentiation of these cells in order to fully understand the causes of NKT cell subset deficiency and dysfunction, particularly of the CD8+ NKT cells. Some investigators hold the opinion that the immature NKT cells undergo extrathymic differentiation in adult blood [27], [28], [33]–[37]. Our studies have demonstrated that the frequency of total NKT cells and the different subsets in thymus, liver and peripheral blood from unchallenged and EBV-challenged chimeras is highly correlated, clearly indicating an intrathymic developmental and differentiation step for human CD8+ Vα24+NKT cells. Moreover, the hu-thy/liv-SCID chimeras have provided an in vivo model to investigate cell development and differentiation of human NKT cells under both physiological and pathophysiological circumstances.
In mice, IL-15 plays an essential role in the maturation and overall population size of NKT cells in the thymus and periphery [21], [22], [30]. On the other hand, IL-7 is critical for the development of NKT cells, but plays a minor role in regulating their maturation and homeostasis [21]. In humans, IL-7 dominates the CD4+ NKT cell development process in the fetus, neonate, and adult [26], [39], whereas IL-15 has a selective and age-specific role in vitro in the expansion and homeostasis of the DN and CD8+ NKT cell subsets [27]. We show here that IL-7 is a major and essential enhancer of EBV-induced development of thymic CD8+ NKT cells in vivo, in the hu-thy/liv-SCID chimeras, and in vitro in FTOCs. We still need to define the role of IL-7 in the continuous NKT cell division in the periphery of adults, if it indeed exists, for instance, in secondary lymphoid organs where IL-7 is available.
Taking the fact that EBV causes asymptomatic life-long infection in ∼90% of adults worldwide into consideration, the experimental design for developmental studies of NKT cells should pay special attention to the EBV status of the donors of any human samples, since we have shown here that EBV-infection status of the hu-thy/liv-SCID chimeras and the human donors can directly affect the frequency of total and co-receptor-expressing populations of NKT cells. More importantly, the present study has raised several interesting questions, such as how the semi-invariant canonical αβTCR is expressed on DP thymocyte precursor before commitment to the CD4 versus CD8 lineage differentiation of NKT cells, as well as what and how the ligand is presented by thymic DCs to the semi-invariant αβTCR-expressing DP thymocyte precursor causing the preferential CD8 differentiation.
Materials and Methods
Patients, cells, tetramers, and other reagents
The latent EBV-infected [referred to as EBV+(La)] or normal control subjects (NS) were healthy EBV seropositive or seronegative individuals, respectively. The patients with EBV-associated acute infectious mononucleosis [lytic phase, referred to as EBV+(IMa)] were diagnosed by a monospot test and the detection of capsid-specific serum IgM [56], and followed-up at 1 year [latent phase, referred to as EBV+(IMy)]. The patients with EBV-associated Hodgkin lymphoma (HL) were diagnosed according to the WHO criteria, and staged according to the Ann Arbor classification (Table S1). All EBV+(La) and NS individuals were healthy volunteers. All patients eligible for this study were in- or out-patients in different Hospitals in Hubei Province in China. HLA typing was performed using the Lymphotype Class I system (Biotest) and an Olerup SSP kit (GenoVision). The clinical information of all patients and healthy EBV-infected and normal control subjects is listed in Table S1. All patients were newly-diagnosed and had no previous treatment before entry into this study. All patients provided informed consent according to the institutional guidelines and protocol titled “The study on the frequency and subset distributions of human peripheral NKT cells in normal and EBV-infected subjects” that was approved by The Wuhan University Ethical Committee. The written informed consent from each patients and subjects was obtained.
Human fetal thymic cells, bone marrow (BM) cells, liver and PBMCs were anonymously obtained from voluntarily elective pregnancy terminations (<24-wk-gestation; HLA typing matched HLA-A2 and HLA-DRB1(*03), the most prevalent HLA-types for Eastern and Southern Chinese populations, and mismatched HLA-A11, -B8 and HLA-DQ5). The mothers were excluded if lytic and latent EBV- and HTLV-1-infections were detected by Q-PCR and serologic determination [57]. Thymic cells, BM cells and PBMCs were isolated, aliquoted, cryopreserved and maintained in the vapor phase of liquid nitrogen for further use. Viability of thawed cells was evaluated by Trypan blue dye exclusion before use. Thymic dendritic cells were separated from the thymocytes by adhesion onto plastic culture dishes. For transplantation, NKT cells were positively depleted from thymic cells by MACS beads based on staining with α-GalCer-loaded CD1d tetramers [58]. For functional studies, NKT cells were purified from human PBMCs or chimeric thymic cells by flow cytometry cell sorting or a MACS bead system based on staining with α-GalCer-loaded CD1d tetramers [58], [59]. Synthesized peptides (proteins) were EBV-epitopes, GLCTLVAML (HLA-A2-restricted, derived from the lytic cycle protein BMLF1), AVFDRKSDAK (HLA-A11-restricted, derived from nuclear antigen EBNA3B), RAKFKQLL (HLA-B8-restricted, derived from the lytic cycle protein BZLF1), TSLYNLRRGTAL (HLA-DRB1-restricted, derived from nuclear antigen EBNA1), SDDELPYIDPNM (HLA-DQ5-restricted, derived from nuclear antigen EBNA3C) [60]. Recombinant peptides (proteins) were verified free of pyrogenicity (endotoxin <10 units/ml, no bacterial or fungal contamination) according to the certifications from the manufacturer. The α-GalCer-loaded CD1d tetramers were synthesized as previously described [58]–[60]. For preparation of viral stocks, a highly productive EBV-producer cell line P3HR-1 (American Type Culture Collection, ATCC, Manassas, VA) was treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 30 ng/ml) for 14 days. The virus was then pelleted from the culture supernatant. The residual TPA in the viral suspension for final use had no significant promoting effect on cell proliferation in the in vivo human-thymus-SCID chimeras, based on our preliminary experiments. Recombinant human (rh) IL-7 (Roche) and rhIL-15 were purchased from R&D Systems. All mouse anti-human monoclonal antibodies were purchased from BD PharMingen, San Diego, CA, USA, except mAbs against human Vα24 or Vβ11, which were from Immunotech, Marseille, France.
Human-thymus/liver-SCID chimeras
To establish the human-thymus/liver-SCID (hu-thy/liv-SCID) chimeras, 8-wk-old female SCID mice (NOD/LtSz-prkdcscid/prkdcscid strain, the Jackson Laboratory) were irradiated (300 cGy/mouse) prior to cell-transplantation. Human fetal thymic cells were depleted of immature and mature NKT cells based on their reactivity with α-GalCer-loaded CD1d tetramers. Then, 1×107 thymocytes, thymocytes: thymic stromal cells including dendritic cells = 1∶1 (Figure S5), were transplanted into the thymus of anaesthetized SCID mice [43], [44], [58], [59]. Syngeneic human fetal liver tissue (equivalent to 1×107 fetal liver cells) was simultaneously implanted under the mouse kidney capsule, unless otherwise noted. The chimeras were then intrathymically challenged with EBV (107 pfu) [60] or HTLV-1 (107 pfu), and the challenge was repeated after 6 days. The chimeras were maintained for 4 wks, unless otherwise stated [43], [44]. In some cases, chimeras were established by transplantation with human fetal thymic cells (thymocytes plus thymic stromal cells), but without implantation of fetal liver tissue referred to as human-thymus-SCID (hu-thy-SCID chimera). The mice were housed in a pathogen-free environment in the Animal Research Institute, Wuhan University. The protocol for animal study titled “The study on the frequency and subset distributions of NKT cells in human-thymus/liver-SCID chimeras” was approved by The Wuhan University Ethical Committee in accordance with the current Chinese laws.
Fetal thymic organ culture (FTOC) and reaggregated thymic organ culture (RTOC)
FTOC was carried out as described previously [61]. Briefly, fetal thymus tissue was dissected into pieces of ∼2 mm3. Three pieces of tissue were placed into 24-well plates with culture medium containing various stimuli as indicated. On day 7, the cultured thymus fragment was dispersed into a single-cell suspension, and cells were stained and analyzed by flow cytometry. RTOC experiments were performed as previously described [61]. Briefly, thymic stromal cells were prepared by disaggregating fetal thymic lobes. DP thymocytes were obtained by gently grinding freshly fetal thymus lobes. The resulting suspensions were sorted for DP thymocytes using CD4 and CD8 labeling. Reaggregates were formed by mixing together the desired thymic stromal cells and DP thymocytes at 1∶1 cell ratio with other stimuli as indicated. After pelleting the cells by centrifugation, the cell mixture was placed as a standing drop on the upper membrane surface, and incubated for 5–12 days.
Flow cytometry
The α-GalCer-loaded CD1d tetramer and αβTCR (Immunotech, clone BMA031) was used to define total NKT cells. For tetramer staining, the cells were incubated with the tetramer labeled with fluorochromes at 37°C for 15 min. The appropriate isotype Ab (αβTCR mAb isotype mouse IgG2b) and empty CD1d tetramer conjugated with a fluorochrome was used to establish negative staining gates. The representative experiments for NKT cell gate negative staining were illustrated in Figure 1 and Figure S6. The αβTCR and other relevant mAbs were used to identify the different subsets of T cells. In some cases, mAb against human CDR3 loop of invariant TCR Vα24 (6B11, Immunotech) and mAb against human Vα24 (including isotype controls) were used for gating NKT cells. For analysis of co-receptor-expressing NKT cells, single cell suspensions were stained with mAbs to human CD4 and CD8α (R&D Systems, clone 11830 and 37006, isotype mouse IgG2a and IgG2b), unless otherwise noted. In some cases, NKT cells were stained with mAbs to CD8α and CD8β (Abcam, clone 2ST8.5H7, isotype mouse IgG2a), simultaneously. In intracellular staining for detection of perforin, different cells were resuspended in cold Dulbecco's PBS, and then permeabilized by Cytofix/Cytoperm solution (15 min, 4°C, in the dark; BD Pharmingen) according to the manufacturer's protocol. These permeabilized cells were stained with mAb specific for human perforin (FITC-conjugated G9, mouse IgG2b, BD Pharmingen), or isotype control, and analyzed by flow cytometry. All analyses were performed with a FACSCalibur (BD Biosciences). Four- and five-color analysis was done using CellQuest software.
Real time quantitative RT-PCR (Q-PCR)
All Q-PCR reactions were performed as described elsewhere [62]. Briefly, total RNA from purified cells (1×104, purity >99%) or cell lines was prepared by using Quick Prep® total RNA extraction kit (Pharmacia Biotech) according to the manufacturer's instructions. RNA was reverse transcribed by using oligo (dT)12-18 and Superscript II reverse transcriptase (Life Technologies, Grand Island, USA). The real time quantitative PCR was performed in special optical tubes in a 96 well microtiter plate (Applied Biosystems, Foster City, CA) with an ABI PRISM® 7700 Sequence Detector Systems (Applied Biosystems). By using the SYBR® Green PCR Core Reagents Kit, fluorescence signals were generated during each PCR cycle via the 5′ to 3′ endonuclease activity of AmpliTaq Gold to provide real time quantitative PCR information. Primers used in Q-PCR are listed in Table S3.
Statistical analysis
Statistical analyses were performed using the Student′s t test. Values of p<0.05 were considered statistically significant.
Supporting Information
Figure S1 EBV-induced in vivo CD8+ NKT cells are mature and activated. Data showed the expression of CD69, CD45RO, CD62L, and CD161 on co-receptor-expressing NKT cells (A) and T cells (B) in thymus from the unchallenged (EBV−) or EBV challenged (EBV+) hu-thy/liv-SCID chimeras, as assessed by flow cytometry. The chimeras were sacrificed at week 5 of post-immune-reconstitution and viral challenge. The various organs and tissues were collected. The experimental and analysis scheme was illustrated in Figure 2. Empty fluorochrome conjugated CD1d tetramers were used as a staining control. ND, no determination since too few cells were detected. Data were representative of each of 5 sets of experiments conducted (n = 7).
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Figure S2 EBV- or HTLV-1-infection of T cells, thymic NKT cells and dendritic cells from hu-thy/liv-SCID chimeras. The chimeras were challenged with either EBV (+) or HTLV-1 (+) for 4 weeks as described in M&M, or left un-challenged (−). The animals were then sacrificed. Human fetal primary (Prim.) thymocytes, total NKT cells or thymic NKT cells, and dendritic cells (DCs) in hu-thy/liv-SCID chimeras were purified according to the αβTCR and co-receptor expression, i.e. CD4+CD8+ (DP), CD4−CD8− (DN), CD4+ and CD8+ cells. The different purified cells were washed extensively after purification. (A) Southern blot (top panel) and Q-PCR (bottom panel) analyses for detections of various EBV genomic DNA and mRNA transcripts, respectively. The primers and probes used were shown in Table S3. The applied methods were detailed in the references listed below [63], [64]. The EBV-transformed Raji B cell line (EBV+ B cell line) was used as a positive control. Data were representative (each n = 6). ND, no determination since too few cells harvested. For simplification, Q-PCR data from various cells were only shown for the representative EBV-exposed NP thymocytes, EBV-exposed CD4+ and CD8+ NKT cells, and EBV-exposed DCs. (B) Q-PCR detection of HTLV-1-infection in genomic DNA extract from various cells from HTLV-1-exposed (+) hu-thy/liv-SCID chimeras as described above. The HTLV-1 primer sets were designed corresponding to the highly conserved HTLV-1 pX region, referred to as SK43 (top panel) and SK44 (bottom panel) [65], [66]. The HTLV-1-infected T-cell lymphoma cell line HUT102 (HTLV-1+ T cell line) was used as a positive control. ND, no determination since too few cells harvested. Data were mean ± s.d. (n = 6).
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Figure S3 EBV-challenge up-regulates IL-7 expression in thymic dendritic cells. (A) Expression of IL-7, IL-7 receptor alpha chain (IL-7Rα) (top panels), IL-15, and IL-15 receptor alpha chain (IL-15Rα) (bottom panels) mRNAs in different thymic cells. The hu-thy/liv-SCID chimeras were established by i.t. challenge with EBV (EBV+). Unchallenged hu-thy/liv-SCID chimeras (EBV−) were used as controls. The chimeras were sacrificed at week 5 post-immune-reconstitution and viral challenge. The indicated cell types were isolated and IL-7, IL-7Rα, IL-15, and IL-15Rα mRNA levels were assessed by Q-PCR. Data were mean ± s.d. (n = 8). *, p<0.001. (B) The time course study of IL-7 and IL-15 mRNA expression in thymic dendritic cells in different chimeras. The hu-thy/liv-SCID chimeras were established by i.t. challenge with EBV (EBV+). Unchallenged hu-thy/liv-SCID chimeras (EBV−) were used as a control. The chimeras were sacrificed at the indicated time points following immune-reconstitution and viral challenge. IL-7 and IL-15 mRNA levels were assessed by Q-PCR. Data were mean ± s.d. (n = 9). *, p<0.001, EBV-challenged chimeras vs. non-challenged chimeras.
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Figure S4 Comparison among the outcomes of the frequencies of NKT cell subsets using two different gating staining by flow cytometry. The comparison was carried out between the counterpart frequencies of total and co-receptor-expressing NKT cells gated by either CD1d tetramers vs. anti-αβTCR mAb or by anti-Vα24 mAb vs. 6B11 mAb (anti-iTCR Vα24JαQ junction CDR3-loop). (A) The experimental and analysis scheme for detecting total and co-receptor-expressing NKT cells in PBMC was illustrated in upper panel. Frequencies of total (left subfigures) and co-receptor-expressing (right subfigures) NKT cells in PBMCs from healthy latent EBV-infected subjects [EBV+(La)], newly-onset acute infectious mononucleosis patients [EBV+(IMa)], IM patients at year 1 post-onset [EBV+(IMy)], EBV-associated HL patients [EBV+(HL)] and EBV-negative normal control subjects (NS) were assessed by flow cytometry using the gating by either CD1d tetramers vs. anti-αβTCR mAb (middle panels) or anti-Vα24 mAb vs. 6B11 mAb (bottom panels). Data were mean ± s.d. (n = 4). *, p<0.001. EBV+(La) or EBV+(IMy) vs, EBV+(IMa), NS or EBV+(HL). (B) The experimental and analysis scheme for detecting total and co-receptor-expressing NKT cells in thymus and liver was illustrated in upper panel. The frequencies of NKT cells in thymus and liver from hu-thy/liv-SCID chimeras challenged i.t. with EBV were assessed by flow cytometry at the indicated timepoints using the gating by either CD1d tetramers vs. anti-αβTCR mAb (left panel) or anti-Vα24 mAb vs. 6B11 mAb (right panel). Data were mean ± s.d. (n = 4). *, p<0.001. EBV+ vs. EBV− hu-thy/liv-SCID chimeras.
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Figure S5 Frequency of Vα24+Vβ11+ NKT cells. The frequency of immature and mature Vα24+Vβ11+ NKT cells in human fetal thymic cells as assessed by flow cytometry (A) and the ratio of rearranged Vα24-Jα18 and Cα DNAs in fetal thymocytes by Q-PCR (B) were examined before and after the positive depletion procedure [58], [59]. In A, thymic cells were stained with Vα24 and Vβ11 mAbs (left panels) or CD1d tetramer and TCRβ (right panels), respectively. Data were representative of three experiments or mean ± s.d. *, p<0.001.
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Figure S6 The staining control data. For accurate quantitation of NKT cells, cells were stained with fluorochrome conjugated empty CD1d tetramers (eCD1d tetramer) and αβTCR isotype mAb (αβTCRIso) to establish the gate for negative controls. Cells were from the thymus and liver of EBV-exposed (A) or HTLV-1-exposed (B) hu-thy/liv-SCID chimeras at week 4 post-challenge as representatives for in vivo hu-thy/liv-SCID mouse experimental models. pt, post-transplantation. Other cells were from EBV-exposed RTOC (C) and FTOC (D) as representatives for in vitro experimental models.
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Table S1 Clinical information of normal control and latent EBV-infected individuals, and patients with IM or HL.
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Table S2 The absolute numbers of NKT cells and αβT cells (thymocytes) in different organs in various hu-thy/liv-SCID chimers.
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Table S3 The sequences of primers and probes for mRNA and DNA detection in Q-PCR and Southern blot assays.
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TJ, HY, XR, XW, LL, and WL have a patent pending on a method to establish the EBV-challenged human-thymus/liver-SCID chimera for NKT cell research and therapeutic usage.
This work was supported by the grants from the National Natural Science Foundation of China (30730054, 30572119, 30670937, 30971279, 30901363), the Hi-tech Research and Development Program of China from the Ministry of Science and Technology (2007AA02Z120), the Ministry of Education (20060486008), the Provincial Department of Science and Technology of Hubei (2007ABC010), the Provincial Department of Health of Hubei (JX4B14), China, and the Chang Jiang Scholars Program from the Ministry of Education, China, and the Li Ka Shing Foundation, Hong Kong, China (Chang Jiang Scholar T.J.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Lung IndiaLILung India : Official Organ of Indian Chest Society0970-21130974-598XMedknow Publications India 20531992LI-26-11410.4103/0970-2113.56344Original ArticleRole of fine-needle aspiration cytology in evaluating mediastinal masses Pandey D. K. Ahmad Zuber Masood I. Singh S. K. Jairajpuri Z. Department of TB and Chest Disease, J.N. Medical College, AMU, Aligarh, Uttar Pradesh, IndiaAddress for correspondence: Dr. Imrana Masood, Department of TB and Chest Disease, J.N. Medical College, AMU Aligarh, Uttar Pradesh, India. E-mail: [email protected] 2009 26 4 114 116 © Lung India2009This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background:
Fine-needle aspiration cytology is an important and useful investigation and is considered next to imaging in the diagnosis of mediastinal lesions. We carried out this study in the Department of TB and respiratory diseases JNMC Aligarh from March 2000 to March 2002 with the following aims.
Objectives:
To make etiological diagnosis of mediastinal lesions, determine the pathological type of the tumor in cases of malignancy and evaluate the role of fine-needle aspiration cytology in staging of bronchogenic carcinoma.
Materials and Methods:
A total of 56 patients were included in this study who had mediastinal mass with or without lung lesions on chest X-ray or computed tomography scan. Of these patients, 36 had mediastinal mass only and 20 had mediastinal mass with parenchymal lesion.
Results:
In the present study, of 56 patients, 36 had mediastinal masses and 20 had pulmonary mass.
Conclusion:
Percutaneous fine-needle aspiration is an easy and reliable method for reaching a quick tissue diagnosis in pulmonary and mediastinal masses.
Fine-needle aspiration cytologymediastinal masstissue diagnosis
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INTRODUCTION
Fine-needle aspiration cytology (FNAC) was first used by Martin and Ellis[1] as a diagnostic tool. It is an important and valuable procedure for the diagnosis of lesion of thyroid, lymph node, bone, etc. FNAC has long been used for the confirmation of metastatic disease and in the diagnosis of primary mediastinal masses.
It can diagnose most lesions by accurate tumor typing. It has few complications and is relatively safe. It is particularly useful for the diagnosis of malignant lesions. The present study was undertaken to assess the role of FNAC in evaluating mediastinal masses. The aim and objective of the study were to cytologically characterize FNAC smears, to cell type the tumor in malignant cases, to correlate the findings and to aspirate mediastinal lymph nodes so as to stage the bronchogenic carcinoma.
MATERIALS AND METHODS
The study was conducted in the department of TB and Respiratory disease, JNMC, Aligarh, from March 2000 to 2002. Fifty-six patients with mediastinal masses confirmed by computed tomography (CT) scan were included in this study. Of these patients, 36 had mediastinal mass only and 20 had lung lesion along with it.
FNAC was performed in each case. Percutaneous FNAC was performed in 20 patients, while CT-guided FNAC was performed in 36 patients. Percutaneous FNAC of mediastinal masses was conducted wherever mass was superficial enough to be approached percutaneously. For FNAC, a 22-gauge spinal needle with 20-ml sterile syringe was used. After fixation, slides were stained by one of the following methods, Hematoxylin and Eosin stain, PAP stain, Eosin Azure stain, Giemsa stain and specia stains such as Gram and Ziehl-Neelsen stain were used whenever necessary. Aspiration smears were studied for the probable diagnosis. Results of FNAC were correlated wherever possible with histological diagnosis.
RESULTS
Maximum of number of cases were in the age group of 51-60 years with a mean age of 52 years. Male preponderance of 85.7% was seen. About 66.1% of total cases were malignant and 19.6% were benign [Table 1]. Benign lesions were inflammatory nonspecific (3.6%) and specific (5.2%). Two cases were diagnosed as specific inflammation, whereas actually there were three such cases. Among these three cases, two were diagnosed as granulomatous. Cytology revealed histiocytes of epitheloid type, forming cohesive clumps that were reminiscent of granuloma, with interspersed lymphocytes seen in one case. One case showed on cytology a cluttered background and necrosis. Numerous polymorphs were also seen. Fungal colonies with broad, folded non-septate hyphae were seen. A diagnosis of fungal abscess was favored.
Table 1 Distribution of cases according to nature of lesion
Nature of lesion N Percentage
Benign 11 19.6
Malignant 37 6.1
Unsatisfactory 8 14.3
Total 56 100
Retrosternal goiter was found in 3.6% cases, while ganglionuroma, mediastinal cyst, cystic teratoma, thymoma and reactive change lymph node comprised 1.8% each. Malignant lesions were cytologically characterized as carcinoma (50%), lymphoma (12.5%) and primitive neuroectodermal tumor (1.8%) [Table 2]. There were no false-positive cases because one case was falsely diagnosed as reactive change lymphadenopathy on cytology, thus giving a false-negative result. Later on, histopathological examination revealed the lesion to be non-Hodgkin lymphoma. FNAC of mediastinal masses was performed in all 56 cases, while histopathological examination was performed in 20 cases wherever required when the diagnosis was not clinically acceptable, hence ruling out the possibility of any false-positive cases. On cell typing, squamous cell was found to comprise 9 cases, adenocarcinoma 7, brochoalveolarcarcinoma 1, small cell carcinoma 10 and poorly differentiated carcinoma 1 [Table 3]. Small cell carcinoma was the most common type of carcinoma in our study. The correlation between cytology and histopathology was made in 20 cases, of which 19 cases were concordant and 1 was discordant [Table 4]. FNAC was inconclusive in 8 (14.3%) cases. Positivity of FNAC was 85.7%. Fortunately, no case of pneumothorax was encountered. This was confirmed in susceptible cases. While complications such as mild hemoptysis were encountered in 2 patients and chest pain in 14 cases.
Table 2 Distribution of cases according to the cytological diagnosis
Cytological diagnosis N Percentage
Benign lesions 12 21.4
Inflammation 5 -
Nonspecific 2 3.6
Specific 3 5.2
Mediastinal cyst 1 1.8
Retrosternal thyroid 2 3.6
Neurogenic tumor 1 1.8
Cystic teratoma 1 1.8
Thymoma 1 1.8
Reactive change 1 1.8
Malignant lesions 36 64.3
Carcinoma 28 50
Lymphoma 7 12.5
primitive neuroectodermal tumor 1 1.8
Unsatisfactory 8 14.3
Total 56 100
Table 3 Cytological types of malignant cases
Types N Percentage
Carcinoma 9 25
Squamous cell Ca 7 19.4
Adenocarcinoma 7 19.4
Bronchoalveolar Ca 1 2.8
Small cell Ca 8 22.2
Poorly differentiated 1 2.8
Combined carcinoma 2 5.6
Lymphoma
Non-Hodgkin's lymphoma 5 13.8
Hodgkin's lymphoma 2 5.6
primitive neuroectodermal tumor 1 2.8
Total 36 100
Ca = Carinoma
Table 4 Cytological and histopathological correlation
Cytological diagnosis N Correlation Concordant Discordant
Benign lesions 12
Inflammation 5 - - -
Nonspecific 2 - - -
Specific 3 1 1 -
Mediastinal cyst 1 - - -
Retrosternal thyroid 2 2 2 -
Neurogenic tumor 1 1 1
Cystic teratoma 1 1 1 -
Thymoma 1 1 1 -
Reactive change 1 1 - 1
Malignant lesions 36 -
Carcinoma 28 5 5 -
Lymphoma 7 7 7 -
PNET 1 1 1 -
Unsatisfactory 8 - - -
Total 56 20 19 1
PNET = Primitive neuroectodermal tumor
DISCUSSION
In our study, 56 FNAC from different compartments of the mediastinum were evaluated over a 2-year period. Of these cases, 19.6% were benign and 66.1% were malignant.
The study showed that the maximum number of cases was seen in the age group of 51-60 years. The increased number of cases in the age group of 51-60 years may be due to increased incidence of malignancies in that group and also because FNAC was mainly used for the diagnosis of neoplasm, which comprises 66.1% of the total cases. The average age in our study was 52 years. Powers et al.,[2] in their study also showed increased prevalence of neoplasm after age 50 years, with average age of 54 years; 71% of total cases were found to be malignant.
Maxcy Rosenau's last[3] study indicated that an experimental increase in the incidence rates with age is observed for most adult malignancies. This is true as increased number of malignant cases is seen in the elderly population.
Our study also showed that mediastinum is the site for a variety of lesions, both benign and malignant. Of 56 cases, 11 (19.6%) were benign and 37 (66.1%) were malignant, while 8 cases (14.3%) were inadequate for reporting. The malignant cases formed the largest category, and in this category metastasis was found in 50% of cases. A study conducted by Blegard et al.[4] also showed an appreciable proportion of malignant mediastinal tumors (30%). Similar results were also shown by Adler et al.[5] and Jareb et al.,[6] who reported a 72% prevalence of malignant disease in their study, which is comparable to 66.1% of the present study [Table 5].
Table 5 Prevalence of malignant tumor in mediastinal biopsies
Cell type Authors
Lange et al. (1972) %[7] Bocking et al. (1995) %[8] Present study%
Squamous cell carcinoma 91 86.6 89
Adenocarcinoma 100 89.5 85.7
Small cell carcinoma 86 94.2 100
As is evident from the above table, squamous cell carcinoma, adenocarcinoma and small cell carcinoma can be effectively typed by cytology. A high degree of accuracy in cytological typing can be of great importance in those cases where no confirming histology is available. The highest typing accuracy was seen with small cell carcinoma and may be attributed to abundant material and characteristic cytological appearance. The carcinomatous lesions represent metastatic disease from thoracic and extrathoracic sites. Maximum cases originated from a lung primary (82%), while larynx, esophagus, stomach, colorectal and cervical primary represented 3.6% cases each. Thirty cases (maximum) were confirmed to the anterior compartment, 16 cases to the middle and 2 cases to the posterior compartment. Percutaneous FNAC, transbronchial needle aspiration (TBNA) and CT-guided FNAC were used to obtain material for cytology. Best results were obtained with CT-guided FNAC with just 1 inadequate yield, while TBNA had 2 and percutaneous FNAC showed a maximum of 5 inadequate samples.
One of the most important factors in the treatment of bronchogenic carcinoma is its curative resection. Because the tumor must be confined to the lung to be resectable and as the initial route of spread of most bronchogenic tumors is to mediastinal lymph nodes, thorough examination of the mediastinum is central to the assessment of resectability. FNAC of mediastinal lymph nodes is a reliable alternative to more invasive surgical staging techniques to prove the presence of mediastinal metastasis. The presence of lymphocytes in the aspirate was considered an essential criterion of specimen adequacy. Five cases of lymph node aspirate with lymphocytes in the background were obtained in our study and all were positive for malignancy, indicating unresectability and no further surgical procedure was undertaken. This highlights the role of FNAC in staging of bronchogenic carcinoma.
Complications of all 3 types of procedures were minor, such as pain and hemoptysis.
Verification of cytological diagnosis was made by histopathological examination wherever possible and therapeutic response to relevant therapy in the remaining cases.
The study concluded that FNAC of mediastinal masses is a relatively safe and easily tolerated procedure, particularly for the diagnosis of mediastinal tumors such as malignant lesions. CT-guided FNAC is the most successful procedure, whereas TBNA is very effective in staging bronchogenic carcinoma. In view of the above study, it is recommended that FNAC should be the first invasive procedure for the patients of mediastinal masses.
Source of Support: Nil
Conflict of Interest: None declared.
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8 Bocking A Klose KC Kyll HJ Hauptmann S Cytologic versus histologic evaluation of needle biopsy of the lung, hilum and mediastinum. Sensitivity, specificity and typing accuracy Acta Cytol 1995 May-Jun 39 3 463 71 7762333 | 20531992 | PMC2876695 | CC BY | 2021-01-04 19:42:03 | yes | Lung India. 2009 Oct-Dec; 26(4):114-116 |
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Indian J AnaesthIJAIndian Journal of Anaesthesia0019-50490976-2817Medknow Publications India 20532074IJA-54-5210.4103/0019-5049.60499Case ReportContinuous cervical epidural analgesia for Isshiki type - I thyroplasty Trivedi Vandana Professor Anaesthesia, M. P. Shah Medical College, Jamnagar - 361 008, Gujarat, IndiaAddress for correspondence: Dr. Vandana Trivedi, “AALAP” 98/A Mahavir ‘C’ Society, Jamnagar, Gujarat, India. E-mail: [email protected] 2010 54 1 52 55 © Indian Journal of Anaesthesia2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Thyroplasty is an operation on the upper airway to improve voice quality in patients with unilateral vocal cord paralysis. It is a difficult anaesthetic procedure that requires sharing the airway with the surgeon. We describe a good anaesthetic technique, which provides a safe airway with excellent operating conditions, using continuous cervical epidural anaesthesia and postoperative analgesia in three patients. The use of a regional anaesthetic technique provides excellent anaesthesia and analgesia while allowing the patient to phonate at the request of the surgeon intraoperatively.
Unilateral vocal cord palsyIshiki type I thyroplastycervical epidural anaesthesiapostoperative analgesia
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INTRODUCTION
Medialisation thyroplasty is a surgical procedure that decreases the incidence of dysphagia and dysphonia in patients with vocal cord paralysis and its subsequent complications like regurgitation and aspiration. This procedure is best performed in a patient who maintains the ability to phonate. It requires access to an uninstrumented larynx and a functional assessment of vocal cord medialisation. Unilateral vocal cord paralysis may occur as a result of intrathoracic disease i.e. tumours of the mediastinum and bronchi, an enlarged left atrium and aortic arch aneurysms, or dysfunction of the central nervous system i.e. neoplastic and infectious processes at meningeal level, neoplasm and vascular lesions within the medulla[1] or following surgery e.g. thyroidectomy. Unilateral vocal cord paralysis is idiopathic in 30-50% of cases. Unilateral vocal cord paralysis causes a hoarse, low pitched, rasping voice. The cause is often the involvement of the vagal nerve from its origin cranially, to its innervation of the muscles responsible for phonation. Complete interruption of the intracranial portion of the vagal nerve results in a paralysis characterised by the loss of the gag reflex on the affected side. The voice is hoarse, slightly nasal and the vocal cord lies immobile in the cadaveric position, i.e. midway between abduction and adduction.[1] The recurrent laryngeal nerves are most often damaged as a result of intrathoracic disease and are much more frequent causes of an isolated vocal cord palsy than are intracranial disorders.
Thyroplasty is a procedure during which a silastic wedge is inserted through a skin incision, at the level of the vocal cords, to move the paralysed vocal cord towards the midline. Assessment of the ideal position is done, by asking the patient to phonate at intervals, during the procedure. This requires the co-operation of the patient and has been achieved with local or regional anaesthesia and sedation. Thyroplasty type 1 is successful for both immediate restoration and long term maintenance of a more normal voice quality[2–5] and speaking pattern.[6] Thyroplasty also decreases aspiration,[2–5] in patients with unilateral vocal cord paralysis. Thyroplasty moves the paralysed anterior membranous vocal cord towards the midline by using a Silastic® implant for external compression of the paralysed vocal cord. A transverse incision is made in the neck at the level of the thyroid lamina. A window is cut into the thyroid cartilage and a preformed Silastic® implant is inserted, pushing the paralysed vocal cord medially.[7] After vocal cord adduction, breathlessness and hoarseness decrease. Closure of the glottic gap may decrease the incidence of pulmonary aspiration.[2–5] Thyroplasty has been performed with local anaesthesia alone or with sedation, using a combination of propofol and fentanyl or midazolam[89] Local anaesthesia, with or without sedation, allows the patient to phonate during surgery which enables the surgeon to assess the correct position of the Silastic wedge.[1011]
Several different anaesthetic techniques have been described for thyroplasty. These include local anaesthesia both alone[12] and combined with midazolam sedation and flumazenil reversal.[13] General anaesthesia has been used for part of the procedure.[14] Such techniques all require the patient to be awake and able to phonate to allow the surgeon to judge optimal vocal cord medialisation. However, it can be difficult to perform precision surgery on the larynx in an ‘awake patient’ if manipulation of the larynx leads to reflex responses such as swallowing and coughing. The technique we describe uses continuous cervical epidural anaesthesia and provides a bloodless operative field where patient can phonate as and when the surgeon requires. This facilitates exact surgery and allows an accurate assessment of vocal cord medialisation.
Written informed consent was taken in all the three cases before documentation of these cases.
CASE REPORTS
Case 1
A 70-year-old male patient weighing 70 kg presented with hoarseness of voice since two months and history of dysphonia along with dysphagia as well as history of recurrent aspiration and coughing. The patient had essential hypertension taking tab enalapril 5 mg twice daily and was controlled. Other systemic examinations were found to be normal. All routine biochemical investigations were within normal limits. Upper nasopharyngo laryngoscopic examination [Figure 1] as well as CT scan images revealed left vocal cord palsy.
Figure 1 Showing fiberoptic laryngoscopic view in case-1. Preoperative laryngoscope images. Left: maximally closed glottis. Right: open glottis on inhalation. Note that the left vocal cord (on the right side in the images) is paralysed
Case 2
A 58-year-old man with chronic obstructive pulmonary disease, bronchiectasis, and a history of treated pulmonary tuberculosis 30 years earlier, complained of hoarseness for three months. He described easy aspiration when swallowing, followed by difficulty coughing out the aspirated materials. There was no concomitant medical illness able to account for his symptoms. On laryngoscopic examination, the left vocal fold was paralysed and there was a persistent glottic gap and inadequate compensation of the contra lateral vocal fold. A thorough head and neck examination and transnasal upper endoscopic examination [Figure 2] were all normal except for the left vocal fold paralysis, confirmed by CT scan.
Figure 2 Fiberoptic laryngoscopic view of vocal cord showing left vocal cord palsy in case-2
Case 3
A 37-year-old woman complained of an 18-year history of hoarseness; a gradual onset at age 19, with deterioration of voice over a period of about one year. Since then, her dysphonia has been very stable. No other head and neck abnormality was noted with the exception of some tympanic membrane scarring, and an early childhood history of otitis. Upper nasopharyngo. laryngoscopy [Figure 3] and CT scan images was done for diagnosis, which revealed left vocal cord palsy.
Figure 3 Showing fiberoptic laryngoscopic view of vocal cord palsy in case-3
Anaesthesia technique
All the three patients were given ASA risk III of anaesthesia for surgery due to major surgery and compromised respiratory system, and written informed consent was taken according to hospital rules and regulations. All three patients were educated regarding postoperative pain assessment using VAS scale 0-10, where 0=no pain and 10=worst possible pain. Patients were premedicated with injection Glycopyrrolate 0.4 mg, injection Pentazocin 30mg and injection promethazine 25 mg I/M 40 minutes prior to operation. Preoperatively, nebulisation was done using ipratropium bromide, budesonide and salbutamol to improve respiratory conditions and prevent postoperative laryngeal oedema. A large bore intravenous cannula was inserted and dextrose normal saline fluid was started. An antiemetic prophylaxis was given in the form of injection ondensetron 4 mg intravenous and injection Dexamethasone 8 mg intravenous to prevent postoperative vocal cord oedema. All routine monitoring was done for ECG, noninvasive blood pressure, SPO2 and urine output measured intraoperatively.
Technique
The patient was made in sitting position, neck flexed and under all aseptic precautions, a continuous cervical epidural catheter was inserted at C6-C7 interspace; loss of resistance technique with 18G toughy epidural needle space was located at about 3-3.5 cm distance from skin and catheter inserted 3cm beyond the tip of the needle in cephalic direction 9. After confirming negative aspiration, 15 ml 0.25% bupivacaine along with clonidine 75 μgm was given as bolus dose followed by infusion of inj bupivacaine 0.25% at the rate of 7 ml/hour with infusion pump. Intravenous medazolam 2 mg given to all patients for intraoperative sedation, no additional other sedation was given in any form intraoperatively. Intraoperative surgeon's satisfaction and patient co-operation was optimum and good. Intraoperative period remained uneventful, Patients haemodynamic stability was maintained, and the patient remained sedated, but co-operative, to be aroused to follow verbal commands of surgeon as and when required for patient's phonation to assess the mobility of vocal cords as well as to know the functional quality outcome of voice at the time of placing implant which is very much important for better prognosis and successful surgical outcome postoperatively. No intraoperative or postoperative complications related to cervical epidural anaesthesia like hypotension, bradycardia, haematoma, infection, urinary retention nausea or vomiting were observed. Patient's preoperative voice and phonation ability were recorded, which was compared with intraoperative voice quality as well as postoperative voice. The postoperative ability to speak was far better in all the three points comparable to normal person's voice.
Postoperative analgesia was given through cervical epidural catheter in the form of injection bupivacaine 0.125% 10 ml along with 50 μgm clonidine every eight-hourly up to 24 hours Postoperative analgesia was assessed using VAS score which remained (no pain) in all three patients up to 24 hours, postoperatively, with patients satisfaction almost 100%. No side-effects or complications related to cervical epidural catheter or anaesthesia were observed in any of the three patients.
DISCUSSION
The surgical impetus for thyroplasty stems from the expectation that it produces better voice quality in unilateral vocal cord paralysis than the conventional para-vocal cord injection of teflon. Surgery on the shared airway is challenging for the anaesthetist. This technique provides a safe anaesthetic airway and optimal operating conditions for the surgeon. Anaesthesia, either local or general, for thyroplasty, presents a challenge for the anaesthesiologist as neither is considered ideal by the patient or surgeon. Several anaesthetic problems may arise during local anaesthesia with or without sedation. As the co-operation of the patient is needed at intervals during the procedure, a balance is required between provision of adequate sedation and anxiolysis, adequate airway control and the ability to reverse sedation rapidly when necessary. The procedure may last for two to three hours and patients may become uncomfortable and restless. Prolonged dissection, more than 30 minutes, and manipulation of the silastic implant may induce oedema, which may lead to overestimation of the degree of voice correction which has been obtained.[3]
Large amounts of sedation may lead to a loss of tone of the unsecured, shared upper airway, causing airway obstruction and respiratory compromise. The drugs available for sedation are limited to midazolam[7] or short-acting drugs such as propofol and fentanyl[9] Anaesthetic problems also arise during general anaesthesia.[15] The surgeon needs to see the larynx to assess the position of the vocal cords as a substitute to phonation. Direct access to the larynx may be difficult in patients with cervical spine problems or other anatomical airway abnormalities. If a tracheal tube is used to maintain the airway, there is a limited view of the vocal cords.
Our surgeons were confident that they could attain a good result with direct peroperative observation of the vocal cords; Continuous cervical epidural anaesthesia was selected to assess the movement of the vocal cords, as spontaneous breathing was required. The continuous cervical epidural anaesthesia enabled good control of the airway at all times along with excellent operative conditions. The surgeons could see the movement of the vocal cords constantly during the procedure.
CONCLUSION
Continuous cervical epidural anaesthesia with a bolus dose of injection bupivacaine 0.25% 15 ml + injection clonidine 75 μgm followed by infusion of bupivacaine 0.25% 7ml/hour, for thyroplasty, can be a very useful technique providing a safe and secure airway as well as free airway access to the surgeon to approach for surgical site. A conscious patient also cooperates for phonation to assess the voice quality intraoperativly as and when the surgeon demands. However, more randomised controlled trial studies can be done.
Source of Support: Nil
Conflict of Interest: None declared
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REFERENCES
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3 Harries ML Unilateral vocal fold paralysis: a review of the current methods of surgical rehabilitation J Laryngol Otol 1996 110 111 6 8729490
4 Koufman JA Laryngoplasty for vocal cord medialization: an alternative to Teflon Laryngoscope 1986 96 726 31 3724322
5 Sasaki CT Driscoll BP Gracco C Eisen R The fate of the medialized cartilage in thyroplasty type I Arch Otolaryngol Head Neck Surg 1994 120 1398 9 7980908
6 Leder SB Sasaki CT Long-term changes in vocal quality following Isshiki thyroplasty type I Laryngoscope 1994 104 275 7 8127182
7 Donnelly M Browne J Fitzpatrick G Anaesthesia for thyroplasty Can J Anaesth 1995 42 813 5 7497565
8 Omori K Slavit DH Kacher A Blaugrund SM Quantitative criteria for predicting thyroplasty type I outcome Laryngoscope 1996 106 689 93 8656952
9 Montgomery WW Blaugrund SM Varvares MA Thyroplasty: a new approach Ann Otol Rhinol Laryngol 1993 102 571 9 8352479
10 Isshiki N Morita H Okamura H Hiramoto M Thyroplasty as a new phonosurgical technique Acta Otolaryngol 1974 78 451 7 4451096
11 Sasaki CT Leder SB Petcu L Friedman CD Longitudinal voice quality changes following Isshiki thyroplasty type I: the Yale experience Laryngoscope 1990 100 849 52 2166192
12 Koufman JA Isaacson G Laryngoplastic phonosurgery Otolaryngol Clin North Am 1991 24 1151 64 1754218
13 Donnelly M Browne J Fitzpatrick G Anaesthesia for thyroplasty Can J Anaesth 1995 42 813 5 7497565
14 Griffin M Russell J Chambers F General anaesthesia for thyroplasty Anaesthesia 1998 53 1199 208 10193224
15 Grundler S Stacey MR Thyroplasty under general anaesthesia using a laryngeal mask airway and fibreoptic bronchoscope Can J Anaesth 1999 46 460 3 10349925 | 20532074 | PMC2876916 | CC BY | 2021-01-04 19:31:19 | yes | Indian J Anaesth. 2010 Jan-Feb; 54(1):52-55 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2057452710-PONE-RA-1743210.1371/journal.pone.0011241Research ArticleOncologyCell Biology/Cell SignalingOncology/Skin CancersEndothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells Endothelin-1 Stabilizes HIF-1αSpinella Francesca
1
Rosanò Laura
1
Del Duca Martina
1
Di Castro Valeriana
1
Nicotra Maria Rita
2
Natali Pier Giorgio
1
Bagnato Anna
1
*
1
Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy
2
Molecular Biology and Pathology Institute, National Research Council, Rome, Italy
Blagosklonny Mikhail V. EditorRoswell Park Cancer Institute, United States of America* E-mail: [email protected] and designed the experiments: FS AB. Performed the experiments: FS LR MDD VDC MRN. Analyzed the data: FS LR PGN AB. Contributed reagents/materials/analysis tools: FS. Wrote the paper: FS AB.
2010 21 6 2010 5 6 e1124125 3 2010 28 5 2010 Spinella et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.
Principal Findings
Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ETBR, enhance the expression and activity of HIF-1α and HIF-2α that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-α stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1α oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ETBR markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1α, ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1α, and HIF-2α expression, and an increase in PHD2 levels.
Conclusions
In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1α stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability.
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Introduction
In melanoma hypoxic setting, the upregulation of hypoxia-inducible factor (HIF)-1α, the main transcriptional factor that allows cellular adaptation to hypoxia, is associated with vascular endothelial growth factor (VEGF) expression, neovascularization, poor prognosis, and resistance to therapy [1]–[4]. Moreover, it has been demonstrated that HIF-1α stabilization is essential for oncogene-driven melanocyte transformation and early stages of melanoma progression [5]. The HIF transcriptional activity is mediated by two distinct heterodimeric complexes composed by a constitutively expressed HIF-β subunit bound to either HIF-1α or HIF-2α [6]–[9]. HIF-α subunit is constantly transcribed and translated, but under normal oxygen conditions, undergoes hydroxylation at two prolyl residues located in the oxygen-dependent degradation domain (ODDD). The hydroxylation allows interaction of HIF-α with the E3-ubiquitin ligase, containing the von Hippen-Lindau protein (pVHL), and subsequently polyubiquitinated, leading to destruction by the proteasome [10], [11].
The increase of HIF-1α subunit is critically dependent on the three prolyl hydroxylase domain proteins termed PHD1, PHD2, and PHD3, that hydroxylate prolines Pro402 and Pro564 in the ODDD of HIF-1α [10]–[13]. Experimental evidences indicate that PHD2 is the major PHD isoform controlling HIF-1α protein stability [14]. In response to hypoxia, HIF-1 binds a conserved DNA consensus sequence known as the hypoxia-responsive element (HRE) on promoters of genes encoding molecules controlling tumor angiogenesis, such as endothelin-1 (ET-1), VEGF, and erythropoietin, in different tumor cells [6], [15], [16].
Recent studies have demonstrated that endothelins (ETs) and endothelin B receptor (ETBR) pathway plays a relevant role in melanocyte transformation and melanoma progression [17], [19]. The ET family consists of three isopeptides, ET-1, ET-2, and ET-3, which bind to two distinct subtypes, ETAR and ETBR, of G protein-coupled receptors [20]. Gene expression profiling of human melanoma biopsies and cell lines indicated ETBR as a tumor progression marker associated with an aggressive phenotype [21], [22]. Activation of ETBR occurs since the early stages of melanoma progression allowing tumor cells to escape growth control, and to invade indicating that ETBR may represent a potential therapeutic target for melanoma [23]–[25]. Among emerging evidences underlining the contribution of ET-1 axis to tumor progression is the finding that ET-1 can influence the accumulation of HIF-1α in different cell types, including melanoma, ovarian and breast cancer and lymphatic endothelial cells [16], [25]–[28]. However the detailed molecular mechanism responsible for the HIF-1α increase remains unknown.
Here we demonstrate that in melanoma cells in normoxic conditions ETBR activation induces HIF-1α and HIF-2α accumulation, activity, and target gene expression by inhibiting HIF-α degradation. These effects are accompanied by inhibition of PHD2 protein levels and promoter activity, associated with increased angiogenic effects and melanoma cell invasion. Finally, we demonstrated that in vivo the inhibition of tumor growth and neovascularization by treatment with a selective ETBR antagonist is associated with an increase in PHD2 protein levels. Therefore, our findings identify the molecular mechanism by which ET-1 axis controls HIF-1α stabilization through the involvement of PHD2 degradation pathway, providing further support to the notion that ETBR blockade may offer a potential tool for melanoma treatment.
Results
ETs induce HIF-1α and HIF-2α accumulation and activity through ETBR
HIF-1α and HIF-2α have been proposed to function as key factors in angiogenesis and their expression has been associated with VEGF expression in human melanoma [4]. In this study we investigated the role of ET-1 axis on both HIF-1α and HIF-2α induction and transcriptional activity in melanoma cells. In primary (1007) and metastatic (SKMel28, M10, Mel120, M14) melanoma cell lines cultured in normoxic conditions ET-1 or ET-3 markedly increased HIF-2α protein levels, that paralleled HIF-1α accumulation, in all cell lines (Figure 1A). Moreover ET-2, similarly to ET-1 and ET-3, was able to induce HIF-1α and HIF-2α protein accumulation (Figure 1B). The inhibitory effect produced by two different ETBR pharmacological inhibitors, BQ788, a peptide antagonist, and A-192621, a nonpeptide ETBR antagonist, as well as by ETBR silencing by specific siRNA showed that ETBR is the relevant receptor that controls HIF-1α and HIF-2α protein accumulation (Figure 1B and Figure S1A). In melanoma cells, ET-1 induced a dose- and time-dependent induction of HIF-1α and HIF-2α reaching the maximum at 100 nM following 16–24 h stimulation (Figure S1B). Similarly, ET-3 stimulated a dose- and time-dependent HIF-1α accumulation, whereas an unrelated peptide not implicated in angiogenesis [29] was unable to induce it (Figure S1C). To determine whether ETs-induced HIF-1α is transcriptionally active, we transfected melanoma cells with a luciferase reporter gene driven by three specific HRE. ET-1 or ET-3 treatment resulted in a significant increase (p<0.005) in HIF-1α-induced luciferase reporter activity, that was blocked by BQ788, as well as by ETBR siRNA (Figure 1C). The ET-1-induced HIF-1α transcriptional activation was further investigated by analyzing the effect of ET-1 or ET-3 on VEGF. The increase in HIF-1α and HIF-2α protein levels in the presence of ET-1 or ET-3 or hypoxia paralleled those of VEGF (Figure 1D). When HIF-1α or HIF-2α were silenced by specific siRNA, ETs- or hypoxia-induced VEGF expression was inhibited (Figure 1D), indicating that either HIF-1α or HIF-2α can regulate target genes, such as VEGF, in melanoma cells.
10.1371/journal.pone.0011241.g001Figure 1 ETs induce HIF-1α and HIF-2α accumulation and activation through ETBR.
HIF-1α or HIF-2α protein expression was analysed in cell lysates from: A. Primary 1007, and metastatic, SKMel28, M10, Mel120, and M14 melanoma cells treated with ET-1 or ET-3; B. 1007 cells treated with ET-1, ET-2 or ET-3 or with BQ788 or A-192621, in combination with ET-1, or transfected with scRNA or ETBR siRNA and treated with ET-1 for 16 h. C. 1007 cells were transiently transfected with HRE-luciferase promoter construct in the presence of either ET-1 or ET-3 or in combination with BQ788, or transfected with ETBR siRNA for 16 h. Luciferase activity was measured and expressed as fold-increase, Bars, ± SD. *, p<0.005 compared to control; **, p<0.001 compared to ET-1 or ET-3. D. 1007 cells transfected with scRNA or with HIF-1α siRNA or HIF-2α siRNA were stimulated with either ET-1 or ET-3 or hypoxia (H) for 16 h, and cell lysates were analyzed for protein expression.
ETs induce HIF-1α stability by impairing HIF-1α hydroxylation
To asses whether ET-1 axis stabilizes HIF-1α protein, we monitored the decay of HIF-1α after blockade of protein synthesis with cyclohexamide (CHX). Melanoma cells were stimulated for 24 h either with hypoxia, or with ET-1 and then treated with CHX under normoxic conditions for the indicated times. In these conditions the decay of HIF-1α protein was observed within 120 min and was completely undetectable by the end of 240 min (Figure 2A). When the cells were treated for 24 h with ET-1 and then with CHX and ET-1, the increased levels of HIF-1α remained constant up to 240 min, demonstrating that ET-1 is able to maintain stability of HIF-1α in normoxia by slowing down its degradation. The proteosome inhibitor MG132 protected the HIF-1α subunit from proteosome degradation and this effect was further increased in the presence of ET-1, indicating that ET-1, similarly to MG132, inhibits HIF-1α degradation (Figure 2B). Because hydroxylation at the 4-position of Pro402 and Pro564 within the ODDD of HIF-1α is responsible for its degradation under normoxia [10], we further investigated the role of ET-1 on the stability of HIF-1α by transfecting melanoma cells with a reporter plasmid expressing HIF-1α ODDD fused with luciferase (CMV-Luc-ODDD). Following the transfection, cells were stimulated for different times with ET-1 or cultured under hypoxia. As shown in Figure 2C, luciferase-ODDD stabilization increased in a time-dependent manner after stimulation with ET-1 or hypoxia, with maximal levels attained at 16h. Dose-response analysis showed that CMV-Luc-ODDD stability increased progressively reaching 3,5 fold induction compared to control at 100 nM ET-1 (Figure S2). ET-1 or ET-3-induced effect on HIF-1α stability was mediated by ETBR, as demonstrated by the inhibitory effect of BQ788 (Figure 2D). Altogether these results indicate that ET-1 axis increases HIF-1α protein stabilization by impairing HIF-1α hydroxylation.
10.1371/journal.pone.0011241.g002Figure 2 ETs induce HIF-1α protein stability by impairing HIFα hydroxylation.
A. 1007 cells were cultured under normoxic conditions (C) or exposed to hypoxia (H) or treated with ET-1 for 24 h. Following stimulation of CHX alone or in combination with ET-1 for the indicated times. B. 1007 cells were treated with MG132 alone or in combination with ET-1 for 24 h. C. 1007 and SKMel28 cells were transfected with CMV-Luc- ODDD construct and stimulated as indicated. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.004 compared to control. D. Cells transfected as in A were treated with ET-1 or ET-3 alone or in combination with BQ788 for 16 h. Bars, ± SD. *, p<0.005, compared to control; **, p<0.001 compared to ET-1 or ET-3.
ETs inhibit PHD2 expression and promoter activity to stabilize HIF-α
To investigate the oxygen sensing mechanism that regulates HIF-1α stability, we evaluated the effect of ET-1 on PHD1, PHD2, and PHD3 protein levels in melanoma cells. While ET-1 produced minor changes on PHD1 and PHD3 expression, this peptide significantly decreased PHD2 protein levels in a time-dependent manner, and this effect was abolished by the presence of BQ788 (Figure 3A,B). Next to assesses how ETBR, HIF-1α, HIF-2α and PHD2 protein expression relate to one another, we examined their expression in five melanoma cell lines in the presence of ET-1. Primary and metastatic melanoma cells with high ETBR activation, following stimulation with ET-1, showed increased HIF-1α and HIF-2α protein associated with decreased PHD2 levels thus indicating that activation of ETBR and PHD2 expression are inversely correlates (Figure 3C). Moreover, to gain further insight into the mechanism through which ETs regulates PHD2 expression, we measured PHD2 mRNA in response to ET-1. As shown in Figure 3D, real-time PCR analysis indicated that ET-1 treatment inhibited PHD2 mRNA expression by ∼50% at the 6 and 8 h time points. To determine whether ETs-suppressed PHD2 mRNA expression is due to an effect on PHD2 transcription, we transfected melanoma cells with a luciferase gene reporter construct driven by the PHD2 promoter. ET-1 and ET-3 induced an inhibitory effect on PHD2 promoter, which after 8 h reached 45% of inhibition compared to the control, while BQ788 blocked this effect (Figure 3E and Figure S3A). To confirm the involvement of PHD2 on ETs-induced HIF-1α protein stability, we performed a reconstitution experiment by overexpressing each of the PHD-cDNA in 1007 cells. The overexpression of PHD1, PHD2 and PHD3 was confirmed by Western blotting (Figure S3B). HIF-1α and HIF-2α accumulation in response to ETs was specifically impaired in PHD2 overexpressing cells, indicating that re-expression of PHD2 is sufficient to counteract the ET-1- or ET-3-induced HIF-α expression (Figure 3F). These results identify the inhibition of PHD2 expression as the mechanism underlying ETs-induced HIF-α stabilization. Concomitantly to the block of HIF-α accumulation, the exogenous expression of PHD2 makes unable ET-1 and ET-3 to increase VEGF protein levels demonstrating a tight link between PHD2/HIF-α and ET-1-dependent VEGF expression (Figure 3F). Moreover, knockdown of PHD2 by inhibiting the prolyl hydroxylases with deferoxamine mesylate (DFO) resulted in a strong induction of HIF-α and VEGF expression. The addition of ET-1 to DFO did not induce a further increase in HIF-α, and VEGF protein, implying that ET-1 primarily regulates HIF-α protein accumulation through inhibition of PHD2 (Figure 3F). Furthermore, the luciferase activity of CMV-Luc-ODDD increased by ET-1 or ET-3 was impaired only in cells overexpressing PHD2 (Figure 3G), demonstrating that the re-expression of PHD2 antagonizes the effect of ET-1 and ET-3 on HIF-α degradation. These results further support the role of PHD2 on ETs-induced HIF-1α stability and angiogenic-related factor expression.
10.1371/journal.pone.0011241.g003Figure 3 ETs decrease PHD2 expression and promoter activity.
A. PHD1, PHD2 and PHD3 expression was analyzed in melanoma cells unstimulated (C) or stimulated with ET-1 for the indicated times. B. PHD2 protein expression was analyzed in cells stimulated as indicated for 24 h. C. Melanoma cells were treated with ET-1 and protein expression was analysed. D. 1007 cells were stimulated as indicated. Results are expressed as copy numbers of PHD2 transcripts over cyclophilin-A. Bars, ± SD. *, p<0.05 compared to the control. Inset shows PCR products for PHD2 and cyclophilin-A (CypA) E. Cells were transfected with the PHD2 promoter construct and stimulated as indicated for 8 h. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.006 compared to control; **, p<0.004 compared to ET-1. F. MOCK- and PHD1-, PHD2-, or PHD3-cDNA-transfected 1007 cells were stimulated with ET-1 or ET-3 for 16 h. Cells were treated with DFO alone or in combination with ET-1 and lysates were analysed for protein expression. G. 1007 cells were cotransfected with the CMV-Luc-ODDD construct and with the construct indicated in F, and stimulated with ET-1 or ET-3 for 16 h. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.001 compared to the control; **, p<0.005 compared to MOCK-transfected cells treated with ET-1 or ET-3.
ETs signal through a PI3K-dependent ILK-AKT-mTOR pathway to induce HIF-1α stability and PHD2 inhibition
It has been reported that ILK, AKT and mTOR signalling are the main pathways controlling HIF-1α expression [6], [30], [31]. ILK is a serine/threonine kinase that plays an important role in linking extracellular signalling to the regulation of melanoma tumor growth and progression [30]–[33]. Therefore we analyzed the signalling pathways involved in ET-1-induced HIF-1α stability. In 1007 cells, ET-1 induced ILK protein expression (Figure 4A). Employing an immunocomplex kinase assay, we documented that ILK kinase activity was upregulated by ET-1 and inhibited by BQ788 demonstrating that ETBR is the relevant receptor in inducing ILK expression and activity (Figure 4A). Moreover, treatment with ET-1 induced phosphorylation of AKT and mTOR, and mTOR-downstream molecule p70S6k and p-4EBP1 (Figure 4A). These effects were blocked by BQ788 (Figure 4A), indicating that this effect occurs via ETBR binding. In 1007 cells treatment with the PI3K inhibitor, LY294002, or with mTOR inhibitor rapamycin, or transfection with a dominant negative ILK mutant (DN-ILK) suppressed the ET-1-induced HIF-1α, HIF-2α, and VEGF expression (Figure 4B), demonstrating that ETBR-induced HIF-1α and HIF-2α accumulation and VEGF expression in melanoma cells are mediated through a PI3K-dependent ILK/AKT/mTOR signalling. We further explored the decay of HIF-1α protein in melanoma cells treated with ET-1 in the presence of these signalling inhibitors. PI3K and mTOR inhibitors, as well as DN-ILK, inhibited the ET-1-mediated HIF-1α stabilization (Figure S4). LY294002, DN-ILK and rapamycin restored also the PHD2 promoter activity and PHD2 protein expression downregulated by ETs (Figure 4C,D). Altogether these results demonstrate that the inhibition of PHD2 progresses through an ETBR-mediated PI3K-dependent ILK/AKT/mTOR pathway to induce HIF-1α stability.
10.1371/journal.pone.0011241.g004Figure 4 ETs-mediated PI3K–dependent ILK/AKT/mTOR pathway induces HIF-1α stability and PHD2 inhibition.
A. Cell lysates from 1007 cells untreated (C), or treated with ET-1 alone or in combination with BQ788 were analyzed for ILK activity and for the indicated protein expression. ILK activity was indicated by the amount of 32P-labeling of MBP (pMBP). B. 1007 cells treated as indicated, were stimulated with ET-1 for 16 h and lysates were examined for indicated protein expression. C. PHD2 promoter activity was measured in cells transfected with the PHD2 promoter and treated as indicated for 8 h. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.001, compared to the control; **, p<0.005, compared to ET-1 or ET-3. D. PHD2 protein levels were analyzed in 1007 cells treated as indicated in B.
PHD2 inhibition induced by ETs regulates angiogenesis and melanoma cell invasion
To determine whether the PHD2 inhibition induced by ETs was functionally involved in ET-1-induced effects regulated by HIF-α, we performed experiments targeting PHD2 in melanoma cells. siRNA against PHD2, similarly to ET-1 or ET-3, completely inhibited PHD2 protein with subsequent stabilization of HIF-1α and HIF-2α and increased VEGF levels that were not further increased by ETs (Figure 5A). To delineate the effect of PHD2 inhibition induced by ETs on angiogenesis, we measured the ability of endothelial cells to sprout forming three-dimensional structures resembling capillaries in response to conditioned medium from ET-1-treated cells silenced for PHD2. Conditioned medium from ET-1-treated 1007 cells promoted capillary branching of endothelial cells compared to untreated cells (Figure 5B). Interestingly, although knockdown of PHD2 enhanced tube formation, ET-1 treatment did not further enhance this angiogenic effect (Figure 5B). Next we determined whether secreted angiogenic factor regulated by PHD2 could explain the angiogenic effects induced by ETs. The secreted VEGF levels were increased by ET-1 or ET-3 as well as by PHD2 silencing, whereas no further increase was observed in ETs-treated PHD2-silenced 1007 cells (Figure 5C).
10.1371/journal.pone.0011241.g005Figure 5 ETs regulate angiogenesis and melanoma cell invasion through inhibition of PHD2.
A. Cell lysates from scRNA or siRNA for PHD2-transfected 1007 cells treated with or without ET-1 or ET-3 for 16 h were analyzed for protein expression. B. The ability of conditioned media from 1007 cells transfected and treated as in A, in inducing in vitro tube formation was analyzed on HUVEC. Results were represented as the number of cells in branch point capillaries. Bars, ± SD. *, p<0.001, compared to the scRNA control. C. Conditioned media from cells treated as in A were analyzed for VEGF secretion by ELISA. Bars, ± SD. *, p<0.001, compared to the scRNA control. D. 1007 cells were treated as in A and cell invasion was measured by chemoinvasion assay. Bars, ± SD. *, p<0.002, compared to the scRNA control.
Because invasive behaviour of melanoma cells is regulated by ETs through HIF-1α [25], we next examined whether PHD2 silencing could affect invasiveness. ETs or PHD2 siRNA promoted invasion in melanoma cells. ETs treatment of silenced PHD2 cells did not further increase cell invasion (Figure 5D), demonstrating that ETs signalling implies HIF-α-dependent angiogenesis and tumor cell invasion through PHD2 inhibition in normoxic conditions.
ETBR blockade inhibits neoangiogenesis in vivo
In malignant melanoma microenvironment, ETBR has been shown to contribute to tumor progression by acting on both tumor and vascular endothelial cells [34], [35]. Indeed, ET-1 through ETBR promotes different steps of angiogenesis in vitro by acting directly on endothelial cells, as well as indirectly through VEGF [35], [36]. Immunostaining with anti-CD31, showed a significant (p = 0,0056) increase of the angiogenic response in the matrigels containing ET-1 (vessel numbers 20±1,4) compared to the control matrigels containing PBS (vessel numbers 1,5±0,3) (Figure 6A). In the plugs containing BQ788 and ET-1, the number of blood vessels was significantly (p = 0,0028) reduced (vessel numbers 1,5±0,2) compared to the matrigels containing ET-1 alone (Figure 6A). These results demonstrate that ET-1 selectively through ETBR promotes neoangiogenesis and that a selective ETBR antagonist can effectively impair angiogenesis in vivo.
10.1371/journal.pone.0011241.g006Figure 6 ETBR blockade results in vivo in neovascularization inhibition, associated with reduced HIF-α and increased PHD2 expression.
A. Matrigel sections containing PBS (C), ET-1, or BQ788+ET-1 were immunostained with anti-CD31 (arrows; original magnification ×160). B. Expression of indicated proteins was analyzed in M10 tumor xenografts by Western blot analysis. C. Tumors removed from control and A-192621-treated M10 xenografts were analyzed for PHD2 expression (original magnification ×250). D. Human metastatic melanoma tissues were analyzed for ETBR, PHD2, and HIF-1α expression (original magnification ×250).
ETBR antagonist-induced decreased neovascularization is associated with reduced HIF-α and increased PHD2 expression in melanoma xenografts
We previously demonstrated that the treatment of nude mice bearing M10 xenograft with an orally active ETBR antagonist, A-192621, produces a significant (p<0,001) reduction of tumor growth [25]. Western blot analysis of tumors from M10 xenografts showed a significant reduction of HIF-1α, HIF-2α expression and an increase of PHD2 expression in A-192621-treated mice compared with the control, whereas no differences were observed in PHD1 and PHD3 expression (Figure 6B). Immunohistochemical evaluation of these tumors revealed a strong and homogenous increase in PHD2 expression levels (Figure 6C) compared to control, which paralleled the ability of A-192621 to reduce tumor vascularization, MMP-2 and VEGF expression [25]. These data underline the relevance of ETBR blockade in the regulation of tumor growth and neovascularization, resulting in down-regulation of VEGF and HIFα expression and increased levels of PHD2.
Decreased PHD2 expression correlates with increased ETBR and HIF-1α expression in human melanomas
To further evaluate the relationship between PHD2, HIF-1α, and ETBR expression, we examined these protein in human primary (n = 6) and metastatic (n = 6) melanoma samples by immunohistochemistry. Of the twelve bioptic samples, eight had low PHD2 levels associated with high ETBR expression, thus indicating that the receptor and PHD2 expression are inversely correlated (p = 0.018). The expression of HIF-1α was very heterogeneous, most likely reflecting the fact that tumor microenvironment comprises areas of highly variable hypoxic and non-hypoxic regions. Figure 6D showed one of the 6 case of metastatic melanoma in which high ETBR expression, that occurs in clinically relevant situation [21], [22], was paralleled by high HIF-1α and low level of PHD2 expression. Taken together, our in vivo analysis suggest that ETBR expression significantly correlates with low PHD2 levels in melanomas, further supporting the potential clinical relevance that ETBR-mediated PHD2 downregulation may contribute to human melanoma tumorigenesis and progression through HIF-dependent pathways.
Discussion
ET-1 axis represents one of the key regulators of tumorigenesis and tumor progression sharing with hypoxia the capacity to induce HIF-1α protein expression [25]–[28]. However, the mechanism underlying the regulation of HIF-1α mediated by ET-1 has been unexplored. In this study we demonstrate that in normoxia, ETs increase both HIF-1α and HIF-2α by preventing HIF-α protein proteosomal degradation through decreased PHD2 expression and that this regulation is critical to induce HIF-α-mediated VEGF expression, angiogenesis and tumor cell invasion. Blockade of ETBR, that inhibits tumor growth [25], results in an increased PHD2 expression concomitantly with a reduction of neovascularization and HIF-α expression in vivo.
Several growth factors, cytokines and hormones upregulate HIF-1α protein levels in normoxia by increasing HIF-1α gene transcription and/or mRNA translation without affecting protein stability [6]. Our results, concordantly with other studies [37], [38], demonstrate that non-hypoxic stimuli as ET-1, share mechanistic similarities with hypoxia regulating post-translational modifications (prolyl hydroxylation) resulting in increased HIF-1α stability. PHD2 is regarded as the main cellular oxygen sensor that regulates HIF-1α degradation in normoxia [10], [14], thereby suggesting that the inactivation of PHD2 may provide a critical mechanism in modulating HIF-1α. Until now very little information is available on the molecular control of PHD2. Our study reveals that ETs reduced PHD2 mRNA and protein expression and promoter activation, results in decreased HIF-1α hydroxylation. In melanoma cells treated with PHD2 inhibitor or in cells silenced for PHD2, ET-1 did not further increases HIF-1α or HIF-2α expression, angiogenesis and invasion, supporting that ET-1 regulates HIF-α-mediated effects through inhibition of PHD2. Moreover, the complete inhibition of ET-1-induced HIF-1α and HIF-2α accumulation observed in PHD2 overexpressing cells indicates that the re-expression of PHD2 is sufficient to counteract the effect of ETs. These results define the HIF-1α hydroxylase pathway as the link between ET-1 axis and the regulation of HIF-1α stabilization. Chan et al. [39] recently demonstrated that the loss of PHD2, observed in different tumor cells including melanoma, accelerates tumor growth and is associated with an induction of angiogenesis, suggesting that PHD2 is at the intersection of multiple complementary pathways regulating tumor growth. In this regard, our analysis of clinical melanoma samples, that express high levels of HIF-1α, reveals that ETBR activation is associated with a reduction of PHD2, further supporting that ETBR-mediated PHD2 downregulation represents a pathway for HIF-1α activation in human melanomas. Accumulating data have established that PHD2 is a direct HIF-1α target gene [40], [41]. Indeed PHD2 promoter contains HRE binding site responsible for the induction of human PHD2 gene by hypoxia [41]–[43]. It was therefore somewhat surprising to observe that ETs, which rapidly increased HIF-1α levels, inhibited PHD2 protein expression. This could be explained by recent results indicating that PHD2 induction generates an autoregulatory loop controlling HIF-1α stability [43]–[45]. Therefore our hypothesis supports the notion that ET-1 axis, similarly to hypoxia, modulates the autoregulatory loop of HIF-1α-PHD2 in melanoma cells through a balance between the inhibitory ET-1 and the stimulatory HIF-1α pathways for PHD2 transcription. In this context, we defined the intracellular signalling pathway that controls ETBR-induced PHD2 regulation in melanoma cells demonstrating that the inhibition of ILK/AKT/mTOR pathway antagonizes the ETs-induced HIF-1α stability and VEGF expression and restores PHD2 promoter activity and protein expression inhibited by ETs (Figure 7). As to whether this pathway is involved in controlling directly or indirectly PHD2, needs to be further characterized. The results demonstrating that knock-down of HIF-1α and HIF-2α makes both ETs and hypoxia unable to induce VEGF expression, implicate HIF as downstream check-point of interconnected signals induced by ET-1 axis and hypoxia, capable of modulating genes involved in tumor angiogenesis. Because the regulation of these factors is critical in the early stage of melanoma progression, one can envision that ET-1 axis, by mimicking hypoxia, can activate HIF-α enhancing the transcription of target genes, such as VEGF. As schematically described in Figure 7, ET-1 through ETBR-mediated signalling, stabilizes HIF-1α and enhances angiogenic factor expression, and hence angiogenesis, by inhibiting PHD2. Consistent with these results, it has been recently demonstrated that silencing of PHD2 induces neoangiogenesis in vivo by regulating the expression of multiple angiogenic factors through the stabilization of HIF-1α [46], [47]. In this regard, we demonstrated that in vitro tube formation of endothelial cells and melanoma cell invasion are regulated by ETBR in a PHD2-dependent manner. Taken together our findings disclose a yet unidentified regulatory mechanism, which relies on the role of ET-1 axis to promote tumor cell invasion, tumor growth and angiogenesis by decreasing PHD2.
10.1371/journal.pone.0011241.g007Figure 7 A diagram of the signalling pathway activated by ET-1/ETBR axis in melanoma cells.
Binding of ET-1 to ETBR leads to activation of PI3K–dependent ILK/AKT/mTOR signalling route, causing the inhibition of PHD2, thereby promoting HIF-1α stability, neovascularization and tumor cell invasion.
We recently identified HIF-1α/VEGF as downstream molecules of ET-1 axis in lymphangiogenesis [28]. In this scenario, it is possible to hypothesize that ET-1 through ETBR can stimulate angiogenesis and lymphangiogenesis via HIF-1α providing an alternative or complementary mechanism to the tumor hypoxic microenvironment. On support of this notion, in melanoma xenografts the reduction of tumor growth by ETBR blockade using the selective ETBR antagonist [25], was accompanied by reduction of tumor microvessel density, HIF-1α, HIF-2α and VEGF expression and a concomitant increase of PHD2 levels. In conclusion we demonstrated that ET-1 promotes melanoma progression by inducing HIF-α-mediated angiogenic signalling, through PHD2 inhibition. Thus ETBR antagonists, which have been shown to induce concomitant antitumor activity and suppression of neovascularization, may therefore represent a targeted therapeutic approach which is warrant to be explored in melanoma treatment.
Materials and Methods
Ethics Statement
The study was reviewed and approved by the ethical committee of Regina Elena National Cancer Institute. Written informed consent for tumor tissue archive collection and use in research was obtained from all melanoma patients prior to tissue acquisition under the auspices of the protocol for the acquisition of human tissues obtained from the Institutional Ethical Committee board (Official statement n.4 March 1st, 2006).
Cells and cell culture conditions
The human cutaneous melanoma cell line 1007 was derived from primary melanoma [48]. The melanoma cell line SKMel28 (ATCC, Rockville, MD, HTB-72), M10, Mel120, and M14 [49] were derived from metastatic lesions. When the cells were exposed to hypoxia, oxygen deprivation was carried out in an incubator with 1%O2, 5%CO2, and 94% N2 and cells were growth for indicated times. Human endothelial cells were isolated from human umbilical vein endothelial (HUVEC), as previously described [34], and grown in complete Endothelial Growth Media. Melanoma cells were starved for 24 h in serum-free medium (SFM) then incubated for indicated times with either ET-1, ET-2 or ET-3 (100 nM; Peninsula Laboratories, Belmont, CA) or with unrelated scramble peptide B3 (IARVSTP) kindly provided by Dr. S. D'Atri [29] or with 100 µM deferoxamine mesylate (DFO; Sigma). The antagonists BQ788 (1 µM; Peninsula Laboratories, Abbott Park, IL) or A-192621 (1 µM; Abbott Laboratories) was added 15 min before agonists, whereas pre-treatment with MG132 (10 µM; Calbiochem, La Jolla, CA), cycloheximide (CHX, 20 µM; Calbiochem), LY294002 (25 µM; Cell Signalling, Beverly, MA), and rapamycin, (10 nM; Cell Signalling) was performed for 30 min before the addition of ETs. Serum-starved melanoma cells were transfected with 100 nM siRNA duplexes against PHD2 (Eurogenetec S.A Explera s.r.l AN, Italy), HIF-1α or HIF-2α or ETBR (ON-TARGETplus SMART pool, Dharmacon, Lafayette, CO) or with scrambled siRNA (scRNA) or positive control siRNA glyceraldehyde 3-phosphate dehydrogenase (GAPDH) obtained commercially (Dharmacon). Cell media were replaced with fresh SFM 48 h later and proteins were then extracted for HIF-1α, HIF-2α, and ETBR expression analysis. Conditioned cell medium containing secreted proteins was collected, centrifuged, filtered and concentrated.
Western blot analysis
Whole cell lysates or homogenized M10 tumor specimens were subjected to SDS-PAGE and analyzed by Western blotting. Blots were developed with the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK). Antibody against HIF-1α was from Transduction Laboratory (Lexington, KY). HIF-2α, PHD1, PHD2 and PHD3 antibodies were from Novus Biologicals (Littleton, CO), VEGF was from Santa Cruz Biotechnology (Santa Cruz, CA), ETBR was from Abcam plc (Cambridge, UK), GAPDH and β-actin, used as loading control, were from Oncogene (CN Biosciences, Inc., Darmastadt, Germany).
Real-time PCR
Total RNA was isolated using the Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. 5 µg of RNA was reversed transcribed using SuperScript® VILO™ cDNA synthesis kit (Invitrogen). Quantitative real-time-PCR was performed by using LightCycler rapid thermal cycler system (Roche Diagnostics, Indianapolis IN) according to the manufacturer's instructions. Reaction was performed in 20 µl volume with 0,3 µM primers, by using LightCycler-FastStart DNA Master Plus SYBR Green mix (Roche Diagnostics) from 1 µl cDNA. Primers used were as follow: PHD2, (forward) 5′-GCACGACACCGGGAAGTT-3′, (reverse) 5′-CCAGCTTCCCGTTACAGT-3′, Cyclophilin-A, (forward) 5′-TTCATCTGCACTGCCAAGAC -3′, (reverse) 5′–TGGAGTTGTCCACAGTCAGC-3′. The number of each gene-amplified product was normalized to the number of cyclophilin-A amplified product and expressed as copy numbers of PHD2 transcripts over cyclophilin-A (×10−3).
Transfectiona and luciferase assay
Transfection experiments employed the LipofectAMINE reagent (Invitrogen) according to the manufacturer's protocol. Plasmid for transfections were used as follow: 1 µg of ILK cDNA (kinase dead, DN-ILK) in pUSEamp (E359K mutant) (Millipore, Billerica, MA) or with pcDNA3-PHD1, pcDNA3-PHD2, or pcDNA3-PHD3 vectors (Dr. J. Geadle, The Henry Wellcome Trust Centre for Human Genetics, Oxford, UK) or empty vector pcDNA3 (MOCK) (Promega Corporation, Madison, WI) as control. 1007, and SKMel28 cells were transfected with different luciferase reporter constructs, including a plasmid encoding CMV-Luc- ODDD (Dr. R. K. Bruick, University of Texas Southwestern Medical Center, TX), or the previously described HRE-Luc construct (Dr. A. Giaccia, Stanford University School of Medicine, Stanford, CA), as well as the human PHD2 proximal promoter construct pGL3b (1454/3172) P2PWT (Dr. E. Metzen, University of Luebeck, Luebeck, Germany). The pCMV-β-galactosidase plasmid (Promega) was used as control for transfection efficiency. The cells were lysed and their luciferase activities were measured (Luciferase assay system, Promega).
ILK Immune Complex Kinase Assay
Integrin linked kinase (ILK) activity was measured as previously described [50]. Briefly cell lysates were immunoprecipitated with anti-ILK (Millipore). Assays were done directly on the protein A-Sepharose (Sigma) beads in the presence of 5 µCi of γ-32P (Amersham Pharmacia Biotech) and 2.5 µg of myelin basic protein (MBP) was used as substrate (Millipore). Phosphorylated MBP bands were visualized by autoradiography of dried SDS-10% PAGE gels.
ELISA
The VEGF protein levels in the conditioned medium were determined in triplicate by ELISA using the Quantikine Human VEGF immunoassay kit (R&D Systems, Minneapolis, MN).
In vitro Angiogenesis Assay
HUVEC were plated on basal membrane extract (10 mg/ml, Cultrex BME; Trevigen Inc. Helgerman, CT) in the presence of conditioned media from scRNA- or siPHD2-transfected 1007 cells. After 24 h, cells were visualized by light microscopy. The amount of angiogenesis was quantified by counting the number of cells in branch point capillaries (≥3 cells per branch) in five random fields per replicate.
Chemoinvasion assay
Chemoinvasion was assessed using a 48-well–modified Boyden's chamber (Neuro Probe Inc. Gaithersburg, MD) and 8 µm pore polyvinyl pyrrolidone–free polycarbonate Nucleopore filters (Costar, New York, NY) as previously described [25]. The filters were coated with an even layer of 0.5 mg/ml Matrigel (Becton Dickinson, Franklin Lakes, NJ). The lower compartment of chamber was filled with chemoattractant (ET-1 or ET-3). 1007 cells (1×106 cells/ml) were harvested and placed in the upper compartment (55 µl per well). After 6 h of incubation at 37°C, the filters were removed, stained with Diff-Quick (Merz-Dade, Dudingen, Switzerland), and the migrated cells in 10 high-power fields were counted. Each experimental point was analyzed in triplicate.
M10 melanoma xenografts
Female athymic (nu+/nu+) mice, 4 to 6 weeks of age (Charles River Laboratories, Milan, Italy), were handled according to the Institutional guidelines under the control of the Italian Ministry of Health (DL 116/92), following detailed internal rules according to: Workamn P., et al. (1998) United Kingdom Coordinating Committee on Cancer Research (Guidelines for the welfare of animals in experimental neoplasia. Br. J. Cancer 77: 1–10). Mice were injected s.c. on one flank with 1.5×106 viable M10 cells expressing ETBR. The mice were randomized in groups (n = 10) to receive treatment i.p. for 21 days with A-192621 (10 mg/kg/d), and controls were injected with 200 µl drug vehicle (0.25 N NaHCO3). The treatments were started 7 days after the xenografts, when the tumor was palpable [25]. Each experiment was repeated thrice, with a total of 20 mice for each experiment. All tumors for each group for each experiment were harvested from M10 xenografts for Western Blot analysis. Immunohistochemical analysis was performed in six samples of each group of the tumors previously analyzed by Western blot.
Matrigel plug assay
Male C57BL/6 mice (Charles River Laboratories) were handled according to the institutional guidelines under the control of the Italian Ministry of Health (DL 116/92), Mice were subcutaneously injected with 0.5 ml matrigel containing PBS (control), 0.8 µM ET-1 alone or in combination with 8µM BQ788, as previously described [34]. The matrigels surrounded by murine tissue were removed 10 days after implantation, and snap frozen in liquid nitrogen for immunohistochemical analysis.
Immunohistochemistry
Indirect immunoperoxidase staining was carried out on acetone-fixed 4 µm tissue sections. The avidin biotin assays were performed using the Vectastatin Elite kit (for nonmurine primary antibodies) and the Vector MOM immunodetection kit (for murine primary antibodies) obtained from Vector Laboratories (Burlingame, CA) on size-matchable tumor tissues from control and A-192621 treated M10 xenografts [25] and on human melanoma samples. Sections incubated with isotype-matched immunoglobulins or normal immunoglobulins served as negative control.
Statistical analysis
Results are representative of at least three independent experiments each performed in triplicate. Statistical analysis was done using the Student t test, Fisher's exact test, as appropriated. All analyses were performed using the SPSS 11 software (SPSS, Inc., Chicago, IL). All statistical tests were two-sided. p<0.05 was considered statistically significant.
Supporting Information
Figure S1 ETs induce HIF-1α and HIF-2α expression in melanoma cells. A. 1007 cells were transfected for 48 h with scRNA or siRNA for ETB R or siRNA for GAPDH, and ETBR or GAPDH protein expression was analyzed by Western blotting. B. Western blotting analysis of HIF-1α and HIF-2α expression was performed in whole cell lysates from 1007 and SKMel28 cells treated with increased concentrations of ET-1 for 16 h or with 100 nM ET-1 for the indicated times. C. Western blotting analysis of HIF-1α expression was performed in whole cell lysates from 1007 cells were treated with increased concentration ET-3 or with unrelated peptide scramble B3 (B3; 30 µM), for 16 h, or with 100 nM ET-3 for the indicated times. Anti-β-actin was used as loading control.
(0.24 MB TIF)
Click here for additional data file.
Figure S2 ET-1 impairs HIF-1α hydroxylation. 1007 and SKMel28 cells transfected with CMV-Luc-ODDD were treated with the indicated concentrations of ET-1 for 16 h. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.004, compared to control.
(0.05 MB TIF)
Click here for additional data file.
Figure S3 ET-3 decreases PHD2 promoter activity. A. 1007 and SKMel28 cells were transfected with the construct containing the PHD2 promoter and treated with 100 nM ET-3 alone or in combination with 1µM BQ788 for 8h. Luciferase activity was expressed as fold induction. Bars, ± SD. *, p<0.006 compared to control, **, p<0.005 compared to ET-1. B. 1007 cells were transfected with each of the pcDNA3-PHDs vectors or with pcDNA3 (empty vector, C). The expression of PHD isoforms was analyzed by Western blotting. Anti-β-actin was used as loading control.
(0.14 MB TIF)
Click here for additional data file.
Figure S4 ET-1-mediated PI3K-dependent ILK/AKT/mTOR pathway induces HIF-1α stability. 1007 or DN-ILK-transfected cells were stimulated with ET-1. Following 24 h, cells were stimulated with CHX for the indicated times with ET-1 alone or in combination with signalling inhibitors and analyzed for protein expression.
(0.12 MB TIF)
Click here for additional data file.
We gratefully acknowledge Valentina Caprara, Danilo Giaccari, Stefano Masi and Aldo Lupo for excellent technical assistance and Maria Vincenza Sarcone for secretarial support. We also thank Dr. J. Geadle, The Henry Wellcome Trust Centre for Human Genetics, Oxford, UK for pcDNA3-PHDs vectors, Dr. R.K. Bruick, University of Texas Southwestern Medical Center, TX, for the plasmid encoding CMV-Luc-HIF-1α ODDD, Dr. A. Giaccia, Stanford University School of Medicine, Stanford, CA for HRE-Luc construct, and Dr. E. Metzen, University of Luebeck, Luebeck, Germany for the human PHD2 promoter construct.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Associazione Italiana Ricerca sul Cancro and Ministero della Salute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2058557610-PONE-RA-16088R110.1371/journal.pone.0011299Research ArticleCell Biology/Gene ExpressionDevelopmental Biology/Plant Growth and DevelopmentGenetics and Genomics/Gene FunctionGenetics and Genomics/Plant Genetics and Gene ExpressionPlant Biology/Plant Genetics and Gene ExpressionPlant Biology/Plant Growth and DevelopmentOsPIE1, the Rice Ortholog of Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, Is Essential for Embryo Development OsPIE1 in Embryo DevelopmentXu Yonghan
1
Deng Minjuan
1
Peng Jianfei
2
Hu Zhanghua
1
Bao Lieming
1
Wang Junming
3
Zheng Zhi-Liang
4
*
1
Province Key Laboratory of Genetic Engineering on Plant Metabolism, Virology and Biotechnology Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
2
College of Biosafety Science and Technology, Hunan Agricultural University, Changsha, China
3
Crop Science and Nuclear Utilization Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
4
Department of Biological Sciences, Lehman College, City University of New York, Bronx, New York, United States of America
Hazen Samuel P. EditorUniversity of Massachusetts Amherst, United States of America* E-mail: [email protected] and designed the experiments: YX ZLZ. Performed the experiments: YX MD JP ZH LB JW. Analyzed the data: YX MD ZLZ. Contributed reagents/materials/analysis tools: YX. Wrote the paper: YX ZLZ.
2010 24 6 2010 5 6 e112995 2 2010 31 5 2010 Xu et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The SWR1 complex is important for the deposition of histone variant H2A.Z into chromatin necessary to robustly regulate gene expression during growth and development. In Arabidopsis thaliana, the catalytic subunit of the SWR1-like complex, encoded by PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1), has been shown to function in multiple developmental processes including flowering time pathways and petal number regulation. However, the function of the PIE1 orthologs in monocots remains unknown.
Methodology/Findings
We report the identification of the rice (Oryza sativa) ortholog, OsPIE1. Although OsPIE1 does not exhibit a conserved exon/intron structure as Arabidopsis PIE1, its encoded protein is highly similar to PIE1, sharing 53.9% amino acid sequence identity. OsPIE1 also has a very similar expression pattern as PIE1. Furthermore, transgenic expression of OsPIE1 completely rescued both early flowering and extra petal number phenotypes of the Arabidopsis pie1-2 mutant. However, homozygous T-DNA insertional mutants of OsPIE1 in rice were embryonically lethal, in contrast to the viable mutants in the orthologous genes for yeast, Drosophila and Arabidopsis (Swr1, DOMINO and PIE1, respectively).
Conclusions/Significance
Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development.
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Introduction
Chromatin remodeling is a dynamic process that controls eukaryotic gene expression critical for various developmental events. Chromatin remodeling involves histone modifications, histone variant deposition and DNA methylation. While the canonical histones are deposited into chromatin strictly during the S phase of cell cycle, histone variants can be incorporated into chromatin during the entire cell cycle. Therefore, histone variants are involved in specialized cellular functions.
Among the currently identified families of the core histones H2A, H2B and H3, the H2A family of histone variants has the largest number of variants (reviewed in [1], [2]). H2A variants include MacroH2A (which is involved in mammalian X chromosome inactivation), H2A.X (important for DNA repair), and the most evolutionary conserved variant H2A.Z (involved in transcriptional control). H2A.Z has been characterized in various species including yeast, animals and plants. While H2A.Z is encoded by single gene in yeast, Drosophila and human, the Arabidopsis genome likely contains four genes (HTA4, HTA8, HTA9 and HTA11) that encode different members of H2A.Z [1], [3], [4]. Knockdown transgenic plants of HTA8, HT9 and HTA11 individually or all three genes together are viable [4], [5], [6]. So far it has not been determine whether H2A.Z is essential for Arabidopsis development through analysis of knockout mutants. In other kingdoms, H2A.Z has been shown not to be essential in yeast, but mutations in H2A.Z lead to lethality in Tetrahymena
[7], Drosophila
[8], and mouse
[9]. Furthermore, although the essential function of H2A.Z remains to be investigated, it has been suggested that H2A.Z plays a critical role in either transcriptional repression or activation by occupying the promoter regions of chromatin [2], [10]. Most recently, Arabidopsis H2A.Z has been shown to protect genes from DNA methylation [11].
The deposition of H2A.Z variants to chromatin is catalyzed by the ATP-dependent SWR1 complex in yeast, dTIP60 complex in Drosophila, SRCAP and TRRAP/TIP60 complexes in humans, or SWR1-like complex in Arabidopsis [12], [13]
[4], [5], [14]. The catalytic subunit of these complexes is Swr1 in yeast [12], [15], Domino in Drosophila
[13], [16], SRCAP in humans [17], [18], and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1) in Arabidopsis [4], [5], [19]. These proteins contain an ATPase domain and belong to the SNF2 family of chromatin remodeling factors. This type of complex also contains other subunits, for example, up to 13 in yeast. Genetic studies reveal that several subunits, Act1, Arp4, Swc4, Rvb1 and Rvb2, are essential for cell viability, while other subunits are not [1], [20], [21].
In Arabidopsis, orthologs for all subunits, except for the two yeast subunits (Swc3 and Swc7), have been identified [1]. However, only a few of them have been functionally characterized. These include PIE1, ARP6/SUF3/ESD1, and SEF/SWC6 [5], [19], [22], [23], [24], [25], [26]. Analysis of mutants of these genes revealed pleiotropic developmental phenotypes, such as changes in plant stature, leaf shape and size, flowers with extra petals, and early flowering under both long- and short-day photoperiods. Furthermore, the early flowering and leaf serration phenotype can also be observed in Arabidopsis H2A.Z knockdown transgenic plants [4], [5], demonstrating a consistent function for the H2A.Z variant and its regulatory SWR1-like complex. Among these phenotypes, flowering time has received the most attention. Several studies have demonstrated that this complex regulates the deposition of H2A.Z variant into chromatin at the FLOWERING LOCUS C (FLC) locus and consequently exerts the transcriptional control of FLC, a MADS box transcription factor that acts as a critical floral repressor of flowering [4], [5], [19], [23], [25]. Arabidopsis is a facultative long-day plant, and for the winter-annual ecotype, vernalization can de-repress FLC, which in turn activates the expression of several flowering time-related genes, such as FT and SOC1, leading to elevated expression of AP1 and LEAFY genes and ultimately flowering [27], [28].
Rice is an economically important crop. Preliminary phylogenetic analyses indicate that the rice genome contains three genes coding for H2A.Z variants, and one ortholog of ARP4 and ARP6 respectively [4], [23]. Except for this information, virtually very little is known regarding the components of the SWR1-like complex and their functions in rice and indeed any other monocot species. As a first step towards dissecting the SWR1-like complex and its function in rice growth and development, we attempted to identify the Arabidopsis PIE1 ortholog (OsPIE1). Surprisingly, homozygous T-DNA insertional mutants of OsPIE1 are embryonically lethal. This suggests that unlike the catalytic subunit of the SWR1 complex in Arabidopsis, yeast and animals, OsPIE1 plays an essential role in rice embryo development.
Results
Identification of the OsPIE1 Gene in Rice
To identify the rice ortholog of PIE1, we used the Arabidopsis PIE1 protein (Accession number: AY279398) encoded by AT3G12810 [19] to perform a BLAST search in the rice genome database (http://signal.salk.edu/cgi-bin/RiceGE). This search resulted in one rice genomic sequence (Os02g46450) and one cDNA clone (AK120785, clone name: J023010H23, with the length of only 3,375 bp). Apparently, this cDNA clone only represents a partial OsPIE1 cDNA encoding the carboxyl-terminus. Therefore, we used RT-PCR to amplify the full-length cDNA. Using both 5′ and 3′ fragments of OsPIE1 cDNA as the mixed probes in Northern blot, we detected a single, strongly hybridized band (data not shown) of 6.7 kb in size, consistent with its predicted length. This indicates that OsPIE1 is likely a single copy gene, encoded by Os02g46450 and located in the upstream of chromosome 2. The OsPIE1 cDNA molecule was sequenced and the sequence has been deposited into the NCBI GenBank with an accession number GQ906768. Sequencing results revealed that there is an additional exon between the predicted ninth and tenth exons (Figure 1A). In addition, we also found that OsPIE1 has a very different genomic structure, with 23 exons and 22 introns, compared to PIE1 which contains 20 exons and 19 introns (Figure 1A).
10.1371/journal.pone.0011299.g001Figure 1 Genomic structure of OsPIE1 gene and sequence alignment of OsPIE1 homologs.
(A) Genomic structure of OsPIE1 and Arabidopsis PIE1 genes. The translation start and stop sites are indicated. Exons are presented as filled black rectangles, and introns or intergenic sequences are presented as solid lines. For OsPIE1, the upper diagram (“predicted”) depicts the predicted structure of the OsPIE1 gene in the RiceGE database with 21 introns and 22 exons. The lower diagram (“cloned”) represents the structure of the OsPIE1 gene cloned and verified in this study, with an extra exon within the predicted 9th intron, corresponding to nucleotides 2368 to 2437 of the genomic fragment. (B) Domains of OsPIE1. Domains are predicted by the SMART program (http://smart.embl-heidelberg.de) and the amino acid numbers of these domains are indicated. The two putative bipartite nuclear localization signals (NLS) at the N-terminal and C-terminal regions of OsPIE1 are KRQKTLEAPKEPRRPKT and KKRDLIVDTDEE KTSKK, respectively. (C) Sequence alignment of the SNF_N domains of OsPIE1, Arabidopsis PIE1, Drosophila DOMINO A, human SRCAP, and yeast SWR1. Numbers indicate the amino acid positions. (D) Sequence alignment of HELICc domain. Amino acids corresponding to the domain are underlined. Numbers indicate the amino acid positions. (E) A phylogenetic tree of OsPIE1 orthologs from several organisms. The names of organisms are indicated in parenthesis, and bootstrap values are provided for the indication of reliability for each node.
Comparative Analysis of the Predicted OsPIE1 Protein and Other PIE1 Homologues
Based on the expressed OsPIE1 cDNA sequence we have obtained, OsPIE1 protein, like Arabidopsis PIE1, has an HAS domain at the N-terminus, two domains highly similar to the SNF2_N and HELICc domains, and a SANT domain in the C-terminus (Figure 1B). Two bipartite nuclear localization signals (NLS) are also present (Figure 1B), indicating that both OsPIE1 and PIE1 are likely localized to the nucleus [4], [19]. The SNF2_N and HELICc domains are present in the SWI2/SNF2 and ISWI class of chromatin remodeling proteins, which are involved in the transcriptional activation or repression of target genes. For these two domains, OsPIE1 and PIE1 share high similarity with DOMINO (Accession number: AF076776) from Drosophila
[16], SRCAP (Accession number: AF143946) from human [17], and SWR1 (Accession number: NP_010621) from yeast [15], with amino acid identity ranging from 58 to 77% (Figure 1, C and D).
A C-terminal region of OsPIE1 also exhibits similarity to the SANT domain (Figure S1). The SANT domain was found originally in SWI3, ADA2, N-CoR, TFIIIB B and ISWI, and it is characteristic of the ISWI family members [29]. Furthermore, it has been shown that the N-terminal half of the SANT domain is required for interactions with histone acetyltransferases or histone deacetylases, while the C-terminal half is required for the interaction with chromatin [30], [31], [32]. Although the amino acid identity of the SANT domains between PIE1 and other proteins, including DOMINO [16], SRCAP [17], and SWR1 from yeast [15], is very low, we found that the conserved and functionally important residues of the SANT domain are still present in this region for the PIE1 orthologs (Figure S1; see also [19]). Additionally, like PIE1, OsPIE1 has a unique 11-amino acid linker, GGAF(AGGA for PIE1) YRGRYRHP), between the N-terminal and C-terminal halves of the SANT domain (Figure S1). This linker is absent in DOMINO, SRCAP and SWR1.
Of particular note, except for the domains described above, other regions of OsPIE1 and PIE1 are quite divergent from the proteins of the SWI2/SNF2 and ISWI family in non-plant species (data not shown). In contrast, OsPIE1 and PIE1, which are from dicotyledon and monocotyledon model plants rice and Arabidopsis, respectively, exhibit a similarity as high as 53.9% at the amino acid level for the full-length proteins (Figure S1). They are almost identical to each other (up to 95% identity) in all of the important functional domains as described above (Figure 1, C and D; Figure S1). Together with phylogenetic analysis using yeast Swr1 and its orthologs in Arabidopsis, rice, human and Drosphila (Figure 1E), these results suggest that OsPIE1 protein is more closely related to Arabidopsis PIE1 than to any other homologues from human, yeast, and Drosophila.
OsPIE1 Exhibits a Similar Expression Pattern as Arabidopsis PIE1
The highly similar amino acid sequences for rice and Arabidopsis PIE1 proteins led us to hypothesize that OsPIE1 has a similar expression pattern as PIE1. To investigate the temporal and spatial expression patterns of OsPIE1, we used both promoter∶reporter assay and reverse transcriptase (RT)-PCR analysis. To analyze OsPIE1 promoter activity, a 1.6 kb genomic fragment upstream of the ATG start codon was used to drive the β-glucuronidase (GUS) reporter expression. A total of 16 independently transformed rice lines were obtained and most of these lines showed similar GUS staining patterns. We observed that GUS activity was detected in all rice plant tissues or organs tested, with a higher expression in the shoot apical meristem, root, young shoot, panicle and spikelet (Figure 2).,High OsPIE1:GUS expression was detected in the divisional and young panicles just prior to heading (Figure 2A). During the heading stage, the highest OsPIE1:GUS expression was detected in the flowering spikelets and the divisional zone (Figure 2 B–E). GUS activity was also present in the mature seed (Figure 2C), but almost absent in the leaf (Figure 2D).
10.1371/journal.pone.0011299.g002Figure 2 Expression patterns of OsPIE1 in rice.
(A) to (F) Histochemical GUS assays in the OsPIE1 promoter:GUS transgenic lines. (A) GUS staining in the primary root with lateral roots. Arrow indicates the lateral root initiation site. (B) to (E) GUS staining in young spikelets (B), the mature spikelet (C), the young leaf (D) and the flower (E). (F) GUS activity in the young shoot (a), including the rapid elongation zones of culm internodes before panicle development (see the enlarged images, b and c), and the elongation zone of peduncle (d). (G) RT-PCR analysis of OsPIE1 mRNA expression in different tissues or organs. Tissues or organs used were: Ca, callus; R,7-day-old roots; SL,7-day-old leaves; ML, mature leaves; FLB, flag leaf blade; FLS, flag leaf sheath; St, stem; P1,1 to 2-cm-long panicles; P2, 3 to 5-cm-long panicles; and P3, 5 to 8-cm-long panicles. The rice Actin gene was used as an internal control.
RT-PCR analysis using RNA extracted from various tissues and organs showed that OsPIE1 is expressed in the growing roots, stems, flowers and preferentially in the shoot apical meristem and the spikelet (Figure 2G). OsPIE1 is also expressed in flag leaf blade and sheath, but at very low level in mature leaves. Furthermore, at the heading stage, flowering panicles and stems accumulated the OsPIE1 transcript at the highest level, followed by panicles before flowering. This result is largely consistent with the promoter:GUS expression patterns (Figure 2, A–F), indicating that the 1.6 kb promoter fragment likely contains all of necessary cis-elements for proper expression of OsPIE1. The preferential expression of OsPIE1 in floral organs compared to leaves is very similar to that of PIE1 in Arabidopsis [19].
Transgenic Expression of OsPIE1 Rescues the Early Flowering Phenotype in the Arabidopsis pie1 Mutant
The highly similar protein sequences and expression patterns between OsPIE1 and PIE1 led us to hypothesize that OsPIE1 also has similar functions as PIE1 in the control of flowering time and petal number. To test this hypothesis, we constructed the Arabidopsis PIE1 promoter:OsPIE1 cDNA expression cassette and investigated whether expression of OsPIE1 could functionally complement the early flowering and iregular petal number phenotype in transgenic plants of Arabidopsis pie1-2, a likely null allele [19]. Among 50 T1 plants, 26 flowered later than pie1-2 and close to wild-type Col. The seeds from these T1 plants were then tested for the segregation of hygromycin resistant versus sensitive seedlings, and we found that 20 lines exhibited 3∶1 segregation ratios. These 20 lines, each with a single T-DNA insertion, were shown to co-segregate the presence of transgene with normal flowering time phenotype (data not shown). As shown in Fig. 3A, two representative lines of transgenic pie1-2 plants did not flower, similar to Col but in sharp contrast to pie1-2 which already flowered. Quantitative analysis of flowering time showed that indeed the transgenic lines had similar number of rosette leaves from seed germination to flowering as Col, under both long-day and short day conditions (Table 1). Furthermore, they also reverted the abnormal petal number (three, five or six) to normally four petals (Figure 3, B–F).
10.1371/journal.pone.0011299.g003Figure 3 Rescue of the pie1-2 mutant phenotypes by transgenic expression of OsPIE1.
(A) to (F) Phenotypes of transgenic Arabidopsis pie1-2 plants transformed with OsPIE1 under the control of the Arabidopsis PIE1 promoter. (A) Plants of two representative transformant (middle), WT (left) and the pie1-2 mutant (right) grown under long-day conditions (16 h light/8 h dark) until pie1-2 plants flowered. Inflorescence of transgenic pie1-2 plant (C) was compared with that of WT (B) and pie1-2 mutant (E). Abnormal floral organ number (with an extra petal indicated by arrowhead in F) in pie1-2 mutant was rescued in transformants of pie1-2 (with 4 petals; D). (G) RT-PCR analysis of PIE1 and OsPIE1 expression patterns. Expression of PIE1 was conducted in Col (wild-type), and OsPIE1 in pie1-2 transformants (Arabidopsis PIE1 promoter:OsPIE1 cDNA). Tissues or organs were collected from adult plants grown in long days. Arabidopsis ACT2 was used as an internal control. (H) Gel blot analysis of FLC expression in various tissues or organs from pie1-2 or one representative pie1-2 transformant (transformed with OsPIE1 under the control of Arabidopsis PIE1 promoter). The blots were probed first with FLC and then re-probed with Arabidopsis UBQ10 as an internal control.
10.1371/journal.pone.0011299.t001Table 1 Rosette leaf number of pie1-2 and pie1-2 transformants at flowering in long and short days.
Light Conditions Wild-type
pie1-2 Transformants
pie1-2
Long days 9.7±1.4 9.4±0.6 6.8±0.5
Short days 40.9±4.9 42.3±3. 17.3±1.5
Values shown are mean rosette leaves numbers±SD at flowering. At least 15 plants were scored for each genotype and treatment.
To confirm that the phenotypic rescue of pie1-2 is the result of proper expression of OsPIE1, RT-PCR analysis was performed. Results showed that expression of OsPIE1 driven by the Arabidopsis native PIE1 promoter was very high in seedlings and flowers but barely detectable in leaves (Figure 3G, lower panel). This pattern was quite similar to PIE1 expression in Col (Figure 3G, upper panel; also see [19]), except that transgenic OsPIE1 expression was also high in roots compared to relatively weak expression of PIE1 in Arabidopsis roots.
Because it has been demonstrated that the early flowering phenotype in pie1 mutants is mainly due to the reduced expression of FLC
[19], we decided to assess whether native FLC expression is restored in OsPIE1 expressing transgenic lines of pie1-2. Results from Northern blot using FLC as a probe showed that in contrast to weak expression of FLC in pie1-2, transgenic expression of OsPIE1 under the control of PIE1 promoter induced the strong FLC expression in the shoot apex but not in other organs tested (Figure 3H). Taken together, we have demonstrated that OsPIE1 can functionally replace PIE1 in the control of flowering time and petal number in Arabidopsis. This suggests that the biochemical function of OsPIE1 and PIE1 are evolutionally conserved [4], [5], [19].
Homozygous Ospie1 T-DNA Insertion Mutants Are Embryonically Lethal
To test whether OsPIE1 may also function in the control of flowering time and petal number regulation in rice, T-DNA knockout mutants of OsPIE1 were isolated and characterized Two independent alleles, Ospie1-1 and Ospie1-2 (corresponding to the PFG_3A-60036 and PFG_1B-21620 stocks, respectively), were obtained from the rice T-DNA insertion mutagenesis collection at the Plant Functional Genomics Laboratory [33]. Both mutants have a T-DNA insertion in the 11th (926 nucleotides downstream of ATG in genomic DNA) and 12th (964 nucleotides downstream of ATG) introns, respectively. The T-DNA insertion sites in OsPIE1 suggest that Ospie1-1 and Ospie1-2 may cause loss of OsPIE1 function. Using PCR- based genotyping of T-DNA insertions in at least 300 segregating plants derived from self-pollinated heterozygous plants (Ospie1-1/+ and Ospie1-2/+), we failed to obtain any homozygous plants for both alleles and thus we were unable to determine whether the mutations are null or leaky. Nevertheless, we found that the progeny from self-fertilized Ospie1-1/+ or Ospie1-2/+ parental plants exhibited a segregation ratio of 2∶1 for heterozygotes and WT plants (Table 2). This genetic analysis indicates that the homozygous T-DNA insertion mutants in OsPIE1 are likely lethal. No obvious phenotype was observed in all of heterozygous plants. However, when the seeds collected from the self-pollinated Ospie1-1/+ and Ospie1-2/+ plants were examined, 21–25% of the seeds were empty (indicated by red arrowheads in Figure 4A and shown in Figure 4B), as compared to WT which had 95% of plump yellowish (filled) seeds (Figure 4C). The percentage of filled grains is consistent with the expected 3∶1 ratio of plump yellowish seeds versus empty seeds for the progeny from self-pollinated Ospie1-1/+ or Ospie1-2/+ plants. Together with the genotyping result, our data suggest that all of the homozygous recessive (Ospie1-1 and Ospie1-2) embryos are inviable.
10.1371/journal.pone.0011299.g004Figure 4 Phenotypic characterization of Ospie1 mutants.
(A) Spikelets of self-pollinated wild-type (WT), Ospie1-1/+, and Ospie1-2/+. Red arrowheads indicate the panicels of abnormal empty seeds for each of the three genotypes. (B) Representative images of normal WT seeds and empty seeds from self-pollinated Ospie1-1/+ and Ospie1-2/+ plants. Half of the husk was removed for visualizing endosperms. No endosperm developed normally in those empty seeds of Ospie1-1/+ and Ospie1-2/+ plants. (C) Quantitative analysis of grain setting rates for heterozygous Ospie1 plants. Grain setting rate is the percent of filled grains over the sum of filled and empty grains for each plant. Data shown are means ± SD of 30 plants for each genotype. The experiment was repeated three times, with a similar result.
10.1371/journal.pone.0011299.t002Table 2 Segregation of T-DNA in Ospie1 heterozygous mutants.
Genotypes of Self-pollinated Plants Progeny with One T-DNA Insertion Progeny with No T-DNA Insertion
X
2-test Result
Ospie1-1/+ 203 97
X
2-value = 0.135 (p-value>0.05)
Ospie1-2/+ 212 98
X
2-value = 0.413 (p-value>0.05)
PCR-based genotyping was performed in the progeny from self-pollinated parental plants. X
2-test was performed to show whether it is consistent with a 2∶1 segregation.
To determine whether the homozygous lethality is due to the defects in gametes or embryos, reciprocal crosses were performed between WT and Ospie1-1/+, and between WT Ospie1-2/+. Due to the abnormal high temperatures in the growing field in tropical Hainan province in China in 2010, the grain setting ratios for the F1 progeny derived from all types of crosses were unexpectedly low (approximately 10%), which were similar to the control cross, WT×WT (Table 3). However, we found no statistical difference in the setting ratios for the F1 progeny derived from the reciprocal crosses involving WT and Ospie1-1 or WT and Ospie1-2. Furthermore, PCR genotyping of those F1 progeny derived from individual crosses showed consistent 1∶1 segregation ratios for Ospie1 heterozygotes and WT in all of the reciprocal crosses (Table 3). These results support the idea that gametes are unaffected by Ospie1-1 and Ospie1-2 mutations and that the homozygous lethality of Ospie1 mutants is likely caused by a defect in embryo development. Indeed, we observed that 10 days after self-pollination, the embryos in the abnormally developed grains from Ospie1-1/+ or Ospie1-2/+ were very small, compared to the normal WT embryos (Figure 5). Those grains with smaller embryos showed endosperm shrinkage (Figure 5), which became empty seeds when mature (Figure 4B).
10.1371/journal.pone.0011299.g005Figure 5 Abnormal embryos in Ospie1 mutants.
Upper panels show the representative seeds (with the hull completely removed) in wild-type (WT) and the abnormally developed grains in self-pollinated Ospie1-1/+ and Ospie1-2/+ plants, respectively. The white rectangular box indicates the approximate position of embryo. The bar represents 1.99 mm for all three images in the upper panels. Lower panels show the embryos collected from the seeds indicated in the upper panels, with the bar representing 0.1 mm.
10.1371/journal.pone.0011299.t003Table 3 Reciprocal crosses between Ospie1 hetrozygotes and wild-type.
Type of Crosses (male × female) Setting Rate (%) Number of Ospie1 Heterozygotes in F1 Number of Wild-type in F1
X
2-test Result
Ospie1-1/+ × WT 6.1±1.8 41 43
X
2-value = 0.048 (p-value>0.05)
Ospie1-2/+ × WT 9.2±4.6 31 34
X
2-value = 0.138 (p-value>0.05)
WT × Ospie1-1/+
10.1±4.8 31 32
X
2-value = 0.016 (p-value>0.05)
WT × Ospie1-2/+
11.0±5.1 19 21
X
2-value = 0.100 (p-value>0.05)
WT × WT 10.8±4.7 0 93
X
2-value = 93.000 (p-value<0.05)
Reciprocal crosses between wild-type and Ospie1-1 or Ospie1-2 heterozygous plants were performed and the setting rates for each cross were scored. Data represented the average ±SD for 5–8 plants in each cross. No statistical significant difference was observed in setting rates between these genotypes. Those filled grains in F1 were then PCR genotyped to determine the number of Ospie1 heterozygotes and wild-type. X2-test was used to determine that they followed the 1∶1 segregation ratio. A cross involving wild-type (WT) was also performed as a control.
Discussion
Several lines of evidence together demonstrate that OsPIE1, encoded by Os02g46450, is the true rice ortholog of Arabidopsis PIE1. First, at the amino acid sequence level, these two proteins share 53.9% identity. Second, protein domain prediction and comparison reveal that OsPIE1 is very similar to PIE1, with almost identical domains. Third, phylogenetic analysis also places OsPIE1 as the most closely related protein of PIE1, compared to other catalytic subunits in yeast and animals. Fourth, OsPIE1 exhibits a very similar tissue and developmental expression pattern as PIE1. Fifth and importantly, transgenic expression of OsPIE1 in Arabidopsis pie1-2 mutant complements the pie1-2 phenotypes with regard to flowering time and petal number. Therefore, the identification of OsPIE1 as the ortholog of the catalytic subunit of the SWR1-like complex in rice provides the basis to dissect the physiological function of OsPIE1 in the future.
We were surprised to observe that homozygous T-DNA insertions lead to lethality in rice. Lethality could be caused by the failure or defects at any stage of reproductive development, such as reduced fertility of male and female gametes, failure of fertilization, or more likely abortion of embryo development. However, we have observed that pollen grains collected from both Ospie1-1/+ and Ospie1-2/+ plants are viable, as indicated by similar kalium iodide staining as WT pollen grains (data not shown). Furthermore, when reciprocal crosses were performed between WT and heterozygous Ospie1-1 or Ospie1-2 mutants, they showed similar F1 setting ratios, and genotyping of the F1 progeny confirmed the 1∶1 segregation ratio for heterozygotes and WT. In addition, we also observed that young embryos (10 days after pollination) in the abnormal grains derived from the self-pollinated Ospie1-1 and Ospie1-2 heterozygous plants were much smaller than the embryos in the normal grains of WT plants. Therefore, homozygous lethality in the T-DNA insertional mutants of OsPIE1 is caused by the abortion of embryos. This demonstrates an essential role for OsPIE1 in proper rice embryo development.
The essential function for OsPIE1 in rice embryo development is a novel finding. Some studies have demonstrated the importance of histone variants in mammalian reproduction (reviewed in [34]). For example, H2A.Z is essential for embryo development in Drosophila and mouse [8], [9]. In addition, the H2A.F/Z variant hv1 has also been shown to be essential in the ciliated protozoan, Tetrahymena thermophila
[7]. However, loss-of-function mutants for the genes encoding the components of the SWR1-like complexes are viable in these organisms. Interestingly, although H2A.Z is not essential in yeast, the H2A.Z mutation is synthetically lethal with an H4 histone mutation [35]. Furthermore, five subunits of the SWR1 complex, Act1, Arp4, Swc4, Rvb1 and Rvb2, play an essential role in yeast cell viability [1], [20], [21]. In Arabidopsis, the mutants or knockdown transgenic plants for the genes encoding H2A.Z variants and the three components of the SWR1-like complex (PIE1, ARP6, SEF) exhibit an obvious early flowering phenotype and the subtle phenotypes in leaf morphology and petal number [4], [5], [6], [19], [22], [23], [24], [25], [26]. The lethal phenotype has not been reported, although the arp6 mutant of Arabidopsis has been shown to reduce female fertility and consequently reduce number of seeds in the silique [23]. Therefore, our finding that the catalytic subunit of the SWR1-like complex can also play an essential role in development is novel. One of the likely explanations for the essential function is that OsPIE1 targets the rice H2A.Z to chromatin at the locus or loci that contain the genes essential for embryo development. Generating OsPIE1 knockdown transgenic rice plants in the future will, if they are not lethal, help us to determine which aspect of cell growth and embryo development is controlled by OsPIE1 and whether OsPIE1 has a similar physiological function in the control of flowering time in rice.
In summary, we have shown that OsPIE1 is the rice ortholog of Arabidopsis PIE1, the catalytic subunit of the SWR1-like complex in Arabidopsis. OsPIE1 can functionally replace PIE1 in the control of flowering time and petal development, supporting that PIE1 orthologs are evolutionarily highly conserved. Most interestingly, we have shown that this catalytic subunit in rice is essential for embryo development, which has not been reported in Arabidopsis, yeast and animals. Future work should further reveal whether OsPIE1 also functions in the control of flowering time in rice and how it remodels the chromatin associated with the genes essential for embryo development.
Materials and Methods
Plant Materials and Growth Conditions
Ospie1/+ T-DNA insertion lines in Dongjin (Oryza sativa spp japonica cv) back- ground were isolated from the POSTECH rice T-DNA insertion mutant bank (http://signal.salk.edu/cgi-bin/RiceGE). The Ospie1/+ plants were propagated vegetatively in the summer in Hangzhou City, China and the winter on Hainan Province (close to South China Sea) under natural conditions. Rice materials were harvested from plants grown in the field.
Arabidopsis plants (ecotype Columbia) were grown routinely under controlled environmental conditions (23°C day/18°C night, white fluorescent lamps with a light intensity of 150 mmol m−2 sec−1, 16 h light/8 h dark, and 65% relative humidity). To isolate T-DNA insertion mutant alleles for Arabidopsis PIE1 (At3g12810), seeds were obtained from either the SALK Collection (http://signal.salk.edu/; pie1-2, which is SALK_003776) or the Syngenta Arabidopsis Insertion Library (http://www.nadii.com/pages/collaborations/).
Cloning of the OsPIE1 Gene and Genotyping of the Ospie1 T-DNA Insertion Mutants
The coding sequence (CDS) of OsPIE1 which encodes 2045 amino acids was PCR-amplified from reverse-transcribed rice cDNA (Invitrogen, Carlsbad, CA, USA), using the following primers: OsPIE1F1, 5′-ATGGCATCAAAAGGTCCTCGATCAAAG-3′; OsPIE1R1, 5′-TCTGGCATTGAG GAGTTGGATCCTCTTAC-3′. Due to the large size of the OsPIE1 transcript, we obtained the full-length cDNA of OsPIE1 by amplifying several fragments. Its 5′ untranslated region (UTR) and 3′ UTR sequence of OsPIE1 were obtained by the method of 5′ rapid amplification of cDNA end (RACE) and 3′ RACE using the kit provided by TaKaRa (TaKaRa, Kyoto, Japan). Primers for 5′-RACE and 3′-RACE were designed according to the manufacturer's instructions. All of the above PCR products were cloned into pMD18-T vector, according to the manufacturer's instructions (TaKaRa, Kyoto, Japan).
Genotyping of the progeny derived from self-pollinated Ospie1-1/+ and Ospie1-2/+ plants or from the reciprocal crosses between WT and Ospie1-1/+ or Ospie1-2/+ was performed by PCR using the following primers. For Ospie1-1: P1LP, 5′-AGTTGTG TACCAGGGCAAGG-3′; P1RP, 5′-GGATCGGACCGATAGTTCAG-3′; 2717LB, 5′-GAACGGCCACAAGTTCAGCGT-3′. For Ospie1-2: P2LP, 5′-TGGACTCT GGGAGAGCTAGG-3′; P2RP, 5′-GAAGGCAGTGGG AAAA ACAG-3′; 2715LB, 5′-CTAGAGTCGAGAATTCAGTACA-3′. The PCR was conducted with an initial step of 94°C incubation for 5 min, followed by 30 cycles of 94°C for 30sec, 56°C for 30 sec, and 72°C for 45 sec.
OsPIE1 Expression Vector Construction and Plant Transformation
The primers OsPIE1F1-1 and OsPIE1R1-1 (with the Sal I and Pml I sites incorporated as to the above described primers OsPIE1F1 and OsPIE1R1; OsPIE1F1-1, 5′-GTCGACATGGCATCAAAAGGTCCTCGATCAAAG-3′; OsPIE1R1-1, 5′-CACGTG TCTGGCATTGAGGA GTTGGATCCTCTTAC-3′) were used to amplify the 6.13 kb CDS that contains the entire OsPIE1 coding region. The PCR product was then inserted into the binary vector pCAMBIA1301 after digestion by Sal I and Pml I, resulting in pPNA109. The 1.5 kb Arabidopsis PIE1 promoter fragment was amplified as previously described [19] and after digestion with EcoR I and Sal I, it was inserted into pPNA109, upstream of OsPIE1 CDS, resulting in the OsPIE1 expression vector pPNA119. The resulting construct was introduced into Agrobacterium tumefaciens starin EHA105 and then transformed into pie1-2 mutant via the floral dip method [36]. Transgenic plants were selected on agar-solidified medium containing 0.65 g/L Peter's Excel 15-5-15 fertilizer and 50 µg/ml hygromycin.
Promoter:GUS Construct and GUS Activity Assay
A 1.6 kb OsPIE1 promoter fragment was amplified by PCR using two primers: OsPIE1PF, 5′-GTGTGCTGCAGTGGGTAGAAGGGTAATTGAGAGC-3′ and OsPIE1PR, 5′-CCATGTCTAGACTTTCCGAT ATTTTGTAGAGAACTATCT-3′
, with the Pst I and Xba I (underlined) restriction sites incorporated, respectively. The PCR product was then digested with Pst I and Xba I and cloned into pCAMBIA1301 to replace the CaMV 35S promoter, resulting in the OsPIE1:GUS reporter construct. The OsPIE1:GUS construct was used to transform Nipponbare (Oryza sativa spp japonica cv) via the Agrobacterium-mediated method as previously described [37]. Histochemical assay for GUS activity in transgenic plants was performed as described [38].
Reverse Transcriptase (RT)-PCR Analysis
The Trizol reagent (Invitrogen) was used to extract total RNA from various tissues or organs of rice and Arabidopsis plants, according to the manufacturer's instructions. RT-PCR was performed using gene-specific primers. OsPIE1: OsPIE1F, 5′-CTGAGCGCAACGAGGAATTGGCTGCt-3′, OsPIE1R, 5′-CCCAGCATGCCT GCAGATTCATGATC-3′; Rice actin: actin1F,5′-GTCAATAACTGGGATGACATGGAG-3′, actin1R, 5′-AGCTTCATGTATGCCAGG AGATT-3′; and PIE1: PIE1F, 5′-GTCCTGAACTCGATGAGGAT-3′, PIE1R,5′-GCAGGGCAATCTCTTCGACATGAT-3′. PCR was performed with 32 cycles for both OsPIE1 and PIE1 and 30 cycles for rice Actin.
RNA Gel Blot Analysis
RNA electrophoresis and subsequent blot and hybridization were performed as described elsewhere [39], [40]. Total RNA of 40 µg was loaded on each lane. Briefly, after hybridization for 20 h at 68°C, the membrane was washed once with 2×SSC plus 0.1% SDS at 68°C for 20min, and then with 13 SSC plus 0.1% SDS at 37°C for 30min. The membrane was exposed to the x-ray film (Kodak, Rochester, NY) at 27°C for 3 to 7 d. The FLC cDNA fragment lacking the conserved MADS-domain sequences was labeled with [32P]dCTP (China Isotope, Beijing) and used as the probe for FLC. The blot was first hybridized with the FLC probe and then re-probed with the UBQ10 probe (a 128 bp fragment of AT4g05320 cDNA). The 128 bp UBQ10 fragment was PCR-amplified using the following primers: sense primer UBQF (5′-TTCACTTGGTCCTGCGTCTTCGTGGTGGTTTC-3′) and antisense primer UBQR (5′-CATCAGGGATTATACAAGGCCCC-3′).
Phylogenetic Analysis
Amino acid sequences of PIE1and its related proteins were obtained from the NCBI database. The retrieved PIE1 homolog sequences were aligned using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and neighbor-joining trees were generated using the MEGA version 3.1 software [41], with bootstrap values obtained from 500 replications.
Microscopic Observation of Embryos
Rice seeds were cut into small pieces (3×3 mm) and fixed with 2.5% glutaraldehyde and 1% osmium tetroxide. After a brief rinse with the phosphate buffer (0.1 M Na2HPO4 and NaH2PO4, pH 7.0), the specimens were dehydrated in a series of ethanol solutions and acetone and then embedded in Spurr resin. Semi-ultra thin sections (2–4 µm) were prepared using a glass knife and stained with the methylene blue for examination under the microscope LEICA (Model DM LB2).
Supporting Information
Figure S1 Aligment of OsPIE1 and Arabidopsis PIE1 protein sequences.
(0.05 MB DOC)
Click here for additional data file.
We thank Dr. An Gynheung in POSTECH Biotech Center, Pohang University of Science and Technology (POSTECH), Korea, for providing the two T-DNA insertional Ospie1 mutants in the study. We are grateful to Dr. Thomas Leustek at Rutgers University for his critical reading of this manuscript. We also appreciate the constructive and helpful comments on the manuscript from the editor and the anonymous reviewers.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors have no support or funding to report.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2058558510-PONE-RA-18066R110.1371/journal.pone.0011305Research ArticleBiochemistry/Biomacromolecule-Ligand InteractionsBiochemistry/Cell Signaling and Trafficking StructuresBiotechnology/Protein Chemistry and ProteomicsCell Biology/Membranes and SortingInfectious Diseases/Bacterial InfectionsA Femtomol Range FRET Biosensor Reports Exceedingly Low Levels of Cell Surface Furin: Implications for the Processing of Anthrax Protective Antigen FRET Biosensor of FurinGawlik Katarzyna
1
Remacle Albert G.
1
Shiryaev Sergey A.
1
Golubkov Vladislav S.
1
Ouyang Mingxing
2
Wang Yingxiao
2
Strongin Alex Y.
1
*
1
Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America
2
Department of Bioengineering and the Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Illinois, United States of America
Buckle Ashley M. EditorMonash University, Australia* E-mail: [email protected] and designed the experiments: KG AR VSG AYS. Performed the experiments: KG AR SS. Analyzed the data: KG AR SS VSG MO YW AYS. Contributed reagents/materials/analysis tools: MO YW. Wrote the paper: AYS.
2010 24 6 2010 5 6 e1130518 4 2010 6 6 2010 Gawlik et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR↓STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.
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Introduction
FRET takes place between a donor and acceptor fluorophore moieties if there is an overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor [1]. In addition to this spectral overlap, the two fluorophores must be properly aligned within a certain distance of each other. There is a close relationship between donor-acceptor distance and efficiency of energy transfer [2], [3], [4]. If the donor and the acceptor are linked by a peptide sequence that spans a proteinase cleavage site, following proteolytic cleavage the donor and the acceptor are separated and are no longer in close proximity. As a result, the level of FRET rapidly and significantly decreases. FRET can be quantified according to a ratio of light emission at the two specific wavelengths which are unique for the donor and the acceptor. These parameters stimulate the use of FRET-based biosensors as the molecular tools in the proteinase research [5], [6], [7], [8], [9], [10], [11], [12].
Multiple cellular proteins including growth factors, hormones, metalloproteinases and cell receptors are synthesized as inactive precursors [13], [14]. These precursors are transformed into functionally active proteins by the cleavage action of proprotein convertases (PCs), specialized serine endoproteinases with the focused cleavage preferences [15], [16]. Seven PCs (furin, PACE4, PC1/3, PC2, PC4, PC5/6, and PC7) have been identified in humans [17]. Furin and other PCs cleave the multibasic motifs Arg-Xxx-Arg/Lys/Xxx-Arg↓, and thus transform proproteins into biologically active proteins and peptides [18], [19], [20], [21]. Furin is currently the most studied enzyme of the PC family. Furin is activated autocatalytically [21]. The autoactivated furin then cleaves latent precursors in the Golgi and the secretory vesicles, and, potentially, also at the cell membrane, post-secretion. In addition to normal cell functions, furin is implicated in many pathogenic states, because it processes to maturity membrane fusion proteins and pro-toxins of a variety of both bacteria and viruses, including anthrax toxin.
Anthrax toxin includes protective antigen and edema and lethal factors (EF and LF, respectively) [22], [23]. It has been suggested that during the process of intoxication, the 83 kDa protective antigen monomer (PA83) binds to the cell surface anthrax toxin receptors. Two receptor types are a capillary morphogenesis protein 2 (CMG2) that is the major receptor mediating lethality of anthrax toxin in vivo and the anthrax toxin receptor/tumor endothelial marker 8 (ATR/TEM8) that plays a minor role. CMG2 is expressed in most human tissues. The expression of TEM8 is restricted to tumor endothelium and cancer cells [24]. The receptor-bound PA83 is believed to be then cleaved by cellular furin and related PCs [25], [26]. This cleavage releases a 20 kDa N-terminal fragment and a cell-bound, C-terminal 63 kDa protein (PA63). The latter oligomerizes into a ring-shaped PA heptamer that exposes the binding sites for EF and LF [27], [28]. The functional heptamer, however, may include both PA63 and PA83 [29]. The N-terminal ends of both EF and LF bind to PA63. The respective C-terminal parts of EF and LF exhibit the adenylate cyclase and the metalloproteinase activity, respectively. A complex formed by the PA63 heptamer and either EF or LF or both is internalized into the cell by receptor-mediated, clathrin-dependent, endocytosis [30]. In the acidified lumen of the endosomes, the heptamer forms a channel through which EF and LF are transported from the endosomal compartment into the cytoplasm of the host cell. In the cytoplasm, EF and LF produce their toxic effects.
To analyze the role of furin in the PA83 processing in more detail and to develop a specific biosensor for furin–like PC activity, we have specifically selected the enhanced CFP (ECFP) and YPet (a variant of YFP) pair [6], [31]. According to our experience, the ECFP/YPet FRET pair allows the development of biosensors with the significantly enhanced sensitivity [31]. The ECFP and YPet moieties were linked by a specially selected peptide sequence that was highly sensitive to furin proteolysis [26], [32]. Experimental evidence demonstrated that, as a result, we developed a highly selective and sensitive furin biosensor which, in contrast to the fluorescent peptide substrates, allowed continuous and accurate monitoring of furin activity both on tumor cell surfaces and in tumor cell extracts.
The use of the FRET biosensor in a combination with other analytical methods determined the presence of exceedingly low levels of active furin on the cancer cell surface. In contrast to the previous concept, our results suggest that anthrax PA83 is processed by the furin activity in the extracellular milieu rather than directly on the cell surface. This parameter provides an opportunity for the design of anthrax inhibitors which would inactivate extracellular furin without interfering with normal physiological functions of cellular furin and furin-like PCs.
Materials and Methods
Materials
Reagents were purchased from Sigma-Aldrich unless indicated otherwise. The fluorescent substrate pyroglutamic acid-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide (Pyr-RTKR-AMC) was from Peptides International. The furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) was from Bachem. Aprotinin was purchased from Serological Corporation. Anthrax PA83 was purchased from List Biological Laboratories. Human myelin basic protein (18.5 kDa isoform) was from Biodesign. The pET directional TOPO Expression kit was obtained from Invitrogen. The Fugene HD transfection reagent was from Roche Diagnostics. The murine MON-148 monoclonal antibody against the catalytic domain of furin was from Axxora. A rabbit polyclonal GFP antibody that cross-reacts with ECFP and YPet was from Abcam. The peroxidase-conjugated donkey anti-mouse and anti-rabbit IgGs were from Jackson ImmunoResearch Laboratories. The NS2B-NS3 proteinase from West Nile virus (WNV) was purified as reported earlier [33].
Cloning and plasmid construction
The ECFP-YPet FRET biosensor (GenBank Accession #EU545473) in the pRSETb vector was initially designed for monitoring the activity of membrane type-1 matrix metalloproteinase [6]. This construct was used as a template for constructing the furin biosensor. The SNSRKKR↓STSAGP sequence of anthrax PA83 was inserted by the PCR mutagenesis in the ECFP-YPet construct using the 5′-AGCAACAGCCGTAAAAAACGTAGTACTAGTGCCGGCCCGATGGTGAGCAAGGGCGAGGAG-3′ and 5′- CGGGCCGGCACTAGTACTACGTTTTTTACGGCTGTTGCTGAGCTCTTTGTACAATTCATT-3′ oligonucleotides as the forward and reverse primers, respectively (the sequence coding for the furin cleavage site is underlined). The amplified sequence was inserted into the pET101/D-TOPO expression vector (Invitrogen) and N-terminally tagged with the Hisx6 and FLAG tags. The authenticity of the constructs was confirmed by DNA sequencing. The constructs encoding the full-length human furin (furWT) and the catalytically inert furin mutant D153N (furD153N) in which Asn replaced the essential active site Asp153 were described earlier [34]. The furin constructs were sub-cloned into the pcDNA 3.1/V5-His-TOPO vector.
Cells
Human glioma TP98G, U373 and U251, fibrosarcoma HT1080, breast carcinoma MCF-7 and colon carcinoma LoVo cells (all from ATCC, Manassas, VA) were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 units/ml and 100 µg/ml, respectively). Sub-confluent MCF-7 and LoVo cells (2×105 and 5×105, respectively) were transfected using Fugene HD reagent (3 µl/1 µg DNA). MCF-7 and LoVo cells were transfected with the furWT and furD153N constructs to obtain the stably transfected MCF-7:furWT, MCF-7:furD153N and LoVo:furWT cells. Transfected cells were grown for 3–4 weeks in the presence of G418 (1 mg/ml). Cell clones with the high expression of furin were identified using Western blotting with the furin MON-148 antibody. Glioma U251 cells stably transfected with α1-anti-trypsin variant Portland (a potent inhibitor of furin; PDX) were constructed and characterized earlier (U251/PDX cells) [35], [36].
Furin biosensor expression and purification
The biosensor construct was expressed in E. coli BL21 (DE3) Codon Plus cells. The expression of the construct was induced for 16 h at 30°C using 1 mM isopropyl β-D-thiogalactoside. The cells were collected by centrifugation and disrupted by sonication on ice in 20 mM Tris-HCl buffer, pH 8.0, containing 200 mM NaCl, a proteinase inhibitor cocktail and lysozyme (5 mg/ml). The biosensor was purified from the supernatant fraction using a HiTrap Co2+-chelating Sepharose FastFlow 1.6×2.5 cm size column (GE Healthcare) equilibrated with 20 mM Tris-HCl buffer, pH 8.0, containing 200 mM NaCl. The biosensor was eluted using a 25–500 mM gradient of imidazole concentrations in 20 mM Tris-HCl buffer, pH 8.0, containing 200 mM NaCl. The peak fractions were combined, dialyzed against 100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2, and frozen at −80°C until use.
Furin expression and purification
The expression of the soluble C-terminally truncated furin construct in Sf9 insect cells (an ovarian cell line from fall armyworm Spodoptera frugiperda) infected with the recombinant baculovirus and purification of furin were described earlier [34].
FRET assay
The biosensor (100 pmol; 6 µg) was co-incubated for 15–240 min with purified furin (10–100 fmol; 0.6–6 ng) at 37°C in 0.1 ml of the assay buffer (100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2 and 1 mM MgCl2) in a well of a 96-well plate. The emission ratio of ECFP/YPet (476 nm/526 nm) at λex = 437 nm was measured by a fluorescence plate reader (FlexStation3, Molecular Devices) to assess the FRET efficiency between ECFP (serving as a donor) and YPet (serving as an acceptor).
Cleavage of protein substrates
The cleavage reactions (22 µl each) were performed in 100 mm HEPES, pH 7.5, containing 1 mm CaCl2, 1 mm β-mercaptoethanol and 0.005% Brij35 (for furin) and 10 mM Tris-HCl, 8.0 containing 20% glycerol (for WNV NS2B-NS3 proteinase). The ECFP/YPet biosensor and PA83 (20 pmol each) were each co-incubated for 1 h at 37°C with the proteinases at a 1∶1-1∶10,000 enzyme-substrate molar ratio. The cleavage reactions were stopped using 1% SDS. The digests were analyzed by SDS-gel electrophoresis followed by Coomassie staining. Where indicated, aprotinin and dec-RVKR-cmk (at a 1∶4-1∶1,100 and a 1∶20 enzyme-inhibitor molar ratio, respectively) were added to the reactions.
Cleavage of the biosensor by cell samples
Cells (5×104) in DMEM-10% FBS were seeded in wells of a 96-well plate for 24 h. After washing, 0.1 ml 100 mM Hepes, pH 7.5, supplemented with 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2, 1% insulin-transferrin-selenium liquid supplement (ITS) and the biosensor (100 pmol, 6 µg) were added to the cells. After incubation for 2–16 h, the emission ratio of ECFP/YPet (476 nm/526 nm) at λex = 437 nm was measured using a FlexStation3 fluorescence plate reader.
For the cleavage of the biosensor by the cell lysates, the cells were detached using 2% EDTA in PBS. After washing, cells were collected by centrifugation and lysed for 1 h at 4°C in 100 mM Hepes, pH 7.5, supplemented with 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 0.1% Triton X-100. The cell lysates (50 µg total protein) were co-incubated for 2 h at 37°C with the biosensor (100 pmol, 6 µg). The emission ratio of ECFP/YPet (476 nm/526 nm) at λex = 437 nm was measured using a FlexStation3 fluorescence plate reader. The samples were also analyzed by Western blotting with the anti-GFP antibody.
Processing of PA83 by the cells
PA83 was labeled for 30 min at 4°C using EZ-Link sulfo-NHS-LC-biotin (Pierce; a 1∶20 protein-biotin molar ratio). Excess biotin was removed using a 0.7-ml spin-column. Where indicated, biotin-labeled PA83 (bPA83) was co-incubated with furin (at a 1∶100 enzyme-substrate molar ratio) to convert bPA83 into biotin-labeled PA63 (bPA63). U251, LoVo-mock and LoVo:furWT cells (3×105) were each incubated for 3 h at 37°C in DMEM supplemented with 25 mM HEPES, pH 7.0, 0.2% BSA and bPA63 or bPA83 (1 µg/ml). Where indicated, dec-RVKR-cmk (25 µM) and aprotinin (100 µM) were added to the cells 20 min before adding bPA83/bPA63. After incubation, cells were lysed in 50 mM octyl-β-D-glucopyranoside (Amresco) in TBS supplemented with 1 mM CaCl2, 1 mM MgCl2, a protease inhibitor mixture set III, 1 mM phenylmethylsulfonyl fluoride (PMSF) and dec-RVKR-cmk (5 µM). To measure cell-associated bPA83 and bPA63, the samples were analyzed by Western blotting with horseradish peroxidase-conjugated ExtrAvidin and a TMB/M substrate (Chemicon). Where indicated, cells were washed for 3 min in 50 mM glycine-100 mM NaCl, pH 3.0, to remove cell surface-associated bPA83/bPA63. The samples were then neutralized using 500 mM Hepes-100 mM NaCl, pH 7.5 [37].
Cell surface biotinylation
Cells (15×106; 90% confluent) were washed twice with an ice-cold Soerensen Buffer (SBS), pH 7.8, containing 14.7 mm KH2PO4, 2 mm Na2HPO4, and 120 mm sorbitol, and then incubated for 10 min in ice-cold SBS. Cell surface-associated furin was biotinylated by incubating cells for 25 min on ice with SBS supplemented with membrane-impermeable EZ-Link NHS-LC-biotin (0.3 mg/ml). Excess biotin was removed by washing the cells in SBS. The residual amounts of biotin were quenched by incubating the cells for 10 min in SBS-100 mm glycine. Quenched cells were lysed in 50 mm N-octyl-β-d-glucopyranoside in 50 mM Tris-HCl, pH 7.4, supplemented with 150 mM NaCl, 1 mm CaCl2, 1 mm MgCl2, a proteinase inhibitor cocktail set III, 1 mm PMSF and 10 µM dec-RVKR-cmk. Biotin-labeled furin was precipitated from the cell lysates using streptavidin-agarose beads. The precipitated samples were analyzed by Western blotting with the MON-148 furin antibody followed by donkey anti-mouse IgG-conjugated with horseradish peroxidase and a SuperSignal West Dura Extended Duration Substrate kit (Pierce).
Peptide substrate cleavage
The cleavage reactions (200 µl each) were performed in 100 mm HEPES, pH 7.5, containing 1 mm CaCl2, 1 mm β-mercaptoethanol and 0.005% Brij-35 (for furin) and 10 mM Tris-HCl, 8.0 containing 20% glycerol (for WNV NS2B-NS3 proteinase). Pyr-RTKR-AMC (25 µM) was used as a substrate. The assays were performed in triplicate in wells of a 96-well plate. The steady-state rate of substrate hydrolysis was monitored continuously at λex = 360 nm and λem = 465 nm at 37°C using a fluorescence plate reader.
Cell viability assay
Cells (5×104) were grown in DMEM-10% FBS for 16 h in wells of a 96-well plate. After washing, the cells were incubated for 2–4 h in 100 mM Hepes, pH 7.5, supplemented with 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1% ITS. Cell viability was determined using an ATP-Lite kit (Perkin-Elmer). The resulting luminescence was measured using a plate reader (SpectroFluor Plus, Tecan). Each datum point represented the results of at least three independent experiments performed in triplicate.
Antibody uptake and immunofluorescence microscopy
Cells were seeded on 13 mm round glass coverslips and grown at 37°C until a 50% confluence. Cells were then washed in PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized for 4 min using 0.1% Triton X-100. Cells were blocked with 3% BSA and incubated for 16 h at 4°C with the primary antibodies (dilution 1∶1,000-1∶1,500) followed by an 1 h incubation with the secondary species–specific IgG conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes). The slides were mounted in the VectaShield anti-fading embedding medium (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole (DAPI) for the nuclear staining.
In the antibody uptake experiments, cells were incubated for 15 min at 4°C in the serum-free, L-15 medium supplemented with 1% ITS. Cells were next incubated for an additional 1 h at 4°C with the MON-148 furin antibody (10 µg/ml). After washing with ice-cold PBS, the cells were next transferred to 37°C for 1 h to stimulate the antibody uptake. Following fixation and permeabilization as above, the cells were stained for 1 h using the Alexa Fluor 594-conjugated secondary antibody, mounted in the VectaShield medium. Images were acquired using a ×400 original magnification on an Olympus BX51 fluorescence microscope equipped with an Olympus MagnaFire digital camera and MagnaFire 2.1C software.
Results
Biosensor design, expression and purification
According the observation by us and others, anthrax PA83 is one of the most sensitive cleavage targets of furin and related PCs [26], [32], [38], [39]. Because the substrate cleavage preferences overlap significantly between furin and other PCs, we refer to furin for simplicity in the text below.
The cleavage of PA83 and its conversion into the N-terminal PA20 and the C-terminal PA63 fragment is a result of the cleavage of the SNSRKKR↓STSAGP sequence by furin [21]. To design an ECFP-YPet biosensor which would be both sensitive to the cleavage by furin and suitable for the FRET-based monitoring of its activity, the C-terminus of YPet and the N-terminus of ECFP were linked by the SNSRKKR↓STSAGP sequence of PA83. To facilitate the isolation of the ECFP-YPet biosensor from the recombinant cells, the construct was N-terminally tagged with the FLAG and Hisx6 tags and expressed in E.coli. After induction with isopropyl β-D-thiogalactoside, the biosensor was produced as a soluble protein. After disruption of E.coli cells by sonication, the soluble protein fraction was loaded onto a Co2+-agarose affinity column. The biosensor protein was eluted with a gradient of imidazole concentrations (Fig. 1).
10.1371/journal.pone.0011305.g001Figure 1 The biosensor and its purification.
Left panel, the ECFP/YPet construct was N-terminally tagged with the Hisx6 and FLAG tags. The furin cleavage sequence (SNSRKKR↓STSAGP) of anthrax PA83 was inserted between the N-terminal Met of ECFP and the C-terminal Leu of YPet. Right panel, SDS-gel electrophoresis of the elution fractions of the biosensor purified by Co2+-chelating chromatography.
The biosensor is cleaved by furin
It is expected that furin would cleave the SNSRKKR↓STSAGP linker sequence and separate YPet and ECFP. These events will decrease FRET and, concomitantly, increase the emission ratio of ECFP/YPet. In agreement, following co-incubation of the biosensor with furin, a decrease in both FRET and the YPet emission was recorded. These events were concomitant with an increase in the ECFP emission. Both the concentration-dependent and time-course studies were consistent with the ratiometric and directly proportional response of the biosensor to furin proteolysis. The levels of furin as low as 10 fmol were sufficient to cause the measurable changes in the ECFP/YPet ratio (Fig. 2).
10.1371/journal.pone.0011305.g002Figure 2 Characterization of the biosensor.
Left panel, the emission spectra (λex = 437 nm) of the purified biosensor (100 pmol) before and after its cleavage for 1 h at 37°C using purified furin (10 fmol and 100 fmol). RFU, relative fluorescence unit. Middle panel, the time course of the ECFP/YPet emission ratio (476 nm/526 nm at λex = 437 nm) of the biosensor (100 pmol) incubated for 4 h at 37°C with or without furin (10 fmol and 100 fmol). Right panel, a ratiometric response of the normalized ECFP/YPet emission ratio to the increasing concentrations of furin. Incubation time, 1 h. These experiments were repeated multiple times with similar results. The representative experiments are shown.
We determined that the biosensor and PA83 were similarly sensitive to furin proteolysis (Fig. 3). There were no cleavage sites in the biosensor additional to the linker and, as a result, only the cleavage products that corresponded to the ECFP and YPet moieties were observed in the digest reactions. Similar to PA83, the biosensor was sensitive to the in vitro proteolysis by PACE4, PC1/3, PC2, PC4, PC5/6 and PC7 (data not shown). In turn, both PA83 and the biosensor were resistant to the proteolysis by WNV NS2B-NS3 proteinase regardless that the latter exhibits the furin-like, albeit less stringent, cleavage preferences [33], [40], [41]. In contrast, NS2B-NS3 proteinase and furin were similarly efficient in the cleavage of the fluorescent Pyr-RTKR-AMC peptide substrate (Fig. 4). From these perspectives, the biosensor appeared to be selective for furin and furin-like PCs.
10.1371/journal.pone.0011305.g003Figure 3 The biosensor is cleaved by furin but it is resistant to WNV NS2B-NS3 proteinase.
Left panels, the biosensor and PA83 were cleaved by furin (1 h; 37°C) at the indicated enzyme-substrate ratio. Right panel, the biosensor was cleaved by WNV NS2B-NS3 proteinase (1 h; 37°C) at the indicated enzyme-substrate ratio. The cleavage reactions were analyzed by SDS-gel electrophoresis followed by Coomassie staining.
10.1371/journal.pone.0011305.g004Figure 4 The similar activity of furin and WNV NS2B-NS3 proteinase against Pyr-RTKR-AMC.
Both furin and WNV NS2B-NS3 proteinase (0.2-4 pmol each) were allowed to cleave the fluorescent peptide for the indicated time. RFU, relative fluorescence unit.
The biosensor is activated by cellular furin
Because of its significant, 500-residue, size, the biosensor is incapable of penetrating the plasma membrane efficiently. As a result, we initially used the biosensor to assess cell surface-associated furin in fibrosarcoma HT1080, breast carcinoma MCF-7, and glioma TP98G, U373 and U251 cells, which naturally express different levels of furin and also in colon carcinoma LoVo cells. Because of the two frame-shift mutations in the furin gene, LoVo cells do not express functionally active furin [42]. We also used MCF-7 cells which were stably transfected with either the wild-type furin (MCF-7:furWT) or the catalytically inert furin mutant (MCF-7:furD153N) and LoVo cells with the reconstituted expression of the wild-type furin (LoVo:furWT).
The activity of cellular furin was readily recorded by using the biosensor. A short, 2-h incubation was sufficient for recording furin activity in MCF-7:furWT cells while 8–16 h were required in the cells which express furin naturally. Because of the expression of the catalytically inert furin, both MCF-7:furD153N and LoVo cells did not activate the biosensor. The naturally expressed furin activity was the most prominent in U251 and HT1080 cells (Fig. 5).
10.1371/journal.pone.0011305.g005Figure 5 Activation of the biosensor by cellular furin.
Adherent glioma TP98G, U373 and U251 cells, fibrosarcoma HT1080 cells, colon carcinoma LoVo cells and breast carcinoma MCF-7, MCF-7:furD153N and MCF-7:furWT cells (5×104) were co-incubated for 2–16 h with the biosensor (100 pmol).
Based on the ratiometric response curve that shows the normalized ECFP/YPet emission ratio of the biosensor co-incubated for 1 h with the increasing concentrations of purified furin (20–100 fmol) (Fig. 2) and on the data of Fig. 6 that show the normalized ECFP/YPet emission ratio of the biosensor co-incubated for 0.5–4 h with 5×104 MCF-7:furWT cells, it is possible to estimate the number of active furin molecules per cell. Thus, the net activity of furin in 5×104 MCF-7:furWT cells roughly corresponded to 10 fmol (∼0.6 ng) furin or ∼100,000 furin molecules/cell. The level of furin in U251 cells was several-fold lower (Fig. 5). The electrophoretic analysis confirmed the specific cleavage of the biosensor by MCF-7:furWT and U251 cells. The predominant cleavage products correlated with the expected ECFP and YPet moieties (Fig. 6).
10.1371/journal.pone.0011305.g006Figure 6 Activation and cleavage of the biosensor by cellular furin.
Left panel, the time course of the biosensor cleavage by U251, MCF-7:furWT and MCF-7:fur:D153N cells (5×104). Right panel, SDS-gel electrophoresis of the cleavage reactions. Cells (5×104) were incubated for 4 h with the biosensor (100 pmol). After centrifugation, the supernatant samples were separated by SDS-gel electrophoresis followed by Coomassie staining. Purified furin (10 fmol and 100 fmol) was used as a control.
Low levels of cell surface furin
Western blotting analysis demonstrated that it was difficult to unambiguously detect furin in cell lysates unless the cells with the enforced expression of furin were used (Fig. 7). To determine the levels of cell surface-associated furin, cells were surface-biotinylated using membrane-impermeable biotin. Biotin-labeled proteins were precipitated using the streptavidin-agarose beads. The resulting samples were analyzed by Western blotting with the MON-148 furin antibody. Surprisingly, despite high amounts of the loaded protein material, which corresponded to 12×106cells/lane, exceedingly low levels of furin were detected in the biotin-labeled MCF-7:furWT cell samples (Fig. 7).
10.1371/journal.pone.0011305.g007Figure 7 Analysis of cellular furin.
Left panel, Western blotting of total cell furin (15 µg total protein that corresponded to 5-7×104 cells depending on a cell type). Right panel, Western blotting of cell surface-associated furin. MCF-7 and MCF-7:furWT cells (15×106) were cell surface biotinylated using membrane-impermeable biotin. Biotin-labeled furin was immunoprecipitated from the total cell lysate (TCL) using streptavidin-agarose beads. The beads were washed in 50 mM Tris-HCl, pH 7.4, supplemented with 50 mm N-octyl-β-d-glucopyranoside, 150 mM NaCl, 1 mm CaCl2, 1 mm MgCl2, a proteinase inhibitor cocktail set III, and 1 mm PMSF (FT, flow through fraction). The immunocaptured proteins (IP, immunoprecipitated protein fraction) were eluted using 1% SDS. The fractions were analyzed by Western blotting with the furin MON-148 antibody followed by donkey anti-mouse IgG-conjugated with horseradish peroxidase and a SuperSignal West Dura Extended Duration Substrate kit. The gels were overexposed to demonstrate the presence of cell-surface furin. Right lane, purified furin (1 ng). WB, Western blotting.
To corroborate these data, we next used the furin antibody uptake. For these purposes, U251 cells and MCF-7:furWT, which express low and high level of furin, respectively, were allowed to bind the MON-148 antibody for 1 h at 4°C. After washings to remove the unbound antibody, the cells were moved to 37°C to stimulate the internalization of the cell surface-associated furin-antibody complex. The cells were next fixed, permeabilized and stained with a secondary antibody to determine the sub-cellular localization of the furin-antibody complex. We, however, did not detect any significant immunoreactivity, thus, suggesting that there was no detectable level of furin on the cell surface (Fig. 8A).
10.1371/journal.pone.0011305.g008Figure 8 Furin is expressed in the trans-Golgi network in MCF-7:furWT cells.
A, left panels, the uptake of the MON-148 furin antibody did not reveal cell surface furin in U251 and MCF-7:furWT cells. Cells were allowed to bind the antibody at 4°C. Cells were then incubated at 37°C to stimulate the antibody uptake, fixed, permeabilized and stained with the Alexa Fluor 594-conjugated secondary antibody (red). Right panels, staining of cellular furin. Cells were fixed, permeabilized and stained using the MON-148 antibody followed by the Alexa Fluor 594-conjugated secondary antibody. B, MCF-7:furWT and MCF-7 cells were fixed, permeabilized and stained using the rabbit polyclonal TGN46 antibody (green) and the MON-148 furin antibody (red). The merged panels show the level of co-localization of furin with TGN46 (a trans-Golgi network marker). Original magnification ×400; the bar, 10 µm. The nuclei were stained with DAPI (blue).
We next use direct immunostaining of the permeabilized MCF-7:furWT and MCF-7 (control) cells with the MON-148 antibody. The presence of the intracellular furin was readily recorded in MCF-7:furWT cells. Co-staining of MCF-7:furWT cells using the MON-148 furin antibody and the TGN46 antibody (a trans-Golgi network marker) demonstrated the presence of furin in the trans-Golgi network and in the intracellular vesicles in the permeabilized cells (Fig. 8B). The staining of MCF-7 cells was clearly negative. Non-permeabilized cells also did not show any furin immunoreactivity in the intracellular compartment and at the cell surface (not shown). Overall, the significant levels of cell surface-furin we detected using the biosensor did not correlate with the results of our other studies.
Proteinases distinct from furin do not cleave PA83
Based on our data, we tested if cellular serine proteinases with PC-like specificity, but distinct from PCs, contributed to the cleavage of both PA83. To exclude this possibility, we used aprotinin, a potent inhibitor of trypsin-like proteinases, in the in vitro and cell-based cleavage tests. Even exceedingly high levels (at a 1∶1000 enzyme-inhibitor molar ratio) of aprotinin did not affect the PA83-converting activity of furin in the cleavage reactions in vitro. In contrast, furin activity was fully repressed by its specific inhibitor, dec-RVKR-cmk (ki = 1 nM), at a low, 1∶20, enzyme-inhibitor molar ratio. Consistent with its inhibitory specificity, aprotinin (a nanomolar range inhibitor of WNV NS2B-NS3 proteinase; ki = 26 nM) [33], [43] blocked this proteinase activity at a low, 1∶20, enzyme-inhibitor molar ratio. Because PA83 is resistant to the viral proteinase, the activity of the latter was determined using myelin basic protein as a substrate (Fig. 9) [33].
10.1371/journal.pone.0011305.g009Figure 9 Aprotinin inhibits WNV NS2B-NS3 proteinase activity but not furin.
PA83 (1 µM) and myelin basic protein (MBP; 11 µM) were incubated for 1 h at 37°C with furin (1–10 nM; 1∶100-1∶1,000 enzyme-substrate molar ratio) and WNV NS2B-NS3 proteinase (1.25 µM; 1∶10 enzyme-substrate molar ratio) in the presence of the indicated enzyme-inhibitor molar ratio.
We next examined if aprotinin and dec-RVKR-cmk affected the proteolytic processing of PA83 in U251 and LoVo:furWT cells. For this purposes, bPA83 was co-incubated with either the intact cells or with the cells co-incubated with aprotinin or dec-RVKR-cmk. The amounts of cell-associated bPA83 and bPA63 were determined by Western blotting. The results showed intact U251 and LoVo:furWT cells readily processed the external bPA83. Because of the binding to the anthrax toxin receptor, bPA63 and the residual amounts of intact bPA83 were detected in cell extracts. Dec-RVKR-cmk (25 µM) caused a near complete inhibition of bPA83 in U251 cells. In turn, aprotinin (100 µM) did not show any effect (Fig. 10A). As a result, we concluded that cell surface-associated proteinases distinct from furin-like PCs, did not significantly contribute to the processing of PA83. Because cell-surface levels of furin are exceedingly low (Fig. 7 and 8), these data also suggest that PA83 is cleaved by furin in the extracellular milieu but not on the cell surface, and that the resulting PA63 would be capable of binding with the cells. Thus, our earlier data directly indicate that the levels of furin in fibrosarcoma HT1080 and glioma U251 cells are sufficient to sustain efficient anthrax toxin intoxication [44].
10.1371/journal.pone.0011305.g010Figure 10 Specific processing of PA83 by cellular furin.
A, LoVo:furWT and U251 cells were co-incubated for 3 h at 37°C with bPA83 (1 µg/ml). Where indicated, cells were pre-incubated for 20 min with dec-RVKR-cmk (25 µM) or aprotinin (100 µM) prior to the addition of bPA83. Cell lysates were examined using Western blotting with horseradish peroxidase-conjugated ExtrAvidin and a TMB/M substrate. B, left panel, U251 cells were incubated for 3 h at 37°C with bPA83 (1 µg/ml) with or without dec-RVKR-cmk (25 µM). Where indicated, cells were exposed to the acid pH treatment to remove the cell surface-associated bPA83 and bPA63. Right panel, conversion of bPA83 into bPA63 using purified furin. The gels were stained with Coomassie. WB, Western blotting. NS, non-specific band.
Both PA83 and PA63 bind the anthrax toxin receptor
To test this suggestion, U251 cells were allowed to bind the equal amounts of bPA83 and of the pre-made bPA63. To generate bPA63, bPA83 was fully processed in the in vitro cleavage reactions using the purified furin (Fig. 10B). The levels of the uptake of bPA83 and bPA63 by the cells were determined using Western blotting of the cell lysates. bPA83 and bPA63 were equally efficiently internalized by the cells. Dec-RVKR-cmk inhibited the processing and uptake of bPA83 by the cells. The inhibitor did not affect the processing and uptake of bPA63. Acid treatment of the cells demonstrated the efficient removal of the cell surface-bound bPA83 while there was no similar effect with bPA63 (Fig. 10B). Taken together, these results indicated that bPA83 was not processed at the cell surface in our cell system. Conversely, these results suggested that bPA83 was processed in the extracellular milieu and that only then the generated bPA63 associated with the anthrax toxin receptor in the cells. These parameters suggested the intracellular furin pool but not the cell surface-associated furin contributed to the measurements in our biosensor cleavage tests.
Intracellular furin pool interfered with the biosensor cleavage
To test if our suggestion was correct, we incubated the biosensor with the adherent MCF-7:furWT and U251 cells. We then determined the normalized ECFP/YPet emission ratio in the supernatant samples (Fig. 11A). In addition, we incubated the cells alone in the assay buffer. We then tested if the supernatant fraction that contained the released cellular proteins was capable of cleaving the biosensor. These tests suggested that the efficiency of the biosensor cleavage by the adherent cells and by the soluble proteins released by the cells was very similar. It appeared that there was a significant release of intracellular furin and, potentially, additional PCs by the cells because the cells did not survive well under our experimental conditions. In agreement, we detected a significant level of apoptosis in the cells. Cell viability tests revealed that 30–35% cells became apoptotic in the course of a short, 4-h, incubation time and that the cell realizate rather than cell surface-associated furin alone contributed to the biosensor cleavage (Fig. 11B). On the other hand, these results also suggested that the cell-impermeable biosensor can be efficiently used to quantify the total cell furin in the cell lysate samples rather than cell surface-associated furin alone using the intact cells.
10.1371/journal.pone.0011305.g011Figure 11 Furin activity was released by the cells.
A, the time course of the biosensor cleavage by cell realizate and by adherent MCF-7:furWT and U251 cells. Cells (5×104) were incubated for 2 h in 100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1% ITS. The cells were then separated by centrifugation and the supernatant (realizate) was co-incubated with the biosensor for 0–120 min. Alternatively, adherent cells (5×104) were directly co-incubated with the biosensor. B, ATP-Lite cell viability assay. Prior to the assay, MCF-7, MCF-7:furWT and U251 cells were incubated for 2–4 h at 37°C in 100 mM Hepes, pH 7.5, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 1% ITS. The level of induced apoptosis was then determined using an ATP-Lite kit.
The biosensor quantifies total cellular furin in cancer cell lysates
Normally, detergents, including Triton X-100, are required for disrupting the cell membrane and for cell protein solubilization. First, we confirmed that the presence of 0.1% Triton in the reactions did not affect the efficiency of the biosensor cleavage by purified furin as determined by both the measurement of the ECFP/YPet ratio and the SDS-gel electrophoresis of the digest samples and (Fig. 12).
10.1371/journal.pone.0011305.g012Figure 12 The biosensor cleavage in cell lysates.
Left panel, Triton X-100 (0.1%) does not affect the efficiency of furin proteolysis of the biosensor. The biosensor was co-incubated with purified furin (5 fmol and 50 fmol) for 0-120 min with or without 0.1% Triton X-100. Right panel, the biosensor was co-incubated 1 h with purified furin at the indicated enzyme-substrate molar ratio. The digests were analyzed by SDS-gel electrophoresis followed by Coomassie staining. Where indicated, reactions contained 0.1% Triton X-100.
We then prepared the total lysates of MCF-7, MCF-7:furWT, MCF-7:furD153N, LoVo, LoVo:furWT, U251 and U251/PDX cells using 0.1% Triton X-100. Following centrifugation to remove the insoluble material, the supernatant aliquots were directly used to cleave the biosensor. Dec-RVKR-cmk was used to inhibit furin in the cleavage reactions. The lysates of MCF-7, MCF-7:furD153N, LoVo and U251/PDX cells did not cleave the biosensor. In contrast, the biosensor cleavage was readily recorded in MCF-7:furWT, LoVo:furWT and U251 cells. Dec-RVKR-cmk fully suppressed the cleavage of the biosensor in these cells (Fig. 13). According to the calibration curve with purified furin (Fig. 2) and the cleavage data using cell lysates, the net levels of furin were 109 fmol, 43 fmol and 24 fmol in MCF-7:furWT, LoVo:furWT and U251 cells, respectively. It is, however, probable that other PCs also contributed to the biosensor cleavage, especially in U251 cells.
10.1371/journal.pone.0011305.g013Figure 13 The biosensor allows to measure reliably furin activity in cell lysates.
The time course of the biosensor cleavage by the MCF-7, LoVo and U251 total cell lysates. The cells were lysed for 1 h at 4°C in the buffer containing 0.1% Triton X-100. The insoluble material was discarded by centrifugation. The supernatant aliquots (50 µg total protein; an equivalent of ∼5×104 cells) were co-incubated for 2 h at 37°C with the biosensor (100 pmol).
In contrast with the biosensor, the fluorescent Pyr-RTKR-AMC peptide substrate cannot be employed with the crude cell samples. Indeed, when Pyr-RTKR-AMC was used, the lysates of U251 and U251/PDX cells were similarly efficient in cleaving Pyr-RTKR-AMC despite the drastically different levels of their furin activity. Similarly, there was no significant difference between of the MCF-7, MCF-7:furWT and MCF-7:furD153N samples if Pyr-RTKR-AMC was used (Fig. 14A).
10.1371/journal.pone.0011305.g014Figure 14 The fluorescent peptide substrate does not allow to measure reliably furin activity in cell lysates.
A, the cleavage of Pyr-RTKR-AMC by the supernatant aliquots (50 µg total protein for MCF-7 and LoVo cells and 5 µg total protein for U251 cells). RFU, relative fluorescence unit. B, the biosensor (100 pmol) was co-incubated for 2 h with the total cell lysates or with the purified furin (100 fmol). The digests were analyzed by Western blotting with the GFP antibody. Where indicated, dec-RVKR-cmk was added to the reactions. WB, Western blotting.
To confirm further that other cellular proteinases which are distinct from PCs did not significantly contribute to the biosensor cleavage, the latter was co-incubated with the totals cell lysates of MCF-7:furWT, LoVo:furWT and U251 cells. The digest samples were then analyzed using Western blotting with a GFP antibody (Fig. 14B). The data confirmed the specific cleavage of the biosensor by the MCF-7:furWT, LoVo:furWT and U231 samples. Dec-RVKR-cmk fully repressed the cleavage. Other cell types did not cleave the biosensor efficiently suggesting that cellular proteinases distinct from furin-like PCs did not contribute significantly to the biosensor cleavage.
Discussion
Multiple physiologically-relevant proteins are synthesized as latent precursors [14], [45]. These precursors are transformed into active proteins by the cleavage action of furin and related PCs [16]. Furin is also implicated in the processing of many pathogens including anthrax [16], [21], [26], [38], [39]. Furin itself is self-activated [21], and upon activation it cleaves de novo synthesized latent precursors in the Golgi compartment and in the secretory vesicles. It was believed that some proportion of the furin molecules cycles between the trans-Golgi compartment and the cell surface, albeit the presence of cell-surface furin was never convincingly demonstrated [46]. Because of the overlapping substrate preferences, there is a redundancy in the PC functionality, albeit distinctive functions of furin have also been reported [47].
Evidence suggests that in multiple cancer types furin overexpression causes an imbalance in the activation of invasion- and proliferation-related cellular substrates leading to acquisition of an advanced malignant phenotype [48], [49], [50], [51], [52]. The multiple effects of furin on cell function have led to a concept that in the course of tumor development and progression furin acts as “a master switch” of the tumorigenic protein functionality. If this concept is valid, furin then could be identified as an important therapeutic target in a number of cancer types. The sensitive and selective read-out technology to reliably monitor furin activity in cells and tissues, however, is not currently available. The absence of a reliable read-out does not allow correlating furin activity with disease progression. It also remains unclear how the activity of furin is coordinated in space and time in the cell compartment.
To shed more light on the furin functionality, we designed and tested a cleavage-activated biosensor to monitor the net activity of furin. The biosensor represents a chimeric protein in which an ECFP and YPet FRET pair is linked by a peptide linker sequence derived from anthrax PA83. We specifically selected this sequence as a linker because the SNSRKKR↓STSAGP furin cleavage site of PA83 is supremely sensitive and selective to the proteolysis by furin and related PCs with the redundant cleavage preferences [26], [32].
The use of the biosensor in a combination with other methodologies have allowed us to determine that the levels of cell surface-associated furin are exceedingly low and that they represent a diminutive fraction of the total cell furin. The detected minuscule cell-surface furin levels are grossly insufficient to accomplish the cell-surface processing of anthrax PA83. Our data directly suggest that, at least in the cell-based systems, PA83 is processed by furin extracellularly. This processing is followed by the binding of the resulting PA63 heptamer or the mixed heptamer that includes both the PA63 and the PA83 subunits to the anthrax receptors [22], [24], [29]. Our data shift a long-term paradigm in the furin biology and anthrax infection [53]. In addition, these novel results provide an opportunity for a design of the specific cell-impermeable furin inhibitors of anthrax. These inhibitors would not interfere with normal cellular function of furin and related PCs in the trans-Golgi compartment [39].
It is now tempting to hypothesize that the release of the intracellular furin pool and, probably, other PCs from the already infected, dying cells is cleverly used by anthrax to support the further avalanche-like propagation of infection. The use of the released PCs instead of cell-surface furin eliminates the spatial constraints that limit the interactions between the membrane-tethered PA83-anthrax receptor complexes and furin at the cell surface.
Our tests demonstrated that the biosensor we designed allowed us to selectively record the cleavage activity using as low as the femtomol levels of furin. This high level of selectivity and sensitivity of the biosensor allowed us to detect furin in the tumor cell lysate samples using as low as thousands of cells, a task that is not possible to accomplish reliably with conventional fluorescent peptide substrates. Because serine proteinases distinct from PCs destroy the biosensor rather than specifically cleave the linker sequence, they do not significantly interfere with the specific action by PCs. The availability of the highly sensitive and selective furin biosensor provides a foundation for the rapid, non-invasive procedures to monitor furin and related proteinases in cancer cells and tumor biopsies. Because furin activity is also important for multiple infectious diseases caused by bacterial and viral pathogens, we suggest that the furin biosensor is applicable for monitoring the activity of furin in a wide range of cells and tissues and in a variety of disease conditions rather than in cancer alone.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The work reported here was supported by National Institutes of Health Grants CA83017 and CA77470 (to AYS), and CA139272 (to YW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC PhysiolBMC Physiology1472-6793BioMed Central 1472-6793-10-72046240210.1186/1472-6793-10-7Research articleThe effect of marathon on mRNA expression of anti-apoptotic and pro-apoptotic proteins and sirtuins family in male recreational long-distance runners Marfe Gabriella [email protected] Marco [email protected] Bruna [email protected] Stefano Carla [email protected] Manuela [email protected] Angela [email protected] Matteo Antonio [email protected] Paola [email protected] Vincenzo [email protected] Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata" Via Montpellier 1, 00133, Rome, Italy2 Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, Via dei Bonacolsi snc, 00163, Rome, Italy3 Human Nutrition Unit, University of Rome "Tor Vergata" Via Montpellier 1, 00133, Rome, Italy4 Department of Experimental Medicine, La Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy5 School of Sport and Exercise Sciences, University of Rome Tor Vergata" Via Columbia s.n.c. 00133 Rome, Italy2010 12 5 2010 10 7 7 20 7 2009 12 5 2010 Copyright ©2010 Marfe et al; licensee BioMed Central Ltd.2010Marfe et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circulating human lymphocytes leading to lipid peroxidation, DNA damage, mitochondrial perturbations, and protein oxidation.
In our research, we investigated the effect of physical load on the extent of apoptosis in primary cells derived from blood samples of sixteen healthy amateur runners after marathon (a.m.).
Results
Blood samples were collected from ten healthy amateur runners peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bcl-2, bax, heat shock protein (HSP)70, Cu-Zn superoxide dismutase (SOD), Mn-SOD, inducible nitric oxide synthase (i-NOS), SIRT1, SIRT3 and SIRT4 (Sirtuins) RNA levels were determined by Northern Blot analysis. Strenuous physical load significantly increased HSP70, HSP32, Mn-SOD, Cu-Zn SOD, iNOS, GADD45, bcl-2, forkhead box O (FOXO3A) and SIRT1 expression after the marathon, while decreasing bax, SIRT3 and SIRT4 expression (P < 0.0001).
Conclusion
These data suggest that the physiological load imposed in amateur runners during marathon attenuates the extent of apoptosis and may interfere with sirtuin expression.
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Background
Apoptosis, or programmed cell death, is a normal physiological function essential for the homeostasis of immuno haemopoietic tissues. This process occurs via specific signaling pathways, eventually leading to DNA fragmentation, nuclear condensation, proteolysis and cell fragmentation [1]. An important regulatory event in the apoptotic process is the activation of caspases, a family of cysteine proteases, which regulate two major and relatively distinct pathways, the extrinsic and intrinsic pathways. The extrinsic pathway known also as mitochondrial pathway involves pro- and anti-apoptotic members of the bcl-2 family of proteins [2,3].
The initiation of apoptosis is dependent on a variety of signals, many of which can be modulated by strenuous exercise [4-7]. Consequently, it has been suggested that apoptosis contributes to the loss of blood lymphocytes after exercise possibly via the cell surface death receptor CD95 (Fas/Apo-1) signaling [8-10], resulting in post-exercise lymphocytopenia, which could lead to lowered immunity in athletes performing frequent and physically demanding training regimens. Studies that have examined the effects of exercise on the extent of apoptosis in blood lymphocyte in humans are few, thus making it difficult to draw any definitive conclusions [8-12].
Exercise increases oxygen consumption and causes a disturbance of intracellular pro-oxidant-antioxidant homeostasis [13]. The mitochondrial electron transport chain [14], and xanthine oxidase [15] have been identified as major sources of intracellular reactive oxygen species (ROS) and free radical generation during exercise [15]. Two recent studies have proven that ROS accumulation readily modifies the activity of a new class of proteins called sirtuins [16,17].
In humans the sirtuins family of proteins is composed of seven members (SIRT1 through 7) that share the catalytic domain with Sir2 [18,19]. In particular, SIRT1 can modulate cellular stress response and survival through regulation of p53 [20-22], NF-kB signaling [23] and FOXO transcription factors [24,25]. Several studies showed that SIRT1 is a key regulator of cellular metabolism [26] and survival in response to external stressors [27]. Furthermore, since skeletal muscle increased levels of oxidative damage with aging [28,29], regular exercise is very useful in increase its antioxidant potential that can be modulated by the activity of SIRT1 [28-31]. Recent studies have reported that others sirtuins, such as SIRT3, SIRT4 and SIRT5, are located in the mitochondria [32,33]. In particular, it has been demonstrated that the mitochondrial NAD-dependent deacetylase SIRT3 plays a role in the maintenance of basal ATP levels and as regulator of mitochondrial electron transport [34]. In fact, SIRT3 decreases mitochondrial membrane potential and reactive oxygen species production, while increasing cellular respiration [35].
The present investigation has been designed to test the effect of an endurance effort, typical in runners participating in standard (42 km) marathon events, on apoptotic cell status in a controlled laboratory setting. Furthermore, we have studied the effect of the same endurance effort on sirtuin proteins in order to understand their role in this kind of exercise.
Results
Marathon race
All subjects successfully completed a marathon (n = 10). Total TRaining IMPulse (TRIMP) score accumulated over the marathon race averaged 523 ± 67 arbitrary unit, equivalent to exercise at 80% of heart rate reserve for 200 to ~220 min.
Exercise and bcl-2 pathway apoptotic signaling
We determined the effects of exercise on bcl-2 family upstream of caspase-3, including caspase-9 and bax: bcl-2 ratio. Figure 1 shows the same procaspase-9 RNA levels in the group before and after marathon (b.m. and a.m.). RNA expression of proapoptotic bax in the group showed a significant decrease after marathon (Figure 2B). By contrast, the antiapoptotic bcl-2 RNA levels increased in the group after marathon (Figure 2B) (Table 1). Thus, we calculated bax: bcl-2 ratio in the same group before and after marathon and such ratio was attenuated after marathon (Figure 2B, C).
Figure 1 Effects of marathon on procaspase-9 mRNA expression. Procaspase-9 mRNA content was estimated by Northern Blot. (A) RNA was extracted, electrophoresed and hybridized with a labelled probe as described under Methods. β-actin was used as loading control. Blot is representative of three separate experiments. (P < 0.0001)
Figure 2 Effects of marathon on bax and bcl-2 mRNA expression. A) bax and bcl-2 RNA content was estimated by Northern Blot. RNA was extracted, electrophoresed and hybridized with a labelled probe for bax and bcl-2 as described under Methods. β-actin was used as loading control. Blots are representative of at least three separate experiments. (P < 0.0001). B) bax: bcl-2 ratio in samples before marathon and after marathon was obtained by densitometry analysis of the blots shown in A. C) Change bax: bcl-2 ratio in samples before and after marathon.
Table 1 Changes of different genes expression before marathon and after marathon.
Before marathon After marathon Mean difference 95% CI Lower - Upper Magnitude of the difference
Mean ± SD Mean ± SD Effect size Interpretation
bax 3.35 ± 0.91 0.60 ± 0.21 -2.75* -3.43 to -2.07 0.90 large
bcl-2 0.91 ± 0.21 3.25 ± 1.17 2.34* 1.56 to 3.12 0.83 large
Cu-Zn SOD 0.69 ± 0.25 3.50 ± 0.74 2.81* 2.25 to 3.37 0.93 large
HSP70 1.27 ± 0.53 3.65 ± 1.31 2.38* 1.73 to 3.02 0.88 large
iNOS 0.95 ± 0.08 4.10 ± 0.66 3.15* 2.67 to 3.63 0.96 large
Mn-SOD 0.52 ± 0.30 5.20 ± 1.11 4.68* 3.73 to 5.63 0.93 large
SIRT1 1.12 ± 0.28 3.60 ± 0.69 2.48* 1.90 to 3.06 0.91 large
SIRT3 0.97 ± 0.26 0.18 ± 0.11 -0.79* -0.98 to -0.59 0.90 large
SIRT4 1.11 ± 0.25 0.20 ± 0.13 -0.91* -1.14 to -0.68 0.90 large
Ratio bax: bcl-2 2.10 ± 0.19 0.87 ± 0.27 -1.23* -1.43 to -1.03 0.96 large
* = Significant difference between before and after marathon (P < 0.0001); 95% CI = 95% confidence intervals of the difference
DNA laddering
Representative DNA fragmentation is shown in Figure 3. A laddering pattern was observed in DNA isolated from samples in which the ratio bax: bcl-2 after marathon was slightly higher than the others. By contrast, we did not observe any apparent laddering pattern in the DNA isolated from those samples in which the ratio bax: bcl-2 was lower than the others.
Figure 3 DNA laddering. Samples 2, 3, 6, 7 and 10 from Figure 2 were collected before and after marathon and processed for DNA fragmentation as described under Methods.
HSP70, HSP32, Cu-Zn-SOD, and Mn-SOD, i-NOS levels
Northern blot analysis demonstrated that the RNA content of HSP70, HSP32 increased substantially in the studied group after marathon (Figure 4). Similarly, the RNA content of Cu-Zn-SOD, Mn-SOD and i-NOS increased after marathon (Figure 4). We have found that the change in HSP70 RNA expression is positively correlated to the change of both Mn-SOD (r = 0.93; P = 0.001; 95% CI: 0.98 to 0.73; ES 0.71) and bcl-2 transcripts (r = 0.84; P = 0.002; 95% CI: 0.96 to 0.45; ES 0.2) after marathon (Fig. 5). In addition, we have also found that the change in HSP32 RNA expression is positively correlated to the change of bcl-2 transcripts (r = 0.82; P = 0.003; 95% CI: 0.96 to 0.41; ES 0.2) (Figure 5).
Figure 4 Effects of marathon on the Cu-Zn-SOD, Mn-SOD, i-NOS, HSP70 and HSP32 mRNA expression. Cu-Zn-SOD, Mn-SOD, i-NOS, HSP70 and HSP32 RNA levels were estimated by Northern Blot. RNA was extracted, electrophoresed and hybridized with a labelled probe as described under Methods β-actin was used as loading control. Blots are representative of at least three separate experiments (P < 0.0001)
Figure 5 Relationships between change in HSP70, HSP32, Mn-SOD and bcl-2 RNA expression after marathon. The HSP70 content is positively correlated to both Mn-SOD and bcl-2 transcript contents after marathon. (P < 0.0001)
SIRT1, SIRT3 and SIRT4 levels
Northern blot analysis demonstrated that the RNA contents of SIRT1 increased substantially in the group after marathon (Figure 6). On the other hand, the RNA contents of SIRT3 and SIRT4 decreased in the group after marathon (Figure 6). Furthermore, we also found a significant positive correlation between a change in SIRT3 RNA levels and TRIMP (r = 0.76; P = 0.03; 95% CI: 0.94 to 0.11; ES 0.99) after marathon (Table 2). A trend of correlation was detected between the TRIMP and change in SIRT4 RNA levels after marathon (r = 0.68, P = 0.06).
Table 2 Pearson correlation (r) between TRIMP, SIRT3 and GADD45
SIRT3 GADD45
r CI (95%) Upper - Lower Effect size r CI (95%) Upper - Lower Effect size
TRIMP 0.76* (0.94 to 0.11) 0.99 0.79** (0.95 to 0.32) 0.99
Abbreviations: TRIMP, Training Impulse; *P < 0.03; **P < 0.006
Figure 6 Effects of marathon on SIRT1, SIRT3 and SIRT4 in mRNA expression. SIRT1, SIRT3 and SIRT4 RNA levels were estimated by Northern Blot. RNA was extracted, electrophoresed and hybridized with a labelled probe as described under Methods β-actin was used as loading control. Blots are representative of at least three separate experiments. (P < 0.0001)
In addition, Northern blot analysis showed that RNA levels of the SIRT1-dependent transcription factor FOXO3A and its target GADD45 were increased in the studied group after marathon (Figure 7). In addition, we found also a significant positive correlation between a change in GADD45 RNA levels and TRIMP (r = 0.79; P = 0.006; 95% CI: 0.94 to 0.32; ES 0.98) after marathon (Table 2).
Figure 7 Effects of marathon on the GADD45 and FOXO3A in mRNA expression. GADD45 and FOXO3A RNA levels were estimated by Northern Blot. RNA was extracted, electrophoresed and hybridized with a labelled probe as described under Methods β-actin was used as loading control. Blots are representative of at least three separate experiments. (P < 0.0001)
Discussion
The present study provides evidence that strenuous physical load attenuates the extent of apoptosis in amateur runners. In particular, we found that both HSP70 and HSP32 RNA expression is positively correlated to bcl-2 transcript content after marathon (Figure 5). We can suppose that HSP70 and HSP32 may play an anti-apoptotic role in modulating the homeostasis of apoptotic factors in amateur runners after exercise. A critical step in the execution of the apoptotic program is cleavage of caspases [36]. The RNA levels of procaspase-9 were not changed in the amateur runners before and after marathon. Combined with DNA fragmentation data, this is the first direct evidence that strenuous physical load endured during marathon provides a physiological protection against the proapoptotic process.
It has also been suggested that ROS production influences apoptosis mainly through the modulation of the mitochondrial mediated pathway [37]. It has been hypothesized that a high oxidative stress level destabilizes the mitochondrial membrane homeostasis and therefore induces the formation of mitochondrial membrane permeability pores releasing pro-apoptotic factors (e.g., cytochrome c). Bcl-2 family proteins are known to be responsible for the modulation of mitochondrial membrane pore formation and therefore regulating mitochondrial-mediated apoptosis. We have shown that RNA levels of proapoptotic bax were consistently decreased after marathon, whereas, RNA levels of antiapoptotic bcl-2 were slightly increased after marathon thereby causing a decreased bax: bcl-2 ratio (Figure 2B, C). Our findings are consistent with the hypothesis that alterations in bax: bcl-2 ratio with exercise regulate downstream caspase-driven apoptosis during the exercise. Furthermore, Ferrer and colleagues (2009) [38] reported a decreased expression of bcl-2 after intense exercise versus the expression obtained previous exercise. The differences with our data could be attributed to differences between both experiments as the type of exercise, duration of the tests, time after exercise of sampling, differences between the moment in which the sampling was made before exercise. We have examined the possibility that the age range and the differences in VO2 max of our subjects could influence the apoptotic parameters considered. However, we did not find any significant correlation between differences in age or VO2 max and all the molecular parameters measured (data not shown).
Several antioxidant enzymes, including Cu-Zn-SOD, catalase, glutathione peroxidase, glutathione reductase, and mitochondrial Mn-SOD have been implicated as crucial endogenous antioxidant enzymes in biological systems. In the present study, we have demonstrated that the RNA content of Mn-SOD increases after marathon (Figure 4). These observations suggest that the answer of the cell may attenuate apoptosis. Our data are consistent with the idea that an increased antioxidant capacity and modulated oxidative stress from strenuous physical load may be involved in reducing pro-apoptotic genes.
HSPs are a group of highly conserved proteins induced by a variety of stresses, including hyperthermia, pH disturbance, and oxidative stress. There is evidence supporting the hypothesis that HSP70 inhibits apoptosis by modulating the mitochondrial-mediated pathway [39-41]. Li and colleagues (2000) [40] reported that HSP70 inhibits apoptosis by suppressing the formation of apoptosomes due to an effect downstream of cytochrome c release and upstream of caspase-3 activation. Beere and colleagues (2000) [39] have shown that HSP70 inhibits apoptosis by preventing the recruitment of procaspase-9 to the apoptosome. Furthermore, Fehrenbach et al. (2003) [42,43] have shown that the protective functions of HSPs include antioxidative and antiapoptotic effects and may prevent damage to DNA. Here, we have found a significant relationship between HSP70 and bcl-2 RNA (Figure 5) levels following marathon, but the underlying cellular and molecular mechanisms involved in this exercise induced adaptations in apoptosis and HSP70 are unknown and require further investigation.
Furthermore, in the present study we demonstrate, for the first time, that SIRT1 mRNA expression increases after marathon (Figure 6). It is therefore very likely that increased SIRT1 expression by endurance exercise results in elevated SIRT1 deacetylase activity as well as causing an allosteric effect of an increased cytosolic NAD+-to-NADH ratio.
In addition, an increase in SIRT1 mRNA levels could exert an antioxidant effect. Brunet et al. [34] demonstrated that in mammalian cells SIRT1 appears to control the cellular response to stress by regulating FOXO transcription factors that function as sensors of the insulin signaling pathway and as regulators of longevity. In particular, these authors showed that SIRT1 and FOXO3A form a complex in response to oxidative stress stimulus [24]. Mammalian FOXO factors control several biological functions, such as cell cycle arrest, detoxification of ROS [44] and repair of damaged DNA [45]. SIRT1 increased FOXO3A ability to induce cell cycle arrest and enhanced expression of a FOXO3A target involved in DNA repair, such as GADD45 [24]. Because it is known that exercise training exerts its beneficial effects particularly on the cardiovascular system, we tested FOXO3A and its targets involvement in group, showing that exercise training enhanced FOXO3A RNA levels. This was associated with an increase in GADD45 mRNAs after marathon. This finding could be related to higher oxidative stress in samples that would induce to choose apoptosis or necrosis rather than repair as mechanism of detoxification.
Furthermore, we found that SIRT3 and SIRT4 RNA levels decreased after marathon and also that there was a positive correlation between SIRT3 RNA levels and training load (Table 2) [38]. A trend of correlation was also detected between the TRIMP and change in SIRT4 RNA levels after marathon, however this trend was not statistically significant (P = 0.06). These two sirtuin proteins are known to localize in the mitochondria; although SIRT3 was reported to change its localization from mitochondrial to nuclear when coexpressed with SIRT5 [46,47]. The recent identification of the first substrates for mitochondrial sirtuins--acetyl coenzyme A synthetase 2 [48,49], and glutamate dehydrogenase (GDH) [32]--as targets of sirtuins 3 and 4, respectively, revealed that these sirtuins control a regulatory network that has implications for energy metabolism and the mechanisms of caloric restriction (CR) and lifespan determination [20]. In particular, SIRT3 has a role for the mitochondrial NAD-dependent deacetylase, for the maintenance of basal ATP levels and as a regulator of mitochondrial electron transport [34]. Considering our preliminary results and the fact that little is known about the role of SIRT3 and SIRT4 in human physiology, the differences in SIRT3 and SIRT4 mRNA expression before and after marathon that we observed may be due to the redox changes in the mitochondria during the marathon stress. Thus, our results support the hypothesis that exercise may interfere with expression of this family of proteins at mitochondrial level. Further studies are under way to study this aspect.
Conclusion
Our data presented in this study show that: 1) the balance between pro and anti-apoptotic genes is shifted to a anti apoptotic state after strenuous exercise 2) strenuous exercise may interfere with expression of SIRT3 and SIRT4, which may be a key regulator of exercise training. Additional studies are under way in order to elucidate the role of the SIRTs family and bcl-2 family during different exercise protocols.
Methods
Study design and ethical approval
Subjects underwent a baseline testing session in an exercise laboratory. Seven days prior to the treadmill runs which were conducted at a local private gymnasium. The study was cleared by the Institute's Ethics committee and informed written consent was obtained from all the participants.
The study protocol conformed to the guidelines of the Helsinki Conference for research on human subjects.
Subject characteristics
Sixteen healthy, well-trained, male recreational long-distance runners were selected from volunteers who offered to take part in this study (additional file 1). The mean physiological and anthropometric characteristics of the ten male subjects are shown in Table 3. Selection criteria included an age range between 30 and 53 years, the absence of clinical signs or symptoms of infection, cardiovascular disease or metabolic disorders and a minimum weekly training distance. Subjects were also asked to refrain from ingesting additional nutrient supplements, analgesics, anti-inflammatory drugs, caffeine or alcohol for at least 24 h prior to the trial run. Six subjects, which did not participate in the marathon, were considered control for transcripts of the genes examined and DNA fragmentation (additional file 1, 2, and 3).
Table 3 Body composition and physiological parameters of the subjects
Variables Mean ± SD
Age (years) 42.00 ± 7.38
Height (m) 1.78 ± 0.06
Body mass (Kg) 74.54 ± 7.70
Fat free mass (Kg) 64.04 ± 4.71
Fat mass (Kg) 10.50 ± 5.68
BMI (Kg/m2) 23.58 ± 2.48
Total body water 46.86 ± 3.43
HRmax (beat·min-1) 183.80 ± 3.82
HRrest (beat·min-1) 51.00 ± 3.40
VO2max (ml·kg-1·min-1) 60.17 ± 3.25
Information on prior race experience, dietary preparation and expected finishing time was obtained with a self-administered questionnaire before the race. The amateur runners were requested to maintain a standardised diet for one month before the marathon and were given an activity diary. The macronutrient composition of the meal provided carbohydrate (65%), protein (15%) and fat (20%). Food and fluid intake during the race was evaluated by direct observation by trained research assistants.
Body composition analysis
Body composition, fat-free mass (FFM), fat mass (FM), total body water (TBW) were assessed using bioelectrical impedance analysis (BIA). BIA measurements were performed in the morning after an overnight fast of at least 12 h, abstinence of alcohol consumption for 48 h and absence of strenuous physical activity for 24 h before the testing day. Participants emptied their bladders within 30 min before undergoing the measurements. All measurements were performed on the dominant side, while participants lied supine on an examination table with their limbs abducted away from the trunk. Four gel electrodes were attached on defined anatomical positions on the hand, wrist, ankle and foot [50,51]. The BIA measurements were performed using an Akern BIA (Florence, Italy) and Littmann 2325VP adhesive electrodes (3M, St Paul, MN, USA).
Fitness assessment and quantification of marathon physical load
In the week before the marathon, maximal oxygen uptake (VO2max) was determined using an incremental running test on a motorized treadmill (Run Race, Technogym, Gambettola, Italy) at an inclination of 1%. All participants were accustomed to treadmill running as it was extensively used during the pre-marathon training period. After 10 min warm-up at 70% of age-predicted maximal heart rate, the work protocol began at 9 km·h-1 and the speed was increased by 1 km·h-1 every minute so that exhaustion was reached in 8-12 min. Maximal oxygen uptake was considered to be the highest oxygen volume recorded during the last minute of exercise.
Achievement of VO2max was considered as the attainment of at least two of the following criteria:
1. a plateau in VO2 despite increasing speeds;
2. a respiratory exchange ratio above 1.10;
3. a heart rate (HR) ± 10 beats·min-1 of age-predicted maximal HR (220--age).
Expired gases were analysed using a breath-by breath automated gas analysis system (VMAX29, Sensor medics, Yorba Linda, CA). The flow, volume, and gas analysers were calibrated before each test according to the manufacturer's instructions. Heart rates were measured during the incremental test using recordable Polar Vantage NV heart rate monitors (Polar Electro Oy, Kempele, Finland).
Individual maximal heart rates were taken as the highest heart rate recorded during the treadmill test.
Resting heart rate (HRrest) was measured after awakening with subjects in rested state (i.e. quiet room, supine position). The HRrest was assumed as the lowest 5 s value out of a 5 min monitoring. During marathon HR were assessed in each subject (Polar Vantage NV heart rate monitors, Polar Electro Oy, Kempele, Finland) and data were downloaded on a portable PC (Acer Aspire 5000, China) and analysed using the specific software (Polar ProTrainer 5, Polar Electro Oy, Kempele, Finland) and an electronic spread-sheet (Excel, Microsoft Corporation, U.S.). To quantify the internal physical load during marathon, we used the method of Banister et al. (1986) [52] for the calculation of the TRIMP. This method multiplies the duration of a training session by the average HR achieved during that session (percentage of heart rate reserve). The heart rate reserve is weighted by a multiplying factor (y), in a manner that reflects the intensity of effort. This y factor is based upon the exponential rise of blood lactate levels with the fractional elevation of exercise above HRrest. Thus, as exercise intensity increases, as indicated by the HR response, the weighting factor increases exponentially.
Participants were asked to refrain from all exercise and the use of alcohol, tobacco, and caffeine in the 48 h before testing. Subjects consumed their last meal at least three hours before treadmill testing and a record of the nutrient content was taken in order to provide the sufficient carbohydrate intake during the week before testing.
Blood sampling
Blood samples were drawn the day before marathon and two hours after marathon from male recreational long-distance runners. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll-Hypaque gradient. Briefly, each 20-mL sample of anticoagulated whole blood was diluted 1:3 in PBS and layered onto Histopaque-1077. Following centrifugation at 1500 g for 20 minutes, the PBMC-containing interface was transferred to a 15-mL conical centrifuge tube and washed once in ice-cold PBS. The viability of the cells before and after marathon was measured using classic trypan blue dye exclusion. The pellets were used to extract DNAs and RNAs.
DNA fragmentation assay
Cells were washed twice with phosphate-buffered saline (PBS) and lysed by addition of a hypotonic solution (1% NP-40 in 20 mM EDTA, 50 mM Tris-HCl pH 7.5). After centrifugation at 1600 ×g for 5 min, the supernatant was collected and the extraction was repeated with the same lysis buffer. The supernatants was brought to 1% SDS and treated with RNase A (final concentration 5 mg/ml) for 2 h at 56°C followed by digestion with proteinase K (final concentration 2.5 mg/ml) at 45°C for at least 6 h. Before hydrolysis, a further cleaning of DNA was performed by phenol-chloroform extraction, followed by three successive ethanol precipitations in 2 M ammonium acetate. Pellets were dried for 30 min and resuspended in 200 µl Tris-EDTA pH 8.0. Aliquots of 20 µl containing 10 µg DNA were electrophoresed in 1.5% agarose gel [53].
Subsequent 3' end-labeling of DNA, gel electrophoresis, and quantitation of DNA fragmentation were performed. Briefly, 500 ng of DNA prepared from samples were end-labeled with [α32P]-ddATP (Amersham) and terminal transferase (Boehringer-Mannheim) for 60 minutes at 37°C. Labeled DNA was loaded onto a 2% agarose gel, separated by electrophoresis and visualized by autoradiography.
RNA isolation and Northern blot
Total RNA was isolated from samples using Trizol reagent (GIBCO) according to the manufacturer's instructions, and separated on 1% (w/v) agarose gel containing 1 × MOPS buffer [20 mM 3-(N-morpholino) propanesulfonic acid, 8 mM sodium acetate and 1 mM EDTA] and 2.2 M formaldehyde. Total RNA was blotted onto nylon membranes (Hybond N, Amersham, Braunschweig, Germany) and hybridized with 32P-different cDNA in a hybridization solution containing 50% formamide at 42°C overnight as previously described [54,55]. The excess 32P-probe was removed by stringent washing three times with 0.1× SSC and 1% SDS at 65°C for 30 min each. Hybridization signals were detected with a PhosphorImager (Biorad). The relative amount of mRNA level was quantified using a Gel-Doc phosphorimager and Quantity One software (Bio-Rad) and normalized by the intensity of β-actin.
Statistical analysis
The results are expressed as means ± standard deviations (SD) and 95% confidence intervals (95% CI). Before using parametric tests, the assumption of normality was verified using the Shapiro-Wilk W-test. Pearson's product moment correlation coefficients were used on all subjects to examine correlations between variables. Student's paired t-test was used to determine any significant differences in physiological variables before and after marathon. The effect size (ES) was calculated to assess meaningfulness of differences. Effect sizes of above 0.8, between 0.8 and 0.5, between 0.5 and 0.2 and lower than 0.2 were considered as large, moderate, small, and trivial respectively [56]. Significance was set at 0.05 (p ≤ 0.05). A Bonferroni correction for the number of paired t-test was used. The resulting p-level was p ≥ 0.005. SPSS statistical software package (SPSS Inc., Version 13.0.1 for Windows Chicago, IL, USA) was used for all statistical calculations.
Authors' contributions
GM carried out the design of the study, drafted and edited the manuscript. GM, MT, MAR reviewed and edited the manuscript. GM, MT, BP, MI, CD carried out Northern Blot. VM, AA PSS participated in the exercise study. VM performed the statistical analysis, and data interpretation. GM and MT conceived of the study and the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
The anti and pro-apoptotic and SIRTs mRNAs in healthy amateur runners who did not participate in the marathon. The results show the DNA fragmentation and the bax, bcl-2 SIRT1, SIRT3 RNA levels in six healthy amateur runners (who did not participate in the marathon) samples before and after marathon.
Click here for file
Additional file 2
Figure 1. DNA laddering
Click here for file
Additional file 3
Figure 2. Bax, bcl-2, SIRT1 and SIRT3 RNA levels were estimated by Northern Blot
Click here for file
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Mol VisMVMolecular Vision1090-0535Molecular Vision 1302010MOLVIS0115Research ArticleThe role of signaling pathways in the expansion of corneal epithelial cells in serum-free B27 supplemented medium Krishnan Sasirekha 1Lakshmanan Shruthi 1Iyer Geetha Krishnan 2UmaMaheswari Krishnan 3Krishnakumar Subramanian 11 L&T Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, India2 Cornea services department, Medical Research Foundation, Sankara Nethralaya, Chennai, India3 Nanobiotechnology Department, CeNTAB, SASTRA University, Tanjore, IndiaCorrespondence to: Subramanian Krishnakumar, Head, L&T Department of Ocular Pathology, Incharge Stem Cell Department, Vision Research Foundation, Sankara Nethralaya, 18, College Road, Nungambakkam, Chennai – 600 006, India; Phone: +91-44 28271616; FAX: +91-44-28254180; email: [email protected]. Sasirekha Krishnan, M.Sc., is a Ph.D. student at SASTRA university, L&T Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, 18, College Road, Chennai – 600 006, India; Phone: +91-44 28271616; FAX: +91-44-28254180; email: [email protected] 25 6 2010 16 1169 1177 29 3 2010 19 6 2010 Copyright © 2010 Molecular Vision.2010This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original
work is properly cited.Purpose
To study the influence of serum-free B27 supplemented culture medium on corneal epithelial cells from limbal explants.
Methods
Human limbal tissues obtained from cadaveric donor eyes were used in this study. The morphological characteristics of cultivated epithelial cells were analyzed by phase contrast microscopy. Growth kinetics, bromodeoxyuridine (BrdU) labeling cell proliferation assay, and reverse transcriptase PCR (RT–PCR) for limbus and corneal markers were studied in serum-dependent and serum-free B27 supplemented corneal epithelial culture. The signaling pathway genes were analyzed by RT2 qPCR profiler array.
Results
The corneal epithelial cells morphology and mRNA expression of markers were similar in both the serum-dependent and serum-free B27 supplemented culture. The growth and proliferation of the serum-free B27 supplemented culture was significantly higher than that of the serum-dependent culture. The wnt, hedgehog, survival, NFkB, Jak-Stat, and calcium protein kinase C pathways were highly expressed in the serum-free B27 supplemented corneal epithelial culture.
Conclusions
Most signaling pathway genes are upfolded by B27 supplementation in the corneal epithelial cell culture; it could be an efficient replacement for serum.
GalleyStatusExport to XMLcorr-authorKrishnakumar
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Introduction
Limbal deficiency or loss of corneal stem cells is associated with ocular surface disease, which is otherwise known as limbal stem cell deficiency (LSCD). The management of the ocular surface using cultured corneal epithelial cells on a human amniotic membrane is preferred. The ex vivo expansion of limbus culture requires unknown factors, such as fetal bovine serum (FBS), autologous serum, feeder layers or bovine pituitary extracts (BPE), as growth factors for the growth of corneal epithelial cells. The usage of these substances raises concern about infection with recognized or unknown-agents [1]. Although there have been successful reports that support the proliferation of corneal epithelial cells using autologous human serum [2], which effectively eliminates the risk of xenogenic contamination during transplantation to LSCD patients, there has been no data supporting the use of corneal epithelial cultures in a serum-free medium condition or showing the important signaling pathways involved.
B27 was originally optimized for culture of hippocampal neurons and used for the growth of neurons from embryonic rat striatum, the substantia nigra, the subiculum, the cerebral cortex, the postnatal dentate granule, the cerebellum, and the dentate gyrus in a serum-free condition [3]. B27 contains vitamins like biotin, DL-alpha-tocopherol, and DL-alpha-tocopherol acetate. It also contains catalase, human recombinant insulin, superoxide dismutase proteins, and other components such as corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, linoleic acid, linolenic acid, triiodo-L-thyronine, etc. It has been reported that corneal endothelial precursors proliferate actively in B27-containing medium with no FBS or feeder cells [4]. Yakoo et al. [1] established a culture technique for human corneal epithelial equivalents with B27 as an alternative for FBS and studied the pututative markers for corneal epithelial cells. However, the signaling pathway that helps to replace serum components and maintain stemness in the corneal culture has not yet been reported in the literature.
Therefore, we have tried to avoid serum, feeder layers, and/or bovine pituitary extract (BPE) in the culturing of corneal limbal stem cells. Instead, we used a serum-free medium supplemented with the growth factor B27 and analyzed the genes involved in the signal transduction pathway by RT2 qPCR profiler array.
Methods
Grading donor eyes
Human cadaveric eyeballs were obtained from the C.U. Shah eye bank of the Medical Research Foundation, Sankara Nethralaya, Chennai, India with the consent of the donor or donor family to be used for medical research in accordance with the principles outlined in the Declaration of Helsinki. In this study, we collected limbus tissues from donors (n=12) aged between 67and 82 years. Corneal limbal tissues of 2 mm in length were collected in Dulbecco’s Modified Eagle Medium (DMEM; Sigma Chemicals, St. Louis, MO) with antibiotics (Sigma Chemicals) and transported to the cell biology laboratory for further processing. The donor blood samples were screened for human immunodeficiency virus (HIV) type 1 and 2, hepatitis B virus (HBV), hepatitis C virus (HCV), and Treponema pallidum infections. Data on age, sex, cause of death, time of death, time of eye donation, and time of biopsy collection were also collected.
Human limbal explant culture
The collected limbal tissue was washed thrice with Hanks balanced salt solution buffer (Sigma Chemicals). After careful removal of excessive sclera and conjunctiva, the tissue was cut into multiple bits using a sharp, sterile Bard-Parker blade (Niraj Industries, Faridabad, India). The tissue bits were placed on a culture plate (BD biosciences, San Jose, CA) using a sterile needle. The plate was incubated at 37 °C and 5% CO2 for 5 min for adhesion. The explants were covered with culture medium containing equal volumes of DMEM and F12 (Sigma Chemicals) containing 5 ng/ml of epidermal growth factor (EGF), 5 μg/ml of insulin, 5 μg/ml of transferrin, 5 ng/ml of sodium selenite, 0.5 mg/ml of hydrocortisone, and 1% antibiotic solution (Sigma Chemicals). Ten percent FBS (Sigma Chemicals) was added to five cultures (serum-dependent culture; n=5) and 1% B27 supplement (Sigma Chemicals) was added to the other five cultures (serum-free B27 supplemented culture; n=5). The control samples were cultured without serum and/or any other supplement replacing serum (control culture; n=2). The plates were incubated at 37 °C and 5% CO2 with 95% humidity. The medium was changed once every two days and growth was monitored daily with an inverted phase contrast microscope (Nikon, Tokyo, Japan). Confluent cells were harvested for further molecular characterization.
Growth kinetics
The outgrowth of all the cultures was photographed every second day; images were transferred to a computer and analyzed using quantity G area measurement software [5]. The mean radius of all the cultures was plotted against each day until they reached confluence.
Cell proliferation assay
Cell proliferation was assessed by measuring 5-bromo-2-deoxyuridine (Qiagen, Santa Clara, CA) incorporation during DNA synthesis in proliferating cells. The detection of BrdU was performed according to the manufacturer’s instruction and chased for 1–21 days. The BrdU labeling indices were assessed by counting the nuclei through a microscope using a 40× objective. The labeling index was expressed as the number of positively labeled nuclei/total number of nuclei×100%.
RNA isolation
The cultures were trypsinised on the 8th day (limbal stem cells) and the 21st day (differentiated corneal cells) from both serum-dependent and serum-free B27 supplemented cultures. The RNA was isolated using the Rneasy (Qiagen) kit according to the manufacturer’s instructions. For RT2 qPCR array, the integrity and purity of the RNA were verified using a bioanalyzer chip (Agilent Technologies Genotypic, Bangalore, India).
Reverse transcriptase PCR
The expression of marker genes (Bangalore Genei, Bangalore, India; Table 1) specific for limbal stem cells and corneal cells was studied by RT–PCR with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.
Table 1 Primer sequence and reaction condition for RT–PCR.
Gene Primer sequence (3′-5′) Annealing temperature (°C) PCR product size (bp)
ABCG2 FP:AGTTCCATGGCACTGGCCATA 62 379
RP:TCAGGTAGGCAATTGTGAAGG
ΔNp63 FP:CAGACTCAATTTAGTGAG 54 440
RP:AGCTCATGGTTGGGGCAC
Connexin 43 FP:CCTTCTTGCTGATCCAGTGGTAC 66 154
RP:ACCAAGGACACCACCAGCAT
Keratin3 FP: GGCAGAGATCGAGGGTGTC 64 145
RP: GTCATCCTTCGCCTGCTGTAG
Keratin12 FP:CATGAAGAAGAACCACGAGGATG 63 150
RP:TCTGCTCAGCGATGGTTTCA
GAPDH FP:GCCAAGGTCATCCATGACAAC 63 498
RP:GTCCACCACCCTGTTGCTGTA
FP: Forward Primer; RP: Reverse Primer; bp: Base Pair.
Signal transduction pathway analysis
The RT2 qPCR profiler Human Signal Transduction Pathway array (catalog number PAHS-014; SABiosciences, Frederick, MD), representing 84 genes involved in signal transduction pathways, plus five housekeeping genes and three controls, was used to analyze the effect of serum on signaling-related gene expression in human limbal and corneal epithelial cells. The total RNA was isolated from the limbus and corneal cells (serum-dependent and serum-free B27 supplemented culture) using the Rneasy Mini Kit (Qiagen). cDNA was generated from 1 µg total RNA using the RT2 qPCR Array First Strand Kit in accordance with the manual. The template was combined with RT2 SYBR Green/Fluorescein PCR master mix. Equal amounts of this mixture (25 μl) were added to each well of the RT2 qPCR profiler plate containing the predispensed gene-specific primer sets, and the reaction was performed using a sequence detector (ABI 7500; Applied Biosystems, LabIndia, Chennai, India) according to the manufacturer’s protocols. Data analysis was based on the ∆∆Ct method with the aid of an Excel (Microsoft Excel; Microsoft, Redmond, WA) spreadsheet containing algorithms provided by the manufacturer. The expression levels of the mRNA of each gene were normalized using the expression of the housekeeping gene GAPDH. A positive value indicates that the gene was upregulated and a negative value indicates that the gene was downregulated.
Statistical analysis
All experiments were performed in triplicate. The summary data were reported as the mean±standard deviation (SD), and were compiled and analyzed on a computer (Microsoft Excel; Microsoft). The mean and SD were calculated for each group using the Student’s t-test. Results were considered to be statistically significant when p<0.01. The results of RT2 qPCR are indicated as “fold increase” (mRNA concentrations of serum-free B27 supplemented cultures divided by mRNA concentrations of serum-dependent controls).
Results
Under microscopic observation, we noted epithelial migration from limbal explants at the end of 48 h in both serum-dependent and serum-free B27 supplemented cultures (Figure 1). By the end of the 15th day, 90%–100% confluent growth was seen. There was no growth in the control samples cultured without serum and/or any other supplement.
Figure 1 Epithelial cell migration from limbal explants. Epithelial cell migration from limbal explants in serum-free B27 supplemented at the end of 48 h (A); serum-dependent culture at the end of 48 h (B); confluent culture of corneal epithelial cells in serum-free B27 supplemented at the end of the 15th day (C); confluent culture of corneal epithelial cells in serum-dependent culture at the end of the 15th day (D).
Growth kinetics
The cells cultured in serum-free B27 supplemented medium showed significantly higher growth after 12 days (Figure 2). The growth rate was faster on cells cultured in a serum-free B27 supplemented culture when compared to a serum-dependent medium (p<0.005).
Figure 2 Growth kinetics of corneal epithelial cultures plotted with area of growth in mm2 (x-axis), against serum-free B27 supplemented and serum-dependent cultures (y-axis).
Cell proliferation
The labeling index was high in serum-free B27 supplemented culture when compared to serum-dependent culture after 24 h. The cultures were reviewed continuously for 7, 14, and 21 days and the labeling indices were 50±7.76, 42±2.24, 20±2.0, and 12±0.2%, respectively, in serum-free B27 supplemented culture. Similarly, in the serum-dependent culture, the labeling indices were 48±3.2, 35±0.33, 17±1.7, and 9±1.1% for 7, 14, and 21 days, respectively (Figure 3).
Figure 3 Cell proliferation index plotted with BrdU labeling indices (x-axis), against serum-free B27 supplemented and serum-dependent cultures (y-axis).
RT–PCR
Semiquantitative RT–PCR results showed similar expressions (Table 2) of various markers such as transformation-related protein 63 - p63, ATP-binding cassette sub-family G member 2 - ABCG2, connexin 43, and Keratin 3/Keratin 12 – K3/K12 of differentiated corneal epithelial cells (21st day) grown in the serum-dependent and serum-free B27 supplemented medium (Figure 4).
Table 2 mRNA expression of cultured corneal cells grown in serum-dependent and serum-free B27 supplemented medium.
Markers Serum-dependent B27-dependent
ABCG2 - -
P63 + +
Connexin 43 + +
Keratin 3 + +
Keratin 12 + +
GAPDH is an internal control; + positive marker; - negative marker.
Figure 4 RT–PCR for mRNA expression of putative limbal/corneal stem cell markers. Lane 1: Negative control; Lane 2: Positive control; Lane 3: serum-free B27 supplemented corneal cells; Lane 4: serum-dependent corneal cells; Lane 5: 100 bp DNA ladder.
Comparison of signal transduction pathway genes supporting the expansion of serum-dependent and serum-free B27 supplemented culture
The array experiment was performed in duplicate. A simple comparison was performed on data to assess the gene expression of a serum-free B27 supplemented culture in relation a serum-dependent culture as a control for limbal stem cells and differentiated corneal epithelial cells (Table 3). The differences in gene expression between the serum-free B27 supplemented culture and the serum-dependent profile of limbal and corneal cells were studied (a more than twofold difference was considered significant). The raw data, i.e., the mean ∆∆Ct values of the genes, were normalized to the housekeeping gene GAPDH. All 84 genes were analyzed thoroughly based on their role in both the serum and serum-free conditions. Among these pathways, the most interesting and highly expressed were wnt, hedgehog, survival, NFkB, Jak-Stat, and the calcium protein kinase C pathways that have been discussed in this study (Figure 5).
Table 3 Signal transduction pathway gene profile supporting the expansion of serum-free B27 supplemented limbus/corneal culture (serum-dependent culture as control).
Symbol Limbus Cornea Description Gene Name
Mitogenic Pathway
EGR1 12.06 1.16 Early growth response 1 AT225/G0S30
FOS 67.78 1.6 V-fos FBJ murine osteosarcoma viral oncogene homolog AP-1/C-FOS
JUN 8.51 1.64 Jun oncogene AP-1/AP1
Wnt Pathway
CCND1 4.28 −1.72 Cyclin D1 BCL1/D11S287E
JUN 8.51 1.64 Jun oncogene AP-1/AP1
LEF1 12.06 1.13 Lymphoid enhancer-binding factor 1 DKFZp586H0919/TCF1ALPHA
MYC 4.28 −3.56 V-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39
PPARG 2.14 −1.33 Peroxisome proliferator-activated receptor gamma CIMT1/NR1C3
TCF7 12.06 1.13 Transcription factor 7 (T-cell specific, HMG-box) TCF-1
VEGFA 1.07 −1.74 Vascular endothelial growth factor A MVCD1/VEGF
WISP1 11.65 1.13 WNT1 inducible signaling pathway protein 1 CCN4/WISP1c
Hedgehog Pathway
BMP2 8.52 −3.57 Bone morphogenetic protein 2 BMP2A
BMP4 4.32 −2.46 Bone morphogenetic protein 4 BMP2B/BMP2B1
EN1 11.76 1.13 Engrailed homeobox 1 Engrailed 1
FOXA2 12.06 1.13 Forkhead box A2 HNF3B/TCF3B
PTCH1 2.01 −2.46 Patched homolog 1 (Drosophila) BCNS/HPE7
WNT1 12.06 1.13 Wingless-type MMTV integration site family, member 1 INT1
WNT2 12.06 1.17 Wingless-type MMTV integration site family member 2 INT1L1/IRP
TGF-Beta Pathway
CDKN1A 3.00 −2.47 Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CAP20/CDKN1
CDKN1B 5.99 1.65 Cyclin-dependent kinase inhibitor 1B (p27, Kip1) CDKN4/KIP1
CDKN2A −1.34 −6.95 Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) ARF/CDK4I
CDKN2B 2.12 −1.75 Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) CDK4I/INK4B
Survival Pathway
PI3 Kinase/AKT Pathway
BCL2 12.06 1.13 B-cell CLL/lymphoma 2 Bcl-2
CCND1 4.28 −1.72 Cyclin D1 BCL1/D11S287E
JUN 8.51 1.64 Jun oncogene AP-1/AP1
MYC 4.28 −3.56 V-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39
Jak/Src Pathway
BCL2 12.06 1.13 B-cell CLL/lymphoma 2 Bcl-2
BCL2L1 6.03 −7.04 BCL2-like 1 BCL-XL/S
NFkB Pathway
BCL2A1 1.50 2.33 BCL2-related protein A1 ACC-1/ACC-2
BIRC2 2.13 3.24 Baculoviral IAP repeat-containing 2 API1/HIAP2
BIRC3 1.06 −2.48 Baculoviral IAP repeat-containing 3 AIP1/API2
NAIP (BIRC1) 2.13 1.14 NLR family, apoptosis inhibitory protein BIRC1/NLRB1
TERT 12.06 1.13 Telomerase reverse transcriptase EST2/TCS1
P53 Pathway
BAX 3.01 −14.1 BCL2-associated X protein BCL2L4
CDKN1A 3.00 −2.47 Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CAP20/CDKN1
Fas −1.33 −1.25 Fas (TNF receptor superfamily, member 6) ALPS1A/APO-1
GADD45A 5.99 2.26 Growth arrest and DNA-damage-inducible, alpha DDIT1/GADD45
IGFBP3 −14.95 −40 Insulin-like growth factor binding protein 3 BP-53/IBP3
MDM2 1.06 −4.93 Mdm2 p53 binding protein homolog (mouse) HDMX/hdm2
TP5313 4.28 −1.22 Tumor protein p53 inducible protein 3 PIG3
Stress Pathway
ATF2 3.01 −2.48 Activating transcription factor 2 CRE-BP1/CREB2
FOS 67.78 1.6 V-fos FBJ murine osteosarcoma viral oncogene homolog AP-1/C-FOS
HSF1 (tcf5) 4.25 1.15 Heat shock transcription factor 1 HSTF1
HSPB1 (hsp27) 4.27 −1.25 Heat shock 27 kDa protein 1 CMT2F/DKFZp586P1322
HSPCA (hsp90) 1.50 −3.48 Heat shock protein 90 kDa alpha (cytosolic), class A member 2 HSP90ALPHA/HSPCA
MYC 4.28 −3.56 V-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39
TP53 1.07 −1.75 Tumor protein p53 LFS1/TRP53
NFkB Pathway
IKBKB 2.11 −2.53 Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta IKK-beta/IKK2
IL1A 8.48 3.27 Interleukin 1, alpha IL-1A/IL1
IL2 11.72 1.17 Interleukin 2 IL-2/TCGF
IL8 1.53 −9.92 Interleukin 8 CXCL8/GCP-1
LTA (TNF beta) 11.75 1.13 Lymphotoxin alpha (TNF superfamily, member 1) LT/TNFB
NOS2A (iNOS) 1.42 −3.52 Nitric oxide synthase 2, inducible HEP-NOS/INOS
PECAM1 8.03 1.09 Platelet/endothelial cell adhesion molecule CD31/PECAM-1
TANK 5.70 −3.63 TRAF family member-associated NFKB activator I-TRAF/TRAF2
TNF 7.99 −1.75 Tumor necrosis factor (TNF superfamily, member 2) DIF/TNF-alpha
VCAM1 12.06 1.13 Vascular cell adhesion molecule 1 CD106/DKFZp779 G2333
NFAT Pathway
CD5 11.65 1.13 CD5 molecule LEU1/T1
FASLG (TNFSF6) 11.69 1.16 Fas ligand (TNF superfamily, member 6) APT1LG1/CD178
IL2 11.72 1.17 Interleukin 2 IL-2/TCGF
CREB Pathway
CYP19A1 11.27 1.13 Cytochrome P450, family 19, subfamily A, polypeptide 1 ARO/ARO1
EGR1 12.06 1.16 Early growth response 1 AT225/G0S30
FOS 67.78 1.6 V-fos FBJ murine osteosarcoma viral oncogene homolog AP-1/C-FOS
Jak-Stat pathway
CXCL9 11.14 1.13 Chemokine (C-X-C motif) ligand 9 CMK/Humig
IL4 11.33 1.13 Interleukin 4 BCGF-1/BCGF1
IL4R 1.51 −3.52 Interleukin 4 receptor CD124/IL4RA
MMP10 3.02 −1.76 Matrix metallopeptidase 10 (stromelysin 2) SL-2/STMY2
NOS2A (iNOS) 1.42 −3.52 Nitric oxide synthase 2, inducible HEP-NOS/INOS
Estrogen Pathway
BCL2 12.06 1.13 B-cell CLL/lymphoma 2 Bcl-2
BRCA1 8.50 1.1 Breast cancer 1, early onset BRCAI/BRCC1
GREB1 11.72 1.16 GREB1 protein KIAA0575
NRIP1 −1.32 −3.51 Nuclear receptor interacting protein 1 RIP140
Androgen Pathway
CDK2 8.55 −1.75 Cyclin-dependent kinase 2 p33(CDK2)
CDKN1A 3.00 −2.47 Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CAP20/CDKN1
KLK2 11.41 1.16 Kallikrein-related peptidase 2 KLK2A2/hK2
TMEPAI −1.87 −1.74 Prostate transmembrane protein, androgen induced 1 STAG1/TMEPAI
Calcium and protein kinase C Pathway
CSF2 11.42 −1.84 Colony stimulating factor 2 (granulocyte-macrophage) GMCSF
FOS 67.78 1.6 V-fos FBJ murine osteosarcoma viral oncogene homolog AP-1/C-FOS
IL2 11.72 1.17 Interleukin 2 IL-2/TCGF
JUN 8.51 1.64 Jun oncogene AP-1/AP1
MYC 4.28 −3.56 V-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39
ODC1 8.54 1.65 Ornithine decarboxylase 1 ODC
PRKCA 5.62 1.13 Protein kinase C, alpha AAG6/PKC-alpha
PRKCE 2.92 −2.48 Protein kinase C, epsilon PKCE/nPKC-epsilon
TFRC −1.30 −1.74 Transferrin receptor (p90, CD71) CD71/TFR
Phospholipase C Pathway
BCL2 12.06 1.13 B-cell CLL/lymphoma 2 Bcl-2
EGR1 12.06 1.16 Early growth response 1 AT225/G0S30
FOS 67.78 1.6 V-fos FBJ murine osteosarcoma viral oncogene homolog AP-1/C-FOS
ICAM1 −2.68 −20.07 Intercellular adhesion molecule 1 BB2/CD54
JUN 8.51 1.64 Jun oncogene AP-1/AP1
NOS2A 1.42 −3.52 Nitric oxide synthase 2, inducible HEP-NOS/INOS
PTGS2 23.98 4.57 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) COX-2/COX2
VCAM1 12.06 1.13 Vascular cell adhesion molecule 1 CD106/DKFZp779G2333
Insulin Pathway
CEBPB 2.90 3.3 CCAAT/enhancer binding protein (C/EBP), beta C/EBP-beta
FASN 4.26 1.14 Fatty acid synthase FAS/OA-519
GYS1 3.03 3.29 Glycogen synthase 1 (muscle) GSY/GYS
HK2 2.99 1.69 Hexokinase 2 DKFZp686M1669/HKII
LEP 12.06 1.13 Leptin OB/OBS
LDL Pathway
CCL2 8.79 1.13 Chemokine (C-C motif) ligand 2 GDCF-2/HC11
CSF2 11.42 −1.84 Colony stimulating factor 2 (granulocyte-macrophage) GMCSF
SELE 11.70 1.13 Selectin E CD62E/ELAM
SELPLG 12.06 1.13 Selectin P ligand CD162/CLA
VCAM1 12.06 1.13 Vascular cell adhesion molecule 1 CD106/DKFZp779 G2333
Retinoic acid Pathway
EN1 11.76 1.13 Engrailed homeobox1 Engrailed 1
HOXA1 12.06 1.13 Homeobox A1 BSAS/HOX1
RBP1 (CRBP1) 1.06 −1.74 Retinol binding protein 1, cellular CRABP-I/CRBP
Figure 5 Six Pathways with relatively high expressions representing corresponding genes by RT2 qPCR profiler array of serum-free B27 supplemented limbus and corneal epithelial cells. Serum-dependent cultured limbus and corneal epithelial cells are the respective controls.
Discussion
We have demonstrated the use of serum-free B27 supplemented medium for the growth of corneal epithelial cells. This serum-free medium supported the proliferation and viability of the cells. The cells expressed presumed limbal stem cell association markers and the cornea phenotype, suggesting that the serum-free B27 supplemented medium retained the stemness of cultured cells. The confluent culture was collected and RNA was isolated to analyze the signaling pathway genes involved in both serum-dependent and serum-free B27 supplemented cultures.
The signal transduction pathway genes involved in the growth of corneal epithelial cells help to determine their role in both serum-dependent and serum-free B27 supplemented corneal epithelial cultures. Among the 17 pathways, six pathways involved in the serum-free B27 supplemented culture were discussed, along with their roles in serum-free limbal stem cell and differentiated corneal epithelial cell cultures.
In the serum-free condition of the corneal epithelial cells, the activation of wnt pathway plays a vital role by activating genes like Homo sapiens jun oncogene (JUN), which codes for a transcription factor called activator protein-1 (AP1) and helps in the differentiation, proliferation, and apoptosis of epithelial cells [6]. Corneal epithelial stem cell proliferation depends on the upregulation of paired box gene 6 (pax6) and downregulation of beta-catenin and lymphoid enhancer-binding factor 1 (Lef-1) [7]. The hedgehog pathway genes were 2 to 8 times upregulated in serum-free B27 supplemented limbal stem cells when compared with differentiated corneal epithelial cells of the same culture. Sonic hedgehog (Shh) is secreted by stem cells, inducing bone morphogenetic protein 4 (BMP4), and is involved in the self-renewal and development of the epithelium [8]. The wingless-type MMTV integration site family, member 1 (wnt1) and Wingless-type MMTV integration site family, member 21 (wnt2) genes of this pathway were found to play an equal role (12 times upregulated in relation to the serum-dependent culture) in the maintenance of stemness in limbal epithelial cells of the serum-free B27 supplemented culture. The cellular survival pathway consists of phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene homolog 1 (PI3K/Akt), Janus kinase/sarcoma proto oncogene (Jak/Src), and nuclear factor kappa-light-chain-enhancer of activated B (NFkB) as three major groups of genes. The cyclin D1 (CCND1) gene is required for cell cycle G1/S transition [9]. Baculoviral Inhibitor of Apoptosis repeat proteins (Birc1) proteins contain BIR domains that can directly bind to active caspases and help in protein–protein interaction [10]. In the stem cell and progenitor cell compartments, the telomerase reverse transcriptase (TERT) gene prevents the adverse consequences of dysfunctional telomeres on cell viability and chromosomal stability [11], and enhances the cell cycle entry of quiescent epidermal stem cells [12]. The NFkB pathway genes in serum-free B27 supplemented cells had a distinct fold increase when compared with the control, and a few genes like interleukin 1 alpha (IL1A), interleukin 2 (IL2), lymphotoxin alpha (LTA), platelet/endothelial cell adhesion molecule 1 (PECAM1), and vascular cell adhesion molecule 1 (VCAM1) exhibited upfolded expression in both limbus and corneal cells. The inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) gene produced an enzyme, IKK2 - inhibitor of nuclear factor kappa-B kinase subunit and activated a transcription factor called NFkB. Interleukin genes like IL1A, interleukin 8 (IL8), and tumor necrosis factor alpha (TNFα) present in the NFkB pathway encode for cytokines and chemokines involved in inflammatory processes [13,14]. They also help in the migration of progenitor and pluripotent stem cells [15]. The chemokine (C-X-C motif) ligand 9 (CXCL9) and interleukin 4 (IL4) genes of the Jak-Stat pathway played an important role in the development and organization of cells, which were upregulated by 12 times in serum-free B27 supplemented limbus culture [16]. Among the other five pathways, the calcium and protein kinase C pathway genes were highly expressed in serum free-B27 supplemented culture when compared to serum-dependent culture. The Homo sapiens V-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) gene of the calcium and protein kinase C pathway belonged to the transcription factor family [17], which is highly upregulated in serum-free B27 supplemented limbal stem cell cultures.
In conclusion, the B27 supplement activated more signaling pathway genes, helping to provide a higher cell number, good capacity for proliferation, better quality, and more functional pieces of engineered corneal equivalents without the support of serum, a feeder layer, and/or BPE.
Acknowledgments
The authors would like to thank the Indian Council of Medical Research (Grant No: 80/7/2003-BMS) for financial support.
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Mediators InflammMediators InflammMIMediators of Inflammation0962-93511466-1861Hindawi Publishing Corporation 2062852210.1155/2010/748919Clinical StudyFurther Increase in the Expression of Activation Markers on Monocyte-Derived Dendritic Cells in Coronary Artery Disease Patients with Ectasia Compared to Patients with Coronary Artery Disease Alone Yildirim Nesligul
1
*Tekin Ishak Ozel
2
Arasli Mehmet
2
Aydin Mustafa
1
1Department of Cardiology, Faculty of Medicine, Zonguldak Karaelmas University, 67600 Zonguldak, Turkey2Department of Immunology, Faculty of Medicine, Zonguldak Karaelmas University, 67600 Zonguldak, Turkey*Nesligul Yildirim: [email protected] Editor: Mark Smith
2010 14 6 2010 2010 74891924 2 2010 12 4 2010 27 4 2010 Copyright © 2010 Nesligul Yildirim et al.2010This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Coronary artery ectasia (CAE) is defined as localized or diffuse dilation of the coronary arteries. There are scarce data about the role of dendritic cells in CAE development. In this study we investigated the activation markers on the surface of monocyte-derived dendritic cells (mDCs) in coronary artery disease (CAD) patients with or without CAE.
Method. The study consisted of 6 patients who had obstructive CAD with CAE, 6 CAD patients without CAE and 6 subjects with angiographically normal coronary arteries. mDCs were cultivated from peripheral blood monocytes. Surface activation markers were detected by flow cytometry.
Results. CAD patients with CAE were detected to have significantly higher mean fluorescence intensities of CD11b, CD11c, CD54 , CD83, CD86 and MHC Class II molecules on mDCs in comparison to CAD patients without CAE and normal controls (P < .001 for all). A significant positive correlation was found between the number of vessels with CAE and the levels of CD11c, CD86, and MHC Class II molecules.
Conclusion. mDCs display an increased cell surface concentration of activation molecules in CAD patients with CAE compared to patients with CAD alone. DC activation may play an important role for CAE development in patients with CAD.
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1. Introduction
Coronary artery ectasia (CAE) has been defined as segmental or diffuse luminal dilatation of the coronary arteries in coronary angiography [1]. CAE is a rare finding among coronary artery anomalies and considered to be congenital in 10–20% of the cases while the remaining are acquired in origin [2]. The common coexistence of CAE with coronary artery disease (CAD) suggests that it may be a variant of atherosclerosis [2, 3]. However it is not clear why some patients with obstructive CAD develop CAE whereas most do not.
Dendritic cells (DCs) are potent antigen presenting and immune modulating cells with the unique ability to initiate a primary immune response to certain antigens by the activation of T lymphocytes [4, 5]. To acquire the ability to contact and activate T cells, DCs must undergo a maturation process with the upregulation of antigen presenting molecules including MHC class I and class II, adhesion molecules (CD11a, CD11b, CD54, CD50, CD58) and costimulatory molecules (CD40, CD80, CD86) [4–6]. In addition to these relatively well-known cell surface molecules; CD83 which is the hallmark of mature DCs and CD11c expressions were also reported to have functions in the regulation of antigen presentation and enhancement of T cell activation in the recent reports [7, 8]. During the development of an adaptive response, T cells form direct contacts with DCs and respond to peptide antigen displayed on MHC Class II and Class I molecules present on DC surfaces. In DC/T cell interactions, the presence of costimulatory molecules is required for T cell activation and differentiation into effector cells [6].
So far the presence of DC was described in atherosclerotic plaques of carotid arteries, aortas, and stenotic aortacoronary vein bypass grafts [9]. The colocalization of DC and T cells as well as the expression of MHC-II and costimulatory molecules on DCs in atherosclerotic plaques suggest that DC initiate an antigen-spesific immune response contributing to the progression of atherosclerosis [9–11]. Until now research on the pathogenesis of CAE has also focused on chronic transmural inflammation [2, 3]. Recently we have shown increased expression of monocyte and lymphocyte adhesion molecules in isolated CAE [12]. Given the pivotal function of DCs in initiating T lymphocyte responses in atherosclerosis and the knowledge that peripheral blood monocytes can be differentiated into DCs by exposure to inflammatory factors [13]; in the present study we investigated the expression levels of MHC Class II, CD54, CD11b, CD11c, CD83, and CD86 on the surface of monocyte-derived DCs (mDCs) in normal subjects and CAD patients with or without CAE. Our aim was to evaluate the role of DCs in CAE development.
2. Material and Methods
2.1. Study Population
The study was designed as a case control prospective study. The study population was selected from a series of 256 consecutive patients who underwent coronary angiography in our hospital between April 2006 and September 2006 due to the presence of chest pain or positive or equivocal results of noninvasive screening tests for myocardial ischemia. Out of 256 patients, 6 consecutive patients with obstructive CAD and CAE (Group 1, 4 male, mean age: 50.1 ± 4.0 years) and accepted to participate our study after giving informed consent, were identified and compared with age and sex matched 6 consecutive subjects with obstructive CAD alone (Group 2, 4 male, mean age: 51.1 ± 4.5) and 6 consecutive subjects who had angiographically shown normal coronary arteries (Group 3, 4 male, mean age: 51.1 ± 4.2). Obstructive CAD was defined as the stenosis of epicardial coronary artery >50% angiographically.
Exclusion criteria were the presence of previous myocardial infarction, acute coronary syndromes, any inflammatory or immunologic disease, active local or systemic infection, history of recent infection (<3 months), left ventricular dysfunction, left ventricular hypertrophy, cardiomyopathies, congenital heart disease, valvular heart disease, any abnormality in thyroid function test, arrhythmias and statin use within the last 6 months. Besides, leukocyte count, erythrocyte sedimentation rate and fibrinogen levels were normal in all patients and control subjects.
2.2. Coronary Angiography
Coronary angiography was routinely performed without the use of nitroglycerin. Selective coronary angiography was performed by means of Judkins technique in multiple projections. We used Iohexol (Omnipaque, Nycomed Ireland Cork, Ireland) as contrast agent during coronary angiography in all patients and control subjects. Coronary angiograms were analyzed by two blinded interventional cardiologists without knowledge of the clinical status and laboratory measurements of the subjects. The definition of CAE was that used in the Coronary Artery Surgery Study (CASS) [1]. According to the angiographic definition of CASS, a vessel is considered to be ectasic when its diameter is ≥1.5 times that of the adjacent normal segment in segmental ectasia. When there was no identifiable adjacent normal segment, the mean diameter of the corresponding coronary segment in the control group served as normal values. The number of epicardial coronary arteries with ectasia was analysed by use of a computerized quantitative coronary angiography analysis system (Philips BH 5000, Netherland).
2.3. Generation of Dendritic Cells and Cell Culture
Heparinized blood samples of the groups were drawn after coronary angiography from an antecubital vein with a 19-gauge needle without venous stasis in the fasting state. mDCs were generated according to an established method with minor modifications [13]. Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll Hypaque 1077 density (PAA laboratories GMBH, Austria). Monocytes were isolated from peripheral blood mononuclear cells by their adherence to plastic. During 9 days of cell culture in the presence of RPMI-1640 medium (Sigma Chemical, Germany) supplemented with 10% FCS (Sigma Chemical) monocytes were differentiated into DC.
2.4. Flow Cytometric Analysis
The technique of flow cytometry involves the staining of blood cells with fluorescence tagged antibodies targeted to specific cell surface associated antigens. This is followed by quantification of fluorescence intensity in the cell population of interest as a measure of the specific antigen abundance and by determination of percentage of cells displaying fluorescence intensity beyond a threshold [4]. DC were incubated with the mouse antihuman, fluorescein isothiocyanate (FITC)-conjugated antibodies against CD14, CD11b, CD11c, CD54, CD83, CD86, and MHC Class II-phycoerythrin (Beckman Coulter, CA, USA) for 15 minutes at room temperature. After immunofluorescence staining, cells were analyzed by Epics Profile II flow cytometer (Beckman Coulter). Appropriate isotype-matched immunoglobulins (Beckman Coulter) were used as negative controls. The mean fluorescence intensity (MFI) was analyzed for at least 5000 cells per sample [4].
2.5. Statistical Analysis
Statistical analysis was made by using SPSS for windows 11.0. Continuous variables were expressed as mean ± SD and categorical variables were expressed as percentage. Comparison of the expression levels of activation markers on mDCs among the groups was performed using one-way anova test. Tukey HSD multivariate analysis was used to determine among which groups the activation marker levels were different. The comparison of categorical variables between the groups were assessed by Fisher's exact or chi-square tests where appropriate. The association between the levels of activation markers and the number of ectasic vessels was calculated by Spearman's rho correlation coefficient. A P-value of <.05 was considered statistically significant.
3. Results
There was statistically no significant difference between the groups with respect to age, gender, body mass index, hypertension, diabetes mellitus, cigarette smoking and hyperlipidemia (P > .05). The distribution of obstructive CAD was also comparable between CAD patients with or without CAE [single vessel involvement 13.3%, two-vessel involvement 26.7%, and three vessel involvement 60% for both groups] (P > .05 for all). The patients in group 1 had diffuse CAE involving the left anterior descending artery in 4 (66.7%), the left circumflex artery in 5 (83.3%) and the right coronary artery in 4 patients (66.7%). One-vessel, two-vessel, and three-vessel ectasia were found to be present in 1 (16.7%), 2 (33.3%), and 3 (50%) patients, respectively. Therefore most of the patients (83.3%) had multivessel CAE. Clinical and coronary angiographic characteristics of the study population were presented in Table 1.
CAD patients with CAE were detected to have significantly higher levels of certain activation markers such as CD11b (44.5 ± 5.0 versus 30.0 ± 3.8 and 20.9 ± 3.6), CD11c, (96.3 ± 10.9 versus 66.1 ± 6.4 and 50.4 ± 5.7) CD54 (45.6 ± 6.7 versus 31.1 ± 4.9 and 20.8 ± 3.2), CD83 (44.6 ± 6.1 versus 30.8 ± 2.4 and 25.6 ± 2.8), CD86 (50.7 ± 5.0 versus 39.2 ± 4.1 and 29.5 ± 4.1) and MHC Class II (112.4 ± 11.3 versus 73.1 ± 9.5 and 54.5 ± 4.5) molecules on the surface of mDCs in comparison to CAD patients without CAE and normal subjects with angiographically normal coronary arteries (Figure 1). MFI of CD14 on mDCs did not significantly differ among Group 1 (13.3 ± 3.1), Group 2 (12.9 ± 2.6), and Group 3 (14 ± 2.9) (P > .05). Furthermore we detected a significant positive correlation between the number of the vessels with CAE and the levels of CD11c (Figure 2), CD86 (Figure 3), and MHC Class II molecules (Figure 4).
4. Discussion
The main findings of the present study are (1) the expression of CD11b, CD11c, CD54, CD83, CD86, and MHC Class II molecules in CAD patients with CAE were higher than control subjects with CAD alone and normal coronary arteries; (2) there was a correlation between the levels of CD11c, MHC Class II, CD86, and the number of coronary vessels with CAE. To our knowledge this is the first study that demonstrate the role of mDCs for CAE development in patients with CAD.
CAE has been defined as localized or diffuse nonobstructive lesions of the epicardial coronary arteries with a luminal dilatation exceeding the 1.5 fold of normal adjacent segment or vessel diameter [1]. It has been suggested that the pathogenesis of abdominal aortic aneurysm and CAE is similar that chronic transmural inflammation with destruction of medial layer of the vessel has a prominent role [2, 14]. Recently we have reported an increase in the plasma levels of tumor necrosis factor-alpha and interleukin-6 in patients with isolated CAE indicating an inflammatory process in the coronary circulation [15]. Furthermore Turhan H et al. showed that levels of soluble CAMs; intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin were increased in patients with isolated CAE in comparison to patients with obstructive CAD and suggested that a more extensive vascular wall inflammation may have a role in the development of isolated CAE [16]. Although the role of inflammation was demonstrated in the pathogenesis of CAE, since inflammation takes part both in CAE and atherosclerosis development, it is still not clear why some patients with obstructive CAD develop CAE whereas most do not.
DCs are a component of the proposed vessel-associated lymphoid tissue and are found in the intima and adventitia of susceptible arteries before atherosclerotic lesion development [6, 17]. In atherosclerotic plaques, the number of DCs increase related to the activation of residing intimal DCs and invasion of adventitial DCs to the plaque [6]. Monocytes that infiltrate the intima from the very early stages of atherosclerosis may differentiate into DCs and contribute to an increased DC population as well [6, 13, 18]. Recent findings suggest that DCs play a role in plaque destabilization through activation of T cells [6]. Yilmaz et al. found that up to70% of DCs in the shoulders of vulnerable carotid plaques express increased level of DC activation marker CD83 [9]. Ranjit et al. reported that CD86 was upregulated in patients with unstable angina in comparison to healthy donors [19]. Antigen processing, presentation, and T cell priming efficacy were shown to be maintained in DCs with increased expression of CD11c under hypercholesterolemic conditions associated with atherosclerosis [8]. Although the role of DCs in atherosclerosis were evaluated in various studies, there is limited data about DCs in CAE which has been suggested as a variant of atherosclerosis.
In the present study we have found increased expression of adhesion, costimulatory and antigen presenting molecules on the surface of mDCs in CAD patients with CAE compared to patients with CAD alone as well as normal subjects. Activated DCs have been shown to exhibit large numbers of adhesion molecules such as CD11b, CD54 which contribute to their ability to adhere to injured endothelium, transmigrate and also interact with T lymphocytes [5, 20, 21]. This interaction is accompanied by the increase in the expression of MHC and costimulatory molecules and the production of cytokines leading to their enhanced ability to prime T lymphocytes [4–6]. Therefore increased expression of the surface markers of mDCs with a positive correlation between CD11c, CD86, and MHC Class II molecules and the number of ectasic vessels observed in the present study may support the concept that a more severe and extensive chronic inflammation modulated by DCs takes place in the coronary circulation of CAD patients with CAE in correlation with the widespread involvement of CAE.
Numerous studies have demonstrated that aneurysm tissue contains high levels of matrix metalloproteinases (MMP) which causes degradation of the extracellular matrix leading to weakening and dilatation of the aortic wall [22]. The histopathological examinations of the coronary arteries in small number of cases with CAE were reported to include inflammatory cells in the medial layer comprising chiefly macrophages which were shown to have immunoreactivity against MMP [23, 24]. Recently we reported increased expression of monocyte adhesion molecules in patients with isolated CAE that may be associated with the medial destruction of the vessel wall and CAE formation in agreement with microscopic examinations [12]. Moreover Dogan et al. reported high plasma levels of MMP-3, MMP-9 and interleukin-6 in CAE patients and suggested that inflammation enhanced MMP secretion in the coronary circulation may cause CAE formation [25]. DCs were also shown to secrete MMP to the same extent as macrophages [26, 27]. Hence our finding of significantly increased DC activation in CAD patients with CAE compared to patients with CAD alone may be associated with weakening of the coronary artery wall by MMP of which secretion may be enhanced by DCs.
5. Study Limitations
It is conceivable that the small number of patients limits the power of our observations. The small number of patients in the present study reflects the small incidence of patients with CAE. The phenotypic characteristics of the most mature DCs have been suggested to be complete loss of CD14, and increased expression of CD83, CD86, and MHC Class II receptor [28]. DCs generated by our methods had low expression of CD14, moderate expression of CD83, CD86, and MHC Class II and thus would be considered as relatively immature DCs compared to DCs generated by the other methods [4, 28, 29]. However since mDCs of the three groups were cultivated in the same environment under the same conditions, the significantly different activation marker levels on the surface of mDCs among the groups detected in our study may be of importance. Further studies with larger samples using additional stimulators for improvement of DC maturation which also investigate the correlation between DC activation and MMP secretion are needed to firmly establish our results.
6. Conclusion
The present study indicates that mDCs display an increased cell surface concentration of adhesion, costimulatory, and antigen presentation molecules with respect to their activation in CAD patients with CAE compared to patients with CAD alone and normal subjects. DC activation may play an important role for CAE development in patients with obstructive CAD.
Figure 1 The graphic showing the mean expression levels of the activation markers on monocyte-derived dendritic cells in each study group. NCA: the group with normal coronary arteries. CAD: the group with coronary artery disease. CAD+: the group with coronary artery disease and coronary artery ectasia. MFI: mean fluorescence intensity.
Figure 2 The graph showing the relationship between the number of ectatic vessels and the expression level of CD11c molecule. r: Spearman's rho correlation coefficient. MFI: mean fluorescence intensity.
Figure 3 The graph showing the relationship between the number of ectatic vessels and the expression level of CD86 molecule. r: Spearman's rho correlation coefficient. MFI: mean fluorescence intensity. MFI: mean fluorescence intensity.
Figure 4 The graph showing the relationship between the number of ectatic vessels and the expression level of MHC Class II. r: Spearman's rho correlation coefficient. MFI: mean fluorescence intensity. MFI: mean fluorescence intensity.
Table 1 Clinical and coronary angiographic findings of the study population.
NCA (n=6)* CAD (n = 6) CAD + CAE (n = 6)
Age (years) 51.1 ± 4.2 51.1 ± 4.5 50.1 ± 4.0
Gender (male/female) 4/2 4/2 4/2
Hypertension 2/6 (33.3%) 2/6 (33.3%) 2/6 (33.3%)
Diabetes mellitus 2/6 (33.3%) 2/6 (33.3%) 2/6 (33.3%)
Hyperlipidemia 3/6 (50%) 3/6 (50%) 3/6 (50%)
Cigarette smoking 4/6 (66.7%) 4/6 (66.7%) 4/6 (66.7%)
CAE distribution
LAD — — 4/6 (66.7%)
Cx — — 5/6 (83.3%)
RCA — — 4/6 (66.7%)
One vessel — — 1/6 (16.7%)
Two vessel — — 2/6 (33.3%)
Three vessel — — 3/15 (50%)
*Indicates no significant difference between all groups for all parameters; NCA: normal coronary artery; CAD: coronary artery disease; CAE: coronary artery ectasia; LAD: left anterior descending artery; Cx: circumflex artery; RCA: right coronary artery.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2063495910-PONE-RA-17729R110.1371/journal.pone.0011548Research ArticleVirology/Emerging Viral DiseasesVirology/Host Antiviral ResponsesVirology/VaccinesInfectious Diseases/Respiratory InfectionsProperly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus HA1 Flu Vaccine Protect FerretKhurana Surender
1
Verma Swati
1
Verma Nitin
1
Crevar Corey J.
2
Carter Donald M.
2
Manischewitz Jody
1
King Lisa R.
1
Ross Ted M.
2
Golding Hana
1
*
1
Division of Viral Products, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, Maryland, United States of America
2
Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
Liu Ding Xiang EditorInstitute of Molecular and Cell Biology, Singapore* E-mail: [email protected] and designed the experiments: SK TMR HG. Performed the experiments: SK SV NV CJC DMC JM LRK. Analyzed the data: SK SV TMR HG. Contributed reagents/materials/analysis tools: SK SV NV CJC DMC JM LRK TMR. Wrote the paper: SK HG.
2010 12 7 2010 5 7 e115487 4 2010 16 6 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.2010This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.Background
In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 “Spanish Flu”. The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.
Methodology/Principal Findings
Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1–330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480). Protein yield for the HA1 (1–330) ranged around 40 mg/Liter, while the HA (1–480) yield was 0.4–0.8 mg/Liter.
Conclusions/Significance
This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.
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Introduction
In April 2009, the Centers for Disease Control and Prevention (CDC) announced the detection of a novel strain of influenza virus in humans. The novel virus derived its genes from viruses circulating in the pig population [1], [2], [3]. Due to sustained human-to-human transmission of this novel virus throughout the world, on June 11th the World Health Organization (WHO) raised the worldwide pandemic alert level to Phase 6.
The most effective way to curtail pandemics is by mass vaccination [4], [5]. At the moment there are two types of licensed vaccines against seasonal influenza in the US: subunit (split) inactivated vaccines (IV) and live cold adapted attenuated influenza vaccine (LAIV) [6]
[7], [8]. Both vaccines are grown in chicken eggs. The process of constructing a new vaccine strain based on newly circulating viruses is quite lengthy. It involves in vivo (in chicken eggs) or in vitro (in cell culture using reverse genetics techniques) reassortment between the internal genes of a donor virus such as A/PR/8/34 with the hemagglutinin (HA) and neuraminidase (NA) of the new influenza strain. The candidate vaccine strains must be further selected based on their high growth capability in eggs before they can be used for production of vaccines. Moreover, the manufacturing process is limited in scalability by the use of eggs and the amount of purified virus that can be produced. This process is used for the production of seasonal influenza vaccines every year, but it may pose a clear impediment to initiation of rapid mass vaccination against spreading pandemic influenza, as was evident for the 2009 H1N1 virus.
Recombinant HA based vaccines provide an alternative that could save several months of manufacturing time, since the HA gene of the newly circulating strain is available shortly after virus isolation. Expression of HA in insect cells and mammalian cells are under development and/or clinical trials [9], [10], [11]. The main challenge to the recombinant technology is to ensure that the HA products resemble the native virion-associated trimeric spike proteins and can elicit robust immune responses targeting protective conformational epitopes in the globular domain of HA.
In previous studies, we constructed H5N1 whole-genome-phage-display libraries (GFPDL) and used them to map the antibody responses following human infection with highly pathogenic H5N1 (A/Vietnam/1203/2004), as well as post-H5N1 vaccination sera. We identified large HA1 fragments, encompassing the receptor binding domain (RBD), that were bound by broadly neutralizing human monoclonal antibodies from H5N1 recovered individuals and by polyclonal convalescent sera. Several HA1 fragments were expressed and purified from E. coli inclusion bodies, and were shown to be properly folded and presented conformational epitopes [12]. The bacterially expressed HA1 proteins were also shown to absorb most of the neutralizing activity in post-H5N1 infection and post-H5N1 vaccination sera [12], [13]. Based on these studies, it was predicted that HA1 fragments that contain most of the neutralizing antibody targets may generate protective immunity against emerging influenza strains.
Compared with insect or mammalian cells, expression of recombinant proteins in bacteria could present a viable alternative in terms of large scale vaccine production and a short time line suitable for rapid response in influenza pandemic. Several studies with bacterially expressed HA proteins based on the H5N1 avian influenza virus (AIV) were reported [14], [15], [16], and one clinical trial with a bacterially expressed fusion protein between the HA fragment and flagellin from Salmonella typhimurium type 2 (STF2), a TLR5 agonist is underway [17]. However, bacterially expressed HA proteins are not subjected to the post-translational modifications that takes place in eukaryotic cells, including step-wise glycosylation process important for proper folding of the HA protein, as well as trimerization and transport to the cell membrane [18], [19], [20]. Indeed it was argued that in the absence of glycosylation, the newly synthesized HA proteins are not likely to fold properly or trimerize like native HA molecules, and may not present native conformational epitopes, which are important for generation of an effective protective immune response. Indeed the majority of the previous studies did not demonstrate proper folding and/or oligomerization of the HA proteins produced in prokaryotic systems [14], [15], [16], [21], [22]. To address this concern, we established multiple assays to monitor the integrity of bacterially expressed HA proteins for proper folding, formation of trimers and oligomers, receptor binding, and agglutination of red blood cells (RBC). Here, we describe the properties of two novel H1N1 swine-like HA proteins, HA1 (1–330) and HA (1–480), expressed in E. coli. Both proteins were properly folded as determined by CD spectra, binding to post-infection and post-vaccination sera, and could adsorbed neutralizing activity from H1N1 immune sera. However, only the bacterially expressed HA1 globular domain (1–330) contained functional trimers and oligomers capable of receptor binding and RBC agglutination. Importantly, vaccination of ferrets with both proteins resulted in reduced viral loads in nasal washes following challenge with novel H1N1 A/California/07/2009. However, after low dose vaccination (important for dose-sparing in pandemic scenario), HA1 (1–330) provided better reduction of morbidity (body temperature elevation and weight loss) compared with HA (1–480) in vivo.
Results
Properties of bacterially expressed H1N1 HA1 (1–330) and HA (1–480)
DNA fragments encoding amino acid sequence 1–330 and 1–480 of HA from A/California/07/2009 were cloned as NotI-PacI inserts in the T7 promoter based expression vector with His6 tag at the C-terminus [13]. Both fragments of H1N1 HA expressed in E. coli Rosetta Gami cells (Novagen) localized to insoluble fraction (inclusion bodies). IBs were refolded in vitro under controlled redox conditions and purified by HisTrap Fast flow chromatography. This process was previously shown to generate highly purified properly folded HA1 fragments from H5N1 [13]. The purified HA1 (1–330) and HA (1–480) proteins ran as a single band on SDS-PAGE with the anticipated MW of approximately 30 and 50 kDa, respectively (Fig. 1A).
10.1371/journal.pone.0011548.g001Figure 1 Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.
(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.
To determine if the bacterially expressed (unglycosylated) HA1 (1–330) and HA (1–480) proteins are properly folded they were analyzed by CD spectroscopy. The change in elipticity at 222 nm, which monitors unfolding of α-helix structures over a range of temperatures (CD melt), confirmed that both HA1 (1–330) and HA (1–480) behaved as properly folded proteins with a melting temperature around 52°C (Fig. 1 B-C).
We next determined if the bacterially expressed proteins oligomerized into higher molecular forms, using gel filtration chromatography on Superdex S200 XK 16/60 column (GE-Healthcare). Surprisingly, the HA1 (1–330) protein contained at least 50% of trimers and oligomers (Fig. 1D), while the larger HA (1–480) contained only monomers (Fig. 1E). To further investigate which HA forms are required for receptor binding we established a fetuin based SPR assay that mimics the simultaneous interactions between the virion spikes and multiple sialic acid moieties[23]. The bacterially expressed HA1 (1–330), HA (1–480), and a mammalian cell derived recombinant full length HA (HA0) (from Immune Technology Corp, NY) were tested for binding to fetuin coated on biosensor chips. As shown in Fig. 1G, only HA1 (1–330) bound efficiently to fetuin (but not to asialo-fetuin; Fig. 1F). In contrast, neither the mammalian cell derived HA0, nor the bacterially expressed HA (1–480) proteins bound to the fetuin in SPR (Fig. 1G).
In addition to receptor binding, hemagglutination of red blood cells (RBC) is a surrogate assay to measure the functionality of the influenza hemagglutinin. The presence of trimers and oligomers (mimicking the virion spikes) is required for the formation of RBC lattice [24]. As seen in Fig. 1H, H1N1 virions (positive control) agglutinated RBC very well at all the dilutions used. Bacterially expressed H1N1 HA1 (1–330) protein (containing trimers and oligomers) also agglutinated human RBC very efficiently, even at the lowest concentration of 45 ng/ml. On the other hand, the bacterially expressed HA (1–480) and mammalian cell derived recombinant HA0 did not agglutinate human RBC. These differences in hemagglutination were in agreement with the results obtained in the fetuin binding SPR assay. The lack of agglutinating capacity most likely reflected the absence of stable trimers and oligomers in the HA (1–480) or the mammalian cell derived HA0 protein preparations.
Bacterially expressed H1N1 HA (1–330) and HA (1–480) and mammalian-expressed HA0 are recognized by sera from ferrets infected with A/California/07/2009 and post vaccination human sera
The differences in the functional properties of HA1 (1–330) compared with the larger proteins containing the HA1+HA2 ectodomain needed further investigation. It was important to confirm that all three proteins expressed conformational “native” antigenic epitopes, recognized by antibodies elicited by H1N1 infection or by immunization with traditional (inactivated) vaccine. Ferrets are a good animal model for influenza virus pathogenesis. Following H1N1 infection, ferrets undergo transient loss of body weight, elevation in body temperature, and extensive viral replication in the upper and lower respiratory track on days 1–5, followed by viral clearance and recovery between Days 7–14 [25]. In the current study, consecutive post-H1N1 infection sera were evaluated for virus neutralizing antibody titers in a microneutralization assay (MN) (Fig. 2A) and for binding to recombinant H1N1 HA proteins by surface plasmon resonance (SPR), using either mammalian cell expressed HA0 or the bacterially expressed H1N1 HA1 (1–330) and HA (1–480) proteins (Fig. 2B–D). MN titers in the ferret sera were <20 during the first 5 days, followed by a rapid rise on days 7 and 14, and started to decline there after (Fig. 2A). On the other hand, using SPR, HA binding antibodies were measured as early as day 5 post infection and peaked on day 14. Importantly, binding of post-H1N1 infection ferret sera to HA0 from mammalian cells and to the bacterially expressed HA1 (1–330) and HA (1–480) proteins demonstrated similar kinetics and binding avidity profiles (Fig. 2 B-C-D), confirming that the bacterially expressed proteins were antigenically similar to the mammalian cell derived HA. The significant increase in binding to H1N1-HA proteins on days 7 and 14 correlated with the appearance of neutralizing antibodies against A/California/07/2009 (Fig. 2A). We also evaluated the binding of pre-and post-vaccination sera from two individuals that were immunized with a licensed inactivated subunit A/California/7/2009 vaccine (Fig. 2 E-F-G). Post-vaccination sera bound to mammalian-expressed HA0 (Fig. 2E) and to bacterially-expressed HA1 (1–330) (Fig. 2F) & HA (1–480) (Fig. 2G) proteins. The SPR results confirmed that all three proteins were properly folded and expressed native conformational epitopes. Interestingly, in both ferret and human studies, antibody binding to the HA1 (1–330) (containing trimers) was superior to that observed with the mammalian cell derived HA0 and the bacterially expressed HA ectodomain proteins (Fig. 2C vs. 2B and 2D, and Fig. 2F vs. 2E and 2G).
10.1371/journal.pone.0011548.g002Figure 2 Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.
(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).
Properly folded bacterial H1N1 HA proteins adsorb neutralizing activity in post-H1N1 vaccination and post-H1N1 infection sera
The functional relevance of binding to properly folded bacterially expressed H1N1-HA proteins was further confirmed in adsorption experiments (Table 1). Both bacterially expressed HA1 (1–330) and HA (1–480) proteins and the mammalian cell derived HA0 adsorbed most of the neutralizing activity of H1N1 hyperimmune sheep sera (NIBSC), reducing the MN titer from 1∶6,400 to <1∶40 (Table 1, top panel). Similar results were obtained with post-H1N1 infection ferret sera from day 21. The H1N1-HA1 (1–330) reduced the neutralizing activity of the convalescent sera from 1∶1,280 to <1∶40. In the case of HA0 (mammalian) and bacterial HA (1–480), residual neutralizing activity (1∶80) was observed after sera adsorption (Table 1, lower panel).
10.1371/journal.pone.0011548.t001Table 1 Adsorption of neutralization activity using HA proteins.
Sheep anti-A/California/07/2009 -HA-sera (NIBSC)
Peptides added TITER*
No peptide 6400
HA 1–330 - FLOW-THROUGH <40
HA 1–480 - FLOW-THROUGH <40
Mammalian HA0 - FLOW-THROUGH <40
GST-His - FLOW-THROUGH 6400
Ferret anti-A/California/07/2009 -infected sera
No peptide 1280
HA 1–330 - FLOW-THROUGH <40
HA 1–480 - FLOW-THROUGH 80
Mammalian HA0- FLOW-THROUGH 80
GST-His - FLOW-THROUGH 1280
*End-point titers (mean of three replicates) using polyclonal rabbit sera in a microneutralization assay performed with A/California/07/2009 (X-179A).
The combined data from the CD melt, SPR-based binding assays, and adsorption studies demonstrated that both bacterially expressed proteins are properly folded and express antigenically relevant conformational neutralizing epitopes. However, only the HA1 globular domain but not the HA ectodomain (or the mammalian cell expressed HA0) contained functional trimers and oligomers required for fetuin binding and RBC agglutination.
Immunization of rabbits and sheep with bacterially expressed H1N1 HA1 (1–330) and HA (1–480) and mammalian HA0 proteins
To evaluate the immunogenicity of the bacterially expressed proteins, we immunized rabbits after mixing of HA1 (1–330) or HA (1–480) with Titermax adjuvant. The pre- and post vaccination sera were evaluated by microneutralization assay. Even after a single immunization with HA1 (1–330), rabbits had a MN titer of 1∶40. After second and third immunizations high MN titers were measured (6,400 and 25,600, respectively) (Table 2, top panel). The HA (1–480) elicited H1N1 neutralizing antibodies only after the second and third boosts, and the peak MN titers (3,200 and 6,400, respectively) were lower compared with the HA1 (1–330) immunized rabbits. In a separate study, sheep were vaccinated with mammalian derived HA0 (Immune Technology Corps) or with the bacterially–expressed HA1 (1–330) (Table 2, bottom panels). Again, the kinetics of immune responses and the peak neutralization titers were significantly higher for the sheep immunized with the bacterially-expressed HA1 (1–330) protein compared with the mammalian HA0 expressed and purified from 293 cells.
10.1371/journal.pone.0011548.t002Table 2 Mean reciprocal neutralizing titers of Rabbit and Sheep anti-HA sera.
RABBIT SERA END-POINT TITERS*
H1N1-HA (1–330)
Pre Vaccine <20
Post- 1
40
Post- 2
6,400
Post- 3
25,600
H1N1-HA (1–480)
Pre Vaccine <20
Post- 1 <20
Post- 2
3,200
Post- 3
6,400
SHEEP
Mammalian H1N1-HA0
Pre Vaccine <20
Post- 1 <20
Post- 2
1,600
Post- 3
6,400
H1N1-HA (1–330)
Pre Vaccine <20
Post- 1
80
Post- 2
12,800
Post- 3
51,200
*End-point titers (mean of three replicates) using polyclonal rabbit sera in a microneutralization assay performed with A/California/07/2009 (X-179A).
Vaccination and challenge studies in ferrets
The protective immunity elicited by bacterially expressed proteins was further evaluated in a ferret challenge model [25]. Female Fitch ferrets (n = 4 in each group) were vaccinated intramuscularly in the quadricep muscle on day 0 and boosted on day 21 with either HA1 (1–330) or HA (1–480) proteins at 7.5 and 30 μg dose combined with Titermax adjuvant. Serum samples were collected after vaccinations and analyzed in HAI (Fig. 3). The 30 μg dose induced 2–4 fold higher titers compared with the 7.5 μg dose for both bacterially expressed proteins (Fig. 3). However, at the lower dose of 7.5 μg, the HA1 (1–330) consistently elicited higher HAI titers compared with the HA (1–480) at the same dose. The observed HAI titers measured in the current study were similar to data from recently reported ferret vaccination studies in which commercially available live attenuated or split-inactivated licensed vaccines were used [26], [27].
10.1371/journal.pone.0011548.g003Figure 3 Hemagglutination-inhibition (HAI) titers in ferrets.
HAI antibody in ferrets (n = 4 per group) vaccinated with either 30 µg or 7.5 µg of influenza H1N1 HA1 or HA. Blood was collected at day 35 (post-dose 2). HAI responses were assessed against A/California/07/2009. Bars indicate geometric mean titer (GMT). The titer from each individual ferret is indicated by symbol. *p = 0.05 HA (1–480) low dose vs. HA (1–480) high dose.
Following second vaccination, ferrets were challenged intranasally with 1×106 50% egg infectious doses (EID50) (∼1×105.75 TCID50/ml) of A/California/07/2009 virus in a volume of one milliliter. To determine viral loads in nasal washes, each ferret was administered each day post-challenge with 1.5 ml of 0.9% saline to each nare and washes were collected for virus titer determinations using the plaque assay.
In unvaccinated animals (naïve), viral loads in the nasal washes were highest on day 1, gradually declining on days 3 and 5 (Fig. 4A) and were back to baseline on day 7 as previously described[25]. Among the vaccinated animals, the high dose groups (30 μg), receiving either HA1 (1–330) or HA (1–480), reduced viral titers by >2 logs as early as day 1 post challenge. In the 7.5 μg vaccinated animals, virus replication on day 1 was observed, followed by a more rapid decline compared with the unvaccinated animals (Fig. 4A). Between day 3 and 5, a more rapid virus clearance was observed in the HA1 (1–330) vaccinated groups compared with the HA (1–480) vaccinated group or the naïve group (Fig. 4A).
10.1371/journal.pone.0011548.g004Figure 4 Viral loads and morbidity following A/California/07/2009 challenge in ferrets.
(A) Viral replication of influenza A/California/07/2009 in nasal washes following intranasal challenge. Average pfu of virus from the nasal washes of each group (4 ferrets per group) on days 1, 3, and 5 post challenges. (B) Change in body temperature and (C) percent body weight.
In terms of morbidity, sustained elevation in body temperatures were measured in the naïve group post H1N1 virus challenge between days 1–4 (Fig. 4B). Inactivity and weight loss were also recorded up to day 7, followed by a slow recovery that did not reach normal weights by day 13 (termination) (Fig. 4C and data not shown). The HA1 (1–330) vaccinated animals that received 30 μg protein showed no temperature elevation and no weight loss (Fig. 4B-C). The 7.5 μg HA (1–330) vaccine dose also showed no weight loss and only a brief mild increase in body temperature on Day 2 (Fig. 4B-C). The HA (1–480) vaccinated animals at the 30 μg dose also showed no weight loss, and a transient elevation in body temperature on days 1–3 (not as high as in the naive group). But the animals that received HA (1–480) at the lower dose (7.5 μg) showed an increase in body temperature similar to the naïve group and some weight loss on days 2–6 post challenge.
Together, these data demonstrate that properly folded bacterially expressed unglycosylated H1N1 HA proteins, elicited high neutralizing antibody titers in ferrets, not different from previously reported antibody responses against licensed influenza vaccines ([26], [27]. Importantly, at the lower vaccine dose of 7.5 μg, the HA1 (1–330) that contained both trimers and oligomers protected ferrets from morbidity more efficiently than the HA (1–480), which only contain monomers. The clinical symptoms correlated with the observed HAI titers prior to challenge.
Discussion
The recent 2009-H1N1 swine-like virus influenza pandemic highlighted the need to rapidly produce enough vaccine doses for global vaccination brought to light the shortcomings of the traditional process of manufacturing influenza vaccines and the need to use alternative approaches for a more rapid generation of vaccine for global immunization in response to impending influenza pandemic. Bacterially expressed HA proteins can be manufactured rapidly and are amenable to mass production that can fulfill global vaccine needs. The main challenge to the prokaryotic production system is to ascertain proper refolding of expressed HA proteins representative of native HA spike structures on influenza virus. In addition to properly folded HA monomers, higher MW structures (i.e., trimers and oligomers) are important and likely to contribute to the optimal immunogenicity of the HA, since all influenza neutralizing antibodies are conformation dependent and some trimer specific antibodies have potent neutralizing activity [28]. In eggs and mammalian cells, post-translational glycosylation contribute to the proper folding, trimerization and transport of the newly synthesized HA molecules to the cell membrane [18]. However, in the case of recombinant HA proteins, trimerization is not always found even in eukaryotic cell substrates [11].
The main findings in the current study are: (a) bacterially expressed H1N1 HA1 (1–330) and HA (1–480) can be purified as properly folded proteins as determined by CD spectroscopy, SPR analyses with H1N1 immune sera, and adsorption of neutralizing activity from post-infection and post vaccination sera; (b) the HA1 (1–330) contained >50% trimeric and oligomeric forms and could bind to fetuin and agglutinated human RBC, while the HA (1–480) and mammalian cell expressed HA0 proteins were predominantly monomeric, did not bind fetuin, and did not agglutinate RBC; (c) the HA1 (1–330) induced higher titers of neutralizing antibodies compared with HA (1–480) or mammalian derived recombinant HA0 in rabbits and sheep; (d) in a ferret H1N1 challenge model, high-dose vaccination (30 μg HA) with both bacterially expressed HA1 (1–330) and HA (1–480) proteins protected animals from morbidity (elevated body temperature and weight loss) following challenge with novel H1N1 A/California/07/2009 virus. However, low-dose vaccination (7.5 μg HA) of ferrets with HA1 (1–330) resulted in lower morbidity and more rapid virus clearance compared with the HA (1–480) vaccinated group.
This study extends our previous reports with the H5N1 highly pathogenic virus, in which we have used whole-genome-phage display libraries (GFPDL) to map the antibody responses following human infection or vaccination. We have identified large HA1 fragments, encompassing the receptor binding domain (RBD), that were bound by broadly neutralizing human monoclonal antibodies from H5N1 recovered individuals and by their polyclonal convalescent sera [12]. In a subsequent study, we found that following vaccination with inactivated H5N1 (A/Vietnam/1203/2004) influenza vaccine the immune sera from the MF59-adjuvanted vaccinated individuals bound with much higher avidity to bacterially expressed properly folded H5 HA1 proteins compared with unadjuvanted vaccine sera [13]. Importantly, the bacterially expressed HA1 proteins were also shown to absorb most of the neutralizing activity in post infection and post vaccination sera [12], [13]. Based on these studies, we hypothesized that bacterially-expressed HA1 fragments if properly folded, could be useful as vaccines against emerging influenza strains.
In the current study, we found that expression and purification of properly folded H1N1 HA1 (1–330) (lacking HA2) in bacterial system was more efficient and gave higher yield compared with the larger HA ectodomain (1–480). Between 39–45 mg of >90% purified HA1 (1–330) protein can be obtained from 1 liter of bacterial batch culture, while the yield for HA (1–480) was only 0.4–0.8 mg/L. In addition to the much lower yield, the HA (1–480) contained only monomers, and as a result, it did not bind to fetuin and did not agglutinate RBC. Both of these functions require the presence of quartenary HA forms (i.e. trimers and oligomers). Mammalian expressed HA0 protein exhibited the same properties as the bacterially expressed HA(1–480) ectodomain. This is in agreement with previous reports on full length HA ectodomain proteins expressed in a variety of cell substrates wherein peptide linkers were introduced to facilitate oligomerization [11]. Similar to our findings, the oligomerized cell-based HA product showed better neutralizing antibodies than its monomeric counterpart [11].
While both proteins were immunogenic in ferrets at the high dose of 30 μg, the HA1 (1–330) was more immunogenic and protected ferrets from H1N1 morbidity more efficiently at a lower dose (7.5 μg) compared with the HA (1–480) protein. In the case of mass vaccination, dose sparing is likely to be of great impact.
The ability of HA1 globular domain to form trimers has not been reported before. We have recently confirmed that bacterially expressed HA1 globular heads from multiple influenza strains (avian H5N1 and seasonal strains), can be produced at high yield. In all cases the HA1 proteins contain trimers and oligomers and agglutinate RBC. Studies to map the trimerization signal of HA1 are ongoing.
In the face of an impending influenza pandemic, HA1 proteins derived from the newly spreading virus can be rapidly expressed in bacterial systems several months before the traditional approach using vaccine strains generated via either gene reassortment or reverse genetics, followed by adaptation to growth in eggs. With appropriate testing methods in place to monitor proper folding and biological activity (hemagglutination assay), this simple and efficient approach may provide an early vaccine for large scale production to fulfill global vaccine needs in a much shorter time frame. Moreover, bacterially produced HA vaccines may also be an alternative for humans with known egg allergies that cannot be immunized with traditional influenza vaccines produced in eggs.
Materials and Methods
Expression vector and cloning of H1N1-HA1 (1–330) and HA (1–480)
cDNA corresponding to the HA gene segment of A/California/07/2009 was generated from RNA isolated from egg-grown virus strain, and was used for cloning. pSK is a T7 promoter based expression vector where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. DNA encoding HA1 (1–330) and HA (1–480) were cloned as NotI-PacI inserts in the pSK expression vector.
Protein expression, refolding and purification
E. coli Rosetta Gami cells (Novagen) were used for expression of H1N1-HA1 (1–330) and HA (1–480). Following expression, inclusion bodies (IB) were isolated by cell lysis and multiple washing steps with 1% Triton X-100. The final IB pellets were resuspended in denaturation buffer containing 6M Guanidine Hydrochloride and dithioerythreitol (DTE) at final protein concentration of 10 mg/ml, and were centrifuged to remove residual debris. For refolding, supernatants were slowly diluted 100-fold in redox folding buffer [13]. The renaturation protein solution was dialyzed against 20 mM Tris HCl pH 8.0 to remove the denaturing agents. The dialysates were filtered through 0.45 μm filters, and were subjected to purification by HisTrap Fast flow chromatography. This process was previously shown to generate highly purified properly folded HA1 fragments from H5N1 [13].
Circular Dichroism (CD)-monitored equilibrium unfolding experiment
To demonstrate that the bacterially expressed HA fragments are properly folded they were analyzed by CD spectroscopy [13]. For CD spectroscopy in solution, H1N1-HA proteins were dissolved in 20 mM PBS, pH 7.4, at 0.1 mg/ml. The change in elipticity at 222 nm (to follow unfolding of α-helices) during unfolding was monitored using a J-715 Circular Dichroism system (JASCO). The unfolding reaction was initiated by subjecting the protein in PBS to 1°C/min increments. The experiments were carried out in triplicate.
Gel filtration Chromatography
H1N1-HA1 (1–330) and HA (1–480) at a concentration of 5 mg/ml were analyzed on Superdex S200 XK 16/60 column (GE-Healthcare) pre-equilibrated with PBS, and the protein elution monitored at 280 nm. Protein molecular weight marker standards (GE healthcare) were used for column calibration and generation of a standard curve to identify the molecular weights of the test protein sample.
Affinity measurements by surface plasmon resonance
Steady-state equilibrium binding of post-H1N1 vaccine or post-H1N1 infection sera was monitored at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). The H1N1-HA proteins were coupled to a GLC sensor chip (BioRad Labs) with amine coupling with 500 resonance units (RU) in the test flow cells. Ten-fold dilution of animal sera (60 µl) was injected at a flow rate of 30 µl/min (120-sec contact time). Flow was directed over a mock surface to which no protein was bound, followed by the HA protein coupled surface. Responses from the protein surface were corrected for the response from the mock surface and for responses from a separate, buffer only, injection. MAb 2D7 (anti-CCR5) and naïve ferret sera were used as a negative control antibody in the experiments. Binding kinetics for the animal sera and the data analysis were performed with BioRad ProteON manager software (version 2.0.1). Similar binding studies were previously conducted with H5N1 HA1 proteins. Human monoclonal antibodies with conformation-dependent epitopes bound only to the properly folded HA proteins that were purified at pH 7.2 (identical to the current study) but not to unfolded HA1 proteins, purified at pH 3.0 [13].
Receptor binding assay using surface plasmon resonance
Binding of different HA1 derivatives to fetuin (natural homolog of sialic acid cell surface receptor proteins) and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Fetuin or Asialo-fetuin (Sigma) were coupled to a GLC sensor chip with amine coupling at 1000 resonance units (RU) in the test flow cells. Samples of 60 µl freshly prepared H1N1-HA1, HA0, and mammalian derived HA0 proteins at 10 µg/ml were injected at a flow rate of 30 µl/min (120-sec contact time). Flow was directed over a mock surface to which no protein was bound, followed by the fetuin or asialo-fetuin coupled surface. Responses from the protein surface were corrected for the response from the mock surface and for responses from a separate, buffer only, injection. Binding kinetics and data analysis were performed with BioRad ProteON manager software (version 2.0.1).
Hemagglutination Assay
Human erythrocytes were separated from whole blood (Lampire Biologicals). After isolation and washing, 30 µl of 1% human RBC suspension (vol/vol in 1% BSA-PBS) was added to 30 µl serial dilutions of HA protein or influenza virus in 1% BSA-PBS in a U-bottom 96-well plate (total volume, 60 µl). Agglutination was read after incubation for 30 min at room temperature.
Neutralizing Antibodies Adsorption with HA proteins
Five-fold diluted post-H1N1 vaccination (NIBSC) sera or post-H1N1 infection ferret sera (500 µl) were added to 0.5 mg of purified HA-His6 or to control GST-His6 protein, and incubated for 1 hr at RT. Nickel-nitrilotriacetic acid (Ni-NTA) magnetic beads (200 µl) (Qiagen) were added for 20 min at RT on end-to-end shaker, to capture the His-tagged proteins and the antibodies bound to them, followed by magnetic separation. Supernatants containing the unbound antibodies were collected. The pre-and post-adsorbed sera were subjected to virus microneutralization assay.
Rabbit immunization and virus neutralization assays
White New Zealand rabbits were immunized three times intramuscularly at 21-day intervals with 100 µg of purified H1N1-HA1 (1–330) or HA 1–480) proteins with Titermax adjuvant (Titermax Inc). Virus-neutralizing titers of pre- and post vaccination rabbit sera were determined in a microneutralization assay based on the methods of the pandemic influenza reference laboratories of the Centers for Disease Control and Prevention (CDC). Low pathogenicity H1N1 virus, generated by reverse genetics, was obtained from CDC (X-179A). The experiments were conducted with three replicates for each serum sample and performed at least twice.
Vaccination of ferrets and blood collection
Ferrets used in the study were tested to be sero-negative for circulating seasonal influenza A (H1N1 and H3N2) and influenza B viruses by HAI. Female Fitch ferrets (n = 4 in each group) were vaccinated intramuscularly in the quadriceps muscle on day 0 and boosted on day 21 and then challenged with virus on day 35. Control animals (n = 4) were mock vaccinated with phosphate buffered saline (PBS; pH 7.2). Each animal was vaccinated with one of two doses (30 μg or 7.5 μg) of recombinant HA in sterile 0.9% saline. Each vaccine was mixed with the adjuvant formulation, TiterMax (TiterMax USA, Inc, Norcross, Georgia, US) at a 1∶1 ratio. The volume for all intra-muscular vaccinations was 0.5 ml. The first and second vaccinations were given in the left and right hind legs, respectively. Blood was collected from anesthetized ferrets via the anterior vena cava. The collected blood was transferred to a tube containing a serum separator and clot activator and allowed to clot at room temperature. Tubes were centrifuged at 6000 rpm for 10 minutes; serum was separated, aliquoted and stored at −80±5°C. All procedures were in accordance with the National Research Council (NRC) Guidelines for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the Centers for Disease Control (CDC)/National Institutes of Health (NIH) Bio- Safety Guidelines in Microbiological and Biomedical Laboratories and approved by the Institutional Animal Care and Use Committee (IACUC).
Infection and monitoring of Ferret
Animal experiments with virus A/California/07/2009 were performed in the AALAC-accredited ABSL-3 enhanced facility. Animals were infected and monitored as previously described[27] except using 5% isofluorane anesthesia. Briefly, ferrets were anesthetized with isofluorane and infected intranasally with 1×106 50% egg infectious doses (EID50) (∼1×105.75 TCID50/ml) of A/California/07/2009 in a volume of one milliliter. Animals were monitored for temperature, weight loss, loss of activity, nasal discharge, sneezing and diarrhea daily following viral challenge. To determine viral load from nasal washes, 1.5 ml of 0.9% saline was administered to each nare and the wash was collected each day post-challenge of each ferret. Temperatures were measured through use of an implantable temperature transponder (BMDS, Sayre, PA) and were recorded at approximately the same time each day. Pre-infection values were averaged to obtain a baseline temperature for each ferret. Clinical signs of sneezing and nasal discharge, inappetence, dyspnea, neurological signs, respiratory distress, and level of activity were assessed daily. A scoring system was used to assess activity level where 0 = alert and playful; 1 = alert but playful only when stimulated; 2 = alert but not playful when stimulated; 3 = neither alert nor playful when stimulated. Based on the daily scores for each animal in a group, a relative inactivity index was calculated [29].
Hemagglutinination Inhibition (HAI) assay
RDE-treated ferret sera were serially diluted in v-bottom 96-well microtiter plates followed by the addition of 8 hemagglutination units (HAU) of influenza virus. Following an incubation of approximately 20 minutes, 0.5% suspension of turkey RBC (TRBC) in PBS (pH 7.2) were added and mixed by agitation. The TRBCs were allowed to settle for 30 minutes at room temperature and HAI titers were determined by the reciprocal value of the last dilution of sera which completely inhibited hemagglutination of TRBC. A negative titer was defined as 1∶10.
Determination of viral loads
Viral loads in nasal washes were determined by the plaque assay. Briefly, MDCK cells plated in 6-well tissue culture plates were inoculated with 0.1 ml of virus-containing sample, serially diluted in Dulbecco's modified Eagle's medium (DMEM). Virus was adsorbed to cells for 1 h, with shaking every 15 min. Wells were overlaid with 1.6% w/v Bacto agar (DIFCO, BD Diagnostic Systems, Palo Alto, CA, USA) mixed 1∶1 with L-15 media (Cambrex, East Rutherford, NJ, USA) containing antibiotics and 0.6 mg/ml trypsin (Sigma, St. Louis, MO, USA). Plates incubated for 5 days. Cells were fixed for 10 minutes using 70% v/v Ethanol and then overlaid with 1% w/v crystal violet. Cells were then washed with deionized water to visualize plaques. Plaques were counted and compared to uninfected cells.
The authors thank Maryna Eichelberger and Vladimir Lugovtsev for thorough review of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was funded in part by an American Recovery and Reinvestment Act supplement to NIH/NIAID grant UO1-AI077771 to TMR. This study was also partly supported by IAA 224-10-1006 from DMID, NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Mol CancerMolecular Cancer1476-4598BioMed Central 1476-4598-9-1482055070810.1186/1476-4598-9-148ResearchLoss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF Johnson Thomas R [email protected] Sweaty [email protected] Binod [email protected] Lakshmipathi [email protected] Sarah [email protected] Paul D [email protected] Randall B [email protected] Hari K [email protected] Program in Urosciences, Division of Urology-Department of Surgery, University of Colorado Denver School of Medicine, Denver Veterans Administrative Medical Center, and University of Colorado Comprehensive Cancer Center, Building P15 or RC2, C-317, 12700 E 19th Avenue, Aurora, CO 80045, USA2010 15 6 2010 9 148 148 18 11 2009 15 6 2010 Copyright ©2010 Johnson et al; licensee BioMed Central Ltd.2010Johnson et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Prostate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer.
Results
We observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer.
Conclusions
These studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.
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Background
Prostate cancer is the second leading cause of cancer death in men. In the United States alone, 192,280 new cases of prostate cancers were diagnosed in 2009 and among them around 27,360 deaths occurred. One of the biggest challenges we face in prostate cancer is determining if the cancer is aggressive. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is no cure once the disease has spread beyond the prostate. Reduction in serum prostate-specific antigen (PSA) levels has been proposed as an endpoint biomarker for human prostate cancer intervention. However, despite being the mainstay of prostate cancer detection, the value of PSA screening is still debated. In particular, there is a growing concern regarding the over diagnosis of potentially indolent disease [1]. Therefore, there remains an urgent need for more accurate biomarkers to diagnose aggressive prostate cancer. Thus, identification of new molecular markers/targets for aggressive prostate cancer is important in order to improve early detection of the aggressive disease and to develop new therapeutic regimens.
Progression of prostate cancer from focal, androgen-dependent lesions to androgen-independent, metastatic cancer requires deregulation of growth control, invasiveness and cell motility. Abundant evidence demonstrates roles for Ets transcription factors in many cancers including prostate. Prostate-derived Ets factor (PDEF), first described nine years ago as preferentially binding to the noncanonical Ets core sequence GGAT [2], has recently received considerable attention due to its potential importance in regulating cell motility and invasion [3-5]. Recently, proteomic analysis of PDEF overexpressing cells revealed 286 proteins in the PDEF-associated protein complex in breast cancer [6]. Thus interaction of PDEF with other partner proteins could help in finding their role in maintenance of malignant phenotype. Published literature concerning experimental manipulation of PDEF expression is paradoxical and limited to tissues of high epithelial content, notably prostate, breast, ovary and colon [7,8]. PDEF expression has been both positively [3,9] and negatively [10] correlated with breast cancer grade at mRNA or protein levels. It is important to note that PDEF mRNA and protein levels do not always correlate, which may have led to different conclusions in some of the studies examining PDEF expression in primary tumors. Turner et al. [4] found that introducing PDEF into invasive breast cancer cell lines reduced their invading ability. Similarly, siRNA-mediated knockdown of PDEF in MCF7 cells increased their ability to migrate in the Transwell assay. Besides its role in cancer metastasis, PDEF expression was also correlated with changes in the actin cytoskeleton and focal adhesion localization, and loss of cellular polarity. Ghadersohi et al. [10] silenced PDEF expression in MCF7 cells, and found that such cells showed greatly accelerated xenograft tumor formation in SCID mice. By contrast, Gunawardane et al. [3] showed that increasing expression of PDEF increased their ability to migrate in a Transwell assay and stimulated colony formation in soft agar. This group also identified a canonical MAP kinase phosphorylation site at T50 (PAT50P) and showed that mutation to alanine at this site abolished all the effects they observed. To date there are few data available formally correlating PDEF expression in maintenance of prostate malignant phenotype. Two published studies, one with a prostate cancer cell-line [5] and another with clinical samples from prostate [11] reached opposite conclusions with respect to the role of PDEF in prostate cancer. Clearly additional studies are necessary to evaluate role of PDEF in prostate cancer biology.
In the current studies, we report here that PDEF expression is lost, whereas MMP9 expression increased with the aggressive behavior of prostate cancer. Overexpression of PDEF in PC3 cells strongly inhibits colony formation, cell migration and invasion, and increased cell adherence. Furthermore, re-introduction of PDEF in PC3 cells led to changes in actin cytoskeleton, altered focal adhesion kinase activity, and reestablished cell polarity in these cultures as indicated by induction of less invasive spheroid-like structures in three-dimensional culture, Moreover, PDEF expression downregulates MMP9 expression, and its promoter activity in PC3 cells. Thus, consideration of both PDEF and MMP9 may have a better prognosis value for determining the aggressive phenotype of prostate cancer.
Materials and methods
Constructs and cell lines
All cell lines (PC3, LNCaP, and C4-2B) were purchased from ATCC and maintained according to ATCC guidelines. Phoenix cells were grown in DMEM containing 10% fetal bovine serum. FLAG tag antibody was purchased from Sigma (St. Louis, USA). PDEF and phospho FAK antibodies were from Santa Cruz Biotechnology (CA, USA). PDEF was cloned from PC3 cDNA with an amino-terminal FLAG tag, and inserted into retroviral vectors pBABE and the bicistronic vector QCXIX (Clontech). The latter vector was modified to contain a wild-type internal ribosome entry site to increase expression from the second multiple cloning site, into which G418 resistance was cloned. Mutations were created using the Quick-Change kit (Stratagene) according to the manufacturer's instructions. Oligonucleotide primers for PCR were purchased from Integrated DNA Technologies.
Retrovirus production and infection
Phoenix cells were transfected with 2 μg DNA using Effectene (Qiagen) according to the manufacturer's instructions, and infection was followed according to Phoenix™ Retrovirus Expression System (Orbigen Inc.). After infection, cells were trypsinized, transferred to 150 mm dishes, and subjected to puromycin or G418 selection after 48 h incubation.
Thymidine incorporation
1 × 105 cells/well were plated in 12-well plates. 48 h later, cells were exposed to medium containing1-3 μCi/ml 3H-thymidine (Perkin Elmer) and incubated for 4 h. After 2 washes with cold PBS, cells were fixed in cold methanol for 5 min followed by an additional methanol wash. Cells were solubilized in 0.1% SDS/0.2 M NaOH and radioactivity determined.
Anchorage independent growth, motility, invasion and attachment assays
Growth in soft agar was performed as described previously [12]. Invasiveness was determined by the method of Repesh LA [13]. Cell migration through Transwell membranes was performed identically, but without the use of Matrigel. Wound healing assays were performed by making a cruciform scratch in a confluent monolayer of cells. Cells were washed, the medium replaced with serum-free medium, and incubated for 48-72 h. Cells were fixed with methanol, stained with Giemsa, and photographed.
Attachment assays were performed essentially by the method of Turner et al. [4] using 96-well plates pretreated overnight with fibronectin, Matrigel, or bovine serum albumin at concentrations of 50 ng/ml, 100 ng/ml, and 10 mg/ml respectively.
Immunohistochemistry for PDEF and MMP9 expression on prostate tissue array slides
Tissue microarray slides containing 9 normal and 40 prostate cancer samples of varying pathological grade were obtained from Imgenex Corporation, San Diego, CA, 92121. Immuno histochemistry for PDEF and MMP9 was performed using the avidin-biotin complex method previously described by Hsu et al. [14]. Expression of PDEF and MMP9 were evaluated by analysis of microscopic scans of each tissue. Expression was considered high if greater than 60% of the scanned area scored positive, while expression was considered moderate if 40-60% area scored positive, and expression was considered low if less than 40% area scored positive.
Reverse transcription PCR
Total RNA was extracted using RNEasy mini kit (Qiagen). 1 μg of RNA was used to prepare cDNA using iScript second strand cDNA synthesis kit (Bio-Rad). 100 ng of synthesized cDNA was used for RT-PCR using forward 5-TTGACAGCGACAAGAAGTGG-3 and reverse 5-TCACGTCGTCCTTATGCAAG-3 for MMP9, forward 5-ACCACAGTCCATGCCATCAC-3 and reverse 5-TCCACCACCCTGTTGCTGTA-3 for GAPDH. Transcripts were separated by agarose gel electrophoresis.
Reporter assay
Cells were transfected with 1 μg of MMP9 luciferase reporter vector along with 10 ng of Renilla luciferase expression plasmid using Effectene (Qiagen, Valencia, CA) according to manufacturer's instructions. Luciferase activity was measured using the Dual luciferase kit (Promega Corporation, Madison, WI) with Monolight 2010 Luminometer (Analytical Luminescence laboratory, San Diego, CA).
MMP Zymography
Zymogram for MMP9 activity was performed according to Bernhard and Muschel [15] using conditioned medium.
Three dimensional cell culture
PDEF overexpressing PC3 cells and respective vector control cells were grown in growth factor reduced Matrigel for 10-12 days and then immunofluorescence staining was performed according to Debnath et al. [16]
Morphology studies
Cell morphology was done on glass chamber slides by immunofluorescence method. Vector control and PDEF expressing cells were seeded on multi-chamber slide, fixed with 4% formalin, permeabilized with 0.1% Triton-X-100, blocked in 2% BSA, and change in actin cytoskeleton was examined by phalloidin staining as per the manufacturer's instructions (Molecular Probes, Eugene, OR). Pictures were taken using Spin Disc Olympus confocal microscope.
Western Blot analysis
Electrophoresis and blotting were performed as described previously [17].
Statistical analysis
Statistical analyses for tissue culture studies were performed using two-dimensional two sample variance T-tests; For data from clinical specimens, statistical analysis was performed using MANN - WHITNEY U: exact test. p ≤ 0.05 was considered significant.
Results
PDEF expression is reduced during the transition from low grade to high grade prostate cancer
PDEF expression was evaluated by immunohistochemical examination in tissue microarray slides containing 40 cores of prostate cancer and 9 cores of normal prostate. Results presented in Figure 1 show that PDEF protein expression is downregulated during the transition to aggressive prostate cancer. As shown in Figure. 1A, high levels of PDEF protein are present in normal prostate epithelial cells as well as early stage prostate cancer. However, in high grade prostate cancer PDEF protein is significantly decreased. Significant reduction in PDEF expression was observed in all cores of prostate tumor tissue with high Gleason grade (Gleason score greater than 7) as compared to normal prostate tissue as well as low grade prostate cancer (Gleason score of 7 or below). Moreover, PDEF protein levels showed graded decrease with increase in pathologically confirmed aggressive disease. Our results show that while 59 ± 3.6% tissue scored positive for PDEF in low to moderate grade (Gleason 6 & 7) prostate cancer, and 33 ± 3.3% of the tissue scored positive for PDEF in moderately high grade (Gleason 8) tumor, there was little or no expression of PDEF in very high grade (Gleason 9 & 10) prostate cancer(Figure. 1B). Antibody specificity for these assays was determined by Immunofluorescence and Western blot analysis using cells with and without PDEF expression (Figure S1, Additional file 1). In addition to clinical samples, we also evaluated PDEF protein expression in established prostate cancer cell lines with low to high aggressive behavior. These results presented in Figure 1C show that PDEF is expressed in LNCaP cells (a less aggressive prostate cancer cell line). However, PDEF expression is reduced in more aggressive lineages of LNCaP cells (C4-2 and C4-2B). Moreover, PDEF expression is completely lost in two widely used aggressive prostate cancer cells (DU145 and PC3 cells). Taken together, results of these studies demonstrate that PDEF expression is decreased or lost in prostate cancer cells with aggressive phenotype, and provide novel insights into the characteristics of PDEF protein expression in progression of prostate cancers. Absence of PDEF protein expression in PC3 cells in our studies is in apparent disagreement with previous report [5] that showed PDEF expression in PC3 cells,. This discrepancy could result from several causes: First, the antibody used in [5] could be more sensitive, such that they were able to detect even negligible amounts of PDEF. Second PC3 cells change over various passages of culture and media conditions, which could explain the differences.
Figure 1 PDEF protein expression in human prostate tissues and prostate cancer cell lines. A, Representative photo-micrographs of Immuno-histochemical analysis of PDEF expression using prostate tissue micro-array slides (containing both normal and tumor samples of different grades) performed as described in Materials and Methods. B, Quantification of percentage staining for PDEF. C, Representative image showing Western blot analysis on prostate cancer cell lines using anti-PDEF antibody (left panel), and quantitation of the same data (right panel).
Re-introduction of PDEF inhibited directional migration, decreased cell migration and anchorage independent growth in prostate cancer cells
To examine the effects of PDEF expression on cell motility, PC3 cells transduced with PDEF or mutant PDEF T50A, or vector alone were assayed for their ability to migrate through Transwell membranes as described in materials and methods. Results presented in Figure 2A indicate that both wild-type and mutant PDEF-transduced cells were significantly inhibited in their ability to migrate through Transwell pores. We also subjected cells to an assay for persistence migratory directionality (in vitro wound healing, Figure. 2B). Expression of either PDEF or the T50A mutant inhibited the ability of PC3 cells to fill in gaps in a monolayer compared to vector alone. These results demonstrated that PDEF significantly interfered with ability of cells to maintain migratory phenotype.
Figure 2 Effect of re-introduction of PDEF on directional migration, trans-well migration and anchorage independent growth in prostate cancer cells. A, Migration of PC3 cells expressing PDEF or PDEF T50A through Transwell membranes as described in Materials and Methods. B, PDEF overexpression decreases directional cell migration (in vitro wound healing migration of these cells). C, Representative photomicrographs from experiments testing colony formation as described in Materials and Methods. D, 3H-thymidine incorporation in PC3 PDEF overexpressing cells measured as described in Materials and Methods. E, Relative expression level of PDEF in these cell lines. Asterisks indicate significance levels of p < 0.05 with respect to controls.
We next examined the effects of PDEF overexpression on the ability of PC3 cells to form colonies in soft agar. Expression of either PDEF or the T50A mutant equally inhibited the ability of PC3 cells to form colonies in soft agar (Figure. 2C) compared to vector alone.
To address the possibility that altered cell proliferation contributed to the results of these assays, we measured DNA synthesis (3H-thymidine incorporation) in control and PDEF transfected PC3. Results of these studies (Figure. 2D) show that decreased clonogenic activity following PDEF expression was not a consequence of decreased DNA synthesis. These results demonstrated that PDEF expression decreased clonogenic ability of the cells independent of DNA synthesis. Moreover, in sharp contrast to the effects of PDEF on anchorage independent growth, PDEF expression did not significantly affect anchorage dependent growth of prostate cancer cells in culture (data not shown).
PDEF expression resulted in increased cell adhesion, altered cell morphology and decreased focal adhesion kinase activity in prostate cancer cells
Immunofluorescence studies of PDEF expressing cells showed a rounded area of cleared fluorescence rather than elongated track as seen on invasive vector control cells (Figure. 3A). These results indicated that PDEF expression resulted in alterations to actin cytoskeleton and altered cell morphology. FAK is a non-receptor protein tyrosine kinase, associated with supramolecular focal adhesion complexes. Focal adhesion complex assembly and disassembly are critical for cell attachment and movement [18]. The lack of morphologic polarity in PDEF expressing cells as shown in Figure 3A raised the possibility that PDEF may affect adhesion complex formation. Moreover, in previous studies we observed that FAK was non-phosphorylated in adherent cultures and FAK phosphorylation was increased in suspension culture [19]. Therefore, we evaluated the effects of re-introduction of PDEF in PC3 cells on FAK phosphorylation in PC3 cells in suspension cultures. Results of these studies revealed a significant reduction in FAK phosphorylation in PDEF expressing cells grown in suspension culture (Figure. 3B). These results demonstrated that PDEF expression in PC3 cells resulted in decreased FAK activity, suggesting decreased focal adhesion formation.
Figure 3 Effects of PDEF expression in prostate cancer cells on cell morphology, FAK phosphorylation and attachment to specific substrates. A, Phalloidin staining of actin cytoskeleton in PDEF expressing and vector control cells. B, Decreased phophorylation of FAK in PDEF expressing cells growing in suspension culture. C, Representative photomicrographs from experiments testing attachment of PC3 cell expressing PDEF to either BSA or fibronectin or matrigel-treated plastic surface as described in Material and Methods; and quantitation of these data. Asterisks indicate significance levels of p < 0.05 with respect to controls.
Focal adhesion formation and its interaction with the ECM play a central role in migration and invasion, since increased adhesion makes cells less motile. To examine this possibility, we directly measured the effects of PDEF expression on adhesion of PC3 cells to various ECM substrates. For these studies, PC3 cells transfected with PDEF or vector alone were assayed for their ability to attach to fibronectin or Matrigel-coated plastic surfaces. Results presented in Figure 3C indicate that attachment of PDEF-expressing cells to fibronectin-coated, Matrigel-coated, or control (BSA treated) plastic was significantly increased compared to vector-transduced cells. These results are in contrast to the effects of PDEF in breast cancer cells, where PDEF was shown to decrease adhesion of the cells to fibronectin and matrigel [4]. Taken together, these results suggest that PDEF mediated inhibition of migration may occur through cytoskeleton disorganization and ECM interaction.
PDEF decreased invasion and inhibited expression of matrix metalloproteinase-9 (MMP9) in prostate cancer cells
To test the effects of PDEF on cell invasion, we examined the effects of PDEF expression on the ability of PC3 cells to invade simulated basement membrane in vitro, a phenotype correlated with aggressive behavior. Results presented in Figure 4A indicate that expression of either PDEF or the T50A mutant inhibited the ability of PC3 cells to invade through Matrigel compared with vector transfected control cells. In addition to transfection of PC3 cells with PDEF, we also performed complementary RNA interference (RNAi) experiments to reduce the endogenous PDEF expression in prostate cancer cells that express PDEF (LNCaP and C4-2B cells), and directly evaluated the effects of decreased PDEF levels on invasion and clonogenic activity of these cells. Results presented in Figure S2, Additional file 1 demonstrated that SiRNA mediated knock-down of PDEF in these cells resulted in an increased ability to form colonies in soft agar and increased invasion through Matrigel basement membrane. Taken together with rest of the results these studies suggest that PDEF may play an important role in prostate cancer metastasis.
Figure 4 Effect of PDEF on invasion through Matrigel Matrix and MMP9 mRNA expression, MMP9 promoter activity and MMP9 enzymatic activity of prostate cancer cells. A, Invasion of PC3 cells expressing PDEF or PDEF T50A through Transwell membranes as described in Methods. B, RT-PCR showing MMP9 mRNA (left panel) and MMP9 enzymatic activity using gelatin Zymography (Right panel) was performed to determine the level of active MMP9. C, MMP9 promoter activity as determined by Luciferase reporter assay. D, Invasion of PC3 cells through a basement membrane matrix after blocking MMP9 using antibodies or overexpressing PDEF. Asterisks indicate significance levels of p < 0.05 with respect to controls.
Matrix metalloproteinases (MMPs) are a family of enzymes whose function primarily relates to degradation of extracellular matrix proteins, and are necessary for cell invasion. Moreover increased MMP activity has been associated with tumor metastasis. In our in vitro studies we observed that only MMP9 was prominently active in PC3 cells. Thus we set out to test the possible role of MMP9 in mediating the effects of PDEF on cell invasion. For these studies we evaluated the effects of PDEF expression on MMP9 mRNA expression, promoter activity and enzymatic activity. As can be seen in Figure 4B &4C, PDEF expression completely abolished MMP9 mRNA expression and enzymatic activity, and significantly reduced MMP9 promoter activity in PC3 cells compared to vector control. These data demonstrate inhibition of MMP9 expression by PDEF, and for the first time demonstrate regulation of MMP9 by any ETS transcription factors. To further confirm the role of MMP9 in modulating invasive behavior of prostate cancer cells, we performed antibody-neutralizing experiments. For these studies, we added MMP9 antibodies to the cells during the invasion assay, and data showed that anti MMP9 antibody inhibited cell invasion in PC3 cells by ~70% as compared to control IgG (Figure. 4D), further supporting the role of MMP9 in mediating the invasive phenotype in prostate cancer cells. Taken together these results demonstrate that PDEF negatively regulates MMP9 expression and provide a possible mechanism of PDEF actions in suppression of the invasive phenotype in prostate cancer.
PDEF expression in metastatic Prostate cancer cells results in phenotypic reversal and decreased migration in three dimensional cultures
To examine the effects of PDEF expression in a context that more closely resembled in vivo settings, we assessed the consequence of PDEF expression in PC3 cells on acinar or spheroid formation in 3 D culture. Results presented in Figure 5 demonstrate that PDEF expressing PC3 cells formed spheroid-like structures in basement membrane cultures, while most of the vector control cells form irregular structures and projections emanating from these structures that readily migrated and invaded the basement membrane by 10-12 days of culture. Several recent studies demonstrated inverse relationship between spheroid formation with cell migration and invasion. Our results show that re-introduction of PDEF in PC3 cells resulted in re-establishment of cellular polarity leading to inhibition of cell migration and reversal to less invasive phenotypes.
Figure 5 Effect of PDEF expression on cells grown in three dimensional cultures. A, representative images of three dimensional matrigel culture of PC3 cells with and without PDEF showing phenotypic reversal. Representative phase image obtained at various days in culture using 20X objective. B, Fluorescence microscopic images of PDEF PC3 cells and vector control cells after phalloidin and DAPI staining in 3 D cultures. C) Schematic depiction of the effects of PDEF expression in Prostate cancer cells.
Phosphorylation of PDEF and PDEF T50A
Since the T50A mutant had little or no effect on many of the phenotypic features associated with aggressive behavior, we asked whether the T50 phosphorylation site was a major contributor to PDEF phosphorylation. PC3 cells were transiently transfected with PDEF, the T50A mutant, or empty vector, and labeled with 32P-phosphoric acid. FLAG-PDEF immunoprecipitates were sequentially analyzed by autoradiography and Western blotting. The results (Figure S3, Additional file 1) indicate that PDEF T50A is phosphorylated, suggesting that amino acids other than T50 in the protein are kinase targets. Of interest, we found in this experiment that elimination of reducing agents during electrophoresis resulted in an approximate doubling of PDEF's apparent molecular weight (under reducing conditions about 45 kd), suggesting that native PDEF exists as a disulfide complex with another molecule(s). Overall, these findings suggest that amino acids other than the MAPK phospho-acceptor site at T50 in the protein could be possible targets for kinase, and they might have essential regulatory mechanism for invasion and migration. Thus, the T50A mutation does not alter the functions of PDEF and PC3 cells can phosphorylate PDEF at other locations.
MMP9 expression is increased in progression from normal to high grade prostate carcinomas and is inversely associated with PDEF expression
To further test whether MMP9 expression correlated with aggressive behavior of prostate cancer cells and to test whether a correlation existed between MMP9 and PDEF expression in human prostate cancer specimens, we evaluated MMP9 expression in the same tissue microarray slides containing 40 cores of prostate cancer and 9 cores of normal prostate that were used for PDEF expression analysis. Results presented in Figure 6A &6B show that MMP9 expression is up-regulated during the transition to high grade prostate cancer. Results presented in Figure. 6B, demonstrate that MMP9 protein staining was observed predominantly (>60-80%) in high grade prostate cancer cells (Gleason 8, 9 & 10), whereas normal prostate cells had very little or undetectable levels of MMP9 protein. Increased MMP9 protein expression was observed in 38 cores of prostate tumor tissue whereas 2 cores of prostate cancer had no positive staining for MMP9. For low grade prostate cancer (Gleason 7 or below) 21 ± 3% tissue scored positive for MMP9 (staining >40-80%) whereas for Gleason 8 to 10 it increased to 60 ± 9.3%. Combining all, these results indicate that there is significant co-relation between the low MMP9 expression in normal tissue with high MMP9 in intermediate to high Gleason prostate carcinoma.
Figure 6 MMP9 protein expression in Human prostate tissues. A, Immunohistochemistry for Representative photo-micrographs of Immuno-histochemical analysis of MMP9 expression using prostate tissue micro-array slides (containing both normal and tumor samples of different grades) performed as described in Materials and Methods. B, Quantification of percentage staining for MMP9.
Inverse relationship between PDEF and MMP9 expression in human prostate tissue
Our analysis of the PDEF and MMP 9 protein expression data presented in Figure 1 and 6 revealed that in human prostate tissue PDEF levels were lower in tumor samples as compared to normal tissue, however this difference reached a statistical significance only in prostate cancer samples form Gleason 8 and above (Normal vs. Gleason 6 & 7 Grade: p = 0.3593; Normal vs. Gleason 8 Grade: p = 0.0176; Normal vs. Gleason 9 & 10 Grade: p < 0.001). By contrast, we observed that MMP9 levels were higher in tumor samples as compared to normal tissue. Again the difference in MMP9 expression reached statistical significance only in prostate cancer samples form Gleason 8 and above (Normal vs. Gleason 6 & 7 Grade: p = 0.1517; Normal vs. Gleason 8 Grade: p = 0.0076; Normal vs. Gleason 9 & 10 Grade: p < 0.001). These data are presented for each individual sample in Figure 7A.
Figure 7 Relationship between PDEF and MMP9 expression in human prostate tissue: A, Expression of PDEF and MMP9 in individual samples B) Regression plot of the data presented in A. Data were collected from the immunohistochemistry performed for PDEF and MMP9 as described for Figure 1 and 6. (Normal = red; Gleason 6 & 7 = blue; Gleason 8 = green; and Gleason 9 and 10 = black). By regression analysis, there is a significant 0.846 (UNITS) drop in MMP9 for every 1.0 (UNIT) increase in PDEF.
Our regression analyses of these data reveal an inverse correlation between PDEF and MMP9 levels (Figure 7B and Table S1, Additional file 1). Thus, a decreased expression of PDEF in prostate cancer is associated with the malignant phenotype, more aggressive tumor behavior, and increased MMP9 expression.
Discussion
This is the first study to demonstrate loss of PDEF protein expression in high grade prostate cancer as compared to normal prostate as well as low grade prostate cancer tissue; and phenotypic reversal of highly migratory, invasive and aggressive prostate cancer cells to adherent polarized and non invasive cells in three dimensional cultures upon re-introduction of PDEF. We also show for the first time regulation of MMP9 by PDEF, and a direct correlation between loss of PDEF and increased expression of MMP9 high grade prostate cancer.
The ETS family is one of the largest families of transcription factors with 27 genes in human chromosome. The ETS family is present throughout the body and is involved in a wide variety of functions including the regulation of cellular differentiation, cell cycle control, cell migration, cell proliferation, apoptosis (programmed cell death) and angiogenesis. Multiple Ets factors have been found to be associated with cancer, such as through gene fusion including prostate cancer (2, 20-25). PDEF is selectively localized to the tissues with high epithelial content including prostate, and like other Ets family members has been shown to have diverse biological functions including tumor suppressor as well as tumor promoter functions.
Our results demonstrate loss of PDEF in high grade prostate cancer as compared to low grade prostate cancer as well as normal prostate tissue. Our results are unique in a way that they demonstrate for the first time loss of PDEF is associated with aggressive phenotype in prostate cancer, and suggest that PDEF might serve as a potential marker for distinguishing aggressive prostate cancer from an indolent disease. These findings are in apparent contrast to the previously published studies that concluded over expression of PDEF in prostate cancer as compared to normal prostate tissue [11]. It is important to point out here that previous studies lumped together all cancer samples and compared them with normal tissue, however, they did not attempt subset analysis of PDEF expression between low grade and high grade disease, which could have resulted in different conclusions. Our conclusion that PDEF expression is a favorable indicator in prostate cancer is, however, in agreement with studies that demonstrated a positive prognostic value of PDEF in ovarian cancer[8].
Our results also show that re-introduction of PDEF in aggressive prostate cancer cells resulted in decreased cell migration, decreased directional migration as well as decrease in clonogenic activity and converse was true when we knocked down PDEF in prostate cancer cells expressing PDEF (Figure S2, Additional file 1). These data suggest PDEF might serve as a suppressor of tumor migration and clonogenic activity. These results are in agreement with the previous studies with various breast and prostate cancer cells [5,7]. Our results are, however in sharp disagreement with studies that suggested that PDEF might promote migratory phenotype in breast cancer cells [3].
In order to become motile, cancer cells establish a defined polarity in the direction of movement through interaction between lamellipodia (a cytoskeletal actin projection on mobile edge of the cells) and focal adhesions that facilitate adhesion and migration of the cells. We also observed distinct changes in cytoskeleton and cell morphology associated with PDEF expression. We also show for the first time in any system that that PDEF expression increased cell adhesion, and resulted in a significant reduction in FAK phosphorylation. In previous studies we have shown an essential role for FAK in aggressive phenotype in prostate cancer cells [19]. Thus modulation of cytoskeleton organization and FAK activity by PDEF expression may provide potential pathways by which PDEF modulates cell behavior. These results extend previous observations in several cell types that show SiRNA mediated knockdown of PDEF was associated with increased cell migration [3-5]. While the results discussed so far point to the possible role of PDEF expression in modulating phenotypic behavior of cancer cells, our studies, are the first to use three dimensional cultures to actually demonstrate directly the effects of PDEF on cellular polarity and spheroid formation. Results clearly demonstrate that re-introduction of PDEF in aggressive prostate cancer cells resulted in phenotypic reversal from a disorganized, migratory and invasive cell growth to an organized, non-migratory and non-invasive phenotype.
Chintala et al. reported that the formation of spheroids is also linked to reduced invasion and expression and activity of MMP9 [26]. Matrix metalloproteinases (MMP) are a family of enzymes whose function primarily relates to the degradation of extracellular matrix proteins, and which are necessary for cell invasion. Our results presented here show that upon PDEF expression, prostate cancer cells lose their ability to invade Matrigel in Boyden chamber assays. These results are similar to the results observed by Turner et al. in invasive breast cancer cells [4]. However, to the best of our knowledge, our results show for the first time that PDEF downregulates MMP9 expression, and its promoter activity in any cell type. We observed that expression of PDEF in PC3 cells resulted in loss of MMP9 mRNA expression, decrease in MMP9 promoter activity and a significant reduction in the gelatinolytic activity. Thus our results again highlight a unique property of PDEF that is distinct from other ETS factors. We carried out additional studies to directly evaluate the functional consequence of MMP9 activity in PC3 cells. Results of these studies demonstrate that antibody mediated neutralization of MMP 9 reduced the invasion of PC3 cells through basement membrane matrix similar to that observed upon PDEF expression. Overall these findings provide for a mechanism by which PDEF expression could modulate cell polarity and other aggressive behavior.
Since we observed negative regulation of MMP9 by PDEF and published results suggested that the activity of MMP9 is associated with the progression and metastasis of prostate cancer [27], we also evaluated MMP9 expression in the tissue microarray slides containing 40 cores of prostate cancer and 9 cores of normal prostate that were used for PDEF expression analysis. Our results demonstrated an increase in MMP9 expression in high grade prostate cancer, which is in agreement with the previous studies [27]. We also observed an inverse correlation between PDEF expression and MMP9 expression in these samples. These results are in agreement with our findings in tissue culture studies that demonstrated negative regulation of MMP9 expression by PDEF. These results also highlight the potential use of loss of PDEF expression and increased MMP9 expression in early detection of aggressive prostate cancer.
Conclusions
In summary results presented herein demonstrate for the first time that PDEF, a member of Ets family, is lost in high grade prostate cancer and decreased PDEF expression is associated with increased MMP9 expression. We also provide direct evidence for the first time demonstrating that PDEF expression results in phenotypic reversal of aggressive prostate cancer cells in three dimensional cultures. Our studies also provide first demonstration in any system of negative regulation of MMP9 expression by PDEF. Taken together, our studies suggest that PDEF, by virtue of suppressing MMP9 expression and by modulating the ability of cancer cells to form a temporal structure required for migration and invasion, may function as suppressor of tumor metastasis in prostate cancer and perhaps other cancers. Our observation of an inverse relationship between PDEF and MMP9 expression suggests that expression of PDEF along with decreased MMP9 could help in early detection of aggressive prostate cancer and may facilitate new approaches to prostate cancer treatment.
Competing interests
A patent application relating in part to the content of the manuscript is being prepared.
No other interests
Authors' contributions
HKK designed research; TJ cloned PDEF, made stable cell lines and performed most of the cell migration, adhesion and invasion assays; S.K. performed all of the experiments with PDEF and MMP 9 expression in clinical samples and MMP 9 gene expression in cell lines. BK performed 3 D culture assays, cytoskeleton imaging, zymograms and the MMP 9 antibody neutralization assays; LK performed phospho-FAK assays and helped with cell culture work; SV performed many colony formation assays and assisted in the retrovirus and cloning work; TJ, BK, SK, PM, RBM and HKK analyzed data; and BK, TJ, LK, SK and HKK wrote the paper. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Supplementary data. Supporting Information. Materials and Methods with respect to following: -Immunofluorescence of cultured cells. -Anchorage independent Growth, and invasion assays. -Western blot analysis. -Metabolic Labeling. Figure S1 PDEF expression in cultured cells with or without PDEF. Figure S2 PDEF knockout increased colony formation and invasion of LNCaP and LNCaP C4-2B cells. Figure S3 Phosphorylation of PDEF and PDEF T50A. Table S1 Relative PDEF and MMP9 gene expression in human prostate tissue.
Click here for file
Acknowledgements
These studies were supported in part by NIH/NCI-P20 CA103680-Schwartz/Byers Program PI's (H Koul, Pilot-Project PI) and the Department of Surgery, School of Medicine Academic Enrichment Funds. We are grateful to Dina Lev for kindly providing MMP9 luciferase reporter construct used in these studies. We also gratefully acknowledge Mr. Colin Odonnell for Statistical analysis on PDEF and MMP 9 expression in human prostate cancer specimen data.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2066147110-PONE-RA-18517R110.1371/journal.pone.0011668Research ArticleCell Biology/Cell Growth and DivisionCell Biology/Cell SignalingCell Biology/Gene ExpressionCo-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation Mechanism of G0/G1 ArrestGopinath Sreelatha
1
Malla Rama Rao
1
Gondi Christopher S.
1
Alapati Kiranmai
1
Fassett Daniel
2
Klopfenstein Jeffrey D.
2
Dinh Dzung H.
2
Gujrati Meena
3
Rao Jasti S.
1
2
*
1
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
2
Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
3
Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
Langsley Gordon EditorINSERM U1016, Institut Cochin, France* E-mail: [email protected] and designed the experiments: SG JR. Performed the experiments: SG RRM CSG KA. Analyzed the data: SG CSG DF JDK DHD MG JR. Contributed reagents/materials/analysis tools: JR. Wrote the paper: SG.
2010 22 7 2010 5 7 e116683 5 2010 24 6 2010 Gopinath et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.
Methodology/Principal Findings
Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.
Conclusion/Significance
Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.
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Introduction
Malignant glioma, a common tumor among the intracranial tumors, remains formidable despite aggressive surgery, radiotherapy and chemotherapy [1]. Cathepsin B and urokinase-type plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas and, as such, are attractive targets for gene therapy. During cancer cell invasion, these proteins, either individually or in combination, function to degrade the extracellular matrix, thereby facilitating metastasis. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied [2]–[4], the effect of these molecules on cell cycle progression has not been thoroughly examined. Moreover, disruption of cell cycle control is a hallmark of cancer [5], [6]. In particular, the reduced expression of p27Kip1, which is a member of the Kip family of cyclin-dependent kinase (Cdk) inhibitors, has been extensively observed in human cancers, and its low levels are often associated with a worse prognosis [7], [8]. Increased susceptibility to cancer and multi-organ hyperplasia have been reported in p27Kip1-null mice [9]. It plays a crucial role in the control of cell proliferation by inhibiting the activities of complexes of G1 cyclins and Cdks and, as such, is an important candidate for therapeutic tumor suppression [10]. Some factors, including accelerated proteolysis, sequestration by cyclin D-Cdk complexes, and phosphorylation events that lead to nuclear export and/or retention in the cytosol, have significant roles in inhibiting the p27Kip1 function in various cancers [11]. Cytoplasmic translocation of p27Kip1 has been increasingly recognized in primary human tumors associated with poor survival whereas nuclear expression confers a more favorable outcome [12].
Another hallmark of most cancers, including glioma, is the increased activity of PI3K/AKT pathway that controls many biological functions like cell proliferation, survival, and insulin response [13]. Constitutive activation of this pathway facilitates tumor formation both by supporting S-phase entry and by conferring resistance to apoptotic signals that normally restrict uncontrolled cell growth [14], [15]. In the presence of growth factors, AKT negatively regulates FOXO proteins by phosphorylating them [16], [17], which results in their binding to 14-3-3 proteins and is followed by their nuclear export [18]. FOXO factors function as transcriptional activators and bind as monomers to the consensus DNA sequence TTGTTTAC [19], [20]. Depending on the cell system studied, forced expression or activation of FOXO factors triggers apoptotic responses or cell cycle arrest [21]. Cell cycle inhibitory effect of FOXO factor through increased transcription of p27kip1 has been reported in gliomas [22], [23]. Several integrins play important roles in promoting cell proliferation, migration and survival in vitro and in vivo. Both uPAR and cathepsin B are known to be associated in close proximity to αVβ3 integrins and has been implicated in their ability to initiate signaling events [24].
In an attempt to elucidate the roles of cathepsin B and uPAR in cell cycle progression, we analyzed the activity of crucial regulators of the G0/G1 transition including p27Kip1 by downregulating cathepsin B and uPAR both individually and simultaneously in SNB19 and U251 glioma cells. Here, we show that shRNA-mediated downregulation of cathepsin B and uPAR results in G0/G1 arrest, prominent increased expression of p27Kip1 and inhibition of p-Rb. This increased expression of p27Kip1 correlates with decreased expression of p-PI3K, p-AKT, cyclin E, cyclin D1, cyclin D2 and increased expression of FOXO3a protein. We also show that increased expression of p27Kip1 is due to the efficient binding of FOXO3a on its promoter, which was analyzed by the luciferase expression.
Results
Knockdown of cathepsin B and uPAR decreases cell proliferation and induces G0/G1 arrest
To gain insight into the molecular roles of cathepsin B and uPAR, we knocked down the expression of these molecules using shRNA in SNB19 and U251 glioma cells and then analyzed the effects on cell proliferation and cell cycle. After 36 hrs of transfection, western blot analysis showed a 80±3% and 82±3% decrease in cathespin B expression in SNB19 and U251 cells, respectively when treated with pC (shRNA construct against cathepsinB). Cells treated with pU (shRNA construct against uPAR) did not show appreciable difference in cathespin B expression when compared to controls (98±2%). pCU-treated (shRNA bicistronic construct against cathepsin B and uPAR) cells showed 86–91% decreased expression of cathepsin B in both SNB19 and U251 cells (p<0.01). Similarly, uPAR expression was reduced by 75–80% in both cell lines when treated with pU. Cells treated with pC did not show any difference in expression when compared to controls (95±3%). uPAR expression in pCU-treated cells was significantly reduced by 80–91% (Fig. 1A) (p<0.01). Immunoblot analysis for GAPDH expression revealed equal loading. Cell proliferation analysis by BrdU incorporation assay showed that the depletion of cathepsin B and uPAR individually and simultaneously resulted in a significant reduction in the proliferation rates by: 37–40% (pU), 34–36% (pC) and 67–68% (pCU) in both cell lines (Fig. 1B). In contrast, untreated control and SV (scrambled vector)-transfected cells showed 100% proliferation in both cell lines. Similarly, MTT assay showed decreased number of cells with the treatments (pU: 38–40%; pC: 33–35% and pCU: 65–68%) compared to the controls (98–100%) in both the cell lines (Fig. S1A). Decreased growth suppression was associated with cell cycle arrest. As shown in Figure 1C, cell cycle analysis showed an increase in the G1 phase fraction with the treatments (pU: 58.34±2%, 66.68±2%; pC: 56.88±2%, 63.84±2%; and pCU: 72.16±1%, 78.45±2% in SNB19 and U251, respectively) and a concomitant decrease in the S phase (pU: 15.23±3%, 7.98±3%; pC: 16±2%, 8.56±1%; and pCU: 8.12±1%, 2.95±2% in SNB19 and U251 cells, respectively) and G2/M phase (pU: 26.43±1%, 25.34±1%; pC: 27.12±1%, 27.6±2%; and pCU: 19.12±1%, 18.6±2% in SNB19 and U251 cells, respectively) fractions. Negligible number of cells were present in sub G0/G1 phase, hence, the data is not included. Cell cycle analysis of untreated control and SV-transfected cells of SNB19 and U251 cells showed 40±5% in G1 phase, 15±10% in S phase and 32±5% in G2/M phase. These results demonstrate that the decrease in cell proliferation is due to the block of progression from G1 to S phase, and the effect was almost the same in both the cell lines. However, cell cycle analysis at 48 hrs of transfection showed significant increase (20–40%) in sub G0/G1 phase with the treatments compared to the controls (7–8%) and concomitant decrease in the G0/G1, S and G2/M phases indicating that the cells were entering into the apoptotic phase (Fig. S1B).
10.1371/journal.pone.0011668.g001Figure 1 RNAi-mediated depletion of cathepsin B and uPAR inhibits SNB19 and U251 cell proliferation and induces G0/G1 arrest.
A. Western Blot analysis of cathepsin B and uPAR in SNB19 and U251 cells 36 hrs after transfection with SV, pU, pC and pCU. GAPDH was used as a loading control. Side panel shows quantitative analysis of cathepsin B and uPAR bands by densitometry. B. We analyzed cell proliferation 36 hrs after transfection using the BrdU incorporation assay and the percent of proliferation is represented graphically. Values are mean ± standard deviation (SD) from three different experiments (p<0.01). C. Propidium iodide-stained SNB19 and U251 cells were analyzed for DNA content using flow cytometry. The graph shows the percentage of cells in G0/G1, S and G2/M phases 36 hrs post transfection. Values are mean ± SD of three different experiments (*p<0.01, in comparison with the control).
Cathepsin B and uPAR depletion affects the p27Kip1 expression and its subcellular localization
It is well known that p27Kip1 plays an important role in G0/G1 arrest. Hence, we checked the expression of p27Kip1 using RT-PCR and western blot analysis. RT-PCR and immunoblot analysis of pC- and pU-treated cell lysates showed increased expression of p27Kip1. The pCU-treated cells showed a further increase in p27Kip1 expression in both the cell lines. Untreated control and SV-treated cells showed very low expression of p27Kip1 (Fig. 2A &B). The p27Kip1 protein is generally phosphorylated at Ser10 and Thr187 positions and its activity depends on its phosphorylation status. Therefore, we checked the phosphorylation status of p27Kip1 by immunoblot analysis and found that the treatments reduced the phosphorylation of p27Kip1 at Ser10 and Thr187 in both the cells lines compared to the controls. Immunoflourescence staining assay revealed that the treatments induced an increase in p27Kip1 localization in the nucleus when compared with control cells (Fig. 2C) and a higher number of cells expressing p27Kip1 in the nuclei was observed with the pCU treatment (Fig. S2A). These results were further confirmed by immunoblot analysis for p27Kip1 protein in cytosolic and nuclear fractions (Fig. S2B).
10.1371/journal.pone.0011668.g002Figure 2 Depletion of cathepsin B and uPAR increases p27Kip1 nuclear localization.
A. Expression of p27Kip1 and p-p27 (Ser10 and Thr187) were studied using immunoblot analysis. GAPDH was used as loading control. B. Total RNA isolated from untreated and treated SNB19 and U251 cells was subjected to semi-quantitative RT-PCR analysis using p27Kip1 primers. Data represents average of triplicates normalized to GAPDH (**p<0.01). C. 36 hrs after transfection with SV, pU, pC and pCU, cells were fixed, immunostained with anti-p27 antibody followed by Texas Red-conjugated anti-mouse secondary antibody. DAPI was used for nuclear staining. Representative images of three independent experiments are shown. D. SNB19 and U251 cells were transfected with siRNA against p27 (p27si) individually and in combination with pU, pC and pCU. The cells were also transfected with control siRNA (C-si) and SV. Thirty six hours post-transfection, cells were lysed and the total lysates were immunoblotted for p27Kip1, p-p27 (Ser10), and p-p27 (Thr187). E. Effect of the above stated treatments on proliferation was assessed using BrdU incorporation assay. The graph represents the percent of proliferating cells and the data represented are the average of three separate experiments (*p<0.05, **p<0.01, in comparison with the control).
To further confirm the role of p27Kip1 in growth arrest induced by the depletion of cathepsin B and uPAR, we knocked down the expression of p27Kip1 alone or in combination with uPAR and/or cathepsin B, and we analyzed cell proliferation using BrdU incorporation assay. As expected, immunoblot analysis showed efficient knockdown of p27Kip1 in p27Kip1 siRNA-treated cells lysates (Fig. 2D). Cell proliferation was also increased (118±2%) with the p27Kip1 siRNA treatment in SNB19 and U251 cells as compared to controls (97–100%) (Fig. 2E). In contrast, immunoblot analysis and BrdU incorporation assay of cells from p27Kip1 and cathepsin B or uPAR co-depleted cells showed a decrease in p27Kip1 expression but the inhibitory effect induced in proliferation by pU, pC and pCU treatments (as described in Fig. 1C) was reverted partially in co-depleted cells (pU+p27si: 82±2%, 79±2%, pC+p27si: 79±1%, 77±2% pCU+p27si: 66±2%, 64±3% in SNB19 and U251 cells, respectively) (Fig. 2E). The results indicate that cooperation from other molecules of G1 phase might be needed for complete growth arrest. When FACS analysis was performed, p27Kip1 knockdown and the co-depletion treatments resulted in a decrease of cells in the G0/G1 phase and a concomitant increase in cells in S and G2/M phases (Fig. S2C).
G0/G1 phase regulators
Cell cycle regulators at the G0/G1 and G1/S phase transition were analyzed after the above mentioned treatments. As p27Kip1 is both an inhibitor and a substrate of cyclin E-Cdk2 complex [25], we analyzed the expression of these molecules using immunoblot analysis and found that the treatments decreased the expression of cyclin E whereas the expression of Cdk2 was unaffected (Fig. 3A). Similarly, expression of αV, β3, αVβ3 integrins decreased with the treatments. Further, Cdk2 was immunoprecipitated from the cell lysates of untreated and SV, pU, pC and pCU treated SNB19 and U251 cells and immunoblotted for cyclin E. The results revealed little or no expression of cyclin E in pU, pC and pCU treated cell lysates compared to control and SV transfected cells indicating that the treatments reduced the cyclinE-Cdk2 complex formation. pCU-treated cells showed significant downregulation of cyclin E as compared to pU and pC treatments. Dimerization of αVβ3 integrin was checked by immunoprecipitating the cell lysate with β3 integrin and immunoblotted for αV integrin and found that the treatments significantly reduced the dimer formation. It was further confirmed by native gel electrophoresis by using the αVβ3 antibody (Fig. 3B).
10.1371/journal.pone.0011668.g003Figure 3 Cathepsin B and uPAR knockdown decreases Cdk2 activity and the expression of αVβ3 integrin.
A. Cell lysates were collected from SNB19 and U251 after transfection with SV, pU, pC or pCU. Western blot analysis of 50 µg of total cell lysates was performed to check the expression of cyclin D1, cyclin D2, Cdk2, cyclin E, Rb, p-Rb, p21, αV, β3, αVβ3 and Ki67. GAPDH was used as a loading control. B. Total lysates from the untreated control and SV, pU, pC or pCU-transfected cells were immunoprecipitated for Cdk2 and β3 individually and then immunoblotted for cyclin E and αV, respectively. The figure also shows the expression of αVβ3 integrin on native gel.
Apart from the cyclinE-Cdk2 complex formation, the treatments also decreased the expression of cyclin D1, p21, cyclin D2 and Ki67, which is an indicator of cell proliferation (Fig. 3A). We also checked for the expression of Rb and p-Rb (Ser780 and Ser249/Thr252) as p-Rb is initially catalyzed by the complexes formed by cyclin D and Cdk4 or Cdk6 and later by cyclin E-Cdk2 [26], [27]. We found that the treatments decreased the phosphorylation status of Rb but did not affect total Rb (Fig. 3A).
Expression of p27Kip1 is influenced by the upregulation of FOXO3a proteins in cathepsin B and uPAR depleted cells
Since we observed p27Kip1 upregulation with decreased cell proliferation and G0/G1 phase arrest with the depletion of cathepsin B and uPAR, we next determined the expression of FOXO proteins, which are important transcriptional regulators of the p27Kip1 promoter. We also checked for the expression of PI3K and AKT molecules, which dictate the phosphorylation status of p27Kip1 and affect the activity of FOXO proteins. Immunoblot analysis revealed that the expression of FOXO3a increased with the treatments, especially with pCU treatment (Fig. 4A); the treatments did not affect the other FOXO forms significantly (Fig. S3A).
10.1371/journal.pone.0011668.g004Figure 4 Cathepsin B and uPAR knockdown induces FOXO3a expression and translocation to the nucleus by inhibiting PI3K activity.
A. After transfection, cell lysates were collected from the untreated control and SV, pU, pC or pCU-treated cells. Equal volume of total protein was blotted for the expression of PI3K, p-PI3K, AKT, p-AKT, FOXO3a, p-FOXO3a (253), and p-FOXO3a (Ser 318). B. SNB19 and U251 cells were treated with either DMSO or LY294002 (Ly 10 µM) for 24 hrs as mentioned in the Materials and Methods. After incubation, the total and nuclear lysates were collected and probed for the expression of p-AKT, p27Kip1 (Nu), FOXO3a (Nu) and lamin B (Nu). Equal loading was confirmed by the GAPDH expression in the total cell lysate. C. After treatments with DMSO- and LY294002- cells were fixed, stained with propidium iodide and subjected to FACS analysis to determine cell cycle status.
For FOXO proteins to be active, newly synthesized FOXO3a must translocate to the nucleus, which is further influenced by its phosphorylation status. To test whether the depletion of uPAR and/or cathepsin B affected localization of the FOXO3a protein, cytosolic and nuclear fractions were immunoblotted for FOXO3a. We found that the nuclear fractions of treated cells expressed more FOXO3a protein than the controls (Fig. S2B). Further, immunoblot analysis of total cell lysates for phospho-FOXO3a revealed that phospho-FOXO3a at Ser318 was decreased significantly more than at Ser253. As expected, the same treatments decreased the expression of phospho-PI3K and phospho-AKT; the total forms were unaffected. The effect of the treatments was the same in SNB19 and U251 cells.
PI3K is a known inhibitor of FOXO proteins. Hence, we checked the effect of the PI3K inhibitor, LY294002 (10 µM) on the expression of p-AKT in the total extracts. Western blotting revealed decreased expression of p-AKT, which is an indicator of PI3K kinase activity, and increased expression of FOXO3a and p27Kip1 in the nuclear extracts (Fig. 4B). FACS analysis showed that the inhibitor induced G0/G1 arrest similar to that induced by the RNAi treatments (Fig. 4C). The expression of p27Kip1 and FOXO3a proteins were also assessed in cathepsin B and uPAR-overexpressing cells, and we found a correlation with the above mentioned results (Fig. S3B). These results indicate that the knockdown of cathespin B and uPAR in SNB19 and U251 cells induced G0/G1 arrest with the increased expression of p27Kip1 and FOXO3a and reduced PI3K activity.
We further analyzed the effect of upregulation of FOXO3a on p27Kip1 expression at the transcriptional level. After transfecting SNB19 and U251 cells with pU, pC and pCU or after treatment with Ly294002 (10 µM), a second transfection was performed with the cDNA constructs containing the luciferase reporter gene controlled by FOXO binding promoter regions of human p27Kip1 [−3125 to 2845 bp (pGL-Kip1-290) and −3507 to −2478 bp (pGL-Kip1-1110)] or the consensus sequence arranged as tandem repeats in triplet (pGL-Kip1-3x) or mutated sequence (pGL-Kip1-M) or with the SV as a control. The p27Kip1 promoter with the normal and mutant versions of the putative FOXO binding sequence and the regions used for PCR amplification are represented in Figure 5A. Expression of luciferase was increased by 2–2.5 and 3 fold in pC- and pU-transfected cells and LY294002-treated cells, respectively when pGL-Kip1-290 (Fig. 5B), pGL-Kip1-1110 (Fig. 5C) and pGL-Kip1-3x (Fig. 5D) vectors were used. In contrast, in pCU-transfected cells, the same treatment increased luciferase expression by 4–4.5 fold. Thus, increased activity of the p27 promoter expressed as luciferase expression with the promoter constructs indicate that the regulation of p27Kip1 protein levels by cathepsin B and uPAR could be, at least partially, explained by the regulation of its promoter activity by increased expression of FOXO3a. The levels of luciferase expression were same with all the constructs used whereas no expression was observed either in mutated sequence driven luciferase construct (Fig. 5E) or in the SV transfected controls.
10.1371/journal.pone.0011668.g005Figure 5 Regulation of p27Kip1 activity in cathepsin B and uPAR-depleted glioma cells occurs through FOXO3a transcription factor.
A. Schematic representation of the p27Kip1 promoter with the normal and mutant versions of the putative FOXO binding site and the regions used for PCR amplification. B–E. SNB19 and U251 cells were initially transfected with SV, pU, pC or pCU, and treated with Ly294002 (Ly 10 µM) separately. 24 hrs after the treatments, a second transfection with the luciferase constructs was performed as described in Materials and Methods. The luciferase expression was quantified using Promega's luciferase assay kit with a Turner Luminometer and is represented graphically. The graphs show luciferase expression when pGL-Kip1-290 (B), pGL-Kip1-1110 (C), pGL-Kip1-3x (D), and pGL-Kip1-M luciferase constructs were used. Assessment for luciferase expression was performed at least in triplicate (*p<0.05, **p<0.01).
Cathepsin B and uPAR shRNA suppresses intracranial tumor growth
The effect of RNAi-mediated inhibition of cathepsin B and uPAR on pre-established tumors was studied. H&E staining revealed a large spread of tumor growth in mock and SV-treated brain sections. Whereas, pre-established intracranial tumor growth was inhibited by 95% when treated with pCU (Fig. 6A). Immunohistochemical analysis of the tumor sections from control mice for cathepsin B and uPAR showed increased expression levels localized to the tumor region while the pCU-treated tumor sections revealed very little or no expression of the cathepsin B and uPAR. When probed for the expression of p27Kip1 and Ki67 proteins, mock and SV-treated brain sections showed very little expression of p27Kip1 and increased expression of Ki67. In contrast, pCU treated brain sections showed high expression of p27Kip1. However, pCU-treated brain sections showed very little or no expression of Ki67 as compared to the controls (Fig. 6B), indicating that cell proliferation is inhibited by these treatments through upregulation of p27Kip1. The effect of the pCU treatment on tumors induced by SNB19 and U251 cells was the same.
10.1371/journal.pone.0011668.g006Figure 6
In vivo inhibition of tumor growth.
Stereotactic implantation of SNB19 and U251 (1×105) tumor cells was performed and, after one week, PBS (mock), SV or pCU was injected into the brain using an Alzet mini osmotic pump. Five animals per group were used. 30 days after implantation, the animals were sacrificed, the brains were removed and fixed, and paraffin sections were prepared. A. Hematoxylin and eosin staining of tissue sections to visualize tumor cells and to examine tumor volumes. Bar: 20 µM (*p<0.01) B. Immunohistochemical analysis of cathepsin B, uPAR, Ki67 and p27Kip1 in paraffin embedded tissue sections. Bar: 200 µM.
Discussion
Various reports have demonstrated that cathepsin B and uPAR levels are overexpressed during glioma progression [28]–[30]. It has been reported that β1 integrins in caveolae bind uPAR and are linked to increased secretion of pro-cathepsin B [31]. An association of cathepsin B and uPAR mediated by active K-RAS in colorectal carcinoma has also been reported [32]. We have previously shown that RNAi-mediated downregulation of cathepsin B and uPAR led to decreased invasion, induction of angiogenesis, increased caspase-mediated apoptosis, and induction of G0/G1 arrest [2], [30], [33]–[37] Data from other reports indicate that inhibition or depletion of cathepsin B prevents cells from entering and leaving the cell cycle, thereby decreasing cell proliferation [38], [39]. However, the molecular mechanisms by which cathepsin B and uPAR regulate cellular proliferation remain poorly understood. The growing body of knowledge of genetic alterations that occur in malignant gliomas has resulted in the development of targeted therapy to restore cell cycle or apoptosis defects in gliomas [22]. In the present study, we show that the co-depletion of cathepsin B and uPAR arrests cells in the G1 phase primarily through the upregulation of p27Kip1 and that this pathway involves the downregulation of p-PI3K, p-AKT, D-type cyclin expression, and cyclin E/Cdk2 complex formation as well as the subsequent upregulation of the FOXO3a protein and its nuclear localization.
In the present study, we have shown that pU, pC and pCU treatments reduced endogenous levels of cathepsin B and uPAR proteins in SNB19 and U251 glioma cells (Fig. 1A) with a 75–78% transformation efficiency as obtained using GFP (Fig. S4). Among the treatments, pCU reduced protein expression more than pU and pC; this same effect was seen in the other experiments. These treatments also led to G1 arrest and decreased cell proliferation. We further investigated the influence of these treatments on p27Kip1 expression and localization. Western blot analysis revealed that p27Kip1 expression increased with the treatments while its phosphorylation at Ser10 and Thr187 decreased, thereby indicating the nuclear localization of increased p27Kip1 protein. This was further confirmed by the immunofluorescence analysis where the treatment showed nuclear presence of p27Kip1. Data from many different studies emphasize the importance of p27Kip1 as a potent inhibitor of cell cycle in human cancers [40]. p27Kip1 phosphorylation at Ser10 or Thr187 facilitates the nuclear-to-cytoplasmic redistribution of p27Kip1
[41], [42], and this sub-cellular localization determines the activity of p27Kip1. Our results suggest that reduced phosphorylation of p27Kip1 at Ser10 and Thr187 increased p27Kip1 nuclear localization, but further experiments using p27Kip1 siRNA and that of decreased expression of cyclin D1, cyclin D2 and cyclin E with the pU, pC and pCU treatments indicate that p27Kip1 alone is partially responsible for cell cycle arrest and decreased Cdk2 kinase activity might be necessary to complete the task. Similar results showing that SHP1 downregulation effected p27Kip1 expression and Cdk2-cyclin E complex formation have been reported [43]. However, we were unable to find any translocation difference of Cdk2 with the treatments as observed by [43]. Our immunoprecipitation results indicate that Cdk2 kinase activity was reduced with the treatments. As a member of the CIP/KIP family, p27Kip1 was found to be associated with and to inhibit the catalytic activities of G1 and S phase-specific Cdk/cyclin complexes [44]. Thus, the decrease in p-p27Kip1 with the treatments could be due to the low Cdk2 kinase activity. Our results also indicate decreased phosphorylation of Rb at Ser780 and Ser249/Thr252. Inactivated retinoblastoma (p-Rb) protein regulates the progression from G1 to S phase through its association with the E2F family of transcription factors [45], [46]. In early and late G1 phase, p-Rb is hyperphosphorylated by D-type Cdks and Cdk2-cyclin E, respectively [26], [47], [48]. These reports suggest conclusively that phosphorylation of p-Rb by Cdk2-cyclin E requires p-Rb to be hypophosphorylated, and thus, the inactivation of p-Rb involves sequential phosphorylation by cyclin D-Cdk4/6 and cyclin E/Cdk2 [26], [27]. Therefore, the G0/G1 arrest induced by the treatments could be due to the combined action of reduced cyclin D1, cyclin D2, and cyclin E-Cdk2 complex formation and increased expression of p27Kip1.
Hyperactivation of the PI3K-AKT pathway is critical in human tumorigenesis because it promotes cell growth, survival and resistance to treatment [49], [50]. In addition, it has been reported that 88% of gliomas show altered PI3K-AKT signaling [51]. Integrins on tumor cells increases tumor cell migration, invasion, proliferation and survival [52]. In this study, we have shown that downregulation of cathepsin B and uPAR significantly decreased the dimer formation of αVβ3 integrins as seen in immunoprecipitation and native gel electrophoresis with the decreased expression of both αV and β3 integrins. The roles of other integrins are being investigated in our laboratory. Transcriptional downregulation of p27kip1 mRNA by AKT occurs through localization and subsequent inhibition of the FOXO protein. Recent data show FOXO3a directly regulates p27kip1 transcription [53], [54], suggesting that reduced p27kip1 levels after a proliferative stimulus may also be associated with FOXO3a. Our results further confirm these findings. In the present study, FOXO3a expression was positively correlated with p27kip1 expression (Fig. 2A) but inversely associated with cell proliferation as identified by Ki67 (Fig. 3A), which is a marker of cell proliferation expressed specifically in the cell nucleus from late G1 to S phase. We observed a decrease in phosphorylation of PI3K, AKT and FOXO3a (Ser318) (Fig. 4A) protein along with decreased activity of Cdk2 (Fig. 3B) with the RNAi treatments. Activated AKT is crucial in preventing FOXO3a displacement to the nucleus [55], [56]. However, AKT-independent and Cdk2-dependent phosphorylation affected FOXO1 nuclear export to different extents in U87 and U251 glioma cells [22]. Our results reveal that LY294002 significantly inhibited the phosphorylation and activation of AKT. More importantly, LY294002-treatment caused FOXO3a nuclear accumulation. These results suggest that LY294002 controlled the activity of FOXO3a by regulating its phosphorylation and subcellular localization. It is known that FOXO3a regulates the transcription of p27kip1 by binding to its promoter [57]. Indeed, we found that p27kip1 expression increased with LY294002 treatment. Moreover, after treatment with pU, pC, pCU and Ly294002, luciferase expression under the influence of FOXO binding promoter region of p27Kip1 increased with the treatments as compared to control and SV-transfected cells. Among the treatments, pCU-treated cells showed more luciferase activity than pU- and pC-treated cells. Notably, luciferase expression was nearly the same irrespective of the vector used. Studies have indicated that other transcription factors, such as Sp1, CRE and NF-κB, regulate p27Kip1 promoter activity [58], [59]. Very recently, Li [60] reported the effect of anti-inflammatory drugs on proliferation of human osteoblasts with the increased activity of FOXO3a by binding to p27Kip1 promoter. Thus, these findings invite the conclusion that the increased p27Kip1 expression with the treatments is due to the increased nuclear expression of FOXO3a, which binds to the -2984 bp region on the p27Kip1 promoter and could be mediated by the low expression of p-AKT. The results obtained were also confirmed by immunoblotting for p-PI3K, p-AKT, FOXO3a and p27Kip1 in the cathepsin B and uPAR-overexpressed SNB19 and U251 cells.
Although overexpression of p27Kip1 can induce protection from apoptosis, other studies have reported pro-apoptotic effects where caspases are able to cleave p27Kip1 in a cell type specific manner [9], [61] reported that the inhibition of cytochrome c release might be a possible mechanism of preventing apoptosis with higher p27Kip1 expression in leukemia cells; this indicates that the regulation of the apoptotic process by p27Kip1 might vary with cell transformation status. In a separate experiment, we observed decreased expression of p27Kip1 in the nucleus at the 72 hours time point (data not shown). Several recent reports have shown that p27Kip1 has cell cycle-independent functions, such as the regulation of cell migration, which might be oncogenic under certain circumstances [25].
The efficiency of in vivo RNAi/adeno virus treatments by targeting proteins like uPA and uPAR [62], cathepsin B and uPAR [3], and uPAR and MMP-9 [63] has been well established in our laboratory. Similarly, the present study demonstrates that the simultaneous downregulation of cathepsin B and uPAR caused the regression of intracranial tumors. Nude mice implanted intracranially with SNB19 and U251 glioma cells became very weak within three to four weeks due to tumor development. In contrast, pCU-treated mice were healthy, and H&E staining of these tissue sections revealed very few to no cancer cells as compared to the controls. Interestingly, we observed high expression of p27Kip1 and very low expression of Ki67 in the tumor sections, indicating the efficiency of treatment both in vitro and in vivo. Moreover, a direct correlation between the low expression of p27Kip1 and FOXO3a and higher expression of Ki67 with the malignant glioma has been reported [23]. Increased expression of p21 and regression of lung tumor growth in vivo with the administration of decorin has been reported [64]. Yu [65] has reported that the downregulation of uPAR induced G0/G1 arrest in vitro but did not affect growth in vivo. Decreased tumor growth and metastasis of malignant melanoma cells in nude mice with the administration of antisense oligonucleotide for uPAR has been described [66]. Likewise, an 88% inhibition of proliferating cancer cells in colorectal carcinoma in vivo when treated with uPAR monoclonal antibody (ATN658) has been reported recently [67]. In contrast, uPAR overexpression inhibited cell growth in murine embryonic fibroblast cells and induced cell growth in keratinocytes [68]. uPAR has been detected as a potential cooperating oncogene in Ink4a KO mice, which are deficient in cell growth control [69]. Thus, the effect of uPAR on growth rate may depend on cell type. Not much has been reported about cathepsin B controlling cell proliferation in vivo. In conclusion, our results demonstrate that Akt/FOXO3a/p27Kip1 signaling contributes to G0/G1 arrest, which was induced by the depletion of cathepsin B and uPAR (Fig. 7). Moreover, our results also demonstrate that the bicistronic construct, pCU, was more effective than the single constructs, pU and pC. Thus, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.
10.1371/journal.pone.0011668.g007Figure 7 Schematic representation of the molecular mechanisms proposed in the regulation of cell proliferation by G0/G1 arrest with the increased expression of FOXO3a and p27Kip1 in cathepsin B and uPAR-depleted glioma cells.
Materials and Methods
Ethics Statement
The Institutional Animal Care and Use Committee of the University Of Illinois College Of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. The consent was written and approved. The approved protocol number is 851, dated November 20, 2009. No de novo cell lines were used.
siRNA constructs, cell culture, transfection and inhibitor treatments
Single shRNA constructs directed against uPAR (pU) and cathepsin B (pC) and the bicistronic construct directed against both cathepsin B and uPAR (pCU) have been described previously [2]. siRNA for p27Kip1 (p27si) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Full length cathepsin B and uPAR over expressing plasmids were purchased from Origene (Rockville, MD). All the antibodies used in this study are from Santa Cruz Biotechnology (Santa Cruz, CA) unless otherwise mentioned.
Human glioma cell lines SNB19 and U251, obtained form American Type Culture Collection (ATCC, Manassas, VA) were cultured in DMEM/high glucose media supplemented with 10% FBS in a humidified atmosphere containing 5% CO2 at 37°C. Cells were grown in 100 mm dishes for all treatment conditions and on two-well chamber slides for immunocytochemistry analysis. Scrambled vector (SV- sequence corresponds to the bicistronic shRNA directed against the cathepisn B and uPAR), pU, pC and pCU vectors were transfected into SNB19 and U251 cells independently with Fugene 2000 reagent as per the manufacturer's instructions (Roche, Indianapolis, IN). For the inhibitor study, cells seeded in six well plate were treated with Ly294002 (10 µM), a potent PI3K inhibitor, for 24 hrs.
Cell proliferation assay and cell cycle analysis
Cell proliferation analysis was performed using Cell Proliferation ELISA (colorimetric) BrdU incorporation assay (Roche diagnostics, Indianapolis, IN), according to the manufacturer's protocol. Cell viability analysis was performed by MTT assay using the Cell Titer 96 colorimetric assay as described previously [3]. Phases of cell cycle were analyzed using flow cytometry after 36 hrs and 48 hrs of transfection. Cells were trypsinized, washed with 1X PBS, fixed and permeabilized with cold 70% ethanol and finally incubated for 30 min with 1 mL of propidium iodide (conatins NP-40) (Biosure, CA) in the dark. The DNA content of these cells was measured based on the presence of propidium iodide (PI)-stained cells. Flow cytometric analysis was done on at least 10,000 cells from each sample, and cell cycle data were analyzed using a FACS Calibur flow cytometer (BD BioSciences, San Jose, CA) with an excitation wavelength of 488 nm and emission wavelength of 530 nm.
RT-PCR, Western blot and immunoprecipitation analysis
36 hrs after transfection, total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, USA) and converted to cDNA using Transcriptor First Strand cDNA synthesis kit (Roche diagnostics, Indianapolis, IN) as per manufacturer's instructions. PCR was performed for p27 mRNA expression using forward 5′TCAAAGCAAGCTCTTCATACCC3′ and reverse 5′GCACATAAACTTTGGGGAAGG3′ primers. For immunoblot analysis, cells were washed with ice-cold DPBS and resuspended in 150 µL of radioimmune precipitation assay buffer. The cell lysates were analyzed by SDS-PAGE followed by western blotting. The following antibodies were used: uPAR, cathepsin B, (Athens Research and Technology, Athens, GA, USA) αV (Millipore, Billerica, MA), β3, αVβ3, PI3K, p-PI3K, AKT, p-AKT (Cell Signaling, Boston, MA), p21, p27Kip1, p-p27Kip1 (Ser10), p-p27Kip1 (Thr187), cyclin D1, cyclin D2, Cdk2, cyclin E, Ki67, FOXO3a, p-FOXO3a (Ser253), FOXO3a (Ser318/321), FOXO1, p-FOXO1 (Ser256) (Cell Signaling, Boston, MA), Rb, p-Rb (Ser780 and Ser249/Thr252) and GAPDH. Signals were detected using Pierce Western Blotting substrate (Pierce, Rockford, IL), and the chemiluminescent images were captured in the Flourchem Q, Alpha Innotech, Gel Documentation system. Also, a native gel electrophoresis (7%) was performed on PAGE in the absence of SDS without denaturing the proteins to check the dimerization of αVβ3 integrin
β3 and Cdk2 was immunoprecipitated from 300 µg of total protein using anti- β3 and -Cdk2 antibody and protein A plus G agarose beads (20 µg). The precipitates were washed five times with lysis buffer and once with PBS. The pellet was then resuspended in sample buffer (50 mM Tris, (pH 6.8), 100 mM bromophenol blue, and 10% glycerol) and incubated at 90°C for 10 min before electrophoresis to release the proteins from the beads and immunoblotted for αV and cyclin E, respectively.
Isolation of nuclear and cytoplasmic cell fractions
Cytoplasmic and nuclear extracts from the treated cells were isolated using Active Motif nuclear extraction kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. Harvested cells were washed once with 1X PBS, the cell pellet was resuspended in 200 µL of hypotonic buffer, incubated for 30 min at 4°C on a rocking platform, and centrifuged. The supernatant was collected as the cytosolic fraction. The nuclear pellet was resuspended, homogenized and incubated in complete lysis buffer provided in the kit for 30 min at 4°C on a rocking platform, and the nuclear fraction was collected after centrifugation. Immunoblot analysis was performed with the cytoplasmic and nuclear fractions for proteins like p27Kip1 and FOXO3a. Nuclear fractions were also tested for the expression of the lamin B.
Immunofluorescence assay
Human glioma cells (SNB19 and U251) grown in two-well chamber slides were treated as described earlier. The cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with ice-cold methanol, and rehydrated with PBS. PBST containing 2% BSA was used for blocking the cells for one hour followed by a two-hour incubation with anti-p27Kip1 antibody (Cell Signaling, Boston, MA) at a dilution of 1∶300 in PBST containing 2% bovine serum albumin, followed by a final incubation with Texas Red conjugated secondary antibody (1∶1000 in PBS/2% bovine serum albumin, 0.5% tween 20) for one hour. Expression was visualized by fluorescence microscopy ((Olympus IX71; Olympus Optical Co, Tokyo, Japan)) and photographed.
Construction of human p27Kip1 promoter reporter vector and luciferase activity
To determine the FOXO influenced promoter activity of p27Kip1, three tandem repeats of FOXO binding consensus sequence (GACTGTAAACAAAAC) comprising a 5′ end phospho modification and SacI and XhoI restriction sites on 5′and 3′ends of upper and bottom strands, respectively, were cloned into the pGL3 basic vector and labeled as pGL-Kip-13x. The consensus sequence is positioned at −2984 to −2992 bp. Another plasmid was constructed by altering the consensus sequence (TTGTTTACAA to TTGTGCGCTA) to serve as a negative control and also to show the specificity of the consensus sequence towards FOXO. Similarly, the human p27Kip1 promoter was amplified from genomic DNA using the following primers:
F- aaaGAGCTCCCCACTTTGCAGAAGGATG and
R-aaaCTCGAGGAGCACCATTTTGTCGCTTT;
F-aaaGAGCTCACCTTCGCAGAAACATTTGG and
R-aaaCTCGAGGCAAGAGGTCTCATCCTCTTTA with SacI and XhoI restriction sites on 5′ and 3′ regions of forward and reverse primers, respectively. These primers amplify a 290 bp (located between −3125 to 2845 bp) and an 1110 bp (located between −3507 to −2478 bp) region that includes the FOXO binding consensus sequence. The PCR product was cloned into the promoter-less luciferase reporter vector, pGL3 basic (Promega), predigested with SacI and XhoI, and labeled as pGL-Kip1-290 and pGL-Kip1-1110, respectively.
Luciferase activity was measured with Promega's luciferase assay kit. Following 24 and 48 hrs of transfection, cells were washed twice with PBS and lysed with 100 mL of reporter lysis buffer. The lysate was shaken at room temperature for 10±15 min, after which 20 µL of each cell lysate was mixed with 100 µL of buffer and measured for luciferase activity in a Turner Luminometer (Turner Designs, Sunnyvale, CA, USA) over an integration period of 15 sec. Values obtained were normalized to GAPDH levels.
Intracranial glioma cell implantation, treatment and immunohistochemistry
Stereotactic implantation of SNB19 and U251 glioma cells (1×105), followed by treatments with mock, SV and pCU using Alzet minipumps at the rate of 0.25 µL/hr, the eventual sacrifice of glioma-bearing mice, and tumor processing were carried out as previously described [62], [70]. Sections were stained with hematoxylin and eosin (H&E) to visualize tumor cells and to examine tumor volume as described earlier [3], [71]. The sections were evaluated by a neuropathologist who was blinded as to the treatment group and scored semiquantitatively for tumor size, as described previously [3], [71]. Five animals were used per treatment. The average tumor area per section integrated to the number of sections where the tumor was visible was used to calculate tumor volume and compared between controls and treated groups. Immunohistochemistry for p27Kip1, cathepsin B, uPAR and Ki67 was performed as described earlier.
Statistical analysis
Values are shown as means ± SD of at least three independent experiments. Results were analysed using a two-tailed Student's t-test to assess statistical significance. p<0.05 was considered significant.
Supporting Information
Figure S1 RNAi-mediated depletion of cathepsin B and uPAR affects cell viability and proliferation in SNB19 and U251. A. We analyzed cell viability 36 hrs after transfection using the MTT assay, and the percent of viable cells are represented graphically. Values are mean ± standard deviation (SD) from three different experiments (p<0.01). B. After 48 hrs of transfection with SV, pC, pU and pCU, cells were collected, stained with propidium iodide and analysed for DNA content using flow cytometry. The graph shows the percentage of cells in sub G0/G1, G0/G1, S and G2/M phases. Values are mean ± SD of three different experiments (*p<0.01).
(0.14 MB TIF)
Click here for additional data file.
Figure S2 Cathepsin B and uPAR knockdown induces p27Kip1 and FOXO3a nuclear translocation. A. The ratio of nuclear vs cytoplasmic distribution of p27Kip1 in SNB19 and U251 cells after immunocyto analysis for p27Kip1 were calculated and represented graphically. The values are an average calculated from ten different fields (*p<0.01). B. After transfection with SV, pU, pC and pCU, SNB19 and U251 cells were collected, and proteins from the cytosolic and nuclear fractions were isolated. Immunoblot analysis was performed for the expression of p27Kip1 and FOXO3a in nuclear fractions. The RNAi treatments increased the expression of the above mentioned molecules. C. Glioma cells were treated with siRNA for p27Kip1 individually and in combination with SV, pU, pC and pCU. The cells were subjected to FACS analysis. The graph shows the percent of cells distributed in G0/G1, S and G2/M phases of cell cycle. Values are mean ± standard deviation (SD) from three different experiments (*p<0.01).
(5.63 MB TIF)
Click here for additional data file.
Figure S3 FOXO1 expression is unaffected by treatment with pU, pC and pCU; p27Kip1 and FOXO3a expression decreases with the upregulation of cathepsin B and uPAR. A. Immunoblot analysis of total protein isolated from transfected SNB19 and U251 cells. Immunoblot analysis was performed for the expression of FOXO1, p-FOXO1 (Thr24), p-FOXO1 (Ser256), p-FOXO1 (Ser319)/FOXO4 (Ser262), p-FOXO4 (Ser262) and GAPDH. B. SNB19 and U251 cells were transfected either with SV or full length uPAR (fl-U) or full length cathepsin B (fl-C). Total cell lysates were collected and immunobloted for the expression of uPAR, cathepsin B, p-PI3K, p-AKT, p27Kip1, FOXO3a and GAPDH.
(0.62 MB TIF)
Click here for additional data file.
Figure S4 Transfection efficiency in glioma cells. Glioma cells were transfected with GFP using Fugene (1∶3, Fugene: plasmid ratio). Shown are the images, after transfections,taken under bright and fluorescent fields.
(0.46 MB TIF)
Click here for additional data file.
We thank Peggy Mankin, Noorjehan Ali for technical assistance, Shellee Abraham for manuscript preparation, and Diana Meister and Sushma Jasti for manuscript review.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This research was supported by a grant from National Institutes of Health, CA116708 (to J.S.R.) The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2066855210-PONE-RA-16190R110.1371/journal.pone.0011772Research ArticleCell Biology/Cell SignalingGenetics and Genomics/Gene FunctionMolecular Biology/Post-Translational Regulation of Gene ExpressionBcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic
Melanoma Cells via the Molecular Chaperone HSP90 HIF-1α Stabilization by
Bcl-2Trisciuoglio Daniela
1
Gabellini Chiara
1
2
Desideri Marianna
1
Ziparo Elio
2
Zupi Gabriella
1
Del Bufalo Donatella
1
*
1
Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute,
Rome, Italy
2
Department of Histology and Medical Embryology, Sapienza University,
Rome, Italy
Vooijs Marc EditorUMC Utrecht, Netherlands* E-mail: [email protected] and designed the experiments: DT DDB. Performed the experiments: DT
CG MD. Analyzed the data: DT CG EZ GZ DDB. Contributed
reagents/materials/analysis tools: EZ GZ DDB. Wrote the paper: DT DDB.
2010 27 7 2010 2 8 2010 5 7 e117729 2 2010 29 6 2010 Trisciuoglio et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a
critical mediator of the cellular response to hypoxia. Enhanced levels of
HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated
with increased tumour angiogenesis, metastasis, therapeutic resistance and
poor prognosis. It is in this context that we previously demonstrated that
under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth
Factor (VEGF)-mediated tumour angiogenesis.
Methodology/Principal Findings
By using human melanoma cell lines and their stable or transient derivative
bcl-2 overexpressing cells, the current study identified HIF-1α
protein stabilization as a key regulator for the induction of HIF-1 by bcl-2
under hypoxia. We also demonstrated that bcl-2-induced accumulation of
HIF-1α protein during hypoxia was not due to an increased gene
transcription or protein synthesis. In fact, it was related to a modulation
of HIF-1α protein expression at a post-translational level, indeed
its degradation rate was faster in the control lines than in bcl-2
transfectants. The bcl-2-induced HIF-1α stabilization in response to
low oxygen tension conditions was achieved through the impairment of
ubiquitin-dependent HIF-1α degradation involving the molecular
chaperone HSP90, but it was not dependent on the prolyl hydroxylation of
HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90
proteins form a tri-complex that may contribute to enhancing the stability
of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic
conditions. Finally, by using genetic and pharmacological approaches we
proved that HSP90 is involved in bcl-2-dependent stabilization of
HIF-1α protein during hypoxia, and in particular the isoform
HSP90β is the main player in this phenomenon.
Conclusions/Significance
We identified the stabilization of HIF-1α protein as a mechanism
through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells
involving the β isoform of molecular chaperone HSP90.
==== Body
Introduction
The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) regulates the expression
of more than 70 genes involved in tumour angiogenesis, metabolic switch to anaerobic
glycolysis, pro-survival, proliferative and apoptotic mechanisms [1].
Overall, the expression of HIF-1 target genes helps cells to adapt to, and thereby
survive in, a stressful microenvironment. The activity of HIF-1 dimer, which is
composed of α and β subunits, is modulated by the availability of
the extremely labile oxygen-sensitive HIF-1α protein subunit. HIF-1 activity
depends on the inhibition of the post-transcriptional hydroxylation of the subunit
α by prolyl hydroxylases PHD1-3 and Factor Inhibiting HIF-1 (FIH-1).
PHDs-mediated hydroxylation targets HIF-1α for proteasomal degradation via
the ubiquitination-dependent Von Hippel-Lindau (VHL) complex, while FIH-1-mediated
hydroxylation leads to the inhibition of HIF-1 transactivation. The activity of
PHD1-3 enzymes is dependent on substrates oxygen and 2-oxoglutarate, a Krebs cycle
intermediate, and cofactor Fe2+; thus, under hypoxic conditions,
PHDs are less active due to the substrate-limiting conditions. The regulation of
HIF-1α stability by an oxygen-independent degradation pathway was also
reported: the molecular chaperone Heat Shock Protein 90 (HSP90) binds and stabilizes
HIF-1α, competing with Receptor of Activated protein Kinase C (RACK1), which
mediates prolyl hydroxylase/VHL-independent ubiquitination and proteasomal
degradation of HIF-1α [2]. Other post-translational modifications of
HIF-1α, such as acetylation, phosphorylation and nitrosylation, were also
reported, despite contradictory results with regard to their effect on
HIF-1α protein stability and transcriptional activity [3]–[6]. Adding
to the complexity of HIF-1α regulation, it has recently been shown that the
SUMOylation of HIF-1α enables the hydroxylation-independent binding and
subsequent degradation of HIF-1α by the VHL-E3 ligase complex [7].
Although hypoxia is considered the main stimulus that drives HIF-1 function, a number
of non-hypoxic stimuli allows the formation of an active HIF-1 complex in many types
of human cancers. Effectors implicated in stimulating or suppressing an immune
response promote HIF-1α transcription [8]–[10], whereas
some autocrine growth factors enhance translation of the HIF-1α protein
[1].
Indeed, the loss of function of tumour suppressors and the gain of function of
oncogenes also regulate different steps that lead to HIF-1 activation [1], [11]. In
this context we also found that overexpression of the anti-apoptotic and
pro-survival protein bcl-2, in human melanoma and breast carcinoma cells, under
hypoxia, enhances HIF-1α protein expression and HIF-1 activity consequently
leading to angiogenesis through vascular endothelial growth factor (VEGF) [12],
[13]. Moreover, the treatment of melanoma cells with a
bcl-2/bcl-xL antisense oligonucleotide exterts antiangiogenic activity [14]. We
also demonstrated that bcl-2 plays a role, in cooperation to hypoxia, in cell
migration and invasion, contributing to tumour progression [15], [16].
Indeed, a significant positive correlation between the expression levels of
HIF-1α and bcl-2 was found in neuroblastoma [17].
This study thoroughly investigated the mechanism by which bcl-2 regulates HIF-1 in
tumour cells exposed to hypoxic conditions. It identified the stabilization of
HIF-1α protein as a mechanism by which bcl-2 induces the activation of HIF-1
in hypoxic melanoma cells, through the impairment of ubiquitin-dependent
HIF-1α degradation with the involvement of the β isoform of the
molecular chaperone HSP90.
Results
bcl-2 modulation regulates HIF-1α protein expression in conditions
strictly dependent on oxygen availability
We have previously reported that bcl-2 overexpression in human breast carcinoma
and melanoma cell lines increases HIF-1 expression and activity and VEGF
secretion under hypoxic conditions [12], [13],
[18]. The ability of bcl-2 to modulate VEGF
expression under hypoxia has been also extended to several other human melanoma
cell lines (Figure
S1A,B). The relevance of HIF-1α as the main mediator of bcl-2 induced
VEGF secretion under hypoxic conditions has been demonstrated using siRNA
directed to HIF-1α in M14 cells stably transfected with bcl-2 expression
vector (Figure
S1C). In fact, the down-regulation of HIF-1α protein reduced VEGF
expression both in control cells and bcl-2 overexpressing clones. Interestingly,
after HIF-1α reduction, VEGF levels secreted by bcl-2 transfectants were
similar to those ones of control cells (Figure
S1D). To evaluate whether down-regulation of bcl-2 shows opposite effect of
bcl-2 overexpression in terms of HIF-1α protein expression, we silenced
the endogenous expression of bcl-2 gene transfecting M14 cells with
siRNA-targeting bcl-2 mRNA (si-bcl-2) and then exposing them to normoxia or
hypoxia for 24 h. Western blot analysis demonstrated that the delivery of
si-bcl-2 reduced expression of bcl-2 protein (
Figure 1A
) while, as expected, the transfection of a scrambled si-RNA (si-contr)
did not have any effect on bcl-2 protein expression when compared to
untransfected parental cell line (data not shown). Then, we evaluated the impact
of reduced bcl-2 expression on HIF-1α protein expression. As expected,
HIF-1α protein was undetectable in all cells under normoxic conditions,
while an increased HIF-1α protein expression was observed in the cells
exposed to si-contr under hypoxia, but not in the cells after down-regulation of
the bcl-2 protein expression (
Figure 1A
).
10.1371/journal.pone.0011772.g001Figure 1 bcl-2 modulation regulates HIF-1α protein expression in
conditions strictly dependent on oxygen avaibility.
(A) Western blot analysis of HIF-1α and bcl-2 protein expression
in total extracts of M14 cells transfected with siRNA targeting bcl-2
mRNA (si-bcl-2) or with a control scrambled si-RNA (si-contr) and then
exposed to normoxia or hypoxia for 24 h. (B) Western blot analysis of
HIF-1α and HIF-1β protein expression in total extracts
of M14 control (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37)
cells plated under low (sparse) or high (dense) cell density conditions,
or cultured under normoxia for 4 days or under hypoxia for 24 h. Western
blot analysis of HIF-1α and HIF-1β protein expression in
total extracts of the cells plated under high cell density conditions
and (C) exposed to 24 h shaking or (D) cultured with different volumes
of medium. (E) Western blot analysis of HIF-1α and
HIF-1β protein expression in total extracts of cells exposed to
Insulin (100 nM) or Epidermal Growth Factor (EGF, 20 ng/ml) for 24 h.
(A–E) β-actin protein amounts are used to check equal
loading and transfer of proteins. Western blot analyses representative
of two independent experiments with similar results are shown.
To further characterize the impact of bcl-2 on HIF-1α expression, we
evaluated whether bcl-2 overexpression was able to cooperate with other stimuli,
beyond hypoxia, known to modulate HIF-1 α expression [1].
Firstly, we verified if increased cell density affected the level of
HIF-1α protein in M14 cells stably transfected with empty vector (puro)
and in their two derivative stably bcl-2 overexpressing clones (Bcl2/5,
Bcl2/37). As shown in
Figure 1B
, while HIF-1α protein is detectable at same extent in all cell
lines plated at low density (sparse), regardless of bcl-2 expression, an
increased HIF-1α protein expression was observed in bcl-2 transfectants,
compared to the control line, either when they were plated at high density
(dense) or when they reached high cell density (4 days of culture) and, as
expected and previously reported [12], [18], in hypoxic conditions. HIF-1β was
constitutively expressed in the cells, and none of those stimuli modulated its
expression. Nuclear translocation of HIF-1α subunit is a necessary step
for HIF-1 transcriptional activity through its association with HIF-1β,
which is constitutively localized in the nucleus [1]. In our
experimental model, high cell density conditions induced the nuclear expression
of HIF-1α in bcl-2 overexpressing clones while its expression was
undetectable in control cells (Figure
S2A). In parallel, control cells and bcl-2 overexpressing clones exhibited
density-dependent induction of the HIF-1-dependent transcriptional activity
under normoxic conditions of about 2.3 fold
(p = 0.039) while HRE-dependent transcriptional
activity was not found to be significantly changed in control cells
(p = 0.49) (Figure
S2B).
To further investigate the induction of HIF-1α protein observed in bcl-2
transfectants under high cell density conditions, we evaluated whether the
creation of a local hypoxic microenvironment could be responsible for
HIF-1α induction. Hence, the cells were cultured at high density and
gently shaked to disrupt any potential oxygen gradient due to the inter-cellular
environment and to ensure a homogenous oxygen concentration within the cell
culture medium. As depicted in
Figure 1C
, the gentle shaking drastically reduced the high density-dependent
HIF-1α induction in bcl-2 transfectants, thus indicating that oxygen
pericellular gradient is an important factor contributing in the increase of
HIF-1α expression by bcl-2 in high cell density conditions. To confirm
these results, we plated cells in high density conditions with decreasing
volumes of medium, to enhance the oxygen exchange rate. As shown in
Figure 1D
, the decrease of culture medium volume from 4 to 1 ml determined a
medium volume-dependent reduction of HIF-1α protein expression in both
bcl-2 transfectants.
Next, we evaluated whether any differences existed between control cells and
bcl-2 overexpressing clones in terms of HIF-1α induction in response to
growth-factor stimulation, another condition that induces hypoxia-independent
HIF-1α expression even in normoxia [19]. As shown in
Figure 1E
, both insulin and the Epidermal Growth Factor (EGF) induced
HIF-1α protein expression in all the cells under normoxia but more
importantly no difference in the levels of HIF-1α protein was observed
in bcl-2 transfectants compared to control cells.
bcl-2 promotes HIF-1α protein stability preventing its
ubiquitin-mediated degradation
Since bcl-2 overexpression in melanoma cells under hypoxia did not alter
HIF-1α mRNA levels [12], we investigated the impact of bcl-2
overexpression on HIF-1α protein stabilization under hypoxia. Firstly,
we performed time course experiments to study the kinetics of HIF-1α
protein induction in control cells and bcl-2 overexpressing clones. As shown in
Figure 2A
(left and right panels), exposure of cells to hypoxia determined a
HIF-1α protein induction, at a greater extent in bcl-2 transfectants
compared to control cells, as previously reported. In particular, HIF-1α
protein level reached the maximum value at 24 h of hypoxia in all cell lines,
but it decreased at later time point of 48 h, slower in bcl-2 overexpressing
clones than in control cells.
10.1371/journal.pone.0011772.g002Figure 2 bcl-2 promotes HIF-1α protein stability preventing its
ubiquitin-mediated degradation.
(A) Western blot analysis (left panel) and quantification (right panel)
of HIF-1α protein expression in M14 control (puro) and bcl-2
stably overexpressing (Bcl2/5, Bcl2/37) clones exposed to hypoxia for
the indicated time. (B) Pulse analysis of HIF-1α protein
synthesis rate in cells exposed to
[35S]–labeled methionine and
cysteine for the indicated time. (C) Western blot analysis (left panel)
and quantification (right panel) of HIF-1α protein expression in
cells exposed to hypoxia for 24 h and then treated with Cyclohexamide
(CHX, 50 µg/ml) for the indicated time. (D) Pulse-chase
analysis of HIF-1α protein (left panel) and quantification
(right panel) in cells plated under dense conditions, pulsed for 45 min
with [35S]–labeled methionine and
cysteine and chased for the indicated time. (B,D) Whole cell lysates
were immunoprecipitated (IP) with anti-HIF-1α antibody and
subjected to SDS-PAGE. (E) Western blot analysis of HIF-1α
ubiquitination in the cells exposed to MG132 (10 µM, 6 h) or
to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP)
with anti-HIF-1α antibody and then the Western blot analysis was
performed using anti-Ubiquitin antibody. (A,C) β-actin protein
amounts are used to check equal loading and transfer of proteins and to
quantify relative HIF-1α protein levels. (A–E) Western
blot, pulse and pulse-chase analyses representative of two independent
experiments with similar results are shown. (A,C,D) Densitometric
analysis (right panel) of the relative Western blot or Pulse-chase
analysis (left panel) was performed using Molecular Analyst Software and
normalized with relative controls.
To verify whether bcl-2 enhances HIF-1α protein expression by affecting
its translational rate, we determined the possible involvement of bcl-2 in the
regulation of HIF-1α protein synthesis using
[35S]-labeled methionine and cysteine in pulse
analysis. As shown in
Figure 2B
, HIF-1α protein synthesis rate was almost identical in control
cells and bcl-2 overexpressing clones, indicating that bcl-2 does not affect
HIF-1α protein synthesis. Therefore, the potential role of bcl-2 in the
regulation of HIF-1α protein turnover was analyzed. As depicted in
Figure 2C
(left and right panels), a time-dependent decrease of HIF-1α
protein level was observed after treatment with the protein synthesis inhibitor
Cyclohexamide (CHX) following hypoxia exposure, both in control cells and bcl-2
transfectants. Particularly under CHX exposure for 60 min, the HIF-1α
protein was still well detectable in bcl-2 transfectants while weakly in the
control cells. Indeed, bcl-2 overexpression increased the HIF-1α
half-life from 15±5 min to 45±5 min under hypoxic
conditions (
Figure 2C
). Similar results were obtained evaluating the effect of bcl-2 on
HIF-1α half-life in high cell density conditions, where the
HIF-1α protein half-life was about 20±10 min in control
cells, and increased to 40±5 min in bcl-2 transfectants (Figure
S3). We confirmed these results performing pulse-chase experiment, in which
a pulse with [35S]-labeled methionine and cysteine
was followed by a chase time of varying length (ranging from 15 to 60 min). As
shown in
Figure 2D
, HIF-1α degradation rate was higher in control cells compared to
bcl-2 transfectants, in fact after 45 min of chase, the HIF-1α protein
was still well detectable in bcl-2 transfectants, but not in the control cells.
Next, we tested by immunoprecipitation experiments whether the effect of bcl-2
on HIF-1α stabilization is due to an impairment of HIF-1α
ubiquitination. As shown in
Figure 2E
, higher levels of ubiquitinated HIF-1α were found in control
cells either treated with the proteasome inhibitor MG132 under normoxia, either
exposed to hypoxia, when compared to levels of ubiquitinated HIF-1α
found in bcl-2 transfectants exposed to the same conditions. Taken together, all
these data demonstrate that under hypoxia bcl-2 overexpression modulates
HIF-1α expression at a post-translational level through the
stabilization of the HIF-1α protein.
bcl-2 protein interacts with HIF-1α protein
To test whether the effect of bcl-2 on the stability of HIF-1α is due to
their functional cooperation, we tested the eventual interaction between bcl-2
and HIF-1α protein by immunoprecipitation experiments. When
immunoprecipiatation was carried out using an antibody against bcl-2 protein and
Western blot analysis was performed using antibodies that specifically
recognizing HIF-1α protein, bcl-2 was found to be immunoprecipitated
with HIF-1α protein in control cells and bcl-2 overexpressing clones
after exposure to hypoxia, even though the bcl-2/HIF-1α immunocomplex
was more evident in bcl-2 transfectants when compared to control cells (
Figure 3A
). To confirm the interaction between endogenous HIF-1α and
bcl-2, the cells were treated with MG132 to accumulate similar levels of
HIF-1α protein in all the cells, then immunoprecipitation experiments
were performed using an anti-HIF-1α antibody and the
bcl-2/HIF-1α immunocomplex were analyzed by Western blot using an
anti-bcl-2 antibody (
Figure 3B
). Under these conditions, in spite of similar levels of
immunoprecipitated HIF-1α, bcl-2 protein was well detectable within the
immunoprecipitates in bcl-2 transfectants but only weakly in control cells,
suggesting that HIF-1α interaction with bcl-2 protein was stronger in
bcl-2 overexpressing clones. Similar results were obtained when
immunoprecipitations were performed using different antibodies recognizing
different epitopes on the bcl-2 and HIF-1α proteins (data not shown).
Immunoprecipitation experiments of HIF-1α protein were also perfomed in
two other melanoma cell lines, JR8 and PLF2, and their bcl-2 derivative stably
clones treated with MG132 obtaining similar results (
Figure 3C,D
) and thus generalizing the ability of bcl-2 protein to interact with
HIF-1α protein.
10.1371/journal.pone.0011772.g003Figure 3 bcl-2 interacts with HIF-1α.
(A) Analysis of HIF-1α/bcl-2 protein interaction in M14 control
(puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37) clones exposed
to hypoxia for 24 h. Whole cell lysates were immunoprecipitated (IP)
with anti-bcl-2 or control (IgG) antibodies and then the Western blot
analysis was performed using anti-HIF-1α and anti-bcl-2
antibodies. Analysis of HIF-1α/bcl-2 protein interaction in (B)
M14 control (puro) and stably bcl-2 overexpressing (Bcl2/5, Bcl2/37)
clones or (C,D) in PLF2 and JR8 control cells (PLF2/puro, JR8/puro) and
stably bcl-2 overexpressing (PLF2/Bcl-2, JR8/Bcl-2) cells, exposed to
MG132 (10 µM, 6 h). Whole cell lysates were immunoprecipitated
with anti-HIF-1α or control (IgG) antibodies and then the
Western blot analysis was performed using anti-HIF-1α and
anti-bcl-2 antibodies. (A–D) β-actin protein amounts
are used to check equal loading and transfer of proteins. Western blot
analyses representative of two independent experiments with similar
results are shown.
bcl-2 protein interacts with HIF-1α protein in the
nucleoplasm
bcl-2 is primarily localized in the outer mitochondrial membrane with minor
expression in the nucleus and in the endoplasmatic reticulum [20]. Recent reports indicate that bcl-2 also resides
in the nuclear membrane and may even function within the nucleus [21]–[24]. On the other
hand, HIF-1α protein induced by hypoxic conditions mainly localizes and
elicits its transcriptional activity in the nucleus [1]. Given that bcl-2
is able to interact with HIF-1α, we examined the effect of hypoxia on
the intracellular localization of HIF-1α and bcl-2 by using biochemical
fractionation and confocal microscopy. As reported in
Figure 4A
, hypoxic conditions induced HIF-1α protein translocation in the
nuclear fraction of both control cells and bcl-2 transfectants, even though
HIF-1α protein expression was higher in bcl-2 transfectants. By
contrast, overexpressed bcl-2 protein was expressed in nuclear and mainly in
cytoplasmic compartments, and hypoxia did not modulate both bcl-2 expression or
its cellular localization. Confocal microscopy (
Figure 4B
) confirmed that bcl-2 protein is mainly cytoplasmic but it is also
localized in the nuclear envelope, and hypoxia does not modify bcl-2
localization. As expected, HIF-1α is mainly localized into the nucleus,
it was found to be organized in spots which co-localized with chromatin,
correlated to an enhanced transcriptional activity of HIF-1α under
hypoxia. Given that hypoxia-induced HIF-1α is mainly localized in the
nuclear compartment, we formulated the hypothesis that bcl-2 may regulate
HIF-1α protein stability through the formation of a protein complex
localized in the nucleus. Immunoprecipitation experiments on isolated nuclear
protein extracts showed that bcl-2 was associated with HIF-1α, while
undetectable levels of HIF-1α/bcl-2 complexes were observed in the
cytosolic fraction, indicating that under hypoxia HIF-1α/bcl-2
interaction may only occur in the nucleus (
Figure 4C
). Thus, the finding of an interaction between HIF-1α/bcl-2
proteins in the nucleus suggests that bcl-2 may act on the stabilization of
HIF-1α in this cellular compartment.
10.1371/journal.pone.0011772.g004Figure 4 bcl-2 interacts with HIF-1α in the nucleus.
(A) Western blot analysis of bcl-2 and HIF-1α protein expression
in nuclear (Nucl) and cytoplasmic (Cyto) protein extracts of M14 control
(puro) and bcl-2 stably overexpressing (Bcl2/5) clones exposed to
hypoxia or to normoxia for 24 h. LaminA/C (Lam A/C) and
β-tubulin were used as markers for nuclear and cytoplasmic
fraction, respectively. β-actin protein amounts are used to
check equal loading and transfer of proteins. (B) Confocal laser
scanning microscopy of immunofluorescence staining performed on Bcl2/5
stably overexpressing clone exposed to hypoxia or to normoxia for 24 h.
Fixed cells were labelled with anti-bcl-2 (green) or
anti-HIF-1α (red) antibodies. Nuclei were
visualized using TO-PRO3® staining (blue). (C)
Analysis of HIF-1α/bcl-2 interaction in Bcl2/5 stably
overexpressing clone exposed to hypoxia for 24 h. Nuclear (Nucl) and
cytoplasmic (Cyto) protein extracts were immunoprecipitated (IP) with
anti-HIF-1α or anti-bcl-2, respectively, or control antibody
(IgG) and then the Western blot analysis was performed using anti-bcl-2
or anti-HIF-1α antibodies. (A–C) Western blot and
confocal analyses representative of two independent experiments with
similar results are shown.
bcl-2 regulates HIF-1α protein stability in a prolyl
hydroxylation-independent manner
Under normoxia, the proline to alanine mutation of residues 402 and 564 of human
HIF-1α makes the protein resistant to PHD-dependent hydroxylation and
subsequent VHL-dependent ubiquitination and degradation [25]. Besides, PHD2 can be
active in the degradation of HIF-1α even under hypoxic conditions [26],
[27]. In order to study the impact of bcl-2 on
PHD-mediated degradation of HIF-1α protein, we generated M14 cell line
stably expressing wild type form of HIF-1α
(HIF-1α wt) or hydroxylation-resistant (P402A/P564A)
form of HIF-1α (HIF-1α PP/AA). These cells were
then transiently transfected with an empty vector or with a vector encoding
bcl-2 protein and HIF-1α expression and transcriptional activity were
analyzed under hypoxic conditions. As depicted in
Figure 5
, bcl-2 overexpression significantly increased the levels of both
exogenous wt and hydroxylation-resistant form of HIF-1α
(
Figure 5A
) and it also enhanced HRE-dependent transcriptional activity (
Figure 5B
). As expected, PHD2 overexpression inhibited the expression of
HIF-1α wt and HRE-dependent transcriptional activity
while it did not abrogate the expression and activation of reporter gene
transcription in cells expressing HIF-1α protein containing the
proline-to-alanine substitutions (
Figure 5B
). The discovery that bcl-2 had similar effects on the
wt and mutant form of HIF-1α indicated that bcl-2
regulates HIF-1α expression independently from prolyl hydroxylation of
HIF-1α. These results are also supported by the findings that forced
expression of bcl-2 had no impact on HIF-1α stabilization when cells
were treated with PHD inhibitors Cobalt Chloride and Desferoxamine, two iron
antagonists known to inhibit hydroxylase activity (Figure
S4).
10.1371/journal.pone.0011772.g005Figure 5 HIF-1α prolyl hydroxylation is not required for bcl-2-induced
increase of HIF-1α expression and HIF-1 activity in
hypoxia.
(A) Western blot analysis of HIF-1α, bcl-2 and PHD2 protein
expression and (B) HRE-dependent transcriptional activity in M14 cells
stably expressing HA-HIF-1α wild-type (HIF1α
wt) or mutated (HIF1α
PP/AA), after transiently transfection with control
vector (empty), bcl-2 or PHD2 expressing vectors, and then exposure to
hypoxia for 24 h. (A) β-actin protein amounts are used to check
equal loading and transfer of proteins. Western blot analyses
representative of two independent experiments with similar results are
shown. (B) Relative luciferase activity of each sample were normalized
to the control vector transfected cells. Results represent the mean
± SD of 3 independent experiments performed in triplicate,
* p≤0.01.
bcl-2 forms a complex with HSP90 and HIF-1α proteins, enhancing their
interaction and protecting HIF-1α from degradation mediated by
17-AAG
HSP90 is a molecular chaperone required for the stability and function of a
number of proteins implicated in cancer cell growth and angiogenesis, including
HIF-1α [28]. In particular, it binds and stabilizes
HIF-1α, and it represents a critical factor in an
O2/PHD/VHL-independent degradation pathway of HIF-1α protein
[2].
To evaluate a possible contribution of HSP90 to bcl-2-induced stabilization of
HIF-1α, we determined whether the pharmacological inhibition of HSP90
with 17-AAG, an inhibitor that can alter the interaction of HSP90 with its
clients [29], modulates HIF-1α expression (
Figure 6A
) and transcriptional activity (
Figure 6B
) in control cells and two bcl-2 transfectants cells under hypoxia.
17-AAG reduced hypoxia-induced HIF-1α accumulation in control cells,
while only a very barely down-regulation of HIF-1α protein expression
was evident in bcl-2 overexpressing clones after 17-AAG treatment (
Figure 6A
). These results suggested that bcl-2 overexpression might confer a
resistance of HIF-1α protein from the degradation induced by the 17-AAG.
On the functional level, 0.05 µM 17-AAG induced about 30%
versus 10% inhibition of HRE-dependent transcriptional activity in
control cells compared with bcl-2 transfectants. The higher dose of 2
µM completely inhibited HRE-dependent transcriptional activity in
control cells, by contrast bcl-2 transfectants cells were resistant to
HRE-dependent transcriptional activity inhibition induced by the same dose of
17-AAG (
Figure 6B
). Most importantly, as shown in
Figure 6C
, HSP90 protein is highly expressed in both control and bcl-2
overexpressing cells, and the impact of either bcl-2 status and either hypoxic
conditions on HSP90 protein expression was not relevant. To provide evidence
that the HSP90 is involved in bcl-2-induced stabilization of HIF-1α, we
investigated the effect of bcl-2 on the interaction between HIF-1α and
HSP90 proteins by immunoprecipitation of HIF-1α and Western blot
analysis of HSP90 protein. As depicted in
Figure 6D
, bcl-2 overexpression under hypoxia enhanced the ability of
HIF-1α to form a complex with HSP90. To confirm the interaction between
HIF-1α and HSP90 proteins, we performed a reverse immunoprecipitation
experiment from total extract of hypoxic cells. Under these conditions, in spite
of similar levels of immunoprecipitated HSP90, a larger amount of HIF-1α
protein within the immunoprecipitate was found in total extracts of bcl-2
transfectants (
Figure 6E
), confirming a stronger interaction between HIF-1α and HSP90
proteins in bcl-2 transfectants. We also studied the interaction between HSP90
and bcl-2 protein under hypoxic conditions and we found that HSP90 was
associated with ectopic bcl-2 protein (
Figure 6E
). Similar results were also observed when immunoprecipitation
experiments were carried out in nuclear extracts (data not shown). These
findings suggest that bcl-2 may promote stabilization of HIF-1α by
increasing its ability to interact with the HSP90 chaperone complex. To gain
insight to these results, we investigated whether the bcl-2/HSP90/HIF-1α
binding could be reversed when exposing the cells to 17-AAG. We found that
17-AAG treatment reduced the binding between HSP90 and HIF-1α only in
control cells and weakly in bcl-2 transfectants, confirming that bcl-2
overexpressing cells were more resistant to 17-AAG-induced degradation of
HIF-1α. Moreover, we found that the interaction of bcl-2 protein with
HIF-1α was not affected by 17-AAG treatment (
Figure 6F
). Because our results showed that both HSP90 and HIF-1α proteins
bind to bcl-2, we investigated the potential formation of a
HSP90/HIF-1α/bcl-2 tri-complex. To address this hypothesis, the cell
lysates were firstly immunoprecipitated with anti-HIF-1α antibody, then
subjected to a second immunoprecipitation with anti-bcl-2 antibody, and the
immunocomplexes were analyzed by Western blot analysis using antibody against
HSP90 protein. As shown in
Figure 6G
, HSP90 could be found in complex with HIF-1α and bcl-2 protein
in cells overexpressing bcl-2, demonstrating the formation of a
HSP90/HIF-1α/bcl-2 tri-complex. Overall these findings suggested that
bcl-2 may promote stabilization of HIF-1α by increasing its ability to
interact with the HSP90 chaperone complex, probably affecting its folding and
maturation.
10.1371/journal.pone.0011772.g006Figure 6 bcl-2 forms a complex with HSP90 and HIF-1α proteins.
(A) Western blot analysis of HIF-1α protein expression in M14
control cells (puro) and bcl-2 stably overexpressing (Bcl2/5, Bcl2/37)
clones treated with 17-AAG under hypoxia or exposed to normoxia for 24
h. (B) HRE-dependent transcriptional activity in the cells treated with
17-AAG from 0.05 to 2 µM under hypoxia or exposed to normoxia
for 24 h. Relative luciferase activity of each sample was normalized to
untreated cells exposed to normoxic conditions. Results represent the
average ± SD of 3 independent experiments performed in
triplicate. p values were calculated relative to untreated cells exposed
to hypoxic conditions, *p≤0.01. (C) Western blot analysis
of HSP90 protein expression in parental M14 cells, control (puro) and
bcl-2 stably overexpressing (Bcl2/5, Bcl2/37) clones. (D) Analysis of
HIF-1α/HSP90 interaction in the cells exposed to hypoxia for 24
h. Whole cell lysates were immunoprecipitated (IP) with
anti-HIF-1α or control (IgG) antibodies and then the Western
blot analysis was performed using anti-HSP90 and anti-HIF-1α
antibodies. (E) Analysis of HSP90/HIF-1α and HSP90/bcl-2
interactions in the cells exposed to hypoxia for 24 h. Cell lysates were
immunoprecipitated (IP) with anti-HSP90 or control (IgG) antibodies and
then the Western blot analysis was performed using anti-HIF-1α,
anti-bcl-2 and anti-HSP90 antibodies. (F) Analysis of
HIF-1α/HSP90 and HIF-1α/bcl-2 interactions in the cells
treated with 0.5 µM 17-AAG for 24 h under hypoxia. Whole cell
lysates were immunoprecipitated (IP) with anti-HIF-1α antibody
and then the Western blot analysis was performed using specific
anti-HSP90 and bcl-2 antibodies. (G) Analysis of
HSP90/HIF-1α/bcl-2 protein complex in the cells exposed to
hypoxia for 24 h. Whole cell lysates were sequentially
immunoprecipitated with anti-HIF-1α (IP1) and anti-bcl-2
antibodies (IP2) and then the Western blot analysis was performed using
anti-HSP90 antibody. (A,C) β-actin protein amounts are used to
check equal loading and transfer of proteins.
HSP90β isoform is the mediator of HIF-1α induction by bcl-2
under hypoxic conditions
The molecular chaperones HSP90 comprise two homologous proteins, HSP90α
and HSP90β, that are encoded by distinct genes [28]. Experiments were
performed to evaluate the impact of bcl-2 overexpression on the expression of
these isoforms and their binding to HIF-1α protein. We found that both
the hypoxic conditions and bcl-2 protein level of the cells did not modulate the
expression of HSP90α and HSP90β proteins (
Figure 7A
). We then investigated the effect of bcl-2 on the interaction between
HIF-1α and HSP90s proteins by immunoprecipitation of HIF-1α
protein. As depicted in
Figure 7B
, Western blot analysis, using antibodies specifically recognizing the
isoform α or β, showed that HSP90β, but not
HSP90α, forms a complex with HIF-1α protein in bcl-2
overexpressing cells exposed to hypoxia. To further validate the involvement of
HSP90 proteins and to confirm the possibility that HSP90β, rather than
α isoform, is involved in HIF-1α stabilization mediated by bcl-2
in hypoxia, HIF-1α protein expression was evaluated in bcl-2
overexpressing cells after transfection with shRNA targeting the α
(shHSP90α) or the β (shHSP90β) isoforms. As control,
cells were transfected with scramble shRNA vector (shNC).
10.1371/journal.pone.0011772.g007Figure 7 HSP90β is the mediator of HIF-1α induction by bcl-2
under hypoxic conditions.
(A) Western blot analysis of HSP90α and HSP90β protein
expression in M14 control (puro) and bcl-2 stably overexpressing
(Bcl2/5, Bcl2/37) clones exposed to hypoxia or to normoxia for 24 h. (B)
Analysis of HSP90α/HIF-1α and
HSP90β/HIF-1α interactions in the cells exposed to
hypoxia for 24 h. Protein extracts were immunoprecipitated (IP) with
anti-HIF-1α and then Western blot analysis was performed using
anti-HSP90α and anti-HSP90β antibodies. (C,D) Western
blot analysis of HIF-1α, HSP90α and HSP90β
protein expression in bcl-2 stably overexpressing cells transiently
transfected with short hairpin construct targeting HSP90β
(shHSP90β), HSP90α (shHSP90α) or with control
vector (shNC) and exposed to hypoxia or to normoxia for 24 h. (A,C,D)
β-actin protein amounts are used to check equal loading and
transfer of proteins. (A–D) Western blot analyses
representative of two independent experiments with similar results are
shown.
Western blot analysis confirmed the effective knockdown of the expression of each
HSP90 target (
Figure 7C,D
). Moreover, the specificity of each shRNA against HSP90 was demonstrated
by the absence of expression modulation of the other HSP90 isoform, verifying
that both shRNAs were highly specific for their respective targets.
Interestingly, Western blot analysis showed that shHSP90β (
Figure 7C
), but not shHSP90α (
Figure 7D
), completely inhibited hypoxic induction of HIF-1α protein in
bcl-2 overexpressing cells.
Discussion
The bcl-2 protein is an inhibitor of apoptosis that has been recognized to play an
important role also in a wide range of other biological processes, among which
autophagy, DNA repair and drug resistance [21], [30]–[32].
Recent studies, including ours, have demonstrated that bcl-2 also promotes tumour
progression and angiogenesis of different tumour histotypes [13], [16],
[33],
[34]. In
this context, we have previously demonstrated that under hypoxic conditions the
overexpression of bcl-2 in tumour cells is able to increase tumor angiogenesis
enhancing the secretion of the pro-angiogenic factor VEGF, through the induction of
HIF-1α protein expression and HIF-1 transcriptional activity [12],
[13].
In the present study, we investigated the mechanism by which bcl-2 regulates
HIF-1α protein expression in M14 melanoma cells under conditions strictly
dependent on oxygen availability, such as hypoxia and high cell density. We
demonstrated that HIF-1α protein is required for bcl-2-induced VEGF
expression under hypoxia by using a small interference approach. Moreover, we
confirmed the capability of bcl-2 to modulate VEGF expression in several melanoma
cells. We showed that also in high cell density conditions, which create a local
pericellular hypoxic microenvironment, bcl-2 overexpression determines an increase
of HIF-1α protein expression and HIF-1 transcriptional activity, similar to
the ones obtained in hypoxia. Alternatively, bcl-2 is not able to cooperate with
insulin or EGF to induce HIF-1α protein expression under normoxia,
highlighting that the capacity of bcl-2 to regulate HIF-1α protein
expression strictly depends on oxygen availability.
We further identified HIF-1α protein stabilization as a key mechanism for
HIF-1 induction by bcl-2 under hypoxia. Our data demonstrated that bcl-2 under this
condition affects HIF-1α protein at the post-translational level, indeed the
degradation rate of HIF-1α protein was faster in the control cells than in
bcl-2 transfectants. Although under normoxia this HIF-1α stabilization is
not sufficient to affect the steady state levels of the protein, it becomes rate
limiting during hypoxia or, in general, in conditions strictly dependent on oxygen
level. In fact, we found that bcl-2 overexpression determines an increase of
HIF-1α protein half-life also in high cell density conditions, as observed
under hypoxia. The stabilization of HIF-1α protein in response to changes in
oxygen concentration is achieved through the impairment of HIF-1α
ubiquitination and subsequent degradation of the protein. Generally, HIF-1α
is degraded in an oxygen-dependent manner through the activity of PHD2 enzyme, which
hydroxylates HIF-1α on proline residues 402 and 564, and this hydroxylated
form is bound by the E3 ubiquitin ligase VHL which promotes HIF-1α
ubiquitination and its subsequent proteasomal degradation [19]. Notwithstanding,
we found that bcl-2 regulates HIF-1α protein stability in a prolyl
hydroxylation-independent manner since bcl-2 overexpression had similar effects on
either wild type protein and the degradation resistant form of
HIF-1α, which contains proline-to-alanine substitutions (P402A/P564A)
triggering a resistance to PHD2-mediated hydroxylation. In agreement with this
finding, in our experimental model PHD2 protein expression was upregulated in
response to hypoxia at comparable levels in parental cells and bcl-2 overexpressing
clones (data not shown). Further, bcl-2 overexpression had no impact on
HIF-1α protein stabilization induced by iron antagonists known to inhibit
hydroxylase activity, such as Cobalt Chloride and Desferoxamine.
Some authors have reported that bcl-2 may reside, and even elicit a function, within
the nucleus [21]–[23], modulating the
transactivity of several transcription factors [35], [36]. Here, we present
evidence that in our experimental model the exogenous bcl-2 protein is also
localized in the nucleus, beyond the cytoplasm. Of note, our results reveal, for the
first time, that bcl-2 protein interacts with HIF-1α in the nucleus, thus
the pro-angiogenic effect of bcl-2 on HIF-1/VEGF axis may result from the nuclear
localization of bcl-2. Since the HIF-1α/bcl-2 complex can be observed in the
nucleus, we can speculate that bcl-2-mediated stabilization of HIF-1α
protein occurs in this cellular compartment. By dissecting the molecular mechanism
of this process, we found that bcl-2 increases HIF-1α protein stability
through the involvement of the molecular chaperone HSP90, which was found to protect
HIF-1α from proteasomal degradation, even in VHL-deficient cells [37], [38].
In this context, our data further indicate that the enhanced levels of
HIF-1α protein in bcl-2 overexpressing clones may be due to a decreased
poly-ubiquitination of HIF-1α by enforcing the interaction between
HIF-1α and HSP90 protein. Moreover, we have shown not only a novel
association of HIF-1α with bcl-2, but we have also observed that bcl-2 is
able to interact with HSP90 itself. Most importantly, we found that the interaction
between bcl-2 and HIF-1α proteins was not dependent on HSP90 inhibition,
because the binding of bcl-2 and HIF-1α was not reversed by the treatment
with 17-AAG. In addition, sequential immunoprecipitation experiments demonstrated
that bcl-2, HIF-1α and HSP90 proteins may form a tri-complex which probably
contributes to enhance HIF-1α protein stability in bcl-2 overexpressing
clones under hypoxia. Here, we investigated the role of HSP90α and
HSP90β isoforms in bcl-2-mediated HIF-1α induction under hypoxic
condition. These two homologous proteins display some differences and elicit
specific functions, such as differential binding to client proteins [28].
Using genetic approaches to specifically knockdown each HSP90 isoform in bcl-2
overexpressing cells, we found that HSP90β, but not HSP90α, is
required for HIF-1α protein stabilization by bcl-2. Moreover, in agreement
with these data, we found that only HSP90β binds HIF-1α protein in
bcl-2 overexpressing cells exposed to hypoxia. These results are in a good
accordance with very recent data demonstrating an association between β
isoform of HSP90 and bcl-2 protein in response to VEGF in leukemia cells [39] or to CpG
oligodeoxynucleotide in macrophages [40]. All together, these results confirm that
HSP90β is an important regulator of HIF-1α stability and indicate
that this molecular chaperone may be one of the mediators of bcl-2 pro-angiogenic
function. A recent report demonstrated that RACK1 protein promotes ubiquitination of
HIF-1α induced by the HSP90 inhibitor 17-AAG and its subsequent
VHL-independent proteasomal degradation competing with HSP90 for binding to PAS
domain of HIF-1α [2]. Notwithstanding, when exposing melanoma cells to
the HSP90 inhibitor 17-AAG, we observed that bcl-2 overexpression counteracts both
HIF-1α protein degradation induced by 17-AAG, and the reduction of
interaction between HIF-1α and HSP90 induced by the inhibitor. Besides, we
did not observe any difference in the HIF-1α binding to RACK1 after forced
expression of bcl-2 under hypoxia even after 17-AAG exposure (data not shown),
suggesting that bcl-2 does not regulates RACK1/Elongin-C dependent HIF-1α
degradation pathways. So far we cannot exclude that other molecular players, such as
HSP70, JNK1 and the COMMD1 proteins [41]–[43], may be
modulated by bcl-2 and play a role in the stabilization process of HIF-1α
protein mediated by bcl-2.
In conclusion, our study establishes a molecular link and highlights the possibility
that bcl-2 is a new HIF-1α-binding protein whose multivalent interactions
are required for the stabilization of HIF-1α, and that nuclear localization
of bcl-2 may have an important role in protecting HIF-1α from ubiquitination
and proteasomal degradation that commences in the nucleus.
Materials and Methods
Cell cultures, hypoxia exposure, transfections and viral infection
Human melanoma cell lines were cultured in complete RPMI medium (Invitrogen,
Carlsbad, CA). JR1, JR8, M14, PLF2 [44], and ASM-SC,
bcl-2 overexpressing clones (Bcl2/5 and Bcl2/37) and a control clone (puro)
derived from the M14 line after stable transfection, bcl-2 overexpressing
(JR8/Bcl-2 and PLF2/Bcl-2) and control (JR8/puro, PLF2/puro) cells derived from
the JR8 and PLF2 line after stable transfection were used. ASM-SC was cloned by
limiting dilution from A375.S2 melanoma cell line (ATCC, Manassas, VA). For
hypoxia exposure, culture dishes were placed in a hypoxia chamber allowing the
formation of a hypoxic environment of 5% CO2,
95% N2. Unless stated otherwise, these hypoxic levels
(1% of oxygen concentration, 24 h) was used in all experiments. For
experiments under low or high cell density conditions, 100 cells/mm2
or 700 cells/mm2 were respectively plated and 24 h later cells were
harvested and subjected to different assays.
The cells were stably or transiently transfected with the expression vector
encoding the human wild type bcl-2. Transfections of expression
vectors or RNA interference were performed as previously reported [44],
using Lipofectamine (Invitrogen). SureSilencing shRNA plasmids against
HSP90α and β isoforms containing the hygromycin resistance gene
were obtained from SABiosciences (Frederick, MD). Polyclonal population of
stably transfected cells were used. Viruses were generated as previously
described [45]. In short, the Phoenix amphotropic packaging
line was transfected with the pBabe-based retroviral expression vectors carrying
wild type (Addgene plasmid 19365) or
hydroxylation-resistant (P402A/P564A) form (Addgene plasmid 19005) of HA-tagged
HIF-1α. Transfected cells were incubated for 48 h at 37°C for
virus production. The virus-containing medium was collected, filtered and used
to infect the target cells. Stable clones or mixed populations were cultured in
the presence of puromycin (1 µg/ml).
Reagents
Cyclohexamide (CHX), Z-leu-leu-leu-CHO (MG132), Cobalt Chloride
(CoCl2), Desferrioxamine (DFO),
17-Allylamino-17-demethoxy-geldanamycin (17-AAG), insulin, Epidermal Growth
Factor (EGF) were purchased from Sigma-Aldrich (St. Louis, MI, USA).
Isolation of nuclear/cytoplasmic fractions
Nuclear and cytoplasmic fractions were prepared as follows:
1–2×106 cells were resuspended in a hypotonic
lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA)
containing protease inhibitors (Boehringer). After resuspension, NP-40 was added
to a final concentration of 0.6% and the nuclei were isolated by
centrifugation at 10,000 r.p.m. for 30 s at 4°C. After removing the
supernatant (i.e. the cytoplasmic extract), the nuclei were re-suspended in a
nuclear extract buffer (20 mM HEPES pH 7.9, 25% glycerol, 0.4 M NaCl,
0.1 mM EDTA, 0.1 mM EGTA), rocked for 15 min at 4°C and then recovered
by centrifugation at 140,00 r.p.m. for 5 min at 4°C.
Immunoprecipitation and Western blot analysis
For immunoprecipitation assays and Western blot analysis, the cells were lysed in
0.3% CHAPS buffer (40 mM HEPES [pH 7.5], 120 mM
NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM
Na3VO4, 0.3% CHAPS, and one tablet
EDTA-free protease inhibitors [Roche] per 10 ml). Followed by
centrifugation, the supernatant was precleared with protein A/G agarose beads
coupled with mouse or rabbit IgG (Pierce, Thermo Fisher Scientific, Rockford,
IL) for >2 h and then was exposed to 1 µg of the antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) or mouse or rabbit IgG, as control,
was added to each of the cellular lysates and incubated overnight at 4°C
followed by incubation with protein A/G-agarose beads (Amersham Biosciences
Europe, Milan, Italy) for 2 h at 4°C. Immunoprecipitates were washed
four times in the lysis buffer before Western blotting analysis. For some
immunoprecipitation experiments we used ExactaCruz™ reagents (Santa
Cruz Biotecnology) to detect the bcl-2 protein without detection of the light
chain of the immunoprecipitation antibody. Immunoprecipitation were also
performed using multiple antibodies recognizing different epitopes on the bcl-2
(Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus
Biologicals, Littleton, CO) protein. Sequential immunoprecipitation experiments
were performed incubating 2 mg of total cell lysate with antibody as for single
immunoprecipitation, after washing the precipitated proteins were released with
1% SDS at 37°C for 30 minutes. Then, the eluate was diluted
to a final concentration of 0.1% SDS with lysis buffer and
immunoprecipitation was repeated with the supernatant with fresh beads and
antibody.
For Western blot analysis, antibodies directed to HIF-1α, HIF-1β,
HSP90 (BD Pharmingen), HA epitope, ubiquitin (Santa Cruz Biotecnology), bcl-2
(Dako, Milan, Italy), β-tubulin (Thermo Scientific), HSP90α,
HSP90β (Abcam, Cambridge, UK), PHD2 (Novus Biologicals), Lamin A/C (Cell
Signaling, Danvers, MA), β-actin (Sigma) were used.
Pulse and pulse-chase assays
In the pulse assay, cells were incubated with methionine/cysteine–free
serum-free DMEM (Invitrogen) for 2 h. [35S]-labeled
methionine-cysteine (88 µCi/ml, EasyTag™
EXPRESS35S Protein Labeling Mix, PerkinElmer, Waltham, MA) was
added to the medium and cells were collected after 15 and 45 min. In the
pulse-chase assay, after 45 min pulse with
[35S]-labeled methionine-cysteine, cells were
washed three times with PBS, chased with DMEM containing 10% FBS and
2.5 mg/mL cold L-methionine and harvested after 15, 30, 45 and 60 min. Total
protein lysates from pulse and pulse-chase assays were immunoprecipitated by
HIF-1α antibody. Radiolabeled HIF-1α protein and the input cell
lysates were subjected to SDS-PAGE. Gels were dried, exposed in phosphorImager
cassette for 1–3 days and imaged using Personal Molecular Imager FX
and Quantity One® software (Biorad Laboratories, Hercules, CA).
ELISA
The supernatants were harvested and assayed for VEGF content by ELISA kit
according to the manufacturer's instructions (R&D Systems,
Minneapolis, MN). VEGF levels were normalized to the number of adherent
cells.
Reporter gene assay
The cells were seeded in 24-well plates and were transfected with a total of 1
µg of DNA/well using Lipofectamine reagent. The evaluation of HIF-1
transcriptional activity was performed as previously described [12]
transfecting cells with a vector expressing luciferase under the control of 4X
Hypoxia Responsive Element (HRE) and another one expressing
β-galactosidase under the control of CMV promoter. The relative
luciferase activity was calculated by luciferase/β- galactosidase ratios
for each sample.
Confocal analysis
After 24 h hypoxic conditions exposure, cells were fixed in 100%
methyl alcohol for 10 min at −20°C and then incubated with
primary antibodies. The cells were incubated with TRITC conjugated Goat
anti-Rabbit and/or FITC conjugated Goat anti-Mouse (Jackson Lab, West Grove,
PA). Nuclei were visualized using TO-PRO3® (Invitrogen). The images were
scanned under a ×40 oil immersion objective and to avoid bleed-through
effects, each dye was scanned independently by a Leica confocal microscope
(laser-scanning TCS SP2) equipped with Ar/ArKr and HeNe lasers. The images were
acquired and electronically merged utilizing the Leica confocal software (Leica
Microsystems Heidelberg GmbH, Mannheim, Germany). Figures were processed using
Adobe PhotoShop software.
Densitometric analysis
Developed films were acquired using GS-700 Imaging Densitometer (Biorad) and
processed with Corel Photo Paint 7.0 to adjust image brightness and contrast.
Densitometric evaluation was performed using Molecular Analyst Software (Biorad)
and normalized with relative controls depending on the analysis performed.
Statistical Analysis
Differences between groups were analyzed with a two-sided paired or unpaired
Student's t test by use of GraphPad Prism 3.00
(GraphPadSoftware, San Diego, CA). Results were considered to be statistically
significant if p<0.05. Experiments were usually repeated three times
unless indicated otherwise.
Supporting Information
Figure S1 HIF-1α protein is required for VEGF induction by bcl-2 in melanoma
cells under hypoxia. (A) Western blot analysis of bcl-2 protein expression
in whole extracts and (B) ELISA assay of VEGF protein in conditioned medium
in several human melanoma cell lines exposed to normoxia and hypoxia for 24
h, after transient transfection with control (empty) or bcl-2 expressing
vector (Bcl-2). (C) Western blot analysis of HIF-1α and
HIF-1β protein expression in total extracts and (D) ELISA assay of
VEGF protein in conditioned medium in M14 cells stably transfected with
control (puro) or bcl-2 expression vector (Bcl2/5) after transfection with
siRNA directed against HIF-1α (siHIF-1α) or unrelated
control mRNA (siNC) and then exposed to normoxia or hypoxia for 24 h. (A,C)
β-actin protein amounts are used to check equal loading and transfer
of proteins. Western blot analyses representative of two independent
experiments with similar results are shown. (B,D) Results represent the mean
± SD of 3 independent experiments performed in triplicate. Fold
induction of secreted VEGF protein relative to normoxia. *
p<0.01
(0.98 MB TIF)
Click here for additional data file.
Figure S2 Bcl-2 cooperates with high cell density conditions to induce nuclear
HIF-1α protein and HIF-1 transactivation activity. (A) Western blot
analysis of HIF-1α and HIF-1β protein expression in
cytoplasmic (Cyto) and nuclear (Nucl) protein extracts of M14 control (puro)
and bcl-2 overexpressing (Bcl2/5, Bcl2/37) cells plated under low (sparse)
or high (dense) cell density condition. β-actin protein amounts are
used to check equal loading and transfer of proteins. Western blot analysis
representative of two independent experiments with similar results are
shown. (B) HRE transcriptional activity of the cells cultured under sparse
or dense conditions. Results represent the mean ±SD of 3
independent experiments performed in triplicate. Fold induction relative to
sparse condition. * p<0.01
(0.88 MB TIF)
Click here for additional data file.
Figure S3 Bcl-2 promotes HIF-1α protein stability in high cell density
conditions. Western blot analysis (panel left) and quantification (panel
right) of HIF-1α protein expression in total lysates of melanoma
control (puro) and bcl-2 overexpressing (Bcl2/5, Bcl2/37) cells cultured
under high cell density conditions (dense) and then treated with
Cyclohexamide (CHX, 50 µg/ml) for the indicated times.
β-actin protein amounts are used to check equal loading and transfer
of proteins. Western blot analysis representative of two independent
experiments with similar results are shown. Densitometric analysis (panel
right) of the relative Western blot (panel left) was performed using
Molecular Analyst Software and normalized with relative controls depending
on the analysis performed.
(0.89 MB TIF)
Click here for additional data file.
Figure S4 Bcl-2 does not cooperate with hypoxic mimetic compounds to induce
HIF-1α protein expression. Western blot analysis of HIF-1α
protein expression in total lysates of M14 control (puro) and bcl-2
overexpressing (Bcl2/5, Bcl2/37) cells exposed to desferrioxamine (DFO, 50
µM) or Cobalt Cloride (CoCl2, 100 µM) for 3 h.
β-actin protein amounts are used to check equal loading and transfer
of proteins. Western blot analyses representative of two independent
experiments with similar results are shown.
(0.39 MB TIF)
Click here for additional data file.
We are grateful to Adele Petricca for secretarial assistance and to Tania Merlino for
English revision of the manuscript. We thank Dr Sergio Anastasi and Dr Fabrizio
Antonangeli for helpful suggestions, and Stefania De Grossi and Carla Ramina for
technical assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by grants from the Italian Association for Cancer
Research, grant number 08/30/c/91 (D.D.B.) and the Italian Ministry of Health,
grant number 08/01/c/48 (D.D.B.) and 08/01/c/1 (G.Z.). The funders had no role
in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2067640210-PONE-RA-1960410.1371/journal.pone.0011817Research ArticleBiochemistry/Transcription and TranslationCell Biology/Gene ExpressionGastroenterology and Hepatology/Inflammatory Bowel DiseaseOxidative Stress and Mitochondrial Functions in the Intestinal Caco-2/15 Cell Line Mitochondrial DysfunctionTaha Rame
1
Seidman Ernest
2
3
Mailhot Genevieve
1
Boudreau François
3
Gendron Fernand-Pierre
3
Beaulieu Jean-François
3
Ménard Daniel
3
Delvin Edgard
4
Amre Devendra
5
Levy Emile
1
3
*
1
Department of Nutrition, Research Center, CHU-Sainte-Justine, Université de Montréal, Montreal, Canada
2
Research Institute, McGill University, Montreal, Canada
3
Canadian Institutes for Health Research Team on the Digestive Epithelium, Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada
4
Department of Biochemistry, Research Center, CHU-Sainte-Justine, Université de Montréal, Montreal, Canada
5
Department of Pediatrics, Research Center, CHU-Sainte-Justine, Université de Montréal, Montreal, Canada
Hansen Immo A. EditorNew Mexico State University, United States of America* E-mail: [email protected] and designed the experiments: RT EL. Performed the experiments: RT EGS GM FPG JFB EL. Analyzed the data: RT JFB EL. Contributed reagents/materials/analysis tools: RT FB DM ED DA EL. Wrote the paper: RT EL.
2010 27 7 2010 5 7 e118177 6 2010 2 7 2010 Taha et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules.
Methodology/Principal Findings
The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 µM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities.
Conclusions/Significance
Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.
==== Body
Introduction
Reactive Oxygen Species (ROS) are by-products of normal aerobic metabolism and are now considered to be important signaling molecules that play a role in gene expression, cell growth and survival as well as oxygen sensing in various cell types [1], [2]. The generation of ROS by a cascade of reactions is efficiently blocked by various endogenous antioxidants to overcome their potentially injurious actions [2], [3]. However, excessive formation of ROS leads to lasting oxidative stress, characterized by an imbalance between oxidant-producing systems and antioxidant defense mechanisms, which can trigger cell damage by oxidizing macromolecular structures (lipids, proteins and DNA) and modifying their biological functions that ultimately causes cell death [4]. Thus, depending on their cell concentrations, ROS can act as either beneficial or harmful biological agents.
The gastrointestinal tract is frequently exposed to noxious stimuli that may cause oxidative stress and injury. In fact, oxygen free radicals are generated both in the lumen and in the intestinal mucosa. Intraluminal pro-oxidants from ingested nutrients, such as alcohol, cholesterol oxides or iron salts and ascorbic acid, frequently consumed together in multiple-vitamin preparations or ingested foods, can build a pro-oxidant milieu [5]–[7]. Moreover, local microbes or infections, ischemia/reperfusion, gastric acid production and non-steroidal anti-inflammatory drugs may promote the formation of reactive radicals [8]–[10]. In addition, the influx of leukocytes, neutrophils and monocytes (associated with inflammation) can produce further ROS via respiratory burst enzymes as well as those involved in prostaglandin and leukotriene metabolism [11]. Clearly, significant oxidative stress has been said to be always associated with mucosal erosions and a causative role in a variety of gastrointestinal diseases such as Crohn's disease and ulcerative colitis [12]–[14].
Despite the frequent occurrence of oxidative stress in the gastrointestinal tract and its involvement in the initiation and propagation of the chronic inflammatory response in chronic bowel diseases [15], little is known about mitochondrion response even though this special organelle is both a major source of oxidants and a target for their damaging effects [16]. We have hypothesized that oxidative stress may affect various mitochondrial functions, including ATP production, calcium (Ca2+) homeostasis, cellular redox state regulation, apoptosis, as well as mtDNA integrity [17], [18]. Therefore, the specific aim of the present study was to characterize the interplay between oxidative stress and mitochondrial dysfunction in the Caco-2/15 cell line using the iron-ascorbate (FE/ASC) oxygen radical-generating system, which participates in lipid peroxidation in inflammatory bowel diseases (IBD) and represents a powerful tool in our hands for the initiation of highly reactive hydroxyl radicals and for the down-regulation of endogenous antioxidants [19]–[26].
Materials and Methods
Caco-2/15 Cell Cultures
The colon carcinoma cell line, Caco-2/15 (ATCC, Rockville, MD), was cultured at subconfluent stages in MEM (GIBCO-BRL, Grand Island, NY) containing 1% penicillin-streptomycin and 1% MEM non-essential amino acids (GIBCO-BRL) and supplemented with 10% decomplemented fetal bovine serum (FBS) (Flow, McLean, VA) as described previously [27]. Briefly, Caco-2/15 cells (passage 20-30) were maintained in T-75-cm2 flasks (Corning Glass Works, Corning, NY). Cultures cells were split (1∶6) when they reached 90% confluence by use of 0.05% trypsin-0.5 mM EDTA (GIBCO-BRL). For individual experiments, cells were plated at a density of 1×106 cells/well on 24.5 mm polyester Transwell filter inserts with 0.4-µm pores (Coster, Cambridge, MA) in MEM supplemented with 5% FBS. Cells were cultured for 21 days post confluence, at which the Caco-2/15 cells are highly differentiated and appropriate for lipid synthesis and metabolism. The medium was refreshed every second day. To determine the implication of oxidative stress per se in alterations in mitochondrial functions, Caco-2/15 cells were incubated with FE/ASC (0.2 mM/2 mM) for 6 h alone and/or with the antioxidant butylated hydoxytoluene (BHT) (2,6-di-t-butyl-p-cresol, Sigma, St-Louis, MA) (50 µM). Caco-2/15 cells were divided into four groups: control (without any addition), oxidative (FE/ASC), antioxidant (BHT), oxidative and antioxidant (FE/ASC + BHT).
Lipid Peroxidation
Caco-2/15 cells were cultured in the presence or absence of (0.2 mM/2 mM) FE/ASC added to the medium. Incubation periods were terminated with 50 µM BHT to measure malondialdehyde (MDA). The level of MDA formed during the oxidative reaction was determined by HPLC, as previously described [19]. Briefly, proteins were first precipitated with a 10% sodium tungstate (Na2WO4) (Aldrich, Milwaukee, WI) solution. The protein-free supernatants were then reacted with an equivalent volume of 0.5% (wt/vol) thiobarbituric acid solution (TBA; Sigma) at 90°C for 60 min. After cooling to room temperature, the pink chromogene [(TBA) 2-MDA] was extracted with 1-butanol and dried over a stream of nitrogen at 37°C. The dry extract was then resuspended in a potassium dehydrogen phosphate (KH2PO4)/methanol mobile phase (70; 30, pH 7.0) before MDA determination by HPLC with fluorescence detection.
Assessment of Intracellular ATP
Intracellular ATP was measured by luciferase driven bioluminescence using ATP Bioluminescence Assay Kit from (Calbiochem, EMD Chemicals, Inc. Gibbstown, NJ) as reported previously [28]. Values were then normalized further with regard to the protein content of the respective sample. All Caco-2/15 culture cells were performed in duplicate.
Calcium Measurements by Confocal
For mitochondrial Ca2+ monitoring, Caco-2/15 cells were trypsinized, transferred from cell culture flasks to 8-well chamber slides (Lab-Tek™ Nunc, Rochester, NY) at a density of 2,5×104 cells in 500 µl of cell culture medium. After a period of three days, cells were serum-starved and incubated with FE/ASC and/or BHT as described above. Cells were rinsed twice in serum-free culture medium and loaded with a mixture of 5 µM Rhod-2/AM (Molecular Probes, Eugene, OR), a fluorescent probe specific for mitochondrial Ca2+, with 0,01% pluronic acid for 30 min at 37°C as described previously [29]–[31]. Medium was removed, replaced with dye-free culture medium and incubated for an additional 60 minutes at 37°C. Thereafter, 1 µl of the fluorescent mitochondria-specific dye MitoTracker™ (green fluorescence, Molecular Probes) was added to each well at the last 30 min of incubation. Cells were visualized using an inverted laser-scanning confocal microscope equipped with a 40× objective (LSM 510, Zeiss). Excitation wavelength was 488 nm and fluorescence emission was recorded at 543 nm (for Rhod-2) and 516 nm (for MitoTracker™). Six to eight fluorescence images were randomly chosen in selected microscopic fields. Fluorescence intensity was quantified using the Image J software (http://rsb.info.nih.gov/ij).
Mitochondrial Preparations
Mitochondria were isolated using standard differential centrifugation techniques [32]. Briefly, Caco-2/15 cells were treated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C. Cells were homogenized with a glass pestle Dounce homogenizer in a buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 0.5% fatty acid-free bovine serum albumin, and 5 mM HEPES, pH 7.2. The homogenate was centrifuged at 1000 x g for 10 min at 4°C. The supernatant was then collected and centrifuged at 10000 x g for 10 min to obtain the pellets containing mitochondria. The pellets were used immediately or stored at −80°C. The protein contents of mitochondrial suspension were determined by the Bradford assay (BioRad, Mississauga, ON) with BSA as a standard.
Evaluation of 8-hydroxy -2-deoxyguanosine
Oxidative DNA damage in whole Caco-2/15 cells, nuclei and mitochondria was evaluated by assessing 8-hydroxy -2-deoxyguanosine (8-OHdG) with high-sensitivity competitive ELISA assays performed with a commercial kit from Genox Corporation (Baltimore, USA). Briefly, 8-OHdG antibody plus sample DNA were added to a 96-well plate percolated with 8-OHdG and incubated overnight at 4°C. After the plate was washed, horseradish peroxidase–conjugated secondary antibody was added for 1 h at room temperature. After washing, 3,3′,5,5′-tetramethylbenzidine was added and incubated for 15 min at room temperature in the dark. The reaction was terminated by the addition of phosphoric acid, and absorbance was measured at 450 nm. All assays were performed in duplicate. Negative controls and 8-OHdG standards (0.125–10 ng/mL) were included in the assay. The average concentration of 8-OHdG was calculated for each sample based on the standard curve.
Mitochondrial Enzyme Assays
The activities of respiratory chain complexes were assayed as previously described in detail [32]–[34]. Briefly, 20–30 µg of mitochondrial protein were used for each complex every 30 sec for 5 min. The activity of complex I (NADH: ubiquinone oxidoreductase) was measured by monitoring the reduction of decylubiquinone. Complex II (succinate:ubiquinone oxidoreductase) activity was examined by monitoring the reduction of dichloroindophenol when coupled to complex II-catalyzed reduction of decylubiquinone. Complex III (ubiquinol:ferricytochrome C oxidoreductase) activity was assayed using oxidized cytochrome C. The activity of complex IV (cytochrome C oxidase) was determined by oxidation of reduced cytochrome C. Enzyme activities were expressed in nanomoles of substrate used per minute per milligram of protein. Enzyme assays for all complexes were performed in duplicate in mitochondrial fraction of Caco-2/15 cell line. Complex I, II, III, IV chemicals were purchased from Sigma Chemical, St Louis, MO.
Western Blots
To assess the protein mass of mitochondrial transcription factors (mt TF): mtTFA, mtTFB1, mtTFB2 and POLRMT, as well as 8-oxoG-DNA glycosylase (OGG1), apoptosis-inducing factor (AIF) and cytochrome C, Caco-2/15 cells were homogenized and adequately prepared for Western blotting as described previously [22], [23], [27], [35]–[39]. The Bradford assay (Bio-Rad) was used to estimate protein concentration. Proteins were denatured in sample buffer containing SDS and β-mercaptoethanol, separated on a 4–20% gradient SDS-PAGE and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked with defatted milk proteins followed by the addition of primary antibodies directed against the different proteins. The relative amount of primary antibody was detected with species-specific horseradish peroxidase-conjugated secondary antibody. Even though identical protein amounts of tissue homogenates were applied, the β-actin protein was used to confirm equal loading on SDS-PAGE (results not shown). Blots were developed and the mass of proteins was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software. Rabbit polyclonal mtTFA Ab was obtained from Santa Cruz Biotechnology Santa Cruz, CA; rabbit polyclonal POLRMT from Abcam, Cambridge, MA; and mouse polyclonal mtTFB1 and mtTFB2 Ab, rabbit polyclonal OGG1 Ab, rabbit polyclonal AIF Ab, and mouse monoclonal cytochrome C Ab from Novus Biologicals, Inc.
RT-PCR
Experiments for mRNA quantification as well as for GAPDH (as a housekeeping gene) were performed in Caco2/15 cells using the UNO II thermocycler (Biometra) as reported previously [35], [40]. Approximately 30–40 cycles of amplification were used at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Amplicons were visualized on standard ethidium bromide-stained agarose gels. Under these experimental conditions related to RT-PCR, the cycles for mtTFA, mtTFB1, mtTFB2, POLRMT, OGG1 and GAPDH were 31, 31,35,31,31, and 30, respectively corresponding to the linear portion of the exponential phase. Fold induction and quantification were determined with the software UN-SCAN-IT gel 6.1.
Primers Used
GAPDH (
F- AGAAGGCTGGGGCTCATT/R-GGGCCATCCACAGTCTTCT)
H-OGG1 (
F-GGGGATTCACAAGGTGAAGA/R-GTAAGCTGGCTTGCATCACA)
POLRMT (
F-CATCACCTACACCCACAACG/R-GTGCACAGAGACGAAGGTCA)
H-mtTFB2 (
F-GTCGCTTTTGCATTTTAGGG/R-GCTGTCCAAGGAACTGCTTC)
h-mtTFB1 (
F-CTCCTGGACTTGAGGCTGAC/R-TTCTCAGTTTCCCAGGTGCT)
h-mtTFA (
F-GGGTTCCAGTTGTGATTGCT/R-TGGACAACTTGCCAAGACAG)
Statistical Analyses
Statistical analyses of data were performed with Prism 4.03 software (GraphPad Software). All values were expressed as the mean ± SEM. The data were evaluated by ANOVA, where appropriate, and the differences between the means were assessed using the Bonferroni's multiple comparison test. A p-value of less than 0.05 was considered to be significant.
Results
MDA Generation after Iron-Ascorbate Exposure
Before evaluating the role of oxidative stress on mitochondrial function, we evaluated the effectiveness of FE/ASC in initiating lipid peroxidation after incubation with Caco-2/15 cells. At the end of a 6-h culture period, the degree of lipid peroxidation was determined by measuring MDA in cells. As illustrated in Figure 1, FE/ASC induced a significant increase in MDA levels above baseline values compared with control cells. The concentration of MDA was 4-fold higher in cells supplemented with FE/ASC compared with untreated cells. Pre-incubation with the strong antioxidant BHT markedly suppressed the production of MDA, providing direct evidence for the ability of the FE/ASC system to provoke profound lipid peroxidation.
10.1371/journal.pone.0011817.g001Figure 1 Malondialdehyde (MDA) concentrations in Caco-2/15 cells challenged with iron/ascorbate and/or BHT.
At 21 days of differentiation, cells were exposed to (0.2 mM/2 mM) FE/ASC, (50 µM) BHT or both for 6 h at 37°C. Oxidative stress was assessed by measuring MDA as an index of lipid peroxidation. Values are means ± SEM for three independent experiments. *P<0.05.
Effect of FE/ASC on Cellular ATP Content
The main function of the mitochondrion is the production of energy in the form of ATP via oxidative phosphorylation and oxygen consumption. We therefore assessed the amount of ATP levels in Caco-2/15 cells exposed to the FE/ASC oxygen radical-generating system. As noted in Figure 2, the administration of FE/ASC led to a four-fold reduction compared with untreated cells. Moreover, pre-incubation with BHT at a concentration of 0.5 mM resulted in a trend of ATP normalization.
10.1371/journal.pone.0011817.g002Figure 2 ATP Levels in Caco-2/15 cells exposed to iron/ascorbate in the presence or absence of BHT.
Caco-2/15 cells were grown on 96-well plates and, after 21 days post confluence, they were treated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C. ATP levels were measured with a bioluminescence assay and corrected for intracellular protein concentrations. Values are expressed as ng of ATP per µg of cellular protein and represent the means ± SEM for three independent experiments. *P<0.001.
Oxidative Phosphorylation Activity
Since mitochondrial oxidative phosphorylation (OXPHOS) is fundamental to all aspects of cell life under aerobic conditions, we evaluated its activity during oxidative stress. The enzymatic activity related to complexes I, II, III, and IV was performed on mitochondrial fraction prepared from Caco-2/15 cells. Our findings documented a significant decrease in the specific activities of complex I, II, III and IV following FE/ASC treatment (Figure 3). Pre-incubation with BHT abrogated the decline in the OXPHOS enzymatic activities.
10.1371/journal.pone.0011817.g003Figure 3 Effect of iron/ascorbate and/or BHT treatment on enzymatic activities of mitochondrial respiratory chain complexes in Caco-2/15 cells.
Enzyme activities of mitochondrial respiratory chain complexes I, II, III, IV were measured by spectrophotometric assays in mitochondrial samples in Caco-2/15 cells treated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C. Enzyme activities are expressed as nmol/min/mg protein. Each value represents the mean ± SEM for 3 separate experiments performed in duplicate. *P<0.05 vs. controls.
Changes in Mitochondrial Calcium Induced by Iron-Ascorbate in Caco-2/15 Cells
We next tested whether FE/ASC caused a change in mitochondrial Ca2+ in Caco-2/15 cells using the positively charged and cell permeant Ca2+ indicator, Rhod-2/AM, which accumulates predominantly in the negatively charged matrix of the mitochondria. The dye Mitotracker™ was used to confirm the mitochondrial localization of Rhod-2. As presented in Figure 4D, FE/ASC treatment of Caco-2/15 cells induced an increase in Rhod-2 fluorescence that appears predominantly located in the mitochondria as demonstrated by the yellow spots of strong intensity found in the merged image (Figure 4F). In contrast, the distribution pattern of colocalized Rhod-2 and Mitotracker™ observed in control cells revealed spots of less intensity characterized by a more diffuse distribution (Figure 4E). Quantification of Rhod-2 fluorescence intensity is shown in Figure 5. Cells exhibited an increase in Rhod-2 fluorescence after FE/ASC treatment whereas pre-incubation with BHT restored fluorescence intensity to control level.
10.1371/journal.pone.0011817.g004Figure 4 Influence of oxidative stress on mitochondrial calcium homeostasis in Caco-2/15 cells.
Representative fluorescence images of control and FE/ASC-treated Caco-2/15 cells loaded with the mitochondrial dye MitoTracker™ (A, B) and the mitochondrial Ca2+ indicator Rhod-2 (C, D). Merged images (E, F) indicate colocalization of the two dyes in the mitochondria. Mitochondrial Ca2+ accumulation is visible upon oxidative stress (arrow). Scale bar-10 µm.
10.1371/journal.pone.0011817.g005Figure 5 Quantification of Rhod-2 fluorescence intensity in Caco-2/15 Cells subjected to iron/ascorbate and/or BHT treatment.
Caco-2/15 cells pretreated for 6 h with (0.2 mM/2 mM) Fe/ASC and/or (50 µM) BHT were loaded with 5 µM of Rhod-2 AM for 30 minutes at 37°C. Fluorescence intensity was quantified by image analysis as described in Material and Methods. Six to eight fluorescence images from three independent experiments were randomly chosen. Results were calculated by dividing the pixel intensity by the area of the spot (µm2). Data illustrated represent the means ± SEM. *P<0.001.
AIF and Cytochrome C Protein Expression
AIF is normally located in the inter-membrane space of mitochondria and is involved in initiating a caspase-independent pathway of apoptosis by causing DNA fragmentation and chromatin condensation. Furthermore, when cell death is triggered by an apoptotic stimulus, cytochrome C is released into the cytosol, and contributes to the caspase-dependent pathway of apoptosis. Western blot analysis revealed a marked (P<0.001) increase in the level of AIF and cytochrome C protein mass in Caco-2/15 cells following FE/ASC compared with controls (Figures 6). Pre-incubation with BHT before the addition of FE/ASC prevented the rise in AIF and cytochrome C protein mass.
10.1371/journal.pone.0011817.g006Figure 6 Cytochrome C and AIF expression levels in Caco-2/15 Cells treated with iron/ascorbate and/or BHT.
Caco-2/15 cells were incubated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C. Gene and protein expression were determined by RT-PCR and Western blotting, respectively. Values are expressed as means ± SEM for three independent experiments. *P<0.001.
Quantification of Oxidative DNA Damage in Caco-2/15
ELISA for 8-OHdG, a recognized marker of oxidative DNA damage, was used to quantify oxidative DNA damage in Caco-2/15 cells. Figure 7 shows the average concentration of 8-OHdG detected in the control and experimental groups. Results clearly indicate that the level of oxidative DNA damage in mitochondria was significantly (P<0.001) higher in Caco-2/15 exposed to FE/ASC-mediated lipid peroxidation (Figure 7A). The oxidative DNA damage was attenuated after pre-incubation with BHT. On the other hand, no significant changes were noted in the homogenate or nucleus (Figure 7B and 7C).
10.1371/journal.pone.0011817.g007Figure 7 Influence of iron/ascorbate treatment in the presence of absence of BHT on 8-hydroxy -2-deoxyguanosine level in Caco-2/15 cells.
The levels of 8-hydroxy -2-deoxyguanosine (8-OHdG) were measured by ELISA kit assay in (A) mitochondrial, (B) homogenate and (C) nucleus samples in Caco-2/15 cells treated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C. Values are means ± SEM for three independent experiments. *P<0.001.
OGG1 Repair Enzyme Level
In mitochondria, the base excision repair pathway is primarily responsible for removing 8-OHdG from DNA [41]. In humans, 8-oxodG is repaired by 8-oxoguanine DNA glycosylase (OGG1), an enzyme that recognizes and hydrolyzes the aberrant base from the DNA backbone. We, therefore, examined its gene expression and protein mass in Caco-2/15 cells. As well illustrated in Figure 8, treatment with FE/ASC resulted in a significant (P<0.001) reduction of OGG1 mRNA and protein mass compared with controls. However, pre-incubation of Caco-2/15 cells with BHT prevented the decline in OGG1 expression.
10.1371/journal.pone.0011817.g008Figure 8 Effect of iron/ascorbate and/or BHT treatment on 8-oxoG-DNA Glycosylase levels in Caco-2/15 cells.
Caco-2/15 cells were incubated with (0.2 mM/2 mM) FE/ASC and/or (50 µM) BHT for 6 h at 37°C to determine the effects of oxidative stress on 8-oxoG-DNA glycosylase (OGG1) gene expression (A) and protein mass (B). Values are expressed as means ± SEM for three independent experiments carried out in triplicate. *P<0.001.
Mitochondrial Transcription Factors
Human mitochondrial transcription requires bacteriophage-related RNA polymerase, POLRMT, mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. These crucial proteins define mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial functions. Given the deleterious effects of FE/ASC, it was mandatory to explore how oxidative stress modulates the core protein components required for mitochondrial transcription. PCR and Western Blot analyses showed a significant (P<0.01) increase in mtTFA, mtTFB1 and mtTFB2 gene expression (Figure 9A) and protein mass (Figure 9B) without any changes in POLRMT in Caco-2/15 cells treated with FE/ASC compared with controls. Pre-incubation with BHT attenuated the modifications of those transcription factors.
10.1371/journal.pone.0011817.g009Figure 9 Effect of iron/ascorbate and/or BHT treatment on gene and protein expression of mitochondrial transcription factors in Caco-2/15 cells.
Effects of (0.2 mM/2 mM) FE/ASC, (50 µM) BHT or both for 6 h at 37°C on gene expression (A) and protein mass (B) of mtTFA, mtTB1, mtTB2, POLRMT. A GAPDH cDNA probe was used as a control for RNA loading; β-actin was used as loading control protein. Data originated from three independent experiments. Values are expressed as means ± SEM. *P<0.01.
Discussion
The Caco-2/15 cell line has been used to examine a variety of intestinal functions. This intestinal model exhibits many of the features of small intestinal epithelial cells. We employed the FE/ASC oxygen radical-generating system to determine how oxidative stress modulates mitochondrial DNA integrity and function in Caco-2/15 cells [20]. Our results show for the first time that FE/ASC can induce lipid peroxidation accompanied by ATP depletion, mitochondrial transport chain complex inhibition, mitochondrial Ca2+ overload, cell apoptosis, mitochondrial DNA lesions and mitochondrial transcription factors alterations.
Iron is the most abundant transition metal in mammalian cells and is essential for the physiological function of multiple proteins [42]. However, excess or non-protein-bound (labile) iron can be detrimental because it can initiate oxygen radical formation and promote ROS [43]. Therefore, iron may cause oxidative damage to biological macromolecules and alter the intracellular redox environment, thereby affecting redox-sensitive cell signaling pathways and transcription factors [44], [45]. Although the mechanisms underlying the cytotoxicity of iron in different organs are not fully delineated, many reports have pointed to the participation of iron-mediated peroxidation in numerous pathological states, including atherosclerosis [46], [47], cancer [48], [49], ischemia-reperfusion injury [50], IBD [51], and conditions of iron overload [52]. Several laboratories [19]–[21], [23]–[26], [52]–[54] have shown the ability of iron to initiate strong lipid peroxidation, whereas ascorbic acid can amplify the oxidative potential of iron by promoting metal ion-induced lipid peroxidation. The data presented here clearly indicate that the FE/ASC system functioned as a producer of lipid peroxidation and, at the same time, altered the DNA integrity and the function of mitochondria. It is noteworthy that the iron dose used in the current study is comparable with normal iron concentration in the gut [11]. The deteriorations resulting from the exposure of Caco-2/15 cells to FE/ASC are probably attributable to oxidative stress, because the addition of the BHT antioxidant simultaneously prevented the occurrence of lipid peroxidation and improved the cellular processes of mitochondrial integrity and functions. BHT was selected as an antioxidant because it represents a powerful agent inhibiting iron-mediated oxidative stress and does not have any toxic effects on Caco-2/15 cell culture [21].
Previous reports observed that an accumulation of peroxidation products in mitochondria leads to a decrease in ATP production and compromises the maintenance of cellular homeostasis [55]. In this study, incubation of Caco-2/15 cells with FE/ASC induced a marked decrease in ATP levels. Our data are consistent with previous investigations showing that ATP decreased in the HT-29 intestinal cell-line after oxidative injury by hydrogen peroxide [56]. The fall in ATP synthesis is probably related to the low mitochondrial metabolic activity resulting from the FE/ASC-mediated lipid peroxidation. In fact, electron movement through complexes I, II, III, and IV enables movement of hydrogen ions across the inner membrane into the inter-membrane space creating an electrochemical gradient, which is harnessed into ATP production by ATP synthase in complex V. We reasonably propose that mitochondrial damage from ROS may lead to a degradation in the efficiency of the mitochondrial respiratory chain enzymes and hence a decline in ATP production. The impairment of mitochondrial complex I, II, III and IV activity noted in our experiments may be attributable to ROS-induced cardiolipin damage that has recently been reported in ischemia/reperfusion rat heart, which ultimately led to a decrease in oxidative phosphorylation [57], [58]. The phospholipid cardiolipin is found almost exclusively in the inner mitochondrial membrane where it promotes the optimal function of numerous enzymes involved in mitochondrial energy metabolism. Finally, inactivation of mitochondrial electron transport chain enzymes and/or ATP-synthase may account for the ATP depletion triggered by the administration of FE/ASC to Caco-2/15 cells.
On top of its ATP generation ability, mitochondria also play a part in modulating the amplitude and spatiotemporal organization of Ca2+ signals through rapidly accumulating and releasing Ca2+
[59], [60]. Indeed, intracellular Ca2+ plays a key role in cellular metabolism. However, excessive mitochondrial Ca2+ overload can trigger ROS overproduction, mitochondrial membrane depolarization and ATP production inhibition, all hallmark events of mitochondrial dysfunction clearly observed in the present work. Additionally, these defective processes may eventually lead to apoptosis [59], [60], which was also documented in our studies. In particular, mitochondrial Ca2+ overload can favor cardiolipin peroxidation, thereby affecting mitochondrial permeability transition, inducing AIF and cytochrome C release, and culminating in mitochondrial dysfunction and apoptosis [61], [62]. Therefore, tools capable of minimizing mitochondrial Ca2+ overload would decrease mitochondrial ROS accumulation and improve mitochondrial energy production, which may impact on mitochondrial-oxidative mediated diseases.
The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase (POLRMT), mtTFA, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2. Both factors seem to interact directly with POLRMT forming a heterodimer that, in addition to mtTFA, is required for the accurate initiation on both H1 and L promoters [63]. The main function of mtTFA is the maintenance of mtDNA replication and transcription during mitochondria biogenesis [64]. In our study, we observed that mtTFA, mtTFB1, mtTFB2 transcriptional level and protein mass were augmented in the presence of Fe/ASC with no marked difference for POLRMT. Currently, we do not know whether the upregulation of mtTFA, mtTFB1, and mtTFB2 in our experiments represent a compensatory mechanism in response to oxidative stress-related reduction in energy metabolism such as defective electron transport chain, incomplete mitochondrion biogenesis or accelerated apoptosis. Accordingly, mtTFA was found upregulated in response to lipopolysaccharide-induced oxidative damage to mitochondria, presumably to enhance mtDNA levels and OXPHOS activity [65]. Furthermore, over-expression of human TFB2M in HeLa cells induced an increase in TFB1M mRNA levels and protein expression [66], suggesting the existence of a retrograde signaling pathway from mitochondria to the nucleus, which precisely regulates the expression of these related factors. Further investigation is needed to examine these important aspects.
In the present study, FE/ASC raised 8-OHdG that represents one of the most frequently generated oxidative base lesions within DNA, owing to guanine, the lowest redox potential among the nucleic acid bases formed in pathological conditions [67]. Similarly, the double immunofluorescence technique revealed that oxidative DNA damage is induced in colon epithelial cells of the IBD mouse model [68]. Furthermore, nuclei were not affected by FE/ASC-mediated oxidative stress, which confirms that mtDNA is more vulnerable than nuclear DNA to oxidative damage given that it is situated much closer to the site of ROS generation and that mitochondria lack protective histones and far fewer mechanisms that prevent reduced base excision repair activity than DNA from nuclei [69]. Our findings confirm that oxidative DNA damage is one of the most common threats to mitochondrial genome stability.
OGG1 is the DNA repair enzyme that recognizes and excises 8-oxodG [70]. The present study shows that incubation of Caco-2/15 with FE/ASC resulted in a marked decrease in OGG1 transcript level and protein mass. Deficiency in DNA repair enzyme OGG1 has likely important functional consequences, compromising the ability of cells to repair DNA. Therefore, intestinal epithelial cells are as sensitive to lipid peroxidation as other cell types, including kidney cortex cells that accumulate 8-OHdG mainly in the mtDNA and to a lesser extent in nuclear DNA under diabetic conditions [71]. We believe that mtDNA damage is linked to the numerous abnormal processes noted in our study, including ATP generation, Ca2+ homeostasis and release of signals for cell death.
In summary, the FE/ASC system in Caco-2/15 appeared to be very effective in promoting lipid peroxidation and, at the same time, altering the mitochondrial function. This mitochondrial dysfunction is probably related to oxidative stress, because the addition of antioxidants prevented the occurrence of lipid peroxidation and improved the mitochondrial function in terms of ATP production, Ca2+ homeostasis and apoptotic protein expression. The pattern of our results using the Caco-2/15 cell line may prove useful in elucidating the molecular mechanisms implicated in IBD. Overall, our data suggest that oxidative-mitochondrial dysfunction is not mediated by a single mechanism, but that it may instead be a consequence of multiple vicious circles organized within a complex functional network.
The authors thank Mrs. Schohraya Spahis for her technical assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was supported by a Canadian Institutes of Health Research team grant (CTP-82942), the J.A. DeSeve Research Chair in Nutrition (EL) and the Fonds de la Recherche en Sante du Quebec(RT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2070048810-PONE-RA-16926R110.1371/journal.pone.0012015Research ArticleGenetics and Genomics/Disease ModelsGenetics and Genomics/Gene FunctionGenetics and Genomics/Genetics of DiseaseGenetics and Genomics/Medical GeneticsHematology/HematopoiesisHematology/Myeloproliferative Disorders, including Chronic Myeloid LeukemiaPathology/Molecular PathologyPediatrics and Child Health/Pediatric HematologyEPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors Primary Polycythemia and EPORPerrotta Silverio
1
Cucciolla Valeria
2
Ferraro Marcella
1
Ronzoni Luisa
3
Tramontano Annunziata
2
Rossi Francesca
1
Scudieri Anna Chiara
2
Borriello Adriana
2
Roberti Domenico
1
Nobili Bruno
1
Cappellini Maria Domenica
3
Oliva Adriana
1
Amendola Giovanni
4
Migliaccio Anna Rita
5
Mancuso Patrizia
6
Martin-Padura Ines
6
Bertolini Francesco
6
Yoon Donghoon
7
Prchal Josef T.
7
Della Ragione Fulvio
2
*
1
Department of Pediatrics, Second University of Naples, Naples, Italy
2
Department of Biochemistry and Biophysics “F. Cedrangolo”, Second University of Naples, Naples, Italy
3
Foundation Ospedale Maggiore Policlinico IRCCS, University of Milan, Milan, Italy
4
Ematologia-Oncologia Pediatrica, Ospedale di Nocera Inferiore, Nocera Inferiore, Italy
5
Mount Sinai School of Medicine, New York, New York, United States of America
6
Laboratory of Hematology-Oncology, European Institute of Oncology, Milan, Italy
7
Hematology Division, School of Medicine, University of Utah and VAH, Salt Lake City, Utah, United States of America
Ng Irene Oi Lin EditorThe University of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: SP DR MDC AO FB JTP FDR. Performed the experiments: VC MF LR AT FR ACS AB DR ARM PM IMP. Analyzed the data: SP VC MF LR AB BN MDC ARM FDR. Contributed reagents/materials/analysis tools: GA. Wrote the paper: SP AO DY JTP FDR.
2010 5 8 2010 5 8 e120159 3 2010 3 7 2010 Perrotta et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined.
Methodology/Principal Findings
We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway.
Conclusions/Significance
Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.
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Introduction
Erythropoietin (EPO) is a key cytokine, produced mainly in peritubular renal cells but also in the liver, that modulates the growth, survival, and differentiation of erythroid progenitor cells, leading to tight control of red blood cell production. Its receptor (EPOR) is a homodimeric transmembrane protein of 508 amino acids in humans (507 in mice) that belongs to a superfamily of cytokine receptors, which includes receptors for GM-CSF and interleukin-3 and -6 [1]. As with other members, the extracellular ligand binding region of EPOR contains four conserved cysteine residues and a WSXWS motif.
The human EPOR gene is located on chromosome 19 and contains eight exons [2 and references therein.] The first five exons encode the extracellular region that embraces the high EPO affinity of A, B, D helix site-1 and the low Epo affinity of A, C helix site-2 interactions, with 7 beta-strand bipartite binding sites in appositioned EPOR dimmers. Exon 6 encodes the membrane spanning domain, while the two intracellular receptor domains are encoded by exons 7 and 8 [2 and references therein].
The receptor does not possess any kinase activity, but its intracellular region binds to JAK2 tyrosine kinase that is essential for EPO signaling. EPOR engagement stimulates JAK2 phosphorylation at Y1007/1008 residues [3]. In turn, activated JAK2 (in concert with other kinases) phosphorylates eight conserved tyrosines in cytoplasmic domain [4]. These phosphotyrosine (PY) sites of EPOR function as docking sites for the binding of molecules containing SH2/SH3 motifs to EPOR. One subset of PY site–recruited factors (PY 402, 430, 432) coordinates negative feedback of EPO signaling. EPO's positive signals are determined by different PY site-recruited factors (PY 344, 426, 461, 465, 480) [4]. It is currently assumed that two boxes at the membrane-proximal region of the EPOR cytoplasmic domain and Y344 are the major positive motifs and that the activation of Stat5 is central for EPOR function [5]. However, since these conclusions have been mainly reached in animal models or in non-erythroid artificial in vitro cell systems, these assumptions await confirmation in the physiological human environment.
In the mouse spleen and bone marrow, EPOR engagement regulates erythropoiesis [6], while the EPO importance in brain development and, possibly, in endothelial precursor mobilization has been suggested [7], [8]. Moreover, EPO/EPOR signaling, at least in the mouse, seems to have a protective role in myocardial ischemia/infarction and in pulmonary hypertension [7], [8]. However, the notion that EPOR plays non-erythropoietic function has been recently strongly challenged [9]. It is also to underscore that erythropoiesis is not equivalent in mice and humans. Thus, a transgenic mouse with the mouse EpoR gene replaced with human polycythemia-causing EPOR, with the truncated deletion in the intracellular region just before tyrosine 410, was polycythemic [10]. Conversely, mice expressing a truncated mouse EpoR were not polycythemic [11]. This emphasizes that results obtained in animal models, albeit important for the understanding of gene function, might not be always extrapolated to human physiology and/or human diseases.
EPOR has been reported to be expressed not only in erythroid precursors but also in other cells and organs, including endothelial cells, the brain, and kidneys [8], [12], [13]. These findings have been obtained by immunohistochemical studies, as well as with highly sensitive reverse transcription-polymerase chain reaction [12], [13]. However, strongly concerns have been raised about the specificity of antibodies used to detect EPOR [14], and the presence of EPOR transcripts is not sufficient to demonstrate the occurrence of an active receptor and its downstream pathways. Therefore, the distribution of functional EPOR in human tissues still remains controversial.
The interest in EPOR has recently exploded following reports of the presence of the receptor on cancer cells with detrimental clinical outcome after EPO treatment [15], [16]. Although the role of EPO in cancer progression remains controversial [17], the possible EPOR-dependent proliferation of neoplastic cells highlights the importance of accurate delineation of human EPOR physiology.
A small number of EPOR mutations have been described, which are associated with primary familial and congenital polycythemias (PFCPs) [18]–[25]. These patients show, low serum EPO levels, normal haemoglobin oxygen affinities, and erythroid progenitors that exhibit EPO hypersensitivity [26]. Clinically, PFCP patients may present with symptoms ranging from headaches, dizziness, epistaxis, exertional dyspnea to pruritis after bathing [27]. Moreover, thrombotic and hemorrhagic events with premature morbidity and mortality have been reported [28], [29]. Clinical symptoms are effectively relieved by phlebotomy, but the increased risk of cardiovascular morbidity is not ameliorated by maintaining a normal hematocrit [30]. However, detailed cellular and molecular explanations of the mutated EPOR pathophysiology of these patients have not been obtained.
We investigated the EPOR gene in a family with dominant polycythemia and found a mutation resulting in a receptor lacking most of the cytoplasmic domain. We demonstrated a marked increase of EPOR protein on the membrane of erythroid progenitors and report its role in deregulating CD34+ cells proliferation and differentiation. We also describe a strong increase of circulating endothelial precursors in the affected subjects.
Results and Discussion
The propositus (P1,
Figure 1A
) was referred from a group of 114 patients included in the Italian registry of congenital erythrocytosis/polycythemia. The index patient is a 14-year-old child who, at 7 years of age, was evaluated for persistent headaches and leg muscle cramps. He had an elevated hemoglobin level and hematocrit (21 g% and 66%, respectively), no splenomegaly, and normal blood pressure. White blood cell and platelet counts were normal. The patient's 41-year-old father (P3) presented at 19 years of age with elevated values for hemoglobin and hematocrit (20.1 g% and 64%, respectively) and normal white-cell and platelet counts. He suffered from persistent headaches and had a mild mitral valve insufficiency and a liver steatosis. Both P1 and P3 were regularly phlebotomized with symptomatic relief. The propositus' paternal grandfather was also reported as “polycythemic”. He died at 55 years of age of liver cirrhosis. The hemoglobin level and hematocrit were also elevated in the paternal uncle (P4) and his daughter (P5), but these patients have scarce clinical symptoms and, thus, were not phlebotomized. The serum EPO was <3 mU/mL (normal range, 4 to 32 mU/mL) in all the four polycythemic family members, while hemoglobin oxygen affinities (p50) were normal.
10.1371/journal.pone.0012015.g001Figure 1 EPOR mutation and pedigree of the polycythemic family.
Panel A. The panel shows nucleotides 1242–1270 (exon 8) of the EPOR gene. A heterozygous G1251
→
T mutation was detected in the propositus P1. The same mutation was verified in all the subjects affected by erythrocytosis (P1, P3, P4, and P5). Panel B. Pedigree of the family with dominant familial erythrocytosis is shown. Squares represent males, circles represent females, and Ps represent the subjects that were genotyped. P1, P3, P4, and P5 are the subjects affected by congenital polycythemia.
All patients were screened for defects in the Von Hippel-Lindau, HIF-1alpha, HIF-2alpha, PHD1-3, JAK2, and EPOR genes. No mutations were found in the Von Hippel-Lindau, HIF-1alpha, HIF-2alpha, PHD1, PHD2, PHD3, and JAK2 genes. However, a heterozygous G→T change at nucleotide 1251 in exon 8 of the EPOR gene was detected in the index patient (
Figure 1A
). This novel mutation (EPOR G1251T) caused the replacement of a glutamic acid residue with a stop codon at amino acid 417, which corresponds to residue 393 of the mature receptor. The EPOR G1251T mutation segregated with the polycythemic status, as demonstrated by the EPOR analysis of other family members (
Figure 1B
). This genetic change resulted in the synthesis of a truncated receptor lacking the cytosolic tyrosines subjected to phosphorylation, except Y344.
Since our patients had a very low serum EPO level, we investigated the growth and differentiation of erythroid precursors in the absence of exogenously added EPO with normal and polycythemia vera (PV) cells used as controls. PV cells were chosen as their erythroid precursors are EPO hypersensitive.
In a first set of experiments, the proliferation of erythroid precursors was investigated by employing liquid cultures of mononuclear cells as previously reported [31]. The concentration of EPO in these experiments, due to the fetal bovine serum present in the culture medium, was calculated to be 0.4 mU/mL, namely about ten-fold lower that the patient EPO serum and 7–8 thousand-fold lower than the amount used in in vitro erythroid precursors cultures (i.e. 3 U/mL). We observed that the peripheral erythroid EPOR G1251T precursors grew 2–3 times faster than cells from PV. Thus, the cells from EPOR G1251T patients increased about 16-fold after 12 days compared to a 6-fold increase of the PV subjects (
Figure 2A
). Under the same condition, normal erythroid precursors underwent apoptosis. Significant growth rate differences between EPOR G1251T and PV patients were evident at all examined time-periods (
Figure 2A
).
10.1371/journal.pone.0012015.g002Figure 2 Phenotypical features of erythroid precursors and CD34+cells from the affected patients.
Panel A. Growth curve of blood erythroid precursors from the the four EPOR G1251T subjects, three subjects affected by PV (JAK2 V617F homozygotes), and three healthy subjects. Erythroid precursors were grown from peripheral mononuclear cells as described (Migliaccio et al., 2002) without adding recombinant EPO. The estimated concentration of EPO in the growth medium was about 0.4 mU/mL. Data represent mean ± SEM (n = 3 per cell type), and are representative of 3 experiments. Panel B. The graphic reports the percentage of BFU-E cells (evaluated by glycophorin A expression after 14 days growth) in liquid cultures of erythroid precursors from the P1 patient and two PV patients (JAK2 V617F homozygotes). The cells were cultured in the absence of exogenously added EPO. Data represent mean ± SEM (n = 3 per cell type), and are representative of 2 experiments. Panel C. Fluorescent activated cell scanner (FACS) analysis of liquid cultures of peripheral purified CD34+ cells grown for 14 days in with minimal EPO. The erythroid precursors were prepared from the P1 subject and from a patient affected by PV associated with a classical JAK2 mutation (JAK2 V617F homozygote). GlyA+ means glycophorin A positive cells. The results are representative of three independent experiments that gave superimposable results. Panel D. FACS analysis of samples shown in panel C. The panel reports the analysis of GlyA levels in cells plotted versus forward scatter. Panel E. Peripheral CD34+ cells were growth on soft agar in the absence of EPO. The images report the features of colonies from the P1 subject (b, d) or from a subject affected by PV (JAK2 V617F homozygote) (a, c). The results are representative of four independent experiments that gave superimposable results.
Glycophorin A expressing cells were analyzed after 14 days of proliferation without exogenously added EPO (i.e. at 0.4 mU/mL EPO concentration) as reported in
Figure 2B
. The percentages of EPOR G1251T cells and PV patients that expressed the erythroid marker were 53±5% and 13±2%, respectively.
When, the same experiment was performed in the presence of 3 U/mL EPO, no significant difference of erythroid maturation was observed (
Figure 2B
).
A second set of experiments was performed employing CD34+ cells prepared from EPOR G1251T and PV patients to highlight phenotypic differences of the early hematopoietic precursors from these two forms of erythrocytoses. As shown in
Figures 2C and 2D
, while a significant percentage (51.5±4.1%) of peripheral CD34+ cells from P1 (grown for 14 days without exogenously added EPO) expressed glycophorin A, the antigen was not detectable in peripheral CD34+ cells from PV patients cultured under the same conditions.
When CD34+ cells were cultured on soft agar with minimal EPO (0.4 mU/mL) marked differences in the number and size of colonies were observed after 14 days. As shown in
Figure 2E
, CD34+ cells of P1 formed large visibly hemoglobinized colonies while only few small, pale, poorly hemoglobinized colonies were seen in CD34+ cells cultures from PV patient (
Figure 2E
). Identical findings were obtained with CD34+ cells from P3, P4 and P5 patients (data not reported). We also prepared the DNA from the largest colonies of P1 and examined the status of the EPOR gene. No gene conversion was observed and the heterozygosity of EPOR G1251T was maintained.
Finally, no difference in the number and size of colonies was observed after 14 days when CD34+ cells from control subjects, PV patients or PFCP patients were cultured on soft agar in the presence of 3 U/mL EPO (data not reported).
It has been suggested, although still debated, that EPO/EPOR pathway plays a role in the endothelial cell homeostasis. Thus, we analyzed the number of circulating endothelial precursors (CEPs) of all PCFP patients by flow cytometry. CEPs were identified as CD45dim/CD34+/VEGFR2+ peripheral cells [32].
Figure 3A
depicts an example of the flow cytometry analysis performed. CEPs were 710±85 cells/mL in the EPOR-mutated subjects (
Figure 3B
), which is about 20-fold higher than the CEPs in the age-matched controls. The CEP level of 3 examined PV patients, all having a classical JAK2 mutation (JAK2V617F), were similar to the CEPs content of EPOR G1251T subjects (
Figure 3B
).
10.1371/journal.pone.0012015.g003Figure 3 Analysis of circulating endothelial precursors from polycythemic patients.
Panel A. Evaluation of circulating endothelial progenitor cells by cell phenotype. The panel reports the flow cytometric analysis of circulating endothelial precursors from patient P1, a) shows the gate made to eliminate platelets and cell debris; b) reports the gate for eliminating apoptotic/necrotic cells (7-AAD positive); c) shows the gate for enumerating CD34+ cells; d) indicates the gate made to enumerate CD34+ and CD45neg/dim cells; e) reports the negative control; and f) is the final gate enumerating CD45neg/dim, CD34+, and VEGFR2+ circulating endothelial precursors. The results are representative of several (>10) independent experiments that gave superimposable results. Panel B. The panel shows the number of circulating endothelial precursors of normal subjects, the four EPOR G1251T patients, and three subjects affected by PV. The results represent the mean (bar, SD) of 4 independent evaluations (each performed in duplicate) of the subjects analyzed. Panel C. The panel reports a semiquantitative evaluation of the expression of EPOR alleles in erythroid precursors from subjects of the family investigated. Total RNA was prepared from erythroid precursors after 7 days of growth (in the presence of 3 U/mL EPO) and retrotranscribed to cDNA. Then, PCR was performed employing primers specific for the two alleles (ie the wild-type and mutated). The antisense primer of the mutated allele inserted a restriction site for NdeI in the amplified product that allowed distinction between the normal and mutated allele. After the PCR reaction, the mixtures were digested with the Ndel enzyme. The 167 bp product is derived from the wild-type transcript while the 144 bp product is from the mutated EpoR mRNA form. The figure is representative of 5 experiments. Panel D. Total RNA (see panel C) was employed to evaluate the content of EPOR alleles (wild type and EPOR G1251T) by realtime PCR. Expression was normalized to beta-actin and expressed as a percentage of wild type EPOR RNA from a control subject. Each bar represents the mean value ±SD. The figure is representative of 4 experiments. Additional details are reported in the text. Panel E. Total RNA were prepared from CD34+ cells (grown 7 days in the presence of EPO) and circulating endothelial precursors. Then, RNA was employed to evaluate the content of EPOR transcript by realtime PCR. Expression was normalized to beta-actin and expressed as a percentage of wild type EPOR RNA from CD34+ cells Each bar represents the mean value ±SD. The figure is representative of 5 experiments.
The growth characteristics of the EPOR G1251T CD34+ cells and the increased levels of peripheral CEPs prompted us to investigate EPOR mRNA and protein levels during CD34+ erythroid differentiation in vitro.
The expression of both EPOR mRNA alleles in erythroid precursors from the four patients was evaluated by semiquantitative PCR (
Figure 3C
) and real-time PCR (
Figure 3D
) after 7 days of cultures in the presence of EPO. Both methods demonstrated that mutant EPOR G1251T and wild type EPOR alleles were expressed at similar levels and that the total EPOR transcript of the EPOR G1251T erythroid precursors corresponds to the EPOR mRNA of erythroid precursors of an healthy subject (
Figure 3D
). This is in accord with results of a previous study on PFCP, although different PCR methods were employed [19].
The realtime PCR approach was also employed to evaluate the expression of the two EPOR alleles of primary cultures of CEPs (P1, P3 and P5 patients). The results obtained are identical to those obtained on the eythroid precursors in that both mutant EPOR G1251T and wild type EPOR alleles were expressed at similar levels (data not shown). It is to underline that CEPs EPOR mRNA is easily detectable and that it corresponds to about 10% of EPOR mRNA of erythroid precursors, the cells that probably express the major amount of this transcript in the human body (
Figure 3E
). In conclusion, the evaluation of levels of the two EPOR mRNA forms indirectly argues that similar amounts of normal and truncated protein receptors may exist in EPOR-expressing cells as reported in previous studies on PCFC.
In order to verify this conclusion, the status of the EPOR protein on the membrane of erythroid precursor cells was investigated. The lack of specific antibodies against the N-terminal region of EPOR has so far hampered direct biochemical analyses of the EPOR peptide. Recently, we have identified and characterized in detail a highly specific antiserum against the extracellular domain of the receptor [33]. To rule out nonspecific crossreactivity by the EPOR antibody and identify the molecular weight of the truncated receptor, we prepared the recombinant wild type and mutated EPOR proteins with an in vitro transcription and translation (IVTT) kit. Moreover, we expressed the native and truncated EPOR in a human EPOR–negative cell line, i.e. K562 cells, for further positive controls. As shown in
Figure 4A
, the antiserum against the extracellular domain recognized the wild type EPOR form at 66–67 kDa and the mutated EPOR G1251T form at about 52–53 kDa.
10.1371/journal.pone.0012015.g004Figure 4 Analyses of EPOR protein in erythroid precursors.
Panel A. Western blot analysis of cellular membranes from the following samples (from the left to right): i) UT-7 cells; ii) untransfected K562 cells (Con); iii) wild-type EPOR transfected K562 cells (WT-EPOR); iv) mutated EPOR transfected K562 cells (MutEPOR); v) in vitro transcription/translation (IVTT) control mixture; vi) in vitro transcribed and translated wild-type EPOR, and vii) in vitro transcribed and translated mutated EPOR. K562 cells were transfected employing pMT21 plasmids encoding wild-type or mutated EPOR, while pcDNA3.1 plasmids were employed in the IVTT experiments. UT-7 cells were employed since these cells contain abundant amounts of wild-type EPOR (Della Ragione et al., 2007). Immunoblotting was performed with the antiserum against the N-end of EPOR. The bottom image is the filter, before immunoblotting, and colored with Red Ponceau. The image confirms equal loading of membrane proteins (lanes 2,3 and 4) and IVTT assay mixtures (lanes 6, 7 and 8). The immunoblotting is representative of 4 experiments. Panel B. The image on the left reports the immunoblotting analysis of membranes from the following samples (from left to right): I) peripheral CD34+ cells from a healthy subject (Con) grown for 7 days with EPO (3 U/mL); ii) peripheral CD34+ cells from patient P1 (MutEPOR) growth for 7 days with EPO (3 U/mL); iii) peripheral CD34+ cells from a healthy subject (Con) grown for 10 days with EPO (3 U/mL); and iv) peripheral CD34+ cells from patient P1 (MutEPOR) grown for 14 days with EPO. The immunoblotting on the right reports cell membranes from K562 cells cotransfected with the wild-type and mutated EPOR pMT21 plasmids as standard for the two EPOR forms. The immunoblotting was performed with the antiserum against the N-end of EpoR. The filter was also re-analyzed by antibodies against glucophorin A (immunoblotting at the center). The bottom image was taken to the filter colored with Red Ponceau before immunoblotting. The image confirms the equal loading of membrane proteins. The image is of 3 experiments. Panel C. The image on the left reports the immunoblotting analysis of membranes from the following samples (from left to right): i) peripheral CD34+ cells from a healthy subject (Con) cultured for 7 days without minimal EPO (0.4 mU/mL); ii) peripheral CD34+ cells from patient P1 (MutEPOR) cultured for 7 days without EPO. The immunoblotting at the right reports cell membranes from K562 cells cotransfected with both the wild-type and mutated EPOR pMT21 plasmids. The immunoblotting was performed using the antiserum against the N-end of EPOR. The bottom image was taken to the filter colored with Red Ponceau before immunoblotting. The image confirms the equal loading of membrane proteins. The immuniblotting is representative of 3 experiments. Panel D. Peripheral CD34+ cells from patient P1 were cultured for 7 days with EPO. Then, the cells were added with (or without) 50 µM LLnL for 4 hours. Finally, cell membranes were prepared and analyzed fot EPOR content. The immunoblotting was performed with the antiserum against the N-end of EpoR. The immunoblotting on the left reports cell membranes from K562 cells cotransfected with the wild-type and mutated EPOR pMT21 plasmids as standard for the two EPOR forms. Panel E. K562 cells were cotransfected with the wild-type and mutated forms of EPOR. Then, K562 cell membranes (Input) were immunoprecipitated with an antiserum directed against the EPOR C-end (IP C-end). Finally, the immunoprecipitated materials (IP) and the supernatant (IP Sup) of the reaction were analyzed with the antiserum directed against the EPOR N-terminus (WB N-end). The blot reports the following samples (from the left): i) IVTT wild-type EPOR; ii) membranes from cotransfected K562 cells (Input); iii) the immunoprecipitated materials (IP); iv) the supernatant of the immunoprecipitation (IP Sup); v) IVTT reaction of mutated EPOR. Note that the asterisk represents the signal due to the heavy chain of antibodies employed in the immunoprecipitation. The signal is immediately up to the band of the truncated receptor. Two different times of exposition are reported (1 minute and 5 minutes) to demonstrate the difference in the ratio of wild type/mutated forms in the input and in the supernatant. The input sample is 1/4 of the supernatant sample. The immunoblotting is representative of 3 experiments.
When the isolated cell membrane fractions from the patient were analyzed, we observed the presence of a strong band at the mutated form and a very faint band at the wild type EPOR. The finding was observed in either CD34+ cells grown for 7 and 10 days in the presence of 3 U/mL EPO (
Figure 4B
) or in cells cultured for 7 days in minimal EPO level (0.4 mU/mL) (
Figure 4C
). The analysis of membrane fractions from control erythroid precursors showed a barely detectable band of the wild type protein.
It is to underline that when CD34+ cells (both from controls or PCFP subjects) were growth in the presence of EPO they showed similar differentiation as demonstrated by their glycophorin A content (
Figure 4B
). Thus, changes in the truncated EPOR protein levels cannot be ascribed to difference in cellular populations due to variable degree of differentiation.
The unanticipated observation that, even in the presence of high EPO, the lack of a large part of the receptor cytoplasmic domain results in the cell membrane accumulation of mutated EPOR, suggested that the truncated receptor has a slower membrane internalization/degradation and an increased half-life due to the absence of sequences required for receptor internalization and degradation.
In order to confirm this hypothesis, we incubated CD34+ cells with a proteasome inhibitor, i.e. LLnL, that has been previously reported to induce membrane EPOR accumulation by hampering the receptor removal [34]. Thus, CD34+ cells were cultured for 7 days with EPO and, then, treated for 4 hours with the proteasome inhibitor. As shown in
Fig. 4D
, LLnL treatment causes the increase of wild-type EPOR while scarce change of mutated EPOR level was observed. The finding suggests that the turn-over time of the two forms was considerably different, being the mutated form remarkably more stable.
The observed difference of receptor turn-over is also in accord with data obtained in UT-7 cells, showing EPOR internalization and removal requires ubiquitination of the receptor's C-terminal region [34]. In particular, members of the beta-Trcp family have been shown to participate in the E3 ligase activity responsible for EPOR ubiquitination [35]. Beta-Trcp binds to Ser 462 within the intracellular part of the receptor and contributes to EPOR ubiquitination in the presence of the hormone. The ubiquitination mediated by beta-Trcp is a crucial signal for the C-terminus degradation of EPOR because the point mutation of Ser 462 to Ala blocks EPOR targeting to the proteasome, thereby leading to sustained activation of EPOR(S462A). It is interesting that BaF3 cells, expressing a mutated EPOR unable to bind beta-Trcp, are hypersensitive to EPO, suggesting beta-Trcp-mediated ubiquitination represents a negative modulator of EPO-induced cellular proliferation [35]. This finding is similar to our observation of a strong EPO hypersensitivity of the erythroid precursors possessing the truncated receptor. In addition, the mechanism is in accord with a recent investigation demonstrating that phosphorylated Y429, Y431, and Y479 in the EPOR cytoplasmic domain bind the p85 subunit of PI3 kinase on EPO stimulation and individually are involved in mediating EPO-dependent EPOR internalization [36].
In order to highlight the biochemical consequences of the truncated receptor accumulation, the possibility that the two EPOR isoforms might interact to form a heterodimer was investigated. Thus, the truncated and wild-type EPORs were overexpressed in K562 cells and membrane proteins were incubated with an antiserum directed against the C-terminus. The immunoprecipitated materials were then analyzed with the antiserum directed against the N-terminus and the occurrence of both the native and the truncated proteins was shown. The result obtained demonstrated that the two EPOR isoforms might undergo heterodimerization (
Figure 4E
). However, although the input material contained equal amounts of the two EPOR isoforms and almost all the wild-type receptor was immunoprecipitated, a large excess of the truncated receptor remained in the supernatant (
Figure 4E
). This argues that the wild-type receptor, and thus the truncated EPOR, preferentially form homodimers.
Therefore, we evaluated whether high levels of homodimers of truncated EPOR might result in excessive EPO signaling and/or in an EPOR autoactivation event. To validate these hypotheses, the truncated EPOR was overexpressed in K562 cells and the effect on receptor autophosphorylation and Stat5 phosphorylation was evaluated. As shown in
Figure 5A
, transfection of the truncated receptor induced a number of biochemical changes (i.e. receptor autophosphorylation and STAT5 phosphorylation), even in the absence of exogenously added EPO, suggesting the possibility of autoactivation and/or hypersensitivity of receptor. Since these events occur at level of truncated EPOR similar to those observed in erythroid precursors cells, it is likely that these receptor features might be observed in these cells.
10.1371/journal.pone.0012015.g005Figure 5 EPOR phosphorylation and STAT5 activation in erythroid cells expressing wild type or mutated EPOR.
Panel A. K562 cells were transfected with the mutated form of EPOR. After 3 days of growth in the absence of exogenously added EPO, K562 cell membranes and total cell extracts were prepared. (On the left) The membranes were analyzed with antibodies against EPOR N-end or against phosphotyrosine. A phosphotyrosine signal occurs only in the transfected cells and at the molecular weight of the mutated EPOR signal. (On the right) Cell extracts was analyzed for STAT5 and phosphoSTAT5 levels by means of specific antibodies. The immunoblotting is representative of 3 experiments. Panel B. Purified CD34+ cells from a control and P1 subject were grown for 7 days in the presence of EPO. Then, cell membranes were prepared and EPOR content and phosphorylated form were evaluated as in panel A. The immunoblotting is representative of 3 experiments. The data are are representative of 3 experiments. Panel C. Purified CD34+ cells from a control and P1 subject were grown for 7 days in the presence of EPO. Then, cell extracts were prepared and STAT5 and its phosphorylated form were analyzed as in panel A. The data are are representative of 3 experiments. Panel D. Purified CD34+ cells from a control and P1 subject were grown for 7 days in minimal EPO (0.4 mU/mL). Then cell extracts were prepared and STAT5 and phosphorylated fprm were analyzed as in panel A. Note: We were unable to evidentiate the phosphorylation of mutated EPOR in CD34+ cells grown in minimal EPO. This was probably due to the scarce amount of material available. However, differences in STAT5 activation between the control cells and cells from the patient were evident in panel D.
Then, we investigated the activity of the EPOR-JAK2-STAT5 pathway in CD34+ from control and EPOR truncated subjects, grown for 7 days with or without exogenously added EPO. As shown in
Figures 5B and 5C
, in the presence of 3 U/mL EPO, we found a clear increase of receptor and STAT5 phosphorylation. Although we were unable to directly demonstrate the phosphorylation of the truncated EPOR in the absence of the cytokine, we observed, under this condition, the activation of EPOR-dependent pathway by detecting STAT5 phosphorylation in CD34+ cells cultured without EPO (
Figure 5D
). The finding was also confirmed by the activation of Erk1/2, which was significantly increased in cells from EPOR truncated subjects, grown for 7 days with EPO (
Figure 6A
). In the presence of minimal amounts EPO, we also found increased Erk1/2 phosphorylation, which suggests greatly augmented activity of the receptor (
Figure 6B
).
10.1371/journal.pone.0012015.g006Figure 6 Transduction pathway status of erythroid precursors.
Panel A. Cytosolic extracts of CD34+ cells, cultured for 7 days in the presence of EPO, were prepared from a healthy subject and patient P1. Then, samples were analyzed for Erk1/2 and phospho Erk1/2 content. Panel B. Cytosolic extracts of of CD34+ cells, cultured for 7 days in the absence of exogenously added EPO (0.4 mU/mL EPO), were prepared from a healthy subject and patient P1. Then the samples were analyzed for Erk1/2 and phospho Erk1/2 content. Panel C. CD34+ cells from a healthy subject were grown for up to 14 days in the presence of EPO. Aliquots of cells at days 0, 7, and 14 were removed and cell extracts were prepared. Then, the samples were analyzed for p27Kip1 content by immunoblotting. Panel D. CD34+ cells from a healthy subject and patient P1 were grown for 7 days in the presence of EPO and cellular extracts were prepared. Then, samples were analyzed for p27Kip1 content.
An increase of p27Kip1 (an inhibitor of cell cycle progression) has been reported during erythroid differentiation of experimental models of erythropoiesis [37]. As reported in
Figure 6C
, we also observed an increase of p27Kip1 on different days of CD34+ growth in the presence of EPO. Intriguingly, in cells grown for 7 days with EPO, we found an increase of p27Kip1 in cells from the EPOR G1251T patient compared with cells of the control subject (
Figure 6D
).
In conclusion, in this study, we report that the gain-of-function EPOR G1251T mutation results in a marked alteration of growth and differentiation of human CD34+ and in an increase of erythroid progenitors' surface EPOR peptide. Moreover, we observed a profound increase of circulating endothelial precursors in the affected subject. Our data elucidate how the heterozygous EPOR gene mutation causes a dominantly inherited polycythemia phenotype and erythroid progenitors' EPO hypersensitivity. We demonstrate for the first time that, although both alleles of the patients were transcribed at similar rates in erythroid precursors and roughly the same as the normal alleles of healthy subjects, the truncated receptor accumulates at very high levels on cellular membranes. This leads to augmented activation of growth and erythroid differentiation of CD34+ observable at EPO levels lower that that necessary to activate CD34+ of PV patients. EPO hypersensitivity might be due to both: i) the remarkable accumulation of truncated EPOR and its autoactivation, and ii) loss of the negative regulatory domain of the EPOR G1251T mutant.
The CD34+ cells proliferation and differentiation, observed without exogenous EPO, also demonstrated the EPOR sequence, including Y344, is sufficient to activate the proliferation and differentiation of human erythroid precursor cells. In the same context, the truncated EPOR-dependent activation of Erk2 suggests Erk2 may not require PY480 or phospholipase C-gamma for its activation, as reported by others [2].
The importance of the EPO-EPOR system in erythropoiesis and vasculogenesis was established using mutant mice lacking either the Epo or EpoR gene [38]. Although these observations are consistent with participation of EPOR in vasculogenesis, the precise contribution of EPOR function in nonhematopoietic tissue remains to be defined, and is the object of intense debate. It has been described that EPO administration can increase the number of CEPs in healthy subjects. It has been postulated that this increase might be due to an EPO-derived stimulation of cytokine production. Our observation that the EPOR G1251T patients with activated EPO-EPOR signaling have an increased number of CEPs suggests the CEPs increase may be due to a constitutive EPO receptor activation in this population. An increase in circulating CD34+ cells has been described in patients carrying JAK2 mutations and suffering from myeloproliferative diseases [39]; to our knowledge, this is the first report demonstrating an increased number of endothelial precursors in the circulation of patients with gain-of-function EPOR mutations. It remains to be established whether this is due to increased CEP production, increased CEP mobilization, or both mechanisms.
Materials and Methods
Mutational analysis and evaluation of EPOR mRNA isoforms
Blood samples were obtained from the index patient, his parents and relatives, from healthy controls, and from four patients with PV (with homozygous JAK2V617F mutation). All patients gave written informed consent on entering the study, which was approved by the research ethics committee of Second University of Naples, Italy, and the study was conducted according to the Declaration of Helsinki. DNA and RNA were prepared by standard methods. To screen for mutations of the EPOR gene, exons 7 and 8 with exon-intron boundaries were amplified by PCR using the following method and primers:
PCR was performed with 30 cycles of denaturation (1 min, 94°C), annealing (1 min, 60°C), and elongation (1 min, 72°C) and the amplified products were analyzed on 2% agarose gels, stained with ethidium bromide. Oligonucleotides used to amplify either exon 7 (362 bp) or 8.1 (301 bp) and exon 8.2 (333 bp) were as follows:
exon 7 forward: 5′-GCCTCTATGACTGGGAGTGG-3′
exon 7 reverse: 5′-GCGCTCTGAGAGGACTTCC-3′
exon 8.1 forward: 5′-GCCTGGGCTTCCCTGCTTCTTGC-3′
exon 8.1 reverse: 5′-TTCGAGGCCAAAGCAGATGAGCA-3′
exon 8.2 forward: 5′-TATCTGGTGCTGGACAAATGGTT-3′
exon 8.2 reverse: 5′-CTGCAGCCTGGTGTCCTAAGAGC-3′
The amplified products were purified and sequenced.
Two independent methods were developed for quantifying the expression of the EPOR alleles (ie wild-type and mutated forms). First, we designed a primer incorporating a mismatched base, corresponding to the allele with the G1251T mutation, to engineer a restriction site for NdeI in the PCR product. After the PCR reaction, the assay mixture was digested with the NdeI enzyme (Invitrogen, Carlsbad, CA, USA) and only the product from the mutated allele was digested. The mutated allele yielded 2 fragments of 144 bp and 23 bp, and wild type an uncut 167 bp band.
Second, a TaqMan assay was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). We synthesized two probes, one specific for the detection of the wild-type allele, labeled with a FAM dye, and one specific for the mutated allele, labeled with a HEX dye. Each incorporated Locked Nucleic Acids (Sigma-Aldrich, Sigma Chemical Company, St. Louis, MO, USA), a nucleic acid analogue that contains a 2′-O, 4′-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the structure into a rigid C3-endo conformation, conferring enhanced hybridization performance and markedly increased stability. The amount of each expressed allele was standardized using wild-type and mutated EPOR pcDNA3.1 plasmids. Further details on the methods, as well as on the primers employed, will be provided on request.
Erythroid precursor cultures
Liquid cultures of erythroid precursors from peripheral blood were prepared by two methodologies. The first utilized peripheral blood mononuclear cells as a source of erythroid progenitors [31], while the second employed the peripheral blood CD34+ cells as a source of progenitors [40]. Soft agar colony assays were performed as described [41]. The colonies were scored after 0, 7, and 14 days and their images captured after 14 days.
Circulating endothelial cell evaluation
Circulating endothelial precursors were measured by six-color flow cytometry as previously described [32]. Primary cultures of circulating endothelial precursors were prepared as previously described [32].
Plasmids, cell lines, and protein analysis
The wild-type coding sequence of human EPOR, cloned into the pMT21 expression vector, was kindly provided by Dr. A. D'Andrea. The sequence was mutagenized at the 1251 (G→T) position, and the wild-type and mutated EPOR sequences were subcloned into the pcDNA3.1 plasmid. These pcDNA3.1 vectors were used to prepare in vitro the respective proteins by TNT kits (Promega Italia, Milan, Italy). pMT21 plasmids containing the wild-type and mutated EPOR coding sequences were transiently transfected in the human K562 cell line (a negative erythroleukemic EPOR cell line) by standard procedures [33], [42]. The treatments with N-Ac-Leu-Leu-norLeucinal (LLnL) (Merck Biosciences, Darmstadt, Germany) were performed as reported in [34]. Cell membranes were prepared with the Qproteome Cell Compartment Kit (Qiagen, Valencia, CA, USA), while cytosol and nuclei were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) [33]. Immunoblotting procedures and immunoprecipitation experiments were essentially performed as described [33], [42].
The following antibodies were employed in the immunoblotting and immunoprecipitation experiments. Goat polyclonal antiserum directed against the extracellular domain of EPOR was from Abcam (Abcam, Cambridge, MA); rabbit polyclonal anti C-end of EPOR, Stat5, and phospho-Erk1/2 and mouse monoclonal antibodies anti Erk1/2 and anti-phosphotyrosine (PY20) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal antibodies anti phospho-Stat5 were from Upstate Biotechnology (Upstate Biotechnology, Charlottesville, VA); monoclonal antibodies against p27Kip1 were from Transduction Laboratories (Transduction Laboratories Lexington, KY).
We sincerely thank Dr. Carmela Migliaccio for mutational analysis of the EPOR gene and Dr. Luciana De Vito for preliminary immunoblotting. We wish to thank the family for their cooperation.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported in part by grants from Progetti di Rilevante Interesse Nazionale (PRIN) to S.P. and F.D.R., Regione Campania, Italy to S.P. and F.D.R., and Associazione Italiana per la Ricerca sul Cancro (AIRC) to F.D.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Virol JVirology Journal1743-422XBioMed Central 1743-422X-7-1692066316210.1186/1743-422X-7-169Case ReportInfluenza or not influenza: Analysis of a case of high fever that happened 2000 years ago in Biblical time Hon Kam LE [email protected] Pak C [email protected] Ting F [email protected] Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China2010 21 7 2010 7 169 169 16 6 2010 21 7 2010 Copyright ©2010 Hon et al; licensee BioMed Central Ltd.2010Hon et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The Bible describes the case of a woman with high fever cured by our Lord Jesus Christ. Based on the information provided by the gospels of Mark, Matthew and Luke, the diagnosis and the possible etiology of the febrile illness is discussed. Infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. If the postulation is indeed correct, the woman with fever in the Bible is among one of the very early description of human influenza disease.
Infectious diseases continue to be a threat to humanity, and influenza has been with us since the dawn of human history. We analysed a case of high fever that happened 2000 years ago in Biblical time and discussed possible etiologies.
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Case
The Bible descrbies the case of a woman with high fever cured by our Lord Jesus Christ. According to Mark 1:29 to 33 and Matthew 8:14-15, the mother-in-law of Simon Peter "lay sick" with a febrile illness [1]. When Jesus took her by the hand and lifted her up, the fever immediately left. The lady began to serve the household and probably prepared a meal. The case is also described in the gospel by Luke (Luke 4:38-39), who was a physician in his days and he specifically mentioned that the fever was high [1].
Discussion
What was the diagnosis of the febrile illness, based on the information provided by the gospels of Mark, Matthew and Luke [1]? It seems that the woman suffered an acute febrile illness with high fever and was sick enough to be bed-ridden. Luke did not quantify the fever as the Fahrenheit temperature scale was not invented until 1724 [2]. No other symptom or chronic illness was described in the three gospels. Possible etiology of her "acute febrile illness" is some sort of infection or inflammation. The Bible describes that when Jesus touched the woman, the fever retreated instantaneously. This implies that the disease was probably not a severe acute bacterial infection (such as septicemia) or subacute endocarditis that would not resolved instantaneously. It was probably not an autoimmune disease such as systemic lupus erythematousus with multiple organ system involvement, as the Bible does not mention any skin rash or other organ system involvement. The instantaneous cure also makes an underlying malignant etiology unlikely. It seems that an acute self-limiting infectious illness is a possible diagnosis. The brief duration, high fever, and abrupt cessation of fever makes influenza disease probable [3]. Shortly following her recovery, presumbly within minutes, it is described that the woman began to serve Jesus and the disciples, thus making influenza illness highly probable. Most miserably sick patients recover without sequlae when the high fevers subside following influenza-like illness [3].
The next question is whether the virus is influenza, avian flu, parainfluenza, or other respiratory viruses such as adenovirus or even SARS-CoV (Severe Acute Respiratory Syndrome-associated Coronavirus) [3-9]. Adenovirus and SARS-CoV are usually associated with pulmonitis, and the pulmonary symptoms may not resolve promptly [3-9]. It is unable to tell if the woman has been in contact with poultry or swine and contracted avian or swine influenza [3,4]. The Bible does not describe if any members of the family including Andrew and Simon developed febrile illness, before or subsequent to her febrile illness. The characteristic features of seasonal influenza include abrupt onset of fever, chills, non-productive cough, myalgias, headache, nasal congestion, sore throat, and fatigue. The diagnosis is mainly clinical. Seasonal influenza would be less likely if no members of the family were affected [3]. Avian influenza and other respiratory viruses may cause isolated infection without efficient human-to-human transmission. In any case, influenza-like illness due to a respiratroy virus would explain her symptomatology and clincial course [3]. Other possibilities include drug fever and poisoning (such as atropine). Naturally-occurring plants containing the belladonna alkaloid atropine could have been consumed but the Bible does not describe unusual food or medicine intake by the woman and her family. The other side effects of anticholinergic agent were absent. The woman would recover spontaneously when the effect of the offending substance wore off.
One final consideration that one might have is whether the illness was inflicted by a demon or devil. The Bible always tells if an illness is caused by a demon or devil (Matthew 9:18-25, 12:22, 9:32-33; Mark 1:23-26, 5:1-15, 9:17-29; Luke 4:33-35, 8:27-35, 9:38-43, 11:14) [1]. The victims often had what sounded like a convulsion when the demon was cast out. In our index case, demonic influence is not stated, and the woman had no apparent convulsion or residual symptomatology.
The Bible has many examples of descriptions of medical diseases. For instance, the first pediatric case of mouth-to-mouth cardiopulmonary resuscitation is vividly described in the Old Testament when the prophet Elisha pressed upon an apparently dead child and breathed into him seven times, and the child was revived (Kings 4:34-35) [1].
Influenza and respiratory viral infections have been documented throughout human history [3]. The current 2009 flu pandemic is a global outbreak of a new strain of H1N1 influenza virus, often referred to colloquially as "swine flu" which began in the state of Veracruz, Mexico in April 2009 and the virus continued to spread globally. The World Health Organization (WHO) and US Centers for Disease Control (CDC) in June escalated the global alert level to phase 6 and declared the outbreak to be a global pandemic since the 1968 Hong Kong flu [4].
Summary
If the postulation is indeed correct, the woman with fever in the Bible is among one of the very early description of human influenza disease.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KLH conceived of the study, and was the principal author. TFL and PCN advised and reviewed the manuscript. All authors read and approved the final manuscript.
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The Holy Bible, New King James Version 1982 Nashville: Broadman & Holman Publishers
Fahrenheit temperature scale. Sizes, Inc 2006 http://www.sizes.com/units/temperature_Fahrenheit.htmRetrieved February 1, 2010
Hon KL Leung AK Severe childhood respiratory viral infections Adv Pediatr 2009 56 47 73 10.1016/j.yapd.2009.08.019 19968942
Hon KL Just like SARS Pediatr Pulmonol 2009 44 1048 9 10.1002/ppul.21085 19774678
Hon KL Leung CW Cheng WT Chan PK Chu WC Kwan YW Li AM Fong NC Ng PC Chiu MC Li CK Tam JS Fok TF Clinical presentations and outcome of severe acute respiratory syndrome in children Lancet 2003 361 1701 3 10.1016/S0140-6736(03)13364-8 12767737
Hon KL Li AM Cheng FW Leung TF Ng PC Personal view of SARS: confusing definition, confusing diagnoses Lancet 2003 361 1984 5 10.1016/S0140-6736(03)13556-8 12801758
Leung TF Wong GW Hon KL Fok TF Severe acute respiratory syndrome (SARS) in children: epidemiology, presentation and management Paediatr Resp Rev 2003 4 334 9 10.1016/S1526-0542(03)00088-5
Li AM Hon KL Cheng WT Ng PC Chan FY Li CK Leung TF Fok TF Severe acute respiratory syndrome: 'SARS' or 'not SARS' J Paediatr Child Health 2004 40 63 5 10.1111/j.1440-1754.2004.00294.x 14718009
Hon KL Leung E Tang J Chow CM Leung TF Cheung KL Ng PC Premorbid factors and outcome associated with respiratory virus infections in a pediatric intensive care unit Pediatr Pulmonol 2008 43 275 80 10.1002/ppul.20768 18219695 | 20663162 | PMC2918564 | CC BY | 2021-01-04 18:17:22 | yes | Virol J. 2010 Jul 21; 7:169 |
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Mol VisMVMolecular Vision1090-0535Molecular Vision 1622010molvis0204Research ArticleCannabidiol protects retinal neurons by preserving glutamine synthetase activity in diabetes El-Remessy A.B. 1234Khalifa Y. 2Ola S. 2Ibrahim A.S. 23Liou G.I. 231 Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA2 Department of Ophthalmology, Medical College of Georgia, Augusta, GA3 Vision Discovery Institute, Medical College of Georgia, Augusta, GA4 Charlie Norwood VA Medical Center, Augusta, GA5 Department of Ophthalmology, King Saud University, Riyadh, SaudiCorrespondence to: Dr. Azza B El-Remessy, Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA, 30912, Phone: (706) 721-6760; FAX: (706) 721-3994; email: [email protected]. Khalifa is now at the University of Rochester Medical Center, Rochester, NY.Dr. Ola is now at King Saud University, Riyadh, Saudi Arabia.2010 04 8 2010 16 1487 1495 21 5 2010 30 7 2010 Copyright © 2010 Molecular Vision.2010This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original
work is properly cited.Purpose
We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process.
Methods
Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, Müller cell activation, GS tyrosine nitration, and GS activity were determined.
Results
Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with Müller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats.
Conclusions
These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration.
GalleyStatusExport to XMLcorr-authorEl-Remessy
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Introduction
Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults, affecting nearly 21 million people in the United States alone (American Diabetes Association). The early clinical features of DR in patients with diabetes as well as observations in experimental diabetes include vascular permeability and vitreoretinal neovascularization secondary to vascular dysfunction as well as retinal glial dysfunction and neuronal cell loss (reviewed in [1,2]). The biochemical mechanisms involved in diabetic retinopathy are complex and include the activation of several cellular pathways (reviewed in [3]). Previously, we and others have shown that an increase in peroxynitrite, as indicated by tyrosine nitration, correlates with accelerated retinal endothelial cell death, breakdown of the blood-retinal barrier (BRB), and accelerated neuronal cell death in experimental models of diabetes, inflammation, and neurotoxicity [4-15]. These studies suggest peroxynitrite plays a key role in mediating different aspects of DR. However, the causal role of diabetes-induced tyrosine nitration in mediating Müller glial cell injury and dysfunction has not been elucidated.
In response to hyperglycemia-induced oxidative stress, both microglial and macroglial cells are activated, and the function of macroglia in transporting glutamate by glutamate transporters and in metabolizing glutamate by glutamine synthetase (GS) may be impaired [16-18]. This may lead to glutamate accumulation, such as that reported in the vitreous humor of diabetic patients [19] and in the retina of diabetic animals [16,20]. Recent studies demonstrated that GS is susceptible to tyrosine nitration, which subsequently can impair the enzyme activity [21,22]. Together, these observations prompted us to study the role of diabetes-induced tyrosine nitration in mediating glial injury and GS dysfunction.
Cannabinoids are known to possess therapeutic properties, including anti-oxidant, anti-inflammatory, and N-methyl-D-aspartic acid (NMDA) receptor-activation blocking activity [23-25]. Non-psychotropic cannabidiol (CBD) has been shown to prevent neuronal damage to the central nervous system in gerbils caused by cerebral ischemia [26]. We recently demonstrated the neuroprotective effect of CBD via antioxidant and anti-inflammatory action in rat models of NMDA-induced retinal neurotoxicity and lipopolysaccharide (LPS)-induced neurotoxicity [9,15,27] as well as the anti-inflammatory and BRB-preserving effects in diabetic rats [12]. However, the mechanism of the neuroprotective effect of CBD via preserving glial function in diabetic retina has not been studied. The present study evaluates the ability of CBD to reduce oxidative and nitrative stress, preserve GS function, and prevent neuronal cell death in experimental diabetes.
Methods
Experimental animals and retina isolation
Eight-week-old male Sprague Dawley rats (≥200 g) were obtained from Charles River (Wilmington, MA) and made diabetic by tail-vein injection of streptozotocin (STZ; Sigma, St. Louis, MO) 65 mg per kg of bodyweight in 0.1 M citrate-buffered saline, pH 4.5. All procedures involving animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with Medical College of Georgia (Augusta, GA) guidelines. Diabetes was confirmed by detection of glucose in the urine and blood of injected animals (>250 mg/dl). Three sets of animals were prepared for a total of 48 rats to study the effects of eight weeks of induced diabetes. The following groups were prepared: untreated controls, controls treated with CBD, untreated diabetics, and diabetics treated with CBD. The CBD-treated groups received intraperitoneal injections of CBD (10 mg/kg) every other day. Cannabidiol was obtained from the National Institute of Drug Abuse (Research Triangle Park, NC), and a fresh solution in 0.25 ml of 1:1:18 alcohol:cremorphol:Ringer solution was prepared. Control groups received vehicle injections at the same time points. Streptozotocin-injected animals had significant increases in blood glucose level (495±35 mg/dl) compared with untreated controls (135±7 mg/dl). Treatment with CBD did not alter blood glucose levels in diabetic animals (455±37 mg/dl) or in treated controls (125±5 mg/dl). After eight weeks of diabetes, eyes were enucleated and retinas were dissected for further analyses.
Glutamine synthetase activity
Frozen retinas were thawed and pulse-sonicated in ice-cold sonication buffer (PBS with 1 mM 2- mercaptoethanol). The ability of an aliquot to convert 14C-glutamate to 14C-glutamine was measured by a modification of a published method [28]. Briefly, 30 µl of sample were reacted with an equivalent volume of reaction buffer (100 mM imidazole HCl, pH 7.2, 30 mM MgCl2, 20 mM ATP, 8 mM NH4Cl, 1 mM 2-mercaptoethanol, and 14 mM 14C-glutamate; Specific Activity, 5×105 CPM/ml) for 20 min at 37 °C. The reaction was stopped by adding 600 µl of 2% perchloric acid (PCA). In the control reactions, PCA was added at the beginning of the incubation period. Glutamine was then separated from glutamate by anion exchange chromatography (AG 1-X8 Resin Acetate form; Bio-Rad, Hercules, CA) and quantified by liquid scintillation counting [29]. Numbers were normalized to proteins measured by DC Protein Assay (Bio-Rad).
Immunoprecipitation and western blot analysis of glutamine synthetase
Individual rat retinas were dissected and homogenized in a Mini-Bead beater with treated Ottawa sand in 250 μl of modified RIPA buffer supplemented with inhibitors for proteases and phosphatases as described previously [12]. Retinal protein extract was determined by DC Protein Assay (Bio-Rad). The supernatants containing 500 μg of protein were combined with 5 µl of polyclonal anti-glutamine synthetase Santa Cruz Biotechnology (Santa Cruz, CA) and 50 µl of protein A/G agarose (Santa Cruz) and mixed overnight at 4 °C. The immunoadsorbents were recovered by centrifugation for 5 min at 700× g and washed three times in modified RIPA buffer and twice in 50 mM Tris (pH 7.5) containing 0.1% (w/v) sodium dodecyl sulfate and 150 mM NaCl. The samples were eluted into 60 µl of sodium dodecyl sulfate loading buffer and subjected to sodium dodecyl sulfate PAGE. The membranes were incubated with polyclonal anti-nitrotyrosine (1:1,000; Upstate Biotechnology, Millipore, Billerica, MA) and then with peroxidase-conjugated goat antirabbit (1:5,000) for detection of immunoreactive bands by ECL advance chemiluminescence (GE Healthcare, Piscataway, NJ).
Terminal dUTP nick end-labeling analysis
Terminal dUTP nick end-labeling (TUNEL) analysis was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore) following the manufacturer’s directions as described previously [9]. Briefly, OCT-frozen eye sections (10 µm) from each group were fixed using paraformaldehyde (PFA) and ethanol:acetic acid (2:1). Then, the samples were incubated with Terminal Deoxynucleotidyl Transferase followed by incubation with anti-digoxigenin conjugate. Propidium iodide 1 µg/ml was added as a nuclear counter stain. On completion of the TUNEL assay, coverslips were applied using VECTASHIELD Mounting Medium for fluorescence (Vector Laboratories, Burlingame, CA). Each section was systematically scanned for positive green fluorescent cells in retinal layers indicating apoptosis. Images were obtained using an AxioObserver.Z1 Microscope (Carl Zeiss, Oberkochen, Germany) with 200× magnification. Four to five animals were used for each group, and the number of TUNEL positive cells were counted in four fields of the mid-peripheral retina and calculated as the number per mm2 of retinal area.
Immunolocalization studies
The distribution of nitrotyrosine, glial fibrillary acidic protein (GFAP), and caspase-3 in frozen eye sections was analyzed using immunolocalization techniques as described previously [12]. Retinal sections were fixed with 4% paraformaldehyde and then reacted with a polyclonal rabbit anti-nitrotyrosine (Millipore), mouse anti-GFAP (Cell Signaling Technology, Danvers, MA) antibody, or rabbit caspase-3 antibody (Cell Signaling Technology) followed by Oregon Green-conjugated goat antirabbit or antimouse antibody (Molecular Probes, Carlsbad, CA). Data (10 fields/retina, n=6 in each group) were analyzed using fluorescence microscopy and UltraVIEW morphometric software to quantify the intensity of immunostaining. For colocalization studies of caspase-3 within retinal ganglion cells, retina sections were stained with mouse Brn-3 antibody (Santa Cruz Biotechnology) followed by Texas Red-conjugated antibody (Molecular Probes).
Dichlorofluorescein assay
Dichlorofluorescein (DCF) is the oxidation product of the reagent 2’,7’-dichlorofluorescin diacetate (H2DCFDA; Molecular Probes, Eugene, OR), a marker of cellular oxidation by hydrogen peroxide and peroxynitrite [12]. Earle’s balanced salt solution containing H2DCFDA was incubated with retina sections, and the fluorescence of DCF was measured and analyzed. The average retinal fluorescence intensity (10 fields/retina, n=6 in each group) was analyzed using AxioObserver.Z1 Microscope and Axio-software (Carl Zeiss).
Data analysis
The results are expressed as mean±SEM. Differences among experimental groups were evaluated by performing an ANOVA (ANOVA), and the significance of differences between groups was assessed by a post-hoc test (Fisher’s PLSD) when indicated. Significance was defined as p<0.05.
Results
Cannabidiol reduces diabetes-induced oxidative and nitrative stress
We have previously shown the neuroprotective effects of CBD in short-term diabetes via inhibiting oxidative and nitrative stress [12]. Here, we tested the antioxidant effect of CBD after eight weeks of diabetes. As shown in Figure 1A, diabetes-induced neurotoxicity involved significant tyrosine nitration within retinal layers with the strongest immunoreactivity in the ganglion cell layer. Quantitative analysis showed that levels of tyrosine nitration increased ~1.6-fold in the diabetic retinas in comparison with the controls (Figure 1B). This tyrosine nitration was almost completely eliminated by CBD (10 mg/kg/2 days). The antioxidant effect of CBD was further confirmed by blocking the fluorescence of DCF, a general marker for both oxidative and nitrative stress in diabetic retinas (Figure 1C,D). The treated control rat retinas were not affected by CBD treatment.
Figure 1 Cannabidiol (CBD) reduces oxidative and nitrative stress in diabetic retinas. A: Representative images show the distribution of nitrotyrosine immunolocalization in different retinal layers, the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), and the outer nuclear layer (ONL), and the retinal pigment epithelium (RPE) (magnification, 200×). B: Morphometric analysis of fluorescence intensity in serial sections of rat eyes shows that diabetic rats had a significant increase in fluorescence compared with controls. Treatment with CBD (10 mg/kg/2 days) inhibited nitrotyrosine formation in the diabetic rats but not in the normal controls. Data shown are the mean±SEM of six or seven animals in each group (*p<0.05). C: Representative images show the distribution of dichlorofluorescein (DCF) fluorescence in different retinal layers, the GCL, the IPL, the INL, and the ONL, and the RPE (magnification, 200×). D: CBD reduces peroxides in the retinas of diabetic rats as represented by morphometric analysis of DCF fluorescence showing that diabetic rats had a significant increase in fluorescence compared with controls. Treatment with CBD (10 g/kg/2 days) inhibited reactive oxygen species formation in diabetic rats but not normal controls. Data shown is the mean±SEM of five or six animals in each group (*p<0.05).
Cannabidiol prevents diabetes-induced Müller glial cell injury
Glial activation, as indicated by GFAP, is a common response to stress conditions. There are two types of glial cells in the retina: astrocytes and Müller cells. Therefore, we assessed glial injury in response to the diabetic insult by immunolocalization of GFAP. Astrocytes were notably positively and equally labeled with GFAP in all groups. As shown in Figure 2, only retinas from the diabetic group demonstrated an increase in the intensity of GFAP immunoreactivity in the filaments of Müller cells that extended from the nerve fiber layer and inner plexiform layer into the outer nuclear layer of retina as compared with controls or the CBD-treated group.
Figure 2 Cannabidiol (CBD) prevents Müller cell activation in diabetic animals. Representative images of glial fibrillary acidic protein (GFAP) showing abundant immunofluorescence at the end-feet of the Müller cells and the radial processes stained intensely throughout both the inner and outer retina in the diabetic retinas compared with normal controls. This effect was blocked by treatment with CBD (10 mg/kg/2days, i.p.). Similar results were obtained from five additional animals per group.
Cannabidiol prevents diabetes-induced glutamine synthetase nitration and restores its activity
Diabetes-induced peroxynitrite formation and its subsequent alteration of protein function via tyrosine nitration are well documented [30]. Recent studies have demonstrated that GS is a susceptible target for tyrosine nitration [21]. Therefore, we evaluated the specific tyrosine nitration levels of GS and the extent to which its activity can be altered in diabetic rat retinas. As shown in Figure 3, diabetes caused significant tyrosine nitration (2.3-fold) of GS that was significantly reduced by treatment with CBD. We next evaluated the effects of tyrosine nitration on GS activity. Indeed, diabetes-induced GS tyrosine nitration was positively correlated with a significant inhibition (40%) of GS activity (Figure 4), and treatment with CBD restored this activity in the diabetic animals. These results suggest a causal role of tyrosine nitration in impairing the function of GS, which can lead to the accumulation of glutamate and possibly cause neurotoxicity.
Figure 3 Cannabidiol (CBD) reduces GS nitration in diabetic (D) animals. Immunoprecipitation with anti-glutamine synthetase (GS) and western blot analysis using anti-nitrotyrosine antibody show that diabetes significantly increased the tyrosine nitration of GS compared with normal retinas. This effect was blocked by treatment with CBD (10 mg/kg/2days, i.p.; n=4–6 retinas/group, *p<0.05, versus control [standard error of mean]).
Figure 4 Cannabidiol (CBD) restores diabetes-impaired glutamine synthetase (GS) activity. Glutamine synthetase activity measured by the ability of the sample to convert 14C-glutamate to 14C-glutamine demonstrated significant inhibition of GS activity in diabetic rat retinas compared with controls. The GS activity was restored by treating the diabetic animals with CBD (10 mg/kg/2days, i.p.; n=4–5 retinas/group, *p<0.05, versus control [standard error of mean]).
Cannabidiol prevents diabetes-induced neuronal cell death and activation of caspase-3
We next evaluated neuronal death after eight weeks of diabetes. Our results demonstrated that diabetic rat retina showed significant increases in TUNEL positive cells (~8-fold) mainly in retinal ganglion cells and inner retinal layers compared with controls. Treatment with CBD blocked neuronal cell death in diabetic animals but did not affect treated controls (Figure 5A,B). Neuronal cell death in diabetic animals was further confirmed by prominent immunostaining of caspase-3, a known marker for apoptosis, within the ganglion cell layer (GCL) as indicated by the specific retinal ganglion cell marker Brn-3. The ganglion cell layer notably includes ~35%–40% displaced amacrine cells in addition to retinal ganglion cells. Treatment with CBD blocked neuronal cell death in diabetic animals but did not affect treated controls (Figure 5C,D).
Figure 5 Retinal neuroprotective effect of cannabidiol (CBD) in experimental diabetes. A: Representative images show the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) labeling of frozen eye sections from the diabetic rats (eight weeks) in different retinal layers. TUNEL-positive cells (arrows) were distributed mainly in the inner retinal layers. B: Statistical analysis of TUNEL-positive nuclei in various groups. At least four fields per mid-peripheral retina were counted for each retina from one animal. (n=4–5 retinas/group, *p<0.05, versus control [standard error of mean]). Treating the diabetic animals with CBD (10 mg/kg/2 days, i.p.) prevented neuronal death. C: Representative images show the localization of the apoptotic marker caspase-3 in the ganglion cell layer and the inner retinal layer in diabetic retina sections but not in other groups. D: Enlarged window of retina sections showing colocalization (yellow) of the apoptotic marker caspase-3 (green) within the retinal ganglion cell layer labeled with Brn-3 (red). The layers shown are the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), and the outer nuclear layer (ONL).
Discussion
Diabetes-induced retinal oxidative and nitrative stress have been well documented in patients and animals and have been positively correlated with neuronal cell death [12,13,31-34]. In response to neuronal injury, glial cells including microglial and macroglial cells are activated. This might be followed by neuroinflammation, during which activated microglial cells release TNF-alpha and migrate toward dying neurons to further exacerbate the damage [35]. However, the effects of diabetes-induced oxidative and nitrative stress on macroglial activation and how this can affect neuronal function have not been fully elucidated. Indeed, our results showed a significant increase in oxidative and nitrative stress as indicated by significant increases in DCF fluorescence and nitrotyrosine as well as prominent Müller glial cell activation compared with controls. Exposure of retinal Müller glial cells to high glucose levels stimulates oxidative stress and peroxynitrite formation ( [36], unpublished data). However, peroxynitrite produced by glial cells is not toxic by itself but causes activation and expression of proinflammatory cytokines [37]. Our previous studies have shown that Müller cells are not among the retinal cell population undergoing apoptosis early in diabetes [13]. Our current study demonstrated that Müller cells are activated as evidenced by an enhanced intensity of GFAP immunoreactivity in the filaments of Müller cells in diabetic retinas that was blocked by CBD treatment.
Previous studies have documented the adverse effects of diabetes on the function of Müller cells in transporting glutamate by glutamate transporter or in metabolizing glutamate by GS [16-18,20]. Although alterations in glutamate transporter activity during diabetes remain controversial, impairment of GS activity has been previously reported [38-40]. Interestingly, recombinant GS enzyme from E. coli, rat liver, or mammalian GS has been reported to be a susceptible target for tyrosine nitration that might reduce its activity [21,22,41]. Therefore, we investigated GS nitration and its impact on GS activity in diabetic rat retinas. Our results showed a 2.3 fold increase in GS tyrosine nitration that was associated with a significant reduction (40%) in GS activity in diabetic retinas compared with controls. Our results lend further support to previous reports showing that diabetes can alter glial function and impair GS activity [38,42,43]. Although the concept of GS nitration and the subsequent impairment of its activity has been demonstrated at the recombinant protein level, we believe our study provides the first experimental evidence in a diabetic model. Further studies of human samples should provide clinical evidence and implications for GS nitration.
Tyrosine nitration and the subsequent loss of protein function have been documented in response to peroxynitrite [11,30,44-46]. Furthermore, the impact of GS nitration and its impairing activity is evidenced by glutamate accumulation, as reported in the vitreous humor of diabetic patients [19] and in the retinas of diabetic animals [16,17,20]. Glutamate excitotoxicity occurs via the activation of NMDA receptors to induce calcium influx and the release of superoxide and nitric oxide, leading to the formation of peroxynitrite and neuronal death [47,48]. Diabetes-impaired GS activity should lead to the accumulation of glutamate and the formation of peroxynitrite, which in turn can sustain tyrosine nitration and the inhibition of GS activity. This vicious cycle of glial dysfunction will result in cell death and the injury of adjacent retinal neurons. Therefore, we next evaluated neuronal cell death in the diabetic animals. Indeed, our results showed significant increases in TUNEL-positive cells that were mainly localized in retinal ganglion cells and inner retinal layers in the retinas of diabetic animals compared with controls. Additional immunolocalization studies using caspase-3, a known apoptotic marker, and Brn-3, the specific retinal ganglion cell marker, confirmed apoptosis of ganglion cells in the diabetic animals. As further support, previous studies have demonstrated retinal ganglion cell loss in response to STZ diabetes within the same time frame, eight weeks [49], that continues to happen later during the progression of the disease [32]. Retinal ganglion cells represent about 60%–65% of neurons in addition to displaced amacrine cells in the ganglion cell layer. These findings suggest a loop where diabetes-induced oxidative and nitrative stress alter the function of Müller cells by impairing GS activity, leading to glutamate neurotoxicity and sustaining retinal neuronal death.
Treating diabetic animals with CBD blocked the increases in oxidative and nitrative stress and significantly reduced the number of apoptotic cells. Neurons are highly susceptible to oxidative stress, which can induce apoptosis; therefore, it is likely that diabetes-induced oxidative stress leads to neuronal injury. Several reports have described the neuroprotective effects of CBD via blocking reactive oxygen species or nitrotyrosine formation in glutamate-induced cell death in neuron cultures and in an NMDA-induced neurotoxicity [9,12,15,25]. Here, we demonstrate a novel role of CBD in restoring GS activity by reducing its tyrosine nitration in diabetic animals. This effect was associated with a significant reduction in Müller glial cell activation, which confirms the preservation of its morphology and function in the diabetic animals. Together, our present findings suggest that CBD represents novel therapeutics in the treatment of diabetes and stress-mediated retinal damage. Furthermore, CBD is an attractive medical alternative to smoked marijuana or plant extract because of its lack of psychoactive effect and because it is well tolerated in humans when administered chronically [50,51]. In addition, CBD has been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans (reviewed in [52]). In conclusion, the data presented here provide experimental evidence that diabetes-activated retinal glial cells represent a central player in retinal neurodegeneration.
Acknowledgments
Grant support from the American Heart Association and Juvenile Diabetes Research Foundation to A.B.E. and from the American Diabetes Association and a pilot grant from the Vision Discovery Institute to G.I.L. is gratefully acknowledged.
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PLoS Biol
PLoS Biol
plos
PLOS Biology
1544-9173
1545-7885
Public Library of Science San Francisco, USA
20808781
09-PLBI-RA-4592R3
10.1371/journal.pbio.1000465
Research Article
Diabetes and Endocrinology/Obesity
Physiology/Neuronal Signaling Mechanisms
IL-6 and IL-10 Anti-Inflammatory Activity Links Exercise to Hypothalamic Insulin and Leptin Sensitivity through IKKβ and ER Stress Inhibition
Exercise Anti-Inflammatory Action in Hypothalamus
Ropelle Eduardo R. 1
Flores Marcelo B. 1
Cintra Dennys E. 1
Rocha Guilherme Z. 1
Pauli José R. 1
Morari Joseane 1
de Souza Claudio T. 1
Moraes Juliana C. 1
Prada Patrícia O. 1
Guadagnini Dioze 1
Marin Rodrigo M. 1
Oliveira Alexandre G. 1
Augusto Taize M. 2
Carvalho Hernandes F. 2
Velloso Lício A. 1
Saad Mario J. A. 1
Carvalheira José B. C. 1 *
1 Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
2 Department of Anatomy, Cell Biology, Physiology and Biophysics, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
Vidal-Puig Antonio J. Academic Editor
University of Cambridge, United Kingdom
* E-mail: [email protected]
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: ERR JBC. Performed the experiments: ERR MBF DEC GZR JRP JM CTDS JCM POP DG RMM AGO TMA HFC. Analyzed the data: ERR DEC LAV MJS JBC. Wrote the paper: ERR JBC.
The authors have declared that no competing interests exist.
8 2010
24 8 2010
8 8 e100046523 10 2009
15 7 2010
© 2010 Ropelle et al
2010
Ropelle et al
https://creativecommons.org/licenses/by/4.0/ Except for the Figure 2F IB: pJak2Tyr1007/8 panel. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
“Mens Sana In Corpore Sano”: Exercise and Hypothalamic ER StressPhysical activity confers beneficial metabolic effects by inducing anti-inflammatory activity in the hypothalamus region of the brain in rodents, resulting in a reorganization of the set point of nutritional balance and reduced insulin and leptin resistance.
Overnutrition caused by overeating is associated with insulin and leptin resistance through IKKβ activation and endoplasmic reticulum (ER) stress in the hypothalamus. Here we show that physical exercise suppresses hyperphagia and associated hypothalamic IKKβ/NF-κB activation by a mechanism dependent upon the pro-inflammatory cytokine interleukin (IL)-6. The disruption of hypothalamic-specific IL-6 action blocked the beneficial effects of exercise on the re-balance of food intake and insulin and leptin resistance. This molecular mechanism, mediated by physical activity, involves the anti-inflammatory protein IL-10, a core inhibitor of IKKβ/NF-κB signaling and ER stress. We report that exercise and recombinant IL-6 requires IL-10 expression to suppress hyperphagia-related obesity. Moreover, in contrast to control mice, exercise failed to reverse the pharmacological activation of IKKβ and ER stress in C3H/HeJ mice deficient in hypothalamic IL-6 and IL-10 signaling. Hence, inflammatory signaling in the hypothalamus links beneficial physiological effects of exercise to the central action of insulin and leptin.
Author Summary
The hypothalamus is a brain region that gathers information on the body's nutritional status and governs the release of multiple metabolic signaling molecules such as insulin and leptin to maintain homeostasis. Overeating and obesity are associated with insulin and leptin resistance in the hypothalamus, and recent studies provide an intriguing link between inflammation and dysfunction of hypothalamic insulin and leptin signaling through activation of IKKβ, a key player in immune response, and endoplasmic reticulum (ER) stress. This means that strategies to reduce the aberrant activation of inflammatory signaling in the hypothalamus are of great interest to improve the central insulin and leptin action and prevent or treat related metabolic diseases. Using a combination of pharmacological, genetic, and physiological approaches, our study indicates that physical activity reorganizes the set point of nutritional balance through anti-inflammatory signaling mediated by interleukin (IL)-6 and IL-10 in the hypothalamus of rodents. Hence, IL-6 and IL-10 are important physiological contributors to the central insulin and leptin action mediated by exercise, linking it to hypothalamic ER stress and inflammation.
This study was supported by grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de desenvolvimento científico e tecnológico (CNPq). The funders had no role in study design, data lection and analysis, decision to publish, or preparation of the manuscript.
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pmcIntroduction
Overnutrition and sedentary lifestyle are among the most important factors that lead to an unprecedented increase in the prevalence of obesity. In mammals, food intake and energy expenditure are tightly regulated by specific neurons localized in the hypothalamus. The hypothalamus can gather information on the body's nutritional status by integrating multiple signals, including potent hormonal signals such as insulin and leptin [1],[2]. The impairment of hypothalamic insulin and leptin signaling pathways is sufficient to promote hyperphagia, obesity, and type 2 diabetes (T2D) in different genetic rodent models with neuronal ablation of insulin and leptin signaling [1],[3],[4]. We and others have proposed that overnutrition induces the central insulin and leptin resistance through the aberrant hypothalamic activation of proinflammatory molecules, including TLR4 and IKK [5]–[7].
IKKβ is a key player in controlling both innate and adaptive immunity. Activation of IKKβ by phosphorylation at S177 and S181 induces phosphorylation, ubiquitination, and subsequent proteosomal degradation of its substrate IκBα. The degradation of IκBα allows NF-κB proteins to translocate to the nucleus and bind their cognate DNA binding sites to regulate the transcription of a large number of genes, including stress-response proteins and cytokines [8]. Growing evidence provides an intriguing link between metabolic inflammation and dysfunction of insulin and leptin signaling via activation of IKKβ and endoplasmatic reticulum (ER) stress [9]–[14]. Examination of ER stress markers in different tissues of dietary (high-fat diet-induced) and genetic (ob/ob) mouse models of obesity demonstrated increased levels of PERK phosphorylation and JNK and IKKβ activity [7],[12]. In addition, a recent study showed the activation of hypothalamic IKKβ/NF-κB, at least in part, through elevated endoplasmic reticulum stress in the hypothalamus and that these phenomena are associated with central insulin and leptin resistance, hyperphagia, and body weight gain in mice [7]. Thus, strategies to reduce the aberrant activation of inflammatory signaling and/or ER stress in hypothalamic neurons are of great interest to improve the central insulin and leptin action and prevent or treat obesity and related diseases.
Physical activity is considered a cornerstone of the treatment for obesity. Exercise has long been reported to reduce body weight and visceral adiposity, increasing the energy expenditure and improving glycaemic control in overweight or T2D patients [15],[16]. Since the discovery of interleukin (IL)-6 releases from contracting skeletal muscle, accumulating evidence indicates that exercise induces metabolic changes in other organs, such as the liver, the adipose tissue, and hypothalamus, in an IL-6 dependent manner. IL-6 is most often classified as a pro-inflammatory cytokine, although consistent data also demonstrate that IL-6 has an anti-inflammatory effect and may negatively regulate the inflammation of acute phase response by increasing IL-10, IL-1 receptor antagonist (IL-1ra), and soluble TNF-receptors (sTNF-R) [17]. Moreover, IL-6 appears to play a central role in the regulation of appetite, energy expenditure, and body composition [18],[19]. However, the effects of physical activity in the metabolic regulatory pathways in the central nervous system (CNS) remain unexplored. Thus, we hypothesized that exercise could exert its effects in the CNS by modulating the specific hypothalamic neurons responsible for the control of food consumption. In the present study, we investigated the effect of the anti-inflammatory response, mediated by IL-6, on hypothalamic IKKβ activation and ER stress, central insulin and leptin sensitivity, and food intake in diet-induced rats after physical activity.
Results
Exercise Suppresses Hyperphagia Mediated by Overnutrition
It has been demonstrated that physical activity may contribute to the energy balance by increasing energy expenditure. Although the energy expenditure aspects of such exercise may contribute to the effects of weight loss, the effect of exercise on the control of energy intake remains unclear. To evaluate the impact of physical activity on food consumption, we measured the 12-h total energy intake in lean and diet-induced obese (DIO) rats after one bout of swimming (SW Exe) and treadmill running (TR Exe) exercise. Neither of the exercise protocols changed the energy intake in lean animals; however, exercise suppressed the hyperphagic response, mediated by chronic overnutrition, restoring the energy intake to the levels of lean animals (Figure 1A). To assess whether the effects of exercise on food intake are dependent on the neuropeptides modulation, we performed a real time PCR assay to determine the mRNA levels of Neuropeptide-Y (NPY) and Proopiomelanocortin (POMC). After 9 h of fasting, we found that chronic overnutrition increased NPY mRNA and reduced POMC mRNA levels, while physical activity restored the NPY (Figure 1B) and POMC mRNA levels (Figure 1C) in obese animals; on the other hand, exercise did not change the NPY and POMC mRNA levels in lean rats (Figure 1B and C).
10.1371/journal.pbio.1000465.g001 Figure 1 Exercise induces appetite-suppressive actions in different models of obesity.
(A) 12 h of food intake (kcal) in lean and diet-induced obesity (DIO) Wistar rats under resting conditions or after swimming exercise (SW Exe) or treadmill running (TR Exe) (n = 20–35 animals per group). Rats were fasted during 9 h and the hypothalamic levels (B) NPY and (C) POMC mRNA were examined using real time PCR assay. (D) Body weight, (E) epididymal fat pad weight, (F) 12-h food intake of leptin-deficient mice (Leptob/ob) and respective wild type group. (G) NPY and (H) POMC mRNA were examined using real time PCR assay. (I) Body weight and (J) epididymal fat pad weight of wild type and leptin-deficient mice under resting conditions or immediately after the exercise protocols (n = 10 animals per group). Data are the means ± SEM. # p<0.05 versus respective lean group at rest; * p<0.05 versus respective obese group at rest. Lean animals (white bars) and obese animals (black bars).
Chronic overnutrition increased body weight, epididymal fat (Figure 1D and E), serum insulin, leptin, triglycerides, and free fatty acid levels (Table 1), compared to age-matched controls. No significant variations were found in body weight, epididymal fat serum leptin, triglycerides, and urinary corticosterone levels between exercised and obese animals under resting conditions (Figure 1D, E and Table 1). The insulin levels were lower in both lean and obese rats after the exercise protocols (Table 1) and exercise increased the free fatty acid in obese animals (Table 1). To determine whether lean and obese rodents were swimming or running in the same fashion, we evaluated lactate production every 15 min during the SW Exe and TR Exe. We did not find any difference in the lactate production between lean and obese rats. Table 1 depicts the final values obtained in this test. These results reinforce the negative relationship between body weight change and stress related with the appetite-suppressive actions mediated by exercise.
10.1371/journal.pbio.1000465.t001 Table 1 Metabolic parameters of lean and DIO rats after acute exercise protocols.
Groups Glucose (mg/dL) Insulin (ng/mL) Leptin (ng/mL) Cholesterol (mg/dL) TG (mg/dL) FFA (mmol/L) Corticost. (ng/mL) Lactate (mmol/L)
Chow rest 97±5 4.0±0.2 2.0±0.2 129.3±8.5 94.0±1,4 0.64±0.2 11.1±0.6 ND
Chow SW exe 108±9 2.8±0.3# 2.1±0.1 123.7±6.4 93,7±7,2 0.81±0.1 11.0±0.4 3.6±0.6
Chow TR exe 118±12 2.9±0.2# 2.1±0.2 121.8±6.3 97.3±7.5 0.79±0.2 11.4±0.5 4.20±0.4
DIO rest 115±5 7.8±0.4# 3.6±0.3# 141.0±10.1 152.5±7.8# 1.75±0.5# 11.2±0.7 ND
DIO SW exe 117±9 6.1±0.3#* 3.7±0.2# 141.6±9.5 141.7±9.5# 2.89±0.3#* 10.4±0.8 4.0±0.5
DIO TR exe 112±15 6.2±0.2#* 3.6±0.3# 145.2±12.5 150.3±8.0# 2.65±0.4#* 10.5±0.7 3.9±0.3
# p<0.05 versus chow rest and * p<0.05 versus DIO rest (n = 8–10).
To extend our hypothesis, we investigated food intake in leptin-deficient mice (ob/ob) after physical activity. Acute SW Exe and TR Exe did not change the food intake in wild type (WT) mice, however the food consumption was reduced in ob/ob mice (Figure 1F). After 9 h of fasting, we found that NPY mRNA was increased and POMC mRNA levels were reduced in ob/ob mice, while physical activity restored the NPY (Figure 1G) and POMC mRNA levels (Figure 1H) in obese animals; on the other hand, exercise did not change the NPY and POMC mRNA levels in control mice (Figure 1G and H). Exercise did not change the total body weight and epididymal fat pad weight in WT and ob/ob mice (Figure 1I and J). In addition, we observed that the exercise protocols did not change the triglycerides and free fatty acid levels but reduced the insulin levels in WT and ob/ob mice (Table 2). The lactate production was similar between lean and obese mice during the respective exercise protocols (Table 2). These exercise protocols did not evoke any significant stressful effect in these animals, as demonstrated by urinary corticosterone levels (Table 2). Thus, our data demonstrate that exercise modulates hypothalamic neuropeptides (NPY and POMC) and suppresses food intake in obese, but not in lean, rodents without changing the adipose tissue content and corticosterone levels.
10.1371/journal.pbio.1000465.t002 Table 2 Metabolic parameters of control and ob/ob mice after acute exercise protocols.
Groups Glucose (mg/dL) Insulin (ng/mL) Leptin (ng/mL) Cholesterol (mg/dL) TG (mg/dL) Corticosterone (ng/mL) Lactate (mmol/L)
WT rest 94±3 3.9±0.4 1.9±0.2 126.7±6.1 73.3±12.6 11.0±0.7 ND
WT SW exe 93±2 2.5±0.3# 2.1±0.3 123.5±3.5 79.0±11.5 11.1±0.5 4.2±0.3
WT TR exe 94±2 2.7±0.3# 2.0±0.3 128.5±3.5 77.33±12.7 11.5±0.8 5.3±1.3
Leptob/ob rest 284±18# 8.0±0.4# ND 156.7±3.8# 194.5±32.9# 11.2±0.4 ND
Leptob/ob SW exe 154±8#* 6.2±0.4#* ND 154.7±2.5# 176.7±14.9# 11.4±0.5 4.9±0.7
Leptob/ob TR exe 175±17#* 6.5±0.3#* ND 153.2±2.7# 173.7±16.5# 11.2±0.6 5.3±0.8
# p<0.05 versus WT rest and *p<0.05 versus Leptob/ob rest (n = 6–8). ND, no detected.
Exercise Restores Insulin and Leptin Sensitivity in the Hypothalamus
Next, we evaluated whether exercise modulates insulin signaling in the hypothalamus. Western blot analysis revealed that IRβ, IRS-1, IRS-2, Akt, and FOXO1 phosphorylation were similar between the groups (Figure 2A and B). Although exercise did not change the basal levels of insulin signaling, we next performed intrahypothalamic insulin (200 mU) or its vehicle injection to evaluated food intake and insulin sensitivity after the SW Exe protocol. Overnutrition markedly reduced the ability of intrahypothalamic insulin infusion to reduce food intake, when compared to chow-fed animals; however, exercise restored the central effects of insulin on reduced food intake (Figure 2C). Using Western blotting analysis, we determined the effects of exercise on the insulin sensitivity in hypothalamic tissue. The high-fat diet impaired insulin-induced tyrosine phosphorylation of insulin receptor β (IRβ), insulin receptor substrate-1 (IRS-1), and IRS-2 in the hypothalamus (Figure 2D). Similar results were observed for the serine phosphorylation of Akt and FOXO1 (Figure 2D). Physical activity was able to restore insulin-induced hypothalamic IRβ, IRS-1, and IRS-2 tyrosine phosphorylation and insulin-induced hypothalamic Akt and FOXO1 serine phosphorylation in DIO rats (Figure 2D). Subcellular fraction of hypothalamic extract was then performed to evaluate the nuclear FOXO1 expression. Intrahypothalamic infusion of insulin reduced the nuclear FOXO1 expression in control rats, but insulin failed to reduce the nuclear FOXO1 expression in rats after overnutrition (Figure 2E). After exercise, insulin reduced the nuclear FOXO1 expression in neuronal cells of obese animals (52%), when compared to DIO at rest (Figure 2E).
10.1371/journal.pbio.1000465.g002 Figure 2 Hypothalamic insulin and leptin signaling after exercise.
Western blots showing hypothalamic lysates from Wistar rats; (A) Hypothalamic IRβ, IRS-1, IRS-2, and Akt phosphorylation, (B) Hypothalamic Foxo1 phosphorylation. (C) 12-h food intake (kcal) after intrahypothalamic infusion of insulin in lean and diet-induced obesity (DIO) Wistar rats under resting conditions or after exercise (n = 6–8 animals per group). Western blots of five independent experiments showing hypothalamic lysates from Wistar rats; (D) Insulin-induced IRβ, IRS-1, IRS-2, Akt, and Foxo1 phosphorylation in the hypothalamus. (E) Subcellular fractionation was performed to evaluate the nuclear Foxo1 expression in the hypothalamus of lean and obese rats at 30 min after insulin infusion. (F) Hypothalamic Jak-2 and (G) STAT-3 tyrosine phosphorylation. (H) 12-h food intake (kcal) after intrahypothalamic infusion of leptin (n = 6–8 animals per group). Western blots showing hypothalamic lysates from Wistar rats; (I) Leptin-induced Jak2, IRS-1, IRS-2, and STAT3 tyrosine phosphorylation in the hypothalamus. (J) Subcellular fractionation was performed to evaluate the nuclear STAT3 expression in the hypothalamic cells of lean and obese rats 30 min after leptin infusion. Data are the means ± SEM. # p<0.05 versus respective lean group at rest; * p<0.05 versus obese group at rest. Lean animals (white bars) and obese animals (black bars). The Figure 2F IB: pJak2Tyr1007/8 panel is excluded from the article's copyright license. See the accompanying retraction notice for more information.
We then explored the effects of exercise on hypothalamic leptin action, monitoring Janus Kinase-2 (Jak-2) and STAT-3 tyrosine phosphorylation. Exercise did not change the Jak-2 and STAT-3 phosphorylation in lean animals; however, overnutrition reduced Jak-2 and STAT-3 phosphorylation when compared to lean animals. Interestingly, physical activity was able to increase the neuronal Jak-2 and STAT-3 tyrosine phosphorylation in obese animals (Figure 2F and G). In addition we investigated the effects of exercise on leptin sensitivity. Intrahypothalamic infusion of leptin markedly reduced the 12-h total energy intake in control rats; however, the anorexigenic effects of leptin were attenuated in obese rats. In contrast, exercise restored the central effects of leptin on reduced food intake (Figure 2H). We noted that leptin modestly promoted the hypothalamic tyrosine phosphorylation of Jak-2, IRS-1, IRS-2, and STAT-3 after high-fat diet treatment. Conversely, exercise restored leptin-induced hypothalamic Jak-2, IRS-1, IRS-2, and STAT-3 tyrosine phosphorylation in obese animals (Figure 2I).
We also evaluated nuclear STAT3 expression after intrahypothalamic leptin infusion. After overnutrition, leptin failed to increase the expression of nuclear STAT3 in the hypothalamus. On the other hand, exercise increased the ability of leptin to increase the nuclear expression of STAT3 (48%) in the hypothalamus of obese animals (Figure 2J).
Increasing Hypothalamic Levels of IL-6 Reverses IKKβ and ER Stress Caused by Obesity
Recently, IL-6 was reported as the first myokine that is produced and released by contracting skeletal muscle fibers, exerting its effects on other organs of the body [20], including the hypothalamus [18],[21]. Thus, we evaluated the central role of IL-6 in the control of food intake. Firstly, the serum level of IL-6 was observed to be slightly up-regulated after high-fat diet treatment and was dramatically increased immediately after SW Exe and TR Exe, but we observed that, in exercised obese animals, the serum levels of IL-6 were higher when compared to exercised lean ones (Figure S1A). Similar results were found when IL-6 protein expression in the hypothalamic tissue was evaluated (Figure S1B). To investigate whether neuronal cells were producing IL-6 in response to exercise, we performed real time PCR to evaluate IL-6 mRNA levels in the hypothalamic tissue. IL-6 mRNA levels were slightly up-regulated after the high-fat diet treatment and were increased by about 53% and 64% immediately after physical activity in lean and obese rats, respectively (Figure 3A). Thus, these data demonstrate that exercise increases the serum and hypothalamic levels of IL-6.
10.1371/journal.pbio.1000465.g003 Figure 3 Anti-hyperphagic response mediated by IL-6.
(A) IL-6 mRNA in the hypothalamus of lean or diet-induced obesity (DIO) rats under resting conditions and lean obese rats immediately after the swimming exercise (SW Exe) or treadmill running (TR Exe). (B) 12 h of food intake in obese rats under resting conditions following intrahypothalamic infusion of different doses of recombinant IL-6. Counter-regulatory effects of anti-IL-6 antibody on food intake in exercised obese rats after (C) insulin or (D) leptin infusion. Western blots of five independent experiments showing hypothalamic lysates from Wistar rats; (E) Expression and activity of protein involved in the inflammatory signaling or ER stress in control animals at rest condition or after acute exercise (F) TLR4 expression, (G) IKKβ phosphorylation, (H) IκBα expression, (I) PERK phosphorylation, (J) CHOP expression, and (K) IRS-1Ser307 phosphorylation from lean, obese, obese injected with recombinant IL-6, obese after exercise, and obese pretreated with anti-IL-6 antibody before the exercise protocol. Data are the means ± SEM. # p<0.05 versus lean group; * p<0.05 versus obese group at rest; ¥ p<0.05 versus respective exercised control rats; ** p<0.01 versus stimulated obese group at rest; § p<0.05 versus obese group injected with recombinant IL-6 and exercised obese rats (n = 8–10 animals per group). Swimming Exercise (SW Exe) or Treadmill Running (TR Exe). Lean animals (white bars) and obese animals (black bars).
Next, we sought to determine whether exercise requires IL-6 to mediate the anti-hyperphagic response. First we showed that the infusion of recombinant IL-6 into the third ventricle of obese animals under resting conditions reduced the food intake in a dose-dependent manner (Figure 3B) and restored the anorexigenic effects of insulin and leptin (Figure S2A and B). Although we used recombinant IL-6 to mimic the effects of exercise, in obese rats, the dose of recombinant IL-6 used (200 ng) is relatively high and this pharmacological approach does not reflect the same physiological conditions observed after exercise. Thus, we hypothesized that if exercise requires hypothalamic IL-6 activity to reduce food intake, inhibiting the hypothalamic effects of this cytokine, under physiological conditions, should diminish the appetite suppressive action mediated by exercise. To address this hypothesis, we developed an experimental strategy aimed at antagonizing the central action of IL-6 in the presence of a systemic elevation in plasma IL-6 concentration after physical activity. For this, we injected an anti-IL-6 antibody into the third-hypothalamic ventricle in obese animals at 15 min before the exercise protocol. Interestingly, pretreatment with anti-IL-6 antibody blocked the anorexigenic effects of insulin and leptin in exercised DIO rats (Figure 3C and D).
We then explored the mechanism by which IL-6 improves insulin and leptin signaling in the hypothalamus, evaluating the pro-inflammatory pathway. Firstly, we demonstrated that acute exercise did not change the expression or activity of the proteins involved in inflammatory signaling and in an ER stress in the hypothalamus of lean rats, when compared to control animals at rest (Figure 3E). However, high-fat diet consumption induced the aberrant activation of the NF-κB pathway components in the hypothalamic tissue, increasing the TLR4 expression, IKKβ serine phosphorylation, and the IκBα degradation (Figure 3F–H). We also monitored PERK phosphorylation and CHOP protein expression in the hypothalamus to evaluate ER stress. High-fat diet also activated ER stress, increasing PERK phosphorylation and CHOP protein expression in the hypothalamus (Figure 3I and J). In addition, high-fat diet increased IRS-1 serine 307 phosphorylation (Figure 3K). Neither acute exercise nor the single injection of recombinant IL-6 was able to reduce the TLR4 expression in the hypothalamic tissue of obese animals (Figure 3F). On the other hand, exercise and the intrahypothalamic injection of recombinant IL-6, in obese rats at rest, markedly reduced the hypothalamic IKKβ serine phosphorylation (∼60%) and prevented IκBα degradation in obese animals (Figure 3G and H). The recombinant IL-6 injection and exercise reduced PERK phosphorylation by about 60% and CHOP protein expression by about 45% (Figure 3I and J) and IRS-1 serine phosphorylation by about 60% (Figure 3K) in the hypothalamic tissue of hyperphagic animals. In addition, recombinant IL-6 and exercise restored insulin-induced Akt and leptin-induced and STAT-3 phosphorylation in the hypothalamus of obese animals (Figure S3A and B). Interestingly, our results show that the intrahypothalamic injection of anti-IL-6 antibody before the exercise protocol attenuated the ability of exercise to reduce the IKKβ/IκBα pathway, ER stress, and IRS1 serine phosphorylation in the hypothalamus (Figure 3G–K). The pretreatment with anti-IL6 antibody also blocked insulin-induced Akt and leptin-induced and STAT-3 phosphorylation, mediated by exercise in the hypothalamus of obese animals (Figure S3A and B).
Immunohistochemistry with an anti-IL-6 Receptor (IL-6R)-specific antibody showed that IL-6R is expressed in a majority of neurons in the arcuate nucleus (Figure 4A). These data were confirmed when we quantified the positive cells in arcuate (Arc), dorsomedial and ventromedial (DMH/VMH), paraventricular (PVN), and lateral (LH) nuclei of hypothalamus (Figure 4B). The in situ hybridization experiment revealed that IL-6R is expressed in both anorexigenic and orexigenic neurons of rats (Figure 4C).
10.1371/journal.pbio.1000465.g004 Figure 4 IL-6R localization in the hypothalamus of rats.
(A) Immunohistochemistry was performed in the hypothalamic tissue of control rats, using IL-6 receptor (IL-6R)-specific antibody (green) and DAPI (blue), with 50× magnification. (B) Positive cells were quantified in different hypothalamic nuclei, § p<0.05 versus the other nuclei. (C) In situ hybridization showing the co-localization of IL-6R (red) with POMC, NPY, and AgRP (green) neuropeptides in the hypothalamus of control rats. Head arrows show neurons and arrows show endothelial cells using 20× and 63× magnification. (D) The dissection of hypothalamic arcuate nucleus of lean and obese rats was obtained as described in Experimental Procedures to evaluate the mRNA of POMC, NPY, and AgRP, using the real time PCR. Data are the means ± SEM. # p<0.05 versus respective control group at rest; * p<0.05 versus obese rats at rest. Lean animals (yellow bars) and obese (blue bars). (E) Confocal microscopy was performed to evaluate the co-localization of IL-6R (green) and IKKβ, PERK, and IRS-1 (red) in the arcuate nuclei of obese rats, with 200× magnification (scale bar, 20 µm).
Since IL-6R is expressed in a majority of neurons in the arcuate nucleus, we dissected this specific hypothalamic region to evaluate the modulation of the neuropeptides in response to exercise in lean and obese rats. We found that exercise did not change the POMC, NPY, and AgRP mRNA in the arcuate nucleus of lean rats but increased the POMC and reduced the NPY mRNA levels in the arcuate nucleus of obese animals (Figure 4D).
Double-staining confocal microscopy showed that most neurons expressing IL-6R in the arcuate nucleus were shown to possess IKKβ, PERK, and IRS-1 in obese rats, showing a possible interaction between these molecules (Figure 4E).
Pharmacological Activation of IKKβ and ER Stress Is Suppressed by IL-6
To further support data indicating that IL-6 may modulate ER stress, we performed an acute intrahypothalamic injection of an ER stress inducer, thapsigargin (TG), in lean rats. Acute intrahypothalamic infusion of thapsigargin did not change food intake in lean animals by itself (Figure 5A). However, our results revealed that intrahypothalamic infusion of thapsigargin blocked the anorexigenic effects mediated by insulin and leptin in lean rats and that the injection of recombinant IL-6 and exercise restored the suppressive appetite action of insulin and leptin (Figure 5B and C). In addition, the infusion of anti-IL6 antibody blocked the improvement in insulin and leptin action mediated by exercise (Figure 5B and C).
10.1371/journal.pbio.1000465.g005 Figure 5 IL-6 reversed pharmacological endoplasmatic reticulum stress induction in the hypothalamus.
(A) 12 h of food intake in lean rats after thapsigargin infusion (3 µg). (B) Anorexigenic effects of insulin in the hypothalamus of lean rats pretreated with thapsigargin. (C) Anorexigenic effects of leptin in the hypothalamus of lean rats pretreated with thapsigargin. Western blots showing hypothalamic lysates from Wistar rats; (D) IKKβ, (E) PERK, and (F) IRS-1Ser307 phosphorylation from lean rats pretreated with thapsigargin. (G) Insulin-induced Akt serine phosphorylation, (H) leptin-induced STAT3 tyrosine phosphorylation in the hypothalamus of lean animals pretreated with thapsigargin, and (I) basal levels of Akt and STAT3 phosphorylation. Data are the means ± SEM. # p<0.05 versus DMSO group; * p<0.05 versus lean plus thapsigargin; § p<0.05 versus thapsigargin plus recombinant IL-6 or thapsigargin plus exercised (n = 8–10 animals per group).
In accordance with previous studies [7],[14],[22], we observed that thapsigargin markedly activated inflammatory signaling and ER stress in lean rats, as reflected by increased levels of hypothalamic IKKβ and PERK phosphorylation, respectively (Figure 5D and E), and induced central insulin and leptin resistance, increasing IRS-1 serine phosphorylation (Figure 5F) and reducing insulin-induced Akt serine phosphorylation and leptin-induced STAT-3 tyrosine phosphorylation (Figure 5G and H). Intrahypothalamic infusion of recombinant IL-6 and physical activity were sufficient to reverse all these phenomena (Figure 5D–H). Conversely, the infusion of intrahypothalamic anti-IL6 antibody before exercise protocol blocked these effects mediated by exercise (Figure 5D–H). There were no differences in the basal levels of Akt and STAT-3 phosphorylation between the groups (Figure 5I).
Low dose TNF-α has been reported to induce insulin and leptin resistance in the hypothalamus [23]. We injected a low dose of TNF-α into the hypothalamus of lean rats to investigate the effects of IL-6 on low-grade inflammation. First we observed that acute intrahypothalamic infusion of TNF-α did not change the food consumption in lean rats (unpublished data); however, TNF-α infusion blocked the anorexigenic actions of insulin and leptin in these animals (Figure S4A and B). The anorexigenic actions of these hormones were restored with the central infusion of recombinant IL-6 or after exercise in lean rats injected with TNF-α. In addition, the pretreatment with anti-IL6 antibody into the third ventricle blocked the improvement in insulin and leptin action mediated by exercise (Figure S4A and B).
The single injection of TNF-α also induced IKKβ serine, PERK threonine, and IRS-1 serine phosphorylation and reduced insulin-induced Akt serine phosphorylation and leptin- induced STAT-3 tyrosine phosphorylation in the hypothalamus of lean rats (Figure S4C–G). Intrahypothalamic infusion of recombinant IL-6 and physical activity were also sufficient to reverse all these phenomena. On the other hand, the central infusion of anti-IL6 antibody before the exercise protocol blocked the effects of physical activity (Figure S4C–G). There were no differences in the basal levels of Akt and STAT-3 phosphorylation between the groups (Figure S4H).
IL-6 Requires IL-10 to Reduce IKKβ and ER Stress in the Hypothalamus
Next, we sought to determine how IL-6 reduces the inflammatory response and ER stress in the hypothalamus after exercise. Several studies have reported that exercise-induced increases in plasma IL-6 levels are followed by increased circulating levels of well-known anti-inflammatory cytokines such as the IL-1ra and IL-10 [24],[25]. We found that the IL-1ra protein level was not changed in the hypothalamus after chronic overnutrition or after acute exercise protocols (Figure 6A); however, IL-10 protein expression was slightly increased in the hypothalamus in obese animals; both of the exercise protocols increased IL-10 expression in a similar fashion, but the induction of IL-10 expression, mediated by exercise, was higher in the hypothalamus of obese when compared to exercised lean animals (Figure 6B). The increase in hypothalamic IL-10 levels mediated by physical activity was confirmed by real time PCR assay (Figure 6C).
10.1371/journal.pbio.1000465.g006 Figure 6 Role of hypothalamic IL-10 in the control of energy intake during obesity.
Western blots showing hypothalamic lysates from Wistar rats; (A) IL-1ra and (B) IL-10 expression in the hypothalamus. (C) IL-10 mRNA in the hypothalamus was examined using real time PCR assay. (D) 12 h food intake (kcal) in obese rats under resting conditions after intrahypothalamic infusion of different doses of recombinant IL-10. Western blots showing hypothalamic lysates from Wistar rats; (E) IL-10 expression after ASO IL-10 treatment in obese animals. (F) Intrahypothalamic treatment with ASO IL-10 blocked the anorexigenic response mediated by (F) insulin and (G) leptin in exercised obese animals or obese animals at rest injected with recombinant IL-6. Western blots showing hypothalamic lysates from Wistar rats; (H) IKKβ, (I) PERK, and (J) IRS-1Ser307 phosphorylation after ASO IL-10 treatment or after acute recombinant IL-10 infusion. (K) Insulin-induced Akt serine phosphorylation and (L) leptin-induced STAT3 tyrosine phosphorylation in the hypothalamus after ASO IL-10 treatment or after acute recombinant IL-10 infusion. (M) Basal levels of Akt serine phosphorylation and (N) STAT3 tyrosine phosphorylation in the hypothalamus after ASO IL-10 treatment or after acute recombinant IL-10 infusion. Data are the means ± SEM. # p<0.05 versus chow group; * p<0.05 versus DIO; ¥ p<0.05 versus exercised control animals; n = 8–10 animals per group. Lean animals (white bars), obese animals (black bars), and exercised obese plus recombinant IL-10 (grey bars). SO, sense oligonucleotide; ASO, antisense oligonucleotide.
We then investigated whether IL-10 reduced the energy intake in rodents. Intrahypothalamic injection of recombinant IL-10 reduced food intake in obese animals in a dose-dependent manner (Figure 6D). To explore whether IL-6 requires IL-10 expression to improve insulin and leptin action in the hypothalamus, we used an IL-10 antisense oligonucleotide (ASO IL-10) in the hypothalamus of obese rats to keep the expression levels of IL-10 low, even in the presence of high levels of IL-6 in the hypothalamus. Three days after ASO IL-10 treatment, IL-10 protein expression was reduced by about 75% in the hypothalamus of obese animals (Figure 6E). Thereafter, exercise and recombinant IL-6 infusion failed to improve the anorexigenic effects of insulin and leptin in obese animals treated with ASO IL-10 (Figure 6F and G).
IL-10 is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines and blocking IKK/NF-κB signaling and ER stress [26],[27]. Thus, we investigated whether exercise and IL-6 requires IL-10 expression to reduce IKKβ activation and ER stress in the hypothalamus of obese animals. As demonstrated above, recombinant IL-6 infusion and exercise reduced IKKβ, PERK, and IRS-1Ser307 phosphorylation (Figure 3G, I, and K) and restored insulin and leptin signaling in the hypothalamus of obese animals (Figure S3), but the intrahypothalamic IL-10 ASO treatment abolished all these parameters mediated by recombinant IL-6 and exercise (Figure 6H–L). Conversely, the injection of recombinant IL-10 in the hypothalamus of obese animals at rest markedly reduced IKKβ, PERK, and IRS-1Ser307 phosphorylation and increased insulin-induced Akt and leptin-induced STAT-3 phosphorylation in the hypothalamic tissue of obese rats (Figure 6H–L). There were no differences in the basal levels of Akt (Figure 6M). However, STAT3 tyrosine phosphorylation was reduced in the hypothalamus of obese rats, but neither exercise nor IL-6 intrahypothalamic injection was able to increase the STAT-3 phosphorylation after IL-10 ASO treatment (Figure 6N).
Attenuating TLR-4-Dependent IL-6 and IL-10 Production Abolishes Exercise Sensitization of Insulin and Leptin in the Hypothalamus
Several studies showed that Toll-like receptor inactivation results in an attenuation of the secretion of several cytokines. TLR4- and MyD88-deficient mice sustain significantly lower levels of serum cytokines such as IL-1β, IL-6, TNFα, and IL-10 after different pro-inflammatory stimuli [28]–[30]. Since TLR4 mediates IL-6 transcriptional responses in myocytes and in the skeletal muscle of C3H/HeJ mice [31], we investigated whether exercise restores insulin and leptin signaling in the hypothalamus of TLR4-deficient mice (C3H/HeJ) injected with thapsigargin (TG, an endoplasmic reticulum stress inducer).
In contrast to WT mice, TLR4-deficient mice were found to sustain significantly lower hypothalamic levels of IL-6 (Figure 7A) and IL-10 (Figure 7B) after exercise. The food consumption was similar between C3H/HeN and C3H/HeJ under basal conditions, and acutely, thapsigargin alone did not affect the food intake in these mice (unpublished data); however, the intrahypothalamic administration of TG impaired the anorexigenic effects of insulin and leptin in WT (C3H/HeN) and in TLR4-deficient mice; while physical activity restored the appetite suppressive actions of insulin and leptin in WT but not in TLR4-deficient mice (Figure 7C and D). Furthermore, the intrahypothalamic injection of either recombinant IL-6 or IL-10 restored the anorexigenic actions of insulin and leptin in both WT and TLR4-deficient mice injected with TG (Figure 7C and D). We also observed that the intrahypothalamic infusion of recombinant IL-6 was able to increase the IL-10 protein expression in the hypothalamus of WT and TLR4-deficient mice (Figure 7E). Moreover, exercise failed to reduce inflammation and ER stress and failed to improve insulin and leptin sensitivity in the hypothalamus of TLR4-deficient mice injected with TG (Figure 7F–J). On the other hand, the intrahypothalamic injection of recombinant IL-6 or IL-10 reduced IKKβ, PERK, and IRS-1Ser307 phosphorylation and restored insulin and leptin signaling in the hypothalamus of TLR4-deficient mice injected with TG (Figure 7F–J). There were no differences in the basal levels of Akt and STAT-3 phosphorylation between the groups (unpublished data). The in situ hybridization experiment revealed that IL-10R is expressed in NPY, POMC, and AgRP neurons of rats (Figure 7K). Finally, immunohistochemistry with anti-IL-6R and anti-IL-10 Receptor (IL-10R)-specific antibodies revealed that IL-6R and IL-10R are expressed in the same specific neuronal subtypes in the arcuate nucleus (Figure 7L).
10.1371/journal.pbio.1000465.g007 Figure 7 The central anti-inflammatory response mediated by exercise requires augmented hypothalamic levels of IL-6 and IL-10.
Western blots showing hypothalamic lysates from C3H/NeN and C3H/HeJ mice under resting conditions or after physical activity; (A) IL-6 and (B) IL-10 expression. Anorexigenic effects of insulin (C) or leptin (D) in C3H/NeN and C3H/HeJ mice under resting conditions, after thapsigargin, thapsigargin plus exercise, and thapsigargin plus recombinant IL-6 or IL-10. Western blots showing hypothalamic lysates from mice; (E) IL-10 expression at 2 h after intrahypothalamic injection of recombinant IL-6 (200 ng) in C3H/NeN and C3H/HeJ mice under resting conditions. (F) IKKβ, (G) PERK, and (H) IRS-1Ser307 phosphorylation and (I) Insulin-induced Akt serine phosphorylation and (J) leptin-induced STAT3 tyrosine phosphorylation in the hypothalamus of C3H/HeJ mice after intrahypothalamic infusion of DMSO, thapsigargin, thapsigargin plus exercise, and thapsigargin plus recombinant IL-6 or IL-10. Data are the means ± SEM. ** p<0.05 versus respective control group at rest; # p<0.05 versus respective control group non-stimulated or stimulated with DMSO; * p<0.05 versus thapsigargin; n = 5–6 animals per group. C3H/NeN (yellow bars) and C3H/HeJ (blue bars). (K) Co-localization of IL-10R (red) with NPY, AgRP, and POMC (green) was evaluated using in situ hybridization technique in the hypothalamus of lean rats, with 20× and 63× magnification. (L) Co-localization of IL-6R (green) and IL-10R (red) in the arcuate nuclei of lean rats, with 200× magnification (scale bar, 10 µm).
Effects of Chronic Exercise on Food Intake and Body Weight
We then investigated the effects of chronic SW Exe on food intake and body weight in lean and obese rats. As observed in acute exercise, the chronic exercise protocol did not change the food consumption in lean animals; however, we observed that the food intake was reduced in obese animals after onset of the chronic exercise protocol, for 3 d, but thereafter, the food intake returned to basal levels on the sixth day and was maintained similar to that of obese rats at rest (Figure 8A). Exercised obese animals showed a significant reduction of the total body weight between the third and the sixth days, but this phenomenon was not observed in control animals (Figure 8B). We also evaluated the weight gain by analyzing the variation of the body weight between the 1st and 24th days. We observed a slight weight gain in control animals at rest, but the chronic exercise protocol did not attenuate the weight gain in lean animals (Figure 8C). On the other hand, overnutrition induced a great weight gain in the group under resting conditions, while chronic exercise attenuated the weight gain in obese animals (Figure 8C). We did not observe a statistical difference in the absolute values of the epididymal fat mass between the exercised obese animals and the obese animals at rest at the end of chronic exercise protocol (Figure 8D).
10.1371/journal.pbio.1000465.g008 Figure 8 Effects of chronic exercise on food consumption, body weight, and IL-6 and IL-10 production.
Evaluation of (A) food intake (kcal) and (B) body weight in control and obese animals during chronic exercise protocol. Chow rest (black square), chow exercise (white square), DIO rest (black ball), and DIO exercise (white ball). (C) Body weight change between the 1st and 24th day. (D) Epididymal fat pad weight after chronic exercise, (E) IL-6 and (F) IL-10 mRNA levels in the hypothalamus of lean and obese rats at rest or after chronic exercise. Western blots showing hypothalamic lysates from lean and obese Wistar rats; (G) IKKβ phosphorylation and IκBα expression and (H) PERK phosphorylation and CHOP expression 1 and 24 d after the chronic exercise protocol. Data are the means ± SEM. * p<0.05 versus chow at rest; § p<0.05 versus DIO at rest; # p<0.05 versus chow group (rest); n = 8–10 animals per group. Lean animals (white bars) and obese animals (black bars).
Chronic overnutrition increased serum insulin, leptin, triglycerides, and free fatty acid levels, compared to age-matched controls; however, chronic exercise reduced serum insulin, triglycerides, and free fatty acid levels in obese animals (Table 3). To determine whether lean and obese rodents were swimming or running in the same fashion, we evaluated lactate production every 15 min during the SW Exe. We did not find any difference in the lactate production between lean and obese rats. Table 3 depicts the final values obtained in this test. We also determined that this exercise protocol did not change the corticosterone levels in lean and obese animals 3 d after the onset of this exercise protocol (Table 3).
10.1371/journal.pbio.1000465.t003 Table 3 Metabolic parameters of lean and DIO rats after chronic exercise.
Groups Glucose (mg/dL) Insulin (ng/mL) Leptin (ng/mL) Cholesterol (mg/dL) TG (mg/dL) FFA (mmol/L) Corticost. (ng/mL) Lactate (mmol/L)
Chow rest 98±4 4.0±0.2 2.0±0.2 132.9±9.3 94.0±1,4 0.64±0.2 11.1±0.6 ND
Chow SW exe 99±8 3.1±0.4# 2.2±0.2 134.5±6.2 92.3±6,3 0.64±0.2 11.6±0.7 5.2±0.5
DIO rest 115±5 7.8±0.4# 3.6±0.3# 149.6±10.8 152.5±7.8# 1.75±0.5# 11.2±0.7 ND
DIO SW exe 114±7 5.1±0.5#* 3.1±0.3# 144.6±10.1 102.3±10.7#* 0.89±0.3#* 11.5±0.9 5.3±0.7
# p<0.05 versus chow rest and *p<0.05 versus DIO rest (n = 8–10).
We also evaluated IL-6 and IL-10 mRNA levels in the hypothalamic tissue during the chronic exercise protocol. Interestingly, we observed that the levels of IL-6 mRNA in the hypothalamus were higher on the first day of exercise, when compared to the 15th and 24th days of exercise; this phenomenon was observed in lean and obese exercised rats (Figure 8E). Similar results were found when we analyzed the levels of IL-10 mRNA during chronic exercise (Figure 8F). Finally, the chronic exercise protocol reduced IKKβ phosphorylation and increased IκBα expression in the hypothalamus of obese rats; however, this anti-inflammatory response was more evident on the first day of exercise (Figure 8G). Similar results were found when we analyzed the ER stress markers, such as PERK phosphorylation and CHOP expression (Figure 8H).
Discussion
Exercise as a Potential Target for Countering Hyperphagia and Obesity
Physical activity is a cornerstone in the prevention of obesity and related diseases. Although the energy expenditure aspects of such exercise may contribute to the effects of weight loss, it has been suggested that physical exercise may also contribute to negative energy balance by altering appetite and reducing food intake in rodents [21],[32] and humans [33],[34]. Our study shows that acute exercise per se did not evoke any meaningful effect, in terms of food intake in lean animals, but interestingly, it was crucial for suppressing hyperphagia mediated by overnutrition, reducing hypothalamic IKKβ/NF-κB activation and ER stress, thus improving insulin and leptin action in an IL-6- and IL-10-dependent manner (Figure 9).
10.1371/journal.pbio.1000465.g009 Figure 9 Schematic diagrams of the proposed role of the hypothalamic anti-inflammatory response mediated by exercise.
(A) Overnutrition induces hypothalamic IKKβ activation and endoplasmatic reticulum stress, leading to central insulin and leptin resistance, hyperphagia, and obesity. (B) We propose that exercise increases the central anti-inflammatory response, increasing hypothalamic IL-6 and IL-10 expression. This phenomenon is crucial for reducing hypothalamic IKKβ activation and endoplasmatic reticulum stress and turn, restoring insulin and leptin signaling, and reorganizing the set point of nutritional balance.
In the absence of obesity, exercise does not affect food behavior, as the anorexigenic or orexigenic pathways remain unchanged in rats. Several experimental studies have demonstrated that physical activity does not activate anorexigenic pathways, such as PI3-K or mTOR/p70S6K [18],[21], and does not inhibit the orexigenic pathways, such as AMPK signaling in the hypothalamus of control rodents [35]. On the other hand, the present study provides substantial evidence that physical activity could help to reorganize the set point of nutritional balance and, therefore, aid in counteracting the energy imbalance induced by overnutrition-related obesity. These data are in accordance with Park and colleagues [36], who showed that exercise improved insulin and leptin signaling, increased STAT3, and reduced AMPK phosphorylation in the cerebral cortex and hypothalamus of diabetic rats, contributing to the regulation of body weight and glucose homeostasis. These data demonstrate that exercise increases the anorexigenic pathways and attenuates the orexigenic signals, only in obese and diabetic animals, changing the anorexigenic and orexigenic signaling pathways in the hypothalamus. We also reported that physical activity reduced the hyperphagic response by reducing NPY mRNA and increasing POMC mRNA predominantly in the arcuate nucleus of obese animals. It is important to emphasize that acute exercise did not change the total body weight or epididymal fat pad weight, showing that physical activity can induce the anorexigenic response in the hypothalamus, independently of the body weight change. Our data showed that the reduction on food intake observed in obese animals after both exercise protocols was not related to stress as demonstrated by costicosterone levels. In opposite fashion, it has been demonstrated that NPY mediates stress-induced exacerbation of diet-induced obesity and metabolic syndrome after different stressor agents such as exposure to cold water or aggression in mice [37]. Thus, we hypothesized that some factors, produced during the exercise session, could be involved in this anorexigenic response.
IL-6 Is a Crucial Cytokine for Exercise to Restore Hypothalamic Insulin and Leptin Signaling
Skeletal muscle is an endocrine organ that, upon contraction, stimulates the production and release of cytokines, also called myokines, which can influence metabolism and modify cytokine production in tissue and organs. IL-6 is the first cytokine present in the circulation during exercise [17]. IL-6 can elicit proinflammatory or anti-inflammatory effects, depending on the in vivo environmental circumstances. Although IL-6 has been associated with low-grade inflammation and insulin resistance, it has been demonstrated that acute IL-6 treatment enhances insulin-stimulated glucose disposal in humans [38].
Centrally acting IL-6 appears to play a role in the regulation of appetite, energy expenditure, and body composition. Wallenius and colleagues elegantly showed that long-term peripheral IL-6 treatment to IL6−/− mice caused a decrease in body weight. In addition to increasing energy expenditure, IL-6 may prevent obesity by inhibiting feeding as obese IL-6−/− mice had increased absolute food intake [39]. In accordance with these data, mice fed on a high-fat diet with sustained circulating human IL-6 secreted predominantly from brain and lung (hIL6tg) had low leptin concentrations, consumed less food, and expended more energy than wild-type mice [40]. In addition, the intercrossing of hIL6tg and ob/ob mice increased the leptin sensitivity in these mice, when compared to ob/ob mice [40]. Recently, we demonstrated that exercise requires IL-6 to increase hypothalamic insulin and leptin sensitivity [18] and increase the effects of leptin on the AMPK/mTOR pathway in the hypothalamus of rodents [21]. Furthermore, IL-6 is also released from the brain during prolonged exercise in humans [41]. In the present study, we showed that the increment of IL-6 expression in the hypothalamus was crucial to exercise for reducing the inflammation and ER stress activation induced by overnutrition. However, these effects, promoted by exercise, were not observed when we used an intrahypothalamic infusion of anti-IL-6 antibody before the exercise protocol. In addition, the infusion of recombinant IL-6 into the third hypothalamic ventricle reduced the energy intake in obese animals under resting conditions, in a dose-dependent manner, and reduced hypothalamic IKKβ and ER stress activation.
In another approach, we used an ER stress inducer in lean rats to evaluate the effects of exercise/IL-6 on hypothalamic ER stress. We demonstrated that acute thapsigargin injection increased IKKβ and PERK phosphorylation and reduced insulin and leptin action in the hypothalamus and that exercise and the infusion of recombinant IL-6 were able to reduce thapsigargin-induced inflammation, ER stress, and insulin and leptin resistance, whereas the IL-6 antibody pretreatment reversed the effects of exercise. Although thapsigargin increased the hypothalamic IKKβ and PERK phosphorylation, we did not observe any difference in the basal levels of Akt serine 473 and STAT3 tyrosine 705 phosphorylation and in food intake in rats injected with thapsigargin alone. These data are in accordance with a previous study that reported that the ER-stress inhibitor, tauroursodeoxycholic acid (TUDCA), acutely reduced the hypothalamic PERK phosphorylation and NF-kB activation but did not change the food intake in mice fed on a high-fat diet [7]. Thus, our data demonstrate that IL-6 plays an important role in the control of the ER stress effects in the hypothalamus of rats.
All these results are significant, since IKKβ and ER stress activation were strongly associated with insulin and leptin resistance in the hypothalamic tissue. Although we showed a consistent anti-inflammatory effect, mediated by IL-6, in the hypothalamus, we cannot exclude the possibility that IL-6 acts directly as an anorexigenic factor.
Hypothalamic IL-10: A Core Anti-Inflammatory Cytokine Induced by IL-6
Although our findings clearly show that IL-6 diminished hypothalamic IKKβ and ER stress activation and restored the central insulin and leptin action in an animal model of obesity, the question remains as to how IL-6 promotes these events in the hypothalamus. Following exercise, the high circulating levels of IL-6 are followed by an increase in two anti-inflammatory molecules, IL-1ra and IL-10 [25]. Therefore, IL-6 induces an anti-inflammatory environment by inducing the production of IL-1ra and IL-10. In our study, we found that exercise increased the hypothalamic levels of IL-10 but did not change IL-1ra expression in this tissue. Thus, we showed that the anti-inflammatory response mediated by IL-6 involves the increase of IL-10 expression in the hypothalamus.
IL-10 is an important immunoregulatory cytokine with multiple biological effects. In the cytoplasm, it has been demonstrated that IL-10 blocks NF-κB activity at two levels: suppressing IKK activity and NF-κB DNA binding activity [26]. Moreover, IL-10 reduced ER stress in intestinal eptithelial cells, whereas IL-10−/− mice demonstrated that the expression of the ER stress response protein grp-78/BiP was increased in intestinal eptithelial cells under conditions of chronic inflammation [27].
In the CNS, the anti-inflammatory role of IL-10 has been extensively studied in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis. The increase in IL-10 expression in the CNS during recovery from brain inflammation and the inability of IL-10 null mice to recover from acute CNS inflammation suggests that the presence of IL-10 within this target organ is required for disease remission [42],[43]. However, the role of hypothalamic IL-10 in the control of low-grade inflammation generated during obesity was unknown. Here, we discovered that intrahypothalamic infusion of recombinant IL-10 blocked IKK/NF-κB signaling and ER stress and restored Akt and STAT3 phosphorylation, promoting a re-balance in the energy intake in obese animals. On the other hand, the selective decrease in IL-10 expression in discrete hypothalamic nuclei of obese animals mediated by ASO treatment blunted the effects of both exercise and the intrahypothalamic infusion of recombinant IL-6 in the restoration of central insulin and leptin actions. In addition, we demonstrated that in mice that sustained significantly lower hypothalamic levels of IL-6 and IL-10 after exercise (C3H/HeJ), there was no reduction in pharmacological ER stress activation, in contrast to WT mice. These data are intriguing as IL-10 represents an important cytokine that may reduce both inflammation and ER stress in the hypothalamus. Thus, the modulation of hypothalamic IL-10 expression could be considered the direct target of exercise/IL-6 and constitutes a promising alternative to reduce hypothalamic inflammation and ER stress related to obesity.
The decrease in food intake induced by IL-10 in obese rats is not in accordance with the effects observed in IL-10 KO. It has been reported that mice with combined deficiency of leptin and IL-10 gain less body weight than mice lacking leptin only [44]. However, these discrepancies may be a consequence of methodological differences related to physiological versus genetic approaches and acute versus chronic situation investigated, and most important it may be consequence of IL-10 effects in the regulation of energy expenditure, likewise observed in mice lacking TNF-α receptor [45]; thus, the role of IL-10 in the control of food intake and energy expenditure deserves further exploration.
The long-term reversal effects on body composition, mediated by exercise alone, are controversial. It should be acknowledged that it is often difficult to find long-term reversal effects on body fat in both experimental animals and humans by exercise alone without restrained diet [46]. In the chronic experiments, we observed that the obese animals lost weight during the same period in which a reduction in food intake was observed. After this period, no significant difference was observed in the body weight of exercised animals, although the obese animals presented a significant improvement in metabolic parameters after the chronic exercise protocol.
Since IKKβ/NF-κB inhibition in the CNS represents a potential target therapy to combat obesity and most anti-inflammatory therapies have limited direct effects on IKKβ/NF-κB and a limited capacity for concentration in the CNS, our study provides substantial evidence that physical activity could help to reorganize the set point of nutritional balance and therefore aid in counteracting the energy imbalance induced by overnutrition through the anti-inflammatory response in hypothalamic neurons. Hence, IL-6 and IL-10 are important physiological contributors to the central insulin and leptin action mediated by physical activity, linking it to hypothalamic ER stress and inflammation.
Materials and Methods
Antibodies and Chemicals
Protein A-Sepharose 6 MB and Nitrocellulose paper (Hybond ECL, 0.45 µm) were from Amersham Pharmacia Biotech United Kingdom Ltd. (Buckinghamshire, United Kingdom). Ketamin was from Parke-Davis (São Paulo, SP, Brazil) and diazepam and thiopethal were from Cristália (Itapira, SP, Brazil). Anti-phospho-JAK2 (rabbit polyclonal, AB3805) antibody was from Upstate Biotechnology (Charlottesville, VA, USA). Anti-JAK2 (rabbit polyclonal, SC-278), anti-STAT3 (rabbit polyclonal, SC-483), anti-phospho-IRβ (rabbit polyclonal, SC-25103), anti-IRβ (rabbit polyclonal, SC-711), anti-phospho-IRS-1 (rabbit polyclonal, SC-17199), anti-IRS-1 (rabbit polyclonal, SC-559), anti-IRS-2 (rabbit polyclonal, SC-1556), anti-phosphotyrosine (mouse monoclonal, SC-508), anti-Foxo1 (rabbit polyclonal, SC-11350), anti-IL-1ra (goat polyclonal, SC-8481), anti-TNF-α (rabbit polyclonal, SC-8301), anti-IKKβ (goat polyclonal, SC-34673), anti-PERK (rabbit polyclonal, SC-13073), anti-phospho-PERK (rabbit polyclonal, SC-32577), anti-CHOP (GADD 153) (rabbit polyclonal, SC-575), anti-IL-10 (goat polyclonal, SC-1783), and anti-IL-6 (rabbit polyclonal, SC-7920) antibodies were from Santa Cruz Biotechnology, Inc. Anti-phospho-STAT3 (rabbit polyclonal, #9131), anti-phospho-Akt (rabbit polyclonal, #9271), anti-phospho-Foxo1 (rabbit polyclonal, #9461), anti-beta tubulin (rabbit polyclonal, #2146), anti-phospho-IKKα/β (rabbit polyclonal, #2687), anti-IκBα (rabbit polyclonal, #9242), anti-TLR4 (rabbit polyclonal, #2219), anti-phospho-IRS-1 307 (rabbit polyclonal, #2381), and anti-Akt (rabbit polyclonal, #9272) were from Cell Signalling Technology (Beverly, MA, USA). Leptin, thapsigargin, and recombinant IL-6 and -10 were from Calbiochem (San Diego, CA, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.
Serum Insulin, Leptin, and IL-6 Quantification
Blood was collected from the cava vein 15 min after the exercise protocols. Plasma was separated by centrifugation (1,100 g) for 15 min at 4 °C and stored at −80 °C until assay. RIA was employed to measure serum insulin. Leptin and IL-6 concentrations were determined using a commercially available Enzyme Linked Immunosorbent Assay (ELISA) kit (Crystal Chem Inc., Chicago, IL). Blood lactate was measured using Accutrend Plus equipment (Roche); sample blood was obtained from the tails every 15 min during the exercise protocols. Serum cholesterol and triglycerides were measured in control and exercised animals after 8 h of fasting using Accutrend Plus equipment (Roche). Serum free fatty acids (FFA) levels were analyzed in rats using the NEFA-kit-U (Wako Chemical GmBH, Neuss, Germany).
Corticosterone levels were determined using urine samples obtained from rats and mice using specific metabolic cage during 24 h after the exercise protocols. The corticosterone level was determined using an EIA kit from Cayman chemical (Ann Arbor, MI).
Animals
Male 4-wk-old Wistar rats were obtained from the University of Campinas Breeding Center. The investigation was approved by the ethics committee and followed the University guidelines for the use of animals in experimental studies and experiments conform to the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication no. 85-23 revised 1996). The animals were maintained on 12h∶12h artificial light-dark cycles and housed in individual cages. Rats were randomly divided into two groups: control, fed on standard rodent chow (3,948 kcal.Kg−1), and DIO, fed a fat-rich chow (5,358 kcal.Kg−1) ad libitum for 3 mo. This diet composition has been previously used [47].
Male (10-wk-old) ob/ob mice and their respective controls C57BL/6J background were obtained from The Jackson Laboratory and provided by the University of São Paulo. The mice were bred under specific pathogen-free conditions at the Central Breeding Center of University of Campinas.
Male C3H/HeJ (10-wk-old) mice and their respective controls C3H/HeN were obtained from The Jackson Laboratory and provided by the University of São Paulo. The mice were bred under specific pathogen-free conditions at the Central Breeding Center of the University of Campinas.
Intracerebroventricular Cannulation
The animals were stereotaxically instrumented under intraperitoneal injection of a mix of ketamin (10 mg) and diazepam (0.07 mg) (0.2 ml/100 g body weight) with a chronic 26-gauge stainless steel indwelling guide cannula aseptically placed into the third ventricle at the midline coordinates of 0.5 mm posterior to the bregma and 8.5 mm below the surface of the skull of rats and 1.8 mm posterior to the bregma and 5.0 mm below the surface of the skull of mice.
Exercise Protocols
Animals were acclimated to swimming for 2 d (10 min per day). Water temperature was maintained at 34–35 °C. Rats performed two 3-h exercise bouts, separated by one 45-min rest period. The rats swam in groups of three in plastic barrels of 45 cm in diameter that were filled to a depth of 50 cm. This protocol was conducted between 11:00 a.m. and 6:00 p.m., as previously described [48], and mice performed four 30-min exercise bouts, separated by one 5-min rest period. The mice swam in groups of four in plastic barrels of 40 cm in diameter that were filled to a depth of 20 cm. This protocol was conducted between 3:00 p.m. and 6:00 p.m. Both exercise protocols finished at 6:00 p.m. for evaluation of food intake and analysis of hypothalamic tissue.
The chronic exercise protocol consisted of daily swimming sessions (1 h/d, 5 d/wk, for 4 wk) with an overload (2.0% of the body weight). The hypothalamic tissues and the metabolic parameter were evaluated 36 h after the last exercise session. Rats also performed a single bout of treadmill (Insight LTDA - Ribeirão Preto, SP) running (60 min, speed of 10–15 m/min at a 5% incline) and mice performed a single bout of treadmill running (90 min, speed of 7–10 m/min at a 5% incline).
Intracerebroventricular Treatments
Rats or mice were deprived of food for 2 h with free access to water and received 3 µl of bolus injection into the third ventricle, as follows:
Insulin and leptin treatments
Animals received intrahypothalamic infusion of vehicle, insulin (200 mU), or leptin (10−6 M) at 6:00 p.m. to evaluate the food intake or insulin and leptin signaling. Food intake was determined by measuring the difference between the weight of chow given and the weight of chow at the end of a 12-h period.
Recombinant IL-6 and IL-10 treatments
Animals received intrahypothalamic infusion of vehicle, or recombinant IL-6 (50, 100, or 200 ng) or recombinant IL-10 (0.5, 1.0, or 3.0 ng) at 6:00 p.m. to evaluate the food intake. For Western blot analysis, we injected recombinant IL-6 or IL-10 2 h after DMSO or thapsigargin into the third ventricle and the hypothalamus was excised 2 h later.
Thapsigargin treatments
Animals received intrahypothalamic infusion of vehicle, or thapsigargin (3.0 µg). To evaluate the energy intake and for Western blot analysis, thapsigargin was infused 40 min before the exercise protocol and 2 h before the recombinant IL-6 infusion. Immediately after exercise or 2 h after IL-6 infusion, animals received intrahypothalamic infusion of insulin (200 mU) or leptin (10−6 M).
IL-6 neutralizing antibody
Animals were randomly selected for treatment with saline, rabbit pre-immune serum (RPIS) or rabbit antiserum against IL-6 (IL-6 Ab) in different doses. IL-6 Ab was injected into the third ventricle of the rats 15 min before the exercise protocol.
ASO IL-10 treatments
Phosphorthioate-modified sense and antisense oligonucleotides (produced by Invitrogen Corp., Carlsbad, CA, USA) were diluted to final concentration of 1 nmol/µl in dilution buffer containing 10 mmol/l Tris–HCl and 1.0 mmol/l EDTA. The oligonucleotides were designed according to the Mus musculus IL-10 sequence deposited at the NIH-NCBI (http://www.ncbi.nlm.nih.gov/entrez) under the designation NM 010548 and were composed of 5′-GCC AGT CAG TAA GAG CAG-3′ (sense) and 5′-TGA GAT CTG CAA TGC A-3′ (antisense). Obese Wistar rats were injected into the third ventricle with two daily doses of 3 µl of dilution buffer containing, or not, sense (Sense IL-10) or antisense oligonucleotides (ASO IL-10) for 3 d. For Western blotting analysis, after ASO IL-10 treatment, obese animals were submitted to the exercise protocol or intrahypothalamic infusion of recombinant IL-6. In some experiments, the rats also received intrahypothalamic infusion of insulin (200 mU) or leptin (10−6 M) for the determination of food intake and Akt and STAT3 phosphorylation.
Recombinant of TNF-α treatments
Animals received intrahypothalamic infusion of vehicle, or TNF-α (10−12). To evaluate the energy intake and for Western blotting analysis, TNF-α was infused 40 min before the exercise protocol and 2 h before the recombinant IL-6 infusion. Immediately after exercise or 2 h after IL-6 infusion, animals received intrahypothalamic infusion of insulin (200 mU) or leptin (10−6 M).
Food Intake Determination
Intrahypothalamic infusions were performed between 5:00 and 6:00 p.m. Thereafter standard chow or high-fat diet was given and food intake was determined by measuring the difference between the weight of chow given and the weight of chow at the end of a 12-h period. Similar studies were carried out in animals after exercise.
Western Blot Analysis
After exercise and/or i.c.v. treatments, the animals were anaesthetized, and the hypothalamus was quickly removed, minced coarsely, and homogenized immediately in a freshly prepared ice-cold buffer (1% Triton X-100, 100 mmol/l Tris pH 7.4, 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium vanadate, 2 mmol/l phenyl methylsulphonyl fluoride, and 0.1 mg aprotinin) suitable for preserving phosphorylation states of enzymes, and Western blot was performed, as previously described [1].
Nuclear Extract
Foxo1 and STAT-3 nuclear expression were obtained as described [49]. Fragments of hypothalamic tissue from untreated rats or rats treated with insulin or leptin were obtained 30 min after insulin or leptin infusion and were minced and homogenized in 2 vol. of STE buffer (0.32 M sucrose, 20 mM Tris–HCl (pH 7.4), 2 mM EDTA, 1 mM DTT, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 1 mM PMSF, and 0.1 mg aprotinin/ml) at 4 °C with a Polytron homogenizer. The homogenates were centrifuged (1,000×g, 25 min, 4 °C) to obtain pellets. The pellet was washed once and suspended in STE buffer (nuclear fraction). The nuclear fraction was solubilized in Triton buffer [1% (v/v) Triton X-100/150 mM NaCl/10 mM Tris/HCl (pH 7.4)/1 mM EGTA/1 mM EDTA/0.2 mM sodium orthovanadate/20 µM leupeptin A/0.2 mM PMSF/50 mM NaF/0.4 nM microcystin LR]. The fraction was centrifuged (15,000 g, 30 min, 4 °C), and the supernatant (nuclear extract) was stored at −80 °C.
Confocal Microscopy
Paraformaldehyde-fixed hypothalami were sectioned (5 µm). The sections were obtained from the hypothalami of six rats per group in the same localization (antero-posterior = −1.78 from bregma) and used in regular single- or double-immunofluorescence staining using DAPI, anti-IL6 receptor alpha (rabbit IgG, SC-13947), anti-IL-10 receptor (rabbit IgG, SC-987), anti-IKKβ (goat IgG, SC-34673), anti-PERK (rabbit IgG, SC-32577), anti-POMC (rabbit IgG, FL-267), and rabbit anti-IRS-1 (rabbit IgG, SC-559) (1∶200; Santa Cruz Biotechnology) antibodies. After incubation with the primary antibody, sections were washed and incubated with specific biotinylated anti-rabbit or anti-goat secondary antibodies (1∶150 dilution) for 2 h at room temperature, followed by incubation with Streptoavidin reagent (containing avidin-conjugated peroxidase) and color reaction using the DAB substrate kit (Vector Laboratories, Burlingame, CA, USA), according to recommendations of the manufacturer. Analysis and photodocumentation of results were performed using a LSM 510 laser confocal microscope (Zeiss, Jena, Germany). The anatomical correlations were made according to the landmarks given in a stereotaxic atlas [50]. The frequency of positive cells was determined in 100 randomly counted cells using Analysis software (Version 2.4).
mRNA Isolation and Real Time PCR
Hypothalamic total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD, USA), according to the manufacturer's recommendations. Total RNA was rendered genomic DNA free by digestion with Rnase-free Dnase (RQ1, Promega, Madison, WI, USA). Rats were deprived of food for 9 h after for real time PCR analysis. Real time PCR and mRNA isolation were performed using a commercial kit, as follows: IL-6: Rn00561420_m1 IL-10: Rn00563409_m1, POMC: Rn00595020_m1, NPY: Rn00561681_m1, AgRP: Rn01431703_g1, GAPD, #4352338E, for rat and RPS-29 (NCBI: NM012876), sense: 5′-AGGCAAGATGGGTCACCAGC-3′, antisense: 5′-AGTCGAATCATCCATTCAGGTCfG-3′.
Dissection of the Arcuate Nucleus
After 9 h of fasting, rats were killed by decapitation and hypothalamic nuclei were quickly dissected and homogenized in Trizol reagent (Life Technologies, Gaithersburg, MD, USA), according to the manufacturer's recommendations. Later on, each region of the hypothalamus was dissected from 1 mm thick sagittal sections of fresh brain. Arcuate nucleus was dissected from the first sections from the midline of the brain. Coordinates for the arcuate nucleus is ventral part of the medial hypothalamus with anterior and dorsal margin and posterior margin (border with mammilary body).
In Situ Double mRNA Hybridization
For mRNA localization all solutions and materials utilized were RNAse free. The probes were determined and designed using the program Gene Runner 3.05 (Hastings Software, Inc., USA) according to mRNA sequences in NCBI: POMC (NM_139326.2), NPY (NM_012614.1), AgRP (XM_574228.2), IL6ra (NM_017020.1), and IL10ra (AJ_305049.1). Two probes were synthesized for each mRNA and were 5′-end labeled with Alexa Fluor 488 or 546 by Invitrogen Life Technologies (Carlsbad CA, USA). See details in the supplemental data (Table S1). Frozen sections were air dried for 30 min at 37 °C, fixed using cold acetone for 10 min, and washed twice in PBS for 5 min and twice in 2× SSC for 2 min. The sections were incubated with Proteinase K (20 µg/mL) for 10 min at room temperature and then washed twice for 5 min with 2× SSC. The sections were incubated in 0.1 M triethanolamine pH 8 (TEA Buffer) for 10 min and then with 0.25% acetic anhydride in TEA buffer for 10 min under magnetic stirring and then washed with 2× SSC. The pre-hybridization solution was composed by 50% formamide, 5× SSC, Denhardt's solution (1× final concentration), and completed with DEPC-treated water. The sections were pre-hybridized for 4 h without the probe at 50 °C in humidified chamber with 50% formamide in SSC. The probe mix (including two probes for each mRNA; i.e., IL6ra or IL10ra with POMC, AgRP, or NPY) was composed (for each tissue section) of 20 µL of pre-hybridization solution plus 500 µg/mL of torula RNA, 500 µg/mL of salmon sperm DNA, and 50 ng of riboprobe mix (anti-sense or sense). The mixture was placed over the sections and incubated at 52 °C overnight in a humidified chamber. After 18 h hybridization, the sections were washed four times with 4× SSC buffer for 10 and 5 min in PBS. The sections were visualized in Zeiss 510 confocal microscope.
Statistical Analysis
All numeric results are expressed as the means ± SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands or spots in autoradiographs and quantified by optical densitometry (Scion Image). Statistical analysis was performed by employing the ANOVA test with Bonferroni post test. Significance was established at the p<0.05 level.
Supporting Information
Figure S1 Serum levels and hypothalamic expression of IL-6. (A) Serum levels of IL-6 and (B) protein expression of IL-6 in the hypothalamic tissue from lean and obese rats under rest condition or after exercise. Data are the means ± SEM. # p<0.05 versus respective control at rest; * p<0.05 versus respective lean plus exercise; § p<0.05 versus control at rest, n = 8 animals per group.
(1.22 MB DOC)
Click here for additional data file.
Figure S2 Effects of IL-6 on leptin and insulin action. Intrahypothalamic infusion of recombinant IL-6 improves the anorexigenic effects of insulin (A) or leptin (B) in obese Wistar rats. Data are the means ± SEM. * p<0.05 versus obese non-stimulated; ** p<0.01 versus obese stimulated with insulin or leptin alone, n = 6–8 animals per group.
(0.88 MB TIF)
Click here for additional data file.
Figure S3 IL-6 improves insulin and leptin signaling. Western blots of five independent experiments showing hypothalamic lysates from Wistar rats; (A) Insulin-induced Akt serine phosphorylation and (B) leptin-induce STAT3 tyrosine phosphorylation in lean, obese, obese plus recombinant IL-6, obese plus exercise, and exercise obese pretreated with anti-IL-6 antibody before the exercise protocol. Data are the means ± SEM. # p<0.05 versus lean group; * p<0.05 versus obese group at rest; § p<0.01 versus exercised obese group; n = 6–8 animals per group.
(1.43 MB TIF)
Click here for additional data file.
Figure S4 IL-6 suppresses TNF-α induced insulin and leptin resistance. Anorexigenic effects of insulin (A) and leptin (B) in the hypothalamus of lean rats injected with TNF-α, TNF-α plus IL-6, TNF-α plus exercise, and TNF-α in exercised lean animals pretreated with anti-IL-6 antibody before the exercise protocol. Western blots showing hypothalamic lysates from Wistar rats; (C) IKKβ, (D) PERK, (E) IRS-1Ser307, (F) insulin-induced Akt serine phosphorylation, and (G) leptin-induced STAT3 tyrosine phosphorylation and (H) basal levels of Akt and STAT3 phosphorylation in the hypothalamus of lean animals injected with TNF-α, TNF-α plus IL-6, TNF-α plus exercise, and TNF-α in exercised lean animals pretreated with anti-IL-6 antibody before the exercise protocol pretreated with TNF-α. Data are the means ± SEM. # p<0.05 versus DMSO group; * p<0.05 versus lean plus TNF-α; § p<0.05 versus TNF-α plus recombinant IL-6 or TNF-α plus exercised; n = 6–8 animals per group.
(2.44 MB TIF)
Click here for additional data file.
Table S1 mRNA and probes sequences used in double mRNA hybridization. The probes were determined and designed according to mRNA sequences in NCBI: POMC (NM_139326.2), NPY (NM_012614.1), AgRP (XM_574228.2), IL6ra (NM_017020.1), and IL10ra (AJ_305049.1). Two probes were synthesized for each mRNA and were 5′-end labeled with Alexa Fluor 488 or 546.
(0.03 MB DOC)
Click here for additional data file.
We thank Mr. Luiz Janeri and Ms. Janine Sabino for the technical assistance and Nicola Conran for the English language editing.
Abbreviations
ASO IL-10 IL-10 antisense oligonucleotide
CNS central nervous system
DIO diet-induced obese
ER endoplasmatic reticulum
IL interleukin
IL-1ra IL-1 receptor antagonist
IL-6R IL-6 Receptor
IL-10R IL-10 Receptor
IRS-1 insulin receptor substrate-1
Jak-2 Janus Kinase-2
NPY Neuropeptide-Y
POMC Proopiomelanocortin
sTNF-R soluble TNF-receptors
SW Exe swimming exercise
T2D type 2 diabetes
TG thapsigargin
TR Exe treadmill running exercise
WT wild type
==== Refs
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Chiropr OsteopatChiropractic & Osteopathy1746-1340BioMed Central 1746-1340-18-232069604010.1186/1746-1340-18-23ResearchA descriptive study of a manual therapy intervention within a randomised controlled trial for hamstring and lower limb injury prevention Hoskins Wayne [email protected] Henry [email protected] Department of Chiropractic, Faculty of Science, Macquarie University, NSW 2109, Australia2010 9 8 2010 18 23 23 1 5 2010 9 8 2010 Copyright ©2010 Hoskins and Pollard; licensee BioMed Central Ltd.2010Hoskins and Pollard; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
There is little literature describing the use of manual therapy performed on athletes. It was our purpose to document the usage of a sports chiropractic manual therapy intervention within a RCT by identifying the type, amount, frequency, location and reason for treatment provided. This information is useful for the uptake of the intervention into clinical settings and to allow clinicians to better understand a role that sports chiropractors offer.
Methods
All treatment rendered to 29 semi-elite Australian Rules footballers in the sports chiropractic intervention group of an 8 month RCT investigating hamstring and lower-limb injury prevention was recorded. Treatment was pragmatically and individually determined and could consist of high-velocity, low-amplitude (HVLA) manipulation, mobilization and/or supporting soft tissue therapies. Descriptive statistics recorded the treatment rendered for symptomatic or asymptomatic benefit, delivered to joint or soft tissue structures and categorized into body regions. For the joint therapy, it was recorded whether treatment consisted of HVLA manipulation, HVLA manipulation and mobilization, or mobilization only. Breakdown of the HVLA technique was performed.
Results
A total of 487 treatments were provided (mean 16.8 consultations/player) with 64% of treatment for asymptomatic benefit (73% joint therapies, 57% soft tissue therapies). Treatment was delivered to approximately 4 soft tissue and 4 joint regions each consultation. The most common asymptomatic regions treated with joint therapies were thoracic (22%), knee (20%), hip (19%), sacroiliac joint (13%) and lumbar (11%). For soft tissue therapies it was gluteal (22%), hip flexor (14%), knee (12%) and lumbar (11%). The most common symptomatic regions treated with joint therapies were lumbar (25%), thoracic (15%) and hip (14%). For soft tissue therapies it was gluteal (22%), lumbar (15%) and posterior thigh (8%). Of the joint therapy, 56% was HVLA manipulation only, 36% high-HVLA and mobilization and 9% mobilization only. Of the HVLA manipulation, 63% was manually performed and 37% mechanically assisted.
Conclusions
The intervention applied was multimodal and multi-regional. Most treatment was for asymptomatic benefit, particularly for joint based therapies, which consisted largely of HVLA manipulation techniques. Most treatment was applied to non-local hamstring structures, in particular the knee, hip, pelvis and spine.
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Background
Hamstring injuries are the most common muscle injury in running based power sports [1]. In Australian Rules football, hamstring injuries are the most prevalent injury, resulting in more missed competition match play than any other injury, whilst other lower-limb muscle strains also feature prominently [2]. The prevention of hamstring and lower limb muscle strains has remained an enigma to the sports clinician. Traditionally, hamstring prevention has focused on local hamstring factors and included warm up, muscle strength and balance, flexibility and fatigue [3]. Orchard has stated that sports medicine dogma counsels that these factors are important in injury prevention, although the scientific evidence for this is sadly lacking [4]. A lack of variety and progression in various prevention and management strategies has been discussed [5], which may be contributing to hamstring injuries remaining a perpetual cause of frustration for athletes and sports clinicians alike.
Whilst the application of manual therapies in the management of hamstring and other sporting injuries has been applied for some time [6,7], its use has remained scarce in more recent scientific literature and research. If used in clinical practice for hamstring injury management, manual therapies typically involve massage and slow velocity spinal mobilizations or slump stretching [8]. Much has been said recently about the role of non-local factors in hamstring injury risk [9,10], and the potential benefits of high velocity spinal and extremity joint manipulation in hamstring injury management [1,5,11]. This has included calls for research incorporating manipulation directed at local and non-local to hamstring areas [5,11]. In addition, despite hands-on therapies being universally used clinically in prevention efforts of sporting injuries, documentation of the various approaches used in the scientific literature is almost non-existent.
There is much controversy [12,13] and little literature describing the use of chiropractic manipulative therapy performed on athletes [14,15]. In particular there is a lack of clinical surveys documenting sports chiropractic treatment techniques and scope of practice. A requirement exists for clinicians of all professions falling under the sports medicine banner to document their clinical practice, as others have done [16-19]. As is the case in the low back pain literature [20], the use of management approaches in sports medicine clinical trials should be documented, particularly if beneficial results are reported. This will assist manual therapists to evolve their management strategies by making treatment decisions based upon the results of clinical trials, allow reproducibility of the study and to allow clinicians to better understand the role that other professions offer to assist in the multidisciplinary management of athletes in an athlete centered approach.
Therefore, we performed a descriptive analysis of the usage of a sports chiropractic manual therapy intervention within a recent randomised controlled trial of semi-elite Australian Rules football players [21]. The study compared the addition of the intervention to the current best practice medical, paramedical and sports science management. It resulted in the significant prevention of lower-limb muscle strains (p = 0.025) with a non-statistically significant trend towards hamstring strains (p = 0.051) and non-contact knee injuries (p = 0.051) [21]. Reductions in overall (p = 0.006) and current low back pain (p = 0.026) were also achieved. A Cochrane systematic review of the literature reviewed the studies methodology and stated that the study exhibited strong external validity [22], whilst a self rated assessment of the trials internal validity using the PEDro criteria rated the study as 'good' [23]. Another strength of the study is the 'missed match' injury definition, which is the only injury definition with proven reliability [24]. The weakness of the study is that it failed to achieve the numbers as determined by the power analysis due to the late withdrawal of two clubs who had previously committed to participation in the study, meaning there is a strong likelihood of a type 2 error in the results [21]. It was the aim of this manuscript to document the type of treatment delivered, whether joint based or soft tissue based, the amount of treatment, the frequency of treatment, to what regions of the body it was directed and to perform a breakdown of the treatment provided into that for symptomatic benefit and that for asymptomatic benefit. Whilst clinical decision making with respect to diagnostic and treatment decisions in the health sciences are often based on previous training, experience and are often considered an art form, publishing of the treatment rendered in this trial is an attempt to allow clinicians to base management decisions upon the results of clinical trials, such that a more scientific component is incorporated into injury management. In particular this information may promote the uptake of newer, non-traditional approaches to injury prevention and management, which may assist in the reduction of hamstring and other lower-limb injuries on a larger scale [25]. Additionally, the publication of these findings may lead to greater awareness of professional roles associated with inter-professional acceptance and optimal standards of care [26].
Methods
Participation and randomization
Full details of participation and randomisation have been published elsewhere [21].
Players were eligible to participate if they were listed players on their respective Victorian Football League (VFL) squad and did not meet the exclusion criteria [21]. Fifty-nine players drawn from two of the thirteen clubs competing in the semi-elite state based (VFL) met the studies entry requirements and were randomised into the intervention (n = 29) or control group (n = 30). The clubs, coaches and medical staff gave permission to participate in the trial. Subjects completed informed consent forms to participate and were informed about the purpose and procedures of the study. The procedures used in this study were in accordance with the ethical standards of the Committee on Human Experimentation of Macquarie University (Ethics Approval Number: HE27AUG2004-RO3066).
Intervention
During the 8 month study, all players from the intervention and control group both continued to receive what can be considered the current best practice management including medication, surgery, manipulative physiotherapy, massage, strength and conditioning and rehabilitation as directed by club staff. All treatment and management from medical, paramedical and sports science staff was independently administered without restriction or interference from the study authors. All medical staff which comprised of at a minimum: doctors, physiotherapists, strength and conditioning staff, trainers and massage therapists were employed by the club and had no limitation in the number of treatments or the type of treatment they could render. The intervention group additionally received a sports chiropractic intervention delivered by a single sports chiropractor (WH). The intervention was pragmatically and individually determined and could involve high velocity, low amplitude (HVLA) manipulation (either manual or mechanically assisted techniques), mobilization (see Table 1[27]) and/or soft tissue therapies: various stretching and soft tissue massage techniques to the spine, pelvis and extremity. Treatment scheduling was also pragmatically and individually determined. During the first 6 weeks of the study players were required to receive one treatment per week minimum. For the next 3 months of the study players were to receive one treatment per fortnight minimum and for the final 3 months of the season (until the completion of the finals series) players were to receive one treatment per month minimum. The study commenced during the pre-season period, 6 weeks prior to round 1 of the regular home and away season.
Table 1 Manual therapy definitions
Manipulation A brief, shallow, sudden carefully administered thrust (high velocity in nature)
Mechanically assisted manipulation Manipulation performed through the assistance of devices (drop tables or portable drop piece units) or instruments (Activator instruments) being non-cavitational but high velocity in intent
Mobilisation When a joint is passively moved within its normal range of motion (usually a slow oscillatory movement)
Data collection
Treatment for the 29 players in the intervention group (mean age 20.2, SD 1.8, range 18-27) for the entirety of the study was continuously recorded by the treating sports chiropractor. Treatment was determined as either being for the purpose of symptomatic benefit for an athlete-reported symptomatic complaint or for asymptomatic functional improvement. Treatment was further broken down as either being joint based (manual or mechanically assisted HVLA manipulation or mobilization) or soft tissue based (soft tissue massage techniques or stretching techniques) and categorized into the various regions of the body to which it was applied (see Table 2). Extremity joints and extremity soft tissue regions were classified as being separate (i.e.: left and right), while spine based treatment was considered as being one on the basis that the effects of manipulation are not limited to a single spinal joint. If multiple treatments were delivered to the same region on the same consultation (e.g. more than one manipulative technique or massage and stretching technique) then this was only recorded once. An analysis of the joint based treatment was conducted to determine the amount of HVLA manipulation only, HVLA manipulation and mobilization, or mobilization only rendered to each joint based region. For the total HVLA manipulation performed, a breakdown was performed to determine the type of technique used, either being manually performed or mechanically assisted.
Table 2 The regions/joints managed with joint based therapy
Region Definition
Foot All joints distal to the talocrural joint
Ankle The talocalcaneal, talonavicular, talo-crural and distal tibial-fibular joints
Knee The patellar-femoral articulation, tibial-femoral articulation and the proximal tibial-fibular joint
Hip The femoral-acetabular articulation
Sacroiliac joint The sacroiliac articulation
Pubic symphysis The public symphysis
Lumbar The articulation of the 5 lumbar vertebrae and lumbo-sacral joint
Thoracic The articulation of the 12 thoracic vertebrae
Cervical The articulation of the 7 cervical vertebrae and the skull
TMJ The temporomandibular joint articulations
Ribs The vertebral-costal articulations posteriorly
Shoulder The gleno-humeral joint, scapulothoracic articulation and acromioclavicualr joint
Chest The manubrio-sternal joint, sternal-costal joint, costal-chondral joints and sternal-clavicular joint
Elbow The ulnar-humeral articulation and the proximal radio-ulnar joint
Wrist The radiocarpal joint, distal radio-ulnar joint and intercarpal joints
Hand All joints distal to the wrist
* Soft tissue structures were defined as surrounding the involved joint as viewed from the anterior, medial, posterior and lateral aspect.
Results
Over the course of the study a total of 487 treatment consultations were provided to the 29 intervention players (average of 16.8 treatment consultations per player), with all players being compliant to the minimum treatment protocol. This resulted in treatment being delivered to 2,000 joint based regions (47.0% total treatment) and 2,258 soft tissue based regions (53.0% of total treatment). On average per treatment consultation players received treatment to approximately 4 joint and 4 soft tissue based regions, which were not necessarily the same. Of the total treatment provided 65.3% was classified as being delivered to asymptomatic regions and 34.7% to symptomatic regions.
Figure 1 demonstrates the breakdown of joint based therapy into that for symptomatic and asymptomatic benefit. Of the total joint based therapy 73.5% was for asymptomatic benefit and 26.5% symptomatic benefit. The most common regions treated for asymptomatic benefit were the thoracic spine (21.3%), knee (20.5%), hip (19.0%), sacroiliac joint (12.5%) and lumbar spine (11.1%). The most common regions treated for symptomatic benefit were the lumbar spine (24.3%), thoracic spine (16.7%), hip (14.0%), cervical spine (13.3%), sacroiliac joint (10.8%) and knee (10.4%). Of interest the following ratios of asymptomatic: symptomatic treatment occurred at the knee (5.5:1), hip (3.8:1), thoracic spine (3.6:1), sacroiliac (3.2:1), and lumbar spine (1.3:1).
Figure 1 Breakdown of joint based therapy into region and as being for symptomatic or asymptomatic benefit.
Of the total joint based therapy delivered to the regions of the body, 55.7% was HVLA manipulation only, 35.9% a combination of HVLA manipulation and mobilization and 8.5% mobilization only. Therefore, 91.6% of the total joint based treatment involved some form of HVLA manipulation technique. When assessing the breakdown of HVLA manipulation techniques performed, 62.9% was manually performed and 37.1% mechanically assisted.
Figure 2 demonstrates the breakdown of soft tissue based therapy into that for symptomatic and asymptomatic benefit. Of the total soft tissue based therapy 58.0% was for asymptomatic benefit and 42.0% was for symptomatic benefit. The most common asymptomatic soft tissue regions treated were the gluteal region (22.0%), hip flexors (13.8%), knee (13.0%) and lumbar spine (10.6%). Only 5.6% of treatment was delivered to the posterior thigh. The most common soft tissue regions treated for symptomatic benefit were the gluteal region (21.5%), lumbar spine (14.2%), thoracic spine (7.6%) and posterior thigh (7.4%). Of interest the following ratios of asymptomatic: symptomatic treatment occurred at the knee (4.3:1), hip flexor (3.3:1), gluteal region (1.4:1) and lumbar spine (1.0:1).
Figure 2 Breakdown of soft tissue based therapy into region and as being for symptomatic or asymptomatic benefit.
Discussion
This study documented that the sports chiropractic intervention applied in a recent RCT [21] comprised an ongoing, multi-region treatment approach incorporating both soft tissue techniques and joint based manipulation and mobilization. A number of joint and soft tissue structures were treated on each consultation, which were not necessarily the same. Whilst not being limited to manipulation only, there was an emphasis on HVLA manipulation techniques, with both manual and mechanically assisted techniques being performed, often in combination with mobilisation. A high proportion of treatment was provided to asymptomatic areas, particularly when joint based therapy was provided. With regards to joint based therapies delivered for asymptomatic benefit, treatment was predominantly delivered to the knee, hip, thoracic spine, sacroiliac joint and lumbar spine. For soft tissue therapies, asymptomatic treatment was predominantly delivered to the knee, hip flexor and gluteal region. When assessing ratios of asymptomatic and symptomatic treatment, the knee, hip and pelvic regions featured prominently for soft tissue and joint based therapies, which are all non-local to hamstring and lower-limb injury. No adverse events were associated with this treatment approach [21]. Based on the findings of the original RCT [21], the addition of this care experience appears to have improved the overall outcome for these players. However, it is important to note the preliminary nature of this research and the pragmatic nature of the study and that conclusions with respect to treatment effectiveness should be made with caution.
The sports chiropractic intervention was pragmatically and individually determined in a patient centered approach. In deciding what treatment to deliver and where to apply it, in particular for the large amount of asymptomatic treatment, several factors were considered. This included the patients current and previous medical history, particularly history of injury. This was combined with examination findings which included a postural assessment, observation of gait and motor patterns, static and motion palpation, range of motion assessment, various orthopaedic and other tests. The information gained from this was pooled together to make a clinical decision, such as occurs in clinical practice of all manual therapy professions. The multimodal and multi-region treatment approach delivered likely reflects the complex multi-factorial aetiology of hamstring and lower-limb injuries which have been said to result from a complex interaction of multiple risk factors and events, of which only a fraction have been identified [28]. In this regard, Dvorak et al. have highlighted the importance of multiple, simultaneous factors to develop a multidimensional predictor score for soccer injuries [29]. This could explain the reason for the amount of soft tissue and joint based treatment delivered on each treatment consultation, as an attempt was made to reduce all possible local and non-local risk factors for hamstring and lower-limb injury. The presence of multiple, simultaneous risk factors could highlight the importance of an effective multi-disciplinary environment providing a multimodal approach to injury prevention. Such an approach has been discussed as being necessary in hamstring injury management [30].
The use of joint based therapy in this study is of interest, as we contend that the biggest difference between the intervention and the best practice management applied to players (which included manipulative physiotherapy), was the addition of high amounts of HVLA chiropractic manipulation to a number of asymptomatic and symptomatic joint regions each treatment consultation. This study documented that 91.6% of joint based treatment involved HVLA manipulation and each treatment consultation involved manipulation or mobilisation to 4 regions. Although data was not included in this study on the management rendered by club staff, from the authors limited knowledge of the treatment provided in the control group, HVLA manipulation was rarely performed whilst the mechanically assisted techniques are exclusive to chiropractic. Previous research has shown that professions falling under the manual therapy banner do in fact have differing treatment methods [31]. Research investigating physiotherapist management of low back pain has shown that high-velocity spinal manipulation is used between 2.8% [32], 3.7% [33], 4.3% [34], 8.9% [35], and more recently in a heavily evidence based education system 36.2% of the time by a group of students [36], figures much lower than in this injury prevention RCT. Alternatively, low velocity mobilization is used between 27.2% [33], 43.8%[35], 58.6% [36], 58.9% [32], and 72% of the time [34]. More relevant to this study is research investigating sports physiotherapy scope of practice. Management provided by sports physiotherapists at international athletics competition has been shown to include asymptomatic treatment [16]. Published literature from the Olympic polyclinic has demonstrated that the most common modalities used are ultrasound (14.2% of total treatment), massage (13.5%), manual therapy techniques (13.4%), therapeutic exercise (12.4%), cryotherapy (9.3%), transcutaneous electrical nerve stimulation (TENS) (8.5%) and taping (7.9%) [18]. The use of manual therapies documented appears significantly lower than in this RCT. Similar literature from the Pan-American Games has also been performed [19]. The most common modalities used were kinesiotherapy (defined as muscle strengthening and/or flexibility exercises) (24.9% of all total treatments), ultrasound (19.4%), cryotherapy (17.2%), superficial heat (12.8%), interferential current (11.1%), TENS (7.3%), with osteopathy rarely used (0.6%) [19].
The findings of this study and the available literature suggest that the sports chiropractic intervention provided, in particular the amount, technique type and reason for HVLA manipulation is different to that of the clinical practice of physiotherapy. Although the management provided appeared to be reflective of published sports chiropractic and modern multimodal (MMM) chiropractic scope of practice [12-14], prospective clinical practice surveys of sports chiropractors do not exist. Such studies are encouraged which would allow assessment of the consistency between this research protocol and clinical practice and comparison with both physiotherapy and chiropractic clinical practice. As discussed by Hurley et al. [20], the results of this study should allow manual therapists to determine how closely the trial design, practitioner and interventions mimic their practice setting. Clinicians can then interpret and perhaps implement the evidence in a more meaningful way and the uptake of a similar treatment approach may have potential for injury prevention benefit as demonstrated in the RCT on a wider scale.
HVLA manipulative techniques are believed to return physiologic and accessory motion to hypomobile structures, correcting deficits in range of motion. Additionally short term strength changes in lower-limb musculature following spinal [37], and lower-limb [38] joint manipulative techniques have been observed. This may have contributed to improved hamstring and lower-limb muscle function and injury prevention in this study. It supports the hypothesis that hamstring and lower limb muscle strain involves a local and distant model [1,11,21]. Further indirect evidence for non-local factors having a role in injury causation exists in that a hamstring flexibility intervention in a military population has been shown to be capable of lowering the number of lower extremity overuse injuries [39], meaning improvements in knee injury in this RCT may have been a direct effect of treatment or through indirect improvements in hamstring function [21].
A large proportion of treatment was directed to the low back and pelvis. This is not surprising considering the incidence of low back injury and pain in Australian footballers [40]. The link between the low back and pelvis and hamstring and lower limb injuries has been discussed for some time [6,41]. Substantial treatment was also directed at the hip and knee. A large amount of treatment directed here was for the aim of asymptomatic or functional improvement. There has only been recent discussion on a possible link between these joints and hamstring and lower limb injury from the perspective of the kinematic chain [1,5,11]. The intricate anatomical attachments of the hamstring muscle to the knee [42], and fascial connections to the peroneus longus at the fibula [43], provide indirect evidence for knee and proximal tibial-fibular joint function to be of importance for hamstring function. This provides indirect support for the contention that non-local factors may play a role in hamstring and lower-limb injury causation [21].
Limitations exist in this study. Firstly, there are limitations in generalizing the results, as all treatment was provided by a single practitioner, who may not be representative of the chiropractic profession or the sports chiropractic subgroup of the profession. As the sports chiropractor in the study was working with the current best practice medical, paramedical and sports science team, it is highly likely that players would have consulted club medical or paramedical staff for treatment of symptomatic tissues, resulting in an under-reporting of symptomatic treatment, which may have occurred in clinical practice. When analysing the results of treatment rendered it should also be noted that some players consulted the sports chiropractor for management of injuries as they may have preferred the sports chiropractic approach for some conditions and because there is also a cultural phenomenon in many of the body contact sports that players do not want to be seen to be receiving treatment for injuries for fear of jeopardising team selection. Because the players were enrolled in an injury prevention study the stigma associated with treatment may have been lifted. Therefore it is not possible to say that all treatment may contribute to an injury prevention benefit, nor may results be entirely reflective of clinical sports chiropractic practice. As a multimodal treatment approach was applied in conjunction with a range of other therapies, it is also not possible to determine what resulted in the injury prevention. Further limitations of this study are that the results are likely biased towards asymptomatic treatment due to the injury prevention focus and again this may not accurately reflect clinical sports chiropractic practice.
The authors recommend this RCT be repeated in other sports with a high prevalence of hamstring and lower-limb injury. Future research would benefit from recording the nature of the control interventions in order to clarify the differences between interventions and to specifically address the role of HVLA based manipulative techniques. Additionally, the actual scope of practice of sports chiropractors needs to be documented and compared to the amount of treatment, the treatment techniques rendered, the location and frequency of treatment in this study to assess whether it is representative. This would allow for a multi-practitioner study to be conducted and allow more meaningful comparisons with both the physiotherapy and chiropractic professions.
Conclusions
An individualized, ongoing multi-region and multimodal sports chiropractic intervention was applied in this cohort of semi-elite athletes. The sports chiropractic intervention aimed to reduce local and non-local risk factors to hamstring and lower limb muscle injury, although it can not be determined whether this occurred. A significant proportion of treatment was delivered to asymptomatic areas, particularly joint based therapies, which consisted largely of HVLA manipulation techniques, often in combination with mobilizations. However, the treatment was not limited to a manipulation only approach. Manual HVLA techniques were most commonly used although mechanically assisted techniques featured prominently. Publication of these findings allows manual therapists to determine how closely the trial design, practitioner and interventions mimic their practice setting. Clinicians not utilizing HVLA manipulation could consider higher utilization rates in a multimodal protocol over other more established interventions that have little to no evidence to support their use in the prevention of hamstring and other lower-limb injuries. However, it is important to note the preliminary nature of the evidence presented and the requisite for future studies to further explore this.
Competing interests
The authors have no conflict of interest that is directly relevant to the content of this manuscript. No source of funding was used in the preparation of this manuscript.
Authors' contributions
WH conceived the idea of the study with study design modified by HP. All treatment provided and recorded was by WH. WH and HP contributed to writing the multiple drafts and the final document. All authors read and approved the final manuscript.
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Indian J NephrolIJNIndian Journal of Nephrology0971-40651998-3662Medknow Publications India IJN-20-6810.4103/0971-4065.65297Original ArticleClinical and laboratory findings and therapeutic responses in children with nephrotic syndrome Safaei A. A. S. L. Maleknejad S. Department of Pediatrics, Guilan University of Medical Science, Rasht, IR IranAddress for correspondence: Dr. Afshin A.S.L. Safaei, Iran-Rasht-17 Shahrivar Hospital Rasht Iran. E-mail: [email protected] 2010 20 2 68 71 © Indian Journal of Nephrology2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Nephrotic syndrome (NS) is a clinical entity characterized by massive loss of urinary protein leading to hypoproteinemia and edema. This prospective cross sectional study was performed on 44 children with idiopathic nephrotic syndrome (INS). The objectives were to study the clinical and biochemical parameters at the time of diagnosis of nephrotic syndrome and to study the histopathological distribution of different subtypes of INS and drug response pattern. There were 29 (66%) males and 15 females (34%). The mean age of NS was 4.87±3.24 years. Facial edema was found in 42 (95%), microscopic hematuria in 10 (23%), gross hematuria in 2 (4.5%), and hypertension in 5 (11.2%) of patients. In 17 children who underwent biopsy, focal segmental glomerulosclerosis was the most common pathologic finding (41%). Other subtypes included minimal change in three (18%), membranoproliferative glomerulonephritis in 1(5.8%), diffuse proliferative glomerulonephritis in 2 (11.6%), membranous glomerulonephritis in 1 (5.8%), and diffuse mesangial proliferation in 3 (17.5%) of cases. At the time of hospital admission, peritonitis were present in five (11.4%), pneumonia and upper respiratory infection (sinusitis) in eight (18%), cellulitis in two (4.5%). Among 44 children with NS, 29 (66%) were steroid sensitive cases, nine (20.5%) were steroid resistant and six (13.5%) were steroid dependent. Among patients with steroid sensitive NS, 37% were without relapsers, 38.8% frequent relapsers and 26.4% were infrequent relapsers. These results suggest that there are differences between season of incidence, response to treatment with corticosteroid and pathologic findings in our study and other studies in Iran and other countries.
Childrencomplicationcorticosteroidnephrotic syndromepathology
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Introduction
Nephrotic syndrome (NS) is characterized by massive loss of urinary protein (primarily albuminuria) leading to hypoproteinemia (hypoalbuminemia) and its result, edema. Hyperlipidemia, hypercholesterolemia, and increased lipiduria are usually associated. Although not commonly thought of as part of the syndrome, hypertension, hematuria, and azotemia may occur. NS is categorized into primary and secondary forms. The primary NS (PNS) occurs without any previous disease and in some circles, the older designation of idiopathic NS (INS), but both terms denote a similar vagueness as to cause. Included are a variety of clinical as well as pathologic states. The term secondary NS relates to some clinical disease such systemic lupus erythematosus, diabetes mellitus, sickle cell disease or syphilis. Secondary NS is rare in children. The overall prevalence of NS in childhood is approximately 2-5 cases per 100,000 children. The cumulative prevalence rate is approximately 15.5 cases per 100,000[12] Minimal change NS is the most common form in children, and its prevalence is inversely proportional to age (i.e., the younger the child, the more likely the histology will show minimal abnormalities on light microscopic evaluation of glomerular histology). Data on differences in racial predilection to NS in children are lacking[13–5] in children younger than eight years at onset, the ratio of males to females varies from 2:1 to 3:2.[125] In older children, adolescents and adults, the male to female prevalence is approximately equal.[4] Histologic variations exist within this category in which some patients demonstrate only fusion and smudging of the epithelial cell podocytes while others may demonstrate mild changes within the glomerular mesangium consisting of either proliferation or sclerosis. Since patients with minimal change NS have the highest rate of responsiveness to standard treatment and best long-term prognosis, the separation of minimal change NS from others is important.[6–8]
Materials and Methods
This prospective cross sectional study was performed on 44 children with idiopathic NS, in 17 Shahrivar hospital (with age at onset up to 14 years) Most patients were referred to our unit for further management. All patients fulfilled the International Study of Kidney Disease in Children (ISKDC) criteria, for the diagnosis of nephrotic syndrome: urinary spot protein/creatinine >2.0, serum albumin <2.5 g/dl, serum cholesterol >200 mg/dl, and edema.[1] The study parameters included age, sex, nationality, presenting symptoms and blood pressure of the patients, complete blood picture, urine analysis and microscopy, 24-hour urinary protein excretion, creatinine clearance, serum electrolytes, serum urea and creatinine levels, serology and immunological studies, serological markers for hepatitis B and C, antibody against the human immunodeficiency virus (HIV), ultrasound, treatment and outcome.
After informed consent, kidney biopsy was performed in the following situations:
age at onset less than one year and more than than 10 years,
no response to eight weeks of prednisolone therapy,
frequent relapser (FR), steroid-dependent (SD), and steroid non-responder (SNR) categories and before cyclosporine therapy, and
unusual clinical features (hypertension and micrscopic hematuria) and/or laboratory abnormalities (abnormal renal function and low c3 and c4).[124]
The biopsy specimens were evaluated histopathologically by light and immunofluorescence microscopy. An adequate biopsy was defined as the presence of at least 10 glomeruli in the specimen on light microscopy.
The response to treatment was classified according to the definitions from ISKDC: (a) steroid sensitive–complete resolution of proteinuria within eight weeks of prednisone therapy; (b) steroid resistance–failure to respond to eight consecutive weeks of treatment with prednisone at 2 mg/kg/day; (c) steroid dependence–recurrence of nephrosis when the dose of corticosteroids is reduced or within two months after the discontinuation of therapy; (d) frequent relapsers–two or more episodes of nephrosis within six months of the initial response or four or more within any 12-month period (not related to changes in prednisone dose)[12] steroid dependent patients, frequent relapsers, steroid resistant patients candidates for alternative agents, particularly, levamisol, cyclophosphamide, cyclosporine, mycofenolate mofetil and tacrolimus.[1910] We obtained data regarding age, sex, and presenting features, laboratory finding, response to treatment and biopsy results using a standardized data–sheath.
Results
There were 29 males (66%) and 15 females (34%); male to female ratio was 1.9/1. The mean of idiopathic NS was 4.87±3.24 years (range of neonate to 14 years). One of these patients had congenital NS. Fifteen patients were admitted to the hospital in spring (34.1%) and other patients were admitted in winter (18.2%), summer (25%) and fall (22.7%). The mean level of serum albumin was 1.75±0.45 g/l, The mean level of 24-hour urinary protein excretion 3344.84±2344.38 mg/dl, mean level serum cholesterol was 473±160 mg/dl and mean serum of triglyceride was 335.4±113.8 mg/dl. The most common presenting signs and symptoms were facial edema in 42 (95%), limb edema 36 (82.2%), scrotal edema 24 (54.5%) anasarca in 18 patient (41%) abdominal pain in eight (18.2%), diarrhea in four (9%) ascitis in 28 (64%) pleurisy in four (9%), anorexia in 24 (54.5%), hypertension in 5 (11.2%) microscopic hematuria in 10 (23%) and gross hematuria in two patients (4.6%). All patients of the studied group were seronegative for HBsAg and HIV. Evaluation for complication of disease was done in all patients. Acute renal failure due to low serum albumin and overenthusiastic diuretic consumption in other centers were seen in eight patients (18%). Acute renal failure was seen in seven patients and renal biopsy was done in one patient because of persistent acute renal failure, and renal pathology was compatible with focal segmental glomerolusclerosis. Regarding hospital admission, peritonitis were present in five (11.4%), pneumonia and upper respiratory infection in eight (18%) and cellulitis in two (4.5%) patients. In follow-up, primary disease progressed toward end stage renal disease (ESRD) in four (9%) patients. Ultimately, renal transplantation was performed in two cases with FSGS. Detail of age and sex distribution of patients are shown in [Table 1].
Table 1 Age and sex distribution of 44 patients with NS
Age (Years)/Sex Male Female Percent Total
<1 1 - 2.2 1
1-5 19 12 70 31
6-10 6 12 18.8 8
>10 3 1 9 4
Kidney biopsy was performed in 17 (38.4%) of 44 patients. FSGS was the most common histopathological subtype in seven of 17 children (41%), Other subtypes included minimal change disease in three (18%), membranoproliferative glomerulonephritis (MPGN) in one (5.8%), diffuse proliferative glomerulonephritis in two (11.6%), membranous glomerulonephritis in one (5.8%), and diffuse mesangial proliferation in three (17.5%). Among 44 children with NS, 29 cases (66%) were steroid sensitive, nine (20.5%) were steroid resistant and six (13.5%) steroid dependent. Of patients with steroid sensitive nephrotic syndrome, 37% were non-relapsers, 38.8% frequent relapsers and 26.4% infrequent relapsers. Among those with steroid resistant NS, seven had focal segmental glomerulosclerosis and 2 had DMP. In this study only one patient with FSGS died because of end stage renal disease.
Discussion
In this study, we analyzed all children with INS who referred to 17 Sharivar Hospital. In our study the male to female ratio was 1.9/1. According to two studies in Turkey, male to female ratio was 1.6/1 and 1.7/1 (6, 7). According to a study in India carried out by Kumar et al. male to female ratio was 2.76/1.[11]
A study by Madani et al. on 502 patients in Center Medical Pediatric showed that 63.7% were male and 36.3% female with a male to female ratio 1.75/1.[8] In the report which was presented by Sorkhi in Babol hospital, male to female ratio was 1.6/1.[12] Considering these results, it can be said that differences in sex predilection to NS and maybe even a more pronounced difference in the types of NS acquired within a geographic area exist although in all of studies male involvement was more. In our report, 31 cases of patients were in the age range 1-5 years (71%). Only one patient had congenital NS, admitted at age 45 days. In this study the mean age of patients at onset of INS was 4.87±3.24 years (range neonate to 14 years). In a study by Kumar et al. in India, the mean age of patients at the onset of NS was 7.9±5.1 years[11]; other studies in New Zealand and and Saudi Arabia showed that the mean age was 5.4±3.9 and 4.3±3.1.[1314] According to a report from Center Medical Pediatric by Madani et al.[8] 337 cases of 502 patients were in the range of 1-5 years (67%). Another study by Sorkhi et al., performed on 75 children, showed that 62.5% patients were in the range of 2-8 years.[12] These results suggest there are difference in age distribution of patients but our findings are relatively similar to results obtained in the two centers in Iran.
In this study, 15 patients were admitted to the hospital in spring season (34.1%), other patients were admitted in winter (18.2%), summer (25%) and (22.7%) in fall. However, according to a report by Sorkhi et al. 38% of cases admitted in winter differed from our results;[12] the most common presenting signs and symptoms were facial edema in 42 (95%), limb edema 36 (82.2%), scrotal edema 24 (54.5%) anasarca in 18 patient (41%) abdominal pain in eight (18.2%), diarrhea in four (9%) ascitis in 28(64%) pleurisy in four(9%) anorexia in 24(54.5%) hypertension in 5(11.2%) microscopic hematuria in one (23%) and gross hematuria in two (4.6%) patients.
The presenting signs and symptoms in our study, differed from others; for example, frequency of microscopic hematuria and hypertension in Indian children was 41 and 26.8% respectively.[914] FSGS was the most common histopathological subtype in seven of 17 children (41%). Other subtypes included minimal change disease in three (18%), membranoproliferative glomerulonephritis (MPGN) in one (5.8%), diffuse proliferative glomerulonephritis in two (11.8%), membranous glomerulonephritis in one (5.8%), and diffuse mesangial proliferation in two (11.8%) and focal and segmental endocapilary proliferation in one patient (5.8%).
Results of renal biopsy in 138 Turkish children showed that in 49% of cases pathologic findings were compatible with mesangial proliferate glomerulonephritis.[67] Another study carried out Kumar et al. in India showed that FSGS was the most common histopathological subtype in 110 of 387 children with NS (38%). According to the Madani et al. study, minimal change disease was the most common histopathological subtype in 67(34/4%) children.[8] This study, in comparison with other studies in other countries and centers, showed variable histology pattern, although at this time it is seems that minimal change disease is the most common variation of nephrotic syndrome in children. These differences may be related to racial, genetic and environmental factors.[36111315–16] Among 44 children with NS, 29 cases (66%) were steroid sensitive and 9(20.5%) steroid resistant and six (13.5%) were steroid dependent. Among patients with steroid sensitive NS, 37% were non-relapsers, 38.8% frequent relapsers and 26.4% infrequent relapsers. Among those with steroid resistant NS seven cases had focal segmental glomerulosclerosis and 2 had DMP.
Other studies have shown that corticosteroid sensitivity in patients is variable:[1718] in Ozkaya report 76%,[6] Madani 79.2%,[8] Ahmadzadeh 87%,[19] Srivastava 77%,[20] Asinobi 78%.[10]
In our study, two patients died - one of them due to congenital NS and the other because of complicatons due to end stage renal disease.
Conclusion
This study suggests there are differences in incidence, responses to treatment with corticosteroid and pathology findings of biopsy among studies conducted in Iran and other countries.
Source of Support: Nil
Conflict of Interest: None declared.
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References
1 Niaudet P Avner ED Harmon WE Niaudet P Steroid-sensitive idiopathic nephrotic syndrome Pediatric Nephrology 2004 5th ed Philadelphia Lippincott Williams and Wilkins 545 73
2 Burgstein JM Behrman RE Kliegman RM Jenson HB Nephrotic syndrome Nelson Textbook of Pediatrics 2008 18th ed Philadelphia Saunders WB 2430 42
3 Churg J Habib R White R Pathology of the nephrotic symdrome in children.A report from the International Study of Kidney Disease in children Lancet 1970 5 1799 802
4 Falk JR Jennette JC Nachman PH Brenner BM Levine SA Primary glomerular disease The Kidney 2004 7th ed Philadelphia Saunders WB 1295 307
5 Hawkins P Millan JA Angelis CD Feigin RD Nephrotic Syndrome Oski pediatrics principles and practice 2000 3rd ed Philadelphia Lippincott Williams and Wilkings 1590 9
6 Ozkaya N Cakar N Ekim M Kara N Akkok N Yalcinkaya F Primary nephrotic syndrome during childhood in Turkey Pediatr Int 2004 46 436 8 15310309
7 Bircan Z Yilmaz AY Katar S Yildrim M Childhood idiopathic nephotic syndrome in turkey Pediatric Int 2002 44 608 13
8 Madani AB Clinicopathologic and drug response in children with idiopathic nephrotic syndrome in pediatric medical center J Tehran University Med Sci 2003 1 71 9
9 Bayazit AK Noyan A Cengiz N Anarat A Mycophenolate Mofetil in the children with Multidrug-resistant nephrotic syndrome Clin Nephrol 2004 61 25 9 14964454
10 Asinobi AO Gbadegesin RA Ogunkunle OO Increased steroid responsiveness of young children with nephrotic syndrome in Nigeria Ann Trop Pediatr 2005 25 199 203
11 Kumar J Gulati S Sharma AP Sharma RK Gupta RK Histopathologial spectrum of childhood nephrotic syndrome in Indian children Pedatr Nephrol 2003 18 660 75
12 Sorkhi HA Steroid response in children with nephrotic syndrome in amirkola hospital J Babol University Med Sci 2002 3 39 42
13 Kari JA Changing trends of histiopathology in childhood nephrotic syndrome in western Saudi Arabia Saudi Med J 2002 23 317 21 11938425
14 Simpson AK Wong W Morris MC Pediatric nephrotic syndrome in Auckland, New Zealand J Ped 1998 16 360 2
15 Craing GC Willis NC Hodson EM Incidence of nephrotic syndrome in children in Australia Ped Nephrol 2000 11 111 6
16 Filler G Treatment of nephrotic syndrome in children and controlled trials Nephrol Dial Transplant 2003 6 75 8
17 International Study of Kidney Disease in Children: Primary nephrotic syndrome in children: clinical significance of histopathologic variants of minimal change and of diffuses mesangial hypercellularity. A Report of the International Study of Kidney Disease in Children Kidney Int 1981 20 765 7 7334749
18 Vehaskari VM High incidence of initial and late steroid resistance in childhood nephrotic syndrome Kidney Int 2005 68 1275 81 16105061
19 Ahmadzzadeh A Derakhshan A Idiopathic nephrotic Syndrome in Iran Indian Ped 2007 45 52 3
20 Srivastava RN Mayekar G Anand R Nephrotic syndrome in children Arch Dis Child 1975 50 62 6 | 20835318 | PMC2931135 | CC BY | 2021-01-04 18:48:43 | yes | Indian J Nephrol. 2010 Apr; 20(2):68-71 |
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Parasit VectorsParasites & Vectors1756-3305BioMed Central 1756-3305-3-552057327810.1186/1756-3305-3-55ResearchTrapping mosquitoes using milk products as odour baits in western Kenya Owino Eunice A [email protected] School of Biological Sciences, University of Nairobi, P.O. Box 30197 - 00100, Nairobi, Kenya2 International Center for Insect Physiology and Ecology, P.O. Box 30772 - 00100, Nairobi, Kenya2010 24 6 2010 3 55 55 23 4 2010 24 6 2010 Copyright ©2010 Owino; licensee BioMed Central Ltd.2010Owino; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Ample evidence has shown that blood seeking mosquitoes locate their hosts by following odours produced by the hosts. Odour baited traps would therefore, provide a solution in controlling diseases spread by mosquitoes. Comparative studies were undertaken to determine the relative efficacies of two odour baits i.e. Limburger cheese and African traditional milk cream in trapping mosquitoes in the field in western Kenya.
Method
Comparative efficacy studies were carried out in the field using Latin square experimental designs. In the first study, a counterflow geometry (CFG) trap (MM-x model; American Biophysics Corp., USA.) baited with Limburger cheese, man landing catches (MLC), Centres for Disease Control (CDC) light trap and an entry trap were compared. In the second study, three CFG traps baited with either Limburger cheese, African traditional milk cream or with no bait were compared and in the third study four CDC traps baited with either Limburger cheese, African traditional milk cream, light or with no bait were compared. Parameters like species, catch size, abdominal status, parity status and size of the collected mosquitoes were compared.
Results
A total of 1,806 mosquitoes were collected (60% An. gambiae s.l and 25% An.funestus, culicines 15%). There was no significant difference in the number of An. funestus trapped by the CFG trap baited with Limburger cheese from those trapped by the MLC (P = 0.351). The Limburger cheese baited CFG trap collected significantly more gravid An. funestus than the MLC (P = 0.022). Furthermore, when the CFG trap baited with Limburger cheese and the CFG trap baited with milk cream were compared, there was no significant difference in the number of An. funestus collected (P = 0.573). The same trend was observed in the comparison of Limburger cheese baited CDC trap and milk cream baited CDC trap.
Conclusions
Limburger cheese and African traditional milk cream have a potential as effective odour baits for sampling/surveillance and as oviposition attractants for the malaria vector, Anopheles funestus.
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Background
Ample evidence has shown that host-seeking female mosquitoes mainly locate their hosts by odours produced by the hosts [1-5]. It could, therefore, be useful if traps baited with host odours would lure mosquitoes as strongly as normal healthy humans [5] or even lure more mosquitoes than healthy human beings [6]. Such traps could then be used in the trapping of mosquitoes in large numbers [7]. This would lead to the control of infection and transmission rates of diseases spread by mosquitoes like malaria, rift valley fever and bancroftian filariasis.
Current efforts centre on searching for new attractants and attractant formulations [6-8] improving on existing ones [9-11], and developing trapping devices [7,12,13]. Efficacy trials under field [14] and semi-field conditions are also on-going [15].
The demonstration that Limburger cheese, which to the human nose has a smell reminiscent of foot odour, was a strong attractant to Anopheles gambiae Giles sensu stricto [16] and to Aedes aegypti [17] in wind tunnel bioassays, together with the successful development and use of synthetic host odour blends in semi-field [8,14] and field conditions [6,15,17] have shown that traps based on synthetic[6-8] host attractants are likely to provide an objective monitoring tool for the host-seeking fraction of mosquito vectors of diseases like malaria and bancroftian filariasis. Such traps can be used to study the vector biology and epidemiology of mosquito-borne diseases, knowledge of which is vital for planning and assessing outcome of intervention strategies. One might even foresee the development of synthetic odour baited mosquito traps [6,7] that might be used to reduce the vector population in a village or in an individual's bedroom to divert mosquitoes away from occupants. Such traps could eventually become part of primary healthcare systems. Therefore, in an effort to look for an effective synthetic odour bait that can be used in sampling and control of disease spreading mosquitoes, comparative efficacy studies using CFG traps and CDC traps baited with either Limburger cheese or African traditional milk cream were conducted in western Kenya.
Methods
Ethics statement
A written institutional ethical clearance to do this study was obtained from the joint University of Nairobi/Kenyatta National Hospital Ethics and Research Committee. Verbal consents from household heads of the houses used in this study were received only after informing them of the rationale and methodology of the research work. Moreover, thick and thin blood smears were taken from the person performing the man-landing catches whenever he complained of fever to examine the presence of malaria parasites. When found positive, he was treated with Coartem® (artemether-lumefantrine).
Study Area
Study sites
The current study was carried out at Lwanda Nyamasari village, located along the southern shores of Lake Victoria in Suba District, western Kenya. Suba district lies at an altitude of 1100 - 1300 metres above sea level and experiences high temperatures (17 - 34°C) throughout the year. The district also experiences two rainy seasons: the long rains occur from March to June, and the short rains from September to November. The annual rainfall ranges between 700 - 1200 mm [18]. Malaria is holoendemic in Suba district and is the leading cause of morbidity, childhood mortality and hospital admissions. Transmission of the disease is maintained by three main vectors: An. gambiae, An. arabiensis and An. funestus which breed in natural and artificial larval habitats [18,19]. Malaria transmission fluctuates throughout the year and reaches its climax in the rainy seasons.
Preliminary investigations
The efficacy of a counter flow geometry (CFG) trap baited with Limburger cheese, an entry trap, a Centers for Disease Control (CDC) light trap and man-landing catches (MLC) performed by a twenty four year-old male in sampling African malaria vectors was evaluated by comparing the species diversity, the number of mosquitoes collected, the abdominal status, the parity status and the sizes of mosquitoes collected by each method. These investigations were carried out during the dry month of January up to mid February because it was important for the CFG traps baited with Limburger cheese to be able to sample mosquitoes in low densities.
A 4 × 4 Latin square experimental design was formulated, whereby four houses were selected after obtaining a verbal consent from the heads of households. All occupants of the experimental houses were provided with untreated mosquito bed nets and asked to sleep under them. All experiments began at 22.00 and concluded at 06.00 hours on each experimental night. The CDC and the CFG traps were suspended 20 cm above the ground. In the case of the Limburger cheese baited CFG trap, 0.6 g of Limburger cheese was wrapped in a small piece of mosquito netting material and suspended inside the thin inner PVC pipe of the CFG trap so that the cheese odour could be pumped to the exterior when the fan in the trap was operated. An entry trap, measuring 0.3 × 0.3 × 0.3 m with a circular aperture of 5 cm in diameter at its center, was fixed on an open window in the sitting room of the experimental house. In the case of the man- landing technique, a 24 year-old man, who acted as both bait and collector, sat upright in the sitting room and exposed his legs. He collected every mosquito that landed on him, albeit before they could bite him.
Trapped mosquitoes were transported to the ICIPE-Mbita laboratories for processing where mosquitoes were killed using chloroform and all non-target insects discarded.
Mosquitoes were identified morphologically into anophelines according to the protocol of Gillies and De Meillon [20] and Gillies and Coetzee [21] and into culicines according to the protocol of Service [22]. The abdominal status of each individual trapped mosquito was assessed into three classes "fed", "unfed" and "gravid (heavy with eggs)" according to the World Health Organization manual [23]. In preliminary studies, Anopheles gambiae complex mosquitoes were identified into species by the Polymerase Chain Reaction (PCR) method according to the protocol of Scott et al. [24]. The mosquito size was determined by pulling off the left wing of each mosquito, mounting it on a glass slide and measuring the wing length from the distal end of the alula to the tip excluding the fringe scales using an ocular micrometer that had been mounted on the eye piece of a light microscope at the magnification of × 40. The reading, which was in micrometers (μm) was then multiplied by 0.025 which was the conversion factor for the micrometer. The product was then taken as the size of the mosquito. Determination of mosquito parity status into parous (mosquitoes that have ever laid eggs) and nulliparous (mosquitoes that have never laid eggs) was performed according to the method of Detinova [25] whereby a small cut was made on the 6th and the 7th segment of the mosquitoes' abdomen to remove the ovaries. The tracheoles of the ovaries were examined under the low power x40 of a compound microscope. When the tracheoles were dilated at the distal end, the mosquito was termed as parous, while when the tracheoles were coiled and not dilated at the distal end; the mosquito was termed as nulliparous.
Comparative efficacy of Limburger cheese and Milk cream as odour baits for sampling mosquitoes
Making Milk cream
Milk cream was made by adding 20 ml of fermented cow urine into a gourd that had been cleaned using water and soap and left in the sun until dry. Milk was then poured into the gourd and left therein for twelve hours. The gourd with the milk was churned until some cream formed on the surface of the milk. This cream is what is referred to as milk cream in this paper. The milk cream was separated from the milk and stored at 24°C in a plastic container filled with water.
Comparative efficacy using CFG traps
The efficacy of a milk cream baited CFG trap, a Limburger cheese baited CFG trap and an unbaited CFG trap was compared by studying the species diversity, the catch size and the abdominal status of trapped mosquitoes. A 3 × 3 Latin square design was formulated whereby three of the houses used in the preliminary investigations were used such that each method of trapping was tested separately in each one of the three houses per night until each method had been applied three times per house. The CFG traps were either baited with milk cream, Limburger cheese or unbaited. When baited, 0.6 g of Limburger cheese or milk cream was wrapped in a small piece of mosquito netting material and suspended inside the thin inner PVC pipe of the Counterflow Geometry (CFG) trap so that odours of the cheese or milk cream could be pumped to the exterior when the fan in each CFG apparatus was operated. All the CFG traps were suspended 20 cm above ground level.
Comparative efficacy using CDC light traps
In the third series of comparative studies, the relative efficacies of a CDC light trap with light on, a CDC light trap with no light, a CDC light trap with no light but baited with Limburger cheese and a CDC light trap with no light but baited with milk cream were compared using a 4 × 4 Latin square experimental design. The four houses used in preliminary investigations were used and each sampling method was rotated four times per house.
In the cases of the CDC light trap baited with Limburger and the CDC light trap baited with milk cream, the light bulbs in each trap were removed and replaced with 0.6 g of Limburger cheese or milk cream. The CDC light traps were then suspended 20 cm above the ground.
All experiments began at 22.00 and concluded at 06.00 hours on each experimental night. All occupants of the experimental houses were provided with untreated mosquito bed nets and asked to sleep under them. Trapped mosquitoes were transported to the ICIPE-Mbita laboratories for processing as explained under the preliminary investigations. However, PCR on anopheline mosquitoes, parity status and mosquito size were not determined for these mosquitoes.
Statistical analysis
Data were analyzed using PASW Statistic 17 (SPSS version 17). The mean mosquito catch per trap per night were first calculated and compared using either bar graphs showing 95% confidence intervals or tables. Further analysis was conducted using General Linear Model (GLM), univariate analysis of variance procedure as follows: mosquito catches were modeled as a function of trapping method as the fixed factor in both the case where the distribution of each mosquito species per trap was analyzed and also in the case where the distribution per trap of each of the three abdominal status (fed, gravid, unfed) for each mosquito species was compared. Each time 'day' was treated as a random variable to reflect daily fluctuations in mosquito numbers. To maintain validity of assumptions for appropriate data analysis, the mosquito catch were log10 (x +1) transformed to normalize prior to statistical analysis. For mosquito wing length comparisons, the wing length was modeled as a function of trap as the fixed factor.
Results
Preliminary investigations
Analysis by mosquito species and sample size
The mean catches for each mosquito species trapped by each method per night are shown in Figure 1. It was observed that the trapping method had significance in the trapping efficacy for the mosquito species An. arabiensis (F = 16.713, df 3, P < 0.001), An gambaie (F = 13.224, df 3, P < 0.001) and An. funestus, (F = 11.522, df 3, P < 0.001). There was no significant difference between the An. arabiensis collected by the CDC light trap and the MLC, (P = 0.113). This same trend was seen for the An. gambiae and An. funestus mosquitoes. However, the CDC light trap and the MLC collected significantly more An. arabiensis than the CFG trap baited with Limburger cheese, (P < 0.001, 0.002) respectively and the Entry trap, (P < 0.001, 0.001) respectively. This same trend was seen for An. gambiae mosquitoes only that the CFG trap baited with Limburger cheese also collected significantly more An. gambiae than the entry trap (P = 0.042). There was no significant difference in the number of An. funestus collected by the CFG trap baited with Limburger cheese from those collected by the MLC (P = 0.351) while the CDC light trap collected significantly more An. funestus than the CFG trap baited with Limburger cheese (P = 0.043). The entry trap did not catch any An. funestus mosquitoes.
Figure 1 Comparison of different trapping methods used in the preliminary investigations. Average number of female anophelines caught per trap per night. The alphabetical symbols, a, b and c are used to represent differences as determined by unianova test. Trapping efficiencies for the different trapping methods in collecting a certain species are not significantly different (P = 0.05), if the bars representing a particular species in each trap share any of these alphabets. The Y-error bars represent 95% confidence intervals. CFG, Counterflow geometry trap baited with Limburger cheese; CDC, standard centers for disease control light trap; ET, Entry trap; MLC, man landing catches.
Day had no significance in the trapping efficacy for An. arabiensis (F = 1.769, df 15, P = 0.071), An. gambiae (F = 1.619, df 15, P = -0.107) and An. funestus (F = 1.454, df 15, P = 0.165).
The mean catches per trap per night for every abdominal status for the different mosquito species are shown in Table 1. The trapping method had significance in the trapping efficacy for fed An. arabiensis (F = 3.575, df 3, P = 0.019) and fed An. funestus (F = 3.000, df 3, P = 0.037) but not for fed An. gambiae (F = 2.277, df 3, P = 0.089). The MLC had significantly more fed An. arabiensis than the CDC light trap (P = 0.002), the CFG trap baited with Limburger cheese, (P = 0.002) and the entry trap (P = 0.002). This same trend was followed for An. funestus mosquitoes while the CFG trap baited with Limburger cheese and the entry trap did not collect any fed An.gambiae mosquitoes.
Table 1 Comparison of the mean catch per trap per night for each of the three abdominal status of every mosquito species collected by the four trapping methods used in the preliminary investigations at Lwanda Nyamasari village.
Anopheles arabiensis
Anopheles gambiae
Anopheles funestus
Total No. of mosquitoes
%
Method N Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean sum
%
CFG 16 0.2b 0.4c 0.0a 10
(9) 0.0a 1.0b 0.1a 16
(15) 0.0b 2.3a 0.4b 44
(23) 70
(17)
CDC 16 0.2b 3.4a 0.1a 59 (51) 0.3a 2.1a 0.1a 40
(36) 0.0b 5.3c 0.2ab 89
(47) 188
(45)
ET 16 0.1b 0.1c 0.0a 3
(3) 0.0a 0.1b 0.0a 2
(2) 0.0b 0.0b 0.0a 0
(0.0) 5 (1)
MLC 16 0.7a
2.0b 0.1a 43 (37) 0.4a 2.8a 0.1a 52
(47) 0.3a 3.2ac 0.1a 57
(30) 152
(37)
Sum of each mosquito species in each trap is also shown with the percentage in parenthesis. CFG, counterflow geometry trap baited with Limburger cheese; CDC, standard centers for disease control light trap; ET, entry trap; MLC, man landing catches; N represents the number of days over which sampling was done. The percentages are calculated for each mosquito species. For example the CFG trap collected 9% of all the An. arabiensis collected by the four trapping methods while the CDC light trap collected 51% of the same. Values following each other in the same column with different letter superscripts are significantly different at (P = 0.05).
The trapping method had significance in the trapping efficacy for gravid An. funestus (F = 3.078, df 3, P = 0.034) mosquitoes whereby the CFG trap baited with Limburger cheese collected significantly more gravid An. funestus than the (MLC P = 0.022). However, the trapping method had no significance in the trapping efficacy for gravid, An. arabiensis (F = 1.279, df 3, P = 0.290) and gravid An. gambiae, (F = 0.699, df 3, P = 0.565).
The trapping method had significance in the trapping efficacy for the unfed An. arabiensis (F = 15.614, df 3, P < 0.001), unfed An. funestus (F = 10.365, df 3, P < 0.001) and unfed An. gambiae (F = 10.374, df 3, P < 0.001). The CDC light trap collected significantly more unfed An. arabiensis than the CFG trap baited with Limburger cheese, (P < 0.001), the MLC (P = 0.019) and the entry trap, (P < 0.001). The MLC also collected significantly more unfed An.arabiensis than the CFG trap baited with Limburger cheese and the entry trap.
The CDC, the CFG trap baited with Limburger cheese and the MLC collected significantly higher numbers of unfed An. gambiae than the Entry trap (P < 0.001, P = 0.021 and P < 0.001) respectively. Furthermore, the MLC collected significantly more unfed An. gambiae mosquitoes than the CFG trap baited with Limburger cheese (P = 0.007). However, there was no significant difference in the number of unfed An. gambiae collected by the CDC trap and the CFG trap baited with Limburger cheese (P = 0.541)
The CDC light trap collected higher numbers of unfed An. funestus than the CFG trap baited with Limburger cheese, (P = 0.021) while there was no significant difference between the unfed An. funestus collected by the MLC and the CFG trap baited with Limburger cheese, (P = 0.255).
Analysis by mosquito parity status
A total of 313 female anopheline mosquitoes were dissected in order to determine their parity status. In general, the total proportion of parous anophelines caught by the CDC light trap, CFG trap baited with Limburger cheese and man-landing catches was proportionally higher than the nulliparous anophelines collected (Table 2). The CFG trap collected higher proportions of parous An. arabiensis, An. gambiae and An. funestus than the CDC light trap, the entry trap and the man landing catches.
Table 2 Comparison of parity rates of dissected mosquitoes for the four trapping methods used in the investigations at Lwanda Nyamasari village.
Anopheles arabiensis
Anopheles gambiae
Anopheles funestus
Method Parous
% Nulli
Parous,
% Not dissected, % Sum Parous, % Nulli
Parous,% Not
dissected, % Sum Parous,% Nulli
Parous, % Not dissected % sum
CFG 80% 0% 20% 10 69% 19% 12% 16 66% 30% 4% 44
CDC 66% 15% 19% 59 63% 15% 22% 40 48% 15% 37% 89
ET 0% 67% 33% 3 50% 0% 50% 2 0% 0% 0% 0
MLC 49% 19% 33% 43 60% 17% 23% 52 55% 18% 27% 57
CFG, counterflow geometry trap baited with Limburger cheese; CDC, standard centers for disease control light trap; ET, entry trap; MLC, man landing catches; The percentages are calculated for each mosquitoes species per trap. For example, of all the An. arabiensis mosquitoes collected by the CFG trap, 80% were parous while of all the An. arabiensis mosquitoes collected by the CDC light trap 66% were parous.
Analysis by mosquito size
Size was determined for all the mosquitoes collected. Trap had a significant influence on the wingsize of An. arabiensis (F = 9.328, df 3, P < 0.001) and An. gambiae (F = 5.231, df 3, P = 0.002) but not for An. funestus (F = 0.413, df 2, P = 0.662) collected by the different trapping methods.
The man-landing catches collected bigger An. arabiensis than the CDC light trap (P < 0.001) and the CFG trap baited with Limburger cheese (P = 0.001) while the CDC light trap had smaller An. gambiae than the CFG trap baited with Limburger cheese, (P = 0.001) and the MLC, (P = 0.002) (Table 3).
Table 3 Comparison of mean wingsize (Measurements in micrometers) for each anopheline species collected by the four sampling methods used in the preliminary investigations at Lwanda Nyamasari village.
Anopheles arabiensis
Anopheles funestus
Anopheles gambiae.
Trap Mean Wingsize/trap ± SD Mean Wingsize/trap ± SD Mean Wingsize/trap ± SD
CDC 2.0(59)b 0.34 2.1(89)a 0.28 2.0(40)a 0.26
CFG 2.0(10)b 0.23 2.1(44)a 0.51 2.4(16)b 0.46
ENTRY 2.3(3)ab 0.46 0 0 2.1(2)ab 0.85
MLC 2.4(43)a 0.36 2.0(57)a 0.35 2.3(52)b 0.36
Sum of mosquitoes is in parenthesis.
CDC, standard centers for disease control light trap; CFG, counterflow geometry trap baited with Limburger cheese; ET, entry trap; MLC, man landing catches.
Values following each other in the same column with different letter superscripts are significantly different at (P = 0.05).
Comparative efficacy studies using CFG traps
The mean catches of mosquitoes for each species trapped by each trapping method per night are shown in Figure 2. The trapping method had significance on the trap efficacy in trapping An. funestus (F = 3.022, df 2, P = 0.055) and the culicine mosquitoes (F = 3.473, df 2, P = 0.036) but it had no significance on trap efficacy in trapping An. gambiae, (F = 2.574, df 2, P = 0.083). There was no significant difference in the number of An.funestus collected by the CFG trap baited with Limburger cheese and the CFG trap baited with milk cream (P = 0.573). This trend was followed for the An. gambiae s.l mosquitoes and the culicine mosquitoes. However, the Limburger cheese baited trap collected significantly higher numbers of An. funestus than the trap with no bait, (P = 0.021) while there was no significant difference in the number of An. funestus collected by the milk cream baited trap, (P = 0.078) and the trap with no bait. Also, the milk cream baited trap had significantly more An. gambiae than the trap with no bait (P = 0.031) while there was no difference in the number of An. gambiae collected by the Limburger cheese baited trap and the no bait trap (P = 0.122). The trap with no bait collected significantly lower numbers of culicines than the trap baited with milk cream P = 0.036 and the trap baited with Limburger cheese P = 0.019
Figure 2 Comparison of different trapping methods used in the comparative studies by counterflow geometry traps. Average number of female mosquitoes caught per trap per night. The alphabetical symbols, a, b are used to represent differences as determined by unianova test. Trapping efficiencies for the different trapping methods in collecting a certain species are not significantly different (P = 0.05), if the bars representing a particular species in each trap share any of these alphabets. The Y-error bars represent 95% confidence intervals. CFG LC, counterflow geometry trap baited with Limburger cheese; CFG MC, counterflow geometry trap baited with milk cream; CFG NB counterflow geometry trap NOT baited.
Day had no significance in the trapping efficacy for An. funestus (F = 0.412, df 8, P = 0.910), An. gambiae, (F = 0.608, df 8, P = 0.768) and culicines (F = 0.265, df 8, P = 0.975).
The mean catches per trap per night for every abdominal status for the different mosquito species are shown in Table 4. The trapping method had no significance on the trap efficacy in trapping fed An. funestus (F = 1.091, df 2, P = 0.352) and culicine mosquitoes (F = 0.600, df 2, P = 0.557), while there were no fed An. gambiae collected by the traps.
Table 4 Comparison of the mean catch per trap per night for each of the three abdominal status of each mosquito species collected by the three CFG traps used in comparative studies using counterflow geometry traps in Lwanda Nyamasari village.
Anopheles gambiae
Anopheles funestus
Culicines
Trap N Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean Sum
% Total No. of mosquitoes
%
MC 9 0.0a 1.4ab 0.0a 13(36) 0.2a 3.4b 0a 33(59) 0.3a 0.9b 0.0a 11(52) 57(50)
LC 9 0.0a 2.4b 0.1a 23(64) 0.1a 2.4ab 0.0a 23 (41) 0.1a 1.0b 0.0a 10(48) 56(50)
NB 9 0.0a 0.0a 0.0a 0 (0) 0.0a 0.0a 0.0a 0(0) 0.0a 0.0a 0.0a 0 (0) 0(0)
Sum of each mosquito species in each trap is also shown with the percentage in parenthesis.
MC, counterflow geometry trap baited with milk cream; LC, counterflow geometry trap baited with Limburger cheese; NB, counterflow geometry trap with no bait. N represents the number of days. The percentages are calculated for each mosquito species. For example the counterflow geometry trap baited with milk cream collected 36% of all the An. gambiae s.l, collected by the three traps while, the counterflow geometry trap baited with Limburger cheese collected 64% of all the same.
Values following each other in the same column with different letter superscripts are significantly different at (P = 0.05).
Also, the trapping method had no significance on the trap efficacy in trapping gravid An. gambiae, (F = 1.000, df 2, P = 0.383), An. funestus and culicines as there were no gravid An. funestus and culicine mosquitoes collected.
The trapping method had significance on the trap efficacy in trapping unfed An. funestus (P = F = 3.654, df 2, P = 0.041) and unfed culicine mosquitoes (F = 5.756, df 2, P = 0.009) but not unfed An. gambiae (F = 3.309, df 2, P = 0.054). There was no significant difference in the number of unfed An. funestus collected by the milk cream baited trap and the Limburger cheese baited trap (P = 0.613). This trend was followed for the An. gambiae and culicine mosquitoes. However, the milk cream baited trap collected significantly higher numbers of unfed An. funestus than the trap with no bait (P = 0.017) while there was no significant difference in the unfed An. funestus collected by the Limburger cheese baited trap (P = 0.052) and the no bait trap. The Limburger cheese baited trap collected a significantly higher number of unfed An. gambiae than the trap with no bait (P = 0.022) while there was no significant difference between the unfed An. gambiae collected by the trap baited with milk cream and the trap with no bait (P = 0.069). Both the milk cream baited trap and the Limburger cheese baited trap collected significantly higher numbers of unfed culicine mosquitoes than the trap with no bait (P = 0.022) and (P = 0.003) respectively.
Comparative efficacy studies using CDC light traps
The mean catches of mosquitoes for each species trapped by each trapping method per night are shown in Figure 3. The trapping method had significance on the trap efficacy in trapping An. funestus (F = 5.169, df 3, P < 0.001), An. gambiae (F = 10.702, df 3, P < 0.001,) and culicine mosquitoes (F = 3.321, df 3, P = 0.021) The CDC light trap with light on collected significantly more An. funestus than the milk cream baited trap (P = 0.002) the Limburger cheese baited trap, (P = 0.018) and the not baited trap (P < 0.001) while there was no significant difference between the milk cream baited trap and the Limburger cheese baited trap with the trap with no bait (P = 0.663 and 0.225) respectively. There was also no significant difference in the number of An. funestus collected by the milk cream baited trap and the Limburger cheese baited trap (P = 0.436). This trend was also followed for the An. gambiae and culicine mosquitoes.
Figure 3 Comparison of different trapping methods used in the comparative studies using centers for disease control light traps. Average number of female mosquitoes caught per trap per night. The alphabetical symbols, a and b are used to represent differences as determined by unianova test. Trapping efficiencies for the different trapping methods in collecting a certain species are not significantly different (P = 0.05), if the bars representing a particular species in each trap share any of these alphabets. The Y-error bars represent 95% confidence intervals. LC, CDC light trap baited with Limburger cheese; LT, CDC light trap baited with light on; MC, CDC light trap baited with milk cream; NB, CDC light trap NOT baited.
Day had no significance in the trapping efficacy for An. funestus (F = 0.502, df 15, P = 0.937), An. gambiae (F = 1.040, df 15, P = 0.417) and culicine mosquitoes (F = 0.292, df 15, P = 996).
The mean catches per trap per night for every abdominal status for the different mosquito species are shown in Table 5. The trapping method had no significance on trap efficacy in trapping fed An. funestus (F = 1.051, df 3, P = 0.377) and fed culicine mosquitoes (F = 2.376, df 3, P = 0.079). However, the trapping method had significance on the trap efficacy in trapping fed An. gambiae, (F = 5.559, df 3, P = 0.002). The trap with light on collected significantly more fed An. gambiae than the trap baited with milk cream (P = 0.001) the trap baited with Limburger cheese (P = 0.003) and the trap with no bait (P = 0.002). It also collected significantly more fed culicines than the trap baited with Limburger cheese (P = 0.016) and the trap with no bait (P = 0.041).
Table 5 Comparison of the mean catch per trap per night for each of the three abdominal status of every mosquito species collected by the four CDC light traps used in the comparative studies using centers for disease control light traps at Lwanda Nyamasari village.
Trap N Anopheles gambiae s.l.
Anopheles funestus
Culicines Total no of
Mosquitoes, %
Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean Sum
% Fed
mean Unfed
mean Gravid
mean Sum
%
MC 16 0.2a 5.7a 0.4a 101
(14) 0.2a 3.1a 0a 53
(16) 0.1ab 2.9ab 0.1ab 50
(20) 204
(16)
LC 16 0.3a 5.3a 0.3a 454
(64) 0.1a 4.8a 0.4ab 144
(45) 0.0a 2.4a 0.1ab 138
(55) 736 (57)
NB 16 0.3a 3a 0.4a 94
(14) 0.1a 2.1a 0.1a 86
(27) 0.1a 1.4a 0.0a 40
(16) 220
(18)
LT 16 3.9b 20.5b 3.9b 59
(8) 0.6a 7.4b 1.1b 38
(12) 0.6b 7.4b 0.6b 23
(9) 120
(9)
Sum of each mosquito species in each trap is also shown with the percentage in parenthesis.
MC, CDC light trap with no light baited with milk cream; LC, CDC light trap with no light baited with Limburger cheese; NB, CDC light trap with no light and no bait; LT, CDC light trap with light on. N represents the number of days the sampling method was set. The percentages are calculated for each mosquito species. For example the CDC light trap with no light baited with milk cream collected 14% of all the An. gambiae s.l, collected by all the four light traps while, the CDC light trap with no light baited with Limburger cheese collected 64% of the same. Values following each other in the same column with different letter superscripts are significantly different at (P = 0.05).
The trapping method had significance on the trap efficacy in trapping gravid An. funestus (F = 4.405, df 3, P = 0.007), gravid An. gambiae (F = 5.162, df 3, P = 0.003) but not gravid culicine mosquitoes (F = 2.041, df 3, P = 0.118). The trap with light on attracted more gravid An. funestus than the trap baited with milk cream (P = 0.001) and the trap with no bait (P = 0.007). The trap with light on also attracted significantly more gravid An. gambiae than the trap baited with milk cream, the trap baited with Limburger cheese and the trap with no bait, (P = 0.004, 0.001 and 0.02) respectively.
The trapping method had significance on the trap efficacy in trapping unfed An. funestus, F = 4.083, df 3, P = 0.011) unfed An. gambiae (F = 5.442, df 3, P = 0.002) and unfed culicine mosquitoes (F = 2.728, df 3, P = 0.052). The trap with light on collected significantly more unfed An. funestus than the milk cream baited trap, the Limburger cheese baited trap and the trap with no bait (P = 0.012, 0.047,0.002) respectively. This same trend was followed for An. gambiae mosquitoes. The trap with light on also collected significantly more unfed culicines than the Limburger cheese baited trap (P = 0.049) and the trap with no bait (P = 0.008).
Discussion
To my knowledge this is the first time that traps baited with Limburger cheese which was observed to be attractive to An. gambiae s.s mosquitoes in a wind tunnel bioassay [16] has successfully trapped An. gambiae s.s, An. arabiensis, An. funestus and culicine mosquitoes in the field.
The current observation made on Limburger cheese contrasts with those of Murphy et al. [26] who found that CDC light traps baited with a synthetic mixture of Limburger cheese compounds did not catch any mosquitoes in the field. This difference in observations could be due to the fact that in their study, Murphy et al. [26] combined the various chemical components of Limburger cheese to bait light traps while in our work we used whole natural Limburger cheese to bait CFG traps.
This is also the first time to my knowledge that traps baited with African traditional milk cream which is also a milk product has trapped An. gambiae s.l, An. funestus and culicine mosquitoes. Considering that Knols et al. [27] suggested that bacteria involved in the ripening of Limburger cheese may have originated from human skin and hence that these bacteria are responsible for the production of 'human-specific' volatile organic compounds (VOCs) that mediate the host-seeking process of malaria mosquitoes as washing the feet with a bactericidal soap significantly altered the selection of biting sites of An. gambiae on a motionless naked volunteer [3]. It would only be sensible to suggest that one of the reasons why the milk cream was able to attract the mosquitoes was probably due to its handling by humans as skin microbes [28] which have been observed to be responsible for the odours produced by the human hosts, could have been transferred into the milk which had been used to make the milk cream.
It was also observed that CFG and CDC light traps baited with milk cream worked just as well as the same traps baited with Limburger cheese in trapping mosquitoes. This observation suggests that milk cream could be used in place of Limburger cheese and vice versa though more studies should be done for reproducible results.
There was no significant difference in the numbers of An. funestus caught by the CFG trap baited with Limburger cheese from the numbers caught by man-landing catches (MLC). This observation suggests that Limburger cheese baited traps would provide an efficient alternative to MLC during surveillance and monitoring of malaria vectors during epidemiological studies. It was also observed that the CFG trap baited with Limburger cheese collected higher proportions of parous An. arabiensis, An. gambiae and An. funestus than the CDC light trap, the entry trap and the MLC. This observation suggests that the CFG trap collects higher proportions of parasite infected mosquitoes as earlier studies have shown that parous mosquitoes have higher sporozoite infections than the nulliparous ones [29].
Furthermore, the Limburger cheese baited CFG trap collected larger An. gambiae than the CDC trap, the entry trap and the MLC. This observation could also suggests that the Limburger cheese baited trap collected An. gambiae mosquitoes that have a higher vector competence and vectorial capacity, as studies before have suggested that larger mosquitoes tend to have more serial feeding than small ones and are therefore likely to infect more hosts than the small ones[30]. Earlier studies also observed that larger mosquitoes have a higher probability of survival, being inseminated and producing more egg batches than smaller ones [31].
It was also observed that the Limburger cheese-baited CFG trap was biased in catching gravid An. funestus when compared with man-landing catches. This indicates that the Limburger cheese would provide a good odour bait in oviposition traps especially for An. funestus as it has been observed that mosquitoes locate their oviposition sites via chemical attractants [32].
The Limburger cheese-baited CFG trap, like the man-landing catches and the CDC light trap attracted higher numbers of An. gambiae, An. arabiensis and An. funestus than the entry trap. This could be explained by the fact that while the entry trap is fixed at an open window some host-seeking mosquitoes could be entering the house through the door and the eaves which are the most important entry points for mosquitoes that spread malaria and a variety of tropical diseases. Some could also be entering into the house earlier before the entry trap is placed onto a window. The entry trap therefore, is not an effective method for sampling Afrotropical malaria vectors according to this study.
Conclusions
In this study, Limburger cheese and milk cream have demonstrated a potential as odour baits that can be used as an alternative to the unethical man-landing catches in sampling and monitoring the African malaria vector An. funestus in the field. Limburger cheese has also shown a potential as an effective odour bait in trapping gravid An. funestus and one could even foresee the use of Limburger cheese as an attractant in oviposition traps for An. funestus mosquitoes. However, it might be prudent to combine odours from Limburger cheese or milk cream with other chemical cues which also play a role in host identification and location mainly components of the human sweat - ammonia, lactic acid and carboxylic acids - [33,34] and breath [2] in order to develop a highly attractive odour blend that can be used in the control of mosquitoes. It would also be important to set up the experiments from 18.00 h to 06.00 h to accommodate the two blood seeking cycles (sunset and sunrise) in mosquitoes [23] unlike in this study where the traps were set at 22.00 h to 06.00 h and therefore only one cycle was covered.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EAO conceived, designed, performed the experiments and analyzed the data.
Acknowledgements
A great many thanks go to the Center for Insect Physiology and Ecology (ICIPE), especially to Professor Ahmed Hassan Ali, who organized use of the laboratory facilities at the Thomas Odhiambo Campus in ICIPE Mbita Point. I would also like to thank Mr. Maurice Otieno who helped in collecting the data. My sincere appreciation goes to Professor Sinclair Mantell of Nakhlatec International, Sweden for helpful comments and suggestions during the editing of the manuscript. This work was funded by the University of Nairobi.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2084458210-PONE-RA-20594R110.1371/journal.pone.0012636Research ArticleMolecular BiologyOncologyMolecular Biology/Post-Translational Regulation of Gene ExpressionOncology/Renal CancerUbiquitin/SUMO Modification Regulates VHL Protein Stability and Nucleocytoplasmic Localization Ub/SUMO Modified VHLCai Qiliang Robertson Erle S.
*
Department of Microbiology and Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America
Jin Dong-Yan EditorUniversity of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: QC ESR. Performed the experiments: QC. Analyzed the data: QC ESR. Wrote the paper: QC ESR.
2010 9 9 2010 5 9 e126362 7 2010 14 8 2010 Cai, Robertson.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is linked to the development of several forms of cancer as well as oncogenic progression like sporadic renal clear-cell carcinomas (RCC). Despite the critical role played by VHL in destruction of hypoxia-inducible factor α (HIFα) via ubiquitin-mediated proteolysis, very little is known about the post-translational modification which regulates VHL activity. Our previous study showed that the SUMO E3 ligase PIASy interacts with VHL and induces VHL SUMOylation on lysine residue 171 (Cai et al, PLoS ONE, 2010, 5(3):e9720). Here we further report that VHL also undergoes ubiquitylation on both lysine residues 171 and 196, which is blocked by PIASy. Moreover, using a VHL-SUMO1 or ubiquitin fusion protein, we found that ubiquitylated VHL is localized predominantly in the cytoplasm, while SUMOylated VHL results in increased VHL protein stability and nuclear redistribution. Interestingly, substitution of lysine 171 and 196 to arginine of VHL abrogates its inhibitory function on the transcriptional activity of HIFα, and tube formation in vitro. This demonstrates that post-translational modifications like ubiquitylation and SUMOylation contributes to VHL protein stability and nucleocytoplasmic shuttling, and that the overall function of VHL in tumor suppression may require a precise and dynamically regulated process which involves protein modification.
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Introduction
The von Hippel-Lindau (VHL) gene was identified as a tumor suppressor in 1993 [1]. Germ-line mutations in this gene causes VHL hereditary cancer syndrome, which is characterized by the development of tumors of the central nervous system (CNS), kidney, eye, and pancreas [2], [3]. Somatic mutations of the VHL gene also has been widely demonstrated in the majority of sporadic renal clear-cell carcinomas (RCCs, the most common form of adult kidney cancer) [4]–[6], and cerebellar hemangioblastomas [7]. Reintroduction of wild type VHL into VHL
−/− renal carcinoma cells (RCC) has been shown to sufficiently suppresses tumor formation in vivo
[8]. In the 3D protein structure, the VHL protein contains two functional domains, α and β. The α domain binds to elongin C and the β domain acts as the substrate-recognition site for targeting proteins [9]. Many studies have demonstrated that VHL is a multi-purpose adaptor protein that engages in regulation of the extracellular matrix [10], [11], cellular differentiation [12], cell cycle [13], cell survival, apoptosis [14]–[16], and senescence [17]. However, the main function of VHL is viewed as an adapter for targeting hypoxia-inducible factor (HIF) α subunit for proteolytic degradation [18], [19].
In the presence of oxygen, HIFα is hydroxylated by prolyl hydroxylase (PHD) and then binds to VHL for proteasome-mediated degradation through the formation of EC2V (Elongin BC-Cul2-VHL) E3 ubiquitin ligase complex [18]–[20]. Under hypoxic environment, this hydroxylation-mediated degradation pathway is blocked, and results in HIFα translocation and accumulation in the nucleus, where it binds with the constitutively expressed HIFβ to form a heterodimer and transactivates hypoxia-responsive genes (including Glut-1 and VEGF) that are implicated in cellular metabolism, angiogenesis, invasion, and metastasis [21], [22]. Thus, loss function of VHL or hypoxic conditions will lead to HIFα accumulation and will also impair several other VHL-modulated biological pathways associated with tumor suppression.
Degradation of nuclear substrates by the ubiquitylation dependent system often requires nuclear-cytoplasmic trafficking of both the E3 ubiquitin ligase and the substrate proteins [23]. As a ubiquitin E3 ligase, it has been viewed that VHL is dynamic in controlling the degradation of HIFα, and several physiological cues can modulate the function of VHL within this setting [24]. For instance, VHL is predominantly nuclear at low cell density and cytoplasmic at high cell density [25]. Additionally, upon transcriptional arrest or low pH, VHL will accumulate in the nucleus [26], [27]. Although the biological significance of this is unclear, the evidence supports the notion that nucleocytoplasmic shuttling of VHL may be important for its antitumor effects. Notably, by using various nuclear import or export sequences fused with VHL, a previous study has indirectly showed that specific subcellular localization affects the antitumor properties of VHL [28].
To date protein posttranslational modification by ubiquitin or other ubiquitin-like molecules (i.e. SUMO, NEDD) has emerged as an important strategy for dynamically regulating target proteins involved in regulation of diverse cellular processes, including protein relocalization, stability and stress response [29]–[31]. We and other groups have previously found that SUMOylation or Neddylation of VHL is able to affect its function in tumor suppression [32]–[34]. To determine whether the posttranslational modification of VHL affects its protein stability and nucleocytoplasmic redistribution, we further investigated the modification of VHL by mutation of specific lysine residues. Our previous finding showed that PIASy (a SUMO E3 ligase) induces VHL SUMOylation [32]. In the present study, we further showed that VHL is also ubiquitylated on both lysines 171 and 196, and that coexpression of PIASy prevents VHL ubiquitylation. Furthermore, we also demonstrated that VHL with ubiquitin or SUMO modification at high cell density exhibited distinct subcellular distribution and protein stability. The mutation of VHL lysine 171/196 which abrogates its ubiquitin and SUMO modification disabled its function related to inhibition of HIFα transcriptional activity and tube formation. Therefore, the ubiquitin/SUMO modification of VHL allows for precise regulation of VHL nucleocytoplasmic trafficking, and disruption of this process can impair its antitumor effects.
Results
Lysine residues 171 and 196 of VHL are targeted for ubiquitylation
Previous studies have demonstrated that VHL is a major player in the ubiquitylation system by acting as an ubiquitin E3 ligase. However, recent studies has shown that VHL is targeted for proteasomal degradation by cellular or viral proteins (like E2-EPF UCP and KSHV LANA) [35], [36]. To investigate whether VHL itself can be ubiquitylated, we overexpressed FLAG-tagged VHL in the presence or absence of HA-tagged ubiquitin in HEK293 cells followed by treatment with or without proteasome inhibitor MG132, and then performed immunoprecipitation and immunoblotting with FLAG antibody. The results showed that VHL had two slower-migrating bands (ubVHL) which appeared when coexpressed with ubiquitin (Figure 1A, compare lane 3 with 2), and the increase intensity of the modified ubVHL bands consistent with higher levels of ubiquitin after proteasomal inhibitor MG132 treatment, further proving that ubVHL is the isoform of VHL with ubiquitin modification (Figure 1A, compare lane 6 with 3). To determine which lysine residues are required for the ubiquitylation of VHL, we generated a series of VHL mutants (K171R, K196R, K171,196R, K159R, and 3KR) based on all the lysine residues with potential modification (like SUMOylation and Neddylation indicated in Figure 1B), and then coexpressed with exogenous HA-tagged ubiquitin in HEK293 cells. Figure 1C showed that among three mutants (K159R, K171R, and K196R) with the single residue mutation, only K171R and K196R showed a significant reduction in the intensity of the modified isoform ubVHL when compared to wild type VHL. Furthermore, the double mutant K171,196R, similar to the triple mutant 3KR, almost completely lost the modified bands (Figure 1C, compare lane 5 with 7). This further confirms that both the lysine 171 and 196 residues are the major residues for ubiquitin modification but not lysine 159. The re-immunoblotting results showing that the double (K171,196R) and triple (3KR) mutants present less association with ubiquitin than wild type of VHL (Figure 1C, middle panel), suggest that lysines 171 and 196 modification contribute to the ubiquitylation function of VHL.
10.1371/journal.pone.0012636.g001Figure 1 VHL can be ubiquitylated on lysines 171 and 196.
(A) The level of ubiquitylated VHL is increased by proteasomal inhibitor treatment. HEK293 cells were individually cotransfected with expression vector encoding the indicated proteins in the top panel. Forty-eight hour posttransfection, the cells were treated with or without 20 µM MG132 for 2 hrs before harvest. Cell extracts were subjected to immunoprecipitated (IP) and immunoblotting (IB) as indicated in the figure. The relative quantitation of VHL ubiquitylation (ubVHL) was presented at the bottom. (B) Schematic representation of VHL α and β domains with three potentially modification lysines K159, K171 and K196. Su, SUMOylation; Ub, ubiquitylation; Nedd, Neddylation; AD, acidic domain; OD, oligomerization domain. (C) Lysine 171 and 196 but not 159 of VHL occurs ubiquitylation in vivo. HEK293 cells cotransfected with expression vector encoding the indicated proteins in the top panel, were treated with 20 µM MG132 for 2 hrs before harvest and subjected to immunoprecipitated (IP) and immunoblotting (IB) as indicated in the figure. (D) PIASy suppresses VHL ubiquitylation. As performed in panel C, HEK293 cells were transfected with indicated plasmids. WCL, whole cell lysate; HC, heavy chain; LC, light chain. The asterisk denotes uncharacterized protein band.
VHL ubiquitylation is blocked by PIASy and its protein stability and nuclear localization is increased
Previously we reported that SUMOylation occurs on lysine 171 of VHL [32]. To determine whether PIASy could prevent VHL ubiquitylation in addition to stimulating SUMOylation, we studied the effects of PIASy on both wild type VHL and the SUMOylation-deficient mutant K171R (that could still be ubiquitylated to some extent on lysine 196). Figure 1D showed that coexpression of PIASy completely suppressed the ubiquitylation of VHL on either lysine 171 or 196. This suggests that PIASy plays a role in stabilization of VHL by blocking ubiquitylation. To further assess the role of PIASy on VHL protein stability, we used cycloheximide to block protein synthesis and examined the stability of wild type VHL when cotransfected with either the plasmid expressing full-length (FL) PIASy or a PIASy mutant which lacks the ability to bind VHL (ΔC) in HEK293 cells. As shown in Figure 2A, VHL stability was significantly increased in the presence of wild type PIASy but not its mutant which lacks VHL-binding ability. The overall role of PIASy on VHL stability was further confirmed by the observation that the half life of endogenous VHL is decreased when endogenous PIASy expression is knocked-down (Figure 2B). Similarly, to determine the effect of ubiquitylation on VHL protein stability, we utilized the same strategy along with or without treatment with the proteasomal inhibitor MG132 to test the stability of wild type VHL and its ubiquitylated-site specific mutants (K171R or K196R). The results showed that MG132 treatment significantly increases the half life of wild type VHL when compared with no MG132 treatment. Consistently, the mutation of ubiquitylated lysine residue 171 or 196 leads to increased stability of wild type VHL (Figure 2A, lower panels). Therefore, these results indicate that in addition to SUMOylation, the lysine 171 of VHL is also targeted for ubiquitylation and degradation, and further supports a role for lysine 196 as an alternative target for VHL ubiquitylation, and that PIASy can function to increase VHL stability by blocking its ubiquitylation.
10.1371/journal.pone.0012636.g002Figure 2 The VHL protein stability is enhanced by lysine 196 mutation and PIASy coexpression.
(A) Proteasomal inhibitor MG132, Lys196 mutation or PIASy coexpression increases VHL protein stability. HEK293 cells were cotransfected with plasmids expressing wild type (WT) VHL, VHL plus full length PIASy or its deletion mutant (ΔC) of VHL-binding domain, or VHL K196R alone. 48 h post-transfection, cell lysates treated with cycloheximide (CHX, 100 µg/ml) for 0, 1, 2 and 4 hours in the presence or absence of MG132 was subjected to immunoblot as indicated. β-Actin immunoblotting was used as the loading control. The relative quantitation of VHL is shown in bottom panel. (B) PIASy knockdown reduces VHL protein stability. HEK293 cells were individually infected by lentivrus expressing small hair RNA against PIASy (shPIASy) or luciferase (shLuc). The infected cells after puromycin (1 µg/ml) selection were treated with cycloheximide, lysated and then subjected to immunoblot as described in panel A.
A number of studies have shown that the process of ubiquitin and ubiquitin-like modification not only increases target protein stability but also contributes to nuclear localization of target proteins [29]–[31]. To explore whether the subcellular localization of VHL is influenced by interaction with PIASy and its deficiency of ubiquitin/SUMO modification, we investigated the subcellular localization of wild type VHL and its K171R mutant in the presence or absence of PIASy coexpression by immunofluorescence assays. In contrast to wild type VHL, the localization pattern of K171R mutant showed a slight increase in nuclear localization (Figure 3A and B, left panels). Moreover, when PIASy was coexpressed, the K171R mutant dramatically localized to the nucleus but lost the punctate foci which overlapped with PIASy in 786-O cells (Figure 3A and B, left panels). Unexpectedly, we also observed that the foci staining pattern of PIASy in the coexpression of wild type VHL is larger and less than in the K171R mutant (Figure 3B, right panels, the enlarged region). Similar results were also seen in the HEK293 cells with less translocation efficiency (Figure 3A). In addition, the evidence of VHL-interacting domain deletion of PIASy lost its effect on the induction of VHL nuclear localization (Figure 3B, right lower panel), further indicating that the punctate foci which represent VHL staining are the dominant modified isoform of VHL on lysine 171 due to PIASy induction. However, SUMO modification of VHL is not a critical prerequisite for PIASy-mediated VHL nuclear localization but may be important for localization in specific subnuclear compartments. To further confirm that PIASy does induce VHL nuclear localization, we individually extracted nucleus and cytoplasm fractions from 786-O cells cotransfected with full length PIASy, its mutant (ΔC) of the VHL-interacting domain deletion or the empty vector alone in the presence of wild type VHL. Consistent with the results from the immunofluorescence assays, the nuclear distribution of wild type VHL was greatly enhanced by full length PIASy coexpression, and disrupted by the deletion of VHL-interacting domain of PIASy (Figure 3C). Moreover, the lentivirus-mediated knockdown inhibition of endogenous PIASy expression in 293 cells dramatically reduced nuclear localization of endogenous VHL (Figure 3D). Taken together, these results further strengthen our hypothesis that PIASy facilitates VHL nuclear localization and that SUMO modification of VHL contributes to its colocalization with PIASy in the subcellular nuclear compartment.
10.1371/journal.pone.0012636.g003Figure 3 PIASy alters VHL subcellular localization.
Immunofluorescence assay of HEK293 (A) or 786-O (B) cells transfected with wild type (WT) HA-VHL, its mutant K171R in the presence and absence of PIASy-RFP (red) coexpression were cultured on coverslips, fixed with 3% paraformaldehyde, and then subjected to immunofluorescence assay followed by mouse anti-HA antibody (green) and nuclear staining (blue) with DAPI as indicated. (C) Immunoblotting analysis. 786-O cells expressing HA-VHL in the presence or absence of PIASy-FLAG coexpression were subjected to nucleus (Nucl) and cytoplasm (Cyto) proteins extract followed by immunoblotting against HA and FLAG antibodies. Nuclear protein Sp1 and cytoplasm protein β-actin were blotted as fraction positive control. FL, full length; ΔC, the carboxyl domain deletion; ns, non-specific band. (D) PIASy knockdown reduces endogenous VHL nuclear distribution. HEK293 cells with constitutively knockdown against PIASy (shPIASy) or luciferase (shLuc) were performed nucleus (Nucl) and cytoplasm (Cyto) fraction assays as described in panel C.
Ubiquitin/SUMO modification distinctly impairs VHL protein stability and subcellular distribution
Despite the fact that some physiological conditions are able to induce the constitutive shuttling of VHL between the nucleus and cytoplasm compartment [26], [27], the underlying molecular mechanism still remains unclear. To explore whether ubiquitin modification of VHL leads to nuclear export and SUMO modification results in nuclear import, as well as whether ubiquitylation and SUMOylation of VHL exhibits distinct protein stability, we engineered the ubiquitin and SUMO modified VHL (VHL-UbΔGG and VHL-SUMO1ΔC4) with a mutation in lysine 171 by utilizing an artificially fused protein strategy [32]. The glycines which are critical for generating the covalent link were removed in both fused ubiquitin and SUMO proteins to exclude the possibility of the fusion protein acting as a ubiquitin or SUMO like protein. The results showed that ubiquitylated VHL (VHL-UbΔGG) was exclusively located in the cytoplasm, while SUMOylated VHL (VHL-SUMO1ΔC4) predominantly existed in the nucleus (Figure 4A). This provides evidence that ubiquitin and SUMO modification of VHL can regulate VHL specific subcellular localization. Consistently, the protein stability of VHL where monitored by ubiquitin or SUMO modification and showed reduced stability of VHL-UbΔGG compared to VHL-SUMO1ΔC4 and may explain why VHL was more stable once lysine 171 was mutated (Figure 4B).
10.1371/journal.pone.0012636.g004Figure 4 Ubiquitin/SUMO1 modification of VHL presents opposite effect on the VHL protein stability and subcellular localization.
(A) The effect of SUMO1 and ubiquitin modification on the VHL subcellular localization. 786-O cells transfected with VHL-SUMO1ΔC4 or VHL-UbΔGG were cultured on coverslips, fixed with 3% paraformaldehyde, and then subjected to immunofluorescent assay followed by mouse anti-FLAG antibody (green) and nuclear staining (blue) with DAPI as indicated. The percentage of cell staining pattern is calculated by total twenty positive staining cells of each sample. (B) The effect of SUMO1 and ubiquitin modification on VHL protein stability. 786-O cells were cotransfected with plasmids expressing VHL-SUMO1ΔC4 or VHL-UbΔGG alone. 48 h post-transfection, cell lysates treated with cycloheximide (CHX, 100 µg/ml) for 0, 1, 2 and 4 hours was subjected to immunoblot as indicated. β-Actin immunoblotting was used as the loading control. The relative quantitation of VHL is shown in bottom panel. (C) Ubiquitin but not SUMO1 modification of VHL induces the ubiquitylation on lysine 196. 786-O cells were individually cotransfected with expression vector encoding the indicated protein in the top panel. Forty-eight hour posttransfection, the cells were treated with 20 µM MG132 for 2 hrs before harvest. Cell extracts were subjected to immunoprecipitated (IP) and immunoblotting (IB) as indicated in the figure. Blank arrows indicate the native VHL-SUMO1ΔC4 or VHL-UbΔGG; ubVHL, ubiquitylated VHL. The position of ubiquitylated VHL is highlighted by asterisks. WCL, whole cell lysate; HC, heavy chain. (D) Immunoblotting analysis of nuclear and cytoplasm fraction of VHL with SUMO1/ubiquitin modification. 786-O cells transfected with VHL-SUMO1ΔC4 or VHL-UbΔGG were subjected to nuclear (Nucl) and cytoplasm (Cyto) proteins extract followed by immunoblotting against FLAG antibody. Nuclear protein Sp1 and cytoplasm protein β-actin were blotted as fraction positive control. ns, non-specific band.
Moreover, to determine whether the SUMO/Ubiquitin modification on lysine 171 of VHL influences the further ubiquitylation on lysine 196 and alters VHL subcellular distribution, we performed the ubiquitylation assays by individually coexpressing ubiquitylated (VHL-UbΔGG) and SUMOylated (VHL-SUMO1ΔC4) form of VHL with or without lysine 196 mutation in the presence of HA-Ub. The results showed that ubiquitin but not SUMO1 modification increases the lysine 196 ubiquitylation which is significantly abolished by lysine 196 mutation (Figure 4C). Coordinately, in the nuclear and cytoplasm fractions, the result showed that mutation of lysine 196 not only increases the nuclear localization of SUMO1-modified VHL (VHL-SUMO1ΔC4) but also ubiquitin-modified VHL (VHL-UbΔGG) (Figure 4D). Interestingly, we also observed that the enhancement of nuclear localization of VHL with either SUMO1 or ubiquitin modification caused by the mutation at lysine 196 occurs with increase oligomerization and are mostly distributed in the cytoplasm compartment (Figure 4D, and the oligomerization definition referred to [32]). This further corroborates the hypothesis that localization in the nuclear compartment is a prerequisite for inducing oligomerization despite the fact that oligomerized VHL eventually goes to cytoplasm.
Mutation of lysine residues 171/196 abolishes the inhibitory function of VHL on the transcriptional activity of HIFα as well as tube formation
VHL functions as a tumor suppressor by targeting specific proteins, including HIFα for degradation [24]. To ascertain if mutation of ubiquitylated (K171 and 196) or SUMOylated (K171) site is sufficient to inactivate the antitumor properties of VHL in cancer cells, we tested whether mutation of lysine 171/196 resulted in disruption of VHL on inhibition of the transcriptional activity of HIFα. The VHL-deficient 786-O cells were transiently transfected with vector, vector expressing wild-type VHL or its mutants along with HIFα responsive element driven luciferase reporter. The results of the reporter assay showed that except for the K159R mutant, both the K171R and K196R mutants disrupted the inhibition of the transcriptional activity of HIFα when compared to WT (Figure 5A). The double mutant of lysine 171/196 (K171,196R) and the triple mutant 159/171/196 (3KR) showed the greatest efficiency of disruption of VHL inhibitory activities (Figure 5A). Concordantly, in vitro endothelial tube formation assay using conditioned medium from the supernatants of the vector, VHL or K171,196R -expressing 786-O cells, showed that the K171,196R mutation markedly reduced the inhibition of tube formation compared to wild type VHL (Figure 5B). Thus, these results demonstrate that modification on lysines 171/196 is critical for VHL to function as a tumor suppressor.
10.1371/journal.pone.0012636.g005Figure 5 The lysine 171/196 mutation abolishes the inhibitory function of VHL as a tumor suppressor.
(A) The lysine 171/196 mutation of VHL attenuates its inhibition on transcriptional activity of HIF-responsive reporter. 786-O cells were transiently transfected with wild type VHL or its mutants in the presence of HRE-luciferase reporter. Empty vector was used as control. Data are presented as means±SD of three independent experiments. The levels of exogenous VHL and endogenous HIF2α were individually detected by immunoblotting against FLAG and HIF2α. RD, relative density. (B) The lysine 171/196 mutation of VHL loss its inhibitory effect on endothelial tube formation in vitro. The conditioned medium from 786-O cells expressing vector, wt VHL, or K171,196R, was tested for tube formation assay in vitro. Photographs were taken at 24 hr post-incubation. The quantitation presents the average of the pattern/value association criterion from 5 random view fields per well. (C) A proposed model for the dynamic regulation of VHL protein stability and nucleocytoplasmic distribution in tumor suppression. In some circumstance, interaction of VHL with PIASy results in VHL nuclear localization, SUMOylation (Su) and stability for blocking ubiquitylation (Ub) of VHL. Meanwhile, PIASy dissociation with VHL or attenuation of VHL SUMOylation facilitates VHL nuclear export, ubiquitylation and instability. This dynamic process of VHL with reversible modification acts in a concert to inhibit HIFα. The ability of VHL with different isoforms on inhibition of HIFα transcriptional activity is indicated at the bottom.
Discussion
Functional inactivation of VHL is not only able to induce the von-Hippel Lindau familial cancer syndrome but can also result in sporadic renal carcinomas, and sporadic hemangioblastomas of CNS [37]. VHL has been shown to associate with the inhibition of several cellular processes including angiogenesis, cell cycle exit [38], [39] and fibronectin matrix assembly [10], [11]. Importantly, the ability of VHL to induce substrate proteolysis as a ubiquitin E3 ligase is critical to its function [18]. Previous studies have shown that the function of a ubiquitin ligase can be regulated by controlling the ligase or its substrate using a number of strategies including posttranslational modifications, interactions with regulatory factors, or subcellular localization [24]. Recently, VHL was shown to be Neddylated on lysine 159 and associated with the fibronectin matrix assembly and suppression of tumor development [33], [34], however, these activities are not related to its E3 ligase activity. Therefore, the posttranslational modifications status in regulation of VHL E3 ligase activity remains understudied. Our previous studies have demonstrated that PIASy interacts with VHL and induces VHL SUMOylation [32], and that the viral protein LANA is able to promote VHL ubiquitylation and degradation [36]. In this work, we further demonstrated that VHL is ubiquitylated on lysine 171 and 196, and that enhanced expression of the SUMO E3 ligase PIASy not only blocks VHL ubiquitylation, but also increases VHL nuclear localization which leads to protein stabilization. By using SUMO1 or ubiquitin fusion VHL proteins, we have further showed that SUMO1 and ubiquitin modified isoform of VHL exhibits reverse nucleocytoplasmic distribution. This suggests that reversible modification of SUMO1 and ubiquitin is a strategy for dynamic regulation of VHL nucleocytoplasmic shuttling. Interestingly, a single or double mutation of lysine 171 and 196 or fusion with ubiquitin or SUMO1 in VHL resulted in a decrease in HIFα inhibition when compared to wild type VHL. The most significant of inactivation was the SUMO1 modification followed by the double lysine 171/196 mutation and the single mutation (Figure 5C). In addition, the evidence of the increased nuclear localization of VHL-UbΔGG with lysine 196 mutation suggested that the regulation of lysine 196 is involved in controlling the process of VHL nucleus –cytoplasm shuttling, and that this regulation may be associated with PIASy interaction since we observed that VHL-UbΔGG interacts with PIASy in our previous studies [32]. These findings are consistent with a prior study [26], and strongly suggest that any steps in this dynamic regulatory process which involves posttranslational modification will contribute to VHL nucleocytoplasmic shuttling (Figure 5C).
To date, one of the difficulties in separating many of the discrete cellular roles of VHL is that individual mutations in the gene result in widespread changes to the protein's function [4]–[6]. Consistent with the previous reports [40], we found that although mutation of ubiquitylated-sites increase VHL stability, the mutants also significantly influenced the ubiquitin ligase activity of VHL toward HIF1α. The explanation of this phenomenon could be attributed to the ubiquitylation of VHL related with proteasomal degradation. The ability of VHL to traffic between nucleus and cytoplasm is decreased, even as the mutation of the ubiquitylated site stabilizes VHL. Recently, Jung et al reported that the cellular protein E2-EPF UCP degrades VHL and stabilizes HIF [35]. Our present study further provides evidence that deficiency of posttranslational modifications at these lysines can disrupt VHL ability to target HIF1α. Meanwhile, we also observed that PIASy expression and SUMO modification can block VHL ubiquitylation, increase its protein stability and nuclear localization. However, it remains to be determined whether the deubiquiylation process is involved in competition between these two modification states. Further experiments will be required to explore these questions in more detail.
Protein ubiquitin and ubiquitin-like posttranslational modification has emerged as an important strategy for reversible modification of many proteins that play regulatory roles in diverse cellular processes, including protein relocalization, stability, transcriptional control and stress response [29]–[31]. Many reports have suggested that posttranslational modifications are an important cellular strategy that enables the cell to react to intracellular or environmental changes caused by exposure to stress factors such as hypoxia [37], [41]. Our previous studies have shown that PIASy expression in response to hypoxic stress negatively regulates VHL function as a tumor suppressor [32]. Here we further show that a single lysine mutation also impairs the ability of VHL in terms of its inhibition of HIFα activity although the level is reduced when compared with the SUMOylated isoform. Therefore, there are many critical steps required for VHL to fully function as a tumor suppressor protein, and the trafficking between the nucleus and cytoplasm could be one of these critical steps. Any block to this step will affect its antitumor activity. This notion has been supported by three points of evidence: 1) Failure of VHL to continuously shuttle between the nuclear and cytoplasmic compartments leads to the stabilization of HIF1α [27]; 2) Oxygen-dependent degradation of nuclear HIF1α is dependent upon the dynamic nuclear-cytoplasmic trafficking of VHL [26]; and 3) Acidosis blocks VHL nuclear-cytoplasmic shuttling [42]. However, the impact of posttranslational modification on VHL nuclear and cytoplasmic localization still remains unclear. Our observation that SUMOylated VHL localizes to the nucleus, while ubiquitylated VHL is cytoplasmic, suggests that posttranslational modification may affect VHL subcellular localization and its inhibitory function on HIFα.
Our data now demonstrate that SUMOylation and not ubiquitylation at the C terminus of VHL contributes to the nuclear import of VHL, and supports a model in which SUMOylation of the C terminus of VHL, in a region close to both the nuclear export and oligomerization sequences, blocks the nuclear export signal and allows interaction of VHL with the nuclear import machinery. Thus, the ability of VHL to shuttle between the nucleus and the cytoplasm may contribute to its antitumor property under specific physiologic conditions.
Materials and Methods
Cell culture and transfection
Human renal carcinoma VHL-null cell lines 786-O and embryonic kidney (HEK) 293 cells were described as previously [36], and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS, Hyclone), 4 µM L-glutamine, penicillin, and streptomycin [36]. Cells were incubated at 37°C in a humidified environmental incubator supplemented with 5% CO2. Ten million cells with 400 µl medium were transfected by electroporation with a Bio-Rad Gene Pulser in 0.4 cm-gap cuvettes at 210 Volts and 975 microfarads.
Plasmids, antibodies and reagents
Plasmid FLAG-VHL was provided individually by Joan W Conaway (Stowers Institute for Medical Research, USA). Plasmids encoding human PIASy with FLAG tag were gifts from Stefan Müller (Max Planck Institute of Biochemistry, Germany). PIASyΔC-FLAG (1-233) and PIASy-myc (1-492) were generated by ligating BamHI/EcoRV PCR fragments into pcDNA-FLAG and pA3M vector, respectively. Full length PIASy-RFP was prepared by ligating KpnI/BamHI PCR fragments into pDs-Red-N1 vector. VHL-UbΔGG and VHL-SUMO1ΔC4 were generated by in-frame ligation of BamHI/XbaI PCR fragments to the downstream of FLAG-VHL(K171R). FLAG-VHL (K171R), (K196R), (K171,196R), (K159R), and (3KR), as well as VHL-UbΔGG (K196R) and VHL-SUMO1ΔC4 (K196R) were individually generated by PCR site-directed mutagenesis. All constructs were confirmed by direct DNA sequencing. Plasmids of pGL2-HRE (HIFα-target reporter), and HA-Ub were previously described [36], [43]. The VHL antibodies were from Cell signaling technology Inc. HIF2α antibodies were from Novus Biogicals Inc. PIASy (I-19) and Sp1 (1C6) was purchased from Santa Cruz Biotech. Inc. Other antibodies used were anti-myc (9E10), anti-FLAG (M2), anti-HA (12CA5), and β-actin (Cell signaling technology). Proteasome inhibitor MG132 was purchased from Biomol International, and Cyclohexamide (C4859, Sigma Inc., St. Louis, MO).
Immunofluorescence assay
Cells on coverslips were washed three times with PBS and then fixed in 3% paraformaldehyde for 20 min at room temperature. After fixation, cells were washed three times in PBS and permeabilized in PBS containing 0.2% fish skin gelatin (G-7765, Sigma), 0.2% Triton X-100 for 5 min, and then incubated for 1 h at room temperature with primary antibodies in blocking solution. Cells were washed three times with PBS and incubated for 30 min at room temperature with fluorescently labeled Alexa fluors-488 or 594 against mouse or rabbit (Molecular Probes Inc., Eugene, OR) in blocking solution. Coverslips were washed three times with PBS and slides were mounted with Prolong antifade mounting medium with 0.5 µM DAPI (4′, 6′-diamidino-2-phenylindole). Fluorescence confocal microscopy was performed with an Olympus microscope using FluoView™ FV300 software (Olympus, Melville, NY).
Immunoblotting
Cells were harvested and washed once with ice-cold phosphate-buffered saline (PBS) and lysed in 1 ml cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.6], 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A) on ice and homogenized. The cell lysates were resuspended in 50 µl of 1× SDS Laemmli buffer and heated 95°C for 5 minutes. The sample was subjected to SDS-PAGE and transferred to a membrane that was probed with specific antibody.
Determination of VHL stability
786-O cells were cotransfected FLAG-VHL with or without FLAG-PIASy as described above. After 24 h transfection, cells were incubated with 100 µM cyclohexamide for 0 to 4 hour and then harvested. Equal amounts of total proteins from each treatment were taken to perform western blot analysis.
Fractionation of nuclear or cytoplasm proteins
Twenty million transfected cells were harvested and washed twice with ice-cold PBS followed by resuspending the cell pellet in a hypotonic buffer A (10 mM HEPES-K+ pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.5 DTT) in the presence of protease inhibitor cocktail (PIC: 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A). Cells were pelleted by spinning at 1000 rpm ×5 min. The cells were lysated in ice-cold 0.5% NP-40 containing buffer A with PIC on ice for 10 min. The nuclei were pelleted by 3,000 rpm ×2 min at 4°C. The supernatant (cytoplasm protein) harvested and frozen at −80°C for use. The nuclear pellets were washed twice with buffer A (without NP-40), followed by resuspending in buffer C (20 mM HEPES-K+ pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 DTT, 25% Glycerol) with PIC. Nuclei were incubated on ice for 30 min, and vortex periodically. Supernatant containing nuclear protein were collected by spinning at 14,500 rpm for 5 min at 4°C and then snap frozen for further use. Antibodies against nuclear protein Sp1 and cytoplasm protein β-Actin were used as markers.
RNA interference
The PIASy shRNA (5′-GTACTTAAACGGACTGGGA-3′) sequence was inserted into lentivirus pGIPz vector according to the manufacturer's instructions (Clonetech). HEK293 cells were individually transduction by lentivirus packaged from Core T which cotransfected with Rev, VSVG and gp expressing plasmids, and selection by 1 µg/ml Puromycin. pGIPz vector with luciferase (shLuc) target (5′-TGCGTTGCTAGTACCAAC-3′) sequence was used as control, and the RNA interfering efficiency was assessed by western blot analysis.
Luciferase reporter assay
The luciferase reporter assays were performed as described previously [36]. After transfection for 48 h, cells were lysed in 200 µl of reporter lysis buffer (Promega, Inc., Madison, WI). Luciferase activities and β-galactosidase were individually measured using luciferase assay reagent (Promega, Inc., Madison, WI) and the OpticompI Luminometer (MGM Instruments, Inc. Hamden, CT) according to the suppliers' instructions. Luciferase activities were normalized with β-galactosidase activities. Relative luciferase activity (RLU) was expressed as fold activation relative to the reporter construct alone. Assays were performed in triplicate.
Endothelial tube formation assay
Human umbilical vascular endothelial cells (HUVEC) were purchased (Cambrex) and maintained in EBM-2 medium supplemented with EGM-2. Tube formation assay on extracellular BD matrigel was performed according to the manufacturer's protocol with minor modifications. Briefly, 2×104 HUVEC resuspended with 500 µl of EBM-2 medium were seeded on Matrixgel solidified in 48-well tissue culture plate. Conditioned medium (1 ml) from the supernatant of each 786-O stable cells was added, respectively. Cells were incubated in a CO2 incubator for 24 hrs at 37°C and then examined for tube formation with a light microscope.
We are grateful to Joan Conaway, Stefan Müeller, Gregg Semenza, Ke Shuai and Volker Haase for providing materials.
Competing Interests: The authors have declared that no competing interests exist.
Funding: National Cancer Institute 5R01CA091792-08, 5R01CA108461-05, 1R01CA137894-01 and 1R01CA138434-01A209; National Institute of Allergy and Infectious Diseases 5R01AI067037-04 and National Institute of Dental and Craniofacial Research 5R01DE017338-03 (to ESR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J Emerg Trauma ShockJETSJournal of Emergencies, Trauma and Shock0974-27000974-519XMedknow Publications India 20930983JETS-3-300a10.4103/0974-2700.66549Case ReportTraumatic hemorrhage of occult phaeochromocytoma in a patient with septic shock Moazzam Mohammad Shahnawaz Ahmed Syed Moied Bano Shahjahan Department of Anaesthesiology & Critical Care, Faculty of Medicine, J.N. Medical College, AMU, Aligarh, U.P.-202 002, IndiaAddress for correspondence: Dr. Md Shahnawaz Moazzam, E-mail: [email protected] 2010 3 3 300 300 23 6 2009 02 1 2010 © Journal of Emergencies, Trauma, and Shock2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Phaeochromocytoma can have a variety of presentations; however, traumatic hemorrhage into a phaeochromocytoma is a very rare presentation. Diagnosing and managing a critically ill, septic patient with a Phaeochromocytoma can be very challenging. We report a case of 53 years old man with a previously undiagnosed Phaeochromocytoma, who presented initially with bowel perforation following an assault. Following a laparotomy for bowel resection and anastomosis, whilst on the intensive care unit, he developed paroxysmal severe hypertension overlying septic shock. Phaeochromocytoma was confirmed using a computed tomography scan and urinary assay of metanephrine and catecholamines. We managed the haemodynamic instability using labetalol and noradrenaline infusions. As his septic state improved he was convention therapy and following control of his symptoms over the next few weeks, he underwent an uncomplicated right sided adrenalectomy. He made a full recovery.
Phaeochromocytomasepsistraumatic haemorrhage
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INTRODUCTION
Phaeochromocytoma is a rare tumour of the chromaffin cells arising most commonly from the adrenal medulla. They typically present with hypertension and a characteristic symptom triad of headache, diaphoresis and palpitations. They are however known to be a great mimic and may have a variety of other presentations. Latent phaeochromocytomas has presented as hypertensive crises during anaesthesia and surgery,[1] acute abdomens,[2] multiple organ failure,[3] cardiac failure, myocardial infarction and stroke.[4] Cases of traumatic haemorrhage of a phaeochromocytoma resulting in its presentation,[5] have also been previously described.
We report a case of an undiagnosed phaeochromocytoma which presented as a hypertensive emergency 5 days after an assault. The patient had already safely undergone an emergency laparotomy during this period, but the tumour had not manifest until he became septic during the postoperative period. We discuss the clinical dilemma of managing a critical ill, septic patient with paroxysmal hypertension because of a stimulated phaeochromocytoma.
CASE HISTORY
A case of a 53 year old man presented to our emergency department with abdominal pain following an assault 16 hours previously. His medical history was unremarkable except for moderate alcohol intake and his surgical history included an uneventful laparoscopic cholecystectomy. A diagnosis of peritonitis followed by bowel perforation was made and he underwent an emergency laparotomy under general anaesthesia. He remained cardiovascularly stable throughout the procedure. The systolic arterial pressure (SAP) range between 90 and 140 mmHg and his heart rate ranged between 90 and 110 per minute. A short segment of perforated ileum was resected and an end-to-end anastomosis performed. Patient was reversed from general anaesthesia and shifted to postoperative ward. After two days, he appeared increasingly unwell with sputum retention and suspected sepsis. He was shifted to intensive care unit (ICU) from surgical ward and a decision was made to re-intubate. Induction of anaesthesia was performed with propofol and rocuronium. Sedation was maintained using midazolam infusions. Following tracheal intubation his arterial pressure increased to 326/142 mmHg. The cardiovascular response was initially presumed to be an exaggerated pressure response to intubation, and further boluses of propofol were administered. Despite adding high dose of propofol his SAP remained in the range of 240-340 mmHg. At this point a glyceryl trinitrate (GTN) infusion was added to manage the hypertensive crises. For the next few hours the mean arterial pressure (MAP) swung between 224 and 44 mm Hg. This raised the possibility of a phaeochromocytoma and septic shock. A labetalol infusion was started and GTN was reduced. The hypotensive episodes required the administration of noradrenaline. A contrast-enhance computed tomography (CECT) scan of the abdomen demonstrated a 5.5 cm lesion originated from the right adrenal gland suggesting the possibility of haemorrhage within an adrenal tumour [Figure 1]. Serum and urinary biochemical test were sent before the exogenous administration of noradrenaline. He remained septic, raising the possibility of an anastomotic breakdown, necessitating a return to the operation theatre. Anaesthetic plans for cardiovascular instability were made but his haemodynamic parameters remained stable throughout the procedure. Anastomotic breakdown was confirmed and end ileostomy was performed. With resolution of his intra-abdominal sepsis his gut function improved and enteral phenoxybenzamine was commenced and labetalol was reduced. He recovered enough to be discharged back to the surgical ward after a 22-day stay in ICU. The phenoxybenzamine dose was slowly increased to 80 mg twice a day and atenolol was also added. Five weeks later the ileostomy closure was done and a right adrenalectomy was performed. He remained cardiovascularly stable throughout the perioperative period. The histopathology of the gland confirmed the presence of haemorrhagic necrosis into a phaeochromocytoma [Figure 2a, 2b].
Figure 1 CECT-scan of abdomen showing tumour arising from the right adrenal gland with streaking of fat posterior to the tumour suggesting haemorrhage (arrow)
Figure 2 Histopatological slides of the excised adrenal mass showing, (a) tumour gland showing large areas of necrosis: (b) tumour showing features of phaeochromocytoma. Note the “Zellballen pattern” composed of rounded nests of cells separated by thin-walled blood vessels and scattered atypical giant cells
DISCUSSION
Phaeochromocytomas are responsible for hypertension in about 0.1% of those presenting with hypertension. Although they typically arise from the adrenal medulla, they may develop from chromaffin cells in or around sympathetic ganglia as well. Ninety percent are unilateral, predominantly right sided. Ten percent of familial cases follow an autosomal dominant pattern of inheritance and occur either alone or in combination with multiple endocrine neoplasia (MEN)-2a, MEN-2b, Von Reck-linghausen’s disease or Von Hipple Lindau retinal cerebellar haemangioblastoma. About 10% of phaeochromocytomas in adult are extra-adrenal and are more likely to be malignant. The other common symptoms include headache, sweating, palpitation, nervousness, nausea and vomiting, abdominal pain and weakness.[6] On questioning this patient following his recovery, he had no such complaints before this hypertensive episode.
Traumatic adrenal haemorrhage, resulting in the manifestations of a phaeochromocytoma, is a very rare. Traumatic adrenal haemorrhage, without a phaeochromocytoma may itself present as a hyper-adrenergic state, but then tends to improve as the haematoma resolves and the gland resumes normal function. Complete destruction of adrenals following injury or haemorrhage is likely to produce adreno-cortical insufficiency resulting in shock.[7]
Faced with severe hypertension, we initially treated the more probable causes in such a situation. The possibility of inadequate sedation or analgesia was managed by increasing the dose of sedation and analgesic agents. In a previously normotensive patient with no underlying renal dysfunction, and improbable drug interaction, the possibility of an underlying endocrine disorder was more likely. The magnitude of the raised blood pressure and the difficulty in controlling it with propofol and GTN; suggested an intense catecholamine response, i.e., a phaeochromocytoma. An α-blocking agent is the drug of choice for hypertensive crisis in these situations with or without a β-blocker added to combat reflex or associated tachycardia. We chose to use labetalol because of it’s combined α- and β-receptor blocking abilities, and also due to the fact that enteral administration of phenoxybenzamine was not possible because of bowel trauma. Labetalol worked well in controlling the hypertensive episodes but the underlying septic shock required a noradrenaline infusion. Our patient had both a noradrenaline and adrenaline secreting tumour with hypertension and tachycardia as well, so we considered combined α- and β-receptor blockade. Subsequently, our patient was started on conventional; oral phenoxybenzamine:- a long acting, non-selective α-receptor blocking drug, with a later addition of atenolol.
The presence of hypotension, requiring noradrenaline infusion raised the possibility of a septic shock. The evidence in favour of sepsis-induced shock included a raised total leukocytes counts (TLC) and acute phase reactants on the background of a recent intra-abdominal pathology requiring surgery. Predominantly adrenaline secreting tumours may present with a “septic shock” kind of picture. However, in our case, the urinary analysis revealed grossly elevated noradrenalin and adrenaline levels suggesting a tumour secreting both the catecholamines. Phaechromocytomas may also cause a hypertensive cardiomyopathy resulting in cardiac failure,[8] but in our case evidence of a hyperdynamic circulation (↑ cardiac index and ↓ systemic vascular resistance) requiring vasopressures rather than inotropes supported the diagnosis of septic shock. The diagnosis of phaeochromocytoma was made following a CECT scan study and assay of the urinary catecholamines and their metabolites. Twenty-four hours urinary catecholamine levels; and total metanephrine level; have consistently proven to be the most specific tests available for the diagnosis of phaeochromocytoma (sensitivity 89% and specificity 99%).[9] The diagnosis in our patient was confirmed by the presence of haemorrhage necrosis of a phaeochromocytoma in the histology of gland.
CONCLUSIONS
Phaeochromocytoma discovered during the management of an unrelated illness is a rare presentation of these tumors. We have reported an unusual case of traumatic hemorrhage of occult pheochromocytoma, presenting with severe labile hypertension and associated with septic shock. Diagnosing and managing a critically ill, septic patient with a Phaeochromocytoma can have very challenging. Because the key to diagnosing and managing the phaeochromocytoma in a septic shock patient,: 1) presence of severe and labile hypertension could not control with the administration of propofol and GTN postoperative period and managed with administration of labetalol; 2) raised TLC and Acute phase reactant with episode of hypotension managed with infusion of noradrenaline. Remarkably, above clinical scenarios were present in our patient in addition to the rare and serious complication of traumatic hemorrhage of adrenal tumour with sepsis.
Source of Support: Nil.
Conflict of Interest: None declared.
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REFERENCES
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9 Harding JL Yeh MW Robinson BG Delbridge LW Sidhu SB Potential pitfalls in the diagnosis of phaeochromocytoma Med J Aust 2005 182 637 9 15963022 | 20930983 | PMC2938503 | CC BY | 2021-01-04 19:39:10 | yes | J Emerg Trauma Shock. 2010 Jul-Sep; 3(3):300a |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2094912510-PONE-RA-17023R110.1371/journal.pone.0013117Research ArticleVirology/Antivirals, including Modes of Action and ResistanceVirology/Effects of Virus Infection on Host Gene ExpressionVirology/Host Antiviral ResponsesVirology/Immune EvasionVirology/Mechanisms of Resistance and Susceptibility, including Host GeneticsSH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells STAT1-CC and HCVPoat Bret
1
Hazari Sidhartha
1
Chandra Partha K.
1
Gunduz Feyza
1
2
Balart Luis A.
2
Alvarez Xavier
3
Dash Srikanta
1
2
*
1
Department of Pathology and Laboratory Medicine, New Orleans, Louisiana, United States of America
2
Department of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana, United States of America
3
Division of Comparative Pathology, Tulane National Primate Research Center, Covington, Louisiana, United States of America
Ott Melanie EditorJ David Gladstone Institutes, University of California San Francisco, United States of America* E-mail: [email protected] and designed the experiments: BP SH SD. Performed the experiments: BP SH PKC FG XA. Analyzed the data: BP SH PKC FG LAB SD. Contributed reagents/materials/analysis tools: BP SH PKC XA SD. Wrote the paper: BP SD. Crtically read the manuscript: LAB.
2010 30 9 2010 5 9 e1311712 3 2010 1 9 2010 Poat et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.
Methodology/Principal Findings
In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.
Conclusions
These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.
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Introduction
Hepatitis C virus (HCV) infection is a major public health concern with a prevalence of approximately 3% of the world population chronically infected by the virus [1]. Approximately 70% of patients that are infected with HCV develop a chronic infection of the liver. Interferon alpha (IFN-α combined with ribavirin is the standard treatment option for chronic HCV infection, however the majority of patients are unable to clear the infection with this therapy [2], [3]. These chronically infected HCV patients experience a slow progressive disease of the liver that can result in end stage liver disease such as liver cirrhosis and hepatocellular carcinoma [4]. In the United States HCV infection is the leading cause of death from liver disease and the number one indication for liver transplant [5]. Currently there are no effective drug therapies available for liver cirrhosis or hepatocellular carcinoma, therefore the development of an antiviral approach to cure chronic HCV infection is essential.
The interferons are a super family of proteins secreted by human cells that manifest multiple functions in the human body such as protection of cells from viral infection, regulation of cell growth, and modulation of the immune system [6]. IFN-α/β also known as type 1 interferon, binds to cell surface receptors consisting of two separate proteins, IFNAR1 and IFNAR2 [7]. High levels of IFNAR1 and IFNAR2 are expressed in human liver cells thus providing a clear rationale for the treatment of chronic HCV infection with IFN-α
[8], [9]. The binding of IFN-α to cell surface receptors activates a cascade of signal transduction reactions that are mediated by two receptor associated tyrosine kinases, Janus kinase 1 (Jak1) and tyrosine kinase 2 (Tyk2). These kinases phosphorylate IFNAR1, which then serve as a docking site for the Src-homology domain 2 of the signal transducer and activator of transcription factor 2 (STAT2), which is then phosphorylated by Tyk2 on tyrosine residue 690. The other STAT proteins including STAT1 are subsequently recruited to the cell membrane for phosphorylation and activation. Activated STAT1 and STAT2 monomers are then disassociate from the receptor and form a heterodimer that interacts with interferon regulatory factor 9 (p48) to form an active transcription complex called IFN-stimulated gene factor 3. This complex translocates into the nucleus and binds to a consensus DNA sequence to initiate antiviral gene transcription. The molecular cascade of events initiated following IFN binding to its receptor in normal cells is called the Jak-STAT pathway [10]. Jak-STAT signaling activates a large number of antiviral genes that are normally quiescent or present at low levels.
Interferon gamma (IFN-γ) is a type II interferon, which binds to a separate receptor consisting of two proteins called IFNGR1 and IFNGR2 [11]. The two kinases that signal through these receptors are called Jak1 and Jak2 tyrosine kinases. The Jak kinases phosphorylate STAT1 protein at tyrosine 701, which then homodimerizes through reciprocal interaction between the phospho-tyrosine at residue 701 and the SH2 domain of another STAT1 molecule. This phospho-STAT1 homodimer referred to as the interferon gamma activated factor complex translocates to the nucleus and binds to a DNA sequence called GAS element in the upstream promoter region of IFN-γ inducible genes [11]. The STAT1 transcription factor is a critical component for both type Type I and Type II IFN-signaling pathways [12], [13].
Our understanding of HCV resistance mechanisms to interferon is possible due to the development of a HCV cell culture system. A number of laboratories have now shown that both type I, and type II interferons inhibit HCV replication in cell culture models [14]–[17]. There have been a number of reviews where IFN resistance mechanisms have been predicted to be related to several viral and host related factors [18]–[20]. To study the role of host cellular factors in the mechanisms of resistance, we have developed resistant stable HCV replicon cells lines for HCV 1b and HCV 2a viruses by prolonged treatment with interferon alpha [21]–[22]. We found that replication of HCV RNA in these cells is totally resistant to IFN-α due to Jak-STAT signaling defects. We have characterized the role of virus and host cellular factor contributions that are responsible for IFN-α resistance in the replicon cell line. We showed that viral factors are not involved in the resistant phenotypes since these cells continue to display defective Jak-STAT signaling even after the elimination of HCV. We showed that due to Jak-STAT signaling defects, the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins are blocked in the IFN-α resistant cell line.
IFN-γ is also important in the innate antiviral immune response against hepatitis C. IFN- γ therapy has not been successful in the treatment of chronic HCV infections that are resistant to IFN-α. The rationale for this study is two fold. Since IFN-γ has been shown to inhibit HCV replication effectively in cell culture first we have asked the question whether or not IFN-γ could inhibit HCV replication in replicon cells that are resistant to IFN-α. Second, we examined whether STAT1 signaling of the host cell could be genetically engineered to improve interferon sensitivity and to overcome resistance in the HCV cell culture model. We found that cells those are resistant to IFN-α survived IFN-γ treatment and formed resistant cell colonies. IFN-γ resistant cell colonies were picked and stable replicon cell lines were developed. In this study, a recombinant STAT1 molecule with a double cysteine-substitution in the SH2 domain called STAT1-CC was utilized to activate the GAS-promoter in the IFN-γ resistant replicon cells. Transient transfection of the STAT1-CC plasmid construct into IFN-resistant replicon cell line inhibited HCV replication and showed enhanced surface expression of HLA-1 in an IFN-γ dependent manner. Results of our study suggest that the engineered STAT1-CC has strong antiviral activity in liver cells that are resistant to IFN-α and IFN-γ. We believe that liver targeted delivery of STAT1-CC can be developed as second line treatment for patients with defective Jak-STAT signaling. STAT1-CC may be able to overcome HCV resistance to IFN and enhance the immune clearance of infected hepatocytes due to high level surface HLA-1 expression.
Materials and Methods
Development of IFN-γ Resistant Huh-7 Cell lines
Interferon resistant replicon cells were generated in our laboratory by a prolonged treatment of low inducer replicon cell lines (Con-15, Con-17, and Con-24) with IFN-α as described previously [21]. A cured Huh-7 cell line with a defective Jak-STAT pathway (R-Huh-7) was prepared from an IFN-αresistant replicon cell line (R-24/1) after repeated treatment with cyclosporine-A (1 µg/ml) as described previously [21]. Interferon sensitive cured Huh-7 cells (S-Huh-7) were prepared using the 5-15 replicon cell line after treatment with IFN-α. Interferon sensitive and interferon resistant phenotypes in the cured S-Huh-7 and R-Huh-7 cells were examined by measuring their ability to activate an ISRE-luciferase promoter in the presence of exogenous IFN-α (Schering, Kenilworth, NJ). The expression of functional Jak-STAT signaling proteins in these cells after IFN-α treatment was examined by western blot analysis of phospho STAT1, and phospho STAT2. All the resistant cell lines displayed defects in the phosphorylation of STAT1, and STAT2 proteins, whereas the S-Huh-7 clone showed high-level phosphorylation of STAT1, and STAT2 proteins within 30 minutes of IFN-α treatment. All Huh-7 cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 µg/ml of streptomycin and 5% fetal bovine serum. IFN-α resistant HCV 1b replicon cell lines were first tested for their ability to activate GAS promoter using GAS-luciferase reporter plasmid obtained from Washington University [23]. Replicon cell lines that showed low activation of the GAS promoter, following IFN-γ treatment were selected by culturing in the presence of 1000 IU/ml IFN-γ (PeproTech Inc. Rocky Hill, NJ) for more than four months. The eight IFN-γ resistant replicon cells were then named GR15-1, GR15-2, GR15-3, GR17-1, GR17-2, GR17-3, GR24-1, and GR24-2. Following the IFN-γ selection, the cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 5% fetal bovine serum and 1 µg/ml of G418 for one month. The IFN-γ sensitive replicon cell line was called S9-13. The IFN-γ resistant nature of the cells was then confirmed by the following methods.
Conformation of IFN-γ Resistance
Analysis of STAT1 dependent gene expression
The first confirmation step involved the transfection of eight IFN-γ resistant and one sensitive cell line with a GAS- Luciferase reporter construct. All plasmid transfections were performed with FuGENE-6 transfection reagent (Roche Diagnostic Corporation, Indianapolis, IN) according to the manufacturer's instructions. The optimal ratio, which was used for all transfection experiments, was 3 µL of Fugene-6 transfection reagent to 1 µg of plasmid DNA. One µg of pGAS-Luc plasmid and 0.5 µg of a renilla luciferase plasmid control was transfected by FuGENE-6 to the sensitive and resistant cells in a 24 well plate according to the manufacturer's specifications. 1000 IU/ml of IFN-γ (PeproTech Inc. Rocky Hill, NJ) was then added at the time of transfection to the appropriate groups. All experiments were performed in triplicate. At 24 hours post-transfection the media was aspirated and 100 µL of 1× reporter lysis buffer (Promega Corporation, Madison, WI) was added to each well and incubated at 37°C for ten minutes. The lysates were then centrifuged at 12,000 rpm for five minutes, and the supernatant was transferred to a new set of tubes. Twenty µg of cell lysate supernatant was added to 100 µL of Firefly-Luciferase assay reagent (Promega Corporation, Madison, WI) and luciferase activity was measured by integrating the total light emission over ten seconds with a luminometer (Luman LB9507, EG&G Bethold, Berlin, Germany).
Ribonuclease Protection Assay for Negative Strand HCV-RNA
The IFN-γ resistance of the two cell lines with the lowest GAS induction following IFN-γ treatment from the previous experiment was then evaluated by the ribonuclease protection assay (RPA). The resistant cell lines GR15-3 and GR17-1 were then plated into two 100 mm plates. At approximately 50% confluence 1000 IU/ml of IFN-γ (PeproTech Inc. Rocky Hill, NJ) was added to one plate from each cell line. At 72 hours after interferon addition the total RNA was isolated via the GITC method. 20 µg of total RNA was added to 1×106 cpm of a sense probe targeting the highly conserved 5′ untranslated region of HCV genotype 1b and incubated at 42°C overnight. The next morning the mixture was treated with RNase A/T1 (1∶200) at 37°C for one hour. This digestion was then terminated by the addition of 2.5 µL of SDS and 10 µL of proteinase K. The digested reaction mixture was extracted with phenol/chloroform, precipitated and analyzed by gel electrophoresis in a 6% denaturing TBE-Urea gel (Invitrogen, Carlsbad, CA). The gel was then dried and exposed on X-Ray film (Kodak Biomax-XAR, Rochester, NY).
Immunocytochemical staining
The GR17-1 cells were seeded at a density of 1×105 in a 12 well plate. The next day the cells were treated with or without 1000 IU/ml IFN-γ. At 72 hours following IFN-γ treatment the replicon cells were mounted onto a glass slide via the cytospin method. The cells were then washed twice with PBS pH 7.4 for five minutes. After air-drying, the cells were fixed in chilled acetone for five minutes. Next, cells were permeabilized by treatment with 0.05% saponin for ten minutes at room temperature. Blocking was then performed utilizing five percent of normal goat serum (Sigma Chemical Company, St. Louis, MO) diluted in DMEM containing 5% FBS for 30 minutes at room temperature. Endogenous biotin was then blocked according to the manufacturer's instructions using the Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA). The cells were then incubated with monoclonal anti-NS3 antibody (Vector Laboratories, Burlingame, CA) at a 1∶50 dilution for two hours at room temperature. Following the primary antibody incubation, the cells were washed three times in PBS and incubated with an anti-mouse biotin conjugated antibody (Vector Laboratories, Burlingame, CA) at a 1∶1000 dilution for one hour at room temperature. Following the secondary antibody incubation, the cells were incubated for 30 minutes with Elite avidin-biotin peroxidase complex (Vector Labs, CA). Next, the cells were treated with diaminobenzidine (DAB) chromogen (Dako Cytomation, Carpinteria, CA) for five minutes. The slides were then counterstained with hematoxylin for one minute, dehydrated, mounted and observed by light microscopy.
HLA-1 Surface Expression in Sensitive and Resistant Cells
Resistant (GR17-1) and sensitive (S9-13) replicon cells were seeded at a density of 1×105 in a six well plate. 24 hours later the cells were transfected according to the previously described method. At 48 hours post- transfection the cells were suspended in 100 µL of phosphate buffered saline (PBS) and 20 µL of phycoerythrin conjugated mouse anti-human HLA-A,B,C [HLA-1(Human Leukocyte Antigen-1)] (Pharmingen, San Jose, CA) and incubated for 15 minutes at 4°C. Following the incubation, the cells were re-suspended in 500 µL of PBS, and analyzed by a BD LSR-II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) using BD FACS Diva software.
Plasmid Constructs and Transfection
Three different STAT1 plasmid constructs were used in a transient transfection assay to study GAS promoter activation in the IFN-γ resistant cells. The first plasmid called the pRC-CMV-STAT1 contains the full-length STAT1 protein under the control of a CMV promoter. The second plasmid, pRC-CMV-STAT1-CC contains the full-length STAT1 coding sequences with Ala-656 to Cys-656 and Asn-658 to Cys-658 substitutions. The third plasmid, pRC-CMV-STAT1-CC-Y701F contains a mutation with Y701F substitution used as control for phosphorylation at the amino acid 701 positions. Three different STAT3 plasmid constructs were also used as control to determine the specificity of STAT1 signaling in the transfected cells. STAT3 contains the full-length wild type STAT3 protein also under the control of a CMV promoter. The STAT3-CC construct contains double cysteine-substituted residues in the SH2 Domain of STAT3 at residues 661 and 663. The STAT3-CC-Y705F also contains the double cysteine substituted residues plus a phenylalanine substitution at residue 705. All six plasmids were obtained as a gift from the laboratory of Dr. David A. Frank (Dana-Farber Cancer Institute, Boston, MA) [24]. To study the role of STAT1-CC nuclear translocation we have used full-length STAT1-GFP clone (Addgene Inc, Cambridge, MA). The plasmids pSTAT1-CC-GFP (pCAGG STAT1-CC-GFP) and pGAS-luciferase plasmids were provided by Michael J Holtzman laboratory at Washington University School of Medicine, St. Louis, Missouri [23]. The pRL-Renila luciferase plasmid was obtained from Promega (Promega Corp, Madison, WI).
Analysis of STAT1 Phosphorylation by Co-immunoprecipitation
The tyrosine residue 701-phosphorylation status of the GFP constructs was analyzed in resistant and sensitive cell lines by co-immunoprecipitation. The cells were transfected via FuGENE-6 transfection reagent in a 10-cm plate at approximately 50% confluence with ten µg of each of the GFP tagged plasmids. At 72 hours post-transfection, the cells were treated with or without IFN-γ. Forty-five minutes after the addition of interferon the cells were washed twice with ice cold PBS. The cells were then lysed by the addition of 500 µL RIPA buffer with proteinase and phosphatase inhibitors (1× PBS, 1% NP-40, 0.5% Deoxycholate, 0.1% SDS, 50 µg/ml PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin). The cell lysate was then sonicated at max energy for three pulses of five seconds each. The lysates were then centrifuged at 12,000 rpm for five minutes and the supernatant was transferred to a new tube. 500 µg of total protein was used for each Co-IP reaction with the final volume adjusted to 1 µg/µl with the addition of deionized water. Four µg of GFP primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to each Co-IP reaction and rotated at 4°C overnight. The next morning 40 µl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added to each sample and rotated at 4°C for three hours. The samples were then centrifuged at 3000 rpm for one minute at 4°C and the supernatants were discarded. The samples were then washed with 500 µl RIPA buffer for ten minutes at 4°C and centrifuged at 3000 rpm for one minute for a total of three cycles. The supernatant was discarded and the samples were resuspended in 25 µl of loading buffer. Next, samples were then boiled for five minutes centrifuged at 12,000 rpm for five minutes and the supernatant was transferred to a new tube. 7.5 µl of 4× NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) and 3 µl of the 10× NuPAGE sample reducing agent (Invitrogen, Carlsbad, CA) were then added to each sample and heated at 70°C for 10 minutes. The samples were then loaded into a NuPAGE Novex 4-12% Bis-Tris gel 1.0 mm with 12 wells (Invitrogen, Carlsbad, CA). The proteins were then transferred to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Pittsburgh, PA). Following the gel transfer, the membrane was stained with five times dilute Poncheau's reagent for ten minutes and thoroughly washed with deionized water until the pink bands clearly appeared.
Western blot analysis
The membrane was blocked in 10 ml of filtered blocking solution [PBS, 0.05% Tween 20, 5% Non fat dried milk, NFDM] for 1–2 hours with gentle shaking at 4°C. Next, membrane was washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) twice for five minutes each. The phospho-STAT1 primary antibody (Cell Signaling Technology, Danvers, MA) was diluted (1∶1000) in blocking reagent, (0.1% Tris buffered saline tween 20, and 5% NFDM) added to the membrane, and incubated at 4°C overnight with gentle shaking. The next day the membrane was washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) three times for five minutes each. The anti-Rabbit IgG HRP labeled secondary antibody (Cell Signaling Technology, Danvers, MA) was diluted (1∶2000) in blocking reagent (0.1% Tris buffered saline tween 20 and 5% NFDM), added to the membrane and incubated at 4°C for two hours with gentle shaking. The membrane was again washed with 15 ml of wash buffer (PBS, 0.05% Tween 20) three times for five minutes each. ECL detection reagent (GE Healthcare Life Sciences, Piscataway, NJ) was then added to the membrane according to the manufacturer's instructions. The membrane was finally exposed on chemiluminescence film (GE Healthcare Life Sciences, Piscataway, NJ) for 30 seconds.
Nuclear Translocation Assay
Cured resistant (GR17-1) and cured sensitive lines were plated in a two well Lab-Tek chamber slide (Electron Microcopy Sciences, Hatfield, PA) at a density of 5×104 cells per ml. Twenty four hours later the cells were transfected with 1 µg of the respective STAT1-GFP plasmid. At 24 hours post- transfection To-Pro3 nuclear marker (Invitrogen, Molecular Probes, Oregon) was added to the samples at 1 µg/ml, and incubated for five minutes in PBS. IFN-γ (1000 IU/ml) was then added to the appropriate groups. Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Optical slices were collected at 512×512 pixel resolution. NIH Image version 1.62 and Adobe Photoshop version 7.0 were used to assign correct colors of channels collected, including the Green Fluorescent Protein (green), To-Pro3 633 (far red), and the differential interference contrast image (DIC) (gray scale). Final magnification is indicated in the figures with a bar.
Infectivity Assay
Stable cell lines were created for STAT1 and STAT1-CC in the IFN-γ resistant cured cell line (GR17-1) and the IFN-γ sensitive cured cell line (S5-15) by treatment with cyclosporine as previously described [21]. The effect of the engineered STAT1 constructs on the production of full length infectious HCV were examined by a multicycle infectivity assay as previously described [22]. Interferon sensitive and resistant stable Huh-7 cell lines containing STAT1 and STAT1-CC were infected with full-length JFH1 HCV at a multiplicity of infection of one. IFN-γ (1000 IU/ml) was added to the appropriate groups at the time of infection. After 96 hours of infection, total RNA from the infected cells was isolated by the GITC method [21]. Two micrograms of total RNA was then reverse-transcribed, and quantified by RT-qPCR utilizing the following primer sets and probe Sense: 5′-TCTTCACGCAGAAAGCGTCTA-3′, Anti-Sense 5′-CGGTTCCGCAGACCACTATG-3′, Taq-man FAM labeled probe 5′-/56-FAM/TGAGTGTCG/ZEN/TGCAGCCTCCAGGA/3IABκFQ/-3′. A CFX96 Real Time instrument with CFX manager software (Bio Rad, Hercules, CA) was used to amplify and analyze the samples.
MTT Assay
The toxicity of each STAT1 construct was evaluated by the MTT assay. 2×104 IFN-γ resistant cells were plated in a 24 well plate. After 24 hours, the media was replaced with 500 µL of DMEM supplemented with 2% FBS. One hour after the media change each well was transfected with 1 µg of STAT1 plasmid, 3 µL of FuGENE-6 transfection reagent (Roche Diagnostics Corporation, Indianapolis, IN), and 30 µL of serum free media according to the manufacturer's recommendations. The experimental controls included cells only, and cells plus FuGENE-6 transfection reagent only. At ten hours post-transfection, 500 µL DMEM containing 10% FBS was added to each well. The MTT solution (Sigma-Aldrich, St. Louis, MO) was then prepared by dissolving 5 mg of the powder (Sigma catalogue #M5655) in 1 mL of distilled water, and filtered through a 0.2 µm filter and stored at 2–8°C until use. At 48 hours post-transfection 100 µL of the MTT solution was added to the media in each well, including an additional control well containing only 1 mL of media without cells. The cells were then incubated at 37°C for three hours. The media was aspirated and 1 ml of acidic isopropanol (0.1N HCL in absolute isopropanol) was added to each well including the cell free media only control well. The absorbance of each sample was then measured at 570 nm utilizing a spectrophotometer. The percent viability was then calculated utilizing the formula (value of sample/mean value of control cells only).
Results
Development of IFN-γ resistant HCV replicon cell line
IFN-α is a key component of the standard treatment for chronic HCV infection. However, the development of resistance to interferon therapy is a major obstacle in curing chronic HCV infection. Previously we have developed IFN-α resistant cell lines in an attempt to understand the contribution of viral and host cellular factors in the mechanisms of IFN resistance. Subsequently we have utilized the IFN-α resistant cell lines as model systems to develop alternative strategies to overcome IFN resistance mechanisms. These cell lines contain defective Jak-STAT signaling due to the expression of a truncated IFNAR1 that leads to impaired STAT1 and STAT2 phosphorylation and an ineffective antiviral response. IFN-γ is also important in the innate antiviral immune response against hepatitis C. IFN-γ therapy has been unsuccessful in the treatment of chronic HCV infections that are resistant to IFN-α
[25]–[27]. The precise molecular mechanism underlying this phenomenon is unclear. Since IFN-γ has been shown to inhibit HCV replication effectively in cell culture first we examined if IFN-γ could inhibit HCV replication in IFN-α resistant replicon cells. It was found that all IFN-α resistant replicon cell lines survived the IFN-γ treatment and formed resistant cell colonies. These experiments suggested that the cells that were IFN-α resistant also remained resistant to IFN-γ treatment. The activity of the GAS promoter in these stable replicon cell lines was determined in a transient transfection assay. The results presented in
Fig. 1A
, suggest that there was significant variation in GAS promoter activation between the sensitive and resistant replicon cells. We also found substantial variation of GAS promoter activation among the nine different HCV 1b replicon cell lines. Among the resistant cell lines the GAS promoter activity of GR15-3 and GR17-1 cells was the lowest. The levels of HCV RNA and protein were examined after IFN-γ treatment to provide a more detailed analysis of the resistant nature of the two cell lines. The GR15-3 and GR17-1 replicon cell lines were treated with IFN-γ for 72 hours and total RNA was probed for HCV RNA levels by RPA. The results presented in
Fig. 1B
, suggest that both of these cell lines displayed no reduction in viral RNA following IFN-γ treatment. Immunocytochemical staining for HCV NS3 protein in GR17-1 cells treated with IFN-γ was used as the final confirmation of IFN-γ resistance. Treatment with IFN-γ had no effect upon viral protein levels thus confirming the resistance of the GR17-1 line (
Fig.1C
). As a result, the GR17-1 cell line was used as the model system for IFN-γ resistance. IFN-γ signaling is mediated by Jak1 and Jak2 tyrosine kinases. IFN-γ binding to the receptor (IFNGR) phosphorylate STAT1 molecule which then subsequently homodimerizes to form the gamma activated factor (GAF) complex. This factor then binds to GAS elements in IFN-γ inducible promoters. Some of the GAF is also formed following IFN-α stimulation, which explains the ability of both types of IFNs to activate genes with GAS sites and their partially overlapping functions [23]. The phosphorylation of Jak1, Jak2 and STAT1 was examined in the sensitive and resistant line by western blot analysis. The results shown in
Fig. 2
suggest a lack of phosphorylation of Jak1, Jak2 and STAT1 in the resistant cell lines compared to the 9-13 sensitive cell line. These results support our conclusion that IFN-γ resistant replicon cells have defective STAT1 phosphorylation and nuclear translocation.
10.1371/journal.pone.0013117.g001Figure 1 Confirmation of the IFN-γ resistance of HCV 1b replicon cell lines.
(A) The GAS-luciferase activity in the sensitive and resistant replicons. One sensitive and eight resistant cell lines were seeded in 24 well plates. The next day the cells were transfected with 1 µg of GAS-firefly luciferase and 500 ng of renilla luciferase plasmid using the FuGENE 6 transfection reagent and then treated with or without IFN-γ (1000 IU/ml). The cells were then assayed for GAS promoter induction 24 hours after the addition of IFN-γ. The GAS firefly luciferase value of each well was normalized with a renilla luciferase control. The values were expressed as fold change in GAS luciferase expression after IFN-γ addition. Error bars represent Standard Error of the Mean (SEM) from six independent experiments. (B) RPA for negative strand RNA showing the replication of HCV in the resistant replicon with and without IFN-γ after 72 hours. Two IFN-γ resistant cell lines were treated with or without IFN-γ (1000 IU/ml). At 72 hours post-transfection total RNA was isolated by the GITC method and subjected to RPA for detection of the positive sense strand of the HCV genotype 1b 5′UTR. (C) Immunostaining of NS3 proteins of HCV using GR17-1 cells treated or not treated with IFN-γ for 72 hours. The IFN-γ resistant GR17-1 cells were treated with or without IFN-γ. At 72 hours the cells were mounted onto a glass slide, stained for HCV NS3 (DAB), and counterstained with hematoxylin.
10.1371/journal.pone.0013117.g002Figure 2 Western blot analysis showing low level p-Jak1 and p-Jak 2 expression in the GR17-1 cells and no STAT1 phosphorylation following IFN-γ treatment.
Resistant and sensitive cell lines were treated with and without IFN-γ. Thirty minutes after IFN-γ addition, protein lysates were obtained and quantified. Ten micrograms of protein lysates were subjected to western blot analysis using phospho specific antibodies. Phosphorylation of Jak1, Jak2 and Stat1 was observed in sensitive Huh-7 (S9-13) cells after treatment with IFN-γ. These proteins were not detected in GR17-1 resistant replicon cell lines after IFN-γ treatment.
STAT1-CC activates GAS promoter in resistant HCV replicon cells in an IFN-γ dependent manner
We tried to determine whether we could overcome the defective Jak-STAT signaling and interferon resistance in HCV cell culture by intracellular expression of a modified STAT1 protein as described previously [23]-[24]. We generated a mutant plasmid clone (STAT1-CC) with double-cysteine substitutions in the C-terminal domains of the STAT1 molecule at the amino acids 656 and 658 as illustrated in
Fig. 3A-i
. This mutation was expected to allow for spontaneous disulfide bonding and STAT1 homodimerization as described for STAT3 [25]. To determine whether the presence of cysteine residues is sufficient to allow for functional activation in the absence of tyrosine phosphorylation, we used a STAT1-CC mutant containing an Y701F substitution. The STAT1 molecule expressed from this construct cannot be phosphorylated at residue 701; therefore this control will determine whether phospho-tyrosine 701 is essential for STAT1-CC dimerization. We also used three different constructs for the STAT3 molecules as a control as shown in
Fig. 3A–ii
, to determine if the defective Jak-STAT signaling in the resistant replicon cell line can be overcome specifically by the modified STAT1 protein. To assess if the disulfide substituted STAT1 construct efficiently translocates to the nucleus, we used three types of STAT1 constructs containing c-terminal green fluorescence protein (GFP) fusions (
Fig. 3A–iii
). The STAT1 GFP fusion constructs were also prepared to study their ability for nuclear translocation in the GR17-1 resistant cell line under a fluorescence microscope. In the first step, we examined whether intracellular expression of STAT1-CC after plasmid DNA transfection could improve the STAT1 signaling in the IFN-γ resistant replicon cells. GR17-1 resistant replicon cells were transfected with the wild type STAT1, STAT1-CC and STAT1-CC (Y701F) mutant plasmid along with GAS-luciferase reporter (
Fig. 3B–D
). After 24 hours, the activity of the GAS reporter in the cell lysates with or without treatment with IFN-α and IFN-γ was determined by the luciferase assay. We found that that intracellular expression of STAT1, STAT1-CC or STAT1-Y701F did not induce GAS promoter in the resistant cells. We then examined activation of the GAS promoter in the transfected cells by the addition of either IFN-γ or IFN-α. The results shown in
Fig. 3B
suggest that GAS promoter activity was substantially increased in the cells after treatment with IFN-γ for STAT1-CC. IFN-α did not increase GAS promoter activity of cells transfected with STAT1-CC suggesting that the activation is IFN-γ dependent. The activation of the GAS-luciferase in the resistant cells is dependent upon tyrosine phosphorylation at residue 701 since no GAS induction activation was observed in cells transfected with the STAT1-CC-Y701F construct. In the second step of our analysis, we asked the question whether the activation of the GAS promoter in the transfected GR-17 resistant cells is specific to the modified STAT1-CC molecule. For this purpose, resistant cells were transfected with three sets of STAT1 constructs (STAT1, STAT1-CC, STAT1-CC-Y701F) and three sets of STAT3 constructs (STAT3, STAT3-CC, STAT3-CC-Y705F) and their activation after IFN-γ treatment was examined. The results presented in
Fig. 3C
suggest that only the engineered STAT1CC could activate GAS-luciferase activity in the resistant cells. The modified STAT3-CC construct did not induce GAS-luciferase activity in resistant Huh-7 cells following IFN-γ treatment. In these experiments we found that the STAT1-CC molecule was able to activate GAS promoter more effectively than the wild type STAT1 protein, but that the activation is IFN-γ treatment dependent. In the third set of experiments, we examined whether the activation of the GAS-promoter in the transfected cells is concentration dependent. The results presented in
Fig. 4D
suggest that the activation of GAS-luciferase is concentration dependent. All of the STAT1 constructs exhibited a dose dependent increase in RLU over the experimental dose range. In the fourth set of experiments we evaluated the kinetics of GAS promoter induction between STAT1-CC and wild type STAT1 at various time points up to 48 hours post-transfection. No noticeable differences were observed between the two groups until the 24 hour time point when the STAT1-CC transfected cells showed a marked increase in GAS promoter induction versus wild type STAT1. In the STAT1-CC transfected cells, an interesting phenomenon occurred at the 48 hours time point when GAS expression had increased from the 24 hour time point whereas the STAT1 cells exhibited lower GAS luciferase expression than the 24 hour time point (
Fig. 3E
). Furthermore, the difference in GAS expression between these two groups reached statistical significance (p<0.05, Students t-test) at the 48-hour time point.
10.1371/journal.pone.0013117.g003Figure 3 IFN-γ dependent activation of GAS promoter by the STAT1-CC molecule in the resistant (GR17-1) replicon.
(A) Summary of different modified STAT plasmid constructs used in this project. (i) Shows pRC-CMV plasmid containing human full-length wild type STAT1 cDNA, STAT1-CC containing two cysteine substitutions in the SH2 Domain of STAT1 at residues 656 and 658 and STAT1-CC-Y701F also contained the double cysteine substituted residues plus a phenylalanine substitution at residue 701. (ii) Shows the pRC-CMV expression plasmid containing the full-length human wild type STAT3 cDNA. STAT3-CC double cysteine substituted residues in the SH2 Domain of STAT3 at residues 661 and 663, STAT3-CC-Y705F also contained the double cysteine substituted residues plus a phenylalanine substitution at residue 705. (iii) STAT1 constructs with a C-terminal GFP fusion, STAT1-CC-GFP and STAT1-CC-Y701F–GFP plasmid. (iv) Firefly luciferase reporter construct driven by GAS promoters. (B) Show the activation of GAS promoter in resistant cell line transfected with Stat1, Stat1CC and Stat1CC Y-F plasmids. IFN-γ resistant cells (GR17-1) were transiently co-transfected with a GAS-firefly luciferase reporter, the indicated STAT1 construct and a control plasmid expressing renilla luciferase. Firefly luciferase activity was normalized with the transfection control renilla luciferase. Each bar represents the fold increase in GAS promoter expression activity at 24 hours after the addition of IFN. The error bars represent the SEM from six independent experiments. GAS-firefly luciferase activity increased in response to IFN-γ treatment. IFN-α treatment did not induce the GAS promoter. (C) Shows that the activation of the GAS promoter is specific to STAT1-CC. Interferon resistant cells (GR17-1) were transiently co-transfected with a GAS luciferase reporter and one microgram of each of the constructs using the FuGENE 6 reagent. Values were normalized with renilla luciferase and presented as the fold increase in GAS promoter induction between IFN naïve and IFN-γ treated cells 24 hours post-transfection are shown. (D) Dose dependent activation of the GAS promoter by STAT1-CC molecules in the GR17-1 cell line. The STAT1 constructs induce the GAS promoter in a dose dependent manner. Different concentrations (0.5, 1, and 2 µg) of the respective STAT1 plasmid and 0.5 µg of GAS-luciferase were co-transfected to GR17-1 cells by FuGENE 6 reagent and then treated with 1000 IU of IFN-γ. At 24 hours, the luciferase activity was measured and normalized with renilla luciferase as a transfection control. Values represent normalized luciferase expression (RLU) from three experiments. (E) Prolonged GAS-luciferase activity in the resistant cells transfected with STAT1-CC compared to wild type STAT1. At the indicated time points, RLU activity was measured, normalized with renilla luciferase, and represented as fold change in GAS induction after IFN-γ addition.
10.1371/journal.pone.0013117.g004Figure 4 Intracellular STAT1-CC upregulates HLA class I surface expression in IFN-γ resistant (GR17-1) cells.
GR17-1 cells were transfected with STAT1, STAT1-CC or STAT1-CC-Y701F plasmid using FuGENE 6 reagent for 72 hours with or without IFN-γ treatment. After 72 hours, the cells were harvested and stained with a phycoerythrin-conjugated anti-human HLA antibody. Surface HLA-1 expression was then quantified by flow cytometry. (A) Each histogram is representative of six independent experiments. The red cell population in each histogram represents IFN-γ naïve cells and the blue cell population represents IFN-γ treated cells. (B) Mean fluorescence intensity of six independent experiments per group. Values represent fold increase in HLA1 expression after the addition of IFN-γ. Mock represents GR17-1 cells without plasmid transfection. Asterisk (*) denotes a significant (p<0.05) increase in HLA1 in STAT1-CC transfected cells plus IFN-γ compared to mock.
Intracellular expression of STAT1-CC significantly up regulates HLA expression in interferon-γ resistant cells
To verify the results of luciferase based promoter activation, we examined the effect of STAT1-CC expression in the resistant cell line on the constitutive expression of a known IFN-γ responsive gene, HLA-1 [28]–[30]. The expression of HLA class I surface expression was analyzed by flow cytometry in the sensitive (S9-13) and resistant (GR17-1) cell line after IFN-γ treatment. The results shown in
Fig. 4
show that HLA-1 surface expression levels remained constant in the resistant cell line after IFN-γ treatment, whereas surface expression of HLA-1 was up-regulated in the sensitive cell line following IFN-γ treatment. Since immune surveillance of the surface-expressed HLA associated peptide complex and presentation to cytotoxic T cells is an important mechanism of viral clearance, we examined the ability of the STAT1-CC constructs to upregulate HLA-1 surface expression in IFN-γ resistant cells. The resistant replicon cell line GR17-1 was transfected separately with either wild type STAT1, STAT1-CC or STAT1-CC-Y-F plasmid. After 72 hours, expression of HLA-1 in the transfected cells was examined after staining with a monoclonal antibody specific to human HLA-1 antigen. The flow analysis results in
Fig. 4
A and B. show that STAT1-CC plus IFN-γ significantly upregulated HLA-1 expression compared to resistant cells alone (Student's t-test p<0.05). The surface expression levels of HLA-1 remained unchanged for the remaining experimental groups.
Phosphorylation of the STAT1-CC molecule in the resistant cells
In the previous experiments we found that intracellular expression of STAT1-CC in the GR17-1 cells after plasmid DNA transfection is not sufficient to cause GAS-luciferase activation. The activation of GAS-luciferase in the STAT1-CC transfected cells is dependent on IFN-γ treatment. Therefore, we examined the phosphorylation of the STAT1-CC molecule in the transfected cells by co-immunoprecipitation experiments. In these experiments we used both wild type STAT1 and mutant STAT1-CC constructs with GFP tags to monitor the extent of phosphorylation. A sensitive Huh-7 replicon (S9-13) and resistant replicon (GR17-1) cell line was transfected with STAT1-GFP, STAT1-CC-GFP or STAT1-CC-Y701F-GFP plasmid. The phosphorylation of each molecule was then examined by co-immunoprecipitation with a GFP antibody followed by a western blot using an antibody against p-STAT1. The results presented in
Fig. 5
suggest that the STAT1-CC molecule was phosphorylated in GR17-1 cells as evidenced by the detection of a distinct band in the immunoblot analysis of STAT1-CC transfected cells after IFN-γ treatment only. Neither the wild type STAT1 nor the STAT1-CC-Y701F construct showed any evidence of phosphorylation in GR17-1 cells. Furthermore, it was found that STAT1-CC was phosphorylated at a very high level in the sensitive 9–13 cells with or without IFN-γ treatment (
Fig. 5
). In the sensitive cells, STAT1-GFP was also phosphorylated after IFN-γ treatment. These findings are consistent with the results of the GAS-luciferase promoter assay induced by the wild type and mutant STAT1 constructs in the resistant cell line.
10.1371/journal.pone.0013117.g005Figure 5 Comparison of spontaneous and IFN-γ dependent phosphorylation of STAT1-CC molecule in sensitive (S9-13) and resistant (GR17-1) cells.
Cells were transfected with individual plasmid constructs of either STAT1-GFP, STAT1-CC-GFP, or STAT1-CC-Y701F–GFP. IFN-γ was added to the appropriate groups at 72 hours post-transfection. Thirty minutes after IFN-γ addition, the protein lysates were immunoprecipitated using an anti-GFP antibody. Immunocomplexes were detected using an antibody to phospho-STAT1 by western blot analysis. Upper panel: Preferential phosphorylation of STAT1-CC over the STAT1 molecule in IFN-γ sensitive Huh-7 cells (S9-13). IFN-γ dependent phosphorylation was seen for both wild type STAT1 molecule as well as Stat1CC molecule. Lower panel: The STAT1-CC showed low level phosphorylation of Stat1CC in the resistant cells in IFN-γ treated cells.
Nuclear translocation of STAT1-CC-GFP in the resistant cells is dependent on tyrosine phosphorylation
We examined whether the low level of phosphorylation of the STAT1-CC construct in the resistant cells was responsible for nuclear translocation of STAT1-CC-GFP molecule in the resistant cell. Both S9-13 and GR17-1 cells were transfected with STAT1-GFP, STAT1-CC-GFP or STAT1CC-Y701F-GFP constructs and their nuclear translocation was examined under a confocal microscope with or without IFN-γ treatment. The results of these experiments are shown in
Fig. 6
. In the sensitive cell line, the STAT1-GFP chimera protein was expressed primarily in the cytoplasm and subsequently translocated to the nucleus 30 minutes following IFN-γ treatment. The STAT1-GFP was unable to localize to the nucleus following IFN-γ treatment in the resistant cell line. In contrast, the STAT1-CC-GFP construct efficiently localized to the nucleus within 30 minutes of IFN-γ addition in both sensitive and resistant cell lines. There were no differences in the nuclear translocation of the STAT1-CC-GFP molecule in the sensitive and resistant cells with the addition of IFN-γ. The translocation of the STAT1-CC-GFP chimera in the sensitive and resistant cell was phosphorylation dependent since we did not observe nuclear translocation of STAT1-CC-GFP protein with Y701F substitution.
10.1371/journal.pone.0013117.g006Figure 6 STAT1-CC-GFP translocates to the nucleus of GR17-1 cells in an IFN-γ dependent manner.
IFN-sensitive (S9-13) and IFN-resistant (GR17-1) cells were transfected with plasmid containing STAT1-GFP, STAT1-CC-GFP and STAT1-CC-Y701F-GFP. The nuclear translocation of each STAT1-GFP construct with or without IFN-γ treatment in the sensitive and resistant cells was examined under a confocal microscope. (A) Shows high resolution picture of Stat1-GFP nuclear translocation after IFN-γ treatment in the sensitive Huh-7 (S9-13) cells. The images are represented as the superimposition of Green Fluorescent Protein (green), To-Pro3 633 (far red), and the differential interference contrast images (DIC) (gray scale). (B) Shows the differential nuclear translocation of STAT1-GFP, STAT1-CC-GFP and STAT1-CC-Y701F-GFP protein in the sensitive (S9-13) versus resistant Huh-7 (GR17-1) cells before and after treatment with IFN-γ. Fluorescence green and red microscopic picture of the same area were taken and superimposed using Abode Photoshop.
Enhanced viral clearance in cell culture by intracellular expression of modified STAT1-CC molecule
The modified STAT1-CC molecule is able to activate the GAS promoter more effectively than the wild type STAT1 molecule in the resistant cells. Therefore, we investigated whether intracellular expression of modified STAT1-CC could overcome IFN-γ resistance and induce an HCV antiviral response. Resistant replicon cells were transfected with STAT1-CC expression plasmid and then treated with IFN-γ for 72 hours. The HCV negative strand RNA level in the transfected cells was then examined by the RPA method. The results of these experiments shown in
Fig. 7
, suggest that STAT1-CC effectively abolished HCV RNA replication in an IFN-γ dependent manner. The inhibition of HCV replication was not observed in cells transfected with either the STAT1 construct or the STAT1-CC construct with an Y701F substitution. GAPDH mRNA levels remained constant in all of the samples tested suggesting that the antiviral effect was due to the intracellular expression of STAT1-CC protein. To verify these results, immunostaining was performed to examine viral NS3 protein levels in the transfected resistant GR17-1 cells at 72 hours post-transfection. These results shown in
Fig. 8
demonstrate potent antiviral action in the IFN-γ treated, STAT1-CC transfected culture, while the controls showed no decrease in viral NS3 protein levels. We also examined if this antiviral strategy can be extended to eliminate cell culture grown full-length infectious virus in the IFN-γ resistant cell line (GR17-1). The antiviral effect of IFN-γ against cell culture grown full-length HCV was also examined using interferon sensitive Huh-7 cells. Cured 5-15 Huh-7 cells were cultured in 6-well plates and infected with cell culture derived full-length HCV at a MOI of one. After 72 hours of infection, the culture was treated with increasing dose of IFN-α or IFN-γ. Antiviral effect was determined after 72 hours by measuring the HCV RNA titer by real-time RT-PCR. The results in
Fig. 9A
demonstrate a dose dependent increase in antiviral activity of IFN-γ against full-length HCV. We then examined the ability of stably expressed STAT1 or STAT1-CC proteins in the cured GR17-1 Huh-7 cells to inhibit replication of full-length infectious virus. The results of the infectivity assay in the engineered STAT1 sensitive (S5-15) and resistant (GR17-1) stable cell lines are shown in
Fig. 9B
. The control GR17-1 cured IFN-γresistant cell line showed no reduction in viral RNA after the addition of IFN-γ (1000 IU/ml). The stable STAT1 expressing GR17-1 cell line showed a modest reduction in HCV RNA with the addition of IFN-γ. In contrast, the stable STAT1-CC expressing GR17-1 cell line showed a significant (p<0.02) reduction in HCV RNA after the addition of IFN-γ. The sensitive STAT1-CC expressing stable cell line also showed a significant (p<0.02) reduction in HCV RNA level after IFN-γ treatment. In order to determine if the viral clearance is due to a toxic effect of intracellular expression of the STAT1-CC molecule, cell viability was determined by the MTT assay. The results in
Fig. 10
show that the viability (as a percentage of control) of the STAT1 transfected resistant cells was 96.5 and the addition of IFN-γ had no significant effect on cell viability. The STAT1-CC transfected cells exhibited intermediate cytotoxicity with 93.7% viability and this number dropped to 86.3 percent with the addition of IFN-γ. The STAT1-CC-Y701F transfected cells exhibited the most toxicity with 88% of cells remaining viable, and 85% of cells remained viable after the addition of interferon. To search for an explanation for the potent antiviral activity of STAT1-CC molecule in the resistant replicon cells, western blot analysis was performed of two targets, p-PKR and p-EIF2α. IFN-γ treatment induced high levels of phosphorylated PKR and phosphorylated eIF-2 alpha in cells expressing STAT1-CC where as STAT1 and STAT1-CC-Y701F expressing cells did not induce these targets (
Fig. 11
). In summary, these results suggest that the intracellular expression of STAT1-CC induced a potential antiviral response in an IFN-γ dependent manner that involves the activation of PKR and eIF-2α phosphorylation.
10.1371/journal.pone.0013117.g007Figure 7 RPA assay demonstrates that STAT1-CC plus IFN-γ eliminates HCV negative strand RNA in resistant replicon cells.
IFN-γ resistant (GR17-1) cells were transfected with the respective plasmid and treated with or without IFN-γ. At 72 hours post-transfection total RNA was isolated by the GITC method and subjected to RPA analysis for HCV negative strand RNA using a probe targeted to the highly conserved 5′-UTR region. The GAPDH mRNA was used as loading control. The nucleotide number in each blot shows the length of the protected fragment.
10.1371/journal.pone.0013117.g008Figure 8 STAT1-CC plus IFN-γ transfected resistant (GR17-1) cells display a marked reduction in HCV NS3 protein.
IFN-γ resistant cells were transfected separately with the respective STAT1 plasmid and treated with or without IFN-γ. At 72 hours the cells were mounted onto a glass slide, stained for with DAB for HCV NS3 (brown), and counterstained with hematoxylin (blue). Huh7 cells without virus were used as a negative control.
10.1371/journal.pone.0013117.g009Figure 9 Effect of STAT1-CC and IFN-γ on cell culture grown full-length HCV.
Stable sensitive (S5-15) and resistant (GR17-1) cell lines with or without STAT1 or STAT1-CC were infected with full length HCV genotype 2a clone JFH1 virus at MOI of 1.0. After 72 hours cells were treated with IFN-γ. At 72 hours post-infection total RNA was isolated, positive strand HCV RNA level was measured by real-time RT-PCR. (A) Antiviral effect of IFN-γ and IFN-α against cell culture grown full-length HCV. (B) Demonstrate the antiviral effect of Stat1-CC expression on cell culture derived full-length HCV RNA in sensitive and resistant cells due to IFN-γ treatment. Bars are representative of three independent experiments and error bars represent standard error of the mean. Significance was considered at values below 0.05 (p<0.05, student t-test). Asterisks (*) represent significant (p<0.02) reductions in HCV RNA.
10.1371/journal.pone.0013117.g010Figure 10 The cytotoxicity of intracellular expression of each STAT1 molecule in the resistant cells.
The GR17-1 cell line was transfected with each of the STAT1 constructs and the cellular toxicity was performed at 48 hours post-transfection by the MTT assay. The values represent viable cells as a percent of cells plus FuGENE-6 control. Bars represent SEM from three experiments.
10.1371/journal.pone.0013117.g011Figure 11 Intracellular expression of STAT1-CC induces p-PKR and p-eIF2α in the resistant cell line affer IFN-γ treatment.
The IFN-γ resistant cell line (GR17-1) was transfected with each STAT1 expression plasmid and treated with or without IFN-γ. At 24 hours post-transfection protein lysates were obtained and examined for p-PKR and p-EIF2α levels by western blot analysis. A protein lysate from the S9-13 cell line was obtained 30 minutes after IFN-γ addition and used as a positive control.
Discussion
IFN-α is the standard treatment for chronic hepatitis C virus infection. More than half of chronic HCV patients are unable to clear the virus infection and develop resistance to combination therapy. We have developed multiple resistance replicon cell lines to understand the mechanisms of HCV resistance to IFN-α. We showed that defects in phosphorylation STAT1 and STAT2 proteins led to their impaired nuclear translocation and IFN-α resistance (21–22). This study was performed to examine effect of IFN-γ treatment on the replication of HCV in IFN-α resistant replicon cells. Although IFN-γ has been shown to have potent antiviral activity against HCV in cell culture but it is not very effective in the treatment of chronic hepatitis C patients who are non-responders to IFN-α
[25]. The reason why IFN-γ treatment is not effective in the chronic HCV patients resistant to IFN-α is unknown. Since the antiviral action of IFN-γ is mediated through separate receptors, we tested here whether IFN-γ can inhibit HCV replication in IFN-γ resistant replicon cells. The results of our study suggest that replicon cells that are resistant to IFN-αalso develop resistant to IFN-γ. Through this approach we have now developed IFN-γ resistant stable replicon cell lines. We describe here a novel approach of how to improve the sustained virologic response of HCV infection using IFN-γ in patients who are non-responders to IFN-α.
As a proof-of-principle, we have utilized these IFN-γ resistant cell lines to develop alternative treatment approaches to overcome HCV resistance to IFN in cell culture. Since STAT1 is activated by both type I and Type II IFN stimulations, we therefore examimed whether intracellular STAT1 signaling could be activated by intracellular expression of the modified STAT1-CC molecule to overcome viral resistance to IFN. We showed that intracellular expression of a STAT1-CC molecule induced GAS promoter activity in an IFN-γ dependent manner. Intracellular expression of the engineered STAT1-CC molecule led to phosphorylation and nuclear translocation in resistant replicon cells in an IFN-γ dependent manner. A mechanism of phospho-STAT1 dephosphorylation has been proposed whereby the phospho-STAT1 homodimer undergoes a molecular rearrangement from a parallel to an antiparallel orientation within the nucleus [31]. This molecular rearrangement then exposes the tyrosine residue at position 701 to the activity of phosphatases. Following dephosphorylation, the STAT1 molecule is exported from the nucleus. Zhong et al [31] was able to demonstrate that STAT1 mutants containing mutations in various STAT1 domains were resistant to tyrosine phosphatases in vitro. The increased activity of the STAT1-CC molecule in the resistant cell is likely as a result of a delay of dephosphorylation when compared to wild type STAT1 [23]. Within the cell the STAT1 molecule undergoes a basal level of phosphorylation and dephosphorylation [31]. The increased stability and delay of dephosphorylation of the STAT1-CC molecule shifts this balance of phosphorylation and dephosphorylation toward the phosphorylated state. As a result, the low level kinase activity of Jak 1 and Jak2 observed in the resistant cell line following IFN-γ treatment may be sufficient to generate pSTAT1 levels that induce the GAS promoter. This may explain the IFN-γ dependence of the STAT1-CC molecule within the resistant cell line. We showed that the increased stability of the STAT1-CC molecule led to prolonged transcriptional activity that resulted in increased antiviral and immunomodulatory activities in the interferon resistant cell line. It was found that HCV RNA replication and viral protein expression were effectively inhibited by intracellular expression of the STAT1-CC molecule. Neither wild type STAT1 nor the STAT1-CC-Y701F mutant transfection resulted in a reduction of HCV RNA levels in the resistant cell line. This suggested that the antiviral effect is specific to the STAT1-CC expression. We also showed that intracellular expression of STAT1-CC has limited cellular toxicity since more than 80% cells remained viable. Intracellular expression of SH2 modified STAT1 protein (called STAT1-CC) improves the defective Jak-STAT signaling and eliminates cell culture derived full-length infectious HCV replication in an IFN-α sensitive and resistant hepatic cell line by IFN-γ. Based on the results, we propose that liver targeted delivery of modified STAT1-CC protein can stimulate the antiviral response as well as HLA-1 expression in hepatocytes in an IFN-γ dependent manner.
The results of this study provide a rationale for an alternative antiviral strategy, which can be explored to overcome IFN-α resistance, and to enhance the immune mediated clearance of virus HCV infected cells. Numerous studies have indicated that cellular Jak-STAT signaling initiated by type I interferon appear to be suppressed in chronic HCV infection [32]. A number of clinical studies including the recent HALT-C trial suggest that impaired expression of IFNAR1 is correlated with the response to IFN-α therapy in chronic hepatitis C. Taniguchi et al [33] indicated that high intrahepatic mRNA levels of IFNAR1 and the ratio of IFNAR1 to IFNAR2 were significantly higher in patients having a sustained virological response to interferon therapy. Katsumi et al [34] found that the expression rate of IFNAR1 and IFNAR2 were significantly higher in responders than non-responders. Fujiwara et al [35] have conducted a study where the expression of IFNAR1 receptor and response to interferon therapy was examined in chronic hepatitis C patients. They found that the IFNAR2 expression level in the liver, but not in the PBMC, is predictive of the response to IFN treatment in chronic hepatitis C patients. Meng et al [36] also examined the expression of interferon alpha/beta receptor in the liver of patients with hepatitis C virus related chronic liver disease between interferon responders and non-responders. In this study, the authors found that the expression of the interferon receptor was higher in the IFN-treatment responsive group than in the non-responsive group. Welzel et al [37] analyzed the relationship between variants in the IFN-α pathway and a sustained virologic response (SVR) among participants in the hepatitis C antiviral long-term treatment against the cirrhosis (HALT-C) trial. They found a statistically significant relationship between IFNAR1 expression and response to antiviral therapy in chronic hepatitis C patients. The results of these clinical studies are supported by a recent cell culture study conducted by Liu et al [38] that suggested that HCV infection can lead to impaired cellular Jak-STAT signaling by down regulation of IFNAR1. These studies provide strong evidence on the contribution of defective cellular Jak-STAT signaling in HCV infected hepatocytes upon the interferon antiviral response. Additional studies have shown that IFN-stimulated genes (ISG) in the liver of HCV infected patients are expressed at higher levels pre-treatment in IFN non-responders compared to IFN responders [39]–[41]. In contrast to these observations another study showed little or no evidence of ISG expression in the liver of chronically infected IFN non-responders [41]. In this study the authors found that IFN-α induced STAT1 phosphorylation and nuclear translocation was stronger in the hepatocytes of responders than in non-responders. The activation of STAT1 in the non-responders was primarily observed in the non-hepatic cells (Kupffer cells) [41]. In this study, we showed that intracellular expression of SH2 modified STAT1 protein (called STAT1-CC) improves defective Jak-STAT signaling and eliminates HCV replication in an IFN-α sensitive and resistant hepatic cell line in an IFN-γ dependent manner. As a result, the subset of patients that contain a functionally inactivated IFNAR1, IFNAR2 or other variants of the Jak-STAT pathway that are adversely associated with a sustained virological response may benefit from a liver targeted STAT1-CC therapy. There are new reports indicating that targeted delivery of therapeutic molecules can be achieved using apolipoprotein conjugated liposomes [42]–[43]. We propose that liver targeted delivery of modified STAT1-CC protein can be used as a second line treatment in patients with defective Jak-STAT signaling in an attempt to stimulate an antiviral response as well as increase HLA-1 expression in hepatocytes in an IFN-γ dependent manner.
The authors acknowledge Mallory Schexnayder, and Dr. Astrid Engel for critically reading this manuscript. The authors are very grateful to Yong Zhang, Michael J Holtzman Department of Medicine and Cell biology, Washington University School of Medicine, St. Louis, Missouri for providing the STAT1-CC-GFP plasmid construct used in this investigation, David Frank and Mousumi Chaudhury, Department of Medical Oncology/Hematologic Neoplasia, Dana-Farber Cancer Institute for STAT1, STAT1-CC and STAT1-CC (Y-F) recombinant plasmids.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by funds received from the National Cancer Institute (CA127481, CA129776), Louisiana Cancer Research Consortium, and Tulane Cancer Center. Louisiana Board of Regents provided a graduate fellowship to BP. This work was also supported partially by National Institutes of Health Grant RR000164 (XAH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Med EthicsBMC Medical Ethics1472-6939BioMed Central 1472-6939-11-152084332710.1186/1472-6939-11-15DebateEnd-of-life discontinuation of destination therapy with cardiac and ventilatory support medical devices: physician-assisted death or allowing the patient to die? Rady Mohamed Y [email protected] Joseph L [email protected] Department of Critical Care Medicine, Mayo Clinic Hospital, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, Arizona, 85054, USA2 Center for Biology and Society, School of Life Sciences, Arizona State University, 300 East University Drive, Tempe, Arizona, 85287, USA3 Department of Physical Medicine and Rehabilitation, Mayo Clinic Hospital, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, Arizona, 85054, USA4 Department of Biomedical Ethics, College of Medicine, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, Arizona, 85054, USA2010 15 9 2010 11 15 15 29 3 2010 15 9 2010 Copyright ©2010 Rady and Verheijde; licensee BioMed Central Ltd.2010Rady and Verheijde; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Bioethics and law distinguish between the practices of "physician-assisted death" and "allowing the patient to die."
Discussion
Advances in biotechnology have allowed medical devices to be used as destination therapy that are designed for the permanent support of cardiac function and/or respiration after irreversible loss of these spontaneous vital functions. For permanent support of cardiac function, single ventricle or biventricular mechanical assist devices and total artificial hearts are implanted in the body. Mechanical ventilators extrinsic to the body are used for permanent support of respiration. Clinical studies have shown that destination therapy with ventricular assist devices improves patient survival compared to medical management, but at the cost of a substantial alteration in end-of-life trajectories. The moral and legal assessment of the appropriateness and permissibility of complying with a patient's request to electively discontinue destination therapy in a life-terminating act in non-futile situations has generated controversy. Some argue that complying with this request is ethically justified because patients have the right to request withdrawal of unwanted treatment and be allowed to die of preexisting disease. Other commentators reject the argument that acceding to an elective request for death by discontinuing destination therapy is 'allowing a patient to die' because of serious flaws in interpreting the intention, causation, and moral responsibility of the ensuing death.
Summary
Destination therapy with cardiac and/or ventilatory medical devices replaces native physiological functions and successfully treats a preexisting disease. We posit that discontinuing cardiac and/or ventilatory support at the request of a patient or surrogate can be viewed as allowing the patient to die if--and only if--concurrent lethal pathophysiological conditions are present that are unrelated to those functions already supported by medical devices in destination therapy. In all other cases, compliance with a patient's request constitutes physician-assisted death because of the pathophysiology induced by the turning off of these medical devices, as well as the intention, causation, and moral responsibility of the ensuing death. The distinction between allowing the patient to die and physician-assisted death is pivotal to the moral and legal status of elective requests for death by discontinuing destination cardiac and/or ventilatory medical devices in patients who are not imminently dying. This distinction also represents essential information that must be disclosed to patients and surrogates in advance of consent to this type of therapy.
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Background
Bioethics and law distinguish between the practices of physician-assisted death and allowing the patient to die, such as by the withdrawal or withholding of life-support treatment in an imminently dying patient. Quill [1] used the term physician-assisted death to describe a spectrum of life-terminating medical acts intended to accelerate the dying process and bring about a quick death. Life-terminating acts include voluntary, involuntary, and nonvoluntary active euthanasia, and assisted suicide. Voluntary active euthanasia is the intentional termination of life at the request of the patient or surrogate. Involuntary active euthanasia is the intentional termination of life against the patient's wish. Nonvoluntary active euthanasia is intentionally terminating a patient's life without a request or consent. Prescribing a lethal dose of medication that is ingested by a patient is assisted suicide, whereas giving a lethal dose of a medication to a competent patient who has voluntarily requested to end his or her life constitutes voluntary active euthanasia.
In an acute life-threatening illness or a progressing incurable disease, lethal pathophysiological conditions are set in motion that eventually culminate in death (ie, irreversible cessation of consciousness, respiration, and circulation). In imminently dying patients, these lethal pathophysiological conditions will progress to death despite life-support treatment. Under such circumstances, life-support treatment becomes an impediment to the natural process of dying and physicians withhold and/or withdraw a harmful, ineffective, or burdensome treatment. The term life-support treatment is used to mean support of vital functions of respiration and/or circulation in contrast to the term life-sustaining-treatment, which includes other treatment avenues such as artificial hydration, nutrition, and hemodialysis. When physicians justifiably withdraw or withhold life-support treatment, they allow patients to die but do not cause, intend, or bear moral responsibility for the patient's death. These medical actions may be perceived as passive euthanasia, but they are not truly euthanasia because there is no intent to terminate life.
The literature demonstrates that there is little agreement on the meaning of the term "euthanasia" [2]. Introducing adjectives (ie, active, voluntary, involuntary or nonvoluntary) to the word euthanasia only adds confusion to the debate on end-of-life decisions. We agree with the opinion that clearly-defined terms should be used to describe exactly what actions are taking place (eg, withdrawal of treatment, continuation of care, relief of pain, deactivation of medical devices, allowing to die, assisting to die) so as to improve our understanding of what really goes on during end-of-life decisions [2]. The descriptive term of physician-assisted death (dying) is widely used in the medical literature, replacing other emotive terms such as physician-assisted suicide and euthanasia [3-8]. However, the labeling of end-of-life interventions by physicians as either physician-assisted death or palliation is not uniform in similar cases [9]. Differences in the labeling of similar acts performed in end-of-life care can impede societal control even where physician-assisted death has been legalized. Methodological difference in end-of-life interventions such as discontinuing a medical device, administering a specific type or dose of medication, and the time to death may not be helpful in distinguishing acts of palliation from physician-assisted death [10].
Advances in biotechnology have expanded the use of medical devices for permanent mechanical support of respiration and cardiac function in a process known as destination therapy when these spontaneous vital functions are irreversibly lost. For example, mechanical ventilators are medical devices used for ventilatory support (VS) of patients with permanent apnea in destination therapy; for the latter purpose, such devices often require a tracheotomy. Single ventricle or biventricular mechanical assist devices and total artificial hearts are medical devices implanted for permanent cardiac support (CS) as destination therapy in patients with end-stage heart failure. We have previously highlighted several ethical challenges in regard to end-of-life care and palliation following destination therapy with the left ventricular assist device (LVAD) [11].
The elective deactivation of mechanical cardiac assist devices has different legal and ethical consequences in non-futile situations compared with futile situations [12]. Physicians often grapple with requests from patients to turn off their LVADs in non-futile situations [13]. In this article, we offer a moral and legal assessment of the decision-making process for discontinuing a constitutive treatment of CS with an LVAD as destination therapy in non-futile situations. We illustrate the similarity to discontinuing, at the patient's request, a constitutive (permanent) treatment of VS with a mechanical ventilator as destination therapy. We argue that compliance with the patient's request to terminate his or her life by discontinuing medical devices for VS or CS is physician-assisted death except under specific circumstances. To distinguish physician-assisted death from allowing a patient to die by deactivation of an LVAD, we evaluate pathophysiological consequences, intention, causation, and moral responsibility for the ensuing death after discontinuation of such a medical device. Responsibility for death is often considered an important factor in the moral distinction between terminating life and letting die [14]. This distinction is not only pivotal to the moral and legal status of requests of discontinuing constitutive VS or CS in patients who are not imminently dying, but it also represents essential information that must be disclosed to patients and surrogates in advance of consent to destination therapy with these medical devices. In assessing the moral and legal status, we differentiate between a constitutive life-support treatment with a medical device as destination therapy and temporary life-support treatment in emergency situations for VS or CS for acute life-threatening illnesses. There is a large body of literature on medical, ethical and legal issues in regard to forgoing temporary or emergent life-support treatment in an acute life-threatening illness. The forgoing of temporary or emergent life-support treatment in an acute life-threatening illness is accepted as morally appropriate, even obligatory, if predicated on true informed consent [15]. In contrast, the literature addressing medical, ethical and legal issues of elective discontinuation of constitutive life-support treatment in non-futile situations is limited and remains controversial [12,14,16-18]. In non-futile situations, the moral significance of acts, omissions and responsibilities in end-of-life interventions continues to be debated in the medical literature [19-21].
Discussion
Hypothetical Scenarios
Consider two hypothetical patients: Adam and David. Adam is a 66-year-old man with traumatic quadriplegia who has been totally dependent on a mechanical ventilator through a tracheotomy because of permanent apnea for two years. David is a 67-year-old man being treated for end-stage heart failure with an LVAD (HeartMate II; Thoratec Corp, Pleasanton, California) implanted two years earlier. Both men live at home with their spouses as their primary caregivers. Adam asks his physician to discontinue VS by turning off the mechanical ventilator and administering medication to ensure a peaceful death. David asks his physician to discontinue CS by deactivating the LVAD and administering medication to induce deep sedation so that he can die a "swift and dignified death." We evaluate whether discontinuing constitutive treatment with VS and CS medical devices, a life-terminating act in these two cases, is either physician-assisted death or death justified by an appeal to the rule of allowing the patient to die. In evaluating both hypothetical scenarios, we focus on the decision and action specifically related to turning off the medical device at the request of the patient. The role of administering medication and pharmacologically inducing deep sedation that results in the deaths of Adam and David will not be discussed. We have argued elsewhere that it is physician-assisted death to administer medication for the purpose of inducing continuous deep sedation to fulfill an elective request for death because of psychological, social, and existential distress [7].
Types of Treatment With Biotechnological Medical Devices
A treatment is a therapeutic intervention intended to restore body functions and health. Treatment can be temporary (ie, discontinued because of spontaneous recovery of a pathologically disordered body function), or it can be permanent if a specific body function is irreversibly lost. A permanent treatment may be either regulative or constitutive [22]. A regulative treatment attempts to regulate body functions, coaxing the body back toward its own homeostatic equilibrium and baseline health [22]. A regulative treatment is distinct from the organism and extrinsic to its function, whether administered internally or externally to the body. Peritoneal dialysis and hemodialysis are examples of regulative treatment because of the permanent loss of intrinsic kidney function necessary for homeostatic equilibrium [22]. An interruption of regulative treatment results in metabolic derangement but does not generally cause abrupt cessation of vital functions (respiration, circulation, and consciousness) or rapid termination of life.
A constitutive treatment takes over a body function that has been permanently lost and that the body can no longer provide for itself. Although a constitutive treatment is distinct from oneself, it replaces or substitutes for a specific body function essential to life. Mechanical ventilators and cardiac assist devices are biotechnological medical devices that provide VS and CS, respectively. The devices that provide these constitutive treatments are used as destination therapy. They permanently replace vital functions of respiration and circulation that the body can no longer maintain spontaneously. For patients who are totally dependent on a medical device for VS or CS, rapid cessation of vital signs and abrupt termination of life will occur if the operation of the medical device is interrupted. Because of the moral burden of discontinuing a medical device that can result in an abrupt termination of life, criteria have been proposed to differentiate between the two subtypes of constitutive treatment: replacement and substitution [22]. Then, it is claimed that it is morally inappropriate to discontinue a replacement treatment because doing so abruptly terminates life and may be viewed as physician-assisted death. In contrast, it is believed to be morally appropriate to discontinue a substitution treatment since such an act is simply a matter of allowing the patient to proceed to die. The criteria for categorizing a treatment as a replacement include: 1) its responsiveness to changes in the organism or its environment; 2) properties such as growth and self-repair; 3) independence from external energy sources or supplies; 4) independence from external control by an expert; 5) immunologic compatibility; and 6) physical integration into the patient's body [22].
The applicability of these criteria in clinical settings appears limited. First, the moral appropriateness or the ethical permissibility of discontinuing constitutive treatment on the premise that it replaces or substitutes for a particular body function is irrelevant regarding end-of-life decisions. There is no absolute standard for judging whether a constitutive treatment with a specific medical device should be considered replacement or substitution of a body function. To morally justify discontinuing these medical devices at the request of patients or surrogates, some physicians consider implantable electronic and mechanical cardiac devices (eg, permanent pacemakers, cardioverter-defibrillators, cardiac resynchronization therapy devices, and ventricular assist devices) as substitution treatment [23,24]. Implantable cardiac devices have also been categorized as life-sustaining treatment to defend the ethical and legal permissibility of device deactivation in patients who are making elective request for death [23]. From a medical perspective, implantable cardiac devices are different from other commonly used life-sustaining treatments, because: (1) these devices can replace native physiological functions of the heart permanently, (2) they control electric and/or mechanical functions of the heart continuously, (3) they are implanted in the body internally, (4) they are responsive to changing body demands intrinsically, and (5) they can induce loss of vital signs rapidly upon deactivation. Deactivating a permanent pacemaker in a pacemaker-dependent patient can induce severe bradycardia or asystole and a rapid cessation of circulation. In a survey of 750 health care providers, 11% of respondents consider that deactivating a permanent pacemaker is euthanasia [25]. In a survey of 185 physicians at a single institution, 9% of physicians characterize the deactivation of a permanent pacemaker in a pacemaker-dependent patient as euthanasia and 19% characterize it as physician-assisted suicide [26]. Deactivating a ventricular assist device can induce a rapid failure or complete arrest of circulation. In a study of end-of-life deactivation of destination LVAD, all patients became unconscious after turning off the device and death followed in < 20 minutes in all cases [27]. The lethal pathophysiology from device deactivation is determined by the type of device and the surgical procedure performed to implant the device in the body and not necessarily by the original pre-existing heart disease (eg. deactivation of a HeartMate II induces an acute aortoventricular regurgitation and deactivating a total artificial heart induces an immediate circulatory arrest). Permanent LVAD support can induce new and irreversible pathological changes in normal heart valves which become the lethal pathophysiology upon device deactivation [28,29].
A careful assessment of the criteria distinguishing a replacement from a substitution treatment unravels inherent clinical inconsistency and confusion about the classification of implantable mechanical cardiac devices. In destination therapy, the physically implanted LVAD becomes an integral part of the body. It is immunologically compatible with the body and does not require immunosuppressive medications to prevent its biological rejection. LVAD settings are responsive to circulatory demands of the body, but the LVAD lacks the ability to self-repair and depends on an external energy source. Thus, an LVAD might be considered replacement rather than substitution treatment. In contrast, a mechanical ventilator displays few of the criteria of replacement treatment; however, in the scenario of a quadriplegic patient with permanent apnea, many physicians consider a mechanical ventilator replacement rather than substitution treatment. Additionally, one might argue that a transplanted heart is not a replacement treatment because of its immunological incompatibility and the requirement for immunosuppressive medications to prevent its biological rejection by the body. Close expert supervision is necessary for monitoring and managing immunosuppressive medications in a transplant recipient. If a recipient refuses to continue on immunosuppressive medications, death ensues because of rejection of the transplanted heart. However, it is argued that a transplanted heart is more likely to be considered replacement rather than substitution of a body function because it cannot be surgically removed without causing a patient's death [22]. The same argument refutes classifying permanent LVAD support as substitution of a body function [24] because the medical device cannot be explanted without causing a patient's death. The arbitrary classification of a constitutive treatment as either replacement or substitution of a body function to ethically permit discontinuation of destination therapy with a medical device in a life-terminating act cannot be substantiated factually but it does give free rein to the construction of what some commentators have referred to as a moral fiction [30].
Second, irrespective of classifying a constitutive treatment of VS or CS as either a replacement or substitution of body function, circumstantial assessment of the request, causation, intention, and moral responsibility of life termination seriously restricts the ethical permissibility of discontinuing such a treatment. This will be illustrated by the discussion of the hypothetical scenarios of Adam and David below.
Finally, it is important to differentiate the discontinuation of a constitutive treatment in the hypothetical scenarios under discussion from that in the situation where either Adam or David would be imminently dying from a lethal pathophysiological condition independent of vital body functions (ie, ventilatory and cardiac functions) supported by respective medical devices. Examples of life-ending pathophysiological conditions, irrespective of continued VS or CS by medical devices used in destination therapy, may include the irreversible loss of consciousness from a catastrophic neurological event, peripheral vascular collapse and shock from an overwhelming infection, and refractory hypoxia from worsening lung disease or disseminated malignancy. Could this difference mark a legally clear and clinically relevant distinction between physician-assisted death and allowing patients to die?
Moral Fiction About Discontinuing Constitutive Medical Devices
Moral fictions are false beliefs upholding entrenched moral positions in the face of a conduct or practice in tension with established moral norms [30]. Moral fictions can be culturally entrenched, even when their falsity is exposed. Moral fictions are created from flawed interpretations of facts or false assumptions about a certain medical practice. When a specific medical practice, viewed candidly, appears to conflict with established moral norms, there is a strong incentive to construe this practice in a way that seems to remove the moral conflict. Moral fictions counteract a cognitive dissonance originating from an inconsistency between the facts about that medical practice and the prevailing moral norms. For critics who recognize the cognitive dissonance, moral fictions appear to be patently false [30].
When the discontinuation of destination therapy with a medical device for VS or CS results in a life-terminating act, moral fiction is often invoked to avoid construing such an act as physician-assisted death. Classifying a constitutive treatment as a substitution instead of the replacement of a body function, and then conveniently assigning an ethical permissibility to discontinuing the former but not the latter is illustrative of a moral fiction. Moral fictions are intended to convert an ethically challenging life-terminating act, whether inducing deep coma with medication and/or discontinuing constitutive VS or CS at the request of a patient or surrogate, into an act that appears congruent with the prevailing moral norm; that is physicians must not kill or assist in killing patients.
The pathophysiological conditions leading to death can distinguish allowing a patient to die from physician-assisted death. Allowing a patient to die is withholding and/or withdrawing life-support treatment for a lethal pathophysiological condition set in motion by a newly developed acute life-threatening illness or by a chronic treatment-refractory advanced disease. Physician-assisted death is creating a new, nontherapeutic, lethal pathophysiological condition in a human being with the intention of thereby causing or hastening that person's death [22]. Can this pathophysiological differentiation adequately categorize a physician's action of discontinuing medical devices for VS or CS?
It is difficult to contend that, in destination therapy, a medical device for VS or CS that replaces basic vital body functions is extrinsic to or separate from the patient's identity. When a mechanical cardiac assist device such as an LVAD is successfully implanted, the recipient perceives this device as part of his or her body image and sense of self [31]. A patient living with an LVAD develops an identity that is physically, emotionally, and psychologically dependent on the implanted device [32-34]. Additionally, a patient with a total artificial heart has no native cardiac function because the left and right ventricles are surgically removed to implant the medical device. A patient's circulation is then totally dependent on normal operation of the medical device. This is not different from a patient's respiration that is totally dependent on normal operation of the mechanical ventilator because of permanent apnea. The medical device becomes a replacement for that body function which is essential for life and thus is an integral part of the restored physiology of the patient. The medical device and its constitutive function are part of the integrated unity of the patient as an intact, living, individual organism. In destination therapy, medical devices permanently replace VS and CS and constitute the vital signs of respiration and circulation for that patient, respectively. From a pathophysiological perspective, turning off a mechanical ventilator or an LVAD creates a new and nontherapeutic lethal pathophysiological condition by interrupting the patient's vital signs and resulting in death through cessation of circulation, respiration, and consciousness. To discontinue such treatment not only discontinues the treatment of preexisting disease but also introduces new and nontherapeutic lethal pathophysiological conditions in the patient. The medical device has successfully treated the underlying preexisting disease (some patients may even mistakenly view it as a cure), which is also the original intent of consent for destination therapy with a medical device. In the absence of a newly developed acute life-threatening illness that sets in motion a lethal pathophysiological condition regardless of VS or CS, turning off a mechanical ventilator or an LVAD would appear to result in a rapid cessation of vital functions, thus leading to death. When regulative and constitutive destination medical devices are discontinued the ensuing lethal pathophysiological conditions can be different. Discontinuing a regulative treatment (eg, hemodialysis) does not generally result in an abrupt interruption of vital signs but, instead, causes a gradual lethal metabolic disorder. Upon discontinuing both regulative and constitutive destination medical devices, new nontherapeutic lethal pathophysiological conditions are set in motion causing death, but with different end-of-life trajectories.
The objective assessment of a patient's request, causation, intention, and moral responsibility reveal two additional moral fictions pertaining to the argument that turning off the medical devices in the hypothetical scenario of Adam and David would not be physician-assisted death. The first moral fiction concerns 1) the nature of the patient's request; 2) the nature of the act that the physician is asked to perform in either case; 3) the causal relationship between the act of treatment withdrawal and the patient's death; and 4) the intention of the physician who accedes to such a request. The second moral fiction is related to erroneous judgments about moral responsibility that are based on these mistaken factual claims. When shorn of these moral fictions, compliance with the request of either Adam or David would more appropriately be defined as physician-assisted death.
Request and Consent for Discontinuing Constitutive Medical Devices
Respect for autonomy and self-determination entails the right of a competent patient to accept or refuse a particular treatment at a specific time. Rescinding a prior "informed" consent to a constitutive treatment or destination therapy with a medical device might be considered to be supported by the patient's right to self-determination. If so, Adam and David would be exercising their rights of autonomy and self-determination when they refuse continued constitutive treatment with a medical device. Bioethicists often argue that withdrawing any life-support treatment is morally and legally similar to withholding life-support treatment in the context of fatal disease at the end of life. The question then becomes whether this ethical argument can be applied to a constitutive treatment with a medical device that is successfully treating a patient's fatal disease without the presence of any new lethal pathophysiological condition. Can this ethical argument uphold the notion that Adam's and David's requests are simply a matter of allowing patients to die rather than a case of physician-assisted suicide or physician-assisted death?
The term suicide, from the Latin words sui caedere, means the intentional killing of oneself. Both Adam and David are making suicidal requests because they believe that depending on artificial machines for basic vital functions has made life too burdensome. Although the concept of rational suicide seems nonsensical to those who believe that it is always irrational to opt for death, the definition of suicide in these two hypothetical cases might also be rejected because neither patient is physically capable of causing his own death. Both must seek a physician's assistance to do so. Both want to die because they believe that only death will free them from an increasingly intolerable condition; thus, it is their requests for assistance in dying that set in motion the causal chain leading to death if their physicians comply with those requests. Logically, however, we might infer that both patients are requesting assisted suicide because they need the help of someone else to actualize their wish to die. Regardless of the suicidal nature of such a request, one could argue that a physician who accedes to it is not engaging in assisted suicide because the immediate death-causing act is performed by the physician rather than the patient. In true assisted suicide, the act causing immediate or imminent death is performed by the patient. Physically capable patients can also disconnect themselves from either mechanical ventilators or LVADs and intentionally cause their own death. But as mentioned previously, the two ethical arguments (ie, self-determination and moral equivalency of withholding or withdrawing treatment) may seem to support the notion that discontinuing medical devices in both cases is simply a matter of allowing the patient to die. However, the assertion that the requests of Adam and David do not involve suicide is grounded in a moral fiction. There is no basis for not describing such life-terminating acts as physician-assisted death. Despite the unequivocal consequence of fulfilling such a request, some physicians might reject the label of assisted death when they comply with it because of the moral fictions regarding causation and intention.
Causation
In these two hypothetical scenarios, both Adam and David have the potential to live for an unknown time--even years--supported by their respective medical devices: a ventilator and an LVAD. What explains Adam's death after withdrawal of VS is not the course of his spinal cord injury but the very act of turning off the ventilator. David's death after withdrawal of CS is not caused by the natural progression of his heart disease, which is being successfully treated by an LVAD, but rather by the act of deactivating the LVAD. Turning off the ventilator or deactivating the LVAD is thus the proximate and immediate cause of death. This conclusion is supported by the proposed pathophysiological differentiation between allowing the patient to die and physician-assisted death. In addition, disconnecting the ventilator or deactivating the LVAD without the patient's consent would constitute an act of nonvoluntary active euthanasia.
The withdrawal of a constitutive VS or CS, when followed immediately by death, is a life-terminating intervention. Indeed, the very fact that a mechanical ventilator can sustain life for patients incapable of spontaneous breathing implies that stopping mechanical ventilation will end life. Similarly, the very fact that a mechanical cardiac assist device can sustain life for patients incapable of spontaneous circulation signifies that stopping such a device will end life. In other words, the power to sustain life by technological means goes hand in hand with the power to end life when those means are withdrawn.
The discontinuation of the ventilator in Adam's case or the deactivation of the LVAD in David's case is what results in dying at the time and in the manner each has chosen for ending his life. They may opt for an elective request to end life because of the belief that suffering is pointless, the fear about future suffering or dependency, tiredness with living, loss of dignity, the wish to die with dignity, a desire to determine the time of death, a desire to avoid being an economic burden on others, concern about family fatigue, or intolerable psychological or social suffering for themselves or their families. These reasons are similar to those of patients requesting physician-assisted death [35]. Hence, we conclude that the causation argument in describing either Adam's death after turning off the ventilator or David's death after deactivating the LVAD as merely allowing death rather than directly causing death can only be made on the basis of moral fiction.
Intention
Withdrawing a life-support treatment is considered legally and ethically permissible when it is based on the valid refusal of treatment by a competent patient or an authorized surrogate decision-maker because of the prior preferences of the patient or the sound judgment of the surrogate about the patient's best interests [15]. These end-of-life decisions are made everyday for critically ill patients with acute life-threatening and nonsurvivable illnesses who are on life-support treatment in an intensive care unit. For an imminently dying patient who is likely to die regardless of life-support treatment, the intention to discontinue that treatment is to remove an impediment to a natural death.
Physicians who view the plans of Adam and David as reasonable given their circumstances, values, and preferences, and who may be prepared to assist with the execution of those plans, intend not only to respect the autonomous choices of the patient but also to cause the patient's death. A survey of end-of-life decisions in 6 European countries (Belgium, Denmark, Italy, the Netherlands, Sweden, and Switzerland) demonstrates that almost half of the physicians reported an explicit intention to hasten death when they elected to withdraw life-support treatment [36]. In a survey of intensive care units in 17 European countries, Sprung et al reported a similar gray area in end-of-life interventions between acts to relieve pain and suffering and acts intended to shorten or hasten the dying process [37]. Neither the type and dose of medication nor the time to death could discriminate between acts intended to palliate symptoms from acts intended to hasten the dying process upon withdrawal of life support. Physicians' true intentions are private and often undisclosed in end-of-life care [38]. Intending to hasten death is the same as intending to cause death because hastening death causes death to occur earlier than it otherwise would.
Moral Responsibility
Once we uncover the moral fictions concerning the causation and intention about discontinuing destination therapy with VS and CS medical devices in the cases of Adam and David, it becomes clear that denying the moral responsibility of the physician for his or her role in their deaths is part of those fictions (Table 1). Specifically, a physician is morally responsible for causing a patient's death either by turning off a ventilator or by deactivating an LVAD when this life-terminating act can be attributed to the physician from a moral perspective. Is causing death something that the physician did voluntarily and knowingly, so that it can be attributed to him? Physicians are morally responsible for what they intend to do, as well as for what they do knowingly or negligently.
Table 1 End-of-Life Discontinuation of Constitutive Medical Devices for Ventilatory and Cardiac Support in Destination Therapy
Consideration Hypothetical Scenario
Discontinuation of Constitutive Ventilatory Support With MV (Adama) Discontinuation of Constitutive Cardiac Support With LVAD
(Davidb)
Pathophysiology
Introducing new, nontherapeutic, lethal conditions Yes Yes
Request/Consent
Is it suicide? Yes Yes
Is it assisted suicide? Yes Yes
Causation
Is the physician causing death? Yes Yes
Is it an active intervention? Yes Yes
Intention
Is the physician intending death? Sometimes Yes,
sometimes No Sometimes Yes,
sometimes No
Moral Responsibility
Is the physician morally responsible for death? Yes Yes
Is it physician-assisted death? Yes Yes
Abbreviations: LVAD, left ventricular assist device; MV, mechanical ventilator.
aAdam is a quadriplegic patient on permanent ventilatory support with a mechanical ventilator (MV) for two years. Adam asks his physician to administer medication to induce deep sedation and to turn off the MV so that he can die peacefully.
bDavid is a patient with end-stage heart failure who has had permanent implantation of a HeartMate II (Thoratec Corp, Pleasanton, California) left ventricular assist device (LVAD) as destination therapy two years earlier. David asks his physician to administer medication to induce deep sedation and to deactivate the LVAD so that he can die quickly and with dignity.
The moral responsibility of assisting in a life-terminating act by discontinuing a destination therapy with a medical device is not lessened by the consent of a competent patient or a legally authorized surrogate decision-maker. The moral responsibility for causing death is shared by the patient or surrogate and the physician. The primary responsibility rests with the patient or surrogate, but prior authorization for treatment withdrawal does not negate the physician's role and moral responsibility for discontinuing a constitutive VS or CS device and thus for causing the ensuing death (Figure 1).
Figure 1 End-of-Life Discontinuation of Destination Therapy with Ventilatory and Cardiac Support Medical Devices. Mechanical ventilators and cardiac assist devices can replace native vital functions of respiration and circulation in destination therapy, respectively. Respiration and circulation become totally dependent on normal operation of these medical devices. Discontinuing mechanical ventilators and cardiac assist devices used as destination therapy can create nontherapeutic and lethal pathophysiological conditions and become the life-terminating events. The absence of concurrent lethal pathophysiological conditions that are set in motion by a newly developed acute life-threatening illness and/or a terminal disease, unrelated to the body function supported by these medical devices, refutes the notion that discontinuing destination therapy at the patient's or surrogate's elective request for death is simply allowing the patient to die. Assessment of intent, causation, and moral responsibility of the ensuing death upon discontinuing the medical device is consistent with physician-assisted death. Discontinuing permanent mechanical ventilators and cardiac assist devices can be viewed as allowing the patient to die if--and only if--concurrent lethal pathophysiological conditions are present that are unrelated to vital functions already supported by these medical devices. Examples of concurrent lethal pathophysiological conditions from an acute life-threatening illness and/or a chronic treatment-refractory advanced disease may include: irreversible loss of consciousness from a catastrophic neurological event, peripheral vascular collapse and shock from an overwhelming infection, multiple organ failures, refractory hypoxia from worsening lung disease or disseminated malignancy.
Moral Assessment of Discontinuing Constitutive Medical Devices
Finally, we come to the differential moral evaluation of physician-assisted death versus allowing the patient to die in response to the patient's request or the patient's refusal of continued treatment by a ventilator or an LVAD. Per the premise of self-determination, patients have both a moral and a legal claim-right to stop unwanted treatment, which physicians and health care institutions are obligated to respect. The US courts have ruled that the right to make decisions about medical treatment is both a common law right based on bodily integrity and self-determination, as well as a constitutional right based on privacy and liberty. Some commentators interpret the court ruling to uphold the patient's right to refuse an ongoing treatment or a successful destination therapy with a permanent medical device implanted in the body [23,24]. On this premise, deactivation of a permanent medical device in a life-terminating act, upon an elective request for death, is considered a patient's right. Compliance with the request, as a respect for this right, is regarded as allowing the patient to die and not assisting in death even though the patient is neither terminally ill nor imminently dying [23]. The validity of this right is not contingent on the physician's endorsement of the patient's reasons for treatment refusal. Does the right to refuse a treatment imply the right to demand a life-terminating act? The right to refuse a treatment, however, is not the same as the right to receive any treatment insisted upon by the patient or a surrogate. Therefore, it can be argued that a patient's right to receive or demand a lethal treatment or intervention (eg, deactivating a medical device) is open to questioning. Although patients arguably have a moral liberty-right of noninterference by others when requesting an abortion or assisted-death, they do not have a claim-right to receive an abortion or assisted-death upon demand if the physician is unwilling to provide such a service. Can a patient's autonomy legitimize a physician's role in assisted death? In Vacco v. Quill, the US Supreme Court "...distinguished between the refusal of lifesaving treatment and assisted suicide [assisted death], by noting that the latter involves the criminal elements of causation and intent. No matter how noble a physician's motives may be, he may not deliberately cause, hasten, or aid a patient's death [39]." In Washington v. Glucksberg, "the Court held that the right to assisted suicide [assisted death] is not a fundamental liberty interest protected by Due Process Clause [of the Fourteenth Amendment of the US Constitution] since its practice has been, and continues to be, offensive to our national traditions and practices. Moreover, employing a rationality test, the Court held that... [state's] ban was rationally related to the state's legitimate interest in protecting medical ethics, shielding disabled and terminally ill people from prejudice which might encourage them to end their lives, and, above all, the preservation of human life [40]."
Allowing a patient to die by an act of omission (eg, withholding a treatment) may have different psychological effects on healthcare providers than allowing a patient to die by an act of commission (eg, turning off a medical device). Furthermore, turning off a VS or CS medical device because of a rapidly progressing fatal illness from which death is imminent may be psychologically different from a situation lacking such an illness. For example, if Adam were to develop a life-threatening kidney infection with septic shock resulting in multiple organ failure, then continuing VS would be unlikely to prevent him from dying. In that scenario, turning off the ventilator is unlikely to be the proximate cause of Adam's death. Likewise, if David were to suffer a catastrophic, bilateral cerebral hemorrhage resulting in an irreversible coma, then he would be unlikely to survive despite continued CS. In such a case, deactivating the LVAD is unlikely to be the proximate cause of death. The psychological challenges of complying with such requests to discontinue the medical device are amplified because they do not differ in intention, causation, and moral responsibility from those inherent in physician-assisted death. Administering lethal doses of medication (an ordinary tool of medicine) to cause a patient's death is active euthanasia. Discontinuing a constitutive VS or CS (ie, turning off a technology tool of medicine) results in death. In both situations, there are no real or meaningful differences in the physician's role. In both cases, the patients are seeking a swift death with physician assistance and there is no relevant difference in the causation and moral responsibility for their deaths.
The Moral Reality of Life-terminating Medical Decisions
The moral norm in the practice of medicine is that physicians must not kill patients or intend their deaths. With few exceptions, such as capital punishment and just war, US law treats the intentional causing of death as criminal homicide. In order for the practice of discontinuing a constitutive treatment of VS or CS to be permitted legally and ethically, moral fictions about life-terminating decisions (in terms of intention, causation, and responsibility) must be accepted as valid justifications. One can choose to perceive the discontinuation of VS or CS as no different from its withdrawal in imminently dying patients, as if it is not suicide or assisted suicide, as if it is passive euthanasia and merely allowing death, and as if death is not necessarily intended. Consequently, when discontinuing a constitutive treatment of VS or CS, physicians would not be considered legally responsible for causing the death of their patients and not be guilty of homicide. Perhaps more importantly, upholding such a perception permits patients and families who are morally opposed to suicide to accept the life-terminating decision to discontinue VS or CS medical devices being used for destination therapy.
Uncovering the plain empirical fact that discontinuing a destination therapy is, in essence, physician-assisted death might also have unintended consequences on medical practice. Physicians may become reluctant to engage patients and family members in conversations about end-of-life care and the anticipated need for discontinuing such devices. Physicians may become resistant to requests from patients or families to discontinue such medical devices in clinical situations when death is imminent from lethal pathophysiological conditions developing from new life-threatening illnesses. Moral and public acceptance of VS and CS medical devices as destination therapies might be imperiled. Recognizing the moral predicament of discontinuing a constitutive treatment might contribute to unease or reluctance on the part of patients and families to consent to destination therapy with medical devices. In addition, the resulting reluctance and declining use of destination therapy with VS and CS medical devices could have negative financial consequences on medical device manufacturers and institutions that provide such specialized services.
The inherent growing pressure within the medical community to expand life-prolonging medical technology introduces the risk of abandonment of one of the most important ethical norms in the practice of medicine: physicians must not harm or kill their patients. Discontinuing certain types of life-sustaining technology [41,42] and medical devices [23] (eg, deactivation of antibradycardia pacing in a pacemaker-dependent patient) in a life-terminating act is prohibited by law in some countries. Some US states have enacted a Death with Dignity Act to alleviate the tension between traditional norms of medical practice and the reality of intentional life-terminating acts by physicians [7]. However, legalizing physician-assisted death may not be the best resolution of this moral conflict. Legalization of physician-assisted death in medical practice can lead to the potential abuse of voluntary active euthanasia because any person can be killed by lethal injection, whereas withdrawing life-support treatment can kill only those who are on life support and require it to continue living. Legalization of physician-assisted death might also expand the withdrawal of life-support treatment with no consent in incompetent and vulnerable patients. Life-ending interventions without explicit consent are already being performed on patients whose diseases have unpredictable end-of-life trajectories [8].
It is difficult to continue to pass off a moral fiction as the truth once the fiction has been exposed. However, indulging in moral fictions may have worse consequences than facing the moral truth and dealing with the conflict about certain life-terminating medical practices. By failing to unravel and abandon moral fictions, we risk perpetuating an unregulated practice that sends the medical profession and society down a slippery slope distant to the fundamental moral values of humanity. Facing the moral truth is essential to preserving society's trust in the integrity of the medical profession and its practice. Confronting the moral truth should be a powerful motive to explore ways of resolving conflict between certain prevailing practices and the moral norms of the medical profession.
Summary
Advances in biotechnology medical devices have produced constitutive (permanent) treatment with mechanical ventilators and cardiac assist devices for VS and CS as destination therapy. The moral and legal assessment of the appropriateness and permissibility of complying with a patient's requests to electively discontinue destination therapy in a life-terminating act in non-futile situations continues to generate controversy. Some argue that complying with this request would be ethically justified because patients have the right to request withdrawal of unwanted treatment and be allowed to die of preexisting disease. Destination therapy with VS or CS should be considered a successful treatment of the original preexisting disease. Discontinuing VS or CS at the request of the patient or surrogate can be viewed as allowing to die, if--and only if--concurrent lethal pathophysiological conditions are present that are unrelated to the functions already supported by the medical devices in destination therapy. In all other cases, acceding with a patient's request constitutes physician-assisted death because of the pathophysiology induced by the turning off of these medical devices, as well as the intention, causation, and moral responsibility of the ensuing death. The distinction between allowing the patient to die and physician-assisted death is not only pivotal to the moral and legal status of elective requests for death by discontinuing destination cardiac and/or ventilatory medical devices in patients who are not imminently dying, but it also represents essential information that must be disclosed to patients and surrogates in advance of consent to this type of therapy.
Abbreviations
CS: cardiac support; LVAD: left ventricular assist device; VS: ventilatory support
Competing interests
The authors have no affiliation or financial involvement to disclose with any organization or entity with a direct financial interest in the subject matter or materials discussed in the manuscript. The authors declare that they have no competing interests.
Authors' contributions
MYR and JLV attest that they have made substantial contributions in drafting the manuscript and revising it critically for important intellectual content, that they have given final approval of the version to be published, and that they have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Both MYR and JLV have read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6939/11/15/prepub
Acknowledgements
We thank Mayo Clinic Scientific Publications and Media Support Services for editorial assistance. No funding was received for this work from any organizations.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2097607110-PONE-RA-18207R210.1371/journal.pone.0013512Research ArticleEcologyInfectious DiseasesMicrobiologyEcology/Conservation and Restoration EcologyEcology/Environmental MicrobiologyEcology/Evolutionary EcologyEvolutionary Biology/Evolutionary EcologyMicrobiology/Environmental MicrobiologyInfectious Diseases/Bacterial InfectionsNatural Cross Chlamydial Infection between Livestock and Free-Living Bird Species Cross Chlamydial InfectionLemus Jesús A.
1
*
Fargallo Juan A.
1
Vergara Pablo
2
Parejo Deseada
3
Banda Eva
4
1
Departamento de Ecología Evolutiva, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain
2
School of Biological Sciences, Aberdeen Centre for Environmental Sustainability (ACES), University of Aberdeen, Aberdeen, United Kingdom
3
Departmento de Ecología Funcional y Evolutiva, Estación Experimental de Zonas Áridas (CSIC), Almería, Spain
4
Oficina de Especies Migratorias, Ministerio de Medio Ambiente y Medio Rural y Marino, Madrid, Spain
Häcker Georg EditorTechnical University Munich, Germany* E-mail: [email protected] and designed the experiments: JAL JAF PV DP EB. Performed the experiments: JAL JAF PV DP EB. Analyzed the data: JAL JAF. Contributed reagents/materials/analysis tools: JAL JAF. Wrote the paper: JAL JAF PV DP EB.
2010 19 10 2010 5 10 e1351222 4 2010 16 9 2010 Lemus et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The study of cross-species pathogen transmission is essential to understanding the epizootiology and epidemiology of infectious diseases. Avian chlamydiosis is a zoonotic disease whose effects have been mainly investigated in humans, poultry and pet birds. It has been suggested that wild bird species play an important role as reservoirs for this disease. During a comparative health status survey in common (Falco tinnunculus) and lesser (Falco naumanni) kestrel populations in Spain, acute gammapathies were detected. We investigated whether gammapathies were associated with Chlamydiaceae infections. We recorded the prevalence of different Chlamydiaceae species in nestlings of both kestrel species in three different study areas. Chlamydophila psittaci serovar I (or Chlamydophila abortus), an ovine pathogen causing late-term abortions, was isolated from all the nestlings of both kestrel species in one of the three studied areas, a location with extensive ovine livestock enzootic of this atypical bacteria and where gammapathies were recorded. Serovar and genetic cluster analysis of the kestrel isolates from this area showed serovars A and C and the genetic cluster 1 and were different than those isolated from the other two areas. The serovar I in this area was also isolated from sheep abortions, sheep faeces, sheep stable dust, nest dust of both kestrel species, carrion beetles (Silphidae) and Orthoptera. This fact was not observed in other areas. In addition, we found kestrels to be infected by Chlamydia suis and Chlamydia muridarum, the first time these have been detected in birds. Our study evidences a pathogen transmission from ruminants to birds, highlighting the importance of this potential and unexplored mechanism of infection in an ecological context. On the other hand, it is reported a pathogen transmission from livestock to wildlife, revealing new and scarcely investigated anthropogenic threats for wild and endangered species.
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Introduction
Cross-species infection is a major cause of emerging infectious diseases [1]-[3]. The economic influence of the animal industry has promoted many investigations regarding the potential of wildlife as a reservoir of cattle and poultry diseases [4], [5]. On the contrary, little is known about the role of domestic species as infectious agents causing diseases in wildlife [5], [6].
Avian chlamydiosis is a well-known human disease caused by the bacterium Chlamydophila psittaci
[7]–[10] and contracted from poultry and wild birds, although pet bird (mainly parrots) are still considered the primary cause [11], [12]. In the wild, isolates have been reported from more than 460 avian species [9] as well as from some mammals, such as hares and muskrats [12], [13]. In birds it is often systemic and infections can be unapparent, severe, acute or chronic with intermittent shedding [12]. Chlamydophila abortus (also identified as Chlamydophila psittaci serovar I) is an abortogenic pathogen in ruminants rarely found in birds [14]. Factors leading to different degrees of symptomatology of this disease may be both internal, such as immune capacity, and external, such as stress [15], [16]. Indeed, adults more often have non-symptomatic infections while young birds frequently have acute disease, probably because adults are able to develop a better immunity response than young birds [15]–[17]. Additionally, stress will commonly trigger the onset of severe symptoms, resulting in rapid deterioration and death [18], [19].
Death outbreaks due to chlamydiosis can be found in wild bird species and are presumed to be due to infection with a strain uncommon to the host or due to secondary infections [11]. Chlamydiosis has been reported to be transferred by translocation of birds of prey, to spread during falconry bird flight or to spread across countries by migratory species [20], [21]. It has also been noted that colonially nesting birds are more likely to spread disease during reproduction than solitary breeders [22]–[24].
Chlamydiosis transmission from mammals to birds has been scarcely investigated, even with the knowledge that parenterally inoculated, polyarthritis-producing chlamydiae of ovine origin affected the leg joints of turkeys, and abortion-producing chlamydiae of ovine origin was infectious for pigeons and fatal for sparrows. Also, several species of small wild birds when inoculated perorally with C. psittaci of turkey origin, seroconverted (36%) and shed the organism (79%) [9]. In this same review, authors also indicated that their aim was to determine whether strains of C. psittaci from domesticated ruminants would infect, multiply in, or be shed by these wild birds, indicating whether or not these species of birds are natural hosts or biologic vectors of these strains. However, considering the heterogeneity of the chlamydial species, certain birds may harbour strains that are associated with naturally occurring infections in some animals. The results are also additional evidence of the more restricted host range of mammalian Chlamydia species when compared with avian isolates.
In this article we present the results of an episode of clinical chlamydiosis in common kestrels (Falco tinnunculus) and lesser kestrels (F. naumanni). During a study about kestrel health status [25], most of the birds in a given area showed a marked gammapathy in the protein electrophoresis pattern. We explored the origin of this abnormality. Gammapathies are well-documented as specific clinical laboratory tools for the study of several infections, including Salmonella and Chlamydophila psittaci
[15], [26]. We show the results of serology, PCR studies and the serovar and genetic clusters of the isolated Chlamydophila psittaci samples, and we explore the possibility of Chlamydophila psittaci cross-species transmission. Additionally, other chlamydial species such as Chlamydia muridarum and Chlamydia suis were tested in spite of the fact that they have not shown to be of major interest in veterinary medicine or as cross-species transmission pathogens. Chlamydia muridarum is a rodent pathogen, especially of laboratory mice and hamsters, causing respiratory disease. No records have been published about its incidence in wild rodents or birds. Chlamydia suis, on the other hand, is a swine pathogen that causes important economic losses in intensive swine production due to digestive disease, and is extremely resistant to most antibiotics. There is no report about its incidence in extensive swine or in birds.
There is some controversy in Chlamydiaceae taxonomy [7], [8], [27], [28], and especially in the psittaci serovars involved in livestock diseases [28]. We followed the taxonomy proposed by Schiller et al (2004) [28].
Results
Protein electrophoresis showed that both kestrel species from LL showed higher levels of γ-globulins than kestrels from CA and LM, being this difference statistically significant (GLMM, F
2,65 = 47.73, P<0.001, Fig. 1). Lesser kestrels showed higher values than common kestrels (GLMM, F
1,65 = 15.47,P<0.001, Fig. 2). This was due to the between-species difference found in LL while no between-species differences were found in CA and LM. This resulted in a significant species x area interaction (GLMM, F
2,65 = 23.89, P<0.001, Fig. 2). In Figure 3 the protein electrophoresis profiles in LL kestrels are represented showing a standard profile and the detected gammapathies.
10.1371/journal.pone.0013512.g001Figure 1 Between-area differences in kestrel gammaglobulin levels.
Differences in gammaglobulin levels (percentage of total proteins) between the three study areas for both Eurasian and Lesser kestrels. Interaction between species and study area is statistically significant.
10.1371/journal.pone.0013512.g002Figure 2 Gammaglobulin levels in infected and uninfected kestrels.
Differences in gammaglobulin levels (percentage of total proteins) between kestrels uninfected and infected by Chlamydophila abortus (Chlamydophila psittaci serovar I). The interaction between infection and species is statistically significant.
10.1371/journal.pone.0013512.g003Figure 3 Protein electrophoretic pattern.
A) Protein electrophoretic profile showing a typical gammapathy found in kestrel individuals infected by Chlamydophila abortus (Chlamydophila psittaci serovar I). B) Normal kestrel protein electrophoretic profile.
Chlamydophila abortus (C.p. serovar I) was the most prevalent of the three species found in kestrel populations (12.6%), followed by Chlamydia suis (5.7%) with the prevalence of Chlamydia muridarum the lowest (3.8%). All individuals infected by Chlamydia suis were also infected by Chlamydophila abortus, while none of the individuals infected by C. muridarum were found to be infected by any other Chlamydiaceae species. In order to explore gammapathies associated with a given Chlamydiaceae species we excluded from the analyses individuals infected with the other two species, thus comparing infected vs. uninfected individuals. Gammapathies in kestrels were found to be associated with Chlamydophila abortus (C.p. serovar I) infection (Table 1), showing significantly higher levels of immunoglobulins in blood in infected compared to uninfected individuals (Fig. 3). The model also showed significant differences between kestrel species and significant infection x species interaction (Table 1, Fig. 3). Similar results were found for Chlamydia suis infection (Table 1). However, note that all of these individuals were also infected by Chlamydophila abortus, for which reason we could not separate the effect of both Chlamydiaceae species. Gammapathies in kestrels were not found to be associated with Chlamydia muridarum (Table 1).
10.1371/journal.pone.0013512.t001Table 1 Effects of kestrel species and Chlamydiaceae infection on immunoglobulin levels.
F
d.f.
P
Chlamydophila abortus
Infection 29.62 1,59 <0.001
Species 39.14 1,59 <0.001
Infection * species 42.09 1,59 <0.001
Chlamydia muridarum
Infection 0.25 1,62 0.624
Species 0.13 1,62 0.653
Infection * species 0.03 1,62 0.877
Chlamydia suis
Infection 6.21 1,57 0.004
Species 5.43 1,57 0.023
Infection * species 6.40 1,57 0.014
Results of general linear mixed models (GLMM) in which immunoglobulin levels are included as a response variable and infection (infected vs. uninfected) and kestrel species are fixed factors. Between-factor interaction is also shown.
Serology analyses showed that all nestlings from LL had Chlamydophila psittaci (C.p.) antibodies, while only a small proportion of kestrels from LM and none from CA had these antibodies (Table 2). Between-area differences were significant for both common (GENMOD, χ2 = 51.18, d.f. = 2, P<0.001) and lesser (GENMOD, χ2 = 44.46, d.f. = 2, P<0.001) kestrels. No other antibodies were found during the serology evaluation. Due to the results obtained from the samples, we prepared a serovar and cluster double blind study in order to establish the Chlamydophila origin.
10.1371/journal.pone.0013512.t002Table 2 Prevalence of Chlamydiaceae species.
Falco tinnunculus
Falco naumanni
CA (n = 19)
LM (n = 8)
LL (n = 17)
CA (n = 6)
LM (n = 28)
LL (n = 13)
Chlamydophila psittaci antibody serology 0% (0)a 25% (2)b 100% (17)c 0% (0)a 7.1% (2)a 100% (13)b
Classical Chlamydophila psittaci PCR 26.3% (5)a 37.5% (3)a 100% (17)b 33.3% (2)a 25% (7)a 100% (13)b
Real time Chlamydophila psittaci PCR 26.3% (5)a 37.5% (3)a 100% (17)b 33.3% (2)a 25% (7)a 100%(13)b
Chlamydophila abortus (Chlamydophila psittaci serovar I) 0% (0)a 0% (0)a 64.7% (11)b 0% (0)a 0 % (0)a 61.5% (8)b
Chlamydophila abortus MLST 0% (0)a 0% (0)a 64.7% (11)b 0% (0)a 0 % (0)a 61.5% (8)b
Chlamydia muridarum
5.3% (1)a 0% (0)a 0% (0)a 16.7% (1) 0 % (0) (0)
Chlamydia suis
0% (0)a 0% (0)a 35.3% (6)b 0% (0)ac 0 % (0)a 23.0% (3)bc
Prevalence of Chlamydiaceae species and strains isolated from both kestrel species in different areas. Prevalence is expressed as percentage of infected individuals. Numbers in brackets represent infected individuals. Different letters indicate between-area significant differences as resulted from between-group contrasts in GENMOD procedure.
First, we performed a classical Chlamydophila psittaci PCR C.p. and real time PCRs. Both PCRs showed the same result, C.p. being identified in all individuals from LL, while only a small proportion of kestrels showed C.p. in the other two areas (Table 2). The difference was significant for both kestrel species (GENMOD, both P<0.001)
We found that a proportion of kestrels from LL, but no kestrels from the other two areas had antibodies for C. abortus (C.p. serovar I) and C. suis. The between-area differences were significant for C. abortus and C. suis in both kestrel species (GENMOD, all P<0.017), while no between-area differences were found in C. muridarum in any of the kestrel species (GENMOD, both P>0.43). MLST analysis showed the same results observed with PCRs (Table 2).
Serovar characterization indicated that both kestrel species from LL showed positive tests for serovars A and C of C.p., while kestrels from CA and LM were positive for serovars F and G (Table 3).
10.1371/journal.pone.0013512.t003Table 3
Chlamydophila psittaci filiation.
Falco tinnunculus
Falco naumanni
Campo Azálvaro (CA) Los Monegros (LM) Los Llanos (LL) Campo Azálvaro (CA) Los Monegros (LM) Los Llanos (LL)
Serovar
A - - 2 - - 6
B - - - - - -
C - - 16 - - 5
D - - - - - -
E - - - - - -
F 1 5 - 2 13 -
G 7 3 - 4 1 -
Clusters
I - - 13 2 2 7
II - 5 - 3 10 -
III 3 3 5 1 2 4
IV 5 - - - - -
Chlamydophila psittaci filiation based in serovars and genetic clusters (ordered following avian phylogenetic origin) of the different kestrel isolates from the three sampled locations.
Genetic cluster analyses for C.p. indicated that kestrel samples from LL were located mainly in cluster I with few samples belonging to cluster III. Lesser kestrels from CA and LM populations mainly “showed clusters” of type II and few of type I and III. Finally, common kestrels from CA and LM populations only showed clusters of type II and III (see Table 3).
In LL, C. abortus (C.p. serovar I) was found in all the possible sources explored: sheep abortions, sheep faeces, sheep stable dust, nest dust of both kestrel species, carrion beetles (Silphidae) and Orthoptera (Table 4). In LM, it was found in lower proportions in sheep stable dust. C.p. was also found in low proportion in samples of stable dust, lesser kestrel nest dust and Orthoptera. In CA, kestrels breed in nest boxes and old buildings, for which reason only Orthoptera invertebrates were checked. We did not find Chlamydophila in these prey species from this locality.
10.1371/journal.pone.0013512.t004Table 4 Presence of Chlamydophila in kestrel environment.
Los Llanos (LL) Campo Azálvaro (CA) Los Monegros (LM)
N
C psittaci
C abortus (C psittaci serovar I)
n
C psittaci
C abortus (C psittaci serovar I)
n
C psittaci
C abortus (C psittaci serovar I)
Sheep abortions 16 0 16
a
- - 3 0 0
Sheep faeces (Facilities) 26 0 9 - - - 14 0 0
Sheep stable dust (Facilities) 14 0 7 - - - 12 1 2
Eurasian kestrel nest dust 25 0 9
b
- - 10 0 0
Lesser kestrel nest dust 22 0 15
c
- - 16 7 0
Carrion beetles 8 0 4 0 - - 4 0 0
Grasshoppers/locust/crickets 60 0 18 60 0 0 60 1 0
Presence of Chlamydophila psittaci and C. abortus (C. psittaci serovar I) in different potential sources of infection for kestrel species.
a No sheep presence in the area.
b Nests in nest-boxes.
c Nest material was not collected.
Discussion
Exploring the health status of common and lesser kestrel populations from three different locations we detected gammapathies in individuals of both species in one of the locations (LL). This gammapathy was found to be associated with infections of Chlamydophila abortus (C. psittaci serovar I). In this same area a Chlamydophila outbreak was observed in sheep, sheep facilities and also in insects, suggesting a cross Chlamydophila infection between livestock and wild insect and bird species.
Chlamydiosis diagnosis is difficult, because there are many false negatives due to the absence of immunological reaction. In our case, common and lesser kestrel nestlings from LL showed a response to infection in protein electrophoresis and serology that was not observed in kestrels from the other two areas. Within the LL area, lesser kestrels showed stronger gammapathies (higher percentage of immunoglobulins) than common kestrels. Between-species differences can be promoted by differences in diet, as lesser kestrels are more insectivorous, thus more prone to ingesting carrion beetles and Orthoptera carrying C.p. serovar I. Furthermore, lesser kestrels tend to use sheep stables as breeding sites in a higher proportion than common kestrels, hence being more exposed to inhaling Chlamydophila fomites, such as dust.
In this study we have tried all diagnostic procedures with the exception of culture. Detection by PCR only isolates genetic material, not pathogens, but allows the detection of Chlamydiaceae exposure. When combining Chlamydiaceae with the determination of pathogen antibodies we can clearly detect those individuals that are clinically infected.
Chlamydophila psittaci is ubiquitous and causes many different diseases and prognoses in birds, and is more aggressive in nestlings [29]. In a previous paper we showed that those kestrels from LL were in poorer condition when compared to CA and LM individuals [25].
Serovar characterization and genetic clusters indicate zone differentiation in the serovars affecting kestrels. While LL typical serovars are A and C of C.p., kestrels from CA and LM were positive for serovars F and G (Table 3), the serovars typical of raptors [9], [27]. Few wildlife studies have described C.p. clusters. Our study also indicates this zone differentiation in C.p. clusters. Isolates from LL were located mainly in cluster I with few samples belonging to cluster III. Lesser kestrel isolates from CA and LM populations were mainly clustered in type II and few of the type I and III. Finally, common kestrels from CA and LM populations only showed clusters of type II and III. Together, these results indicate the origin of all isolates and permit the linkage of isolates to their original host. With the exception of the ruminant-hosted C.p. serovar I, the remaining Chlamydophila isolated from kestrels were avian-hosted Chlamydophila. Serovar A (found in LL) is naturally hosted by psittacines, columbids and several corvids [9], [27], while serovar C is naturally hosted by storks [9], [27]. Raptors are not natural hosts for either serovar. On the contrary, kestrels from CA and LM were infected with typical F or G serovars that are only susceptible to disease in case of immunological disruption, since these serovars are considered to be moderately pathogenic in their natural hosts [9], [27].
Enzootic abortion (the denomination of C.p. serovar I in sheep) is endemic in Spanish locations, including the Extremadura region where the Chlamydophila outbreak was found [30]. Abortions and mothers remain uncontrolled in the field with no assistance. We have identified potential infectious agents that can act through the two known Chlamydophila transmission routes: ingestion and inhalation. Invertebrates can be infected by direct consumption of sheep abortions, carcasses and faeces. Apart from these routes, vertebrates, as in the case of kestrels, can also be infected through the ingestion of infected insects. The presence of C. p. in dust from sheep facilities (also in kestrel nests) suggests that both vertebrates and invertebrates can contract the disease through inhalation in the surroundings of sheep stables. Measures including 1) vaccination [31] of all the sheep at risk or in enzootic areas and 2) increasing the frequency of health controls should be mandatory to minimize the risk of transmission to wildlife. To our knowledge this is the first study in which Chlamydophila psittaci is detected in livestock remains and in the environment. This isolation reflects the infective potential of this pathogen and the environmental dependence of prophylactic measures in order to avoid cross-species transmission. It is important to be aware of the potential of zoonotic transmission of C. psittaci from poultry to men [32]–[34], and also the zoonotic potential to pregnant women [35].
Similarly, Chlamydia suis and C. muridarum have never been recorded in birds. They typically appear in swine and rodents, respectively [12]. In principle, this suggests two more cases of cross-species pathogen transmission found in this study, which would be expected to provoke a conspicuous immune reaction. However, we have only actually detected the genetic material of these two species, because no antibody reactions have occurred in the serology panel. This was observed in the case of C. suis. However, due to the fact that individuals infected by C. suis were infected by Chlamydophila abortus as well, we could not disentangle its true effect on immunoglobulin levels. In the case of C. muridarum we did not detect gammapathies in infected individuals. The paucity of knowledge about Chlamydiaceae pathology in wildlife makes it difficult to explain this lack of immunological reaction. One possibility is that we are only measuring one component of the immune system, and that other immunological branches, such as a cell-mediated immune response, could be acting without our detection. A second possibility is that C. muridarum could be a common pathogen in kestrels, as they usually prey on rodents. In this sense, our study highlights the interest of investigating this aspect in future studies.
The lesser kestrel is considered as a “Vulnerable” species throughout its range (www.iucnredlist.org, 25). Farmlands and grasslands are the most common habitats for this species [36]. Extremadura possesses up to 25% of the lesser kestrel Spanish population and its numbers have shown a positive trend over the last several years [37]. The common kestrel, on the other hand, is the most common diurnal raptor species in Spain, however with negative population trends in Europe [38]. The changes in land-use practices (agricultural intensification and pesticide use) and direct persecution have traditionally been the causes proposed to explain population declines in both kestrel species [37]–[40]. However, other problems more subtle to identify, such as infectious disease episodes, call into question the conservation efforts, especially those devoted to the lesser kestrel. Epizooties can operate in wild species causing population declines at a local scale [41]–[43]. Wildlife populations are immunologically prepared for many of the pathogens in the environment, but changes in the serovars usually imply mortality episodes [3], [4], [6]. Our study emphasizes the necessity of wildlife veterinary controls as useful tools for conservation plans and detection of risks in wild species.
Materials and Methods
Samples examined
We tested for Chlamydophila psittaci in a total of 91 common (n = 44) and lesser (n = 47) kestrel nests present in three study areas located in Los Llanos (Cáceres province, 39° 28′ N, 6° 22′ W), Campo Azálvaro (Segovia province, 40° 40′ N, 4° 20′ W) and Los Monegros (Zaragoza province, 41° 20′ N, 0° 11′ W). The three locations are subjected to high extensive livestock pressure, with extensive ovine livestock in Los Llanos (LL) and Los Monegros (LM) and extensive bovine livestock in Campo Azálvaro (CA); see Vergara et al. (2008) [25] for more study area characteristics. Ovine livestock receive no veterinary interference except legal controls in LL, and receive some veterinary assistance and prophylactic treatments in LM. Sample size for each kestrel species and area is shown in Table 2. One chick per nest in each kestrel species was randomly selected for blood samples.
All the nestlings were sampled at about three weeks old. One ml of blood was taken from the brachial vein, centrifuged and the pellet was separated from plasma and both were frozen until analyses.
Protein electrophoresis
As a part of the health status design, protein electrophoresis was performed in all checked specimens. Plasma protein electrophoresis fractions were run on commercial agarose gels (Hydragel Protein (E), Sebia Hispania S.A., Barcelona, Spain) using a semi-automated Hydrasys System (Sebia Hispania S.A., Barcelona, Spain) with manufacturer's reagents to determine the concentration of albumin and globulins (α, β and γ-globulins) in percent, that were used in the analyses. Total plasma proteins were determined by the Biuret method [44]. Total plasma protein concentrations (g/dl), which were also used in the analyses, were calculated by the multiplication of each protein fraction with the total protein value.
Chlamydophila psittaci serology
A serology panel that included Salmonella and Chlamydophila psittaci serology was performed using plasma samples. A whole blood-plate agglutination test was used to detect the Salmonella antigen presence Difco (TM) Salmonella O Group B Antigen (1-4-5-13) (Becton Dickinson and Company, Maryland, USA). The test was conducted by using the manufacturer's standard instructions [45]. Chlamydophila psittaci antibodies were determined by using Rida-Screen antibody ELISA (R-Biopharm, Darmstadt, Germany)
Chlamydophila psittaci PCR, real time PCR and Chlamydophila abortus (Chlamydophila psittaci serovar I) PCR
Blood PCRs were performed following Hewinson et al, 1997, for conventional PCR for Chlamydophila psittaci, Sachse et al, 2009, for real time PCR for Chlamydophila psittaci, and Laroucau et al, 2001 were used to Chlamydophila abortus conventional PCR [46]–[48]. We have considered Chlamydophila psittaci serovar I as Chlamydophila abortus, following Kaleta & Taday (2003) [9] and Schiller et al. (2004) [28]. This technique has been demonstrated to be successful when showing pathogen exposure in common and lesser kestrels [49].
Due to the presence of extensive livestock in the area, and the occurrence of enzootic chlamydial abortion, we also performed a chlamydial serovar characterization to establish the serovar involved in the epizootic episode. In addition, we also obtained the genetic cluster of the same isolates according to Chahota et al, 2006 [14]. We explored the presence of Chlamydia species, Chlamydia suis and Chlamydia muridarum. For Chlamydia suis we used the specification of Laroucau et al, 2001, and Robertson et al, 2009 [48], [50] whilst for C. muridarum we used the specifications of Pantchev et al and Robertson et al, 2009 [50], [51]
Serovar characterization
For serovar characterization the isolates were either grown directly in Buffalo green monkey (BGM) cells or in 6-day-old specific pathogen-free embryonated chicken eggs as is indicated in Vanrompay et al, 1993 [27]. The six serovar- specific MAbs were designated VS-1 (serovar A specific; psittacine group), CP3 (serovar B specific; pigeon I group), GR-9 (serovar C specific; duck group), NJ-1 (serovar D specific; turkey group), MP (serovar E specific; pigeon II group), NJ-1D3 (serovar F) and serovar G [52]. The microimmunofluorescence test was also performed following Vanrompay et al. (1993) [27].
Chlamydophila genetic diversity
Genetic diversity and epizootiology of Chlamydophila psittaci was based on the VD2 region of the ompA gene. DNA was extracted, a nested PCR was performed followed by cloning of the PCR product and sequencing [14]. The sequence analyses were performed following Chahota et al. (2006) [14].
We also tested for Chlamydophila psittaci type I in sheep abortions, sheep faeces, sheep stable dust, kestrel nest dust, necrophilous beetles and orthoptera (grasshoppers, locusts, crickets) in the study areas. Beetles and orthoptera are common prey species of common and lesser kestrels in Spain [36], [53]. Arthropods were collected close to nests (50 m away from carcasses in the case of beetles and 200 m away from nests in the case of orthoptera), and were euthanized by congelation.
Sheep abortion samples were processed following Schiller et al, 2004 [28], whilst sheep faeces and dust preparation was performed following Tanaka et al, 2005 [54], and arthropods were prepared by homogenization [55].
Chlamydophila abortus MLST analysis
Because of the difficulty to discriminate between C. psittaci and C. abortus and not possible on the basis of the major outer membrane protein A, we additionally carried out a. MLST analyses as described by Pannekoek et al.(http://www.pubmlst.org/chlamydiales).
Statistical procedures
Nestlings share genes and environments within the nest for which reason these cannot be considered independent samples. We attempted to analyse between-location and between-species differences in nestling infection (infected vs. uninfected) by using Generalized Mixed Models, in which the nest was included as a random factor and species as a fixed factor. This procedure avoids pseudoreplication considering the nestling as the sampling unit. Due to the fact that some chlamydial isolates where absent in some locations our data were unbalanced and most of the models did not converge. For this reason we randomly selected one nestling from each nest and analysed frequencies of infection in different locations and species by using GENMOD procedure with logit link function and binomial distribution in SAS statistical software (SAS 9.0, 2002, Institute Inc., Cary, NC, USA). Differences in protein electrophoresis between kestrel species and populations were analysed using General Linear Mixed Models with GLMM procedure in. The percentage of γ-globulins was arcsine transformed. Nest was included in the model as a random factor and location and species as fixed factors.
Ethics Statement
Our study followed ethical guidelines proposed for the Spanish Royal Decree 1205/2005 about the protection of animals used in experiments and scientific research and was approved by the Spanish Ministry of Science and Innovation (CGL2007-61395/BOS).
L. de Neve, J.I. Aguirre, A. Gajón, P. Laiolo, J.C. Núñez and M. Kauffman helped in the field. Regional Governments from Extremadura, Castilla y León and Aragón provided the necessary licenses for sampling kestrels. Sarah Young revised the English.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The Spanish Ministerio de Ciencia e Innovación (Project CGL2007-61395/BOS) financed the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J Exp Clin Cancer ResJournal of Experimental & Clinical Cancer Research : CR0392-90781756-9966BioMed Central 1756-9966-29-1332093711110.1186/1756-9966-29-133ResearchThe candidate tumor suppressor gene ECRG4 inhibits cancer cells migration and invasion in esophageal carcinoma Li Linwei [email protected] Chunpeng [email protected] Xiaoyan [email protected] ShihHsin [email protected] Yun [email protected] Oncology Department, Henan Provincial People's Hospital, Zhengzhou 450003, PR China2 State Key Laboratory of Molecular Oncology and Department of Etiology and Carcinogenesis, Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, PR China2010 11 10 2010 29 1 133 133 4 8 2010 11 10 2010 Copyright ©2010 Li et al; licensee BioMed Central Ltd.2010Li et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The esophageal cancer related gene 4 (ECRG4) was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no.AF325503). ECRG4 was a new tumor suppressor gene in esophageal squamous cell carcinoma (ESCC) associated with prognosis. In this study, we investigated the novel tumor-suppressing function of ECRG4 in cancer cell migration, invasion, adhesion and cell cycle regulation in ESCC.
Methods
Transwell and Boyden chamber experiments were utilized to examined the effects of ECRG4 expression on ESCC cells migration, invasion and adhesion. And flow cytometric analysis was used to observe the impact of ECRG4 expression on cell cycle regulation. Finally, the expression levels of cell cycle regulating proteins p53 and p21 in human ESCC cells transfected with ECRG4 gene were evaluated by Western blotting.
Results
The restoration of ECRG4 expression in ESCC cells inhibited cancer cells migration and invasion (P < 0.05), which did not affect cell adhesion capacity (P > 0.05). Furthermore, ECRG4 could cause cell cycle G1 phase arrest in ESCC (P < 0.05), through inducing the increased expression of p53 and p21 proteins.
Conclusion
ECRG4 is a candidate tumor suppressor gene which suppressed tumor cells migration and invasion without affecting cell adhesion ability in ESCC. Furthermore, ECRG4 might cause cell cycle G1 phase block possibly through inducing the increased expression of p53 and p21 proteins in ESCC.
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Introduction
Esophageal carcinoma ranks 7th and 6th in terms of cancer incidence and mortality rate worldwide, respectively [1]. Moreover, nearly 50% of esophageal carcinoma cases in the world occurred in China [2]. Esophageal squamous cell carcinoma (ESCC), which is the most common histological subtype, accounts for ~90% of all esophageal cancers diagnosed in China each year. Despite advances in clinical comprehensive treatment, ESCC prognosis remains poor due to its diffuse and invasive nature. To date, the molecular pathogenesis of ESCC is still unclear [3,4].
The ECRG4 gene (GenBank accession no. AF325503) was initially identified and cloned in our laboratory from human normal esophageal epithelium [5,6]. Our previous results demonstrated that ECRG4 protein was an independent prognostic factor for ESCC, and the low expression of ECRG4 protein in patients with ESCC was associated with poor prognosis [7,8]. Furthermore, overexpression of ECRG4 gene in ESCC cells inhibited tumor cells growth in vitro and in vivo [7,8].
In the present study, we further examined the tumor-suppressing function of ECRG4 gene, in terms of cell migration and invasion, and explored possible molecular mechanism in ESCC.
Materials and methods
Construction of eukaryotic expression vector and stable transfection
The coding region of ECRG4 cDNA was subcloned into constitutive mammalian expression vector pcDNA3.1 (Invitrogen). The cDNA was then fully sequenced to ensure that no mutation was introduced during the PCR amplification. The resulting plasmid construct was named pcDNA3.1-ECRG4. The human esophageal squamous cell line EC9706 was established and studied by Han et al [9]. EC9706 cells were seeded in 6-cm dishes at 5×105 cells/dish and transfected with pcDNA3.1-ECRG4 and pcDNA3.1 using lipofectamine™2000 (Invitrogen), according to the manufacturer's protocol. After culturing in medium containing 400 μg/ml of geneticin (Invitrogen) for 3 weeks, individual clones were isolated. Clones that expressed the ECRG4 cDNA coding region were maintained in medium containing 200 μg/ml of geneticin and used for further experiments.
Cell proliferation assay
EC9706 cells (pcDNA3.1 and pcDNA3.1-ECRGR4) were seeded into 96-well plates (1.5 × 103 cells/well). After culturing for various durations, cell proliferation was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay, according to the manufacturer's protocol (Sigma-Aldrich Co., St. Louis, MO, USA). In brief, 10 μl MTT solution (5 mg/ml) was added to each well, then the cells were cultured for another 4 hours at 37°C, and 100 μl DMSO was added to each well and mix vigorously to solubilize colored crystals produced within the living cells. The absorbance at 570 nm was measured by using a multi-well scanning spectrophotometer Victor 3.
In vitro cell migration and invasion assay
Cells growing in the log phase were treated with trypsin and re-suspended as single-cell solutions. A total of 1 × 105 cells in 0.5 ml of serum-free RPMI 1640 medium were seeded on a 8 μm-pore polycarbonate membrane Boyden chambers insert in a transwell apparatus(Costar, Cambridge, MA), either coated with or without Matrigel(BD Biosciences, San Jose, CA). 600 μl RPMI1640 containing 20% FBS was added to the lower chamber. After the cells were incubated for 12-24 hours at 37°C in a 5% CO2 incubator, cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 minutes, stained in 0.5% crystal violet for 2 min, rinsed in PBS and then subjected to microscopic inspection (×200). Values for invasion and migration were obtained by counting five fields per membrane and represent the average of three independent experiments.
Cell adhesion assay
Cells were plated on 100 ng/μl Matrigel-coated 96-well plates at a density of 5 × 104 per well. The cells were incubated at 37°C for 30, 60, and 90 minutes in a 5% CO2 incubator, respectively. Nonattached cells were removed by PBS washings for three times. Attached cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Promega, Madison, WI) assay according to the user manual. The mean absorbance values for statistical analysis represent the average of three independent experiments.
Western blot analysis
Whole-cell lysates of EC9706 cells were prepared by incubating cells in RIPA buffer (1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris-HCl [pH 7.5]) containing protease inhibitors. Cell lysates were centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was collected, and the protein concentration was measured using the BCA ™ Protein Assay Kit (Pierce). Proteins (40 ug) in cell lysates or culture media were separated by 10-15% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane. The membranes were blocked in TBST (0.2 M NaCl; 10 mM Tris pH7.4; 0.2% Tween20)/5% skim milk for 2 hours at room temperature and then incubated with primary antibodies in TBST/5% skim milk. The primary antibodies used for Western blot analysis were polyclonal rabbit anti-ECRG4 (1:2000) [8], polyclonal rabbit anti-p21(1:4000; Santa Cruz, CA), polyclonal rabbit anti-p53 (1:4000; Santa Cruz, CA), and monoclonal mouse anti-β-actin (1:4000; Santa Cruz, CA). The membranes were then washed three times with TBST, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:4000) in TBST/5% skim milk. Bound antibody was visualized using ECL detection reagent.
RT-PCR analysis
Cells were washed with PBS and collected for RT-PCR. The primers designed for ECRG4 were 5'-GGT TCT CCC TCG CAG CAC CT-3' as forward and 5'-CAG CGT GTG GCA AGT CAT GGT TAG-3' as reverse. Thermal cycles were: at 95°C for 2 min, then 30 cycles at 95°C for 30 sec, at 62°C for 30 sec, at 72°C for 1 min followed by extension at 72°C for 7 min [7].
Flow cytometric analysis of cell cycle
The transfected cells (pcDNA3.1-ECRG4 and pcDNA3.1) were seeded at a density of 106 cells/100-mm dish in RPMI-1640 medium with 10% FBS for 48 hours. Then cells were washed with ice-cold PBS, harvested and fixed in 70% ethanol for 30 minutes. Cells were treated with RNase A and stained with 25 μg/ml propidium iodide (PI). Samples were analyzed using a FACScan flow cytometer (Becton Dickinson), according to the manufacturer's protocol. Experiments were performed three times in triplicate. The mean values for statistical analysis represent the average of three independent experiments.
Statistical analysis
All statistical analysis was performed with the SPSS statistical program (version 13.0). Statistical significance was determined using Student's t-tests and analysis of variance. P < 0.05 was considered statistically significant.
Results
ECRG4 overexpression suppressed cell migration and invasion
The stable-transfected EC9706/pcDNA3.1-ECRG4 cells exhibited detectable ECRG4 mRNA (Figure 1A) and ECRG4 protein expression (Figure 1B), compared with EC9706/pcDNA3.1 cells and EC9706 cells. And the cell growth curve of EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1 was plotted for further migration-invasion analysis (Figure 1C). To measure the effect of ECRG4 overexpression on tumor cell migration, cells growing in the log phase were collected and cultured on Transwell apparatus. After 12 h incubation, cell migration was significantly decreased in EC9706/pcDNA3.1-ECRG4 group than in control group (P < 0.05) (Figure 2). Using Boyden chamber precoated with Matrigel, we examined the effect of ECRG4 overexpression on tumor cell invasion. After 24 h incubation, EC9706/pcDNA3.1-ECRG4 cells showed significantly decreased invasiveness, compared with the EC9706/pcDNA3.1 cells (P < 0.05) (Figure 3). These results demonstrated that ECRG4 overexpression reduced the migration and invasion of ESCC cells.
Figure 1 Evaluation of ECRG4 gene expression and cell growth curve of EC9706/pcDNA3.1 and EC9706/pcDNA3.1-ECRG4. (A) ECRG4 mRNA was detected in EC9706/pcDNA3.1-ECRG4 cells by RT-PCR. M: Marker; Lane 1: EC9706/pcDNA3.1; Lane 2: EC9706/pcDNA3.1-ECRG4; Lane 3: EC9706 cells. (B) ECRG4 protein (17 KD) was detected in EC9706/pcDNA3.1-ECRG4 cells by Western blot. Lane 1: EC9706 cells; Lane 2: EC9706/pcDNA3.1; Lane 3: EC9706/pcDNA3.1-ECRG4. (C) Cell growth curve of EC9706/pcDNA3.1 and EC9706/pcDNA3.1-ECRG4 by MTT assay (P < 0.05).
Figure 2 Effect of ECRG4 overexpression on tumor cells migration. Representative photos and statistic plots of migration assay in EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1 cells (×200). The number of EC9706/pcDNA3.1-ECRG4 cells transversed the Transwell membrane was decreased compared with that of EC9706/pcDNA3.1 cells (P < 0.05). Error bars represent standard deviation from mean value.
Figure 3 Effect of ECRG4 overexpression on tumor cells invasion. Representative photos and statistic plots of invasion assay in EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1 cells (×200). The number of EC9706/pcDNA3.1-ECRG4 cells transversed the Transwell membrane was decreased compared with that of EC9706/pcDNA3.1 cells (P < 0.05). Error bars represent standard deviation from mean value.
The impact of ECRG4 overexpression on cell adhesion capacity
As the apparent ECRG4-induced decrease in migration and invasion could be the result of reduction in adhesion of tumor cells to the substrate, we evaluated cell adhesive ability by measuring the number of cells attached to Matrigel. No significant difference was detected between the two groups by MTS adhesion assay (P > 0.05) (Table 1). Therefore, ECRG4 overexpression in EC9706 cells drastically suppressed cancer cells mobility without affecting cell adhesion capacity.
Table 1 ECRG4 exerted no significant effect on tumor cells adhesion capacity
Group 30 min 60 min 90 min
EC9706/pcDNA3.1-ECRG4
* 1.268 ± 0.293 1.988 ± 0.341 2.564 ± 0.537
EC9706/pcDNA3.1 1.274 ± 0.247 2.040 ± 0.360 2.531 ± 0.524
* P > 0.05, compared with EC9706/pcDNA3.1
ECRG4 overexpression blocked cell cycle progression
The stable-transfected EC9706/pcDNA3.1-ECRG4 cells exhibited detectable ECRG4 protein expression compared with EC9706/pcDNA3.1 cells, as shown in Figure 1B. The percentages of cells in the G1, S and G2/M phase of cell cycle demonstrated that overexpression of ECRG4 in EC9706 cells resulted in an accumulation of cells in G1 phase and a decrease in S and G2/M phase compared with EC9706/pcDNA3.1 control cells (P < 0.05) (Table 2). Flow cytometric analysis suggested that ECRG4 overexpression could arrest EC9706 cells at the G1/S checkpoint and delay cell cycle into S phase. Consequently, ECRG4 overexpression slowed down cell cycle progression and caused cell cycle G1 phase block.
Table 2 ECRG4 overexpression caused cell cycle G1 phase block
Group G1 S G2/M
EC9706/pcDNA3.1-ECRG4* 73.7 ± 1.86 14.8 ± 1.13 11.5 ± 0.92
EC9706/pcDNA3.1 59.8 ± 2.06 25.0 ± 1.39 15.2 ± 1.64
* P < 0.05, compared with EC9706/pcDNA3.1
ECRG4 may be involved in p53 pathway
In exploring the molecular mechanism of cell cycle G1 phase block caused by ECRG4 overexpression in EC9706 cells, we found that p53 and p21 protein expression levels were increased in EC9706/pcDNA3.1-ECRG4 cells compared with in EC9706/pcDNA3.1 cells (Figure 4). It indicated that ECRG4 may be involved in p53 pathway in ESCC. ECRG4 might induce p21 upregulation through p53 pathway to block cell cycle progression in ESCC.
Figure 4 ECRG4 may be involved in p53 pathway. Representative photos and statistic plots of relative protein expression levels in EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1. Analysis of cell's total proteins by Western blot showed that p53 and p53 target gene p21 expressions were increased in EC9706/pcDNA3.1-ECRG4 cells compared with in EC9706/pcDNA3.1 cells (P < 0.05). Lane 1: EC9706/pcDNA3.1-ECRG4; Lane 2: EC9706/pcDNA3.1. *, P < 0.05, compared with EC9706/pcDNA3.1.
Discussion
ESCC is a highly invasive and clinically challenging cancer in China, and its molecular basis remains poorly understood. ECRG4 is a novel gene identified and cloned in our laboratory [5,6]. ECRG4 gene is highly conserved among various species, suggesting an important role for ECRG4 in eukaryotic cells [10]. However, its exactly biological function in carcinogenesis is still unclear. Our previous study demonstrated that ECRG4 gene promoter hypermethylation accounted for decreased expression in ESCC, and the low expression of ECRG4 protein in patients with ESCC was associated with poor prognosis [7,8]. These findings were also supported by similar studies of other research groups [11,12]. Furthermore, restoration of ECRG4 expression in ESCC cells inhibited tumor cells growth in vitro and in vivo [7,8].
In this study, we revealed a novel function of ECRG4 that suppressed tumor cells migration and invasion without affecting cell adhesion ability in ESCC, implicating its potential involvement in tumor development. Li et al [13] observed the similar result in glioma consistent with ours. Enhancement in motility and loss of adhesion capacity are advantageous to tumor invasion, which is one main mechanism to cause cancer metastasis. Transformed cells acquire a series of additional malignant traits, such as invasion and metastasis abilities, during tumorigenesis and progression.
It is now generally accepted that transcription factor NF-κB and COX-2 pathway plays a central role between inflammation and carcinogenesis [14,15]. Recently, NF-κB and COX-2 were approved to promote tumor cells migration and invasion [16-23]. Our previous results showed that ECRG4 attenuated NF-κB expression and nuclear translocation and reduced NF-κB target gene COX-2 expression in ESCC [8]. Li et al [13] also observed that ECRG4 transfection decreased NF-κB expression in glioma. Therefore, we speculated that NF-κB pathway might be involved in ECRG4-induced decrease of tumor cells migration and invasion in ESCC. However, the detailed molecular mechanism remained to be clarified in subsequent research.
The cell cycle alteration plays a major role in carcinogenesis. Once the cell cycle regulation balance was broken, it might result in tumorigenesis. Evidence has revealed that many oncogenes and tumor suppressor genes are directly involved in regulation of cell cycle events [24]. In the present research, we discovered for the first time that ECRG4 inhibited cancer cells proliferation and induced cell cycle G1 phase block by up-regulating p21 expression level through p53 mediated pathway in ESCC. It is well known that p21, the critical cyclin-dependent kinase inhibitor, is able to block the cell cycle at G1 phase [25,26]. So the p21 expression upregulation could be the molecular mechanism for the ECRG4-induced cell cycle G1 phase block in ESCC.
Taken together, ECRG4 is a candidate tumor suppressor gene which suppressed cancer cells migration and invasion in ESCC. Furthermore, ECRG4 could also cause cell cycle G1 phase block through the upregulation of p53 and p21 expression levels. Our study indicated that loss of ECRG4 function might play a pivotal role in ESCC carcinogenesis and implied that ECRG4 could be an important therapeutic target for ESCC.
Abbreviations
ECRG4: esophageal cancer related gene 4; ESCC: esophageal squamous cell carcinoma; NF-κB: NF-kappaB; COX-2: cyclooxygenase 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PBS: phosphate-buffered saline; RT-PCR: reverse transcriptase-polymerase chain reaction.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LL carried out cell culture, gene transfection, gene functional assays, RT-PCR and Western blotting. CZ and XL analyzed and interpreted data. YZ and SL supervised experimental and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the Chinese State Key Projects for Basic Research (2002CB513101 and 2004CB518701) and the Henan Province Science Research Key Project (0624410058). We thank professor Wei Jing of Burnham Institute Cancer Center (La Jolla, CA92037, USA) for helpful comments on this manuscript. We also thank Dr Xiao-chun Wang and Dr Hong-yan Chen for the technical assistance.
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography cf221310.1107/S1600536808028389ACSEBHS1600536808028389Metal-Organic Papers
catena-Poly[[(2,2′-bipyridine-κ2
N,N′)nickel(II)]-μ-oxalato-κ4
O
1,O
2:O
1′,O
2′] [Ni(C2O4)(C10H8N2)]Li Sheng aYan Xing-Lian bWang Shou-Bin cMa Yuan-Fang a*a Institute of Immunology, Key Laboratory of Natural Drugs and Immunological Engineering of Henan Province, College of Medicine, Henan University, Kaifeng 475003, People’s Republic of Chinab First Affiliated Hospital, Henan University, Kaifeng 475003, People’s Republic of Chinac College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475003, People’s Republic of ChinaCorrespondence e-mail: [email protected] 10 2008 13 9 2008 13 9 2008 64 Pt 10 e081000m1258 m1258 04 8 2008 04 9 2008 © Li et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Ni(C2O4)(C10H8N2)]n, is isostructural with its MnII, FeII, CuII and ZnII analogues. Each NiII atom is chelated by two oxalate ligands and one 2,2′-bipyridine, forming a slightly distorted octahedral geometry. Oxlate acts as a bridge to link neighbouring pairs of NiII cations, forming a one-dimensional wave-like chain. The crystal showed partial inversion twinning.
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Related literature
For related literature, see: Hong & Do (1997 ▶); Eddaoudi et al. (2001 ▶); Liang et al. (2004 ▶); Shi et al. (2005 ▶). For the isostructural MnII, FeII, CuII and ZnII complexes, see: Li et al. (2006 ▶); Deguenon et al. (1990 ▶); Fun et al. (1999 ▶); Luo et al. (2001 ▶); Yu et al. (2006 ▶); Lin et al. (2006 ▶).
Experimental
Crystal data
[Ni(C2O4)(C10H8N2)]
M
r = 302.91
Orthorhombic,
a = 9.6486 (14) Å
b = 9.2627 (14) Å
c = 13.883 (2) Å
V = 1240.7 (3) Å3
Z = 4
Mo Kα radiation
μ = 1.57 mm−1
T = 296 (2) K
0.12 × 0.10 × 0.06 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.834, T
max = 0.912
6146 measured reflections
2114 independent reflections
1810 reflections with I > 2σ(I)
R
int = 0.030
Refinement
R[F
2 > 2σ(F
2)] = 0.047
wR(F
2) = 0.154
S = 1.00
2114 reflections
173 parameters
1 restraint
H-atom parameters constrained
Δρmax = 0.44 e Å−3
Δρmin = −0.43 e Å−3
Absolute structure: Flack (1983 ▶), 971 Friedel pairs
Flack parameter: 0.20 (3)
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808028389/cf2213sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808028389/cf2213Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: CF2213).
The authors are grateful for financial support from the Scientific Research Foundation of Outstanding Talented Persons of Henan Province (grant No. 74200510014).
supplementary crystallographic
information
Comment
The design of coordination compounds has attracted long-lasting research
interest not only because of their appealing structural and topological
novelty but also due to their unusual optical, electronic, magnetic and
catalytic properties, and their further potential medical value derived
from their antiviral properties and the inhibition of angiogenesis.
To date, much of the work has been focused on coordination polymers with
organic acid ligands (Hong et al. 1997; Eddaoudi et al.
2001;
Liang et al. 2004; Shi et al.2005).
Here we report the synthesis and X-ray crystal structure analysis of the
title compound, (I), with a bridging oxalate ligand. It is isostructural
with its MnII, FeII, CuII, and ZnII analogues (Li et al.,
2006;
Deguenon et al., 1990; Fun et al., 1999; Luo
et al., 2001;
Yu et al., 2006; Lin et al., 2006).
As shown in Fig. 1, the Ni(II) atom is chelated by two oxlates and one
2,2'-bipyridine, forming a slightly distorted octahedral geometry. Oxalate acts
as a bridge to link neighboring pairs of Ni(II) cations, forming a
one-dimensional wave-like chain (Fig. 2). The Ni—N and Ni—O bond lengths
are in the ranges 2.239 (5)–2.243 (5) and 2.161 (4)–2.166 (4) Å, respectively.
Experimental
A mixture of nickel(II) nitrate hexahydrate (0.1 mmol), oxalic acid (0.2 mmol),
2,2'-bipyridine (0.1 mmol), and water (16 ml) in a 25 ml Teflon-lined
stainless steel autoclave was kept at 473 K for three days. Green crystals
were obtained after cooling to room temperature, with a yield of 6%. Anal.
Calc. for C12H8N2NiO4: C 47.54, H 2.64, N 9.24%; Found: C 47.51, H
2.58, N 9.16%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93 Å and
refined as riding with Uiso(H) = 1.2Ueq(C).
Figures
Fig. 1. The coordination of the Ni atom in the title structure, drawn with 30% probability displacement ellipsoids. [Symmetry code: (I) 1/2+x, 1/2-y, z.]
Fig. 2. The chain of the title compound, viewed along the [010] direction.
Crystal data
[Ni(C2O4)(C10H8N2)] F(000) = 616
Mr = 302.91 Dx = 1.622 Mg m−3
Orthorhombic, Pna21 Mo Kα radiation, λ = 0.71073 Å
Hall symbol: P 2c -2n Cell parameters from 1779 reflections
a = 9.6486 (14) Å θ = 2.6–21.5°
b = 9.2627 (14) Å µ = 1.57 mm−1
c = 13.883 (2) Å T = 296 K
V = 1240.7 (3) Å3 Block, green
Z = 4 0.12 × 0.10 × 0.06 mm
Data collection
Bruker APEXII CCD area-detector diffractometer 2114 independent reflections
Radiation source: fine-focus sealed tube 1810 reflections with I > 2σ(I)
graphite Rint = 0.030
φ and ω scans θmax = 25.1°, θmin = 2.6°
Absorption correction: multi-scan (SADABS; Bruker, 2001) h = −11→5
Tmin = 0.834, Tmax = 0.912 k = −11→11
6146 measured reflections l = −16→16
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.047 H-atom parameters constrained
wR(F2) = 0.154 w = 1/[σ2(Fo2) + (0.118P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
2114 reflections Δρmax = 0.44 e Å−3
173 parameters Δρmin = −0.44 e Å−3
1 restraint Absolute structure: Flack (1983), 971 Friedel pairs
Primary atom site location: structure-invariant direct methods Flack parameter: 0.20 (3)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Ni1 0.88396 (7) 1.09402 (7) 0.25107 (8) 0.04352 (14)
O1 1.0093 (4) 1.2363 (5) 0.1613 (3) 0.0503 (11)
O2 1.0673 (4) 1.1297 (4) 0.3368 (3) 0.0415 (9)
O3 0.7686 (4) 1.2511 (5) 0.3347 (3) 0.0452 (10)
O4 0.7033 (4) 1.1389 (4) 0.1640 (3) 0.0416 (9)
N1 0.8006 (5) 0.9059 (5) 0.3352 (4) 0.0401 (11)
N2 0.9510 (5) 0.8925 (5) 0.1747 (4) 0.0395 (11)
C1 1.1555 (6) 1.2124 (6) 0.3004 (4) 0.0380 (12)
C2 1.1199 (4) 1.2745 (6) 0.1993 (4) 0.0305 (12)
C3 0.7196 (8) 0.9174 (8) 0.4115 (5) 0.0535 (17)
H3 0.6964 1.0099 0.4320 0.064*
C4 0.6668 (8) 0.8030 (10) 0.4630 (5) 0.070 (2)
H4 0.6106 0.8163 0.5168 0.084*
C5 0.7033 (9) 0.6629 (9) 0.4291 (6) 0.073 (2)
H5 0.6728 0.5809 0.4613 0.087*
C6 0.7820 (8) 0.6507 (7) 0.3504 (6) 0.0633 (19)
H6 0.8054 0.5598 0.3269 0.076*
C7 0.8286 (6) 0.7736 (6) 0.3038 (4) 0.0405 (13)
C8 0.9159 (6) 0.7661 (7) 0.2154 (4) 0.0408 (13)
C9 0.9646 (9) 0.6356 (7) 0.1766 (6) 0.0627 (19)
H9 0.9447 0.5484 0.2067 0.075*
C10 1.0418 (10) 0.6383 (10) 0.0936 (7) 0.081 (3)
H10 1.0713 0.5519 0.0663 0.097*
C11 1.0758 (8) 0.7647 (9) 0.0510 (5) 0.066 (2)
H11 1.1292 0.7669 −0.0048 0.080*
C12 1.0277 (8) 0.8924 (8) 0.0937 (5) 0.0601 (19)
H12 1.0495 0.9802 0.0651 0.072*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Ni1 0.0387 (2) 0.0456 (2) 0.0463 (2) 0.0000 (2) −0.0004 (3) 0.0045 (3)
O1 0.042 (2) 0.065 (3) 0.044 (3) −0.012 (2) −0.0101 (19) 0.022 (2)
O2 0.0384 (19) 0.052 (2) 0.034 (2) −0.0078 (19) −0.0056 (16) 0.0150 (18)
O3 0.039 (2) 0.061 (2) 0.036 (2) 0.0098 (19) −0.0144 (18) −0.0128 (19)
O4 0.042 (2) 0.048 (2) 0.035 (2) −0.0017 (18) −0.0036 (17) −0.0095 (18)
N1 0.043 (3) 0.045 (3) 0.032 (2) −0.0063 (18) 0.006 (2) −0.0005 (19)
N2 0.045 (3) 0.038 (2) 0.036 (3) 0.0049 (19) 0.008 (2) 0.002 (2)
C1 0.038 (3) 0.036 (3) 0.040 (3) 0.007 (3) −0.004 (3) 0.004 (3)
C2 0.028 (3) 0.034 (3) 0.029 (3) −0.002 (2) −0.0031 (19) 0.005 (2)
C3 0.062 (4) 0.058 (4) 0.041 (4) −0.005 (3) 0.016 (3) 0.005 (3)
C4 0.068 (4) 0.098 (6) 0.043 (4) −0.024 (5) 0.015 (4) 0.011 (4)
C5 0.087 (5) 0.071 (5) 0.060 (4) −0.034 (4) 0.011 (4) 0.021 (4)
C6 0.088 (5) 0.039 (3) 0.063 (4) −0.022 (3) 0.004 (4) −0.002 (3)
C7 0.040 (3) 0.045 (3) 0.037 (3) −0.008 (3) 0.000 (3) 0.005 (2)
C8 0.045 (3) 0.046 (3) 0.031 (3) 0.008 (3) 0.000 (3) −0.005 (2)
C9 0.089 (5) 0.041 (3) 0.058 (4) 0.012 (3) −0.002 (4) 0.005 (3)
C10 0.096 (7) 0.073 (5) 0.073 (5) 0.040 (5) 0.009 (5) −0.017 (5)
C11 0.091 (5) 0.070 (5) 0.037 (4) 0.028 (4) 0.012 (4) 0.003 (3)
C12 0.069 (5) 0.064 (4) 0.047 (4) 0.023 (3) 0.014 (3) 0.012 (3)
Geometric parameters (Å, °)
Ni1—O4 2.162 (4) C3—C4 1.376 (10)
Ni1—O2 2.157 (4) C3—H3 0.930
Ni1—O1 2.180 (4) C4—C5 1.425 (13)
Ni1—O3 2.169 (4) C4—H4 0.930
Ni1—N2 2.242 (5) C5—C6 1.335 (12)
Ni1—N1 2.246 (5) C5—H5 0.930
O1—C2 1.242 (6) C6—C7 1.385 (9)
O2—C1 1.251 (7) C6—H6 0.930
O3—C1i 1.238 (6) C7—C8 1.491 (8)
O4—C2i 1.237 (6) C8—C9 1.404 (9)
N1—C3 1.322 (8) C9—C10 1.372 (12)
N1—C7 1.328 (7) C9—H9 0.930
N2—C8 1.343 (8) C10—C11 1.353 (13)
N2—C12 1.346 (9) C10—H10 0.930
C1—O3ii 1.238 (6) C11—C12 1.402 (10)
C1—C2 1.554 (6) C11—H11 0.930
C2—O4ii 1.237 (6) C12—H12 0.930
O4—Ni1—O2 160.07 (14) N1—C3—C4 125.0 (7)
O4—Ni1—O1 90.65 (14) N1—C3—H3 117.5
O2—Ni1—O1 76.55 (13) C4—C3—H3 117.5
O4—Ni1—O3 75.90 (14) C3—C4—C5 115.9 (7)
O2—Ni1—O3 91.31 (15) C3—C4—H4 122.0
O1—Ni1—O3 100.66 (17) C5—C4—H4 122.0
O4—Ni1—N2 97.39 (17) C6—C5—C4 119.3 (7)
O2—Ni1—N2 98.72 (18) C6—C5—H5 120.4
O1—Ni1—N2 94.20 (18) C4—C5—H5 120.4
O3—Ni1—N2 163.69 (17) C5—C6—C7 119.8 (7)
O4—Ni1—N1 98.71 (17) C5—C6—H6 120.1
O2—Ni1—N1 97.23 (17) C7—C6—H6 120.1
O1—Ni1—N1 164.72 (18) N1—C7—C6 122.7 (6)
O3—Ni1—N1 93.35 (18) N1—C7—C8 115.3 (5)
N2—Ni1—N1 72.74 (17) C6—C7—C8 122.0 (6)
C2—O1—Ni1 114.0 (3) N2—C8—C9 120.3 (6)
C1—O2—Ni1 115.4 (4) N2—C8—C7 116.6 (5)
C1i—O3—Ni1 115.4 (3) C9—C8—C7 123.0 (6)
C2i—O4—Ni1 115.3 (3) C8—C9—C10 119.2 (7)
C3—N1—C7 117.2 (5) C8—C9—H9 120.4
C3—N1—Ni1 124.5 (4) C10—C9—H9 120.4
C7—N1—Ni1 118.2 (4) C11—C10—C9 121.0 (7)
C8—N2—C12 119.3 (5) C11—C10—H10 119.5
C8—N2—Ni1 117.0 (4) C9—C10—H10 119.5
C12—N2—Ni1 123.7 (4) C10—C11—C12 117.7 (7)
O2—C1—O3ii 127.7 (5) C10—C11—H11 121.2
O2—C1—C2 116.2 (5) C12—C11—H11 121.2
O3ii—C1—C2 116.2 (5) N2—C12—C11 122.4 (7)
O4ii—C2—O1 125.1 (5) N2—C12—H12 118.8
O4ii—C2—C1 117.0 (4) C11—C12—H12 118.8
O1—C2—C1 117.8 (4)
O4—Ni1—O1—C2 162.4 (4) O1—Ni1—N2—C12 −7.8 (6)
O2—Ni1—O1—C2 −2.1 (4) O3—Ni1—N2—C12 147.8 (6)
O3—Ni1—O1—C2 86.7 (4) N1—Ni1—N2—C12 −179.8 (6)
N2—Ni1—O1—C2 −100.1 (4) Ni1—O2—C1—O3ii −179.5 (5)
N1—Ni1—O1—C2 −69.5 (8) Ni1—O2—C1—C2 −0.7 (6)
O4—Ni1—O2—C1 −49.9 (7) Ni1—O1—C2—O4ii −175.8 (5)
O1—Ni1—O2—C1 1.4 (4) Ni1—O1—C2—C1 2.5 (6)
O3—Ni1—O2—C1 −99.2 (4) O2—C1—C2—O4ii 177.1 (6)
N2—Ni1—O2—C1 93.7 (4) O3ii—C1—C2—O4ii −3.9 (7)
N1—Ni1—O2—C1 167.2 (4) O2—C1—C2—O1 −1.3 (7)
O4—Ni1—O3—C1i 3.6 (4) O3ii—C1—C2—O1 177.7 (6)
O2—Ni1—O3—C1i 168.2 (4) C7—N1—C3—C4 2.7 (11)
O1—Ni1—O3—C1i 91.6 (4) Ni1—N1—C3—C4 179.3 (6)
N2—Ni1—O3—C1i −63.7 (8) N1—C3—C4—C5 −0.5 (11)
N1—Ni1—O3—C1i −94.5 (4) C3—C4—C5—C6 −1.4 (12)
O2—Ni1—O4—C2i −52.8 (7) C4—C5—C6—C7 1.0 (12)
O1—Ni1—O4—C2i −102.2 (4) C3—N1—C7—C6 −3.1 (9)
O3—Ni1—O4—C2i −1.3 (4) Ni1—N1—C7—C6 −179.9 (5)
N2—Ni1—O4—C2i 163.5 (4) C3—N1—C7—C8 177.8 (6)
N1—Ni1—O4—C2i 89.9 (4) Ni1—N1—C7—C8 0.9 (6)
O4—Ni1—N1—C3 −80.8 (6) C5—C6—C7—N1 1.3 (11)
O2—Ni1—N1—C3 87.1 (6) C5—C6—C7—C8 −179.6 (7)
O1—Ni1—N1—C3 152.0 (6) C12—N2—C8—C9 3.2 (9)
O3—Ni1—N1—C3 −4.6 (6) Ni1—N2—C8—C9 −174.3 (5)
N2—Ni1—N1—C3 −175.9 (6) C12—N2—C8—C7 −178.8 (6)
O4—Ni1—N1—C7 95.7 (4) Ni1—N2—C8—C7 3.6 (7)
O2—Ni1—N1—C7 −96.3 (4) N1—C7—C8—N2 −3.0 (7)
O1—Ni1—N1—C7 −31.5 (9) C6—C7—C8—N2 177.8 (6)
O3—Ni1—N1—C7 171.9 (4) N1—C7—C8—C9 174.9 (6)
N2—Ni1—N1—C7 0.6 (4) C6—C7—C8—C9 −4.3 (9)
O4—Ni1—N2—C8 −99.2 (5) N2—C8—C9—C10 −3.7 (11)
O2—Ni1—N2—C8 92.6 (4) C7—C8—C9—C10 178.5 (7)
O1—Ni1—N2—C8 169.6 (4) C8—C9—C10—C11 2.4 (13)
O3—Ni1—N2—C8 −34.7 (9) C9—C10—C11—C12 −0.9 (13)
N1—Ni1—N2—C8 −2.3 (4) C8—N2—C12—C11 −1.7 (11)
O4—Ni1—N2—C12 83.4 (6) Ni1—N2—C12—C11 175.7 (6)
O2—Ni1—N2—C12 −84.9 (6) C10—C11—C12—N2 0.4 (12)
Symmetry codes: (i) x−1/2, −y+5/2, z; (ii) x+1/2, −y+5/2, z.
==== Refs
References
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Flack, H. D. (1983). Acta Cryst A39, 876–881.
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Li, L. L., Lin, K. J., Ho, C. J., Sun, C. P. & Yang, H. D. (2006). Chem. Commun. pp. 1286–1287.
Liang, M., Sun, Y. Q., Liao, D. Z., Jiang, Z. H., Yan, S. P. & Cheng, P. (2004). J. Coord. Chem.57, 275–280.
Lin, X.-R., Ye, B.-Z., Liu, J.-S., Wei, C.-X. & Chen, J.-X. (2006). Acta Cryst. E62, m2130–m2132.
Luo, J.-H., Hong, M.-C., Liang, Y.-C. & Cao, R. (2001). Acta Cryst. E57, m361–m362.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Shi, J. M., Yin, H. L. & Wu, C. J. (2005). J. Coord. Chem.58, 915–920.
Yu, J. H., Hou, Q., Bi, M. H., Lu, Z. L., Zhang, X., Qu, X. J., Lu, J. & Xu, J. Q. (2006). J. Mol. Struct.800, 69–75. | 21201013 | PMC2959270 | CC BY | 2021-01-04 18:54:11 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Sep 13; 64(Pt 10):m1258 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography gk217010.1107/S1600536808035150ACSEBHS1600536808035150Metal-Organic Papers
catena-Poly[[aqua(2,2′-bipyridine-κ2
N,N′)copper(II)]-μ-5-nitroisophthalato-κ3
O
1,O
1′:O
3] [Cu(C8H3NO6)(C10H8N2)(H2O)]Hao Lujiang a*Liu Xia ba College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Insitute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 12 2008 08 11 2008 08 11 2008 64 Pt 12 e081200m1500 m1500 28 9 2008 28 10 2008 © Hao and Liu 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the asymmetric unit of the title compound, [Cu(C8H3NO6)(C10H8N2)(H2O)]n, there are two symmetry-independent one-dimensional coordination polymers related by a non-crystallographic inversion center. Within the polymers, the CuII atoms, coordinated by the water molecule and the chelating 2,2′-bipyridine ligands, are bridged by 5-nitrobenzene-1,3-dicarboxylate dianions which act as tridentate ligands; the carboxylate groups exhibit monodentate and symmetric bidentate coordination modes. The CuII atoms show a strongly distorted octahedral coordination geometry. In the crystal structure, the two symmetry-independent coordination polymers form another one-dimensional polymeric structure via O—H⋯O hydrogen bonds between coordinated water molecules and carboxylate groups.
==== Body
Related literature
For the uses of carboxylic acids in materials science, see: Church & Halvorson (1959 ▶), and in biological systems, see: Okabe & Oya (2000 ▶); Kim et al. (2001 ▶).
Experimental
Crystal data
[Cu(C8H3NO6)(C10H8N2)(H2O)]
M
r = 446.85
Monoclinic,
a = 10.1326 (10) Å
b = 23.263 (3) Å
c = 15.6087 (15) Å
β = 97.28 (2)°
V = 3649.6 (7) Å3
Z = 8
Mo Kα radiation
μ = 1.25 mm−1
T = 293 (2) K
0.12 × 0.10 × 0.08 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.865, T
max = 0.907
18862 measured reflections
6694 independent reflections
5089 reflections with I > 2σ(I)
R
int = 0.025
Refinement
R[F
2 > 2σ(F
2)] = 0.042
wR(F
2) = 0.123
S = 1.00
6694 reflections
535 parameters
6 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.83 e Å−3
Δρmin = −0.40 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808035150/gk2170sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808035150/gk2170Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: GK2170).
This work was supported by the Natural Science Foundation of Shandong Province (grant No. Y2007D39).
supplementary crystallographic
information
Comment
In recent years, carboxylic acids have been widely used as polydentate ligands,
which can coordinate to transition or rare earth ions yielding complexes with
interesting properties that are useful in materials science (Church &
Halvorson, 1959) and in biological systems (Okabe & Oya, 2000).
For example,
Kim et al. (2001) focused on the syntheses of transition metal
complexes containing benzene carboxylate and rigid aromatic pyridine ligands
in order to study their electronic conductivity and magnetic properties. The
importance of transition metal dicarboxylate complexes motivated us to pursue
synthetic strategies for these compounds, using 5-nitroisophthalic acid as a
polydentate ligand. Here we report the synthesis and X-ray crystal structure
analysis of the title compound.
The molecular structure of the title compound is shown in Fig. 1. The title
compound, [Cu(C8H3NO6)(C10H8N2)(H2O)]n is a one-dimensional
coordination polymer (Fig. 2). There are two symmetry independent 1D polymers
in the crystal. The Cu(II) atom shows a strongly disordered coordination
geometry. It is coordinated by two carboxylate groups from two different
5-nitroisophthalate ligands, 2,2'-bipyridyl and water molecule. The
carboxylate groups act in a monodentate and bidentate coordination modes.The
symmetry independent polymeric chains are linked via O-H···O hydrogen bonds
(Table 1).
Experimental
A mixture of copper dichloride (0.5 mmol), 2,2'-bipyridine (0.5 mmol), and
5-nitroisophthalic acid (0.5 mmol) in H2O (8 ml) and ethanol (8 ml) was
sealed in a 25 ml Teflon-lined stainless steel autoclave and kept at 413 K for
three days. Blue crystals were obtained after cooling to room temperature
(yield 27%). Anal. Calc. for C18H13CuN3O7: C 48.34, H 2.91, N
10.74%; Found: C 48.30, H 2.84, N 10.69%.
Refinement
The H atoms of water molecule were located from difference Fourier maps and were
refined with distance restraints: d(H–H) = 1.38 (2) Å, d(O–H) = 0.88 (2) Å, and with a fixed Uiso of 0.080 Å2. All other H atoms were
placed in calculated positions with a C—H bond distance of 0.93 Å and
refined in the riding model approximation with Uiso(H) =
1.2Ueq of the carrier atom.
Figures
Fig. 1. A view of the title structure showing the atomic numbering scheme and 30% probability displacement ellipsoids.
Fig. 2. One of the symmetry-independent coordination polymers
Crystal data
[Cu(C8H3NO6)(C10H8N2)(H2O)] F000 = 1816
Mr = 446.85 Dx = 1.627 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 6694 reflections
a = 10.1326 (10) Å θ = 1.8–25.5º
b = 23.263 (3) Å µ = 1.25 mm−1
c = 15.6087 (15) Å T = 293 (2) K
β = 97.28 (2)º Block, blue
V = 3649.6 (7) Å3 0.12 × 0.10 × 0.08 mm
Z = 8
Data collection
Bruker APEXII CCD area-detector diffractometer 6694 independent reflections
Radiation source: fine-focus sealed tube 5089 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.025
T = 293(2) K θmax = 25.5º
φ and ω scans θmin = 1.8º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −12→10
Tmin = 0.865, Tmax = 0.907 k = −28→22
18862 measured reflections l = −18→18
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.042 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.124 w = 1/[σ2(Fo2) + (0.075P)2 + 2.4671P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.003
6694 reflections Δρmax = 0.83 e Å−3
535 parameters Δρmin = −0.40 e Å−3
6 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Cu1 1.16168 (4) 0.154431 (15) 0.85573 (3) 0.02711 (13)
Cu2 0.12372 (4) 0.354405 (16) 0.93350 (3) 0.03053 (13)
C1 1.1959 (4) 0.07213 (16) 1.0079 (2) 0.0457 (9)
H1 1.2145 0.1048 1.0416 0.055*
C2 1.2039 (5) 0.0193 (2) 1.0471 (3) 0.0635 (13)
H2 1.2268 0.0161 1.1065 0.076*
C3 1.1773 (5) −0.02890 (18) 0.9970 (3) 0.0634 (13)
H3 1.1810 −0.0651 1.0224 0.076*
C4 1.1452 (4) −0.02334 (16) 0.9089 (3) 0.0481 (10)
H4 1.1282 −0.0556 0.8741 0.058*
C5 1.1388 (3) 0.03117 (13) 0.8732 (2) 0.0291 (7)
C6 1.1072 (3) 0.04138 (13) 0.7785 (2) 0.0266 (7)
C7 1.0796 (3) −0.00183 (15) 0.7180 (2) 0.0376 (8)
H7 1.0788 −0.0401 0.7354 0.045*
C8 1.0527 (4) 0.01249 (16) 0.6306 (2) 0.0408 (9)
H8 1.0328 −0.0160 0.5891 0.049*
C9 1.0562 (4) 0.06932 (16) 0.6068 (2) 0.0415 (9)
H9 1.0383 0.0800 0.5491 0.050*
C10 1.0866 (3) 0.11017 (14) 0.6700 (2) 0.0337 (8)
H10 1.0889 0.1486 0.6536 0.040*
C11 0.3761 (3) 0.19406 (14) 0.8105 (2) 0.0284 (7)
C12 0.8756 (3) 0.17568 (14) 0.8380 (2) 0.0286 (7)
C13 0.7500 (3) 0.20954 (13) 0.80617 (19) 0.0232 (6)
C14 0.6258 (3) 0.18759 (13) 0.8202 (2) 0.0252 (6)
H14 0.6213 0.1526 0.8484 0.030*
C15 0.5081 (3) 0.21767 (13) 0.79223 (19) 0.0228 (6)
C16 0.5143 (3) 0.27058 (14) 0.7520 (2) 0.0287 (7)
H16 0.4375 0.2912 0.7332 0.034*
C17 0.6391 (3) 0.29176 (14) 0.7409 (2) 0.0300 (7)
C18 0.7573 (3) 0.26273 (13) 0.7665 (2) 0.0268 (7)
H18 0.8388 0.2784 0.7574 0.032*
C19 0.0858 (4) 0.43908 (16) 0.7844 (3) 0.0419 (9)
H19 0.0717 0.4066 0.7497 0.050*
C20 0.0710 (4) 0.49282 (18) 0.7464 (3) 0.0506 (10)
H20 0.0493 0.4965 0.6869 0.061*
C21 0.0891 (4) 0.54045 (17) 0.7982 (3) 0.0530 (11)
H21 0.0801 0.5770 0.7741 0.064*
C22 0.1206 (3) 0.53393 (15) 0.8859 (3) 0.0437 (9)
H22 0.1312 0.5660 0.9217 0.052*
C23 0.1365 (3) 0.47885 (13) 0.9207 (2) 0.0331 (8)
C24 0.1701 (3) 0.46692 (14) 1.0141 (2) 0.0312 (7)
C25 0.1994 (4) 0.50930 (15) 1.0773 (3) 0.0450 (10)
H25 0.1976 0.5480 1.0620 0.054*
C26 0.2313 (4) 0.49311 (18) 1.1632 (3) 0.0505 (10)
H26 0.2502 0.5209 1.2059 0.061*
C27 0.2346 (4) 0.43618 (19) 1.1844 (3) 0.0502 (10)
H27 0.2573 0.4245 1.2414 0.060*
C28 0.2036 (4) 0.39641 (17) 1.1200 (2) 0.0423 (9)
H28 0.2048 0.3577 1.1348 0.051*
C29 0.4118 (3) 0.33262 (14) 0.9445 (2) 0.0276 (7)
C30 0.5355 (3) 0.29913 (13) 0.9800 (2) 0.0245 (7)
C31 0.6609 (3) 0.31950 (13) 0.9659 (2) 0.0247 (6)
H31 0.6668 0.3529 0.9339 0.030*
C32 0.7773 (3) 0.29078 (12) 0.9988 (2) 0.0230 (6)
C33 0.7701 (3) 0.24090 (13) 1.0462 (2) 0.0281 (7)
H33 0.8466 0.2213 1.0687 0.034*
C34 0.6445 (3) 0.22111 (13) 1.0589 (2) 0.0274 (7)
C35 0.5281 (3) 0.24928 (14) 1.0278 (2) 0.0273 (7)
H35 0.4461 0.2350 1.0387 0.033*
C36 0.9110 (3) 0.31370 (13) 0.9804 (2) 0.0265 (7)
N1 1.1618 (3) 0.07821 (11) 0.92194 (18) 0.0308 (6)
N2 1.1133 (2) 0.09711 (10) 0.75448 (17) 0.0256 (6)
N3 0.1197 (3) 0.43222 (12) 0.86995 (18) 0.0310 (6)
N4 0.1716 (3) 0.41090 (11) 1.03653 (19) 0.0315 (6)
N5 0.6350 (3) 0.16771 (13) 1.1080 (2) 0.0421 (8)
N6 0.6481 (4) 0.34942 (14) 0.7038 (2) 0.0526 (9)
O1 0.0918 (2) 0.30549 (9) 0.81941 (17) 0.0320 (5)
O2 0.3006 (2) 0.31188 (9) 0.96166 (16) 0.0358 (6)
O3 0.4241 (3) 0.37683 (12) 0.90419 (19) 0.0532 (7)
O4 0.9139 (2) 0.35925 (10) 0.93797 (18) 0.0392 (6)
O5 1.0167 (2) 0.28721 (10) 1.00723 (16) 0.0360 (6)
O6 0.5265 (3) 0.14511 (15) 1.1064 (3) 0.0852 (13)
O7 0.7369 (3) 0.14842 (12) 1.1487 (2) 0.0579 (8)
O8 0.5457 (3) 0.37104 (19) 0.6687 (4) 0.128 (2)
O9 0.7554 (3) 0.37294 (13) 0.7091 (2) 0.0734 (10)
O10 0.9862 (2) 0.19817 (9) 0.82626 (16) 0.0328 (5)
O11 0.8637 (3) 0.12791 (13) 0.8686 (2) 0.0653 (9)
O12 1.1948 (2) 0.20384 (9) 0.97015 (16) 0.0319 (5)
O13 0.2693 (2) 0.22031 (10) 0.78406 (16) 0.0369 (6)
O14 0.3736 (2) 0.14837 (10) 0.85273 (17) 0.0390 (6)
H1W 1.245 (4) 0.2303 (13) 0.960 (3) 0.080*
H2W 1.132 (3) 0.2151 (17) 0.994 (3) 0.080*
H3W 0.157 (3) 0.2969 (18) 0.795 (3) 0.080*
H4W 0.042 (4) 0.2781 (13) 0.824 (3) 0.080*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Cu1 0.0215 (2) 0.0234 (2) 0.0363 (2) 0.00148 (15) 0.00314 (16) 0.00163 (15)
Cu2 0.0210 (2) 0.0245 (2) 0.0466 (3) 0.00113 (15) 0.00654 (18) 0.00113 (16)
C1 0.065 (3) 0.041 (2) 0.031 (2) 0.0004 (19) 0.0076 (18) 0.0047 (16)
C2 0.096 (4) 0.060 (3) 0.033 (2) 0.006 (3) 0.007 (2) 0.014 (2)
C3 0.092 (4) 0.040 (2) 0.059 (3) 0.004 (2) 0.012 (3) 0.031 (2)
C4 0.063 (3) 0.0273 (18) 0.053 (3) −0.0023 (17) 0.005 (2) 0.0101 (16)
C5 0.0230 (16) 0.0246 (16) 0.0396 (19) −0.0009 (12) 0.0039 (14) 0.0044 (13)
C6 0.0201 (15) 0.0235 (15) 0.0361 (18) −0.0006 (12) 0.0037 (13) 0.0017 (13)
C7 0.0313 (18) 0.0270 (17) 0.054 (2) −0.0024 (14) 0.0052 (16) −0.0048 (16)
C8 0.036 (2) 0.043 (2) 0.042 (2) −0.0019 (16) 0.0012 (16) −0.0122 (16)
C9 0.041 (2) 0.048 (2) 0.034 (2) 0.0023 (17) −0.0006 (16) −0.0037 (16)
C10 0.0362 (19) 0.0319 (17) 0.0323 (19) 0.0054 (14) 0.0015 (15) 0.0049 (14)
C11 0.0197 (16) 0.0332 (17) 0.0332 (18) −0.0034 (13) 0.0071 (13) −0.0116 (14)
C12 0.0172 (16) 0.0347 (18) 0.0332 (18) 0.0028 (13) 0.0008 (13) 0.0056 (14)
C13 0.0147 (14) 0.0287 (16) 0.0257 (16) 0.0017 (12) 0.0008 (12) 0.0000 (12)
C14 0.0219 (16) 0.0261 (15) 0.0283 (16) −0.0010 (12) 0.0054 (13) −0.0006 (13)
C15 0.0156 (14) 0.0280 (15) 0.0252 (16) −0.0013 (12) 0.0041 (12) −0.0061 (12)
C16 0.0173 (15) 0.0372 (18) 0.0314 (18) 0.0093 (13) 0.0027 (13) 0.0019 (14)
C17 0.0269 (17) 0.0298 (16) 0.0343 (18) 0.0035 (13) 0.0078 (14) 0.0110 (13)
C18 0.0156 (14) 0.0337 (17) 0.0314 (18) −0.0016 (12) 0.0039 (12) 0.0049 (13)
C19 0.040 (2) 0.040 (2) 0.049 (2) 0.0029 (16) 0.0137 (17) 0.0071 (17)
C20 0.043 (2) 0.058 (3) 0.052 (2) 0.0069 (19) 0.0135 (19) 0.024 (2)
C21 0.040 (2) 0.034 (2) 0.088 (3) 0.0026 (17) 0.021 (2) 0.027 (2)
C22 0.031 (2) 0.0237 (17) 0.078 (3) 0.0018 (14) 0.0160 (19) 0.0050 (18)
C23 0.0160 (15) 0.0232 (16) 0.062 (2) 0.0000 (12) 0.0137 (15) 0.0010 (15)
C24 0.0149 (15) 0.0292 (17) 0.051 (2) −0.0005 (12) 0.0090 (14) −0.0043 (15)
C25 0.032 (2) 0.0279 (18) 0.077 (3) −0.0019 (15) 0.0121 (19) −0.0106 (18)
C26 0.034 (2) 0.059 (3) 0.058 (3) −0.0019 (18) 0.0037 (19) −0.021 (2)
C27 0.039 (2) 0.064 (3) 0.048 (2) 0.0050 (19) 0.0075 (18) −0.006 (2)
C28 0.037 (2) 0.043 (2) 0.047 (2) 0.0056 (17) 0.0058 (17) −0.0007 (17)
C29 0.0166 (15) 0.0327 (17) 0.0326 (18) 0.0031 (13) −0.0002 (13) −0.0046 (14)
C30 0.0155 (15) 0.0300 (16) 0.0279 (17) 0.0005 (12) 0.0024 (12) −0.0048 (12)
C31 0.0192 (15) 0.0241 (15) 0.0312 (17) −0.0012 (12) 0.0053 (12) −0.0004 (12)
C32 0.0156 (15) 0.0266 (15) 0.0276 (16) −0.0004 (11) 0.0059 (12) −0.0043 (12)
C33 0.0187 (15) 0.0345 (17) 0.0305 (18) 0.0020 (13) 0.0017 (13) 0.0005 (13)
C34 0.0216 (16) 0.0316 (16) 0.0288 (17) −0.0020 (13) 0.0027 (13) 0.0043 (13)
C35 0.0175 (15) 0.0342 (17) 0.0307 (18) −0.0059 (13) 0.0050 (13) 0.0015 (13)
C36 0.0140 (15) 0.0294 (16) 0.0370 (18) 0.0009 (12) 0.0073 (13) −0.0085 (14)
N1 0.0305 (15) 0.0280 (14) 0.0347 (16) −0.0011 (11) 0.0068 (12) 0.0048 (11)
N2 0.0213 (13) 0.0233 (13) 0.0320 (15) 0.0026 (10) 0.0030 (11) −0.0005 (11)
N3 0.0216 (14) 0.0277 (14) 0.0446 (18) 0.0022 (11) 0.0075 (12) 0.0052 (12)
N4 0.0239 (14) 0.0268 (14) 0.0445 (18) 0.0028 (11) 0.0071 (12) −0.0015 (12)
N5 0.0325 (17) 0.0448 (18) 0.0488 (19) −0.0039 (14) 0.0042 (14) 0.0181 (14)
N6 0.044 (2) 0.049 (2) 0.068 (2) 0.0143 (16) 0.0185 (17) 0.0321 (17)
O1 0.0243 (12) 0.0261 (12) 0.0455 (14) 0.0024 (9) 0.0044 (10) −0.0001 (10)
O2 0.0148 (11) 0.0301 (12) 0.0621 (16) 0.0023 (9) 0.0033 (10) −0.0029 (11)
O3 0.0327 (14) 0.0511 (17) 0.075 (2) 0.0086 (12) 0.0035 (13) 0.0290 (15)
O4 0.0241 (12) 0.0291 (12) 0.0669 (17) −0.0021 (9) 0.0153 (12) 0.0085 (11)
O5 0.0157 (11) 0.0366 (12) 0.0564 (16) 0.0029 (9) 0.0072 (10) −0.0003 (11)
O6 0.0464 (19) 0.086 (2) 0.116 (3) −0.0290 (17) −0.0150 (19) 0.065 (2)
O7 0.0401 (16) 0.0511 (17) 0.083 (2) 0.0105 (13) 0.0080 (15) 0.0336 (15)
O8 0.046 (2) 0.117 (3) 0.226 (5) 0.037 (2) 0.036 (3) 0.131 (4)
O9 0.061 (2) 0.0534 (18) 0.102 (3) −0.0163 (16) −0.0027 (19) 0.0388 (18)
O10 0.0121 (10) 0.0274 (11) 0.0583 (15) 0.0017 (8) 0.0015 (10) −0.0021 (10)
O11 0.0313 (15) 0.0648 (19) 0.101 (2) 0.0135 (13) 0.0130 (15) 0.0564 (18)
O12 0.0267 (12) 0.0261 (11) 0.0415 (14) 0.0039 (9) −0.0003 (10) −0.0027 (10)
O13 0.0157 (11) 0.0404 (13) 0.0550 (16) 0.0031 (10) 0.0066 (10) −0.0012 (11)
O14 0.0223 (12) 0.0378 (14) 0.0583 (16) −0.0027 (10) 0.0103 (11) 0.0048 (11)
Geometric parameters (Å, °)
Cu1—O10 2.049 (2) C19—N3 1.345 (5)
Cu1—N1 2.052 (3) C19—C20 1.384 (5)
Cu1—N2 2.078 (3) C19—H19 0.9300
Cu1—O12 2.115 (2) C20—C21 1.371 (6)
Cu1—O14i 2.158 (2) C20—H20 0.9300
Cu1—O13i 2.258 (2) C21—C22 1.374 (6)
Cu2—O2 2.046 (2) C21—H21 0.9300
Cu2—N3 2.062 (3) C22—C23 1.393 (5)
Cu2—N4 2.086 (3) C22—H22 0.9300
Cu2—O1 2.104 (2) C23—N3 1.341 (4)
Cu2—O4ii 2.139 (2) C23—C24 1.481 (5)
Cu2—O5ii 2.294 (2) C24—N4 1.349 (4)
C1—N1 1.350 (5) C24—C25 1.400 (5)
C1—C2 1.372 (6) C25—C26 1.391 (6)
C1—H1 0.9300 C25—H25 0.9300
C2—C3 1.373 (6) C26—C27 1.364 (6)
C2—H2 0.9300 C26—H26 0.9300
C3—C4 1.379 (6) C27—C28 1.373 (6)
C3—H3 0.9300 C27—H27 0.9300
C4—C5 1.383 (5) C28—N4 1.346 (5)
C4—H4 0.9300 C28—H28 0.9300
C5—N1 1.337 (4) C29—O3 1.220 (4)
C5—C6 1.491 (5) C29—O2 1.284 (4)
C6—N2 1.353 (4) C29—C30 1.520 (4)
C6—C7 1.383 (5) C30—C35 1.386 (4)
C7—C8 1.398 (5) C30—C31 1.400 (4)
C7—H7 0.9300 C31—C32 1.395 (4)
C8—C9 1.375 (5) C31—H31 0.9300
C8—H8 0.9300 C32—C33 1.382 (4)
C9—C10 1.375 (5) C32—C36 1.518 (4)
C9—H9 0.9300 C33—C34 1.391 (4)
C10—N2 1.347 (4) C33—H33 0.9300
C10—H10 0.9300 C34—C35 1.383 (4)
C11—O14 1.253 (4) C34—N5 1.470 (4)
C11—O13 1.265 (4) C35—H35 0.9300
C11—C15 1.506 (4) C36—O4 1.252 (4)
C12—O11 1.222 (4) C36—O5 1.260 (4)
C12—O10 1.271 (4) N5—O6 1.216 (4)
C12—C13 1.525 (4) N5—O7 1.226 (4)
C13—C18 1.390 (4) N6—O9 1.211 (4)
C13—C14 1.401 (4) N6—O8 1.218 (4)
C14—C15 1.404 (4) O1—H3W 0.83 (3)
C14—H14 0.9300 O1—H4W 0.82 (3)
C15—C16 1.387 (4) O4—Cu2i 2.139 (2)
C16—C17 1.388 (5) O5—Cu2i 2.294 (2)
C16—H16 0.9300 O12—H1W 0.82 (4)
C17—C18 1.389 (4) O12—H2W 0.82 (4)
C17—N6 1.468 (4) O13—Cu1ii 2.258 (2)
C18—H18 0.9300 O14—Cu1ii 2.158 (2)
O10—Cu1—N1 119.16 (10) N3—C19—C20 122.2 (4)
O10—Cu1—N2 91.92 (10) N3—C19—H19 118.9
N1—Cu1—N2 79.26 (10) C20—C19—H19 118.9
O10—Cu1—O12 87.71 (9) C21—C20—C19 118.5 (4)
N1—Cu1—O12 93.12 (10) C21—C20—H20 120.7
N2—Cu1—O12 171.06 (10) C19—C20—H20 120.7
O10—Cu1—O14i 149.84 (9) C20—C21—C22 119.7 (3)
N1—Cu1—O14i 90.99 (10) C20—C21—H21 120.1
N2—Cu1—O14i 94.65 (10) C22—C21—H21 120.1
O12—Cu1—O14i 90.11 (10) C21—C22—C23 119.4 (4)
O10—Cu1—O13i 90.66 (9) C21—C22—H22 120.3
N1—Cu1—O13i 150.04 (10) C23—C22—H22 120.3
N2—Cu1—O13i 98.11 (10) N3—C23—C22 120.9 (4)
O12—Cu1—O13i 90.83 (9) N3—C23—C24 115.2 (3)
O14i—Cu1—O13i 59.29 (8) C22—C23—C24 123.8 (3)
O2—Cu2—N3 119.41 (10) N4—C24—C25 120.2 (3)
O2—Cu2—N4 91.57 (10) N4—C24—C23 115.5 (3)
N3—Cu2—N4 78.83 (11) C25—C24—C23 124.3 (3)
O2—Cu2—O1 87.52 (9) C26—C25—C24 119.4 (4)
N3—Cu2—O1 94.37 (10) C26—C25—H25 120.3
N4—Cu2—O1 171.62 (10) C24—C25—H25 120.3
O2—Cu2—O4ii 149.99 (9) C27—C26—C25 119.5 (4)
N3—Cu2—O4ii 90.60 (9) C27—C26—H26 120.3
N4—Cu2—O4ii 94.38 (10) C25—C26—H26 120.3
O1—Cu2—O4ii 90.54 (10) C26—C27—C28 118.7 (4)
O2—Cu2—O5ii 91.14 (9) C26—C27—H27 120.6
N3—Cu2—O5ii 149.09 (9) C28—C27—H27 120.6
N4—Cu2—O5ii 96.95 (10) N4—C28—C27 123.0 (4)
O1—Cu2—O5ii 91.40 (9) N4—C28—H28 118.5
O4ii—Cu2—O5ii 58.96 (8) C27—C28—H28 118.5
N1—C1—C2 122.1 (4) O3—C29—O2 125.0 (3)
N1—C1—H1 119.0 O3—C29—C30 119.1 (3)
C2—C1—H1 119.0 O2—C29—C30 115.9 (3)
C1—C2—C3 118.7 (4) C35—C30—C31 118.7 (3)
C1—C2—H2 120.6 C35—C30—C29 121.8 (3)
C3—C2—H2 120.6 C31—C30—C29 119.5 (3)
C2—C3—C4 119.7 (4) C32—C31—C30 121.5 (3)
C2—C3—H3 120.1 C32—C31—H31 119.3
C4—C3—H3 120.1 C30—C31—H31 119.3
C3—C4—C5 118.8 (4) C33—C32—C31 120.0 (3)
C3—C4—H4 120.6 C33—C32—C36 120.3 (3)
C5—C4—H4 120.6 C31—C32—C36 119.7 (3)
N1—C5—C4 121.7 (3) C32—C33—C34 117.7 (3)
N1—C5—C6 115.7 (3) C32—C33—H33 121.1
C4—C5—C6 122.6 (3) C34—C33—H33 121.1
N2—C6—C7 121.3 (3) C35—C34—C33 123.2 (3)
N2—C6—C5 114.6 (3) C35—C34—N5 118.3 (3)
C7—C6—C5 124.1 (3) C33—C34—N5 118.4 (3)
C6—C7—C8 119.4 (3) C34—C35—C30 118.9 (3)
C6—C7—H7 120.3 C34—C35—H35 120.5
C8—C7—H7 120.3 C30—C35—H35 120.5
C9—C8—C7 118.9 (3) O4—C36—O5 121.0 (3)
C9—C8—H8 120.5 O4—C36—C32 118.6 (3)
C7—C8—H8 120.5 O5—C36—C32 120.4 (3)
C8—C9—C10 118.8 (3) O4—C36—Cu2i 57.03 (16)
C8—C9—H9 120.6 O5—C36—Cu2i 64.09 (16)
C10—C9—H9 120.6 C32—C36—Cu2i 174.0 (2)
N2—C10—C9 123.0 (3) C5—N1—C1 119.0 (3)
N2—C10—H10 118.5 C5—N1—Cu1 115.6 (2)
C9—C10—H10 118.5 C1—N1—Cu1 125.1 (2)
O14—C11—O13 120.5 (3) C10—N2—C6 118.5 (3)
O14—C11—C15 119.1 (3) C10—N2—Cu1 126.8 (2)
O13—C11—C15 120.4 (3) C6—N2—Cu1 114.8 (2)
O14—C11—Cu1ii 58.05 (16) C23—N3—C19 119.2 (3)
O13—C11—Cu1ii 62.60 (17) C23—N3—Cu2 115.6 (2)
C15—C11—Cu1ii 174.8 (2) C19—N3—Cu2 124.6 (2)
O11—C12—O10 124.3 (3) C28—N4—C24 119.1 (3)
O11—C12—C13 118.5 (3) C28—N4—Cu2 126.4 (2)
O10—C12—C13 117.1 (3) C24—N4—Cu2 114.5 (2)
C18—C13—C14 119.6 (3) O6—N5—O7 123.5 (3)
C18—C13—C12 121.0 (3) O6—N5—C34 118.3 (3)
C14—C13—C12 119.4 (3) O7—N5—C34 118.2 (3)
C13—C14—C15 121.0 (3) O9—N6—O8 123.4 (3)
C13—C14—H14 119.5 O9—N6—C17 119.1 (3)
C15—C14—H14 119.5 O8—N6—C17 117.6 (4)
C16—C15—C14 119.8 (3) Cu2—O1—H3W 118 (3)
C16—C15—C11 120.1 (3) Cu2—O1—H4W 112 (3)
C14—C15—C11 120.0 (3) H3W—O1—H4W 113 (4)
C15—C16—C17 117.8 (3) C29—O2—Cu2 122.8 (2)
C15—C16—H16 121.1 C36—O4—Cu2i 93.58 (18)
C17—C16—H16 121.1 C36—O5—Cu2i 86.30 (19)
C16—C17—C18 123.9 (3) C12—O10—Cu1 121.3 (2)
C16—C17—N6 118.4 (3) Cu1—O12—H1W 106 (3)
C18—C17—N6 117.6 (3) Cu1—O12—H2W 121 (3)
C13—C18—C17 117.9 (3) H1W—O12—H2W 112 (4)
C13—C18—H18 121.0 C11—O13—Cu1ii 87.6 (2)
C17—C18—H18 121.0 C11—O14—Cu1ii 92.45 (19)
Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O12—H1W···O2i 0.83 (4) 1.98 (2) 2.742 (3) 153 (4)
O1—H4W···O10ii 0.82 (3) 1.95 (2) 2.724 (3) 158 (4)
O12—H2W···O5 0.82 (4) 2.07 (3) 2.760 (3) 141 (4)
O1—H3W···O13 0.83 (3) 2.13 (3) 2.778 (3) 135 (4)
Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O12—H1W⋯O2i 0.83 (4) 1.98 (2) 2.742 (3) 153 (4)
O1—H4W⋯O10ii 0.82 (3) 1.95 (2) 2.724 (3) 158 (4)
O12—H2W⋯O5 0.82 (4) 2.07 (3) 2.760 (3) 141 (4)
O1—H3W⋯O13 0.83 (3) 2.13 (3) 2.778 (3) 135 (4)
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (2001). SADABS and SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Church, B. S. & Halvorson, H. (1959). Nature (London), 183, 124–125.
Kim, Y., Lee, E. & Jung, D. Y. (2001). Chem. Mater.13, 2684–2690.
Okabe, N. & Oya, N. (2000). Acta Cryst. C56, 1416–1417.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21581121 | PMC2959803 | CC BY | 2021-01-04 18:54:31 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Nov 8; 64(Pt 12):m1500 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb284210.1107/S1600536808038440ACSEBHS1600536808038440Metal-Organic PapersBis[2,4-pentanedionato(1−)]bis[4,4,5,5-tetramethyl-2-(4-pyridyl)-imidazoline-1-oxyl 3-oxide]manganese(II) [Mn(C5H7O2)2(C12H16N3O2)]Liu Ying a*He Qingpeng aZhang Xianxi aXue Zechun aLv Chunyan aa College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 12 2008 22 11 2008 22 11 2008 64 Pt 12 e081200m1604 m1604 09 11 2008 18 11 2008 © Liu et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Mn(C5H7O2)2(C12H16N3O2)], the manganese(II) cation (site symmetry ) is hexacoordinated by four O and two N atoms in a distorted trans-MnN2O4 octahedral geometry. The four O atoms belonging to two 2,4-pentanedionate anions lie in the equatorial plane and the two N atoms occupy the axial coordination sites.
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Related literature
For related structures, see: Caruso et al. (2005 ▶); Iskander et al. (2001 ▶); Rajak et al. (2000 ▶); Sangeetha et al. (2000 ▶); Sutradhar et al. (2006 ▶).
Experimental
Crystal data
[Mn(C5H7O2)2(C12H16N3O2)]
M
r = 721.71
Triclinic,
a = 7.277 (3) Å
b = 9.7167 (15) Å
c = 13.2643 (15) Å
α = 97.978 (10)°
β = 103.342 (10)°
γ = 92.869 (10)°
V = 900.4 (4) Å3
Z = 1
Mo Kα radiation
μ = 0.42 mm−1
T = 293 (2) K
0.12 × 0.10 × 0.08 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.951, T
max = 0.970
6210 measured reflections
3264 independent reflections
2511 reflections with I > 2σ(I)
R
int = 0.033
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.118
S = 1.00
3264 reflections
229 parameters
H-atom parameters constrained
Δρmax = 0.58 e Å−3
Δρmin = −0.51 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808038440/hb2842sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808038440/hb2842Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB2842).
The authors thank the National Ministry of Science and Technology of China (grant No. 2001CB6105-07).
supplementary crystallographic
information
Comment
To design different kinds of metal-based coordination architectures with
appropriate organic radicals and co-ligands has been an important subject
during the last decade because of its potential usages for molecule-based
magnetic materials and optical devices. Varying the organic units, such as
tridentate nitronyl nitroxide
radical, and bidentate nitroxide radical could results in a large number of
building blocks with the potentional applications. In this paper, we report the
structure of the title compound, (I).
As shown in Fig. 1, the manganese(II) cation is hexacoordinated with four O and
two N atoms showing a slightly distorted octahedral geometry. The Mn(II)
cation lies on an inversion centre. The four oxygen atoms belonging to two
2,4-pentanedionate lie in the equatorial plane and the two nitrogen atoms lie
in the axial coordination sites (Table 1).
Experimental
A mixture of manganese(II) acetylacetonate (1 mmol) and
2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (1 mmol) in 20 ml
methanol was refluxed for several hours. The above cooled solution was
filtered and the filtrate was kept in an ice box. One week later, brown
blocks of (I) were obtained with a yield of ca 3%. Anal. Calc. for
C34H46N6MnO8: C 56.48, H 6.31, N 11.55%; Found: C 56.53, H 6.37, N
11.64%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93–0.96 Å
and refined as riding with Uiso(H) = 1.2Ueq(C)
or 1.5Ueq(methyl C).
Figures
Fig. 1. The molecular structure of (I) around Mn(II), drawn with 30% probability displacement ellipsoids for the non-hydrogen atoms. The unlabelled atoms are generated by the symmetry operation (-x, -y, -z).
Crystal data
[Mn(C5H7O2)2(C12H16N3O2)] Z = 1
Mr = 721.71 F000 = 381
Triclinic, P1 Dx = 1.331 Mg m−3
Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å
a = 7.277 (3) Å Cell parameters from 3264 reflections
b = 9.7167 (15) Å θ = 2.9–25.5º
c = 13.2643 (15) Å µ = 0.42 mm−1
α = 97.978 (10)º T = 293 (2) K
β = 103.342 (10)º Block, brown
γ = 92.869 (10)º 0.12 × 0.10 × 0.08 mm
V = 900.4 (4) Å3
Data collection
Bruker APEXII CCD area-detector diffractometer 3264 independent reflections
Radiation source: fine-focus sealed tube 2511 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.033
T = 293(2) K θmax = 25.5º
φ and ω scans θmin = 2.9º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −6→8
Tmin = 0.951, Tmax = 0.970 k = −11→11
6210 measured reflections l = −11→16
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.041 H-atom parameters constrained
wR(F2) = 0.118 w = 1/[σ2(Fo2) + (0.07P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.007
3264 reflections Δρmax = 0.58 e Å−3
229 parameters Δρmin = −0.51 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Mn1 0.0000 0.0000 0.0000 0.0236 (6)
C1 −0.0486 (4) 0.2828 (3) 0.0582 (2) 0.0237 (6)
C2 0.2048 (4) 0.2371 (3) −0.0432 (2) 0.0249 (6)
C3 0.3726 (4) 0.2936 (3) −0.0812 (2) 0.0343 (7)
H3A 0.4838 0.2496 −0.0522 0.052*
H3B 0.3946 0.3926 −0.0589 0.052*
H3C 0.3449 0.2741 −0.1564 0.052*
C4 0.1139 (4) 0.3210 (3) 0.0232 (2) 0.0264 (6)
H4 0.1672 0.4122 0.0467 0.032*
C5 −0.1320 (4) 0.3856 (3) 0.1294 (2) 0.0319 (7)
H5A −0.2665 0.3830 0.1019 0.048*
H5B −0.0754 0.4781 0.1327 0.048*
H5C −0.1066 0.3607 0.1985 0.048*
C6 0.1703 (4) 0.1166 (3) 0.2387 (2) 0.0220 (6)
H6 0.0417 0.1238 0.2338 0.026*
C7 0.4138 (4) 0.0539 (3) 0.1578 (2) 0.0232 (6)
H7 0.4539 0.0187 0.0984 0.028*
C8 0.5526 (4) 0.0956 (3) 0.2535 (2) 0.0246 (6)
H8 0.6802 0.0871 0.2560 0.030*
C9 0.2971 (4) 0.1612 (3) 0.3371 (2) 0.0222 (6)
H9 0.2526 0.1967 0.3950 0.027*
C10 0.4944 (4) 0.1510 (3) 0.3461 (2) 0.0222 (6)
C11 0.6315 (4) 0.1986 (3) 0.4507 (2) 0.0222 (6)
C12 0.9181 (3) 0.2690 (3) 0.58642 (19) 0.0210 (6)
C13 0.7430 (4) 0.2984 (3) 0.6343 (2) 0.0232 (6)
C14 0.7504 (4) 0.2549 (3) 0.7443 (2) 0.0307 (7)
H14A 0.6361 0.2770 0.7652 0.046*
H14B 0.8576 0.3046 0.7948 0.046*
H14C 0.7620 0.1564 0.7404 0.046*
C15 0.6888 (4) 0.4434 (3) 0.6352 (2) 0.0328 (7)
H15A 0.6878 0.4715 0.5685 0.049*
H15B 0.7786 0.5045 0.6894 0.049*
H15C 0.5648 0.4483 0.6483 0.049*
C16 1.0020 (4) 0.1369 (3) 0.6128 (2) 0.0254 (6)
H16A 0.9033 0.0623 0.5969 0.038*
H16B 1.0634 0.1492 0.6861 0.038*
H16C 1.0930 0.1147 0.5722 0.038*
C17 1.0770 (4) 0.3830 (3) 0.6102 (2) 0.0260 (6)
H17A 1.1689 0.3565 0.5711 0.039*
H17B 1.1363 0.3975 0.6838 0.039*
H17C 1.0271 0.4677 0.5908 0.039*
N1 0.2243 (3) 0.0629 (2) 0.14911 (16) 0.0214 (5)
N2 0.5856 (3) 0.2134 (2) 0.54863 (17) 0.0250 (5)
N3 0.8188 (3) 0.2417 (2) 0.46660 (17) 0.0221 (5)
O1 −0.1368 (2) 0.16513 (17) 0.03794 (14) 0.0254 (4)
O2 0.9120 (3) 0.25072 (19) 0.39302 (14) 0.0277 (4)
O3 0.1567 (2) 0.11183 (18) −0.07693 (14) 0.0259 (4)
O4 0.4265 (3) 0.1723 (2) 0.56899 (15) 0.0351 (5)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Mn1 0.0270 (15) 0.0192 (14) 0.0214 (15) 0.0067 (11) 0.0002 (11) 0.0001 (11)
C1 0.0267 (14) 0.0198 (13) 0.0197 (14) 0.0041 (10) −0.0057 (11) 0.0045 (10)
C2 0.0244 (14) 0.0214 (13) 0.0246 (15) 0.0003 (10) −0.0055 (11) 0.0087 (11)
C3 0.0301 (16) 0.0276 (15) 0.0445 (19) −0.0007 (12) 0.0041 (14) 0.0122 (13)
C4 0.0316 (15) 0.0180 (13) 0.0256 (15) 0.0005 (11) −0.0013 (12) 0.0040 (11)
C5 0.0373 (17) 0.0220 (14) 0.0327 (17) 0.0074 (12) 0.0012 (13) 0.0017 (12)
C6 0.0193 (13) 0.0234 (13) 0.0245 (15) 0.0035 (10) 0.0056 (11) 0.0061 (11)
C7 0.0246 (14) 0.0242 (13) 0.0212 (15) 0.0045 (10) 0.0052 (11) 0.0045 (11)
C8 0.0208 (14) 0.0270 (14) 0.0258 (16) 0.0026 (10) 0.0051 (11) 0.0044 (11)
C9 0.0238 (14) 0.0234 (13) 0.0199 (14) 0.0033 (10) 0.0055 (11) 0.0038 (10)
C10 0.0233 (14) 0.0217 (13) 0.0209 (15) 0.0006 (10) 0.0035 (11) 0.0045 (10)
C11 0.0230 (14) 0.0260 (13) 0.0178 (14) 0.0022 (10) 0.0051 (11) 0.0030 (11)
C12 0.0201 (13) 0.0256 (14) 0.0151 (14) 0.0009 (10) 0.0007 (10) 0.0026 (10)
C13 0.0224 (14) 0.0268 (14) 0.0185 (14) 0.0038 (11) 0.0009 (11) 0.0029 (11)
C14 0.0270 (15) 0.0405 (17) 0.0236 (16) 0.0033 (12) 0.0035 (12) 0.0060 (12)
C15 0.0292 (16) 0.0346 (16) 0.0330 (18) 0.0106 (12) 0.0046 (13) 0.0017 (13)
C16 0.0225 (14) 0.0250 (14) 0.0284 (16) 0.0034 (10) 0.0040 (12) 0.0058 (11)
C17 0.0254 (14) 0.0254 (14) 0.0254 (16) 0.0013 (11) 0.0031 (12) 0.0034 (11)
N1 0.0236 (12) 0.0185 (11) 0.0221 (13) 0.0025 (8) 0.0051 (10) 0.0039 (9)
N2 0.0175 (12) 0.0340 (13) 0.0225 (13) 0.0003 (9) 0.0038 (9) 0.0040 (10)
N3 0.0192 (11) 0.0260 (11) 0.0206 (12) 0.0014 (9) 0.0038 (10) 0.0041 (9)
O1 0.0261 (10) 0.0207 (9) 0.0268 (11) 0.0038 (7) 0.0006 (8) 0.0039 (8)
O2 0.0235 (10) 0.0367 (11) 0.0239 (11) 0.0013 (8) 0.0087 (8) 0.0033 (8)
O3 0.0280 (10) 0.0210 (9) 0.0258 (11) −0.0002 (7) 0.0011 (8) 0.0031 (8)
O4 0.0205 (10) 0.0555 (14) 0.0309 (12) −0.0011 (9) 0.0076 (9) 0.0113 (10)
Geometric parameters (Å, °)
Mn1—O1i 1.9964 (17) C9—C10 1.422 (4)
Mn1—O1 1.9964 (17) C9—H9 0.9300
Mn1—O3 2.0597 (17) C10—C11 1.508 (4)
Mn1—O3i 2.0597 (17) C11—N3 1.365 (3)
Mn1—N1 2.242 (2) C11—N2 1.405 (3)
Mn1—N1i 2.242 (2) C12—C16 1.498 (3)
C1—O1 1.246 (3) C12—C17 1.507 (3)
C1—C4 1.417 (4) C12—N3 1.566 (3)
C1—C5 1.524 (4) C12—C13 1.571 (4)
C2—O3 1.241 (3) C13—C15 1.482 (4)
C2—C4 1.415 (4) C13—N2 1.527 (3)
C2—C3 1.530 (4) C13—C14 1.564 (4)
C3—H3A 0.9600 C14—H14A 0.9600
C3—H3B 0.9600 C14—H14B 0.9600
C3—H3C 0.9600 C14—H14C 0.9600
C4—H4 0.9300 C15—H15A 0.9600
C5—H5A 0.9600 C15—H15B 0.9600
C5—H5B 0.9600 C15—H15C 0.9600
C5—H5C 0.9600 C16—H16A 0.9600
C6—N1 1.379 (3) C16—H16B 0.9600
C6—C9 1.412 (4) C16—H16C 0.9600
C6—H6 0.9300 C17—H17A 0.9600
C7—N1 1.364 (3) C17—H17B 0.9600
C7—C8 1.421 (4) C17—H17C 0.9600
C7—H7 0.9300 N2—O4 1.304 (3)
C8—C10 1.434 (4) N3—O2 1.320 (3)
C8—H8 0.9300
O1i—Mn1—O1 180.0 N3—C11—N2 108.1 (2)
O1i—Mn1—O3 87.80 (7) N3—C11—C10 126.2 (2)
O1—Mn1—O3 92.20 (7) N2—C11—C10 125.6 (2)
O1i—Mn1—O3i 92.20 (7) C16—C12—C17 108.1 (2)
O1—Mn1—O3i 87.80 (7) C16—C12—N3 106.9 (2)
O3—Mn1—O3i 180.0 C17—C12—N3 110.9 (2)
O1i—Mn1—N1 90.40 (7) C16—C12—C13 112.5 (2)
O1—Mn1—N1 89.60 (7) C17—C12—C13 117.4 (2)
O3—Mn1—N1 89.56 (7) N3—C12—C13 100.45 (18)
O3i—Mn1—N1 90.44 (7) C15—C13—N2 103.7 (2)
O1i—Mn1—N1i 89.60 (7) C15—C13—C14 109.2 (2)
O1—Mn1—N1i 90.40 (7) N2—C13—C14 112.0 (2)
O3—Mn1—N1i 90.44 (7) C15—C13—C12 114.0 (2)
O3i—Mn1—N1i 89.56 (7) N2—C13—C12 100.01 (19)
N1—Mn1—N1i 180.0 C14—C13—C12 116.9 (2)
O1—C1—C4 125.9 (2) C13—C14—H14A 109.5
O1—C1—C5 112.3 (2) C13—C14—H14B 109.5
C4—C1—C5 121.8 (2) H14A—C14—H14B 109.5
O3—C2—C4 123.9 (2) C13—C14—H14C 109.5
O3—C2—C3 113.3 (2) H14A—C14—H14C 109.5
C4—C2—C3 122.8 (2) H14B—C14—H14C 109.5
C2—C3—H3A 109.5 C13—C15—H15A 109.5
C2—C3—H3B 109.5 C13—C15—H15B 109.5
H3A—C3—H3B 109.5 H15A—C15—H15B 109.5
C2—C3—H3C 109.5 C13—C15—H15C 109.5
H3A—C3—H3C 109.5 H15A—C15—H15C 109.5
H3B—C3—H3C 109.5 H15B—C15—H15C 109.5
C2—C4—C1 127.8 (2) C12—C16—H16A 109.5
C2—C4—H4 116.1 C12—C16—H16B 109.5
C1—C4—H4 116.1 H16A—C16—H16B 109.5
C1—C5—H5A 109.5 C12—C16—H16C 109.5
C1—C5—H5B 109.5 H16A—C16—H16C 109.5
H5A—C5—H5B 109.5 H16B—C16—H16C 109.5
C1—C5—H5C 109.5 C12—C17—H17A 109.5
H5A—C5—H5C 109.5 C12—C17—H17B 109.5
H5B—C5—H5C 109.5 H17A—C17—H17B 109.5
N1—C6—C9 124.4 (2) C12—C17—H17C 109.5
N1—C6—H6 117.8 H17A—C17—H17C 109.5
C9—C6—H6 117.8 H17B—C17—H17C 109.5
N1—C7—C8 123.1 (2) C7—N1—C6 116.8 (2)
N1—C7—H7 118.5 C7—N1—Mn1 124.50 (17)
C8—C7—H7 118.5 C6—N1—Mn1 118.67 (16)
C7—C8—C10 119.5 (2) O4—N2—C11 127.6 (2)
C7—C8—H8 120.2 O4—N2—C13 120.6 (2)
C10—C8—H8 120.2 C11—N2—C13 111.6 (2)
C6—C9—C10 118.6 (2) O2—N3—C11 126.1 (2)
C6—C9—H9 120.7 O2—N3—C12 122.78 (18)
C10—C9—H9 120.7 C11—N3—C12 110.91 (19)
C9—C10—C8 117.6 (2) C1—O1—Mn1 118.40 (17)
C9—C10—C11 119.2 (2) C2—O3—Mn1 119.22 (17)
C8—C10—C11 123.2 (2)
Symmetry codes: (i) −x, −y, −z.
Table 1 Selected bond lengths (Å)
Mn1—O1 1.9964 (17)
Mn1—O3 2.0597 (17)
Mn1—N1 2.242 (2)
==== Refs
References
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Caruso, U., Centore, R., Panunzi, B., Roviello, A. & Tuzi, A. (2005). Eur. J. Inorg. Chem. pp. 2747–2758.
Iskander, M. F., Khalil, T. E., Haase, W., Werner, R., Svoboda, I. & Fuess, H. (2001). Polyhedron, 20, 2787–2792.
Rajak, K. K., Baruah, B., Rath, S. P. & Chakravorty, A. (2000). Inorg. Chem.39, 1598–1605.
Sangeetha, N. R. & Pal, S. (2000). Bull. Chem. Soc. Jpn, 73, 357–361.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sutradhar, M., Mukherjee, G., Drew, M. G. B. & Ghosh, S. (2006). Inorg. Chem.45, 5150–5158. | 21581201 | PMC2959951 | CC BY | 2021-01-04 18:54:34 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Nov 22; 64(Pt 12):m1604 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb284410.1107/S1600536808038178ACSEBHS1600536808038178Metal-Organic Papers
catena-Poly[[aqua(2,2′-bipyridyl)cobalt(II)]-μ-5-nitroisophthalato] [Co(C8H3NO6)(C10H8N2)(H2O)]Liu Ying a*He Qingpeng aZhang Xianxi aXue Zechun aLv Chunyan aa College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 12 2008 22 11 2008 22 11 2008 64 Pt 12 e081200m1605 m1606 13 11 2008 17 11 2008 © Liu et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the crystal structure of the title compound, [Co(C8H3NO6)(C10H8N2)(H2O)]n, there are two symmetry-independent one-dimensional coordination polymers, which are approximately related by noncrystallographic inversion symmetry. Each zigzag chain is constructed from one CoII ion, one O-monodentate 5-nitroisophthalate (ndc) dianion, one N,N′-bidentate 2,2′-bipyridyl ligand and one water molecule. A symmetry-generated O,O′-bidentate ndc dianion completes the cobalt coordination environment, which could be described as very distorted cis-CoN2O4 octahedral. The bridging ndc ligands result in parallel chains running along the a direction, and O—H⋯O hydrogen bonds arising from the water molecules complete the structure.
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Related literature
For uses of carboxylic acids in materials science, see: Church & Halvorson (1959 ▶); and in biological systems, see: Okabe & Oya (2000 ▶).
Experimental
Crystal data
[Co(C8H3NO6)(C10H8N2)(H2O)]
M
r = 442.24
Monoclinic,
a = 10.0125 (10) Å
b = 23.575 (2) Å
c = 15.403 (2) Å
β = 97.28 (1)°
V = 3606.3 (7) Å3
Z = 8
Mo Kα radiation
μ = 1.00 mm−1
T = 293 (2) K
0.43 × 0.28 × 0.20 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.673, T
max = 0.825
18893 measured reflections
6672 independent reflections
5103 reflections with I > 2σ(I)
R
int = 0.025
Refinement
R[F
2 > 2σ(F
2)] = 0.037
wR(F
2) = 0.106
S = 1.01
6672 reflections
535 parameters
6 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.95 e Å−3
Δρmin = −0.29 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808038178/hb2844sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808038178/hb2844Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB2844).
The authors thank the Natural Science Foundation of China (grant No. 20501011) and are grateful for financial support from Liaocheng University (grant No. X071011).
supplementary crystallographic
information
Comment
In recent years, carboxylic acids have been widely used as polydentate ligands,
which can coordinate to transition or rare earth ions yielding complexes with
interesting properties that are useful in materials science (Church &
Halvorson, 1959) and in biological systems (Okabe & Oya, 2000).
The importance
of transition metal dicarboxylate complexes motivated us to pursue synthetic
strategies for these compounds, using 5-nitroisophthalic acid as a polydentate
ligand. Here we report the synthesis and X-ray crystal structure analysis of
the title compound, (I), (Fig. 1).
Compound (I) is constructed from two zigzag chains, each containing one CoII
atom, one O-monodentate 5-nitroisophthalato (ndc) dianion, one N,N-bidentate
2,2'-bipyridyl ligand and one water molecule. A symmetry-generated,
O,O-bidentate ndc dianion completes the cobalt coordination, which could be
described as very distorted cis-CoN2O4 octahedral (Table 1). The bridging
ndc ligands result in parallel chains running along the a direction (Fig. 2)
and O—H···O hydrogen bonds arising from the water molecules (Table 2)
complete the structure (Fig. 3).
Experimental
A mixture of cobalt dichloride (0.5 mmol), 2,2'-bipyridine (0.5 mmol), and
5-nitroisophthalic acid (0.5 mmol) in H2O (8 ml) and ethanol (8 ml) sealed
in a 25 ml Teflon-lined stainless steel autoclave was kept at 413 K for three
days. Red blocks of (I) were obtained after cooling to room temperature with a
yield of 27%. Anal. Calc. for C18H13CoN3O7: C 48.34, H 2.91, N 10.74%;
Found: C 48.30, H 2.84, N 10.69%.
Refinement
The H atoms of the water molecules were located from difference density maps
and were refined with distance restraints of H···H = 1.38 (2) Å, O—H =
0.88 (2) Å, and with a fixed Uiso of 0.80 Å2. All other H atoms
were placed in calculated positions with C—H = 0.93 Å and refined as
riding with Uiso(H) = 1.2Ueq(carrier).
Figures
Fig. 1. The asymmetric unit of (I), extended to show the Co coordination spheres, showing 30% probability displacement ellipsoids (arbitrary spheres for the H atoms). Symmetry codes: O5A, O6A; A = (1+x, y, z), O9A, O10A, A = (x-1, y, z).
Fig. 2. Part of a one-dimensional polymeric chain in (I)
Fig. 3. The packing diagram of (I) formed with the hydrogen bonds.
Crystal data
[Co(C8H3NO6)(C10H8N2)(H2O)] F000 = 1800
Mr = 442.24 Dx = 1.629 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 6672 reflections
a = 10.0125 (10) Å θ = 1.7–25.5º
b = 23.575 (2) Å µ = 1.00 mm−1
c = 15.403 (2) Å T = 293 (2) K
β = 97.28 (1)º Block, red
V = 3606.3 (7) Å3 0.43 × 0.28 × 0.20 mm
Z = 8
Data collection
Bruker APEXII CCD diffractometer 6672 independent reflections
Radiation source: fine-focus sealed tube 5103 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.025
T = 293(2) K θmax = 25.5º
ω scans θmin = 1.7º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −12→10
Tmin = 0.673, Tmax = 0.825 k = −28→22
18893 measured reflections l = −18→18
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.037 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.106 w = 1/[σ2(Fo2) + (0.0548P)2 + 2.8058P] where P = (Fo2 + 2Fc2)/3
S = 1.01 (Δ/σ)max = 0.032
6672 reflections Δρmax = 0.95 e Å−3
535 parameters Δρmin = −0.29 e Å−3
6 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Co1 1.16169 (4) 0.154445 (14) 0.85573 (2) 0.02466 (11)
Co2 0.12374 (4) 0.354444 (15) 0.93350 (3) 0.02797 (12)
C1 0.0858 (3) 0.43912 (15) 0.7845 (2) 0.0469 (8)
H1 0.0715 0.4073 0.7489 0.056*
C2 0.0708 (4) 0.49272 (17) 0.7466 (3) 0.0555 (10)
H2 0.0482 0.4963 0.6864 0.067*
C3 0.0893 (4) 0.54031 (16) 0.7979 (3) 0.0570 (10)
H3 0.0810 0.5763 0.7731 0.068*
C4 0.1204 (3) 0.53368 (14) 0.8864 (3) 0.0474 (9)
H4 0.1307 0.5651 0.9231 0.057*
C5 0.1365 (3) 0.47893 (12) 0.9205 (2) 0.0363 (7)
C6 0.1704 (3) 0.46688 (12) 1.0143 (2) 0.0361 (7)
C7 0.1997 (3) 0.50925 (14) 1.0774 (3) 0.0490 (9)
H7 0.1978 0.5474 1.0616 0.059*
C8 0.2313 (3) 0.49307 (17) 1.1631 (3) 0.0557 (10)
H8 0.2506 0.5203 1.2067 0.067*
C9 0.2341 (4) 0.43613 (17) 1.1843 (3) 0.0559 (10)
H9 0.2569 0.4248 1.2421 0.067*
C10 0.2034 (3) 0.39623 (15) 1.1200 (2) 0.0468 (8)
H10 0.2049 0.3580 1.1351 0.056*
C11 0.5280 (3) 0.24937 (12) 1.02748 (19) 0.0320 (6)
H11 0.4449 0.2352 1.0380 0.038*
C12 0.6449 (3) 0.22104 (12) 1.05893 (19) 0.0321 (6)
C13 0.7703 (3) 0.24074 (12) 1.04616 (19) 0.0327 (7)
H13 0.8479 0.2213 1.0685 0.039*
C14 0.7776 (3) 0.29080 (11) 0.99863 (18) 0.0271 (6)
C15 0.9110 (3) 0.31378 (12) 0.9803 (2) 0.0312 (6)
C16 0.6606 (3) 0.31947 (12) 0.96577 (18) 0.0286 (6)
H16 0.6666 0.3527 0.9339 0.034*
C17 0.5354 (3) 0.29914 (12) 0.98002 (18) 0.0288 (6)
C18 0.4121 (3) 0.33266 (13) 0.9446 (2) 0.0325 (7)
C19 1.1953 (4) 0.07221 (15) 1.0079 (2) 0.0512 (9)
H19 1.2137 0.1044 1.0424 0.061*
C20 1.2036 (5) 0.01944 (18) 1.0474 (3) 0.0684 (12)
H20 1.2268 0.0164 1.1076 0.082*
C21 1.1774 (5) −0.02879 (17) 0.9971 (3) 0.0688 (12)
H21 1.1813 −0.0645 1.0230 0.083*
C22 1.1455 (4) −0.02303 (14) 0.9087 (2) 0.0526 (9)
H22 1.1289 −0.0547 0.8731 0.063*
C23 1.1386 (3) 0.03108 (12) 0.8732 (2) 0.0327 (7)
C24 1.1073 (3) 0.04138 (12) 0.7783 (2) 0.0314 (6)
C25 1.0794 (3) −0.00192 (13) 0.7180 (2) 0.0411 (8)
H25 1.0787 −0.0396 0.7357 0.049*
C26 1.0526 (3) 0.01263 (15) 0.6307 (2) 0.0456 (8)
H26 1.0329 −0.0152 0.5883 0.055*
C27 1.0556 (3) 0.06936 (15) 0.6071 (2) 0.0459 (8)
H27 1.0365 0.0798 0.5486 0.055*
C28 1.0866 (3) 0.11017 (13) 0.6701 (2) 0.0381 (7)
H28 1.0889 0.1480 0.6532 0.046*
C29 0.8750 (3) 0.17566 (13) 0.8380 (2) 0.0336 (7)
C30 0.7500 (3) 0.20949 (12) 0.80607 (18) 0.0274 (6)
C31 0.7569 (3) 0.26263 (12) 0.76643 (19) 0.0314 (6)
H31 0.8396 0.2780 0.7575 0.038*
C32 0.6258 (3) 0.18764 (12) 0.81981 (19) 0.0297 (6)
H32 0.6210 0.1529 0.8477 0.036*
C33 0.6392 (3) 0.29172 (12) 0.7409 (2) 0.0347 (7)
C34 0.5084 (3) 0.21757 (12) 0.79203 (18) 0.0273 (6)
C35 0.5143 (3) 0.27061 (13) 0.75206 (19) 0.0332 (7)
H35 0.4363 0.2910 0.7336 0.040*
C36 0.3756 (3) 0.19401 (13) 0.8104 (2) 0.0331 (7)
H1W 1.243 (3) 0.2304 (11) 0.958 (3) 0.080*
H2W 1.126 (2) 0.2153 (14) 0.989 (3) 0.080*
H3W 0.164 (2) 0.2956 (15) 0.803 (3) 0.080*
H4W 0.042 (3) 0.2778 (10) 0.823 (3) 0.080*
N1 1.1134 (2) 0.09709 (10) 0.75443 (15) 0.0302 (5)
N2 1.1617 (3) 0.07823 (10) 0.92182 (16) 0.0349 (6)
N3 0.1199 (2) 0.43207 (10) 0.87001 (17) 0.0349 (6)
N4 0.1713 (2) 0.41090 (10) 1.03641 (17) 0.0361 (6)
N5 0.6352 (3) 0.16753 (12) 1.10790 (19) 0.0477 (7)
N6 0.6479 (3) 0.34937 (13) 0.7036 (2) 0.0578 (9)
O1 0.8634 (2) 0.12806 (12) 0.8691 (2) 0.0705 (9)
O2 0.98644 (19) 0.19824 (8) 0.82627 (15) 0.0379 (5)
O3 0.7556 (3) 0.37273 (12) 0.7088 (2) 0.0801 (10)
O4 0.5459 (3) 0.37066 (17) 0.6681 (3) 0.1342 (19)
O5 0.3733 (2) 0.14822 (9) 0.85254 (16) 0.0440 (6)
O6 0.26919 (19) 0.22039 (9) 0.78407 (15) 0.0422 (5)
O7 0.5264 (3) 0.14506 (14) 1.1063 (2) 0.0932 (12)
O8 0.7366 (3) 0.14819 (11) 1.14856 (19) 0.0642 (8)
O9 1.0167 (2) 0.28717 (9) 1.00726 (15) 0.0411 (5)
O10 0.9137 (2) 0.35927 (9) 0.93795 (16) 0.0442 (6)
O11 0.4241 (2) 0.37689 (11) 0.90426 (18) 0.0586 (7)
O12 0.30031 (19) 0.31194 (9) 0.96161 (15) 0.0409 (5)
O1W 1.1948 (2) 0.20370 (8) 0.97017 (15) 0.0371 (5)
O2W 0.0917 (2) 0.30554 (8) 0.81966 (16) 0.0374 (5)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Co1 0.01864 (19) 0.02268 (19) 0.0325 (2) 0.00142 (14) 0.00270 (15) 0.00151 (15)
Co2 0.01822 (19) 0.0237 (2) 0.0425 (2) 0.00121 (14) 0.00602 (16) 0.00113 (16)
C1 0.0428 (19) 0.048 (2) 0.052 (2) 0.0030 (15) 0.0148 (16) 0.0066 (16)
C2 0.046 (2) 0.065 (3) 0.057 (2) 0.0082 (18) 0.0139 (18) 0.024 (2)
C3 0.043 (2) 0.042 (2) 0.090 (3) 0.0027 (16) 0.020 (2) 0.026 (2)
C4 0.0326 (18) 0.0322 (17) 0.080 (3) 0.0017 (14) 0.0173 (17) 0.0057 (17)
C5 0.0193 (14) 0.0299 (15) 0.062 (2) −0.0004 (12) 0.0141 (14) 0.0001 (14)
C6 0.0188 (14) 0.0342 (16) 0.057 (2) −0.0007 (12) 0.0101 (13) −0.0045 (14)
C7 0.0352 (18) 0.0352 (18) 0.078 (3) −0.0024 (14) 0.0127 (18) −0.0098 (17)
C8 0.038 (2) 0.067 (3) 0.062 (3) −0.0018 (17) 0.0042 (18) −0.022 (2)
C9 0.043 (2) 0.073 (3) 0.052 (2) 0.0057 (18) 0.0078 (17) −0.0072 (19)
C10 0.0398 (19) 0.051 (2) 0.050 (2) 0.0067 (16) 0.0065 (16) 0.0001 (17)
C11 0.0204 (14) 0.0411 (16) 0.0348 (17) −0.0058 (12) 0.0051 (12) 0.0010 (13)
C12 0.0267 (15) 0.0373 (16) 0.0323 (16) −0.0020 (12) 0.0039 (12) 0.0048 (12)
C13 0.0210 (14) 0.0419 (17) 0.0347 (17) 0.0023 (12) 0.0012 (12) 0.0005 (13)
C14 0.0194 (14) 0.0317 (14) 0.0313 (15) −0.0005 (11) 0.0069 (11) −0.0043 (12)
C15 0.0185 (14) 0.0359 (16) 0.0403 (17) 0.0013 (12) 0.0077 (12) −0.0087 (13)
C16 0.0228 (14) 0.0299 (14) 0.0336 (16) −0.0011 (11) 0.0054 (12) −0.0007 (12)
C17 0.0194 (14) 0.0358 (16) 0.0310 (16) 0.0001 (11) 0.0023 (11) −0.0047 (12)
C18 0.0209 (15) 0.0387 (17) 0.0373 (17) 0.0023 (12) 0.0010 (12) −0.0054 (13)
C19 0.070 (3) 0.048 (2) 0.0355 (19) 0.0013 (18) 0.0076 (17) 0.0048 (15)
C20 0.102 (4) 0.065 (3) 0.038 (2) 0.005 (2) 0.009 (2) 0.0146 (19)
C21 0.096 (3) 0.046 (2) 0.063 (3) 0.002 (2) 0.008 (2) 0.030 (2)
C22 0.068 (3) 0.0338 (18) 0.056 (2) −0.0038 (17) 0.0053 (19) 0.0100 (16)
C23 0.0258 (15) 0.0308 (15) 0.0415 (18) −0.0007 (12) 0.0043 (12) 0.0044 (13)
C24 0.0230 (14) 0.0304 (15) 0.0408 (17) −0.0015 (12) 0.0042 (12) 0.0016 (13)
C25 0.0343 (17) 0.0329 (16) 0.056 (2) −0.0028 (13) 0.0052 (15) −0.0044 (15)
C26 0.0389 (19) 0.051 (2) 0.046 (2) −0.0031 (15) 0.0035 (15) −0.0147 (16)
C27 0.045 (2) 0.055 (2) 0.0367 (18) 0.0019 (16) −0.0003 (15) −0.0041 (15)
C28 0.0405 (18) 0.0378 (17) 0.0353 (17) 0.0061 (14) 0.0027 (14) 0.0054 (13)
C29 0.0221 (15) 0.0413 (17) 0.0369 (17) 0.0018 (13) 0.0017 (12) 0.0045 (13)
C30 0.0185 (14) 0.0351 (15) 0.0285 (15) 0.0014 (11) 0.0021 (11) −0.0006 (12)
C31 0.0192 (14) 0.0402 (16) 0.0351 (16) −0.0014 (12) 0.0045 (12) 0.0051 (13)
C32 0.0256 (15) 0.0311 (15) 0.0328 (15) −0.0014 (12) 0.0060 (12) −0.0004 (12)
C33 0.0303 (16) 0.0368 (16) 0.0379 (17) 0.0033 (13) 0.0078 (13) 0.0108 (13)
C34 0.0193 (13) 0.0346 (15) 0.0284 (15) −0.0007 (11) 0.0047 (11) −0.0063 (12)
C35 0.0215 (14) 0.0439 (17) 0.0340 (16) 0.0086 (12) 0.0030 (12) 0.0023 (13)
C36 0.0240 (15) 0.0401 (17) 0.0361 (17) −0.0022 (13) 0.0070 (12) −0.0110 (13)
N1 0.0251 (12) 0.0298 (12) 0.0358 (14) 0.0034 (10) 0.0039 (10) 0.0004 (10)
N2 0.0335 (14) 0.0349 (14) 0.0369 (15) −0.0008 (11) 0.0067 (11) 0.0042 (11)
N3 0.0247 (13) 0.0352 (14) 0.0458 (17) 0.0018 (10) 0.0085 (11) 0.0055 (11)
N4 0.0264 (13) 0.0343 (14) 0.0483 (16) 0.0041 (10) 0.0079 (11) −0.0007 (11)
N5 0.0348 (16) 0.0546 (17) 0.0532 (18) −0.0040 (14) 0.0034 (13) 0.0203 (14)
N6 0.0458 (19) 0.0573 (19) 0.073 (2) 0.0145 (16) 0.0191 (16) 0.0331 (16)
O1 0.0359 (14) 0.0722 (18) 0.105 (2) 0.0136 (13) 0.0152 (14) 0.0578 (17)
O2 0.0161 (10) 0.0346 (11) 0.0623 (14) 0.0004 (8) 0.0022 (9) −0.0024 (10)
O3 0.065 (2) 0.0644 (18) 0.108 (2) −0.0176 (15) −0.0012 (17) 0.0410 (17)
O4 0.0504 (19) 0.129 (3) 0.228 (5) 0.039 (2) 0.036 (2) 0.134 (3)
O5 0.0274 (12) 0.0441 (13) 0.0621 (15) −0.0034 (9) 0.0117 (10) 0.0051 (11)
O6 0.0187 (10) 0.0491 (13) 0.0594 (15) 0.0039 (9) 0.0073 (10) −0.0021 (11)
O7 0.0504 (18) 0.100 (2) 0.123 (3) −0.0321 (16) −0.0136 (18) 0.068 (2)
O8 0.0439 (15) 0.0619 (17) 0.087 (2) 0.0111 (12) 0.0070 (14) 0.0357 (14)
O9 0.0185 (10) 0.0453 (12) 0.0603 (14) 0.0035 (9) 0.0079 (10) −0.0007 (10)
O10 0.0276 (11) 0.0363 (12) 0.0711 (16) −0.0028 (9) 0.0161 (11) 0.0094 (11)
O11 0.0374 (14) 0.0576 (16) 0.0799 (18) 0.0077 (11) 0.0040 (13) 0.0303 (14)
O12 0.0190 (10) 0.0378 (12) 0.0658 (15) 0.0009 (9) 0.0052 (10) −0.0032 (10)
O1W 0.0303 (12) 0.0335 (11) 0.0465 (13) 0.0030 (9) 0.0009 (10) −0.0036 (9)
O2W 0.0287 (11) 0.0335 (11) 0.0502 (13) 0.0025 (9) 0.0050 (10) 0.0004 (10)
Geometric parameters (Å, °)
Co1—N2 2.065 (2) C19—C20 1.382 (5)
Co1—N1 2.075 (2) C19—H19 0.9300
Co1—O2 2.0369 (19) C20—C21 1.382 (6)
Co1—O1W 2.102 (2) C20—H20 0.9300
Co1—O5i 2.131 (2) C21—C22 1.365 (5)
Co1—O6i 2.257 (2) C21—H21 0.9300
Co2—N3 2.073 (2) C22—C23 1.386 (4)
Co2—N4 2.078 (3) C22—H22 0.9300
Co2—O12 2.031 (2) C23—N2 1.344 (4)
Co2—O2W 2.089 (2) C23—C24 1.475 (4)
Co2—O10ii 2.116 (2) C24—N1 1.367 (4)
Co2—O9ii 2.294 (2) C24—C25 1.385 (4)
C1—N3 1.329 (4) C25—C26 1.380 (5)
C1—C2 1.392 (5) C25—H25 0.9300
C1—H1 0.9300 C26—C27 1.387 (5)
C2—C3 1.372 (6) C26—H26 0.9300
C2—H2 0.9300 C27—C28 1.374 (4)
C3—C4 1.367 (6) C27—H27 0.9300
C3—H3 0.9300 C28—N1 1.329 (4)
C4—C5 1.395 (4) C28—H28 0.9300
C4—H4 0.9300 C29—O1 1.231 (4)
C5—N3 1.349 (4) C29—O2 1.270 (3)
C5—C6 1.470 (5) C29—C30 1.513 (4)
C6—N4 1.362 (4) C30—C32 1.386 (4)
C6—C7 1.398 (5) C30—C31 1.399 (4)
C7—C8 1.373 (5) C31—C33 1.377 (4)
C7—H7 0.9300 C31—H31 0.9300
C8—C9 1.381 (5) C32—C34 1.391 (4)
C8—H8 0.9300 C32—H32 0.9300
C9—C10 1.372 (5) C33—C35 1.377 (4)
C9—H9 0.9300 C33—N6 1.482 (4)
C10—N4 1.332 (4) C34—C35 1.398 (4)
C10—H10 0.9300 C34—C36 1.500 (4)
C11—C12 1.381 (4) C35—H35 0.9300
C11—C17 1.389 (4) C36—O6 1.256 (3)
C11—H11 0.9300 C36—O5 1.261 (4)
C12—C13 1.376 (4) C36—Co1ii 2.515 (3)
C12—N5 1.479 (4) N5—O7 1.209 (4)
C13—C14 1.396 (4) N5—O8 1.212 (3)
C13—H13 0.9300 N6—O3 1.204 (4)
C14—C16 1.390 (4) N6—O4 1.205 (4)
C14—C15 1.501 (4) O5—Co1ii 2.131 (2)
C15—O9 1.255 (3) O6—Co1ii 2.257 (2)
C15—O10 1.257 (4) O9—Co2i 2.294 (2)
C16—C17 1.386 (4) O10—Co2i 2.116 (2)
C16—H16 0.9300 O1W—H1W 0.830 (10)
C17—C18 1.508 (4) O1W—H2W 0.830 (10)
C18—O11 1.228 (4) O2W—H3W 0.835 (10)
C18—O12 1.278 (3) O2W—H4W 0.831 (10)
C19—N2 1.334 (4)
O2—Co1—N2 119.80 (9) C20—C19—H19 119.1
O2—Co1—N1 92.92 (9) C19—C20—C21 119.9 (4)
N2—Co1—N1 77.83 (9) C19—C20—H20 120.1
O2—Co1—O1W 86.94 (8) C21—C20—H20 120.1
N2—Co1—O1W 94.42 (9) C22—C21—C20 118.7 (3)
N1—Co1—O1W 171.00 (9) C22—C21—H21 120.6
O2—Co1—O5i 149.41 (9) C20—C21—H21 120.6
N2—Co1—O5i 90.78 (9) C21—C22—C23 118.6 (3)
N1—Co1—O5i 94.31 (9) C21—C22—H22 120.7
O1W—Co1—O5i 90.33 (9) C23—C22—H22 120.7
O2—Co1—O6i 89.58 (8) N2—C23—C22 123.0 (3)
N2—Co1—O6i 150.47 (9) N2—C23—C24 114.6 (2)
N1—Co1—O6i 99.27 (9) C22—C23—C24 122.4 (3)
O1W—Co1—O6i 89.73 (8) N1—C24—C25 122.7 (3)
O5i—Co1—O6i 59.93 (8) N1—C24—C23 114.4 (2)
O12—Co2—N3 119.99 (9) C25—C24—C23 122.9 (3)
O12—Co2—N4 92.54 (9) C26—C25—C24 117.9 (3)
N3—Co2—N4 77.48 (10) C26—C25—H25 121.0
O12—Co2—O2W 86.76 (9) C24—C25—H25 121.0
N3—Co2—O2W 95.71 (9) C25—C26—C27 119.2 (3)
N4—Co2—O2W 171.71 (10) C25—C26—H26 120.4
O12—Co2—O10ii 149.51 (9) C27—C26—H26 120.4
N3—Co2—O10ii 90.50 (9) C28—C27—C26 120.0 (3)
N4—Co2—O10ii 94.22 (9) C28—C27—H27 120.0
O2W—Co2—O10ii 90.54 (9) C26—C27—H27 120.0
O12—Co2—O9ii 90.11 (8) N1—C28—C27 121.8 (3)
N3—Co2—O9ii 149.55 (8) N1—C28—H28 119.1
N4—Co2—O9ii 98.13 (9) C27—C28—H28 119.1
O2W—Co2—O9ii 90.14 (8) O1—C29—O2 124.4 (3)
O10ii—Co2—O9ii 59.50 (8) O1—C29—C30 119.5 (3)
N3—C1—C2 122.0 (4) O2—C29—C30 116.0 (3)
N3—C1—H1 119.0 C32—C30—C31 119.6 (2)
C2—C1—H1 119.0 C32—C30—C29 118.4 (3)
C3—C2—C1 120.1 (4) C31—C30—C29 121.9 (2)
C3—C2—H2 120.0 C33—C31—C30 118.9 (3)
C1—C2—H2 120.0 C33—C31—H31 120.6
C4—C3—C2 118.6 (3) C30—C31—H31 120.6
C4—C3—H3 120.7 C30—C32—C34 120.3 (3)
C2—C3—H3 120.7 C30—C32—H32 119.9
C3—C4—C5 118.8 (3) C34—C32—H32 119.9
C3—C4—H4 120.6 C35—C33—C31 122.8 (3)
C5—C4—H4 120.6 C35—C33—N6 118.6 (3)
N3—C5—C4 122.7 (3) C31—C33—N6 118.5 (3)
N3—C5—C6 113.9 (3) C32—C34—C35 120.5 (3)
C4—C5—C6 123.4 (3) C32—C34—C36 119.2 (3)
N4—C6—C7 121.6 (3) C35—C34—C36 120.2 (2)
N4—C6—C5 115.2 (3) C33—C35—C34 117.9 (3)
C7—C6—C5 123.2 (3) C33—C35—H35 121.0
C8—C7—C6 118.2 (3) C34—C35—H35 121.0
C8—C7—H7 120.9 O6—C36—O5 121.3 (3)
C6—C7—H7 120.9 O6—C36—C34 119.4 (3)
C7—C8—C9 119.5 (3) O5—C36—C34 119.3 (3)
C7—C8—H8 120.2 O6—C36—Co1ii 63.61 (16)
C9—C8—H8 120.3 O5—C36—Co1ii 57.85 (15)
C10—C9—C8 120.0 (4) C34—C36—Co1ii 174.8 (2)
C10—C9—H9 120.0 C28—N1—C24 118.4 (3)
C8—C9—H9 120.0 C28—N1—Co1 125.6 (2)
N4—C10—C9 121.6 (3) C24—N1—Co1 115.95 (19)
N4—C10—H10 119.2 C19—N2—C23 118.0 (3)
C9—C10—H10 119.2 C19—N2—Co1 124.5 (2)
C12—C11—C17 119.6 (3) C23—N2—Co1 117.12 (19)
C12—C11—H11 120.2 C1—N3—C5 117.8 (3)
C17—C11—H11 120.2 C1—N3—Co2 124.4 (2)
C13—C12—C11 122.3 (3) C5—N3—Co2 117.2 (2)
C13—C12—N5 118.7 (3) C10—N4—C6 119.1 (3)
C11—C12—N5 119.0 (3) C10—N4—Co2 125.1 (2)
C12—C13—C14 118.0 (3) C6—N4—Co2 115.9 (2)
C12—C13—H13 121.0 O7—N5—O8 122.6 (3)
C14—C13—H13 121.0 O7—N5—C12 118.7 (3)
C16—C14—C13 120.3 (3) O8—N5—C12 118.7 (3)
C16—C14—C15 119.0 (3) O3—N6—O4 122.7 (3)
C13—C14—C15 120.7 (2) O3—N6—C33 119.3 (3)
O9—C15—O10 121.7 (3) O4—N6—C33 118.0 (3)
O9—C15—C14 119.4 (3) C29—O2—Co1 120.23 (19)
O10—C15—C14 118.9 (2) C36—O5—Co1ii 92.08 (17)
C17—C16—C14 120.8 (3) C36—O6—Co1ii 86.49 (18)
C17—C16—H16 119.6 C15—O9—Co2i 85.29 (18)
C14—C16—H16 119.6 C15—O10—Co2i 93.31 (17)
C16—C17—C11 119.0 (3) C18—O12—Co2 121.93 (19)
C16—C17—C18 118.5 (3) Co1—O1W—H1W 105 (3)
C11—C17—C18 122.5 (3) Co1—O1W—H2W 115 (3)
O11—C18—O12 124.9 (3) H1W—O1W—H2W 111.4 (18)
O11—C18—C17 120.0 (3) Co2—O2W—H3W 111 (3)
O12—C18—C17 115.1 (3) Co2—O2W—H4W 114 (3)
N2—C19—C20 121.7 (3) H3W—O2W—H4W 111.0 (17)
N2—C19—H19 119.1
Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O1W—H1W···O12i 0.830 (10) 2.01 (2) 2.771 (3) 153 (3)
O2W—H4W···O2ii 0.831 (10) 1.957 (17) 2.747 (3) 159 (3)
O1W—H2W···O9 0.830 (10) 2.05 (2) 2.763 (3) 143 (3)
O2W—H3W···O6 0.835 (10) 2.10 (3) 2.781 (3) 138 (3)
Symmetry codes: (i) x+1, y, z; (ii) x−1, y, z.
Table 1 Selected bond lengths (Å)
Co1—N2 2.065 (2)
Co1—N1 2.075 (2)
Co1—O2 2.0369 (19)
Co1—O1W 2.102 (2)
Co1—O5i 2.131 (2)
Co1—O6i 2.257 (2)
Co2—N3 2.073 (2)
Co2—N4 2.078 (3)
Co2—O12 2.031 (2)
Co2—O2W 2.089 (2)
Co2—O10ii 2.116 (2)
Co2—O9ii 2.294 (2)
Symmetry codes: (i) ; (ii) .
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O1W—H1W⋯O12i 0.830 (10) 2.01 (2) 2.771 (3) 153 (3)
O2W—H4W⋯O2ii 0.831 (10) 1.957 (17) 2.747 (3) 159 (3)
O1W—H2W⋯O9 0.830 (10) 2.05 (2) 2.763 (3) 143 (3)
O2W—H3W⋯O6 0.835 (10) 2.10 (3) 2.781 (3) 138 (3)
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (2001). SADABS and SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Church, B. S. & Halvorson, H. (1959). Nature (London), 183, 124–125.
Okabe, N. & Oya, N. (2000). Acta Cryst. C56, 1416–1417.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21581202 | PMC2960127 | CC BY | 2021-01-04 18:54:26 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Nov 22; 64(Pt 12):m1605-m1606 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography er205510.1107/S1600536808023507ACSEBHS1600536808023507Metal-Organic PapersTetra-μ-2,5-difluorobenzoato-bis[(2,2′-bipyridine)(2,5-difluorobenzoato)gadolinium(III)] [Gd2(C7H3F2O2)6(C10H8N2)2]Li Sheng aZhang Fu-Li bTang Kun bMa Yuan-Fang a*a The Institute of Immunology, Key Laboratory of Natural Drugs and Immunological Engineering of Henan Province, College of Medicine, Henan University, Kaifeng 475003, People’s Republic of Chinab College of Medicine, Henan University, Kaifeng 475003, People’s Republic of ChinaCorrespondence e-mail: [email protected] 9 2008 09 8 2008 09 8 2008 64 Pt 9 e080900m1142 m1142 28 4 2008 25 7 2008 © Li et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the centrosymmetric title compound, [Gd2(C7H3F2O2)6(C10H8N2)2], the asymmetric unit comprises one cation chelated by two 2,5-difluorobenzoate and one 2,2′-bipyridine. Two cations are linked into dimers via three bridging carboxylate groups from three 2,5-difluorobenzoic acid units. The GdIII ion is nine-coordinated by seven O atoms and two N atoms.
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Related literature
For related literature, see: Church & Halvorson (1959 ▶); Chung et al. (1971 ▶); Okabe & Oya (2000 ▶); Okabe et al. (2002 ▶); Serre et al. (2005 ▶); Pocker & Fong (1980 ▶); Scapin et al. (1997 ▶).
Experimental
Crystal data
[Gd2(C7H3F2O2)6(C10H8N2)2]
M
r = 1569.43
Triclinic,
a = 11.4012 (10) Å
b = 12.1890 (10) Å
c = 12.588 (2) Å
α = 103.99 (2)°
β = 102.90 (2)°
γ = 113.58 (2)°
V = 1451.6 (3) Å3
Z = 1
Mo Kα radiation
μ = 2.37 mm−1
T = 293 (2) K
0.44 × 0.26 × 0.20 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.422, T
max = 0.648
8233 measured reflections
5557 independent reflections
4813 reflections with I > 2σ(I)
R
int = 0.021
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.110
S = 1.00
5557 reflections
416 parameters
H-atom parameters constrained
Δρmax = 2.22 e Å−3
Δρmin = −0.62 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808023507/er2055sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808023507/er2055Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: ER2055).
The authors are grateful for financial support from Henan University (grant No. 05YBGG013)
supplementary crystallographic
information
Comment
In recent years, carboxylic acids have been widely used as polydentate ligands,
which can coordinate to transition or rare earth ions yielding complexes with
interesting properties that are useful in materials science (Church &
Halvorson, 1959; Chung et al., 1971) and in biological
systems (Okabe &
Oya, 2000; Serre et al., 2005; Pocker & Fong,
1980; Scapin et
al., 1997). Herein, we report the synthesis and X-ray crystal
structure
analysis of the title compound, hexa(2,5-difluorobenzoato)
bis(2,2'-bipyridine) bisgadolinium(III).
The molecular structure of the title compound is shown in Fig.1, GdIII is
chelated by two 2,5-difluorobenzoate and one 2,2'-bipyridine. Two cations are
linked into a dimer via bridging carboxylate groups from four
2,5-difluorobenzoate ions.
The GdIII ion is nine-coordinated with seven O atoms
and two N atoms. The Gd—N and Gd—O bond lengths are in the range of
2.567 (4)–2.585 (5) Å and 2.364 (4)–2.495 (4) Å, respectively.
Experimental
A mixture of gadolinium chloride (0.5 mmol), 2,5-difluorobenzoic acid (1 mmol),
Sodium hydroxide(1 mmol), 2,2'-bipyridine(0.5 mmol), H2O (8 ml) and Ethanol
(8 ml) in a 25 ml Teflon-lined stainless steel autoclave was kept at 433 K for
three days. Colorless crystals were obtained after cooling to room
temperature.
Refinement
All H atoms on C atoms were generated geometrically and refined as riding atoms
with C—H= 0.93Å and Uiso(H)= 1.2 times Ueq(C).
Figures
Fig. 1. A view of the structure of (I), showing 30% probability displacement ellipsoids. Atoms labeled with i at the symmetry positions (-x + 1,-y + 2,-z + 1).
Crystal data
[Gd2(C7H3F2O2)6(C10H8N2)2] Z = 1
Mr = 1569.43 F000 = 766
Triclinic, P1 Dx = 1.795 Mg m−3
Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å
a = 11.4012 (10) Å Cell parameters from 5557 reflections
b = 12.1890 (10) Å θ = 1.8–26.0º
c = 12.588 (2) Å µ = 2.37 mm−1
α = 103.99 (2)º T = 293 (2) K
β = 102.90 (2)º Block, colorless
γ = 113.58 (2)º 0.44 × 0.26 × 0.20 mm
V = 1451.6 (3) Å3
Data collection
Bruker APEXII CCD area-detector diffractometer 5557 independent reflections
Radiation source: fine-focus sealed tube 4813 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.021
T = 293(2) K θmax = 26.0º
φ and ω scans θmin = 1.8º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −10→14
Tmin = 0.422, Tmax = 0.649 k = −15→12
8233 measured reflections l = −15→14
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.041 H-atom parameters constrained
wR(F2) = 0.110 w = 1/[σ2(Fo2) + (0.071P)2 + 0.5036P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.030
5557 reflections Δρmax = 2.22 e Å−3
416 parameters Δρmin = −0.62 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Gd1 0.39616 (2) 0.80450 (2) 0.43978 (2) 0.03327 (12)
C1 0.5204 (7) 0.6201 (6) 0.2986 (6) 0.0566 (16)
H1 0.5606 0.6949 0.2835 0.068*
C2 0.5504 (8) 0.5214 (7) 0.2568 (7) 0.071 (2)
H2 0.6112 0.5310 0.2168 0.086*
C3 0.4902 (9) 0.4138 (7) 0.2755 (7) 0.074 (2)
H3 0.5067 0.3456 0.2468 0.089*
C4 0.4041 (8) 0.4025 (6) 0.3367 (6) 0.067 (2)
H4 0.3630 0.3274 0.3511 0.080*
C5 0.3784 (6) 0.5033 (5) 0.3773 (5) 0.0470 (15)
C6 0.6740 (5) 0.9487 (5) 0.6191 (5) 0.0369 (11)
C7 0.8263 (6) 1.0068 (5) 0.6801 (5) 0.0424 (13)
C8 0.9145 (7) 1.0683 (6) 0.6300 (7) 0.0626 (18)
H8 0.8813 1.0778 0.5601 0.075*
C9 1.0543 (8) 1.1162 (7) 0.6852 (9) 0.079 (2)
C10 1.1059 (8) 1.1078 (8) 0.7849 (9) 0.088 (3)
H10 1.2004 1.1430 0.8197 0.105*
C11 1.0194 (8) 1.0459 (9) 0.8400 (8) 0.084 (3)
H11 1.0547 1.0395 0.9110 0.100*
C12 0.8802 (6) 0.9953 (7) 0.7841 (6) 0.0538 (15)
C13 0.1577 (6) 0.6867 (5) 0.2396 (5) 0.0403 (12)
C14 0.0390 (6) 0.6516 (5) 0.1350 (5) 0.0457 (13)
C15 −0.0965 (7) 0.5954 (7) 0.1250 (6) 0.0602 (17)
C16 −0.2024 (7) 0.5661 (9) 0.0290 (7) 0.079 (2)
H16 −0.2919 0.5269 0.0273 0.094*
C17 −0.1783 (8) 0.5937 (8) −0.0639 (7) 0.077 (2)
H17 −0.2498 0.5751 −0.1299 0.092*
C18 −0.0444 (9) 0.6503 (8) −0.0579 (6) 0.074 (2)
C19 0.0612 (7) 0.6787 (6) 0.0364 (6) 0.0595 (17)
H19 0.1501 0.7167 0.0365 0.071*
C20 0.6082 (5) 0.9901 (5) 0.3314 (5) 0.0379 (11)
C21 0.6108 (6) 0.9679 (5) 0.2087 (5) 0.0440 (13)
C22 0.7087 (7) 1.0531 (6) 0.1808 (6) 0.0552 (16)
C23 0.7022 (9) 1.0268 (7) 0.0655 (7) 0.068 (2)
H23 0.7709 1.0843 0.0480 0.082*
C24 0.5951 (9) 0.9165 (8) −0.0237 (7) 0.0695 (19)
H24 0.5889 0.9002 −0.1014 0.083*
C25 0.5001 (8) 0.8335 (7) 0.0047 (6) 0.0660 (18)
C26 0.5039 (6) 0.8539 (6) 0.1199 (6) 0.0528 (15)
H26 0.4374 0.7935 0.1369 0.063*
C27 0.2142 (7) 0.6084 (7) 0.5643 (7) 0.0621 (18)
H27 0.2169 0.6855 0.6019 0.075*
C28 0.1338 (8) 0.5019 (8) 0.5805 (8) 0.078 (2)
H28 0.0841 0.5069 0.6292 0.094*
C29 0.1264 (9) 0.3904 (8) 0.5264 (9) 0.091 (3)
H29 0.0694 0.3160 0.5350 0.109*
C30 0.2040 (9) 0.3852 (7) 0.4569 (8) 0.084 (3)
H30 0.2005 0.3077 0.4192 0.101*
C31 0.2871 (6) 0.4973 (5) 0.4440 (5) 0.0527 (16)
F1 0.8152 (5) 1.1601 (4) 0.2644 (4) 0.0831 (13)
F2 0.3947 (6) 0.7242 (5) −0.0810 (4) 0.1036 (18)
F3 0.7955 (5) 0.9343 (6) 0.8322 (4) 0.0982 (17)
F4 1.1395 (6) 1.1740 (7) 0.6349 (8) 0.139 (3)
F5 −0.1314 (7) 0.5674 (10) 0.2087 (7) 0.179 (4)
F6 −0.0098 (10) 0.6785 (11) −0.1474 (8) 0.217 (5)
N1 0.2898 (5) 0.6091 (4) 0.4975 (5) 0.0485 (12)
N2 0.4380 (5) 0.6129 (4) 0.3585 (4) 0.0456 (11)
O1 0.1425 (4) 0.6937 (4) 0.3365 (3) 0.0488 (10)
O2 0.2724 (4) 0.7142 (4) 0.2302 (4) 0.0522 (10)
O3 0.6255 (4) 1.0193 (3) 0.5933 (3) 0.0384 (8)
O4 0.5990 (4) 0.8317 (3) 0.5899 (4) 0.0526 (11)
O5 0.5617 (4) 0.8922 (3) 0.3554 (3) 0.0421 (8)
O6 0.3488 (4) 0.8965 (3) 0.6021 (4) 0.0478 (9)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Gd1 0.03152 (16) 0.02485 (15) 0.04068 (18) 0.01156 (11) 0.00743 (11) 0.01632 (11)
C1 0.053 (4) 0.044 (3) 0.067 (4) 0.025 (3) 0.016 (3) 0.014 (3)
C2 0.080 (5) 0.068 (5) 0.063 (4) 0.051 (4) 0.012 (4) 0.006 (4)
C3 0.099 (6) 0.047 (4) 0.060 (4) 0.049 (4) −0.006 (4) 0.000 (3)
C4 0.084 (5) 0.035 (3) 0.056 (4) 0.030 (3) −0.009 (4) 0.006 (3)
C5 0.054 (3) 0.029 (3) 0.041 (3) 0.020 (2) −0.010 (3) 0.009 (2)
C6 0.034 (3) 0.034 (3) 0.039 (3) 0.015 (2) 0.005 (2) 0.017 (2)
C7 0.038 (3) 0.036 (3) 0.052 (3) 0.023 (2) 0.007 (2) 0.013 (2)
C8 0.051 (4) 0.059 (4) 0.089 (5) 0.027 (3) 0.026 (4) 0.044 (4)
C9 0.050 (4) 0.068 (5) 0.130 (8) 0.026 (4) 0.037 (5) 0.054 (5)
C10 0.042 (4) 0.079 (5) 0.133 (8) 0.031 (4) 0.009 (5) 0.042 (5)
C11 0.050 (4) 0.103 (6) 0.088 (6) 0.040 (4) −0.001 (4) 0.038 (5)
C12 0.044 (3) 0.070 (4) 0.049 (3) 0.032 (3) 0.008 (3) 0.024 (3)
C13 0.041 (3) 0.026 (2) 0.043 (3) 0.013 (2) 0.005 (2) 0.011 (2)
C14 0.048 (3) 0.043 (3) 0.037 (3) 0.019 (3) 0.005 (2) 0.017 (2)
C15 0.042 (3) 0.084 (5) 0.042 (3) 0.020 (3) 0.012 (3) 0.026 (3)
C16 0.035 (3) 0.109 (7) 0.066 (5) 0.025 (4) −0.001 (3) 0.026 (4)
C17 0.064 (5) 0.079 (5) 0.056 (4) 0.030 (4) −0.017 (4) 0.019 (4)
C18 0.083 (6) 0.080 (5) 0.046 (4) 0.029 (4) 0.011 (4) 0.031 (4)
C19 0.056 (4) 0.058 (4) 0.051 (4) 0.012 (3) 0.015 (3) 0.030 (3)
C20 0.035 (3) 0.035 (3) 0.048 (3) 0.018 (2) 0.017 (2) 0.020 (2)
C21 0.049 (3) 0.046 (3) 0.054 (3) 0.031 (3) 0.025 (3) 0.026 (3)
C22 0.066 (4) 0.045 (3) 0.069 (4) 0.027 (3) 0.040 (3) 0.029 (3)
C23 0.099 (6) 0.073 (5) 0.085 (5) 0.059 (5) 0.066 (5) 0.052 (4)
C24 0.092 (6) 0.079 (5) 0.057 (4) 0.051 (5) 0.038 (4) 0.027 (4)
C25 0.078 (5) 0.065 (4) 0.046 (4) 0.032 (4) 0.023 (3) 0.010 (3)
C26 0.050 (3) 0.056 (4) 0.060 (4) 0.023 (3) 0.031 (3) 0.027 (3)
C27 0.053 (4) 0.061 (4) 0.089 (5) 0.028 (3) 0.030 (4) 0.050 (4)
C28 0.065 (5) 0.075 (5) 0.107 (6) 0.025 (4) 0.032 (4) 0.068 (5)
C29 0.085 (6) 0.058 (5) 0.107 (7) 0.006 (4) 0.019 (5) 0.060 (5)
C30 0.092 (6) 0.037 (4) 0.083 (6) 0.007 (4) −0.004 (5) 0.033 (4)
C31 0.056 (4) 0.028 (3) 0.052 (3) 0.011 (2) −0.007 (3) 0.021 (2)
F1 0.078 (3) 0.061 (2) 0.087 (3) 0.004 (2) 0.048 (2) 0.025 (2)
F2 0.111 (4) 0.098 (4) 0.057 (3) 0.024 (3) 0.033 (3) 0.000 (3)
F3 0.070 (3) 0.169 (5) 0.069 (3) 0.055 (3) 0.025 (2) 0.071 (3)
F4 0.072 (3) 0.148 (5) 0.253 (8) 0.052 (3) 0.089 (4) 0.135 (6)
F5 0.092 (5) 0.272 (10) 0.123 (6) 0.029 (5) 0.033 (4) 0.099 (6)
F6 0.151 (7) 0.295 (12) 0.128 (7) 0.030 (7) 0.016 (5) 0.120 (8)
N1 0.038 (3) 0.038 (2) 0.065 (3) 0.013 (2) 0.009 (2) 0.031 (2)
N2 0.043 (3) 0.033 (2) 0.055 (3) 0.018 (2) 0.005 (2) 0.018 (2)
O1 0.042 (2) 0.055 (2) 0.044 (2) 0.0152 (18) 0.0094 (17) 0.0274 (19)
O2 0.040 (2) 0.058 (3) 0.047 (2) 0.0221 (19) 0.0079 (18) 0.013 (2)
O3 0.0357 (18) 0.0307 (17) 0.048 (2) 0.0166 (15) 0.0074 (16) 0.0200 (16)
O4 0.044 (2) 0.0254 (18) 0.068 (3) 0.0106 (16) −0.0058 (19) 0.0200 (18)
O5 0.041 (2) 0.0355 (19) 0.053 (2) 0.0166 (16) 0.0211 (17) 0.0215 (17)
O6 0.057 (2) 0.034 (2) 0.052 (2) 0.0167 (18) 0.0257 (19) 0.0200 (18)
Geometric parameters (Å, °)
Gd1—O6 2.364 (4) C13—C14 1.489 (8)
Gd1—O3i 2.378 (3) C14—C15 1.377 (9)
Gd1—O5 2.380 (4) C14—C19 1.409 (8)
Gd1—O2 2.419 (4) C15—F5 1.279 (9)
Gd1—O4 2.482 (4) C15—C16 1.357 (9)
Gd1—O1 2.495 (4) C16—C17 1.347 (12)
Gd1—N1 2.567 (4) C16—H16 0.9300
Gd1—N2 2.585 (5) C17—C18 1.375 (12)
Gd1—O3 2.692 (4) C17—H17 0.9300
Gd1—C13 2.806 (5) C18—C19 1.345 (10)
Gd1—C6 2.942 (5) C18—F6 1.350 (11)
Gd1—Gd1i 4.0615 (12) C19—H19 0.9300
C1—N2 1.319 (8) C20—O5 1.239 (6)
C1—C2 1.395 (9) C20—O6i 1.253 (6)
C1—H1 0.9300 C20—C21 1.511 (8)
C2—C3 1.322 (12) C21—C22 1.369 (8)
C2—H2 0.9300 C21—C26 1.397 (9)
C3—C4 1.363 (12) C22—F1 1.335 (8)
C3—H3 0.9300 C22—C23 1.386 (10)
C4—C5 1.382 (9) C23—C24 1.380 (11)
C4—H4 0.9300 C23—H23 0.9300
C5—N2 1.342 (7) C24—C25 1.339 (10)
C5—C31 1.467 (10) C24—H24 0.9300
C6—O4 1.237 (6) C25—F2 1.352 (8)
C6—O3 1.261 (6) C25—C26 1.399 (9)
C6—C7 1.505 (7) C26—H26 0.9300
C7—C8 1.371 (9) C27—N1 1.330 (9)
C7—C12 1.383 (9) C27—C28 1.352 (9)
C8—C9 1.389 (10) C27—H27 0.9300
C8—H8 0.9300 C28—C29 1.324 (13)
C9—C10 1.309 (13) C28—H28 0.9300
C9—F4 1.335 (9) C29—C30 1.384 (14)
C10—C11 1.409 (13) C29—H29 0.9300
C10—H10 0.9300 C30—C31 1.389 (9)
C11—C12 1.380 (9) C30—H30 0.9300
C11—H11 0.9300 C31—N1 1.352 (8)
C12—F3 1.325 (8) O3—Gd1i 2.378 (3)
C13—O1 1.258 (7) O6—C20i 1.253 (6)
C13—O2 1.254 (7)
O6—Gd1—O3i 75.83 (13) C9—C8—C7 118.9 (7)
O6—Gd1—O5 132.95 (13) C9—C8—H8 120.5
O3i—Gd1—O5 74.27 (13) C7—C8—H8 120.5
O6—Gd1—O2 132.75 (15) C10—C9—F4 118.7 (8)
O3i—Gd1—O2 78.28 (14) C10—C9—C8 122.7 (8)
O5—Gd1—O2 74.17 (14) F4—C9—C8 118.6 (8)
O6—Gd1—O4 84.95 (15) C9—C10—C11 120.5 (7)
O3i—Gd1—O4 123.14 (12) C9—C10—H10 119.8
O5—Gd1—O4 81.99 (14) C11—C10—H10 119.8
O2—Gd1—O4 142.06 (15) C12—C11—C10 117.1 (8)
O6—Gd1—O1 84.39 (15) C12—C11—H11 121.4
O3i—Gd1—O1 81.23 (13) C10—C11—H11 121.4
O5—Gd1—O1 125.10 (13) F3—C12—C7 119.0 (5)
O2—Gd1—O1 52.89 (14) F3—C12—C11 118.8 (6)
O4—Gd1—O1 149.72 (13) C7—C12—C11 122.2 (7)
O6—Gd1—N1 79.37 (15) O1—C13—O2 121.3 (5)
O3i—Gd1—N1 144.99 (15) O1—C13—C14 119.4 (5)
O5—Gd1—N1 140.06 (15) O2—C13—C14 119.2 (5)
O2—Gd1—N1 101.82 (16) O1—C13—Gd1 62.8 (3)
O4—Gd1—N1 78.17 (14) O2—C13—Gd1 59.3 (3)
O1—Gd1—N1 72.04 (14) C14—C13—Gd1 168.3 (4)
O6—Gd1—N2 138.74 (15) C15—C14—C19 114.5 (6)
O3i—Gd1—N2 145.41 (15) C15—C14—C13 125.5 (6)
O5—Gd1—N2 78.19 (14) C19—C14—C13 120.0 (6)
O2—Gd1—N2 74.34 (15) F5—C15—C16 114.6 (7)
O4—Gd1—N2 72.15 (14) F5—C15—C14 121.2 (6)
O1—Gd1—N2 98.28 (15) C16—C15—C14 124.2 (7)
N1—Gd1—N2 62.87 (17) C17—C16—C15 120.1 (7)
O6—Gd1—O3 70.83 (13) C17—C16—H16 119.9
O3i—Gd1—O3 73.72 (12) C15—C16—H16 119.9
O5—Gd1—O3 66.40 (12) C16—C17—C18 117.8 (6)
O2—Gd1—O3 136.31 (13) C16—C17—H17 121.1
O4—Gd1—O3 49.42 (11) C18—C17—H17 121.1
O1—Gd1—O3 148.18 (13) C19—C18—F6 115.1 (8)
N1—Gd1—O3 120.33 (13) C19—C18—C17 122.5 (7)
N2—Gd1—O3 113.49 (13) F6—C18—C17 122.4 (8)
O6—Gd1—C13 108.05 (16) C18—C19—C14 120.8 (7)
O3i—Gd1—C13 76.10 (14) C18—C19—H19 119.6
O5—Gd1—C13 98.99 (15) C14—C19—H19 119.6
O2—Gd1—C13 26.47 (16) O5—C20—O6i 126.5 (5)
O4—Gd1—C13 159.67 (14) O5—C20—C21 115.8 (5)
O1—Gd1—C13 26.63 (15) O6i—C20—C21 117.8 (5)
N1—Gd1—C13 88.73 (16) C22—C21—C26 119.4 (6)
N2—Gd1—C13 88.09 (15) C22—C21—C20 124.0 (5)
O3—Gd1—C13 149.07 (13) C26—C21—C20 116.6 (5)
O6—Gd1—C6 80.23 (15) F1—C22—C23 118.8 (6)
O3i—Gd1—C6 98.81 (13) F1—C22—C21 120.7 (6)
O5—Gd1—C6 69.55 (14) C23—C22—C21 120.4 (7)
O2—Gd1—C6 142.89 (15) C24—C23—C22 120.9 (7)
O4—Gd1—C6 24.53 (12) C24—C23—H23 119.6
O1—Gd1—C6 164.08 (15) C22—C23—H23 119.6
N1—Gd1—C6 100.90 (14) C25—C24—C23 118.1 (7)
N2—Gd1—C6 90.66 (15) C25—C24—H24 120.9
O3—Gd1—C6 25.36 (12) C23—C24—H24 120.9
C13—Gd1—C6 168.47 (16) F2—C25—C24 119.3 (6)
O6—Gd1—Gd1i 68.84 (9) F2—C25—C26 117.5 (7)
O3i—Gd1—Gd1i 39.52 (8) C24—C25—C26 123.2 (7)
O5—Gd1—Gd1i 64.90 (9) C25—C26—C21 117.9 (6)
O2—Gd1—Gd1i 111.12 (11) C25—C26—H26 121.0
O4—Gd1—Gd1i 83.62 (8) C21—C26—H26 121.0
O1—Gd1—Gd1i 118.30 (10) N1—C27—C28 123.7 (8)
N1—Gd1—Gd1i 144.52 (12) N1—C27—H27 118.1
N2—Gd1—Gd1i 138.26 (11) C28—C27—H27 118.1
O3—Gd1—Gd1i 34.20 (7) C29—C28—C27 119.4 (9)
C13—Gd1—Gd1i 115.34 (11) C29—C28—H28 120.3
C6—Gd1—Gd1i 59.38 (10) C27—C28—H28 120.3
N2—C1—C2 123.4 (7) C28—C29—C30 119.6 (7)
N2—C1—H1 118.3 C28—C29—H29 120.2
C2—C1—H1 118.3 C30—C29—H29 120.2
C3—C2—C1 118.2 (8) C29—C30—C31 119.2 (8)
C3—C2—H2 120.9 C29—C30—H30 120.4
C1—C2—H2 120.9 C31—C30—H30 120.4
C2—C3—C4 120.1 (7) N1—C31—C30 120.0 (8)
C2—C3—H3 119.9 N1—C31—C5 116.6 (5)
C4—C3—H3 119.9 C30—C31—C5 123.4 (7)
C3—C4—C5 119.6 (7) C27—N1—C31 118.1 (5)
C3—C4—H4 120.2 C27—N1—Gd1 120.0 (4)
C5—C4—H4 120.2 C31—N1—Gd1 120.8 (4)
N2—C5—C4 121.1 (7) C1—N2—C5 117.6 (5)
N2—C5—C31 116.7 (5) C1—N2—Gd1 121.2 (4)
C4—C5—C31 122.2 (6) C5—N2—Gd1 121.2 (4)
O4—C6—O3 120.8 (5) C13—O1—Gd1 90.6 (3)
O4—C6—C7 119.9 (4) C13—O2—Gd1 94.2 (3)
O3—C6—C7 119.2 (4) C6—O3—Gd1i 163.0 (3)
O4—C6—Gd1 56.4 (3) C6—O3—Gd1 88.5 (3)
O3—C6—Gd1 66.2 (3) Gd1i—O3—Gd1 106.28 (12)
C7—C6—Gd1 162.8 (4) C6—O4—Gd1 99.0 (3)
C8—C7—C12 118.6 (6) C20—O5—Gd1 135.6 (3)
C8—C7—C6 119.9 (5) C20i—O6—Gd1 133.6 (3)
C12—C7—C6 121.4 (5)
O6—Gd1—O1—C13 153.0 (3) O5—Gd1—C6—O4 −117.5 (4)
O3i—Gd1—O1—C13 76.5 (3) O2—Gd1—C6—O4 −104.8 (4)
O5—Gd1—O1—C13 12.9 (4) O1—Gd1—C6—O4 84.1 (6)
O2—Gd1—O1—C13 −5.4 (3) N1—Gd1—C6—O4 22.1 (4)
O4—Gd1—O1—C13 −137.2 (4) N2—Gd1—C6—O4 −40.3 (4)
N1—Gd1—O1—C13 −126.4 (4) O3—Gd1—C6—O4 164.6 (6)
N2—Gd1—O1—C13 −68.5 (3) C13—Gd1—C6—O4 −123.9 (7)
O3—Gd1—O1—C13 114.6 (3) Gd1i—Gd1—C6—O4 170.3 (4)
C6—Gd1—O1—C13 167.9 (4) O6—Gd1—C6—O3 −65.4 (3)
Gd1i—Gd1—O1—C13 90.7 (3) O3i—Gd1—C6—O3 8.5 (4)
O6—Gd1—O2—C13 −24.6 (4) O5—Gd1—C6—O3 77.9 (3)
O3i—Gd1—O2—C13 −82.4 (3) O2—Gd1—C6—O3 90.6 (4)
O5—Gd1—O2—C13 −159.2 (4) O4—Gd1—C6—O3 −164.6 (6)
O4—Gd1—O2—C13 147.7 (3) O1—Gd1—C6—O3 −80.5 (6)
O1—Gd1—O2—C13 5.4 (3) N1—Gd1—C6—O3 −142.5 (3)
N1—Gd1—O2—C13 61.8 (4) N2—Gd1—C6—O3 155.1 (3)
N2—Gd1—O2—C13 118.9 (4) C13—Gd1—C6—O3 71.5 (8)
O3—Gd1—O2—C13 −133.2 (3) Gd1i—Gd1—C6—O3 5.7 (3)
C6—Gd1—O2—C13 −171.5 (3) O6—Gd1—C6—C7 −177.5 (12)
Gd1i—Gd1—O2—C13 −104.8 (3) O3i—Gd1—C6—C7 −103.6 (12)
O6—Gd1—O3—C6 108.4 (3) O5—Gd1—C6—C7 −34.2 (12)
O3i—Gd1—O3—C6 −171.3 (4) O2—Gd1—C6—C7 −21.5 (13)
O5—Gd1—O3—C6 −91.7 (3) O4—Gd1—C6—C7 83.3 (12)
O2—Gd1—O3—C6 −119.1 (3) O1—Gd1—C6—C7 167.4 (10)
O4—Gd1—O3—C6 8.3 (3) N1—Gd1—C6—C7 105.4 (12)
O1—Gd1—O3—C6 149.2 (3) N2—Gd1—C6—C7 43.0 (12)
N1—Gd1—O3—C6 43.8 (4) O3—Gd1—C6—C7 −112.1 (13)
N2—Gd1—O3—C6 −27.4 (3) C13—Gd1—C6—C7 −40.6 (16)
C13—Gd1—O3—C6 −158.3 (4) Gd1i—Gd1—C6—C7 −106.4 (12)
Gd1i—Gd1—O3—C6 −171.3 (4) O4—C6—C7—C8 120.0 (7)
O6—Gd1—O3—Gd1i −80.30 (16) O3—C6—C7—C8 −56.7 (8)
O3i—Gd1—O3—Gd1i 0.0 Gd1—C6—C7—C8 47.2 (14)
O5—Gd1—O3—Gd1i 79.60 (15) O4—C6—C7—C12 −57.5 (8)
O2—Gd1—O3—Gd1i 52.2 (2) O3—C6—C7—C12 125.8 (6)
O4—Gd1—O3—Gd1i 179.6 (2) Gd1—C6—C7—C12 −130.3 (11)
O1—Gd1—O3—Gd1i −39.5 (3) C12—C7—C8—C9 0.6 (10)
N1—Gd1—O3—Gd1i −144.87 (17) C6—C7—C8—C9 −177.0 (6)
N2—Gd1—O3—Gd1i 143.94 (16) C7—C8—C9—C10 −2.1 (13)
C13—Gd1—O3—Gd1i 13.0 (4) C7—C8—C9—F4 178.9 (7)
C6—Gd1—O3—Gd1i 171.3 (4) F4—C9—C10—C11 −179.4 (8)
O6—Gd1—O4—C6 −77.6 (4) C8—C9—C10—C11 1.6 (15)
O3i—Gd1—O4—C6 −8.2 (4) C9—C10—C11—C12 0.3 (14)
O5—Gd1—O4—C6 57.1 (4) C10—C11—C12—F3 178.5 (8)
O2—Gd1—O4—C6 108.1 (4) C10—C11—C12—C7 −1.8 (13)
O1—Gd1—O4—C6 −147.3 (4) C8—C7—C12—F3 −178.9 (6)
N1—Gd1—O4—C6 −157.8 (4) C6—C7—C12—F3 −1.4 (10)
N2—Gd1—O4—C6 137.2 (4) C8—C7—C12—C11 1.4 (11)
O3—Gd1—O4—C6 −8.6 (3) C6—C7—C12—C11 178.9 (7)
C13—Gd1—O4—C6 151.4 (5) Gd1—O2—C13—O1 −10.1 (6)
Gd1i—Gd1—O4—C6 −8.4 (4) Gd1—O2—C13—C14 166.6 (4)
O6—Gd1—O5—C20 −43.1 (6) Gd1—O1—C13—O2 9.7 (5)
O3i—Gd1—O5—C20 9.3 (5) Gd1—O1—C13—C14 −166.9 (4)
O2—Gd1—O5—C20 91.3 (5) O6—Gd1—C13—O2 161.3 (3)
O4—Gd1—O5—C20 −118.5 (5) O3i—Gd1—C13—O2 91.5 (3)
O1—Gd1—O5—C20 76.2 (5) O5—Gd1—C13—O2 20.3 (3)
N1—Gd1—O5—C20 −179.1 (5) O4—Gd1—C13—O2 −71.0 (6)
N2—Gd1—O5—C20 168.1 (5) O1—Gd1—C13—O2 −170.3 (5)
O3—Gd1—O5—C20 −69.5 (5) N1—Gd1—C13—O2 −120.3 (3)
C13—Gd1—O5—C20 82.0 (5) N2—Gd1—C13—O2 −57.5 (3)
C6—Gd1—O5—C20 −96.7 (5) O3—Gd1—C13—O2 78.6 (4)
Gd1i—Gd1—O5—C20 −31.8 (5) C6—Gd1—C13—O2 26.3 (9)
O3i—Gd1—O6—C20i −43.7 (5) Gd1i—Gd1—C13—O2 86.7 (3)
O5—Gd1—O6—C20i 8.2 (6) O6—Gd1—C13—O1 −28.4 (3)
O2—Gd1—O6—C20i −102.4 (5) O3i—Gd1—C13—O1 −98.2 (3)
O4—Gd1—O6—C20i 82.3 (5) O5—Gd1—C13—O1 −169.4 (3)
O1—Gd1—O6—C20i −126.0 (5) O2—Gd1—C13—O1 170.3 (5)
N1—Gd1—O6—C20i 161.2 (5) O4—Gd1—C13—O1 99.4 (6)
N2—Gd1—O6—C20i 137.8 (5) N1—Gd1—C13—O1 50.0 (3)
O3—Gd1—O6—C20i 33.7 (5) N2—Gd1—C13—O1 112.8 (3)
C13—Gd1—O6—C20i −113.7 (5) O3—Gd1—C13—O1 −111.0 (4)
C6—Gd1—O6—C20i 58.1 (5) C6—Gd1—C13—O1 −163.3 (6)
Gd1i—Gd1—O6—C20i −2.8 (5) Gd1i—Gd1—C13—O1 −103.0 (3)
O6—Gd1—N1—C27 18.7 (5) O6—Gd1—C13—C14 75 (2)
O3i—Gd1—N1—C27 −26.7 (6) O3i—Gd1—C13—C14 6(2)
O5—Gd1—N1—C27 167.5 (5) O5—Gd1—C13—C14 −66 (2)
O2—Gd1—N1—C27 −113.1 (5) O2—Gd1—C13—C14 −86 (2)
O4—Gd1—N1—C27 105.7 (5) O4—Gd1—C13—C14 −157 (2)
O1—Gd1—N1—C27 −68.8 (5) O1—Gd1—C13—C14 104 (2)
N2—Gd1—N1—C27 −178.5 (5) N1—Gd1—C13—C14 154 (2)
O3—Gd1—N1—C27 78.9 (5) N2—Gd1—C13—C14 −143 (2)
C13—Gd1—N1—C27 −90.0 (5) O3—Gd1—C13—C14 −7(2)
C6—Gd1—N1—C27 96.5 (5) C6—Gd1—C13—C14 −60 (3)
Gd1i—Gd1—N1—C27 45.0 (6) Gd1i—Gd1—C13—C14 1(2)
O6—Gd1—N1—C31 −174.0 (4) O2—C13—C14—C15 166.7 (7)
O3i—Gd1—N1—C31 140.6 (4) O1—C13—C14—C15 −16.6 (9)
O5—Gd1—N1—C31 −25.2 (5) Gd1—C13—C14—C15 −114 (2)
O2—Gd1—N1—C31 54.2 (4) O2—C13—C14—C19 −15.2 (8)
O4—Gd1—N1—C31 −87.0 (4) O1—C13—C14—C19 161.6 (6)
O1—Gd1—N1—C31 98.5 (4) Gd1—C13—C14—C19 64 (2)
N2—Gd1—N1—C31 −11.1 (4) C19—C14—C15—F5 −178.7 (9)
O3—Gd1—N1—C31 −113.8 (4) C13—C14—C15—F5 −0.4 (13)
C13—Gd1—N1—C31 77.4 (4) C19—C14—C15—C16 0.9 (12)
C6—Gd1—N1—C31 −96.2 (4) C13—C14—C15—C16 179.1 (8)
Gd1i—Gd1—N1—C31 −147.7 (3) F5—C15—C16—C17 178.4 (10)
O6—Gd1—N2—C1 −148.1 (4) C14—C15—C16—C17 −1.2 (15)
O3i—Gd1—N2—C1 34.4 (6) C15—C16—C17—C18 0.8 (14)
O5—Gd1—N2—C1 −3.3 (4) C16—C17—C18—C19 −0.3 (14)
O2—Gd1—N2—C1 73.4 (5) C16—C17—C18—F6 178.4 (10)
O4—Gd1—N2—C1 −88.6 (5) F6—C18—C19—C14 −178.7 (9)
O1—Gd1—N2—C1 121.0 (5) C17—C18—C19—C14 0.1 (13)
N1—Gd1—N2—C1 −174.1 (5) C15—C14—C19—C18 −0.3 (11)
O3—Gd1—N2—C1 −60.8 (5) C13—C14—C19—C18 −178.7 (7)
C13—Gd1—N2—C1 96.3 (5) Gd1—O5—C20—O6i 48.8 (8)
C6—Gd1—N2—C1 −72.2 (5) Gd1—O5—C20—C21 −130.3 (5)
Gd1i—Gd1—N2—C1 −31.0 (5) O5—C20—C21—C22 −147.5 (6)
O6—Gd1—N2—C5 30.7 (5) O6i—C20—C21—C22 33.3 (8)
O3i—Gd1—N2—C5 −146.7 (4) O5—C20—C21—C26 34.0 (7)
O5—Gd1—N2—C5 175.5 (4) O6i—C20—C21—C26 −145.3 (5)
O2—Gd1—N2—C5 −107.8 (4) C26—C21—C22—F1 −177.0 (6)
O4—Gd1—N2—C5 90.3 (4) C20—C21—C22—F1 4.5 (9)
O1—Gd1—N2—C5 −60.1 (4) C26—C21—C22—C23 −0.4 (10)
N1—Gd1—N2—C5 4.7 (4) C20—C21—C22—C23 −178.9 (6)
O3—Gd1—N2—C5 118.0 (4) F1—C22—C23—C24 179.0 (7)
C13—Gd1—N2—C5 −84.8 (4) C21—C22—C23—C24 2.3 (11)
C6—Gd1—N2—C5 106.7 (4) C22—C23—C24—C25 −2.3 (11)
Gd1i—Gd1—N2—C5 147.8 (3) C23—C24—C25—F2 −179.5 (7)
C5—N2—C1—C2 −1.8 (9) C23—C24—C25—C26 0.3 (12)
Gd1—N2—C1—C2 177.1 (5) C22—C21—C26—C25 −1.5 (9)
N2—C1—C2—C3 2.5 (11) C20—C21—C26—C25 177.1 (6)
C1—C2—C3—C4 −2.2 (11) C24—C25—C26—C21 1.6 (11)
C2—C3—C4—C5 1.4 (11) F2—C25—C26—C21 −178.6 (6)
C1—N2—C5—C4 0.9 (8) C31—N1—C27—C28 −0.6 (10)
Gd1—N2—C5—C4 −178.0 (4) Gd1—N1—C27—C28 167.0 (6)
C1—N2—C5—C31 −179.9 (5) N1—C27—C28—C29 −1.3 (13)
Gd1—N2—C5—C31 1.2 (6) C27—C28—C29—C30 2.2 (13)
C3—C4—C5—N2 −0.7 (9) C28—C29—C30—C31 −1.2 (13)
C3—C4—C5—C31 −179.9 (6) C27—N1—C31—C30 1.5 (9)
Gd1—O4—C6—O3 16.4 (6) Gd1—N1—C31—C30 −166.0 (5)
Gd1—O4—C6—C7 −160.3 (5) C27—N1—C31—C5 −176.1 (6)
Gd1i—O3—C6—O4 −165.2 (9) Gd1—N1—C31—C5 16.4 (6)
Gd1—O3—C6—O4 −14.9 (6) C29—C30—C31—N1 −0.6 (11)
Gd1i—O3—C6—C7 11.4 (16) C29—C30—C31—C5 176.8 (7)
Gd1—O3—C6—C7 161.8 (5) N2—C5—C31—N1 −11.4 (7)
Gd1i—O3—C6—Gd1 −150.3 (12) C4—C5—C31—N1 167.8 (5)
O6—Gd1—C6—O4 99.2 (4) N2—C5—C31—C30 171.1 (6)
O3i—Gd1—C6—O4 173.1 (4) C4—C5—C31—C30 −9.7 (9)
Symmetry codes: (i) −x+1, −y+2, −z+1.
==== Refs
References
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Church, B. S. & Halvorson, H. (1959). Nature (London), 183, 124–125.
Okabe, N., Kyoyama, H. & Fujimoto, A. (2002). Acta Cryst. E58, m354–m356.
Okabe, N. & Oya, N. (2000). Acta Cryst. C56, 1416–1417.
Pocker, Y. & Fong, C. T. O. (1980). Biochemistry, 19, 2045–2049.
Scapin, G., Reddy, S. G., Zheng, R. & Blanchard, J. S. (1997). Biochemistry, 36, 15081–15088.
Serre, C., Marrot, J. & Ferey, G. (2005). Inorg. Chem.44, 654–658.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21201598 | PMC2960618 | CC BY | 2021-01-04 18:55:11 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Aug 9; 64(Pt 9):m1142 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hg241510.1107/S1600536808024616ACSEBHS1600536808024616Organic Papers2,2′-Dimethyl-7,7′-(methylenediimino)di-1,8-naphthyridin-1-ium bis(perchlorate) C19H20N62+·2ClO4−Mo Juan a*Liu Jian-Hua aPan Yu-Shan aZhang Su-Mei aDu Xiang-Dang aa College of Animal Husbandry and Veterinary Studies, Henan Agricultural University, Zhengzhou, Henan Province 450002, People’s Republic of ChinaCorrespondence e-mail: [email protected] 9 2008 06 8 2008 06 8 2008 64 Pt 9 e080900o1702 o1702 14 6 2008 31 7 2008 © Mo et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title salt, C19H20N6
2+·2ClO4
−, the two planar 1,8-naphthyridine systems are linked by a methylenediamine group with a dihedral angle of 60.6 (1)° between the two systems. The crystal structure involves extensive N—H⋯O and C—H⋯O hydrogen bonding.
==== Body
Related literature
For related literature, see: Baker & Norman (2004 ▶); Gavrilova & Bosnich (2004 ▶); Nakatani et al. (2000 ▶, 2001 ▶); Stadie et al. (2007 ▶); Ferrarini et al. (1997 ▶).
Experimental
Crystal data
C19H20N6
2+·2ClO4
−
M
r = 531.31
Orthorhombic,
a = 8.191 (1) Å
b = 19.325 (2) Å
c = 27.885 (2) Å
V = 4413.9 (5) Å3
Z = 8
Mo Kα radiation
μ = 0.36 mm−1
T = 113 (2) K
0.34 × 0.16 × 0.14 mm
Data collection
Bruker SMART CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Sheldrick, 1996 ▶) T
min = 0.908, T
max = 0.952
31220 measured reflections
3882 independent reflections
3598 reflections with I > 2σ(I)
R
int = 0.061
Refinement
R[F
2 > 2σ(F
2)] = 0.060
wR(F
2) = 0.134
S = 1.16
3882 reflections
308 parameters
16 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.35 e Å−3
Δρmin = −0.41 e Å−3
Data collection: SMART (Bruker, 1997 ▶); cell refinement: SAINT (Bruker, 1997 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: XP in SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: XP in SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808024616/hg2415sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808024616/hg2415Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HG2415).
We thank Henan Agricultural University for the generous support of this study.
supplementary crystallographic
information
Comment
1,8-Naphthyridine and its derivatives are used for binding of mismatched guanine
or used as versatile ligands which are able to form metal aggregates with
monodentates fashion or chelating bidentate fashion(Nakatani et al.,
2000; Nakatani et al., 2001; Ferrarini et al.,
1997; Gavrilova &
Bosnich, 2004; Baker & Norman, 2004; Stadie et al.,
2007). We report
here a new 1,8-Naphthyridine compound (Fig. 1).
The title compound reveals 1,8-naphthyridine rings are linked by
methenediamine with a dihedral angle between two
1,8-naphthyridine rings of 60.6 (1)°. Each 1,8-naphthyridine ring is an almost
planar in which the ten atoms forming the 1,8-naphthyridine ring have
mean deviation of 0.03Å from the least-squares plane calculated using the
ten atoms. To balance hydrogen ion charge of two 1,8-naphthyridine rings,
there are two perchlorate groups in crystal cell. From the packing diagram
(Fig. 2), it seems that the intramolecular N–H···O and C–H···O and
hydrogen bonds are effective in the stabilization of the crystal structure.
Experimental
To the solution of 2-amino-7-methyl-1,8-naphthyridine (3.18 g, 0.02 mol)
in mixed solvent of water (28 mL) and ethanol (2 mL), 37% formadehyde
solution (0.86 mL, 0.01 mol) was added dropwise at 0°C and the reaction
mixture was stirred at room temperature for 24h. The white precipitate
formed was filtered, washed several times with water and then with
diethyl ether and dried. Yield: 55% (1.81 g). FTIR (KBr)cm-1: νNH
3389, 3266; νCH 3026. Anal. Calc. For C19H18N6: C, 69.07;
H, 5.49; N, 25.44. Found: C, 68.86; H, 5.56; N, 25.37. Single crystals
of (I) suitable for an X-ray study were obtained by slow evaporation
of an aqueous ethanol solution (30% v/v) under the
conditions in the presence of perchloric acid at 293 K over a period of
one month.
Refinement
Hydrogen atoms of NH (naphthyridine and amine) were located in a Fourier map
and refined freely. All the other hydrogen atoms were generated geometrically
(C—H bond lengths of methyl group fixed at 0.98Å, C—H bond lengths of
naphthyridine fixed at 0.95 Å) assigned appropriated isotropic thermal
parameters, Uiso(H) = 1.2Ueq(C). Each perchlorate anion
is disordered over two different orientations. The Cl–O distances
were restrained to 1.43 (4)Å.
Figures
Fig. 1. Molecular structure of the cation of the title compound showing the atom-numbering scheme and displacement ellipsoids drawn at the 40% probability level.
Fig. 2. Unit-cell packing diagram as viewed down the c-direction. Hydrogen bonds are shown as dashed lines.
Crystal data
C19H20N62+·2ClO4– F000 = 2192
Mr = 531.31 Dx = 1.599 Mg m−3
Orthorhombic, Pbca Mo Kα radiation λ = 0.71070 Å
Hall symbol: -P 2ac 2ab Cell parameters from 7263 reflections
a = 8.1910 (5) Å θ = 2.1–28.0º
b = 19.3250 (12) Å µ = 0.36 mm−1
c = 27.8850 (19) Å T = 113 (2) K
V = 4413.9 (5) Å3 Prism, colorless
Z = 8 0.34 × 0.16 × 0.14 mm
Data collection
Bruker SMART CCD area-detector diffractometer 3882 independent reflections
Radiation source: fine-focus sealed tube 3598 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.061
Detector resolution: 7.31 pixels mm-1 θmax = 25.0º
T = 113(2) K θmin = 2.1º
φ and ω scans h = −9→9
Absorption correction: multi-scan(SADABS; Sheldrick, 1996) k = −22→22
Tmin = 0.909, Tmax = 0.952 l = −33→32
31220 measured reflections
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.060 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.134 w = 1/[σ2(Fo2) + (0.0498P)2 + 5.3196P] where P = (Fo2 + 2Fc2)/3
S = 1.16 (Δ/σ)max = 0.003
3882 reflections Δρmax = 0.35 e Å−3
308 parameters Δρmin = −0.41 e Å−3
16 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq Occ. (<1)
Cl1 0.24246 (10) 0.22574 (4) 0.56855 (3) 0.0360 (2)
Cl2 1.00163 (9) 0.01682 (3) 0.69459 (3) 0.0323 (2)
O1 0.1693 (6) 0.1730 (2) 0.59780 (16) 0.0534 (14) 0.747 (5)
O2 0.3894 (5) 0.2014 (2) 0.5483 (2) 0.0758 (17) 0.747 (5)
O3 0.1302 (6) 0.2438 (2) 0.53084 (12) 0.0738 (15) 0.747 (5)
O4 0.2651 (6) 0.28664 (19) 0.59698 (15) 0.0700 (14) 0.747 (5)
O5 0.8509 (4) 0.02894 (17) 0.67091 (14) 0.0664 (12) 0.870 (6)
O6 0.9850 (5) −0.03196 (14) 0.73264 (10) 0.0657 (13) 0.870 (6)
O7 1.1129 (3) −0.01123 (15) 0.65972 (10) 0.0490 (10) 0.870 (6)
O8 1.0661 (6) 0.0802 (2) 0.7134 (2) 0.0444 (13) 0.870 (6)
O1' 0.1358 (14) 0.1676 (5) 0.5766 (4) 0.039 (3) 0.253 (5)
O2' 0.3710 (11) 0.2233 (6) 0.6037 (3) 0.062 (4) 0.253 (5)
O3' 0.3135 (14) 0.2178 (6) 0.5220 (3) 0.062 (4) 0.253 (5)
O4' 0.1558 (13) 0.2875 (4) 0.5708 (4) 0.052 (3) 0.253 (5)
O5' 0.8301 (12) 0.0031 (10) 0.7058 (7) 0.053 (6) 0.130 (6)
O6' 1.0870 (19) −0.0453 (6) 0.7069 (7) 0.048 (6) 0.130 (6)
O7' 1.012 (3) 0.0307 (10) 0.6446 (4) 0.062 (7) 0.130 (6)
O8' 1.056 (3) 0.0738 (10) 0.7228 (8) 0.039 (10) 0.130 (6)
N1 0.1466 (3) 0.16498 (13) 0.27420 (9) 0.0328 (6)
N2 0.1460 (3) 0.16453 (12) 0.35633 (8) 0.0293 (6)
N3 0.1396 (3) 0.16601 (15) 0.43819 (10) 0.0353 (6)
N6 0.3295 (3) 0.04505 (14) 0.58097 (9) 0.0336 (6)
N5 0.2041 (3) 0.04253 (13) 0.50731 (9) 0.0327 (6)
N4 0.0922 (4) 0.04231 (16) 0.43264 (11) 0.0417 (7)
C2 0.1739 (4) 0.19127 (16) 0.23034 (10) 0.0377 (8)
C3 0.2463 (5) 0.25570 (18) 0.22665 (11) 0.0435 (9)
H3 0.2634 0.2759 0.1960 0.052*
C4 0.2935 (4) 0.29061 (16) 0.26746 (11) 0.0390 (8)
H4 0.3453 0.3344 0.2647 0.047*
C5 0.2662 (4) 0.26239 (14) 0.31277 (10) 0.0285 (7)
C6 0.1858 (4) 0.19774 (14) 0.31595 (10) 0.0268 (6)
C7 0.1866 (4) 0.19636 (15) 0.39716 (10) 0.0288 (7)
C8 0.2786 (4) 0.25928 (15) 0.39855 (10) 0.0314 (7)
H8 0.3132 0.2779 0.4284 0.038*
C9 0.3159 (4) 0.29193 (15) 0.35722 (10) 0.0324 (7)
H9 0.3748 0.3343 0.3578 0.039*
C1 0.1243 (6) 0.14783 (18) 0.18868 (12) 0.0518 (10)
H1A 0.2123 0.1154 0.1808 0.078*
H1B 0.1026 0.1776 0.1610 0.078*
H1C 0.0253 0.1219 0.1968 0.078*
C16 0.4320 (4) 0.02351 (16) 0.61578 (11) 0.0360 (8)
C17 0.5112 (4) −0.03943 (17) 0.60916 (12) 0.0402 (8)
H17 0.5862 −0.0556 0.6326 0.048*
C18 0.4812 (4) −0.07835 (17) 0.56878 (12) 0.0398 (8)
H18 0.5322 −0.1222 0.5653 0.048*
C14 0.3773 (4) −0.05443 (15) 0.53293 (12) 0.0357 (7)
C15 0.3015 (4) 0.01043 (16) 0.53928 (11) 0.0316 (7)
C11 0.1802 (4) 0.00934 (17) 0.46592 (11) 0.0368 (8)
C12 0.2463 (4) −0.05808 (16) 0.45608 (12) 0.0424 (8)
H12 0.2221 −0.0808 0.4267 0.051*
C13 0.3428 (4) −0.08878 (16) 0.48880 (12) 0.0411 (8)
H13 0.3878 −0.1332 0.4825 0.049*
C19 0.4554 (5) 0.06921 (19) 0.65809 (11) 0.0446 (8)
H19A 0.4863 0.1156 0.6473 0.067*
H19B 0.5418 0.0503 0.6785 0.067*
H19C 0.3533 0.0718 0.6763 0.067*
C10 0.0224 (4) 0.10976 (18) 0.43970 (12) 0.0431 (8)
H10A −0.0336 0.1106 0.4712 0.052*
H10B −0.0612 0.1178 0.4147 0.052*
H3A 0.163 (4) 0.1865 (17) 0.4619 (12) 0.035 (10)*
H1 0.101 (5) 0.124 (2) 0.2764 (13) 0.055 (11)*
H4A 0.081 (5) 0.0269 (19) 0.4097 (13) 0.040 (12)*
H6 0.281 (4) 0.0828 (18) 0.5853 (12) 0.035 (9)*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Cl1 0.0433 (5) 0.0317 (4) 0.0331 (4) −0.0003 (3) 0.0046 (4) −0.0008 (3)
Cl2 0.0360 (5) 0.0256 (4) 0.0353 (4) −0.0007 (3) 0.0018 (3) −0.0012 (3)
O1 0.064 (3) 0.046 (2) 0.050 (3) 0.016 (2) 0.026 (2) 0.022 (2)
O2 0.055 (3) 0.049 (2) 0.124 (4) −0.002 (2) 0.053 (3) −0.011 (3)
O3 0.118 (4) 0.059 (2) 0.045 (2) 0.031 (2) −0.025 (2) 0.0033 (18)
O4 0.089 (3) 0.052 (2) 0.070 (3) −0.001 (2) 0.002 (3) −0.027 (2)
O5 0.0421 (19) 0.063 (2) 0.095 (3) 0.0139 (15) −0.0291 (19) −0.019 (2)
O6 0.126 (4) 0.0396 (16) 0.0313 (15) −0.0408 (19) −0.0042 (19) 0.0050 (12)
O7 0.0410 (17) 0.0542 (18) 0.0519 (18) −0.0075 (14) 0.0127 (14) −0.0213 (14)
O8 0.055 (3) 0.0232 (18) 0.055 (2) −0.0038 (16) −0.002 (2) −0.0076 (17)
O1' 0.036 (6) 0.030 (5) 0.051 (7) −0.003 (4) 0.014 (5) 0.002 (5)
O2' 0.049 (6) 0.086 (7) 0.052 (6) −0.022 (5) −0.013 (5) 0.031 (5)
O3' 0.056 (7) 0.087 (7) 0.041 (6) −0.016 (6) 0.023 (5) −0.020 (5)
O4' 0.063 (6) 0.027 (4) 0.065 (6) 0.017 (4) 0.012 (5) 0.007 (4)
O5' 0.030 (8) 0.067 (9) 0.061 (10) 0.008 (7) 0.004 (7) 0.003 (8)
O6' 0.046 (9) 0.033 (8) 0.064 (10) 0.009 (7) 0.000 (8) 0.003 (7)
O7' 0.085 (11) 0.060 (10) 0.041 (9) −0.012 (8) 0.014 (8) −0.003 (7)
O8' 0.053 (14) 0.032 (13) 0.032 (12) 0.002 (8) −0.006 (8) −0.010 (8)
N1 0.0448 (17) 0.0233 (13) 0.0302 (14) −0.0046 (12) −0.0070 (12) 0.0000 (10)
N2 0.0321 (14) 0.0275 (12) 0.0283 (13) −0.0027 (11) −0.0009 (11) 0.0035 (10)
N3 0.0381 (16) 0.0419 (15) 0.0260 (14) 0.0023 (13) −0.0004 (12) 0.0032 (12)
N6 0.0353 (15) 0.0344 (15) 0.0310 (14) 0.0059 (13) 0.0054 (12) 0.0103 (12)
N5 0.0314 (14) 0.0360 (14) 0.0306 (13) −0.0023 (11) 0.0053 (11) 0.0101 (11)
N4 0.0479 (19) 0.0467 (18) 0.0306 (16) −0.0128 (14) −0.0024 (15) 0.0084 (14)
C2 0.053 (2) 0.0331 (16) 0.0266 (16) 0.0011 (15) −0.0066 (15) −0.0008 (13)
C3 0.064 (2) 0.0381 (18) 0.0287 (16) −0.0036 (17) −0.0008 (16) 0.0063 (14)
C4 0.050 (2) 0.0317 (16) 0.0349 (17) −0.0098 (15) 0.0017 (15) 0.0043 (13)
C5 0.0341 (17) 0.0212 (14) 0.0302 (15) −0.0031 (12) −0.0020 (13) −0.0004 (12)
C6 0.0325 (16) 0.0223 (13) 0.0257 (14) −0.0012 (12) −0.0021 (13) −0.0003 (11)
C7 0.0299 (16) 0.0301 (15) 0.0264 (15) 0.0067 (13) 0.0012 (13) 0.0022 (12)
C8 0.0359 (18) 0.0293 (15) 0.0290 (15) 0.0050 (13) −0.0035 (13) −0.0066 (12)
C9 0.0374 (18) 0.0262 (15) 0.0335 (16) −0.0018 (13) −0.0029 (14) −0.0030 (12)
C1 0.083 (3) 0.0408 (19) 0.0316 (18) −0.0031 (19) −0.0128 (19) −0.0052 (15)
C16 0.0290 (17) 0.0429 (18) 0.0362 (17) −0.0019 (14) 0.0067 (14) 0.0172 (14)
C17 0.0313 (18) 0.0449 (19) 0.0444 (19) 0.0015 (15) 0.0056 (15) 0.0222 (16)
C18 0.0337 (18) 0.0324 (17) 0.053 (2) 0.0026 (14) 0.0174 (16) 0.0202 (15)
C14 0.0355 (18) 0.0285 (15) 0.0430 (18) −0.0041 (14) 0.0150 (15) 0.0115 (14)
C15 0.0287 (17) 0.0350 (16) 0.0309 (16) −0.0028 (13) 0.0097 (13) 0.0104 (13)
C11 0.0381 (19) 0.0409 (18) 0.0314 (16) −0.0138 (15) 0.0053 (15) 0.0099 (14)
C12 0.052 (2) 0.0331 (17) 0.0417 (19) −0.0172 (16) 0.0116 (17) 0.0006 (15)
C13 0.048 (2) 0.0273 (16) 0.048 (2) −0.0080 (15) 0.0165 (17) 0.0061 (14)
C19 0.042 (2) 0.058 (2) 0.0341 (17) −0.0008 (17) 0.0011 (16) 0.0121 (16)
C10 0.0343 (19) 0.061 (2) 0.0343 (18) −0.0038 (17) 0.0030 (15) 0.0176 (16)
Geometric parameters (Å, °)
Cl1—O4' 1.390 (7) C2—C3 1.383 (5)
Cl1—O2 1.410 (4) C2—C1 1.490 (4)
Cl1—O3' 1.430 (7) C3—C4 1.378 (4)
Cl1—O4 1.431 (3) C3—H3 0.9500
Cl1—O1 1.436 (4) C4—C5 1.394 (4)
Cl1—O2' 1.439 (7) C4—H4 0.9500
Cl1—O3 1.440 (3) C5—C6 1.415 (4)
Cl1—O1' 1.441 (8) C5—C9 1.424 (4)
Cl2—O5 1.420 (3) C7—C8 1.431 (4)
Cl2—O7' 1.423 (9) C8—C9 1.349 (4)
Cl2—O8' 1.424 (10) C8—H8 0.9500
Cl2—O6 1.426 (3) C9—H9 0.9500
Cl2—O6' 1.432 (9) C1—H1A 0.9800
Cl2—O8 1.433 (3) C1—H1B 0.9800
Cl2—O7 1.439 (3) C1—H1C 0.9800
Cl2—O5' 1.464 (9) C16—C17 1.390 (5)
N1—C2 1.343 (4) C16—C19 1.486 (5)
N1—C6 1.364 (4) C17—C18 1.376 (5)
N1—H1 0.88 (4) C17—H17 0.9500
N2—C7 1.336 (4) C18—C14 1.392 (5)
N2—C6 1.336 (4) C18—H18 0.9500
N3—C7 1.342 (4) C14—C15 1.410 (4)
N3—C10 1.451 (4) C14—C13 1.427 (5)
N3—H3A 0.80 (3) C11—C12 1.437 (5)
N6—C16 1.350 (4) C12—C13 1.345 (5)
N6—C15 1.360 (4) C12—H12 0.9500
N6—H6 0.84 (3) C13—H13 0.9500
N5—C11 1.335 (4) C19—H19A 0.9800
N5—C15 1.348 (4) C19—H19B 0.9800
N4—C11 1.337 (4) C19—H19C 0.9800
N4—C10 1.437 (5) C10—H10A 0.9900
N4—H4A 0.71 (4) C10—H10B 0.9900
O4'—Cl1—O2 137.8 (4) N1—C2—C3 118.7 (3)
O4'—Cl1—O3' 109.9 (6) N1—C2—C1 116.8 (3)
O4'—Cl1—O4 48.3 (4) C3—C2—C1 124.5 (3)
O2—Cl1—O4 112.7 (3) C4—C3—C2 120.0 (3)
O3'—Cl1—O4 122.6 (5) C4—C3—H3 120.0
O4'—Cl1—O1 111.8 (5) C2—C3—H3 120.0
O2—Cl1—O1 110.3 (3) C3—C4—C5 120.8 (3)
O3'—Cl1—O1 127.5 (5) C3—C4—H4 119.6
O4—Cl1—O1 108.9 (3) C5—C4—H4 119.6
O4'—Cl1—O2' 111.7 (6) C4—C5—C6 118.5 (3)
O2—Cl1—O2' 68.8 (5) C4—C5—C9 125.9 (3)
O3'—Cl1—O2' 108.5 (6) C6—C5—C9 115.6 (3)
O4—Cl1—O2' 63.6 (5) N2—C6—N1 116.0 (2)
O1—Cl1—O2' 84.0 (4) N2—C6—C5 126.2 (3)
O4'—Cl1—O3 59.9 (5) N1—C6—C5 117.8 (3)
O2—Cl1—O3 109.5 (3) N2—C7—N3 117.0 (3)
O3'—Cl1—O3 67.9 (5) N2—C7—C8 123.0 (3)
O4—Cl1—O3 106.7 (2) N3—C7—C8 120.0 (3)
O1—Cl1—O3 108.7 (3) C9—C8—C7 119.6 (3)
O2'—Cl1—O3 166.5 (4) C9—C8—H8 120.2
O4'—Cl1—O1' 110.7 (6) C7—C8—H8 120.2
O2—Cl1—O1' 108.6 (6) C8—C9—C5 119.4 (3)
O3'—Cl1—O1' 107.7 (6) C8—C9—H9 120.3
O4—Cl1—O1' 129.3 (5) C5—C9—H9 120.3
O2'—Cl1—O1' 108.2 (6) C2—C1—H1A 109.5
O3—Cl1—O1' 85.2 (5) C2—C1—H1B 109.5
O5—Cl2—O7' 64.3 (8) H1A—C1—H1B 109.5
O5—Cl2—O8' 113.6 (11) C2—C1—H1C 109.5
O7'—Cl2—O8' 112.1 (10) H1A—C1—H1C 109.5
O5—Cl2—O6 111.8 (2) H1B—C1—H1C 109.5
O7'—Cl2—O6 149.3 (8) N6—C16—C17 117.7 (3)
O8'—Cl2—O6 97.5 (11) N6—C16—C19 117.9 (3)
O5—Cl2—O6' 132.4 (7) C17—C16—C19 124.4 (3)
O7'—Cl2—O6' 111.3 (8) C18—C17—C16 120.2 (3)
O8'—Cl2—O6' 111.4 (10) C18—C17—H17 119.9
O6—Cl2—O6' 46.6 (7) C16—C17—H17 119.9
O5—Cl2—O8 110.5 (2) C17—C18—C14 121.0 (3)
O7'—Cl2—O8 100.1 (8) C17—C18—H18 119.5
O6—Cl2—O8 109.1 (3) C14—C18—H18 119.5
O6'—Cl2—O8 116.7 (8) C18—C14—C15 118.3 (3)
O5—Cl2—O7 107.38 (19) C18—C14—C13 125.9 (3)
O7'—Cl2—O7 50.9 (8) C15—C14—C13 115.8 (3)
O8'—Cl2—O7 117.9 (11) N5—C15—N6 116.0 (3)
O6—Cl2—O7 108.3 (2) N5—C15—C14 125.9 (3)
O6'—Cl2—O7 62.4 (8) N6—C15—C14 118.1 (3)
O8—Cl2—O7 109.6 (3) N5—C11—N4 116.8 (3)
O5—Cl2—O5' 45.2 (7) N5—C11—C12 123.1 (3)
O7'—Cl2—O5' 107.7 (8) N4—C11—C12 120.2 (3)
O8'—Cl2—O5' 108.7 (10) C13—C12—C11 119.5 (3)
O6—Cl2—O5' 68.2 (8) C13—C12—H12 120.3
O6'—Cl2—O5' 105.3 (8) C11—C12—H12 120.3
O8—Cl2—O5' 115.5 (8) C12—C13—C14 119.8 (3)
O7—Cl2—O5' 133.2 (8) C12—C13—H13 120.1
C2—N1—C6 124.2 (3) C14—C13—H13 120.1
C2—N1—H1 118 (2) C16—C19—H19A 109.5
C6—N1—H1 117 (2) C16—C19—H19B 109.5
C7—N2—C6 115.9 (2) H19A—C19—H19B 109.5
C7—N3—C10 122.8 (3) C16—C19—H19C 109.5
C7—N3—H3A 115 (2) H19A—C19—H19C 109.5
C10—N3—H3A 121 (2) H19B—C19—H19C 109.5
C16—N6—C15 124.6 (3) N4—C10—N3 114.4 (3)
C16—N6—H6 117 (2) N4—C10—H10A 108.7
C15—N6—H6 118 (2) N3—C10—H10A 108.7
C11—N5—C15 116.0 (3) N4—C10—H10B 108.7
C11—N4—C10 123.5 (3) N3—C10—H10B 108.7
C11—N4—H4A 120 (3) H10A—C10—H10B 107.6
C10—N4—H4A 117 (3)
C6—N1—C2—C3 −0.1 (5) C15—N6—C16—C19 176.9 (3)
C6—N1—C2—C1 179.5 (3) N6—C16—C17—C18 −1.5 (4)
N1—C2—C3—C4 2.1 (5) C19—C16—C17—C18 179.8 (3)
C1—C2—C3—C4 −177.4 (4) C16—C17—C18—C14 2.8 (5)
C2—C3—C4—C5 −1.4 (6) C17—C18—C14—C15 −0.8 (4)
C3—C4—C5—C6 −1.3 (5) C17—C18—C14—C13 177.1 (3)
C3—C4—C5—C9 177.0 (3) C11—N5—C15—N6 178.3 (3)
C7—N2—C6—N1 179.2 (3) C11—N5—C15—C14 −0.7 (4)
C7—N2—C6—C5 0.2 (4) C16—N6—C15—N5 −175.3 (3)
C2—N1—C6—N2 178.4 (3) C16—N6—C15—C14 3.8 (4)
C2—N1—C6—C5 −2.5 (5) C18—C14—C15—N5 176.7 (3)
C4—C5—C6—N2 −177.9 (3) C13—C14—C15—N5 −1.5 (4)
C9—C5—C6—N2 3.7 (5) C18—C14—C15—N6 −2.3 (4)
C4—C5—C6—N1 3.1 (4) C13—C14—C15—N6 179.5 (3)
C9—C5—C6—N1 −175.3 (3) C15—N5—C11—N4 −176.0 (3)
C6—N2—C7—N3 176.2 (3) C15—N5—C11—C12 3.0 (4)
C6—N2—C7—C8 −4.9 (4) C10—N4—C11—N5 −0.9 (5)
C10—N3—C7—N2 −12.6 (4) C10—N4—C11—C12 −179.9 (3)
C10—N3—C7—C8 168.5 (3) N5—C11—C12—C13 −2.9 (5)
N2—C7—C8—C9 5.7 (5) N4—C11—C12—C13 176.0 (3)
N3—C7—C8—C9 −175.5 (3) C11—C12—C13—C14 0.5 (5)
C7—C8—C9—C5 −1.4 (5) C18—C14—C13—C12 −176.5 (3)
C4—C5—C9—C8 178.9 (3) C15—C14—C13—C12 1.5 (4)
C6—C5—C9—C8 −2.8 (4) C11—N4—C10—N3 74.2 (4)
C15—N6—C16—C17 −1.9 (4) C7—N3—C10—N4 85.6 (4)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
N1—H1···O6i 0.88 (4) 1.92 2.794 (4) 168
N3—H3A···O3 0.80 (3) 2.23 2.990 (6) 159
N4—H4A···O5i 0.71 (4) 2.56 3.233 (5) 160
N4—H4A···O7i 0.71 (4) 2.52 3.133 (5) 145
N6—H6···O1 0.84 (3) 2.00 2.838 (8) 178
C1—H1A···O7ii 0.98 2.54 3.501 (3) 167
C1—H1B···O4iii 0.98 2.33 3.078 (4) 132
C4—H4···O8iv 0.95 2.52 3.392 (7) 152
C10—H10B···O4iv 0.99 2.35 3.082 (5) 130
C13—H13···O2i 0.95 2.41 3.259 (3) 149
C19—H19B···O5 0.98 2.57 3.351 (6) 136
C19—H19C···O7v 0.98 2.58 3.207 (7) 122
C19—H19C···O8v 0.98 2.57 3.548 (4) 172
Symmetry codes: (i) −x+1, −y, −z+1; (ii) −x+3/2, −y, z−1/2; (iii) x, −y+1/2, z−1/2; (iv) x−1/2, −y+1/2, −z+1; (v) x−1, y, z.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
N1—H1⋯O6i 0.88 (4) 1.92 2.794 (4) 168
N3—H3A⋯O3 0.80 (3) 2.23 2.990 (6) 159
N4—H4A⋯O5i 0.71 (4) 2.56 3.233 (5) 160
N4—H4A⋯O7i 0.71 (4) 2.52 3.133 (5) 145
N6—H6⋯O1 0.84 (3) 2.00 2.838 (8) 178
C1—H1A⋯O7ii 0.98 2.54 3.501 (3) 167
C1—H1B⋯O4iii 0.98 2.33 3.078 (4) 132
C4—H4⋯O8iv 0.95 2.52 3.392 (7) 152
C10—H10B⋯O4iv 0.99 2.35 3.082 (5) 130
C13—H13⋯O2i 0.95 2.41 3.259 (3) 149
C19—H19B⋯O5 0.98 2.57 3.351 (6) 136
C19—H19C⋯O7v 0.98 2.58 3.207 (7) 122
C19—H19C⋯O8v 0.98 2.57 3.548 (4) 172
Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) .
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References
Baker, R. S. & Norman, R. E. (2004). Acta Cryst. E60, m1761–m1763.
Bruker (1997). SMART and SAINT Bruker AXS Inc., Madison, Wisconsin, USA.
Ferrarini, P. L., Mori, C., Badawneh, M., Manera, C., Martinelli, A., Miceli, M., Ramagnoli, F. & Saccomanni, G. (1997). J. Heterocycl. Chem.34, 1501–1504.
Gavrilova, E. L. & Bosnich, B. (2004). Chem. Rev.104, 349–383.
Nakatani, K., Sando, S. & Saito, I. (2000). J. Am. Chem. Soc.122, 2172–2178.
Nakatani, K., Sando, S. & Saito, I. (2001). Nat. Biotechnol.19, 51–55.
Sheldrick, G. M. (1996). SADABS University of Göttingen, Germany.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Stadie, N. P., Sanchez-Smith, R. & Groy, T. L. (2007). Acta Cryst. E63, m2153–m2154. | 21201691 | PMC2960735 | CC BY | 2021-01-04 18:55:11 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Aug 6; 64(Pt 9):o1702 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography lh260410.1107/S160053680800809XACSEBHS160053680800809XMetal-Organic PapersBis(2-ethoxy-6-formylphenolato-κ2
O
1,O
6)nickel(II) [Ni(C9H9O3)2]Han Zhen-Quan a*a Applied Technical College, Qiqihar University, Qiqihar 161006, People’s Republic of ChinaCorrespondence e-mail: [email protected] 4 2008 29 3 2008 29 3 2008 64 Pt 4 e080400m592 m592 18 3 2008 25 3 2008 © Zhen-Quan Han 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Ni(C9H9O3)2], was synthesized by the reaction of 3-ethoxysalicylaldehyde with nickel(II) nitrate in methanol solution. The asymmetric unit onsists of two half-molecules; each Ni atom lies on a centre of symmetry. The NiII ions are coordinated by four O atoms from two deprotonated 3-ethoxysalicylaldehyde ligands in a slightly distorted square-planar coordination environment.
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Related literature
For related literature, see: Carlsson et al. (2004 ▶); Li & Chen (2006 ▶); Mounts & Fernando (1974 ▶); Volkmer et al. (1996 ▶).
Experimental
Crystal data
[Ni(C9H9O3)2]
M
r = 389.03
Triclinic,
a = 8.448 (2) Å
b = 10.123 (2) Å
c = 11.919 (3) Å
α = 111.175 (2)°
β = 97.377 (2)°
γ = 102.431 (3)°
V = 904.1 (4) Å3
Z = 2
Mo Kα radiation
μ = 1.10 mm−1
T = 298 (2) K
0.32 × 0.32 × 0.30 mm
Data collection
Bruker SMART CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Sheldrick, 1996 ▶) T
min = 0.719, T
max = 0.733
5465 measured reflections
3993 independent reflections
3187 reflections with I > 2σ(I)
R
int = 0.013
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.116
S = 1.02
3993 reflections
231 parameters
H-atom parameters constrained
Δρmax = 0.65 e Å−3
Δρmin = −0.66 e Å−3
Data collection: SMART (Bruker, 1998 ▶); cell refinement: SAINT (Bruker, 1998 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S160053680800809X/lh2604sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S160053680800809X/lh2604Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: LH2604).
The author acknowledges Qiqihar University for a research grant.
supplementary crystallographic
information
Comment
The authors interest in nickel(II) complexes arises from the fact that Ni(II)
is the active center of the urease enzyme (Carlsson et al.,
2004;
Volkmer et al., 1996). The author reports herein the crystal
structure
of the title nickel(II) complex.
In the asymmetric unit of the title compound, there are two independent complex
(Fig. 1). Each NiII ion lies on an inversion center and is coordinated by
four O atoms from two deprotonated 3-ethoxysalicylaldehyde ligands.
The coordinate bond values (Table 1) in each molecule are
comparable to each other between the two independent complex molecules.
The structure is similar
to other nickel(II) complexes derived from the derivatives of salicylaldehyde
(Li & Chen, 2006; Mounts & Fernando, 1974).
Experimental
All chemicals were of AR grade. 3-Ethoxysalicylaldehyde (33.2 mg, 0.2 mmol)
and nickel(II) nitrate hexahydrate (29.0 mg, 0.1 mmol) were refluxed for 30 min in 10 ml methanol solution. The mixture was cooled to room temperature
and filtered. Keeping the filtrate in air for a week, yielded red block
crystals suitable for X-ray analysis.
Refinement
H atoms were placed in idealized positions and constrained to ride on their
parent atoms with C–H distances in the range 0.93–0.97 Å, and with
Uiso(H) set at 1.2Ueq(C) and 1.5Ueq(methyl C).
Although no significant density was located in the
solvent accessible VOIDS of 47.00 Å3, these might be
able to accommodate disordered water molecules.
Figures
Fig. 1. The molecular structures of the two centrosymmetric independent molecules, showing 30% probability displacement ellipsoids and the atom-numbering scheme. The unlabeled atoms are related by the symmetry operators (-x, -y+1, -z) and (-x, -y, -z) for the molecules containing Ni1 and Ni2 respectively.
Crystal data
[Ni(C9H9O3)2] Z = 2
Mr = 389.03 F000 = 404
Triclinic, P1 Dx = 1.429 Mg m−3
Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å
a = 8.448 (2) Å Cell parameters from 2386 reflections
b = 10.123 (2) Å θ = 2.2–27.9º
c = 11.919 (3) Å µ = 1.10 mm−1
α = 111.175 (2)º T = 298 (2) K
β = 97.377 (2)º Block, red
γ = 102.431 (3)º 0.32 × 0.32 × 0.30 mm
V = 904.1 (4) Å3
Data collection
Bruker SMART CCD area-detector diffractometer 3993 independent reflections
Radiation source: fine-focus sealed tube 3187 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.013
T = 298(2) K θmax = 27.5º
ω scans θmin = 2.3º
Absorption correction: multi-scan(SADABS; Sheldrick, 1996) h = −10→8
Tmin = 0.719, Tmax = 0.733 k = −13→13
5465 measured reflections l = −11→15
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.041 H-atom parameters constrained
wR(F2) = 0.116 w = 1/[σ2(Fo2) + (0.0596P)2 + 0.672P] where P = (Fo2 + 2Fc2)/3
S = 1.02 (Δ/σ)max < 0.001
3993 reflections Δρmax = 0.65 e Å−3
231 parameters Δρmin = −0.66 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Ni1 0.0000 0.5000 0.0000 0.02805 (13)
Ni2 0.0000 0.0000 0.0000 0.02920 (14)
O1 0.0505 (2) −0.0121 (2) 0.15045 (16) 0.0363 (4)
O2 −0.0576 (3) 0.1741 (2) 0.0663 (2) 0.0506 (5)
O3 0.1596 (3) −0.0689 (2) 0.33683 (18) 0.0484 (5)
O4 0.0529 (3) 0.3494 (3) −0.1190 (2) 0.0513 (5)
O5 0.1989 (2) 0.63719 (19) 0.02863 (17) 0.0350 (4)
O6 0.4541 (3) 0.8659 (2) 0.1053 (2) 0.0552 (6)
C1 0.3232 (3) 0.4873 (3) −0.1220 (3) 0.0366 (6)
C2 0.3219 (3) 0.6192 (3) −0.0278 (2) 0.0328 (5)
C3 0.4641 (3) 0.7438 (3) 0.0107 (3) 0.0416 (7)
C4 0.5959 (4) 0.7345 (4) −0.0462 (3) 0.0534 (8)
H4 0.6872 0.8170 −0.0214 0.064*
C5 0.5940 (4) 0.6031 (4) −0.1404 (3) 0.0583 (9)
H5 0.6837 0.5983 −0.1779 0.070*
C6 0.4608 (4) 0.4814 (4) −0.1777 (3) 0.0508 (8)
H6 0.4606 0.3938 −0.2403 0.061*
C7 0.1866 (3) 0.3570 (3) −0.1613 (3) 0.0387 (6)
H7 0.1946 0.2713 −0.2220 0.046*
C8 0.5934 (4) 0.9930 (4) 0.1562 (4) 0.0630 (10)
H8A 0.6162 1.0304 0.0939 0.076*
H8B 0.6910 0.9687 0.1859 0.076*
C9 0.5528 (5) 1.1062 (5) 0.2603 (4) 0.0745 (11)
H9A 0.4523 1.1251 0.2308 0.112*
H9B 0.6424 1.1959 0.2933 0.112*
H9C 0.5375 1.0706 0.3238 0.112*
C10 0.0426 (4) 0.2306 (3) 0.2817 (3) 0.0403 (6)
C11 0.0741 (3) 0.0937 (3) 0.2595 (2) 0.0344 (6)
C12 0.1373 (4) 0.0681 (3) 0.3646 (3) 0.0408 (6)
C13 0.1716 (5) 0.1762 (4) 0.4812 (3) 0.0603 (9)
H13 0.2143 0.1582 0.5484 0.072*
C14 0.1433 (6) 0.3135 (4) 0.5010 (3) 0.0737 (12)
H14 0.1689 0.3866 0.5806 0.088*
C15 0.0782 (5) 0.3389 (4) 0.4027 (3) 0.0604 (10)
H15 0.0571 0.4292 0.4158 0.073*
C16 −0.0311 (4) 0.2596 (3) 0.1806 (3) 0.0390 (6)
H16 −0.0617 0.3467 0.1996 0.047*
C17 0.2484 (5) −0.0970 (4) 0.4322 (3) 0.0552 (8)
H17A 0.1831 −0.0992 0.4930 0.066*
H17B 0.3528 −0.0202 0.4733 0.066*
C18 0.2804 (6) −0.2441 (5) 0.3720 (4) 0.0832 (14)
H18A 0.1769 −0.3208 0.3414 0.125*
H18B 0.3527 −0.2606 0.4315 0.125*
H18C 0.3322 −0.2449 0.3047 0.125*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Ni1 0.0239 (2) 0.0277 (2) 0.0327 (2) 0.00622 (17) 0.00861 (17) 0.01208 (19)
Ni2 0.0308 (2) 0.0285 (2) 0.0283 (2) 0.00865 (18) 0.00732 (18) 0.01098 (18)
O1 0.0484 (11) 0.0311 (9) 0.0290 (9) 0.0129 (8) 0.0067 (8) 0.0113 (8)
O2 0.0517 (13) 0.0484 (12) 0.0514 (13) 0.0158 (10) 0.0160 (10) 0.0173 (10)
O3 0.0639 (14) 0.0438 (12) 0.0361 (10) 0.0201 (10) 0.0001 (10) 0.0152 (9)
O4 0.0470 (12) 0.0502 (13) 0.0537 (13) 0.0132 (10) 0.0147 (10) 0.0164 (11)
O5 0.0277 (9) 0.0310 (9) 0.0459 (11) 0.0057 (7) 0.0132 (8) 0.0149 (8)
O6 0.0374 (11) 0.0432 (12) 0.0731 (16) −0.0024 (9) 0.0148 (11) 0.0170 (11)
C1 0.0303 (13) 0.0484 (16) 0.0388 (14) 0.0144 (12) 0.0129 (11) 0.0225 (12)
C2 0.0246 (12) 0.0412 (14) 0.0404 (14) 0.0104 (10) 0.0093 (10) 0.0238 (12)
C3 0.0300 (13) 0.0469 (17) 0.0530 (18) 0.0070 (12) 0.0110 (12) 0.0272 (15)
C4 0.0316 (15) 0.061 (2) 0.072 (2) 0.0021 (14) 0.0163 (15) 0.0362 (18)
C5 0.0380 (17) 0.079 (3) 0.069 (2) 0.0179 (17) 0.0286 (16) 0.036 (2)
C6 0.0414 (16) 0.065 (2) 0.0521 (18) 0.0205 (15) 0.0228 (14) 0.0239 (16)
C7 0.0367 (14) 0.0426 (15) 0.0386 (14) 0.0161 (12) 0.0154 (12) 0.0132 (12)
C8 0.0436 (18) 0.055 (2) 0.076 (3) −0.0064 (16) 0.0034 (17) 0.0253 (19)
C9 0.073 (3) 0.060 (2) 0.064 (2) −0.008 (2) 0.005 (2) 0.0134 (19)
C10 0.0472 (16) 0.0387 (15) 0.0354 (14) 0.0168 (13) 0.0131 (12) 0.0114 (12)
C11 0.0318 (13) 0.0374 (14) 0.0314 (13) 0.0086 (11) 0.0091 (10) 0.0110 (11)
C12 0.0423 (15) 0.0421 (15) 0.0341 (14) 0.0127 (12) 0.0064 (12) 0.0115 (12)
C13 0.084 (3) 0.064 (2) 0.0297 (15) 0.030 (2) 0.0049 (16) 0.0120 (15)
C14 0.114 (3) 0.064 (2) 0.0347 (17) 0.041 (2) 0.0089 (19) 0.0036 (16)
C15 0.093 (3) 0.0484 (19) 0.0391 (17) 0.0340 (19) 0.0151 (17) 0.0092 (14)
C16 0.0474 (16) 0.0345 (14) 0.0392 (15) 0.0176 (12) 0.0166 (12) 0.0139 (12)
C17 0.062 (2) 0.065 (2) 0.0424 (17) 0.0247 (17) 0.0015 (15) 0.0249 (16)
C18 0.118 (4) 0.077 (3) 0.062 (2) 0.057 (3) 0.000 (2) 0.027 (2)
Geometric parameters (Å, °)
Ni1—O5 1.837 (2) C6—H6 0.9300
Ni1—O5i 1.837 (2) C7—H7 0.9300
Ni1—O4 1.852 (2) C8—C9 1.491 (5)
Ni1—O4i 1.852 (2) C8—H8A 0.9700
Ni2—O1 1.843 (2) C8—H8B 0.9700
Ni2—O1ii 1.843 (2) C9—H9A 0.9600
Ni2—O2ii 1.851 (2) C9—H9B 0.9600
Ni2—O2 1.851 (2) C9—H9C 0.9600
O1—C11 1.309 (3) C10—C11 1.405 (4)
O2—C16 1.282 (3) C10—C15 1.406 (4)
O3—C12 1.365 (3) C10—C16 1.438 (4)
O3—C17 1.429 (3) C11—C12 1.430 (4)
O4—C7 1.294 (3) C12—C13 1.369 (4)
O5—C2 1.319 (3) C13—C14 1.402 (5)
O6—C3 1.367 (4) C13—H13 0.9300
O6—C8 1.417 (4) C14—C15 1.362 (5)
C1—C2 1.404 (4) C14—H14 0.9300
C1—C6 1.412 (4) C15—H15 0.9300
C1—C7 1.432 (4) C16—H16 0.9300
C2—C3 1.426 (4) C17—C18 1.502 (5)
C3—C4 1.380 (4) C17—H17A 0.9700
C4—C5 1.391 (5) C17—H17B 0.9700
C4—H4 0.9300 C18—H18A 0.9600
C5—C6 1.364 (5) C18—H18B 0.9600
C5—H5 0.9300 C18—H18C 0.9600
O5—Ni1—O5i 180 O6—C8—H8B 110.2
O5—Ni1—O4 94.16 (9) C9—C8—H8B 110.2
O5i—Ni1—O4 85.84 (9) H8A—C8—H8B 108.5
O5—Ni1—O4i 85.84 (9) C8—C9—H9A 109.5
O5i—Ni1—O4i 94.16 (9) C8—C9—H9B 109.5
O4—Ni1—O4i 180 H9A—C9—H9B 109.5
O1—Ni2—O1ii 180 C8—C9—H9C 109.5
O1—Ni2—O2ii 86.30 (9) H9A—C9—H9C 109.5
O1ii—Ni2—O2ii 93.70 (9) H9B—C9—H9C 109.5
O1—Ni2—O2 93.70 (9) C11—C10—C15 120.7 (3)
O1ii—Ni2—O2 86.30 (9) C11—C10—C16 120.0 (2)
O2ii—Ni2—O2 180 C15—C10—C16 119.3 (3)
C11—O1—Ni2 126.59 (17) O1—C11—C10 125.3 (2)
C16—O2—Ni2 127.6 (2) O1—C11—C12 117.4 (2)
C12—O3—C17 118.6 (2) C10—C11—C12 117.3 (2)
C7—O4—Ni1 127.6 (2) O3—C12—C13 125.1 (3)
C2—O5—Ni1 127.62 (17) O3—C12—C11 114.3 (2)
C3—O6—C8 118.6 (3) C13—C12—C11 120.5 (3)
C2—C1—C6 120.1 (3) C12—C13—C14 121.2 (3)
C2—C1—C7 120.5 (2) C12—C13—H13 119.4
C6—C1—C7 119.4 (3) C14—C13—H13 119.4
O5—C2—C1 125.0 (2) C15—C14—C13 119.4 (3)
O5—C2—C3 117.0 (2) C15—C14—H14 120.3
C1—C2—C3 118.0 (2) C13—C14—H14 120.3
O6—C3—C4 125.7 (3) C14—C15—C10 120.9 (3)
O6—C3—C2 114.0 (2) C14—C15—H15 119.6
C4—C3—C2 120.3 (3) C10—C15—H15 119.6
C3—C4—C5 120.8 (3) O2—C16—C10 124.7 (3)
C3—C4—H4 119.6 O2—C16—H16 117.6
C5—C4—H4 119.6 C10—C16—H16 117.6
C6—C5—C4 120.2 (3) O3—C17—C18 107.2 (3)
C6—C5—H5 119.9 O3—C17—H17A 110.3
C4—C5—H5 119.9 C18—C17—H17A 110.3
C5—C6—C1 120.6 (3) O3—C17—H17B 110.3
C5—C6—H6 119.7 C18—C17—H17B 110.3
C1—C6—H6 119.7 H17A—C17—H17B 108.5
O4—C7—C1 125.0 (3) C17—C18—H18A 109.5
O4—C7—H7 117.5 C17—C18—H18B 109.5
C1—C7—H7 117.5 H18A—C18—H18B 109.5
O6—C8—C9 107.6 (3) C17—C18—H18C 109.5
O6—C8—H8A 110.2 H18A—C18—H18C 109.5
C9—C8—H8A 110.2 H18B—C18—H18C 109.5
Symmetry codes: (i) −x, −y+1, −z; (ii) −x, −y, −z.
Table 1 Selected geometric parameters (Å, °)
Ni1—O5 1.837 (2)
Ni1—O4 1.852 (2)
Ni2—O1 1.843 (2)
Ni2—O2 1.851 (2)
O5—Ni1—O5i 180
O5—Ni1—O4 94.16 (9)
O5i—Ni1—O4 85.84 (9)
O4—Ni1—O4i 180
O1—Ni2—O1ii 180
O1—Ni2—O2 93.70 (9)
O1ii—Ni2—O2 86.30 (9)
O2ii—Ni2—O2 180
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (1998). SMART (Version 5.628) and SAINT (Version 6.02). Bruker AXS Inc., Madison, Wisconsin, USA.
Carlsson, H., Haukka, M., Bousseksou, A., Latour, J.-M. & Nordlander, E. (2004). Inorg. Chem.43, 8252–8262.
Li, Y.-G. & Chen, H.-J. (2006). Acta Cryst. E62, m1038–m1039.
Mounts, R. D. & Fernando, Q. (1974). Acta Cryst. B30, 542–543.
Sheldrick, G. M. (1996). SADABS University of Göttingen, Germany.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Volkmer, D., Hommerich, B., Griesar, K., Haase, W. & Krebs, B. (1996). Inorg. Chem.35, 3792–3803. | 21202039 | PMC2960901 | CC BY | 2021-01-04 18:55:23 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Mar 29; 64(Pt 4):m592 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography at254910.1107/S1600536808006958ACSEBHS1600536808006958Metal-Organic Papers{μ-6,6′-Dimethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato}-μ-nitrato-dinitratoterbium(III)zinc(II) [TbZn(C18H18N2O4)(NO3)3]Chen Jing-Rong aSui Yan b*Chen Li cWen Ji-Wu bYin Li-Yang ba College of Chemistry & Chemical Engineering, Southwest University, 400715 Beibei, Chongqing, People’s Republic of Chinab JiangXi Province Key Laboratory of Coordination Chemistry, College of Chemistry & Chemical Engineering, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of Chinac College of Education, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of ChinaCorrespondence e-mail: [email protected] 4 2008 20 3 2008 20 3 2008 64 Pt 4 e080400m562 m563 08 3 2008 12 3 2008 © Chen et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title heteronuclear ZnII—TbIII complex (systematic name: {6,6′-dimethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato-1κ4
O
6,O
1,O
1′,O
6′}:2κ4
O
1,N,N′,O
1′-μ-nitrato-1:2κ2
O:O′-dinitrato-1κ4
O,O′-terbium(III)zinc(II)), [TbZn(C18H18N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bis(3-methoxysalicylidene)ethylenediamine (H2
L), the Tb and Zn atoms are triply bridged by two phenolate O atoms of the Schiff base ligand and one nitrate ion. The five-coordinate Zn atom is in a square-pyramidal geometry with the donor centers of two imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms. The TbIII center has a ninefold coordination environment of O atoms, involving the phenolate O atoms, two methoxy O atoms, two O atoms from two nitrate ions and one from the bridging nitrate ion. Weak intermolecular C—H⋯O interactions generate a two-dimensional layer structure.
==== Body
Related literature
For related literature, see: Baggio et al. (2000 ▶); Caravan et al. (1999 ▶); Edder et al. (2000 ▶); Knoer et al. (2005 ▶); Sui et al. (2006 ▶, 2007 ▶).
Experimental
Crystal data
[TbZn(C18H18N2O4)(NO3)3]
M
r = 736.66
Monoclinic,
a = 10.6818 (4) Å
b = 16.5022 (6) Å
c = 14.9546 (6) Å
β = 99.618 (1)°
V = 2599.04 (17) Å3
Z = 4
Mo Kα radiation
μ = 3.69 mm−1
T = 293 (2) K
0.33 × 0.22 × 0.12 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2004 ▶) T
min = 0.375, T
max = 0.666
15507 measured reflections
4431 independent reflections
3722 reflections with I > 2σ(I)
R
int = 0.022
Refinement
R[F
2 > 2σ(F
2)] = 0.023
wR(F
2) = 0.068
S = 1.00
4431 reflections
345 parameters
2 restraints
H-atom parameters constrained
Δρmax = 0.56 e Å−3
Δρmin = −0.52 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: APEX2; data reduction: APEX2; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: APEX2; software used to prepare material for publication: APEX2 and publCIF (Westrip, 2008 ▶).
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808006958/at2549sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808006958/at2549Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2549).
We gratefully acknowledge financial support from the Department of Education, JiangXi Province (No. 2007317) and the Natural Science Foundation of JiangXi Province (No: 2007GZH1667).
supplementary crystallographic
information
Comment
The potential applications of trivalent lanthanide complexes as contrast agent
for magnetic resonance imaging and stains for fluorescence imaging have
prompted considerable interest in the preparation, magnetic and optical
properties of 3 d-4f hetorometallic dinuclear complexes (Baggio et al.,
2000; Caravan et al., 1999; Edder et al., 2000; Knoer et
al., 2005). As part of our investigations into the structure and
applications of 3 d-4f hetorometallic Schiff base complexes (Sui et al.
2006; Sui et al. 2007), we report here the synthesis and X-ray crystal
structure analysis of the title complex, (I), a new ZnII—TbIII complex
with salen-type Schiff base N,N'-bis(3-methoxysalicylidene)
ethylenediamine (H2L).
Complex (I) crystallizes in the space group P21/n, with zinc
and terbium triply bridged by two phenolate O atoms provided by the Schiff
base ligand and one nitrate ion. The inner salen-type cavity is occupied by
zinc(II), while terbium(III) is present in the open and larger portion of the
dinucleating compartmental Schiff base ligand.
The TbIII center has a ninefold coordination environment of O atoms,
involving the phenolate O atoms, two methoxy O atoms, two O atoms from two
nitrate ions and one from the bridging nitrate ion. The four kinds of Tb—O
bond distances are significantly different, the longest being the Tb—O
(methoxy) separations and the shortest being the Tb—O (phenolate) and
Tb—O5 (bridging nitrate).
The ZnII is in a square-pyramidal geometry and is five-coordinated by two
imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms.
The Zn atom is 0.6062 (4) Å above the mean N2O2 plane with an average
deviation from the plane of 0.0383 (3) Å, which construct the bottom of
square-pyramid. The Zn—O6 (bridging nitrate) separation is 1.978 (3)Å and
the angles of this Zn—O vector with the Zn—N or Zn—O bonds lie between
102.5 (5)° and 112.7 (6)°, which suggesting that the ZnII is in a slightly
distorted square-pyramidal conformation.
Adjacent molecules are held together by weak interactions (C5—H5···O11i =
3.376 (5) Å and C9—H9A···O13ii = 3.487 (6) Å; symmetry codes: (i) 1/2 +
x, 1/2 - y, -1/2 + z; (ii) 1 - x, -y, 1 -
z). These link the molecules into a two-dimensional layer structure
(Fig 2).
Experimental
H2L was prepared by the 2:1 condensation of 3-methoxysalicylaldehyde and
ethylenediamine in methanol. Complex (I) was obtained by the treatment of
zinc(II) acetate dihydrate (0.188 g, 1 mmol) with H2L (0.328 g, 1 mmol) in
methanol solution (80 ml) under reflux for 3 h and then for another 3 h after
the addition of terbium(III) nitrate hexahydrate (0.453 g, 1 mmol). The
reaction mixture was cooled and the resulting precipitate was filtered off,
washed with diethyl ether and dried in vacuo. Single crystals of (I)
suitable for X-ray analysis were obtained by slow evaporation at room
temperature of a methanol solution. Analysis calculated for
C18H18N5O13TbZn: C 29.35 H 2.46, N 9.51, Tb 21.57, Zn 8.88%; found: C
29.40, H 2.45, N 9.53, Tb 21.60, Zn 8.906%. IR (KBr, cm-1): 1640 (C=N),
1386,1490(nitrate).
Refinement
The H atoms were positioned geometrically and treated as riding on their parent
atoms, with C—H distances of 0.97 (methylene), 0.96 Å (methyl) and 0.93 Å (aromaticmethyl), and with Uiso(H) = 1.5Ueq(C) for
methyl H atoms and 1.2Ueq(C) for other H atoms. The main directions
of movement of covalently bonded atoms N4, O5 and O6 are enforced to be the
same.
Figures
Fig. 1. The molecular structure of (I), showing 30% probability displacement ellipsoids. All the H atoms on carbon have been omitted for clarity.
Fig. 2. The packing diagram of (I), viewed along the b axis; hydrogen bonds are shown as dashed lines.
Crystal data
[TbZn(C18H18N2O4)(NO3)3] F000 = 1440
Mr = 736.66 Dx = 1.883 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 8984 reflections
a = 10.6818 (4) Å θ = 1.9–25.0º
b = 16.5022 (6) Å µ = 3.69 mm−1
c = 14.9546 (6) Å T = 293 (2) K
β = 99.6180 (10)º Block, yellow
V = 2599.04 (17) Å3 0.33 × 0.22 × 0.12 mm
Z = 4
Data collection
Bruker APEXII area-detector diffractometer 4431 independent reflections
Radiation source: fine-focus sealed tube 3722 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.022
T = 293(2) K θmax = 25.0º
φ and ω scan θmin = 1.9º
Absorption correction: multi-scan(SADABS; Bruker, 2004) h = −12→12
Tmin = 0.375, Tmax = 0.666 k = −19→18
15507 measured reflections l = −17→16
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.023 H-atom parameters constrained
wR(F2) = 0.068 w = 1/[σ2(Fo2) + (0.044P)2 + 0.9659P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.001
4431 reflections Δρmax = 0.56 e Å−3
345 parameters Δρmin = −0.52 e Å−3
2 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Tb1 0.637703 (16) 0.109788 (10) 0.776551 (11) 0.04217 (8)
Zn1 0.77995 (4) 0.03607 (2) 0.61063 (3) 0.04267 (12)
C1 0.7286 (3) 0.2144 (2) 0.6077 (2) 0.0439 (8)
O3 0.6387 (3) 0.26176 (14) 0.72934 (18) 0.0516 (6)
O2 0.7099 (2) 0.14261 (15) 0.64411 (17) 0.0503 (6)
N2 0.8021 (3) 0.0844 (2) 0.4882 (2) 0.0527 (8)
O12 0.4281 (3) 0.12967 (19) 0.6805 (2) 0.0638 (8)
O4 0.5108 (2) −0.02009 (15) 0.80424 (17) 0.0504 (6)
C16 0.5882 (3) −0.0716 (2) 0.6797 (2) 0.0422 (8)
O11 0.4437 (3) 0.16395 (19) 0.8203 (2) 0.0628 (7)
O6 0.9420 (2) 0.02850 (16) 0.69642 (19) 0.0533 (6)
O1 0.6478 (2) −0.00117 (14) 0.68386 (17) 0.0494 (6)
N1 0.7505 (3) −0.06699 (19) 0.5360 (2) 0.0488 (7)
C15 0.5114 (3) −0.0853 (2) 0.7453 (3) 0.0455 (8)
N3 0.3756 (3) 0.1600 (2) 0.7427 (3) 0.0645 (10)
C11 0.5965 (3) −0.1320 (2) 0.6144 (3) 0.0473 (9)
C7 0.8020 (4) 0.1592 (3) 0.4688 (3) 0.0543 (10)
H7 0.8186 0.1729 0.4116 0.065*
N4 0.9460 (3) 0.0491 (2) 0.7789 (3) 0.0664 (9)
O5 0.8517 (3) 0.07391 (19) 0.81165 (19) 0.0608 (7)
C6 0.7784 (4) 0.2255 (2) 0.5279 (3) 0.0505 (9)
C10 0.6736 (4) −0.1246 (2) 0.5428 (3) 0.0514 (10)
H10 0.6658 −0.1650 0.4988 0.062*
O13 0.2669 (3) 0.1838 (3) 0.7280 (3) 0.1126 (15)
O7 1.0729 (4) 0.0402 (3) 0.8425 (4) 0.1448 (19)
C12 0.5286 (4) −0.2039 (2) 0.6189 (3) 0.0612 (11)
H12 0.5315 −0.2438 0.5754 0.073*
O8 0.6731 (3) 0.07585 (17) 0.94168 (19) 0.0623 (7)
O9 0.7154 (3) 0.19769 (16) 0.90722 (18) 0.0606 (7)
N5 0.7195 (4) 0.1437 (2) 0.9680 (2) 0.0634 (9)
C2 0.6904 (3) 0.2826 (2) 0.6531 (2) 0.0457 (8)
C17 0.4344 (4) −0.0290 (3) 0.8748 (3) 0.0648 (12)
H17A 0.3470 −0.0361 0.8478 0.097*
H17B 0.4427 0.0187 0.9122 0.097*
H17C 0.4627 −0.0754 0.9113 0.097*
C9 0.8243 (4) −0.0616 (3) 0.4619 (3) 0.0580 (11)
H9A 0.7976 −0.1036 0.4175 0.070*
H9B 0.9138 −0.0692 0.4854 0.070*
C3 0.7059 (4) 0.3594 (2) 0.6228 (3) 0.0563 (10)
H3 0.6813 0.4038 0.6540 0.068*
C5 0.7933 (4) 0.3058 (3) 0.4989 (3) 0.0651 (12)
H5 0.8278 0.3147 0.4466 0.078*
C14 0.4478 (4) −0.1572 (2) 0.7495 (3) 0.0594 (11)
H14 0.3985 −0.1658 0.7943 0.071*
C13 0.4583 (4) −0.2167 (3) 0.6858 (4) 0.0724 (13)
H13 0.4169 −0.2659 0.6886 0.087*
C8 0.8030 (4) 0.0213 (3) 0.4178 (3) 0.0604 (11)
H8A 0.8700 0.0327 0.3831 0.073*
H8B 0.7227 0.0218 0.3764 0.073*
C4 0.7583 (4) 0.3706 (3) 0.5456 (3) 0.0631 (12)
H4 0.7699 0.4228 0.5251 0.076*
C18 0.5764 (5) 0.3264 (2) 0.7697 (3) 0.0668 (12)
H18A 0.6343 0.3708 0.7842 0.100*
H18B 0.5497 0.3071 0.8241 0.100*
H18C 0.5037 0.3443 0.7277 0.100*
O10 0.7648 (4) 0.1569 (2) 1.0465 (2) 0.1072 (14)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Tb1 0.04948 (12) 0.04348 (12) 0.03743 (12) −0.00214 (7) 0.01852 (8) −0.00262 (7)
Zn1 0.0471 (2) 0.0457 (2) 0.0388 (2) 0.00041 (18) 0.01728 (18) −0.00323 (17)
C1 0.0430 (19) 0.048 (2) 0.042 (2) −0.0022 (16) 0.0099 (16) 0.0065 (16)
O3 0.0677 (16) 0.0417 (14) 0.0496 (16) 0.0050 (12) 0.0221 (13) −0.0013 (11)
O2 0.0664 (17) 0.0405 (13) 0.0517 (16) 0.0042 (12) 0.0320 (13) 0.0052 (12)
N2 0.0564 (19) 0.066 (2) 0.0399 (19) 0.0074 (16) 0.0209 (15) −0.0010 (15)
O12 0.0532 (16) 0.092 (2) 0.0479 (18) 0.0077 (15) 0.0122 (14) −0.0148 (15)
O4 0.0574 (15) 0.0521 (15) 0.0474 (16) −0.0096 (12) 0.0253 (12) −0.0004 (12)
C16 0.0383 (18) 0.0426 (19) 0.046 (2) −0.0018 (15) 0.0084 (16) −0.0010 (16)
O11 0.0608 (17) 0.085 (2) 0.0482 (18) 0.0038 (15) 0.0240 (15) −0.0143 (14)
O6 0.0474 (14) 0.0574 (15) 0.0565 (15) 0.0035 (12) 0.0126 (12) −0.0068 (12)
O1 0.0631 (16) 0.0427 (14) 0.0500 (16) −0.0133 (11) 0.0315 (13) −0.0112 (11)
N1 0.0485 (17) 0.0564 (19) 0.0447 (19) 0.0008 (15) 0.0172 (14) −0.0107 (15)
C15 0.045 (2) 0.0453 (19) 0.047 (2) −0.0030 (16) 0.0111 (17) 0.0012 (16)
N3 0.053 (2) 0.076 (3) 0.069 (3) 0.0059 (18) 0.022 (2) −0.0088 (19)
C11 0.045 (2) 0.044 (2) 0.054 (2) −0.0018 (16) 0.0091 (18) −0.0069 (17)
C7 0.054 (2) 0.071 (3) 0.043 (2) −0.002 (2) 0.0225 (18) 0.0113 (19)
N4 0.062 (2) 0.067 (2) 0.071 (2) 0.0024 (17) 0.0139 (18) −0.0074 (18)
O5 0.0519 (15) 0.081 (2) 0.0492 (17) 0.0075 (14) 0.0077 (12) −0.0072 (14)
C6 0.052 (2) 0.056 (2) 0.044 (2) −0.0040 (18) 0.0114 (18) 0.0131 (17)
C10 0.052 (2) 0.052 (2) 0.050 (2) 0.0066 (18) 0.0060 (18) −0.0182 (17)
O13 0.059 (2) 0.178 (4) 0.103 (3) 0.033 (2) 0.020 (2) −0.033 (3)
O7 0.105 (3) 0.164 (5) 0.147 (4) 0.014 (3) −0.031 (3) −0.013 (4)
C12 0.057 (2) 0.049 (2) 0.078 (3) −0.0045 (19) 0.013 (2) −0.016 (2)
O8 0.087 (2) 0.0564 (17) 0.0460 (17) −0.0187 (15) 0.0187 (15) −0.0008 (13)
O9 0.085 (2) 0.0527 (16) 0.0463 (17) −0.0165 (14) 0.0161 (14) −0.0020 (13)
N5 0.077 (2) 0.072 (2) 0.045 (2) −0.023 (2) 0.0195 (18) −0.0063 (18)
C2 0.051 (2) 0.0432 (19) 0.043 (2) −0.0007 (16) 0.0092 (17) 0.0035 (16)
C17 0.070 (3) 0.074 (3) 0.061 (3) −0.014 (2) 0.039 (2) 0.001 (2)
C9 0.050 (2) 0.073 (3) 0.053 (3) 0.003 (2) 0.0170 (19) −0.022 (2)
C3 0.065 (3) 0.045 (2) 0.056 (3) −0.0018 (19) 0.001 (2) 0.0043 (18)
C5 0.068 (3) 0.077 (3) 0.051 (3) −0.013 (2) 0.012 (2) 0.022 (2)
C14 0.053 (2) 0.058 (2) 0.071 (3) −0.0129 (19) 0.021 (2) 0.007 (2)
C13 0.070 (3) 0.051 (2) 0.101 (4) −0.023 (2) 0.025 (3) −0.008 (2)
C8 0.065 (3) 0.081 (3) 0.040 (2) 0.005 (2) 0.0209 (19) −0.010 (2)
C4 0.080 (3) 0.052 (2) 0.056 (3) −0.011 (2) 0.006 (2) 0.016 (2)
C18 0.088 (3) 0.048 (2) 0.069 (3) 0.012 (2) 0.029 (2) −0.008 (2)
O10 0.156 (4) 0.118 (3) 0.043 (2) −0.058 (3) 0.005 (2) −0.0054 (19)
Geometric parameters (Å, °)
Tb1—O1 2.310 (2) C11—C12 1.398 (5)
Tb1—O2 2.307 (2) C11—C10 1.462 (5)
Tb1—O3 2.606 (2) C7—C6 1.456 (6)
Tb1—O4 2.606 (2) C7—H7 0.9300
Tb1—O5 2.335 (3) N4—O5 1.260 (4)
Tb1—O8 2.498 (3) N4—O7 1.528 (5)
Tb1—O9 2.464 (3) C6—C5 1.411 (5)
Tb1—O11 2.444 (3) C10—H10 0.9300
Tb1—O12 2.472 (3) C12—C13 1.363 (6)
Zn1—O1 2.021 (2) C12—H12 0.9300
Zn1—O2 2.007 (2) O8—N5 1.261 (4)
Zn1—O6 1.978 (3) O9—N5 1.269 (4)
Zn1—N1 2.030 (3) N5—O10 1.212 (5)
Zn1—N2 2.046 (3) C2—C3 1.367 (5)
C1—O2 1.333 (4) C17—H17A 0.9600
C1—C6 1.398 (5) C17—H17B 0.9600
C1—C2 1.409 (5) C17—H17C 0.9600
O3—C2 1.390 (4) C9—C8 1.520 (6)
O3—C18 1.442 (4) C9—H9A 0.9700
N2—C7 1.267 (5) C9—H9B 0.9700
N2—C8 1.483 (5) C3—C4 1.377 (6)
O12—N3 1.266 (4) C3—H3 0.9300
O4—C15 1.391 (4) C5—C4 1.363 (6)
O4—C17 1.446 (4) C5—H5 0.9300
C16—O1 1.322 (4) C14—C13 1.386 (6)
C16—C15 1.399 (5) C14—H14 0.9300
C16—C11 1.408 (5) C13—H13 0.9300
O11—N3 1.264 (4) C8—H8A 0.9700
O6—N4 1.273 (4) C8—H8B 0.9700
N1—C10 1.271 (5) C4—H4 0.9300
N1—C9 1.466 (5) C18—H18A 0.9600
C15—C14 1.373 (5) C18—H18B 0.9600
N3—O13 1.211 (5) C18—H18C 0.9600
O2—Tb1—O1 67.35 (9) C14—C15—O4 125.8 (3)
O2—Tb1—O5 78.32 (10) C14—C15—C16 121.6 (4)
O1—Tb1—O5 77.97 (10) O4—C15—C16 112.6 (3)
O2—Tb1—O11 124.32 (10) O13—N3—O11 122.5 (4)
O1—Tb1—O11 125.26 (9) O13—N3—O12 121.6 (4)
O5—Tb1—O11 151.00 (10) O11—N3—O12 116.0 (3)
O2—Tb1—O9 115.22 (9) C12—C11—C16 118.3 (4)
O1—Tb1—O9 154.06 (10) C12—C11—C10 117.8 (3)
O5—Tb1—O9 77.49 (10) C16—C11—C10 123.8 (3)
O11—Tb1—O9 76.16 (10) N2—C7—C6 125.9 (3)
O2—Tb1—O12 82.53 (10) N2—C7—H7 117.0
O1—Tb1—O12 83.41 (10) C6—C7—H7 117.0
O5—Tb1—O12 157.24 (10) O5—N4—O6 124.3 (3)
O11—Tb1—O12 51.74 (9) O5—N4—O7 118.2 (4)
O9—Tb1—O12 122.37 (10) O6—N4—O7 117.4 (4)
O2—Tb1—O8 152.16 (10) N4—O5—Tb1 144.2 (3)
O1—Tb1—O8 113.64 (9) C1—C6—C5 117.7 (4)
O5—Tb1—O8 74.94 (10) C1—C6—C7 123.3 (3)
O11—Tb1—O8 79.19 (10) C5—C6—C7 118.6 (4)
O9—Tb1—O8 51.15 (9) N1—C10—C11 124.9 (3)
O12—Tb1—O8 125.24 (10) N1—C10—H10 117.6
O2—Tb1—O4 125.94 (9) C11—C10—H10 117.6
O1—Tb1—O4 61.39 (8) C13—C12—C11 121.4 (4)
O5—Tb1—O4 105.75 (10) C13—C12—H12 119.3
O11—Tb1—O4 76.80 (9) C11—C12—H12 119.3
O9—Tb1—O4 118.27 (8) N5—O8—Tb1 95.5 (2)
O12—Tb1—O4 75.93 (10) N5—O9—Tb1 97.0 (2)
O8—Tb1—O4 69.84 (8) O10—N5—O8 122.4 (4)
O2—Tb1—O3 61.59 (8) O10—N5—O9 121.9 (4)
O1—Tb1—O3 126.69 (8) O8—N5—O9 115.8 (3)
O5—Tb1—O3 104.88 (10) C3—C2—O3 126.0 (4)
O11—Tb1—O3 76.27 (9) C3—C2—C1 121.4 (4)
O9—Tb1—O3 68.42 (9) O3—C2—C1 112.6 (3)
O12—Tb1—O3 76.06 (10) O4—C17—H17A 109.5
O8—Tb1—O3 118.47 (9) O4—C17—H17B 109.5
O4—Tb1—O3 149.37 (8) H17A—C17—H17B 109.5
O6—Zn1—O2 102.47 (11) O4—C17—H17C 109.5
O6—Zn1—O1 104.10 (11) H17A—C17—H17C 109.5
O2—Zn1—O1 78.92 (10) H17B—C17—H17C 109.5
O6—Zn1—N1 110.00 (12) N1—C9—C8 108.8 (3)
O2—Zn1—N1 147.27 (12) N1—C9—H9A 109.9
O1—Zn1—N1 89.14 (11) C8—C9—H9A 109.9
O6—Zn1—N2 112.68 (12) N1—C9—H9B 109.9
O2—Zn1—N2 89.16 (12) C8—C9—H9B 109.9
O1—Zn1—N2 143.00 (12) H9A—C9—H9B 108.3
N1—Zn1—N2 82.26 (13) C2—C3—C4 119.4 (4)
O2—C1—C6 124.7 (3) C2—C3—H3 120.3
O2—C1—C2 116.0 (3) C4—C3—H3 120.3
C6—C1—C2 119.2 (3) C4—C5—C6 121.6 (4)
C2—O3—C18 115.8 (3) C4—C5—H5 119.2
C2—O3—Tb1 118.7 (2) C6—C5—H5 119.2
C18—O3—Tb1 125.1 (2) C15—C14—C13 118.9 (4)
C1—O2—Zn1 126.1 (2) C15—C14—H14 120.6
C1—O2—Tb1 130.8 (2) C13—C14—H14 120.6
Zn1—O2—Tb1 101.57 (10) C12—C13—C14 120.8 (4)
C7—N2—C8 121.4 (3) C12—C13—H13 119.6
C7—N2—Zn1 126.1 (3) C14—C13—H13 119.6
C8—N2—Zn1 112.2 (3) N2—C8—C9 110.0 (3)
N3—O12—Tb1 95.4 (2) N2—C8—H8A 109.7
C15—O4—C17 116.5 (3) C9—C8—H8A 109.7
C15—O4—Tb1 118.6 (2) N2—C8—H8B 109.7
C17—O4—Tb1 124.9 (2) C9—C8—H8B 109.7
O1—C16—C15 116.3 (3) H8A—C8—H8B 108.2
O1—C16—C11 124.7 (3) C5—C4—C3 120.7 (4)
C15—C16—C11 119.0 (3) C5—C4—H4 119.7
N3—O11—Tb1 96.8 (2) C3—C4—H4 119.7
N4—O6—Zn1 119.7 (2) O3—C18—H18A 109.5
C16—O1—Zn1 128.1 (2) O3—C18—H18B 109.5
C16—O1—Tb1 130.9 (2) H18A—C18—H18B 109.5
Zn1—O1—Tb1 101.02 (9) O3—C18—H18C 109.5
C10—N1—C9 122.8 (3) H18A—C18—H18C 109.5
C10—N1—Zn1 128.7 (3) H18B—C18—H18C 109.5
C9—N1—Zn1 108.1 (2)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C5—H5···O11i 0.93 2.45 3.376 (5) 173
C9—H9A···O13ii 0.97 2.54 3.487 (6) 165
Symmetry codes: (i) x+1/2, −y+1/2, z−1/2; (ii) −x+1, −y, −z+1.
Table 1 Selected bond lengths (Å)
Tb1—O1 2.310 (2)
Tb1—O2 2.307 (2)
Tb1—O3 2.606 (2)
Tb1—O4 2.606 (2)
Tb1—O5 2.335 (3)
Tb1—O8 2.498 (3)
Tb1—O9 2.464 (3)
Tb1—O11 2.444 (3)
Tb1—O12 2.472 (3)
Zn1—O1 2.021 (2)
Zn1—O2 2.007 (2)
Zn1—O6 1.978 (3)
Zn1—N1 2.030 (3)
Zn1—N2 2.046 (3)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C5—H5⋯O11i 0.93 2.45 3.376 (5) 173
C9—H9A⋯O13ii 0.97 2.54 3.487 (6) 165
Symmetry codes: (i) ; (ii) .
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References
Baggio, R., Garland, M. T., Moreno, Y., Pena, O., Perec, M. & Spodine, E. (2000). J. Chem. Soc. Dalton Trans. pp. 2061–2066.
Bruker (2004). APEX2 and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Caravan, P., Ellison, J. J., McMurry, T. J. & Lauffer, R. B. (1999). Chem. Rev.99, 2293–2352.
Edder, C., Piguet, C., Bernardinelli, G., Mareda, J., Bochet, C. G., Bunzli, J.-C. G. & Hopfgartner, G. (2000). Inorg. Chem.39, 5059–5073.
Knoer, R., Lin, H.-H., Wei, H.-H. & Mohanta, S. (2005). Inorg. Chem.44, 3524–3536.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sui, Y., Fang, X.-N., Xiao, Y.-A., Luo, Q.-Y. & Li, M.-H. (2006). Acta Cryst. E62, m2230–m2232.
Sui, Y., He, D.-Y., Fang, X.-N., Chen, L. & Peng, J.-L. (2007). Acta Cryst. E63, m2013–m2014.
Westrip, S. P. (2008). publCIF. In preparation. | 21202018 | PMC2961047 | CC BY | 2021-01-04 18:55:22 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Mar 20; 64(Pt 4):m562-m563 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hk245010.1107/S1600536808010507ACSEBHS1600536808010507Metal-Organic PapersDiaquabis(pyridine-2-carboxylato-κ2
N,O)cobalt(II) [Co(C6H4NO2)2(H2O)2]Huang G. S. a*a College of Chemistry & Chemical Engineering, Provincial Key Laboratory of Coordination Chemistry, Jinggangshan University, Jian 343009, People’s Republic of ChinaCorrespondence e-mail: [email protected] 5 2008 18 4 2008 18 4 2008 64 Pt 5 e080500m685 m686 07 4 2008 16 4 2008 © G. S. Huang 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the molecule of the title compound, [Co(C6H4NO2)2(H2O)2], the coordination environment around the CoII atom is distorted octahedral; two N and two O atoms of the pyridine-2-carboxylate ligands lie in the equatorial plane and the two water O atoms in the axial positions. In the crystal structure, intermolecular O—H⋯O hydrogen bonds link the molecules, forming a supramoleular network structure.
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Related literature
For general background, see: Desiraju (1997 ▶); Braga et al. (1998 ▶); McCann et al. (1996 ▶); Wai et al. (1990 ▶); Yaghi et al. (1996 ▶); Min & Lee (2002 ▶); Maira et al. (2001 ▶). For bond-length data, see: Allen et al. (1987 ▶).
Experimental
Crystal data
[Co(C6H4NO2)2(H2O)2]
M
r = 339.17
Monoclinic,
a = 11.7401 (3) Å
b = 8.9994 (6) Å
c = 14.9211 (3) Å
β = 105.985 (2)°
V = 1515.52 (11) Å3
Z = 4
Mo Kα radiation
μ = 1.16 mm−1
T = 273 (2) K
0.24 × 0.18 × 0.08 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Sheldrick, 1996 ▶) T
min = 0.770, T
max = 0.918
9384 measured reflections
2926 independent reflections
2065 reflections with I > 2σ(I)
R
int = 0.042
Refinement
R[F
2 > 2σ(F
2)] = 0.060
wR(F
2) = 0.227
S = 1.07
2926 reflections
200 parameters
6 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.83 e Å−3
Δρmin = −0.62 e Å−3
Data collection: APEX2 (Bruker, 2005 ▶); cell refinement: SAINT (Siemens, 1996 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808010507/hk2450sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808010507/hk2450Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HK2450).
We thank the Youth Program of Jinggangshan University for financial support of this work.
supplementary crystallographic
information
Comment
In the synthesis of crystal structures by design, the assembly of molecular
units in predefined arrangements is a key goal (Desiraju, 1997; Braga
et al., 1998). Due to carboxyl groups are one of the most
important classes of
biological ligands, the coordination of metal-carboxyl groups complexes are of
critical importance in biological systems, organic materials and coordination
chemistry. Recently, carboxyl groups with variable coordination modes have
been used to construct metal-organic supramolecular structures (McCann et
al., 1996; Wai et al., 1990; Yaghi et al.,
1996; Min & Lee 2002; Maira et al., 2001). We
report herein the crystal structure of the title compound, (I).
In the molecule of (I) (Fig. 1), the ligand bond lengths (Allen et al.,
1987)
and angles are within normal ranges. The two N and the two O atoms of the two
pyridine-2-carboxylato ligands in the equatorial plane around the CoII atom
form a distorted square-planar arrangement, while the distorted octahedral
coordination is completed by the two O atoms of water molecules in the axial
positions (Table 1 and Fig. 1). The Co-O bonds [average 2.154 (3) Å] are
somewhat shorter than the Co-N distances [average 2.279 (3) Å].
In the crystal structure, intermolecular O-H···O hydrogen bonds (Table 2) link
the molecules to form a supramoleular network structure (Fig. 2), in which
they may be effective in the stabilization of the structure.
Experimental
The title compound was synthesized using hydrothermal method in a 23 ml
Teflon-lined Parr bomb. Cobalt(II) chloride hexahydrate (47.6 mg, 0.2 mmol),
pyridine-2-carboxylic acid (49.2 mg, 0.4 mmol) and distilled water (6 g) were
placed into the bomb and sealed. The bomb was then heated under autogenous
pressure up to 413 K over the course of 7 d and allowed to cool at room
temperature for 24 h. Upon opening the bomb, a clear colorless solution was
decanted from small pink crystals. These crystals were washed with distilled
water followed by ethanol, and allowed to air-dry at room temperature.
Refinement
H1B and H2B (for H2O) were located in difference syntheses and refined
isotropically [O-H = 0.784 (18) and 0.771 (16) Å, Uiso(H) = 0.065 (16) and
0.035 (12) Å2]. The remaining H1A and H2A (for H2O) and aromatic H atoms
were positioned geometrically, with O-H = 0.82 Å (for H2O) and C-H =
0.93 Å for aromatic H, and constrained to ride on their parent atoms, with
Uiso(H) = xUeq(C,O), where x = 1.2 for aromatic H atoms and x = 1.5 for
all other H atoms.
Figures
Fig. 1. The molecular structure of the title molecule, with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Hydrogen bond is shown as dashed line.
Fig. 2. A packing diagram of (I). Hydrogen bonds are shown as dashed lines.
Crystal data
[Co(C6H4NO2)2(H2O)2] F000 = 692
Mr = 339.17 Dx = 1.486 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 2863 reflections
a = 11.7401 (3) Å θ = 2.6–23.8º
b = 8.9994 (6) Å µ = 1.16 mm−1
c = 14.9211 (3) Å T = 273 (2) K
β = 105.985 (2)º Plate, pink
V = 1515.52 (11) Å3 0.24 × 0.18 × 0.08 mm
Z = 4
Data collection
Bruker APEXII area-detector diffractometer 2926 independent reflections
Radiation source: fine-focus sealed tube 2065 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.042
T = 273(2) K θmax = 26.0º
φ and ω scans θmin = 2.0º
Absorption correction: multi-scan(SADABS; Sheldrick, 1996) h = −14→14
Tmin = 0.770, Tmax = 0.918 k = −11→11
9384 measured reflections l = −18→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.060 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.227 w = 1/[σ2(Fo2) + (0.152P)2 + 0.1958P] where P = (Fo2 + 2Fc2)/3
S = 1.07 (Δ/σ)max < 0.001
2926 reflections Δρmax = 0.83 e Å−3
200 parameters Δρmin = −0.62 e Å−3
6 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s.
planes) are estimated using the full covariance matrix. The cell e.s.d.'s
are taken into account individually in the estimation of e.s.d.'s in
distances, angles and torsion angles; correlations between e.s.d.'s in cell
parameters are only used when they are defined by crystal symmetry. An
approximate (isotropic) treatment of cell e.s.d.'s is used for estimating
e.s.d.'s involving l.s. planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and
goodness of fit S are based on F2, conventional R-factors R are based
on F, with F set to zero for negative F2. The threshold expression of
F2 > 2sigma(F2) is used only for calculating R-factors(gt) etc. and is
not relevant to the choice of reflections for refinement. R-factors based
on F2 are statistically about twice as large as those based on F, and R-
factors based on ALL data will be even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Co1 0.74558 (5) 0.85905 (7) 0.62775 (4) 0.0500 (3)
O1 0.8193 (3) 0.7460 (4) 0.7578 (2) 0.0528 (8)
H1A 0.7662 0.7036 0.7737 0.079*
H1B 0.8854 (17) 0.719 (5) 0.773 (3) 0.065 (16)*
O2 0.5921 (3) 0.8896 (4) 0.6792 (3) 0.0562 (9)
H2A 0.5826 0.8156 0.7083 0.084*
H2B 0.546 (3) 0.953 (3) 0.673 (3) 0.035 (12)*
O3 0.6510 (3) 0.9270 (3) 0.48901 (19) 0.0497 (8)
O4 0.5390 (3) 0.8577 (3) 0.3503 (2) 0.0560 (9)
O5 0.8183 (3) 1.0767 (4) 0.6673 (2) 0.0512 (8)
O6 0.9716 (3) 1.2287 (4) 0.6898 (3) 0.0724 (11)
N1 0.9333 (3) 0.8598 (4) 0.6113 (3) 0.0504 (9)
N2 0.6905 (3) 0.6452 (4) 0.5461 (2) 0.0406 (8)
C1 0.6032 (3) 0.8310 (5) 0.4295 (3) 0.0423 (9)
C2 0.6267 (3) 0.6693 (5) 0.4583 (3) 0.0392 (9)
C3 0.5838 (4) 0.5535 (5) 0.3968 (3) 0.0533 (11)
H3 0.5424 0.5722 0.3351 0.064*
C4 0.6040 (4) 0.4115 (6) 0.4293 (3) 0.0552 (11)
H4 0.5727 0.3319 0.3906 0.066*
C5 0.6700 (4) 0.3862 (5) 0.5184 (4) 0.0552 (12)
H5 0.6864 0.2897 0.5404 0.066*
C6 0.7120 (4) 0.5054 (5) 0.5755 (3) 0.0495 (10)
H6 0.7569 0.4880 0.6364 0.059*
C7 0.9224 (4) 1.1095 (5) 0.6665 (3) 0.0513 (11)
C8 0.9904 (4) 0.9869 (5) 0.6340 (3) 0.0489 (11)
C9 1.1060 (4) 1.0058 (7) 0.6312 (4) 0.0704 (15)
H9 1.1443 1.0961 0.6485 0.085*
C10 1.1635 (6) 0.8917 (7) 0.6029 (6) 0.091 (2)
H10 1.2403 0.9039 0.5983 0.109*
C11 1.1064 (6) 0.7581 (8) 0.5811 (6) 0.102 (2)
H11 1.1445 0.6768 0.5639 0.123*
C12 0.9906 (5) 0.7476 (6) 0.5856 (5) 0.0772 (17)
H12 0.9510 0.6578 0.5698 0.093*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Co1 0.0509 (5) 0.0450 (5) 0.0472 (5) −0.0007 (3) 0.0018 (3) −0.0005 (2)
O1 0.0397 (16) 0.060 (2) 0.0502 (18) 0.0013 (15) −0.0015 (13) 0.0150 (14)
O2 0.056 (2) 0.0450 (19) 0.070 (2) 0.0125 (16) 0.0209 (17) 0.0143 (16)
O3 0.0515 (17) 0.0412 (18) 0.0466 (17) −0.0020 (13) −0.0031 (13) 0.0045 (13)
O4 0.0550 (19) 0.052 (2) 0.0467 (18) 0.0079 (14) −0.0106 (14) 0.0083 (13)
O5 0.0467 (17) 0.0448 (18) 0.0613 (19) −0.0019 (14) 0.0135 (14) −0.0096 (15)
O6 0.078 (2) 0.059 (2) 0.089 (3) −0.0299 (19) 0.037 (2) −0.031 (2)
N1 0.049 (2) 0.046 (2) 0.059 (2) −0.0010 (17) 0.0189 (18) −0.0046 (17)
N2 0.0418 (18) 0.037 (2) 0.0366 (18) −0.0012 (14) 0.0011 (14) 0.0024 (13)
C1 0.0331 (19) 0.049 (2) 0.040 (2) 0.0023 (17) 0.0035 (16) 0.0025 (18)
C2 0.0334 (19) 0.041 (2) 0.039 (2) −0.0012 (16) 0.0032 (15) −0.0034 (17)
C3 0.051 (2) 0.053 (3) 0.046 (2) 0.005 (2) −0.0033 (18) −0.008 (2)
C4 0.056 (3) 0.044 (3) 0.061 (3) 0.002 (2) 0.008 (2) −0.012 (2)
C5 0.064 (3) 0.041 (3) 0.064 (3) 0.001 (2) 0.023 (2) −0.001 (2)
C6 0.057 (2) 0.044 (3) 0.044 (2) 0.001 (2) 0.0068 (19) 0.0031 (19)
C7 0.059 (3) 0.055 (3) 0.039 (2) −0.011 (2) 0.0124 (19) −0.0056 (19)
C8 0.050 (2) 0.055 (3) 0.042 (2) 0.006 (2) 0.0146 (18) 0.0039 (19)
C9 0.058 (3) 0.073 (4) 0.086 (4) −0.008 (3) 0.027 (3) −0.001 (3)
C10 0.069 (4) 0.085 (5) 0.134 (6) −0.001 (4) 0.054 (4) 0.006 (4)
C11 0.085 (4) 0.079 (5) 0.165 (8) 0.016 (4) 0.074 (5) −0.007 (5)
C12 0.076 (4) 0.052 (3) 0.114 (5) −0.001 (3) 0.043 (3) −0.012 (3)
Geometric parameters (Å, °)
Co1—O1 2.150 (3) C1—C2 1.521 (6)
Co1—O2 2.162 (3) C2—C3 1.389 (6)
Co1—O3 2.151 (3) C3—C4 1.365 (7)
Co1—O5 2.153 (3) C3—H3 0.9300
Co1—N1 2.284 (4) C4—C5 1.361 (7)
Co1—N2 2.274 (3) C4—H4 0.9300
O1—H1A 0.8200 C5—C6 1.374 (6)
O1—H1B 0.784 (18) C5—H5 0.9300
O2—H2A 0.8200 C6—H6 0.9300
O2—H2B 0.771 (16) C7—C8 1.517 (6)
O3—C1 1.255 (5) C8—C9 1.380 (7)
O4—C1 1.238 (5) C9—C10 1.359 (8)
O5—C7 1.261 (5) C9—H9 0.9300
O6—C7 1.222 (5) C10—C11 1.371 (9)
N1—C8 1.321 (6) C10—H10 0.9300
N1—C12 1.327 (6) C11—C12 1.383 (8)
N2—C6 1.334 (5) C11—H11 0.9300
N2—C2 1.335 (5) C12—H12 0.9300
O1—Co1—O2 84.68 (13) N2—C2—C1 116.2 (3)
O1—Co1—O3 167.36 (12) C3—C2—C1 121.8 (4)
O1—Co1—O5 98.78 (12) C4—C3—C2 118.2 (4)
O2—Co1—O3 92.63 (13) C4—C3—H3 120.9
O2—Co1—O5 95.35 (13) C2—C3—H3 120.9
O3—Co1—O5 93.75 (12) C5—C4—C3 120.0 (4)
O1—Co1—N1 86.44 (14) C5—C4—H4 120.0
O2—Co1—N1 163.96 (15) C3—C4—H4 120.0
O3—Co1—N1 98.83 (14) C4—C5—C6 119.1 (5)
O5—Co1—N1 72.84 (12) C4—C5—H5 120.5
O1—Co1—N2 93.86 (12) C6—C5—H5 120.5
O2—Co1—N2 98.99 (14) N2—C6—C5 122.0 (4)
O3—Co1—N2 74.33 (12) N2—C6—H6 119.0
O5—Co1—N2 161.68 (13) C5—C6—H6 119.0
N1—Co1—N2 94.91 (13) O6—C7—O5 125.9 (5)
Co1—O1—H1A 109.5 O6—C7—C8 118.7 (4)
Co1—O1—H1B 122 (3) O5—C7—C8 115.4 (4)
H1A—O1—H1B 123.0 N1—C8—C9 122.2 (4)
Co1—O2—H2A 109.5 N1—C8—C7 116.0 (4)
Co1—O2—H2B 132 (2) C9—C8—C7 121.9 (5)
H2A—O2—H2B 118.0 C10—C9—C8 119.5 (5)
C1—O3—Co1 119.8 (3) C10—C9—H9 120.2
C7—O5—Co1 121.5 (3) C8—C9—H9 120.2
C8—N1—C12 118.1 (4) C9—C10—C11 119.0 (5)
C8—N1—Co1 114.3 (3) C9—C10—H10 120.5
C12—N1—Co1 127.5 (3) C11—C10—H10 120.5
C6—N2—C2 118.6 (4) C10—C11—C12 118.1 (6)
C6—N2—Co1 128.5 (3) C10—C11—H11 120.9
C2—N2—Co1 112.8 (3) C12—C11—H11 120.9
O4—C1—O3 125.3 (4) N1—C12—C11 123.0 (6)
O4—C1—C2 118.2 (4) N1—C12—H12 118.5
O3—C1—C2 116.6 (4) C11—C12—H12 118.5
N2—C2—C3 122.0 (4)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O1—H1B···O4i 0.784 (18) 1.98 (3) 2.733 (4) 161 (4)
O1—H1A···O5ii 0.82 1.88 2.679 (4) 164
O2—H2B···O4iii 0.771 (16) 1.959 (16) 2.712 (4) 166 (3)
O2—H2A···O6ii 0.82 1.96 2.699 (5) 149
Symmetry codes: (i) x+1/2, −y+3/2, z+1/2; (ii) −x+3/2, y−1/2, −z+3/2; (iii) −x+1, −y+2, −z+1.
Table 1 Selected geometric parameters (Å, °)
Co1—O1 2.150 (3)
Co1—O2 2.162 (3)
Co1—O3 2.151 (3)
Co1—O5 2.153 (3)
Co1—N1 2.284 (4)
Co1—N2 2.274 (3)
O1—Co1—O2 84.68 (13)
O1—Co1—O3 167.36 (12)
O1—Co1—O5 98.78 (12)
O2—Co1—O3 92.63 (13)
O2—Co1—O5 95.35 (13)
O3—Co1—O5 93.75 (12)
O1—Co1—N1 86.44 (14)
O2—Co1—N1 163.96 (15)
O3—Co1—N1 98.83 (14)
O5—Co1—N1 72.84 (12)
O1—Co1—N2 93.86 (12)
O2—Co1—N2 98.99 (14)
O3—Co1—N2 74.33 (12)
O5—Co1—N2 161.68 (13)
N1—Co1—N2 94.91 (13)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O1—H1B⋯O4i 0.784 (18) 1.98 (3) 2.733 (4) 161 (4)
O1—H1A⋯O5ii 0.82 1.88 2.679 (4) 164
O2—H2B⋯O4iii 0.771 (16) 1.959 (16) 2.712 (4) 166 (3)
O2—H2A⋯O6ii 0.82 1.96 2.699 (5) 149
Symmetry codes: (i) ; (ii) ; (iii) .
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References
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Yaghi, O. M., Li, H. & Groy, T. L. (1996). J. Am. Chem. Soc.118, 9096–9101. | 21202222 | PMC2961307 | CC BY | 2021-01-04 18:55:30 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Apr 18; 64(Pt 5):m685-m686 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hg240110.1107/S1600536808013755ACSEBHS1600536808013755Metal-Organic Papers{μ-6,6′-Diethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato}trinitratoholmium(III)nickel(II) [HoNi(C20H22N2O4)(NO3)3]Xiao Yi-An aFu Xiang-Kai bSui Yan c*Wu Qing cXiong Shao-Hui ca College of Life Sciences, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of Chinab College of Chemistry & Chemical Engineering, Southwest University, 400715 Beibei, Chongqing, People’s Republic of Chinac JiangXi Province Key Laboratory of Coordination Chemistry, College of Chemistry & Chemical Engineering, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of ChinaCorrespondence e-mail: [email protected] 6 2008 14 5 2008 14 5 2008 64 Pt 6 e080600m806 m807 06 5 2008 08 5 2008 © Xiao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title heteronuclear NiII–HoIII complex (systematic name: {μ-6,6′-diethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato-1κ4
O
1,O
1′,O
6,O
6′:2κ4
O
1,N,N′,O
1′}trinitrato-1κ6
O,O′-holmium(III)nickel(II)), [HoNi(C20H22N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bis(3-ethoxysalicylidene)ethylenediamine (H2
L), the Ho and Ni atoms are doubly bridged by two phenolate O atoms of the Schiff base ligand. The coordination of Ni is square-planar with the donor centers of two imine N atoms and two phenolate O atoms. The holmium(III) center has a tenfold coordination environment of O atoms, involving the phenolate O atoms, two ethoxy O atoms and two O atoms each from the three nitrates. Weak C—H⋯O and O⋯Ni [3.383 (4) Å] interactions generate a two-dimensional zigzag sheet.
==== Body
Related literature
For related literature, see: Baggio et al. (2000 ▶); Caravan et al. (1999 ▶); Edder et al. (2000 ▶); Knoer et al. (2005 ▶); Sui et al. (2006 ▶).
Experimental
Crystal data
[HoNi(C20H22N2O4)(NO3)3]
M
r = 764.07
Orthorhombic,
a = 8.5825 (8) Å
b = 13.7028 (14) Å
c = 21.203 (2) Å
V = 2493.6 (4) Å3
Z = 4
Mo Kα radiation
μ = 3.98 mm−1
T = 293 (2) K
0.17 × 0.16 × 0.13 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2004 ▶) T
min = 0.559, T
max = 0.625
18705 measured reflections
5970 independent reflections
4299 reflections with I > 2σ(I)
R
int = 0.055
Refinement
R[F
2 > 2σ(F
2)] = 0.043
wR(F
2) = 0.113
S = 1.04
5970 reflections
364 parameters
H-atom parameters constrained
Δρmax = 2.00 e Å−3
Δρmin = −0.61 e Å−3
Absolute structure: Flack (1983 ▶), 2455 Friedel pairs
Flack parameter: −0.003 (18)
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: APEX2; data reduction: APEX2; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: APEX2; software used to prepare material for publication: APEX2 and publCIF (Westrip, 2008 ▶).
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808013755/hg2401sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808013755/hg2401Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HG2401).
We gratefully acknowledge financial support from the Department of Education, JiangXi Province (No. 2007317) and the Natural Science Foundation of JiangXi Province (No. 2007GZH1667).
supplementary crystallographic
information
Comment
The potential applications of trivalent lanthanide complexes as contrast agent
for magnetic resonance imaging and stains for fluorescence imaging have
prompted considerable interest in the preparation, magnetic and optical
properties of 3 d-4f hetorometallic dinuclear complexes (Baggio et al.,
2000; Caravan et al., 1999; Edder et al.,
2000; Knoer et
al., 2005). As part of our investigations into the structure and
applications of 3 d-4f hetorometallic Schiff base complexes(Sui et al.
2006), we report here the synthesis and X-ray crystal structure
analysis of
the title complex, (I), a new NiII—HoIII complex with salen-type Schiff
base N,N'-bis(3-ethoxysalicylidene) ethylenediamine(H2L).
Complex (I) crystallizes in the space group P212121, with nickel
and holmium doubly bridged by two phenolate O atoms provided by a salen-type
Schiff base ligand. The inner salen-type cavity is occupied by nickel(II),
while holmium(III) is present in the open and larger portion of the
dinucleating compartmental Schiff base ligand. The dihedral angles between the
mean planes of Ni1/O1/O2 and Ho1/O1/O2 is 6.97 (26)° suggesting that the
bridging moiety is almost planar; the deviation of atoms from the least
squares Ni1/O1/O2/Ho1 plane being -0.0583 (2)Å for Ni, -0.0397 (3)Å for Ho,
0.0483 (2)Å for O1 and 0.0497 (2)Å for O2.
The holmium(III) center in (I) has a decacoordination environment of O atoms.
In addition to the phenolate ligands, two ethoxy O atoms coordinate to this
metal center, two O atoms from each of the three nitrates chelate to holmium
to complete the decacoordination. The three kinds of Ho—O bond distances are
significantly different, the shortest being the Ho—O(phenolate) and longest
being the Ho—O(ethoxy) separations.
The coordination of nickel(II) is approximately square planar. The donor
centers are alternatively above and below the mean N2O2 plane with an
average deviation from the plane of 0.0698 (2) Å, while Ni1 is 0.0022 (2)Å
above this square plane.
Adjacent molecules are held together by weak interactions (O13···Ni1=3.383 (4) Å, C10—H10A···O10i=3.301 (9), and C12—H12···O10ii=3.286 (8); symmetry
codes:(i)x - 1, y, z; (ii)-x, 1/2 + y, 1/2
- z.) these link the molecules into a two-dimensional zigzag sheet(Fig
2).
Experimental
H2L was prepared by the 2:1 condensation of 3-ethoxysalicylaldehyde and
ethylenediamine in methanol. Complex (I) was obtained by the treatment of
nickel(II) acetate tetrahydrate (0.217 g, 1 mmol) with H2L(0.356 g, 1 mmol)
in methanol solution (80 ml) under reflux for 3 h and then for another 3 h
after the addition of holmium(III) nitrate hexahydrate (0.459 g, 1 mmol). The
reaction mixture was cooled and the resulting precipitate was filtered off,
washed with diethyl ether and dried in vacuo. Single crystals of (I)
suitable for X-ray analysis were obtained by slow evaporation at room
temperature of a methanol solution. Analysis calculated for
C20H22HoN5NiO13: C 31.44, H 2.90, Ho 21.59, N 9.17, Ni 7.68%; found: C
31.85, H 2.95, Ho 21.55, N 9.24, Ni 7.78. IR(KBr, cm-1): 1645(C=N),
1385,1491(nitrate).
Refinement
The H atoms were positioned geometrically and treated as riding on their parent
atoms, with C—H distances of 0.97 (methylene) and 0.96 Å (methyl), and
with Uiso(H) = 1.5Ueq(C) for methyl H atoms and
1.2Ueq(C) for other H atoms.
Figures
Fig. 1. The molecular structure of (I), showing 30% probability displacement ellipsoids.
Fig. 2. The packing diagram of (I), viewed along the b axis; hydrogen bonds are shown as dashed lines.
Crystal data
[HoNi(C20H22N2O4)(NO3)3] F000 = 1504
Mr = 764.07 Dx = 2.035 Mg m−3
Orthorhombic, P212121 Mo Kα radiation λ = 0.71073 Å
Hall symbol: P 2ac 2ab Cell parameters from 6504 reflections
a = 8.5825 (8) Å θ = 1.9–28.3º
b = 13.7028 (14) Å µ = 3.98 mm−1
c = 21.203 (2) Å T = 293 (2) K
V = 2493.6 (4) Å3 Block, red
Z = 4 0.17 × 0.16 × 0.13 mm
Data collection
Bruker APEXII area-detector diffractometer 5970 independent reflections
Radiation source: fine-focus sealed tube 4299 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.055
T = 293(2) K θmax = 28.3º
φ and ω scans θmin = 1.9º
Absorption correction: multi-scan(SADABS; Bruker, 2004) h = −11→11
Tmin = 0.559, Tmax = 0.626 k = −17→18
18705 measured reflections l = −27→27
Refinement
Refinement on F2 H-atom parameters constrained
Least-squares matrix: full w = 1/[σ2(Fo2) + (0.0473P)2] where P = (Fo2 + 2Fc2)/3
R[F2 > 2σ(F2)] = 0.043 (Δ/σ)max = 0.001
wR(F2) = 0.113 Δρmax = 2.00 e Å−3
S = 1.04 Δρmin = −0.61 e Å−3
5970 reflections Extinction correction: SHELXL97 (Sheldrick, 2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
364 parameters Extinction coefficient: 0.0058 (4)
Primary atom site location: structure-invariant direct methods Absolute structure: Flack (1983), 2455 Friedel pairs
Secondary atom site location: difference Fourier map Flack parameter: −0.003 (18)
Hydrogen site location: inferred from neighbouring sites
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
C5 −0.1509 (7) 0.3317 (5) 0.3044 (3) 0.0427 (15)
Ho1 0.25263 (3) 0.49982 (2) 0.400886 (12) 0.04143 (11)
Ni1 −0.06665 (9) 0.56000 (6) 0.31450 (4) 0.03838 (19)
O2 0.0323 (4) 0.4496 (3) 0.34434 (19) 0.0399 (10)
O1 0.0808 (5) 0.6237 (3) 0.36270 (19) 0.0411 (10)
N1 −0.1998 (6) 0.4934 (4) 0.2621 (2) 0.0434 (12)
N2 −0.1729 (6) 0.6703 (4) 0.2905 (3) 0.0423 (13)
N3 0.1352 (8) 0.5100 (5) 0.5279 (3) 0.0588 (16)
O6 0.0496 (6) 0.5277 (4) 0.4813 (2) 0.0647 (14)
C12 −0.1460 (7) 0.7577 (5) 0.3087 (3) 0.0470 (16)
H12 −0.2126 0.8065 0.2945 0.056*
O3 0.1551 (5) 0.3283 (3) 0.4234 (2) 0.0422 (10)
C18 0.0875 (7) 0.7189 (4) 0.3736 (3) 0.0386 (13)
O4 0.3135 (5) 0.6799 (3) 0.4295 (2) 0.0473 (11)
O5 0.2787 (6) 0.4948 (4) 0.5150 (2) 0.0609 (13)
C15 0.1149 (8) 0.9161 (5) 0.4034 (4) 0.0556 (18)
H15 0.1222 0.9816 0.4147 0.067*
C4 −0.0255 (7) 0.3594 (4) 0.3442 (3) 0.0387 (14)
O7 0.0834 (8) 0.5055 (4) 0.5815 (3) 0.0841 (17)
C19 0.4500 (8) 0.7097 (5) 0.4648 (3) 0.0533 (17)
H19A 0.4917 0.6538 0.4872 0.064*
H19B 0.4203 0.7583 0.4957 0.064*
C1 0.3381 (9) 0.1918 (6) 0.4400 (4) 0.059 (2)
H1A 0.2848 0.1544 0.4084 0.089*
H1B 0.3793 0.1487 0.4716 0.089*
H1C 0.4219 0.2275 0.4208 0.089*
C17 0.2135 (7) 0.7529 (4) 0.4098 (3) 0.0422 (15)
C13 −0.0195 (8) 0.7860 (4) 0.3498 (3) 0.0434 (15)
C14 −0.0046 (9) 0.8868 (5) 0.3667 (3) 0.0515 (17)
H14 −0.0773 0.9320 0.3524 0.062*
C2 0.2241 (8) 0.2632 (5) 0.4706 (3) 0.0487 (16)
H2A 0.1421 0.2270 0.4917 0.058*
H2B 0.2784 0.3017 0.5021 0.058*
O9 0.3184 (6) 0.5422 (4) 0.2897 (2) 0.0581 (13)
N4 0.3530 (6) 0.4577 (5) 0.2744 (3) 0.0517 (16)
O8 0.3482 (6) 0.3918 (3) 0.3167 (3) 0.0573 (12)
C9 −0.2254 (8) 0.3989 (5) 0.2623 (3) 0.0447 (15)
H9 −0.2964 0.3741 0.2333 0.054*
O12 0.5289 (6) 0.5328 (4) 0.3902 (3) 0.0593 (13)
N5 0.5831 (8) 0.4621 (5) 0.4212 (3) 0.0605 (16)
C16 0.2267 (9) 0.8516 (5) 0.4248 (3) 0.0510 (17)
H16 0.3099 0.8738 0.4489 0.061*
C6 −0.2073 (8) 0.2359 (5) 0.3070 (3) 0.0506 (17)
H6 −0.2863 0.2162 0.2798 0.061*
C3 0.0358 (7) 0.2907 (5) 0.3862 (3) 0.0417 (14)
C11 −0.3100 (8) 0.6507 (5) 0.2514 (3) 0.0516 (17)
H11A −0.3272 0.7036 0.2218 0.062*
H11B −0.4022 0.6434 0.2774 0.062*
C10 −0.2754 (8) 0.5578 (5) 0.2171 (3) 0.0513 (16)
H10A −0.3710 0.5285 0.2016 0.062*
H10B −0.2073 0.5702 0.1815 0.062*
O11 0.4839 (6) 0.4045 (4) 0.4421 (3) 0.0728 (17)
C20 0.5726 (10) 0.7506 (6) 0.4231 (4) 0.071 (2)
H20A 0.6009 0.7031 0.3919 0.106*
H20B 0.6624 0.7672 0.4478 0.106*
H20C 0.5336 0.8081 0.4026 0.106*
C8 −0.0229 (7) 0.1970 (5) 0.3887 (4) 0.0484 (16)
H8 0.0192 0.1514 0.4164 0.058*
O10 0.3939 (7) 0.4348 (5) 0.2204 (2) 0.0808 (18)
O13 0.7234 (6) 0.4536 (7) 0.4307 (3) 0.099 (2)
C7 −0.1465 (9) 0.1717 (5) 0.3492 (3) 0.0563 (19)
H7 −0.1884 0.1092 0.3518 0.068*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
C5 0.040 (3) 0.041 (3) 0.048 (4) −0.006 (3) −0.003 (3) −0.002 (3)
Ho1 0.04247 (16) 0.03543 (16) 0.04639 (17) −0.00007 (18) −0.00389 (12) 0.00217 (13)
Ni1 0.0375 (4) 0.0340 (4) 0.0436 (4) −0.0005 (3) −0.0050 (4) 0.0047 (4)
O2 0.038 (2) 0.033 (2) 0.049 (2) −0.0065 (18) −0.0099 (18) 0.004 (2)
O1 0.045 (2) 0.026 (2) 0.052 (2) 0.0024 (19) −0.014 (2) 0.004 (2)
N1 0.037 (2) 0.051 (3) 0.043 (3) −0.005 (3) −0.002 (2) 0.002 (3)
N2 0.040 (3) 0.038 (3) 0.049 (3) −0.001 (2) 0.001 (2) 0.010 (3)
N3 0.085 (5) 0.037 (3) 0.054 (3) 0.003 (3) 0.007 (3) −0.005 (3)
O6 0.066 (3) 0.076 (4) 0.052 (3) 0.003 (3) −0.001 (3) −0.001 (3)
C12 0.041 (3) 0.046 (4) 0.054 (4) 0.011 (3) 0.009 (3) 0.018 (4)
O3 0.045 (2) 0.027 (2) 0.054 (3) −0.0026 (19) −0.004 (2) 0.008 (2)
C18 0.039 (3) 0.034 (3) 0.043 (3) −0.002 (3) 0.000 (3) 0.006 (3)
O4 0.049 (2) 0.037 (2) 0.056 (3) −0.004 (2) −0.016 (2) 0.000 (2)
O5 0.070 (3) 0.061 (3) 0.051 (3) 0.005 (3) −0.008 (2) 0.002 (3)
C15 0.063 (4) 0.034 (4) 0.069 (5) −0.007 (3) 0.008 (4) 0.003 (4)
C4 0.041 (3) 0.036 (3) 0.039 (3) −0.004 (3) 0.001 (3) −0.001 (3)
O7 0.120 (5) 0.077 (4) 0.055 (3) 0.004 (4) 0.013 (4) 0.003 (3)
C19 0.048 (4) 0.052 (4) 0.060 (4) −0.002 (3) −0.010 (4) −0.006 (3)
C1 0.057 (4) 0.044 (4) 0.077 (5) 0.010 (3) −0.016 (4) −0.002 (4)
C17 0.053 (4) 0.031 (3) 0.043 (3) 0.004 (3) 0.010 (3) 0.003 (3)
C13 0.052 (4) 0.032 (3) 0.046 (3) 0.001 (3) 0.005 (3) 0.004 (3)
C14 0.061 (4) 0.032 (3) 0.062 (4) 0.011 (3) 0.001 (3) 0.008 (3)
C2 0.061 (4) 0.042 (4) 0.043 (3) 0.002 (3) −0.010 (3) 0.008 (3)
O9 0.069 (3) 0.051 (3) 0.055 (3) 0.002 (3) −0.004 (3) 0.012 (3)
N4 0.043 (3) 0.063 (4) 0.049 (3) −0.018 (3) −0.006 (3) −0.003 (3)
O8 0.064 (3) 0.051 (3) 0.056 (3) −0.007 (2) 0.001 (3) −0.009 (3)
C9 0.047 (4) 0.041 (3) 0.046 (3) −0.006 (3) −0.003 (3) 0.000 (3)
O12 0.053 (3) 0.052 (3) 0.073 (3) 0.000 (2) 0.000 (3) 0.005 (3)
N5 0.055 (4) 0.068 (4) 0.059 (3) −0.002 (3) 0.002 (3) 0.000 (3)
C16 0.054 (4) 0.047 (4) 0.053 (3) −0.011 (3) 0.005 (3) −0.001 (3)
C6 0.047 (4) 0.041 (4) 0.064 (4) −0.006 (3) 0.002 (4) −0.007 (4)
C3 0.042 (3) 0.034 (3) 0.049 (3) −0.001 (3) 0.001 (3) 0.001 (3)
C11 0.046 (4) 0.056 (5) 0.053 (4) 0.005 (3) −0.008 (3) 0.011 (4)
C10 0.049 (4) 0.051 (4) 0.054 (3) −0.007 (3) −0.019 (3) 0.004 (3)
O11 0.054 (3) 0.081 (4) 0.083 (4) 0.005 (3) −0.003 (3) 0.029 (3)
C20 0.066 (5) 0.058 (5) 0.087 (5) −0.016 (5) 0.006 (5) −0.012 (5)
C8 0.047 (4) 0.034 (4) 0.064 (4) 0.001 (3) 0.005 (3) 0.008 (3)
O10 0.080 (4) 0.115 (5) 0.048 (3) −0.034 (4) 0.013 (3) −0.018 (4)
O13 0.044 (3) 0.138 (6) 0.114 (5) 0.010 (4) −0.012 (3) 0.022 (5)
C7 0.063 (4) 0.034 (4) 0.072 (5) −0.013 (3) 0.000 (4) −0.002 (4)
Geometric parameters (Å, °)
Ho1—O1 2.390 (4) C15—H15 0.9300
Ho1—O2 2.343 (4) C4—C3 1.398 (9)
Ho1—O3 2.540 (4) C19—C20 1.484 (10)
Ho1—O4 2.594 (4) C19—H19A 0.9700
Ho1—O5 2.430 (5) C19—H19B 0.9700
Ho1—O6 2.468 (5) C1—C2 1.529 (10)
Ho1—O8 2.460 (5) C1—H1A 0.9600
Ho1—O9 2.492 (5) C1—H1B 0.9600
Ho1—O11 2.531 (5) C1—H1C 0.9600
Ho1—O12 2.425 (5) C17—C16 1.394 (9)
Ni1—O1 1.846 (4) C13—C14 1.433 (9)
Ni1—O2 1.847 (4) C14—H14 0.9300
Ni1—N1 1.837 (5) C2—H2A 0.9700
Ni1—N2 1.837 (5) C2—H2B 0.9700
C5—C6 1.400 (9) O9—N4 1.239 (8)
C5—C4 1.419 (8) N4—O10 1.238 (7)
C5—C9 1.433 (9) N4—O8 1.272 (8)
O2—C4 1.332 (7) C9—H9 0.9300
O1—C18 1.327 (7) O12—N5 1.259 (8)
N1—C9 1.313 (8) N5—O13 1.227 (8)
N1—C10 1.453 (8) N5—O11 1.242 (8)
N2—C12 1.280 (8) C16—H16 0.9300
N2—C11 1.465 (9) C6—C7 1.359 (10)
N3—O7 1.221 (7) C6—H6 0.9300
N3—O6 1.255 (8) C3—C8 1.380 (9)
N3—O5 1.279 (8) C11—C10 1.496 (10)
C12—C13 1.445 (9) C11—H11A 0.9700
C12—H12 0.9300 C11—H11B 0.9700
O3—C3 1.392 (7) C10—H10A 0.9700
O3—C2 1.466 (7) C10—H10B 0.9700
C18—C13 1.393 (8) C20—H20A 0.9600
C18—C17 1.406 (9) C20—H20B 0.9600
O4—C17 1.383 (7) C20—H20C 0.9600
O4—C19 1.449 (8) C8—C7 1.396 (10)
C15—C14 1.348 (10) C8—H8 0.9300
C15—C16 1.382 (10) C7—H7 0.9300
C6—C5—C4 119.3 (6) C19—O4—Ho1 123.5 (4)
C6—C5—C9 118.2 (6) N3—O5—Ho1 96.9 (4)
C4—C5—C9 122.4 (6) C14—C15—C16 121.8 (6)
O2—Ho1—O1 62.44 (14) C14—C15—H15 119.1
O2—Ho1—O12 142.78 (15) C16—C15—H15 119.1
O1—Ho1—O12 116.07 (17) O2—C4—C3 118.9 (5)
O2—Ho1—O5 125.12 (15) O2—C4—C5 122.1 (6)
O1—Ho1—O5 114.48 (17) C3—C4—C5 118.9 (5)
O12—Ho1—O5 90.49 (18) O4—C19—C20 111.9 (6)
O2—Ho1—O8 73.78 (15) O4—C19—H19A 109.2
O1—Ho1—O8 112.76 (15) C20—C19—H19A 109.2
O12—Ho1—O8 73.62 (17) O4—C19—H19B 109.2
O5—Ho1—O8 132.44 (19) C20—C19—H19B 109.2
O2—Ho1—O6 80.15 (16) H19A—C19—H19B 107.9
O1—Ho1—O6 71.83 (16) C2—C1—H1A 109.5
O12—Ho1—O6 136.61 (17) C2—C1—H1B 109.5
O5—Ho1—O6 51.82 (17) H1A—C1—H1B 109.5
O8—Ho1—O6 146.11 (17) C2—C1—H1C 109.5
O2—Ho1—O9 76.56 (16) H1A—C1—H1C 109.5
O1—Ho1—O9 69.75 (16) H1B—C1—H1C 109.5
O12—Ho1—O9 69.29 (18) O4—C17—C16 125.6 (6)
O5—Ho1—O9 157.98 (18) O4—C17—C18 113.8 (5)
O8—Ho1—O9 51.54 (17) C16—C17—C18 120.6 (6)
O6—Ho1—O9 141.01 (17) C18—C13—C14 119.1 (6)
O2—Ho1—O11 131.15 (18) C18—C13—C12 122.4 (6)
O1—Ho1—O11 165.27 (17) C14—C13—C12 118.5 (6)
O12—Ho1—O11 50.32 (17) C15—C14—C13 119.9 (6)
O5—Ho1—O11 64.50 (19) C15—C14—H14 120.0
O8—Ho1—O11 71.28 (19) C13—C14—H14 120.0
O6—Ho1—O11 113.28 (17) O3—C2—C1 111.0 (5)
O9—Ho1—O11 105.60 (18) O3—C2—H2A 109.4
O2—Ho1—O3 63.79 (14) C1—C2—H2A 109.4
O1—Ho1—O3 121.17 (14) O3—C2—H2B 109.4
O12—Ho1—O3 120.80 (16) C1—C2—H2B 109.4
O5—Ho1—O3 79.44 (17) H2A—C2—H2B 108.0
O8—Ho1—O3 71.93 (16) N4—O9—Ho1 94.8 (4)
O6—Ho1—O3 77.33 (17) O10—N4—O9 123.1 (7)
O9—Ho1—O3 117.91 (17) O10—N4—O8 118.8 (7)
O11—Ho1—O3 73.51 (16) O9—N4—O8 118.1 (6)
O2—Ho1—O4 124.19 (15) N4—O8—Ho1 95.5 (4)
O1—Ho1—O4 61.83 (14) N1—C9—C5 124.2 (6)
O12—Ho1—O4 69.37 (16) N1—C9—H9 117.9
O5—Ho1—O4 77.05 (16) C5—C9—H9 117.9
O8—Ho1—O4 132.43 (16) N5—O12—Ho1 99.7 (4)
O6—Ho1—O4 80.41 (17) O13—N5—O11 123.6 (8)
O9—Ho1—O4 87.35 (17) O13—N5—O12 121.4 (7)
O11—Ho1—O4 104.59 (17) O11—N5—O12 114.9 (6)
O3—Ho1—O4 154.49 (18) C15—C16—C17 119.3 (7)
N1—Ni1—N2 86.1 (3) C15—C16—H16 120.3
N1—Ni1—O1 175.1 (2) C17—C16—H16 120.3
N2—Ni1—O1 96.0 (2) C7—C6—C5 120.0 (6)
N1—Ni1—O2 94.9 (2) C7—C6—H6 120.0
N2—Ni1—O2 175.7 (2) C5—C6—H6 120.0
O1—Ni1—O2 83.27 (17) C8—C3—O3 126.2 (6)
C4—O2—Ni1 126.1 (4) C8—C3—C4 120.9 (6)
C4—O2—Ho1 125.1 (4) O3—C3—C4 112.9 (5)
Ni1—O2—Ho1 107.79 (18) N2—C11—C10 105.8 (5)
C18—O1—Ni1 126.2 (4) N2—C11—H11A 110.6
C18—O1—Ho1 127.8 (4) C10—C11—H11A 110.6
Ni1—O1—Ho1 105.95 (17) N2—C11—H11B 110.6
C9—N1—C10 121.8 (5) C10—C11—H11B 110.6
C9—N1—Ni1 126.3 (5) H11A—C11—H11B 108.7
C10—N1—Ni1 111.9 (4) N1—C10—C11 106.7 (5)
C12—N2—C11 119.2 (6) N1—C10—H10A 110.4
C12—N2—Ni1 126.7 (5) C11—C10—H10A 110.4
C11—N2—Ni1 113.9 (4) N1—C10—H10B 110.4
O7—N3—O6 122.0 (7) C11—C10—H10B 110.4
O7—N3—O5 122.8 (7) H10A—C10—H10B 108.6
O6—N3—O5 115.3 (6) N5—O11—Ho1 95.0 (4)
N3—O6—Ho1 95.8 (4) C19—C20—H20A 109.5
N2—C12—C13 124.7 (6) C19—C20—H20B 109.5
N2—C12—H12 117.7 H20A—C20—H20B 109.5
C13—C12—H12 117.7 C19—C20—H20C 109.5
C3—O3—C2 117.3 (5) H20A—C20—H20C 109.5
C3—O3—Ho1 118.6 (3) H20B—C20—H20C 109.5
C2—O3—Ho1 124.0 (4) C3—C8—C7 119.0 (6)
O1—C18—C13 123.9 (6) C3—C8—H8 120.5
O1—C18—C17 117.0 (5) C7—C8—H8 120.5
C13—C18—C17 119.1 (6) C6—C7—C8 121.7 (6)
C17—O4—C19 117.0 (5) C6—C7—H7 119.1
C17—O4—Ho1 119.5 (4) C8—C7—H7 119.1
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C20—H20A···O12 0.96 2.41 3.088 (9) 127
C12—H12···O10i 0.93 2.37 3.286 (8) 169
C10—H10A···O10ii 0.97 2.42 3.301 (9) 150
C2—H2B···O11 0.97 2.59 3.015 (9) 107
C1—H1C···O11 0.96 2.52 3.173 (10) 125
Symmetry codes: (i) −x, y+1/2, −z+1/2; (ii) x−1, y, z.
Table 1 Selected bond lengths (Å)
Ho1—O1 2.390 (4)
Ho1—O2 2.343 (4)
Ho1—O3 2.540 (4)
Ho1—O4 2.594 (4)
Ho1—O5 2.430 (5)
Ho1—O6 2.468 (5)
Ho1—O8 2.460 (5)
Ho1—O9 2.492 (5)
Ho1—O11 2.531 (5)
Ho1—O12 2.425 (5)
Ni1—O1 1.846 (4)
Ni1—O2 1.847 (4)
Ni1—N1 1.837 (5)
Ni1—N2 1.837 (5)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C12—H12⋯O10i 0.93 2.37 3.286 (8) 169
C10—H10A⋯O10ii 0.97 2.42 3.301 (9) 150
Symmetry codes: (i) ; (ii) .
==== Refs
References
Baggio, R., Garland, M. T., Moreno, Y., Pena, O., Perec, M. & Spodine, E. (2000). J. Chem. Soc. Dalton Trans. pp. 2061–2066.
Bruker (2004). APEX2 and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Caravan, P., Ellison, J. J., McMurry, T. J. & Lauffer, R. B. (1999). Chem. Rev.99, 2293–2352.
Edder, C., Piguet, C., Bernardinelli, G., Mareda, J., Bochet, C. G., Bunzli, J.-C. G. & Hopfgartner, G. (2000). Inorg. Chem.39, 5059–5073.
Flack, H. D. (1983). Acta Cryst. A39, 876–881.
Knoer, R., Lin, H.-H., Wei, H.-H. & Mohanta, S. (2005). Inorg. Chem.44, 3524–3536.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sui, Y., Fang, X.-N., Xiao, Y.-A., Luo, Q.-Y. & Li, M.-H. (2006). Acta Cryst. E62, m2230–m2232.
Westrip, S. P. (2008). publCIF. In preparation. | 21202492 | PMC2961363 | CC BY | 2021-01-04 18:55:37 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 May 14; 64(Pt 6):m806-m807 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography at256710.1107/S1600536808013743ACSEBHS1600536808013743Metal-Organic Papers{μ-6,6′-Dimethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato}-μ-nitrato-dinitratoholmium(III)zinc(II) [HoZn(C18H18N2O4)(NO3)3]Xiao Yi-An aSui Yan b*Yi Xiu-Guang bWu Jian-Hong bZhang Li-Ping ba College of Life Science, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of Chinab JiangXi Province Key Laboratory of Coordination Chemistry, College of Chemistry & Chemical Engineering, JingGangShan University, 343009 Ji’an, JiangXi, People’s Republic of ChinaCorrespondence e-mail: [email protected] 6 2008 14 5 2008 14 5 2008 64 Pt 6 e080600m804 m805 06 5 2008 08 5 2008 © Xiao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title heteronuclear ZnII–HoIII complex (systematic name: {μ-6,6′-dimethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato-1κ4
O
1,O
1′,O
6,O
6′:2κ4
O
1,N,N′,O
1′)-μ-nitrato-1:2κ2
O:O′-dinitrato-1κ4
O,O′-holmium(III)zinc(II)), [HoZn(C18H18N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bis(3-methoxysalicylidene)ethylenediamine (H2
L), the Ho and Zn atoms are triply bridged by two phenolate O atoms of the Schiff base ligand and one nitrate ion. The five-coordinate Zn atom is in a square-pyramidal geometry with the donor centers of two imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms. The HoIII center has a ninefold coordination environment of O atoms, involving the phenolate O atoms, two methoxy O atoms, two O atoms from two nitrate ions and one from the bridging nitrate ion. Weak intermolecular C—H⋯O interactions generate a two-dimensional double-layer structure.
==== Body
Related literature
For related literature, see: Baggio et al. (2000 ▶); Caravan et al. (1999 ▶); Edder et al. (2000 ▶); Knoer et al. (2005 ▶); Sui et al. (2006 ▶, 2007 ▶).
Experimental
Crystal data
[HoZn(C18H18N2O4)(NO3)3]
M
r = 742.67
Monoclinic,
a = 10.694 (4) Å
b = 16.481 (7) Å
c = 14.921 (6) Å
β = 99.667 (6)°
V = 2592.4 (18) Å3
Z = 4
Mo Kα radiation
μ = 4.03 mm−1
T = 293 (2) K
0.16 × 0.16 × 0.10 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2004 ▶) T
min = 0.565, T
max = 0.689
15217 measured reflections
4499 independent reflections
3377 reflections with I > 2σ(I)
R
int = 0.037
Refinement
R[F
2 > 2σ(F
2)] = 0.034
wR(F
2) = 0.093
S = 1.02
4499 reflections
345 parameters
2 restraints
H-atom parameters constrained
Δρmax = 0.60 e Å−3
Δρmin = −1.18 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: APEX2; data reduction: APEX2; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: APEX2; software used to prepare material for publication: APEX2 and publCIF (Westrip, 2008 ▶).
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808013743/at2567sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808013743/at2567Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2567).
We gratefully acknowledge financial support from the Department of Education, JiangXi Province (No. 2007317) and the Natural Science Foundation of JiangXi Province (No. 2007GZH1667).
supplementary crystallographic
information
Comment
The potential applications of trivalent lanthanide complexes as contrast agent
for magnetic resonance imaging and stains for fluorescence imaging have
prompted considerable interest in the preparation, magnetic and optical
properties of 3d-4f hetorometallic dinuclear complexes (Baggio et al.,
2000; Caravan et al., 1999; Edder et al.,
2000; Knoer et
al., 2005). As part of our investigations into the structure and
applications of 3d-4f hetorometallic Schiff base complexes (Sui et al.
2006; Sui et al. 2007), we report here the synthesis and
X-ray crystal
structure analysis of the title complex, (I), a new ZnII—HoIII complex
with salen-type Schiff base N,N'-bis(3-methoxysalicylidene)
ethylenediamine (H2L).
Complex (I) crystallizes in the space group P21/n, with zinc
and holmium triply bridged by two phenolate O atoms provided by the Schiff
base ligand and one nitrate ion. The inner salen-type cavity is occupied by
zinc(II), while holmium(III) is present in the open and larger portion of the
dinucleating compartmental Schiff base ligand.
The HoIII center has a ninefold coordination environment of O atoms,
involving the phenolate O atoms, two methoxy O atoms, two O atoms from two
nitrate ions and one from the bridging nitrate ion. The four kinds of Ho—O
bond distances are significantly different, the longest being the Ho—O
(methoxy) separations and the shortest being the Ho—O (phenolate).
The ZnII is in a square-pyramidal geometry and is five-coordinated by two
imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms.
The Zn atom is 0.6067 (4) Å below the mean N2O2 plane with an average
deviation from the plane of 0.0380 (3) Å, which construct the bottom of
square-pyramid. The Zn—O5 (bridging nitrate) separation is 1.979 (4) Å and
the angles of this Zn—O vector with the Zn—N or Zn—O bonds lie between
102.5 (4)° and 112.6 (4)°, which suggesting that the ZnII is in a slightly
distorted square-pyramidal conformation.
Adjacent molecules are held together by weak interactions [C5(H5)···O11i =
3.377 (7) Å and C10(H10A)···O13ii = 3.483 (8) Å; symmetry codes: (i) -1/2
+ x, 1/2 - y, 1/2 + z; (ii) 1 - x, -y, 2 -
z]. These link the molecules into a two-dimensional double layer
structure (Fig 2).
Experimental
H2L was prepared by the 2:1 condensation of 3-methoxysalicylaldehyde and
ethylenediamine in methanol. Complex (I) was obtained by the treatment of
zinc(II) acetate dihydrate (0.188 g, 1 mmol) with H2L (0.328 g, 1 mmol) in
methanol solution (80 ml) under reflux for 3 h and then for another 3 h after
the addition of holmium(III) nitrate hexahydrate (0.459 g, 1 mmol). The
reaction mixture was cooled and the resulting precipitate was filtered off,
washed with diethyl ether and dried in vacuo. Single crystals of (I)
suitable for X-ray analysis were obtained by slow evaporation at room
temperature of a methanol solution. Analysis calculated for
C18H18HoN5O13Zn: C 29.11 H 2.44, Ho 22.21, N 9.43, Zn 8.80%; found: C
29.20, H 2.45, Ho 22.30, N 9.50, Zn 8.90%. IR (KBr, cm-1): 1640 (C=N),
1386,1490 (nitrate).
Refinement
The H atoms were positioned geometrically and treated as riding on their parent
atoms, with C—H distances of 0.93 (aromatic), 0.97 (methylene) and 0.96 Å
(methyl), and with Uiso(H) = 1.5Ueq(C) for methyl H atoms
and 1.2Ueq(C) for other H atoms. The main directions of movement of
covalently bonded atoms N3, O5 and O6 are enforced to be the same.
Figures
Fig. 1. The molecular structure of (I), showing 30% probability displacement ellipsoids. All the H atoms on carbon have been omitted for clarity.
Fig. 2. The packing diagram of (I), viewed along the b axis; hydrogen bonds are shown as dashed lines.
Crystal data
[HoZn(C18H18N2O4)(NO3)3] F000 = 1448
Mr = 742.67 Dx = 1.903 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 5632 reflections
a = 10.694 (4) Å θ = 2.2–25.3º
b = 16.481 (7) Å µ = 4.03 mm−1
c = 14.921 (6) Å T = 293 (2) K
β = 99.667 (6)º Block, yellow
V = 2592.4 (18) Å3 0.16 × 0.16 × 0.10 mm
Z = 4
Data collection
Bruker APEXII area-detector diffractometer 4499 independent reflections
Radiation source: fine-focus sealed tube 3377 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.037
T = 293(2) K θmax = 25.0º
φ and ω scans θmin = 2.2º
Absorption correction: multi-scan(SADABS; Bruker, 2004) h = −12→12
Tmin = 0.565, Tmax = 0.689 k = −19→19
15217 measured reflections l = −17→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.034 H-atom parameters constrained
wR(F2) = 0.093 w = 1/[σ2(Fo2) + (0.0558P)2 + 0.1192P] where P = (Fo2 + 2Fc2)/3
S = 1.02 (Δ/σ)max < 0.001
4499 reflections Δρmax = 0.61 e Å−3
345 parameters Δρmin = −1.18 e Å−3
2 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Ho1 0.36187 (2) 0.109293 (15) 0.723858 (16) 0.04439 (11)
Zn1 0.21999 (6) 0.03550 (4) 0.88927 (4) 0.04303 (17)
O1 0.2906 (4) 0.1419 (2) 0.8559 (2) 0.0493 (9)
O12 0.5712 (4) 0.1290 (3) 0.8198 (3) 0.0640 (12)
O2 0.3521 (4) −0.0016 (2) 0.8158 (2) 0.0497 (9)
O4 0.4894 (3) −0.0201 (2) 0.6956 (2) 0.0495 (9)
C17 0.4119 (5) −0.0722 (3) 0.8204 (3) 0.0413 (12)
O8 0.2843 (4) 0.1975 (2) 0.5945 (2) 0.0582 (10)
N4 0.2806 (5) 0.1437 (3) 0.5329 (4) 0.0624 (14)
O6 0.1491 (4) 0.0740 (3) 0.6881 (3) 0.0613 (11)
C3 0.2935 (6) 0.3590 (4) 0.8767 (4) 0.0567 (15)
H3 0.3175 0.4036 0.8452 0.068*
N5 0.6224 (5) 0.1590 (4) 0.7564 (4) 0.0659 (15)
O5 0.0581 (3) 0.0283 (2) 0.8031 (3) 0.0543 (10)
C16 0.4896 (5) −0.0846 (3) 0.7542 (4) 0.0460 (13)
O9 0.3274 (4) 0.0751 (3) 0.5596 (3) 0.0624 (11)
C2 0.3092 (5) 0.2813 (3) 0.8465 (3) 0.0448 (13)
C12 0.4039 (5) −0.1325 (3) 0.8854 (4) 0.0457 (13)
N3 0.0556 (5) 0.0490 (3) 0.7206 (4) 0.0695 (14)
O11 0.5537 (4) 0.1634 (3) 0.6792 (3) 0.0612 (11)
O10 0.2350 (6) 0.1567 (3) 0.4552 (3) 0.107 (2)
N1 0.1978 (4) 0.0839 (3) 1.0120 (3) 0.0506 (12)
N2 0.2494 (4) −0.0675 (3) 0.9634 (3) 0.0488 (11)
C11 0.3278 (5) −0.1246 (4) 0.9578 (4) 0.0513 (15)
H11 0.3374 −0.1641 1.0030 0.062*
C7 0.2716 (5) 0.2135 (3) 0.8916 (3) 0.0428 (12)
C8 0.1985 (5) 0.1588 (4) 1.0310 (4) 0.0573 (16)
H8 0.1823 0.1728 1.0884 0.069*
C10 0.1754 (6) −0.0615 (4) 1.0385 (4) 0.0590 (16)
H10A 0.2017 −0.1037 1.0830 0.071*
H10B 0.0859 −0.0688 1.0151 0.071*
C6 0.2222 (5) 0.2248 (3) 0.9720 (4) 0.0500 (14)
C18 0.5665 (6) −0.0292 (4) 0.6251 (4) 0.0638 (17)
H18A 0.6543 −0.0328 0.6524 0.096*
H18B 0.5544 0.0168 0.5852 0.096*
H18C 0.5421 −0.0777 0.5910 0.096*
C5 0.2056 (6) 0.3045 (4) 1.0014 (4) 0.0631 (17)
H5 0.1699 0.3130 1.0533 0.076*
O13 0.7311 (5) 0.1830 (4) 0.7710 (4) 0.112 (2)
C4 0.2412 (7) 0.3697 (4) 0.9550 (4) 0.0643 (18)
H4 0.2302 0.4219 0.9762 0.077*
C15 0.5530 (6) −0.1572 (4) 0.7500 (4) 0.0569 (15)
H15 0.6023 −0.1659 0.7052 0.068*
C9 0.1972 (6) 0.0203 (4) 1.0823 (4) 0.0612 (17)
H9A 0.1307 0.0317 1.1174 0.073*
H9B 0.2777 0.0205 1.1234 0.073*
C14 0.5419 (6) −0.2169 (4) 0.8140 (5) 0.0701 (19)
H14 0.5833 −0.2662 0.8113 0.084*
C13 0.4716 (6) −0.2043 (4) 0.8802 (4) 0.0623 (17)
H13 0.4683 −0.2446 0.9235 0.075*
O7 −0.0708 (6) 0.0407 (5) 0.6557 (5) 0.142 (3)
O3 0.3629 (4) 0.2616 (2) 0.7706 (2) 0.0502 (9)
C1 0.4246 (7) 0.3260 (4) 0.7307 (4) 0.0661 (17)
H1A 0.3638 0.3673 0.7090 0.099*
H1B 0.4611 0.3051 0.6808 0.099*
H1C 0.4903 0.3486 0.7755 0.099*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Ho1 0.05102 (19) 0.04515 (17) 0.04112 (16) 0.00162 (12) 0.01960 (11) 0.00255 (11)
Zn1 0.0470 (4) 0.0447 (4) 0.0411 (3) −0.0003 (3) 0.0182 (3) 0.0028 (3)
O1 0.067 (3) 0.036 (2) 0.052 (2) −0.0004 (19) 0.034 (2) −0.0054 (18)
O12 0.050 (2) 0.091 (3) 0.053 (2) −0.010 (2) 0.0144 (19) 0.013 (2)
O2 0.064 (3) 0.042 (2) 0.051 (2) 0.0077 (19) 0.0327 (19) 0.0102 (17)
O4 0.060 (2) 0.048 (2) 0.047 (2) 0.0107 (19) 0.0264 (18) 0.0023 (18)
C17 0.035 (3) 0.037 (3) 0.051 (3) 0.003 (2) 0.006 (2) 0.000 (3)
O8 0.081 (3) 0.049 (2) 0.045 (2) 0.013 (2) 0.014 (2) 0.0020 (19)
N4 0.076 (4) 0.063 (3) 0.053 (3) 0.018 (3) 0.024 (3) 0.004 (3)
O6 0.058 (3) 0.074 (3) 0.052 (2) −0.010 (2) 0.0101 (18) 0.007 (2)
C3 0.068 (4) 0.039 (3) 0.060 (4) 0.001 (3) 0.003 (3) 0.001 (3)
N5 0.056 (4) 0.079 (4) 0.068 (4) −0.008 (3) 0.024 (3) 0.003 (3)
O5 0.047 (2) 0.055 (2) 0.064 (2) −0.0028 (19) 0.0182 (19) 0.0082 (19)
C16 0.044 (3) 0.047 (3) 0.048 (3) 0.004 (3) 0.010 (2) −0.002 (3)
O9 0.088 (3) 0.055 (3) 0.047 (2) 0.015 (2) 0.021 (2) 0.000 (2)
C2 0.053 (3) 0.035 (3) 0.045 (3) 0.003 (3) 0.004 (2) −0.001 (2)
C12 0.041 (3) 0.044 (3) 0.053 (3) −0.002 (3) 0.009 (3) 0.002 (3)
N3 0.065 (3) 0.072 (4) 0.072 (3) −0.007 (3) 0.015 (3) 0.010 (3)
O11 0.060 (3) 0.079 (3) 0.048 (2) −0.007 (2) 0.021 (2) 0.014 (2)
O10 0.154 (5) 0.118 (5) 0.043 (3) 0.058 (4) 0.003 (3) 0.004 (3)
N1 0.052 (3) 0.062 (3) 0.044 (3) −0.005 (2) 0.024 (2) −0.001 (2)
N2 0.047 (3) 0.052 (3) 0.049 (3) 0.001 (2) 0.015 (2) 0.010 (2)
C11 0.049 (4) 0.053 (4) 0.050 (3) −0.007 (3) 0.003 (3) 0.011 (3)
C7 0.043 (3) 0.049 (3) 0.037 (3) −0.001 (3) 0.008 (2) −0.012 (3)
C8 0.055 (4) 0.078 (5) 0.044 (3) 0.000 (3) 0.025 (3) −0.013 (3)
C10 0.056 (4) 0.070 (4) 0.055 (3) −0.006 (3) 0.021 (3) 0.024 (3)
C6 0.058 (4) 0.048 (3) 0.045 (3) 0.001 (3) 0.012 (3) −0.013 (3)
C18 0.071 (4) 0.068 (4) 0.063 (4) 0.011 (3) 0.040 (3) 0.003 (3)
C5 0.069 (4) 0.072 (5) 0.051 (3) 0.010 (4) 0.016 (3) −0.022 (3)
O13 0.064 (3) 0.173 (6) 0.100 (4) −0.036 (4) 0.021 (3) 0.026 (4)
C4 0.085 (5) 0.051 (4) 0.056 (4) 0.013 (4) 0.009 (3) −0.016 (3)
C15 0.053 (4) 0.055 (4) 0.065 (4) 0.014 (3) 0.018 (3) −0.007 (3)
C9 0.065 (4) 0.077 (5) 0.046 (3) 0.000 (3) 0.023 (3) 0.007 (3)
C14 0.067 (4) 0.046 (4) 0.102 (5) 0.021 (3) 0.025 (4) 0.006 (4)
C13 0.062 (4) 0.045 (4) 0.080 (4) 0.009 (3) 0.013 (3) 0.020 (3)
O7 0.099 (5) 0.175 (7) 0.138 (6) −0.011 (5) −0.026 (4) −0.001 (5)
O3 0.067 (3) 0.038 (2) 0.049 (2) −0.0051 (19) 0.0216 (18) 0.0003 (18)
C1 0.092 (5) 0.048 (4) 0.063 (4) −0.013 (4) 0.026 (3) 0.005 (3)
Geometric parameters (Å, °)
Ho1—O1 2.293 (3) C2—C7 1.398 (7)
Ho1—O2 2.298 (3) C12—C13 1.396 (8)
Ho1—O3 2.604 (4) C12—C11 1.463 (8)
Ho1—O4 2.604 (4) N3—O7 1.531 (8)
Ho1—O6 2.323 (4) N1—C8 1.267 (8)
Ho1—O8 2.448 (4) N1—C9 1.484 (7)
Ho1—O9 2.481 (4) N2—C11 1.273 (7)
Ho1—O11 2.430 (4) N2—C10 1.480 (6)
Ho1—O12 2.468 (4) C11—H11 0.9300
Zn1—O1 2.005 (4) C7—C6 1.401 (7)
Zn1—O2 2.022 (3) C8—C6 1.449 (8)
Zn1—O5 1.979 (4) C8—H8 0.9300
Zn1—N1 2.047 (4) C10—C9 1.498 (9)
Zn1—N2 2.021 (5) C10—H10A 0.9700
O1—C7 1.324 (6) C10—H10B 0.9700
O12—N5 1.269 (6) C6—C5 1.406 (8)
O2—C17 1.325 (6) C18—H18A 0.9600
O4—C16 1.376 (6) C18—H18B 0.9600
O4—C18 1.449 (6) C18—H18C 0.9600
C17—C12 1.402 (7) C5—C4 1.367 (9)
C17—C16 1.408 (7) C5—H5 0.9300
O8—N4 1.273 (6) C4—H4 0.9300
N4—O10 1.199 (6) C15—C14 1.390 (9)
N4—O9 1.272 (6) C15—H15 0.9300
O6—N3 1.251 (6) C9—H9A 0.9700
C3—C2 1.379 (8) C9—H9B 0.9700
C3—C4 1.389 (9) C14—C13 1.355 (9)
C3—H3 0.9300 C14—H14 0.9300
N5—O13 1.213 (7) C13—H13 0.9300
N5—O11 1.260 (6) O3—C1 1.431 (6)
O5—N3 1.274 (6) C1—H1A 0.9600
C16—C15 1.382 (8) C1—H1B 0.9600
C2—O3 1.390 (6) C1—H1C 0.9600
O1—Ho1—O2 67.54 (12) C15—C16—C17 120.6 (5)
O1—Ho1—O6 78.56 (14) N4—O9—Ho1 95.4 (3)
O2—Ho1—O6 78.26 (14) C3—C2—O3 124.9 (5)
O1—Ho1—O11 124.55 (14) C3—C2—C7 121.6 (5)
O2—Ho1—O11 125.51 (14) O3—C2—C7 113.4 (4)
O6—Ho1—O11 150.31 (13) C13—C12—C17 118.0 (5)
O1—Ho1—O8 114.92 (13) C13—C12—C11 118.3 (5)
O2—Ho1—O8 154.15 (14) C17—C12—C11 123.7 (5)
O6—Ho1—O8 77.22 (14) O6—N3—O5 125.0 (5)
O11—Ho1—O8 75.84 (14) O6—N3—O7 117.5 (5)
O1—Ho1—O12 82.58 (14) O5—N3—O7 117.4 (5)
O2—Ho1—O12 83.46 (14) N5—O11—Ho1 96.7 (3)
O6—Ho1—O12 157.61 (13) C8—N1—C9 122.1 (5)
O11—Ho1—O12 52.07 (13) C8—N1—Zn1 125.6 (4)
O8—Ho1—O12 122.24 (14) C9—N1—Zn1 111.8 (4)
O1—Ho1—O9 152.44 (15) C11—N2—C10 122.7 (5)
O2—Ho1—O9 113.29 (14) C11—N2—Zn1 129.0 (4)
O6—Ho1—O9 74.88 (14) C10—N2—Zn1 107.7 (4)
O11—Ho1—O9 78.74 (14) N2—C11—C12 124.6 (5)
O8—Ho1—O9 51.77 (13) N2—C11—H11 117.7
O12—Ho1—O9 124.92 (14) C12—C11—H11 117.7
O1—Ho1—O4 126.16 (12) O1—C7—C2 116.2 (4)
O2—Ho1—O4 61.54 (11) O1—C7—C6 124.6 (5)
O6—Ho1—O4 106.10 (14) C2—C7—C6 119.1 (5)
O11—Ho1—O4 76.52 (13) N1—C8—C6 126.2 (5)
O8—Ho1—O4 118.41 (12) N1—C8—H8 116.9
O12—Ho1—O4 75.69 (14) C6—C8—H8 116.9
O9—Ho1—O4 69.45 (12) N2—C10—C9 109.0 (5)
O1—Ho1—O3 62.03 (12) N2—C10—H10A 109.9
O2—Ho1—O3 127.20 (12) C9—C10—H10A 109.9
O6—Ho1—O3 105.25 (14) N2—C10—H10B 109.9
O11—Ho1—O3 75.75 (13) C9—C10—H10B 109.9
O8—Ho1—O3 67.95 (12) H10A—C10—H10B 108.3
O12—Ho1—O3 75.82 (14) C7—C6—C5 118.5 (6)
O9—Ho1—O3 118.46 (12) C7—C6—C8 123.4 (5)
O4—Ho1—O3 148.63 (12) C5—C6—C8 117.8 (5)
O5—Zn1—O1 102.49 (16) O4—C18—H18A 109.5
O5—Zn1—N2 110.09 (18) O4—C18—H18B 109.5
O1—Zn1—N2 147.12 (18) H18A—C18—H18B 109.5
O5—Zn1—O2 104.16 (16) O4—C18—H18C 109.5
O1—Zn1—O2 78.64 (14) H18A—C18—H18C 109.5
N2—Zn1—O2 89.09 (16) H18B—C18—H18C 109.5
O5—Zn1—N1 112.60 (18) C4—C5—C6 121.1 (6)
O1—Zn1—N1 89.23 (17) C4—C5—H5 119.5
N2—Zn1—N1 82.44 (19) C6—C5—H5 119.5
O2—Zn1—N1 143.00 (18) C5—C4—C3 120.8 (6)
C7—O1—Zn1 126.1 (3) C5—C4—H4 119.6
C7—O1—Ho1 130.6 (3) C3—C4—H4 119.6
Zn1—O1—Ho1 101.65 (15) C16—C15—C14 118.9 (6)
N5—O12—Ho1 94.7 (3) C16—C15—H15 120.5
C17—O2—Zn1 127.8 (3) C14—C15—H15 120.5
C17—O2—Ho1 131.3 (3) N1—C9—C10 110.3 (5)
Zn1—O2—Ho1 100.93 (15) N1—C9—H9A 109.6
C16—O4—C18 116.2 (4) C10—C9—H9A 109.6
C16—O4—Ho1 118.3 (3) N1—C9—H9B 109.6
C18—O4—Ho1 125.5 (3) C10—C9—H9B 109.6
O2—C17—C12 125.0 (5) H9A—C9—H9B 108.1
O2—C17—C16 115.3 (5) C13—C14—C15 121.0 (6)
C12—C17—C16 119.7 (5) C13—C14—H14 119.5
N4—O8—Ho1 96.9 (3) C15—C14—H14 119.5
O10—N4—O9 122.4 (5) C14—C13—C12 121.8 (6)
O10—N4—O8 122.1 (5) C14—C13—H13 119.1
O9—N4—O8 115.4 (5) C12—C13—H13 119.1
N3—O6—Ho1 144.0 (4) C2—O3—C1 116.6 (4)
C2—C3—C4 118.8 (6) C2—O3—Ho1 117.3 (3)
C2—C3—H3 120.6 C1—O3—Ho1 125.8 (3)
C4—C3—H3 120.6 O3—C1—H1A 109.5
O13—N5—O11 122.6 (5) O3—C1—H1B 109.5
O13—N5—O12 120.9 (6) H1A—C1—H1B 109.5
O11—N5—O12 116.5 (5) O3—C1—H1C 109.5
N3—O5—Zn1 119.1 (3) H1A—C1—H1C 109.5
O4—C16—C15 126.0 (5) H1B—C1—H1C 109.5
O4—C16—C17 113.3 (5)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C1—H1B···O11 0.96 2.54 3.167 (8) 123
C5—H5···O11i 0.93 2.45 3.377 (7) 174
C10—H10A···O13ii 0.97 2.54 3.483 (8) 165
C18—H18B···O9 0.96 2.58 3.100 (8) 114
Symmetry codes: (i) x−1/2, −y+1/2, z+1/2; (ii) −x+1, −y, −z+2.
Table 1 Selected bond lengths (Å)
Ho1—O1 2.293 (3)
Ho1—O2 2.298 (3)
Ho1—O3 2.604 (4)
Ho1—O4 2.604 (4)
Ho1—O6 2.323 (4)
Ho1—O8 2.448 (4)
Ho1—O9 2.481 (4)
Ho1—O11 2.430 (4)
Ho1—O12 2.468 (4)
Zn1—O1 2.005 (4)
Zn1—O2 2.022 (3)
Zn1—O5 1.979 (4)
Zn1—N1 2.047 (4)
Zn1—N2 2.021 (5)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C5—H5⋯O11i 0.93 2.45 3.377 (7) 174
C10—H10A⋯O13ii 0.97 2.54 3.483 (8) 165
Symmetry codes: (i) ; (ii) .
==== Refs
References
Baggio, R., Garland, M. T., Moreno, Y., Pena, O., Perec, M. & Spodine, E. (2000). J. Chem. Soc. Dalton Trans. pp. 2061–2066.
Bruker (2004). APEX2 and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Caravan, P., Ellison, J. J., McMurry, T. J. & Lauffer, R. B. (1999). Chem. Rev.99, 2293–2352.
Edder, C., Piguet, C., Bernardinelli, G., Mareda, J., Bochet, C. G., Bunzli, J.-C. G. & Hopfgartner, G. (2000). Inorg. Chem.39, 5059–5073.
Knoer, R., Lin, H.-H., Wei, H.-H. & Mohanta, S. (2005). Inorg. Chem.44, 3524–3536.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sui, Y., Fang, X.-N., Xiao, Y.-A., Luo, Q.-Y. & Li, M.-H. (2006). Acta Cryst. E62, m2230–m2232.
Sui, Y., He, D.-Y., Fang, X.-N., Chen, L. & Peng, J.-L. (2007). Acta Cryst. E63, m2013–m2014.
Westrip, S. P. (2008). publCIF In preparation. | 21202491 | PMC2961445 | CC BY | 2021-01-04 18:56:00 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 May 14; 64(Pt 6):m804-m805 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography kp217710.1107/S1600536808018539ACSEBHS1600536808018539Metal-Organic PapersDiazidobis(2,2′-biimidazole)iron(II) [Fe(N3)2(C6H6N4)2]Hao Lujiang a*Mu Chunhua bKong Binbin aa College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Insitute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2008 25 6 2008 25 6 2008 64 Pt 7 e080700m956 m956 16 6 2008 19 6 2008 © Hao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Fe(N3)2(C6H6N4)2], the Fe atom is bonded to two azide ions located in axial positions and to two equatorially positioned bidentate biimidazole ligands, forming a slightly distorterd octahedron. The non-H atoms of the equatorial plane are coplanar, with a mean deviation of 0.0355 (2) Å. The FeII cation lies on an inversion centre. Thus, the asymmetric unit comprises one half-molecule.
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Related literature
For related literature, see: Caneschi et al. (1989 ▶); Tsukuda et al. (2002 ▶); Vostrikova et al. (2000 ▶); Kuchar et al. (2003 ▶).
Experimental
Crystal data
[Fe(N3)2(C6H6N4)2]
M
r = 404.17
Monoclinic,
a = 12.487 (3) Å
b = 9.012 (2) Å
c = 14.222 (3) Å
β = 91.91 (3)°
V = 1599.6 (6) Å3
Z = 4
Mo Kα radiation
μ = 0.98 mm−1
T = 293 (2) K
0.14 × 0.12 × 0.10 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.875, T
max = 0.909
1964 measured reflections
1504 independent reflections
1250 reflections with I > 2σ(I)
R
int = 0.022
Refinement
R[F
2 > 2σ(F
2)] = 0.038
wR(F
2) = 0.118
S = 1.00
1504 reflections
124 parameters
H-atom parameters constrained
Δρmax = 0.65 e Å−3
Δρmin = −0.26 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808018539/kp2177sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808018539/kp2177Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: KP2177).
The authors thank the National Ministry of Science and Technology of China for support (grant No. 2001CB6105–07).
supplementary crystallographic
information
Comment
Different kinds of metal-radical coordination architectures with
appropriate organic radicals and coligands have been an important subject
during the last decade because of their potential use for molecule-based
magnetic materials and optical devices (Caneschi et al., 1989;
Tsukuda
et al., 2002; Vostrikova et al., 2000; Kuchar
et al.,
2003). The organic species, such as tridentate nitronyl nitroxide
radical,
and bidentate nitroxide radical could result in a large number of building
blocks with the potential applications. In this paper, we report the
structure of the title compound, (I).
The Fe atom, located at the inversion centre, is bonded to two azide ions
and the two bidentate biimidizole ligands, forming a slightly distorterd
octahedron (Fig. 1). The four nitrogen atoms belonging to two biimidizole
ligands lie in the equatorial plane and the two nitrogen atoms from azide
groups lie at the axial coordination sites. In the equatorial plane
the Fe—N(imidzole) bond lengths are in the range of
2.095 (2)–2.113 (2) /%A (Table 1).
Experimental
A mixture of iron(II) dichloride anhydrous (1 mmoL), 2,2'-biimidazole(1 mmoL),
and sodium azide (2 mmol) in 20 mL methanol was refluxed for several h.
The above cooled solution was filterated and the filtrate was kept in the ice
box. One week later, colourless blocks of (I) were obtained with the yield of
ca 8%. Anal. Calc. for C12H8FeN14: C 35.63, H 1.98, N 48.49%;
Found: C 35.58, H 1.96, N 48.45%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93Å and refined
as riding with Uiso(H) = 1.2Ueq(carrier).
Figures
Fig. 1. The molecular structure of (I) around FeII drawn with the 30% probability displacement ellipsoids for the non-hydrogen atoms.
Crystal data
[Fe(N3)2(C6H6N4)2] F000 = 816
Mr = 404.17 Dx = 1.678 Mg m−3
Monoclinic, C2/c Mo Kα radiation λ = 0.71073 Å
Hall symbol: -C 2yc Cell parameters from 1504 reflections
a = 12.487 (3) Å θ = 2.8–25.7º
b = 9.012 (2) Å µ = 0.98 mm−1
c = 14.222 (3) Å T = 293 (2) K
β = 91.91 (3)º Block, colourless
V = 1599.6 (6) Å3 0.14 × 0.12 × 0.10 mm
Z = 4
Data collection
Bruker APEXII CCD diffractometer 1504 independent reflections
Radiation source: fine-focus sealed tube 1250 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.022
T = 293(2) K θmax = 25.7º
φ and ω scans θmin = 2.8º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −1→15
Tmin = 0.875, Tmax = 0.909 k = −1→10
1964 measured reflections l = −17→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.038 H-atom parameters constrained
wR(F2) = 0.118 w = 1/[σ2(Fo2) + (0.075P)2 + 1.004P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.006
1504 reflections Δρmax = 0.65 e Å−3
124 parameters Δρmin = −0.25 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Fe1 0.7500 1.2500 0.5000 0.0610 (9)
C1 0.6773 (3) 1.0317 (4) 0.3216 (2) 0.0554 (7)
H1 0.6279 1.0905 0.2883 0.067*
C2 0.7058 (3) 0.8903 (4) 0.2969 (2) 0.0583 (8)
H2 0.6805 0.8374 0.2446 0.070*
C3 0.9629 (2) 0.9250 (4) 0.6284 (2) 0.0555 (8)
H3 1.0126 0.8825 0.6704 0.067*
C4 0.9159 (2) 1.0612 (4) 0.63750 (19) 0.0543 (7)
H4 0.9290 1.1270 0.6869 0.065*
C5 0.7896 (2) 0.9564 (3) 0.42568 (19) 0.0456 (6)
C6 0.8539 (2) 0.9633 (3) 0.51069 (18) 0.0454 (6)
N1 0.60420 (19) 1.0322 (3) 0.57863 (17) 0.0519 (6)
N2 0.6173 (2) 1.1634 (3) 0.57318 (18) 0.0544 (6)
N3 0.5894 (2) 0.9020 (3) 0.5859 (2) 0.0697 (8)
N4 0.73154 (18) 1.0735 (3) 0.40187 (16) 0.0499 (6)
N5 0.84709 (18) 1.0851 (3) 0.56302 (15) 0.0491 (6)
N6 0.92402 (18) 0.8623 (3) 0.54644 (16) 0.0498 (6)
N7 0.7786 (2) 0.8422 (3) 0.36365 (16) 0.0517 (6)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Fe1 0.064 (2) 0.067 (2) 0.0510 (18) −0.0005 (18) −0.0087 (15) 0.0092 (16)
C1 0.0573 (17) 0.0577 (18) 0.0505 (15) 0.0058 (14) −0.0099 (13) −0.0031 (14)
C2 0.0618 (18) 0.064 (2) 0.0489 (16) −0.0009 (16) −0.0092 (14) −0.0094 (14)
C3 0.0499 (16) 0.069 (2) 0.0472 (15) 0.0174 (15) −0.0050 (12) 0.0040 (14)
C4 0.0513 (16) 0.0661 (19) 0.0448 (14) 0.0160 (15) −0.0081 (12) −0.0049 (13)
C5 0.0440 (14) 0.0452 (15) 0.0473 (14) 0.0058 (12) 0.0004 (11) −0.0026 (12)
C6 0.0454 (14) 0.0474 (16) 0.0435 (13) 0.0084 (12) 0.0018 (11) −0.0004 (11)
N1 0.0492 (14) 0.0514 (16) 0.0547 (14) 0.0132 (11) −0.0061 (11) −0.0075 (11)
N2 0.0528 (14) 0.0487 (16) 0.0614 (15) 0.0104 (12) −0.0006 (11) −0.0037 (12)
N3 0.0706 (18) 0.0500 (17) 0.088 (2) 0.0088 (14) −0.0093 (15) −0.0077 (15)
N4 0.0497 (13) 0.0521 (15) 0.0474 (12) 0.0085 (11) −0.0060 (10) −0.0052 (11)
N5 0.0462 (13) 0.0545 (15) 0.0463 (12) 0.0117 (11) −0.0042 (10) −0.0045 (11)
N6 0.0476 (13) 0.0525 (14) 0.0491 (12) 0.0130 (11) 0.0005 (10) 0.0034 (11)
N7 0.0553 (14) 0.0513 (15) 0.0482 (12) 0.0048 (12) −0.0012 (10) −0.0068 (11)
Geometric parameters (Å, °)
Fe1—N5 2.100 (2) C3—C4 1.368 (4)
Fe1—N5i 2.100 (2) C3—H3 0.9300
Fe1—N4i 2.123 (2) C4—N5 1.359 (3)
Fe1—N4 2.123 (2) C4—H4 0.9300
Fe1—N2i 2.134 (3) C5—N4 1.318 (4)
Fe1—N2 2.134 (3) C5—N7 1.360 (4)
C1—N4 1.361 (4) C5—C6 1.430 (4)
C1—C2 1.373 (4) C6—N5 1.330 (4)
C1—H1 0.9300 C6—N6 1.351 (4)
C2—N7 1.363 (4) N1—N3 1.193 (4)
C2—H2 0.9300 N1—N2 1.197 (3)
C3—N6 1.369 (4)
N5—Fe1—N5i 180.000 (1) N6—C3—H3 126.0
N5—Fe1—N4i 101.60 (9) C4—C3—H3 126.0
N5i—Fe1—N4i 78.40 (9) N5—C4—C3 109.3 (3)
N5—Fe1—N4 78.40 (9) N5—C4—H4 125.4
N5i—Fe1—N4 101.60 (9) C3—C4—H4 125.4
N4i—Fe1—N4 180.000 (1) N4—C5—N7 113.3 (2)
N5—Fe1—N2i 91.16 (10) N4—C5—C6 118.1 (2)
N5i—Fe1—N2i 88.84 (10) N7—C5—C6 128.6 (3)
N4i—Fe1—N2i 88.71 (10) N5—C6—N6 113.4 (2)
N4—Fe1—N2i 91.29 (10) N5—C6—C5 117.7 (2)
N5—Fe1—N2 88.84 (10) N6—C6—C5 128.9 (3)
N5i—Fe1—N2 91.16 (10) N3—N1—N2 178.3 (3)
N4i—Fe1—N2 91.29 (10) N1—N2—Fe1 120.2 (2)
N4—Fe1—N2 88.71 (10) C5—N4—C1 104.4 (2)
N2i—Fe1—N2 180.0 C5—N4—Fe1 112.58 (18)
N4—C1—C2 110.2 (3) C1—N4—Fe1 143.0 (2)
N4—C1—H1 124.9 C6—N5—C4 104.8 (2)
C2—C1—H1 124.9 C6—N5—Fe1 113.05 (17)
N7—C2—C1 106.8 (3) C4—N5—Fe1 141.8 (2)
N7—C2—H2 126.6 C6—N6—C3 104.4 (2)
C1—C2—H2 126.6 C5—N7—C2 105.2 (2)
N6—C3—C4 108.0 (2)
Symmetry codes: (i) −x+3/2, −y+5/2, −z+1.
Table 1 Selected geometric parameters (Å, °)
Fe1—N5 2.100 (2)
Fe1—N4 2.123 (2)
Fe1—N2 2.134 (3)
N5—Fe1—N5i 180
N5—Fe1—N4 78.40 (9)
N5—Fe1—N2 88.84 (10)
N4—Fe1—N4i
N4—Fe1—N2 88.71 (10)
N2i—Fe1—N2 180
Symmetry code: (i) .
==== Refs
References
Bruker (2001). SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Caneschi, A., Gatteschi, D., Renard, J. P., Rey, P. & Sessoli, R. (1989). J. Am. Chem. Soc.111, 785–786.
Kuchar, J., Cernak, J., Zak, Z. & Massa, W. (2003). Monogr. Ser. Int. Conf. Coord. Chem.6, 127–132.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Tsukuda, T., Suzuki, T. & Kaizaki, S. (2002). J. Chem. Soc. Dalton Trans. pp. 1721–1726.
Vostrikova, K. E., Luneau, D., Wernsdorfer, W., Rey, P. & Verdaguer, M. (2000). J. Am. Chem. Soc.122, 718–719. | 21202805 | PMC2961650 | CC BY | 2021-01-04 18:56:04 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jun 25; 64(Pt 7):m956 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography kp217610.1107/S1600536808018552ACSEBHS1600536808018552Metal-Organic PapersBis(pentane-2,4-dionato)bis[2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide]nickel(II) [Ni(C5H7O2)2(C12H16N3O2)]Hao Lujiang a*Mu Chunhua bKong Binbin aa College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Insitute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2008 25 6 2008 25 6 2008 64 Pt 7 e080700m957 m957 12 6 2008 19 6 2008 © Hao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Ni(C5H7O2)2(C12H16N3O2)], the NiII cation is hexacoordinated by four O and two N atoms, showing a slightly distorted octahedral geometry. The NiII cation lies on an inversion centre, as a consequence of which the asymmetric unit comprises one half-molecule. The four O atoms belonging to two pentane-2,4-dionate ligands lie in the equatorial plane and two pyridyl N atoms occupy the axial coordination sites.
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Related literature
For related literature, see: Caneschi et al. (1989 ▶); Tsukuda et al. (2002 ▶); Vostrikova et al. (2000 ▶); Kuchar et al. (2003 ▶).
Experimental
Crystal data
[Ni(C5H7O2)2(C12H16N3O2)]
M
r = 725.48
Triclinic,
a = 6.9862 (10) Å
b = 10.121 (3) Å
c = 12.735 (3) Å
α = 98.20 (2)°
β = 103.21 (2)°
γ = 93.08 (2)°
V = 864.1 (3) Å3
Z = 1
Mo Kα radiation
μ = 0.62 mm−1
T = 293 (2) K
0.43 × 0.28 × 0.22 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.776, T
max = 0.875
5805 measured reflections
2968 independent reflections
2356 reflections with I > 2σ(I)
R
int = 0.032
Refinement
R[F
2 > 2σ(F
2)] = 0.039
wR(F
2) = 0.101
S = 1.00
2968 reflections
229 parameters
H-atom parameters not refined
Δρmax = 0.38 e Å−3
Δρmin = −0.51 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808018552/kp2176sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808018552/kp2176Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: KP2176).
The authors thank the National Ministry of Science and Technology of China for support (grant No. 2001CB6105-07).
supplementary crystallographic
information
Comment
Design of different kinds of metal-radical coordination architectures with
appropriate organic radicals and coligands has been an important subject
during the last decade because of their potential use for molecule-based
magnetic materials and optical devices (Caneschi et al., 1989;
Tsukuda
et al., 2002; Vostrikova et al., 2000; Kuchar
et al.,
2003). The organic species, such as tridentate nitronyl nitroxide
radical,
and bidentate nitroxide radical could results in a large number of building
blocks with the potential applications. In this paper, we report the
structure of the title compound, (I).
The NiII cation is hexacoordinated with four O and
two N atoms showing the slightly distorted octahedral geometry (Fig. 1).
The NiII cation lies on an inversion centre. The four oxygen atoms belonging
to two pentane-2,4-dionate lie in the equatorial plane and the two
nitrogen atoms occupy the axial coordination sites. The Ni—N and
Ni—O bond lengths are in the range of 2.154 (2)–2.154 (2) and
2.0239 (17)–2.0292 (16) /%A, respectively (Table 1).
Experimental
A mixture of nickel(II) acetylacetonate (1 mmoL) and
2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (1 mmoL) in 20 mL
methanol was refluxed for several h. The above cooled solution was
filterated and the filtrate was kept in the ice box. One week later, green
blocks of (I) were obtained with yield of ca 3%. Anal. Calc. for
C34H46N6NiO8: C 56.24, H 6.34, N 11.58%; Found: C 56.19, H 6.28, N
11.47%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93Å and refined
as riding with Uiso(H) = 1.2Ueq(carrier).
Figures
Fig. 1. The molecular structure of (I) around NiII, drawn with the 30% probability displacement ellipsoids for the non-hydrogen atoms.
Crystal data
[Ni(C5H7O2)2(C12H16N3O2)] Z = 1
Mr = 725.48 F000 = 384
Triclinic, P1 Dx = 1.394 Mg m−3
Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å
a = 6.9862 (10) Å Cell parameters from 2968 reflections
b = 10.121 (3) Å θ = 3.1–25.0º
c = 12.735 (3) Å µ = 0.62 mm−1
α = 98.20 (2)º T = 293 (2) K
β = 103.21 (2)º Block, green
γ = 93.08 (2)º 0.43 × 0.28 × 0.22 mm
V = 864.1 (3) Å3
Data collection
Bruker APEXII CCD area-detector diffractometer 2968 independent reflections
Radiation source: fine-focus sealed tube 2356 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.032
T = 293(2) K θmax = 25.0º
φ and ω scans θmin = 3.1º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −6→8
Tmin = 0.776, Tmax = 0.876 k = −12→12
5805 measured reflections l = −11→15
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.039 H-atom parameters not refined
wR(F2) = 0.101 w = 1/[σ2(Fo2) + (0.0577P)2] where P = (Fo2 + 2Fc2)/3
S = 1.01 (Δ/σ)max < 0.001
2968 reflections Δρmax = 0.38 e Å−3
229 parameters Δρmin = −0.51 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Ni1 0.0000 0.0000 0.0000 0.01842 (16)
C1 0.6315 (4) 0.1982 (3) 0.4503 (2) 0.0171 (5)
C2 0.4941 (4) 0.1511 (2) 0.3460 (2) 0.0157 (5)
C3 0.5524 (4) 0.0953 (3) 0.2533 (2) 0.0182 (5)
H3 0.6852 0.0863 0.2562 0.022*
C4 0.4134 (4) 0.0538 (2) 0.1578 (2) 0.0169 (5)
H4 0.4551 0.0175 0.0965 0.020*
C5 0.1699 (4) 0.1168 (2) 0.2385 (2) 0.0163 (5)
H5 0.0360 0.1243 0.2334 0.020*
C6 0.2972 (3) 0.1614 (2) 0.33692 (19) 0.0159 (5)
H6 0.2511 0.1980 0.3967 0.019*
C7 0.9190 (3) 0.2686 (2) 0.58605 (18) 0.0148 (5)
C8 0.7425 (4) 0.2983 (3) 0.6346 (2) 0.0181 (5)
C9 0.6895 (4) 0.4438 (3) 0.6355 (2) 0.0260 (6)
H9A 0.6955 0.4711 0.5671 0.039*
H9B 0.7816 0.5013 0.6938 0.039*
H9C 0.5585 0.4500 0.6462 0.039*
C10 0.7513 (4) 0.2543 (3) 0.7443 (2) 0.0232 (6)
H10A 0.6333 0.2747 0.7674 0.035*
H10B 0.8640 0.3008 0.7971 0.035*
H10C 0.7623 0.1595 0.7378 0.035*
C11 1.0773 (3) 0.3828 (3) 0.6102 (2) 0.0186 (6)
H11A 1.1758 0.3599 0.5708 0.028*
H11B 1.1372 0.3998 0.6872 0.028*
H11C 1.0201 0.4616 0.5882 0.028*
C12 1.0024 (4) 0.1370 (3) 0.6126 (2) 0.0186 (5)
H12A 0.8965 0.0672 0.5967 0.028*
H12B 1.0684 0.1480 0.6886 0.028*
H12C 1.0945 0.1133 0.5690 0.028*
C13 −0.1318 (4) 0.3856 (3) 0.1296 (2) 0.0236 (6)
H13A −0.2720 0.3832 0.1017 0.035*
H13B −0.0716 0.4731 0.1295 0.035*
H13C −0.1042 0.3661 0.2028 0.035*
C14 −0.0496 (4) 0.2828 (2) 0.05844 (19) 0.0169 (5)
C15 0.1142 (4) 0.3210 (2) 0.0228 (2) 0.0193 (6)
H15 0.1681 0.4094 0.0447 0.023*
C16 0.2042 (4) 0.2369 (2) −0.0434 (2) 0.0183 (6)
C17 0.3724 (4) 0.2937 (3) −0.0815 (2) 0.0257 (6)
H17A 0.4877 0.2489 −0.0560 0.039*
H17B 0.3981 0.3877 −0.0531 0.039*
H17C 0.3400 0.2812 −0.1599 0.039*
N1 0.2240 (3) 0.06298 (19) 0.14880 (16) 0.0147 (4)
N2 0.5853 (3) 0.2130 (2) 0.54884 (17) 0.0188 (5)
N3 0.8188 (3) 0.2419 (2) 0.46714 (16) 0.0154 (5)
O1 0.4272 (2) 0.1725 (2) 0.56916 (14) 0.0276 (5)
O2 0.9117 (2) 0.25109 (18) 0.39294 (14) 0.0212 (4)
O3 −0.1365 (2) 0.16536 (16) 0.03800 (13) 0.0173 (4)
O4 0.1568 (2) 0.11205 (17) −0.07687 (13) 0.0183 (4)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Ni1 0.0180 (3) 0.0196 (3) 0.0157 (3) 0.00152 (18) 0.00049 (19) 0.00216 (19)
C1 0.0170 (13) 0.0237 (14) 0.0112 (13) 0.0031 (10) 0.0052 (10) 0.0011 (10)
C2 0.0157 (13) 0.0186 (13) 0.0127 (13) 0.0000 (10) 0.0026 (10) 0.0040 (10)
C3 0.0149 (13) 0.0234 (14) 0.0164 (14) 0.0020 (10) 0.0039 (11) 0.0028 (11)
C4 0.0173 (13) 0.0198 (13) 0.0143 (13) 0.0030 (10) 0.0041 (10) 0.0039 (10)
C5 0.0133 (12) 0.0192 (13) 0.0180 (14) 0.0022 (10) 0.0045 (10) 0.0069 (10)
C6 0.0166 (13) 0.0217 (13) 0.0100 (13) 0.0024 (10) 0.0039 (10) 0.0026 (10)
C7 0.0139 (12) 0.0242 (14) 0.0047 (12) 0.0012 (10) 0.0009 (10) 0.0000 (10)
C8 0.0134 (13) 0.0279 (15) 0.0114 (13) 0.0040 (10) 0.0003 (10) 0.0018 (11)
C9 0.0217 (14) 0.0332 (16) 0.0218 (15) 0.0111 (12) 0.0025 (11) 0.0012 (12)
C10 0.0186 (14) 0.0393 (17) 0.0112 (14) 0.0027 (11) 0.0023 (11) 0.0046 (12)
C11 0.0155 (13) 0.0246 (14) 0.0144 (14) 0.0020 (10) 0.0016 (10) 0.0017 (11)
C12 0.0148 (13) 0.0241 (14) 0.0170 (14) 0.0024 (10) 0.0039 (10) 0.0034 (11)
C13 0.0270 (15) 0.0192 (14) 0.0214 (15) 0.0067 (11) 0.0002 (11) 0.0001 (11)
C14 0.0179 (13) 0.0193 (14) 0.0094 (13) 0.0045 (10) −0.0062 (10) 0.0029 (10)
C15 0.0221 (14) 0.0155 (13) 0.0168 (14) 0.0003 (10) −0.0029 (11) 0.0035 (10)
C16 0.0165 (13) 0.0207 (14) 0.0144 (13) −0.0002 (10) −0.0061 (10) 0.0087 (10)
C17 0.0190 (14) 0.0257 (15) 0.0308 (16) −0.0023 (11) 0.0009 (12) 0.0093 (12)
N1 0.0183 (11) 0.0148 (11) 0.0113 (11) 0.0021 (8) 0.0037 (9) 0.0019 (8)
N2 0.0099 (11) 0.0332 (13) 0.0128 (12) 0.0012 (9) 0.0023 (9) 0.0030 (9)
N3 0.0096 (10) 0.0243 (12) 0.0112 (11) −0.0003 (8) 0.0012 (9) 0.0014 (8)
O1 0.0109 (9) 0.0545 (13) 0.0181 (10) −0.0025 (8) 0.0046 (8) 0.0083 (9)
O2 0.0145 (9) 0.0369 (11) 0.0126 (9) 0.0002 (8) 0.0058 (7) 0.0010 (8)
O3 0.0157 (9) 0.0184 (9) 0.0159 (9) 0.0034 (7) −0.0005 (7) 0.0022 (7)
O4 0.0172 (9) 0.0214 (10) 0.0140 (9) −0.0006 (7) −0.0002 (7) 0.0027 (7)
Geometric parameters (Å, °)
Ni1—O3i 2.0239 (17) C9—H9B 0.9600
Ni1—O3 2.0239 (17) C9—H9C 0.9600
Ni1—O4i 2.0292 (16) C10—H10A 0.9600
Ni1—O4 2.0292 (16) C10—H10B 0.9600
Ni1—N1 2.154 (2) C10—H10C 0.9600
Ni1—N1i 2.154 (2) C11—H11A 0.9600
C1—N3 1.318 (3) C11—H11B 0.9600
C1—N2 1.355 (3) C11—H11C 0.9600
C1—C2 1.450 (3) C12—H12A 0.9600
C2—C6 1.364 (3) C12—H12B 0.9600
C2—C3 1.390 (4) C12—H12C 0.9600
C3—C4 1.366 (4) C13—C14 1.506 (3)
C3—H3 0.9300 C13—H13A 0.9600
C4—N1 1.312 (3) C13—H13B 0.9600
C4—H4 0.9300 C13—H13C 0.9600
C5—N1 1.337 (3) C14—O3 1.269 (3)
C5—C6 1.361 (3) C14—C15 1.380 (4)
C5—H5 0.9300 C15—C16 1.388 (4)
C6—H6 0.9300 C15—H15 0.9300
C7—N3 1.495 (3) C16—O4 1.275 (3)
C7—C11 1.503 (3) C16—C17 1.488 (3)
C7—C8 1.526 (3) C17—H17A 0.9600
C7—C12 1.533 (3) C17—H17B 0.9600
C8—N2 1.491 (3) C17—H17C 0.9600
C8—C10 1.515 (3) N2—O1 1.252 (3)
C8—C9 1.537 (4) N3—O2 1.273 (3)
C9—H9A 0.9600
O3i—Ni1—O3 180.00 (12) C8—C10—H10A 109.5
O3i—Ni1—O4i 87.77 (7) C8—C10—H10B 109.5
O3—Ni1—O4i 92.23 (7) H10A—C10—H10B 109.5
O3i—Ni1—O4 92.23 (7) C8—C10—H10C 109.5
O3—Ni1—O4 87.77 (7) H10A—C10—H10C 109.5
O4i—Ni1—O4 180.00 (9) H10B—C10—H10C 109.5
O3i—Ni1—N1 91.68 (7) C7—C11—H11A 109.5
O3—Ni1—N1 88.33 (7) C7—C11—H11B 109.5
O4i—Ni1—N1 91.41 (7) H11A—C11—H11B 109.5
O4—Ni1—N1 88.59 (7) C7—C11—H11C 109.5
O3i—Ni1—N1i 88.32 (7) H11A—C11—H11C 109.5
O3—Ni1—N1i 91.67 (7) H11B—C11—H11C 109.5
O4i—Ni1—N1i 88.59 (7) C7—C12—H12A 109.5
O4—Ni1—N1i 91.41 (7) C7—C12—H12B 109.5
N1—Ni1—N1i 180.00 (8) H12A—C12—H12B 109.5
N3—C1—N2 107.4 (2) C7—C12—H12C 109.5
N3—C1—C2 127.1 (2) H12A—C12—H12C 109.5
N2—C1—C2 125.4 (2) H12B—C12—H12C 109.5
C6—C2—C3 117.6 (2) C14—C13—H13A 109.5
C6—C2—C1 119.1 (2) C14—C13—H13B 109.5
C3—C2—C1 123.3 (2) H13A—C13—H13B 109.5
C4—C3—C2 119.6 (2) C14—C13—H13C 109.5
C4—C3—H3 120.2 H13A—C13—H13C 109.5
C2—C3—H3 120.2 H13B—C13—H13C 109.5
N1—C4—C3 123.0 (2) O3—C14—C15 125.1 (2)
N1—C4—H4 118.5 O3—C14—C13 116.2 (2)
C3—C4—H4 118.5 C15—C14—C13 118.7 (2)
N1—C5—C6 124.5 (2) C14—C15—C16 124.7 (2)
N1—C5—H5 117.8 C14—C15—H15 117.7
C6—C5—H5 117.8 C16—C15—H15 117.7
C5—C6—C2 118.5 (2) O4—C16—C15 126.3 (2)
C5—C6—H6 120.7 O4—C16—C17 114.7 (2)
C2—C6—H6 120.7 C15—C16—C17 118.9 (2)
N3—C7—C11 110.84 (19) C16—C17—H17A 109.5
N3—C7—C8 100.11 (18) C16—C17—H17B 109.5
C11—C7—C8 114.5 (2) H17A—C17—H17B 109.5
N3—C7—C12 105.69 (19) C16—C17—H17C 109.5
C11—C7—C12 112.2 (2) H17A—C17—H17C 109.5
C8—C7—C12 112.4 (2) H17B—C17—H17C 109.5
N2—C8—C7 98.88 (19) C4—N1—C5 116.8 (2)
N2—C8—C10 109.8 (2) C4—N1—Ni1 124.33 (16)
C7—C8—C10 115.3 (2) C5—N1—Ni1 118.92 (16)
N2—C8—C9 106.9 (2) O1—N2—C1 127.2 (2)
C7—C8—C9 113.5 (2) O1—N2—C8 121.2 (2)
C10—C8—C9 111.4 (2) C1—N2—C8 111.4 (2)
C8—C9—H9A 109.5 O2—N3—C1 125.5 (2)
C8—C9—H9B 109.5 O2—N3—C7 122.47 (18)
H9A—C9—H9B 109.5 C1—N3—C7 111.82 (19)
C8—C9—H9C 109.5 C14—O3—Ni1 122.70 (15)
H9A—C9—H9C 109.5 C16—O4—Ni1 121.69 (15)
H9B—C9—H9C 109.5
Symmetry codes: (i) −x, −y, −z.
Table 1 Selected geometric parameters (Å, °)
Ni1—O3 2.0239 (17)
Ni1—O4 2.0292 (16)
Ni1—N1 2.154 (2)
O3i—Ni1—O3 180
O3—Ni1—O4 87.77 (7)
O4i—Ni1—O4 180
O3—Ni1—N1 88.32 (7)
O4—Ni1—N1 88.59 (7)
N1—Ni1—N1i 180
Symmetry code: (i) .
==== Refs
References
Bruker (2001). SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Caneschi, A., Gatteschi, D., Renard, J. P., Rey, P. & Sessoli, R. (1989). J. Am. Chem. Soc.111, 785–786.
Kuchar, J., Cernak, J., Zak, Z. & Massa, W. (2003). Monogr. Ser. Int. Conf. Coord. Chem., 6, 127-132.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Tsukuda, T., Suzuki, T. & Kaizaki, S. (2002). J. Chem. Soc. Dalton Trans. pp. 1721–1726.
Vostrikova, K. E., Luneau, D., Wernsdorfer, W., Rey, P. & Verdaguer, M. (2000). J. Am. Chem. Soc.122, 718–719. | 21202806 | PMC2961770 | CC BY | 2021-01-04 18:56:08 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jun 25; 64(Pt 7):m957 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography cf220610.1107/S1600536808018540ACSEBHS1600536808018540Metal-Organic PapersTris[2-(propyliminomethyl)phenolato-κ2
N,O]iron(III) [Fe(C10H12NO)3]Hao Lujiang a*Mu Chunhua bKong Binbin aa College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Insitute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2008 25 6 2008 25 6 2008 64 Pt 7 e080700m955 m955 14 6 2008 19 6 2008 © Hao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Fe(C10H12NO)3], is isostructural with its CoIII-containing analogue. The FeIII cation is chelated by three Schiff base ligands via three N and three O atoms, and exhibits a slightly distorted octahedral geometry. The longest Fe—O and Fe—N bonds lie trans to each other and may be regarded as axial bonds, while the equatorial plane contains two mutually trans O and two trans N atoms.
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Related literature
For related literature, see: Iskander et al. (2001 ▶); Caruso et al. (2005 ▶); Sangeetha & Pal (2000 ▶); Rajak et al. (2000 ▶); Sutradhar et al. (2006 ▶). For the isostructural Co complex, see: Li et al. (2008 ▶).
Experimental
Crystal data
[Fe(C10H12NO)3]
M
r = 542.47
Tetragonal,
a = 19.369 (2) Å
c = 30.216 (3) Å
V = 11336 (2) Å3
Z = 16
Mo Kα radiation
μ = 0.57 mm−1
T = 293 (2) K
0.12 × 0.10 × 0.08 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.935, T
max = 0.956
41740 measured reflections
5198 independent reflections
3125 reflections with I > 2σ(I)
R
int = 0.073
Refinement
R[F
2 > 2σ(F
2)] = 0.047
wR(F
2) = 0.099
S = 1.00
5198 reflections
337 parameters
H-atom parameters constrained
Δρmax = 0.33 e Å−3
Δρmin = −0.27 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808018540/cf2206sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808018540/cf2206Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: CF2206).
The authors thank the National Ministry of Science and Technology of China (grant No. 2001CB6105–07).
supplementary crystallographic
information
Comment
The design and construction of novel discrete Schiff-basd metal complexes has
attracted long-lasting research interest, not only because of their appealing
structural and topological features, but also due to their unusual optical,
electronic, magnetic and catalytic properties, and their further potential
medical value derived from their antiviral properties and inhibition of
angiogenesis (Iskander et al. 2001; Caruso et al.
2005;
Sangeetha & Pal, 2000; Rajak et al. 2000; Sutradhar
et al.
2006). Here we report the synthesis and X-ray crystal structure
analysis of
the title compound, which is isostructural with its CoIII-containing
analogue (Li et al., 2008).
As shown in Figure 1, the FeIII cation is chelated by three Schiff base
ligands via three N and three O atoms, and exhibits a slightly
distorted octahedral geometry. The Fe—N and Fe—O bond lengths are in the
ranges 1.917 (3)–1.969 (3) and 1.846 (2)–1.913 (2) Å, respectively. The
Fe1—O2 and Fe1—N2 bonds are much longer than the other related ones. Thus
the atoms O1, O3, N1, and N3 may be considered to lie in the equatorial plane,
and O2 and N2 in the axial coordination sites.
Experimental
A mixture of iron(III) acetylacetonate (0.5 mmol) and
2-(propyliminomethyl)phenol (0.5 mmol) in 20 ml methanol was refluxed for
several hours. The filtrate obtained from this soution was allowed to
evaporate at room temperature for three days. Brown crystals were obtained
with a yield of 5%. Anal. Calc. for C30H36FeN3O3: C 65.36, H 6.64 N
7.74%; Found: C 65.21, H 6.59, N 7.67%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93Å and
refined as riding with Uiso(H) = 1.2Ueq(C).
Figures
Fig. 1. The molecular structure of (I), drawn with 30% probability displacement ellipsoids for the non-hydrogen atoms.
Crystal data
[Fe(C10H12N1O1)3] Z = 16
Mr = 542.47 F000 = 4592
Tetragonal, I41/a Dx = 1.271 Mg m−3
Hall symbol: -I 4ad Mo Kα radiation λ = 0.71073 Å
a = 19.369 (2) Å Cell parameters from 5198 reflections
b = 19.369 (2) Å θ = 1.3–25.5º
c = 30.216 (3) Å µ = 0.57 mm−1
α = 90º T = 293 (2) K
β = 90º Block, green
γ = 90º 0.12 × 0.10 × 0.08 mm
V = 11336 (2) Å3
Data collection
Bruker APEXII CCD diffractometer 5198 independent reflections
Radiation source: fine-focus sealed tube 3125 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.073
T = 293(2) K θmax = 25.5º
φ and ω scans θmin = 1.3º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −23→22
Tmin = 0.935, Tmax = 0.956 k = −23→23
41740 measured reflections l = −36→36
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.047 H-atom parameters constrained
wR(F2) = 0.099 w = 1/[σ2(Fo2) + (0.0375P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
5198 reflections Δρmax = 0.33 e Å−3
337 parameters Δρmin = −0.27 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Fe1 0.22632 (2) 0.98149 (2) 0.992992 (13) 0.05735 (17)
C1 0.14742 (16) 0.94922 (15) 0.89961 (9) 0.0579 (8)
C2 0.10654 (19) 0.94259 (17) 0.86092 (10) 0.0703 (9)
H2 0.1202 0.9650 0.8352 0.084*
C3 0.04851 (19) 0.90467 (18) 0.86056 (12) 0.0783 (10)
H3 0.0213 0.9020 0.8353 0.094*
C4 0.03008 (16) 0.86948 (17) 0.89893 (12) 0.0755 (9)
H4 −0.0097 0.8426 0.8989 0.091*
C5 0.06917 (16) 0.87330 (15) 0.93694 (10) 0.0647 (8)
H5 0.0554 0.8488 0.9619 0.078*
C6 0.12904 (15) 0.91330 (14) 0.93872 (10) 0.0548 (7)
C7 0.09561 (16) 0.97163 (15) 1.05726 (9) 0.0577 (8)
C8 0.03352 (16) 0.95695 (16) 1.07764 (10) 0.0693 (9)
H8 −0.0047 0.9843 1.0713 0.083*
C9 0.02656 (19) 0.90334 (17) 1.10688 (11) 0.0745 (9)
H9 −0.0158 0.8943 1.1202 0.089*
C10 0.0822 (2) 0.86399 (18) 1.11603 (10) 0.0749 (9)
H10 0.0774 0.8266 1.1351 0.090*
C11 0.14583 (19) 0.87783 (16) 1.09771 (10) 0.0706 (9)
H11 0.1836 0.8506 1.1053 0.085*
C12 0.15458 (16) 0.93302 (15) 1.06737 (9) 0.0561 (8)
C13 0.34061 (18) 1.0494 (2) 1.03341 (11) 0.0738 (9)
C14 0.3673 (2) 1.1078 (2) 1.05590 (13) 0.1009 (12)
H14 0.3404 1.1474 1.0587 0.121*
C15 0.4327 (2) 1.1063 (3) 1.07356 (14) 0.1233 (17)
H15 0.4494 1.1449 1.0885 0.148*
C16 0.4732 (2) 1.0498 (3) 1.06961 (15) 0.1199 (16)
H16 0.5172 1.0495 1.0819 0.144*
C17 0.4490 (2) 0.9930 (3) 1.04737 (13) 0.1051 (13)
H17 0.4772 0.9544 1.0445 0.126*
C18 0.38262 (17) 0.9922 (2) 1.02892 (11) 0.0764 (10)
C19 0.36133 (19) 0.9318 (2) 1.00561 (11) 0.0781 (10)
H19 0.3945 0.8974 1.0026 0.094*
C20 0.20828 (17) 0.98920 (16) 0.89763 (11) 0.0678 (9)
H20 0.2235 1.0028 0.8698 0.081*
C21 0.09974 (16) 1.02899 (16) 1.02768 (10) 0.0660 (8)
H21 0.0631 1.0600 1.0285 0.079*
C22 0.14568 (17) 1.10734 (17) 0.97424 (12) 0.0844 (10)
H22A 0.1397 1.0951 0.9433 0.101*
H22B 0.1903 1.1297 0.9769 0.101*
C23 0.0919 (2) 1.15896 (17) 0.98566 (12) 0.0889 (11)
H23A 0.0971 1.1730 1.0163 0.107*
H23B 0.0465 1.1386 0.9821 0.107*
C24 0.0987 (2) 1.22091 (17) 0.95597 (13) 0.1048 (13)
H24A 0.1464 1.2340 0.9539 0.157*
H24B 0.0725 1.2586 0.9680 0.157*
H24C 0.0814 1.2098 0.9270 0.157*
C25 0.3097 (2) 1.0451 (3) 0.92058 (14) 0.1238 (15)
H25A 0.3341 1.0515 0.9483 0.149*
H25B 0.3373 1.0136 0.9029 0.149*
C26 0.3106 (3) 1.1023 (3) 0.9010 (2) 0.126 (3)
H26A 0.2831 1.1346 0.9182 0.240*
H26B 0.2878 1.0966 0.8727 0.240*
C27 0.3813 (2) 1.1356 (2) 0.89256 (15) 0.1331 (17)
H27A 0.3935 1.1643 0.9173 0.200*
H27B 0.3792 1.1632 0.8662 0.200*
H27C 0.4154 1.1001 0.8890 0.200*
C28 0.29461 (18) 0.85217 (19) 0.96588 (12) 0.0870 (11)
H28A 0.3396 0.8358 0.9565 0.104*
H28B 0.2665 0.8584 0.9396 0.104*
C29 0.2615 (2) 0.7984 (2) 0.99533 (14) 0.1041 (13)
H29A 0.2164 0.8152 1.0043 0.125*
H29B 0.2541 0.7569 0.9779 0.125*
C30 0.3005 (2) 0.7793 (2) 1.03570 (18) 0.1528 (19)
H30A 0.3439 0.7591 1.0274 0.229*
H30B 0.2743 0.7465 1.0526 0.229*
H30C 0.3087 0.8198 1.0532 0.229*
N1 0.30265 (14) 0.91895 (14) 0.98831 (8) 0.0679 (7)
N2 0.24462 (14) 1.00853 (13) 0.93127 (9) 0.0697 (7)
N3 0.14842 (12) 1.04268 (13) 0.99995 (8) 0.0635 (7)
O1 0.27883 (11) 1.05426 (11) 1.01689 (7) 0.0753 (6)
O2 0.21526 (10) 0.94675 (11) 1.05183 (6) 0.0651 (6)
O3 0.16501 (10) 0.91380 (10) 0.97551 (6) 0.0622 (5)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Fe1 0.0520 (3) 0.0673 (3) 0.0527 (3) 0.0034 (2) 0.0019 (2) −0.0006 (2)
C1 0.068 (2) 0.0595 (19) 0.0458 (18) 0.0098 (17) 0.0056 (16) −0.0015 (15)
C2 0.092 (3) 0.071 (2) 0.049 (2) 0.010 (2) 0.0032 (18) −0.0026 (16)
C3 0.086 (3) 0.086 (3) 0.063 (2) 0.001 (2) −0.011 (2) −0.0124 (19)
C4 0.064 (2) 0.079 (2) 0.084 (3) 0.0000 (18) −0.007 (2) −0.021 (2)
C5 0.066 (2) 0.062 (2) 0.066 (2) 0.0029 (17) 0.0066 (17) −0.0042 (16)
C6 0.060 (2) 0.0522 (18) 0.0517 (19) 0.0087 (15) 0.0042 (16) −0.0050 (15)
C7 0.064 (2) 0.0522 (18) 0.0571 (19) 0.0062 (16) 0.0055 (16) −0.0028 (15)
C8 0.070 (2) 0.064 (2) 0.074 (2) 0.0055 (17) 0.0133 (18) −0.0013 (18)
C9 0.085 (3) 0.068 (2) 0.070 (2) −0.005 (2) 0.0171 (19) −0.0010 (19)
C10 0.104 (3) 0.064 (2) 0.057 (2) −0.006 (2) 0.013 (2) 0.0015 (16)
C11 0.095 (3) 0.062 (2) 0.055 (2) 0.0135 (19) −0.0056 (19) −0.0051 (17)
C12 0.067 (2) 0.0582 (19) 0.0436 (17) 0.0072 (17) 0.0007 (16) −0.0091 (15)
C13 0.057 (2) 0.095 (3) 0.069 (2) −0.010 (2) 0.0062 (18) −0.006 (2)
C14 0.072 (3) 0.126 (3) 0.105 (3) −0.012 (2) 0.004 (2) −0.034 (3)
C15 0.074 (3) 0.184 (5) 0.112 (3) −0.033 (3) −0.001 (3) −0.050 (3)
C16 0.065 (3) 0.183 (5) 0.112 (4) −0.007 (3) −0.016 (3) −0.023 (4)
C17 0.061 (3) 0.153 (4) 0.101 (3) 0.001 (3) 0.000 (2) −0.001 (3)
C18 0.053 (2) 0.106 (3) 0.070 (2) 0.004 (2) 0.0030 (18) −0.002 (2)
C19 0.066 (2) 0.095 (3) 0.074 (2) 0.019 (2) 0.013 (2) 0.002 (2)
C20 0.074 (2) 0.077 (2) 0.052 (2) 0.0004 (19) 0.0096 (17) 0.0060 (17)
C21 0.060 (2) 0.064 (2) 0.074 (2) 0.0074 (17) 0.0052 (17) 0.0079 (17)
C22 0.075 (2) 0.079 (2) 0.099 (3) 0.005 (2) 0.015 (2) 0.027 (2)
C23 0.104 (3) 0.074 (2) 0.089 (3) 0.007 (2) −0.005 (2) 0.002 (2)
C24 0.130 (3) 0.065 (2) 0.120 (3) 0.001 (2) −0.008 (3) 0.021 (2)
C25 0.127 (4) 0.144 (4) 0.100 (3) −0.035 (3) −0.002 (3) 0.037 (3)
C26 0.148 (8) 0.122 (7) 0.110 (7) −0.007 (6) −0.014 (6) −0.005 (6)
C27 0.124 (4) 0.118 (3) 0.157 (4) −0.063 (3) 0.028 (3) 0.000 (3)
C28 0.080 (3) 0.092 (3) 0.089 (3) 0.021 (2) 0.007 (2) −0.024 (2)
C29 0.108 (3) 0.076 (3) 0.129 (4) 0.014 (2) 0.000 (3) 0.007 (3)
C30 0.139 (4) 0.138 (4) 0.182 (5) 0.003 (3) −0.040 (4) 0.040 (4)
N1 0.0608 (17) 0.083 (2) 0.0596 (16) 0.0104 (15) 0.0072 (14) −0.0056 (14)
N2 0.0630 (17) 0.0785 (19) 0.0675 (18) −0.0054 (15) 0.0064 (15) 0.0089 (15)
N3 0.0560 (15) 0.0691 (17) 0.0653 (17) 0.0024 (13) 0.0007 (13) 0.0120 (14)
O1 0.0568 (14) 0.0766 (15) 0.0925 (17) 0.0006 (12) −0.0033 (12) −0.0077 (12)
O2 0.0572 (13) 0.0848 (15) 0.0532 (12) 0.0147 (11) −0.0012 (10) −0.0047 (11)
O3 0.0650 (13) 0.0710 (14) 0.0506 (12) −0.0013 (10) −0.0026 (10) 0.0063 (10)
Geometric parameters (Å, °)
Fe1—O3 1.846 (2) C17—C18 1.402 (5)
Fe1—O1 1.882 (2) C17—H17 0.930
Fe1—O2 1.913 (2) C18—C19 1.425 (5)
Fe1—N1 1.917 (3) C19—N1 1.276 (4)
Fe1—N3 1.930 (2) C19—H19 0.930
Fe1—N2 1.969 (3) C20—N2 1.292 (4)
C1—C20 1.412 (4) C20—H20 0.930
C1—C2 1.418 (4) C21—N3 1.289 (3)
C1—C6 1.417 (4) C21—H21 0.930
C2—C3 1.343 (4) C22—N3 1.475 (4)
C2—H2 0.930 C22—C23 1.484 (4)
C3—C4 1.392 (4) C22—H22A 0.970
C3—H3 0.930 C22—H22B 0.970
C4—C5 1.378 (4) C23—C24 1.504 (4)
C4—H4 0.930 C23—H23A 0.970
C5—C6 1.396 (4) C23—H23B 0.970
C5—H5 0.930 C24—H24A 0.960
C6—O3 1.312 (3) C24—H24B 0.960
C7—C8 1.381 (4) C24—H24C 0.960
C7—C12 1.399 (4) C25—C26 1.257 (5)
C7—C21 1.428 (4) C25—N2 1.481 (5)
C8—C9 1.370 (4) C25—H25A 0.970
C8—H8 0.930 C25—H25B 0.970
C9—C10 1.348 (4) C26—C27 1.535 (6)
C9—H9 0.930 C26—H26A 0.970
C10—C11 1.378 (4) C26—H26B 0.970
C10—H10 0.930 C27—H27A 0.960
C11—C12 1.418 (4) C27—H27B 0.960
C11—H11 0.930 C27—H27C 0.960
C12—O2 1.293 (3) C28—N1 1.469 (4)
C13—O1 1.300 (4) C28—C29 1.514 (5)
C13—C18 1.382 (5) C28—H28A 0.970
C13—C14 1.418 (5) C28—H28B 0.970
C14—C15 1.376 (5) C29—C30 1.482 (5)
C14—H14 0.930 C29—H29A 0.970
C15—C16 1.352 (6) C29—H29B 0.970
C15—H15 0.930 C30—H30A 0.960
C16—C17 1.372 (5) C30—H30B 0.960
C16—H16 0.930 C30—H30C 0.960
O3—Fe1—O1 171.58 (9) N2—C20—C1 125.5 (3)
O3—Fe1—O2 86.80 (8) N2—C20—H20 117.2
O1—Fe1—O2 88.13 (9) C1—C20—H20 117.2
O3—Fe1—N1 91.50 (11) N3—C21—C7 127.4 (3)
O1—Fe1—N1 94.86 (11) N3—C21—H21 116.3
O2—Fe1—N1 86.14 (9) C7—C21—H21 116.3
O3—Fe1—N3 87.97 (10) N3—C22—C23 118.4 (3)
O1—Fe1—N3 85.46 (10) N3—C22—H22A 107.7
O2—Fe1—N3 91.56 (9) C23—C22—H22A 107.7
N1—Fe1—N3 177.67 (10) N3—C22—H22B 107.7
O3—Fe1—N2 91.93 (10) C23—C22—H22B 107.7
O1—Fe1—N2 93.83 (11) H22A—C22—H22B 107.1
O2—Fe1—N2 173.65 (9) C22—C23—C24 109.7 (3)
N1—Fe1—N2 87.67 (10) C22—C23—H23A 109.7
N3—Fe1—N2 94.62 (10) C24—C23—H23A 109.7
C20—C1—C2 118.8 (3) C22—C23—H23B 109.7
C20—C1—C6 121.0 (3) C24—C23—H23B 109.7
C2—C1—C6 120.2 (3) H23A—C23—H23B 108.2
C3—C2—C1 121.6 (3) C23—C24—H24A 109.5
C3—C2—H2 119.2 C23—C24—H24B 109.5
C1—C2—H2 119.2 H24A—C24—H24B 109.5
C2—C3—C4 118.4 (3) C23—C24—H24C 109.5
C2—C3—H3 120.8 H24A—C24—H24C 109.5
C4—C3—H3 120.8 H24B—C24—H24C 109.5
C5—C4—C3 121.8 (3) C26—C25—N2 122.4 (5)
C5—C4—H4 119.1 C26—C25—H25A 106.7
C3—C4—H4 119.1 N2—C25—H25A 106.7
C4—C5—C6 121.2 (3) C26—C25—H25B 106.7
C4—C5—H5 119.4 N2—C25—H25B 106.7
C6—C5—H5 119.4 H25A—C25—H25B 106.6
O3—C6—C5 118.6 (3) C25—C26—C27 117.4 (5)
O3—C6—C1 124.7 (3) C25—C26—H26A 108.0
C5—C6—C1 116.7 (3) C27—C26—H26A 107.9
C8—C7—C12 120.2 (3) C25—C26—H26B 107.9
C8—C7—C21 119.2 (3) C27—C26—H26B 107.9
C12—C7—C21 120.5 (3) H26A—C26—H26B 107.2
C9—C8—C7 122.0 (3) C26—C27—H27A 109.5
C9—C8—H8 119.0 C26—C27—H27B 109.5
C7—C8—H8 119.0 H27A—C27—H27B 109.5
C10—C9—C8 118.8 (3) C26—C27—H27C 109.5
C10—C9—H9 120.6 H27A—C27—H27C 109.5
C8—C9—H9 120.6 H27B—C27—H27C 109.5
C9—C10—C11 121.5 (3) N1—C28—C29 112.3 (3)
C9—C10—H10 119.2 N1—C28—H28A 109.1
C11—C10—H10 119.2 C29—C28—H28A 109.1
C10—C11—C12 120.9 (3) N1—C28—H28B 109.1
C10—C11—H11 119.6 C29—C28—H28B 109.1
C12—C11—H11 119.6 H28A—C28—H28B 107.9
O2—C12—C7 123.5 (3) C30—C29—C28 116.0 (4)
O2—C12—C11 119.9 (3) C30—C29—H29A 108.3
C7—C12—C11 116.5 (3) C28—C29—H29A 108.3
O1—C13—C18 124.2 (3) C30—C29—H29B 108.3
O1—C13—C14 117.5 (4) C28—C29—H29B 108.3
C18—C13—C14 118.3 (3) H29A—C29—H29B 107.4
C15—C14—C13 120.3 (4) C29—C30—H30A 109.5
C15—C14—H14 119.9 C29—C30—H30B 109.5
C13—C14—H14 119.9 H30A—C30—H30B 109.5
C14—C15—C16 121.2 (4) C29—C30—H30C 109.5
C14—C15—H15 119.4 H30A—C30—H30C 109.5
C16—C15—H15 119.4 H30B—C30—H30C 109.5
C15—C16—C17 119.6 (4) C19—N1—C28 117.1 (3)
C15—C16—H16 120.2 C19—N1—Fe1 122.2 (2)
C17—C16—H16 120.2 C28—N1—Fe1 120.6 (2)
C16—C17—C18 121.2 (4) C20—N2—C25 115.5 (3)
C16—C17—H17 119.4 C20—N2—Fe1 124.7 (2)
C18—C17—H17 119.4 C25—N2—Fe1 119.1 (2)
C17—C18—C13 119.5 (4) C21—N3—C22 119.4 (3)
C17—C18—C19 118.1 (4) C21—N3—Fe1 121.1 (2)
C13—C18—C19 122.4 (3) C22—N3—Fe1 119.5 (2)
N1—C19—C18 128.3 (3) C13—O1—Fe1 126.2 (2)
N1—C19—H19 115.8 C12—O2—Fe1 120.76 (18)
C18—C19—H19 115.8 C6—O3—Fe1 126.11 (18)
==== Refs
References
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Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Caruso, U., Centore, R., Panunzi, B., Roviello, A. & Tuzi, A. (2005). Eur. J. Inorg. Chem. pp. 2747–2758.
Iskander, M. F., Khalil, T. E., Haase, W., Werner, R., Svoboda, I. & Fuess, H. (2001). Polyhedron, 20, 2787–2792.
Li, S., Wang, S.-B., Tang, K. & Ma, Y.-F. (2008). Acta Cryst. E64, m823.
Rajak, K. K., Baruah, B., Rath, S. P. & Chakravorty, A. (2000). Inorg. Chem.39, 1598–1605.
Sangeetha, N. R. & Pal, S. (2000). Bull. Chem. Soc. Jpn, 73, 357–361.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sutradhar, M., Mukherjee, G., Drew, M. G. B. & Ghosh, S. (2006). Inorg. Chem.45, 5150–5158. | 21202804 | PMC2961833 | CC BY | 2021-01-04 18:56:08 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jun 25; 64(Pt 7):m955 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography cf220510.1107/S1600536808017893ACSEBHS1600536808017893Metal-Organic PapersDiaqua-1κO,3κO-di-μ-cyanido-1:2κ2
N:C;2:3κ2
C:N-dicyanido-2κ2
C-bis{4,4′-dibromo-2,2′-[propane-1,2-diylbis(nitrilomethylidyne)]diphenolato}-1κ4
O,N,N′,O′;3κ4
O,N,N′,O′-1,3-diiron(III)-2-nickel(II) [Fe2Ni(C17H14Br2N2O2)2(CN)4(H2O)2]Zhang Xiutang aWei Peihai a*Li Bin aa Department of Chemistry and Chemical Engineering, ShanDong Institute of Education, Jinan 250013, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2008 19 6 2008 19 6 2008 64 Pt 7 e080700m926 m926 09 6 2008 12 6 2008 © Zhang et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Fe2Ni(C17H14Br2N2O2)2(CN)4(H2O)2] or [{Fe(C17H14Br2N2O2)(H2O)}2(μ-CN)2{Ni(CN)2}], is isostructural with its MnIII-containing analogue. Each FeIII atom is chelated by a Schiff base ligand via two N and two O atoms and is additionally coordinated by a water molecule, forming a slightly distorted octahedral geometry. The two FeIII centres are bridged by a square-planar Ni(CN)4 unit, which lies on an inversion centre. A two-dimensional network is formed via O—H⋯O and O—H⋯N hydrogen bonds.
==== Body
Related literature
For related literature, see: Kuang et al. (2002 ▶); Kuchar et al. (2003 ▶); Yang et al. (2003 ▶). For the isostructural MnIII-containing compound, see: Sun et al. (2008 ▶).
Experimental
Crystal data
[Fe2Ni(C17H14Br2N2O2)2(CN)4(H2O)2]
M
r = 1186.71
Monoclinic,
a = 11.599 (2) Å
b = 13.538 (3) Å
c = 14.715 (3) Å
β = 112.04 (3)°
V = 2141.8 (7) Å3
Z = 2
Mo Kα radiation
μ = 4.89 mm−1
T = 293 (2) K
0.10 × 0.10 × 0.10 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.449, T
max = 0.641
13404 measured reflections
3699 independent reflections
2263 reflections with I > 2σ(I)
R
int = 0.085
Refinement
R[F
2 > 2σ(F
2)] = 0.066
wR(F
2) = 0.181
S = 1.00
3699 reflections
276 parameters
3 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.96 e Å−3
Δρmin = −0.64 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL).
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808017893/cf2205sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808017893/cf2205Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: CF2205).
The authors thank the National Ministry of Science and Technology of China (grant No. 2001CB6105–07).
supplementary crystallographic
information
Comment
Cyanide-bridged oligonuclear complexes with chain-like arrangements of metal
ions and cyanide ligands have been studied for a long time due to the good
electronic conductivity between the metallic groups (Kuang
et al., 2002; Kuchar et al., 2003; Yang et
al., 2003). In
this context, bulk properties such as magnetism, luminescence, electrical
conductivity resulting from metal-metal charge transfer like multi-redox steps,
mixed valence and long-range electronic interactions prompted us to
report our research work on cyanide-bridged complexes. In this paper, we
report the structure of the title compound, (I). It is isostructural with
its MnIII-containing analogue (Sun et al., 2008).
As shown in Fig. 1, each FeIII atom is chelated by a Schiff base ligand
via two N and two O atoms and is additionally coordinated by a water
molecule, forming a slightly distorted octahedral geometry. The
Schiff base lies in the equatorial plane, and the cyanido and aqua ligands lie
in the axial coordination sites. The Fe—N and Fe—O axial bond lengths are
much longer than the equatorial ones. A centrosymmetric square-planar
Ni(CN)4 unit links two FeIII centres. With O—H···O and O—H···N hydrogen
bonds, a two-dimensional network is formed, as shown in Fig. 2.
Experimental
A mixture of iron(III) acetylacetonate (1 mmol),
N,N'-bis(2-hydroxy-5-bromobenzyl)-1,2-diaminopropane (1 mmol),
and dipotassium tetracyanidonickelate(II) (1 mmol) in 20 ml methanol was
refluxed for several hours. The cooled solution was filtered and the filtrate
was kept in an ice box. One week later, brown blocks of (I) were obtained
with a yield of 5%. Anal. Calc. for C38H32Br4Fe2N8NiO6: C 38.43, H
2.70, N 9.44%; Found: C 38.40, H 2.63, N 9.39.
Refinement
All C-bound H atoms were placed in calculated positions with C—H = 0.93 Å
and refined as riding with Uiso(H) = 1.2Ueq(C). H atoms on
the aqua ligand were located in a difference density map and were refined with
the distance restraint O—H = 0.82 (1) Å.
Figures
Fig. 1. The molecular structure of (I), drawn with 30% probability displacement ellipsoids for the non-hydrogen atoms. [Symmetry code for unlabelled atoms: -x, 2-y, -z.]
Fig. 2. Two-dimensional network formed by hydrogen bonds (dashed lines).
Crystal data
[Fe2Ni(C17H14Br2N2O2)2(CN)4(H2O)2] F000 = 1168
Mr = 1186.71 Dx = 1.840 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 3699 reflections
a = 11.599 (2) Å θ = 3.0–25.1º
b = 13.538 (3) Å µ = 4.89 mm−1
c = 14.715 (3) Å T = 293 (2) K
β = 112.04 (3)º Block, brown
V = 2141.8 (7) Å3 0.10 × 0.10 × 0.10 mm
Z = 2
Data collection
Bruker APEXII CCD diffractometer 3699 independent reflections
Radiation source: fine-focus sealed tube 2263 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.085
T = 293(2) K θmax = 25.1º
φ and ω scans θmin = 3.0º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −13→12
Tmin = 0.449, Tmax = 0.641 k = −16→15
13404 measured reflections l = −17→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.066 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.181 w = 1/[σ2(Fo2) + (0.09P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
3699 reflections Δρmax = 0.96 e Å−3
276 parameters Δρmin = −0.64 e Å−3
3 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Fe1 0.29547 (11) 0.95323 (8) 0.36728 (8) 0.0341 (4)
Ni1 0.0000 1.0000 0.0000 0.0334 (4)
Br1 −0.07330 (10) 1.37221 (7) 0.43825 (7) 0.0569 (4)
Br2 0.75936 (10) 0.61244 (8) 0.30711 (8) 0.0621 (4)
C1 0.1210 (8) 0.9936 (5) 0.1261 (6) 0.035 (2)
C2 −0.0637 (8) 0.8804 (6) 0.0276 (6) 0.037 (2)
C3 0.2234 (8) 1.1459 (6) 0.4134 (6) 0.034 (2)
C4 0.2476 (8) 1.2492 (5) 0.4247 (5) 0.033 (2)
H4 0.3242 1.2728 0.4277 0.039*
C5 0.1608 (9) 1.3146 (6) 0.4311 (6) 0.042 (2)
H5 0.1780 1.3819 0.4362 0.050*
C6 0.0471 (9) 1.2807 (6) 0.4303 (6) 0.042 (2)
C7 0.0185 (9) 1.1818 (6) 0.4197 (6) 0.045 (2)
H7 −0.0581 1.1603 0.4187 0.054*
C8 0.1029 (8) 1.1136 (5) 0.4104 (6) 0.037 (2)
C9 0.0680 (8) 1.0105 (6) 0.3966 (6) 0.035 (2)
H9 −0.0079 0.9939 0.4004 0.042*
C10 0.0874 (10) 0.8350 (7) 0.3700 (9) 0.067 (3)
H10A 0.1154 0.8044 0.4343 0.080*
H10B −0.0028 0.8331 0.3424 0.080*
C11 0.1355 (9) 0.7815 (7) 0.3082 (9) 0.067 (3)
H11 0.0893 0.8088 0.2429 0.080*
C12 0.1048 (10) 0.6739 (6) 0.2961 (8) 0.060 (3)
H12A 0.1567 0.6390 0.3537 0.091*
H12B 0.1188 0.6491 0.2400 0.091*
H12C 0.0191 0.6646 0.2869 0.091*
C13 0.3443 (8) 0.7546 (5) 0.3198 (5) 0.032 (2)
H13 0.3196 0.6893 0.3047 0.039*
C14 0.4688 (8) 0.7786 (6) 0.3302 (5) 0.033 (2)
C15 0.5437 (9) 0.7030 (6) 0.3193 (5) 0.038 (2)
H15 0.5141 0.6384 0.3116 0.046*
C16 0.6591 (9) 0.7209 (7) 0.3197 (6) 0.049 (3)
C17 0.7053 (9) 0.8158 (7) 0.3289 (6) 0.048 (2)
H17 0.7829 0.8280 0.3262 0.058*
C18 0.6337 (8) 0.8932 (6) 0.3422 (6) 0.039 (2)
H18 0.6657 0.9570 0.3509 0.047*
C19 0.5155 (8) 0.8770 (6) 0.3428 (5) 0.033 (2)
N1 0.1906 (7) 0.9903 (4) 0.2063 (5) 0.0368 (18)
N2 −0.0938 (7) 0.8039 (5) 0.0441 (5) 0.046 (2)
N3 0.1306 (6) 0.9396 (5) 0.3796 (5) 0.0400 (18)
N4 0.2649 (6) 0.8131 (4) 0.3289 (4) 0.0294 (16)
O1 0.4524 (5) 0.9530 (3) 0.3561 (4) 0.0309 (13)
O2 0.3095 (5) 1.0870 (4) 0.4047 (4) 0.0308 (13)
O3 0.3783 (5) 0.9024 (4) 0.5250 (4) 0.0352 (14)
H1W 0.433 (5) 0.942 (3) 0.547 (6) 0.042*
H2W 0.397 (6) 0.8444 (16) 0.530 (6) 0.042*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Fe1 0.0365 (8) 0.0271 (7) 0.0247 (6) 0.0005 (5) −0.0045 (5) −0.0009 (5)
Ni1 0.0368 (9) 0.0254 (8) 0.0201 (7) −0.0002 (6) −0.0101 (6) 0.0006 (6)
Br1 0.0723 (8) 0.0473 (6) 0.0454 (6) 0.0242 (5) 0.0155 (5) −0.0023 (5)
Br2 0.0537 (7) 0.0690 (8) 0.0549 (7) 0.0211 (5) 0.0105 (5) −0.0137 (5)
C1 0.054 (6) 0.012 (4) 0.029 (5) −0.002 (4) 0.003 (4) 0.000 (3)
C2 0.036 (5) 0.032 (5) 0.024 (4) 0.002 (4) −0.010 (4) 0.000 (4)
C3 0.037 (5) 0.029 (4) 0.022 (4) 0.004 (4) −0.007 (4) −0.003 (3)
C4 0.039 (5) 0.031 (4) 0.018 (4) −0.008 (4) −0.001 (4) 0.001 (3)
C5 0.060 (7) 0.028 (5) 0.031 (5) 0.011 (5) 0.010 (5) −0.004 (4)
C6 0.056 (6) 0.026 (5) 0.032 (5) 0.006 (4) 0.005 (4) 0.001 (4)
C7 0.052 (6) 0.054 (6) 0.022 (4) 0.011 (5) 0.005 (4) −0.006 (4)
C8 0.044 (6) 0.030 (5) 0.024 (4) 0.009 (4) −0.002 (4) 0.001 (3)
C9 0.031 (5) 0.038 (5) 0.030 (4) 0.001 (4) 0.004 (4) −0.004 (4)
C10 0.064 (7) 0.043 (6) 0.104 (9) −0.016 (5) 0.045 (7) −0.025 (6)
C11 0.047 (7) 0.040 (6) 0.112 (10) −0.004 (5) 0.030 (7) −0.028 (6)
C12 0.059 (7) 0.039 (5) 0.076 (8) −0.008 (5) 0.017 (6) −0.008 (5)
C13 0.040 (5) 0.019 (4) 0.027 (4) 0.000 (4) −0.001 (4) −0.001 (3)
C14 0.034 (5) 0.034 (5) 0.020 (4) 0.009 (4) −0.003 (4) −0.008 (3)
C15 0.047 (6) 0.038 (5) 0.019 (4) 0.001 (4) −0.001 (4) 0.000 (3)
C16 0.053 (6) 0.052 (6) 0.025 (5) 0.020 (5) −0.004 (4) −0.009 (4)
C17 0.043 (6) 0.054 (6) 0.043 (6) −0.001 (5) 0.013 (5) −0.011 (5)
C18 0.042 (6) 0.045 (5) 0.025 (4) −0.003 (4) 0.006 (4) −0.004 (4)
C19 0.035 (5) 0.043 (5) 0.010 (4) 0.010 (4) −0.005 (3) −0.003 (3)
N1 0.042 (4) 0.026 (4) 0.024 (4) −0.007 (3) −0.009 (3) 0.000 (3)
N2 0.055 (5) 0.029 (4) 0.037 (4) −0.008 (4) −0.001 (4) −0.005 (3)
N3 0.038 (4) 0.033 (4) 0.043 (4) −0.003 (3) 0.007 (4) −0.011 (3)
N4 0.028 (4) 0.026 (4) 0.026 (4) 0.000 (3) 0.000 (3) −0.001 (3)
O1 0.031 (3) 0.028 (3) 0.023 (3) 0.002 (2) −0.001 (2) 0.001 (2)
O2 0.031 (3) 0.028 (3) 0.025 (3) 0.001 (2) 0.001 (2) 0.001 (2)
O3 0.040 (4) 0.025 (3) 0.025 (3) −0.004 (3) −0.004 (3) −0.003 (3)
Geometric parameters (Å, °)
Fe1—O2 1.882 (5) C9—H9 0.930
Fe1—O1 1.888 (6) C10—C11 1.430 (13)
Fe1—N4 1.973 (6) C10—N3 1.490 (11)
Fe1—N3 1.996 (7) C10—H10A 0.970
Fe1—O3 2.261 (5) C10—H10B 0.970
Fe1—N1 2.276 (6) C11—N4 1.478 (11)
Ni1—C1i 1.862 (8) C11—C12 1.494 (11)
Ni1—C1 1.862 (8) C11—H11 0.980
Ni1—C2 1.886 (9) C12—H12A 0.960
Ni1—C2i 1.886 (9) C12—H12B 0.960
Br1—C6 1.903 (9) C12—H12C 0.960
Br2—C16 1.924 (9) C13—N4 1.260 (9)
C1—N1 1.154 (10) C13—C14 1.431 (11)
C2—N2 1.148 (9) C13—H13 0.930
C3—O2 1.322 (9) C14—C15 1.390 (11)
C3—C4 1.423 (10) C14—C19 1.424 (11)
C3—C8 1.449 (12) C15—C16 1.359 (13)
C4—C5 1.371 (11) C15—H15 0.930
C4—H4 0.930 C16—C17 1.378 (12)
C5—C6 1.393 (13) C17—C18 1.396 (12)
C5—H5 0.930 C17—H17 0.930
C6—C7 1.374 (11) C18—C19 1.392 (12)
C7—C8 1.389 (12) C18—H18 0.930
C7—H7 0.930 C19—O1 1.318 (9)
C8—C9 1.445 (10) O3—H1W 0.80 (6)
C9—N3 1.284 (10) O3—H2W 0.81 (2)
O2—Fe1—O1 92.7 (2) N3—C10—H10B 109.6
O2—Fe1—N4 174.5 (3) H10A—C10—H10B 108.2
O1—Fe1—N4 92.8 (3) C10—C11—N4 109.3 (8)
O2—Fe1—N3 92.5 (2) C10—C11—C12 115.9 (10)
O1—Fe1—N3 174.6 (2) N4—C11—C12 119.0 (8)
N4—Fe1—N3 82.0 (3) C10—C11—H11 103.5
O2—Fe1—O3 92.1 (2) N4—C11—H11 103.5
O1—Fe1—O3 92.1 (2) C12—C11—H11 103.5
N4—Fe1—O3 87.8 (2) C11—C12—H12A 109.5
N3—Fe1—O3 86.1 (3) C11—C12—H12B 109.5
O2—Fe1—N1 92.7 (2) H12A—C12—H12B 109.5
O1—Fe1—N1 93.8 (2) C11—C12—H12C 109.5
N4—Fe1—N1 86.9 (2) H12A—C12—H12C 109.5
N3—Fe1—N1 87.6 (3) H12B—C12—H12C 109.5
O3—Fe1—N1 172.3 (2) N4—C13—C14 126.5 (7)
C1i—Ni1—C1 180.0 (4) N4—C13—H13 116.8
C1i—Ni1—C2 92.6 (3) C14—C13—H13 116.8
C1—Ni1—C2 87.4 (3) C15—C14—C19 118.8 (8)
C1i—Ni1—C2i 87.4 (3) C15—C14—C13 118.0 (7)
C1—Ni1—C2i 92.6 (3) C19—C14—C13 123.0 (7)
C2—Ni1—C2i 180.000 (1) C16—C15—C14 121.7 (8)
N1—C1—Ni1 176.0 (9) C16—C15—H15 119.2
N2—C2—Ni1 174.3 (8) C14—C15—H15 119.2
O2—C3—C4 118.7 (8) C15—C16—C17 120.9 (9)
O2—C3—C8 124.7 (7) C15—C16—Br2 119.5 (7)
C4—C3—C8 116.5 (7) C17—C16—Br2 119.6 (8)
C5—C4—C3 121.7 (8) C16—C17—C18 118.9 (9)
C5—C4—H4 119.2 C16—C17—H17 120.5
C3—C4—H4 119.2 C18—C17—H17 120.5
C4—C5—C6 120.3 (8) C19—C18—C17 121.5 (8)
C4—C5—H5 119.9 C19—C18—H18 119.2
C6—C5—H5 119.9 C17—C18—H18 119.2
C7—C6—C5 120.6 (8) O1—C19—C18 118.8 (8)
C7—C6—Br1 119.4 (7) O1—C19—C14 123.0 (8)
C5—C6—Br1 119.9 (6) C18—C19—C14 118.2 (8)
C6—C7—C8 120.7 (9) C1—N1—Fe1 165.6 (7)
C6—C7—H7 119.7 C9—N3—C10 122.3 (8)
C8—C7—H7 119.7 C9—N3—Fe1 125.4 (6)
C7—C8—C9 119.0 (9) C10—N3—Fe1 112.3 (6)
C7—C8—C3 120.2 (8) C13—N4—C11 121.5 (7)
C9—C8—C3 120.8 (7) C13—N4—Fe1 125.1 (6)
N3—C9—C8 126.9 (8) C11—N4—Fe1 113.4 (5)
N3—C9—H9 116.6 C19—O1—Fe1 128.4 (5)
C8—C9—H9 116.6 C3—O2—Fe1 128.5 (5)
C11—C10—N3 110.1 (8) Fe1—O3—H1W 100 (6)
C11—C10—H10A 109.6 Fe1—O3—H2W 112 (6)
N3—C10—H10A 109.6 H1W—O3—H2W 118 (4)
C11—C10—H10B 109.6
Symmetry codes: (i) −x, −y+2, −z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O3—H1W···O1ii 0.81 (2) 2.09 (4) 2.859 (7) 159 (8)
O3—H2W···N2iii 0.81 (2) 2.02 (2) 2.813 (9) 167 (7)
Symmetry codes: (ii) −x+1, −y+2, −z+1; (iii) x+1/2, −y+3/2, z+1/2.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O3—H1W⋯O1i 0.81 (2) 2.09 (4) 2.859 (7) 159 (8)
O3—H2W⋯N2ii 0.81 (2) 2.02 (2) 2.813 (9) 167 (7)
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Kuang, S. M., Fanwick, P. E. & Walton, R. A. (2002). Inorg. Chem.41, 147–151.
Kuchar, J., Cernak, J., Zak, Z. & Massa, W. (2003). Monogr. Ser. Int. Conf. Coord. Chem.6, 127–132.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Sun, Z.-H., Yang, G.-B., Meng, L.-B. & Chen, S. (2008). Acta Cryst. E64, m783.
Yang, J. Y., Shores, M. P., Sokol, J. J. & Long, J. R. (2003). Inorg. Chem.42, 1403–1408. | 21202781 | PMC2961845 | CC BY | 2021-01-04 18:56:07 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jun 19; 64(Pt 7):m926 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb274410.1107/S1600536808017984ACSEBHS1600536808017984Metal-Organic PapersDiazidobis(2,2′-biimidazole)manganese(II) [Mn(N3)2(C6H6N4)2]Zhang Xiutang aWei Peihai a*Li Bin aa Department of Chemistry and Chemical Engineering, ShanDong Institute of Education, Jinan 250013, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2008 19 6 2008 19 6 2008 64 Pt 7 e080700m934 m934 09 6 2008 12 6 2008 © Zhang et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Mn(N3)2(C6H6N4)2], the Mn atom (site symmetry ) is bonded to two azide ions and two bidentate biimidizole ligands, resulting in a slightly distorted octahedral MnN6 geometry for the metal ion. In the crystal structure, N—H⋯N hydrogen bonds help to consolidate the packing.
==== Body
Related literature
For a related structure, see: Hester et al. (1997 ▶).
Experimental
Crystal data
[Mn(N3)2(C6H6N4)2]
M
r = 407.30
Monoclinic,
a = 12.5097 (10) Å
b = 8.9728 (5) Å
c = 14.1416 (10) Å
β = 91.883 (10)°
V = 1586.50 (19) Å3
Z = 4
Mo Kα radiation
μ = 0.87 mm−1
T = 293 (2) K
0.40 × 0.26 × 0.20 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2004 ▶) T
min = 0.723, T
max = 0.846
1966 measured reflections
1505 independent reflections
1250 reflections with I > 2σ(I)
R
int = 0.022
Refinement
R[F
2 > 2σ(F
2)] = 0.038
wR(F
2) = 0.131
S = 1.00
1505 reflections
131 parameters
2 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.49 e Å−3
Δρmin = −0.25 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2004 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808017984/hb2744sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808017984/hb2744Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB2744).
The authors thank the National Ministry of Science and Technology of China (grant No. 2001CB6105-07).
supplementary crystallographic
information
Comment
The study of coordination compounds including one-, two- and three-dimensional
infinite frameworks has been expanding rapidly because of their fascinating
structural diversity and potential application as functional materials. To
date, much of the work has been focused on coordination polymers with
semi-rigid ligands, such as 4,4'-bipyridine, pyrazine and their analogues. In
this paper, we report the structure of the molecular title compound, (I), with
the use of the 2,2'-biimidazole bridging ligand (Hester et al.,
1997).
As shown in Fig. 1, the Mn ion in (I) occupies an inversion centre, and is
hexacoordinated by six N atoms from two chelating ligands of H2bim
(biimidizole; C6H6N4) and two azide ions, showing a slightly distorted
MnN6 octahedral geometry (Table 1).
In the crystal of (I), N—H···N hydrogen bonds, one of which
is bifurcated (Table 2), help to consolidate the packing.
Experimental
A mixture of manganese(II) perchlorate hexahydrate (1 mmol), 2,2'-biimidazoline
(2 mmol) and Na3N3 (2 mmol) in 20 ml ethanol was reflued for several hours.
The cooled solution was filtered and the filtrate was kept in an ice box for
about one week. Yellow blocks of (I) were obtained with a yield of 10%. Anal.
Calc. for C12H12MnN14: C 35.35, H 2.95, N 48.12%; Found: C 35.31, H 2.92,
N 48.06%.
Refinement
The N-bound H atoms were located in a difference map and their positions were
freely refined with Uiso(H) = 1.2Ueq(N). The C-bound H atoms
were placed in calculated positions (C—H = 0.93 Å) and refined as riding
with Uiso(H) = 1.2Ueq(C).
Figures
Fig. 1. The molecular structure of (I), drawn with 30% probability displacement ellipsoids for the non-hydrogen atoms. Symmetry code: (i) 3/2-x, 3/2-y, 1-z.
Crystal data
[Mn(N3)2(C6H6N4)2] F000 = 828
Mr = 407.30 Dx = 1.705 Mg m−3
Monoclinic, C2/c Mo Kα radiation λ = 0.71073 Å
Hall symbol: -C 2yc Cell parameters from 1505 reflections
a = 12.5097 (10) Å θ = 2.8–25.9º
b = 8.9728 (5) Å µ = 0.87 mm−1
c = 14.1416 (10) Å T = 293 (2) K
β = 91.883 (10)º Block, yellow
V = 1586.50 (19) Å3 0.40 × 0.26 × 0.20 mm
Z = 4
Data collection
Bruker APEXII CCD diffractometer 1505 independent reflections
Radiation source: fine-focus sealed tube 1250 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.022
T = 293(2) K θmax = 25.9º
φ and ω scans θmin = 2.8º
Absorption correction: multi-scan(SADABS; Bruker, 2004) h = −1→15
Tmin = 0.723, Tmax = 0.846 k = −1→10
1966 measured reflections l = −17→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.038 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.131 w = 1/[σ2(Fo2) + (0.081P)2 + 1.7249P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max = 0.024
1505 reflections Δρmax = 0.49 e Å−3
131 parameters Δρmin = −0.25 e Å−3
2 restraints Extinction correction: none
Primary atom site location: structure-invariant direct methods
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Mn1 0.7500 0.7500 0.5000 0.0486 (5)
C1 0.5842 (3) 0.5617 (4) 0.3628 (2) 0.0572 (8)
H1 0.5711 0.6280 0.3132 0.069*
C2 0.5366 (3) 0.4245 (4) 0.3721 (2) 0.0585 (9)
H2 0.4865 0.3819 0.3303 0.070*
C3 0.6462 (2) 0.4631 (4) 0.4891 (2) 0.0482 (7)
C4 0.7106 (2) 0.4562 (4) 0.5744 (2) 0.0489 (7)
C5 0.8223 (3) 0.5322 (4) 0.6783 (2) 0.0580 (8)
H5 0.8713 0.5915 0.7118 0.070*
C6 0.7940 (3) 0.3903 (4) 0.7030 (2) 0.0610 (9)
H6 0.8195 0.3369 0.7554 0.073*
N1 0.5763 (2) 0.3633 (3) 0.45300 (18) 0.0534 (7)
H1A 0.546 (3) 0.270 (3) 0.474 (3) 0.064*
N2 0.6529 (2) 0.5851 (3) 0.43706 (17) 0.0520 (7)
N3 0.7688 (2) 0.5733 (3) 0.59802 (18) 0.0525 (7)
N4 0.7220 (2) 0.3432 (3) 0.63640 (19) 0.0551 (7)
H4 0.680 (3) 0.255 (3) 0.634 (3) 0.066*
N5 0.8833 (2) 0.6634 (3) 0.4268 (2) 0.0529 (7)
N6 0.8963 (2) 0.5320 (3) 0.4212 (2) 0.0552 (7)
N7 0.9109 (3) 0.4018 (3) 0.4143 (2) 0.0723 (9)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Mn1 0.0504 (12) 0.0400 (13) 0.0550 (13) 0.0096 (10) −0.0016 (10) −0.0034 (10)
C1 0.0542 (18) 0.069 (2) 0.0481 (17) −0.0156 (16) −0.0081 (14) 0.0038 (15)
C2 0.0551 (18) 0.070 (2) 0.0495 (17) −0.0171 (16) −0.0056 (14) −0.0031 (15)
C3 0.0485 (16) 0.0500 (18) 0.0462 (15) −0.0087 (13) 0.0012 (12) −0.0003 (13)
C4 0.0481 (15) 0.0480 (17) 0.0503 (16) −0.0051 (13) 0.0012 (13) 0.0026 (13)
C5 0.0598 (19) 0.061 (2) 0.0527 (17) −0.0041 (16) −0.0095 (14) 0.0033 (15)
C6 0.064 (2) 0.067 (2) 0.0510 (18) −0.0005 (18) −0.0087 (15) 0.0092 (16)
N1 0.0539 (15) 0.0539 (16) 0.0525 (15) −0.0158 (13) 0.0020 (12) −0.0012 (12)
N2 0.0512 (14) 0.0569 (17) 0.0477 (14) −0.0117 (13) −0.0035 (11) 0.0045 (12)
N3 0.0519 (15) 0.0543 (16) 0.0508 (14) −0.0088 (13) −0.0063 (11) 0.0042 (12)
N4 0.0600 (16) 0.0524 (16) 0.0524 (14) −0.0079 (13) −0.0021 (12) 0.0077 (12)
N5 0.0585 (16) 0.0552 (17) 0.0444 (14) −0.0006 (14) −0.0080 (12) −0.0089 (12)
N6 0.0525 (15) 0.0540 (18) 0.0585 (16) −0.0129 (13) −0.0071 (12) 0.0069 (13)
N7 0.073 (2) 0.0512 (18) 0.092 (2) −0.0078 (15) −0.0108 (17) 0.0073 (16)
Geometric parameters (Å, °)
Mn1—N2 2.094 (3) C3—C4 1.430 (4)
Mn1—N2i 2.094 (3) C4—N3 1.315 (4)
Mn1—N3i 2.114 (3) C4—N4 1.345 (4)
Mn1—N3 2.114 (3) C5—N3 1.350 (4)
Mn1—N5 2.138 (3) C5—C6 1.370 (5)
Mn1—N5i 2.138 (3) C5—H5 0.9300
C1—N2 1.351 (4) C6—N4 1.350 (4)
C1—C2 1.375 (5) C6—H6 0.9300
C1—H1 0.9300 N1—H1A 0.966 (18)
C2—N1 1.349 (4) N4—H4 0.952 (19)
C2—H2 0.9300 N5—N6 1.193 (4)
C3—N2 1.323 (4) N6—N7 1.187 (4)
C3—N1 1.340 (4)
N2—Mn1—N2i 180.0) N3—C4—N4 113.0 (3)
N2—Mn1—N3i 101.59 (10) N3—C4—C3 118.2 (3)
N2i—Mn1—N3i 78.41 (10) N4—C4—C3 128.8 (3)
N2—Mn1—N3 78.41 (10) N3—C5—C6 110.1 (3)
N2i—Mn1—N3 101.59 (10) N3—C5—H5 125.0
N3i—Mn1—N3 180.0 C6—C5—H5 125.0
N2—Mn1—N5 89.31 (11) N4—C6—C5 106.5 (3)
N2i—Mn1—N5 90.69 (11) N4—C6—H6 126.7
N3i—Mn1—N5 91.53 (11) C5—C6—H6 126.7
N3—Mn1—N5 88.47 (11) C3—N1—C2 105.6 (3)
N2—Mn1—N5i 90.69 (11) C3—N1—H1A 135 (3)
N2i—Mn1—N5i 89.31 (11) C2—N1—H1A 119 (3)
N3i—Mn1—N5i 88.47 (11) C3—N2—C1 104.7 (3)
N3—Mn1—N5i 91.53 (11) C3—N2—Mn1 113.26 (19)
N5—Mn1—N5i 180.0 C1—N2—Mn1 141.7 (2)
N2—C1—C2 109.3 (3) C4—N3—C5 104.5 (3)
N2—C1—H1 125.3 C4—N3—Mn1 112.6 (2)
C2—C1—H1 125.3 C5—N3—Mn1 142.9 (2)
N1—C2—C1 107.3 (3) C4—N4—C6 105.8 (3)
N1—C2—H2 126.4 C4—N4—H4 124 (3)
C1—C2—H2 126.4 C6—N4—H4 130 (3)
N2—C3—N1 113.1 (3) N6—N5—Mn1 120.1 (2)
N2—C3—C4 117.4 (3) N7—N6—N5 178.6 (4)
N1—C3—C4 129.5 (3)
Symmetry codes: (i) −x+3/2, −y+3/2, −z+1.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
N1—H1A···N7ii 0.966 (18) 2.26 (3) 3.031 (4) 136 (3)
N1—H1A···N5iii 0.966 (18) 2.33 (4) 3.021 (4) 127 (3)
N4—H4···N7ii 0.952 (19) 1.92 (2) 2.834 (4) 160 (4)
Symmetry codes: (ii) −x+3/2, −y+1/2, −z+1; (iii) x−1/2, y−1/2, z.
Table 1 Selected bond lengths (Å)
Mn1—N2 2.094 (3)
Mn1—N3 2.114 (3)
Mn1—N5 2.138 (3)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
N1—H1A⋯N7i 0.966 (18) 2.26 (3) 3.031 (4) 136 (3)
N1—H1A⋯N5ii 0.966 (18) 2.33 (4) 3.021 (4) 127 (3)
N4—H4⋯N7i 0.952 (19) 1.92 (2) 2.834 (4) 160 (4)
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (2004). APEX2, SADABS and SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Hester, C. A., Baughman, R. G. & Collier, H. L. (1997). Polyhedron, 16, 2893–2895.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21202788 | PMC2961868 | CC BY | 2021-01-04 18:56:05 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jun 19; 64(Pt 7):m934 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography bi229310.1107/S1600536808021892ACSEBHS1600536808021892Metal-Organic PapersBis{μ-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato}bis[(thiocyanato-κN)iron(III)] [Fe2(C16H14N2O2)2(NCS)2]Hao Lujiang a*Mu Chunhua bKong Binbin aa College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Insitute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 8 2008 16 7 2008 16 7 2008 64 Pt 8 e080800m1034 m1034 28 6 2008 14 7 2008 © Hao et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Fe2(C16H14N2O2)2(NCS)2], is isostructural with the MnIII-containing analogue. Each FeIII atom is chelated by a tetradentate 2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolate ligand and by the N atom of a thiocyanate anion, in a square-pyramidal arrangement. The complex molecules form centrosymmetric dimers, with an Fe—O contact of 2.549 (3) Å, trans to each thiocyanate anion, completing a distorted octahedral coordination geometry.
==== Body
Related literature
For related literature, see: Garnovskii et al. (1993 ▶); Huang et al. (2002 ▶); Bhadbhade & Srinivas (1993 ▶); Bunce et al. (1998 ▶). For the isostructural MnIII-containing compound, see: Wang et al. (2008 ▶).
Experimental
Crystal data
[Fe2(C16H14N2O2)2(NCS)2]
M
r = 380.23
Monoclinic,
a = 8.9231 (10) Å
b = 14.0779 (10) Å
c = 14.9716 (10) Å
β = 106.844 (1)°
V = 1800.0 (3) Å3
Z = 4
Mo Kα radiation
μ = 0.97 mm−1
T = 293 (2) K
0.12 × 0.11 × 0.09 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.893, T
max = 0.918
12796 measured reflections
3191 independent reflections
2535 reflections with I > 2σ(I)
R
int = 0.023
Refinement
R[F
2 > 2σ(F
2)] = 0.058
wR(F
2) = 0.180
S = 1.00
3191 reflections
217 parameters
H-atom parameters constrained
Δρmax = 1.12 e Å−3
Δρmin = −0.33 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808021892/bi2293sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808021892/bi2293Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BI2293).
This work is supported by the Natural Science Foundation of Shandong Province (grant No. Y2007D39).
supplementary crystallographic
information
Comment
The design of Schiff-base complexes has received long-lasting research interest
not only because of their appealing structural and topological novelty but
also due to their potential medical value derived from their antiviral and the
inhibition of angiogenesis (Garnovskii et al., 1993; Huang et
al., 2002). The related Fe complexes with multidentate Schiff-base
ligands
have aroused particular interest because this metal can exhibit several
oxidation states and may provide the basis of models for active sites of
biological systems (Bhadbhade & Srinivas, 1993; Bunce et al.,
1998).
The title compound is isostructural with its MnIII-containing analogue (Wang
et al., 2008). Each FeIII atom is chelated by a tetradentate
2,2'-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolate ligand and by the N
atom of a thiocyanate anion, in a square-pyramidal arrangement. The maximum
atomic deviation from the least-square plane of the equatorially located
atoms, Fe1, N1, N2, O1 and O2, is 0.077 Å. The Fe—N(isothiocyanato) bond
length (2.178 (4) Å) is longer than the other two Fe—N bonds (1.985 (4) and
1.988 (4) Å). The complexes form centrosymmetric dimers, with an Fe—O
contact of 2.549 (3)Å trans to each thiocyanate anion, completing a
distorted octahedral coordination geometry.
Experimental
A mixture of iron(III) 2,4-pentanedionate (0.5 mmol),
N,N'-disalicylidene-ethylenediamine (0.5 mmoL), and sodium
isothiocyanate (1 mmoL) in 20 ml methanol was refluxed for two hours. The
resulting solution was cooled and filtered and the filtrate was evaporated
naturally at room temperature to yield brown blocks after a few days with a
yield of 11%. Elemental analysis calculated: C 53.65, H 3.68, N 11.05%; found:
C 53.60, H 3.64, N 11.02%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93 Å and
refined as riding with Uiso(H) = 1.2Ueq(C).
Figures
Fig. 1. The asymmetric unit drawn with 30% probability displacement ellipsoids for the non-H atoms.
Crystal data
[Fe2(C16H14N2O2)2(NCS)2] F000 = 780
Mr = 380.23 Dx = 1.403 Mg m−3
Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 4497 reflections
a = 8.9231 (10) Å θ = 2.4–24.4º
b = 14.0779 (10) Å µ = 0.97 mm−1
c = 14.9716 (10) Å T = 293 (2) K
β = 106.844 (1)º Block, brown
V = 1800.0 (3) Å3 0.12 × 0.11 × 0.09 mm
Z = 4
Data collection
Bruker APEXII CCD diffractometer 3191 independent reflections
Radiation source: fine-focus sealed tube 2535 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.023
T = 293(2) K θmax = 25.3º
φ and ω scans θmin = 2.4º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −10→10
Tmin = 0.893, Tmax = 0.918 k = −16→16
12796 measured reflections l = −18→17
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.058 H-atom parameters constrained
wR(F2) = 0.180 w = 1/[σ2(Fo2) + (0.114P)2 + 1.289P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
3191 reflections Δρmax = 1.12 e Å−3
217 parameters Δρmin = −0.33 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Fe1 0.64494 (7) 0.01030 (4) 0.10343 (4) 0.0586 (3)
C1 0.9422 (5) −0.0874 (3) 0.2499 (3) 0.0612 (10)
C2 0.5250 (4) −0.1763 (3) 0.0554 (3) 0.0563 (9)
C3 0.5503 (5) −0.2621 (3) 0.0151 (4) 0.0690 (11)
H3 0.6005 −0.2622 −0.0314 0.083*
C4 0.5012 (7) −0.3464 (4) 0.0441 (5) 0.0905 (15)
H4 0.5190 −0.4032 0.0171 0.109*
C5 0.4258 (7) −0.3475 (4) 0.1126 (5) 0.103 (2)
H5 0.3939 −0.4049 0.1318 0.124*
C6 0.3974 (7) −0.2631 (4) 0.1529 (4) 0.0895 (16)
H6 0.3438 −0.2638 0.1977 0.107*
C7 0.4493 (5) −0.1778 (3) 0.1262 (3) 0.0665 (11)
C8 0.4150 (6) −0.0914 (4) 0.1685 (3) 0.0757 (13)
H8 0.3416 −0.0948 0.2016 0.091*
C9 0.4311 (10) 0.0777 (5) 0.2043 (5) 0.117 (2)
H9A 0.3827 0.0625 0.2528 0.141*
H9B 0.3577 0.1145 0.1563 0.141*
C10 0.5833 (10) 0.1335 (4) 0.2450 (4) 0.109 (2)
H10A 0.5603 0.1986 0.2577 0.130*
H10B 0.6451 0.1046 0.3027 0.130*
C11 0.7504 (8) 0.2036 (4) 0.1629 (4) 0.0936 (18)
H11 0.7469 0.2578 0.1977 0.112*
C12 0.8444 (6) 0.2076 (3) 0.1012 (4) 0.0801 (13)
C13 0.8633 (5) 0.1309 (3) 0.0459 (3) 0.0684 (11)
C14 0.9632 (6) 0.1410 (4) −0.0104 (4) 0.0900 (16)
H14 0.9784 0.0901 −0.0464 0.108*
C15 1.0394 (7) 0.2261 (6) −0.0128 (7) 0.125 (3)
H15 1.1052 0.2319 −0.0507 0.150*
C16 1.0191 (9) 0.3026 (6) 0.0405 (7) 0.136 (3)
H16 1.0706 0.3596 0.0382 0.163*
C17 0.9219 (9) 0.2942 (5) 0.0971 (5) 0.123 (3)
H17 0.9076 0.3457 0.1326 0.148*
N1 0.8171 (5) −0.0695 (3) 0.2098 (3) 0.0785 (10)
N2 0.6685 (6) 0.1309 (3) 0.1752 (3) 0.0796 (11)
N3 0.4771 (5) −0.0113 (3) 0.1639 (3) 0.0721 (10)
O1 0.5684 (3) −0.09420 (17) 0.02262 (18) 0.0553 (6)
O2 0.7880 (3) 0.0489 (2) 0.0418 (2) 0.0660 (7)
S1 1.11963 (15) −0.11760 (11) 0.30804 (10) 0.0858 (4)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Fe1 0.0672 (4) 0.0522 (4) 0.0613 (4) 0.0061 (2) 0.0262 (3) −0.0013 (2)
C1 0.075 (3) 0.048 (2) 0.065 (2) 0.0050 (19) 0.028 (2) 0.0093 (17)
C2 0.053 (2) 0.050 (2) 0.066 (2) 0.0008 (16) 0.0163 (17) 0.0063 (17)
C3 0.064 (2) 0.051 (2) 0.090 (3) 0.0037 (18) 0.020 (2) 0.005 (2)
C4 0.087 (3) 0.055 (3) 0.120 (4) 0.000 (2) 0.016 (3) 0.011 (3)
C5 0.104 (4) 0.074 (4) 0.120 (5) −0.018 (3) 0.014 (4) 0.040 (3)
C6 0.084 (3) 0.096 (4) 0.087 (3) −0.019 (3) 0.023 (3) 0.034 (3)
C7 0.056 (2) 0.079 (3) 0.064 (2) −0.002 (2) 0.0153 (19) 0.021 (2)
C8 0.075 (3) 0.095 (4) 0.068 (3) 0.011 (3) 0.038 (2) 0.021 (2)
C9 0.149 (6) 0.116 (5) 0.122 (5) 0.046 (5) 0.093 (5) −0.003 (4)
C10 0.177 (7) 0.081 (4) 0.078 (3) 0.032 (4) 0.052 (4) −0.008 (3)
C11 0.127 (5) 0.056 (3) 0.070 (3) 0.007 (3) −0.015 (3) −0.014 (2)
C12 0.084 (3) 0.067 (3) 0.071 (3) −0.011 (2) −0.006 (2) −0.001 (2)
C13 0.051 (2) 0.065 (3) 0.079 (3) −0.0061 (18) 0.002 (2) 0.012 (2)
C14 0.059 (3) 0.089 (4) 0.120 (4) −0.006 (2) 0.023 (3) 0.024 (3)
C15 0.075 (4) 0.118 (6) 0.164 (7) −0.038 (4) 0.006 (4) 0.045 (5)
C16 0.104 (5) 0.109 (6) 0.156 (7) −0.057 (5) −0.024 (5) 0.032 (5)
C17 0.129 (6) 0.082 (4) 0.117 (5) −0.037 (4) −0.032 (4) −0.001 (3)
N1 0.077 (3) 0.075 (2) 0.082 (3) 0.017 (2) 0.020 (2) 0.012 (2)
N2 0.113 (3) 0.063 (2) 0.057 (2) 0.019 (2) 0.015 (2) −0.0085 (16)
N3 0.080 (2) 0.079 (3) 0.069 (2) 0.0157 (19) 0.040 (2) 0.0052 (17)
O1 0.0636 (15) 0.0482 (13) 0.0608 (14) 0.0017 (11) 0.0284 (12) 0.0024 (11)
O2 0.0625 (16) 0.0566 (16) 0.084 (2) −0.0024 (13) 0.0289 (14) −0.0006 (14)
S1 0.0658 (7) 0.0956 (9) 0.0968 (9) 0.0082 (6) 0.0248 (6) 0.0267 (7)
Geometric parameters (Å, °)
Fe1—O2 1.860 (3) C9—N3 1.500 (7)
Fe1—O1 1.902 (3) C9—C10 1.534 (11)
Fe1—N3 1.985 (4) C9—H9A 0.970
Fe1—N2 1.988 (4) C9—H9B 0.970
Fe1—N1 2.178 (4) C10—N2 1.459 (7)
C1—N1 1.132 (5) C10—H10A 0.970
C1—S1 1.627 (5) C10—H10B 0.970
C2—O1 1.355 (4) C11—N2 1.302 (7)
C2—C7 1.412 (6) C11—C12 1.418 (8)
C2—C3 1.397 (6) C11—H11 0.930
C3—C4 1.378 (7) C12—C13 1.401 (7)
C3—H3 0.930 C12—C17 1.412 (8)
C4—C5 1.380 (9) C13—O2 1.328 (5)
C4—H4 0.930 C13—C14 1.400 (7)
C5—C6 1.388 (9) C14—C15 1.383 (9)
C5—H5 0.930 C14—H14 0.930
C6—C7 1.387 (7) C15—C16 1.384 (13)
C6—H6 0.930 C15—H15 0.930
C7—C8 1.445 (7) C16—C17 1.382 (12)
C8—N3 1.267 (6) C16—H16 0.930
C8—H8 0.930 C17—H17 0.930
O2—Fe1—O1 94.72 (12) H9A—C9—H9B 108.7
O2—Fe1—N3 171.11 (14) N2—C10—C9 106.7 (5)
O1—Fe1—N3 89.55 (14) N2—C10—H10A 110.4
O2—Fe1—N2 92.21 (17) C9—C10—H10A 110.4
O1—Fe1—N2 165.26 (15) N2—C10—H10B 110.4
N3—Fe1—N2 81.92 (19) C9—C10—H10B 110.4
O2—Fe1—N1 94.22 (14) H10A—C10—H10B 108.6
O1—Fe1—N1 96.51 (14) N2—C11—C12 125.9 (5)
N3—Fe1—N1 93.03 (16) N2—C11—H11 117.0
N2—Fe1—N1 95.94 (16) C12—C11—H11 117.0
N1—C1—S1 177.6 (4) C13—C12—C17 119.7 (6)
O1—C2—C7 122.3 (4) C13—C12—C11 123.4 (4)
O1—C2—C3 118.8 (4) C17—C12—C11 116.9 (6)
C7—C2—C3 118.9 (4) O2—C13—C14 117.6 (5)
C2—C3—C4 120.2 (5) O2—C13—C12 123.4 (4)
C2—C3—H3 119.9 C14—C13—C12 119.0 (5)
C4—C3—H3 119.9 C13—C14—C15 120.5 (7)
C5—C4—C3 120.7 (5) C13—C14—H14 119.8
C5—C4—H4 119.6 C15—C14—H14 119.7
C3—C4—H4 119.6 C16—C15—C14 120.8 (8)
C4—C5—C6 120.2 (5) C16—C15—H15 119.6
C4—C5—H5 119.9 C14—C15—H15 119.6
C6—C5—H5 119.9 C17—C16—C15 119.8 (6)
C7—C6—C5 119.8 (5) C17—C16—H16 120.1
C7—C6—H6 120.1 C15—C16—H16 120.1
C5—C6—H6 120.1 C16—C17—C12 120.2 (8)
C2—C7—C6 120.1 (5) C16—C17—H17 119.9
C2—C7—C8 121.6 (4) C12—C17—H17 119.9
C6—C7—C8 118.2 (4) C1—N1—Fe1 151.7 (4)
N3—C8—C7 125.0 (4) C11—N2—C10 120.8 (5)
N3—C8—H8 117.5 C11—N2—Fe1 124.8 (4)
C7—C8—H8 117.5 C10—N2—Fe1 114.4 (4)
N3—C9—C10 105.9 (5) C8—N3—C9 123.1 (5)
N3—C9—H9A 110.6 C8—N3—Fe1 124.1 (3)
C10—C9—H9A 110.6 C9—N3—Fe1 112.8 (4)
N3—C9—H9B 110.6 C2—O1—Fe1 121.1 (2)
C10—C9—H9B 110.6 C13—O2—Fe1 130.0 (3)
==== Refs
References
Bhadbhade, M. M. & Srinivas, D. (1993). Inorg. Chem.32, 6122–6130.
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Bunce, S., Cross, R. J., Farrugia, L. J., Kunchandy, S., Meason, L. L., Muir, K. W., Donnell, M., Peacock, R. D., Stirling, D. & Teat, S. J. (1998). Polyhedron, 17, 4179–4187.
Garnovskii, A. D., Nivorozkhin, A. L. & Minkin, V. (1993). Coord. Chem. Rev.126, 1–69.
Huang, D. G., Zhu, H. P., Chen, C. N., Chen, F. & Liu, Q. T. (2002). Chin. J. Struct. Chem.21, 64–66.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Wang, S.-B., Tang, K., Yang, B.-H. & Li, S. (2008). Acta Cryst. E64, m543. | 21203025 | PMC2961955 | CC BY | 2021-01-04 18:56:20 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jul 16; 64(Pt 8):m1034 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography bx216010.1107/S1600536808022952ACSEBHS1600536808022952Metal-Organic PapersBis(pentane-2,4-dionato-κ2
O,O′)bis[4,4,5,5-tetramethyl-2-(4-pyridyl)imidazoline-1-oxyl 3-oxide-κN
2]manganese(II) [Mn(C5H7O2)2(C12H16N3O2)2]Liu Ying a*Zhang Xianxi aXue Zechun aHe Qingpeng aZhang Yu aa College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 8 2008 26 7 2008 26 7 2008 64 Pt 8 e080800m1077 m1077 10 7 2008 21 7 2008 © Liu et al. 20082008This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, [Mn(C5H7O2)2(C12H16N3O2)2], is isostructural with its NiII-containing analogue [Hao, Mu & Kong (2008 ▶). Acta Cryst. E64, m957]. The asymmetric unit comprises one-half of the molecule and the MnII ion is located on an inversion centre. The coordination geometry around the MnII ion is slightly distorted octahedral, comprised of four O and two N atoms, in which the four O atoms in the equatorial plane come from two pentane-2,4-dionate ligands and the two N atoms in the axial coordination sites from 4,4,5,5-tetramethyl-2-(4-pyridyl)imidazoline-1-oxyl 3-oxide.
==== Body
Related literature
For related literature, see: Eddaoudi et al. (2000 ▶); Hye & Myunghyun (1998 ▶); Li et al. (1999 ▶); Tabares et al. (2001 ▶). For the isostructural NiII-containing compound, see: Hao et al. (2008 ▶).
Experimental
Crystal data
[Mn(C5H7O2)2(C12H16N3O2)2]
M
r = 721.71
Triclinic,
a = 7.107 (2) Å
b = 10.018 (2) Å
c = 12.786 (2) Å
α = 98.16 (3)°
β = 103.20 (3)°
γ = 92.76 (3)°
V = 874.3 (3) Å3
Z = 1
Mo Kα radiation
μ = 0.44 mm−1
T = 298 (2) K
0.39 × 0.28 × 0.17 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.848, T
max = 0.929
6447 measured reflections
3371 independent reflections
2590 reflections with I > 2σ(I)
R
int = 0.033
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.114
S = 1.00
3371 reflections
229 parameters
H-atom parameters constrained
Δρmax = 0.56 e Å−3
Δρmin = −0.50 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808022952/bx2160sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808022952/bx2160Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BX2160).
The authors thank the Natural Science Foundation of China (grant No. 20501011) and Liaocheng University (grant No. X071011) for financial support.
supplementary crystallographic
information
Comment
Due to the interesting structures from supramolecular assemblies as well as
potential applications on smart optoelectronic, magnetic and porous materials,
the design and synthesis of metal–organic coordination polymers have attracted
considerable attention (Eddaoudi et al., 2000; Hye & Myunghyun,
1998;
Li et al., 1999; Tabares et al., 2001). In this
paper, we report
the structure of the title compound, (I).
As shown in Fig. 1, the asymmetric unit comprises a half of the molecule and
MnII ion locates on an inversion centre. The coordination geometry around
MnII is slightly distorted octahedral, comprised of four O and two N atoms.
In which, the four oxygen atoms in the equatorial plane come from two
pentane-2,4-dionate and the two nitrogen atoms in the axial coordination sites
from 2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The Mn—N
and Mn—O bond lengths are in the range of 2.178 (2)–2.178 (2) and
2.0151 (17)–2.0386 (17) Å, respectively.
Experimental
A mixture of Manganese(II) acetylacetonate (0.5 mmol) and
2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (1 mmol) in 20 ml
methanol was refluxed for one day. The resulted solution was filtered. The
filtrate was kept in the open flask and evaporated naturally at room
temperature. Several days later, pink blocks of (I) were obtained with a high
yield of ca 67% based on MnII. Anal. Calc. for C34H46MnN6O8:
C 56.53, H 6.37, N 11.64%; Found: C 56.45, H 6.29, N 11.58%.
Refinement
All H atoms were placed in calculated positions with C—H = 0.93 Å and C—H
= 0.96 distances and refined as riding with Uiso(H) = 1.2 and 1.5
Ueq(carrier).
Figures
Fig. 1. The molecular structure of (I) around MnII, drawn with 30% probability displacement ellipsoids for the non-hydrogen atoms.
Crystal data
[Mn(C5H7O2)2(C12H16N3O2)2] Z = 1
Mr = 721.71 F000 = 381
Triclinic, P1 Dx = 1.371 Mg m−3
Hall symbol: -P 1 Mo Kα radiation λ = 0.71073 Å
a = 7.107 (2) Å Cell parameters from 3371 reflections
b = 10.018 (2) Å θ = 3.0–26.1º
c = 12.786 (2) Å µ = 0.44 mm−1
α = 98.16 (3)º T = 298 (2) K
β = 103.20 (3)º Block, pink
γ = 92.76 (3)º 0.39 × 0.28 × 0.17 mm
V = 874.3 (3) Å3
Data collection
Bruker APEXII CCD area-detector diffractometer 3371 independent reflections
Radiation source: fine-focus sealed tube 2590 reflections with I > 2σ(I)
Monochromator: graphite Rint = 0.033
T = 298(2) K θmax = 26.1º
φ and ω scans θmin = 3.0º
Absorption correction: multi-scan(SADABS; Bruker, 2001) h = −6→8
Tmin = 0.848, Tmax = 0.930 k = −12→12
6447 measured reflections l = −11→15
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.041 H-atom parameters constrained
wR(F2) = 0.114 w = 1/[σ2(Fo2) + (0.066P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
3371 reflections Δρmax = 0.56 e Å−3
229 parameters Δρmin = −0.50 e Å−3
Primary atom site location: structure-invariant direct methods Extinction correction: none
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Mn1 0.0000 0.0000 0.0000 0.0281 (7)
C1 −0.1322 (4) 0.3856 (3) 0.1293 (2) 0.0310 (6)
H1A −0.2696 0.3835 0.1006 0.046*
H1B −0.0731 0.4742 0.1305 0.046*
H1C −0.1069 0.3644 0.2019 0.046*
C2 −0.0486 (3) 0.2828 (2) 0.05829 (19) 0.0231 (5)
C3 0.1138 (4) 0.3209 (3) 0.0230 (2) 0.0257 (6)
H3 0.1665 0.4100 0.0456 0.031*
C4 0.2049 (3) 0.2369 (2) −0.0433 (2) 0.0241 (6)
C5 0.3726 (4) 0.2934 (3) −0.0814 (2) 0.0327 (6)
H5A 0.4879 0.2528 −0.0507 0.049*
H5B 0.3914 0.3896 −0.0585 0.049*
H5C 0.3458 0.2740 −0.1593 0.049*
C6 0.1702 (3) 0.1166 (2) 0.23876 (19) 0.0219 (5)
H6 0.0386 0.1239 0.2339 0.026*
C7 0.2971 (3) 0.1613 (2) 0.3372 (2) 0.0220 (5)
H7 0.2520 0.1980 0.3969 0.026*
C8 0.5527 (4) 0.0957 (3) 0.2535 (2) 0.0243 (6)
H8 0.6833 0.0870 0.2560 0.029*
C9 0.4137 (3) 0.0539 (2) 0.1579 (2) 0.0229 (5)
H9 0.4544 0.0177 0.0964 0.027*
C10 0.4945 (3) 0.1510 (2) 0.3462 (2) 0.0214 (5)
C11 0.6317 (3) 0.1984 (3) 0.45046 (19) 0.0220 (5)
C12 0.7429 (3) 0.2985 (3) 0.63433 (19) 0.0228 (5)
C13 0.9183 (3) 0.2690 (2) 0.58648 (18) 0.0206 (5)
C14 1.0771 (3) 0.3830 (3) 0.6102 (2) 0.0254 (6)
H14A 1.1718 0.3589 0.5696 0.038*
H14B 1.1380 0.3985 0.6866 0.038*
H14C 1.0221 0.4638 0.5896 0.038*
C15 1.0021 (4) 0.1370 (3) 0.6128 (2) 0.0248 (6)
H15A 0.8995 0.0657 0.5955 0.037*
H15B 1.0644 0.1474 0.6888 0.037*
H15C 1.0952 0.1146 0.5706 0.037*
C16 0.7505 (4) 0.2552 (3) 0.7443 (2) 0.0304 (6)
H16A 0.6337 0.2761 0.7667 0.046*
H16B 0.8603 0.3025 0.7970 0.046*
H16C 0.7622 0.1595 0.7388 0.046*
C17 0.6888 (4) 0.4436 (3) 0.6351 (2) 0.0320 (6)
H17A 0.6873 0.4698 0.5656 0.048*
H17B 0.7823 0.5025 0.6907 0.048*
H17C 0.5626 0.4502 0.6496 0.048*
N1 0.5857 (3) 0.2135 (2) 0.54865 (16) 0.0248 (5)
N2 0.8187 (3) 0.2418 (2) 0.46656 (16) 0.0217 (5)
N3 0.2243 (3) 0.06280 (19) 0.14902 (16) 0.0206 (4)
O1 0.1568 (2) 0.11188 (17) −0.07682 (13) 0.0249 (4)
O2 −0.1367 (2) 0.16521 (17) 0.03802 (13) 0.0245 (4)
O3 0.4265 (2) 0.1723 (2) 0.56890 (14) 0.0353 (5)
O4 0.9121 (2) 0.25076 (18) 0.39302 (14) 0.0274 (4)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Mn1 0.0255 (17) 0.0255 (17) 0.0309 (19) −0.0021 (13) 0.0044 (15) −0.0037 (14)
C1 0.0354 (15) 0.0245 (14) 0.0295 (15) 0.0070 (11) 0.0010 (12) 0.0017 (11)
C2 0.0253 (13) 0.0220 (13) 0.0173 (13) 0.0039 (10) −0.0057 (10) 0.0043 (10)
C3 0.0297 (14) 0.0208 (13) 0.0232 (14) 0.0009 (10) −0.0009 (11) 0.0043 (10)
C4 0.0228 (13) 0.0245 (13) 0.0215 (14) 0.0001 (10) −0.0047 (10) 0.0090 (10)
C5 0.0275 (14) 0.0306 (14) 0.0399 (17) 0.0000 (11) 0.0039 (12) 0.0122 (12)
C6 0.0186 (12) 0.0260 (13) 0.0220 (14) 0.0034 (10) 0.0049 (10) 0.0061 (10)
C7 0.0218 (13) 0.0276 (13) 0.0171 (13) 0.0031 (10) 0.0051 (10) 0.0035 (10)
C8 0.0196 (13) 0.0308 (14) 0.0223 (14) 0.0033 (10) 0.0045 (11) 0.0037 (11)
C9 0.0229 (13) 0.0268 (13) 0.0192 (13) 0.0041 (10) 0.0046 (10) 0.0044 (10)
C10 0.0213 (13) 0.0240 (12) 0.0184 (13) 0.0009 (10) 0.0033 (10) 0.0042 (10)
C11 0.0210 (13) 0.0293 (13) 0.0155 (13) 0.0018 (10) 0.0046 (10) 0.0025 (10)
C12 0.0204 (13) 0.0301 (14) 0.0162 (13) 0.0044 (10) 0.0004 (10) 0.0032 (10)
C13 0.0190 (12) 0.0291 (13) 0.0119 (12) 0.0009 (10) 0.0007 (10) 0.0023 (10)
C14 0.0231 (13) 0.0284 (14) 0.0233 (14) 0.0013 (10) 0.0030 (11) 0.0033 (11)
C15 0.0209 (13) 0.0279 (13) 0.0253 (14) 0.0037 (10) 0.0037 (11) 0.0057 (11)
C16 0.0259 (14) 0.0444 (17) 0.0200 (14) 0.0032 (12) 0.0030 (11) 0.0062 (12)
C17 0.0271 (14) 0.0388 (16) 0.0284 (16) 0.0112 (12) 0.0039 (12) 0.0015 (12)
N1 0.0158 (11) 0.0388 (13) 0.0188 (12) 0.0004 (9) 0.0032 (9) 0.0036 (9)
N2 0.0179 (11) 0.0298 (11) 0.0171 (11) 0.0011 (8) 0.0038 (9) 0.0039 (9)
N3 0.0222 (11) 0.0214 (10) 0.0184 (11) 0.0028 (8) 0.0049 (9) 0.0036 (8)
O1 0.0256 (9) 0.0244 (9) 0.0219 (10) 0.0001 (7) 0.0009 (7) 0.0030 (7)
O2 0.0238 (9) 0.0245 (9) 0.0230 (10) 0.0043 (7) 0.0005 (7) 0.0037 (7)
O3 0.0183 (10) 0.0617 (14) 0.0272 (11) −0.0016 (9) 0.0067 (8) 0.0113 (10)
O4 0.0218 (9) 0.0406 (11) 0.0204 (10) 0.0010 (8) 0.0078 (8) 0.0032 (8)
Geometric parameters (Å, °)
Mn1—O2i 2.0151 (17) C9—N3 1.333 (3)
Mn1—O2 2.0151 (17) C9—H9 0.9300
Mn1—O1i 2.0386 (17) C10—C11 1.461 (3)
Mn1—O1 2.0386 (17) C11—N2 1.339 (3)
Mn1—N3i 2.178 (2) C11—N1 1.358 (3)
Mn1—N3 2.178 (2) C12—N1 1.503 (3)
C1—C2 1.509 (3) C12—C16 1.520 (3)
C1—H1A 0.9600 C12—C17 1.520 (3)
C1—H1B 0.9600 C12—C13 1.533 (3)
C1—H1C 0.9600 C13—N2 1.514 (3)
C2—O2 1.271 (3) C13—C14 1.513 (3)
C2—C3 1.388 (4) C13—C15 1.526 (3)
C3—C4 1.395 (4) C14—H14A 0.9600
C3—H3 0.9300 C14—H14B 0.9600
C4—O1 1.268 (3) C14—H14C 0.9600
C4—C5 1.502 (3) C15—H15A 0.9600
C5—H5A 0.9600 C15—H15B 0.9600
C5—H5B 0.9600 C15—H15C 0.9600
C5—H5C 0.9600 C16—H16A 0.9600
C6—N3 1.342 (3) C16—H16B 0.9600
C6—C7 1.372 (3) C16—H16C 0.9600
C6—H6 0.9300 C17—H17A 0.9600
C7—C10 1.391 (3) C17—H17B 0.9600
C7—H7 0.9300 C17—H17C 0.9600
C8—C9 1.382 (3) N1—O3 1.279 (3)
C8—C10 1.394 (3) N2—O4 1.279 (2)
C8—H8 0.9300
O2i—Mn1—O2 180.00 (10) N2—C11—N1 108.0 (2)
O2i—Mn1—O1i 89.02 (7) N2—C11—C10 127.1 (2)
O2—Mn1—O1i 90.98 (7) N1—C11—C10 124.8 (2)
O2i—Mn1—O1 90.98 (7) N1—C12—C16 109.9 (2)
O2—Mn1—O1 89.02 (7) N1—C12—C17 105.8 (2)
O1i—Mn1—O1 180.00 (9) C16—C12—C17 110.7 (2)
O2i—Mn1—N3i 89.00 (7) N1—C12—C13 99.99 (19)
O2—Mn1—N3i 91.00 (7) C16—C12—C13 115.7 (2)
O1i—Mn1—N3i 88.10 (7) C17—C12—C13 113.8 (2)
O1—Mn1—N3i 91.90 (7) N2—C13—C14 110.88 (19)
O2i—Mn1—N3 91.00 (7) N2—C13—C15 105.76 (19)
O2—Mn1—N3 89.00 (7) C14—C13—C15 110.9 (2)
O1i—Mn1—N3 91.90 (7) N2—C13—C12 99.71 (17)
O1—Mn1—N3 88.10 (7) C14—C13—C12 115.9 (2)
N3i—Mn1—N3 180.00 (8) C15—C13—C12 112.7 (2)
C2—C1—H1A 109.5 C13—C14—H14A 109.5
C2—C1—H1B 109.5 C13—C14—H14B 109.5
H1A—C1—H1B 109.5 H14A—C14—H14B 109.5
C2—C1—H1C 109.5 C13—C14—H14C 109.5
H1A—C1—H1C 109.5 H14A—C14—H14C 109.5
H1B—C1—H1C 109.5 H14B—C14—H14C 109.5
O2—C2—C3 126.0 (2) C13—C15—H15A 109.5
O2—C2—C1 114.5 (2) C13—C15—H15B 109.5
C3—C2—C1 119.5 (2) H15A—C15—H15B 109.5
C4—C3—C2 125.6 (2) C13—C15—H15C 109.5
C4—C3—H3 117.2 H15A—C15—H15C 109.5
C2—C3—H3 117.2 H15B—C15—H15C 109.5
O1—C4—C3 125.0 (2) C12—C16—H16A 109.5
O1—C4—C5 114.8 (2) C12—C16—H16B 109.5
C3—C4—C5 120.1 (2) H16A—C16—H16B 109.5
C4—C5—H5A 109.5 C12—C16—H16C 109.5
C4—C5—H5B 109.5 H16A—C16—H16C 109.5
H5A—C5—H5B 109.5 H16B—C16—H16C 109.5
C4—C5—H5C 109.5 C12—C17—H17A 109.5
H5A—C5—H5C 109.5 C12—C17—H17B 109.5
H5B—C5—H5C 109.5 H17A—C17—H17B 109.5
N3—C6—C7 124.0 (2) C12—C17—H17C 109.5
N3—C6—H6 118.0 H17A—C17—H17C 109.5
C7—C6—H6 118.0 H17B—C17—H17C 109.5
C6—C7—C10 118.9 (2) O3—N1—C11 127.0 (2)
C6—C7—H7 120.5 O3—N1—C12 121.8 (2)
C10—C7—H7 120.5 C11—N1—C12 111.0 (2)
C9—C8—C10 119.1 (2) O4—N2—C11 126.4 (2)
C9—C8—H8 120.5 O4—N2—C13 121.89 (18)
C10—C8—H8 120.5 C11—N2—C13 111.45 (18)
N3—C9—C8 123.4 (2) C9—N3—C6 116.9 (2)
N3—C9—H9 118.3 C9—N3—Mn1 124.82 (16)
C8—C9—H9 118.3 C6—N3—Mn1 118.27 (16)
C7—C10—C8 117.7 (2) C4—O1—Mn1 121.54 (16)
C7—C10—C11 119.6 (2) C2—O2—Mn1 121.01 (16)
C8—C10—C11 122.7 (2)
Symmetry codes: (i) −x, −y, −z.
Table 1 Selected geometric parameters (Å, °)
Mn1—O2 2.0151 (17)
Mn1—O1 2.0386 (17)
Mn1—N3 2.178 (2)
O2i—Mn1—O2 180
O2i—Mn1—O1 90.98 (7)
O2—Mn1—O1 89.02 (7)
O1i—Mn1—O1 180
O2—Mn1—N3i 91.00 (7)
O1—Mn1—N3i 91.90 (7)
O2—Mn1—N3 89.00 (7)
O1—Mn1—N3 88.10 (7)
N3i—Mn1—N3 180
Symmetry code: (i) .
==== Refs
References
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Eddaoudi, M., Li, H. & Yaghi, O. M. (2000). J. Am. Chem. Soc.122, 1391–1397.
Hao, L., Mu, C. & Kong, B. (2008). Acta Cryst. E64, m957.
Hye, J. C. & Myunghyun, P. S. (1998). J. Am. Chem. Soc.120, 10622–10628.
Li, H., Eddaoudi, M., O’Keeffe, M. & Yaghi, O. M. (1999). Nature (London), 402, 276–279.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Tabares, L. C., Navarro, J. A. R. & Salas, J. M. (2001). J. Am. Chem. Soc.123, 383–387. | 21203057 | PMC2961987 | CC BY | 2021-01-04 18:56:15 | yes | Acta Crystallogr Sect E Struct Rep Online. 2008 Jul 26; 64(Pt 8):m1077 |
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Indian J DermatolIJDIndian Journal of Dermatology0019-51541998-3611Medknow Publications India IJD-55-30110.4103/0019-5154.70695CorrespondenceNAIL CHANGES AND NAIL DISORDERS IN THE ELDERLY Rao Sudhakar Banerjee Sabyasachi Ghosh Sadhan Kumar Gangopadhyay Dwijendra Nath Jana Sukumar Mridha Kakali From the Department of Dermatology, Calcutta National Medical College, Kolkata, India. E-mail: [email protected] 2010 55 3 301 304 © Indian Journal of Dermatology2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Sir,
Long life is much desired but not all people are destined to enjoy it. The elderly population in India aged 60 years and above[1] is steadily growing and its absolute size is already quite large. As per SRS estimation in 2003, this population comprised 7.2% of the total population.[2]
Nail changes associated with ageing are common in the elderly and include characteristic modifications of color, contour, growth, surface, thickness, and histology. No cutaneous examination is complete without a careful evaluation of the nails. The calcium content of the ageing nail increases whereas the iron content of the ageing nail decreases.[3] Histologically, the keratinocytes of the nail plate are increased in size with increased number of ‘pertinax bodies’ (remnants of keratinocyte nuclei).[4] The nail bed dermis also shows thickening of the blood vessels and elastic tissue, especially beneath the pink part of the nail.[5] Nail growth decreases by approximately 0.5% per year between 20 and 100 years of age.[6]
This study has been undertaken for nail changes and nail disorders in the elderly because of the scarcity of such studies in our country.
Lots were drawn to randomly select every second elderly OPD patient aged 60 years and above for this study, irrespective of their presenting symptoms. A total of 100 patients were studied by this method to eliminate observers’ bias in the study.
A detailed history of each elderly patient was taken along with a detailed nail examination, an examination of the hair, and a general survey as per the proforma. We examined KOH preparations of nail clippings microscopically for all the suspected cases of onychomycosis to detect fungal elements.
The following table shows the age- and sexwise breakup of patients enrolled for the study. The details of age and sex distribution has been shown in Table 1.
Table 1 Age and sex distribution
Age (in years) Male Female Total Percentage
60–70 39 21 60 60
70–80 20 12 32 32
80–90 03 02 05 05
90–100 02 00 02 02
> 100 01 00 01 01
The youngest patient in this study was 60 years old while the oldest patient was 101 years old; 6% had the habit of occasional nail biting.
The majority of the patients were not aware about their nail changes or nail diseases. However, nail disease was the presenting complaint in 14/100 cases.
A general survey showed that 44 patients had mild conjunctival pallor which raised the clinical suspicion of mild anemia. It was also seen that they suffered from various system diseases like hypertension (19%), diabetes mellitus (5%), COPD and bronchial asthma (5%), ischemic heart disease (4%), arthritis (2%), prostatic hypertrophy (1%), and Parkinsonism (1%).
Out of 100 patients, 98 showed at least one change due to ageing although two patients, both incidentally female, didn’t show any age-related changes.
Regarding age-related color change of the nail plate, 73% of the patients showed pale, dull, and lusterless nail plates, followed by opacity in 8% and grey color in 6%.
Lunular visibility decreases with increasing age and out of the 31 cases in whom the lunula was visible, 23 were 60–70 years old. In these 23 patients, lunular visibility was maximal (100%) in LF (first left finger = left thumb) and RF1 (first left great toe) followed by 67.8 and 64.5% for RT1 and RT2 respectively. Lunular visibility was very low or absent in other fingers and toes.
Among changes in the nail surface due to ageing, prominent longitudinal ridges were the most common change (85%) followed by rough nails in 33% of the patients, transverse ridges in 23%, and lamellar split in 15% of the cases.
Brittleness of the nail is a common condition related to ageing. Twenty-six males (40%) and eight females (26%) showed brittle nails. Toe nails were more likely to be brittle in both sexes. Among males, 25 had brittle toenails, five had brittle fingernails, and four had brittle finger- and toenails. Among females, eight had brittle toenails, two had brittle fingernails, and two patients had brittle finger- and toenails.
Onychauxis which is an age-associated thickening of the nail plate, was noticed in 23% of the patients and its prevalence was 10% in the left great toe and 13% in the right great toe, followed by 7% in the right 5th toe and 4% in the left 4th toe.
Among changes in nail contour, increased transverse curvature was seen in five cases, pincer nail in two, and platyonychia in one case.
Table 2 shows the prevalence of nail disorders in the elderly. Out of 33 cases of acquired disorders, the majority of them were infective disease. Onychomycosis was seen in 16%, followed by chronic paronychia in 9%; 31% showed a single disorder, 4% showed two disorders, and 1% showed three disorders.
Table 2 Prevalence of nail disorders
Disease Number Percentage
A) Congenital
Raquette nail 01 01
B) Acquired
Onychomycosis 16 16
Chronic paronychia 09 09
Traumatic nail 08 08
Psoriatic nail disease 04 04
Periungual wart 01 01
Acute paronychia 01 01
The prevalence of onychomycosis was 22% in women and 12% in men. The most common change in onychomycosis is loss of luster, subungual hyperkerotosis, onycholysis, brittleness, and color change with blackish, brownish, or yellowish discoloration.
Of 16 cases of onychomycosis, fungal element could be found in ten cases (62.5%) in the KOH preparations. Diagnosis was made in the remaining cases when there was a high index of clinical suspicion, presence of dermatophytic lesions in some other body part, and absence of any cutaneous disorder that could explain similar nail changes.
Fingernails were involved in ten patients and toenails were involved in 12 patients.
Out of the nine patients who were suffering from chronic paronychia, five were male. Of these five males, three were retired, one a farmer, and another jobless. Of the four female patients, three were housewives and one a maid-servant. The right thumb, index, and ring fingers were affected in four cases each, the left thumb and the right middle finger in three cases while the left index, middle, ring and little fingers, right little finger and left toe were involved in two cases each. Thus, the right hand was found to be more commonly affected. Common changes seen in chronic paronychia were loss of cuticle, nail fold erythema, and edema. Common nail plate changes were transverse furrows, loss of luster, and thickening.
Traumatic nail disorders were the third most common disorder seen in the study in eight patients. Among these, subungual hematoma was seen in three cases, followed by nail loss in two and onycholysis in two cases. One case of splinter hemorrhage was also seen.
In our study, we found that senile changes in the elderly were studied under four headings:
Change in color with emphasis on lunular color change
Change in contour of nail
Change in the surface of the nail including brittle nail and
Gross change in thickness of the nail with presence of onychauxis.
The most common ageing change in the nail was a pale, dull, and lusterless appearance of the nail in 73% of the patients. The color of the ageing nail may vary from yellow to grey with a dull opaque appearance.[7] The senile nail may appear pale, dull, and opaque, with its color varying from white or yellow to brown to grey.[5]
Although the lunula is often not visible in all fingers and toes, as in our study, it is most consistently observed on the thumb, the index finger, and the great toe.[8]
Lunular size decreased with age and has been noted as an ageing-related nail change in elderly persons.[4] In our study, we found that the lunula was not visible in 69 cases and its visibility decreased consistently with age.
Ageing-related nail changes can be seen in the form of increased longitudinal furrowing or ridges and increased friability and fissuring.[9] Ageing is the most common cause of onychorrhexis or superficial longitudinal ridges.[10] Transverse furrows/ridges are also found very frequently. The nails may be rough (trachyonychia with lamellar splitting and fissuring). In our study, we found prominent/increased longitudinal ridges in 85% of the cases with no significant difference in the percentage of finger- and toenails. Transverse ridges/furrows (22%) were seen in the toenails of all 22 patients (mainly in the great toe nails) and in the fingernails of two cases. Rough nails (33%) were also seen, mostly in the toes.
Repeated cycles of hydration and dehydration occurring in excessive domestic wet work or overuse of dehydrating agents, nail enamel and nail enamel removers, and cuticle removers may cause brittleness of the nails. Brittle nails are a common finding in the elderly.[311] Consistent with the findings of Lubach et al. (31% in males and 36% in females ≥ 60 years and above),[11] we found the prevalence of brittle nails to be 34% in our patient sample. The first three fingers of the dominant hand are particularly susceptible to brittle nails. The incidence of brittle nails was, however, higher in the toenails in our study population because these patients were from poorer socioeconomic strata who walk barefoot or use ill-fitting shoes and sandals. Constant low-grade trauma hastens the brittle nail change that is seen in elderly patients.
Senile nails may have an increased transverse curvature and a decreased longitudinal curvature.[7] Flattening of the nail plate (platyonchia), spooning (koilonychia), and pincer nail deformity are found more frequently in the elderly.[5] In our study, we observed increased transverse curvature in five cases. We also found two cases of pincer nail and one case of platyonychia; no case of koilonychia was seen in our study.
The prevalence of onychomycosis increases with age and reaches nearly 20% in patients over 60 years of age.[12] In our study, we found the prevalence of onychomycosis to be 15%. Onychomycosis has been reported to be more common in elderly men than in elderly women.[13] In our study, we found that the prevalence of onychomycosis was 22% in women and 12% in men. The most common type of onychomycosis observed in our study was distal and lateral subungual onychomycosis, as also observed earlier.[14] Chronic paronychia (9%) was also not uncommon in the present study. The right hand, being the working hand of the majority, was found to be predominantly affected. Most of the patients in our study were from the poorer sections of the society. Many of them walk barefoot most of the time and due to their unsanitary dwelling habits, their feet are usually exposed to dirty and wet conditions. The above factors have been seen as inducing and hastening factors in causing brittle nails. Occupation as well as household work of many patients, especially the women, involve repeated minor trauma, and hand and foot exposure to water, chemicals, and irritants for long durations. These factors may be contributory to ageing-related changes in our study group as well as to onychomycosis and paronychia.
In our study, we found six cases of psoriasis, out of which four presented with nail changes. Prevalence of nail involvement in psoriasis was found to be 67%. Nail involvement is common in psoriasis and has been reported between 50[15] and 56%.[16] It is estimated that between 80 and 90% of psoriatics will suffer from nail disease during their lifetime.[17] Pitting was seen in all the four cases as the most common finding, as has been described by others.[18] Pitting was more common on the fingernails than on the toenails and was scattered rather than a regular pattern. Other common findings were subungual keratin deposits and onycholysis. Yellowish discoloration and loss of texture were also common findings. Although loss of cuticle has been a common finding,[3] it was not found in the present study.
With improved socioeconomic condition and awareness, more and more geriatric patients will visit dermatologists with nail-related problems in the future. Hence, dermatologists will have to brace themselves up beforehand to handle such common geriatric problems of this organ.
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1 WHO Scientific group, Health of elderly, TRS NO. 779, WHO, General 1989
2 Park K Preventive Medicine in Obstetrics Paediatrics and Geriatrics In: Park’s Text Book of Preventive and Social Medicine 2007 9th ed Jabalpur India Banarasidas Bhanot 471
3 Baran R Dawber RPR Baran R Dawber RPR The nail in childhood and old age Diseases of the nails and their management 1994 2nd ed Oxford Blackwell Science 81 96
4 Lewis BL Montgomery H The senile nail J Invest Dermatol 1955 24 11 8 13233575
5 Cohen PR Scher RK Hordinsky MK Sawaya ME Scher RK Aging Atlas of hair and nails 2000 Philadelphia Churchill Livingstone 213 25
6 Singh G Haneef NS Uday A Nail changes and disorders among the elderly Indian J Dermatol, Veneveol Leprol 2005 71 386 92
7 Baran R Dawber RPR Fry L The ageing nail Skin problems in the elderly 1985 Edinburgh Churchill Livingstone 15 30
8 Flukman P Anatomy and physiology of the nail Dermatol Clin 1986 3 373 81
9 Baran R Dawber RPR Baran R Dawber RPR The nail in childhood and old age Diseases of the nails and their management 1984 Oxford Backwell Scientific 105 20
10 Holzberg M Hordinsky MK Sawaya ME Scher RK Nail signs of systemic disease Atlasof hair and nails 2000 Philadelphia Churchill Livingstone 59 70
11 Lubach D Cohrs W Wurzinger R Incidence of brittle nails Dermatologics 1986 172 144 7
12 Loo DS Cutaneous fungal infections in the elderly Dermatol Clin 2004 22 33 50 15018008
13 Weinberg JW Vafaie J Scheinfeld NS Skin infections in the elderly Dermatol Clin 2004 22 51 61 15018009
14 Gupta AK Lynde CW Jain HC Sibbald RG Elewski BE Daniel CR 3rd Watteel GN Summerbell RC A higher prevalence of onychomycosis in psoriatics compared with non- psoriatics: a multicentre study, Br J Dermatol 1997 136 786 9
15 Zaias N Psoriasis of the nail, a clinical-pathology study Archives of Dermatalogy 1969 99 567
16 Kaur I Handa S Kumar B Natural history of psoriasis: a study from the Indian subcontinent J Dermatol 1997 24 230 4 9164063
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2106083310-PONE-RA-21075R110.1371/journal.pone.0013731Research ArticleCell BiologyMolecular BiologyPharmacologyDownregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas uPAR and Cathepsin B SilencingMalla Ramarao
1
Gopinath Sreelatha
1
Alapati Kiranmai
1
Gondi Christopher S.
1
Gujrati Meena
2
Dinh Dzung H.
3
Mohanam Sanjeeva
1
Rao Jasti S.
1
3
*
1
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
2
Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
3
Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America
Hotchin Neil A. EditorUniversity of Birmingham, United Kingdom* E-mail: [email protected] and designed the experiments: RRM JR. Performed the experiments: RRM SG KA CSG. Analyzed the data: RRM CSG MG DHD JR. Contributed reagents/materials/analysis tools: SM JR. Wrote the paper: RRM. Provided discussion and revision of critically important intellectual content: JR.
2010 29 10 2010 5 10 e1373114 7 2010 7 10 2010 Malla et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.
Methodology/Principal Findings
In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.
Conclusions/Significance
In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
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Introduction
Apoptosis is a tightly regulated form of programmed cell death involving a series of biochemical events that leads to a variety of morphological changes including membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [1], [2]. The components of the apoptotic signaling network are genetically encoded in an inactive form and are activated by various external and internal stimuli including DNA damage, drugs, or irradiation [3], [4]. Mitochondria play a central part in cellular survival and apoptotic death [5]. The key events of mitochondrial apoptosis include the release of cytochrome C, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and anti-apoptotic Bcl-2 family proteins [6].
The Bcl-2 family of genes is known to be involved in the regulation of the cell death process [7], [8]. Bcl-2 and Bcl-xL are anti-apoptotic members predominantly localized in mitochondria that regulate mitochondrial membrane integrity and cytochrome C release. Pro-apoptotic members, such as Bax (Bcl-2–associated X protein) and BAD (Bcl-2-associated death promoter), mainly reside in the cytoplasm and redistribute into mitochondria in response to death stimuli [6], [9]–[11]. Bcl-2 family proteins are able to undergo homodimerization and heterodimerization, and the ratio of pro- to anti-apoptotic proteins determines the fate of cells [12], [13]. The hallmark of glioma is increased activity of the PI3K/Akt pathway that controls the expression of pro-survival proteins, including NF-κB (nuclear factor-kappaB), CREB (cAMP response element binding) (CREB) and Bcl-2 as well as pro-apoptotic molecules such as Bax and BAD [14]–[18]. CREB plays a key role in regulating neuronal survival and differentiation [19], and it promotes a pro-survival effect by regulating the transcription of several pro-survival factors, including Bcl-2 [20], [21]. In addition, in some populations of neurons, the loss of CREB imparts a Bax-dependent form of apoptosis [22].
In the present study, we demonstrate for the first time that either individual or simultaneous downregulation of uPAR and cathepsin B using siRNA decreased Bcl-2 expression and increased Bax expression in U251 glioma cells and 5310 glioma xenograft cells (in vitro), and in brain tumor tissue sections and tissue lysates (in vivo). We also show that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued apoptosis in pCU-treated U251 cells, but not in 5310 cells. In addition, uPAR and cathepsin B downregulation decreased the expression levels of PDGFR-β, PI3K, and Akt and also affected the promoter (DNA) binding activity of CREB. Moreover, CREB siRNA decreased the expression of Bcl-2, but it did not affect the expression of Bax. In the present study, we also observed that PDGFR tyrosine kinase inhibitor IV decreased the expression of p-PDGFR-β and p-PI3K. Both PDGFR tyrosine kinase inhibitor IV and a PI3K inhibitor (Wortmannin) decreased the expression of p-Akt, pCREB, and Bcl-2 while increasing the expression of Bax. Taken together, the findings of the present study suggest a novel mechanism underlying uPAR and cathepsin B-mediated regulation of Bcl-2 and Bax in gliomas.
Results
Downregulation of uPAR and cathepsin B induces apoptosis by collapsing mitochondrial membrane potential
To gain insight into the molecular roles of uPAR and cathepsin B, we knocked down the expression of these molecules using siRNA in U251 and 5310 glioma cells and then analyzed the effect on apoptosis. Western blot analysis showed that uPAR and cathepsin B expression was significantly decreased with pU, pC and pCU (Figs. 1A–B). Quantification of protein bands revealed that uPAR expression decreased by 88% in U251 cells and 68% in 5310 cells with pU (p<0.01). Similarly, cathepsin B expression was reduced by 76% in both cell lines when treated with pC (p<0.01). Cells treated with pU did not show appreciable difference in cathespin B expression as compared to controls. Similarly, cells treated with pC did not show any significant difference in expression of uPAR as compared to controls (90%). pCU-treated cells showed 95% decreased expression of uPAR in both U251 and 5310 cells. Cathepsin B expression in pCU-treated cells was significantly reduced by 90% (Fig. 1C). Immunoblot analysis for GAPDH expression revealed equal loading. Analysis of apoptotic cell distribution by FACS analysis showed that silencing of uPAR and cathepsin B expression significantly increased the apoptotic cell population in both U251 and 5310 cells (Fig. 1D). Cell cycle distribution in histograms showed that the percentage of apoptotic cell population was 2.8% in untreated U251 cells, 2.2% in SV-treated U251 cells, 70% in pU-treated U251 cells, 61% in pC-treated U251 cells, 79% in pCU-treated U251 cells, 2.5% in untreated 5310 cells, 3.0% in SV-treated 5310 cells, 72% in pU-treated 5310 cells, 65% in pC-treated 5310 cells, and 76% in pCU-treated 5310 cells (Fig. 1E).
10.1371/journal.pone.0013731.g001Figure 1 RNAi-mediated depletion of cathepsin B and uPAR induces mitochondrial apoptosis in U251 and 5310 cells.
(A–B) Western blot analysis of uPAR and cathepsin B expression in U251 and 5310 cells 72 hrs after transfection with SV, pU, pC and pCU. (C) Quantitative analysis of uPAR and cathepsin B expression by densitometry. Results from three independent experiments are shown as mean ± SD (**p<0.001). GAPDH was used as a loading control. (D) Cell cycle distribution of U251 and 5310 cells. Propidium iodide-stained cells were analyzed for DNA content using flow cytometry. (E) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of three independent experiments. Values are mean ± SD of three different experiments (**p<0.001). (F) Cells were stained for apoptosis using TdT-mediated dUTP nick end-labeling (TUNEL) assay. Data shown are from a representative experiment. (G) Quantification of apoptotic cells expressed as percent of DAPI-stained cells. Bars represent the mean ± SD of three experiments (*p<0.05; **p<0.001). (H) Analysis of mitochondrial membrane potential using MitoLight kit. Cells were collected, incubated with MitoLight dye for 20 min at room temperature, and observed for fluorescence; red fluorescence indicates healthy cells while green fluorescence indicates apoptotic cells.
In addition, TUNEL assay results for cells at 72 hrs post-transfection indicated a significant increase in TUNEL-positive cells in both U251 and 5310 cells (Fig. 1F). Quantification of apoptotic cells revealed that in 5% untreated, 6% SV-treated, 30% pU-treated, 25% pC-treated, and 58% pCU-treated U251 cells were apoptotic. 5% untreated, 6% SV-treated, 48% pU-treated, 38% pC-treated, and 64% pCU-treated 5310 cells were apoptotic as compared to DAPI stained cells (100%)(Fig. 1G). DNA fragmentation in treated samples further confirmed apoptosis (Supplementary Fig. S1). Analysis of mitochondrial membrane potential collapse by fluorescence microscopy using MitoLight dye indicated that pU, pC and pCU caused significant loss of mitochondrial membrane potential (Δψm) as indicated by a decrease in red fluorescence as compared to controls (Fig. 1H). Taken together, the data indicate that uPAR and cathepsin B play pivotal roles as anti-apoptotic molecules in glioma, and targeting uPAR and cathepsin B activity led to the induction of apoptosis.
RNAi-mediated downregulation of uPAR and cathepsin B increases the expression of pro-apoptotic genes and decreases the expression of anti-apoptotic genes
To evaluate the expression of apoptosis-related proteins, SV- and pCU-transfected U251 and 5310 cell lysates were analyzed using RayBio Human Apoptosis Antibody Array kit. A comparison of pCU- and SV-transfected cells indicated that expression of Bax, caspase-3 and p27 increased while expression of Bcl-2 decreased with pCU in U251 cells. In pCU-treated 5310 cells, the expression of BAX increased while Bcl-2 decreased (Fig. 2A). Quantification of protein signals by densitometry revealed that both BAX and capsase-3 increased 2.5-fold and p27 increased 5.4-fold. In contrast, Bcl-2 decreased 8.6-fold in pCU-treated U251 cells. In pCU-treated 5310 cells, Bax increased 3.6-fold; however, Bcl-2 decreased 3.8-fold (Fig. 2B). To confirm these results, we determined the expression of Bcl-2 and Bax by western blot analysis using antibodies specific to Bcl-2 and Bax. The results showed that the expression of Bcl-2 was significantly decreased with pU, pC, and pCU in both U251 and 5310 cells. In contrast, expression of Bax significantly increased with these treatments (Fig. 2C). Densitometric analysis reveals that the Bcl-2/Bax ratio decreased by 65% with pU, 60% with pC, 75% with pCU in U251 as compared to controls. Similarly, in 5310 cells, the Bcl-2/Bax ratio decreased by 85% with pU, 80% with pC, and 90% with pCU as compared to controls (Fig. 2D). To determine whether decreased Bcl-2 expression and increased Bax expression were caused by gene transcription, we examined the transcript levels of Bcl-2 and Bax using semi-quantitative RT-PCR. The results show that expression of Bcl-2 mRNA was significantly decreased by pU, pC, and pCU in both U251 and 5310 cells (Fig. 2E). Furthermore, quantification by densitometry revealed that the Bcl-2/Bax ratio (at the mRNA level) decreased by 80%, 85%, and 90% with pU-, pC- and pCU-treatments. The results were similar in both U251 and 5310 cells compared to the controls (Fig. 2F).
10.1371/journal.pone.0013731.g002Figure 2 Effect of uPAR and cathepsin B downregulation on the expression of pro- and anti-apoptotic molecules.
(A) Expression of pro- and anti-apoptotic molecules in U251 and 5310 cells 72 hrs after transfection with SV or pCU. Human apoptosis antibody arrays were exposed to cell lysates and processed as per the manufacturer's instructions. (B) Densitometric analysis and graphical representation of fold change of pro- and anti-apoptotic molecules. (C) Western blot analysis of Bcl-2 and Bax expression in U251 and 5310 cells 72 hrs transfection. The blots were stripped and re-probed with GAPDH antibody to verify equal loading. The experiments were repeated three times and representative blots are shown. (D) Densitometric analysis showing the Bcl-2/Bax ratio in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; **p<0.001. (E) Semi-quantitative RT-PCR analysis of Bcl-2 and Bax mRNA expression in pU-, pC- and pCU-transfected U251 and 5310 cells. Total RNA was extracted 72 hrs after transfection, and cDNA was synthesized as described in Materials and Methods. PCR was set up using first-strand cDNA as the template for Bcl-2, Bax and GAPDH. (F) Densitometric analysis showing the Bcl-2/Bax mRNA ratio. Columns: mean of triplicate experiments; bars: SE; **p<0.001, significant difference from untreated control or SV-transfected control.
Caspase dependent and independent apoptosis
To reveal the involvement of caspases, we used a broader caspase inhibitor, Z-Asp-2, 6-dichlorobenzoyloxymethylketone, to examine its ability to prevent apoptosis caused by uPAR and cathepsin B gene silencing. Analysis of cell distribution by FACS showed that pretreatment with the broad caspase inhibitor at 40 µM rescued the apoptosis in pCU-transfected U251 cells, but failed to prevent apoptosis in 5310 cells (Fig. 3A). Cell cycle distribution in histogram showed that the percentage of apoptotic cell population (G0–G1) was 4% in untreated, 5% in SV-treated, and 13% in pCU-treated U251 cells; in contrast, the apoptotic population was 7% in pCU-transfected cells pretreated with the broad caspase inhibitor. In 5310 cells, the percentage of the apoptotic cell population was 4% and 6% in untreated and SV-transfected cells, respectively. However, the percentage of the apoptotic cell population was 27% in both pCU-transfected cells and pCU-transfected cells pretreated with the broad caspase inhibitor (Fig. 3B). The above results indicate caspase-dependent apoptosis in U251 cells and caspase-independent apoptosis in 5310 cells.
10.1371/journal.pone.0013731.g003Figure 3 Downregulation of uPAR and cathepsin B induced caspase-dependent apoptosis in U251 cells and caspase-independent apoptosis in 5310 cells.
(A) FACS analysis of pCU-transfected U251 and 5310 cells pretreated with 40 µM broad caspase inhibitor (Z-Asp-2, 6-dichlorobenzoylmethylketone). (B) Histograms represent the percentage of cells in G0/G1, S and G2/M phases. The data represent one of the three independent experiments. Values are mean ± SD of three different experiments (*p<0.05, **p<0.001). uPAR and cathepsin B downregulation induced activation of caspase-9, caspase-3, ICAD, CAD in pU-, pC- and pCU-treated U251 cells. (C) Western blot analysis for active caspase-9, caspase-3, ICAD and CAD in U251 cells. (D) Expression of cytochrome c in mitochondrial and cytosolic fractions was determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions (MF); GAPDH was used for cytosolic fractions (CF). (E) Immunoprecipitation of Apaf-1 from U251 cell lysates. Total cell lysates were subjected to immunoprecipitation using anti-caspase-9 antibody and then immunoblotted for Apaf-1. Total lysates from SV-, pU-, pC- and pCU-treated U251 cells were separated into mitochondrial and nuclear fractions as per standard protocols and immunoblotted for AIF. (F) Expression levels of AIF in mitochondrial (MF) and nuclear fractions (NF) were determined by western blot analysis. Cytochrome oxidase IV was used as a marker for mitochondrial fractions; laminin B was used for nuclear fractions. Total lysates from SV-, pU-, pC- and pCU-treated 5310 cells were fractionated into mitochondrial and cytosolic fractions as per standard protocols and immunoblotted for cytochrome c. Immunoblots are representative of three experiments.
Downregulation of uPAR and cathepsin B induces release of cytochrome c from mitochondria into cytosol and activation of caspase-3, caspase-9 and CAD in U251 cells
We performed a colorimetric assay for caspase activity in uPAR and cathepsin B siRNA-induced apoptosis in U251 cells to explore the roles of activated caspase-3 and caspase-9. We observed substantial increases in the activity of caspase-3 and caspase-9 with pU and pC and a significant increase with pCU when compared to controls (Supplementary 2). These results were confirmed by western blot analysis. Figure 3C shows that pU and pC resulted in the substantial cleavage of caspase-3, caspase-9 and CAD as compared to controls. Notably, pCU significantly increased cleaved caspase-3, caspase-9 and CAD. The cleavage of caspases is aided by the release of caspase-activating factors, particularly cytochrome c, from the mitochondrial membrane into the cytosol [23]. Hence, we determined cytochrome c levels in both mitochondrial and cytosolic fractions using western blot analysis. In the present study, we found significantly increased signal for cytochrome c in the cytosolic fractions of pU-, pC- and pCU-treated U251 cells as compared to controls. In contrast, a decreased signal for cytochrome c was noticed in mitochondrial fractions (Fig. 3D). Once released into the cytosol, cytochrome c activates apoptotic protease-activating factor (Apaf-1), which together with caspase-9, forms an active holoenzyme complex known as apoptosome [23]. To test whether uPAR and cathepsin B knockdown interferes with the formation of apoptosome complex, we performed immunoprecipitation of caspase-9 with anti-caspase-9 antibody, followed by immunoblot analysis with anti-Apaf-1 antibody. The results indicate that Apaf-1 was significantly immunoprecipitated with caspase-9 in pU-, pC- and pCU-treated samples as compared to controls (Fig. 3E).
Downregulation of uPAR and cathepsin B activates the nuclear translocation of apoptosis-inducing factor (AIF) in 5310 glioma xenograft cells
To determine the nuclear localization of AIF, the expression levels of AIF were determined by western blot analysis of the cytoplasmic, mitochondrial and nuclear extracts of untreated and treated 5310 cells. From the results, we observed that AIF levels were remain same in the cytoplasmic extract of all treatments (data not shown). AIF levels were decreased in mitochondrial fractions of pU, pC and pCU treated 5310cells. However, nuclear localization of AIF was increased significantly when uPAR and cathepsin B were downregulated either individually or simultaneously (Fig. 3F).
Downregulation of uPAR and cathepsin B through siRNA treatment interferes with intracellular signaling events
To further elucidate uPAR and cathepsin B-mediated molecular signaling in the regulation of Bcl-2 and Bax expression, we investigated the involvement of the PI3K/Akt pathway by western blot analysis. Total PI3K (data not shown) and Akt expression levels remained more or less unchanged whereas the levels of p-PI3K p85α (Y 508) and p-Akt (S473) were significantly decreased with pCU transfection in both U251 and 5310 cells (Figs. 4A–B). Activation of PDGFR-β has been shown to induce the activation of PI3K and mediate uPA/uPAR signaling, so we next investigated the hypothesis that PDGFR-β serves as the accessory transmembrane adaptor molecule of uPAR [24]. Figures 4A–B show that expression of total PDGFR-β remained unchanged in both U251 and 5310, cells but p-PDGFR-β (Y751) significantly decreased with pCU transfection. To confirm these results, we used PI3K inhibitor (Wortmannin) at 20 and 40 µg/mL and PDGFR tyrosine kinase inhibitor (PTKI) at 5 and 10 nM concentrations. At a concentration of 40 µg/mL, the specific inhibitor of PI3K significantly inhibited basal expression of p-Akt, p-CREB (S133) and Bcl-2 and induced the expression of Bax in both U251 and 5310 cells (Figs. 4C&D). At a concentration of 10 nM, PDGFR tyrosine kinase inhibitor IV significantly inhibited the expression of p-PDGFR-β, p-PI3K, p-Akt, p-CREB and Bcl-2 and increased the expression of Bax (Figs. 4E&F).
10.1371/journal.pone.0013731.g004Figure 4 Downregulation of uPAR and cathepsin B using siRNA interferes with intracellular signaling events.
U251 and 5310 cells were transfected with SV, pU, pC or pCU. Untreated cells served as the control. After 72 hrs, cells were collected, and total cell lysates were prepared and western blotted as per standard protocol using normal and phosphorylated forms of PI3K, Akt and PDGFR-β. (A–B) Expression of normal and phosphorylated forms of PI3K, Akt and PDGFR-β in U251 and 5310 cells. Columns: mean of triplicate experiments; bars: SE; *p<005 and **p<0.001, significant difference from untreated control or SV-transfected control. Effect of PI3K inhibitor (Wortmannin) and PDGFR tyrosine kinase inhibitor (PTKI) on expression of p-PDGFR-β, p-PI3K p85α, Bcl-2 and Bax. U251 and 5310 cells were treated with 20 and 40 µg/mL of PI3K inhibitor (Wortmannin;WN) for 48 hrs, and the expression levels of p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (C–D) Western blot analysis of p-AKT, p-CREB, Bcl-2 and Bax expression levels after treatment with the PI3K inhibitor. Separately, U251 and 5310 cells were treated with 5 nM and 10 nM PDGFR tyrosine kinase inhibitor (PTKI) for 48 hrs. Expression levels of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax were determined by western blotting using appropriate antibodies. (E–F) Western blot analysis of p-PDGFR-β, p-PI3K, p-Akt, p-CREB, Bcl-2 and Bax expression levels in U251 and 5310 cells treated with PDGFR tyrosine kinase inhibitor.
uPAR and cathepsin B downregulation retards nuclear translocation and affects DNA binding activity of CREB
It has been shown that Bcl-2 expression might be regulated by the transcription factor CREB [25], so we investigated expression and phosphorylation of CREB in nuclear extracts of U251 and 5310 cells using western blotting. The results depicted in Figures 5A–B show that expression of total CREB was not altered; however expression of p-CREB was significantly decreased in the nuclear fraction of pCU-transfected U251 and 5310 cells as compared to controls. To further investigate the effect of uPAR and cathepsin B downregulation on CRE binding activity of CREB, we used antibody induced supershift analysis in EMSA. Nuclear extracts from SV- and pCU- treated cells showed supershift with strong binding when subjected to antibody induced supershift in EMSA. However, the intensity of the band showing supershift in pCU-treated cells was significantly decreased (Figs. 5C–D, lane 5). In contrast, with either no antibody or normal IgG antibody the CREB DNA binding mobility was not affected (Figs. 5C–D, lanes1, 3, 4 and 6). Taken together, these results strongly support that pCU-treatment specifically decreased the DNA binding activity of CREB.
10.1371/journal.pone.0013731.g005Figure 5 uPAR and cathepsin B downregulation retards nuclear translocation and decreases DNA binding activity of CREB.
U251 and 5301 cells were transfected with SV, pU, pC or pCU for 72 hrs. Cell lysates were separated into nuclear (NF) and cytoplasmic fractions (CF) and immunoblotted for p-CREB. Total cell lysates were probed for CREB. (A–B) Western blot analysis of p-CREB in nuclear and cytoplasmic fractions of U251 and 5310 cells. Nuclear extracts were prepared from SV- (control) and pCU-transfected U251 and 5310 cells and supershift analysis of CREB binding activity was carried out using EMSA. For supershift analysis, nuclear extracts were incubated with supershift specific CREB antibody (2.0 µg) or IgG (2.0 µg) prior to incubation with CREB binding buffer. (C–D) Supershift analysis of CREB DNA binding activity in U251 and 5310 cells. The experiments were performed three times and representative blots are shown. Nuclear extracts were prepared from SV-, pU-, pC- and pCU-transfected U251 and 5310 cells, and DNA binding activity of CREB was determined by colorimetric assay using the TransAM ELISA kit. To test specificity, DNA binding activity was tested in the presence of an excess of oligonucleotide containing a wild-type or mutated CREB consensus binding site. (E–F) Inhibition of DNA binding activity of CREB by nuclear extracts of pU-, pC- and pCU-treated U251 and 5310 cells. The bars represent the mean ± SD of three different experiments. *Statistically different compared to controls and pU-, pU- and pCU-treated groups (**p<0.001).
DNA-binding activity of CREB was also determined by colorimetric assay using the TransAM ELISA kit. To test the specificity of DNA binding, the assay was also performed in the presence of excess oligonucleotide containing a wild-type or mutated CREB consensus binding site. At 20X excess, the wild-type oligonucleotide prevents CREB binding to the probe immobilized on the plate. Conversely, the mutated oligonucleotide has little effect on CREB binding. The results show that without probe, optical density (OD) at 450 nm was 2.55 in nuclear extracts of untreated U251 and 5310 cells (control). In contrast, the OD was 1.25 in both pU- and pC-treated nuclear extracts of U251 cells and 1.3 and 1.5 in pU- and pC-treated nuclear extracts of 5310 cells. However, OD of all nuclear extracts with wild-type and mutated consensus oligonucleotides was 3, which indicates specific binding of CREB with CREB responsive elements (Figs. 5E–F). To further confirm CREB-mediated regulation of Bcl-2 and Bax, we treated both U251 and 5310 cells with CREB siRNA for 48 hrs and determined the expression of CREB, Bcl-2 and Bax using western blotting. The results indicate that expression levels of CREB and Bcl-2 were significantly decreased with CREB gene silencing in both U251 and 5310 cells. However, expression of BAX remains same in controls and siRNA treated U251 and 5310 cells (Supplementary Fig. S3).
pCU induces apoptosis, inhibits expression of uPAR, cathepsin B and Bcl-2, and enhances expression of BAX in vivo
To correlate the in vitro results with in vivo experiments, we further investigated the effect of uPAR and cathepsin B downregualtion on apoptosis using U251 and 5310 cells in nude mice. After the mice were implanted with U251 and 5310 cells as explained in Materials and Methods, the mice were observed for 30 days. Tumor samples were then taken and paraffin-embeded sections were prepared for immunohistopathological examination. These in vivo experiments would be helpful to confirm our in vitro data showing that uPAR and cathepsin B downregulation induces apoptosis in the glioma cells. To check the apoptosis in in vivo, we have carried out TUNEL assay on paraffin-embedded brain tissue sections. The results demonstrated significant DNA fragmentation in pCU-treated brain tissue sections as compared to control sections (Figs. 6A–B). Next, we determined expression of uPAR, cathepsin B, Bcl-2 and Bax in brain tissue lysates and tissue sections. Immunohistochemical analysis revealed that expression levels of uPAR, cathepsin B and Bcl-2 were decreased in pCU-treated brain tissue sections of both U251 and 5310 as compared to controls. In contrast, expression of Bax was increased in pCU-treated brain tissue sections as compared to controls (Figs. 6C–D). Western blot analysis further confirmed that pCU significantly inhibited the expression of uPAR, cathepsin B and Bcl-2 while the expression of BAX increased in both U251 and 5310 brain tissue lysates as compared to controls (Figs. 6E–F).
10.1371/journal.pone.0013731.g006Figure 6 RNAi-mediated downregulation of uPAR and cathepsin B induces apoptosis in pre-established intracranial tumors.
Intracranial tumors were established in nude mice by injecting U251 and 5310 cells that were treated with SV and pCU as described in Materials and Methods. (A–B) The brains were embedded in paraffin, sectioned and stained for apoptosis by TdT-mediated nick end-labeling (TUNEL) followed by DAB staining. Nuclei were counterstained with methyl green. Data shown are representative of five fields. Brown stain around green nuclei indicates apoptotic cells. (C–D) Immunohistochemical analysis of uPAR, cathepsin B, Bcl-2 and Bax was performed in paraffin-embedded U251 and 5310 tumor sections. Appropriate protein-specific antibodies were used. Fields with brown staining as a result of DAB interaction were scored for protein expression. (E–F) uPAR and cathepsin B, Bcl-2 and Bax expression was detected in tumor tissue lysates from intracranial tumors of mice that received SV and pCU. Results are representative of three separate experiments.
Discussion
uPAR and cathepsin B are overexpresssed in high-grade glioma, and this overexpression strongly correlates with invasive cancer phenotype and poor prognosis [26]–[29]. As stated earlier, RNAi has emerged as potent technology for exploiting target genes in glioma. As a preliminary assessment of the potential of RNAi against uPAR and cathepsin B as a therapeutic agent in growth inhibition, we assessed its in vitro apoptotic activity against U251 glioma and 5310 glioma xenograft cells. Transfection of glioma cells with siRNA for uPAR and/or cathepsin B strongly inhibited the expression of both proteins as previously reported [30]. When glioma cells were transfected with the bicistronic construct pCU, the morphology of cells became rounded and growth was inhibited (data not shown), which is suggestive of apoptosis. Further, our investigation using flow cytometric analysis and TUNEL assay confirmed that cell death induced by pU, pC and pCU transfection was due to induction of apoptosis.
Mitochondria are the central integrators and coordinators of both intracelluar and extracellular signals that mediate caspase-dependent and caspase-independent cell death [31]. Recently, it has been reported that induction of mitochondrial apoptosis requires the involvement of the Bcl-2 family, including apoptosis inhibiting gene products (e.g., Bcl-2, Bcl-xL) and apoptosis accelerating gene products (e.g., Bax, Bak, Bcl-xS, Bim) [32]. Loss of mitochondrial membrane potential is a prerequisite for mitochondrial-mediated apoptosis as it is associated with the reshuffling of Bcl-2 family members between the cytoplasm and mitochondria. The Bcl-2 family is comprised of proteins, which share a Bcl-2 homology (BH) region and undergo either heterodimerization or homodimerizaton [33]. The ratio between anti- and pro-apoptotic proteins is said to be a determinant for tissue homeostasis because it influences the sensitivity of cells to inducers of apoptosis [34], [35]. Overexpression of Bcl-2 is reported to cause inhibition of programmed cell death in many cell types, and this anti-apoptotic function appears to be modulated by its ability to heterodimerize with other members of the family, especially the apoptosis-inducing Bax protein [36].
In this study, we have demonstrated that the downregulation of uPAR and cathepsin B induced mitochondrial-mediated apoptosis in the U251 glioma cell line and 5310 glioma xenograft cells and was accompanied by the collapse of mitochondrial membrane potential. We also noticed that downregulation of uPAR and cathepsin B decreased the Bcl-2/Bax ratio. An earlier study reported that downregulation of uPAR and cathepsin B initiated partial extrinsic apoptotic cascade accompanied by the collapse of mitochondrial membrane potential in SNB19 glioma cells [37]. Moreover, Kin et al. [38] reported that downregulation of uPAR was associated with increased expression of the pro-apoptotic protein Bax in glioma cells. Taken together, these results suggest that uPAR and cathepsin B downregulation induced apoptosis by modulating the Bcl-2/Bax ratio accompanied by collapse of mitochondrial membrane potential.
In the present study, significant increases in caspase-3, Apaf-1 and cytochrome c were also observed with the downregulation of uPAR and cathepsin B in U251 cells but not in 5310 cells (data not shown). In contrast, in 5310 cells, the downregulation of uPAR and cathepsin B was accompanied by the nuclear translocation of AIF from the mitochondria. Cytochrome c combines with Apaf-1, caspase-9 and ATP to form the apoptosome in the cytoplasm [39]. AIF is another mitochondrial protein that is translocated to the cytosol and nucleus during apoptosis [40]. In the nucleus, AIF is thought to increase chromatin condensation and large-scale DNA fragmentation [41]. Interestingly, a broad caspase inhibitor, Z-Asp-2-6-dichlorobenzoylmethylketone, prevented pCU-induced apoptosis in U251 cells but fail to reduce the apoptotic percentage of 5310 xenograft cells, which indicates caspase-dependent apoptosis in U251 cells and caspase-independent apoptosis in 5310 cells. Similarly, Shih et al. [42] reported that cadmium induced a caspase-independent apoptotic pathway involving mitochondria-mediated translocation of AIF in normal human lung cells. Further, Tae-Jin Lee et al. [43] also reported caspase-dependent and caspase-independent apoptosis in human leukemic U937 cells.
The translocation of AIF and release of cytochrome c would occur after the collapse of mitochondrial membrane potential. However, mechanisms of membrane permeabilization are still matter of debate. Irrespective of the exact mechanisms, the anti-apoptotic members of the Bcl-2 family tend to stabilize the barrier function of mitochondrial membranes, whereas pro-apoptotic Bcl-2 family proteins destabilize it [44]. uPAR is a GPI-anchored cell surface protein known to be associated with pro-uPA and cathepsin B [45]. uPAR relies on cell surface and transmembrane proteins, including platelet-derived growth factor receptor (PDGFR) for its non-proteolytic functions [46]. Recently, a direct interaction of uPAR with PDGFR beta in macrophages [24] and vascular smooth muscle cells [44] by the immunefluorescence approach, immunoprecipitation and chemical cross linking experiments have been reported. Further, the effect of cathepsin B on PDGFR beta mediated signaling might be indirect and possibly through uPAR.
In the current study, western blot analysis using phospho-specific antibodies confirmed that expression of p-PDGFR-β, p-PI3K and p-Akt were significantly decreased with the downregulation of uPAR and cathepsin B in both U251 and 5310 cells. However, this protein phosphorylation was inhibited by pre-treatment with PDGFR tyrosine kinase inhibitor IV and PI3K inhibitor. It is well known that tyrosine kinase activity of PDGFR-β was found to be decisive for mediating downstream signaling. Duronio [47] recently reported that PDGFR-β is associated with PI3K/Akt-mediated cell survival. These results further confirm that uPAR-initiated signaling is mediated though PDGFR-β and the PI3K/Akt pathway.
Recently, CREB was reported to be activated as a transcription factor by phosphorylation at Ser-133 through several signaling pathways, including PI3K/Akt [48]. Pugazhenthi et al. [49] reported that Akt/protein kinase B upregulates Bcl-2 expression through cAMP-response element-binding protein. CREB plays a key role in regulating neuronal survival and appears to be a primary transcription activator of the anti-apoptotic gene, Bcl-2 [50], [51]. CREB mediates regulation of gene expression via its binding to a CRE in the promoter region [52].
In this study, western blot analysis of cytoplasmic and nuclear fractions showed that uPAR and cathepsin B downregulation retarded the translocation of p-CREB in both U251 and 5310 cells. In addition, analysis of DNA binding activity by EMSA and ELISA revealed that uPAR and cathepsin B downregulation also affected DNA binding activity of CREB. To our knowledge, this is the first report on the uPAR and cathepsin B-mediated regulation of DNA binding activity of CREB in glioma. Moreover, knockdown of CREB using siRNA significantly decreased expression of Bcl-2 but not Bax. Further, use of a PI3K inhibitor significantly decreased the expression of both Bcl-2 and Bax. These results suggest that uPAR and cathepsin B downregulation suppressed Bcl-2 expression possibly though inhibition of the PI3/Akt pathway and regulation of DNA binding activity (transcriptional) of CREB. However, Bax levels might be enhanced by retaining Bax in the cytoplasm. This is an accordance with an earlier study that reported PI3K/AKT activity was essential for retaining Bax in the cytoplasm [18].
It has been previously demonstrated that in vivo treatment of pre-established intracranial tumors with plasmids expressing siRNA for uPAR and cathepsin B significantly inhibited tumor growth in glioma [53]. In this study, we observed significant cell death in pCU-treated U251 and 5310 brain tissue sections. In addition, immunohistochemical analysis for Bcl-2 and Bax in tumor brain sections treated with pCU and western blot analysis of Bcl-2 and Bax in brain tissue lysates strongly confirmed our in vitro results. Further, simultaneous downregulation of two molecules using bicistronic vectors proved to be more effective than specifically targeting a single gene/molecule [54]. However, the mechanism of cell cycle and apoptosis is highly complex and involves coordinated regulation of several molecules. Each of these molecules might have a specific and independent role in apoptosis; hence the effect is not always synergistic [55]. Studies have also shown that the cellular proteases can either act independently or coordinate with other proteases in the process of invasion, angiogenesis and apoptosis [56], [57]. In conclusion, downregulation of uPAR and/or cathepsin B induced mitochondrial-mediated apoptosis by modulating the Bcl-2/Bax ratio via inhibition of PDGFR-β and the PI3K/Akt pathway (Fig. 7). Taken together, our results suggest uPAR and cathepsin B are promising potential therapeutic targets for glioma.
10.1371/journal.pone.0013731.g007Figure 7 Schematic representation of proposed molecular mechanisms involved in the regulation of mitochondrial-mediated apoptosis via PDGFR β and the PI3K/Akt pathway in uPAR and cathepsin B-depleted glioma cells.
Materials and Methods
Ethics Statement
The Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. The consent was written and approved. The approved protocol number is 851, dated November 20, 2009.
Cell culture and transfection conditions
We used the U251 glioblastoma cancer cell line (obtained from ATCC) and 5310 glioblastoma xenograft cells (kindly provided by Dr. David James, University of California, San Francisco) in this study. U251 and 5310 cells were grown in DMEM/F12 medium and RPMI 1640 medium, respectively and supplemented with 10% FBS and 1% penicillin/streptomycin. After overnight culturing in serum-free medium, cells were transfected with scrambled vector (SV), puPAR (pU), pCathepsin B (pC), or a bicistronic construct comprised of puPAR and pCathepsin B (pCU) for 72 hrs using FuGene according to manufacturer's instructions (Roche Applied Science, IN). For CREB knockdown, U251 and 5310 cells were transfected with CREB siRNA for 48 hrs using FuGene according to manufacturer's instructions (Cell Signaling Tech., Danvers, MA).
Western blotting and immunoprecipitation
U251 and 5310 glioma cells or brain tissues were harvested as previously described [58]. Equivalent amounts of total protein (30–50 µg per lane) were loaded onto 6–14% acrylamide gels, resolved by electrophoresis, and electrotransferred onto nitrocellulose membranes. Membranes were blocked and subsequently incubated overnight at 4°C with primary antibodies. We obtained all of the antibodies used in this study from Santa Cruz Biotechnology (Santa Cruz, CA) except for anti-PDGFR-β, anti-p-PDGFR-β, anti-Akt, anti-p-Akt, anti-PI3K, anti-p-PI3K, anti-CREB, anti-p-CREB (S133), anti-Bcl-2, and anti-Bax, which were supplied by Cell Signaling (Boston, MA). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibody raised against the corresponding species for 1 hr at room temperature. Proteins on membranes were developed using Pierce ECL Western Blotting Substrate according to manufacturer's instructions (Thermo Scientific Inc, Rockford, IL). Protein content was normalized against the GAPDH level, which was used as a loading control.
Caspase-9 was immunoprecipitated from 300 µg of total protein using anti-caspase-9 antibody and protein A coupled with G agarose beads (20 µg). The precipitates were washed five times with lysis buffer and once with PBS. The pellet was then resuspended in 1X sample buffer [50 mM Tris, (pH 6.8), 100 mM bromophenol blue, and 10% glycerol], incubated at 90°C for 10 min before electrophoresis to release the proteins from the beads, and then immunoblotted for Apaf-1.
Cell cycle analysis
Phases of cell cycle were analyzed using flow cytometry as described previously [59]. Cells transfected with SV, pU, pC, pCU or pCU + pretreatment with broad caspase inhibitor were trypsinized, washed with 1X PBS, and incubated with 1 mL of propidium iodide for 30 min in the dark. The DNA content of these cells was measured based on the presence of propidium iodide (PI)-stained cells (Sigma Aldrich, St. Louis, MO). Flow cytometric analysis was carried out on at least 10,000 cells from each sample, and cell cycle data were analyzed using a FACS Calibur flow cytometer (BD BioSciences, San Jose, CA) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
TUNEL assay
TUNEL assay was performed with adherent cells [40] and paraffin-embedded tissue sections [60] using In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Indianapolis, IL). After transfection with SV, pU, PC, or pCU, cells were fixed with buffered formaldehyde (pH 7.4) and incubated with a reaction mixture containing biotin-dUTP and terminal deoxynucleotidyl transferase for 1 hr. The slides were stained with DAPI, mounted with cover slips, and positively fluorescein-labeled cells were visualized with fluorescence microscopy, quantified, and expressed as percent compared to DAPI stained cells. For tissue sections, after deparaffinization and rehydration, tissue sections were pretreated with sodium chloride-sodium citrate buffer (pH 7.0) at 80°C for 20 min, followed by thorough washing in distilled water. Subsequently, sections were digested with Proteinase K for 1 hr with gentle agitation at 37°C and digestion was stopped by washing in running water. After protease treatment, tissue sections were treated as described above and subsequently stained with DAB via peroxidase-conjugated avidin and counterstained with 0.5% methyl green.
Visualization of mitochondrial permeability transition
Mitochondrial membrane potential changes were assayed with MitoLight dye according to manufacturer's instructions (Millipore, Danvers, MA). After transfection with SV, pU, pC or pCU, cells were incubated with pre-diluted MitoLight solution for 30 min. Cells were washed twice with 1X incubation buffer, mounted with cover slips, and observed immediately under a fluorescence microscope.
Apoptosis array
Cell lysates from SV- and pCU-treated U251 and 5310 cells were analyzed using a human apoptosis antibody array (RayBiotech, Norcross, GA) according to the manufacturer's instructions. Signal intensities were quantified by densitometry, and fold change was calculated by comparing with controls.
Reverse transcription PCR (RT-PCR)
Total RNA was extracted from the transfected cells using TRIZOL reagent (Invitrogen, Carlsbad, CA) as per standard protocol. DNase-treated RNA was used as a template for reverse transcription (Invitrogen) followed by PCR analysis using specific primers for Bcl-2, Bax and GAPDH (Table 1). The PCR conditions were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 58°C for 45 sec, and 72°C for 45 sec. GAPDH was used as an internal control.
10.1371/journal.pone.0013731.t001Table 1 Genes analyzed by RT-PCR.
Gene Forward Primer Reverse Primer
Bcl-2
5′TTCCACGCCGAAGGACAGCG 3′
5′GGCACTTGTGGCGGCCTGAT 3′
BAX
5′AGTGGCAGCTGACATGTTTT 3′
5′GGAGGAAGTCCAATGTCCAG 3′
GAPDH
5′CGGAGTCAACGGATTTGGTCGTAT 3′
5′AGCCTTCTCCATGGTGGTGAAGAC3′
DNA binding activity of CREB
The interaction of CREB protein with DNA present in the nuclear extracts from SV- and pCU-treated U251 and 5310 cells was determined by supershift analysis experiments in EMSA using Pamomics kit according to manufacturer's instructions Affymetrix Inc, Fremont, CA). Briefly, nuclear extracts were incubated with supershift specific antibody (2.0 µg) (Santa Cruz Biotechnology, SC:271x) and normal IgG antibody (2.0 µg) for 30 min prior to incubation with biotin-labeled CREB transcription probe. The protein/DNA complexes were separated on a 6% non-denaturing polyacrylamide gel, and then they were transferred to a nylon membrane and detected using streptavidin-HRP and a chemiluminescent substrate.
DNA-binding activity of CREB was confirmed by ELISA (Active Motif, which is based on multi-well plates coated with an oligonucleotide containing the consensus binding site of the transcription factor), according to the manufacturer's recommendations. Briefly, 10 µg of nuclear proteins were incubated for 2 hrs in a 96-well plate pre-coated with a double-stranded oligonucleotide containing the consensus CRE site (TGACGTCA). Specificity of binding was also determined using wild-type and mutated consensus oligonucleotides. Binding of p-CREB was determined with an anti-Ser133-pCREB rabbit antibody. Colorimetric reaction was then performed with a HRP-conjugated, anti-rabbit IgG antibody, and absorbance was measured at 450 nm in a spectrophotometer.
Subcellular fractionation
Fractionation of nuclear and cytosolic proteins was carried out as previously described [40]. Fractionation of mitochondrial proteins was done using a mitochondrial isolation kit (Sigma, St. Louis, MO) according to manufacturer's instructions. Briefly, cells were suspended in cell lysis buffer and homogenized on ice using a Dounce homogenizer (10–30 strokes) and centrifuged at 600×g for 10 min at 4°C. The supernatant was carefully transferred to a fresh tube and centrifuged at 11,000×g for 10 min at 4°C. The supernatant was discarded and the pellet was resuspended in CellLytic M cell lysis reagent with a protease inhibitor cocktail (1∶100 [v/v]) and used to analyze mitochondrial proteins.
Immunohistochemical analysis of brain tumor sections for Bcl-2 and Bax
Stereotactic implantation of untreated, SV-, or pCU-treated U251 and 5310 cells (1×105) was performed using Alzet minipumps at the rate of 0.25 µL/hr. Sacrifice of glioma-bearing mice and tumor processing were done as previously described [61], [62]. Five animals were used per treatment condition. Immunohistochemical analysis for Bcl-2 and Bax was performed using standard protocols.
Caspase activity assay
The activity of caspase-3 and caspase-9 was determined using colorimetric assay according to manufacturer's instructions (Chemicon International Inc., Temecula, CA). The lysates were transferred to a 96-well plate and treated with the respective peptide substrate for each caspase conjugated with p-nitroaniline (Ac-DEVD-pNA). After overnight incubation, OD was measured at 405 nm using a microplate reader.
DNA laddering assay
DNA laddering assay was carried out as described previously [63]. Briefly, untreated and treated cells were resuspended in PBS, fixed in 70% ethanol, and incubated overnight at −20°C. Ethanol was removed by centrifugation at 5000 rpm for 3 min, and the pellet was resuspended in 0.2 M phosphate-citrate buffer (pH 7.8) and kept at room temperature for 30 min. The pellet was then spun down at 10,000 rpm for 5 min, mixed with 3 µL of NP-40 and 3 µL of RNase A, and then incubated at 37°C for 30 min. After adding 3 µL of Proteinase K solution, the mixture was incubated for an additional 30 min. An aliquot of each DNA extract was then mixed with loading buffer and separated on a 1.5% agarose gel. DNA fragmentation was visualized under UV light after staining with ethidium bromide.
Supporting Information
Figure S1 RNAi-mediated downregulation of uPAR and cathepsin B induces DNA fragmentation in U251 and 5320 cells. Untreated and treated cells were fixed in 70% ethanol overnight at −20°C. Ethanol was removed, and DNA was extracted and separated on 1.5% agarose gels as described in Materials and Methods. DNA fragmentation was visualized under UV light after staining with ethidium bromide. Agarose gel electrophoresis pattern of DNA obtained from U251 and 5310 cells after transfection with SV, pU, pC or pCU. Arrow indicates 180 bp laddering.
(0.60 MB TIF)
Click here for additional data file.
Figure S2 RNAi-mediated downregulation of uPAR and cathepsin B enhances activity of caspase-3 and caspase-9 in U251 and 5310 cells. Cell lysates were prepared from U251 cells transfected with SV, pU, pC or pCU and treated overnight with the respective peptide substrate for each caspase conjugated with p-nitroaniline (Ac-DEVD-pNA). Activity of these proteases was measured at 405 nm using a microplate reader. Bars represent the mean ± SD of three experiments (*p<0.05, **p<0.001).
(0.03 MB TIF)
Click here for additional data file.
Figure S3 Effect of CREB knockdown on Bcl-2 and Bax expression. U251(A) and 5310(B) cells were transfected with CREB siRNA for 48 hrs, and the expression levels of total CREB, p-CREB, Bcl-2 and Bax were determined by western blot analysis using appropriate antibodies.
(0.14 MB TIF)
Click here for additional data file.
We thank Noorjehan Ali for technical assistance, Shellee Abraham for manuscript preparation, and Diana Meister and Sushma Jasti for manuscript review.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This research was supported by a grant from National Institutes of Health, CA116708 and CA75557 (to J.S.R.). The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central 1471-2474-11-2412095897310.1186/1471-2474-11-241Research ArticleLow back pain in junior Australian Rules football: a cross-sectional survey of elite juniors, non-elite juniors and non-football playing controls Hoskins Wayne [email protected] Henry [email protected] Chris [email protected] Andrew [email protected] Peter [email protected] Andrew [email protected] Kate [email protected] George [email protected] Department of Chiropractic, Macquarie University, Sydney, NSW 2109, Australia2 Harbord Village Chiropractic, Freshwater, NSW 2096, Australia3 Norwest Orthopaedic and Sports Physiotherapy, Norwest, NSW 2153, Australia4 Enhance Chiropractic and Massage Sports Injury Centre, Ngunnawal, ACT 2913, Australia2010 19 10 2010 11 241 241 11 11 2009 19 10 2010 Copyright ©2010 Hoskins et al; licensee BioMed Central Ltd.2010Hoskins et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Low back pain in junior Australian Rules footballers has not been investigated despite findings that back pain is more prevalent, severe and frequent in senior footballers than non-athletic controls and findings that adolescent back pain is a strong predictor for adult back pain. The aim of this study was to determine the prevalence, intensity, quality and frequency of low back pain in junior Australian Rules footballers and a control group and to compare this data between groups.
Methods
A cross-sectional survey of male non-elite junior (n = 60) and elite junior players (n = 102) was conducted along with a convenience sample of non-footballers (school children) (n = 100). Subjects completed a self-reported questionnaire on low back pain incorporating the Quadruple Visual Analogue Scale and McGill Pain Questionnaire (short form), along with additional questions adapted from an Australian epidemiological study. Linear Mixed Model (Residual Maximum Likelihood) methods were used to compare differences between groups. Log-linear models were used in the analysis of contingency tables.
Results
For current, average and best low back pain levels, elite junior players had higher pain levels (p < 0.001), with no difference noted between non-elite juniors and controls for average and best low back pain. For low back pain at worst, there were significant differences in the mean pain scores. The difference between elite juniors and non-elite juniors (p = 0.040) and between elite juniors and controls (p < 0.001) was significant, but not between non-elite juniors and controls. The chance of suffering low back pain increases from 45% for controls, through 55% for non-elite juniors to 66.7% for elite juniors. The chance that a pain sufferer experiences chronic pain is 16% for controls and 41% for non-elite junior and elite junior players. Elite junior players experienced low back pain more frequently (p = 0.002), with no difference in frequency noted between non-elite juniors and controls. Over 25% of elite junior and non-elite junior players reported that back pain impacted their performance some of the time or greater.
Conclusions
This study demonstrated that when compared with non-elite junior players and non-footballers of a similar age, elite junior players experience back pain more severely and frequently and have higher prevalence and chronicity rates.
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Background
There has been increased awareness of low back pain (LBP) in children and adolescents with several studies showing that LBP is highly prevalent in the early years of life [1,2]. LBP increases with age during the first decades [3], with prevalence increasing significantly following sexual maturity [4]. It has been theorized that LBP in childhood may have important consequences for chronic LBP in adulthood [5]. This theory has been validated with clear correlations between LBP in childhood and adolescence and LBP in adulthood [6]. Hestbaek et al. in a large longitudinal study found LBP in adolescence to be a significant risk factor for LBP in adulthood with odds ratios as high as four [6]. A dose-response association was also demonstrated: the more days with LBP, the higher the risk of future LBP. These findings are supported by research which demonstrates that 90% of schoolchildren with LBP suffer from LBP 25 years later [7].
Questions have been raised regarding LBP at the junior level of sporting competition given that participation in adolescent sports has been found to be a risk factor for LBP [8] and sport participation produces higher LBP prevalence than in non-athletes [9], particularly in males [1]. It is believed that adolescent athletes with less musculoskeletal maturity may be at a heightened risk for more severe and permanent skeletal damage and structural abnormalities, particularly when exposed to years of intense athletic training [10].
There are no specific studies investigating LBP in junior Australian Rules footballers. This is despite the popularity of the sport and recent evidence showing that elite senior and semi-elite footballers experience LBP more severely and frequently than non-athletic controls, with this trend being more evident in elite players [11]. Thus, the primary objective of the present study was to determine the prevalence, intensity, quality and frequency of LBP in junior Australian Rules footballers. The secondary objective was to compare this data between non-elite junior and elite junior participants and with that of a control group of non-footballers.
Methods
The study was designed as a cross-sectional survey of male junior Australian Rules football participants. Players were drawn from local underage competitions (classified as non-elite junior), the Victorian state based under 18 TAC Cup (classified as elite junior) and a convenience sample of high school students (classified as controls). The study was approved by the Macquarie University Human Ethics Committee.
The Victorian Football League (VFL) was approached by the authors to participate in the study. They agreed and randomly selected 3 TAC Cup teams to provide players for the elite junior group. Non-elite junior players were selected from a convenience sample of junior football clubs from different states. For clubs to consent to participate it was required that they provide access to their entire player rosters to ensure 100% compliance which would assist in providing an accurate reflection of the status of LBP in the participating clubs. The survey was completed during the 2006 season. All players on the playing lists of the selected clubs were invited to participate and complete the survey with responses remaining confidential. Controls were drawn from a convenience sample of male school students, with the specification that they not participate in Australian Rules football.
The study was presented to the clubs and players as a LBP survey. Questionnaires were either administered by an author of the study or by an official representing the participating club, at the preference of the club. In the case of the club wishing to administer the survey, the questionnaires were mailed out along with consent forms and instructions describing the purposes and procedures of the study and how the instruments were to be administered. This was followed by a telephone call to confirm that all procedures would be correctly administered, to ensure players completed every question and to make certain the club officials were qualified to answer questions of the players. None of the assessors were involved in the analysis of the data. Analysis was provided by a person independent to each of the group allocations.
The questionnaire was developed using the validated and reliable Quadruple Visual Analogue Scale (QVAS) [12], the McGill Pain Questionnaire (short-form) (MPQ-SF) [13], along with a series of questions either adapted from an Australian LBP epidemiological study with permission of the author [14] or that the authors developed themselves and thoroughly pilot tested.
The additional questions were:
• How old were you when you had your first ever attack or episode of LBP?
• If applicable, was this a result of your sporting commitments and activities or not related to this?
• When did your current episode of LBP commence?
• If applicable, was this a result of your sporting commitments and activities or not related to this?
• How often do you experience LBP?
• Do you feel that your low back pain negatively effects or impacts your performance?
To assist with answering the questions a diagram of a mannequin that defined the anatomical boundaries of the low back as a shaded area between the last ribs and the gluteal folds was provided. For the purposes of this survey the shaded area represented the low back and subjects were told to focus only on LBP and not other sources of pain. This area was found to be the most commonly used in a review of methodologically sound LBP prevalence studies [15].
The forms were manually entered using Microsoft Excel® and analyzed using GenStat. Descriptive statistics are used to report player characteristics. Linear Mixed Model (Residual Maximum Likelihood) methods were used to compare differences between groups because of imbalance in replication. Log-linear models were used in the analysis of contingency tables. For all tests a p value <0.05 was considered significant, however actual p values are reported in the study.
Results
Three of the 12 TAC Cup teams, 3 non-elite junior clubs and 2 high schools participated. The subject characteristics of the different groups are shown in table 1. The results of the QVAS questionnaire are presented in table 2. For LBP now, pain levels were significantly different between groups (p < 0.001). For the LBP average and LBP at best, there were significant differences across the three groups (p < 0.001), with no difference between non-elite junior players and controls, but strong differences between them and elite players. For LBP at worst, there were significant differences in the mean pain scores across the three groups (p < 0.001). The difference between elite juniors and non-elite juniors (p = 0.040) and between elite juniors and controls (p < 0.001) was significant, but not between non-elite juniors and controls.
Table 1 Descriptive statistics and distribution for the ages of subjects in the study
Elite junior Non-elite junior Control
Number 102 60 100
Mean (SD) 17.2 (0.576) 15.8 (0.676) 15.8 (1.237)
Median 17 16 16
Range 16-18 14-17 14-18
Frequency Percentage
Age Elite junior Non-elite junior Control Elite junior Non-elite junior Control
14 0 1 24 0 1.7 24.0
15 0 17 12 0 28.3 12.0
16 10 34 26 9.8 56.7 26.0
17 66 8 35 64.7 13.3 35.0
18 26 0 3 25.5 0 3.0
Table 2 Analysis of the Quadruple Visual Analogue Scale (QVAS) questionnaire and results of the overall pain question from the MPQ-SF and frequency of LBP episodes.
QVAS Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100)
LBP now mean (SE) 21.32 (2.24) 11.87 (2.02) 6.57 (1.18)
LBP average mean (SE) 21.68 (1.92) 11.90 (1.60) 11.21 (1.28)
LBP best mean (SE) 6.50 (1.13) 2.97 (0.94) 1.67 (0.36)
LBP worst mean (SE) 48.63 (2.83) 39.03 (3.69) 31.71 (2.86)
Frequency Percentage
Overall pain Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100) Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100)
No pain 29 24 41 28.4 40.0 41.0
Mild 43 23 40 42.2 38.3 40.0
Discomforting 24 12 15 23.5 20.0 15.0
Distressing 5 1 4 4.9 1.7 4.0
Horrible 1 0 0 1.0 0 0
Frequency of pain Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100) Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100)
No pain 8 6 6 7.8 10.0 6.0
Daily 30 7 12 29.4 11.7 12.0
Weekly 9 7 9 8.8 11.7 9.0
Fortnightly 12 8 7 11.8 13.3 7.0
Monthly 11 4 9 10.8 6.7 9.0
3 Monthly 1 4 8 1.0 6.7 8.0
6 monthly 5 5 15 4.9 8.3 15.0
Yearly 26 19 34 25.5 31.7 34.0
The results of the overall pain question are presented in table 2. There were few players with distressing or horrible LBP and the percentage of elite juniors having no pain appeared low.
The results of the MPQ-SF are presented in table 3. Analysing the MPQ-SF, there were no significant differences in the mean sensory values across the three groups (p = 0.068). The difference between elite juniors and non-elite juniors just failed to be significant (p = 0.057). This difference may have been significant had the study had larger subject numbers. There were no significant differences in the mean affective values (p = 0.575) and MPQ-SF total questions (p = 0.112) across the three groups.
Table 3 Analysis of the McGill Pain Questionnaire (short form) (MPQ-SF).
MPQ-SF Elite junior
(n = 102) Non-elite junior
(n = 60) Control
(n = 100) P value between groups
Sensory questions (SE) 14.97 (1.28) 10.95 (1.67) 11.28 (1.29) 0.068
Affective questions (SE) 8.66 (1.38) 6.81 (1.31) 7.05 (1.23) 0.575
Total questions (SE) 13.29 (1.21) 9.84 (1.58) 10.16 (1.22) 0.112
There were a substantial number of players who could not recall when they first experienced LBP (36.3%, n = 37 of elite juniors, 51.7%, n = 31 of non-elite juniors and 41.0%, n = 41 of controls). The mean (SE) age of first onset LBP were: elite juniors 15.18 (0.20), non-elite juniors 13.68 (0.31), and controls 13.88 (0.24). Of the elite juniors, 8.8%, n = 9 said that they had not experienced back pain, 6.7%, n = 4 of non-elite juniors and 18.0%, n = 18 of controls. Using the players who could recall first onset of LBP as being representative of the population, the mean ages of first onset LBP was different across groups (p < 0.001), but there was no difference between non-elite junior players and controls (p = 0.612). The age of first onset LBP for elite juniors was significantly older, by 1.3 years (p < 0.001). When asked whether their first onset LBP was caused by sport or not 71.0%, n = 66 of elite juniors, 66.1%, n = 37 of non-elite juniors and 69.5%, n = 57 of controls indicated that their first onset of LBP was due to sport, although this was non-significant (X2 = 0.39, df = 2, p = 0.821).
With regards to the commencement of the current episode of LBP the probability of a player suffering LBP varied significantly across groups (X2 = 9.71, df = 2, p = 0.008). The chance of suffering LBP increases from 45% for controls), through 55% for non-elite juniors to 66.7% for elite juniors. Defining acute LBP as pain commencing within the last three months, and chronic LBP as pain commencing beyond the last three months then for players who suffer LBP, the probability of LBP being acute is significantly different across groups (X2 = 9.64, df = 2, p = 0.008). The chance that a pain sufferer experiences chronic pain is 16% for controls, and 41% for non-elite juniors and elite juniors. When asked if their current episode of LBP was due to sporting commitments or activities the percentages across groups was not significant (X2 = 5.36, df = 2, p = 0.069). There was no difference in probability that sport was the cause of the current LBP for non-elite juniors and controls (X2 = 0.261, df = 1, p = 0.609) but was significantly more likely for elite juniors (X2 = 5.094, df = 1, p = 0.024).
The frequency of LBP is presented in table 2. The distribution of episodes was different across groups (X2 = 27.27, df = 14, p = 0.018). The difference between non-elite juniors and controls was not significant (X2 = 4.41, df = 7, p = 0.732), while elite juniors were strongly significantly different by comparison (X2 = 22.86, df = 7, P = 0.002).
When the elite junior and non-elite junior players were asked whether LBP impacted their performance there was no statistical significance in distributions (X2 = 3.24, df = 3, p = 0.356). For the elite juniors 33.3%, n = 34 said LBP did not impact their performance, 40.2%, n = 42 said it did little of the time and 21.6%, n = 22 some of the time. For the non-elite junior players 43.3%, n = 26 said LBP did not impact their performance, 26.7%, n = 16 said it did little of the time and 25.0%, n = 15 some of the time.
Discussion
This study demonstrated that when compared with non-elite junior players and non-football playing school children of a similar age, elite junior Australian Rules football players experience LBP more severely and frequently and have higher prevalence and chronicity rates. Although affected by recall bias and low response rate, the elite junior players experience first time onset LBP at an older age at an age typical of entry into underage elite training programs. The elite juniors did not attribute first time LBP to be due to sporting commitments or activities more so than non-elite junior players or school children. However, sporting commitments or activities were attributed to cause a high percentage of first time LBP in all groups. Elite juniors did attribute sporting commitments or activities to be more likely to cause their current LBP episode. Although there was no difference between the elite junior and non-elite junior players in their belief of whether LBP impacted their performance, both groups had over 25% report that LBP affected their performance some of the time or greater. Of interest, the elite juniors have a prevalence and overall LBP rating the same as semi-elite senior Australian football code participants, using the same research methodology [11].
Strengths of our study include the use of validated questionnaires to quantify the intensity (QVAS) and quality of LBP (MPQ-SF). Functional disability associated with LBP was not determined. This important aspect was not investigated as it was felt that validated questionnaires in use to determine these parameters are not created for elite athletic populations. As far as we are aware, this is the first study to investigate LBP in junior Australian Rules footballers.
Limitations exist in the study conducted. Firstly, the convenience sample taken for the non-football playing school children and non-elite junior players is not a random population sample and may not be representative. However, random sampling not producing a 100% response rate has been discussed as potentially leading to overestimates of LBP in similar research [11,16]. Our controls were used because of their likely non-elite junior sporting participation and likelihood to be matched for age. It should be noted that at school age most children participate in some form of recreational or organised sport either at school or externally. That is why they were termed non-footballers, with the important finding of our study being that their LBP profile largely does not differ from junior footballers. Future study would benefit from documenting what sporting participation, if any, is performed by the control group. Secondly, there was a difference in numbers between the groups, with a larger number of non-elite junior players being preferred. Future study would also benefit from larger total numbers. Thirdly, although ages were close, it was difficult to achieve a complete match between groups, largely due to junior football clubs often finishing before the under 18 level and school children finishing their eduction at an earlier mean age than the elite juniors. Fourthly, as some questions asked were retrospective in nature, there is likely an element of recall bias in the answers to some questions. This is in particular for questions regarding first time onset of LBP, with questions regarding the current episode of LBP being less affected. Lastly, there may be issues with reliability with adolescents competing the questionnaires, although this would have been affected equally between groups.
The older age of onset in LBP of elite juniors is interesting given the increased prevalence, severity, frequency and chronicity in their LBP. A large cross sectional survey has found that adolescents are at a greater risk of LBP if they have low isometric muscle endurance in the back extensors, with no associations found for aerobic fitness, functional strength, flexibility, or physical activity level after adjustment for muscle endurance [17]. It may be that elite juniors are initially protected from LBP due to their increased physical fitness, but this is lost following the excessive spinal loading [9] and high training duration [18] these players face when they enter the elite junior pathway. To support this, sporting participation in the general population is known to result in less frequent LBP, although once LBP is established, sporting participation contributes to increased severity of pain [19]. Elite juniors also face pressure to play and train with LBP (and other injuries) given injuries can affect future selection to professional senior clubs in the Australian Football League (AFL). However, future research would benefit from the inclusion of training and competition volume to more clearly identify its role in the increased incidence of LBP occurring in elite junior footballers. Other potential reasons for the increased incidence of LBP in elite adolescent Australian Rules footballers includes the likely increased prevalence of weight lifting training and the effects that increased loading and training volume may have on the developing skeleton [10].
It has previously been documented that LBP is rarely a self-limiting disorder but characterized by unpredictable variations in pain status, with temporary, rather than permanent remissions [20]. Although these studies are based on adult populations, given that the natural history of LBP in adolescence involves a significantly increased risk of adult LBP, it has been suggested that it might be counterproductive to postpone treatment/prevention until the problems become more severe and chronic [6]. This would require a greater understanding of the knowledge of risk factors for LBP in elite junior Australian Rules footballers along with research investigating prevention and treatment interventions. Hestbaek et al. have suggested a change in focus from the adult to the young population in relation to research, prevention, and treatment of LBP [6]. However, it remains to be seen whether a greater focus on prevention and treatment can eliminate the risk and consequences of future LBP episodes and minimise future chronicity. Future study should assess the potential that LBP has for later career and end of career injury occurring in elite junior players.
Although most LBP is non-specific in nature [1], adolescent athletes presenting with LBP may have a pathologic cause for their symptoms [21]. For this reason, it is important for those caring for younger athletes to maintain a high index of suspicion for some of the more common pathologic causes of LBP in this population. Sports-related diagnoses that must be considered include disc-related back pain, atypical Scheuermann's kyphosis, spondylolysis, and spondylolisthesis [22]. It is unclear whether football code players have a greater prevalence of radiographic lumbar spine abnormalities, including spondylolysis and spondylolisthesis, as age-matched controls [23]. This casts doubts on the usefulness of routine radiographic screening. Other research has documented that junior athletes with chronic LBP form a population of adolescents who have degenerative disc disease (DDD) identified on MRI [24]. For adolescent athletes with DDD, the relative risk of reporting recurrent LBP up to the age of 23 years is 16 compared with those having no disc degeneration [25]. Furthermore, disc protrusion and Scheuermann-type changes also contribute to the risk of persistently recurrent LBP at a later age [25]. LBP in adolescent athletes is a problem that should not be ignored but instead fully evaluated.
Future study should target elite junior footballers to determine whether LBP renders them more susceptible to developing other injuries given LBP in senior footballers produces a 29% increased risk of other injury [26]. Already evidence exists documenting that LBP produces changes in the neuromuscular control of the lumbopelvis [27] and in athletes it produces altered muscle response patterns required for lumbopelvic stabilization during sudden trunk loading following clinical recovery from LBP [28]. These changes in lumbopelvic stabilisation and neuromuscular control could explain the high rates of injuries such as hamstring injuries and groin injuries, which occur in elite junior players [29]. If LBP is determined to be a risk factor for injuries, it should be assessed whether prevention or effective treatment reduces this risk.
Considering that a high percentage of elite junior players stated that LBP impacted their performance some of the time or greater, this should be further investigated. Evidence exists that college level athletes with a history of low back injury with resolved LBP demonstrate significantly diminished athletic performance in a 20 m shuttle run test compared with a healthy group [30]. This is of immense consideration for the elite juniors who get tested for sprint speed and endurance (through a beep test) at the AFL draft camp, results which can affect future career prospects.
Conclusions
This study demonstrated that elite junior Australian Rules footballers strongly experience LBP more severely, frequently and with higher prevalence and chronicity than non-elite junior Australian Rules footballers and non-football playing school children, who share a similar pain profile. This suggests that Australian Rules football participation is not a risk for adolescent LBP, but elite junior participation is. Future research is required to investigate the consequences of these findings, to determine whether LBP produces a greater risk of other injuries, leads to later or post-career LBP or impacts player performance. In addition, risk factors for elite junior LBP need to be identified along with best practices for prevention and treatment.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
WH conceived the idea of the study. All authors were involved in recruitment of subjects and data entry. WH and HP contributed to writing an initial draft document. All authors contributed to the re-writing of this paper. All of the authors participated in the editing and revisions of the multiple drafts that existed between the initial and final draft. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2474/11/241/prepub
Acknowledgements
No source of funding was used in the preparation of this manuscript. The authors would like to acknowledge Mick O'Neil for the statistical analysis and the following people who contributed to the study: David Code (Victorian Football League), Lilian Hoskins (Ramsgate RSL), Rob Young (Research), Michael Hoskins (Sorrento-Duncraig), Ron Webb and Karen Davies (Broughton Anglican College) and Alan McManus and John Tannous (Magdalene Catholic College).
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb288710.1107/S1600536808043948ACSEBHS1600536808043948Metal-Organic PapersBis(6-methoxy-2-{[tris(hydroxymethyl)methyl]iminomethyl}phenolato)copper(II) dihydrate [Cu(C12H16NO5)2]·2H2OZhang Xiutang ac*Wei Peihai bDou Jianmin cLi Bin bHu Bo ba Advanced Materials Institute of Research, Department of Chemistry and Chemical Engineering, Shandong Institute of Education, Jinan 250013, People’s Republic of Chinab Department of Chemistry and Chemical Engineering, Shandong Institute of Education, Jinan 250013, People’s Republic of Chinac College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 2 2009 08 1 2009 08 1 2009 65 Pt 2 e090200m151 m152 24 12 2008 25 12 2008 © Zhang et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Cu(C12H16NO5)2]·2H2O, the CuII ion adopts a trans-CuN2O4 octahedral geometry arising from two N,O,O′-tridentate 6-methoxy-2-{[tris(hydroxymethyl)methyl]iminomethyl}phenolate ligands. The Jahn–Teller distortion of the copper centre is unusally small. In the crystal structure, O—H⋯O hydrogen bonds, some of which are bifurcated, link the component species.
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Related literature
For the ligand synthesis, see: Wang et al. (2007 ▶). For background on Schiff base complexes, see: Ward (2007 ▶).
Experimental
Crystal data
[Cu(C12H16NO5)2]·2H2O
M
r = 608.09
Monoclinic,
a = 11.9421 (9) Å
b = 11.0238 (9) Å
c = 20.6706 (17) Å
β = 97.462 (1)°
V = 2698.2 (4) Å3
Z = 4
Mo Kα radiation
μ = 0.88 mm−1
T = 293 (2) K
0.12 × 0.10 × 0.08 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.902, T
max = 0.933
13183 measured reflections
4912 independent reflections
4397 reflections with I > 2σ(I)
R
int = 0.061
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.117
S = 1.01
4912 reflections
352 parameters
8 restraints
H-atom parameters constrained
Δρmax = 0.47 e Å−3
Δρmin = −0.48 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808043948/hb2887sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808043948/hb2887Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB2887).
The authors thank the National Ministry of Science and Technology of China (grant No. 2001CB6105-07) for support.
supplementary crystallographic
information
Comment
Transition metal-Schiff based complexes have been intensely focused on owing to
their excellent physical and chemical properties including magnetic, optics
and catalysis (Ward, 2007). Herein, we report the crystal structure of
the
title compound, (I), based on a Schiff base ligand, L,
(E)-2-(2-hydroxy-3-methoxybenzylideneamino)-2-(hydroxymethyl)propane-1,3-diol, (Fig. 1).
The CuII ion in (I) is surrounded by two L-1 ligands and
hexa-coordinated by four oxygen atoms and two nitrogen atoms, with a slightly
distorted octahedral coordination sphere (Table 1). The metal–ligand bond
distances are similar to those in a related structure (Wang et al.,
2007). In the crystal, a network of O—H···O hydrogen bonds (Table 2)
help to
establish the packing.
Experimental
The ligand (HL) was synthesized according to the literature method (Wang et
al., 2007). HL1 (0.050 g, 0.2 mmol) and Cu(OAc)2.4H2O
(0.0498 g, 0.2 mmol) were refluxed in a mixed solvent solution (CH3OH:H2O = 4:1
v/v) until all solid was dissolved. The solution was cooled to
room temperature and filtrated and blue blocks of (I) slowly grew by allowing
slow evaporation of the solution. Anal. Calc. for C24H36CuN2O12: C
47.36, H 5.92, N 4.60%; Found: C 47.25, H 5.78, N 4.54%.
Refinement
The non-water H atoms were geometrically placed (C—H = 0.93–0.97 Å, O—H =
0.82 Å) and refined as riding with Uiso(H) =
1.2Ueq(carrier) or 1.5Ueq(methyl C). The water H atoms were
located in a difference map and reifned with restraints of O—H = 0.82 (2)Å
and H···H = 1.37 (2)Å and with Uiso(H) = 1.5Ueq(O).
Figures
Fig. 1. A view of (I) with Displacement ellipsoids drawn at the 30% probability level.
Crystal data
[Cu(C12H16NO5)2]·2H2O F(000) = 1276
Mr = 608.09 Dx = 1.497 Mg m−3
Monoclinic, P21/c Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2ybc Cell parameters from 4912 reflections
a = 11.9421 (9) Å θ = 2.1–25.5°
b = 11.0238 (9) Å µ = 0.88 mm−1
c = 20.6706 (17) Å T = 293 K
β = 97.462 (1)° Block, blue
V = 2698.2 (4) Å3 0.12 × 0.10 × 0.08 mm
Z = 4
Data collection
Bruker APEXII CCD diffractometer 4912 independent reflections
Radiation source: fine-focus sealed tube 4397 reflections with I > 2σ(I)
graphite Rint = 0.061
ω scans θmax = 25.5°, θmin = 2.1°
Absorption correction: multi-scan (SADABS; Bruker, 2001) h = −14→11
Tmin = 0.902, Tmax = 0.933 k = −13→13
13183 measured reflections l = −25→21
Refinement
Refinement on F2 Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.041 Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.117 H-atom parameters constrained
S = 1.01 w = 1/[σ2(Fo2) + (0.076P)2 + 1.4852P] where P = (Fo2 + 2Fc2)/3
4912 reflections (Δ/σ)max = 0.010
352 parameters Δρmax = 0.47 e Å−3
8 restraints Δρmin = −0.48 e Å−3
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Cu1 0.76750 (2) 0.15964 (2) 0.742179 (13) 0.02060 (12)
C1 0.8354 (4) 0.4629 (4) 0.52078 (18) 0.0604 (10)
H1A 0.9090 0.4980 0.5316 0.091*
H1B 0.7847 0.5226 0.4997 0.091*
H1C 0.8400 0.3953 0.4919 0.091*
C2 0.6893 (2) 0.3686 (2) 0.57268 (13) 0.0304 (6)
C3 0.6096 (3) 0.3782 (3) 0.51893 (14) 0.0448 (8)
H3 0.6257 0.4235 0.4833 0.054*
C4 0.5048 (3) 0.3213 (3) 0.51666 (16) 0.0490 (9)
H4 0.4504 0.3303 0.4805 0.074*
C5 0.4835 (3) 0.2521 (3) 0.56846 (14) 0.0386 (7)
H5 0.4141 0.2132 0.5668 0.058*
C6 0.5636 (2) 0.2380 (2) 0.62432 (12) 0.0250 (5)
C7 0.6687 (2) 0.3017 (2) 0.62939 (11) 0.0212 (5)
C8 0.5359 (2) 0.1518 (2) 0.67278 (13) 0.0245 (5)
H8 0.4620 0.1230 0.6679 0.029*
C9 0.5630 (2) 0.0167 (2) 0.76438 (12) 0.0241 (5)
C10 0.4561 (2) −0.0514 (2) 0.73731 (14) 0.0328 (6)
H10A 0.4384 −0.1111 0.7689 0.039*
H10B 0.3935 0.0052 0.7301 0.039*
C11 0.5411 (2) 0.0769 (3) 0.82836 (14) 0.0348 (6)
H11A 0.5217 0.0157 0.8587 0.042*
H11B 0.6088 0.1184 0.8480 0.042*
C12 0.6611 (2) −0.0728 (2) 0.77953 (13) 0.0290 (5)
H12A 0.6453 −0.1287 0.8135 0.035*
H12B 0.6706 −0.1195 0.7408 0.035*
C13 0.6631 (3) 0.4397 (3) 0.97678 (15) 0.0459 (8)
H13A 0.5841 0.4585 0.9684 0.069*
H13B 0.7058 0.5136 0.9827 0.069*
H13C 0.6773 0.3912 1.0156 0.069*
C14 0.8076 (2) 0.3405 (2) 0.92680 (12) 0.0286 (6)
C15 0.8870 (3) 0.3615 (3) 0.97939 (13) 0.0369 (6)
H15 0.8664 0.4006 1.0159 0.044*
C16 0.9992 (3) 0.3245 (3) 0.97888 (13) 0.0362 (6)
H16 1.0529 0.3381 1.0149 0.043*
C17 1.0282 (2) 0.2683 (2) 0.92462 (12) 0.0290 (5)
H17 1.1027 0.2446 0.9240 0.035*
C18 0.9483 (2) 0.2451 (2) 0.86945 (11) 0.0211 (5)
C19 0.8334 (2) 0.2797 (2) 0.86871 (11) 0.0209 (5)
C20 0.9936 (2) 0.1931 (2) 0.81378 (11) 0.0207 (5)
H20 1.0715 0.1824 0.8180 0.025*
C21 0.9988 (2) 0.1161 (2) 0.70679 (11) 0.0205 (5)
C22 1.1144 (2) 0.0571 (2) 0.72962 (12) 0.0261 (5)
H22A 1.1434 0.0190 0.6929 0.031*
H22B 1.1679 0.1186 0.7473 0.031*
C23 1.0162 (2) 0.2241 (2) 0.66108 (11) 0.0254 (5)
H23A 1.0629 0.1980 0.6287 0.031*
H23B 0.9436 0.2487 0.6383 0.031*
C24 0.9245 (2) 0.0216 (2) 0.66761 (12) 0.0241 (5)
H24A 0.9509 0.0087 0.6257 0.029*
H24B 0.9288 −0.0549 0.6909 0.029*
N1 0.60220 (16) 0.11103 (17) 0.72146 (10) 0.0209 (4)
N2 0.93778 (16) 0.16042 (16) 0.75959 (9) 0.0176 (4)
O1 0.79480 (18) 0.4224 (2) 0.57864 (10) 0.0417 (5)
O2 0.74271 (14) 0.30303 (15) 0.68132 (8) 0.0215 (3)
O3 0.76136 (14) −0.00642 (16) 0.80055 (9) 0.0287 (4)
H3A 0.8136 −0.0541 0.8032 0.043*
O4 0.46929 (17) −0.11041 (18) 0.67767 (11) 0.0403 (5)
H4A 0.4108 −0.1462 0.6640 0.060*
O5 0.4506 (2) 0.16184 (19) 0.81536 (13) 0.0504 (6)
H5A 0.4388 0.1942 0.8496 0.076*
O6 0.69604 (17) 0.3742 (2) 0.92296 (9) 0.0424 (5)
O7 0.75320 (14) 0.26443 (15) 0.82097 (8) 0.0237 (4)
O8 0.81044 (14) 0.06366 (16) 0.65805 (8) 0.0279 (4)
H8A 0.7676 0.0108 0.6433 0.042*
O9 1.10148 (16) −0.03065 (18) 0.77788 (10) 0.0378 (5)
H9 1.1627 −0.0622 0.7901 0.057*
O10 1.06809 (16) 0.32536 (16) 0.69565 (9) 0.0316 (4)
H10 1.0280 0.3489 0.7225 0.047*
O1W 0.6645 (2) 0.8968 (2) 0.60223 (11) 0.0496 (6)
H1W 0.6872 0.8234 0.6027 0.074*
H2W 0.6038 0.8985 0.6197 0.074*
O2W 0.2699 (2) 0.1555 (2) 0.87731 (13) 0.0553 (6)
H3W 0.2413 0.0889 0.8885 0.083*
H4W 0.3253 0.1387 0.8565 0.083*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Cu1 0.02012 (18) 0.01953 (18) 0.02172 (18) −0.00072 (10) 0.00112 (12) −0.00073 (10)
C1 0.086 (3) 0.052 (2) 0.051 (2) −0.0176 (19) 0.0366 (19) 0.0000 (16)
C2 0.0429 (15) 0.0226 (12) 0.0255 (13) −0.0034 (11) 0.0034 (11) 0.0012 (10)
C3 0.069 (2) 0.0355 (16) 0.0269 (14) −0.0041 (15) −0.0042 (14) 0.0095 (12)
C4 0.062 (2) 0.0405 (17) 0.0370 (17) −0.0055 (15) −0.0241 (15) 0.0080 (13)
C5 0.0390 (15) 0.0282 (14) 0.0432 (16) −0.0034 (12) −0.0149 (13) 0.0016 (12)
C6 0.0266 (12) 0.0197 (12) 0.0268 (12) 0.0017 (10) −0.0039 (10) −0.0009 (9)
C7 0.0263 (12) 0.0148 (10) 0.0220 (11) −0.0004 (9) 0.0015 (9) −0.0013 (9)
C8 0.0208 (12) 0.0193 (12) 0.0322 (13) −0.0017 (9) −0.0008 (10) −0.0027 (9)
C9 0.0240 (12) 0.0182 (11) 0.0307 (12) −0.0052 (10) 0.0059 (9) 0.0033 (10)
C10 0.0236 (13) 0.0249 (13) 0.0496 (16) −0.0065 (10) 0.0040 (11) 0.0050 (12)
C11 0.0392 (15) 0.0314 (14) 0.0372 (15) −0.0034 (12) 0.0180 (12) 0.0028 (12)
C12 0.0283 (13) 0.0195 (12) 0.0387 (14) −0.0024 (10) 0.0024 (11) 0.0053 (10)
C13 0.0513 (18) 0.0533 (19) 0.0356 (15) 0.0175 (15) 0.0151 (13) −0.0120 (14)
C14 0.0338 (14) 0.0300 (14) 0.0223 (12) 0.0064 (11) 0.0047 (10) −0.0028 (10)
C15 0.0461 (17) 0.0422 (16) 0.0221 (13) 0.0031 (13) 0.0032 (12) −0.0104 (11)
C16 0.0391 (16) 0.0463 (17) 0.0207 (13) −0.0032 (13) −0.0056 (11) −0.0057 (11)
C17 0.0278 (13) 0.0338 (14) 0.0240 (12) 0.0004 (11) −0.0019 (10) 0.0005 (10)
C18 0.0243 (12) 0.0198 (11) 0.0193 (11) −0.0007 (9) 0.0028 (9) 0.0002 (9)
C19 0.0280 (12) 0.0176 (11) 0.0168 (10) −0.0012 (9) 0.0016 (9) 0.0009 (9)
C20 0.0192 (11) 0.0184 (11) 0.0237 (11) −0.0006 (9) 0.0002 (9) 0.0008 (9)
C21 0.0219 (11) 0.0188 (11) 0.0213 (11) −0.0001 (9) 0.0044 (9) −0.0028 (9)
C22 0.0234 (12) 0.0234 (12) 0.0324 (13) 0.0041 (10) 0.0065 (10) −0.0009 (10)
C23 0.0303 (13) 0.0243 (12) 0.0230 (11) −0.0016 (10) 0.0082 (10) 0.0010 (9)
C24 0.0275 (12) 0.0187 (11) 0.0257 (12) 0.0003 (10) 0.0022 (9) −0.0062 (9)
N1 0.0182 (9) 0.0171 (9) 0.0274 (10) −0.0011 (8) 0.0031 (8) −0.0003 (8)
N2 0.0189 (9) 0.0155 (9) 0.0189 (9) 0.0008 (7) 0.0040 (7) 0.0006 (7)
O1 0.0490 (12) 0.0433 (12) 0.0344 (10) −0.0155 (10) 0.0118 (9) 0.0073 (9)
O2 0.0230 (8) 0.0178 (8) 0.0226 (8) −0.0040 (7) −0.0009 (6) 0.0020 (6)
O3 0.0240 (9) 0.0226 (9) 0.0384 (10) 0.0017 (7) −0.0004 (7) 0.0041 (7)
O4 0.0328 (10) 0.0305 (10) 0.0542 (13) −0.0085 (8) −0.0070 (9) −0.0069 (9)
O5 0.0451 (13) 0.0364 (12) 0.0763 (17) 0.0023 (9) 0.0325 (12) −0.0080 (11)
O6 0.0365 (11) 0.0621 (14) 0.0288 (10) 0.0173 (10) 0.0040 (8) −0.0155 (9)
O7 0.0216 (8) 0.0273 (9) 0.0217 (8) 0.0019 (7) 0.0005 (7) −0.0058 (7)
O8 0.0248 (9) 0.0262 (9) 0.0313 (9) −0.0020 (7) −0.0019 (7) −0.0107 (7)
O9 0.0278 (10) 0.0307 (10) 0.0543 (12) 0.0112 (8) 0.0035 (9) 0.0136 (9)
O10 0.0318 (10) 0.0247 (9) 0.0398 (11) −0.0078 (7) 0.0103 (8) −0.0018 (8)
O1W 0.0575 (14) 0.0423 (13) 0.0482 (13) −0.0186 (11) 0.0043 (10) −0.0024 (10)
O2W 0.0421 (13) 0.0466 (14) 0.0814 (18) 0.0021 (10) 0.0241 (12) −0.0010 (12)
Geometric parameters (Å, °)
Cu1—N1 2.0367 (19) C13—H13B 0.9600
Cu1—N2 2.0185 (19) C13—H13C 0.9600
Cu1—O2 2.0180 (16) C14—C15 1.366 (4)
Cu1—O3 2.1989 (18) C14—O6 1.376 (3)
Cu1—O7 2.0220 (16) C14—C19 1.443 (3)
Cu1—O8 2.1537 (17) C15—C16 1.401 (4)
C1—O1 1.419 (4) C15—H15 0.9300
C1—H1A 0.9600 C16—C17 1.365 (4)
C1—H1B 0.9600 C16—H16 0.9300
C1—H1C 0.9600 C17—C18 1.412 (3)
C2—C3 1.370 (4) C17—H17 0.9300
C2—O1 1.383 (3) C18—C19 1.422 (3)
C2—C7 1.433 (4) C18—C20 1.452 (3)
C3—C4 1.396 (5) C19—O7 1.294 (3)
C3—H3 0.9300 C20—N2 1.279 (3)
C4—C5 1.365 (5) C20—H20 0.9300
C4—H4 0.9300 C21—N2 1.472 (3)
C5—C6 1.409 (4) C21—C24 1.530 (3)
C5—H5 0.9300 C21—C22 1.544 (3)
C6—C7 1.430 (3) C21—C23 1.551 (3)
C6—C8 1.450 (4) C22—O9 1.412 (3)
C7—O2 1.299 (3) C22—H22A 0.9700
C8—N1 1.278 (3) C22—H22B 0.9700
C8—H8 0.9300 C23—O10 1.423 (3)
C9—N1 1.481 (3) C23—H23A 0.9700
C9—C10 1.524 (3) C23—H23B 0.9700
C9—C12 1.533 (4) C24—O8 1.428 (3)
C9—C11 1.532 (4) C24—H24A 0.9700
C10—O4 1.421 (4) C24—H24B 0.9700
C10—H10A 0.9700 O3—H3A 0.8115
C10—H10B 0.9700 O4—H4A 0.8200
C11—O5 1.428 (4) O5—H5A 0.8200
C11—H11A 0.9700 O8—H8A 0.8085
C11—H11B 0.9700 O9—H9 0.8200
C12—O3 1.422 (3) O10—H10 0.8200
C12—H12A 0.9700 O1W—H1W 0.8520
C12—H12B 0.9700 O1W—H2W 0.8511
C13—O6 1.424 (3) O2W—H3W 0.8541
C13—H13A 0.9600 O2W—H4W 0.8538
O2—Cu1—N2 99.83 (7) H13A—C13—H13C 109.5
O2—Cu1—O7 91.93 (7) H13B—C13—H13C 109.5
N2—Cu1—O7 92.43 (7) C15—C14—O6 124.6 (2)
O2—Cu1—N1 90.88 (7) C15—C14—C19 122.7 (2)
N2—Cu1—N1 164.82 (7) O6—C14—C19 112.8 (2)
O7—Cu1—N1 97.97 (7) C14—C15—C16 120.7 (2)
O2—Cu1—O8 84.98 (7) C14—C15—H15 119.7
N2—Cu1—O8 78.82 (7) C16—C15—H15 119.7
O7—Cu1—O8 170.05 (7) C17—C16—C15 118.9 (2)
N1—Cu1—O8 91.54 (7) C17—C16—H16 120.6
O2—Cu1—O3 168.97 (6) C15—C16—H16 120.6
N2—Cu1—O3 90.59 (7) C16—C17—C18 121.9 (2)
O7—Cu1—O3 91.22 (7) C16—C17—H17 119.0
N1—Cu1—O3 78.22 (7) C18—C17—H17 119.0
O8—Cu1—O3 93.58 (7) C17—C18—C19 120.7 (2)
O1—C1—H1A 109.5 C17—C18—C20 115.4 (2)
O1—C1—H1B 109.5 C19—C18—C20 123.8 (2)
H1A—C1—H1B 109.5 O7—C19—C18 126.2 (2)
O1—C1—H1C 109.5 O7—C19—C14 118.6 (2)
H1A—C1—H1C 109.5 C18—C19—C14 115.2 (2)
H1B—C1—H1C 109.5 N2—C20—C18 126.8 (2)
C3—C2—O1 124.6 (3) N2—C20—H20 116.6
C3—C2—C7 121.9 (3) C18—C20—H20 116.6
O1—C2—C7 113.5 (2) N2—C21—C24 108.05 (19)
C2—C3—C4 121.2 (3) N2—C21—C22 114.98 (19)
C2—C3—H3 119.4 C24—C21—C22 108.01 (19)
C4—C3—H3 119.4 N2—C21—C23 108.37 (18)
C5—C4—C3 118.9 (3) C24—C21—C23 108.33 (19)
C5—C4—H4 120.6 C22—C21—C23 108.92 (19)
C3—C4—H4 120.6 O9—C22—C21 109.19 (19)
C4—C5—C6 121.9 (3) O9—C22—H22A 109.8
C4—C5—H5 119.0 C21—C22—H22A 109.8
C6—C5—H5 119.1 O9—C22—H22B 109.8
C5—C6—C7 120.1 (2) C21—C22—H22B 109.8
C5—C6—C8 116.5 (2) H22A—C22—H22B 108.3
C7—C6—C8 123.3 (2) O10—C23—C21 112.36 (19)
O2—C7—C6 124.2 (2) O10—C23—H23A 109.1
O2—C7—C2 120.0 (2) C21—C23—H23A 109.1
C6—C7—C2 115.8 (2) O10—C23—H23B 109.1
N1—C8—C6 126.9 (2) C21—C23—H23B 109.1
N1—C8—H8 116.6 H23A—C23—H23B 107.9
C6—C8—H8 116.6 O8—C24—C21 109.23 (18)
N1—C9—C10 116.2 (2) O8—C24—H24A 109.8
N1—C9—C12 106.37 (19) C21—C24—H24A 109.8
C10—C9—C12 109.9 (2) O8—C24—H24B 109.8
N1—C9—C11 108.37 (19) C21—C24—H24B 109.8
C10—C9—C11 107.6 (2) H24A—C24—H24B 108.3
C12—C9—C11 108.3 (2) C8—N1—C9 120.4 (2)
O4—C10—C9 111.2 (2) C8—N1—Cu1 123.88 (17)
O4—C10—H10A 109.4 C9—N1—Cu1 115.57 (15)
C9—C10—H10A 109.4 C20—N2—C21 119.4 (2)
O4—C10—H10B 109.4 C20—N2—Cu1 123.80 (16)
C9—C10—H10B 109.4 C21—N2—Cu1 116.77 (14)
H10A—C10—H10B 108.0 C2—O1—C1 117.8 (2)
O5—C11—C9 109.3 (2) C7—O2—Cu1 122.38 (14)
O5—C11—H11A 109.8 C12—O3—Cu1 110.35 (14)
C9—C11—H11A 109.8 C12—O3—H3A 107.2
O5—C11—H11B 109.8 Cu1—O3—H3A 119.7
C9—C11—H11B 109.8 C10—O4—H4A 109.5
H11A—C11—H11B 108.3 C11—O5—H5A 109.5
O3—C12—C9 108.8 (2) C14—O6—C13 117.1 (2)
O3—C12—H12A 109.9 C19—O7—Cu1 123.85 (15)
C9—C12—H12A 109.9 C24—O8—Cu1 111.75 (13)
O3—C12—H12B 109.9 C24—O8—H8A 111.3
C9—C12—H12B 109.9 Cu1—O8—H8A 117.0
H12A—C12—H12B 108.3 C22—O9—H9 109.5
O6—C13—H13A 109.5 C23—O10—H10 109.5
O6—C13—H13B 109.5 H1W—O1W—H2W 107.6
H13A—C13—H13B 109.5 H3W—O2W—H4W 108.2
O6—C13—H13C 109.5
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O3—H3A···O10i 0.81 1.94 2.748 (3) 176
O4—H4A···O6ii 0.82 2.08 2.681 (3) 130
O4—H4A···O7ii 0.82 2.25 2.997 (3) 152
O5—H5A···O2W 0.82 2.21 2.649 (4) 114
O8—H8A···O1Wiii 0.81 1.88 2.689 (3) 175
O9—H9···O2i 0.82 1.91 2.670 (3) 153
O10—H10···O9iv 0.82 2.04 2.685 (3) 135
O1W—H1W···O2Wv 0.85 1.95 2.790 (3) 168
O1W—H2W···O4vi 0.85 2.13 2.969 (3) 170
O2W—H3W···O1ii 0.85 2.02 2.866 (3) 169
O2W—H4W···O5 0.85 1.83 2.649 (4) 159
Symmetry codes: (i) −x+2, y−1/2, −z+3/2; (ii) −x+1, y−1/2, −z+3/2; (iii) x, y−1, z; (iv) −x+2, y+1/2, −z+3/2; (v) −x+1, y+1/2, −z+3/2; (vi) x, y+1, z.
Table 1 Selected bond lengths (Å)
Cu1—N1 2.0367 (19)
Cu1—N2 2.0185 (19)
Cu1—O2 2.0180 (16)
Cu1—O3 2.1989 (18)
Cu1—O7 2.0220 (16)
Cu1—O8 2.1537 (17)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O3—H3A⋯O10i 0.81 1.94 2.748 (3) 176
O4—H4A⋯O6ii 0.82 2.08 2.681 (3) 130
O4—H4A⋯O7ii 0.82 2.25 2.997 (3) 152
O5—H5A⋯O2W 0.82 2.21 2.649 (4) 114
O8—H8A⋯O1Wiii 0.81 1.88 2.689 (3) 175
O9—H9⋯O2i 0.82 1.91 2.670 (3) 153
O10—H10⋯O9iv 0.82 2.04 2.685 (3) 135
O1W—H1W⋯O2Wv 0.85 1.95 2.790 (3) 168
O1W—H2W⋯O4vi 0.85 2.13 2.969 (3) 170
O2W—H3W⋯O1ii 0.85 2.02 2.866 (3) 169
O2W—H4W⋯O5 0.85 1.83 2.649 (4) 159
Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) ; (vi) .
==== Refs
References
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Wang, Q., Li, X., Wang, X. & Zhang, Y. (2007). Acta Cryst. E63, m2537.
Ward, M. D. (2007). Coord. Chem. Rev.251, 1663–1677. | 21581764 | PMC2968260 | CC BY | 2021-01-04 18:57:39 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Jan 8; 65(Pt 2):m151-m152 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb281210.1107/S1600536808043936ACSEBHS1600536808043936Metal-Organic PapersTetrakis(μ-2,4-difluorobenzoato)bis[(2,2′-bipyridine)(2,4-difluorobenzoato)terbium(III)] [Tb2(C7H3F2O2)6(C10H8N2)2]Hao Lujiang a*Liu Xia ba College of Food and Biological Engineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of Chinab Maize Research Institute, Shandong Academy of Agricultural Science, Jinan 250100, People’s Republic of ChinaCorrespondence e-mail: [email protected] 2 2009 08 1 2009 08 1 2009 65 Pt 2 e090200m150 m150 29 9 2008 25 12 2008 © Hao and Liu 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the centrosymmetric dinuclear title compound, [Tb2(C7H3F2O2)6(C10H8N2)2], the TbIII ion is coordinated by an N,N′-bidentate 2,2′-bipyridine molecule, and two O,O′-bidentate 2,4-difluorobenzoate (dfb) anions. One of the latter also bonds to the second TbIII centre through one of its O atoms. The third dfb anion bonds to one Tb atom from each of its O atoms. Thus, the three dfb species have three different coordination modes. This results in an irregular TbN2O7 coordination sphere for the metal ion. The F atoms and their associated H atoms in the simple bidentate dfb anion are disordered over two sets of sites in a 0.672 (10):0.328 (10) ratio.
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Related literature
For related literature on the biological applications of carboxylates as ligands, see, for example: Serre et al. (2005 ▶).
Experimental
Crystal data
[Tb2(C7H3F2O2)6(C10H8N2)2]
M
r = 1572.77
Triclinic,
a = 11.401 (1) Å
b = 12.189 (1) Å
c = 12.588 (2) Å
α = 103.99 (2)°
β = 102.90 (2)°
γ = 113.58 (2)°
V = 1451.5 (3) Å3
Z = 1
Mo Kα radiation
μ = 2.52 mm−1
T = 293 (2) K
0.44 × 0.26 × 0.20 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.403, T
max = 0.632
8233 measured reflections
5557 independent reflections
4813 reflections with I > 2σ(I)
R
int = 0.0210
Refinement
R[F
2 > 2σ(F
2)] = 0.034
wR(F
2) = 0.078
S = 1.03
5557 reflections
434 parameters
H-atom parameters constrained
Δρmax = 0.60 e Å−3
Δρmin = −0.57 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808043936/hb2812sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536808043936/hb2812Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB2812).
This work was supported by the Natural Science Foundation of Shandong Province (grant No. Y2007D39).
supplementary crystallographic
information
Comment
In recent years, carboxylic acids have been widely used as polydentate ligands,
which can coordinate to transition or rare earth ions yielding complexes with
interesting biological properties (e.g. Serre et al., 2005).
Herein, we report the synthesis and X-ray crystal structure
analysis of the centrosymetric title compound, (I), Fig. 1.
The TbIII is
chelated by two 2,4-difluorobezoate anions and one
4,4'-bipyridine molecule. Two cations are
linked into dimer via three bridging carboxylate groups from three
2,4-difluorobezoic acid. As a result, the TbIII ion is
nine-coordinated with seven O atoms and two N atoms (Table 1).
Experimental
A mixture of terbium(III) chloride (0.5 mmol), 2,4-difluorobezoic acid (1 mmol),
sodium hydroxide (1 mmol), 4,4'-bipyridine (0.5 mmol), H2O (8 ml) and ethanol
(8 ml) in a 25 ml Teflon-lined stainless steel autoclave was kept at 433 K for
three days. Colourless blocks of (I)
were obtained after cooling to room temperature
with a yield of 16%. Anal. Calc. for C62H34F12Tb2N4O12: C 47.41, H
2.17, N 3.57%; Found: C 47.38, H 2.19, N 3.55%.
Refinement
The H atoms
were placed in calculated positions (C—H = 0.93 Å) and refined as riding
with Uiso(H) = 1.2Ueq(C). The F atoms of the bidenate
dfb anion are disordered over two sets of sites in a
0.672 (10):0.328 (10) ratio.
Figures
Fig. 1. A view of the molecular structure of (I), showing 30% probability displacement ellipsoids. Symmetry code: (i) –x +1, –y + 2, –z + 1.
Crystal data
[Tb2(C7H3F2O2)6(C10H8N2)2] Z = 1
Mr = 1572.77 F(000) = 768
Triclinic, P1 Dx = 1.799 Mg m−3
Hall symbol: -P 1 Mo Kα radiation, λ = 0.71073 Å
a = 11.401 (1) Å Cell parameters from 5557 reflections
b = 12.189 (1) Å θ = 1.8–26.0°
c = 12.588 (2) Å µ = 2.52 mm−1
α = 103.99 (2)° T = 293 K
β = 102.90 (2)° Block, colorless
γ = 113.58 (2)° 0.44 × 0.26 × 0.20 mm
V = 1451.5 (3) Å3
Data collection
Bruker APEXII CCD diffractometer 5557 independent reflections
Radiation source: fine-focus sealed tube 4813 reflections with I > 2σ(I)
graphite Rint = 0.021
φ and ω scans θmax = 26.0°, θmin = 1.8°
Absorption correction: multi-scan (SADABS; Bruker, 2001) h = −14→13
Tmin = 0.403, Tmax = 0.632 k = −15→14
8233 measured reflections l = 0→15
Refinement
Refinement on F2 Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.034 Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.078 H-atom parameters constrained
S = 1.03 w = 1/[σ2(Fo2) + (0.0345P)2 + 0.822P] where P = (Fo2 + 2Fc2)/3
5557 reflections (Δ/σ)max = 0.001
434 parameters Δρmax = 0.60 e Å−3
0 restraints Δρmin = −0.57 e Å−3
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq Occ. (<1)
Tb1 0.39615 (2) 0.804496 (17) 0.439773 (18) 0.03337 (8)
F1 0.8156 (4) 1.1600 (3) 0.2648 (3) 0.0809 (11)
F2 0.3952 (4) 0.7245 (4) −0.0812 (3) 0.1009 (13)
F3 0.7957 (4) 0.9340 (4) 0.8322 (3) 0.0947 (13)
F4 1.1389 (4) 1.1727 (5) 0.6340 (6) 0.136 (2)
F5 −0.1318 (6) 0.5659 (8) 0.2091 (5) 0.131 (3) 0.672 (10)
F6 −0.0100 (8) 0.6768 (9) −0.1480 (6) 0.155 (4) 0.672 (10)
F7 0.1797 (11) 0.7301 (13) 0.0303 (10) 0.100 (5) 0.328 (10)
F8 −0.3317 (10) 0.5145 (17) 0.0353 (11) 0.126 (7) 0.328 (10)
O1 0.1421 (3) 0.6937 (3) 0.3366 (3) 0.0459 (8)
O2 0.2722 (3) 0.7141 (3) 0.2304 (3) 0.0497 (8)
O3 0.6259 (3) 1.0197 (2) 0.5936 (3) 0.0361 (7)
O4 0.5990 (3) 0.8317 (3) 0.5898 (3) 0.0492 (9)
O5 0.5614 (3) 0.8920 (3) 0.3552 (3) 0.0392 (7)
O6 0.6504 (3) 1.1034 (3) 0.3975 (3) 0.0449 (8)
N1 0.2901 (4) 0.6091 (3) 0.4973 (4) 0.0459 (10)
N2 0.4379 (4) 0.6128 (3) 0.3584 (3) 0.0431 (9)
C1 0.5204 (5) 0.6201 (5) 0.2990 (5) 0.0539 (13)
H1 0.5613 0.6955 0.2850 0.065*
C2 0.5503 (7) 0.5218 (6) 0.2559 (5) 0.0681 (17)
H2 0.6097 0.5311 0.2146 0.082*
C3 0.4902 (7) 0.4132 (5) 0.2760 (5) 0.0704 (18)
H3 0.5076 0.3455 0.2486 0.085*
C4 0.4042 (7) 0.4023 (5) 0.3364 (5) 0.0638 (17)
H4 0.3626 0.3270 0.3504 0.077*
C5 0.3780 (5) 0.5031 (4) 0.3773 (4) 0.0446 (12)
C6 0.6742 (4) 0.9489 (4) 0.6192 (4) 0.0345 (9)
C7 0.8260 (4) 1.0066 (4) 0.6802 (4) 0.0385 (10)
C8 0.9148 (5) 1.0685 (5) 0.6301 (5) 0.0592 (14)
H8 0.8820 1.0786 0.5605 0.071*
C9 1.0537 (6) 1.1153 (6) 0.6853 (7) 0.0772 (19)
C10 1.1062 (6) 1.1066 (6) 0.7881 (8) 0.086 (2)
H10 1.2005 1.1417 0.8242 0.103*
C11 1.0191 (6) 1.0454 (7) 0.8389 (6) 0.0792 (19)
H11 1.0532 1.0379 0.9095 0.095*
C12 0.8806 (5) 0.9955 (5) 0.7837 (5) 0.0525 (13)
C13 0.1575 (5) 0.6867 (4) 0.2397 (4) 0.0385 (10)
C14 0.0384 (5) 0.6523 (4) 0.1333 (4) 0.0400 (10)
C15 −0.0962 (5) 0.5956 (6) 0.1252 (5) 0.0591 (14)
H15 −0.1166 0.5754 0.1877 0.071* 0.328 (10)
C16 −0.2018 (6) 0.5664 (7) 0.0283 (6) 0.0759 (18)
H16 −0.2913 0.5274 0.0267 0.091* 0.672 (10)
C17 −0.1771 (6) 0.5947 (6) −0.0640 (5) 0.0720 (18)
H17 −0.2484 0.5771 −0.1298 0.086*
C18 −0.0453 (7) 0.6497 (6) −0.0581 (5) 0.0716 (17)
H18 −0.0263 0.6684 −0.1216 0.086* 0.328 (10)
C19 0.0606 (6) 0.6780 (5) 0.0380 (5) 0.0569 (14)
H19 0.1495 0.7160 0.0381 0.068* 0.672 (10)
C20 0.6086 (4) 0.9903 (4) 0.3307 (4) 0.0363 (10)
C21 0.6107 (5) 0.9677 (4) 0.2092 (4) 0.0418 (11)
C22 0.7081 (6) 1.0524 (5) 0.1800 (5) 0.0531 (13)
C23 0.7024 (7) 1.0276 (6) 0.0660 (6) 0.0652 (16)
H23 0.7709 1.0859 0.0489 0.078*
C24 0.5965 (7) 0.9176 (6) −0.0230 (5) 0.0681 (16)
H24 0.5903 0.9012 −0.1007 0.082*
C25 0.5015 (6) 0.8340 (6) 0.0064 (5) 0.0636 (15)
C26 0.5055 (5) 0.8547 (5) 0.1189 (4) 0.0491 (12)
H26 0.4385 0.7939 0.1352 0.059*
C27 0.2154 (5) 0.6091 (5) 0.5643 (6) 0.0599 (15)
H27 0.2191 0.6867 0.6022 0.072*
C28 0.1331 (6) 0.5017 (6) 0.5811 (6) 0.0762 (19)
H28 0.0830 0.5066 0.6294 0.091*
C29 0.1267 (7) 0.3900 (6) 0.5266 (7) 0.087 (2)
H29 0.0705 0.3154 0.5355 0.105*
C30 0.2036 (7) 0.3854 (5) 0.4570 (6) 0.080 (2)
H30 0.1997 0.3077 0.4189 0.095*
C31 0.2873 (5) 0.4976 (4) 0.4440 (4) 0.0496 (13)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Tb1 0.03168 (12) 0.02483 (11) 0.04084 (14) 0.01158 (9) 0.00750 (9) 0.01636 (9)
F1 0.075 (2) 0.059 (2) 0.086 (2) 0.0040 (17) 0.046 (2) 0.0247 (18)
F2 0.109 (3) 0.095 (3) 0.055 (2) 0.024 (2) 0.030 (2) 0.000 (2)
F3 0.070 (2) 0.161 (4) 0.069 (2) 0.053 (2) 0.0260 (19) 0.071 (3)
F4 0.070 (3) 0.148 (4) 0.246 (6) 0.052 (3) 0.088 (3) 0.135 (4)
F5 0.054 (4) 0.219 (8) 0.078 (4) 0.016 (4) 0.019 (3) 0.078 (5)
F6 0.113 (6) 0.211 (9) 0.079 (5) 0.015 (5) 0.008 (4) 0.089 (5)
F7 0.051 (7) 0.170 (13) 0.094 (9) 0.037 (7) 0.027 (6) 0.104 (9)
F8 0.032 (6) 0.229 (17) 0.083 (9) 0.030 (8) 0.006 (5) 0.073 (10)
O1 0.0388 (18) 0.0500 (19) 0.0408 (19) 0.0129 (15) 0.0084 (14) 0.0241 (15)
O2 0.0381 (19) 0.057 (2) 0.0442 (19) 0.0216 (16) 0.0075 (15) 0.0124 (16)
O3 0.0339 (16) 0.0284 (14) 0.0453 (18) 0.0156 (12) 0.0066 (13) 0.0184 (13)
O4 0.0407 (18) 0.0239 (15) 0.064 (2) 0.0096 (13) −0.0056 (16) 0.0193 (15)
O5 0.0390 (17) 0.0323 (16) 0.0500 (19) 0.0158 (13) 0.0196 (14) 0.0202 (14)
O6 0.0522 (19) 0.0304 (16) 0.051 (2) 0.0140 (14) 0.0240 (16) 0.0179 (15)
N1 0.037 (2) 0.035 (2) 0.062 (3) 0.0114 (17) 0.0091 (19) 0.0296 (19)
N2 0.042 (2) 0.0300 (19) 0.051 (2) 0.0176 (17) 0.0046 (19) 0.0161 (17)
C1 0.052 (3) 0.043 (3) 0.063 (3) 0.025 (2) 0.017 (3) 0.015 (2)
C2 0.077 (4) 0.068 (4) 0.058 (4) 0.051 (3) 0.011 (3) 0.006 (3)
C3 0.094 (5) 0.045 (3) 0.061 (4) 0.048 (3) −0.002 (3) 0.002 (3)
C4 0.084 (4) 0.033 (3) 0.052 (3) 0.030 (3) −0.008 (3) 0.006 (2)
C5 0.052 (3) 0.027 (2) 0.038 (3) 0.020 (2) −0.011 (2) 0.0080 (19)
C6 0.033 (2) 0.033 (2) 0.034 (2) 0.0145 (18) 0.0048 (18) 0.0158 (18)
C7 0.034 (2) 0.032 (2) 0.048 (3) 0.0206 (19) 0.007 (2) 0.013 (2)
C8 0.050 (3) 0.054 (3) 0.084 (4) 0.026 (3) 0.025 (3) 0.041 (3)
C9 0.046 (3) 0.066 (4) 0.132 (6) 0.026 (3) 0.037 (4) 0.053 (4)
C10 0.036 (3) 0.069 (4) 0.138 (7) 0.024 (3) 0.009 (4) 0.037 (4)
C11 0.055 (4) 0.099 (5) 0.080 (4) 0.041 (4) 0.002 (3) 0.040 (4)
C12 0.042 (3) 0.067 (3) 0.050 (3) 0.030 (3) 0.009 (2) 0.024 (3)
C13 0.039 (3) 0.023 (2) 0.043 (3) 0.0114 (18) 0.005 (2) 0.0113 (19)
C14 0.038 (2) 0.036 (2) 0.035 (2) 0.013 (2) 0.0032 (19) 0.0131 (19)
C15 0.042 (3) 0.078 (4) 0.045 (3) 0.019 (3) 0.010 (2) 0.024 (3)
C16 0.040 (3) 0.103 (5) 0.059 (4) 0.025 (3) −0.001 (3) 0.023 (4)
C17 0.057 (4) 0.076 (4) 0.052 (4) 0.027 (3) −0.016 (3) 0.017 (3)
C18 0.080 (5) 0.075 (4) 0.043 (3) 0.025 (3) 0.009 (3) 0.028 (3)
C19 0.051 (3) 0.053 (3) 0.050 (3) 0.011 (2) 0.010 (2) 0.025 (3)
C20 0.031 (2) 0.035 (2) 0.048 (3) 0.0168 (19) 0.015 (2) 0.020 (2)
C21 0.045 (3) 0.043 (3) 0.055 (3) 0.029 (2) 0.025 (2) 0.026 (2)
C22 0.062 (3) 0.043 (3) 0.066 (4) 0.025 (3) 0.036 (3) 0.027 (3)
C23 0.092 (5) 0.068 (4) 0.082 (4) 0.052 (4) 0.063 (4) 0.048 (4)
C24 0.093 (5) 0.079 (4) 0.053 (4) 0.054 (4) 0.038 (3) 0.026 (3)
C25 0.074 (4) 0.060 (3) 0.055 (4) 0.030 (3) 0.029 (3) 0.014 (3)
C26 0.048 (3) 0.051 (3) 0.053 (3) 0.022 (2) 0.026 (2) 0.023 (2)
C27 0.051 (3) 0.054 (3) 0.089 (4) 0.024 (3) 0.028 (3) 0.047 (3)
C28 0.063 (4) 0.076 (4) 0.104 (5) 0.024 (3) 0.033 (4) 0.068 (4)
C29 0.081 (5) 0.054 (4) 0.103 (5) 0.003 (3) 0.015 (4) 0.059 (4)
C30 0.092 (5) 0.034 (3) 0.079 (4) 0.010 (3) 0.000 (4) 0.030 (3)
C31 0.052 (3) 0.028 (2) 0.050 (3) 0.011 (2) −0.004 (2) 0.022 (2)
Geometric parameters (Å, °)
Tb1—N1 2.565 (3) C5—C31 1.463 (8)
Tb1—N2 2.586 (4) C6—C7 1.501 (6)
Tb1—O6i 2.364 (3) C7—C8 1.377 (7)
Tb1—O3i 2.377 (3) C7—C12 1.377 (7)
Tb1—O5 2.379 (3) C8—C9 1.382 (8)
Tb1—O2 2.418 (3) C8—H8 0.9300
Tb1—O4 2.481 (3) C9—C10 1.347 (10)
Tb1—O1 2.498 (3) C10—C11 1.367 (10)
Tb1—O3 2.696 (3) C10—H10 0.9300
F1—C22 1.347 (6) C11—C12 1.373 (8)
F2—C25 1.365 (7) C11—H11 0.9300
F3—C12 1.331 (6) C13—C14 1.508 (6)
F4—C9 1.344 (7) C14—C19 1.363 (7)
F5—C15 1.294 (7) C14—C15 1.374 (7)
F5—H15 0.3691 C15—C16 1.362 (7)
F6—C18 1.353 (9) C15—H15 0.9300
F6—H18 0.4290 C16—C17 1.346 (9)
F7—C19 1.286 (11) C16—H16 0.9300
F7—H19 0.3654 C17—C18 1.354 (9)
F8—C16 1.392 (13) C17—H17 0.9300
F8—H16 0.4706 C18—C19 1.362 (8)
O1—C13 1.258 (6) C18—H18 0.9300
O2—C13 1.254 (6) C19—H19 0.9300
O3—C6 1.259 (5) C20—C21 1.495 (6)
O3—Tb1i 2.377 (3) C21—C22 1.372 (6)
O4—C6 1.239 (5) C21—C26 1.393 (7)
O5—C20 1.248 (5) C22—C23 1.375 (7)
O6—C20 1.252 (5) C23—C24 1.373 (9)
O6—Tb1i 2.364 (3) C23—H23 0.9300
N1—C27 1.325 (7) C24—C25 1.351 (8)
N1—C31 1.347 (6) C24—H24 0.9300
N2—C1 1.315 (7) C25—C26 1.364 (7)
N2—C5 1.345 (6) C26—H26 0.9300
C1—C2 1.394 (7) C27—C28 1.371 (7)
C1—H1 0.9300 C27—H27 0.9300
C2—C3 1.342 (9) C28—C29 1.332 (10)
C2—H2 0.9300 C28—H28 0.9300
C3—C4 1.354 (9) C29—C30 1.378 (10)
C3—H3 0.9300 C29—H29 0.9300
C4—C5 1.385 (7) C30—C31 1.394 (7)
C4—H4 0.9300 C30—H30 0.9300
O6i—Tb1—O3i 75.94 (10) C10—C9—C8 122.7 (6)
O6i—Tb1—O5 132.93 (10) C9—C10—C11 119.2 (6)
O3i—Tb1—O5 74.31 (10) C9—C10—H10 120.4
O6i—Tb1—O2 132.91 (12) C11—C10—H10 120.4
O3i—Tb1—O2 78.22 (11) C10—C11—C12 118.9 (6)
O5—Tb1—O2 74.15 (11) C10—C11—H11 120.5
O6i—Tb1—O4 84.77 (12) C12—C11—H11 120.5
O3i—Tb1—O4 123.24 (9) F3—C12—C11 118.8 (5)
O5—Tb1—O4 82.03 (11) F3—C12—C7 118.7 (4)
O2—Tb1—O4 142.07 (12) C11—C12—C7 122.4 (6)
O6i—Tb1—O1 84.52 (12) O2—C13—O1 121.4 (4)
O3i—Tb1—O1 81.13 (10) O2—C13—C14 118.7 (4)
O5—Tb1—O1 125.07 (10) O1—C13—C14 119.8 (4)
O2—Tb1—O1 52.89 (11) O2—C13—Tb1 59.2 (2)
O4—Tb1—O1 149.70 (10) O1—C13—Tb1 62.9 (2)
O6i—Tb1—N1 79.45 (12) C14—C13—Tb1 167.8 (3)
O3i—Tb1—N1 145.03 (12) C19—C14—C15 115.9 (5)
O5—Tb1—N1 139.96 (12) C19—C14—C13 120.2 (4)
O2—Tb1—N1 101.74 (13) C15—C14—C13 123.9 (4)
O4—Tb1—N1 78.13 (11) F5—C15—C16 115.0 (6)
O1—Tb1—N1 72.07 (11) F5—C15—C14 122.3 (5)
O6i—Tb1—N2 138.65 (12) C16—C15—C14 122.7 (5)
O3i—Tb1—N2 145.39 (12) F5—C15—H15 3.3
O5—Tb1—N2 78.18 (11) C16—C15—H15 118.3
O2—Tb1—N2 74.34 (12) C14—C15—H15 119.0
O4—Tb1—N2 72.15 (11) C17—C16—C15 120.4 (6)
O1—Tb1—N2 98.32 (12) C17—C16—F8 123.5 (7)
N1—Tb1—N2 62.76 (14) C15—C16—F8 115.9 (7)
O6i—Tb1—O3 70.68 (10) C17—C16—H16 120.1
O3i—Tb1—O3 73.82 (10) C15—C16—H16 119.5
O5—Tb1—O3 66.47 (10) F8—C16—H16 4.6
O2—Tb1—O3 136.36 (10) C16—C17—C18 117.8 (5)
O4—Tb1—O3 49.42 (9) C16—C17—H17 121.1
O1—Tb1—O3 148.14 (10) C18—C17—H17 121.1
N1—Tb1—O3 120.33 (11) C17—C18—F6 122.6 (6)
N2—Tb1—O3 113.49 (10) C17—C18—C19 122.0 (6)
C15—F5—H15 8.4 F6—C18—C19 115.3 (7)
C18—F6—H18 8.0 C17—C18—H18 119.2
C19—F7—H19 11.1 F6—C18—H18 3.7
C16—F8—H16 9.1 C19—C18—H18 118.8
C13—O1—Tb1 90.5 (3) F7—C19—C18 114.6 (7)
C13—O2—Tb1 94.3 (3) F7—C19—C14 124.2 (6)
C6—O3—Tb1i 163.2 (3) C18—C19—C14 121.1 (5)
C6—O3—Tb1 88.4 (2) F7—C19—H19 4.4
Tb1i—O3—Tb1 106.18 (10) C18—C19—H19 119.0
C6—O4—Tb1 99.1 (2) C14—C19—H19 119.9
C20—O5—Tb1 136.0 (3) O5—C20—O6 125.8 (4)
C20—O6—Tb1i 134.3 (3) O5—C20—C21 115.8 (4)
C27—N1—C31 118.3 (4) O6—C20—C21 118.4 (4)
C27—N1—Tb1 119.6 (3) C22—C21—C26 117.6 (5)
C31—N1—Tb1 121.0 (3) C22—C21—C20 124.3 (4)
C1—N2—C5 117.7 (4) C26—C21—C20 118.1 (4)
C1—N2—Tb1 121.2 (3) F1—C22—C21 119.9 (5)
C5—N2—Tb1 121.1 (3) F1—C22—C23 118.5 (5)
N2—C1—C2 124.0 (5) C21—C22—C23 121.5 (5)
N2—C1—H1 118.0 C24—C23—C22 120.5 (5)
C2—C1—H1 118.0 C24—C23—H23 119.7
C3—C2—C1 117.5 (6) C22—C23—H23 119.7
C3—C2—H2 121.2 C25—C24—C23 117.6 (5)
C1—C2—H2 121.2 C25—C24—H24 121.2
C2—C3—C4 120.0 (5) C23—C24—H24 121.2
C2—C3—H3 120.0 C24—C25—C26 123.2 (6)
C4—C3—H3 120.0 C24—C25—F2 118.2 (5)
C3—C4—C5 120.1 (5) C26—C25—F2 118.5 (5)
C3—C4—H4 120.0 C25—C26—C21 119.4 (5)
C5—C4—H4 120.0 C25—C26—H26 120.3
N2—C5—C4 120.7 (6) C21—C26—H26 120.3
N2—C5—C31 116.6 (4) N1—C27—C28 124.0 (6)
C4—C5—C31 122.6 (5) N1—C27—H27 118.0
O4—C6—O3 120.9 (4) C28—C27—H27 118.0
O4—C6—C7 119.8 (4) C29—C28—C27 118.4 (7)
O3—C6—C7 119.2 (4) C29—C28—H28 120.8
O4—C6—Tb1 56.3 (2) C27—C28—H28 120.8
O3—C6—Tb1 66.3 (2) C28—C29—C30 119.8 (5)
C7—C6—Tb1 162.8 (3) C28—C29—H29 120.1
C8—C7—C12 118.1 (5) C30—C29—H29 120.1
C8—C7—C6 119.9 (4) C29—C30—C31 119.7 (6)
C12—C7—C6 122.0 (4) C29—C30—H30 120.2
C7—C8—C9 118.6 (5) C31—C30—H30 120.2
C7—C8—H8 120.7 N1—C31—C30 119.9 (6)
C9—C8—H8 120.7 N1—C31—C5 116.7 (4)
F4—C9—C10 119.3 (6) C30—C31—C5 123.4 (5)
F4—C9—C8 118.0 (6)
O6i—Tb1—O1—C13 153.0 (3) O1—Tb1—C6—O3 −80.4 (4)
O3i—Tb1—O1—C13 76.4 (2) N1—Tb1—C6—O3 −142.7 (2)
O5—Tb1—O1—C13 12.8 (3) N2—Tb1—C6—O3 155.0 (3)
O2—Tb1—O1—C13 −5.5 (2) C13—Tb1—C6—O3 70.9 (7)
O4—Tb1—O1—C13 −137.2 (3) O6i—Tb1—C6—C7 −178.3 (10)
N1—Tb1—O1—C13 −126.3 (3) O3i—Tb1—C6—C7 −104.4 (9)
N2—Tb1—O1—C13 −68.6 (3) O5—Tb1—C6—C7 −34.8 (9)
O3—Tb1—O1—C13 114.7 (3) O2—Tb1—C6—C7 −22.2 (10)
C6—Tb1—O1—C13 167.7 (3) O4—Tb1—C6—C7 82.4 (10)
O6i—Tb1—O2—C13 −24.4 (3) O1—Tb1—C6—C7 166.9 (8)
O3i—Tb1—O2—C13 −82.2 (3) N1—Tb1—C6—C7 104.6 (9)
O5—Tb1—O2—C13 −159.0 (3) N2—Tb1—C6—C7 42.3 (9)
O4—Tb1—O2—C13 147.7 (2) O3—Tb1—C6—C7 −112.7 (10)
O1—Tb1—O2—C13 5.5 (2) C13—Tb1—C6—C7 −41.8 (13)
N1—Tb1—O2—C13 62.0 (3) O4—C6—C7—C8 119.8 (5)
N2—Tb1—O2—C13 119.1 (3) O3—C6—C7—C8 −56.7 (6)
O3—Tb1—O2—C13 −133.1 (2) Tb1—C6—C7—C8 47.9 (11)
C6—Tb1—O2—C13 −171.4 (2) O4—C6—C7—C12 −57.6 (6)
O6i—Tb1—O3—C6 108.2 (3) O3—C6—C7—C12 126.0 (5)
O3i—Tb1—O3—C6 −171.4 (3) Tb1—C6—C7—C12 −129.5 (9)
O5—Tb1—O3—C6 −91.8 (3) C12—C7—C8—C9 0.5 (8)
O2—Tb1—O3—C6 −119.2 (3) C6—C7—C8—C9 −176.9 (5)
O4—Tb1—O3—C6 8.2 (2) C7—C8—C9—F4 178.5 (5)
O1—Tb1—O3—C6 149.0 (2) C7—C8—C9—C10 −2.2 (10)
N1—Tb1—O3—C6 43.6 (3) F4—C9—C10—C11 −178.5 (6)
N2—Tb1—O3—C6 −27.5 (3) C8—C9—C10—C11 2.2 (11)
C13—Tb1—O3—C6 −158.5 (3) C9—C10—C11—C12 −0.4 (10)
O6i—Tb1—O3—Tb1i −80.42 (12) C10—C11—C12—F3 178.8 (6)
O3i—Tb1—O3—Tb1i 0.0 C10—C11—C12—C7 −1.2 (10)
O5—Tb1—O3—Tb1i 79.59 (12) C8—C7—C12—F3 −178.9 (5)
O2—Tb1—O3—Tb1i 52.25 (19) C6—C7—C12—F3 −1.5 (7)
O4—Tb1—O3—Tb1i 179.60 (19) C8—C7—C12—C11 1.1 (8)
O1—Tb1—O3—Tb1i −39.5 (2) C6—C7—C12—C11 178.6 (5)
N1—Tb1—O3—Tb1i −144.96 (13) Tb1—O2—C13—O1 −10.3 (4)
N2—Tb1—O3—Tb1i 143.93 (13) Tb1—O2—C13—C14 166.2 (3)
C13—Tb1—O3—Tb1i 12.9 (3) Tb1—O1—C13—O2 9.9 (4)
C6—Tb1—O3—Tb1i 171.4 (3) Tb1—O1—C13—C14 −166.5 (3)
O6i—Tb1—O4—C6 −77.4 (3) O6i—Tb1—C13—O2 161.4 (2)
O3i—Tb1—O4—C6 −8.0 (3) O3i—Tb1—C13—O2 91.6 (3)
O5—Tb1—O4—C6 57.3 (3) O5—Tb1—C13—O2 20.4 (3)
O2—Tb1—O4—C6 108.4 (3) O4—Tb1—C13—O2 −70.9 (5)
O1—Tb1—O4—C6 −147.1 (3) O1—Tb1—C13—O2 −170.2 (4)
N1—Tb1—O4—C6 −157.7 (3) N1—Tb1—C13—O2 −120.1 (3)
N2—Tb1—O4—C6 137.4 (3) N2—Tb1—C13—O2 −57.3 (3)
O3—Tb1—O4—C6 −8.4 (3) O3—Tb1—C13—O2 78.8 (3)
C13—Tb1—O4—C6 151.6 (4) C6—Tb1—C13—O2 27.0 (7)
O6i—Tb1—O5—C20 −43.3 (5) O6i—Tb1—C13—O1 −28.4 (3)
O3i—Tb1—O5—C20 9.4 (4) O3i—Tb1—C13—O1 −98.2 (3)
O2—Tb1—O5—C20 91.3 (4) O5—Tb1—C13—O1 −169.4 (2)
O4—Tb1—O5—C20 −118.5 (4) O2—Tb1—C13—O1 170.2 (4)
O1—Tb1—O5—C20 76.2 (4) O4—Tb1—C13—O1 99.3 (4)
N1—Tb1—O5—C20 −179.2 (4) N1—Tb1—C13—O1 50.1 (3)
N2—Tb1—O5—C20 168.2 (4) N2—Tb1—C13—O1 112.8 (3)
O3—Tb1—O5—C20 −69.5 (4) O3—Tb1—C13—O1 −111.0 (3)
C13—Tb1—O5—C20 82.0 (4) C6—Tb1—C13—O1 −162.9 (5)
C6—Tb1—O5—C20 −96.6 (4) O6i—Tb1—C13—C14 77.8 (17)
O6i—Tb1—N1—C27 18.5 (4) O3i—Tb1—C13—C14 8.0 (17)
O3i—Tb1—N1—C27 −27.2 (5) O5—Tb1—C13—C14 −63.2 (17)
O5—Tb1—N1—C27 167.3 (3) O2—Tb1—C13—C14 −83.6 (17)
O2—Tb1—N1—C27 −113.4 (4) O4—Tb1—C13—C14 −154.5 (15)
O4—Tb1—N1—C27 105.4 (4) O1—Tb1—C13—C14 106.2 (17)
O1—Tb1—N1—C27 −69.0 (4) N1—Tb1—C13—C14 156.3 (17)
N2—Tb1—N1—C27 −178.8 (4) N2—Tb1—C13—C14 −140.9 (17)
O3—Tb1—N1—C27 78.6 (4) O3—Tb1—C13—C14 −4.8 (18)
C13—Tb1—N1—C27 −90.2 (4) C6—Tb1—C13—C14 −57 (2)
C6—Tb1—N1—C27 96.1 (4) O2—C13—C14—C19 −14.5 (6)
O6i—Tb1—N1—C31 −173.7 (4) O1—C13—C14—C19 162.0 (4)
O3i—Tb1—N1—C31 140.5 (3) Tb1—C13—C14—C19 62.3 (18)
O5—Tb1—N1—C31 −24.9 (4) O2—C13—C14—C15 166.1 (5)
O2—Tb1—N1—C31 54.3 (3) O1—C13—C14—C15 −17.4 (7)
O4—Tb1—N1—C31 −86.9 (3) Tb1—C13—C14—C15 −117.2 (16)
O1—Tb1—N1—C31 98.7 (3) C19—C14—C15—F5 179.9 (7)
N2—Tb1—N1—C31 −11.0 (3) C13—C14—C15—F5 −0.6 (10)
O3—Tb1—N1—C31 −113.6 (3) C19—C14—C15—C16 0.1 (9)
C13—Tb1—N1—C31 77.5 (3) C13—C14—C15—C16 179.5 (5)
C6—Tb1—N1—C31 −96.1 (3) F5—C15—C16—C17 178.9 (7)
O6i—Tb1—N2—C1 −147.6 (3) C14—C15—C16—C17 −1.2 (10)
O3i—Tb1—N2—C1 34.8 (4) F5—C15—C16—F8 2.9 (13)
O5—Tb1—N2—C1 −3.0 (4) C14—C15—C16—F8 −177.2 (10)
O2—Tb1—N2—C1 73.6 (4) C15—C16—C17—C18 1.6 (10)
O4—Tb1—N2—C1 −88.3 (4) F8—C16—C17—C18 177.3 (11)
O1—Tb1—N2—C1 121.2 (4) C16—C17—C18—F6 177.0 (8)
N1—Tb1—N2—C1 −173.9 (4) C16—C17—C18—C19 −1.0 (10)
O3—Tb1—N2—C1 −60.6 (4) C17—C18—C19—F7 −179.9 (10)
C13—Tb1—N2—C1 96.6 (4) F6—C18—C19—F7 1.9 (12)
C6—Tb1—N2—C1 −72.0 (4) C17—C18—C19—C14 −0.1 (10)
O6i—Tb1—N2—C5 31.1 (4) F6—C18—C19—C14 −178.3 (7)
O3i—Tb1—N2—C5 −146.5 (3) C15—C14—C19—F7 −179.7 (10)
O5—Tb1—N2—C5 175.7 (3) C13—C14—C19—F7 0.9 (12)
O2—Tb1—N2—C5 −107.7 (3) C15—C14—C19—C18 0.6 (8)
O4—Tb1—N2—C5 90.3 (3) C13—C14—C19—C18 −178.9 (5)
O1—Tb1—N2—C5 −60.1 (3) Tb1—O5—C20—O6 47.9 (6)
N1—Tb1—N2—C5 4.8 (3) Tb1—O5—C20—C21 −130.2 (4)
O3—Tb1—N2—C5 118.1 (3) Tb1i—O6—C20—O5 −24.7 (7)
C13—Tb1—N2—C5 −84.8 (3) Tb1i—O6—C20—C21 153.4 (3)
C6—Tb1—N2—C5 106.7 (3) O5—C20—C21—C22 −147.8 (5)
C5—N2—C1—C2 −0.7 (7) O6—C20—C21—C22 33.9 (7)
Tb1—N2—C1—C2 178.1 (4) O5—C20—C21—C26 33.5 (6)
N2—C1—C2—C3 0.4 (9) O6—C20—C21—C26 −144.8 (4)
C1—C2—C3—C4 0.0 (9) C26—C21—C22—F1 −176.8 (5)
C2—C3—C4—C5 0.0 (9) C20—C21—C22—F1 4.6 (7)
C1—N2—C5—C4 0.7 (7) C26—C21—C22—C23 0.4 (8)
Tb1—N2—C5—C4 −178.1 (3) C20—C21—C22—C23 −178.3 (5)
C1—N2—C5—C31 179.7 (4) F1—C22—C23—C24 178.5 (5)
Tb1—N2—C5—C31 1.0 (5) C21—C22—C23—C24 1.3 (9)
C3—C4—C5—N2 −0.4 (7) C22—C23—C24—C25 −1.9 (9)
C3—C4—C5—C31 −179.3 (5) C23—C24—C25—C26 0.8 (9)
Tb1—O4—C6—O3 16.1 (5) C23—C24—C25—F2 179.6 (5)
Tb1—O4—C6—C7 −160.3 (3) C24—C25—C26—C21 0.9 (9)
Tb1i—O3—C6—O4 −165.0 (7) F2—C25—C26—C21 −178.0 (5)
Tb1—O3—C6—O4 −14.6 (4) C22—C21—C26—C25 −1.5 (7)
Tb1i—O3—C6—C7 11.5 (12) C20—C21—C26—C25 177.3 (5)
Tb1—O3—C6—C7 161.8 (4) C31—N1—C27—C28 −1.3 (8)
Tb1i—O3—C6—Tb1 −150.4 (10) Tb1—N1—C27—C28 166.8 (4)
O6i—Tb1—C6—O4 99.4 (3) N1—C27—C28—C29 −0.4 (9)
O3i—Tb1—C6—O4 173.3 (3) C27—C28—C29—C30 1.1 (10)
O5—Tb1—C6—O4 −117.2 (3) C28—C29—C30—C31 −0.2 (10)
O2—Tb1—C6—O4 −104.6 (3) C27—N1—C31—C30 2.2 (7)
O1—Tb1—C6—O4 84.5 (5) Tb1—N1—C31—C30 −165.7 (4)
N1—Tb1—C6—O4 22.3 (3) C27—N1—C31—C5 −175.9 (4)
N2—Tb1—C6—O4 −40.1 (3) Tb1—N1—C31—C5 16.1 (5)
O3—Tb1—C6—O4 164.9 (5) C29—C30—C31—N1 −1.5 (8)
C13—Tb1—C6—O4 −124.1 (6) C29—C30—C31—C5 176.5 (5)
O6i—Tb1—C6—O3 −65.6 (2) N2—C5—C31—N1 −11.0 (6)
O3i—Tb1—C6—O3 8.3 (3) C4—C5—C31—N1 168.0 (4)
O5—Tb1—C6—O3 77.9 (2) N2—C5—C31—C30 170.9 (5)
O2—Tb1—C6—O3 90.5 (3) C4—C5—C31—C30 −10.1 (7)
O4—Tb1—C6—O3 −164.9 (5)
Symmetry codes: (i) −x+1, −y+2, −z+1.
Table 1 Selected bond lengths (Å)
Tb1—N1 2.565 (3)
Tb1—N2 2.586 (4)
Tb1—O6i 2.364 (3)
Tb1—O3i 2.377 (3)
Tb1—O5 2.379 (3)
Tb1—O2 2.418 (3)
Tb1—O4 2.481 (3)
Tb1—O1 2.498 (3)
Tb1—O3 2.696 (3)
Symmetry code: (i) .
==== Refs
References
Bruker (2001). SADABS and SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Serre, C., Marrot, J. & Ferey, G. (2005). Inorg. Chem.44, 654–658.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21581763 | PMC2968370 | CC BY | 2021-01-04 18:57:43 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Jan 8; 65(Pt 2):m150 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography at272510.1107/S1600536809005765ACSEBHS1600536809005765Metal-Organic PapersDiaquabis(pyridine-2-carboxylato-κ2
N,O)iron(II) [Fe(C6H4NO2)2(H2O)2]Xia Guohua a*Sun Zexi ba Institute of Applied Materials, College of Resource & Environmental Management, Jiangxi University of Finance & Economics, Nanchang, Jiangxi 330013, People’s Republic of Chinab Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, People’s Republic of ChinaCorrespondence e-mail: [email protected] 3 2009 25 2 2009 25 2 2009 65 Pt 3 e090300m315 m316 15 2 2009 18 2 2009 © Xia and Sun 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The FeII atom in the title complex, [Fe(C6H4NO2)2(H2O)2], exists in a distorted octahedral coordination geometry defined by two O and two N atoms from two pyridine-2-carboxylate ligands and two O atoms of two water molecules. In the crystal structure, molecules are linked into a three-dimensional framework by O—H⋯O hydrogen bonds.
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Related literature
For the design and construction of metal-organic supramolecular structures, see: Desiraju (1997 ▶); Braga et al. (1998 ▶); Mccann et al. (1996 ▶); Wai et al. (1990 ▶); Yaghi et al. (1996 ▶); Min & Lee (2002 ▶); Maira et al. (2001 ▶). For bond-length data, see: Allen et al. (1987 ▶).
Experimental
Crystal data
[Fe(C6H4NO2)2(H2O)2]
M
r = 336.09
Monoclinic,
a = 11.6255 (3) Å
b = 9.0247 (4) Å
c = 14.9724 (2) Å
β = 105.568 (2)°
V = 1513.22 (8) Å3
Z = 4
Mo Kα radiation
μ = 1.02 mm−1
T = 293 K
0.23 × 0.19 × 0.07 mm
Data collection
Bruker SMART APEX area-detector diffractometer
Absorption correction: multi-scan (SADABS; Sheldrick, 1996 ▶) T
min = 0.796, T
max = 0.928
10191 measured reflections
3283 independent reflections
2158 reflections with I > 2σ(I)
R
int = 0.043
Refinement
R[F
2 > 2σ(F
2)] = 0.051
wR(F
2) = 0.166
S = 1.08
3283 reflections
201 parameters
6 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.72 e Å−3
Δρmin = −0.47 e Å−3
Data collection: SMART (Siemens, 1996 ▶); cell refinement: SAINT (Siemens, 1996 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809005765/at2725sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809005765/at2725Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2725).
We thank the Youth Program of Jiangxi University of Finance and Economics for financial support of this work.
supplementary crystallographic
information
Comment
In the synthesis of crystal structures by design, the assembly of molecular
units in predefined arrangements is a key goal (Desiraju, 1997; Braga
et
al., 1998). Due to carboxyl groups are one of the most important
classes
of biological ligands, the coordination of metal-carboxyl groups complexes are
of critical importance in biological systems, organic materials and
coordination chemistry. Recently, carboxyl groups with variable coordination
modes have been used to construct metal-organic supramolecular structures
(Mccann et al., 1996; Wai et al., 1990; Yaghi
et al.,
1996; Min & Lee 2002; Maira et al., 2001). We
report here in the
crystal structure of the title compound, (I).
In the molecule of (I) (Fig. 1), the ligand bond lengths and angles are within
normal ranges (Allen et al., 1987). In the title complex, each
FeII
atom is axially coordinated by water molecules and consists of an equatorial
plane of two oxygen donors and two nitrogen donors from two
pyridine-2-carboxylato ligands with a distorted octahedral coordination
geometry. The Fe—O bonds [average 2.152 (4) Å] are somewhat shorter than
the Fe—N distances [average 2.270 (8) Å].
In the crystal structure, O—H···O hydrogen bonds (Fig. 2 and Table 2) seem to
be effective in the stabilization of the structure, resulting in the formation
of a supramolecular network structure.
Experimental
Crystals of the title compound were synthesized using hydrothermal method in a
23 ml Teflon-lined Parr bomb, which was then sealed. Iron(II) chloride
tetrahydrate (198.71 mg, 1 mmol), pyridine-2-carboxylic acid (246 mg, 2 mmol)
and distilled water (10 g) were placed into the bomb and sealed. The bomb was
then heated under autogenous pressure up to 433 K over the course of 7 d and
allowed to cool at room temperature for 24 h. Upon opening the bomb, a clear
colourless solution was decanted from small purple crystals. These crystals
were washed with distilled water followed by ethanol, and allowed to air-dry
at room temperature.
Refinement
H1B and H2B (for two water molecules) were located in difference syntheses and
refined isotropically [O—H = 0.805 (18) and 0.82 (5) Å, Uiso(H) =
0.093 (15) and 0.18 (3) Å2]. The remaining H atoms were positioned
geometrically, with O—H = 0.82 Å (for H2O) and C—H = 0.93 Å for
aromatic H, and constrained to ride on their parent atoms, with
Uiso(H) = xUeq(C,O), where x = 1.2 for
aromatic H atoms and x = 1.5 for all other H atoms.
Figures
Fig. 1. View of the molecule of (I), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level.
Fig. 2. A packing diagram of (I). Hydrogen bonds are shown as dashed lines.
Crystal data
[Fe(C6H4NO2)2(H2O)2] F(000) = 688
Mr = 336.09 Dx = 1.475 Mg m−3
Monoclinic, P21/n Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 2834 reflections
a = 11.6255 (3) Å θ = 2.4–24.8°
b = 9.0247 (4) Å µ = 1.02 mm−1
c = 14.9724 (2) Å T = 293 K
β = 105.568 (2)° Plane, purple
V = 1513.22 (8) Å3 0.23 × 0.19 × 0.07 mm
Z = 4
Data collection
Bruker SMART APEX area-detector diffractometer 3283 independent reflections
Radiation source: fine-focus sealed tube 2158 reflections with I > 2σ(I)
graphite Rint = 0.043
φ and ω scans θmax = 27.3°, θmin = 2.0°
Absorption correction: multi-scan (SADABS; Sheldrick, 1996) h = −14→14
Tmin = 0.796, Tmax = 0.928 k = −11→11
10191 measured reflections l = −19→18
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.051 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.166 w = 1/[σ2(Fo2) + (0.089P)2 + 0.0485P] where P = (Fo2 + 2Fc2)/3
S = 1.08 (Δ/σ)max = 0.001
3283 reflections Δρmax = 0.72 e Å−3
201 parameters Δρmin = −0.47 e Å−3
6 restraints Extinction correction: SHELXL97 (Sheldrick, 2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.0048 (16)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Fe1 0.74557 (4) 0.85895 (5) 0.62770 (3) 0.0492 (2)
O1 0.8189 (2) 0.7456 (3) 0.75783 (16) 0.0565 (6)
H1A 0.7646 0.7075 0.7752 0.085*
O2 0.5913 (2) 0.8895 (3) 0.6788 (2) 0.0606 (7)
H2A 0.5961 0.8351 0.7235 0.091*
O3 0.65069 (19) 0.9271 (3) 0.48892 (15) 0.0527 (6)
O4 0.5389 (2) 0.8582 (3) 0.35014 (17) 0.0592 (7)
O5 0.8181 (2) 1.0762 (3) 0.66750 (16) 0.0540 (6)
O6 0.9719 (3) 1.2298 (3) 0.6897 (2) 0.0772 (8)
N1 0.9337 (3) 0.8594 (3) 0.6119 (2) 0.0534 (7)
N2 0.6907 (2) 0.6454 (3) 0.54619 (18) 0.0435 (6)
C1 0.6030 (3) 0.8306 (4) 0.4297 (2) 0.0454 (7)
C2 0.6264 (3) 0.6693 (3) 0.4578 (2) 0.0418 (7)
C3 0.5833 (3) 0.5536 (4) 0.3976 (2) 0.0561 (9)
H3 0.5407 0.5724 0.3367 0.067*
C4 0.6042 (3) 0.4112 (4) 0.4288 (3) 0.0587 (9)
H4 0.5742 0.3320 0.3897 0.070*
C5 0.6703 (3) 0.3863 (4) 0.5188 (3) 0.0580 (9)
H5 0.6864 0.2900 0.5407 0.070*
C6 0.7123 (3) 0.5052 (4) 0.5758 (2) 0.0530 (8)
H6 0.7568 0.4880 0.6365 0.064*
C7 0.9225 (3) 1.1079 (4) 0.6662 (2) 0.0538 (8)
C8 0.9906 (3) 0.9877 (4) 0.6345 (2) 0.0514 (8)
C9 1.1059 (3) 1.0068 (5) 0.6312 (3) 0.0731 (11)
H9 1.1448 1.0967 0.6481 0.088*
C10 1.1632 (4) 0.8908 (6) 0.6027 (4) 0.0974 (17)
H10 1.2406 0.9023 0.5974 0.117*
C11 1.1060 (4) 0.7585 (6) 0.5820 (4) 0.108 (2)
H11 1.1452 0.6771 0.5659 0.130*
C12 0.9911 (4) 0.7474 (5) 0.5854 (3) 0.0813 (13)
H12 0.9510 0.6583 0.5686 0.098*
H1B 0.8859 (15) 0.715 (5) 0.778 (3) 0.093 (15)*
H2B 0.539 (5) 0.953 (5) 0.673 (4) 0.18 (3)*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Fe1 0.0490 (3) 0.0448 (4) 0.0474 (3) −0.0005 (2) 0.0020 (2) −0.00062 (19)
O1 0.0430 (12) 0.0648 (16) 0.0542 (14) 0.0009 (12) 0.0002 (10) 0.0158 (11)
O2 0.0602 (15) 0.0518 (15) 0.0723 (18) 0.0102 (12) 0.0223 (13) 0.0148 (12)
O3 0.0536 (13) 0.0450 (14) 0.0507 (13) −0.0018 (10) −0.0013 (10) 0.0043 (10)
O4 0.0566 (14) 0.0563 (16) 0.0512 (14) 0.0076 (11) −0.0091 (10) 0.0080 (10)
O5 0.0489 (13) 0.0466 (13) 0.0659 (15) −0.0013 (10) 0.0144 (10) −0.0088 (11)
O6 0.0798 (18) 0.0646 (18) 0.096 (2) −0.0277 (15) 0.0388 (15) −0.0312 (15)
N1 0.0535 (16) 0.0470 (18) 0.0624 (18) −0.0004 (13) 0.0205 (13) −0.0032 (13)
N2 0.0437 (13) 0.0411 (15) 0.0403 (14) 0.0004 (11) 0.0018 (10) 0.0027 (10)
C1 0.0345 (14) 0.054 (2) 0.0428 (17) 0.0025 (13) 0.0016 (12) 0.0015 (14)
C2 0.0361 (14) 0.0442 (18) 0.0418 (16) −0.0003 (12) 0.0045 (11) −0.0028 (13)
C3 0.0540 (19) 0.057 (2) 0.0473 (18) 0.0048 (16) −0.0028 (14) −0.0067 (16)
C4 0.059 (2) 0.047 (2) 0.065 (2) 0.0017 (17) 0.0074 (17) −0.0142 (17)
C5 0.064 (2) 0.043 (2) 0.070 (2) 0.0023 (16) 0.0226 (18) −0.0010 (16)
C6 0.0591 (18) 0.047 (2) 0.0489 (18) 0.0017 (16) 0.0080 (14) 0.0045 (15)
C7 0.059 (2) 0.057 (2) 0.0445 (18) −0.0115 (16) 0.0121 (15) −0.0065 (15)
C8 0.0509 (17) 0.058 (2) 0.0456 (17) 0.0039 (16) 0.0140 (13) 0.0021 (15)
C9 0.061 (2) 0.075 (3) 0.088 (3) −0.008 (2) 0.029 (2) −0.001 (2)
C10 0.070 (3) 0.095 (4) 0.143 (5) 0.002 (3) 0.055 (3) 0.007 (3)
C11 0.090 (3) 0.077 (3) 0.183 (6) 0.013 (3) 0.081 (4) −0.007 (3)
C12 0.076 (3) 0.056 (3) 0.124 (4) 0.003 (2) 0.047 (3) −0.007 (2)
Geometric parameters (Å, °)
Fe1—O1 2.163 (2) C1—C2 1.520 (4)
Fe1—O2 2.148 (3) C2—C3 1.382 (4)
Fe1—O3 2.164 (2) C3—C4 1.366 (5)
Fe1—O5 2.154 (2) C3—H3 0.9300
Fe1—N1 2.262 (3) C4—C5 1.379 (5)
Fe1—N2 2.279 (2) C4—H4 0.9300
O1—H1A 0.8200 C5—C6 1.377 (5)
O1—H1B 0.805 (18) C5—H5 0.9300
O2—H2A 0.8200 C6—H6 0.9300
O2—H2B 0.82 (5) C7—C8 1.494 (5)
O3—C1 1.260 (4) C8—C9 1.365 (5)
O4—C1 1.249 (4) C9—C10 1.369 (6)
O5—C7 1.251 (4) C9—H9 0.9300
O6—C7 1.247 (4) C10—C11 1.361 (7)
N1—C12 1.329 (5) C10—H10 0.9300
N1—C8 1.332 (4) C11—C12 1.353 (6)
N2—C6 1.342 (4) C11—H11 0.9300
N2—C2 1.351 (4) C12—H12 0.9300
O1—Fe1—O2 84.52 (10) N2—C2—C1 115.8 (3)
O1—Fe1—O3 167.30 (9) C3—C2—C1 122.4 (3)
O1—Fe1—O5 98.68 (10) C4—C3—C2 119.2 (3)
O2—Fe1—O3 92.66 (10) C4—C3—H3 120.4
O2—Fe1—O5 95.00 (10) C2—C3—H3 120.4
O3—Fe1—O5 93.89 (9) C3—C4—C5 119.2 (3)
O1—Fe1—N1 86.39 (10) C3—C4—H4 120.4
O2—Fe1—N1 163.77 (12) C5—C4—H4 120.4
O3—Fe1—N1 99.03 (10) C6—C5—C4 119.4 (3)
O5—Fe1—N1 73.12 (9) C6—C5—H5 120.3
O1—Fe1—N2 93.92 (9) C4—C5—H5 120.3
O2—Fe1—N2 99.14 (10) N2—C6—C5 121.7 (3)
O3—Fe1—N2 74.26 (9) N2—C6—H6 119.1
O5—Fe1—N2 161.88 (9) C5—C6—H6 119.1
N1—Fe1—N2 94.86 (10) O6—C7—O5 124.9 (3)
Fe1—O1—H1A 109.5 O6—C7—C8 119.1 (3)
Fe1—O1—H1B 128 (3) O5—C7—C8 116.0 (3)
H1A—O1—H1B 119.0 N1—C8—C9 121.7 (3)
Fe1—O2—H2A 109.5 N1—C8—C7 116.3 (3)
Fe1—O2—H2B 136 (3) C9—C8—C7 122.0 (3)
H2A—O2—H2B 112.5 C8—C9—C10 118.6 (4)
C1—O3—Fe1 119.6 (2) C8—C9—H9 120.7
C7—O5—Fe1 120.9 (2) C10—C9—H9 120.7
C12—N1—C8 118.8 (3) C11—C10—C9 119.5 (4)
C12—N1—Fe1 127.4 (3) C11—C10—H10 120.2
C8—N1—Fe1 113.8 (2) C9—C10—H10 120.2
C6—N2—C2 118.7 (3) C12—C11—C10 118.9 (4)
C6—N2—Fe1 128.3 (2) C12—C11—H11 120.5
C2—N2—Fe1 113.04 (19) C10—C11—H11 120.5
O4—C1—O3 124.8 (3) N1—C12—C11 122.3 (4)
O4—C1—C2 118.1 (3) N1—C12—H12 118.8
O3—C1—C2 117.0 (3) C11—C12—H12 118.8
N2—C2—C3 121.8 (3)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O1—H1B···O4i 0.81 (2) 1.93 (2) 2.726 (3) 169 (4)
O1—H1A···O5ii 0.82 1.87 2.661 (3) 161
O2—H2B···O4iii 0.82 (5) 1.92 (5) 2.704 (3) 159 (6)
O2—H2A···O6ii 0.82 1.94 2.697 (4) 153
Symmetry codes: (i) x+1/2, −y+3/2, z+1/2; (ii) −x+3/2, y−1/2, −z+3/2; (iii) −x+1, −y+2, −z+1.
Table 1 Selected geometric parameters (Å, °)
Fe1—O1 2.163 (2)
Fe1—O2 2.148 (3)
Fe1—O3 2.164 (2)
Fe1—O5 2.154 (2)
Fe1—N1 2.262 (3)
Fe1—N2 2.279 (2)
O1—Fe1—O2 84.52 (10)
O1—Fe1—O3 167.30 (9)
O1—Fe1—O5 98.68 (10)
O2—Fe1—O3 92.66 (10)
O2—Fe1—O5 95.00 (10)
O3—Fe1—O5 93.89 (9)
O1—Fe1—N1 86.39 (10)
O2—Fe1—N1 163.77 (12)
O3—Fe1—N1 99.03 (10)
O5—Fe1—N1 73.12 (9)
O1—Fe1—N2 93.92 (9)
O2—Fe1—N2 99.14 (10)
O3—Fe1—N2 74.26 (9)
O5—Fe1—N2 161.88 (9)
N1—Fe1—N2 94.86 (10)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O1—H1B⋯O4i 0.805 (18) 1.93 (2) 2.726 (3) 169 (4)
O1—H1A⋯O5ii 0.82 1.87 2.661 (3) 161
O2—H2B⋯O4iii 0.82 (5) 1.92 (5) 2.704 (3) 159 (6)
O2—H2A⋯O6ii 0.82 1.94 2.697 (4) 153
Symmetry codes: (i) ; (ii) ; (iii) .
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References
Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19.
Braga, D., Grepioni, F. & Desiraju, G. R. (1998). Chem. Rev.98, 1375–1386.
Desiraju, G. R. (1997). J. Chem. Soc. Chem. Commun. pp. 1475–1476.
Maira, S. M., Galetic, I., Brazil, D. P., Decech, S., Ingley, E., thelen, M. & Hemmings, B. A. (2001). Science, 294, 374–380.
Mccann, M., Casey, M. T., Devereux, M., Curran, M. & Cardin, C. (1996). Polyhedron, 15, 2117–2120.
Min, D. & Lee, S. M. (2002). Inorg. Chem. Commun.5, 978–983.
Sheldrick, G. M. (1996). SADABS University of Göttingen, Germany.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Siemens (1996). SMART and SAINT Siemens Analytical X-ray Instruments Inc., Madison, Wisconsin, USA.
Wai, H. Y., Ru, J. W. & Mark, T. C. W. (1990). J. Crystallogr. Spectrosc. Res.20, 307–312.
Yaghi, O. M., Li, H. & Groy, T. L. (1996). J. Am. Chem. Soc.118, 9096–9101. | 21582090 | PMC2968519 | CC BY | 2021-01-04 18:57:47 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Feb 25; 65(Pt 3):m315-m316 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography bi234710.1107/S1600536809008836ACSEBHS1600536809008836Metal-Organic Papers
catena-Poly[[[diaquathulium(III)]-μ-6-carboxynicotinato-μ-pyridine-2,5-dicarboxylato] dihydrate] [Tm(C7H3NO4)(C7H4NO4)(H2O)2]·2H2OLi Sheng aChen Yue bHe Hong-Mei bMa Yuan-Fang a*a Institute of Immunology, Key Laboratory of Natural Drugs and Immunological Engineering of Henan Province, College of Medicine, Henan University, Kaifeng 475003, People’s Republic of Chinab College of Medicine, Henan University, Kaifeng 475003, People’s Republic of ChinaCorrespondence e-mail: [email protected] 4 2009 19 3 2009 19 3 2009 65 Pt 4 e090400m411 m411 13 1 2009 10 3 2009 © Li et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, {[Tm(C7H3NO4)(C7H4NO4)(H2O)2]·2H2O}n, is isotypic with the analogous TbIII compound [Li et al. (2009 ▶). Acta Cryst. E65, m410]. All interatomic distances and angles and the hydrogen-bond geometries are very similar for the two structures. The refined Flack parameter of 0.49 (2) suggests inversion twinning.
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Related literature
For the isotypic TbIII compound, see Li et al. (2009 ▶). For other related structures, see: Huang et al. (2007 ▶).
Experimental
Crystal data
[Tm(C7H3NO4)(C7H4NO4)(H2O)2]·2H2O
M
r = 572.21
Tetragonal,
a = 15.1286 (12) Å
c = 14.849 (2) Å
V = 3398.6 (6) Å3
Z = 8
Mo Kα radiation
μ = 5.30 mm−1
T = 298 K
0.12 × 0.11 × 0.09 mm
Data collection
Bruker APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.569, T
max = 0.647
7093 measured reflections
3085 independent reflections
2977 reflections with I > 2σ(I)
R
int = 0.031
Refinement
R[F
2 > 2σ(F
2)] = 0.043
wR(F
2) = 0.111
S = 1.07
3085 reflections
263 parameters
H-atom parameters constrained
Δρmax = 6.96 e Å−3
Δρmin = −1.18 e Å−3
Absolute structure: Flack (1983 ▶), with 1444 Friedel pairs
Flack parameter: 0.49 (2)
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809008836/bi2347sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809008836/bi2347Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BI2347).
The authors are grateful for financial support from Henan University (grant No. 05YBGG013)
supplementary crystallographic
information
Comment
The asymmetric unit of the title compound is shown in Fig. 1. Atom Tm1 displays
octa-coordination through two water molecules, four carboxylate O atoms and
two pyridyl N atoms from two 2,5-pydc and two 2,5-Hpydc ligands (2,5-pydc =
2,5-pyridinedicarboxylate). The 2,5-pydc and 2,5-Hpydc ligands bridge between
TmIII atoms to generate helical coordination polymers along [001] (Fig. 2).
An extensive network of O—H···O hydrogen bonds is formed between the
coordination polymers and the lattice water molecules (Table 1 and Fig. 3).
Experimental
A mixture of thulium oxide (0.5 mmol), 2,5-pyridinedicarboxylic acid (0.5 mmol), in H2O (8 ml) and ethanol (8 ml) was sealed in a 25 ml Teflon-lined
stainless steel autoclave and kept at 413 K for three days. Colourless
crystals were obtained after cooling to room temperature with a yield of 27%.
Elemental analysis calculated: C 28.90, H 2.75, N 4.82%; Found: C 28.75, H
2.72, N 4.79%.
Refinement
H atoms bound to C atoms were placed in calculated positions with C—H = 0.93 Å and refined as riding with Uiso(H) = 1.2Ueq(C). H atoms
of the water molecules were placed so as to form a reasonable H-bond network
and refined as riding with Uiso(H) = 1.5Ueq(O). The Flack
parameter was refined as a full least-squares parameter, and the refined value
of 0.49 (2) suggests inversion twinning. Two relatively large peaks remain in
the residual electron density (5.5–7.0 eÅ-3) on the special positions
(0,0,0) and (0.5,0,0.25), which may indicate further lattice water molecules.
The refinement as a dihydrate is consistent with the isomorphous TbIII
compound (Li et al., 2009).
Figures
Fig. 1. Aysymmetric unit of the title compound, showing 50% probability displacement ellipsoids for non-H atoms.
Fig. 2. One-dimensional coordination polymer running along [001].
Fig. 3. Projection along [001], showing the tetragonal arrangement of coordination polymers. O—H···O hydrogen bonds are shown as dashed lines.
Crystal data
[Tm(C7H3NO4)(C7H4NO4)(H2O)2]·2H2O Dx = 2.237 Mg m−3
Mr = 572.21 Mo Kα radiation, λ = 0.71073 Å
Tetragonal, I4 Cell parameters from 3085 reflections
Hall symbol: I -4 θ = 1.9–25.5°
a = 15.1286 (12) Å µ = 5.30 mm−1
c = 14.849 (2) Å T = 298 K
V = 3398.6 (6) Å3 Block, colorless
Z = 8 0.12 × 0.11 × 0.09 mm
F(000) = 2224
Data collection
Bruker APEXII CCD diffractometer 3085 independent reflections
Radiation source: fine-focus sealed tube 2977 reflections with I > 2σ(I)
graphite Rint = 0.031
φ and ω scans θmax = 25.5°, θmin = 1.9°
Absorption correction: multi-scan (SADABS; Bruker, 2001) h = −18→16
Tmin = 0.569, Tmax = 0.647 k = −18→15
7093 measured reflections l = −17→16
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.043 H-atom parameters constrained
wR(F2) = 0.111 w = 1/[σ2(Fo2) + (0.0703P)2 + 51.4546P] where P = (Fo2 + 2Fc2)/3
S = 1.07 (Δ/σ)max < 0.001
3085 reflections Δρmax = 6.96 e Å−3
263 parameters Δρmin = −1.18 e Å−3
0 restraints Absolute structure: Flack (1983), 1444 Friedel pairs
Primary atom site location: structure-invariant direct methods Flack parameter: 0.49 (2)
Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell esds are taken
into account individually in the estimation of esds in distances, angles
and torsion angles; correlations between esds in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell esds is used for estimating esds involving l.s. planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and
goodness of fit S are based on F2, conventional R-factors R are based
on F, with F set to zero for negative F2. The threshold expression of
F2 > 2sigma(F2) is used only for calculating R-factors(gt) etc. and is
not relevant to the choice of reflections for refinement. R-factors based
on F2 are statistically about twice as large as those based on F, and R-
factors based on ALL data will be even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Tm1 0.30232 (3) 0.22523 (3) 0.22714 (3) 0.01768 (14)
C1 0.1831 (5) 0.4107 (6) 0.1942 (6) 0.0111 (18)
C2 0.1327 (6) 0.4765 (6) 0.1551 (6) 0.0164 (19)
H2A 0.1115 0.5229 0.1900 0.020*
C3 0.1140 (6) 0.4732 (6) 0.0642 (6) 0.0156 (18)
H3A 0.0816 0.5185 0.0376 0.019*
C4 0.1428 (6) 0.4034 (6) 0.0126 (6) 0.0132 (17)
C5 0.1880 (6) 0.3365 (6) 0.0577 (7) 0.0170 (19)
H5A 0.2041 0.2864 0.0253 0.020*
C6 0.2156 (6) 0.4129 (6) 0.2916 (5) 0.0115 (19)
C7 0.1298 (6) 0.3968 (6) −0.0877 (7) 0.017 (2)
C8 0.1188 (6) 0.1237 (6) 0.1724 (6) 0.0133 (18)
C9 0.0969 (5) 0.1481 (5) 0.2698 (7) 0.0091 (15)
C10 0.0232 (6) 0.1189 (6) 0.3171 (7) 0.0160 (18)
H10A −0.0196 0.0856 0.2876 0.019*
C11 0.0128 (6) 0.1380 (6) 0.4051 (7) 0.0160 (19)
H11A −0.0358 0.1162 0.4364 0.019*
C12 0.0768 (6) 0.1922 (6) 0.4506 (6) 0.0103 (16)
C13 0.1453 (6) 0.2202 (6) 0.3982 (6) 0.0136 (18)
H13A 0.1868 0.2571 0.4251 0.016*
C14 0.0689 (6) 0.2110 (7) 0.5496 (6) 0.016 (2)
N1 0.1582 (5) 0.1994 (5) 0.3117 (6) 0.0152 (16)
N2 0.2099 (5) 0.3404 (5) 0.1464 (5) 0.0117 (15)
O1 0.2739 (4) 0.3555 (4) 0.3098 (5) 0.0170 (14)
H1 0.3059 0.3647 0.3559 0.025*
O2 0.1850 (6) 0.4687 (6) 0.3432 (5) 0.0315 (18)
O3 0.1614 (5) 0.3285 (4) −0.1275 (5) 0.0191 (14)
O4 0.0916 (5) 0.4563 (5) −0.1268 (5) 0.0221 (15)
O5 0.1938 (4) 0.1458 (4) 0.1461 (5) 0.0177 (14)
O6 0.0627 (5) 0.0860 (5) 0.1274 (5) 0.0221 (15)
O7 0.0051 (5) 0.1854 (6) 0.5920 (5) 0.0281 (17)
O8 0.1335 (4) 0.2552 (5) 0.5835 (5) 0.0215 (15)
O9 0.3709 (5) 0.0809 (4) 0.2010 (4) 0.0189 (14)
H91 0.3827 0.0691 0.2556 0.028*
H92 0.4227 0.0840 0.1790 0.028*
O10 0.4516 (4) 0.2745 (5) 0.2619 (5) 0.0217 (15)
H101 0.4653 0.2877 0.2080 0.033*
H102 0.4887 0.2336 0.2727 0.033*
O11 0.2679 (7) 0.0199 (6) 0.0226 (6) 0.042 (2)
H111 0.2463 0.0571 0.0594 0.063*
H112 0.2823 0.0234 −0.0326 0.063*
O12 0.1257 (6) −0.0911 (6) 0.0638 (6) 0.041 (2)
H121 0.0724 −0.0900 0.0821 0.061*
H122 0.1146 −0.0831 0.0083 0.061*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Tm1 0.0186 (2) 0.0188 (2) 0.0156 (2) −0.00011 (16) −0.00025 (17) 0.00017 (17)
C1 0.006 (4) 0.014 (4) 0.013 (5) 0.002 (3) −0.006 (3) 0.001 (3)
C2 0.018 (4) 0.014 (4) 0.017 (5) 0.000 (4) 0.004 (4) −0.006 (4)
C3 0.012 (4) 0.016 (4) 0.019 (4) 0.008 (3) −0.002 (4) 0.002 (4)
C4 0.011 (4) 0.016 (4) 0.013 (4) −0.004 (3) −0.005 (3) 0.001 (3)
C5 0.022 (5) 0.012 (4) 0.017 (5) 0.010 (4) −0.001 (4) −0.004 (4)
C6 0.016 (4) 0.015 (4) 0.003 (5) −0.001 (3) 0.002 (3) −0.006 (3)
C7 0.017 (5) 0.019 (5) 0.017 (5) −0.001 (4) 0.001 (4) −0.009 (4)
C8 0.024 (5) 0.013 (4) 0.003 (4) 0.006 (4) −0.004 (4) −0.005 (3)
C9 0.013 (4) 0.010 (4) 0.004 (4) −0.002 (3) −0.005 (4) 0.005 (3)
C10 0.016 (4) 0.019 (4) 0.013 (4) 0.000 (3) −0.002 (4) −0.002 (4)
C11 0.013 (4) 0.015 (4) 0.021 (5) 0.000 (3) −0.001 (4) 0.002 (4)
C12 0.012 (4) 0.012 (4) 0.007 (4) 0.005 (3) 0.004 (3) 0.000 (3)
C13 0.016 (4) 0.015 (4) 0.009 (4) −0.002 (3) 0.000 (3) −0.003 (3)
C14 0.014 (4) 0.024 (5) 0.011 (5) 0.001 (4) −0.003 (4) −0.001 (4)
N1 0.017 (4) 0.017 (4) 0.011 (4) −0.002 (3) 0.000 (3) −0.007 (3)
N2 0.015 (4) 0.014 (4) 0.006 (4) 0.003 (3) −0.002 (3) 0.000 (3)
O1 0.022 (3) 0.015 (3) 0.015 (3) −0.003 (3) −0.005 (3) 0.000 (3)
O2 0.047 (5) 0.036 (4) 0.012 (3) 0.021 (4) −0.004 (3) −0.005 (3)
O3 0.026 (4) 0.017 (3) 0.014 (3) 0.009 (3) −0.001 (3) −0.004 (3)
O4 0.031 (4) 0.022 (4) 0.013 (3) 0.014 (3) −0.007 (3) 0.000 (3)
O5 0.018 (3) 0.017 (3) 0.018 (4) −0.003 (3) 0.006 (3) 0.000 (3)
O6 0.019 (3) 0.025 (4) 0.022 (4) −0.008 (3) −0.003 (3) −0.006 (3)
O7 0.030 (4) 0.038 (4) 0.016 (4) −0.012 (3) 0.007 (3) −0.007 (3)
O8 0.016 (3) 0.034 (4) 0.015 (3) −0.008 (3) 0.003 (3) −0.007 (3)
O9 0.021 (3) 0.022 (3) 0.014 (3) 0.003 (3) 0.007 (3) 0.002 (3)
O10 0.016 (3) 0.032 (4) 0.017 (4) 0.001 (3) 0.001 (3) −0.001 (3)
O11 0.061 (6) 0.036 (5) 0.029 (4) 0.002 (4) 0.015 (4) −0.007 (4)
O12 0.044 (5) 0.041 (5) 0.036 (5) 0.019 (4) 0.002 (4) 0.012 (4)
Geometric parameters (Å, °)
Tm1—O1 2.361 (7) C8—C9 1.529 (13)
Tm1—O8i 2.363 (7) C9—N1 1.360 (12)
Tm1—O5 2.364 (7) C9—C10 1.390 (13)
Tm1—O3ii 2.371 (7) C10—C11 1.347 (15)
Tm1—O10 2.434 (7) C10—H10A 0.930
Tm1—O9 2.448 (7) C11—C12 1.438 (13)
Tm1—N2 2.536 (8) C11—H11A 0.930
Tm1—N1 2.546 (8) C12—C13 1.363 (13)
C1—N2 1.341 (12) C12—C14 1.502 (13)
C1—C2 1.381 (13) C13—N1 1.338 (13)
C1—C6 1.528 (12) C13—H13A 0.930
C2—C3 1.380 (14) C14—O7 1.215 (13)
C2—H2A 0.930 C14—O8 1.287 (12)
C3—C4 1.376 (13) O1—H1 0.850
C3—H3A 0.930 O3—Tm1i 2.371 (7)
C4—C5 1.392 (13) O8—Tm1ii 2.363 (7)
C4—C7 1.506 (14) O9—H91 0.850
C5—N2 1.359 (13) O9—H92 0.850
C5—H5A 0.930 O10—H101 0.850
C6—O2 1.230 (12) O10—H102 0.850
C6—O1 1.268 (11) O11—H111 0.850
C7—O4 1.217 (13) O11—H112 0.850
C7—O3 1.284 (12) O12—H121 0.850
C8—O6 1.221 (12) O12—H122 0.850
C8—O5 1.246 (12)
O1—Tm1—O8i 116.1 (3) O1—C6—C1 114.3 (8)
O1—Tm1—O5 124.3 (2) O4—C7—O3 123.6 (9)
O8i—Tm1—O5 83.6 (2) O4—C7—C4 119.1 (9)
O1—Tm1—O3ii 81.6 (2) O3—C7—C4 117.4 (9)
O8i—Tm1—O3ii 140.4 (2) O6—C8—O5 126.0 (9)
O5—Tm1—O3ii 116.7 (2) O6—C8—C9 118.6 (9)
O1—Tm1—O10 78.6 (2) O5—C8—C9 115.4 (8)
O8i—Tm1—O10 76.8 (2) N1—C9—C10 119.8 (9)
O5—Tm1—O10 155.2 (2) N1—C9—C8 115.0 (8)
O3ii—Tm1—O10 72.4 (2) C10—C9—C8 125.1 (8)
O1—Tm1—O9 154.7 (2) C11—C10—C9 121.0 (9)
O8i—Tm1—O9 78.1 (2) C11—C10—H10A 119.5
O5—Tm1—O9 76.1 (2) C9—C10—H10A 119.5
O3ii—Tm1—O9 75.0 (2) C10—C11—C12 120.0 (9)
O10—Tm1—O9 85.1 (2) C10—C11—H11A 120.0
O1—Tm1—N2 64.7 (2) C12—C11—H11A 120.0
O8i—Tm1—N2 73.3 (3) C13—C12—C11 114.9 (8)
O5—Tm1—N2 74.1 (3) C13—C12—C14 124.1 (8)
O3ii—Tm1—N2 142.5 (2) C11—C12—C14 120.9 (8)
O10—Tm1—N2 113.6 (3) N1—C13—C12 125.9 (9)
O9—Tm1—N2 140.5 (2) N1—C13—H13A 117.0
O1—Tm1—N1 73.5 (3) C12—C13—H13A 117.0
O8i—Tm1—N1 144.7 (3) O7—C14—O8 124.5 (9)
O5—Tm1—N1 65.1 (3) O7—C14—C12 120.7 (9)
O3ii—Tm1—N1 72.3 (3) O8—C14—C12 114.8 (8)
O10—Tm1—N1 137.5 (3) C13—N1—C9 118.3 (8)
O9—Tm1—N1 107.7 (2) C13—N1—Tm1 124.2 (6)
N2—Tm1—N1 82.3 (3) C9—N1—Tm1 116.5 (7)
N2—C1—C2 121.0 (8) C1—N2—C5 118.3 (8)
N2—C1—C6 114.9 (7) C1—N2—Tm1 117.5 (6)
C2—C1—C6 124.0 (8) C5—N2—Tm1 124.2 (6)
C3—C2—C1 119.9 (8) C6—O1—Tm1 126.0 (6)
C3—C2—H2A 120.0 C6—O1—H1 117.1
C1—C2—H2A 120.0 Tm1—O1—H1 116.9
C4—C3—C2 120.4 (8) C7—O3—Tm1i 141.4 (6)
C4—C3—H3A 119.8 C8—O5—Tm1 127.5 (6)
C2—C3—H3A 119.8 C14—O8—Tm1ii 136.9 (6)
C3—C4—C5 116.5 (8) Tm1—O9—H91 97.2
C3—C4—C7 124.0 (9) Tm1—O9—H92 113.8
C5—C4—C7 119.5 (8) H91—O9—H92 100.6
N2—C5—C4 123.6 (8) Tm1—O10—H101 95.7
N2—C5—H5A 118.2 Tm1—O10—H102 115.4
C4—C5—H5A 118.2 H101—O10—H102 100.8
O2—C6—O1 126.8 (8) H111—O11—H112 132.5
O2—C6—C1 119.0 (8) H121—O12—H122 96.9
N2—C1—C2—C3 −4.1 (14) O3ii—Tm1—N1—C9 −127.0 (7)
C6—C1—C2—C3 173.5 (8) O10—Tm1—N1—C9 −162.2 (6)
C1—C2—C3—C4 2.0 (14) O9—Tm1—N1—C9 −59.8 (7)
C2—C3—C4—C5 2.3 (13) N2—Tm1—N1—C9 80.9 (6)
C2—C3—C4—C7 −176.5 (9) C2—C1—N2—C5 1.6 (13)
C3—C4—C5—N2 −5.0 (14) C6—C1—N2—C5 −176.2 (8)
C7—C4—C5—N2 173.9 (9) C2—C1—N2—Tm1 179.9 (7)
N2—C1—C6—O2 −170.7 (9) C6—C1—N2—Tm1 2.1 (10)
C2—C1—C6—O2 11.5 (14) C4—C5—N2—C1 3.1 (14)
N2—C1—C6—O1 10.3 (11) C4—C5—N2—Tm1 −175.1 (7)
C2—C1—C6—O1 −167.5 (8) O1—Tm1—N2—C1 −7.8 (6)
C3—C4—C7—O4 0.1 (14) O8i—Tm1—N2—C1 −138.3 (7)
C5—C4—C7—O4 −178.7 (9) O5—Tm1—N2—C1 133.7 (7)
C3—C4—C7—O3 178.9 (9) O3ii—Tm1—N2—C1 20.3 (9)
C5—C4—C7—O3 0.2 (13) O10—Tm1—N2—C1 −71.3 (7)
O6—C8—C9—N1 −172.2 (8) O9—Tm1—N2—C1 176.1 (6)
O5—C8—C9—N1 7.3 (11) N1—Tm1—N2—C1 67.5 (7)
O6—C8—C9—C10 10.7 (13) O1—Tm1—N2—C5 170.3 (8)
O5—C8—C9—C10 −169.9 (9) O8i—Tm1—N2—C5 39.8 (8)
N1—C9—C10—C11 −2.3 (13) O5—Tm1—N2—C5 −48.2 (8)
C8—C9—C10—C11 174.7 (8) O3ii—Tm1—N2—C5 −161.6 (7)
C9—C10—C11—C12 2.3 (14) O10—Tm1—N2—C5 106.8 (8)
C10—C11—C12—C13 −0.2 (13) O9—Tm1—N2—C5 −5.7 (10)
C10—C11—C12—C14 −177.1 (9) N1—Tm1—N2—C5 −114.3 (8)
C11—C12—C13—N1 −2.1 (14) O2—C6—O1—Tm1 161.1 (8)
C14—C12—C13—N1 174.7 (9) C1—C6—O1—Tm1 −20.0 (11)
C13—C12—C14—O7 178.6 (9) O8i—Tm1—O1—C6 69.8 (7)
C11—C12—C14—O7 −4.8 (14) O5—Tm1—O1—C6 −30.8 (8)
C13—C12—C14—O8 −1.2 (13) O3ii—Tm1—O1—C6 −147.5 (7)
C11—C12—C14—O8 175.4 (8) O10—Tm1—O1—C6 138.9 (7)
C12—C13—N1—C9 2.2 (14) O9—Tm1—O1—C6 −170.2 (6)
C12—C13—N1—Tm1 −166.1 (7) N2—Tm1—O1—C6 15.6 (7)
C10—C9—N1—C13 0.1 (13) N1—Tm1—O1—C6 −73.6 (7)
C8—C9—N1—C13 −177.2 (8) O4—C7—O3—Tm1i 14.3 (17)
C10—C9—N1—Tm1 169.3 (6) C4—C7—O3—Tm1i −164.4 (7)
C8—C9—N1—Tm1 −8.0 (9) O6—C8—O5—Tm1 176.6 (7)
O1—Tm1—N1—C13 −44.8 (7) C9—C8—O5—Tm1 −2.8 (11)
O8i—Tm1—N1—C13 −156.8 (7) O1—Tm1—O5—C8 −46.8 (8)
O5—Tm1—N1—C13 173.4 (8) O8i—Tm1—O5—C8 −164.2 (8)
O3ii—Tm1—N1—C13 41.5 (7) O3ii—Tm1—O5—C8 51.6 (8)
O10—Tm1—N1—C13 6.3 (9) O10—Tm1—O5—C8 158.1 (7)
O9—Tm1—N1—C13 108.7 (7) O9—Tm1—O5—C8 116.5 (8)
N2—Tm1—N1—C13 −110.6 (8) N2—Tm1—O5—C8 −89.7 (7)
O1—Tm1—N1—C9 146.7 (7) N1—Tm1—O5—C8 −0.9 (7)
O8i—Tm1—N1—C9 34.7 (9) O7—C14—O8—Tm1ii 23.9 (16)
O5—Tm1—N1—C9 4.9 (6) C12—C14—O8—Tm1ii −156.4 (6)
Symmetry codes: (i) −x+1/2, −y+1/2, z−1/2; (ii) −x+1/2, −y+1/2, z+1/2.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O1—H1···O12iii 0.85 1.97 2.788 (11) 162
O9—H91···O4ii 0.85 1.83 2.679 (10) 180
O9—H92···O4iv 0.85 1.99 2.842 (10) 180
O10—H101···O7i 0.85 1.83 2.675 (11) 179
O10—H102···O9iii 0.85 2.14 2.996 (10) 179
O11—H111···O5 0.85 2.02 2.872 (11) 179
O11—H112···O2i 0.85 1.91 2.763 (11) 180
O12—H121···O6v 0.85 2.15 3.004 (12) 179
O12—H122···O6vi 0.85 2.08 2.933 (12) 179
Symmetry codes: (iii) y+1/2, −x+1/2, −z+1/2; (ii) −x+1/2, −y+1/2, z+1/2; (iv) −y+1, x, −z; (i) −x+1/2, −y+1/2, z−1/2; (v) −x, −y, z; (vi) y, −x, −z.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O1—H1⋯O12i 0.85 1.97 2.788 (11) 162
O9—H91⋯O4ii 0.85 1.83 2.679 (10) 180
O9—H92⋯O4iii 0.85 1.99 2.842 (10) 180
O10—H101⋯O7iv 0.85 1.83 2.675 (11) 179
O10—H102⋯O9i 0.85 2.14 2.996 (10) 179
O11—H111⋯O5 0.85 2.02 2.872 (11) 179
O11—H112⋯O2iv 0.85 1.91 2.763 (11) 180
O12—H121⋯O6v 0.85 2.15 3.004 (12) 179
O12—H122⋯O6vi 0.85 2.08 2.933 (12) 179
Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) ; (vi) .
==== Refs
References
Bruker (2001). SADABS and SAINT-Plus Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Flack, H. D. (1983). Acta Cryst. A39, 876–881.
Huang, Y. G., Wu, B. L., Yuan, D. Q., Xu, Y. Q., Jiang, F. L. & Hong, M. C. (2007). Inorg. Chem.46, 1171–1176.
Li, S., Zhang, F.-L., Wang, S.-B. & Bai, H.-L. (2009). Acta Cryst.E65, m410.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21582354 | PMC2968913 | CC BY | 2021-01-04 18:58:00 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Mar 19; 65(Pt 4):m411 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography bx223210.1107/S160053680903236XACSEBHS160053680903236XOrganic PapersTerephthalic acid–4,4′-bipyridine (2/1) 2C8H6O4·C10H8N2Wang Suwen aYang Tianyu bLi Zhongfang a*Yu Xianjin aa College of Chemical Engineering, Shandong University of Technology, Zibo 255049, People’s Republic of Chinab The College of Life Sciences, Northwest University, Xi-An 710069, People’s Republic of ChinaCorrespondence e-mail: [email protected] 9 2009 22 8 2009 22 8 2009 65 Pt 9 e090900o2198 o2198 04 8 2009 15 8 2009 © Wang et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, 2C8H6O4·C10H8N2, the 4,4′-bipyridine molecule is located on an inversion centre. In the crystal structure, strong intermolecular O—H⋯N hydrogen bonds between the terephthalic acid and 4,4′-bipyridine molecules lead to the formation of chains with graph-set motif C
2
2(8) along the diagonal of the bc plane.
==== Body
Related literature
For the potential applications of metal-organic frameworks, see: Zhang et al. (2007 ▶); Zhang et al. (2009 ▶) For hydrogen-bond motifs, see: Bernstein et al. (1995 ▶).
Experimental
Crystal data
2C8H6O4·C10H8N2
M
r = 488.44
Monoclinic,
a = 7.788 (10) Å
b = 6.814 (8) Å
c = 20.77 (3) Å
β = 92.25 (2)°
V = 1102 (2) Å3
Z = 2
Mo Kα radiation
μ = 0.11 mm−1
T = 296 K
0.27 × 0.19 × 0.18 mm
Data collection
Bruker APEXII CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2001 ▶) T
min = 0.971, T
max = 0.980
5164 measured reflections
1930 independent reflections
1192 reflections with I > 2σ(I)
R
int = 0.028
Refinement
R[F
2 > 2σ(F
2)] = 0.054
wR(F
2) = 0.178
S = 1.00
1930 reflections
169 parameters
2 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.20 e Å−3
Δρmin = −0.23 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT-Plus (Bruker, 2001 ▶); data reduction: SAINT-Plus; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S160053680903236X/bx2232sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S160053680903236X/bx2232Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BX2232).
The authors thank the NSFC (grant No. 20776081) and the Natural Science Foundation of Shandong Province (grant No. Y2006B37).
supplementary crystallographic
information
Comment
Design and construction of metal-organic frameworks (MOFs) have attracted
considerable attention in recent years, not only for their intriguing
structural motifs but also for their potential applications in the areas of
catalysis, separation, gas adsorption, molecular recognition, nonlinear
optics, and magnetochemistry (Zhang et al. (2007); Zhang et
al.
(2009)). The title compound was not the intended product of a reaction
to make
a MOFs. We report here the crystal and molecular structure of (I). The
asymmetric unit of the title compound contains one terephthalic acid molecule
and half 4,4'-bipyridine molecule, Fig. 1. The crystal structure is stabilized
by strong intermolecular O—H··· N hydrogen bonds between terephthalic acid
and 4,4'-bipyridine molecules, this interaction lead to the formation chains
C22(8) (Bernstein, et al., 1995) along the diagonal of the
bc-plane
, Fig 2, Table 1.
Experimental
A mixture of terephthalic acid (1 mmoL), 4,4'-bipyridine (1 mmoL, 0.156 g), and
iron trichloride (1 mmoL, 0.162 g) in 10 ml distilled water sealed in a 25 ml
Teflon-lined stainless steel autoclave was kept at 433 K for three days.
Colorless crystals suitable for the X-ray experiment were obtained. Anal.
Calc. for C26H20N2O8: C 63.88, H 4.09, N 5.73%; Found: C 63.70, H
3.98, N 5.62%.
Refinement
The H2 atom was refined isotropically. All other H atoms were placed in
calculated positions with C—H = 0.93 and O1—H1 = 0.80 Å and refined as
riding with Uiso(H) = 1.2Ueq(C) and Uiso(H) = 1.5(O).
Figures
Fig. 1. A view of the structure of (I), showing the atomic numbering scheme and 30% probability displacement ellipsoids.
Fig. 2. Packing diagram of (I). Dotted lines show hydrogen bonding.
Crystal data
2C8H6O4·C10H8N2 F(000) = 508
Mr = 488.44 Dx = 1.473 Mg m−3
Monoclinic, P21/n Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 1066 reflections
a = 7.788 (10) Å θ = 2.8–23.0°
b = 6.814 (8) Å µ = 0.11 mm−1
c = 20.77 (3) Å T = 296 K
β = 92.25 (2)° Block, colorless
V = 1102 (2) Å3 0.27 × 0.19 × 0.18 mm
Z = 2
Data collection
Bruker APEXII CCD area-detector diffractometer 1930 independent reflections
Radiation source: fine-focus sealed tube 1192 reflections with I > 2σ(I)
graphite Rint = 0.028
φ and ω scans θmax = 25.0°, θmin = 2.0°
Absorption correction: multi-scan (SADABS; Bruker, 2001) h = −9→9
Tmin = 0.971, Tmax = 0.980 k = −8→6
5164 measured reflections l = −24→16
Refinement
Refinement on F2 Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.054 Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.178 H atoms treated by a mixture of independent and constrained refinement
S = 1.00 w = 1/[σ2(Fo2) + (0.098P)2 + 0.1551P] where P = (Fo2 + 2Fc2)/3
1930 reflections (Δ/σ)max = 0.001
169 parameters Δρmax = 0.20 e Å−3
2 restraints Δρmin = −0.23 e Å−3
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
C1 1.1228 (3) 1.5441 (4) 0.21096 (13) 0.0521 (7)
C2 1.0571 (3) 1.3616 (4) 0.18496 (13) 0.0568 (7)
C3 1.0878 (4) 1.3136 (4) 0.12209 (14) 0.0652 (8)
H3 1.1497 1.3981 0.0966 0.078*
C4 1.0260 (3) 1.1400 (4) 0.09756 (13) 0.0615 (7)
H4 1.0458 1.1057 0.0552 0.074*
C5 0.9345 (3) 1.0161 (4) 0.13570 (12) 0.0531 (7)
C6 0.8676 (3) 0.8295 (4) 0.10696 (13) 0.0594 (7)
C7 0.9048 (3) 1.0691 (4) 0.19886 (13) 0.0616 (8)
H7 0.8420 0.9859 0.2245 0.074*
C8 0.9666 (3) 1.2420 (4) 0.22379 (13) 0.0615 (7)
H8 0.9475 1.2771 0.2662 0.074*
C9 0.6969 (4) 0.3813 (4) 0.02420 (16) 0.0782 (9)
H9 0.7712 0.4676 0.0046 0.094*
C10 0.6398 (4) 0.2208 (4) −0.00991 (15) 0.0758 (9)
H10 0.6744 0.2015 −0.0518 0.091*
C11 0.5323 (3) 0.0887 (3) 0.01718 (13) 0.0519 (7)
C12 0.4889 (3) 0.1285 (4) 0.07984 (14) 0.0655 (8)
H12 0.4178 0.0426 0.1012 0.079*
C13 0.5501 (4) 0.2936 (4) 0.11059 (14) 0.0688 (8)
H13 0.5178 0.3167 0.1525 0.083*
N1 0.6518 (3) 0.4205 (3) 0.08396 (11) 0.0632 (7)
O1 1.2080 (3) 1.6465 (4) 0.17755 (14) 0.0971 (8)
O2 1.0894 (3) 1.5957 (3) 0.26370 (13) 0.1001 (8)
O3 0.9023 (3) 0.7783 (3) 0.05288 (10) 0.0876 (7)
O4 0.7709 (3) 0.7296 (3) 0.14372 (10) 0.0740 (6)
H1 1.2411 1.7424 0.1966 0.146*
H2 0.732 (3) 0.634 (3) 0.1242 (13) 0.080*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
C1 0.0583 (16) 0.0482 (15) 0.0498 (16) −0.0067 (12) 0.0016 (13) −0.0059 (13)
C2 0.0580 (16) 0.0590 (16) 0.0531 (16) 0.0056 (12) −0.0013 (13) −0.0056 (13)
C3 0.0746 (18) 0.0663 (17) 0.0554 (17) −0.0053 (14) 0.0115 (14) −0.0004 (14)
C4 0.0753 (18) 0.0649 (17) 0.0452 (16) −0.0029 (14) 0.0132 (13) −0.0048 (13)
C5 0.0594 (15) 0.0560 (15) 0.0439 (15) 0.0040 (12) 0.0033 (12) −0.0003 (12)
C6 0.0703 (17) 0.0568 (15) 0.0515 (17) −0.0001 (13) 0.0079 (14) 0.0009 (13)
C7 0.0730 (18) 0.0671 (17) 0.0454 (16) −0.0052 (13) 0.0106 (13) 0.0006 (13)
C8 0.0702 (17) 0.0695 (18) 0.0451 (15) 0.0023 (14) 0.0052 (13) −0.0081 (13)
C9 0.103 (2) 0.0695 (19) 0.064 (2) −0.0242 (17) 0.0162 (18) 0.0023 (16)
C10 0.106 (2) 0.0689 (18) 0.0530 (18) −0.0249 (17) 0.0161 (17) −0.0045 (15)
C11 0.0536 (15) 0.0519 (14) 0.0501 (15) 0.0024 (11) −0.0001 (12) 0.0001 (12)
C12 0.0717 (18) 0.0655 (17) 0.0603 (18) −0.0128 (14) 0.0139 (15) −0.0041 (14)
C13 0.0780 (19) 0.0737 (19) 0.0556 (18) −0.0069 (15) 0.0138 (15) −0.0087 (15)
N1 0.0732 (15) 0.0586 (14) 0.0579 (15) −0.0049 (11) 0.0043 (12) 0.0007 (11)
O1 0.1010 (18) 0.0836 (17) 0.105 (2) −0.0092 (14) −0.0137 (17) −0.0178 (15)
O2 0.1216 (19) 0.0888 (16) 0.0897 (19) −0.0073 (13) 0.0038 (15) −0.0325 (14)
O3 0.1339 (18) 0.0751 (14) 0.0559 (13) −0.0234 (12) 0.0306 (12) −0.0190 (11)
O4 0.0982 (15) 0.0655 (13) 0.0594 (13) −0.0208 (11) 0.0183 (11) −0.0096 (10)
Geometric parameters (Å, °)
C1—O2 1.189 (3) C8—H8 0.9300
C1—O1 1.202 (4) C9—N1 1.330 (4)
C1—C2 1.441 (4) C9—C10 1.368 (4)
C2—C8 1.362 (4) C9—H9 0.9300
C2—C3 1.376 (4) C10—C11 1.366 (4)
C3—C4 1.368 (4) C10—H10 0.9300
C3—H3 0.9300 C11—C12 1.384 (4)
C4—C5 1.375 (3) C11—C11i 1.482 (5)
C4—H4 0.9300 C12—C13 1.370 (4)
C5—C7 1.389 (4) C12—H12 0.9300
C5—C6 1.491 (4) C13—N1 1.309 (3)
C6—O3 1.217 (3) C13—H13 0.9300
C6—O4 1.288 (3) O1—H1 0.8000
C7—C8 1.367 (4) O4—H2 0.82 (2)
C7—H7 0.9300
O2—C1—O1 120.4 (3) C2—C8—C7 118.3 (3)
O2—C1—C2 120.9 (3) C2—C8—H8 120.9
O1—C1—C2 118.7 (3) C7—C8—H8 120.8
O2—C1—H1 94.0 N1—C9—C10 123.4 (3)
C2—C1—H1 145.0 N1—C9—H9 118.3
C8—C2—C3 122.2 (3) C10—C9—H9 118.3
C8—C2—C1 118.6 (3) C11—C10—C9 120.4 (3)
C3—C2—C1 119.2 (3) C11—C10—H10 119.8
C4—C3—C2 119.2 (3) C9—C10—H10 119.8
C4—C3—H3 120.4 C10—C11—C12 115.8 (2)
C2—C3—H3 120.4 C10—C11—C11i 122.8 (3)
C3—C4—C5 119.9 (3) C12—C11—C11i 121.4 (3)
C3—C4—H4 120.0 C13—C12—C11 120.4 (3)
C5—C4—H4 120.0 C13—C12—H12 119.8
C4—C5—C7 119.6 (3) C11—C12—H12 119.8
C4—C5—C6 118.3 (2) N1—C13—C12 123.4 (3)
C7—C5—C6 122.1 (2) N1—C13—H13 118.3
O3—C6—O4 123.5 (3) C12—C13—H13 118.3
O3—C6—C5 121.8 (2) C13—N1—C9 116.6 (2)
O4—C6—C5 114.6 (2) C13—N1—H2 123.3 (10)
C8—C7—C5 120.8 (2) C9—N1—H2 120.1 (10)
C8—C7—H7 119.6 C1—O1—H1 111.00
C5—C7—H7 119.6 C6—O4—H2 110 (2)
C13—N1—C9—C10 1.7 (5) C1—C2—C3—C4 179.7 (2)
N1—C9—C10—C11 −0.9 (5) C2—C3—C4—C5 −0.2 (4)
C9—C10—C11—C12 −0.6 (4) C3—C4—C5—C7 0.4 (4)
C9—C10—C11—C11i −179.2 (3) C3—C4—C5—C6 179.4 (2)
C10—C11—C12—C13 1.3 (4) C4—C5—C6—O3 5.4 (4)
C11i—C11—C12—C13 179.9 (3) C7—C5—C6—O3 −175.6 (3)
C9—N1—C13—C12 −1.0 (4) C4—C5—C6—O4 −174.3 (2)
C11—C12—C13—N1 −0.5 (4) C7—C5—C6—O4 4.7 (4)
O2—C1—C2—C8 −5.1 (4) C4—C5—C7—C8 −0.5 (4)
O1—C1—C2—C8 177.3 (3) C6—C5—C7—C8 −179.5 (2)
O2—C1—C2—C3 175.2 (3) C3—C2—C8—C7 −0.2 (4)
O1—C1—C2—C3 −2.6 (4) C1—C2—C8—C7 −179.8 (2)
C8—C2—C3—C4 0.1 (4) C5—C7—C8—C2 0.4 (4)
Symmetry codes: (i) −x+1, −y, −z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O4—H2···N1 0.82 (2) 1.78 (2) 2.598 (4) 178 (1)
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O4—H2⋯N1 0.82 (2) 1.78 (2) 2.598 (4) 177.8 (14)
==== Refs
References
Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem. Int. Ed. Engl 34, 1555–1573.
Bruker (2001). SAINT-Plus and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Bruker (2004). APEX2 Bruker AXS Inc., Madison, Wisconsin, USA.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Zhang, X. T., Dou, J. M., Wang, D. Q., Zhou, Y., Zhang, Y. X., Li, R. J., Yan, S. S., Ni, Z. H. & Jiang, J. Z. (2007). Cryst. Growth Des.7, 1699–1705.
Zhang, X. T., Dou, J. M., Wei, P. H., Li, D. C., Li, B., Shi, C. W. & Hu, B. (2009). Inorg. Chim. Acta, 362, 3325–3332. | 21577601 | PMC2969865 | CC BY | 2021-01-04 18:59:23 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Aug 22; 65(Pt 9):o2198 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb506010.1107/S1600536809034874ACSEBHS1600536809034874Metal-Organic PapersTris(ethylenediamine)manganese(II) sulfate [Mn(C2H8N2)3]SO4Lu Jing a*a School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 10 2009 09 9 2009 09 9 2009 65 Pt 10 e091000m1187 m1187 21 8 2009 31 8 2009 © Jing Lu 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, [Mn(C2H8N2)3]SO4, the metal atom (site symmetry 3.2) is coordinated by six N atoms from three ethylenediamine (en) ligands in a slightly distorted octahedral geometry. The en ligands are generated from one half-molecule in the asymmetric unit. The O atoms of the sulfate anion (S site symmetry 3.2) are disordered over four orientations in a 0.220 (12):0.210 (13):0.203 (14):0.10 (2) ratio, with one of the O atoms having site symmetry 3. In the crystal, the ions are connected by N—H⋯O hydrogen bonds, forming a three-dimensional network.
==== Body
Related literature
For a structure containing MnII and aromatic amine ligands, see: Shang et al. (2009 ▶). For other compounds containing transition metals coordinated by ethylenediamine, see: Cullen & Lingafelter (1970 ▶); Daniels et al. (1995 ▶); Jameson et al. (1982 ▶).
Experimental
Crystal data
[Mn(C2H8N2)3]SO4
M
r = 331.31
Trigonal,
a = 8.9460 (13) Å
c = 9.6230 (19) Å
V = 666.96 (19) Å3
Z = 2
Mo Kα radiation
μ = 1.17 mm−1
T = 293 K
0.35 × 0.30 × 0.28 mm
Data collection
Bruker SMART CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2003 ▶) T
min = 0.686, T
max = 0.736
2640 measured reflections
402 independent reflections
386 reflections with I > 2σ(I)
R
int = 0.031
Refinement
R[F
2 > 2σ(F
2)] = 0.058
wR(F
2) = 0.159
S = 1.55
402 reflections
44 parameters
2 restraints
H-atom parameters constrained
Δρmax = 0.72 e Å−3
Δρmin = −0.63 e Å−3
Data collection: SMART (Bruker, 2003 ▶); cell refinement: SAINT (Bruker, 2003 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809034874/hb5060sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809034874/hb5060Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB5060).
This work was supported by the National Natural Science Foundation of China (No. 20671048) and the Doctoral Foundation of Liaocheng University (No. 31805).
supplementary crystallographic
information
Comment
Ethylenediamine (en) ligand has been seen
in a number of
coordination compound (Cullen et al., 1970; Daniels et
al., 1995
and Jameson et al., 1982), because it can not only chelate metal
center
by two nitrogen atoms, but also offer hydrogen atoms to form
N—H···X hydrogen bonds.
In this paper, we report the structure of the title compound, (I).
In the title compound (Fig. 1), [Mn(C2H8N2)3]SO4, the cation and
anion are situated on a sixfold rotation axis. The Mn(II) is coordinated by
six N atoms from three en ligands in a distorted octahedral geometry. The
Mn—N bond length is 2.129 Å, which is shorter than the distance between
Mn(II) and aromatic nitrogen atom (Shang et al., 2009). The O
atoms of
the sulfate anions are disordered. The disordered anions hydrogen bond with
the coordination cations by N—H···O hydrogen bonds, forming
three-dimensional supramolecular network. The hydrogen bond is listed in table
1.
Experimental
Manganese sulfate (0.2 mmol) and malic acid (0.4 mmol) were added to
water (15 ml). The pH value was adjusted to 9 by en. Violet blocks of (I)
were obtained after several days in 30% yield. Elemental
analysis, Found:C, 21.73; H, 7.24; N, 25.35%.
Calc. for C6H24N6MnSO4: C, 21.20; H, 7.00; N, 24.93%.
Refinement
All H atoms were positioned geometrically and treated as riding on their parent
atoms, with C—H 0.970 and N—H 0.900 Å, and with Uiso(H) =
1.2Ueq(C,N). The O atoms are resolved into four positions by
PART instructions. The geometries and anisotropic displacement parameters of
disordered atoms were refined with soft restraints using the SHELXL
commands SUMP, SIMU and EADP.
Figures
Fig. 1. The molecular structure of (I) with 50% probability displacement ellipsoids. The symmetry codes for N and C atoms: A -y+1, x-y+1, z; B -x+y, -x+1, z; C -y+1, -x+1, -z+3/2; D -x+y, y, -z+3/2; E x, x-y+1, -z+3/2.
Crystal data
[Mn(C2H8N2)3]SO4 Dx = 1.650 Mg m−3
Mr = 331.31 Mo Kα radiation, λ = 0.71073 Å
Trigonal, P31c Cell parameters from 2640 reflections
a = 8.9460 (13) Å θ = 2.6–25.0°
c = 9.6230 (19) Å µ = 1.17 mm−1
V = 666.96 (19) Å3 T = 293 K
Z = 2 Block, violet
F(000) = 350 0.35 × 0.30 × 0.28 mm
Data collection
Bruker SMART CCD diffractometer 402 independent reflections
Radiation source: fine-focus sealed tube 386 reflections with I > 2σ(I)
graphite Rint = 0.031
ω scans θmax = 25.0°, θmin = 2.6°
Absorption correction: multi-scan (SADABS; Bruker, 2003) h = −8→10
Tmin = 0.686, Tmax = 0.736 k = −10→4
2640 measured reflections l = −11→11
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.058 H-atom parameters constrained
wR(F2) = 0.159 w = 1/[σ2(Fo2) + (0.0637P)2 + 1.2291P] where P = (Fo2 + 2Fc2)/3
S = 1.55 (Δ/σ)max = 0.001
402 reflections Δρmax = 0.72 e Å−3
44 parameters Δρmin = −0.63 e Å−3
2 restraints Extinction correction: SHELXS97 (Sheldrick, 2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.051 (15)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq Occ. (<1)
Mn1 0.3333 0.6667 0.7500 0.0200 (8)
S1 0.3333 0.6667 0.2500 0.0332 (10)
O1 0.489 (3) 0.813 (4) 0.186 (2) 0.051 (4) 0.220 (12)
O2 0.489 (3) 0.721 (6) 0.163 (3) 0.051 (4) 0.210 (13)
O3 0.373 (6) 0.799 (3) 0.146 (3) 0.051 (4) 0.203 (14)
O4 0.3333 0.6667 0.114 (9) 0.051 (4) 0.10 (2)
N1 0.3136 (7) 0.4590 (8) 0.8712 (6) 0.0427 (14)
H1A 0.4072 0.4479 0.8581 0.051*
H1B 0.3070 0.4791 0.9621 0.051*
C1 0.1570 (9) 0.3007 (9) 0.8275 (8) 0.0491 (18)
H1C 0.0561 0.2960 0.8692 0.059*
H1D 0.1626 0.2002 0.8578 0.059*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Mn1 0.0208 (9) 0.0208 (9) 0.0184 (12) 0.0104 (5) 0.000 0.000
S1 0.0327 (13) 0.0327 (13) 0.034 (2) 0.0163 (7) 0.000 0.000
O1 0.045 (8) 0.042 (12) 0.047 (7) 0.008 (10) 0.007 (6) 0.000 (8)
O2 0.045 (8) 0.042 (12) 0.047 (7) 0.008 (10) 0.007 (6) 0.000 (8)
O3 0.045 (8) 0.042 (12) 0.047 (7) 0.008 (10) 0.007 (6) 0.000 (8)
O4 0.045 (8) 0.042 (12) 0.047 (7) 0.008 (10) 0.007 (6) 0.000 (8)
N1 0.041 (3) 0.051 (3) 0.040 (3) 0.026 (3) 0.008 (2) 0.000 (2)
C1 0.051 (4) 0.053 (4) 0.054 (4) 0.035 (3) 0.002 (3) −0.006 (3)
Geometric parameters (Å, °)
Mn1—N1i 2.125 (6) S1—O2iii 1.48 (3)
Mn1—N1ii 2.125 (6) S1—O2vii 1.48 (3)
Mn1—N1iii 2.125 (6) S1—O2viii 1.48 (3)
Mn1—N1iv 2.125 (6) S1—O2ii 1.48 (3)
Mn1—N1v 2.125 (6) O1—O1viii 1.74 (5)
Mn1—N1 2.125 (6) O2—O2viii 1.72 (6)
S1—O4 1.30 (9) O3—O3iii 1.82 (4)
S1—O4vi 1.30 (9) O3—O3ii 1.82 (4)
S1—O3vii 1.45 (3) N1—C1 1.471 (9)
S1—O3iii 1.45 (3) N1—H1A 0.9000
S1—O3 1.45 (3) N1—H1B 0.9000
S1—O3vi 1.45 (3) C1—C1iv 1.496 (15)
S1—O3ii 1.45 (3) C1—H1C 0.9700
S1—O3viii 1.45 (3) C1—H1D 0.9700
N1i—Mn1—N1ii 81.6 (3) O3ii—S1—O2iii 99.3 (13)
N1i—Mn1—N1iii 93.5 (3) O3viii—S1—O2iii 170.3 (17)
N1ii—Mn1—N1iii 92.7 (2) O4—S1—O2vii 124.3 (10)
N1i—Mn1—N1iv 92.7 (2) O4vi—S1—O2vii 55.7 (10)
N1ii—Mn1—N1iv 93.5 (3) O3vii—S1—O2vii 62.4 (15)
N1iii—Mn1—N1iv 171.8 (3) O3iii—S1—O2vii 97 (2)
N1i—Mn1—N1v 92.7 (2) O3—S1—O2vii 94 (3)
N1ii—Mn1—N1v 171.8 (3) O3vi—S1—O2vii 30.1 (12)
N1iii—Mn1—N1v 81.6 (3) O3ii—S1—O2vii 170.3 (17)
N1iv—Mn1—N1v 92.7 (2) O3viii—S1—O2vii 99.3 (13)
N1i—Mn1—N1 171.8 (3) O2iii—S1—O2vii 71 (2)
N1ii—Mn1—N1 92.7 (2) O4—S1—O2viii 124.3 (11)
N1iii—Mn1—N1 92.7 (2) O4vi—S1—O2viii 55.7 (10)
N1iv—Mn1—N1 81.6 (3) O3vii—S1—O2viii 30.1 (12)
N1v—Mn1—N1 93.5 (3) O3iii—S1—O2viii 170.3 (18)
O4—S1—O4vi 180.000 (19) O3—S1—O2viii 97 (2)
O4—S1—O3vii 133.7 (10) O3vi—S1—O2viii 99.3 (13)
O4vi—S1—O3vii 46.3 (10) O3ii—S1—O2viii 94 (3)
O4—S1—O3iii 46.3 (10) O3viii—S1—O2viii 62.4 (15)
O4vi—S1—O3iii 133.7 (10) O2iii—S1—O2viii 116 (3)
O3vii—S1—O3iii 156 (4) O2vii—S1—O2viii 91.3 (15)
O4—S1—O3 46.3 (10) O4—S1—O2ii 55.7 (10)
O4vi—S1—O3 133.7 (10) O4vi—S1—O2ii 124.3 (10)
O3vii—S1—O3 90 (2) O3vii—S1—O2ii 170.3 (17)
O3iii—S1—O3 77.5 (15) O3iii—S1—O2ii 30.1 (12)
O4—S1—O3vi 133.7 (10) O3—S1—O2ii 99.3 (13)
O4vi—S1—O3vi 46.3 (10) O3vi—S1—O2ii 97 (2)
O3vii—S1—O3vi 77.5 (15) O3ii—S1—O2ii 62.4 (15)
O3iii—S1—O3vi 90 (2) O3viii—S1—O2ii 94 (3)
O3—S1—O3vi 121 (3) O2iii—S1—O2ii 91.3 (15)
O4—S1—O3ii 46.3 (10) O2vii—S1—O2ii 116 (3)
O4vi—S1—O3ii 133.7 (10) O2viii—S1—O2ii 147 (4)
O3vii—S1—O3ii 121 (3) S1—O1—O1viii 54.1 (11)
O3iii—S1—O3ii 77.5 (15) S1—O2—O2viii 54.5 (12)
O3—S1—O3ii 77.5 (15) S1—O3—O3iii 51.2 (7)
O3vi—S1—O3ii 156 (4) S1—O3—O3ii 51.2 (7)
O4—S1—O3viii 133.7 (10) O3iii—O3—O3ii 60.000 (1)
O4vi—S1—O3viii 46.3 (10) C1—N1—Mn1 107.9 (4)
O3vii—S1—O3viii 77.5 (15) C1—N1—H1A 110.1
O3iii—S1—O3viii 121 (3) Mn1—N1—H1A 110.1
O3—S1—O3viii 156 (4) C1—N1—H1B 110.1
O3vi—S1—O3viii 77.5 (15) Mn1—N1—H1B 110.1
O3ii—S1—O3viii 90 (2) H1A—N1—H1B 108.4
O4—S1—O2iii 55.7 (10) N1—C1—C1iv 108.9 (5)
O4vi—S1—O2iii 124.3 (10) N1—C1—H1C 109.9
O3vii—S1—O2iii 97 (2) C1iv—C1—H1C 109.9
O3iii—S1—O2iii 62.4 (15) N1—C1—H1D 109.9
O3—S1—O2iii 30.1 (12) C1iv—C1—H1D 109.9
O3vi—S1—O2iii 94 (3) H1C—C1—H1D 108.3
O4—S1—O1—O1viii −129 (2) O3vii—S1—O3—O3iii −159 (3)
O4vi—S1—O1—O1viii 51 (2) O3vi—S1—O3—O3iii −83 (2)
O3vii—S1—O1—O1viii 65.8 (19) O3ii—S1—O3—O3iii 79.8 (10)
O3iii—S1—O1—O1viii −140 (3) O3viii—S1—O3—O3iii 141.8 (18)
O3—S1—O1—O1viii 179 (3) O2iii—S1—O3—O3iii −55 (3)
O3vi—S1—O1—O1viii 100 (3) O2vii—S1—O3—O3iii −96 (3)
O3ii—S1—O1—O1viii −77 (2) O2viii—S1—O3—O3iii 172 (3)
O3viii—S1—O1—O1viii 0(2) O2ii—S1—O3—O3iii 20.9 (16)
O2iii—S1—O1—O1viii 171 (2) O4—S1—O3—O3ii −39.9 (5)
O2vii—S1—O1—O1viii 112.2 (18) O4vi—S1—O3—O3ii 140.1 (5)
O2viii—S1—O1—O1viii 34.5 (19) O3vii—S1—O3—O3ii 122 (4)
O2ii—S1—O1—O1viii −108 (3) O3iii—S1—O3—O3ii −79.8 (10)
O4—S1—O2—O2viii 163 (4) O3vi—S1—O3—O3ii −163 (2)
O4vi—S1—O2—O2viii −17 (4) O3viii—S1—O3—O3ii 62 (2)
O3vii—S1—O2—O2viii 20 (2) O2iii—S1—O3—O3ii −135 (3)
O3iii—S1—O2—O2viii 179 (4) O2vii—S1—O3—O3ii −176 (3)
O3—S1—O2—O2viii 109 (4) O2viii—S1—O3—O3ii 92 (3)
O3vi—S1—O2—O2viii −3(22) O2ii—S1—O3—O3ii −58.9 (16)
O3ii—S1—O2—O2viii −136 (5) N1i—Mn1—N1—C1 32.2 (4)
O3viii—S1—O2—O2viii −58 (2) N1ii—Mn1—N1—C1 78.5 (5)
O2iii—S1—O2—O2viii 117 (4) N1iii—Mn1—N1—C1 171.4 (4)
O2vii—S1—O2—O2viii 61 (2) N1iv—Mn1—N1—C1 −14.7 (3)
O2ii—S1—O2—O2viii −151 (4) N1v—Mn1—N1—C1 −106.9 (4)
O4—S1—O3—O3iii 39.9 (5) Mn1—N1—C1—C1iv 41.5 (7)
O4vi—S1—O3—O3iii −140.1 (5)
Symmetry codes: (i) x, x−y+1, −z+3/2; (ii) −x+y, −x+1, z; (iii) −y+1, x−y+1, z; (iv) −x+y, y, −z+3/2; (v) −y+1, −x+1, −z+3/2; (vi) −y+1, −x+1, −z+1/2; (vii) −x+y, y, −z+1/2; (viii) x, x−y+1, −z+1/2.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
N1—H1A···O1ix 0.90 2.28 3.16 (3) 166
N1—H1A···O1x 0.90 2.43 3.15 (3) 138
N1—H1B···O1xi 0.90 2.17 3.06 (2) 170
N1—H1B···O2xi 0.90 2.11 3.00 (2) 167
N1—H1A···O2xii 0.90 2.14 2.95 (5) 148
N1—H1A···O3ix 0.90 1.92 2.80 (3) 166
N1—H1B···O3xi 0.90 2.15 2.96 (3) 150
N1—H1B···O3xiii 0.90 2.36 3.18 (3) 152
N1—H1B···O4xiv 0.90 2.15 2.94 (7) 146
Symmetry codes: (ix) x−y+1, x, −z+1; (x) −x+1, −x+y, z+1/2; (xi) −x+y, −x+1, z+1; (xii) −x+1, −y+1, −z+1; (xiii) −y+1, x−y+1, z+1; (xiv) x, y, z+1.
Table 1 Selected geometric parameters (Å, °)
Mn1—N1 2.125 (6)
N1i—Mn1—N1 81.6 (3)
Symmetry code: (i) .
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
N1—H1A⋯O1ii 0.90 2.28 3.16 (3) 166
N1—H1A⋯O1iii 0.90 2.43 3.15 (3) 138
N1—H1B⋯O1iv 0.90 2.17 3.06 (2) 170
N1—H1B⋯O2iv 0.90 2.11 3.00 (2) 167
N1—H1A⋯O2v 0.90 2.14 2.95 (5) 148
N1—H1A⋯O3ii 0.90 1.92 2.80 (3) 166
N1—H1B⋯O3iv 0.90 2.15 2.96 (3) 150
N1—H1B⋯O3vi 0.90 2.36 3.18 (3) 152
N1—H1B⋯O4vii 0.90 2.15 2.94 (7) 146
Symmetry codes: (ii) ; (iii) ; (iv) ; (v) ; (vi) ; (vii) .
==== Refs
References
Bruker (2003). SMART, SAINT and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Cullen, D. L. & Lingafelter, E. C. (1970). Inorg. Chem.9, 1858–1864.
Daniels, L. M., Murillo, C. A. & Rodriguez, K. G. (1995). Inorg. Chim. Acta, 229, 27–32.
Jameson, G. B., Schneider, R., Dubler, E. & Oswald, H. R. (1982). Acta Cryst. B38, 3016–3020.
Shang, S.-M., Ren, C.-X., Wang, X., Lu, L.-D. & Yang, X.-J. (2009). Acta Cryst. E65, m1023–m1024.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21577720 | PMC2970169 | CC BY | 2021-01-04 18:58:55 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Sep 9; 65(Pt 10):m1187 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography at286210.1107/S1600536809033558ACSEBHS1600536809033558Metal-Organic Papers{6,6′-Dimethoxy-2,2′-[ethane-1,2-diylbis(nitrilomethylidyne)]diphenolato-1κ4
O
1,O
1′,O
6,O
6′:2κ4
O
1,N,N′,O
1′}(ethanol-1κO)-μ-nitrato-1:2κ2
O:O′-dinitrato-1κ4
O,O′-samarium(III)zinc(II) [SmZn(C18H18N2O4)(NO3)3(C2H6O)]Huang Qiang a*Sui Yu-Hua bZhang Guo-Xiang ca College of Mechanical and Materials Engineering, Jiujiang University, 332005 Jiujiang, JiangXi, People’s Republic of Chinab Vacational Education Center of JiLin Oil Feild, 131200 SongYuan, JiLin, People’s Republic of Chinac The Pipeline Tools Subsidiary, Well Drilling Technology and Service Corporation of Jilin Oil Field, 131200 SongYuan, JiLin, People’s Republic of ChinaCorrespondence e-mail: [email protected] 10 2009 05 9 2009 05 9 2009 65 Pt 10 e091000m1161 m1162 13 8 2009 23 8 2009 © Huang et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title heteronuclear ZnII–SmIII complex, [SmZn(C18H18N2O4)(NO3)3(CH3CH2OH)], with the hexadentate Schiff base compartmental ligand N,N′-bis(3-methoxysalicylidene)ethylenediamine (H2
L), the SmIII and ZnII ions are triply bridged by two phenolate O atoms from the Schiff base ligand and one nitrate anion. The five-coordinate ZnII ion is in a square-pyramidal geometry formed by the donor centers of two imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms. The SmIII center is in a ten-fold coordination of O atoms, involving the phenolate O atoms, two methoxy O atoms, one ethanol O atom, and two O atoms from two nitrate anions and one from the bridging nitrate anion. In the crystal, intermolecular O—H⋯O and C—H⋯O interactions generate a layer structure extending parallel to (101).
==== Body
Related literature
For the preparation, magnetic and optical properties of 3d-4f hetorometallic dinuclear complexes, see: Baggio et al. (2000 ▶); Caravan et al. (1999 ▶); Edder et al. (2000 ▶); Knoer et al. (2005 ▶).
Experimental
Crystal data
[SmZn(C18H18N2O4)(NO3)3(C2H6O)]
M
r = 774.16
Monoclinic,
a = 9.975 (3) Å
b = 13.780 (4) Å
c = 19.889 (6) Å
β = 91.745 (4)°
V = 2732.4 (13) Å3
Z = 4
Mo Kα radiation
μ = 3.08 mm−1
T = 293 K
0.26 × 0.23 × 0.19 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2004 ▶) T
min = 0.501, T
max = 0.592
16112 measured reflections
4741 independent reflections
4175 reflections with I > 2σ(I)
R
int = 0.021
Refinement
R[F
2 > 2σ(F
2)] = 0.027
wR(F
2) = 0.079
S = 1.08
4741 reflections
377 parameters
3 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.67 e Å−3
Δρmin = −0.70 e Å−3
Data collection: APEX2 (Bruker, 2004 ▶); cell refinement: SAINT (Bruker, 2004 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXL97; software used to prepare material for publication: SHELXL97 and publCIF (Westrip, 2009 ▶).
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809033558/at2862sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809033558/at2862Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2862).
We gratefully acknowledge financial support from the Educational Commission of Jiangxi Province of China (GJJ08448) and the Natural Science Foundation of Jiangxi Province of China (2008GQC0002).
supplementary crystallographic
information
Comment
The potential applications of trivalent lanthanide complexes as contrast agent
for magnetic resonance imaging and stains for fluorescence imaging have
prompted considerable interest in the preparation, magnetic and optical
properties of 3 d-4f hetorometallic dinuclear complexes (Baggio et al.,
2000; Caravan et al., 1999; Edder et al.,
2000; Knoer et
al., 2005). we report here the synthesis and X-ray crystal
structure
analysis of the title complex, (I), a new ZnII—SmIII complex with
salen-type Schiff base N,N'-bis(3-methoxysalicylidene)
ethylenediamine(H2L).
Complex (I) crystallizes in the space group P21/n, with zinc
and samarium triply bridged by two phenolate O atoms provided by a salen-type
Schiff base ligand and one nitrate. The inner salen-type cavity is occupied by
zinc(II), while samarium(III) is present in the open and larger portion of the
dinucleating compartmental Schiff base ligand.
The SmIII center has a decacoordination environment of O atoms, involving the
phenolate O atoms, two methoxy O atoms, one ethanol O atom, two O atoms from
two nitrates and one from the bridging nitrate. The five kinds of Sm—O bond
distances are significantly different, the longest being the
Sm—O(methoxy) separations and the shortest being the Sm—O5(bridging
nitrate).
The ZnII is in a square-pyramidal geometry and is five-coordinated by two
imine N atoms, two phenolate O atoms and one of the bridging nitrate O atoms.
Adjacent molecules are held together by typical O—H···O hydrogen bonds and
weak C—H···O interactions. these link the molecules into a two-dimensional
layer structure(Fig 2).
Experimental
H2L was prepared by the 2:1 condensation of 3-methoxysalicylaldehyde
and
ethylenediamine in methanol. Complex (I) was obtained by the treatment of
zinc(II) acetate dihydrate (0.188 g, 1 mmol) with H2L(0.328 g, 1 mmol)
in
ethanol solution (80 ml) under reflux for 3 h and then for another 3 h after
the addition of samarium(III) nitrate hexahydrate(0.444 g, 1 mmol). The
reaction mixture was cooled and the resulting precipitate was filtered off,
washed with diethyl ether and dried in vacuo. Single crystals of (I)
suitable for X-ray analysis were obtained by slow evaporation at room
temperature of a ethanol solution. Analysis calculated for
C20H24N5O14SmZn: C 31.03, H 3.12, N 9.05, Sm 19.42, Zn 8.45%; found: C
31.10, H 2.98, N 8.99, Sm 20.01, Zn 8.40%. IR(KBr, cm-1): 1640(C=N),
1386,1490(nitrate).
Refinement
The H atoms were positioned geometrically and treated as riding on their parent
atoms, with C—H distances of 0.97 (methylene), 0.96 Å (methyl) and 0.93 Å (aromatic), and with Uiso(H) = 1.5Ueq(C) for methyl H
atoms and 1.2Ueq(C) for other H atoms. The hydroxyl H atom, H14s, was
located in a difference Fourier map and the O14—H14s was restrained to 0.88 Å.
Figures
Fig. 1. The molecular structure of (I), showing 30% probability displacement ellipsoids.
Fig. 2. The packing diagram of (I), viewed along the c axis; hydrogen bonds are shown as dashed lines.
Crystal data
[SmZn(C18H18N2O4)(NO3)3(C2H6O)] F(000) = 1532
Mr = 774.16 Dx = 1.882 Mg m−3
Monoclinic, P21/n Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2yn Cell parameters from 5725 reflections
a = 9.975 (3) Å θ = 2.5–28.2°
b = 13.780 (4) Å µ = 3.08 mm−1
c = 19.889 (6) Å T = 293 K
β = 91.745 (4)° Block, yellow
V = 2732.4 (13) Å3 0.26 × 0.23 × 0.19 mm
Z = 4
Data collection
Bruker APEXII area-detector diffractometer 4741 independent reflections
Radiation source: fine-focus sealed tube 4175 reflections with I > 2σ(I)
graphite Rint = 0.021
φ and ω scans θmax = 25.0°, θmin = 1.8°
Absorption correction: multi-scan (SADABS; Bruker, 2004) h = −11→11
Tmin = 0.501, Tmax = 0.592 k = −15→16
16112 measured reflections l = −23→23
Refinement
Refinement on F2 Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.027 Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.079 H atoms treated by a mixture of independent and constrained refinement
S = 1.08 w = 1/[σ2(Fo2) + (0.045P)2 + 4.1668P] where P = (Fo2 + 2Fc2)/3
4741 reflections (Δ/σ)max < 0.001
377 parameters Δρmax = 0.67 e Å−3
3 restraints Δρmin = −0.70 e Å−3
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
C1 0.5387 (4) 0.1513 (4) 0.1046 (2) 0.0435 (11)
H1A 0.6226 0.1846 0.1108 0.065*
H1B 0.5050 0.1609 0.0594 0.065*
H1C 0.5517 0.0832 0.1126 0.065*
C2 0.4878 (4) 0.1904 (3) 0.21745 (19) 0.0287 (8)
C3 0.4091 (4) 0.2491 (3) 0.25846 (18) 0.0248 (8)
C4 0.4475 (4) 0.2570 (3) 0.32728 (19) 0.0309 (9)
C5 0.5626 (4) 0.2059 (3) 0.3513 (2) 0.0405 (11)
H5 0.5894 0.2119 0.3963 0.049*
C6 0.6343 (5) 0.1489 (4) 0.3106 (2) 0.0468 (12)
H6 0.7083 0.1151 0.3280 0.056*
C7 0.5978 (4) 0.1404 (3) 0.2430 (2) 0.0416 (10)
H7 0.6473 0.1011 0.2150 0.050*
C8 0.3727 (4) 0.3097 (3) 0.37649 (19) 0.0325 (9)
H8 0.4042 0.3070 0.4209 0.039*
C9 0.1883 (5) 0.4013 (3) 0.4187 (2) 0.0419 (11)
H9A 0.1913 0.4716 0.4174 0.050*
H9B 0.2236 0.3797 0.4621 0.050*
C10 0.0444 (5) 0.3652 (4) 0.4069 (2) 0.0432 (11)
H10A 0.0380 0.2976 0.4201 0.052*
H10B −0.0162 0.4027 0.4340 0.052*
C11 −0.1155 (4) 0.3718 (3) 0.3160 (2) 0.0376 (10)
H11 −0.1793 0.3652 0.3488 0.045*
C12 −0.1630 (4) 0.3768 (3) 0.2471 (2) 0.0337 (9)
C13 −0.3008 (4) 0.3975 (3) 0.2340 (3) 0.0454 (11)
H13 −0.3576 0.4041 0.2700 0.054*
C14 −0.3520 (4) 0.4080 (3) 0.1702 (3) 0.0482 (12)
H14 −0.4427 0.4212 0.1631 0.058*
C15 −0.2698 (4) 0.3991 (3) 0.1159 (2) 0.0419 (11)
H15 −0.3044 0.4081 0.0724 0.050*
C16 −0.1355 (4) 0.3769 (3) 0.1268 (2) 0.0327 (9)
C17 −0.0801 (4) 0.3635 (3) 0.1915 (2) 0.0291 (8)
C18 −0.0918 (5) 0.3868 (4) 0.0095 (2) 0.0523 (13)
H18A −0.1594 0.3406 −0.0040 0.078*
H18B −0.0188 0.3835 −0.0207 0.078*
H18C −0.1295 0.4509 0.0085 0.078*
C19 0.0334 (4) 0.0595 (3) 0.1448 (3) 0.0453 (11)
H19A 0.0360 0.0054 0.1761 0.054*
H19B 0.0421 0.0337 0.0998 0.054*
C20 −0.0964 (5) 0.1103 (4) 0.1492 (3) 0.0640 (15)
H20A −0.1040 0.1376 0.1934 0.096*
H20B −0.1683 0.0651 0.1409 0.096*
H20C −0.1014 0.1612 0.1163 0.096*
H14S 0.189 (4) 0.093 (3) 0.1907 (18) 0.045 (14)*
N1 0.2671 (4) 0.3598 (3) 0.36409 (16) 0.0334 (8)
N2 0.0069 (4) 0.3757 (3) 0.33567 (17) 0.0365 (8)
N3 0.2742 (4) 0.5201 (3) 0.18223 (18) 0.0429 (8)
N4 0.3575 (4) 0.3693 (3) −0.00107 (19) 0.0461 (10)
N5 0.1636 (4) 0.1430 (3) −0.00124 (17) 0.0380 (8)
O1 0.3020 (3) 0.29131 (19) 0.22943 (13) 0.0285 (6)
O2 0.0480 (2) 0.3360 (2) 0.19722 (13) 0.0310 (6)
O3 0.4443 (3) 0.1886 (2) 0.15072 (13) 0.0340 (6)
O4 −0.0437 (3) 0.3650 (2) 0.07643 (14) 0.0378 (7)
O5 0.2537 (3) 0.4574 (2) 0.13814 (15) 0.0460 (8)
O6 0.2370 (3) 0.5111 (2) 0.24316 (13) 0.0334 (6)
O7 0.3498 (5) 0.6110 (3) 0.1638 (3) 0.0920 (15)
O8 0.4194 (3) 0.3382 (2) 0.05096 (16) 0.0441 (7)
O9 0.2306 (3) 0.3687 (2) −0.00015 (14) 0.0422 (7)
O10 0.4160 (5) 0.3958 (4) −0.0499 (2) 0.0997 (17)
O11 0.0688 (3) 0.1928 (2) 0.02181 (14) 0.0383 (7)
O12 0.2775 (3) 0.1549 (2) 0.02628 (15) 0.0408 (7)
O13 0.1458 (4) 0.0871 (3) −0.04800 (19) 0.0694 (11)
O14 0.1442 (3) 0.1242 (2) 0.16021 (15) 0.0361 (7)
Sm1 0.209482 (19) 0.287372 (15) 0.113944 (9) 0.02816 (9)
Zn1 0.17006 (4) 0.38157 (3) 0.27338 (2) 0.02711 (12)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
C1 0.035 (2) 0.060 (3) 0.036 (2) 0.016 (2) 0.0029 (18) −0.011 (2)
C2 0.0245 (19) 0.034 (2) 0.027 (2) −0.0018 (16) −0.0040 (15) 0.0050 (16)
C3 0.0228 (19) 0.028 (2) 0.0233 (19) −0.0031 (15) −0.0020 (14) 0.0067 (15)
C4 0.028 (2) 0.038 (2) 0.027 (2) −0.0031 (17) −0.0074 (16) 0.0051 (17)
C5 0.035 (2) 0.058 (3) 0.028 (2) 0.001 (2) −0.0093 (18) 0.0101 (19)
C6 0.036 (2) 0.058 (3) 0.046 (3) 0.012 (2) −0.012 (2) 0.015 (2)
C7 0.036 (2) 0.045 (3) 0.044 (3) 0.0095 (19) 0.0013 (19) 0.007 (2)
C8 0.036 (2) 0.041 (2) 0.0203 (19) −0.0063 (18) −0.0052 (16) 0.0038 (17)
C9 0.056 (3) 0.049 (3) 0.021 (2) 0.003 (2) 0.0044 (18) −0.0073 (19)
C10 0.052 (3) 0.053 (3) 0.026 (2) 0.004 (2) 0.0140 (19) −0.0007 (19)
C11 0.035 (2) 0.036 (2) 0.043 (3) −0.0011 (18) 0.0172 (19) −0.0053 (19)
C12 0.026 (2) 0.031 (2) 0.045 (2) −0.0022 (16) 0.0072 (17) −0.0053 (18)
C13 0.028 (2) 0.041 (3) 0.068 (3) −0.0012 (19) 0.015 (2) −0.008 (2)
C14 0.022 (2) 0.049 (3) 0.074 (4) 0.0056 (19) −0.004 (2) −0.007 (2)
C15 0.029 (2) 0.038 (3) 0.057 (3) 0.0059 (18) −0.010 (2) −0.005 (2)
C16 0.029 (2) 0.027 (2) 0.042 (2) 0.0032 (16) −0.0028 (17) −0.0083 (17)
C17 0.025 (2) 0.024 (2) 0.038 (2) 0.0009 (15) 0.0006 (16) −0.0061 (16)
C18 0.051 (3) 0.066 (3) 0.039 (3) 0.017 (2) −0.013 (2) −0.001 (2)
C19 0.038 (3) 0.038 (3) 0.060 (3) −0.002 (2) −0.002 (2) 0.006 (2)
C20 0.036 (3) 0.059 (3) 0.097 (5) −0.002 (2) 0.009 (3) −0.003 (3)
N1 0.043 (2) 0.0359 (19) 0.0215 (16) −0.0051 (16) 0.0014 (14) −0.0033 (14)
N2 0.040 (2) 0.041 (2) 0.0291 (18) −0.0007 (16) 0.0111 (15) 0.0003 (15)
N3 0.058 (2) 0.038 (2) 0.0329 (14) 0.0040 (17) 0.0048 (15) −0.0028 (12)
N4 0.053 (3) 0.048 (2) 0.038 (2) 0.0035 (18) 0.0141 (18) 0.0109 (18)
N5 0.047 (2) 0.038 (2) 0.0287 (18) −0.0019 (17) −0.0023 (16) −0.0059 (16)
O1 0.0244 (14) 0.0385 (16) 0.0221 (14) 0.0051 (11) −0.0042 (10) −0.0010 (11)
O2 0.0226 (13) 0.0421 (16) 0.0282 (14) 0.0061 (12) −0.0005 (11) −0.0067 (12)
O3 0.0251 (14) 0.0489 (17) 0.0279 (15) 0.0091 (12) −0.0016 (11) −0.0054 (12)
O4 0.0317 (15) 0.0516 (18) 0.0298 (15) 0.0093 (13) −0.0054 (12) −0.0056 (13)
O5 0.071 (2) 0.0360 (17) 0.0308 (15) −0.0057 (15) 0.0056 (15) −0.0015 (11)
O6 0.0410 (16) 0.0288 (15) 0.0303 (12) −0.0026 (12) 0.0015 (11) −0.0031 (11)
O7 0.114 (4) 0.069 (3) 0.095 (4) −0.018 (3) 0.033 (3) −0.002 (2)
O8 0.0332 (16) 0.053 (2) 0.0460 (19) −0.0035 (14) −0.0014 (13) 0.0090 (15)
O9 0.0420 (18) 0.054 (2) 0.0309 (16) 0.0052 (14) −0.0023 (13) 0.0070 (14)
O10 0.085 (3) 0.143 (5) 0.074 (3) 0.015 (3) 0.042 (3) 0.058 (3)
O11 0.0355 (16) 0.0480 (18) 0.0310 (16) 0.0025 (13) −0.0073 (12) −0.0040 (13)
O12 0.0341 (17) 0.0488 (19) 0.0393 (17) 0.0041 (13) −0.0037 (13) −0.0085 (14)
O13 0.082 (3) 0.069 (3) 0.056 (2) −0.001 (2) −0.013 (2) −0.036 (2)
O14 0.0323 (16) 0.0368 (17) 0.0386 (17) −0.0058 (12) −0.0078 (13) 0.0112 (13)
Sm1 0.02985 (13) 0.03342 (14) 0.02110 (13) 0.00080 (8) −0.00085 (8) −0.00187 (8)
Zn1 0.0286 (2) 0.0328 (3) 0.0200 (2) −0.00050 (18) 0.00205 (17) −0.00135 (17)
Geometric parameters (Å, °)
C1—O3 1.430 (5) C17—O2 1.334 (5)
C1—H1A 0.9600 C18—O4 1.433 (5)
C1—H1B 0.9600 C18—H18A 0.9600
C1—H1C 0.9600 C18—H18B 0.9600
C2—C7 1.379 (6) C18—H18C 0.9600
C2—O3 1.384 (5) C19—O14 1.446 (5)
C2—C3 1.406 (6) C19—C20 1.477 (7)
C3—O1 1.333 (4) C19—H19A 0.9700
C3—C4 1.414 (5) C19—H19B 0.9700
C4—C5 1.418 (6) C20—H20A 0.9600
C4—C8 1.444 (6) C20—H20B 0.9600
C5—C6 1.348 (7) C20—H20C 0.9600
C5—H5 0.9300 N1—Zn1 2.043 (3)
C6—C7 1.387 (6) N2—Zn1 2.077 (3)
C6—H6 0.9300 N3—O5 1.244 (5)
C7—H7 0.9300 N3—O6 1.284 (4)
C8—N1 1.277 (5) N3—O7 1.512 (6)
C8—H8 0.9300 N4—O10 1.205 (5)
C9—N1 1.474 (5) N4—O8 1.264 (5)
C9—C10 1.530 (7) N4—O9 1.267 (5)
C9—H9A 0.9700 N5—O13 1.217 (5)
C9—H9B 0.9700 N5—O12 1.257 (4)
C10—N2 1.462 (5) N5—O11 1.265 (5)
C10—H10A 0.9700 O1—Zn1 2.028 (3)
C10—H10B 0.9700 O1—Sm1 2.450 (3)
C11—N2 1.272 (6) O2—Zn1 2.014 (3)
C11—C12 1.438 (6) O2—Sm1 2.439 (3)
C11—H11 0.9300 O3—Sm1 2.787 (3)
C12—C17 1.413 (6) O4—Sm1 2.822 (3)
C12—C13 1.420 (6) O5—Sm1 2.429 (3)
C13—C14 1.362 (7) O6—Zn1 2.004 (3)
C13—H13 0.9300 O8—Sm1 2.570 (3)
C14—C15 1.380 (7) O9—Sm1 2.545 (3)
C14—H14 0.9300 O11—Sm1 2.621 (3)
C15—C16 1.385 (6) O12—Sm1 2.627 (3)
C15—H15 0.9300 O14—Sm1 2.523 (3)
C16—O4 1.387 (5) O14—H14S 0.855 (19)
C16—C17 1.398 (6)
O3—C1—H1A 109.5 O5—N3—O7 118.5 (4)
O3—C1—H1B 109.5 O6—N3—O7 118.0 (3)
H1A—C1—H1B 109.5 O10—N4—O8 121.8 (4)
O3—C1—H1C 109.5 O10—N4—O9 121.5 (4)
H1A—C1—H1C 109.5 O8—N4—O9 116.7 (3)
H1B—C1—H1C 109.5 O13—N5—O12 121.7 (4)
C7—C2—O3 124.6 (4) O13—N5—O11 121.8 (4)
C7—C2—C3 121.7 (4) O12—N5—O11 116.6 (3)
O3—C2—C3 113.7 (3) C3—O1—Zn1 127.1 (2)
O1—C3—C2 117.0 (3) C3—O1—Sm1 132.1 (2)
O1—C3—C4 125.1 (3) Zn1—O1—Sm1 100.77 (10)
C2—C3—C4 117.8 (3) C17—O2—Zn1 122.1 (2)
C3—C4—C5 118.7 (4) C17—O2—Sm1 132.1 (2)
C3—C4—C8 124.4 (4) Zn1—O2—Sm1 101.52 (10)
C5—C4—C8 116.8 (4) C2—O3—C1 115.4 (3)
C6—C5—C4 121.8 (4) C2—O3—Sm1 118.7 (2)
C6—C5—H5 119.1 C1—O3—Sm1 124.9 (2)
C4—C5—H5 119.1 C16—O4—C18 115.8 (3)
C5—C6—C7 120.1 (4) C16—O4—Sm1 117.3 (2)
C5—C6—H6 119.9 C18—O4—Sm1 126.7 (3)
C7—C6—H6 119.9 N3—O5—Sm1 146.6 (3)
C2—C7—C6 119.9 (4) N3—O6—Zn1 118.6 (2)
C2—C7—H7 120.1 N4—O8—Sm1 96.1 (2)
C6—C7—H7 120.1 N4—O9—Sm1 97.2 (2)
N1—C8—C4 125.5 (4) N5—O11—Sm1 97.6 (2)
N1—C8—H8 117.3 N5—O12—Sm1 97.6 (2)
C4—C8—H8 117.3 C19—O14—Sm1 132.6 (2)
N1—C9—C10 106.2 (3) C19—O14—H14S 103 (3)
N1—C9—H9A 110.5 Sm1—O14—H14S 125 (3)
C10—C9—H9A 110.5 O5—Sm1—O2 73.74 (10)
N1—C9—H9B 110.5 O5—Sm1—O1 74.45 (10)
C10—C9—H9B 110.5 O2—Sm1—O1 66.10 (9)
H9A—C9—H9B 108.7 O5—Sm1—O14 146.58 (10)
N2—C10—C9 109.1 (3) O2—Sm1—O14 79.28 (10)
N2—C10—H10A 109.9 O1—Sm1—O14 76.81 (9)
C9—C10—H10A 109.9 O5—Sm1—O9 74.48 (10)
N2—C10—H10B 109.9 O2—Sm1—O9 123.96 (10)
C9—C10—H10B 109.9 O1—Sm1—O9 141.92 (10)
H10A—C10—H10B 108.3 O14—Sm1—O9 138.33 (10)
N2—C11—C12 125.3 (4) O5—Sm1—O8 71.83 (11)
N2—C11—H11 117.4 O2—Sm1—O8 145.17 (10)
C12—C11—H11 117.4 O1—Sm1—O8 99.31 (9)
C17—C12—C13 118.0 (4) O14—Sm1—O8 130.09 (10)
C17—C12—C11 123.7 (4) O9—Sm1—O8 49.82 (10)
C13—C12—C11 118.2 (4) O5—Sm1—O11 135.09 (10)
C14—C13—C12 121.6 (4) O2—Sm1—O11 105.13 (9)
C14—C13—H13 119.2 O1—Sm1—O11 147.66 (9)
C12—C13—H13 119.2 O14—Sm1—O11 70.91 (9)
C13—C14—C15 120.4 (4) O9—Sm1—O11 69.69 (10)
C13—C14—H14 119.8 O8—Sm1—O11 102.80 (10)
C15—C14—H14 119.8 O5—Sm1—O12 138.80 (10)
C14—C15—C16 119.5 (4) O2—Sm1—O12 146.17 (9)
C14—C15—H15 120.3 O1—Sm1—O12 122.69 (9)
C16—C15—H15 120.3 O14—Sm1—O12 72.40 (10)
C15—C16—O4 124.8 (4) O9—Sm1—O12 71.60 (10)
C15—C16—C17 121.8 (4) O8—Sm1—O12 68.60 (10)
O4—C16—C17 113.5 (3) O11—Sm1—O12 48.25 (9)
O2—C17—C16 117.8 (3) O5—Sm1—O3 105.83 (10)
O2—C17—C12 123.5 (4) O2—Sm1—O3 121.53 (8)
C16—C17—C12 118.6 (4) O1—Sm1—O3 58.34 (8)
O4—C18—H18A 109.5 O14—Sm1—O3 72.15 (9)
O4—C18—H18B 109.5 O9—Sm1—O3 110.93 (9)
H18A—C18—H18B 109.5 O8—Sm1—O3 64.54 (9)
O4—C18—H18C 109.5 O11—Sm1—O3 111.62 (9)
H18A—C18—H18C 109.5 O12—Sm1—O3 66.62 (8)
H18B—C18—H18C 109.5 O5—Sm1—O4 80.93 (10)
O14—C19—C20 111.1 (4) O2—Sm1—O4 58.08 (8)
O14—C19—H19A 109.4 O1—Sm1—O4 123.23 (8)
C20—C19—H19A 109.4 O14—Sm1—O4 101.26 (9)
O14—C19—H19B 109.4 O9—Sm1—O4 72.24 (9)
C20—C19—H19B 109.4 O8—Sm1—O4 120.25 (9)
H19A—C19—H19B 108.0 O11—Sm1—O4 63.13 (9)
C19—C20—H20A 109.5 O12—Sm1—O4 109.57 (9)
C19—C20—H20B 109.5 O3—Sm1—O4 173.01 (9)
H20A—C20—H20B 109.5 O6—Zn1—O2 104.55 (12)
C19—C20—H20C 109.5 O6—Zn1—O1 100.96 (11)
H20A—C20—H20C 109.5 O2—Zn1—O1 82.54 (10)
H20B—C20—H20C 109.5 O6—Zn1—N1 104.02 (13)
C8—N1—C9 121.5 (3) O2—Zn1—N1 151.32 (13)
C8—N1—Zn1 128.0 (3) O1—Zn1—N1 89.69 (12)
C9—N1—Zn1 110.2 (3) O6—Zn1—N2 119.22 (13)
C11—N2—C10 120.6 (4) O2—Zn1—N2 88.27 (13)
C11—N2—Zn1 125.5 (3) O1—Zn1—N2 139.80 (13)
C10—N2—Zn1 113.6 (3) N1—Zn1—N2 79.99 (14)
O5—N3—O6 123.4 (4)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C1—H1B···O12 0.96 2.35 2.995 (5) 125
C18—H18B···O9 0.96 2.52 3.237 (6) 132
O14—H14S···O6i 0.86 (2) 1.87 (2) 2.718 (4) 171 (5)
C8—H8···O11ii 0.93 2.55 3.440 (5) 160
Symmetry codes: (i) −x+1/2, y−1/2, −z+1/2; (ii) x+1/2, −y+1/2, z+1/2.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C18—H18B⋯O9 0.96 2.52 3.237 (6) 132
O14—H14S⋯O6i 0.855 (19) 1.87 (2) 2.718 (4) 171 (5)
C8—H8⋯O11ii 0.93 2.55 3.440 (5) 160
Symmetry codes: (i) ; (ii) .
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References
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Knoer, R., Lin, H.-H., Wei, H.-H. & Mohanta, S. (2005). Inorg. Chem.44, 3524–3536.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Westrip, S. P. (2009). publCIF. In preparation. | 21577700 | PMC2970192 | CC BY | 2021-01-04 18:58:58 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Sep 5; 65(Pt 10):m1161-m1162 |
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography hb511910.1107/S1600536809040227ACSEBHS1600536809040227Organic Papers1-Phenyl-3-(2,4,6-trimethoxyphenyl)prop-2-en-1-one C18H18O4Liu Ying a*Zhang Xianxi aXue Zechun aLv Chunyan aa College of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, People’s Republic of ChinaCorrespondence e-mail: [email protected] 11 2009 13 10 2009 13 10 2009 65 Pt 11 e091100o2724 o2724 25 9 2009 02 10 2009 © Liu et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the title compound, C18H18O4, the dihedral angle between the mean planes of the aromatic rings is 7.39 (6)°. The dihedral angles between the linking C—C=C—C plane and the phenyl and benzene rings are 11.27 (5) and 4.20 (5)°, respectively.
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Related literature
For background to the properties and applications of chalcones, see: Satish et al., (1995 ▶), Meng et al., (2004 ▶), Indira et al., (2002 ▶). For the synthesis, see: Migrdichian (1957 ▶).
Experimental
Crystal data
C18H18O4
M
r = 298.32
Monoclinic,
a = 8.8921 (10) Å
b = 15.114 (3) Å
c = 11.618 (3) Å
β = 104.289 (10)°
V = 1513.1 (5) Å3
Z = 4
Mo Kα radiation
μ = 0.09 mm−1
T = 293 K
0.12 × 0.10 × 0.05 mm
Data collection
Bruker SMART CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2005 ▶) T
min = 0.989, T
max = 0.995
7545 measured reflections
2581 independent reflections
2037 reflections with I > 2σ(I)
R
int = 0.037
Refinement
R[F
2 > 2σ(F
2)] = 0.036
wR(F
2) = 0.108
S = 1.01
2581 reflections
203 parameters
H-atom parameters constrained
Δρmax = 0.13 e Å−3
Δρmin = −0.13 e Å−3
Data collection: SMART (Bruker, 2005 ▶); cell refinement: SAINT (Bruker, 2005 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: XP in SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXL97.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809040227/hb5119sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809040227/hb5119Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB5119).
The work was supported by Liaocheng University (grant No. X071011) and the National Ministry of Science and Technology of China (grant No. 20501011).
supplementary crystallographic
information
Comment
In recent years, chalcones consisting of –C=C—C(O)- group have been widely
researched due to their interesting properties, such as photoreaction (Satish
et al., 1995), biological activity (Meng et al.,
2004) and
non-linear optical properties (Indira et al., 2002). Herein, we
report
the synthesis and structure of the title compound.
As shown in figure 1, the C(1)—C(6) phenyl ring is taken as plane 1, another
C(10)—C(15) one as plane 2 and the central C(7)—C(8)=C(9)—C(10) as plane
3, with the dihedral angles between them, A12, A13 and A23, of 7.39, 11.27 and
4.20 °, respectively, showing the two phenyl rings are rotated oppositely
with respect to the central part of plane 3. The torsional angle
C(7)—C(8)=C(9)—C(10) is 177.5 ° and the phenone O(1) atom deviates from
plane 3 by 0.13 Å, suggesting C=O is not coplanar with this plane.
Experimental
The synthesis of the title compound was according to the related literature
(Migrdichian et al., (1957)). An aqueous solution of sodium
hydroxide (10%, 10 ml) was added to the mixture of acetophenone (0.02 mol) and
2,4,6-trimethoxyphenylaldehyde (0.02 mol) in 95% ethanol (30 ml). The reaction
mixture was stirred at room temperature for 5 h, yielding light yellow solid
neutralized by hydrochloric acid (10%) and water. Colourless blocks of (I)
were
obtained by slow evaporation from dry ethanol. Elemental Analysis. Calc. for
C18H18O4: C 72.41, H 6.03%; Found: C 72.38, H 6.01%.
Refinement
The H atoms were placed in calculated positions (C—H = 0.93–0.96Å)
and refined as riding with Uiso(H) = 1.2Ueq(C) or
1.5Ueq(methyl C).
Figures
Fig. 1. A view of the structure of (I), showing 30% probability displacement ellipsoids for the non-hydrogen atoms.
Crystal data
C18H18O4 F(000) = 632
Mr = 298.32 Dx = 1.310 Mg m−3
Monoclinic, P21/c Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -P 2ybc Cell parameters from 2581 reflections
a = 8.8921 (10) Å θ = 2.4–25.0°
b = 15.114 (3) Å µ = 0.09 mm−1
c = 11.618 (3) Å T = 293 K
β = 104.289 (10)° Block, colourless
V = 1513.1 (5) Å3 0.12 × 0.10 × 0.05 mm
Z = 4
Data collection
Bruker SMART CCD diffractometer 2581 independent reflections
Radiation source: fine-focus sealed tube 2037 reflections with I > 2σ(I)
graphite Rint = 0.037
ω scans θmax = 25.0°, θmin = 2.4°
Absorption correction: multi-scan (SADABS; Bruker, 2005) h = −10→10
Tmin = 0.989, Tmax = 0.995 k = −17→16
7545 measured reflections l = −13→13
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.036 H-atom parameters constrained
wR(F2) = 0.108 w = 1/[σ2(Fo2) + (0.0725P)2] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
2581 reflections Δρmax = 0.13 e Å−3
203 parameters Δρmin = −0.13 e Å−3
0 restraints Extinction correction: SHELXL97 (Sheldrick, 2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.329 (18)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
C1 −0.14370 (15) 0.24856 (10) 0.22992 (12) 0.0564 (4)
H1 −0.1511 0.2141 0.2946 0.068*
C2 −0.25419 (15) 0.31225 (11) 0.18742 (14) 0.0645 (4)
H2 −0.3356 0.3200 0.2236 0.077*
C3 −0.24594 (16) 0.36410 (11) 0.09297 (14) 0.0653 (4)
H3 −0.3202 0.4076 0.0659 0.078*
C4 −0.12883 (17) 0.35171 (10) 0.03889 (13) 0.0654 (4)
H4 −0.1228 0.3865 −0.0258 0.078*
C5 −0.01819 (15) 0.28718 (9) 0.08018 (12) 0.0550 (4)
H5 0.0607 0.2784 0.0417 0.066*
C6 −0.02275 (13) 0.23540 (9) 0.17773 (10) 0.0463 (3)
C7 0.09868 (14) 0.16867 (9) 0.23099 (11) 0.0505 (3)
C8 0.22237 (14) 0.15192 (9) 0.17349 (11) 0.0505 (4)
H8 0.2182 0.1777 0.1001 0.061*
C9 0.34224 (14) 0.10042 (8) 0.22297 (10) 0.0452 (3)
H9 0.3361 0.0743 0.2941 0.054*
C10 0.47889 (13) 0.07856 (8) 0.18512 (10) 0.0405 (3)
C11 0.51540 (14) 0.11331 (8) 0.08352 (10) 0.0431 (3)
C12 0.64572 (14) 0.08891 (8) 0.05099 (10) 0.0474 (3)
H12 0.6667 0.1122 −0.0175 0.057*
C13 0.74710 (13) 0.02941 (8) 0.11969 (11) 0.0460 (3)
C14 0.72008 (13) −0.00523 (8) 0.22196 (10) 0.0457 (3)
H14 0.7900 −0.0443 0.2688 0.055*
C15 0.58727 (13) 0.01938 (8) 0.25291 (10) 0.0422 (3)
C16 0.65272 (16) −0.07565 (10) 0.42236 (11) 0.0592 (4)
H16A 0.6572 −0.1275 0.3757 0.089*
H16B 0.6140 −0.0913 0.4898 0.089*
H16C 0.7548 −0.0509 0.4491 0.089*
C17 0.44269 (17) 0.21138 (9) −0.08152 (12) 0.0616 (4)
H17A 0.5398 0.2424 −0.0602 0.092*
H17B 0.3610 0.2521 −0.1157 0.092*
H17C 0.4479 0.1661 −0.1383 0.092*
C18 0.97451 (18) −0.05696 (11) 0.14022 (14) 0.0733 (5)
H18A 1.0180 −0.0392 0.2209 0.110*
H18B 1.0565 −0.0657 0.1009 0.110*
H18C 0.9181 −0.1112 0.1393 0.110*
O1 0.55255 (10) −0.01251 (6) 0.35230 (8) 0.0566 (3)
O2 0.41252 (10) 0.17263 (6) 0.02034 (7) 0.0574 (3)
O3 0.87224 (10) 0.01005 (7) 0.08024 (8) 0.0639 (3)
O4 0.09522 (11) 0.13301 (8) 0.32405 (9) 0.0787 (4)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
C1 0.0473 (7) 0.0676 (9) 0.0567 (8) −0.0009 (6) 0.0171 (6) −0.0055 (7)
C2 0.0418 (7) 0.0773 (10) 0.0776 (10) 0.0068 (7) 0.0208 (7) −0.0089 (8)
C3 0.0514 (8) 0.0694 (10) 0.0725 (10) 0.0144 (7) 0.0104 (7) −0.0030 (8)
C4 0.0649 (9) 0.0683 (9) 0.0623 (9) 0.0123 (7) 0.0144 (7) 0.0046 (7)
C5 0.0487 (7) 0.0644 (9) 0.0541 (8) 0.0055 (6) 0.0166 (6) −0.0045 (6)
C6 0.0375 (6) 0.0555 (7) 0.0446 (7) −0.0005 (5) 0.0076 (5) −0.0105 (6)
C7 0.0428 (7) 0.0661 (8) 0.0415 (7) 0.0028 (6) 0.0083 (5) −0.0058 (6)
C8 0.0442 (7) 0.0645 (8) 0.0422 (7) 0.0074 (6) 0.0093 (5) −0.0014 (6)
C9 0.0439 (7) 0.0519 (7) 0.0395 (6) 0.0012 (6) 0.0097 (5) −0.0049 (5)
C10 0.0392 (6) 0.0437 (7) 0.0379 (6) 0.0010 (5) 0.0081 (5) −0.0025 (5)
C11 0.0436 (7) 0.0430 (7) 0.0402 (6) 0.0021 (5) 0.0059 (5) 0.0008 (5)
C12 0.0513 (7) 0.0519 (7) 0.0413 (7) 0.0007 (6) 0.0158 (6) 0.0063 (5)
C13 0.0420 (6) 0.0506 (7) 0.0484 (7) 0.0031 (5) 0.0169 (5) 0.0025 (6)
C14 0.0428 (7) 0.0478 (7) 0.0475 (7) 0.0064 (5) 0.0131 (6) 0.0073 (5)
C15 0.0428 (7) 0.0467 (7) 0.0380 (6) −0.0013 (5) 0.0115 (5) 0.0022 (5)
C16 0.0574 (8) 0.0705 (9) 0.0505 (8) 0.0117 (7) 0.0149 (6) 0.0197 (7)
C17 0.0695 (9) 0.0633 (9) 0.0503 (8) 0.0047 (7) 0.0117 (7) 0.0161 (6)
C18 0.0631 (9) 0.0874 (11) 0.0802 (10) 0.0323 (8) 0.0385 (8) 0.0277 (9)
O1 0.0515 (5) 0.0748 (6) 0.0479 (5) 0.0156 (5) 0.0209 (4) 0.0201 (4)
O2 0.0562 (6) 0.0661 (6) 0.0504 (5) 0.0162 (4) 0.0140 (4) 0.0177 (4)
O3 0.0564 (6) 0.0799 (7) 0.0647 (6) 0.0207 (5) 0.0328 (5) 0.0226 (5)
O4 0.0661 (7) 0.1169 (9) 0.0584 (6) 0.0289 (6) 0.0255 (5) 0.0239 (6)
Geometric parameters (Å, °)
C1—C6 1.3733 (18) C11—C12 1.3556 (17)
C1—C2 1.3770 (19) C12—C13 1.3797 (17)
C1—H1 0.9300 C12—H12 0.9300
C2—C3 1.365 (2) C13—O3 1.3362 (14)
C2—H2 0.9300 C13—C14 1.3727 (17)
C3—C4 1.355 (2) C14—C15 1.3682 (15)
C3—H3 0.9300 C14—H14 0.9300
C4—C5 1.3843 (19) C15—O1 1.3554 (14)
C4—H4 0.9300 C16—O1 1.4172 (15)
C5—C6 1.3860 (18) C16—H16A 0.9600
C5—H5 0.9300 C16—H16B 0.9600
C6—C7 1.4954 (18) C16—H16C 0.9600
C7—O4 1.2153 (16) C17—O2 1.4044 (15)
C7—C8 1.4431 (17) C17—H17A 0.9600
C8—C9 1.3305 (17) C17—H17B 0.9600
C8—H8 0.9300 C17—H17C 0.9600
C9—C10 1.4292 (16) C18—O3 1.4232 (16)
C9—H9 0.9300 C18—H18A 0.9600
C10—C11 1.4014 (16) C18—H18B 0.9600
C10—C15 1.4051 (16) C18—H18C 0.9600
C11—O2 1.3594 (14)
C6—C1—C2 120.67 (13) C11—C12—C13 119.83 (11)
C6—C1—H1 119.7 C11—C12—H12 120.1
C2—C1—H1 119.7 C13—C12—H12 120.1
C3—C2—C1 120.96 (13) O3—C13—C14 123.49 (11)
C3—C2—H2 119.5 O3—C13—C12 115.14 (11)
C1—C2—H2 119.5 C14—C13—C12 121.36 (11)
C4—C3—C2 119.55 (14) C15—C14—C13 118.08 (11)
C4—C3—H3 120.2 C15—C14—H14 121.0
C2—C3—H3 120.2 C13—C14—H14 121.0
C3—C4—C5 119.89 (14) O1—C15—C14 121.34 (11)
C3—C4—H4 120.1 O1—C15—C10 115.78 (10)
C5—C4—H4 120.1 C14—C15—C10 122.88 (10)
C4—C5—C6 121.29 (12) O1—C16—H16A 109.5
C4—C5—H5 119.4 O1—C16—H16B 109.5
C6—C5—H5 119.4 H16A—C16—H16B 109.5
C1—C6—C5 117.61 (12) O1—C16—H16C 109.5
C1—C6—C7 118.63 (12) H16A—C16—H16C 109.5
C5—C6—C7 123.73 (11) H16B—C16—H16C 109.5
O4—C7—C8 121.57 (12) O2—C17—H17A 109.5
O4—C7—C6 119.54 (12) O2—C17—H17B 109.5
C8—C7—C6 118.80 (11) H17A—C17—H17B 109.5
C9—C8—C7 121.57 (12) O2—C17—H17C 109.5
C9—C8—H8 119.2 H17A—C17—H17C 109.5
C7—C8—H8 119.2 H17B—C17—H17C 109.5
C8—C9—C10 130.82 (12) O3—C18—H18A 109.5
C8—C9—H9 114.6 O3—C18—H18B 109.5
C10—C9—H9 114.6 H18A—C18—H18B 109.5
C11—C10—C15 116.18 (10) O3—C18—H18C 109.5
C11—C10—C9 124.32 (11) H18A—C18—H18C 109.5
C15—C10—C9 119.49 (10) H18B—C18—H18C 109.5
O2—C11—C12 122.44 (11) C15—O1—C16 119.06 (9)
O2—C11—C10 115.92 (10) C11—O2—C17 119.14 (10)
C12—C11—C10 121.64 (11) C13—O3—C18 118.25 (10)
==== Refs
References
Bruker (2005). SMART, SAINT and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Indira, J., Prakash Karat, P. & Sarojini, B. K. J. (2002). J. Cryst. Growth, 242, 209–214.
Meng, C. Q., Zheng, X. S., Ni, L., Ye, Z. H., Simpson, J. E., Worsencroft, K. J., Hotema, M. R., Weingarten, J. W., Gilmore, J. M., Hoong, L. K., Hill, R. R., Marino, E. M., Suen, K. L., Kunsch, C., Wasserman, M. A. & Sikorski, J. A. (2004). Bioorg. Med. Chem. Lett., 14, 1513–1517.
Migrdichian, V. (1957). Org. Synth.1, 171–173.
Satish, G. B., Kaliyamoorthy, P. Z. D. E. & Desiraju, G. R. (1995). J. Chem. Soc. Perkin Trans. 2, pp. 325–330.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. | 21578321 | PMC2971127 | CC BY | 2021-01-04 18:59:36 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Oct 13; 65(Pt 11):o2724 |
==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography at276410.1107/S1600536809014482ACSEBHS1600536809014482Metal-Organic PapersDi-μ-chlorido-bis[chlorido(1,10-phenanthroline-κ2
N,N′)zinc(II)] [Zn2Cl4(C12H8N2)2]Yang Xiao-Mao a*Leng Qing-Bo aChen Yi aHe You-Gang aLuo Shi-Wu aa Institute of Applied Materials, College of Resource & Environment Management, Jiangxi University of Finance and Economics, Nanchang 330013, People’s Republic of ChinaCorrespondence e-mail: [email protected] 5 2009 25 4 2009 25 4 2009 65 Pt 5 e090500m567 m567 12 4 2009 18 4 2009 © Yang et al. 20092009This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the crystal structure of the title complex, [Zn2Cl4(C12H8N2)2], each of the two five-coordinated ZnII atoms displays a strongly distorted trigonal-bipyramidal geometry defined by two N atoms from the chelate ligand and by one terminal and two bridging chloride anions. The crystal structure is stabilized by C—H⋯Cl interactions. There is intermolecular π–π stacking between adjacent phenanthroline ligands, with a centroid–centroid distance of 3.151 (3) Å.
==== Body
Related literature
For the use of metal complexes of phenanthroline and its derivatives with π-π stacking to study the hydrolysis of biologically important phosphate diesters with poor leaving groups, see: Wall et al. (1999 ▶). For the structures of a series of metal complexes, see: Wu et al. (2003 ▶); Pan & Xu (2004 ▶); Li et al. (2005 ▶). For bond-length data, see: Allen et al. (1987 ▶).
Experimental
Crystal data
[Zn2Cl4(C12H8N2)2]
M
r = 632.95
Monoclinic,
a = 9.8537 (12) Å
b = 17.873 (2) Å
c = 13.3798 (12) Å
β = 106.502 (3)°
V = 2259.3 (4) Å3
Z = 4
Mo Kα radiation
μ = 2.62 mm−1
T = 293 K
0.19 × 0.16 × 0.12 mm
Data collection
Bruker APEXII area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2000 ▶) T
min = 0.636, T
max = 0.744
7229 measured reflections
4218 independent reflections
3453 reflections with I > 2σ(I)
R
int = 0.033
Refinement
R[F
2 > 2σ(F
2)] = 0.033
wR(F
2) = 0.073
S = 1.00
4218 reflections
307 parameters
2 restraints
H-atom parameters constrained
Δρmax = 0.47 e Å−3
Δρmin = −0.43 e Å−3
Absolute structure: Flack (1983 ▶), 1983 ▶ Friedel pairs
Flack parameter: 0.079 (12)
Data collection: SMART (Bruker, 2000 ▶); cell refinement: SAINT (Bruker, 2000 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXTL (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809014482/at2764sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536809014482/at2764Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2764).
We thank the Youth Program of Jiangxi University of Finance and Economics for financial support of this work.
supplementary crystallographic
information
Comment
Simple metal complexes of phenanthroline and its derivatives with π-π stacking
have attracted great interest because they can be used to study the hydrolysis
of biologically important phosphate diesters with poor leaving groups (Wall
et al., 1999). A series of metal complexes incorporating
different
aromatic ligands such as phenanthroline(phen), benzimidazole and quinoline
have been prepared and their crystal structures provide useful information
about π-π stacking (Wu et al., 2003; Pan & Xu, 2004;
Li et
al., 2005). We report herein the crystal structure of the title
compound,
(I).
In the molecule of (I) (Fig. 1), the ligand bond lengths and angles are within
normal ranges (Allen et al., 1987). In the crystal structure of
the
title complex, each of the two five-coordinated ZnII atoms displays a
strongly distorted trigonalbipyramidal geometry, defined by two N atom from
the organic ligand, and by one terminal and two bridging chloride anions
(Table 1).
The crystal structure is stabilized by C—H···Cl interactions (Table 1).
There is intermolecular π-π stacking between adjacent phenanthrolines, with
a centroid-centroid distance of 3.151 (3) Å (symmetry code: -1/2 + x,
1/2 + y, z). These π-π stacking interactions lead to a
supramolecular network structure (Fig. 2).
Experimental
Crystals of the title compound were synthesized using hydrothermal method in a
23 ml Teflon-lined Parr bomb, which was then sealed. Zinc(II) chloride (136.3 mg, 1 mmol), phen (396 mg, 2 mmol) and distilled water (10 g) were placed into
the bomb and sealed. The bomb was then heated under autogenous pressure up to
453 K over the course of 7 d and allowed to cool at room temperature for 24 h.
Upon opening the bomb, a clear colourless solution was decanted from small
colourless crystals. These crystals were washed with distilled water followed
by ethanol, and allowed to air-dry at room temperature.
Refinement
H atoms were positioned geometrically, with C—H = 0.93 Å and constrained to
ride on their parent atoms, with Uiso(H) = 1.2Ueq(C).
Figures
Fig. 1. The molecular structure of the title complex, with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level.
Fig. 2. A packing diagram of (I). Hydrogen bonds are shown as dashed lines.
Crystal data
[Zn2Cl4(C12H8N2)2] F(000) = 1264
Mr = 632.95 Dx = 1.861 Mg m−3
Monoclinic, Cc Mo Kα radiation, λ = 0.71073 Å
Hall symbol: C -2yc Cell parameters from 3822 reflections
a = 9.8537 (12) Å θ = 2.3–27.3°
b = 17.873 (2) Å µ = 2.62 mm−1
c = 13.3798 (12) Å T = 293 K
β = 106.502 (3)° Plane, colourless
V = 2259.3 (4) Å3 0.19 × 0.16 × 0.12 mm
Z = 4
Data collection
Bruker APEXII area-detector diffractometer 4218 independent reflections
Radiation source: fine-focus sealed tube 3453 reflections with I > 2σ(I)
graphite Rint = 0.033
φ and ω scans θmax = 26.0°, θmin = 2.3°
Absorption correction: multi-scan (SADABS; Bruker, 2000)) h = −12→11
Tmin = 0.636, Tmax = 0.744 k = −22→21
7229 measured reflections l = −16→16
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.033 H-atom parameters constrained
wR(F2) = 0.073 w = 1/[σ2(Fo2) + (0.03P)2 + 0.16P] where P = (Fo2 + 2Fc2)/3
S = 1.00 (Δ/σ)max < 0.001
4218 reflections Δρmax = 0.47 e Å−3
307 parameters Δρmin = −0.43 e Å−3
2 restraints Absolute structure: Flack (1983), 1983 Freidel pairs
Primary atom site location: structure-invariant direct methods Flack parameter: 0.079 (12)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Zn1 0.69851 (5) 0.33467 (3) 0.27140 (4) 0.03385 (16)
Zn2 0.99585 (6) 0.30433 (3) 0.49316 (5) 0.03673 (16)
Cl3 0.76987 (13) 0.34082 (7) 0.48194 (10) 0.0360 (3)
Cl2 0.91611 (14) 0.29488 (8) 0.27124 (10) 0.0366 (4)
Cl1 0.60068 (15) 0.21961 (8) 0.23650 (12) 0.0457 (4)
Cl4 1.08005 (15) 0.42235 (7) 0.50920 (12) 0.0478 (4)
N1 0.5066 (4) 0.3839 (2) 0.2553 (3) 0.0315 (10)
N2 0.7513 (4) 0.4444 (2) 0.2596 (3) 0.0321 (10)
C11 0.6385 (5) 0.4924 (3) 0.2474 (3) 0.0277 (11)
C12 0.5088 (5) 0.4583 (3) 0.2469 (4) 0.0303 (12)
C1 0.3845 (6) 0.3509 (4) 0.2559 (4) 0.0489 (16)
H1 0.3828 0.2993 0.2635 0.059*
C2 0.2584 (6) 0.3918 (4) 0.2455 (5) 0.0515 (16)
H2 0.1752 0.3675 0.2461 0.062*
C8 0.7830 (7) 0.5967 (3) 0.2373 (4) 0.0468 (16)
H8 0.7944 0.6476 0.2278 0.056*
C7 0.6504 (6) 0.5691 (3) 0.2363 (4) 0.0367 (13)
C10 0.8756 (6) 0.4746 (3) 0.2632 (4) 0.0366 (12)
H10 0.9538 0.4433 0.2739 0.044*
C3 0.2615 (6) 0.4669 (4) 0.2345 (4) 0.0495 (15)
H3 0.1788 0.4943 0.2266 0.059*
C4 0.3884 (6) 0.5051 (3) 0.2347 (4) 0.0401 (14)
C6 0.5230 (6) 0.6132 (3) 0.2241 (4) 0.0447 (14)
H6 0.5271 0.6646 0.2145 0.054*
C5 0.4016 (7) 0.5830 (3) 0.2260 (4) 0.0497 (16)
H5 0.3238 0.6136 0.2215 0.060*
N4 0.9434 (4) 0.1943 (2) 0.4907 (3) 0.0284 (9)
N3 1.1864 (4) 0.2591 (2) 0.4971 (3) 0.0357 (10)
C13 0.8177 (6) 0.1637 (3) 0.4836 (4) 0.0408 (13)
H13 0.7405 0.1948 0.4789 0.049*
C23 1.1830 (5) 0.1816 (3) 0.4989 (4) 0.0342 (13)
C22 1.3047 (6) 0.2925 (3) 0.4985 (4) 0.0403 (15)
H22 1.3085 0.3445 0.4992 0.048*
C24 1.0533 (6) 0.1489 (3) 0.4961 (4) 0.0309 (12)
C16 1.0413 (7) 0.0691 (3) 0.4961 (4) 0.0429 (15)
C17 1.1629 (7) 0.0258 (4) 0.5004 (4) 0.0515 (17)
H17 1.1565 −0.0261 0.5016 0.062*
C15 0.9031 (7) 0.0404 (3) 0.4884 (4) 0.0477 (15)
H15 0.8885 −0.0110 0.4873 0.057*
C18 1.2876 (7) 0.0575 (4) 0.5027 (4) 0.0545 (18)
H18 1.3657 0.0274 0.5057 0.065*
C14 0.7984 (7) 0.0854 (3) 0.4831 (4) 0.0428 (15)
H14 0.7094 0.0660 0.4788 0.051*
C19 1.3008 (6) 0.1378 (3) 0.5006 (4) 0.0450 (15)
C20 1.4266 (7) 0.1761 (4) 0.5006 (5) 0.063 (2)
H20 1.5086 0.1500 0.5016 0.075*
C21 1.4242 (7) 0.2522 (5) 0.4989 (5) 0.0599 (19)
H21 1.5059 0.2779 0.4980 0.072*
C9 0.8946 (7) 0.5507 (3) 0.2517 (5) 0.0473 (16)
H9 0.9834 0.5695 0.2540 0.057*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Zn1 0.0277 (3) 0.0256 (3) 0.0473 (3) 0.0018 (3) 0.0090 (2) 0.0012 (3)
Zn2 0.0278 (3) 0.0222 (3) 0.0593 (4) 0.0017 (3) 0.0111 (3) −0.0001 (3)
Cl3 0.0305 (8) 0.0301 (7) 0.0480 (8) 0.0072 (5) 0.0121 (6) −0.0017 (6)
Cl2 0.0308 (8) 0.0313 (8) 0.0493 (8) 0.0074 (6) 0.0141 (6) −0.0001 (6)
Cl1 0.0413 (9) 0.0267 (8) 0.0675 (10) −0.0035 (7) 0.0127 (7) −0.0038 (7)
Cl4 0.0398 (8) 0.0239 (7) 0.0723 (10) −0.0047 (6) 0.0037 (7) 0.0008 (6)
N1 0.025 (3) 0.033 (3) 0.036 (2) 0.0032 (19) 0.0084 (18) −0.0008 (18)
N2 0.029 (2) 0.027 (2) 0.042 (2) 0.009 (2) 0.0108 (18) 0.0046 (18)
C11 0.030 (3) 0.022 (3) 0.030 (2) 0.002 (2) 0.006 (2) 0.0006 (19)
C12 0.029 (3) 0.029 (3) 0.032 (3) 0.009 (2) 0.006 (2) −0.003 (2)
C1 0.047 (4) 0.043 (4) 0.058 (4) 0.000 (3) 0.016 (3) 0.007 (3)
C2 0.026 (3) 0.066 (5) 0.065 (4) 0.004 (3) 0.016 (3) 0.004 (3)
C8 0.067 (5) 0.020 (3) 0.049 (3) −0.008 (3) 0.010 (3) −0.002 (2)
C7 0.048 (3) 0.022 (3) 0.038 (3) 0.012 (2) 0.008 (2) 0.001 (2)
C10 0.029 (3) 0.027 (3) 0.052 (3) 0.002 (2) 0.009 (2) 0.003 (2)
C3 0.033 (3) 0.060 (4) 0.056 (4) 0.016 (3) 0.013 (3) 0.002 (3)
C4 0.031 (3) 0.049 (4) 0.039 (3) 0.014 (3) 0.008 (2) −0.002 (3)
C6 0.062 (4) 0.028 (3) 0.042 (3) 0.016 (3) 0.011 (3) 0.005 (2)
C5 0.045 (4) 0.047 (4) 0.056 (4) 0.027 (3) 0.011 (3) −0.002 (3)
N4 0.025 (2) 0.021 (2) 0.040 (2) −0.0010 (18) 0.0112 (17) 0.0033 (17)
N3 0.030 (3) 0.034 (3) 0.042 (2) 0.007 (2) 0.0083 (19) 0.0000 (19)
C13 0.031 (3) 0.034 (3) 0.054 (3) −0.004 (3) 0.007 (2) 0.009 (3)
C23 0.034 (3) 0.039 (4) 0.029 (3) 0.010 (2) 0.007 (2) 0.001 (2)
C22 0.021 (3) 0.049 (4) 0.052 (4) −0.009 (3) 0.011 (2) 0.000 (3)
C24 0.038 (3) 0.022 (3) 0.033 (3) 0.010 (2) 0.010 (2) 0.001 (2)
C16 0.071 (4) 0.025 (3) 0.032 (3) 0.012 (3) 0.013 (3) 0.002 (2)
C17 0.081 (5) 0.037 (3) 0.036 (3) 0.027 (4) 0.016 (3) −0.001 (2)
C15 0.073 (5) 0.021 (3) 0.049 (3) −0.008 (3) 0.016 (3) −0.004 (2)
C18 0.066 (5) 0.047 (4) 0.050 (4) 0.032 (4) 0.015 (3) 0.004 (3)
C14 0.047 (4) 0.021 (3) 0.059 (4) −0.008 (3) 0.012 (3) 0.002 (2)
C19 0.045 (4) 0.054 (4) 0.034 (3) 0.025 (3) 0.009 (2) 0.005 (3)
C20 0.031 (3) 0.098 (6) 0.059 (4) 0.019 (4) 0.012 (3) −0.010 (4)
C21 0.022 (3) 0.085 (6) 0.074 (5) 0.000 (4) 0.015 (3) 0.001 (4)
C9 0.042 (4) 0.031 (3) 0.070 (4) −0.007 (3) 0.017 (3) 0.007 (3)
Geometric parameters (Å, °)
Zn1—Cl1 2.2629 (16) C4—C5 1.406 (8)
Zn1—Cl2 2.2596 (15) C6—C5 1.318 (8)
Zn1—Cl3 2.7049 (14) C6—H6 0.9300
Zn1—N1 2.041 (4) C5—H5 0.9300
Zn1—N2 2.046 (4) N4—C13 1.332 (6)
Zn2—Cl2 2.8525 (15) N4—C24 1.338 (6)
Zn2—Cl3 2.2839 (14) N3—C22 1.305 (7)
Zn2—Cl4 2.2545 (14) N3—C23 1.387 (7)
Zn2—N3 2.031 (4) C13—C14 1.412 (7)
Zn2—N4 2.032 (4) C13—H13 0.9300
N1—C12 1.335 (6) C23—C19 1.395 (7)
N1—C1 1.341 (7) C23—C24 1.397 (7)
N2—C10 1.328 (6) C22—C21 1.380 (9)
N2—C11 1.376 (6) C22—H22 0.9300
C11—C7 1.388 (7) C24—C16 1.431 (7)
C11—C12 1.414 (7) C16—C17 1.413 (7)
C12—C4 1.423 (7) C16—C15 1.431 (8)
C1—C2 1.413 (8) C17—C18 1.345 (8)
C1—H1 0.9300 C17—H17 0.9300
C2—C3 1.353 (8) C15—C14 1.294 (9)
C2—H2 0.9300 C15—H15 0.9300
C8—C9 1.342 (8) C18—C19 1.443 (9)
C8—C7 1.393 (8) C18—H18 0.9300
C8—H8 0.9300 C14—H14 0.9300
C7—C6 1.452 (7) C19—C20 1.416 (9)
C10—C9 1.386 (8) C20—C21 1.359 (10)
C10—H10 0.9300 C20—H20 0.9300
C3—C4 1.424 (8) C21—H21 0.9300
C3—H3 0.9300 C9—H9 0.9300
Cl1—Zn1—Cl2 93.58 (6) C5—C6—H6 118.8
Cl1—Zn1—Cl3 102.79 (5) C7—C6—H6 118.8
Cl2—Zn1—Cl3 92.74 (5) C6—C5—C4 120.8 (5)
N1—Zn1—Cl1 92.42 (13) C6—C5—H5 119.6
N2—Zn1—Cl1 163.25 (11) C4—C5—H5 119.6
N1—Zn1—Cl2 170.64 (13) C13—N4—C24 118.5 (5)
N2—Zn1—Cl2 92.27 (12) C13—N4—Zn2 128.7 (4)
N1—Zn1—Cl3 92.93 (11) C24—N4—Zn2 112.8 (3)
N2—Zn1—Cl3 92.59 (11) C22—N3—C23 118.8 (5)
N1—Zn1—N2 80.04 (17) C22—N3—Zn2 129.3 (4)
Cl3—Zn2—Cl4 93.71 (6) C23—N3—Zn2 111.8 (3)
N3—Zn2—Cl3 172.83 (13) N4—C13—C14 121.8 (5)
N4—Zn2—Cl3 92.09 (12) N4—C13—H13 119.1
N3—Zn2—Cl4 93.28 (14) C14—C13—H13 119.1
N4—Zn2—Cl4 172.95 (12) N3—C23—C19 122.5 (5)
N3—Zn2—N4 81.04 (16) N3—C23—C24 116.4 (4)
Zn2—Cl3—Zn1 90.98 (4) C19—C23—C24 121.1 (5)
C12—N1—C1 118.3 (5) N3—C22—C21 121.3 (6)
C12—N1—Zn1 113.6 (3) N3—C22—H22 119.4
C1—N1—Zn1 128.0 (4) C21—C22—H22 119.4
C10—N2—C11 117.2 (4) N4—C24—C23 117.9 (4)
C10—N2—Zn1 129.8 (4) N4—C24—C16 122.6 (5)
C11—N2—Zn1 113.1 (3) C23—C24—C16 119.4 (5)
N2—C11—C7 122.6 (5) C17—C16—C15 125.8 (5)
N2—C11—C12 115.5 (4) C17—C16—C24 118.5 (6)
C7—C11—C12 122.0 (5) C15—C16—C24 115.7 (5)
N1—C12—C11 117.7 (4) C18—C17—C16 121.9 (6)
N1—C12—C4 124.2 (5) C18—C17—H17 119.1
C11—C12—C4 118.0 (5) C16—C17—H17 119.1
N1—C1—C2 122.5 (6) C14—C15—C16 120.6 (5)
N1—C1—H1 118.8 C14—C15—H15 119.7
C2—C1—H1 118.8 C16—C15—H15 119.7
C3—C2—C1 118.5 (6) C17—C18—C19 120.4 (6)
C3—C2—H2 120.7 C17—C18—H18 119.8
C1—C2—H2 120.7 C19—C18—H18 119.8
C9—C8—C7 120.8 (5) C15—C14—C13 120.8 (6)
C9—C8—H8 119.6 C15—C14—H14 119.6
C7—C8—H8 119.6 C13—C14—H14 119.6
C11—C7—C8 117.2 (5) C23—C19—C20 117.0 (6)
C11—C7—C6 116.8 (5) C23—C19—C18 118.6 (6)
C8—C7—C6 126.0 (5) C20—C19—C18 124.4 (6)
N2—C10—C9 123.2 (5) C21—C20—C19 118.2 (6)
N2—C10—H10 118.4 C21—C20—H20 120.9
C9—C10—H10 118.4 C19—C20—H20 120.9
C2—C3—C4 121.5 (5) C20—C21—C22 122.2 (7)
C2—C3—H3 119.3 C20—C21—H21 118.9
C4—C3—H3 119.3 C22—C21—H21 118.9
C5—C4—C12 119.9 (5) C8—C9—C10 119.1 (6)
C5—C4—C3 125.2 (5) C8—C9—H9 120.5
C12—C4—C3 114.9 (5) C10—C9—H9 120.5
C5—C6—C7 122.4 (5)
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C1—H1···Cl1 0.93 2.68 3.229 (7) 118
C6—H6···Cl2i 0.93 2.77 3.525 (6) 139
C10—H10···Cl2 0.93 2.68 3.235 (6) 119
C13—H13···Cl3 0.93 2.62 3.200 (6) 121
C17—H17···Cl3ii 0.93 2.67 3.500 (7) 149
C18—H18···Cl4ii 0.93 2.82 3.742 (7) 173
C22—H22···Cl4 0.93 2.68 3.238 (6) 119
Symmetry codes: (i) x−1/2, y+1/2, z; (ii) x+1/2, y−1/2, z.
Table 1 Selected geometric parameters (Å, °)
Zn1—Cl1 2.2629 (16)
Zn1—Cl2 2.2596 (15)
Zn1—Cl3 2.7049 (14)
Zn1—N1 2.041 (4)
Zn1—N2 2.046 (4)
Zn2—Cl2 2.8525 (15)
Zn2—Cl3 2.2839 (14)
Zn2—Cl4 2.2545 (14)
Zn2—N3 2.031 (4)
Zn2—N4 2.032 (4)
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C1—H1⋯Cl1 0.93 2.68 3.229 (7) 118
C6—H6⋯Cl2i 0.93 2.77 3.525 (6) 139
C10—H10⋯Cl2 0.93 2.68 3.235 (6) 119
C13—H13⋯Cl3 0.93 2.62 3.200 (6) 121
C17—H17⋯Cl3ii 0.93 2.67 3.500 (7) 149
C18—H18⋯Cl4ii 0.93 2.82 3.742 (7) 173
C22—H22⋯Cl4 0.93 2.68 3.238 (6) 119
Symmetry codes: (i) ; (ii) .
==== Refs
References
Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1–19.
Bruker (2000). SMART, SAINT and SADABS Bruker AXS Inc., Madison, Wisconsin, USA.
Flack, H. D. (1983). Acta Cryst. A39, 876–881.
Li, H., Yin, K.-L. & Xu, D.-J. (2005). Acta Cryst. C61, m19–m21.
Pan, T.-T. & Xu, D.-J. (2004). Acta Cryst. E60, m56–m58.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Wall, M., Linkletter, B., Williams, D., Lebuis, A.-M., Hynes, R. C. & Chin, J. (1999). J. Am. Chem. Soc.121, 4710–4711.
Wu, Z.-Y., Xue, Y.-H. & Xu, D.-J. (2003). Acta Cryst. E59, m809–m811. | 21583798 | PMC2977612 | CC BY | 2021-01-04 19:00:57 | yes | Acta Crystallogr Sect E Struct Rep Online. 2009 Apr 25; 65(Pt 5):m567 |
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Indian J UrolIJUIndian Journal of Urology : IJU : Journal of the Urological Society of India0970-15911998-3824Medknow Publications India 21116370IJU-26-43410.4103/0970-1591.70589Case ReportSuccessful microsurgical penile replantation following self amputation in a schizophrenic patient Gyan Saurabh Sushma Sagar Maneesh Singhal Rajesh Sagar 1Misra MC Department of Surgical Disciplines, JPN Apex Trauma Centre, India1 Department of Psychiatry, All India Institute of Medical Sciences, IndiaFor correspondence: Dr. Gyan Saurabh, Department of Surgical Disciplines, JPN Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi – 110 029, India. E-mail: [email protected] 2010 26 3 434 437 © Indian Journal of Urology2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Amputation of the penis is a rare condition reported from various parts of the world as isolated cases or small series of patients; the common etiology is self-mutilating sharp amputation or an avulsion or crush injury in an industrial accident. A complete reconstruction of all penile structures should be attempted in one stage which provides the best chance for full rehabilitation of the patient. We report here a single case of total amputation of the penis in an acute paranoid schizophrenic patient. The penis was successfully reattached using a microsurgical technique. After surgery, near-normal appearance and function including a good urine flow and absence of urethral stricture, capabilities of erection and near normal sensitivity were observed.
Genital self mutilationpenile replantationschizophreniamanagement
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INTRODUCTION
Total penile amputation is an uncommon injury.[1–6] About 87% of the patients reported had psychiatric problems. Self-amputation of external genitals is also known as Klingsor syndrome.[4–7] A few patients had poor gender identity feeling themselves inadequate as males. Some cases arise from felonious assault by jealous homosexual lovers.[1–6] In 1970 in Thailand, an epidemic was seen, of penile amputation as punishment for philandering by humiliated wives.[2–6] Microvascular penile replantation offers the best prospect for restoration of micturition function, return of sensations and erectile functions. This case highlights the management of such a patient not only in the operative room but also in the emergency resuscitation room.
CASE REPORT
We report the case of a 25-year-old man with acute paranoid schizophrenia who presented in our emergency with an alleged history of cutting his penis with a shaving blade 2 cm distal from the mons pubis. Immediately after the self-mutilation the amputated penis was kept in a clean plastic bag by the patient’s brother. The patient presented at our institution four hours later. Bleeding from the proximal penile stump was stopped by a tourniquet. The patient was prepared for general anesthesia. Intravenous administration of 2 gm Cefoperazone+Sulbactum along with 500 mg metronidazole was given. The patient was given 1500 units of anti-tetanus serum and 2 ml of tetanus toxoid. Four units each of red blood cells (RBC), fresh frozen plasma (FFP) and platelets were arranged. As the patient had lost blood before coming to our trauma center, we transfused one unit each, of RBC and FFP, in the emergency room. We found a clear cut through all penile structures without major lacerations at approximately 2 cm from the mons pubis [Figures 1 and 2]. Gross cleaning of the wound was followed by meticulous debridement using an operating microscope with assistant optic. After identification of all main vessels, the distal and proximal ends of both dorsal arteries were clipped and both ends of the urethra, together with the corpus spongiosum, mobilized. The amputate was then put on an 18F silicone Foley catheter [Figure 3], which was passed into the patient’s bladder. To achieve a stable basis, the tunica albuginea of both corpora cavernosa and the septum were attached by running suture using 3-0 vicryl [Figure 4]. The deep corporeal arteries were identified but not anastomozed. After irrigation with heparinized saline, both deep dorsal arteries were anastomozed with 9-0 prolene sutures [Figure 5]. Next the deep dorsal vein and the two nerves were anastomozed using 9-0 prolene for the vein and 10-0 prolene for the nerves. The urethra was repaired by spatulated end to end anastomosis with interrupted 4-0 vicryl sutures. Buck’s fascia was closed with 4-0 vicryl [Figure 6] and then the superficial vein was anastomozed with 9-0 prolene. Finally the skin was closed [Figure 7]. Next, a gentle pressure dressing was applied and the penis was fixed in an upright fashion. Transurethral catheter was kept for 21 days. Suprapubic cystostomy was done [Figure 8]. Five hundred ml per day, of low molecular dextran, was given for three days to reduce blood viscosity, decrease platelet adhesion, and promote antithrombotic property. On the first postoperative day, swelling at the anastomozed area was seen. Patient was taken to the operation theatre (OT) and removal of skin sutures, evacuation of hematoma and fasciotomy on anterior and lateral aspect of penis was performed. On the fourth postoperative day, patient was again taken to the OT for debridement of necrotic penile skin and application of split thickness skin graft over the raw area. After the first two post-operative weeks the patient developed a mummification of the tip of the glans that had to be resected. More than 80% of the glans remained intact. Retrograde urethrography is performed and Suprapubic cystostomy was removed after six weeks.
Figure 1 Self amputated distal part of penis
Figure 2 Proximal penile stump
Figure 3 Passage of Foley’s Catheter through urethral meatus of amputated penis into transected urethral opening in the proximal stump
Figure 4 Anastomosis of corpora cavernosa of proximal and distal penile stump
Figure 5 Anastomosis of dorsal artery and dorsal vein of penis
Figure 6 Muscular and subcutaneous repair
Figure 7 Final repair: Post aspect
Figure 8 Final repair: Anterior aspect
Postoperative psychiatric consultation was done and Olanzapine 10mg and Clonazepam 0.5mg were started.
At three-month follow-up examination [Figure 9], he reported infrequent nocturnal penile tumescence. At one-year follow-up [Figure 10], retrograde urethrography showed normal urethra with no stricture formation. Penile Color Doppler showed normal blood flow in the dorsal vessels and a normal Doppler waveform changes were seen in the cavernosal arteries during the onset of erection suggesting a good collateral formation in the unanastomozed cavernosal arteries as well.
Figure 9 At follow-up of 3 months
Figure 10 At one-year follow-up
Penile sensations showed recovery with appreciation of fine touch. The patient reported the restoration of his penile erection and ejaculation during sexual intercourse.
DISCUSSION
The first documented case of macroscopic penile replantation was reported in 1929 by Ehrlich.[4] Cohen et al. reported the first microvascular replantation of penis in 1977.[8] A review of the literature revealed that 80 cases underwent penile replantation, of which 30 cases underwent microsurgical replantation since 1970. These 30 cases have been reported to be of higher quality in terms of both functional and aesthetic result.[45] Many factors contribute to favorable final outcomes.[9] Analysis of our case revealed that the cleanly incised injury with a short duration of cold ischemia was an important factor that influenced the outcome. Another factor was the concept of microsurgical reapproximation. The macrosurgical replantation of the penis depends on corporal sinusoidal blood flow with the distal amputated part as a composite graft leading to high complication rates of skin necrosis, fistula formation, loss of sensations and erectile dysfunction. In contrast, the microsurgical approximation of the penile shaft structures provides early restoration of blood flow with the best prospects for graft survival, normal erectile function and optimal benefits with fewer complications.[4] Another critical factor for the success of replantation was the adequacy of venous outflow and the sequence of microsurgical anastomosis. Due to the dual vascular drainage in the penis, the superficial and deep dorsal veins, tributaries of saphenous and santorini plexus respectively, were both anastomozed for good venous return. The return of penile sensations over the glans was as expected in the yearly follow-up of our case with a distal amputated length of approximately 8 cm of total penile length. In our opinion, another important factor was the critical postoperative monitoring of the replantation. Timely intervention was done in the form of release incisions to relieve edema and maintain vascularity of the penis. The initial raw areas may appear as disfiguring but the final result was satisfactory, with near uniform girth of the penile shaft. We suggest similar measures to protect the anastomosis and prevent failure. Prophylactic release incisions can be an option when regular monitoring is not contemplated.
The adverse effect seen in our case was the skin loss due to necrosis of the proximal part of the penile skin, probably because we had anastomozed only the deep dorsal arteries, which are branches of the internal iliac artery. The external pudendal vessels were not anastomozed. It may be advisable to anastomoze the superficial system also to avoid skin necrosis. The microsurgical restoration of penile vascularity provides early restoration of blood flow with the best prospects for graft survival, normal erectile function and optimal benefits due to fewer complications.
CONCLUSION
The current concept of microvascular replantation for penile amputation is the treatment of choice with the best prospects for cosmetic restoration, physiological micturition and preservation of sensation and erectile function.
Source of Support: Nil
Conflict of Interest: None declared.
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REFERENCES
1 Jordan GH Gilbert DA Management of amputation injuries of the male external genitalia Urol Clin North Am 1989 16 359 67 2652860
2 Bhangananda K Chaiyavatana T Pongnumkul C Surgical management of an epidemic of penile amputations in Siam Am J Surg 1983 146 376 82 6614331
3 Kochakarn W Muangman V Krauwit A Traumatic penile amputation: Results with primary reattachment J Urol 1997 157 857
4 Babaei AR Safarinejad RM Penile replantation-Science or myth? A systematic review J Urol 2007 4 62 5
5 Volker BG Maier S Successful penile replantation following auto amputation twice Int J Impot Res 2002 14 197 8 12058248
6 Kochakarn W Traumatic amputation of penis Braz J Urol 2000 26 385 9
7 Schweitzer I Genital self amputation and the klingsor syndrome Aust Nz J Psychiatry 1990 24 566 9
8 Cohen BE May JW Dalsy JS Young HH Successful clinical replantation of an acute amputated penis by microvascular repair Plast Reconstr Surg 1997 59 276 80 834785
9 Darewicz B Galek L Darewicz Malezyk E Successful microsurgical replantation of an amputated penis Int Urol Nephrol 2001 33 385 6 12092662 | 21116370 | PMC2978450 | CC BY | 2021-01-04 19:38:14 | yes | Indian J Urol. 2010 Jul-Sep; 26(3):434-437 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2110335910-PONE-RA-20622R110.1371/journal.pone.0014020Research ArticleNeuroscience/Neurobiology of Disease and RegenerationNeuroscience/Neuronal and Glial Cell BiologyNeurological Disorders/Movement DisordersAlterations in mGluR5 Expression and Signaling in Lewy Body Disease and in Transgenic Models of Alpha-Synucleinopathy – Implications for Excitotoxicity mGluR5 in A-SynucleinopathyPrice Diana L.
1
3
¤
Rockenstein Edward
1
Ubhi Kiren
1
Phung Van
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4
MacLean-Lewis Natalie
3
4
Askay David
1
3
Cartier Anna
1
Spencer Brian
1
Patrick Christina
1
Desplats Paula
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Ellisman Mark H.
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3
4
Masliah Eliezer
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2
*
1
Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America
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Department of Pathology, University of California San Diego, La Jolla, California, United States of America
3
National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California, United States of America
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Center for Research in Biological Systems, University of California San Diego, La Jolla, California, United States of America
Okazawa Hitoshi EditorTokyo Medical and Dental University, Japan* E-mail: [email protected] and designed the experiments: DLP ER PD EM. Performed the experiments: DLP ER NML DA AC CP PD EM. Analyzed the data: DLP ER KU VP NML DA BS CP PD EM. Contributed reagents/materials/analysis tools: DLP ER VP NML DA BS MHE EM. Wrote the paper: DLP KU EM. Interpretation of data, drafting, and final approval and preparation of manuscript for publication: KU.
¤ Current address: ACADIA Pharmaceuticals Inc., San Diego, California, United States of America
2010 16 11 2010 5 11 e140204 7 2010 19 10 2010 Price et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are neurodegenerative disorders of the aging population characterized by the abnormal accumulation of alpha-synuclein (alpha-syn). Previous studies have suggested that excitotoxicity may contribute to neurodegeneration in these disorders, however the underlying mechanisms and their relationship to alpha-syn remain unclear. For this study we proposed that accumulation of alpha-syn might result in alterations in metabotropic glutamate receptors (mGluR), particularly mGluR5 which has been linked to deficits in murine models of PD. In this context, levels of mGluR5 were analyzed in the brains of PD and DLB human cases and alpha-syn transgenic (tg) mice and compared to age-matched, unimpaired controls, we report a 40% increase in the levels of mGluR5 and beta-arrestin immunoreactivity in the frontal cortex, hippocampus and putamen in DLB cases and in the putamen in PD cases. In the hippocampus, mGluR5 was more abundant in the CA3 region and co-localized with alpha-syn aggregates. Similarly, in the hippocampus and basal ganglia of alpha-syn tg mice, levels of mGluR5 were increased and mGluR5 and alpha-syn were co-localized and co-immunoprecipated, suggesting that alpha-syn interferes with mGluR5 trafficking. The increased levels of mGluR5 were accompanied by a concomitant increase in the activation of downstream signaling components including ERK, Elk-1 and CREB. Consistent with the increased accumulation of alpha-syn and alterations in mGluR5 in cognitive- and motor-associated brain regions, these mice displayed impaired performance in the water maze and pole test, these behavioral alterations were reversed with the mGluR5 antagonist, MPEP. Taken together the results from study suggest that mGluR5 may directly interact with alpha-syn resulting in its over activation and that this over activation may contribute to excitotoxic cell death in select neuronal regions. These results highlight the therapeutic importance of mGluR5 antagonists in alpha-synucleinopathies.
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Introduction
Movement disorders with parkinsonism and cognitive impairment continue to be a significant neurological problem in the aging population. While patients with classical Parkinson's Disease (PD) present with tremor, motor deficits and autonomic dysfunction(s), others patients develop cognitive alterations including dementia. Patients that present first with cognitive impairments followed by development of parkinsonism are denominated dementia with Lewy bodies (DLB) to distinguish them from patients with PD dementia (PDD). Jointly this heterogeneous group of disorders is referred to as Lewy body disease (LBD) [1]. These conditions are associated with progressive and selective loss of dopaminergic and non-dopaminergic cells [2] and the formation of Lewy bodies (LBs) and Lewy neurites containing fibrillar alpha-synuclein (alpha-syn) [3], [4], [5], [6], [7], [8] in cortical and subcortical regions [9], [10], [11]. Previous studies have suggested that excitotoxicity may contribute to neurodegeneration in these disorders however the underlying mechanisms and their relationship to alpha-syn remain unclear.
Synucleins are a family of related proteins including alpha-, beta-, and gamma-synuclein. Alpha-syn is a 14 kDa 'naturally unfolded protein' [12], [13] abundant at the presynaptic terminal [14] and likely plays a role in modulating vesicular synaptic release [15]. Abnormal accumulation of alpha-syn is thought to be centrally involved in the pathogenesis of both sporadic and inherited forms of parkinsonism as mutations and multiplications in the alpha-syn gene have been associated with rare familial forms of PD [4], [8], [16]. In addition, over expression of alpha-syn in transgenic (tg) mice [17], [18], [19] and Drosophila
[20] recreates several pathological and dysfunctional motor performance features of PD. Recent studies have shown that accumulation of oligomeric, rather than polymeric (fibrilar) forms of alpha-syn in the synapses and axons may be responsible for neuronal dysfunction and degeneration [21], [22], [23].
In addition to the modulation of vesicular synaptic release, alpha-syn has been shown to regulate dopaminergic neurotransmission (reviewed by [24]) and to be involved in dopamine release [25], [26] whilst dopamine in turn, has been reported to promote alpha-syn oligomerization [27], [28]. These interactions between dopamine and alpha-syn may help explain the selective vulnerability of the dopaminergic system in PD. Recent studies have demonstrated that, in addition to well-documented dopaminergic alterations, other neurotransmitter systems are also dysregulated in PD and DLB. For example, altered glutamatergic neurotransmission within basal ganglia circuitry is thought to contribute to the clinical presentation of parkinsonian-related motor symptoms, though the mechanisms underlying this are not yet fully understood. Abnormal activation of group I metabotropic receptors (mGluR1 and mGluR5) within the basal ganglia circuitry has been proposed to account for cognitive and motor alterations in patients with DLB [29], [30], [31]. mGluR5 in particular has attracted considerable interest because of its potential involvement in Alzheimer's Disease (AD) [32] and PD [33], [34], its role in learning and memory [35], [36], and its abundant expression in the frontal cortex, limbic system, and caudoputamen [37]—brain regions selectively affected in AD and PD.
Further support for a role of group I mGluR receptors in the pathogenesis of PD and DLB steams from studies showing that mGluR5 antagonists ameliorate the behavioral alterations in animal model of parkinsonism [38], [39], [40], [41] and are neuroprotective against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in animals [42], [43]. Mice lacking the mGluR5 receptors also display reduced MPTP toxicity [43].
Although extensive behavioral pharmacology studies with mGluR5 antagonists in PD-like animal models have been performed, surprisingly very limited information is available as to the potential role of alterations in mGluR's in the pathogenesis of the excitotoxicity in PD or DLB patients, or alpha-syn tg animal models. Here we describe studies of mGluR5 expression the brains of patients with DLB or PD and in alpha-syn over-expressing tg mice, and discuss a role for altered mGluR5 expression in excitotoxicity. Furthermore, we present evidence that mGluR5 receptor antagonism is capable of ameliorating the behavioral deficits observed in alpha-syn tg mice. Taken together these results support the notion that mGluRs play an important role in the pathogenesis of disorders with alpha-syn accumulation and that mGluR5 receptors may be an important target for therapeutic intervention.
Materials and Methods
Human specimens, Neuropathological Evaluation and Criteria for diagnosis
A total of 24 cases (n = 8 non-demented controls; n = 8 DLB and n = 8 PD cases) were included for the present study. Autopsy material was obtained from patients (Table 1) studied neurologically and psychometrically at the Alzheimer Disease Research Center/University of California, San Diego (ADRC/UCSD). The last neurobehavioral evaluation was performed within 12 months before death and included Blessed score, Mini Mental State Examination (MMSE) and dementia-rating scale (DRS) [44], [45]. Brains were processed and evaluated according to standard methods [46]. At autopsy, brains were divided sagitally, the left hemibrain was fixed in formalin of 4% paraformaldehyde (PFA) for neuropathological analysis and the right frozen at −70°C for subsequent neurochemical analysis. Paraffin sections from 10% buffered formalin-fixed neocortical, limbic system and subcortical material stained with hematoxylin and eosin (H&E), thioflavine-S, ubiquitin (Dako, Carpinteria, CA) and α-syn (Millipore, Temecula, CA) were used for routine neuropathological analysis that included assessment of plaques, tangles, Lewy bodies and Braak stage [46]. The diagnosis of DLB was based in the initial clinical presentation with dementia followed by parkinsonism and the presence of alpha-syn and ubiquitin-positive LBs in cortical and subcortical regions [47], [48]. The diagnosis of PD was based on the initial presentation with parkinsonism and presence of alpha-syn and ubiquitin positive LBs in subcortical regions.
10.1371/journal.pone.0014020.t001Table 1 Summary of clinical neurobehavioral and post-mortem features for human samples.
Group N Age(years, mean ± SEM) GenderM/F PMT(hours, mean ± SEM) Braak stage Blessed score(mean ± SEM) Brain weight (grams, mean ± SEM) LBs neocortex(score) LBs midbrain(score)
Control 8 76 (±10) 2/3 8 (±3) 0 0 1200 (±97) 0 0
Dementia with Lewy bodies 8 83 (±5) 4/1 9 (±2) III-IV 28 (±3) 1216 (±85) 3+ 2+
Parkinson's Disease 8 77 (±8) 3/2 6 (±2) 0 5 (±2) 1276 (±128) 0 3+
LBs = Lewy bodies; PMT = postmortem time, SEM = standard error of the mean.
Transgenic mouse lines
Transgenic mice over expressing wildtype human (h) alpha-syn under the control of the platelet-derived growth factor (PDGF) (D-line; [19]) and the mThy1 (line 61; [49]) promoters were used. The PDGF-alpha-syn tg mice were selected because they display accumulation of α-syn in the frontal cortex and limbic system similar to DLB accompanied by behavioral deficits, early motor alterations, loss of dopaminergic terminals and formation of inclusion bodies [19]. The mThy1-alpha-syn tg mice were selected because they display more extensive α-syn accumulation in the frontal cortex, limbic system and subcortical regions including the basal ganglia and the substantia nigra. For the immunoblot and immunochemical studies a total of 24 mice were used (n = 8 non-tg; n = 8 PDGF-alpha-syn and n = 8 mThy1-alpha-syn tg) age 6 months old. For the behavioral and therapeutic studies a total of 36 animals were utilized (n = 12 non-tg; n = 12 PDGF-alpha-syn and n = 12 mThy1-alpha-syn tg) at 9 months of age. From each of the groups half of the mice (n = 6) were treated with vehicle only and the other half (n = 6) were treated with MPEP (2-Methyl-6-(phenylethynyl)-pyridine).
Treatment with mGluR5 antagonist and Behavioral testing
For these experiments MPEP, a selective and systemically active mGluR5 receptor antagonist, was used to test the effects of mGluR5 receptor antagonism on behavioral performance [50]. MPEP HCl was obtained from Tocris Biosciences (Ellisville, MO; Catalog #1212), and dissolved in 0.9% saline solution at a dose of 20 mg/ml. This dose was chosen based on reports in the literature demonstrating effective dose ranges for behavioral studies in mice without adverse effects [51], [52]. The MPEP solution was sonicated to ensure complete dilution and the solution was used the same day. MPEP was administered by intraperitoneal (IP) injection, the animals were weighed on the day of testing and the appropriate volume was determined and recorded for each animal (approximately 0.2 ml/animal). A 30-minute pre-injection time was used, and administration times were staggered to account for this time prior to testing. The behavioral effects of MPEP in the α-syn over-expressing tg mice were tested in the vertical pole test (motor test) and the Morris water maze (learning and memory test).
The pole test was divided into 3 phases, including a pretest (with saline injection), a challenge with MPEP administration, and a reassessment (no injections) to determine performance one week after MPEP administration. The percentage change in performance from Test 1 to Test 2 was determined by calculating the difference between success ratios on these two separate test days. Animals underwent testing on a vertical pole apparatus to evaluate their ability to negotiate and descend the apparatus. The testing procedures were derived from previous reports [53], [54]. The apparatus consists of a rough-surfaced pole (diameter 1 cm; height 50 cm) inserted perpendicular to a circular platform. The pole was lightly roughened with medium grade sandpaper in between animals to ensure that all animals were presented with a comparable pole surface. The circular platform was covered with multilayered padding to avoid injury to the animals in the event of a fall from the apparatus. The top padded surface was disposable and frequently changed to avoid a soiled surface. Non-tg mice easily complete this task, and to date, our group has noted performance deficits in the alpha-syn tg mice as early as 4 months of age. The pole testing session consisted of 2 training trials followed by 5 test trials (7 trials total). The first 2 untimed trials served to acquaint the animal with the apparatus. The 5 test trials were timed to a maximum of 120 sec. Two timed measures were recorded including: (1) the time until the animal completely turned around and re-oriented towards platform base (T-Turn), and (2) the total time spent on apparatus (T-Total). This procedure was repeated a total of 5 times. The ratio of success for each subject based on the number of times (out of 5 total trials) the apparatus was successfully negotiated was noted.
To evaluate the effects of MPEP on learning and memory in the alpha-syn over-expressing tg mice they were tested in the Morris water maze as previously described [55]. Mice were treated for 28 days with saline vehicle solution of MPEP as a daily IP injection (20 mg/ml, 0.2 ml per animal per day). At day 20 of the treatments mice were tested in the water maze for 8 days. For this purpose a pool (diameter 180 cm) was filled with opaque water (24°C) and mice were first trained to locate a visible platform (days 1–3) and then a submerged hidden platform (days 4–7) in three daily trials 2–3 min apart. Mice that failed to find the hidden platform within 90 seconds were placed on it for 30 seconds. The same platform location was used for all sessions and all mice. The starting point at which each mouse was placed into the water was changed randomly between two alternative entry points located at a similar distance from the platform. On day 7, another visible platform trial was performed to exclude differences in motivation and fatigue. On day 8 the platform was removed (probe test) and mice were tested to evaluate the numbers of entrances and time expended in the target quadrant. Time to reach the platform (latency), path length, and swim speed were recorded with a Noldus Instruments EthoVision video tracking system (San Diego Instruments, San Diego, CA) set to analyze two samples per second. UCSD is an Institutional Animal Care and Use Committee accredited institution and the UCSD Animal Subjects Committee approved the experimental protocol followed in all studies according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.
Immunoblot analysis
The immunoblot procedures for human and transgenic mouse samples were performed as previously described [19]. Briefly, dissected frozen tissues were homogenized, and processed and separated into cytosolic and particulate fractions. The nuclear fraction was obtained by resuspending the pellet (from the 5000 g spin after homogenization) in buffer +1%SDS +1% TritonX. Incubated on ice for 15 minutes, vortexing every 3 minutes, then centrifuged for 10 minutes at 14,000 g at 4°C. The protein concentrations of individual samples were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Approximately 20 ug of each fraction were loaded onto either 3–8% Tris-acetate (mGluR5 human) or 4–12% Bis-Tris (all others) SDS polyacrylamide gel electrophoresis (SDS/PAGE) gels (Invitrogen, Carlsbad, CA), transferred into Immobilon membranes (Millipore, Temecula, CA), blocked with either 3% BSA (for animal samples) or 5% Milk/1% BSA (for human samples) in PBS, and incubated overnight at 4°C with antibodies against mGluR5 (rabbit polyclonal, 1∶1000, Millipore, Temecula, CA), beta-arrestin (rabbit polyclonal, 1∶1000, Cell Signaling, Boston, MA), extracellular signal-regulated kinase (ERK, mouse monoclonal, 1∶1000, Cell Signaling, Boston, MA), phospho-ERK (pERK, mouse monoclonal, 1∶1000 Cell Signaling, Boston, MA), Elk-1 member of ETS oncogene family (Elk-1, rabbit polyclonal, 1∶1000, Cell Signaling, Boston, MA, phospho-Elk-1 (pElk-1, rabbit polyclonal, 1∶1000, Cell Signalling, Boston, MA), cAMP response element-binding (CREB, mouse monoclonal, 1∶1000 Cell Signaling, Boston, MA) and phospho-CREB (pCREB, rabbit polyclonal, 1∶1000 Cell Signaling, Boston, MA). The next day, blots were rinsed several times and incubated with the appropriate secondary antibody on a shaker for 1 hour at room temperature. Membranes were processed using a chemiluminescence kit (Western Lightning Chemiluminescence Reagent Plus; Perkin Elmer, Boston, MA) and imaged using a Versadoc system (Biorad, Hercules, CA). Following imaging, the membranes were processed for further immunolabeling using an antibody re-probing kit (Chemicon, Temecula, CA). Actin loads (Beta-actin, mouse monoclonal, Sigma, St. Louis, MO) were then determined for each membrane as a control for protein loading. All mGluR5 protein levels were normalized using the actin immunoreactivity values. The Quality One™ image analysis program (Biorad, Hercules, CA) was used for quantitative assessment of bands. Statistical analyses were performed using one-way ANOVAs and the level for significance set at p<0.05. Resultant data were graphed using SigmaPlot 9.0 (Systat Software Inc., Richmond, CA). Graphed data are presented as the mean optical density (group means mGluR5/actin immunolevels) ± the standard error of the mean (SEM).
Tissue processing, immunocytochemical analysis and laser scanning confocal microsocopy
Briefly, as previously described [56], free-floating 40 µm thick vibratome sections were washed with Tris buffered saline (TBS, pH 7.4), pre-treated in 3% H2O2, and blocked with 10% serum (Vector Laboratories, Burlingame, CA), 3% bovine serum albumin (Sigma), and 0.2% gelatin in TBS-Tween (TBS-T). For human brains, sections from the frontal cortex, hippocampus and putamen were used; for the mice sagittal sections from the complete brain were studied. Sections were incubated at 4°C overnight with the monoclonal antibodies against mGluR5 (Millipore) and beta-arrestin (Cell Signaling) or the polyclonal antibody against α-syn (Millipore). Sectionswere then incubated in secondary antibody (1∶75, Vector), followed by Avidin D horseradish peroxidase (HRP, ABC Elite, Vector) and reacted with diaminobenzidine (DAB, 0.2 mg/ml) in 50 mM Tris (pH 7.4) with 0.001% H2O2. Control experiments consisted of incubation with pre-immune rabbit serum. To investigate the effects of postmortem delay and fixation on the levels of mGluR5 immunoreactivity, preliminary studies were performed in a subset of cases (n = 5) with postmortem delay ranging from 4–48 h. Immunostained sections were imaged with a digital Olympus microscope and assessment of levels of mGluR5 and arrestin immunoreactivity was performed utilizing the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD). For each case a total of three sections (10 digital images per section at 400x) were analyzed in order to estimate the average number of immunolabeled cells per unit area (mm2) and the average intensity of the immunostaining (corrected optical density).
Double-immunocytochemical analyses was performed utilizing the Tyramide Signal Amplification™-Direct (Red) system (NEN Life Sciences, Boston, MA) to detect alpha-syn. Specificity of this system was tested by deleting each primary antibody. For this purpose, sections were double-labeled with the monoclonal antibodies against alpha-syn (1∶20,000, Millipore) detected with Tyramide Red, and mGluR5 detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1∶75, Vector). All sections were processed simultaneously under the same conditions and experiments were performed twice for reproducibility. Sections were imaged with laser scanning confocal microscope BioRad Radiance 2000 (Hercules, CA) equipped with a Nikon E600FN Ellipse microscope (Japan) and using a Nikon Plan Apo 60x oil objective (NA 1.4; oil immersion).
Tissue section acquisition and immunocytochemistry for large scale mosaic
An additional group of non-tg and PDGF- alpha-syn tg mice (n = 6 per group; 6 month old) were deeply anesthetized with Nembutal™ (pentobarbital) and perfused via intracardiac catheterization. Perfusion with oxygenated Ringer's solution containing 250 U/ml heparin, 0.2 mg/ml xylocaine and 1% dextrose was followed 4% paraformadehyde in 0.1 M phosphate buffer solution (PBS) (both at 37 degrees Celsius). The brains were carefully removed from the skull and postfixed for 1 hour in the same fixative used in the perfusion. The brain was blocked and cut into 2 mm thick sections using an acrylic brain matrix (David Kopf; Tujunga, CA) to facilitate reproducibility of sections. These thick sections were then sectioned into 80 micron thick coronal sections using a Vibratome (VT1000E, Leica Microsystems, Wetzlar, Germany).
Tissue sections were incubated with monoclonal anti-α-syn (1∶250; BD Transduction Laboratories, San Diego, CA) and rabbit anti-mGluR5 (1∶250; Millipore, Temecula, CA) followed by incubation with donkey anti-mouse Alexa Red (1∶100, Molecular Probes, Carlsbad, CA) and donkey anti-rabbit FITC (1∶100, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) overnight at 4C. The immunolabeling procedure consisted of the following steps: (1) 6×5 min rinses in 0.1 M PBS; (2) 1 hr blocking step in PBS containing 3% normal donkey (NDS), 0.1% Triton X-100, 1% fish gelatin, and 1% BSA; (3) 48 hr incubation in primary antibodies diluted in working buffer (PBS, 1% NDS) at 20 degrees C; (4) 6×5 minute rinsed in working buffer; (5) 24 hr incubation in working buffer containing donkey anti-mouse Alexa Fluor 488 (Molecular Probes, Carlsbad, CA) and donkey anti-rabbit RRX (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). (6) 6×10 min rinses in working buffer; (7) 3×10 min rinses in PBS; (8) the sections were free floated onto slides and coverslipped using ProLong mounting media (Invitrogen Molecular Probes, Carlsbad, CA) with DAPI nuclear stain. Controls for the mGluR5 antibody experiments included both preabsorption with the control peptide (Chemicon,Temecula, CA), as well as primary omission studies, which both revealed a lack of non-specific staining (data not shown). Controls for other antibodies used were performed via omission of primary antibodies, and revealed no non-specific staining (data not shown). All steps were conducted at 4°C, on wet ice and with ice-cold solutions.
Acquisition of large-scale mosaic survey images
The wide-field mosaic image data sets were acquired with the multiphoton scanning system described previously [57], [58] and these were used to survey brain regions for further targeted imaging and analyses. For these purpose, sections double labeled with antibodies against mGluR5 and α-syn as described above were used. Representative large scale images were acquired using a customized video-rate multiphoton microscope [59] equipped with a custom automated high precision motorized stage (Applied Precision LLC, Issaquah, WA), which allows for the automatic acquisition of ultra-large field image mosaics in 2 and 3 dimensions [57], [58]. A Nikon Plan Apo TIRF (60×1.45) oil immersion objective was used. These mosaic images are acquired by rastering the specimen along the X, Y, and Z axes, introducing a prescribed amount of overlap between acquired images (in this case 10%) to aid alignment. Unprocessed image data acquired on the high-speed multiphoton microscope is subsequently stored as a single stack of images. The image stack is analyzed using the JAVA-based ImageJ, a freely available software package, using plugins developed at NCMIR for processing, aligning, and assembling these massive datasets. Briefly, each file is separated into three separate.tiff stacks, one for each channel. Each tile of the image mosaic is normalized to eliminate shading gradients, followed by the automatic alignment of individual tiles to form a full size mosaic image of the data for each channel. A “globally optimized,” normalized cross-correlation algorithm is used to achieve sub-pixel alignment accuracy. The assembled mosaics are then combined into one full-scale color image. For 3D imaging, the process is repeated for each wide field image plane in Z. The large-scale images were used to identify regions of interest for confocal imaging studies.
Statistical analyses
One-way ANOVAs were performed for water maze and pole test data using the Statview statistical package (version 5.0.1, Cary, NC). Behavioral and image data were graphed using SigmaPlot 9.0 (Systat Software Inc., Richmond, CA). All data are presented as the mean group value ± the standard error of the mean. The criterion for significance was set at p<0.05.
Results
Regionally selective increases in the levels of mGluR5 in DLB and PD cases
Given the abundant expression of mGluR5 in brain regions involved in AD and PD such as the frontal cortex, limbic system, and caudoputamen [37], it has been proposed that alterations in mGluR5 may be involved in the pathogenesis of AD [32] and PD [33], [34], [35], [36]. Nonetheless the relative levels of mGluR5 expression and alterations in DLB or PD have not been studied. For this purpose, patterns of distribution for mGluR5 and the downstream regulators were evaluated by immunocytochemistry and immunoblot analysis.
mGluR5 immunoreactivity was associated with pyramidal neurons in layers 2–3 of the frontal cortex (Figure 1), in pyramidal neurons in the CA3 region of the hippocampus and in mid-spiny neurons in the caudo-putamen (Figure S1). Compared to control cases, in DLB there was a significant increase in the levels of mGluR5 immunoreactivity in neurons in the frontal cortex (Figure 1A, B, D), hippocampus (Figure S1, A, B, D) and caudo-putamen region (Figure S1E, F, analyzed in H) Compared to controls, in the PD cases, levels of mGluR5 were increased in the caudo-putamen (Figure S1E, G, H). However levels in the frontal cortex and hippocampus were comparable to controls (frontal cortex, Figure 1A, C, D; hippocampus, Figure S1A, C, D). Beta-arrestin is a downstream regulator of mGluR5 recycling. Group I mGluRs undergo rapid internalization after agonist exposure, this internalization is strongly inhibited by the expression of both beta-arrestin and dynamin dominant-negative mutants [60], [61], [62]. Similar to mGluR5, beta-arrestin was associated with pyramidal neurons in layers 2–3 of the frontal cortex (Figure 1). Compared to controls, beta-arrestin immunoreactivity was increased in the DLB cases in the frontal cortex (Figure 1E, F, H). Compared to controls, PD cases displayed no differences in beta-arrestin immunoreactivity in the frontal cortex (Figure 1E, G, H). As expected, compared to control cases, neocortical alpha-syn levels were much higher in DLB and were associated with LBs in these cases as opposed to the neuritic, punctate staining observed in the controls (Figure 1I, J, L). There was no significant difference in neocortical alpha-syn immunoreactivity in the PD cases compared to controls (Figure 1I, K, L).
10.1371/journal.pone.0014020.g001Figure 1 Immunohistochemical analysis of mGluR5, beta-arrestin and alpha-syn in the frontal cortex of Control, DLB and PD cases.
(A, B, C) Representative bright field microscopy images of mGluR5 immunoreactivity in the frontal cortex of control, DLB and PD cases respectively. (D) Analysis of mGluR5 immunoreactivity in the frontal cortex of control, DLB and PD cases. (E, F, G) Representative bright field microscopy images of beta-arrestin immunoreactivity in the frontal cortex of control, DLB and PD cases respectively. (H) Analysis of beta-arrestin immunoreactivity in the frontal cortex of control, DLB and PD cases. (I, J, K) Representative bright field microscopy images of alpha-syn immunoreactivity in the frontal cortex of control, DLB and PD cases respectively. (L) Analysis of alpha-syn immunoreactivity in the frontal cortex control, DLB and PD cases. Scale bar = 30 µM. * Indicates a significant difference between DLB or PD cases compared to control cases (p<0.05, one-way ANOVA and post hoc Fisher). # Indicates a significant difference between DLB and PD cases (p<0.05, one-way ANOVA and post hoc Fisher) (n = 8 cases per group).
By immunoblot analysis, mGluR5 was identified in the membrane fractions from athe frontal cortex, hippocampus and caudate of controls, DLB and PD cases as a double band with an approximate MW of 132 kDa (Figure 2A, C and E). Compared to controls, in the DLB cases, there was a significant increase in mGluR5 levels in the membrane fractions from the frontal cortex (Figure 2A, B), hippocampus (Figure 2C, D) and caudo-putamen (Figure 2E, F). In PD cases, there was an increase in the levels of mGluR5 in the membrane fraction of the caudo-putamen (Figure 2E, F), but levels in the frontal cortex and hippocampus were comparable to controls (Figure 2A–D). No mGluR5 was detected in the cytosolic fractions. In a subset of the PD cases there appeared to be an inverse relationship between monomeric alpha-syn and mGluR5 levels in the membrane fraction from the frontal cortex. This might be driven by the increased oligomerization of alpha-syn in these cases, which might correlate with the increase of mGluR5. Beta-arrestin was identified as a double band both in the membrane (Figure 2A, C and E) and cytosolic (Figure 2G, I, K) fractions with an approximate MW of 50 kDa. Compared to controls, in the DLB cases, there was a significant increase in beta-arrestin levels in the membrane fractions from frontal cortex (Figure 2A, B), hippocampus (Figure 2C, D) and caudo-putamen (Figure 2E, F). In PD cases, compared to controls, there was an increase in the levels of beta-arrestin in the membrane fractions from the hippocampus (Figure 2C, D) and caudo-putamen (Figure 2E, F), but levels in the frontal cortex were comparable to controls (Figure 2A, B). In the cytoplasmic fractions from the frontal cortex beta-arrestin levels were reduced in both DLB and PD cases compared to controls (Figure 2G, H). In cytoplasmic fractions from the hippocampus, beta-arrestin levels were increased in both DLB and PD cases compared to controls (Figure 2I, J). In cytoplasmic fractions from the caudo-putamen region beta-arrestin levels, in comparison to controls, were unchanged in DLB, but higher in the PD cases (Figure 2K, L).
10.1371/journal.pone.0014020.g002Figure 2 mGluR5, beta-arrestin and alpha-syn expression in frontal cortex, hippocampus and caudate of Control, DLB and PD cases.
(A) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the membrane fraction of the frontal cortex from control, DLB and PD cases. (B) Analysis of mGluR5 and beta-arrestin levels in the membrane fraction of the frontal cortex. (C) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the membrane fraction of the hippocampus from control, DLB and PD cases. (D) Analysis of mGluR5 and beta-arrestin levels in the membrane fraction of the hippocampus. (E) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the membrane fraction of the caudate from control, DLB and PD cases. (F) Analysis of mGluR5 and beta-arrestin levels in the membrane fraction of the caudate. (G) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the cytosolic fraction of the frontal cortex from control, DLB and PD cases. (H) Analysis of mGluR5 and beta-arrestin levels in the cytosolic fraction of the frontal cortex. (I) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the cytosolic fraction of the hippocampus from control, DLB and PD cases. (J) Analysis of mGluR5 and beta-arrestin levels in the cytosolic fraction of the hippocampus. (K) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the cytosolic fraction of the caudate from control, DLB and PD cases. (L) Analysis of mGluR5 and beta-arrestin levels in the cytosolic fraction of the caudate. * Indicates a significant difference between DLB or PD cases compared to control cases (p<0.05, one-way ANOVA and post hoc Fisher). # Indicates a significant difference between DLB and PD cases (p<0.05, one-way ANOVA and post hoc Fisher).
Levels of mGluR5 immunolabeling are elevated in the brains of α-syn transgenic mice
Two different lines of tg mice were used, the first express alpha-syn under the PDGF promoter which favors alpha-syn accumulation in the frontal cortex and limbic system with a distributions similar to DLB. The second express alpha-syn under the mThy-1 promoter, which results in greater accumulation of alpha-syn in subcortical nuclei including the basal ganglia and midbrain, analogous to PD. In both non-tg and tg mice lines, mGluR5 immunoreactivity was associated with pyramidal neurons in layers 2–3 of the frontal cortex (Figure 3A–C), in pyramidal neurons in the CA3 region of the hippocampus (Figure S2A–C) and mid-spiny neurons in the caudo-putamen (Figure S2E–G). Compared to non-tg mice, in PDGF-alpha-syn tg mice there was a significant increase in the levels of mGluR5 immunoreactivity in neurons in the frontal cortex (Figure 3A, B, D), hippocampus (Figure S2A, B, D) and caudo-putamen (Figure S2E, F, H). In the mThy1-alpha-syn tg mice levels of mGluR5 immunoreactivity were similarly increased in the frontal cortex (Figure 3A, C, D), hippocampus (Figure S2A, C, D) and caudo-putamen (Figure S2E, G, H). In the control non-tg mice mild levels of beta-arrestin immunoreactivity were detected in pyramidal neurons in layers 2–3 and 5 of the frontal cortex (Figure 3E). Levels of beta-arrestin immunoreactivity were increased in the frontal cortex of PDGF- and mThy1-alpha-syn tg mice, compared to non-tg mice (Figure 3E–H).
10.1371/journal.pone.0014020.g003Figure 3 Immunohistochemical Analysis of mGluR5, beta-arrestin and alpha-syn in the frontal cortex of alpha-syn transgenic mice.
(A, B, C) Representative bright field microscopy images of mGluR5 immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice, respectively. (D) Analysis of mGluR5 immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (E, F, G) Representative bright field microscopy images of beta-arrestin immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice, respectively. (H) Analysis of beta-arrestin immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (I, J, K) Representative bright field microscopy images of alpha-syn immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice, respectively. (L) Analysis of alpha-syn immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. Scale bar = 30 µM. * Indicates a significant difference between alpha-syn tg mice compared to non-tg controls (p<0.05, one-way ANOVA and post hoc Fisher) (n = 8 cases per group).
By immunoblot analysis, mGluR5 was detected in the membrane (Figure 4A) and cytosolic (Figure 4B) fractions as a doublet with an estimated MW of 132 kDa. Consistent with the immunocytochemical studies, levels of mGluR5 in the frontal cortex were elevated in the membrane fractions of the alpha-syn tg mice compared to non-tg controls (Figure 4A, C). Beta-arrestin was detected as a single band at 50 kDa in both the cytosolic and membrane fractions. Compared to non-tg controls, in the PDGF- and mThy1-alpha-syn tg mice the levels of beta-arrestin immunoreactivity were increased the neocotex in both the membrane (Figure 4A, C) and cytoplasmic (Figure 4B, D) fractions.
10.1371/journal.pone.0014020.g004Figure 4 mGluR5, beta-arrestin and alpha-syn expression in frontal cortex of alpha-syn transgenic mice.
(A) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the membrane fraction of the frontal cortex from non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (B) Representative immunoblot of mGluR5, beta-arrestin and alpha-syn expression levels in the cytoplasmic fraction of the frontal cortex from non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (C) Analysis of mGluR5 and beta-arrestin levels in the membrane fraction of the frontal cortex. (D) Analysis of mGluR5 and beta-arrestin levels in the cytoplasmic fraction of the frontal cortex. * Indicates a significant difference between alpha-syn tg mice and non-tg controls p<0.05, one-way ANOVA and post hoc Fisher). # Indicates a significant difference between PDGF-alpha-syn and mThy1-alpha-syn tg mice (p<0.05, one-way ANOVA and post hoc Fisher)(n = 8 mice per group).
mGluR5 interacts with α-syn in the brains of transgenic animals
In order to identify whether the alterations in mGluR5 protein levels were related to transcription changes at the mRNA level, qPCR analysis was conducted. By qPCR, levels of mRNA were comparable between DLB, PD and control cases (Figure S3A) non-tg and PDGF- and mThy1-alpha-syn tg mice (Figure S3B), suggesting that transcriptional events are not involved and that the interaction between alpha-syn and mGluR5 occurs post-transcription/post-translationally. Since, mGluR5 alterations by immunoblot were primarily detected in the membrane rather than cytosolic fractions and were accompanied by increased expression of beta-arrestin suggesting that mGluR5 localization might be altered by alpha-syn. Immunohistochemical double labeling with antibodies against alpha-syn and mGluR5 showed a small degree of alpha-syn co-localization with mGluR5 within the frontal cortex of controls (Figure 5A-C) however the degree of co-localization was greatly increased in the DLB (Figure 5D–F) and PD (Figure 5G–I) cases. Consistent with the human cases, non-tg mice showed a small degree of alpha-syn co-localization with mGluR5 within pyramidal neurons in the frontal cortex of (Figure 5J–L) however the degree of co-localization was greatly increased in the PDGF-alpha-syn tg (Figure 5M–O) and mThy1-alpha-syn tg (Figure 5P–R) mice.
10.1371/journal.pone.0014020.g005Figure 5 Co-immunoprecipitation and co-localization of alpha-syn and mGluR5.
(A, D, G) Representative confocal images of alpha-syn immunolabeling in the frontal cortex of control, DLB and PD cases, respectively. (B, E, H) Representative confocal images of mGluR5 immunolabeling in the frontal cortex of control, DLB and PD cases, respectively. (C, F, I) Co-localization of alpha-syn and mGluR5 immunoreactivity in the frontal cortex of control, DLB and PD cases, respectively. (J, M, P) Representative confocal images of alpha-syn immunolabeling in the frontal cortex of non-tg, PDGF-alpha-syn tg and mThy1-alpha-syn tg mice, respectively. (K, N, Q) Representative confocal images of mGluR5 immunolabeling in the frontal cortex of non-tg, PDGF-alpha-syn tg and mThy1-alpha-syn tg mice, respectively. (L, O, R) Co-localization of alpha-syn and mGluR5 immunoreactivity in the frontal cortex of non-tg, PDGF-alpha-syn tg and mThy1-alpha-syn tg mice, respectively. (S) Representative immunoblot of the co-immunoprecipitation of mGluR5 and alpha-syn using the anti-mGluR5 antibody for the pull-down and the alpha-syn antibody for the detection. (T) Representative immunoblot of the co-immunoprecipitation of mGluR5 and alpha-syn using the alpha-syn antibody for the pull-down and the anti-mGluR5 antibody for the detection. Scale bar = 20 µM.
In order to examine whether mGluR5 and alpha-syn interact (directly or indirectly) co-immunoprecipitation (co-IP) studies were conducted. When samples from the mouse brains of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice were immunoprecipitated with an antibody against mGluR5 and then analyzed by immunoblot with an antibody against alpha-syn, the strongest interaction was observed in the alpha-syn tg mice when compared to non-tg controls (Figure 5S). No interacting bands were detected in control experiments with samples immunoprecipitated with a non-immune IgG or when the tissue sample was excluded. Similarly when the reverse IP was performed, by immunoprecipitating with an antibody against alpha-syn and then analyzing by immunoblot with an antibody against mGluR5, the strongest interaction was again observed in the alpha-syn tg mice when compared to non-tg controls (Figure 5T). No interacting bands were detected in control experiments with samples immunoprecipitated with a non-immune IgG. To further validate the IP, samples immunoprecipitated with the antibody against alpha-syn were analyzed with an antibody against alpha-syn. This study confirmed that in the samples from the alpha-syn tg mice higher levels of alpha-syn immunoreactivity were detected than in the non-tg mice (data not shown).
Wide field mosaic analysis of alpha-syn and mGluR5 distribution in alpha-syn transgenic mice
Wide field mosaic imaging was conducted to examine in a more comprehensive manner the relationship between the distribution of alpha-syn and mGluR5 immunolabeling in several brain regions associated with rodent spatial memory and motor functions. (Figure S4). Patterns of alpha-syn and mGluR5 immunolabeling were examined within the motor cortex, piriform cortex, four sub-regions of striatum, three regions within the hippocampal formation, and the substantia nigra. Alpha-syn immunolabeling in non-tg samples was punctate in nature, and no labeled cell bodies or neurites were observed in any samples. In contrast, alpha-syn immunolabeling in tg mice was noticeably increased, more diffuse in nature, and immunopositive cell bodies, dendrites and other elements in the neuropil were observed in many regions. In comparison, increased mGluR5 immunolabeling was noted in nearly identical regions. The regional statistics are provided in Table 2. Within the motor cortex (Figure S4B) an increase in alpha-syn was noted in tg versus non-tg samples.
10.1371/journal.pone.0014020.t002Table 2 Regional statistics and percentages for α-syn and mGluR5 immunolabeling for data presented in Figure S4.
Mouse CNS region
alpha-syn Immunolabeling
mGluR5 Immunolabeling
Motor Cortex
+95.7%
p<0.05, F(1,11) = 8.99
+120.8%
p<0.01, F(1,11) = 20.64
Piriform Cortex
+5.6%n.s., F(1,9) = 0.28
+53.13%
P<0.01, F(1,9) = 51.59
Dorsomedial Striatum
+275.6%
p<0.01, F(1,14) = 17.60
+159.6%
p<0.01, F(1,14) = 55.63
Dorsolateral Striatum
+199.6%
p<0.01, F(1,14) = 17.22
+150.6%
p<0.01, F(1,14) = 100.54
Ventromedial Striatum
+42.7%
p<0.05, F(1,14) = 8.57
+62.3%
p<0.05, F(1,14) = 4.77
Ventrolateral Striatum
+112.2%
p<0.01, F(1,14) = 8.04
+98.5%
p<0.01, F(1,14) = 38.27
Substantia Nigra
+55.5%
p<0.01, F(1,11) = 7.41
+59.26%
p<0.05, F(1,11) = 7.41
CA1 region
+178.5
p<0.01, F(1,10) = 103.95
+51.52%n.s., F(1,10) = 4.57
CA3 region
+97.8%
p<0.05, F(1,7) = 8.97
+259.3%
p<0.01, F(1,7) = 42.53
Dentate Gyrus
+51.4%
p<0.05, F(1,17) = 6.70
+80.7%
p<0.01, F(1,17) = 32.59
The mGluR5 labeling was diffuse in nature and included immunopositive cell bodies present within the inner granular cell layer. In non-tg samples we observed apparent axonal mGluR5 immunolabeling through the cortical layers presumably on pyramidal cell axons. This pattern was also seen in the tg samples, with a noticeable increase in the amount of staining, as well as an extension of staining to varicosities along the axonal projections through the outer granular layer. Alpha-syn immunolabeling within tg piriform cortex (Figure S4B, C) was also more diffuse, with immunopositive cell bodies and neurites present within the cell body layers II and III. mGluR5 immunolabeling within the piriform cortex of non-tg animals consisted of punctate staining throughout the cell body layer. The levels were increased in tg samples and also included increased axonal staining through the piriform cortical cell body layers II and III. In contrast with the immunostaining patterns observed within the aforementioned cortical regions, the pattern of alpha-syn immunolabeling within tg striatal regions (dorsomedial, dorsolateral, ventromedial and ventrolateral) was punctate, and synaptic like in nature. No immunopositive cell bodies or neurites were observed in any striatal region (Figure S4B, D). Striatal mGluR5 immunolabeling in tg samples was increased within the neuropil, and also included perineuronal staining not observed in the non-tg. Sparse, small, punctate alpha-syn immunolabeling was found in the substantia nigra pars compacta (SNc) in non-tg samples. SNc immunolabeling was increased in tg samples, with the addition of tyrosine hydroxylase-like labeled projections innervating the substantia nigra pars reticulata (Figure S4B). mGluR5 immunolabeling was increased in the SNc and consisted of labeled fibers and neurites, in a pattern similar to TH immunoreactivity.
Within the hippocampal formation we examined alpha-syn and mGluR5 immunolabeling within CA1, CA3 and dentate gyrus. Within the CA1 field of non-tg samples, alpha-syn immunolabeling within the neuropil was largely confined to the area immediately surrounding the pyramidal cell layer. Within the pyramidal cell layer of CA1, alpha-syn immunolabeling was present on axonal projections and surrounding cell bodies. mGluR5 labeling within the CA1 largely followed the pattern of alpha-syn immunoreactivity. Axonal mGluR5 staining was also evident through the extent of the pyramidal cell layer with labeled axonal projections towards the stratum radiatum (Figure S4B). Within the CA3 region of non-tg samples, punctate alpha-syn immunolabeling was largely confined to the area adjacent to the stratum lucidum. An expansion of alpha-syn immunoreactivity was seen in tg samples with an increase in staining within the neuropil, and including many immunopositive cell bodies within the stratum lucidum (Figure S4B). In non-tg samples, the pattern of larger punctate mGluR5 immunolabeling corresponded to the distribution of alpha-syn immunoreactivity. Like alpha-syn immunoreactivity in tg animals, mGluR5 immunolabeling was increased in tg mice throughout the neuropil and surrounded cell bodies within the stratum lucidum.
Alpha-syn immunolabeling was sparse within all sub-regions of non-tg dentate gyrus, and was substantially increased within molecular layers, hilus and supra- and infrapyramidal blades of the dentate gyrus (Figure S4B). This included punctate labeling in the neuropil surrounding cell bodies and cytoplasmic labeling of cells within the hilus. mGluR5 labeling of non-tg dentate gyrus included labeling of collateral varicosities within the hilus, diffuse labeling of the neuropil in the molecular layers, and minimal axonal-like labeling through cell body layers of the suprapyramidal and infrapyramidal blades. The intensity of mGluR5 labeling was increased in tg samples and included labeling of intracytoplasmic regions of large neuronal cell bodies, with an approximate shape and size appropriate for interneurons and main apical dendrites within the hilus.
Increased mGluR5 signaling in DLB/PD and alpha-syn transgenic mice
In order to investigate whether the increased mGluR5 level in the DLB and PD cases and in the alpha-syn tg mice was associated with a concomitant increase in the levels of downstream signaling components, levels of total and phosphorylated ERK, Elk–1 and CREB were examined in the cytoplasmic and membrane fractions from the frontal cortex. In the membrane fraction ERK activation (ratio of pERK/tERK) was increased in the DLB and PD cases in comparison to the control cases (Figure 6A, E). Elk-1 activation (ratio of pElk-1/tElk-1) was increased in the membrane fraction from the DLB cases but not PD cases, when compared to controls. (Figure 6A, G). CREB activation (ratio of pCREB/tCREB) was increased in the nuclear fraction from the DLB and PD cases when compared to controls (Figure 6C, I), In the cytoplasmic fraction, both DLB and PD cases displayed increased ERK activation (ratio pERK/totalERK) compared to control cases (Figure 6B, F). No significant differences in Ekl-1 activation were noted between DLB or PD cases and controls (Figure 6B, H). CREB activation was decreased in the soluble fraction from both DLB and PD when compared to control cases (Figure 6D, J). A similar immunoblot to investigate downstream signaling components was conducted on the membrane, cytosolic and nuclear fractions from the frontal cortex of non-tg and alpha-syn tg mice (Figure 7).
10.1371/journal.pone.0014020.g006Figure 6 Immunoblot analysis of ERK, Elk-1 and CREB activity in Control, DLB and PD cases.
(A) Representative immunoblot of phospho-ERK (pERK), total ERK (tERK) phospho-ELK-1 (pELK-1) and total ELK-1 (tELK-1) expression levels in the membrane fraction from the frontal cortex of control, DLB and PD cases. (B) Representative immunoblot of pERK, tERK, pELK-1 and tELK-1 expression in the cytoplasmic fraction from the frontal cortex of control, DLB and PD cases. (C) Analysis of ERK activity (pERK/tERK ratio) in the membrane fraction. (D) Analysis of ERK activity (pERK/tERK ratio) in the cytosolic fraction. (E) Analysis of Elk-1 activity (pElk-1/tElk-1 ratio) in the membrane fraction. (F) Analysis of Elk-1 activity (pElk-1/tElk-1 ratio) in the cytosolic fraction. (G) Representative immunoblot of phospho-CREB (pCREB) and total CREB (tCREB) in the nuclear fraction from the frontal cortex of control, DLB and PD cases. (H) Representative immunoblot of pCREB and tCREB in the soluble fraction from the frontal cortex of control, DLB and PD cases. (I) Analysis of CREB activity in the nuclear fraction. (J) Analysis of CREB activity in the soluble fraction. * Indicates a significant difference between DLB or PD cases compared to control cases. (p<0.05, one-way ANOVA and post hoc Fisher) (n = 8 cases per group).
10.1371/journal.pone.0014020.g007Figure 7 Immunoblot Analysis of ERK, Elk-1 and CREB activity in alpha-syn transgenic mice.
(A) Representative immunoblot of phospho-ERK (pERK), total ERK (tERK) phospho-ELK-1 (pELK-1) and total ELK-1 (tELK-1) expression levels in the membrane fraction from the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (B) Representative immunoblot of pERK, tERK, pELK-1 and tELK-1 expression in the cytoplasmic fraction from the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (C) Analysis of ERK activity (pERK/tERK ratio) in the membrane fraction. (D) Analysis of ERK activity (pERK/tERK ratio) in the cytosolic fraction. (E) Analysis of Elk-1 activity (pElk-1/tElk-1 ratio) in the membrane fraction. (F) Analysis of Elk-1 activity (pElk- 1/tElk-1 ratio) in the cytosolic fraction. (G) Representative immunoblot of phospho- CREB (pCREB) and total CREB (tCREB) in the nuclear fraction from the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (H) Representative immunoblot of pCREB and tCREB in the soluble fraction from the frontal cortex of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice. (I) Analysis of CREB activity in the nuclear fraction. (J) Analysis of CREB activity in the soluble fraction. * Indicates a significant difference between alpha-syn tg mice and non-tg controls p<0.05, one-way ANOVA and post hoc Fisher). # Indicates a significant difference between PDGF-alpha-syn and mThy1-alpha-syn tg mice (p<0.05, one-way ANOVA and post hoc Fisher)(n = 8 mice per group).
The ERK activation was increased in the mThy-1-alpha-syn tg mice in both the membrane (Figure 7A, E) and cytoplasmic (Figure 7B, F) fractions in comparison to non-tg controls, however it did not differ between PDGF-alpha-syn tg mice and non-tg controls on either fraction (Figure 7A, B, E, F). The membrane fraction both the PDGF-alpha-syn and mThy1-alpha-syn tg mice displayed increased levels of Elk-1 activation in comparison to non-tg controls (Figure 7A, G). No Elk-1 activation was detected in the cytoplasmic fraction (Figure 7B, H). CREB activation was increased in the PDGF-alpha-syn and mThy1-alpha-syn tg mice in the nuclear fraction compared to non-tg controls (Figure 7C, I), however in the soluble fraction CREB activation was only increased in the mThy1-alpha-syn tg mice when compared to non-tg controls (Figure 7D, J).
Motor deficits in the pole test apparatus in alpha-syn transgenic mice are ameliorated by the mGluR5 inhibitor MPEP
In the tg mice there is considerable accumulation of alpha-syn in the caudo-putamen region that is accompanied by motor deficits in the pole test. Since it is possible that increased expression of mGluR5 in mid-spine neurons as shown here might play a role in the motor deficits, non-tg and alpha-syn tg mice were treated with the mGluR5 antagonist MPEP and tested in the pole test. This behavioral test requires the subjects to grip and traverse the pole requiring motor strength and coordination [54], [63]. The stringent test parameters help to ensure that tg deficits are not obscured by compensatory responses (e.g. incomplete turns prior to descending the pole apparatus). Compared with non-tg mice, alpha-syn tg mice were impaired in ability to negotiate the pole apparatus as evidenced by a significant longer T-Turn time (Baseline, Figure 8A).
10.1371/journal.pone.0014020.g008Figure 8 Motor and learning/memory deficits in alpha-syn transgenic mice are ameliorated by MPEP administration.
(A) Pole test performance (T-Turn) of the non-tg and PDGF-alpha-syn tg mice, at baseline, following MPEP treatment and at re-test (no treatment). (B) Morris water maze performance of vehicle-treated non-tg and PDGF-alpha-syn tg mice. (C) Morris water maze performance of MPEP- treated PDGF-alpha-syn tg mice. (D) Morris water maze performance of MPEP-treated non-tg mice. * Indicates a significant difference between groups examined (p<0.05, one-way ANOVA and post hoc Fisher) (n = 8 per group).
To assess whether mGluR5 antagonism could ameliorate the pole test behavioral deficit observed in the tg animals, mice were treated with the mGluR5 antagonist MPEP. Following treatment with MPEP T-Turn was comparable between the non-tg and alpha-syn tg mice (MPEP Treatment, Figure 8A). Analysis of the difference between the first and second test days revealed an improvement in T-Turn times of the alpha-syn tg mice as indicated by significant improvement in the success ratio from the first test to second test session (F(1,6) = 6.40; p = 0.0447). When the mice were tested a day after the MPEP treatment (Re-test, no treatment, Figure 8A), once more alpha-syn tg mice displayed impaired performance in the pole test when compared to the non-tg controls (F(1,6) = 18.498; p = 0.0051).
The spatial memory deficits in alpha-syn transgenic mice are ameliorated by the mGluR5 inhibitor MPEP
Though motor deficits are more often highlighted, cognitive deficits associated with alpha-syn accumulation in the limbic system have also been reported in patients with Parkinson's disease dementia and dementia with LB's [56]. These alterations might be associated with the increased expression of mGluR5 in the frontal cortex and hippocampus of the alpha-syn tg mice. To investigate this possibility, mice were tested in the water maze and treated with the mGluR5 antagonist MPEP. During the training period of the test, repeated measures ANOVA revealed no significant differences among the groups path length (F(1,12) = 2.294, p = 0.1557 and F(1,6) = 0.500, p = 0.8060). During the spatial learning period of the test with the platform submerged, vehicle treated alpha-syn tg mice displayed performance deficits when compared to the vehicle treated non-tg controls (Figure 8B). In contrast, MPEP treatment improved the behavioral performance of the alpha-syn tg compared to the vehicle treated alpha-syn tg mice (Figure 8C). The MPEP treated alpha-syn tg mice performance was similar to the non-tg controls. In the last day of testing the platform was removed. The probe test indicated a significant main effect of the genotype on entrances and passes into target zone (F(1,12) = 6.649, p = 0.0242). In contrast, alpha-syn tg mice treated with MPEP exhibited a similar number of entrances and passes into target zone when compared to the non-tg controls. MPEP treatment had no effect on the performance of the non-tg mice (Figure 8D).
A subsequent trial in which the platform was visible, confirmed that both groups were able to locate the platform via visual sighting. These results indicate that the deficit in ability to locate the correct zone, seen in the alpha-syn tg mice, is not attributable to a visual or gross motor deficit, but is instead likely to be a consequence of a memory or spatial navigation defect.
Discussion
Excitoxicity has been proposed to play a role in the mechanisms of neurodegeneration in DLB and PD. The present study showed that alterations in the levels of mGluR5 in selected brain regions in patients with DLB or PD and in alpha-syn transgenic mice might be involved. Specifically, mGluR5 was increased in the frontal cortex, hippocampus and caudate in DLB in and in the caudate in PD, these areas correspond closely with areas displaying increased alpha-syn accumulation. The results of this study are consistent with previous studies that have shown increased expression or activation of glutamate receptors in the acute neurotoxicity rodent [43] and primate [64] models of PD and in other neurodegenerative disorders with protein accumulation such as Amyotrophic lateral sclerosis (mGluR5; [65]), Down's syndrome (mGluR5; [66]), and AD (mGluR2/3; [32], [67], [68], [69]). However, our study is the first to document alterations in mGluR5 in human DLB and PD cases and in alpha-syn tg mice.
The important of metabotropic glutamate receptors, particularly mGluR5, in neurodegenerative disorders is underlined by recent work suggesting that excitotoxicity mediated by metabotropic receptors may be a key mechanism underlying neurodegeneration [70], [71] and by recent experimental therapies based in the development of mGluR5 antagonists [71], [72], [73], [74]. Consistent with this proposed role of mGluR5 in excitotoxicity and the potential therapeutic use of mGluR5 antagonists we show that when the alpha-syn tg mice were treated with the mGluR5 antagonist MPEP their motor and learning/memory deficits were ameliorated. This is in agreement with previous studies showing that mGluR5 inhibitors reduce the motor alterations in rodent models challenged with MPTP or 6-OH DOPA [75], [76], [77], [78] and recent studies in primates showing that MPEP and another mGluR5 antagonist such as MTEP (3-((2-Methyl-4-thiazolyl)ethynyl)pyridine) has beneficial anti-dyskinetic effects in L-Dopa-treated MPTP monkeys [79], [80]. Moreover, co-administration of adenosine 2A and mGluR5 antagonists reverses the behavioral deficits in a reserpinized mouse model of PD [51]. In addition to the effects at reducing motor alterations, MPEP has been shown to reduce the visuo-spatial discrimination deficit induced by bilateral dopamine lesion of the striatum [81]. This same treatment increased contralateral turning induced by L-DOPA in mice bearing unilateral 6-OHDA lesion suggesting that mGluR5 blockade may also have beneficial effects on cognitive deficits induced by dopamine depletion [81]. Therefore, both neurotoxic and transgenic models of parkinsonism support the possibility that antagonism of mGluR5 might be potentially beneficial in the treatment of DLB and PD patients.
mGluR5 has attracted considerable interest due to its abundant expression in the frontal cortex, limbic system, and caudo-putamen [37]—brain regions selectively affected in PD. The vulnerability of selected neuronal populations in PD and DLB patients has been linked to glutamate-mediated excitotoxicity [82]. Consistent with this, the double labeling and mosaic analysis showed that there was some regional specificity to the increased levels of mGluR5 that corresponded to the areas of greater α-syn accumulation and neurodegeneration. For example, in the DLB cases mGluR5 was elevated in the frontal cortex, limbic system and putamen, while in the PD cases the increase was more prominent in the putamen. In the PDGF-alpha-syn tg mice mGluR5 increases were more prominent in the frontal cortex and limbic system while in the mThy1-alpha-syn tg mice mGluR5 was also elevated in the striatum. Of the affected areas mGluR5 was found in pyramidal neurons in the deeper layers of the frontal cortex, CA3 region of the hippocampus and mid-spiny neurons in the striatum. These are all neuronal populations and brain regions selectively affected in DLB and PD patients [11], [83], [84], [85] suggesting that mGluR5 might play a role in the mechanisms of selective neuronal vulnerability in disorders with alpha-syn accumulation.
The precise mechanisms by which alpha-syn accumulation in DLB, PD and tg models might lead to the increase mGluR5 is not completely clear. However the alterations in mGluR5 are most likely post-translational since levels of mGluR5 mRNA did not differ between control and DLB or PD cases or between non-tg and alpha-syn transgenic mice. Thus, it is possible that alpha-syn interacts with mGluR5 to alter the distribution of these receptors at the cell membrane of particular cell populations, which may lead to an increase in mGluR5 activity and glutamate-mediated excitotoxicity in specific brain regions. In support of this interaction hypothesis we showed a greater degree of mGluR5 and alpha-syn co-immunoprecipitation in the alpha-syn tg mice compared to non-tg mice and a greater accumulation of mGluR5 in the membrane fractions from DLB or PD cases and in alpha-syn tg mice compared to control cases or non-tg mice. Moreover the increase in mGluR5 was accompanied by an increase in beta-arrestin, a protein involved in the internalization and recycling of the receptor. Taken together, these results suggest that accumulation of alpha-syn might alter mGluR5 localization at the cell surface, allowing a greater proportion to remain membrane-associated rather than being recycled.
If this is the case, then it is also possible that increased presence of the receptor at the cell surface may lead to over activation of the downstream mGluR5 signaling pathway. mGluR5 is a Group I metabotropic glutamate receptor that couples with phospholipase C (PLC) via Gq-like G-proteins. PLC activates protein kinase-C (PKC) activation via diacylglycerol and PKC goes on to phosphorylate and activate ERK, which activates ElK-1. PLC additionally activates inositol 1,4,5-trisphosphate (IP3) resulting in the release of calcium from intracellular stores such as the endoplasmic reticulum. This calcium in turn activates a number of calcium-responsive proteins such as those involved in the calcium/calmodulin/calmodulin kinase (CaM kiinase) cascade including CaMKinase I and IV which phosphorylate cyclic-AMP response element-binding (CREB) and activate gene expression [86].
We demonstrate that the increased expression of mGluR5 in DLB, PD and alpha-syn tg mice is accompanied by an increase activation of down-stream signaling pathway components including ERK, Elk-1 and CREB. Dysregulated calcium homeostasis has been linked to a number of neurodegenerative diseases including PD and AD [87] and as over activation of the mGluR5 signaling pathway may also contribute to increases in intracellular calcium it might play a role in mGluR5-mediated selective vulnerability.
In addition to the increased expression and over activation of mGluR5, alpha-syn has been reported to interact with individual components of the mGluR5 signaling pathway including ERK and Elk-1 [88], [89], this is supported by the detection of ERK, Elk-1 and alpha-syn in Lewy Body aggregates. Iwata and colleagues have demonstrated that in vitro over expression of alpha-syn reduces ERK-1/2 phosphorylation and activation of ERK-1/2 signaling, eventually resulting in cell death [90]. Therefore it is possible that alpha-syn may modulate mGluR5 activity both directly - by increasing cell surface concentrations of the receptor itself, as evidenced by the concomitant increase in levels of beta-arrestin that may serve to keep greater proportions of the receptor at the cell surface rather than being recycled and indirectly - by binding to the downstream signaling components of the mGluR5 pathway.
Taken together the results from study this support the notion that alpha-syn may directly interact with mGluR5 resulting in its over activation and suggest that this over activation may contribute to excitotoxic cell death in selected neuronal populations expressing higher levels of mGluR5. These results also highlight the potential therapeutic importance of mGluR5 antagonists in disorders characterized by alpha-syn accumulation.
Supporting Information
Figure S1 Hippocampal and caudate levels of mGluR5 in Control, DLB and PD cases. (A–C) Representative images of mGluR5 immunoreactivity in the CA2/3 of the hippocampus from control, DLB and PD cases respectively, analyzed in (D). (E–G) Representative images of mGluR5 immunoreactivity in the caudate from control, DLB and PD cases respectively, analyzed in (H). Scale bar = 50 µM * Indicates a significant difference between DLB or PD cases compared to control cases (p<0.05, one-way ANOVA and post hoc Fisher). # Indicates a significant difference between DLB and PD cases (p<0.05, one-way ANOVA and post hoc Fisher) (n = 8, case per group).
(2.38 MB TIF)
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Figure S2 Hippocampal and caudate levels of mGluR5 in alpha-syn transgenic mice. (A–C) Representative images of mGluR5 immunoreactivity in the CA2/3 of the hippocampus of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice respectively, analyzed in (D). (E–G) Representative images of mGluR5 immunoreactivity in the caudate of non-tg, PDGF-alpha-syn and mThy1-alpha-syn tg mice respectively, analyzed in (H). * Indicates a significant difference between alpha-syn tg mice compared to non-tg controls (p<0.05, one-way ANOVA and post hoc Fisher).
(2.34 MB TIF)
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Figure S3 mGluR5 mRNA levels in Control, DLB and PD cases and alpha-syn transgenic mice. (A) Quantitative real-time PCR (qPCR) analysis of mGluR5 mRNA levels in Control, DLB and PD cases. (B) qPCR analysis of mGluR5 mRNA levels in non-tg, PDGF-alpha-syn tg and mThy1-alpha-syn tg mice.
(0.27 MB TIF)
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Figure S4 High-resolution, large-scale maps of alpha-syn and mGluR5 in alpha-syn transgenic mice. (A) Representative confocal image depicting localization of alpha-syn (green) and mGluR5 (red) immunoreactivity in an alpha-syn tg mouse brain - inset at higher magnification in the right panel. (B) Representative confocal image depicting localization of alpha-syn and mGluR5 immunoreactivity in the motor cortex (mCtx), piriform cortex (pCtx), CA1 region of hippocampus (CA1), CA3 region of hippocampus, (CA3), dentate gyrus (DG), dorsomedial striatum, (dmSTR) and substantia nigra (SN) in non-tg and alpha-syn tg mice respectively. (C) Semi-quantitative analysis of mGluR5 immunoreactivity in the mCtx, pCtx, CA1, CA3, and DG of non-tg and alpha-syn tg mice. (D) Semi-quantitative analysis of mGluR5 immunoreactivity in the dmSTR, dorsolateral striatum (dlSTR), ventromedial striatum (vmSTR), ventrolateral striatum (vlSTR) and SN of non-tg and alpha-syn tg mice. Imaging parameters were kept consistent within regions and data are presented as mean pixel intensity ± SEM. * Indicates a significant difference between non-tg and alpha-syn tg mice (p<0.05, one-way ANOVA and post hoc Fisher).
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The authors thank Drs. Neil Smalheiser, Vetle Torvik, Philip Kahle, and Sheila Fleming for helpful discussions during preparation of the manuscript. We also thank Mr. Mike Mante for animal husbandry, Ms. Amy Paulino for technical training for immunoblot experimental procedures, Ms. Chandra Inglis for assistance in conducting behavioral studies and Mr. Sunny Chow for development of data processing plugins for Image J during the course of these studies.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by The Branfman Family and MJ Fox Foundations, National Center for Research Resources RR004050, National Institute on Deafness and Other Communication Disorders DC03192 (CCDB), RR043050 (Mouse BIRN), National Institute on Aging AG18840, AG022074, AG10435 and National Institutes of Health LM07292. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2115240510-PONE-RA-20666R210.1371/journal.pone.0014163Research ArticleEcology/Conservation and Restoration EcologyEcology/Environmental MicrobiologyEcology/Population EcologyLivestock Drugs and Disease: The Fatal Combination behind Breeding Failure in Endangered Bearded Vultures Failure in Bearded VulturesBlanco Guillermo
*
Lemus Jesús A.
¤
Departamento de Ecología Evolutiva, Museo de Ciencias Naturales (CSIC), Madrid, Spain
Brown Justin EditorUniversity of Georgia, United States of America* E-mail: [email protected] and designed the experiments: GB JAL. Performed the experiments: GB JAL. Analyzed the data: GB JAL. Contributed reagents/materials/analysis tools: GB JAL. Wrote the paper: GB JAL.
¤ Current address: Department of Conservation Biology, Estación Biológica de Doñana, CSIC, Sevilla, Spain
2010 30 11 2010 5 11 e141635 7 2010 29 10 2010 Blanco, Lemus.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health, especially of threatened avian scavengers feeding upon medicated livestock carcasses. We conducted a comprehensive study of failed eggs and dead nestlings in bearded vultures (Gypaetus barbatus) to attempt to elucidate the proximate causes of breeding failure behind the recent decline in productivity in the Spanish Pyrenees. We found high concentrations of multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and nestlings, associated with multiple internal organ damage and livestock pathogens causing disease, especially septicaemia by swine pathogens and infectious bursal disease. The combined impact of drugs and disease as stochastic factors may result in potentially devastating effects exacerbating an already high risk of extinction and should be considered in current conservation programs for bearded vultures and other scavenger species, especially in regards to dangerous veterinary drugs and highly pathogenic poultry viruses.
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Introduction
Environmental pollutants are increasingly documented as a driver of wildlife endangerment due to their roles in organ damage, hormonal disruption and alteration of the immune system [1], [2]. Disease may also facilitate endangerment and extinction at global and local scales, especially when pathogens interact with other drivers such as pollutants [3]. There is increasing concern about the impact of veterinary drugs and livestock pathogens as factors damaging wildlife health [4]–[6], and even causing declines approaching extinction [7]. These threats may be especially detrimental to wildlife as they increasingly concur and interact as a consequence of the elimination of livestock residues containing veterinary pharmaceuticals and resistant pathogens due to growing intensive livestock operations worldwide [6], [8], [9]. In particular, the ingestion of antimicrobials, primarily fluoroquinolones, has been recently related to immunodepression-mediated acquisition of opportunistic pathogens and disease, as well as to organ damage in nestling vultures [6], [10], [11]. Fluoroquinolone residues have also been found in avian scavenger eggs and are associated with severe alterations in the development of embryo cartilage and bones that could preclude embryo movement and subsequently normal development, pre-hatch position and successful hatching [12]. Therefore, antimicrobials and other drugs may negatively affect embryo and nestling health with potentially devastating consequences on breeding success and conservation of vultures and other threatened avian scavengers.
The bearded vulture (Gypaetus barbatus) is one of the most endangered birds in Europe, with a main stronghold in the Pyrenees. Increasing declines in productivity (average number of fledglings raised per territorial pair) have recently been reported in the Spanish Pyrenees associated with habitat saturation processes [13], [14]. Given that bearded vultures may raise only one fledgling per breeding attempt, this productivity decline should be linked to increasing breeding failure when the proportion of territorial pairs that are breeding does not greatly vary with time [15]. The proximate mechanisms by which density can affect productivity have been investigated, including habitat heterogeneity, with progressively poorer territories being used, territory shrinkage and interference with breeders and floaters [13]. However, the proximate causes of breeding failure are poorly known despite the long-term interests in the conservation of this species [16]. To evaluate these causes, the examination of failed eggs and dead nestlings is imperative, including the study of the presence and impact of injury, developmental problems, poor nutritional condition, pollutants, organ damage, pathogens causing disease, etc. in order to determine the most likely cause of breeding failure.
Here, we conducted a comprehensive study of failed eggs and dead nestling bearded vultures collected during recent years in the Pyrenees. Both the productivity and survival rates of adults and young birds have reached the lowest values since the bovine spongiform encephalopathy (BSE) crisis [13], [14], [17]. This temporal decline could be related to illegal poisoning [17] and recent changes in the abundance, distribution and quality of carrion available to avian scavengers as a consequence of EU regulations derived from the BSE crisis [6], [18]–[20]. In particular, the BSE crisis caused the lack or scarcity of unstabled livestock available to scavengers and their subsequent increase in the consumption of carrion from stabled livestock, which is intensively medicated [21]. Therefore, we specifically focused on determining whether breeding failure in bearded vultures is related to the ingestion of veterinary drugs from stabled livestock carrion, as documented in other avian scavenger species [12]. We also assessed the potential effects of veterinary drugs on embryo damage and immunodepression increasing the probability of acquisition and proliferation of pathogens causing fatal disease [6], [10]–[12], [21]. Because veterinary drugs should be exclusively acquired from the ingestion of carrion from livestock medicated to combat disease, we predict that their presence should be associated with that of pathogens acquired from the same livestock, especially poultry pathogens more likely transmitted between avian species [22]. Alternatively, if the temporal decline in productivity was primarily associated with breeding failure due to the effects of habitat saturation processes [13], [17], we should expect egg and nestling mortality to be directly related to developmental and nutritional problems indicating progressively lower quality territories (e.g. embryo emaciation, nestling starvation) and interference by both conspecifics and heterospecifics (e.g. incubation failure, injury due to predation attempts or disturbance).
Materials and Methods
Failed eggs (n = 5) and dead nestlings (n = 4) were collected from bearded vulture nests located in the Spanish Pyrenees between 2005 and 2008. The study of this material did not require of the approval of an ethics committee because it was collected after breeding failure (egg or nestling death) was confirmed in the field. Three of the specimens (two nestlings and one egg) were collected in 2005, 2007 and 2008 from a particular territory. Eggs and nestlings were collected after breeding failure and frozen. Necropsies were performed on all specimens according to standard protocols [12]. The age of embryos and nestlings were estimated according to size and development. Samples of liver, kidney, spleen, large and small intestines, lungs, brain, lymphoid organs (thymus, bursa of Fabricius, Peyer's patches) and knee joints were fixed in 10% buffered formalin, sectioned at 4 µm and stained for histopathological analysis [10], [12].
Liver (dead nestlings and failed embryos) and yolk (failed embryos) were used for the determination of the presence of veterinary drugs, including fluoroquinolones (enrofloxacin and ciprofloxacin), other antimicrobials (amoxicillin and oxytetracycline), non-steroideal anti-inflamatories (NSAIDs) such as diclofenac, flunixin meglumine, ketoprofen, ibuprofen, meloxicam, sodium salicylate, acetaminophen, and antiparasitics (metronidazole, diclazuril, fenbendazole, ivermectin) as described previously [12]. The limits of quantification, percentage recoveries, and inter- and intra-assay reproducibility were adequate [10], [12].
Other contaminants potentially affecting eggs and embryos were determined in liver, including heavy metals (Cd, Zn, Pb and Hg), following Blanco et al. [23], dithiocarbamate thiram, disulfuram, polybrominated diphenyl ethers, organochlorines and brominated flame retardants, following Lemus et al. [12] and carbamate and organophosphate pesticides (carbofuran, aldicarb and fenthion) following Elliot et al. [24]. We measured brain cholinesterase activity to assess early exposure to anticholinesterase pesticides [25]. Potential contamination was assessed by comparison with levels from apparently normal wild birds of other species [26] in the absence of basal levels for bearded vultures.
Determination of bacterial and fungal pathogens were conducted by sampling oropharynx, lung, liver, kidney, spleen, and intestine with sterile swabs and cultured using standard microbiology protocols [10], [12], [27], [28]. Salmonella serotypes and phage types were determined in the Spanish Reference Laboratory (Laboratorio Central Veterinario, Algete, Madrid). For confirmation of the identification of the alpha hemolytic Streptococcus pneumoniae we used a specific identification test (Accuprobe, Salem, MA) based on the detection of specific ribosomal RNA sequences. Samples of lesions found in internal organs and tissues during necropsies were taken with sterile swabs and cultured using the same standard microbiology protocols. In addition, we determined the presence of selected avian pathogens, including bacterial, viral, fungal, and protozoan pathogens by means of PCR-based methods (see Table S1 for details). The presence of Chlamyophila psittaci and Mycoplasma sp. in blood was determined as described previously [29], [30]. The presence of poxvirus, the paramyxovirus causing Newcastle disease, the serotypes H5, H7 and H9 of avian influenza, falcon adenovirus, circovirus, herpesvirus, polyomavirus, reovirus and West Nile virus were determined following the PCR-based methods available in the literature [31]–[39]. We also searched for helminths and protozoans in the gastrointestinal tract by macroscopic and microscopic observations using standard protocols [40].
Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43]) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44]. In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid. All immunohistochemistry analyses were conducted at the Department of Veterinary Anatomy, Veterinary Faculty, Universidad Complutense de Madrid, Spain and at the Pathology Department of the Veterinary Faculty, University of Utrecht, The Netherlands. The presence of these viruses was also determined by PCR-based methods [43], [46]–[48].
Results
All dead nestlings and three of five unhatched embryos showed two to six different veterinary drugs in liver (nestlings) and egg yolk (embryos). In addition, the two embryos with fluoroquinolones in the yolk also had them in the liver (Table 1). Fluoroquinolones were the most prevalent drugs and showed the highest concentrations (Table 1). Other drugs such as NSAIDs and antiparasitics were found in most nestlings at variable concentrations, but in no eggs (Table 1). Other toxic compounds were detected in lower prevalence and concentrations (see Table 1 for those more relevant values; all insecticides were found at concentrations <0.001 ppb), which was further supported by basal levels of brain cholinesterase (Table 1).
10.1371/journal.pone.0014163.t001Table 1 Presence and concentration (between brackets) of veterinary drugs, tissue damage and pathogens found in failed embryo and nestling bearded vultures.
Sample (Age) Tissue for toxicology Veterinary drugs1
Other toxicants2
Brain cholinesterase3
Pathology Pathogen determination5
Tissue Damage4
Immunohistochemistry5
Microbiology PCR
Nestling (35d) Liver EN (0.14), CI (0.03), OX (0.17), FL (32.48), AS (62.7), IV (5.4) nondetected 17.15 UD, LE, LI, FN, BH, PK, GN, GO, MI, WP, IDp, JD nondetected CA, EC, SA* (septicaemia) CH, WN
Nestling* (10d) Liver EN (0.11), CI (0.06), AS (47.9) nondetected 16.24 UD, LE, BH, PK, GN, GO, MI, WP, IDb,t,p, JD IBD CA, EC IBD
Nestling* (7d)) Liver EN (0.08), CI (0.07), AS (37.4) Pb (18.9) 18.42 UD, LE, BH, FN, PK, GN, MI, WP, IDb,t, JD IBD CA, EC, PM IBD, WN
Embryo* (prehatch) Liver nondetected OR (0.21), Pb (48.1) 15.37 UD, PK,MI, IDp
nondetected CA nondetected
Nestling (7d) Liver EN (0.03), CI (0.04), AS (52.3) OR (4.9) 15.21 UD, LE, PK, GN, MI, WP, IDb, JD IBD CA, EC IBD, WN
Embryo (prehatch) Liver nondetected OR (0.88) 17.22 Endocarditis, leptomeningitis, PK, MI BR SS, SP (septicaemia) BR
Embryo (mid incub.) Liver Egg yolk EN (0.06), CI (0.03) EN (0.04), CI (0.02) nondetected 16.58 BH, FN, PK, GN, MI, WP, IDb,p, JD IBD SA**
IBD
Embryo (mid incub.) Liver Egg yolk EN (0.08), CI (0.03) EN (0.04), CI (0.05) Pb (21.3) 18.11 BH, FN, PK, GN, MI, WP, IDb,t, JD IBD SA**
IBD
Embryo (mid incub.) Liver Egg yolk EN (0.05), CI (0.07) EN (0.07), CI (0.04) nondetected 16.22 BH, FN, PK, GN, MI, WP, IDb,t, JD IBD SA**
IBD
Table 1 (cont.)
*Samples from the same territory in different years.
1 Veterinary drugs. EN: enrofloxacin (µg/g), CI: ciprofloxacin (µg/g), OX: oxytetracyclin (µg/g), FL: flunixin meglumine (µg/g), AS: sodium salicylate (ng/g), IV: Ivermectin (µg/g).
2 Other toxicants. OR: organochlorines (ng/g), Pb: lead (ng/g).
3 µmol/min/g
4 Tissue Damage. UD: upper digestive tract swelling, LI: liver lymphocytic infiltration, FN: focal liver necrosis, LE: liver enlarged, BH: bile duct hyperplasia, PK: pinkish kidney, GN: glomerullonephritis, GO: glomerullonephrosis, MI: mononuclear kidney infiltrates, WP: white kidney precipitates, ID: immunological tissue damage (b = damage in Bursa of Fabricius, t = damage in thymus, p = damage in Peyer's patches), JD: joint damage.
5 Pathogens. CA: Candida albicans, EC: Escherichia coli enterotoxigenic, PM: Pasteurella multocida, SA: Salmonella (Salmonella enterica enteritidis 4, 5, 12: i: 1, 2, LT DT 104,
**Salmonella enterica enterica serotype Brancaster 4, 12. z29. SS: Streptococcus suis, SP: Streptococcus pneumoniae, CH: Chlamydophila psittaci, IBD: infectious bursal disease virus, BR: chicken infectious bronchitis virus, WN: West Nile virus.
Dead embryos and nestlings showed a moderate to good nutritional state. Major histopathological lesions were primarily located in the kidney, including glomerulonephritis and/or glomerulonephrosis present in all individuals with fluoroquinolones, but not in those without drugs (Table 1). All individuals with fluoroquinolones also showed joint lesions, including arthritis and/or arthrosis of the long bone articulations, as well as massive osseous stroma of the spongeous bones.
The fungi Candida albicans was isolated from the oral cavity of five individuals. All individuals showed non-specific mixed-bacterial flora. Enterotoxigenic Escherichia coli and Salmonella spp. were isolated in four cases (Table 1). Salmonella typing determined the presence of Salmonella enterica enteritidis 4, 5, 12: i: 1, 2, LT DT 104 (one case) and Salmonella enterica enterica serotype Brancaster 4, 12. z29 (three cases, see Table 1). One individual showed infection by Salmonella enterica enteritidis (see above) and enterotoxigenic Escherichia coli O86 in all examined organs (septicaemia) except brain, which rejected the possibility of post-mortem contamination. Pasteurella multocida was isolated in a single individual that also showed enterotoxigenic Escherichia coli O86 (Table 1); all of these individuals contained fluoroquinolones. One of the failed embryos without veterinary drugs showed suppurative myocarditis, multiple microabscesses in head muscles, suppurative leptomeningitis, as well as lower jaw gangrenous inflammation with loss of the osseous stroma due to a mixed infection with Streptococcus suis and Streptococcus pneumoniae in brain, meninges and neck muscles; this embryo also showed infection by chicken infectious bronchitis (Table 1). Both immunocytochemistry for the detection of poultry viruses and PCR pathogen survey were positive to IBDV in six individuals with fluoroquinolones (Table 1). Immunocytochemical procedures failed to detect West Nile virus antigens in individuals in which PCR for this virus had been positive. Parasitology was negative for all helminths, helminth eggs and protozoans.
Discussion
We found multiple veterinary drugs, primarily fluoroquinolones, in most failed eggs and dead nestling bearded vultures from the Pyrenees. They also showed multiple internal organ damage and pathogens potentially acquired from medicated livestock carrion, especially viruses often infecting poultry. Recorded drug concentrations were among the highest reported in avian scavengers [6], [10]–[12], [21]. NSAIDs and antiparasitics were found in lower prevalence than fluoroquinolones, but at higher concentrations than those found in other avian scavengers, especially for flunixin meglumine and sodium salicylate [6], [12], [21]. On the contrary, we found no sterile eggs, poor nutritional conditions or injury in any failed embryo or nestling. Other pollutants were found in low prevalence and concentrations posing low risk to embryo and nestling health.
Fluoroquinolones may cause generalized direct developmental damage precluding embryo hatching, physiological alterations due to their impact on liver and kidney and immunodepression reducing resistance to opportunistic pathogens [6], [10]–[12], [21]. These pathogens may be acquired at the same time that drugs used to treat diseased livestock are ingested, as indicated by their high prevalence in embryos and nestlings. Therefore, despite the relatively small sample size resulting from low abundance, endangerment and logistic difficulties in reaching nests in this species, the results provide evidence of a combined impact of veterinary drugs and livestock disease as the primary cause of breeding failure in the sampled individuals.
The presence of West Nile virus is not likely to be associated with nestling disease or mortality because the lack of lesions in target tissues and viral antigen particles in the immunohistochemistry study. Fatal septicaemia caused by Streptococcus suis, one of the most important swine pathogens worldwide [49], in combination with septicaemia from Streptococcus pneumoniae and infection by chicken infectious bronchitis virus were found in a single embryo. This concentration of livestock pathogens has not been reported before and, to our knowledge, this is the first report of the three pathogens causing disease in a wild bird. Other pathogens recorded in embryos and nestlings, including Salmonella serotypes and phages typical of livestock [50], and enterotoxigenic Escherichia coli O86 causing septicaemia, were potentially transmitted by consumption of carcasses of infected poultry and other livestock [22], [27], [28]. In addition, we found that the IBD virus infected most individuals alone or together with other pathogens also potentially acquired from livestock carrion. This virus causes a highly contagious immunosuppressive bursal disease in poultry [51] and may be transmitted to wildlife in contact with poultry waste or by ingestion of carcasses [22], [52]. Nestlings are especially susceptible to IBD because of the primary role of bursa of Fabricius in immune function development at this age. In fact, immunosuppression due to IBD was indicated by the inflammation, necrosis and loss of lymphocytes in the bursa of Fabricius together with the presence of viral antigens recorded by means of immunocytochemical procedures. The potential impact of highly pathogenic and contagious poultry viruses has been previously recognized as a threat to wildlife health due to the increasing contact of wildlife with livestock operations in general, and poultry farms and their residues in particular, in natural areas worldwide [52]–[55]. However, damage from IBD virus on the bursa of Fabricius represents, to our knowledge, the first evidence of clinical disease compatible with death caused by this poultry virus in wildlife. The presence of IBD has been not previously recorded in embryos of wild birds, probably because vertical transmission has been ruled out in poultry and, as consequence, it has probably not been evaluated in other species until now. This striking and concerning result could be related to the longer egg development and incubation periods of bearded vultures compared with poultry, and/or due to contrasting environmental conditions during incubation between bearded vultures and poultry. Thus, embryo infection with IBD may occurs via the female or during incubation as a consequence of egg contact between the egg and poultry remains in the nests of bearded vultures, which requires more research.
Despite their potential effects on population dynamics and conservation through a reduction of productivity and changes in mating behaviour [13], [14], habitat saturation processes were apparently not directly related to particular proximate causes of egg and nestling failure in this study or in these sampled individuals. As an alternative non-mutually exclusive explanation, we suggest that the recent decline in productivity could also be linked to the increasing ingestion of veterinary drugs and acquisition of pathogens from medicated stabled livestock carcasses due to decreasing availability of unstabled livestock carcasses - the traditional primary food of bearded vultures [16]- since the BSE crisis [21], accompanied by a possible increasing use of antibiotics in stabled livestock operations. In this sense, it is remarkable that bearded vultures primarily feed upon livestock bones, which are one of the major target tissues of fluoroquinolones in medicated animals [56], therefore, rendering this species especially sensitive to the consequences of an increase in the consumption of stabled intensively medicated livestock. The presence of veterinary drugs in eggs implies their previous presence at least in breeding females [12], but also probably in breeding males and non-breeders frequently using artificial feeding sites and livestock carcass dumps [17], where veterinary drugs may be ingested from medicated livestock carcasses [10], [21]. Therefore, further research is required to determine the impact of veterinary drugs and livestock disease on fitness of full-grown individuals, including the potentially subtle, sublethal or indirect effects of these factors on population dynamics.
The link between veterinary drugs and livestock disease should be further investigated in scavenger species, because both threats may concur in food and because the immunodepressive effects and other physiological alterations caused by drugs may facilitate the acquisition and proliferation of pathogens [6], [11], [21]. Given that both threats acting together may greatly contribute to breeding failure decreasing productivity, their potential as stochastic factors with potentially devastating effects increasing the risk of extinction should be not overlooked in current conservation programs of bearded vultures and other scavenger species, especially regarding dangerous veterinary drugs and highly pathogenic viruses frequently infecting poultry. In addition, restricted geographic distribution and low genetic variability [57] common to many threatened species may favour pathogen transmission and reduce the ability of a naïve immune system to fight against novel pathogens [3], [28], [58], making them especially vulnerable to the potential cross-species transmission of highly virulent virus strains able to cause important outbreaks, as reported in poultry [59]–[61].
The association of pollution and disease may further increase extinction risk if it interacts with the effects of habitat saturation processes [13], [14], [17]. These processes may facilitate conspecific contact and interactions also likely to increase intra- and inter-specific pathogen transmission rates in breeding and feeding areas, especially of highly contagious poultry diseases [22]. This could be further enhanced by the artificially high numbers of bearded vultures and other scavengers attracted to feeding points and carcass refuse dumps, both as a result of management and due to the scarcity of unstabled livestock carcasses since the BSE crisis [17], [21]. Whatever the potential contribution of underlying ultimate mechanisms reducing productivity, our findings highlight the need to determine the proximate causes of breeding failure and mortality in wildlife populations in order to understand the processes regulating demography from an ecological framework perspective.
Supporting Information
Table S1 (0.05 MB DOC)
Click here for additional data file.
We thank Gobierno de Aragón and D. Campión (Comunidad Foral de Navarra) for providing samples. We thank M. Carrete and an anonymous referee for comments on the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The study was funded by the project CGL2007-61395/BOS of Spanish Ministerio de Educacion y Ciencia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2115193210-PONE-RA-20909R210.1371/journal.pone.0014229Research ArticleMolecular Biology/Chromatin StructureMolecular Biology/DNA MethylationMolecular Biology/Histone ModificationMolecular Biology/Transcription Initiation and ActivationGastroenterology and Hepatology/Colon and RectumOncology/Gastrointestinal CancersEpigenetic Silencing of Peroxisome Proliferator-Activated Receptor γ Is a Biomarker for Colorectal Cancer Progression and Adverse Patients' Outcome PPAR γ in CRCPancione Massimo
1
Sabatino Lina
1
Fucci Alessandra
1
Carafa Vincenzo
2
Nebbioso Angela
2
Forte Nicola
4
Febbraro Antonio
4
Parente Domenico
4
Ambrosino Concetta
1
5
Normanno Nicola
6
Altucci Lucia
2
3
Colantuoni Vittorio
1
5
*
1
Department of Biological and Environmental Sciences, University of Sannio, Benevento, Italy
2
Department of General Pathology, Second University of Naples, Napoli, Italy
3
CNR-IGB, Napoli, Italy
4
Departments of Medical Oncology and Clinical Pathology, Fatebenefratelli Hospital, Benevento, Italy
5
Biogem “G. Salvatore” Genetic Research Institute, Ariano Irpino, Italy
6
Pharmacogenomic Laboratory, Center for Oncology Research, Mercogliano, Italy
Wong Chun-Ming EditorUniversity of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: MP LA VC. Performed the experiments: MP LS A. Fucci VC AN CA NN. Analyzed the data: MP LS A. Fucci VC AN LA VC. Contributed reagents/materials/analysis tools: LS A. Fucci AN NF A. Febbraro DP CA NN. Wrote the paper: MP LA VC.
2010 3 12 2010 5 12 e1422910 7 2010 9 11 2010 Pancione et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The relationship between peroxisome proliferator-activated receptor γ (PPARG) expression and epigenetic changes occurring in colorectal-cancer pathogenesis is largely unknown. We investigated whether PPARG is epigenetically regulated in colorectal cancer (CRC) progression. PPARG expression was assessed in CRC tissues and paired normal mucosa by western blot and immunohistochemistry and related to patients' clinicopathological parameters and survival. PPARG promoter methylation was analyzed by methylation-specific-PCR and bisulphite sequencing. PPARG expression and promoter methylation were similarly examined also in CRC derived cell lines. Chromatin immunoprecipitation in basal conditions and after epigenetic treatment was performed along with knocking-down experiments of putative regulatory factors. Gene expression was monitored by immunoblotting and functional assays of cell proliferation and invasiveness. Methylation on a specific region of the promoter is strongly correlated with PPARG lack of expression in 30% of primary CRCs and with patients' poor prognosis. Remarkably, the same methylation pattern is found in PPARG-negative CRC cell lines. Epigenetic treatment with 5′-aza-2′-deoxycytidine can revert this condition and, in combination with trichostatin A, dramatically re-activates gene transcription and receptor activity. Transcriptional silencing is due to the recruitment of MeCP2, HDAC1 and EZH2 that impart repressive chromatin signatures determining an increased cell proliferative and invasive potential, features that can experimentally be reverted. Our findings provide a novel mechanistic insight into epigenetic silencing of PPARG in CRC that may be relevant as a prognostic marker of tumor progression.
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Introduction
Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-dependent transcription factors belonging to the nuclear hormone receptor superfamily [1]. Three different PPAR isoforms, α, β and γ have been isolated so far, each with a distinct pattern of tissue expression and ability to interact with diverse classes of compounds. Specifically, the PPARγ isoform is implicated in a wide range of physiological processes [2]: it integrates the control of energy, lipid and glucose homeostasis and plays a pivotal role in adipogenesis, inflammatory response and differentiation of many epithelial cells [3]. Consistently, variations in PPARG expression or gene mutations have been associated with tumorigenesis [4]–[6]. However, conflicting results have been reported so far, raising the question as to whether PPARγ facilitates or suppresses tumorigenesis [7], [8]. Recently, we have shown that sporadic colorectal cancers (CRCs) presenting reduced PPARγ expression levels are significantly associated with patients' worse prognosis; in the same type of tumours, PPARG has been shown to be an independent prognostic factor [9], [10], suggesting the possibility to target this gene with drugs in clinical applications [10]. The molecular mechanisms underlying PPARG expression regulation in CRC progression are still unknown [9].
It is becoming increasingly clear that, in addition to genetic alterations, epigenetic modifications contribute to tumorigenesis [11]. Epigenetic regulation involves heritable modifications that do not change the DNA sequences but provide “extra” layers of control to regulate chromatin organization and gene expression [12]. Aberrant DNA methylation at CpG-rich sequences, also known as “CpG islands”, located in the promoter regions of approximately half of the known genes, leads to epigenetic silencing of gene expression [11], [12]. In CRC, extensive DNA methylation has been detected at several loci, specifically at the promoter regions of tumor suppressor genes (TSG), a characteristic of a subgroup of tumours presenting the so-called “CpG island methylator phenotype” (CIMP) [13]. Other epigenetic events, such as repressive histone modifications, cooperate to establish stable gene silencing. A “histone code” has been suggested to provide a signature on specific amino acid residues that correlates with active or repressed gene expression [11], [12]. The link between DNA methylation and histone modifications seems to be mediated by Methyl CpG DNA binding proteins, a member of which MeCP2 plays an important role to establish this interaction [14]. DNA methylated regions, usually enriched in modified histones, generate a more tightly packed chromatin where the access of specific transcription factors to their cognate binding sites is greatly impaired [12]. How DNA methylation and the pattern of histone modifications on promoter regions of specific genes are associated with cancer initiation and progression, in particular in sporadic CRC, remains to be elucidated [15]. In this report, we analyzed one-hundred and fifty-two primary CRCs and paired normal mucosa in order to correlate PPARG expression variations mediated by epigenetic events with tumor progression and patients' survival. We extended the analysis to CRC derived cell lines as a system to investigate the molecular mechanisms underlying PPARG silencing due to epigenetic variations.
Materials and Methods
Ethics Statement
This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of Fatebenefratelli Hospital in Benevento. All patients provided written informed consent for the collection of samples and subsequent analysis.
Tumor samples
One hundred and fifty-two patients diagnosed primary sporadic CRC and surgically treated at the Department of Surgery, Fatebenefratelli Hospital, Benevento, Italy, between 1999–2004, were investigated in this study. Fifty-two cases comprise both liquid nitrogen snap-frozen specimens, obtained immediately after surgical resection, and paraffin blocks. Each sample was matched with the adjacent apparently normal mucosa (at 20 cm distance from the tumor mass) removed during the same surgery. None of the patients had a familial history of intestinal dysfunction or CRC, had received chemotherapy or radiation prior to resection nor had taken non steroidal anti-inflammatory drugs on a regular basis. Conventional postoperative treatments were provided to all patients, depending upon the severity of the disease. The clinico-pathological features of the patients investigated are reported (
Table 1
). The follow-up was available for all patients, with a median post-operative duration of 59.5±26.5 months. Overall length of survival was calculated starting from the first surgery. Patients were prospectively followed until death or their most recent medical examination.
10.1371/journal.pone.0014229.t001Table 1 Correlation between PPARG promoter methylation and several clinico- pathological parameters of the patients.
Parameters
N M3 PPARG methylation P
Met Umet
Gender
0.719
Male 95 31 64
Female 57 17 40
Age(Years)
0.001**
≤60 32 18 14
>60 120 30 90
Location
a
0.230
Proximal 56 21 35
Distal 96 27 69
Tumor size
0.683
≤5 cm 51 15 36
>5 cm 101 33 68
Differentiation
0.069
Well/Mod 129 37 92
Por 23 11 12
Histology
b
0.110
AD 123 36 87
AD-MUC 26 10 16
MUC 3 2 1
T stage
0.05*
pT1 9 2 7
pT2 12 5 7
pT3 125 37 88
pT4 6 4 2
N stage
0.002**
N0 111 26 85
N1 25 14 11
N2 16 8 8
Distant Metastasis
0.0001**
M0 107 23 84
M1 45 25 20
Total
152
48
104
a Proximal: caecum, ascending and transverse colon; Distal: descending and sigmoid colon, rectum;
b AD = adenocarcinoma; AD-MUC = adenocarcinoma with a mucinous component below 50%; MUC = adenocarcinoma with a mucinous component above 50%. Abbreviations: Well = well-differentiated; Mod = moderately differentiated; Por = poorly differentiated adenocarcinoma. χ2 test;
*Significant at 0.05 level;
**Significant at 0.01 level. Patients' mean age was 71.1±12.3 years old. The classification of the tumours was based on the TNM (Tumor-Node-Metastasis) system according to the criteria of the International Union Against Cancer (UICC).
Cell lines and 5′-aza-2′-deoxycytidine and Trichostatin A treatment
Twelve CRC derived cell lines were used in this study and were obtained from the ATCC and cultured as suggested. For DNA demethylation, cells were treated with 1 or 5 µM 5′-aza-2′-deoxycytidine (AZA) for 72 hs or 300 nM Trichostatin A (TSA) (Sigma-Aldrich, St. Louis, USA) for 24 hs, alone or in combination. After the treatments, cells were harvested for DNA, RNA or protein extraction.
DNA extraction, bisulphite treatment, methylation analysis and sequencing
Genomic DNA was isolated from frozen tissues or from paraffin embedded samples using a standard procedure [6], or the FFPE tissue kit (56404, Qiagen, Hilden, Germany), respectively. One µg of each DNA sample was bisulphite modified according to the manufacturer's protocol (59104, Qiagen, Hilden, Germany). Both universally unmethylated (59665) and CpGenome universally methylated DNA (59655, Qiagen, Hilden, Germany) were used in each reaction as unmethylated or methylated control, respectively. The search for CpG content in the PPARG promoter was performed using the Methprimer software according to CpG island definition. PCR primers for methylation specific PCR (MS-PCR) were designed using Methyl Primer Express software v1. Both unmethylated (U) and methylated (M) specific sets of primers were designed based on the positive strand of the bisulfite-converted DNA covering the CpG islands within the PPARG promoter region. MS-PCR reactions were performed using the MS-PCR kit (59305, Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR products were loaded onto non-denaturing 3% agarose gels, stained with ethidium bromide, and visualized under an UV transilluminator. Primer sequences are listed (Table S1). Bisulphite sequencing (BS) was automatically carried out on the PCR amplification product obtained by using a primer set not containing CpG sites within their sequences and designed on bisulphite modified DNA (Applied Biosystems, Applera, Foster City, USA).
ChIP and MeDIP assay
ChIP assays and q-PCR amplification (Biorad, Hercules, USA) were performed as described [16]. The primers used are described (Table S1). MeDIP assay was carried out as recommended by the supplier (Diagenode, Liège, Belgium). Antibodies raised against: AcH3K9, H3K4me3, HDAC1 and MeCP2 (Abcam, Cambridge, UK), H3K27me3, (Millipore, Billerica, USA), RNA pol II and P-RNA pol II (Covance, Dallas, USA), ZAC and purified IgG (Santa Cruz Biotechnology, Santa Cruz, USA) were used in ChIP assays.
Western blot and immunohistochemical analysis
Western blot analysis was performed on protein extracts from tumor tissues and adjacent normal mucosa taken during surgery and CRC cell lines, as previously reported [6], [9]. Immunohistochemical analysis on tumors and distant non-neoplastic mucosa was performed as described [9]. The following antibodies were used: anti-PPARγ (E-8), anti-ZAC (H-253), anti-ERK 1/2 (MK1) and anti-p-ERK (E-4) (Santa Cruz Biotechnology, Santa Cruz, USA); anti-E-cadherin (610405) (BD Transduction Laboratories, Lexington, USA); anti-MeCP2 (ab55538), anti-HDAC1 (ab19845) (Abcam, Cambridge, UK); anti-EZH2 (4905) (Cell Signaling, Boston, USA); anti-β-actin (A5441) (Sigma-Aldrich, St. Louis, USA).
MTT and apoptosis assays
Cells were seeded in triplicate in 24 or 96 well-plates and the MTT assay (Sigma-Aldrich, St. Louis, USA) was performed according to the manufacturer's protocol at 0, 24, 48, 72 and 96 hs after reaching confluence, as indicated. The growth curves were set up taking into account the average results obtained from three independent experiments. To analyze chemo-sensitivity to PPARγ agonists cells were treated with 5 µM troglitazone (TZD). Apoptosis assay was performed by flow cytometric analysis (FCA) using propidium iodide (PI) staining. Briefly, after incubation with TZD, cells were harvested and fixed in 70% ice-cold ethanol/PBS and stored at 4°C overnight. The suspended cells were then washed with PBS, incubated in a PI solution for 15 min. at 37°C and immediately analyzed with a FAC scan flow cytometer (Becton Dichinson, San Jose, USA).
Migration and invasion assays
For the wound-healing assay, cells were plated in 60-mm plates and grown to confluence. After a 6 hs long serum starvation, a wound was made by using a micropipette tip to scrape off the cells. Cell motility was studied after 24 and 48 hs, following cells from different microscope fields. Finally, the corresponding wound area at each time point was digitalized and quantified using Metamorph Imaging System Software version 6.0 for Microsoft Windows. An average percentage of wound closure was calculated from three independent experiments. To determine invasiveness a transwell assay was carried out using a 24 well cell culture insert, 8 µm pore (3097, Falcon-Becton Dickinson, USA). Following hydration of the matrigel in the upper compartment, cells were seeded and incubated. Twenty-four and forty-eight hs later the cells of the upper surface of the filter were removed with a cotton swab; those underneath were fixed with 4% paraformaldehyde, stained with 0,2% crystal violet and counted. Quantification was obtained by counting at least 10 lower power fields from three independent experiments.
RNA extraction and semi-quantitative reverse transcription-PCR
Total RNA was isolated from cell lines and tissues with Trizol with minor modifications (Invitrogen Carlsbad, USA). Reverse transcription-PCR (RT-PCR) was made using Super-script II (Invitrogen, Carlsbad, USA) and PCR amplification using specific primers for PPARG. The RT-PCR conditions and the primers used have previously been reported [5]. In all PCR reactions, GAPDH served as an internal control for normalization. The amplified products were run on a 2% agarose gel and stained with ethidium bromide.
Plasmids and transfections
The PPRE-TK driven luciferase reporter plasmid, the pcDNA3 carrying the wild type PPARG cDNA and the transfection conditions have already been described [6]. For gene re-activation assays, cells were seeded in 6-well plates, treated with AZA and TSA alone or in combination and transiently transfected with the reporter plasmid. After 12 hs, 1 µM TZD or the vehicle alone were added to the medium. When indicated, GW9662, a selective PPARγ antagonist, was used.
siRNA
A retroviral vector PSM2C (clone ID VH2-203345) that carries a short hairpin DNA (shRNA, catalog number RHS1764-9494331) for targeting PPARγ mRNA was used. HT29 cells were stably transfected with the shRNA-PPARγ vector and the positive clones selected by using 1 µM puromycin. A vector carrying non-specific shRNAs or an empty vector were used as controls. The siRNA designed for targeting human MeCP2 mRNA (code: HS-MECP2-7HP SI02664893, Qiagen, Hilden, Germany) was kindly provided by Prof. Chiariotti. HCT116 cells were seeded in 6-well plates and transiently transfected with the MeCP2 siRNA or non-specific oligos, according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). To silence EZH2, a retroviral vector PSM2C carrying an EZH2-shRNA (code: RHS1764-9100483, CN: V2HS-63033) scrambled shRNAs or an empty vector were used, according to the manufacturer's instructions (Invitrogen, Carlsbad, USA). In both cases, cells were harvested for western blot analysis 56 hs later.
Loss of heterozygosity, BRAF and KRAS mutations and microsatellite instability analysis
Loss of heterozygosity (LOH) was assessed as previously described, using the microsatellite markers D31259 and D3S3701, which flank PPARG
[6]. Microsatellite instability (MSI) was performed as reported [17]. MLH1 promoter methylation was also assessed on some representative tumour samples [18]. BRAF and KRAS mutations at codon 600 in exon 15 and codons 12/13 in exon 2, respectively, were evaluated by PCR/sequencing and Real-Time PCR using primers previously described [18].
Statistical analysis
All statistical analyses were carried out with the SPSS (version 15.0) for Windows (SPSS Inc., Chicago, USA). Association between PPARG promoter methylation, other markers and clinico-pathological parameters was assessed using the χ2 test or the Spearman rank test, as indicated. The Kaplan-Meier method was used to estimate survival; survival differences were analyzed with the log-rank test. Data were reported as mean ± SD, and mean values were compared using the Student's t test or Mann-Whitney test. Results were considered statistically significant when a P≤0.05 was obtained.
Results
PPARG promoter methylation in CRCs correlates with gene expression and is associated with patients' outcome
To assess the role that PPARG plays in colorectal tumorigenesis in vivo, we analyzed 152 primary sporadic CRCs and the matched adjacent non-neoplastic mucosa for PPARG expression (
Figure 1
, panel A). About 60% of tumors showed PPARG over-expression and 5% of cases showed not significant differences between tumour tissues and the matched normal mucosa. In contrast, 35% of the tumours showed lower PPARγ levels than the normal mucosa and a significant association with distant metastases and reduced patients' survival, in line with our previous data (
Figure 1
, panels B and C) [9]. We have already reported that reduced PPARG expression in sporadic CRCs is not associated with LOH [6]. Thus, to determine whether PPARG reduced expression is correlated with DNA methylation, we examined the entire promoter region. Inspection of the human PPARG promoter showed that the core region, from −474 to +600 with respect to the transcription start site, is particularly enriched in “CpG islands” that might be target of DNA methylation (
Figure 1
, panel D). A shorter CpG-rich DNA tract located upstream (from −793 to −580) has been found stably methylated in a study evaluating the epigenetic risk factor associated with the early onset of adult metabolic syndrome (
Figure 1
, panel D) [19]. To investigate whether these two regions are differentially methylated in primary CRCs and paired normal mucosa, we performed MS-PCR on four promoter segments (M1 to M4 starting from the more downstream) (
Figure 1
, panel D). Segments M4 (from−746 to −616) and M1 (from −123 to +49) were always methylated or unmethylated in normal and tumor samples and were not correlated with PPARG expression levels (
Figure 1
, panel E). M2 (from −235 to −151) was variably methylated and, finally, M3 (from −359 to −260) was methylated in about 30% of tumours as compared to 8% of paired normal mucosas (n = 80) and correlated with reduction/loss of PPARG expression (
Figure 1
, panel E and
Table 1
). A closer inspection of the M3 segment identified 9 CpG sites, the methylation status of which was analyzed by bisulphite sequencing (
Figure 1
, panel F). The CpG islands methylation level was significantly higher in PPARG-negative than PPARG-positive tumours and paired normal mucosas (
Figure 1
, panel G). Consistently, the M3 region methylation correlated with patient's age, deep invasion, Duke's C and D stages, whereas no association was detected with tumour location (proximal or distal colon) and gender (
Figure 1
, panel H and
Table 1
). PPARG methylation was more frequently observed in a subgroup of microsatellite high (MSI-H) than in microsatellite stable (MSS) tumours. Moreover, these cases were not related with KRAS or BRAF mutations (Figure S1). All together these results indicate that PPARG promoter methylation, specifically at the M3 region, is significantly correlated with tumour progression and patients' poor outcome (
Figure 1
, panel I).
10.1371/journal.pone.0014229.g001Figure 1 A specific PPARG promoter methylation is associated with protein expression and patients' poor prognosis.
(A) Fifty µg of total protein extracts from tumour tissues (T) and matched non-neoplastic mucosa (N) were analyzed by Western blot. In the panel only some representative samples (from 1 to 8) are shown. The histogram reports the quantification to β-actin, used as internal control. (B) Correlation of PPARG expression levels in the absence = M0 or presence = M1 of distant metastases. (C) Kaplan Meier survival analysis related to PPARG expression levels. The P value is reported in each graph. (D) Schematic structure of the PPARG promoter and identification of the CpG-enriched regions encompassing the transcription start site of the human gene. The MS-PCR regions analyzed (M1–M4) and the positions of the primers used are depicted as rectangles. (E) Representative MS-PCRs show a correlation between methylation of the M3 region and reduced PPARG expression in tumour tissues vs. matched normal mucosa, C+ indicates methylated (M) and unmethylated (U) controls. (F) The M3 nucleotide sequence is reported; the CpG islands are highlighted. The chromatograms show which CpG dinucleotide is methylated in some representative samples after BS. Black or white circles indicate the methylated or unmethylated cytosines, respectively. (G) Methylation levels detected by BS in tumour and normal mucosa specimens. The M3 region methylation is related to tumour' stage (Duke's from A to D) (H) and patients' survival (I). The P value is reported in each graph.
PPARG silencing in CRC cell lines correlates with promoter methylation
To investigate the molecular mechanism(s) underlying this relationship, we sought to use an in vitro cell culture system. We screened a series of human CRC cell lines (Table S2) for PPARG expression levels and correlated these with possible silencing events. PPARγ expression was investigated at the mRNA and protein level by RT-PCR and western blot analysis, respectively (
Figure 2
, panel A). PPARγ mRNA mirrored protein levels with a wide range of variations from the highest in HT29 to the lowest in HCT116 cells. The differences detected were attributed to transcription variations; a reduced protein expression not related to mRNA levels was observed only in Caco-2 cells, likely due to post-translational mechanism(s). Among these cell lines, we selected HT29 and HCT116 for further investigations. We did not detect loss of heterozygosity at the PPARG locus in both cell lines. Similarly, we did not correlate the different PPARG levels observed in HCT116 and HT29 cells with post-translational modifications caused by an active mitogen-activated protein kinase (MAPK/ERK) pathway (Figure S2) [6], [20]. To investigate whether PPARG expression correlates with promoter methylation in the CRC cell lines, we performed MS-PCR (
Figure 2
, panel B). The M4 and M1 segments were stably methylated or unmethylated in all cell lines, regardless of PPARγ expression. The M2 segment was methylated in 5 out of 8 cell lines, including HCT116. Interestingly, the M3 segment was methylated only in HCT116 out of the eight CRC cell lines analyzed (
Figure 2
, panel B). By examining four additional CRC cell lines, we found that also RKO cells were negative for PPARG expression, due to aberrantly methylated M3 region (Table S2 and data not shown). MeDIP assays confirmed these results: the PPARG promoter DNA (from −368 to −166) was three-fold more methylated in HCT116 than in HT29 cells, indicating a more tightly packed chromatin structure (Figure S2). 90% of the CpG sites contained in the M3 segment were methylated in HCT116, whereas only few or none were methylated in other cell lines as assessed by bisulphite sequencing (
Figure 2
, panel C). These findings suggest that promoter methylation could play a role in silencing PPARG expression in the CRC cell lines analyzed.
10.1371/journal.pone.0014229.g002Figure 2 Promoter methylation is associated with reduced PPARG expression levels in CRC cell lines.
(A) PPARG mRNA and protein levels detected by RT-PCR ad western blot analysis in a panel of eight CRC cell lines. GAPDH and β-actin were used as controls, respectively. (B) MS-PCR results obtained on the M1–M4 promoter regions analyzed; M, methylated; U, unmethylated; C+ indicates methylated and unmethylated controls. The methylation status of all CRC cell lines analyzed is summarized, with HCT116 and HT29 cells depicted in grey. (C) CpG dinucleotides methylation was assessed by BS. The results of some representative cell lines are reported. Methylated or unmethylated CpG dinucleotides are depicted as black or white circles, respectively. (D) Pharmacologic demethylation induced PPARG expression in HCT116 while no significant difference was found in HT29 cells used as control. Cells were exposed to 1 and 5 µM AZA for 72 hs, MS-PCR was carried out on the M2 and M3 regions, before and after treatment, **P<0.01.
PPARG expression is re-activated by pharmacologic demethylation
To verify whether PPARG expression can be re-activated by pharmacologic demethylation, we treated HCT116 cells with AZA, a well-known inhibitor of DNA methylation. RT-PCR analysis from HCT116 exposed to 1 and 5 µM AZA showed a dose-dependent increase of the PPARγ mRNA whereas did not show significant variations in HT29 cells, as expected (
Figure 2
, panel D). MS-PCR performed on the M3 region, that is methylated only in HCT116 cells, showed loss of methylation following the treatment; the M2 region, that in basal conditions is methylated in both cell lines, got demethylated after the treatment (
Figure 2
, panel D). All together these data demonstrate that extensive promoter methylation is associated with reduced PPARG expression in CRC cell lines.
Co-operative effect of AZA and TSA on PPARG re-activation
That specific regions of the PPARG promoter are differentially methylated points out that epigenetic mechanism(s) are involved in its deregulated expression in CRC cells. To verify this hypothesis, we treated HCT116 with AZA, alone or in combination with TSA, a known histone deacetylase inhibitor (HDACi). HT29 cells were used as a control. PPARG expression was synergistically induced by the combined treatment with AZA and TSA in HCT116 cells while was not affected in HT29 cells, when the drugs were used either alone or in combination (Figure S3). These data indicate that chromatin-associated histone enzymes may contribute to gene silencing. To determine whether the re-activated PPARγ behaves as a bona fide functional transcriptional factor, we treated HCT116 cells with AZA or TSA alone or in combination and subsequently transfected with a PPRE-driven luciferase reporter gene. Luciferase activity determined in cell extracts increased upon AZA and/or TSA treatment and, strikingly, even further upon addition of troglitazone, a specific PPARγ ligand (Figure S3). To demonstrate that the increase in luciferase activity was really dependent upon the re-activated receptor, we exposed the transfected cells to the PPARγ antagonist, GW9662. A significant reduction of reporter gene activity was observed due to GW9662 ability to irreversibly interfere with the transactivating ability of the mature protein (Figure S3). All together these data show that DNA promoter methylation and histone modifications likely co-operate to down-regulate PPARG expression, suggesting that epigenetic treatments re-establish gene transcription and activity.
Specific repressive chromatin marks and DNA methylation are associated with PPARG transcription
To provide insights into the mechanism(s) by which DNA methylation and histone modifications affect PPARG expression, quantitative ChIP assays were performed investigating the promoter segment extending from −368 to −166 in HCT116 cells. Consistent with the MeDIP data, HDAC1 was tightly bound to PPARG promoter, whereas RNA-Polymerase II (RNAPol-II) and its phosphorylated form (P-RNAPol-II) were barely present in untreated HCT116 cells (
Figure 3
, panel A). Upon exposure to AZA and TSA in combination, HDAC1 was remarkably depleted along with a reduced DNA methylation (Figure S2), whereas RNAPol-II and P-RNAPol-II were greatly enhanced, indicating transcription recovering (
Figure 3
, panel A). Accordingly, trimethylated H3K4 and acetylated H3K9, that are histone modifications normally associated with transcriptional activity, were almost reduced in untreated HCT116 cells and significantly increased with TSA, AZA or their combination (
Figure 3
, panel B). Of note, trimethylated H3K9, a marker of silenced chromatin, was enriched at the PPARG promoter and progressively depleted after epigenetic treatments, while trimethylated H3K27 was significantly reduced only by the AZA/TSA combined treatment (
Figure 3
, panel C). This analysis suggests that DNA methylation is closely associated with repressive chromatin marks at the PPARG promoter to impair gene expression in CRC cells.
10.1371/journal.pone.0014229.g003Figure 3
PPARG transcriptional repression is due to specific repressive chromatin marks and recruitment of HDAC1.
(A) Quantitative ChIP analysis in HCT116 cells was performed before and after the treatment with AZA and TSA alone or in combination. Native chromatin was incubated with antibodies directed against the indicated proteins. The immunoprecipitated DNA was used as template in qPCR reactions using specific primers for the PPARG promoter region *P<0.05, **P<0.01. (B) ChIP assays were carried out as described above against acetylated H3K9 and trimethylated H3K4 *P<0.05, **P<0.01 or (C) trimethylated H3K9 and H3K27 *P<0.05. The time-points for co-treatments were 72 hs for 5 µM AZA and 24 hs for 300 nM TSA, alone or in combination; CC indicates untreated control cells. Results are the mean values ± SD of three independent experiments, each performed in duplicate.
siRNA mediated knock-down of MeCP2 and EZH2 rescues PPARG expression in HCT116 colon cancer cells
The link between DNA methylation and histone modifications appears to be mediated by a group of proteins with methyl DNA binding activity that includes methyl CpG binding protein 2 (MeCP2) [14]. These proteins localize to methylated promoter regions and recruit protein complexes containing HDACs, especially HDAC1, and histone methyltransferases (HMTs). Enhancer of zeste 2 (EZH2) is a member of the polycomb repressor complex 2 (PRC2) with histone methyl-transferase activity at specific H3K27 sites [21]. Both MeCP2 and EZH2 have been reported to be involved in Pparg repression in mouse stellate cells undergoing liver fibrogenesis [22]. PPARG transcriptional activation, on the other hand, appears to be modulated by the zinc-finger protein (ZAC) likely together with other still unknown factors [23]. MeCP2, EZH2 and HDAC1 were more expressed in HCT116 than HT29 cells, thus inversely correlating with PPARγ, whereas ZAC levels directly associated with PPARγ (
Figure 4
, panels A and B and Figure S4). In line with this, ChIP analysis showed that ZAC was more recruited in HT29 than HCT116 cells. (
Figure 4
, panel B). Importantly, in PPARG-negative cells following epigenetic treatment, ZAC became highly enriched at the promoter correlating with PPARG transcription recovery (
Figure 4
, panel B). ChIP assays performed in the same setting showed that MeCP2 was highly recruited at the PPARG promoter in basal conditions and depleted after treatment with AZA or TSA alone or in combination (
Figure 4
, panel C). Accordingly, silencing MeCP2 caused a marked increase of PPARG expression (
Figure 4
, panel D). In the same HTC116 cells, the PPARG promoter was particularly enriched in H3K27me3 (
Figure 4
, panel E). Knocking-down EZH2 associated with a three-fold increase of PPARγ and reduction of H3K27me3 at the PPARG promoter as compared with control cells (
Figure 4
, panel F). Finally, MeCP2, HDAC1 and EZH2 levels were examined in a subset of CRCs. HDAC1 and EZH2 levels were more expressed in tumour tissues than the paired normal mucosas and directly correlated with advanced Duke's stages (Figure S4). MeCP2, in contrast, was unchanged or slightly increased in the same subset of tumours (Figure S4). Altogether these results indicate that MeCP2, HDAC1 and EZH2 are involved in PPARG repression in colon tumorigenesis both in vivo and in vitro.
10.1371/journal.pone.0014229.g004Figure 4 MeCP2 and EZH2 are negative regulators of PPARG expression in CRC cell lines.
(A) Western-blot analysis shows higher levels of MeCP2 and EZH2 in HCT116 than HT29 cells *P<0.01. (B) In contrast, ZAC is more expressed in HT29 than HCT116 cells, thus directly correlating with PPARγ levels. ChIP assays performed in basal conditions show a ZAC enrichment in HT29 cells. In HCT116, ZAC is highly recruited at the PPARG promoter after epigenetic treatments correlating with PPARG transcription recovery *P<0.05; **P = 0.004; ***P = 0.0001. (C) qChIP analysis shows enrichment of MeCP2 at the PPARG promoter that is lost after pharmacological treatments. (D) A specific MeCP2-siRNA transfected into HCT116 cells determines complete silencing of its own gene and PPARγ re-expression, as assessed by Western blot, relatively to controls *P = 0.01. (E) qChIP analysis shows that trimethylated H3K27 is enriched in HCT116 as compared to HT29 cells. After AZA/TSA addition, H3K27 is unchanged in HT29 and reduced in HCT116 cells *P<0.05. (F) A specific EZH2-shRNA introduced in HCT116 efficiently silences its own gene and induces PPARγ expression, as illustrated by Western-blot analysis. This coincides with reduced H3K27me3 levels analyzed by qChIP. *P<0.05. The time-points for co-treatments were 72 hs for 5 µM AZA and 24 hs for 300 nM TSA, alone or in combination; CC indicates untreated control cells. Error bars indicate the standard deviation of the mean.
PPARG silencing is associated with an increased growth rate and higher invasiveness of CRC cells
PPARγ seems to play a role in cell proliferation and invasiveness [6], [24]. To investigate whether CRC cells growth and invasiveness correlate with PPARγ levels, we compared the HCT116 and HT29 proliferation index. HCT116 showed a proliferation rate two-fold higher than HT29 cells (
Figure 5
, panel A). We also assessed the motility and invasiveness of the two cell lines by performing the wound-healing and migration assays. Interestingly, HCT116 showed a higher motility and invasive potential than HT29 cells (
Figure 5
, panels B and C). To investigate whether these HCT116 growth characteristics rely on PPARG expression, we stably silenced PPARG in HT29 cells. Among the HT29 clones tested for PPARγ levels, the clone shPPARG displayed a reduction of more than 60% than cells transfected with the empty vector or with a vector carrying non-specific shRNA (
Figure 5
, panel D). According to our hypothesis, the shPPARG clone showed a higher cell proliferation index, lower apoptotic rate and increased invasion potential than HT29 transfected with a non-specific shRNA (
Figure 5
, panels E and F and Figure S4). Regarding the apoptotic rate, administration of TZD had no effect on the HT29 shPPARG clone, as compared to parental cells (Figure S4). Furthermore, we generated HCT116 cells stably over-expressing a transfected wild type PPARγ (
Figure 5
, panel G). Consistently, in the presence of TZD these clones were inhibited in their growth and displayed a higher apoptotic index than the parental cells transfected with an empty vector (
Figure 5
, panel H and Figure S4). These clones showed also a reduced invasiveness and increased E-cadherin expression than the parental cells (
Figure 5
, panels G–I). Collectively these results suggest that PPARG differential expression and a TZD-dependent activity accounts for the different growth and motility properties of the CRC cell lines analyzed.
10.1371/journal.pone.0014229.g005Figure 5
PPARG silencing increases proliferation and migration/invasiveness of CRC cells.
(A) MTT assays on HCT116 and HT29 cells were carried out at different time-points *P = 0.004; **P = 0.001. (B) A wound-healing migration assay was carried out comparing and measuring the “wound area” at 24 and 48 hs *P = 0.045; **P = 0.0051; ***P = 0.0001. (C) Transwell migration assay was performed counting the run-through cells in 10 microscopic fields *P = 0.024; **P<0.01. The symbols represent the mean values of three independent experiments (mean ± SD). (D) Specific PPARG- or scrambled-shRNAs were stably transfected into HT29 cells to generate the shPPARG or control clones, respectively; the extent of PPARγ knock-down was documented by Western blot and referred to β-actin. (E) shPPARG cells showed higher proliferation than control clones and parental cells *P = 0.022; **P = 0.012. (F) The wound-healing migration and transwell migration assays were performed on the HT29 parental, the shPPARG and the control clone, respectively. The measurements were done as above. In both cases, cells were fixed after 48 hs and stained with hematoxylin & eosin or crystal violet, respectively. Magnification: 100×. Quantification of the wound-area after 24 and 48 hs is reported in the histogram where the control was set at 100%, *P = 0.016, **P = 0.001. Bars represent mean values ± SD of three independent experiments. (G) HCT116 cells were stably transfected with an empty expression vector or a vector carrying the PPARγ cDNA to generate control or the HCT116-PPARγ clones, respectively. Western blot analysis of the transfected PPARγ and activated target E-cadherin referred to β-actin. (H) The HCT116-PPARγ cells showed lower proliferation than control clones and parental cells in the presence of TZD *P = 0.012. (I) The wound-healing migration assay in HCT116 parental, the HCT116-PPARγ and the control clone. Cells were fixed after 48 hs and stained with hematoxylin & eosin. Magnification: 100×. The histogram shows quantification of the wound-area, measured as above, with the control set at 100%. Bars represent mean values ± SD of three independent experiments *P<0.05; **P<0.01.
Discussion
Colorectal cancer is one of the most frequent malignancies in western countries and the third most common cause of cancer-related death worldwide [10], [13]. Among genetic alterations, chromosomal and microsatellite instability (CIN and MSI) have been invoked in CRC tumorigenesis. Several lines of evidence suggest that also epigenetic modifications contribute to the establishment and/or to the progression of a tumour [12], [13]. DNA hypermethylation is the most common epigenetic change observed in human cancers, particularly in CRC, where it is associated with TSGs silencing [12], [13]. In colorectal tumorigenesis, the precise role played by PPARG has been questioned because of the conflicting results reported [4]–[10]. PPARG mutations alone do not fully explain the frequent variations in expression detected in tumours [9], [10], [25]. Here, we provide evidence that epigenetic alterations at the PPARG promoter are related with gene repression that occurs in 30% of CRCs. Dissecting the PPARG proximal promoter, we demonstrate that a specific DNA segment (M3) is differentially methylated and PPARG expression is directly correlated with its methylation status (
Figs. 1
and
2
). Consistent with this observation, only 8% of the paired normal mucosa is methylated in the same region, probably due to the so-called “field defect” [13]. An association with patients' age at diagnosis was observed also in our CRC samples.
We also demonstrate that a reduced PPARG expression due to specific promoter methylation is associated with advanced tumour stages (Duke's stages C–D), deep invasion, and, ultimately, shorter survival. Other molecular alterations such as KRAS and BRAF mutations do not seem to be associated with PPARG methylation, while a correlation with the microsatellite instability status was found (Figure S1) [10]. These data imply that PPARG promoter methylation could be associated with CRC progression, providing a molecular basis to our previous data and to a recent proposal of PPARG as a favourable prognostic marker for CRC survival [9], [10]. It is poorly understood whether other genetic and epigenetic events contribute not only to PPARG silencing but also to overexpression detected in about 60% of CRCs. Likewise it is not clear whether this epigenetic change is a cause or a consequence of tumor progression. A subset of CRCs characterized by wide-spread methylation at CpG islands in the promoter regions of several genes is recognized as CIMP. This group appears not to be directly correlated with PPARG hypermethylation (our unpublished data), suggesting that methylation at this specifc gene promoter is not caused by an aberrant spread of methylation over extended genomic regions. Consistently, LINE methylation levels, a surrogate marker of global DNA methylation, does not correlate with PPARG expression in CRC [10]. It is worth of note that methylation at the M3 segment of the PPARG promoter occurs not only in tumours in vivo but also in CRC derived cell lines. This is the first report that shows promoter methylation to play a role in PPARG repression in tumorigenesis. The only analysis reported so far, refers to the Pparg2 promoter in a mouse model of diabetes related to the adult metabolic syndrome [26]. The results obtained in 3T3-L1 preadipocytes and extended to the human gene, for structure and sequence similarities, suggest that also PPARG2 is regulated by DNA methylation [26]. More recently, epigenetic regulation of Pparg has been invoked as an important step in mouse myofibroblast transdifferentiation of hepatic stellate cells that promotes liver fibrogenesis [22].
The link between DNA methylation and histone modifications is mediated by a group of proteins with methyl-CpG-binding activity. MeCP2 recruits co-repressor complexes including HDACs and HMTs [27]. Its role in tumorigenesis is, however, still debated [22], [27], [28]. EZH2 is also recruited and appears to be involved in the maintenance of the repressed status [21]. Both MeCP2 and EZH2 have recently been shown to be key regulators of Pparg repression [22]. Consistently, MeCP2 and EZH2 levels inversely correlate with PPARG expression in the CRC cells investigated. In the silenced state, as in PPARG-negative HCT116 cells, the promoter is significantly enriched in HDAC1, MeCP2 and chromatin repressive marks such as H3K9me3 and H3K27me3. This latter suggests the presence of EZH2-containing repressive complexes. Differently, exposure to AZA and TSA causes replacement with active chromatin marks such as H3K9Ac and H3K4me3, accompanied by a complete loss of HDAC1 and MeCP2. Recruitment of RNAPol-II and P-RNAPol-II under these conditions fully correlates with the ability of the newly synthesized receptor to transactivate a reporter gene. MeCP2 and EZH2 silencing re-activate PPARγ, confirming their crucial role in PPARG epigenetic repression. These conclusions are supported by the enhanced growth properties of HT29 cells carrying a silenced PPARG and by the reduced growth rate and migration properties of HCT116 over-expressing a transfected wild type PPARγ. Collectively these functional analyses suggest that PPARG silencing may actively contribute to colon tumor progression. Thus, we propose a novel regulatory model in which an unmethylated (or partially methylated) PPARG core promoter region is normally recognized by unknown transcriptional activators, among which only the zinc-finger protein ZAC has been identified so far in CRC cells [23]. Upon promoter methylation, HDAC1 and MeCP2 repressive complexes are recruited to form a condensed chromatin structure that suppresses transcription initiation. In this context, EZH2-containing repressive complexes are further recruited, fully “marking” the histones via H3K27 methylation to establish a stable PPARG silencing by blocking transcription elongation (
Figure 6
). PPARG has been shown to potentiate the effects of a variety of chemotherapeutic regimens on the assumption that the addition of a specific ligand would render the receptor more efficient in transactivating target genes [29]. Only few evidences in the literature support the notion of adding a specific PPARγ agonist to well-established chemotherapeutic regimens for the treatment of PPARG-positive CRCs [30]. On the basis of our data, it is tempting to speculate a possible intervention for the treatment of PPARG-negative CRCs, based on the combination of a conventional chemotherapy with epigenetic drugs and a specific PPARγ agonist [31]. This regimen would re-establish PPARγ expression and activity, sensitize the tumour to the therapy, overcome possible resistance to the agonist and result in a better outcome with possibly longer survival.
10.1371/journal.pone.0014229.g006Figure 6 Schematic drawing of the proposed molecular mechanism(s) of PPARG silencing.
(I) The unmethylated or partially methylated PPARG core promoter is activated by unknown transcriptional factors, among which only the zinc-finger protein ZAC that induces apoptosis and cell-cycle arrest has been identified. (II) Upon extensive promoter DNA methylation, MeCP2, HDAC1 and EZH2 containing repressive complexes are recruited to form a condensed chromatin structure, inhibiting RNA polymerase II and impairing gene transcription.
In conclusion, we demonstrate that epigenetic events play a role in PPARG expression. DNA methylation and the associated chromatin repressive marks are responsible for PPARG silencing in a proportion of sporadic CRCs and derived cell lines. Larger epidemiological studies are required to support this hypothesis and to translate these results into clinical practice.
Supporting Information
Figure S1 Correlation between K-RAS and B-RAF mutations, microsatellite instability and PPARG methylation in CRCs. A subset of our colorectal cancer series was analyzed for K-RAS mutations at codons 12, 13 and B-RAF mutation at codon 600. CRCs were stratified based on Microsatellite stability (MSS) or instability (MSI) and related to PPARG methylation status (in grey).
(2.85 MB TIF)
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Figure S2 Loss of PPARG expression in CRC cell lines is due to DNA promoter methylation. (A) PPARG Loss Of Heterozigosity (LOH) was tested in HCT116 and HT29 cells using two DNA markers flanking the PPARG locus at the 5′ and 3′ end, respectively. No differences were appreciated, indicating that the locus had not been rearranged. Size marker = SM. (B) Activation of the MAPK/ERK signalling pathway did not correlate with the loss of PPARG expression. Basal and phosphorylated ERK levels were lower in PPARG-negative HCT116 than in PPARG-positive HT29 cells. (C) Quantitative ChIP analysis demonstrated enrichment of 5-methyl-cytosine (5-MeC) at the PPARG promoter in HCT116 with respect to HT29 cells. Epigenetic treatment significantly reduced 5-methyl-cytosine only in HCT116 cells *P<0.01. Error bars indicate the standard deviation of the mean.
(1.90 MB TIF)
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Figure S3 Synergistic effect of AZA and Trichostatin A (TSA) on PPARG re-activation. (A) HCT116 were treated with 1- 5 μM AZA for 72 hs, with 300 nM TSA for 24 hs alone or in combination with 1 or 5 μM AZA. HT29 cells served as control. PPARγ levels were analyzed by western-blot and quantified referring to β-actin. The histograms show that AZA/TSA in combination induce a synergistic PPARG re-activation only in HCT116 cells *P<0.05, **P<0.01, ***P = 0.0001, whereas no significant differences were observed in treated HT29 as compared to untreated control cells. (B) To confirm the synergistic effect on PPARγ activity, HCT116 were treated with AZA and TSA alone or in combination and transiently transfected with the PPRE-TK-luciferase reporter gene. After twelve hours the cells were treated with 1μM TZD or GW9662, a selective antagonist, or the vehicle alone (V). TZD administration increased the luciferase reporter gene activity, while exposure to GW9662 drastically reduced it even if compared with the vehicle alone. Luciferase activity was determined and normalized to β-galactosidase for transfection efficiency. Results are the mean values ± SD of three independent experiments, each performed in duplicate and compared with the corresponding controls; CC indicates untreated control cells *P<0.01; **P = 0.002.
(2.61 MB TIF)
Click here for additional data file.
Figure S4 HDAC1, MeCP2 and EZH2 expression levels in CRCs samples and cell lines. (A) Western blot analysis for HDAC1 was carried out in HCT116 and in the indicated CRC cell lines. (B) Protein extracts from representative tumour tissues (T) and matched adjacent normal mucosa (N) were analyzed for HDAC1, EZH2 and MeCP2. β-actin was used as internal control in both cases. (C) Immunohistochemical analysis of some representative tumour samples expressing high and low HDAC1 and PPARγ levels, respectively. (D) HDAC1, EZH2 and MeCP2 expression levels in a subset of CRCs (N = 20) and paired normal mucosa are represented by box-plot. The edges of the boxes are the interquartile range box, lines in the boxes represent the median value; the P value in each graph was obtained by the Mann-Whitney test (E). In some representative tumour samples (n = 52), HDAC1 and EZH2 high expression was directly related with C-D Duke's tumour stages. The same relationship was not found for MeCP2. P value was calculated by the Spearman correlation. (F) To assess the apoptotic rate induced by the PPARγ ligand troglitazone (TZD), flow cytometrical analysis (FCA) was carried out in HT29 parental cells transfected with a control plasmid (CC) or with an shPPARG. Alternatively, FCA was performed in HCT116 transfected with an empty vector (EV) or with an expression vector for PPARγ HCT116+PPARGγ.
(1.24 MB PDF)
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Table S1 (0.05 MB DOC)
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Table S2 (0.04 MB DOC)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work is supported by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro) to V.C. and L.A.; EU project to L.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2115190810-PONE-RA-20325R110.1371/journal.pone.0014232Research ArticleDiabetes and EndocrinologyMolecular BiologyCell Biology/Cell SignalingCell Biology/Cellular Death and Stress ResponsesCell Biology/Developmental Molecular MechanismsCell Biology/Leukocyte Signaling and Gene ExpressionAtorvastatin Improves Survival in Septic Rats: Effect on Tissue Inflammatory Pathway and on Insulin Signaling Atorvastatin Improves SurvivalCalisto Kelly Lima Carvalho Bruno de Melo Ropelle Eduardo Rochete Mittestainer Francine Cappa Camacho Angélica Costa Aranha Guadagnini Dioze Carvalheira José Barreto Campelo Saad Mario José Abdalla
*
Department of Internal Medicine, FCM, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
Calbet Jose A. L. EditorUniversity of Las Palmas de Gran Canaria, Spain* E-mail: [email protected] and designed the experiments: KLC MJAS. Performed the experiments: KLC BdMC FCM ACAC DG MJAS. Analyzed the data: KLC BdMC ERR JBCC MJAS. Contributed reagents/materials/analysis tools: JBCC MJAS. Wrote the paper: KLC ERR MJAS.
2010 6 12 2010 5 12 e1423227 6 2010 13 11 2010 Calisto et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The aim of the present study was to investigate whether the survival-improving effect of atorvastatin in sepsis is accompanied by a reduction in tissue activation of inflammatory pathways and, in parallel, an improvement in tissue insulin signaling in rats. Diffuse sepsis was induced by cecal ligation and puncture surgery (CLP) in male Wistar rats. Serum glucose and inflammatory cytokines levels were assessed 24 h after CLP. The effect of atorvastatin on survival of septic animals was investigated in parallel with insulin signaling and its modulators in liver, muscle and adipose tissue. Atorvastatin improves survival in septic rats and this improvement is accompanied by a marked improvement in insulin sensitivity, characterized by an increase in glucose disappearance rate during the insulin tolerance test. Sepsis induced an increase in the expression/activation of TLR4 and its downstream signaling JNK and IKK/NF-κB activation, and blunted insulin-induced insulin signaling in liver, muscle and adipose tissue; atorvastatin reversed all these alterations in parallel with a decrease in circulating levels of TNF-α and IL-6. In summary, this study demonstrates that atorvastatin treatment increased survival, with a significant effect upon insulin sensitivity, improving insulin signaling in peripheral tissues of rats during peritoneal-induced sepsis. The effect of atorvastatin on the suppression of the TLR-dependent inflammatory pathway may play a central role in regulation of insulin signaling and survival in sepsis insult.
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Introduction
Sepsis is one of the most prevalent diseases and one of the main causes of death among hospitalized patients [1]. During the onset of sepsis, the inflammatory system becomes hyperactive, leading to an over-production of pro-inflammatory mediators [2], which contribute to septic shock, multiple organ failure, and death. Hyperglycemia and insulin resistance occur during sepsis, as a consequence of the metabolic effects of stress hormone and cytokine production [3], [4], [5], [6]. Although in the past years there has been considerable progress in our understanding of the pathological pathways that contribute to sepsis and septic shock, pharmacological interventions are currently limited to insulin and activated protein C [7]. Insulin is infused in septic patients with hyperglycemia to normalize glucose levels [7], [8], and it is hypothesized that this reduction in glycaemia is associated with decreased inflammation and endothelial cell damage [4], [5], [6], [9], [10].
Conversely, results from animal studies indicate that insulin may have direct anti-inflammatory effects, independent of its effect on hyperglycemia [11], [12], [13]. However, the mechanism by which insulin reduces inflammation in the absence of hyperglycemia is unknown. Recent data demonstrate that insulin reduces inflammation by activating anti-inflammatory signaling pathways, such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Moreover, it is now well established that this pathway negatively regulates LPS-induced signaling and pro-inflammatory cytokine production [14], [15], [16], [17] and activation of PI3K/Akt pathway enhanced survival, whereas inhibition of PI3K reduced survival of endotoxemic mice [15], [18]. In animal models of sepsis, there is a down regulation of the PI3K/Akt pathway that may be the consequence of TLR4 activation and downstream in activation of well established inducers of insulin resistance as JNK, IKK-β and iNOS. Taken together, these data indicate that this insulin signaling pathway, which is reduced in sepsis, may be activated by insulin to mediate the protective effects of insulin in endotoxemia. However, insulin-induced hypoglycemia may counteract the beneficial effects of aggressive insulin therapy in patients with severe sepsis [19].
These data suggest that the ideal drug to improve survival in sepsis should reduce the over-reaction of the inflammatory response and, in parallel, should improve insulin signaling in the PI3K/Akt pathway, without inducing hypoglycemia. Recently, knowledge about statins, a class of powerful hypolipemic drugs with pleiotropic effects, such as anti-inflammatory, antioxidative, immunomodulatory properties, suggest that these agents may offer a novel therapeutic or prophylactic strategy to sepsis [1], [2], [20]. Relevant epidemiological evidence suggests that use of statins may decrease progression to severe sepsis [21], reduce mortality rates [22], [23], [24], [25], [26], [27], [28], [29] and reduce the risk of infections and infection-related complications [30], [31], [32], [33], [34]. However, the mechanisms by which statins induce this protective effect is not well established. The aim of the present study was to investigate whether the ability of atorvastatin to improve survival in sepsis is accompanied by a reduction in tissue activation of inflammatory pathways and, in parallel, an improvement in tissue insulin signaling.
Results
Atorvastatin Improves Survival and Protects Against Insulin Resistance in Septic Rats
In the first part of the study, we examined the effect of atorvastatin on the survival curve in animals in which sepsis was induced via CLP. Atorvastatin (10 mg/kg) or placebo was administered by gavage 3 h after surgery or the procedure in sham-operated animals. No deaths occurred in the sham-operated animals, whether or not they had been treated with atorvastatin. The survival curves for rats, in which CLP was performed, are shown in Figure 1A and clearly delineate the benefit sustained from atorvastatin treatment due to improved survival (P<0.0001). As shown in Fig. 1B and 1C, septic animals were more insulin resistant than sham rats; fasting plasma glucose and insulin levels were higher in septic rats than in the control group, and atorvastatin treatment reduced these levels. As depicted by Fig. 1D the plasma glucose disappearance rates measured during the insulin tolerance test (Kitt) were lower in septic animals and atorvastatin treatment reversed these alterations. This improvement is also suggested by the HOMA-IR index, calculated from fasting glycaemia and insulinemia, which is increased in septic animals and significantly decreased in those treated with atorvastatin (Fig. 1E). Atorvastatin treatment had no effect on insulin tolerance in the sham group. Taken together, these data suggest that atorvastatin reversed the sepsis-induced insulin resistance.
10.1371/journal.pone.0014232.g001Figure 1 Effect of atorvastatin on survival in CLP sepsis model.
Male Wistars rats, 8 weeks old, were given saline (Sepsis/Sal, n = 20) or atorvastatin 10 mg/kg (Sepsis/Ator, n = 20), 3 h and once a day after CLP. Survival of the rats was monitored at intervals of 12 h for 15 days. The overall difference in survival rate between the groups with and without atorvastatin was significant (P<0.0001) (A). Fasting blood glucose (B). Fasting insulin levels (C). Glucose disappearance rate (D). HOMA-IR index (E). Serum levels of TNF-α (F) and IL-6 (G). Data are presented as means and S.E. of six to eight rats per group. *P<0.05 (Sepsis saline vs. all others groups).
Effect of Atorvastatin on Serum Levels of IL-6 and TNF-α
The serum levels of IL-6 and TNF-α were also higher in septic animals compared with sham-operated rats. After atorvastatin treatment there was a significant decrease in IL-6 (Fig. 1F) and TNF-α (Fig. 1G) circulating levels.
Atorvastatin Improves Insulin Signaling in Liver, Muscle and Adipose Tissue of Septic Animals
In the sepsis group, insulin-induced IR and IRS-1 tyrosine phosphorylation were decreased in liver, muscle and adipose tissue when compared with sham rats and these alterations were reversed by atorvastatin (Fig. 2A–F). In parallel, there was a decrease in insulin-induced Akt serine phosphorylation in the liver, muscle and adipose tissue of septic animals when compared with sham rats and atorvastatin was able to reverse these reductions in Akt phosphorylation (Fig. 2G–I). The modulation in IR, IRS-1 and Akt phosphorylation, induced by sepsis and reversed by atorvastatin, was independent of changes in tissue protein expression (Fig. 2A–I). The protein concentration of IR, IRS-1, and Akt did not change between the groups. Equal protein loading in the gels was confirmed by reblotting the membranes with an anti-β-actin antibody (lower panels).
10.1371/journal.pone.0014232.g002Figure 2 Effects of atorvastatin treatment on insulin signaling in the CLP rat.
Representative blots show insulin-induced tyrosine phosphorylation of Insulin Receptor β (IRβ) in liver (A), muscle (B) and adipose (C) of sham and septic rats. Total protein expression of IRβ (A–C, lower panels). Insulin-induced tyrosine phosphorylation of Insulin Receptor Substrate 1 (IRS1) in liver (D), muscle (E) and adipose tissue (F) of sham and septic rats. Total protein expression of IRS1 (D–F, lower panels). Insulin-induced serine phosphorylation of Akt in liver (G), muscle (H) and adipose (I) of sham and septic rats. Insulin-induced threonine phosphorylation and total protein expression of Akt (G–I, lower panels). In this case, blots were stripped and reprobed with β-actin (A–I, lower panels) to confirm equal loading of proteins. Data are presented as means +/− S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.
Atorvastatin Attenuates Sepsis-Induced Inflammatory Changes
Toll-like receptor 4 (TLR4) is a transmembrane receptor that participates in pathogen recognition during the inflammatory response, and leads to cytokine and other immune-related gene expression [35], [36]. During sepsis, the activation of TLR4 signaling induces upregulation of intracellular inflammatory pathways, such as the IκB kinase β (IKK-β)/nuclear factor kappa B (NFκ-B) pathway.
Next, we examined the immunomodulatory effects of atorvastatin on TLR4 activation in liver, muscle and adipose tissue of septic animals. Fig. 3 shows that sepsis induced TLR4 protein levels and activation, as demonstrated by an increase in TLR4/MyD88 interaction in the three tissues investigated, and atorvastatin reduced this early step of TLR4 activation and also TLR4 expression (Fig. 3A–C).
10.1371/journal.pone.0014232.g003Figure 3 To evaluate the association of TLR4 with MyD88, immunoprecipitations were performed with MyD88 antibody followed by immunoblotting with TLR4 specific antibody.
Representative blots show TLR4 activation (upper panels) and expression (lower panels) in liver (A), muscle (B) and adipose tissue (C) of sham and septic rats. IKKβ phosphorylation in liver (D), muscle (E) and adipose (F) of sham and septic rats. Total protein expression of IKKβ (D–F, lower panels). Phosphorylation of IκBα in liver (G), muscle (H) and adipose (I) of sham and septic rats. Data are presented as means ±S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all other groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.
Downstream of TLR4 activation, we examined the IKK–NF-κB pathway, an important regulator of inflammation and insulin resistance. The main function of the IKK complex is the activation of NFκB through phosphorylation and degradation of IκBα [37], [38]. NFκB activity was monitored using IKKβ and IκBα phosphorylation, as described previously [39]. As expected, IKKβ and IκBα phosphorylation were increased in liver, muscle and adipose tissue of septic animals and atorvastatin decreased these phosphorylations in the tissues investigated (Fig. 3D–I).
JNK activation was determined by monitoring phosphorylation of JNK (Thr183 and Tyr185) and c-Jun (Ser63), which is a substrate of JNK. JNK phosphorylation in liver, muscle and adipose tissue were increased in septic animals and atorvastatin induced a downmodulation in the phosphorylation of this serine kinase (Fig. 4A–C). Consistent with JNK activation, c-Jun phosphorylation was induced by sepsis and reversed by atorvastatin in the tissues investigated (Fig. 4D–F).
10.1371/journal.pone.0014232.g004Figure 4 Representative blots show the JNK phosphorylation in liver (A), muscle (B) and adipose tissue (C) of sham and septic rats (upper panels).
Total protein expression of JNK (A–C, lower panels). Phosphorylation of c-jun in liver (D), muscle (E) and adipose tissue (F) of sham and septic rats. Serine 307 Phosphorylation of IRS1 in liver (G), muscle (H) and adipose tissue (I) of sham and septic rats (upper panels). Total protein expression of IRS-1 (G–I, lower panels). Data are presented as means ± S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.
We also investigated Ser307 phosphorylation of IRS-1 in liver, muscle, and adipose tissue in the four groups of rats. Ser307 phosphorylation was induced by sepsis in the three tissues and the treatment with atorvastatin reversed this alteration (Fig. 4G–I). NFκB nuclear subunit p50 expression was determined in nuclear extracts and we found an increase in this subunit in nuclear extracts of septic animals, but there was a clear decrease in the three tissues after atorvastatin treatment (Fig. 5A–C). The tissue expressions of iNOS (Fig. 5D–F) and IL-6 (Fig. 5G–I) were higher in liver, muscle and adipose tissue of septic rats that received saline, and were significantly reduced by atorvastatin treatment.
10.1371/journal.pone.0014232.g005Figure 5 Representative blots show the NFkB activation in nuclear fractions of liver (A), muscle (B) and adipose tissue (C) of sham and septic rats.
In this case blots were stripped and reprobed with actin (A–C, lower panels) to confirm equal loading of proteins. Tissue levels of iNOS (D–F) and IL-6 (G–I) expression in liver, muscle and adipose tissue of sham and septic rats. Data are presented as means ± S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups); **P<0.001 (Sepsis/Sal vs. control); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.
Previous studies have shown that sepsis is also characterized by endoplasmic reticulum stress. It is clear that ER stress can also induce activation of JNK and IKKβ. We then investigated the effect of sepsis (treated or not with atorvastatin) on proteins that reflect ER stress. Our data showed that sepsis induced ER-stress, as determined by the increased phosphorylation of the ER membrane kinase, PERK (PKR-like endoplasmic reticulum kinase) and its substrate (Fig. 6A–C), eIF2α (eukaryotic translation initiation factor 2α) (Fig. 6D–F) and increased the expression of ATF6α (Fig. 6G–I). Treatment with atorvastatin significantly reduced the expression of all markers of ER-stress. (Fig. 6A and D)
10.1371/journal.pone.0014232.g006Figure 6 Representative blots show the PERK phosphorylation in liver (A), muscle (B) and adipose tissue (C) of sham and septic rats.
eIF2α phosphorylation (D–F) and ATF6 (G–I) expression in liver, muscle and adipose tissue of sham and septic rats. In this case, blots were stripped and reprobed with actin (A–I, lower panels) to confirm equal loading of proteins. Data are presented as means ± S.E.M from 6–8 rats per group. *P<0.05 (Sepsis/Sal vs. all others groups); #P<0.05 (Sepsis/Sal vs. Sepsis/Ator). IB, immunoblot; CLT: Sham/Saline; ShT: Sham/Atorvastatin; SAL: saline; ATOR: atorvastatin.
Discussion
In sepsis, the acute immune response is organized and executed by innate immunity. This response starts with sensing of danger by pattern-recognition receptors on the immune competent cells and endothelium. The pattern-recognition receptors, mainly toll-like receptor (TLR), are also activated in other tissues such as liver, muscle and adipose tissue [40]. The sensed danger signals, through specific signaling pathways, activate transcription factors and gene regulatory systems, which up-regulate the expression of pro-inflammatory mediators. However, the over-reaction of this pro-inflammatory response has an important role in the development of multiple organ failure and death. The combinations of the activation of pattern recognition receptors, specifically TLR4, and the cytokine storm of sepsis have an important role in insulin resistance. In this regard, tissue insulin resistance may be used as an important indicator of the resultant actions of pattern recognition receptors and the induction of the pro-inflammatory cytokines, being a tissue marker of severity of sepsis, before organ failure. In addition, this insulin resistance may also aggravate sepsis, as previously described [3], [4], [41]. In this regard, we believe that the evaluation of insulin signaling pathway through PI3K/Akt in liver, muscle and adipose tissue may be important indicators of this over-reaction at the tissue level, and the improvement in this signaling pathway, induced by some treatments, in parallel with a decrease in tissue inflammation, may predict the effectiveness of this treatment.
In the present study, we demonstrated that atorvastatin improves survival in septic rats, decreases circulating inflammatory cytokines and improves insulin resistance, in parallel with a decrease in TLR4 signaling and an improvement in insulin signaling in liver, muscle and adipose tissue. The improvement in survival of septic animals when using statins has been previously described, and was related to a complete preservation of cardiac function and hemodynamic status and also a reversion of increased monocyte adhesion to the endothelium, all of which were altered in septic animals without treatment [42], [43].
Our data show that atorvastatin, administered 3 hours after the induction of sepsis, is able to improve the survival curve, attenuating higher levels of IL-6 and TNF-α and reduce insulin resistance, as demonstrated during the insulin tolerance test. In septic animals, insulin resistance was accompanied by a reduction in insulin-induced IR, IRS-1 tyrosine phosphorylation and in insulin-induced Akt phosphorylation, in liver, muscle and adipose tissue. Since Akt has a critical role in protection from apoptosis, it is possible that the reduced insulin signaling through this IRSs/PI3K/Akt pathway, in sepsis, may contribute to multi-organ failure by preventing or delaying apoptosis [14], [15], [44]. In this study, we demonstrate that pretreatment with atorvastatin inhibits sepsis-induced insulin resistance by improving insulin signaling via the IR–IRS-1–Akt pathway in target tissues. This restoration of insulin signaling in these tissues would allow the animal to have an appropriate control of hepatic glucose output and of the peripheral glucose uptake and storage. In addition, skeletal muscle and adipose tissue contribute to IL-6 expression during sepsis [45], [46], [47], and since the anti-inflammatory effect of insulin is mediated through the PI3K pathway [14], [15], [18], we can speculate that the restoration of this pathway in the insulin-dependent tissues, induced by atorvastatin in septic animals, may have also contributed to the anti-inflammatory effect of this drug.
During the past ten years, accumulating evidence shows a clear molecular interaction between metabolic and immune signaling systems in different situations of insulin resistance [40], [48], [49]. We and others have previously demonstrated that TLR4 signaling is activated in diet induced obesity (DIO), and that this activation culminates in an increase in the activation of downstream effectors such as IKKβ, JNK and iNOS, which have critical roles in insulin resistance [40]. In septic animals, there was an increase in TLR4 activation and also in downstream effectors that may have an important role in the insulin resistance of these animals [50]. Our data show that atorvastatin decreases TLR4 expression/activation, a modulation that might have a role in the attenuated expression of inflammatory mediators in response to a septic insult. The reduction in TLR4 expression/activation, observed in our study, is in accordance with a previous study that also observed this effect on TLR4 expression/activation with different statins and cell types [51].
The immune modulator activity of atorvastatin was evident downstream from TLR4, at the level of IKK/IκB/NF-κB pathway activation. Atorvastatin induced a significant decrease in IKK phosphorylation and, as expected, an increase in IκB phosphorylation, suggesting a deactivation of this pathway. NF-κB has been documented to play a major role in sepsis induced inflammatory cytokine expression [52], [53], [54]. Our findings suggest that NF-κB, which is normally translocated from the cytoplasm to the nucleus after sepsis insult, was strongly inhibited by atorvastatin in the three target tissues studied. This result suggests that the atorvastatin-mediated inhibition of cytokine production may be the consequence of the modulation of the IKK/IκB/NF-κB pathway by this drug.
The activation of NF-κB with its translocation to the nucleus is able to induce the increase in TNF-α, IL-6 and in iNOS in septic animals [37], [38], [55]. TNF-α is one of the crucial pro-inflammatory cytokines; however, when over produced by deregulation or persistent infection, TNF-α may induce septic shock and contributes to insulin resistance, and its levels are drastically elevated in a number of forms of human sepsis, in turn correlating with increased mortality [56]. Specifically, iNOS and IL-6 have been shown to be produced early in the response and have been suggested to play critical roles in driving physiological/pathological responses that lead to septic shock [57]. We further investigated the effects of atorvastatin on the production of these inflammatory proteins in tissues and serum of septic rats. In accordance with previous data, atorvastatin treatment inhibited iNOS, TNF-α and IL-6 expression in the muscle, liver and adipose tissues of septic rats, and also the circulating levels of TNF-α and IL-6. [58]. Previous studies have demonstrated that skeletal muscle and adipose tissue contribute to IL-6 production during endotoxemia [45], [46], [47], and the anti-inflammatory effects of atorvastatin on IL-6 production in sepsis may be the result of the direct activation of PI3K pathway in these tissues. The atorvastatin-mediated reduction of these negative modulators of insulin signaling may have an important role in the improvement of insulin signaling observed in the three tissues of septic animals.
Another TLR4 downstream pathway by which atorvastatin could attenuate the inflammatory response induced by sepsis is through JNK, a serine kinase that is responsible for activation of the inflammatory pathway by phosphorylation of the c-Jun and ATF2 transcription factors [59], [60]. Several studies suggest that JNK contributes to insulin resistance by phosphorylating IRS-1 at serine 307, and this phosphorylation leads to inhibition of IRS-1 function [26], [27], [30], [33], [61], [62], [63], [64], although this has very recently been questioned [65]. Here, we observed that sepsis led to serine phosphorylation of IRS-1 and that atorvastatin reversed this phenomenon in three target tissues, in parallel with a reduction in JNK activity. Our data showing that atorvastatin inhibits JNK phosphorylation/activation in septic rats indicate that the beneficial effect of this drug in improving survival and reducing insulin resistance is mediated by different pathways.
Besides being activated by TLR4, JNK activity is induced in different pathophysiological states including infection, inflammation, obesity and hyperlipidemia, also as a consequence of ER stress [66], [67], [68]. The cellular response to ER stress, referred to as UPR, results in the activation of three linked signal transduction pathways emanating from three principle ER stress sensors: IRE1α, double-stranded RNA–dependent protein kinase–like kinase (PERK) and ATF6α [49], [69]. Mechanistically, activation of the UPR contributes to the decrease in insulin sensitivity through IRE1α-dependent activation of c-Jun N-terminal kinase (JNK) [70], [71]. Recently, it was demonstrated that statins are able to prevent ER stress [72]. In the present study, we confirm this finding by showing that atorvastatin strongly inhibited phosphorylation of IRE1α and PERK and ATF6α expression, suggesting that this drug can attenuate the ER-stress induced by sepsis.
Overall, our data demonstrate that atorvastatin exerts direct regulatory effects on TLR4 expression/activation in an animal model of sepsis that influences TLR4 signaling. Atorvastatin reduces TLR4 surface expression on liver, muscle and adipode tissues, causing downregulation of proinflammatory pathways such as IKK and JNK, leading to a decrease in NF-κB activation and cytokine expression. In addition, atorvastatin also decreases ER stress and, consequently, the activation of JNK and IKK. Thus, we suggest that the effects of atorvastatin on TLR4 expression/activation and on ER stress are mechanistically relevant to improve the sepsis-induced insulin resistance. There are several potential limitations to the present study.
To date, pharmacological interventions in sepsis have usually been limited to insulin and Protein C [7]. The beneficial effects of activated protein C are partially independent from its anticoagulant activity and may be related to anti-inflammatory and anti-apoptotic effects, but its effect on insulin signaling is unknown. Insulin has anti-inflammatory effects [11], [12], [13], [19], which are dependent on PI3K signaling, and the infusion of this hormone induces the PI3K/Akt pathway. However, the beneficial effect of insulin may be overcome by hypoglycemia. It is important to mention that, in sepsis, many inflammatory pathways are activated in parallel with a reduction in the PI3K/Akt pathway, thus, merely blocking a single component of the inflammatory pathways or inducing the activation of PI3K may be insufficient to arrest the process. In this regard, our data show that atorvastatin is able to modulate entire families of inflammatory mediators, associated with a clear improvement in tissue insulin signaling and in insulin sensitivity, suggesting mechanisms for its efficiency in sepsis. Additionally, our data may suggest that the investigation of drugs in sepsis should take into account tissue measurements of inflammatory pathways and insulin signaling, or other early tissue markers that indicate the severity of sepsis.
In summary, this study demonstrated that treatment with atorvastatin increased survival with a significant effect upon insulin sensitivity, improving insulin signaling in peripheral tissues of the rat during peritoneal-induced sepsis. This drug reduces TLR4 activation, in association with downstream JNK and IKK/NF-κB activation and downregulated the serum levels of cytokine release. The effect of atorvastatin on TLR-dependent inflammatory pathway suppression may play a central role in the regulation of insulin signaling and survival following the sepsis insult.
Materials and Methods
Materials
Anti-IR-β (α-IR), anti-IRS-1, anti-Akt, anti-p-JNK, anti-iNOS, anti-NFκB, anti-IL6, anti-TLR4, anti-IKKβ, anti-pIKKβ, anti-pIκBα, anti-MyD88, anti-p-cjun, anti-pJNK, anti-pPERK anti-PERK, anti-ATF6α and anti-IRE1α antibodies were from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-pAkt was from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-IRS-1ser307 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Anti-peIF2α was from Abcam (Cambridge, MA, USA). Atorvastatin was obtained from Pfizer (Loughbeg, County Cork, Ireland). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, Indiana, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless specified elsewhere.
Animal Care and Experimental Procedures
All experiments were approved by the Ethics Committee at the University of Campinas, CEEA/Unicamp 1267-1. Male Wistar- Hannover (8 wks old) were maintained in a room with 12-hour day/night cycles and room temperature of 21°C, with food and water ad libitum. Wistar rats were randomly divided into four groups; atorvastatin-treated sepsis (Sepsis/Ator), saline-treated sepsis (Sepsis/Sal), atorvastatin-treated sham (Sham), and saline-treated sham (Control).
Cecal ligation and puncture (CLP) was performed, as previously described [73], and is a commonly-used surgical technique in rodents and thought to be a clinically relevant animal model of sepsis. Anesthesia was induced by IP administration of ketamine (80 mg/kg BW) and xylazine (15 mg/kg). Through a 1-cm abdominal midline incision, the cecum was ligated below the ileocecal valve with careful attention to avoid obstruction of the ileum or colon. The cecum was subjected to a four “through-and-through” perforations (20-gauge needle). The abdominal incision was closed in layers. Sham-operated rat underwent the same procedure, except for ligation and perforation of the cecum. All procedures were performed under sterile conditions. Animals were allowed to recover and were observed twice a day. Three hours after the induction of sepsis and every 24 hours rats received atorvastatin (10 mg/kg) or an equivalent volume of saline by oral gavage. The dose of atorvastatin was chosen on the basis of previous findings [74] and was consistent with the maximum human dose (1.1 mg/kg per day), and the higher metabolic rate of the drug in rodents [75].
Homeostasis Model Assessment
Insulin resistance was assessed from fasting insulin and glucose levels, using the previously validated homeostasis model of assessment (HOMA-IR), as previously described [76], [77]. HOMA-IR was calculated by the formula: fasting plasma glucose (mmol/l) x fasting plasma insulin (mU/l)/22.5. Fasting blood glucose was measured by the glucose oxidase method. Plasma insulin was assayed using commercial rat-specific radioimmunoassay kits (Linco Research Inc, St. Louis, MO, USA).
Insulin tolerance Test (ITT)
The insulin tolerance test (ITT) was performed on these rats at 24 hours after sepsis, as previously described [78]. Insulin (1.5 U/kg) was administered by i.p. injection and blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min to determine serum glucose. The constant rate for glucose disappearance (Kitt) was calculated using the formula 0.693/t1/2. Glucose t1/2 was calculated from the slope of the least-squares analysis of plasma glucose concentrations during the linear decay phase [76].
Cytokines Assays
IL-6 and TNF-α were determined using commercially available ELISA kits (Pierce Biotechnology Inc., Rockford, IL, USA), following the instructions of the manufacturer.
Tissue Extraction, Immunoprecipitation and Immunoblotting
Rats were anaesthetized by intraperitoneal injection of sodium thiopental and were used 10–15 min later, i.e. as soon as anesthesia was assured by the loss of pedal and corneal reflexes. Five minutes after saline (0.2 ml) or insulin injection (3.8 U/Kg ip), liver, muscle and adipose tissue were removed, minced coarsely and homogenized immediately in extraction buffer, as described elsewhere. Extracts were used for immunoprecipitation with MyD88 and Protein A-Sepharose 6MB (Pharmacia, Uppsala, Sweden). NFκB p50 activation was determined in nuclear extracts from liver, muscle and adipose tissue, as previously described [79]. The precipitated proteins and/or whole tissue extracts were subjected to SDS-PAGE and immunoblotting, as previously described [40], [78].
Statistical Analysis
The overall difference in survival rate was determined by the Kaplan–Meier test followed by log–rank test. Specific protein bands present in the blots were quantified by digital densitometry (ScionCorp Inc., Frederick, MD, USA). Means ± S.E.M. obtained from densitometric scans, area measurements, and the values for blood IL-6, TNF-α and glucose were compared by ANOVA with post hoc test (Bonferroni). A P value of p<0.05 was accepted as statistically significant.
We acknowledge the excellent technical assistance of Luis Janieri, Jósimo Pinheiro and Antonio Calixto for their technical assistance. We thank Dr. N. Conran for English grammar review.
Competing Interests: The authors have declared that no competing interests exist.
Funding: K.L.C. holds a graduate fellowship from CAPES, www.capes.gov.br. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Int J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 2115146410.3390/ijms11114687ijms-11-04687ArticleHybrid Endovascular Repair in Aortic Arch Pathologies: A Retrospective Study Ma Xiaohui Guo Wei *Liu Xiaoping Yin Tai Jia Xin Xiong Jiang Zhang Hongpeng Wang Lijun Department of Vascular Surgery, Clinical Division of Surgery, Chinese People Liberation Army (PLA) General Hospital and Postgraduate Medical School. 28 Fuxing Road, Beijing 100853, China; E-Mails: [email protected] (X.M.); [email protected] (X.L.); [email protected] (T.Y.); [email protected] (X.J.); [email protected] (J.X.); [email protected] (H.Z.); [email protected] (L.W.)* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-010-669-383-49; Fax: +86-010-681-769-94.18 11 2010 2010 11 11 4687 4696 19 10 2010 7 11 2010 10 11 2010 © 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.2010This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The aortic arch presents specific challenges to endovascular repair. Hybrid repair is increasingly evolving as an alternative option for selected patients, and promising initial results have been reported. The aim of this study was to introduce our experiences and evaluate mid-term results of supra aortic transpositions for extended endovascular repair of aortic arch pathologies. From December 2002 to January 2008, 25 patients with thoracic aortic aneurysms and dissections involving the aortic arch were treated with hybrid endovascular treatment in our center. Of the 25 cases, 14 were atherosclerotic thoracic aortic aneurysms and 11 were thoracic aortic dissection. The hybrid repair method included total-arch transpositions (15 cases) or hemi-arch transpositions (10 cases), and endovascular procedures. All hybrid endovascular procedures were completed successfully. Three early residual type-I endoleaks and one type-II endoleak were observed. Stroke occurred in three patients (8%) during the in-hospital stage. The perioperative mortality rate was 4%; one patients died post-operatively from catheter related complications. The average follow-up period was 15 ± 5.8 months (range, 1–41 months). The overall crude survival rate at 15 months was 92% (23/25). During follow-up, new late endoleaks and stent-raft related complications were not observed. One case (4%) developed a unilateral lower limb deficit at 17 days and was readmitted to hospital. In conclusion, the results are encouraging for endovascular aortic arch repair in combination with supra-aortic transposition in high risk cases. Aortic endografting offers good mid-term results. Mid-term results of the hybrid approach in elderly patients with aortic arch pathologies are satisfying.
aortic archendovascular repairaneurysmdissection
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1. Introduction
The conventional surgical repair of thoracic aortic aneurysms and dissections remains a high risk procedure [1,2]. Based on the eight largest recent researches published (with over 40 patients) [3–10], the 30-day stroke/death rate after aortic arch surgical repair is up to 25.6% (mean 17.5%). The introduction of endovascular stent graft technology has reached an evolutionary threshold for the treatment of complex aortic diseases. The aortic arch presents specific challenges to endovascular repair, which mainly arise from the involvement of the supra-aortic branches and the tight inner curve. Inoue et al. reported one case of triple-branched stengraft [11] and Chuter and colleagues reported a branched stentgraft to the innominate artery [12,13]. However, these new designs are still at an experimental stage. Hybrid repair, which constitutes a combination of open supra-aortic branch revascularization and endovascular aortic repair, has increasingly evolved as an alternative option for selected patients, and promising initial results have been reported [14–18]. The aim of this study was to introduce our expriences and evaluate mid-term results of supra aortic transpositions for extended endovascular repair of aortic arch pathologies.
2. Methods
From December 2002 to January 2008, 25 patients with thoracic aortic aneurysms and dissections involving the aortic arch were treated with hybrid endovascular treatment in our center. Preoperative planning was at the discretion of the operating surgeon and was based upon contrast enhanced CT scanning with 1.5 mm cuts and three-dimensional reconstruction that allowed accurate, centerline measurements of the aorta. As a prerequisite for successful stent-graft placement, a proximal landing zone of at least 1.5 cm along the curvature of the aortic arch was necessary. All patients underwent risk evaluation according to EuroSCORE guidelines [19]. Patients who were not suitable for endografting and those at low risk for surgery were treated by open surgery (three patients during the same period).
The average age of the patients was 71.5 ± 9.9 years (range, from 50 to 83 years), and the ratio (male:female) was 5.5:1. Risk factors of the patients are shown in Table 1. Among 25 cases, 14 (56%) were atherosclerotic thoracic aortic aneurysms, the average length of aneurysms was 242.33 ± 82.34 mm; 11 (44%) were aortic dissection, three (12%) were type A thoracic aortic dissection, 8 (32%) were type B thoracic aortic dissection, of which seven were chronic phase and one was acute phase. The acute phase was defined as within two weeks after symptom onset; the subacute phase as the following two-month period; and the chronic phase as anything thereafter. The interval from onset of type B thoracic aortic dissection to treatment was 2–7 months. The average maximum aortic diameter was 64 ± 11.3 mm. None of the pathologies were a result of trauma.
In order to distinguish from Ishimaru’s anatomical aortic classification [20] using antegrade numbering, we propose a retrograde landing zone classification [21]. This classification is based on pathophysiology and reflects the extension of the disease and case complexity, with respect to the need for transposition. We define four proximal landing zones as seen in Figure 1. An endografting procedure at Zone 3 is an ideal situation and requires no surgical complementary step for both aneurysms and dissections. Starting at Zone 2 requires either coverage or transposition of the left subclavian artery (LSA). If the origin of the left common carotid artery (CCA) (Zone 1) is involved, transposition to the right CCA via a carotido-carotid bypass must be performed. We call this adjunctive procedure a hemiarch transposition. If the disease extends the full length of the aortic arch, requiring coverage of the innominate artery (IA), a bypass to the IA and left CCA is performed through a median sternotomy from the ascending aorta. We refer to this as total-arch transposition (Figure 2). The terminology of hemi-arch and total-arch transposition is used in order to simplify the discussion, thus avoiding repetition of the different bypasses performed.
We performed 15 total-arch transpositions and 10 hemi-arch transpositions. For the total-arch transpositions, the endografts were deployed at a second step: 1 week following the creation of the proximal landing zone. We always used a femoral percutaneous access and an additional percutaneous humeral approach was used in some instances to mark the origin of the native IA and LSA. Carotid and vertebral artery circulation were assessed before operation. During the transposition procedure, the stump pressure was checked before clamping the arch vessels.
Hemi-arch transposition was performed via a vertical 4 cm cervical approach to both CCAs. Then an 8 mm Dacron graft (Braun Unigraft, Melsungen) was implanted between two CCAs in a U shape anterior to the trachea. The strategy of this procedure was to perform an end-to-side anastomosis between the left CCA and the brachiocephalic trunk. Afterwards, an end-to-side anastomosis was performed between the LSA and the already transposed LCCA. Total arch transposition is performed through a median sternotomy. A 12 mm Dacron bifurcated graft (Braun Unigraft, Melsungen) was implanted on the ascending aorta as proximal as possible, using lateral clamping. An 8 mm branch anastomosed end-to-end to the IA, while another 8 mm branch was anastomosed the same way to the right CCA. The proximal stumps of these vessels were clamped during the anastomosis and sutured with a 5/0 prolene (Ethicon, Inc, Somerville, NJ, USA) suture after the bypass was finished in order to reduce the clamping time. Depending on the patient’s anatomy, the graft was passed in front or behind the innominate vein, which can be divided or reconstructed if necessary. The LSA was not bypassed unless the vertebral artery was dominating, since it is often hard to reach through a standard sternotomy. Moreover, a patent LSA may serve as access to the aneurysm, when coiling was necessary to treat a residual type I endoleak. A retrograde type II endoleak will appear only if there was an outflow from the sac, such as created by patent intercostal arteries, which are normally thrombosed. In only one case we observed a type II endoleak that was easily treated by percutaneous occlusion of the LSA (Figure 3). Endoleaks are defined as follows: type I include leaks from the proximal and distal seal zones, type II are secondary to patency of aortic branches (intercostal arteries, lumbars, etc.) [22].
Following total-arch transposition, markers (metal clips) were placed at the proximal anastomosis to the arch, to define the proximal extent of the proximal landing zone.
Three different commercially available stent-graft systems were used, as shown in Table 2. Endovascular procedures were performed under general anesthesia. In the majority of patients, a transfemoral approach was chosen. If the diameter of the external iliac artery was not large enough, the common iliac artery was used for arterial access. Stent-graft deployment was routinely performed under hypotonic conditions (systolic pressure < 90 mmHg). We did not use adenosine induced transient cardiac asystole. Endografts were oversized by 20% for aneurysms and 10% for dissections. The distal diameter of the endograft was initially slightly reduced with non-tapered devices. Since this series, we have been treating dissections with a tapered endograft whose 24 mm distal diameter better fits the distal landing zone. Stent-graft related data are shown in Table 3. We avoided using dilatation balloons unless it was necessary due to a residual endoleak. This was especially true for dissections.
Following hospital discharge, patients were regularly contacted either by mail or telephone and they were asked to undergo both CT-scan and plain X-ray examinations at 3 (in case of post-operative residual minor type 1 endoleak), 6, 12, 18 and 24 months post-operatively, and yearly there after. Data such as pre-operative size, patients’ condition, risk factors, and post-operative control information, etc., were collected during regular working meetings, and were put together in a single Excel file. We calculated crude rates of survival, neurological complications and endoleaks because the study was not large enough to carry out a life-table analysis.
3. Results
3.1. Duration of Hospital Stay
3.1.1. Surgical Procedure
All patients recovered uneventfully without any serious neurologic injury after aortic debranching. One patient in the hemi-arch transposition group suffered a minor stroke, but was eventually discharged successfully. In the total arch transposition group, one proximal dissection occurred at the site of lateral clamping, which sealed spontaneously.
3.1.2. Stent-Graft Placement
All endovascular procedures were completed successfully. One worsening minor stroke was observed in the hemi-arch transposition group, while no neurological complication occurred in the group of total arch exclusion. During the deployment, the stent graft had not misplacement. We observed three early residual type-I endoleaks (12%), which were left untreated since they may thrombose spontaneously in the post-operative course. The first residual endoleak thrombosed spontaneously and the second was successfully treated by graft extension. The third was due to an uncovered entry tear in the ascending aorta and would have required total arch transposition, which was rejected by the patient. We also had one type-II endoleak in an aneurysm from LSA, which was successfully coiled after one week. We had no case of early paraplegia.
Stroke occurred in three patients (8%) during the in-hospital stage; one patient had a minor stroke within 48 h due to the occlusion of the left CCA bypass, which was resolved by a cervical carotid-carotid bypass. The perioperative mortality rate was 4%; one patient died post-operatively from catheter related complications: The patient died at three days from multiorgan failure after rupture of the descending of the aorta.
3.2. Follow-up Period
The average follow-up period was 15 ± 5.8 months (range from 1–41 months), and all patients adopted follow-up regularly. Three-dimensional CT-scan and X-ray examinations were obtained for all the patients before their discharge to act as control images. The overall crude survival rate at 15 months was 92% (23/25). Another patient with chronic obstructive pulmonary disease (COPD) died three months after the procedure because of acute respiratory failure.
During follow-up, new late endoleaks were not observed. The aneurysmal sac exclusion rate was 100%. The rate of occlusion in thoracic false lumen was 91%, while we observed seven cases (28%) patent abdominal false lumens, of which the maximum aortic diameter was <50 mm. Endograft migration, fracture, and stent-raft related complications such as aorto-esophageal fistula were also not observed. None of the patients had new cerebral neurological adverse events. One case (4%) developed a unilateral lower limb deficit at 17 days and was readmitted to hospital. According to independent neurological assessment, this deficit could be due to medullar ischemia, based on cerebral and medullar MRI findings.
4. Discussion
Since the first description of revascularization of the left carotid and sbuclavian artery from the ascending aorta prior to stent grafting, only case reports and small case series have been published [24–30]. No comparative randomized or non-randomized studies of combined open debranching and endovascular procedures with other conventional treatment strategies for aortic arch repair have been identified. Furthermore, mid-term and long-term results are still awaited. Our mid-term results of alternative treatment approaches for aortic arch pathologies are satisfying. This hybrid approach provides safe and effective treatment for patients at high risk for conventional repair.
We recommend additional transposition of LSA when it supplies coronary circulation through the left internal mammary artery, when the contralateral vertebral artery (VA) is stenosed or in a diseased vertabro-basilar system. We also recommend transposing the LSA in association with the left CCA when they are included in the aneurysm, except during total transpositions since the LSA is difficult to reach by median sternotomy. In all other cases, LSA transposition is only required later if the coverage becomes symptomatic. Great vessel transposition appears to be safe. There were no major strokes or deaths related to transposition. There was one early death (4%) after the endovascular step, which was either access or guide-wire related.
In the total arch exclusion group, no immediate neurological complications occurred during either surgical or endovascular steps. On the other hand, in the hemi-arch exclusion group, we observed one major stroke. This may be due to catheter manipulation in front of a patent innominate artery ostium, in a patient with an atherosclerotic aorta. A possible way to reduce embolic complications may be to perform pre-operative trans-esophageal echography to better select the patients. We should also pay attention to reduce cross-clamping times of the brain supplying vessels by as much as possible, and furthermore, the absence of substantial atherosclerotic disease in the wall of the asending aorta in total arch rerouting procedures. Without doubt, the risk of embolism is present in all these procedures and careful manipulation of central vessels as well as minimizing the cross-clamp times, in order to not exceed the ischemic frame of cerebral tissue, is mandatory for success [31].
Total arch transposition allows availability of a longer proximal landing zone, easily reaching 3 cm in length for a better anchoring of the endograft. It also avoids stentgraft deployment within the arch curvature, which may cause endoleaks and migration. In selected cases of conical or larger aortas exceeding 40 mm in diameter, the banding technique may be useful in association with total arch transposition to allow a better proximal landing zone.
We prefer a staged procedure for the following reasons: the operating time is decreased; bleeding volume is lowered; the risk of graft infection may be lowered, since endovascular and imaging manoeuvers are not performed in front of an open chest. Considering our encouraging results, we have decided in our department to extend the use of total arch transposition with acute type A aortic dissection. We are combining the replacement of the ascending aorta with the transposition of the IA to the ascending aortic graft. This allows secondary arch coverage for recalcitrant dissection.
The future of this challenging approach is dependent on whether the endografting technology will be reliable or not [32]. Improvement of stent-grafting is needed in terms of flexibility to improve aortic arch navigation and reduce the embolic risk.
In summary, this study analyzed the mid-term results of endovascular repair of aortic arch aneurysm and dissection. The results are encouraging for endovascular aortic arch repair in combination with supra-aortic transposition in high risk cases. Combined treatment for high risk cases offers as good results as seen for conventional surgery for low risk patients. Aortic endografting offers good mid-term results. The mid-term results of the treatment approach in elderly patients with aortic arch pathologies at high risk are satisfying. Nevertheless, meticulous technique is mandatory in order to avoid diverse complications. Thus, the long-term utility of this technology awaits further investigation, although intermediate-term results are encouraging in high-risk patients.
Figure 1. The proximal landing zone classification.
Figure 2. The intra-operative view of the implanted endograft (left) and the Angiogram (right) demonstrating the reconstruction of the total-arch transposition.
Figure 3. Coiling of the left subclavian artery after total arch repair [23].
Table 1. Risk factors in patients.
Risk factors Number of patients Percentage (%)
Age over 70 17 68
Severe cardiac impairment: cardiac valvulopathy, previous coronary bypass and/or MI 12 48
Chronic pulmonary disease: (FEV1 ≤ 1l) 10 40
Neurological dysfunction 3 12
Surgery on thoracic aorta 7 28
Table 2. The relevant characteristics of the three proximal device implants used.
Device Patients treated, %(n) Proximal bare spring Deployment strategy
Talent (Medtronic, minneapolis, Minn) 32 (8) With pullback
Ankura (Lifetech, Shenzhen, China) 24 (6) With pullback
Zenith TX2 (Cook, Bjaeverskov, Denmark) 44 (11) without Pullback and then release of trigger wire
Table 3. Stent-graft related data.
Covered length Proximal diameters Distal diameters Graft number
aneurysm 280.33 ± 82.34 (184–388) 43.33 ± 2.07 38.00 ± 2.19 2.5 ± 1.05
dissection 223.33 ± 111.01 (113–335) 43.33 ± 2.31 42.00 ± 2.00 2 ± 1
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21179203PONE-D-10-0166110.1371/journal.pone.0015137Research ArticleBiologyAnatomy and PhysiologyMusculoskeletal SystemMuscleMuscle FunctionsBiochemistryCytochemistryCell MembraneMembrane ProteinsMetabolismBiological TransportProteinsTransmembrane Transport ProteinsMedicineAnatomy and PhysiologyMusculoskeletal SystemMuscleMuscle BiochemistryMuscle FunctionsElectrophysiologyT Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle Sarcolemmal and T-Tubular Lactic Acid TransportHallerdei Janine
1
Scheibe Renate J.
2
Parkkila Seppo
3
Waheed Abdul
4
Sly William S.
4
Gros Gerolf
1
*
Wetzel Petra
1
Endeward Volker
1
*
1
Molecular and Cell Physiology, Vegetative Physiologie, Medizinische Hochschule Hannover, Hannover, Germany
2
Abteilung Physiologische Chemie, Medizinische Hochschule Hannover, Hannover, Germany
3
Institute of Medical Technology, Tissue Biology, University of Tampere, Tampere, Finland
4
Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, Missouri, United States of America
Deli Maria A. EditorBiological Research Center of the Hungarian Academy of Sciences, Hungary* E-mail: [email protected] (VE); [email protected] (GG)Conceived and designed the experiments: VE PW GG. Performed the experiments: JH RJS VE. Analyzed the data: JH RJS VE PW GG. Contributed reagents/materials/analysis tools: AW WSS SP. Wrote the paper: VE GG. Performed the electrophysiological measurements: JH. Performed the immunocytochemistry and confocal microscopy: RJS. All authors critically read the manuscript and approved its final version. Contributed equally to the experimental part of the work: JH RJS. Mainly responsible for its concept, supervision and interpretation: VE PW GG.
2010 13 12 2010 5 12 e151376 9 2010 27 10 2010 Hallerdei et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3
- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.
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Introduction
Fast skeletal muscles can, during phases of maximal work – for example during a sprint -, accumulate intracellular lactic acid concentrations of up to 40–50 mM, which may be accompanied by a fall in intracellular pH to as low as 6.4 [1], [2]. At this point anaerobic glycolysis breaks down and the muscle looses its energy supply. On the other hand, slow skeletal muscles and heart muscle can take up lactic acid from the blood and use it as an important substrate for aerobic energy metabolism. Thus, the mechanisms responsible for the transport of lactic acid across the sarcolemmal membrane are decisive for the endurance of glycolytically operating skeletal muscles as well as for the extent of blood lactacidosis. Indeed, arguments have been presented to show that these mechanisms are limiting for the shuttling of lactic acid between fast muscles, erythrocytes, slow muscles and heart, respectively [2].
We have previously presented evidence from measurements of intracellular and surface pH in rat skeletal muscles showing that an extracellular membrane-bound carbonic anhydrase (CA) facilitates lactic acid transfer across the sarcolemma [3]. During lactic acid influx, for example, the presence of a CA at the extracellular surface of the muscle fiber provides a rapid source of protons by catalysing the CO2 hydration reaction, protons which are essential for lactate influx since the lactate-transporting monocarboxylate transporter (MCT) is a lactate-H+ cotransporter with a stoichiometry of 1∶1 [2]. During lactic acid efflux, the role of the extracellular CA is then to rapidly buffer the protons appearing at the membrane surface in order to avoid severe acidosis in an environment essentially lacking non-bicarbonate buffers. These functions of extracellular CA become apparent in surface pH (pHS) transients, alkaline during lactic acid influx and acidic during lactic acid efflux, which are small when functional CA is present and become very large when CA is inhibited. The large pHS transients indicate a severe disequilibrium of the CO2-H+-HCO3
− system, which reduces lactate fluxes to about ½. The fluxes of lactic acid have been quantitated by Wetzel et al. [3] by measurements of intracellular pH (pHi), whose change with time during lactic acid flux can be converted to a change in intracellular lactic acid concentration by multiplication with intracellular buffer capacity. Thus, combined measurements of pHS and pHi with microelectrodes can be employed to assess lactate transport rates and the role of the CO2 hydration-dehydration reaction in this process. This experimental approach is also used in the present paper.
Our aim in the present study was a) to study which of the muscular membrane-bound CAs are involved in lactate transport and b) to assign precise functional roles to each of these CAs in the transport process. To this end, we have performed lactic acid flux measurements in fast EDL muscles of wildtype (WT), CA IV-, CA IX- and CA XIV-single, CA IV-CA XIV-double and triple knockout mice. We also have performed further subcellular localization studies of the membrane-bound CA isozymes detected in skeletal muscle, and we interpret here the flux measurements in the light of these and previous morphological results. It turns out that indeed novel specific molecular roles in sarcolemmal lactic acid transport can be attributed to each of the three isozymes. CA XIV is homogeneously distributed across the surface sarcolemma and involved in lactic acid transfer across the surface membrane. Sarcolemmal CA IX is localized only in the transverse (T) tubules and involved in lactic acid transport across the T tubular membrane. In determining this, CA IX knockout has been a unique tool allowing us to provide the first demonstration of a T tubular pathway for lactic acid. CA IV, which to a considerable part is concentrated at the T tubular openings in the surface membrane, is likely to play an important role in the diffusional transport of lactic acid out of the T tubular lumen towards the interstitial space. In the case of CA IX, the present observation represents the first physiological function that has been possible to define for this isozyme. It is interesting that it is cellular lactic acid efflux, in which CA IX is involved physiologically, since lactic acid removal is also of great importance pathophysiologically in tumors, many of which have been shown to drastically upregulate CA IX. In fact, it is well known that both MCT4 and CA IX, which we propose here to cooperate closely in eliminating lactic acid from the muscle cell via T tubules, are both up-regulated under hypoxia through HIF-1α in normal and tumour cells [4], [5].
Results
Membrane-bound carbonic anhydrases in mouse EDL
We have shown previously by Western Blotting that CA IV, IX and XIV are present in mouse skeletal muscle membranes, while CA XII is absent [6]. Here, we have studied the distribution the three CA isoforms in fast-twitch EDL. For that reason, single mouse EDL fibers were isolated, permeabilized with Triton X-100 and stained with primary antibodies raised against mouse CA IV, CA IX or CA XIV and fluorescently labelled secondary antibodies. We have shown previously that the antibodies against CA IV, CA IX and CA XIV are highly specific and that immunocytochemical staining is absent in muscle fibers of mice in which the CA isozyme considered had been knocked out [6], [7]. In addition, omission of the primary antibodies confirmed specificity of the CA as well as of the MCT4 and ryanodine receptor (RyR) staining. Fig. 1 shows immunocytochemical images obtained by confocal laser scanning microscopy (CLSM). In order to investigate the distribution of the three CA isozymes in the plasma membrane the microscope first was focused on the plane of the surface membrane (Fig. 1a). It is apparent that CA IV exhibits a homogeneously distributed membrane staining, but is much more concentrated at rows of dots whose pattern suggests they represent openings of T tubules. The homogeneous surface membrane staining, as well as the staining of T-tubular openings, both clearly exceed the very low background staining in CA IV-ko muscle, as it has been reported previously [6]. Using antibodies recognizing MCT4, the isoform of the lactate-H+ cotransporter predominantly expressed in fast muscle [8], exactly the same pattern of staining was observed. The merged images of these stainings show a perfect colocalization of CA IV and MCT4 at the T tubular openings but not on the remaining membrane surface. CA IX, although present in preparations of sarcolemmal fractions [6], is entirely absent from the sarcolemmal surface membrane (Fig. 1a), confirming a previous report [6]. CA XIV, on the other hand, shows clear homogeneous staining of the entire surface membrane and no systematic colocalization with MCT4.
10.1371/journal.pone.0015137.g001Figure 1 Immunocytochemical CLSM images from fibers of mouse EDL muscle.
a) Simultaneous exposure to antibodies against CA IV, CA IX or CA XIV and MCT4. The microscope was focussed on the plane of the fiber surface membrane. CA IV, basal homogeneous membrane staining and enrichment at entrances to T tubuli. CA IX, absence from the surface membrane. CA XIV, homogeneous surface membrane staining. b) Simultaneous staining with antibodies against CA IV, CA IX or CA XIV and RyR. The microscope was focussed on a plane inside the cell exhibiting triads. CA IV, staining of entire SR. CA IX, staining of triads (T tubules) but not SR. CA XIV, staining of light SR but not the triads (terminal cisternae of the SR and T tubules). c) Simultaneous staining with antibody against CA IX and RyR or MCT4. In the upper lane, the microscope was focussed on a plane inside the fiber free of triads; nevertheless, CA IX staining exihibits the same staining pattern as in Fig. 1b, middle lane, indicating it is associated with T tubules. In the middle lane a plane is shown, in which RyR are fully, or partially or not at all visible, causing an incomplete RyR pattern compared to that seen in Fig. 1b. Nevertheless, the staining pattern of CAIX is as complete and regular as in the lane above. This confirms that CAIX staining is associated with T tubules but not necessarily with RyR. In the lowest lane it is seen that CA IX and MCT4 are perfectly colocalized in T tubules.
In Fig. 1b single mouse EDL fibers were exposed to anti-RyR antibodies and the CLSM was focussed on a plane inside the muscle fiber. Staining of the RyR reveals a pattern typical of the arrangement of triads in skeletal muscle. With antibodies recognizing CA IV, a punctate but largely homogeneously distributed staining is obtained, suggesting staining of the entire sarcoplasmic reticulum (SR), light as well as heavy SR. This is in perfect agreement with the previously reported presence of CA IV in SR membrane fractions [9] and the earlier observation of an only partial colocalization of SR Ca++-ATPase (SERCA) and CA IV staining that was interpreted to indicate association of CA IV not only with terminal but also with longitudinal SR [6]. The colocalization with ryanodine receptors is highly incomplete, which is again consistent with CA IV staining of the light SR in addition to staining of the terminal SR. The minimal colocalization of ryanodine receptors and CA IV is clearly not caused by staining of T tubules, which should result in an entirely different pattern of CA IV staining, such as it is seen in the lane below for CA IX. Rather, it appears that the density of stained spots is reduced at the sites where T tubules are expected. Clearly, an increased staining intensity is not present at these sites, as would be the case if T tubules were also stained for CA IV (Fig. 1b, upper lane, left-hand picture). In accordance with this, we have observed in similar sections (unpublished) that there is also only minor overlap of intracellular CA IV and MCT4 staining. Since intracellularly only T tubules appear stained for MCT4 (see below), this confirms that the partial overlap is due to the close proximity of CA IV-stained terminal cisternae and MCT4-stained T tubules at the triads, and again argues against the presence of CA IV in T tubular membranes. In contrast to this, an example of a perfect T-tubular CA and MCT4 colocalization is shown below in Fig. 1c (lowest lane) for the case of CA IX. Fig. 1b, middle lane, shows CA IX staining in rows of high intensity dots, a pattern characteristic of T tubules, similar to what is seen for CA IV in the surface membrane, and exhibits no staining of the space in between these rows (Figs. 1b and c). The latter indicates that CA IX is not expressed in SR, which is compatible with the previous report [6] that >90% of the muscle CA IX is associated with the sarcolemmal fraction (that includes the T-tubular membranes), while the minor fraction of CA IX appearing with SR likely constitutes a contamination with T tubules rather than a genuine SR staining. The exact colocalization of CA IX with ryanodine receptors is compatible with a specific CA IX staining of T tubules that are closely attached to the triads visualized by the RyR staining. This interpretation is strongly confirmed by the data shown in Fig. 1c (see below). CA XIV in Fig. 1b (lower lane) shows punctate staining, which, as also seen in the merged image, is completely interrupted wherever triads are located. This is most easily explained by a staining of the longitudinal or light SR but not of the terminal cisternae and is compatible with the previous observation of complete colocalization of CA XIV and SERCA staining [7]. Another clear-cut implication of these pictures is the complete absence of CA XIV from T tubules. While the results presented in Figs. 1a and b largely confirm recent observations [6], [7], the following results of Fig. 1c lead to an even clearer interpretation of these data.
In Fig. 1c (upper lane), additional images have been obtained in the intracellular space of the fiber, but in a plane where triads are absent, as demonstrated by the lack of specific ryanodine receptor staining. Nevertheless, CA IX staining shows the same pattern as it does in the plane of the triads (Fig. 1b), clearly indicative of exclusive T tubular staining for CA IX. T-tubular CA IX staining is further supported by the middle lane of Fig. 1c, which shows that the pattern of CA IX staining remains complete and regular even when the intracellular plane observed exhibits an incomplete and irregular RyR pattern when compared to that seen in Fig. 1b. The association between T tubules and CA IX is further confirmed by the perfect colocalization of CA IX and MCT4 staining (Fig. 1 c, lower lane), in agreement with the findings of Bonen et al. [8].
The subcellular localization of MCT4 in mouse EDL as evident from Fig. 1 can be summarized as follows: a) there is MCT4 staining of T tubules and openings to T tubules (Fig. 1 c, lower lane, figure in the center; Fig. 1 a, all figures in the center), and b) there is surface membrane background staining between the rows of T-tubular openings (Fig. 1 a, all figures in the center). These observations verify the finding of Bonen et al. [8] of an association of MCT4 with isolated surface membranes as well as with T tubuli.
Summarizing the findings on CA localization in the sarcolemmal surface and T tubular membranes, CA IX is exclusively found in association with T tubules and is neither expressed in the surface membrane nor in the SR. CA XIV is homogeneously distributed across the sarcolemmal surface membrane, but absent in T tubuli. In contrast, CA IV exhibits some basal homogeneous staining of the surface membrane with an enriched expression at the openings of the T tubules, but is absent from the T tubules further down inside the cell.
Lactate transport in CA IV- and CA XIV-knockout EDL muscles
Fig. 2a illustrates that lactate influx leads to an intracellular acidification, which we measure here with an intracellular pH microelectrode, and at the same time to an alkalinization on the surface of the cell, which we measure with a pH microelectrode positioned on the surface of the muscle fiber. The extracellular alkalinization occurs, because lactate influx requires the mobilization of protons, and this is strongly dependent on extracellular CA, because in the extracellular space – in contrast to the intracellular space – almost only the CO2-H+-HCO3
− system is available as a H+ buffer. During lactate efflux the reverse phenomena occur, with the consequence that both influx and efflux are equally dependent on CA.
10.1371/journal.pone.0015137.g002Figure 2 Lactic acid fluxes in mouse EDL fibers.
a) Schematic representation of the mechanisms of H+ production and H+ buffering associated with lactic acid influx and efflux in skeletal muscle. Fluxes can be quantitated by following the changes in intracellular pH. The changes in surface pH illustrate the associated processes of proton consumption or production on the surface membrane. b) pHS and pHI during exposure to and after subsequent withdrawal of lactate in the bathing solution in a WT EDL fiber. c) pHS and pHI during the same maneuver as in 2b in a CA IV-CA XIV double ko mouse EDL fiber. Lactate fluxes are decreased in comparison to 2b and pHS transients are greatly enhanced. Note that pHS curves in b and c are shifted on the pH ordinate by + 0.3 units to improve visibility. The standard bathing solution is Krebs-Henseleit solution, pH 7.4, at room temperature and equilibrated with 5% CO2/95%O2.
Figs. 2b and c show original recordings of pHS and pHi in mouse EDL fibers from WT animals (Fig. 2b) and from CA IV – CA XIV double knockout animals (Fig. 2c). Upon exposure of the fiber to 20 mM lactate at time 0, pHS rises and pHi falls as expected. The alkaline shift of pHS is much more pronounced in the double ko muscle, in which all surface membrane CAs are lacking, than in WT-EDL. This increased alkaline pH shift indicates a strong limitation of H+ supply to the lactic acid transporter, and consequently the initial slope dpHi/dt is shallower in double ko than in WT muscle. The flux of lactic acid, which we define here as initial change of intracellular lactic acid concentration with time, is obtained by multiplying initial dpHi/dt by the total intracellular H+ buffer capacity. It follows therefore that lactic acid influx is reduced in double ko EDL compared to the WT muscle. In the following, we present mostly lactic acid influx values, because usually the initial slope dHi/dt is better defined during influx than during efflux. It should be noted that pHi under normal conditions of equilibration with 5% CO2 is around 7.22 and identical in all knockout animals – single, double as well as triple – studied here.
Figs. 3a and b give a summary of the experimental results with CA IV and CA XIV ko mouse EDL muscle fibers. Fig. 3a shows that lactic acid influx is just below 2 mM/min in WT- EDL, is not significantly reduced in CA IV single ko EDL, but is significantly reduced in CA XIV single ko muscle. The further decrease of flux to a value below 1 mM/min seen in double ko EDL shows that lack of CA IV does have an effect on lactate transport, when CA XIV has been eliminated. Benzolamide, an extracellular CA inhibitor in muscle [3], does not further reduce lactate flux beyond that of the double ko EDL when either applied to WT fibers or to double ko fibers.
10.1371/journal.pone.0015137.g003Figure 3 Lactate influxes and amplitudes of surface pH transients.
Influxes (a) and amplitudes (b) are shown for WT fibers and for fibers lacking CA IV, CA XIV or both. On the right, both figures show the effects of the extracellular CA inhibitor benzolamide.
Fig. 3b illustrates that the changes in amplitudes of the alkaline surface pH shifts roughly correspond with the changes in fluxes. Although the pHS measurement is compromised by the difficulty of positioning the electrode at a reproducible distance from the cell membrane, it is apparent that the three lowest fluxes in Fig. 3a, obtained in the absence of functional surface CAs, are associated with the three highest pHS amplitudes in Fig. 3b, which is in line with the causal relation expected to exist between pHS disequilibrium and flux reduction.
Lactate transport in CA IX-knockout EDL muscles
Fig. 4a shows a comparison of the effects of CA IV, CA XIV and CA IX single ko on lactate influx in EDL fibers. It is apparent that lack of CA IX causes a marked and statistically significant reduction of flux from about 1.8 mM/min in WT to ∼1.2 mM/min, almost identical to the reduction caused by lack of CA XIV. Fig. 4b is an example showing that qualitatively identical effects are observed for lactic acid efflux, as calculated from the dpHi/dt seen after withdrawal of lactate from the bathing solution (Fig. 2 b, c). Both sets of data show that CA IX makes a substantial contribution to the CA-dependent lactic acid flux. Fig. 4c adds a surprising feature to this observation: while CA IX contributes significantly to flux when CA IV and CA XIV are present (two leftmost columns), it has no significant effect on flux anymore after CA IV and CA XIV have been eliminated in the double ko EDL (comparison of double and triple ko EDL in the two columns in the middle of Fig. 4c). This must be interpreted to mean that CA IX can only exert its facilitation of lactic acid flux when CA IV and CA XIV are present, i.e. the action of CA IX requires a cooperation with CA IV and CA XIV. A second information from Fig. 4c is the finding that addition of the membrane-permeable CA inhibitor ethoxzolamide to either WT or to triple ko EDL has no further effect on flux in comparison to the triple ko without inhibitor. This indicates that no other CA isoenzymes, cytosolic or membrane-bound, are involved in lactic acid transport besides CA IV, CA IX and CA XIV. The three rightmost columns in Fig. 2c indicate that the flux in WT-EDL, 1.8 mM/min, falls to ∼0.8 mM/min, when all functional CAs have been eliminated, i.e. about ½ of the total flux is dependent on CA, while the remainder does not require CA activity.
10.1371/journal.pone.0015137.g004Figure 4 Lactate influx and efflux measurements in EDL fibers from various knockout mice.
a) Lactate influxes in WT, CA IV ko, CA XIV ko and CA IX ko fibers. b) Lactate effluxes from the same combination of fibers as in 4a. c) Comparisons of lactate influxes in WT vs. CA IX ko fibers (left), in CA IV-CA XIV double ko vs. CA IV-CA IX-CA XIV triple ko (middle), and WT vs. triple ko, both in the presence of the membrane-permeable CA inhibitor ethoxzolamide (right). Columns for lactate influxes in WT, CA IV ko, CA XIV ko, and CA IV/CA XIV double ko are the same as in Fig. 3. Stars indicate significant differences from WT (* P<0.05; ** P<0.01). #(s) indicates a significant difference between the CA IX ko and the triple ko fluxes (P<0.05), &(ns) indicates that there is no significant difference between double ko and triple ko fluxes.
Discussion
Subcellular distribution of membrane-bound carbonic anhydrases and of MCT4
Table 1 in its first column shows previously reported activities of CA IV, CA IX and CA XIV [6]. The numbers were obtained from measurements of CA activities in preparations of sarcolemmal membrane vesicles from mouse WT, CA IV ko and CA IV-CA XIV double ko skeletal muscle. Accordingly, CA XIV is responsible for 55% of all sarcolemmal CA activity and CA IV for 30%; CA IX with 15% makes the smallest contribution. It should be noted that Western Blots of all three isozymes in WT, CA IV, CA IX and CA XIV ko mice showed no significant upregulation of any of the three CA isozymes in the ko muscles [10]. Also, the numbers given in Table 1 are compatible with those obtained by studying the CA activities of WT and CA XIV ko muscle sarcolemma [7] applying an inhibitory CA XIV antibody [11]. It follows from the immunocytochemical results described above that all sarcolemmal CA IV and CA XIV is localized in the sarcolemmal surface membrane, while all CA IX activity is associated with the T tubular membranes. This leads to the presentation of the numbers given in the 3rd and 4th column of Table 1.
10.1371/journal.pone.0015137.t001Table 1 Sarcolemmal (SL) carbonic anhydrases in mouse skeletal muscle.
CA activity of SL fraction (surface membrane +T tubuli) CA activity of surface membranes CA activity of T tubuli
U • ml/mg
U • ml/mg
U • ml/mg
CA IV
1.9 (30%) 1.9* (34%) -
CA IX
1.0 (15%) - 1.0
CA XII
- - -
CA XIV
3.6 (55%) 3.6** (66%) -
The activities are given in enzyme units per protein concentration in the sarcolemmal membrane fraction. They are derived from activity measurements in WT, CA IV- and CA IV-CA XIV-double-ko mouse muscle [6]. The immunoctyochemical results presented here allow us to attribute CA IV and CA XIV to the surface membrane alone and CA IX exclusively to the T tubules.
*CA IV is localized on the entire surface membrane, but greatly enriched at the openings of T tubules,
**CA XIV is homogeneously distributed over the entire surface membrane.
The subcellular distribution of MCT4 has been described by Bonen et al. [8]. Quantification of MCT4 protein in separated fractions of surface membranes, T tubuli and triads revealed similar amounts of MCT4 in surface membranes and in T tubuli, provided that the amounts associated with triads are mainly attributed also to T tubuli. This suggests that perhaps one half of the lactate transported out of fast muscles is transferred across the T tubular membranes in addition to the lactate released via the surface membranes. It seems surprising that the T tubular pathway should be efficient in comparison to the pathway across the surface membrane, and this has so far not been analysed functionally.
Lactic acid transport across EDL surface membrane
Using the localizations of CA IV and XIV given in Table 1 and described in detail in the section Results, we can analyse the mechanism of lactic acid transport across the surface membrane in a straightforward fashion. As illustrated in Fig. 5, the MCT4 present in the surface membrane will interact with the surface carbonic anhydrases CA IV and CA XIV, which – in the absence of non-bicarbonate buffers - act to buffer the H+ appearing on the cell surface during efflux (as indicated in Fig. 5 by the interaction between CA XIV and MCT4) and to provide H+ to the transporter during lactate acid influx (as indicated in Fig. 2a, left hand side). This mechanism is suggested by the experimental finding of a reduced lactate influx in CA XIV single and CA IV-CA XIV double ko EDL (Fig. 3a) and by the increased amplitudes of surface pH transients observed in the double ko EDL (Fid. 3b). It is possible that CA XIV is more important for the surface MCT4, as both proteins are homogenously distributed across the cell surface, and CA IV may be more important for the MCT4 localized at the T tubular openings. However, since lack of CA IV alone has no significant effect on lactate flux (Fig. 3a), it appears that CA XIV, due to its presence everywhere on the surface and its contribution of 2/3 of surface CA activity (Table 1), can functionally compensate the absence of CA IV. The reverse is not true, CA IV with its more specialized localization and its contribution of only 1/3 to total surface activity, cannot compensate the lack of CA XIV as apparent from the CA XIV single ko column in Fig. 3a.
10.1371/journal.pone.0015137.g005Figure 5 Schematic representation of the cooperation of the MCT4 and the three membrane-bound CAs in lactic acid transport across the sarcolemma.
About half of lactic acid transport (in this scheme efflux) occurs via the surface membrane, supported by the buffering action of CA XIV and CA IV. The other half occurs via the T tubular membrane and is supported by the buffering action of CA IX. CA IX and half of the total sarcolemmal MCT4 are colocalized in the T tubule. The removal of lactic acid from the T tubules occurs by outward diffusion of lactate, while the H+ are transported out by an inward diffusion of HCO3
− in combination with an outward diffusion of CO2, a CO2- HCO3
− shuttle. This removal mechanism operates effectively in spite of the long diffusion distance from the T tubule interior to the extracellular space due to very large concentration gradients of lactate and HCO3
− that can build up along the T tubule. These gradients and the mobility of protons are much smaller in the sarcoplasm of the fiber.
Lactic acid transport across the T tubular membrane
Due to the present finding of an exclusive localization of CA IX in T tubules, we can analyse here the mechanism of T tubular lactic acid transport. Fig. 5 illustrates our postulate that T tubular CA IX and MCT4, which microscopically are perfectly co-localized (Fig. 1c), cooperate in a fashion analogous to that of surface CA XIV and MCT4. CA IX accordingly mediates buffering of H+ in the T tubular lumen during lactic acid efflux and provides H+ to the transporter during influx. That this pathway is important, follows from the major reduction of lactate influx as well as efflux seen in the CA IX-deficient EDL (Figs. 4a, b). The marked effect is surprising in view of the small overall contribution of CA IX of only 15% to total sarcolemmal CA activity (Table 1), only half of the CA IV activity, whose knockout is of no consequence for lactate flux (Fig. 3a), and only ¼ of the CA XIV activity, whose knockout causes a loss of lactic acid flux similar to that by CA IX (Fig. 4b). This observation supports the idea that CA IX is involved in lactic acid transport in a way entirely different from CA IV and CA XIV. One question raised by the T-tubular transport mechanism is: how can lactate and H+ be removed efficiently from the T tubule, in which both ions have to overcome diffusion distances of up to half the muscle fiber diameter or more [12]?
In view of the "transport metabolon" consisting of MCT1 and CA II proposed by Becker and Deitmer [13], it may be asked whether the functional interactions of the membrane-bound CA isozymes are due to a physical interaction of the CA and the MCT4 molecules. In some cases this can be ruled out, namely when not even microsopic co-localization is evident as in the case of CA XIV and MCT4 (Fig. 1a), or in the case of that part of CA IV that is homogeneously distributed on the surface membrane and there shows no co-localization either. Very good co-localizations with MCT4, on the other hand, are seen in the case of the CA IV that is concentrated at the T tubular openings (Fig. 1a) and in the case of CA IX in the cross-sectioned T tubules (Fig. 1c). In these latter cases, physical interaction would be conceivable, but of course the microscopic co-localization is no proof for this.
Lactic acid transport in the T tubular lumen
The present experimental data appear to suggest a mechanism for this process. Consideration of Fig. 4c shows that CA IX is quite effective in facilitating lactate flux as long as CA IV and CA XIV are present (two columns on the left), but after the latter two isozymes are removed, CA IX looses its effect on lactate flux (two columns in the middle). We hypothesize that this observation is related to the mechanism of lactic acid removal from the T tubule. Deeply inside the T tubule lactate concentration may become as high as it can become in the sarcoplasm, up to 40–50 mM [1], [2]. Although protons are buffered by HCO3
−, pH inside the T tubule can be expected to be low, and thus we assume here for the purpose of a rough quantitative approximation that the concentration of HCO3
− deep in the T tubule is equal to the CO2 concentration of ∼1.3 mM (corresponding to pH 6.1) or even less. On the surface of the cell, close to the capillary, the situation will resemble more the conditions in the blood, which under conditions of exhaustive exercise in the study of Sahlin et al. [1] are characterized by a lactate concentration of ∼12 mM, a pH of 7.2, and accordingly a HCO3
− concentration of ∼16 mM. In this example, one would have concentration differences between deep inside the T tubule and the surface of the fiber of 40 to 50–12 = 28 to 38 mM for lactate, and of 1–16 = −15 mM for HCO3
−. This is the situation to which the intraluminal mechanisms depicted in Fig. 5 refer to. The gradient for lactate drives lactate out of the T tubule towards the interstitium. The protons cannot diffuse as free protons because their gradient will be far too small. Mobile buffers that could mediate facilitated H+ diffusion [14], [15] are not present. The only known mechanism applicable in this situation is the one shown in Fig. 5: H+ transport out of the T tubular lumen is achieved by an inward diffusion of HCO3
− (which will partly or fully electrically compensate outward lactate diffusion) in combination with an outward diffusion of CO2, i.e. a CO2-HCO3
− shuttle for H+ transport. This shuttle requires that inside the T tubule HCO3
− and H+ react rapidly to produce CO2, and that at the opening of the T tubule to the interstitial space CO2 reacts back to produce the HCO3
−, which then diffuses again down into the T tubule to supply HCO3
− for H+ buffering to the sites where MCT4 and CA IX are located. Thus a CA, i.e. CA IX, is needed deep in the T tubule to buffer H+ and produce CO2, and again, at the opening of the T tubule, a CA, i.e. CA IV and probably also CA XIV, is required to regenerate HCO3
−. The arguments for this mechanism are: 1) it explains why CA IX can only facilitate lactic acid transport in conjunction with the surface CAs CA IV and probably CA XIV, 2) it convincingly explains the specific enrichment of CA IV at the openings of the T tubules (Fig. 1 a), a location ideally suited for this purpose as visualized in Fig. 5. Finally, two properties of CA IX make it appear especially suited for its proposed function in the T tubule. CA IX exhibits a greater tolerance towards acidic conditions in comparison to other membrane-bound CAs [16], which will allow it to remain active when pH inside the T tubule falls to much lower values than on the cell surface. Another notable property of CA IX is its resistance towards inhibition by lactate (KI>150 mM; [17]), which makes it especially suited for the expected high intraluminal lactate concentrations in T tubules. This situation is in contrast to the surface membrane, where much lower lactate concentrations are expected physiologically under intense exercise and where CA IV as well as CA XIV, like many other CA isoforms, have KI values towards lactate in the low millimolar range ([17]; Dr. C.T. Supuran, Florence, personal communication). In concluding this section, it should be noted that the net effect of all T tubular transport mechanisms is a release of lactate and H+ into the interstitial space.
Physiological significance of T tubular vs. surface membrane lactic acid transport
The relative contributions of these two pathways can be derived from the present data. Fig. 4c shows the WT level of lactate influx, ∼1.8 mM/min, and the basal CA-independent flux level, in triple ko EDL and in EDL exposed to ethoxzolamide, of 0.8 mM/min. In CA IX ko EDL, in which the CA-dependent T tubular pathway should be completely suppressed, a flux of 1.25 mM/min has been measured, suggesting that about ½ of the CA-dependent lactic acid transport occurs via the T tubule, the other half via the surface membrane. Such a distribution of the quantitative roles of flux pathways is in excellent agreement with the finding of Bonen et al. [8] of an equal allocation of the sarcolemmal MCT4 to surface membranes and T tubuli.
What may be the advantage of additionally using the T tubular pathway over only using the route through the sarcoplasm and the surface membrane? Peachey and Eisenberg [12] have shown that the T tubular system forms a close-mesh network extending all over the interior of the skeletal muscle fiber. Thus, T tubules can take up lactic acid everywhere in the cell at very short distances, and thus rapidly remove lactic acid from the sarcoplasm right where it is produced. The overall distance that has to be overcome by intracellular radial diffusion and by diffusion through the T tubule may be considered similar, and the diffusivities of lactate and HCO3
−, even if considering tortuosity effects in the T tubules and in the sarcoplasm [18], are also expected to be similar. However, the volume fraction of the T system is 0.3% [19] and thus the total diffusional cross section of the T tubules will be small. In fact, there has been a long-standing speculation and discussion on whether ions are exchanged to a significant extent between the T-tubular lumen and the extracellular space [20], [21], but clear-cut evidence to our knowledge has not been obtained so far. This disadvantage of the small volume and diameter of T tubules in the present case may be overcome by the large gradients of lactate as well as HCO3
− concentration along the diffusion path through the T tubules, as explained above. These gradients are expected to be much smaller in the sarcoplasm. During acid loading of enterocytes maximal intracellular pH gradients have been measured in the order of ∼0.1 unit [22]. The intra-tubular pH gradient in the above hypothetical example would be 7.2–6.1 = 1.1 units, i.e. ten times greater than the possible intracellular pH gradient.
If we express the transport properties of sarcoplasm vs. T tubular lumen by the product D · Δc, we find that not only the concentration gradients but also the effective proton mobilities are greater within the T tubule than in the sarcoplasm. We consider here proton transport rather than lactate transport, because it is likely that in both cases proton flux constitutes the limiting step:
1) Intracellular transport of protons: D is interpreted according to the concept proposed by Junge and McLaughlin [23] as an apparent diffusion coefficient of bound together with free protons. This quantity was reported to assume a value of Dapp = 3.8 · 10−7 cm2s−1 in cardiomyocytes [24], when buffering by HCO3
− is of little importance, as is the case for the low intracellular pH value considered here. Δc represents the combined intracellular concentration difference of bound and free protons, which is given by the product of intracellular pH difference, ΔpH, times the intracellular non-bicarbonate buffer capacity BF of 24 mM/ΔpH (see Methods). With an assumed ΔpH of 0.1 we obtain a proton transport rate of Dapp · ΔpH · BF = 3.8 · 10−7 cm2 s−1 · 0.1 · 24 mM = 9.1 · 10−7 µM cm−1 s−1.
2) Intratubular transport of protons: In this case, we can approximate the H+ transport rate by the transport rate of bicarbonate in opposite direction, assuming that CO2 diffusion, due to the greater diffusivity of CO2 compared to HCO3
−, is not rate-limiting. D · Δc is then obtained as follows. DHCO3- is taken to be 1.2 · 10−5 cm2 s−1
[25], and for Δ[HCO3
−] we use the above hypothetical value of 15 mM. With this we estimate the intratubular proton transport rate to be DHCO3- · Δ[HCO3
−] = 1.2 · 10−5 cm2 s−1 · 15 mM = 1,800·10−7 µM cm−1 s−1.
These estimates indicate that T tubular proton transport can be ∼200 times more efficient than intracellular proton transport. This is so because a) due to the highly different pH gradients, the gradient of the molecules directly or indirectly mediating proton transport is 15/2.4 = 6.3 times greater in the T tubule than intracellularly, and b) the effective diffusion coefficient of these molecules is 32 times greater inside the T tubule than in the cytoplasm of the muscle cell. It should be noted that the intracellular pH gradients occurring while the muscle cell produces and releases lactic acid have not been measured directly and may be less than 0.1, making the intracellular proton transport pathway even less effective in comparison to the T tubular pathway. In conclusion, the small volume fraction taken up by the T tubules is counterbalanced approximately quantitatively by the properties of the proton transport inside the T tubules. This latter transport mechanism is so much more efficient than the route across the sarcoplasm that comparable contributions of both pathways to the elimination of lactic acid from the muscle cell can be expected. This constitutes a satisfactory physicochemical explanation of the experimental finding of identical sizes of lactic acid fluxes across the surface membrane and across the T tubular membrane.
It may be hypothesized in addition that during contraction T tubuli are alternatingly compressed and decompressed, thereby producing a convective flow of water and solutes along the T tubular lumina, which would further improve lactic acid transport out of the T tubuli, although at present there is no experimental evidence for such a phenomenon.
Methods
Ethical approval
All experiments were done according to the guidelines of the Bezirksregierung Hannover and approved by this institution (Approval ID 42500/1H).
Animals and muscle fiber preparation
The CA IV, CA IX and CA XIV ko mice have been characterized earlier [26], [27]. They were bred on a C57BL/6J background and crossbred to obtain CA IV-CA XIV double ko and CA IV-CA IX-CA XIV triple ko animals. Mice were sacrificed by cervical dislocation and the extensor digitorum muscle (EDL) was dissected out. From the muscles, fiber bundles were prepared under a microscope as described [3].
Electrophysiology
The membrane potential electrodes (2 MΩ), the intracellular pH microelectrode (20–30 GΩ), the surface pH microelectrode (5–10 GΩ), the latter two filled at the tip with Hydrogen Ionophore Cocktail A (Fluka), and the reference electrode were constructed as described earlier [3]. The muscle fiber bundles were subjected to moderate passive tension and placed in an open bathing chamber that was continuously perfused with Krebs-Henseleit solution equilibrated with 5%CO2/95%O2 at room temperature. One superficial single fiber of the bundle was chosen to position the intracellular pH (pHi) and the membrane potential electrode intracellulary and the surface pH microelectrode (pHS) on the cell surface. pHi values in EDL fibers under control conditions were 7.22 (S.D. ±0.10, n = 23). Control pHi values in muscle fibers from knockout animals and/or in the presence of benzolamide or ethoxzolamide were not significantly different from this value. pHS values in control EDL fibers were on average 7.31 (S.D. ±0.09, n = 23), and again pHS values of knockout fibers and fibers in the presence of CA inhibitors were not significantly different from this value. Intracellular non-bicarbonate buffer capacity was determined by exposing the muscle fibers to three different CO2 partial pressures: Krebs-Henseleit solution (98 mM NaCl) with 5% CO2, 25 mM NaHCO3 and 27 mM methane sulfonic acid, or with 2% CO2, 10 mM NaHCO3 and 42 mM methane sulfonic acid, or with 14% CO2 and 52 mM NaHCO3. pHi was measured at these three pCO2 values, used to calculate intracellular [HCO3
−], and Δ[HCO3
−]/ΔpHi yielded the intracellular non-bicarbonate buffer capacity (24 mM/ΔpH for mouse EDL, with no significant differences between wildtype and knockout muscles). For a given experimental ΔpHi the bicarbonate buffer capacity was calculated applying the Henderson-Hasselbalch equation for a constant pCO2. The sum of both buffering factors was used to convert the initial dpHi/dt observed during lactate influx and efflux into lactate fluxes in mM/min. Lactate flux experiments were conducted by rapidly switching between a Krebs-Henseleit solution with 100 mM NaCl, 20 mM methane sulfonic acid (5%CO2) and one with 100 mM NaCl, 20 mM Na-lactate (5%CO2). The switch led to a decrease in pHi with time, whose initial kinetics was determined as described [3]. The pHi reached a plateau during continued exposure to Na-lactate and returned gradually to the control value after the perfusion was switched back to the lactate-free solution (see Fig. 2). pH of all Krebs-Henseleit solutions employed was 7.36 at room temperature. Further details of this method to measure lactate fluxes have been reported previously [3].
CA Inhibitors
Benzolamide (2-benzenesulfonamido-1,3,4-thiadiazole-5-sulfonamide) was a kind gift from Dr. Erik Swenson (Seattle, USA) and was used at a final concentration of 1· 10−5 M. Ethoxzolamide (6-ethoxy-2-benzothiazolesulfonamide) was from Sigma Aldrich (Seelze, Germany) and was used at a final concentration of 1·10−4 M.
Immunocytochemistry
EDL muscle fiber bundles were fixed while subjected to passive tension with 3% paraformaldehyde and 100% methanol and permeabilized in 0.1% Triton X-100 for 5 min, and stained with primary and secondary antibodies as described [6]. Fibers were incubated with primary antibodies for 30 min. The polyclonal rabbit anti-mouse CA IV and CA XIV [11], [28], [29] and the polyclonal rabbit anti-mouse CA IX antibody (Santa Cruz Biotechnology sc-25600; Santa Cruz, CA, USA) were diluted 1∶400. Goat anti-mouse ryanodine receptor RyR (sc-8170) and goat anti-mouse MCT4 (sc-14934) antibodies were also from Santa Cruz Biotechnology and diluted 1∶200. Incubation with FITC-labelled anti-rabbit and TRITC-labelled anti-goat IgG secondary antibodies (Santa Cruz Biotechnology) was performed for additional 30 min. The subcellular localization was examined by CLSM (Leica DMIRRE, Wetzlar, Germany) and analysed with Image Span software (Leica TCS-NT).
Statistics
The mean values of experimental results in Figs. 3 and 4 are given together with S.D. values (bars) and n ( = number of fibers investigated). Significance of differences between groups of data and WT controls were performed using One-Way ANOVA followed by Dunnett's Multiple Comparison Post Test using the program Prism 3.0. The Bonferoni Post Test for selected pairs of data groups was used to test two pairs of data groups of special functional interest (Prism 3.0).
We are indebted to Mr. Werner Zingel for expert technical assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Supported by the Deutsche Forschungsgemeinschaft WE 1962/4-1,2 (website: www.dfg.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 2119709310.1155/2011/724743Research ArticlePsorinum Therapy in Treating Stomach, Gall Bladder, Pancreatic, and Liver Cancers: A Prospective Clinical Study Chatterjee Aradeep
1
*Biswas Jaydip
2
Chatterjee Ashim
1
Bhattacharya Sudin
2
Mukhopadhyay Bishnu
3
Mandal Syamsundar
2
1Critical Cancer Management Research Centre & Clinic, 381 S K Deb Road, West Bengal, Kolkata 700 048, India2Chittaranjan National Cancer Institute, Kolkata 700 026, India3National Institute of Technology, Durgapur 713209, India*Aradeep Chatterjee: [email protected] 8 12 2010 2011 72474327 11 2009 14 10 2010 27 10 2010 Copyright © 2011 Aradeep Chatterjee et al.2011Copyright © 2011 Aradeep Chatterjee et al.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.We prospectively studied the clinical efficacy of an alternative cancer treatment “Psorinum Therapy” in treating stomach, gall bladder, pancreatic and liver cancers. Our study was observational, open level and single arm. The participants' eligibility criteria included histopathology/cytopathology confirmation of malignancy, inoperable tumor, and no prior chemotherapy or radiation therapy. The primary outcome measures of the study were (i) to assess the radiological tumor response (ii) to find out how many participants survived at least 1 year, 2 years, 3 years, 4 years and finally 5 years after the beginning of the study considering each type of cancer. Psorinum-6x was administered orally to all the participants up to 0.02 ml/Kg body weight as a single dose in empty stomach per day for 2 years along with allopathic and homeopathic supportive cares. 158 participants (42 of stomach, 40 of gall bladder, 44 of pancreatic, 32 of liver) were included in the final analysis of the study. Complete tumor response occurred in 28 (17.72%) cases and partial tumor response occurred in 56 (35.44%) cases. Double-blind randomized controlled clinical trial should be conducted for further scientific exploration of this alternative cancer treatment.
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1. Introduction
Although, great advances have been made in the treatment of some forms of cancer and new advances in surgery, radiotherapy, and chemotherapy leading to an increase in cure rates have been achieved, such interventions are often too much expensive and beyond the reach of many cancer patients of the developing as well as of the developed countries [1–3]. In developing countries, majority of the cancer patients have inadequate access to the mainstream cancer treatments due to lack of proper medical infrastructures, skills, and above all limited financial resources [4, 5]. Some types of cancer (i.e., liver, gall bladder, pancreatic, and stomach) are still associated with poor prognosis to conventional cancer treatments [6–9]. Side effects of the chemotherapy and radiation therapy are also intolerable to many cancer patients [10–12]. In most of the situations, elderly cancer patients cannot be provided with conventional cancer treatments because of old age-related problems [13, 14]. As a result, alternative cancer treatments have become an important feature of oncology regardless of geographic region and they appear to exist in greater abundance through out the world. Many alternative cancer therapeutic modalities are now being practiced in India, and one of them which has gained significant popularity is called Psorinum Therapy [15–17]. The investigational anticancer drug used in this alternative cancer therapy is “Psorinum” which is derived from the sphere of homeopathy. The supportive treatments of Psorinum Therapy are adopted both from the spheres of allopathy and homeopathy. Psorinum is an alcoholic extract of the scabies, slough, and pus cells. According to the pre-clinical data, “Psorinum-6x” (“x” stands for decimal potency of homeopathy) activates different immune effector cells (e.g., T cells, and accessory cells like, macrophages, dendritic cells, and natural killer cells) which can trigger a complex antitumor immune response [18, 19]. In a rat model study, daily oral administration of Psorinum 6x at doses up to 0.5 ml/Kg body weight/day for 2 weeks resulted in no adverse side effect [19]. Published retrospective and prospective studies also support the efficacy of Psorinum Therapy in treating patients with various malignancies [20–28]. The prospective observational clinical trial, reported here, was conducted to evaluate the efficacy of the Psorinum Therapy in treating stomach, gall bladder, pancreatic, and liver cancers and to assess the side effects of the drug Psorinum if any [29].
2. Materials and Methods
2.1. Settings
The study was conducted by the Critical Cancer Management Research Centre and Clinic (CCMRCC) situated in Kolkata of West Bengal, India. The study started from June 2001 and completed in July 2009. The study protocol was approved by the Institutional Review Board (IRB approval Number: 2001–05) of the CCMRCC in conformity with the World Medical Association (WMA) declaration of Helsinki and it is subsequent amendments and the ethical guidelines of the Indian Council of Medical Research (ICMR) for the biomedical research on human participants.
2.2. Study Design
The study was prospective, observational, open level, and single arm.
2.3. Inclusion and Exclusion Criteria
Only the patients of confirmed malignancy (by histopathological examination of endoscopic biopsy, cytopathological exam of CT guided FNAC) involving stomach, gall bladder, pancreatic, and liver cancers of both sexes were enrolled. The participants' eligibility criteria included (i) histopathology/cytopathology confirmation of malignancy, (ii) inoperable tumors, and (iii) no prior chemotherapy or radiation therapy. The lower age limit was 18 years and there was no upper age limit for the eligibility. Patients who were unable to understand English, Hindi, or Bengali or resided outside India were excluded from the study. The patients who reported the cancer centre from the period of June 2001 to November 2003 and fulfilled the eligibility criteria were recruited. Written informed signed consent was taken from each patient before starting the study.
2.4. Intervention
Psorinum-6x was administered orally to all the participants up to 0.02 ml/Kg body weight as a single dose in empty stomach per day for complete course duration of 2 years.
2.5. Supportive Treatments
In this study, the supportive cares were taken both from the spheres of allopathy and homeopathy. Supportive cares for control of infection, pain, electrolytic balance, bleeding, nutritional deficiencies were taken, and blood transfusion, abdominal or plural paracentesis, analgesic, bronchodilator, stenting of the hepato-pancreato-biliary system, and bypass were done as and when required to improve the survival and the quality of life of the participants. The frequently used homeopathic medicines for the purpose of the supportive cares were Chelidonium majus, Carduus marianus, Baryta carbonica, Conium maculatum, Carbo animalis, Bryonia alba, Medorrhinum, Thuja occidentalis, Cholesterinum, and Lycopodium clavatum (Table 1). Less frequently used homeopathic medicines for the purpose of the supportive cares were mother tincture of the Berberis vulgaris, mother tincture of the Calendula officinalis, mother tincture of the Hamamelis virginiana, mother tincture of the Symphytum officianale, mother tincture of the Syzygium jambolanum, Gelsemium 200c, Cantharides 200c, Sulphur 200c, Arsenicum album 200c, and Causticum 200c.
2.6. Outcome Measures
Primary outcome measures of the study were (i) to assess the radiological tumor response and (ii) to find out in each type of cancer how many participants survived at least 1 year, 2 years, 3 years, 4 years, and finally, after 5 years since the beginning of the study. To assess the radiological tumor response, CT Scans were done at the beginning of the study, repeated every 3–6 months during the 1st year of the study and repeated every 6–8 months during the next 2 years of the study. Radiological tumor response was defined by Response Evaluation Criteria in Solid Tumors (RECIST). A complete response was defined as complete disappearance of all targeted lesions without disease progression or any new lesion, and a partial response was defined as at least 30% regression in the sum of the longest diameter of the targeted lesions as reference to the baseline sum LD without disease progression or any new lesion. To assess the survival, the investigators followed up the participants via personal meetings, phone calls, and mails at least for 5 years (where applicable) after the study began. Secondary outcome measure of the study was to assess the side effects of the Psorinum. The investigators asked the participants and also examined them clinically to assess if they had any side effect. Apart from these, participants were also followed up to know if they were taking any other conventional or investigational cancer treatments.
3. Results
10 (5.95%) participants were dropped out from the study as they opted for conventional cancer treatments, among them 4 of stomach, 2 of gall bladder, 3 of pancreatic, and 1 of liver cancers. 158 participants (42 of stomach, 40 of gall bladder, 44 of pancreatic, and 32 of liver) were included in the final analysis at the end of the study. In these participants, the diagnosis of malignancies was confirmed by histopathological examination of endoscopic biopsies and cytopathological examination of CT-guided FNAC. In case of stomach, gall bladder, and pancreatic cancers, the histology type was adenocarcinoma, and in case of liver cancer the histology type was hepato cellular carcinoma (HCC). Among the 158 participants, 84 (53.16%) were male and 74 (46.84%) were female. According to the AJCC TNM staging system, 39 (24.68%) were diagnosed at stage-III, and 112 (70.89%) were diagnosed at stage-IV. The participants' Karnofsky status was between 40–70%, and Eastern Cooperative Oncology Group (ECOG) status was between 2-3. Among the 39 participants (24.68%) who were diagnosed at stage-III, 13 (33.33%) had complete response and 16 (41.03%) had radiological partial response. Among the 112 (70.89%) participants who were diagnosed at stage-IV, 12 (10.71%) had radiological complete response and 38 (33.93%) had radiological partial response (Tables 2 and 3, Figures 1, 2, and 3). In this study, no adverse side effects were observed from the drug Psorinum. However, very few patients reported to have mild oral irritation and skin itching which were successfully controlled by the supportive cares. Psorinum Therapy was also effective in improving the disease symptoms and the quality of life of the participants. At least 60% participants of stage-III and at least 45% participants of stage-IV reported that the therapy was effective in reducing their cancer-related pain, cough, dysponea, nausea and vomiting, fatigue, constipation and improving appetite, and weakness. These were also confirmed after examining the participants clinically. Improvements were also observed in the lab investigations like Complete Blood Count (CBC), Liver Function Test (LFT), Kidney function test, AFP level, and CA 19.9. These lab investigations were done as a part of their routine clinical check ups. Among the 158 participants, 98 (62.03%) were aged 65 years or more. Better outcomes were observed among the participants below 65 years of age than the participants who were over the age of 65. The outcomes did not vary significantly while considering gender. Figures 4(a) and 4(b) show complete tumor response in one stomach and one gall bladder cancer patients, respectively, who were treated through Psorinum Therapy.
4. Discussion and Conclusion
Many studies were published on the role of complementary and alternative medicines in treating cancer patients. Some studies support the CAM therapies to be beneficial for palliative cancer cares [30–35]. However, very few of the published reports support their efficacy with regard to the primary care of cancer. According to our knowledge, the clinical study, reported here, is the only prospective study that intrigued a fair number of complete and partial tumor responses along with impressive survival outcomes in treating patients with stomach, gall bladder, pancreatic, and liver cancers through psorinum therapy. Previously, interviews were conducted on 300 biopsy-proved cancer patients of Psorinum Therapy. The primary purpose of the study was to ascertain the patients' and/or their caregivers' view on this CAM therapy. The survey showed the patients had tried Psorinum Therapy mainly due to no other available treatment options, financial constraints, frustration with the conventional cancer treatments, and belief in the efficacy of the Psorinum Therapy. According to the survey, among the 300 cancer patients, 195 (65%) had consulted their oncologists before trying the therapy [17]. This therapy can be easily replicated by other practitioners in different clinical centers due to the following advantages.
The reagent to prepare the drug Psorinum is available. The specific dosing and the medicinal power are established. The medicine administration technique is easy as it can be taken orally.
The supportive treatments are adopted from the allopathic streams. The supportive treatments with homeopathic medicines are done by specific ailment versus specific medicine concept instead of the concept of specific patient versus specific medicine, making the homeopathic supportive cares easier to replicate. In a nutshell, we should remember that, 158 participants of histopathology or cytopathology confirmed stomach, gall bladder, pancreatic, and liver cancers were included in the final analysis at the end of the study. According to the AJCC TNM staging system, 39 (24.68%) were diagnosed at stage-III and 112 (70.89%) were diagnosed at stage-IV. The participants Karnofsky status was between 30–60% and ECOGstatus was between 2-3. The participants received the drug Psorinum along with allopathic and homeopathic supportive treatments without trying conventional or any other investigational cancer treatments. According to the RECIST criteria, radiological complete response occurred in 28 (17.72%) and partial response occurred in 56 (35.44%) participants. The limitation of this study is that it did not have any placebo or treatment control arm; therefore, it cannot be concluded that Psorinum Therapy is effective in improving the survival and the quality of life of the participants due to the academic rigours of the scientific clinical trials. This study also cannot rule out the effects of the implemented allopathic and homeopathic supportive measures in the observed results. However, the results of the study showed a fair number of complete and partial tumor responses along with impressive survival outcomes in difficult to treat cancer types. Therefore, randomized double-blind clinical trial, detailed molecular, pharmacokinetics, and pharmacodynamics studies should be conducted for further scientific exploration of this alternative cancer treatment to determine if it can be integrated into the mainstream oncology.
Funding
Dr. Rabindranath Chatterjee Memorial Cancer Trust provided funding for this study.
Conflict of Interests
The authors declare that they have no conflict of interests.
Acknowledgments
The authors would like to acknowledge the cooperation rendered by the pathologists, radiologists, oncologists, gastroenterologists, general physicians, nurses, and other technical and nontechnical persons to carry out the study. The statistical analysis was done by Ms. Moumita Mukherjee and Ms. Rituparna Mukherjee of CCMRCC. The whole study was presented at the 2009 Annual Meeting of the American Society of Clinical Oncology (ASCO).
Figure 1 Distribution of partial and complete tumor response rates in different cancer types.
Figure 2 Lorenz Analysis: Distribution of tumor response in different cancer types.
Figure 3 Kaplan Meier survival analysis in different cancer types.
Figure 4 (a) Showing complete tumor response of a stomach cancer patient who underwent Psorinum Therapy. (b) Showing complete tumor response of a gall bladder cancer patient who underwent PsorinumTherapy.
Table 1 Details of the frequently used homeopathic medicines for the purpose of the supportive cares.
Name Origin Dosing Power Used to control ailments
(1) Chelidonium majus
Herb-Chelidonium majus
Up to 0.04 ml/Kg body weight/day orally Mother tincture (1) Abnormal liver functions
(2) Dysponea
(2) Carduus marianus
Herb-Carduus marianus
Up to 0.04 ml/Kg body weight/day orally Mother tincture (1) Abnormal liver function
(2) Cholestasis
(3) Baryta carbonica
Barium carbonate Up to 0.02 ml/Kg body weight/day orally 200c (1) Anaemia
(2) Cancer-related pain
(4) Conium maculatum
Herb-Conium maculatum
Up to 0.02 ml/Kg body weight/day orally 200c (1)Heart troubles
(2) Abnormal blood pressure
(5) Carbo animalis
Animal charcoal Up to 0.02 ml/Kg body weight/day orally 200c (1) Cough
(2) Constipation
(6) Bryonia alba
Herb-Bryonia alba
Up to 0.02 ml/Kg body weight/day orally 200c (1) Dysponea
(2) Cancer-related pain
(7) Medorrhinum
Gonorrhoeal cocci
Up to 0.02 ml/Kg body weight/day orally 200c (1) Abnormal blood sugar
(2) Cancer-related pain
(8) Thuja occidentalis
Herb-Thuja occidentalis
Up to 0.02 ml/Kg body weight/day orally Mother tincture (1) Abdominal distension
(2) Electrolytic imbalance
(9) Cholesterinum
Cholesterine Up to 0.02 ml/Kg body weight/day orally 200c (1) Abnormal liver function
(2) Cholestasis
(10) Lycopodium clavatum
Herb-Lycopodium clavatum
Up to 0.02 ml/Kg body weight/day orally 200c (1) Abdominal distension
(2) Cancer-related pain
c → Centesimal potency of homeopathy.
Table 2 TNM Staging, partial and complete tumor response in each cancer type.
Primary cancer types No. of participants TNM Staging of the participants No. of patients: Complete tumor response occurred No. of patients: Partial tumor response occurred
Diagnosed at stage-II and stage-III Diagnosed at stage-IV
Stomach 42 11 31 6 (14.29%) 16 (38.1%)
G. Bladder 40 13 27 7 (17.5%) 17 (42.5%)
Pancreas 44 9 35 8 (18.18%) 13 (29.55%)
Liver 32 13 19 7 (21.87%) 10 (31.25%)
Table 3 Survival outcomes in each cancer type.
Primary organ affected No. of Patients Male Female Survived at least 1 year Survived at least 2 years Survived at least 3 years Survived at least 4 years Survived at least 5 years
Stomach 42 22 20 34 24 21 20 16 (38.1%)
G. Bladder 40 21 19 32 25 20 18 15 (37.5%)
Pancreas 44 24 20 34 28 27 21 17 (38.64%)
Liver 32 17 15 26 22 19 17 14 (43.75%)
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21203472PONE-D-10-0263710.1371/journal.pone.0015773Research ArticleBiologyMicrobiologyBacterial PathogensGram NegativeMicrobial PathogensPlant MicrobiologyPlant SciencePlant PathologyPlant PathogensDetection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD N-Terminal Extension in the Xcv T3E Effector XopDCanonne Joanne
1
Marino Daniel
1
Noël Laurent D.
1
Arechaga Ignacio
3
Pichereaux Carole
2
Rossignol Michel
2
Roby Dominique
1
Rivas Susana
1
*
1
Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR CNRS-INRA 2594/441, Castanet Tolosan, France
2
Institut Fédératif de Recherche (IFR40), Plateforme protéomique Génopole Toulouse Midi-Pyrénées, Institut de Pharmacologie et Biologie Structurale, Université de Toulouse, Toulouse, France
3
Departamento de Biología Molecular, Universidad de Cantabria (UC) and Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), Santander, Spain
Bendahmane Mohammed EditorEcole Normale Superieure, France* E-mail: [email protected] and designed the experiments: JC DM SR. Performed the experiments: JC DM CP SR. Analyzed the data: JC DM LDN IA MR DR SR. Contributed reagents/materials/analysis tools: LDN. Wrote the paper: SR.
2010 22 12 2010 5 12 e1577328 9 2010 26 11 2010 Canonne et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.
==== Body
Introduction
Plants have developed a complex defence network to fight off invading pathogens. The first layer of plant defence involves recognition of PAMPs (Pathogen-Associated Molecular Patterns), defined as invariant epitopes within molecules that are fundamental to pathogen fitness and widely distributed among different microbes [1]. This recognition, previously known as basal defence, is now referred to as PAMP-triggered immunity (PTI) [2]. PTI is associated to the production of reactive oxygen species and antimicrobial compounds, the induction of mitogen-activated protein kinase (MAPK) cascades, the modulation of host gene transcription and the deposition of lignin and callose at the plant cell wall [3]–[6]. Some bacterial pathogens evolved to suppress PTI and promote successful infection by injecting T3Es (type III effectors) into plant cells using the type III protein secretion system (T3SS) [7]–[9]. In turn, plants acquired the ability to recognize directly or indirectly effectors through resistance (R) proteins. This recognition response is associated with the long-standing gene-for-gene hypothesis, and more recently with the guard hypothesis [10], and is now known as effector-triggered immunity (ETI). Several T3Es from Gram-negative phytopathogenic bacteria have also been shown to suppress ETI [11], suggesting that T3Es may have multiple targets or that they target shared components between PTI and ETI.
The coevolutionary arms race between plants and pathogens has generated highly polymorphic repertoires of R proteins and T3Es. A significant number of T3Es from different bacteria has been identified during the last few years. Known biochemical activities of T3Es include manipulation of host protein turnover, either through protease activity [12]–[14] or protein degradation via the 26S proteasome [15]–[17], modification of host transcription or RNA stability [18]–[21] and alteration of the phosphorylation state of plant proteins [22]–[27].
Gram-negative pathogenic bacteria of the genus Xanthomonas infect a wide range of host plants and are responsible for important crop plant diseases. Xanthomonas campestris pathovar vesicatoria (Xcv, also known as Xanthomonas axonopodis pathovar vesicatoria or Xanthomonas euvesicatoria
[28], [29]) is the causal agent of bacterial spot on tomato (Solanum lycopersicum) and pepper (Capsicum annum) [30]. The T3SS of Xcv is encoded by the chromosomal hrp (HR and pathogenicity) gene cluster, which contains 25 genes [31], [32]. hrp gene expression is activated during plant infection or when bacteria are incubated in special minimal media by two regulatory proteins, HrpG and HrpX. HrpG is a member of the OmpR family of two-component system response regulators and controls the expression of a genome-wide regulon including hrpX
[33], [34]. HrpX is an AraC-type transcriptional activator that binds to a conserved DNA motif [plant-inducible promoter (PIP) box; consensus: TTCGC-N15-TTCGC], which is present in the promoter regions of most hrpX-regulated genes [33], [35].
T3SS-dependent secretion of a protein is mediated by an N-terminal secretion signal within its first 15–20 residues. The presence of hydrophilic amino acids, absence of acidic residues in the first 12 amino acids, amphipacity and a bias towards serine and glutamine in the first 50 residues are common features of the N-terminal sequence of T3Es [36]–[39]. In addition to the secretion signal, effector proteins contain a 1–50 to 1–100 amino acid region at their N-terminus that is required for translocation across the eukaryotic plasma membrane [40].
The presence of a variety of putative structural motifs in the primary sequence of Xanthomonas T3Es has provided insights into their putative biochemical function [41]. The XopD T3E from Xcv 85-10 has been classified as a member of the C48 protease family, and it has been shown to release SUMO (small ubiquitin-like modifier) from SUMO-modified plant proteins [42], [43]. XopD is a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs [L/FDLNL/F(X)P] [44], previously described in plant transcriptional repressors that negatively regulate gene transcription during stress and defence responses [45], and (iii) a C-terminal cysteine protease domain with structural similarity with the yeast ubiquitin-like protease 1 (ULP1) [42]. XopD is localized in nuclear foci indicating that host targets are likely nuclear SUMOylated proteins [43]. Intriguingly, consistent with its protein structure, XopD has been reported to be a non-specific DNA-binding protein that represses plant gene transcription, delaying the onset of symptom development and altering basal defence and cell death responses, which in turn promotes pathogen growth in infected leaves [46]. However, as for many of the known bacterial effectors, XopD direct plant targets remain unknown.
XopD was previously predicted to code for a protein of 612 amino acids [47]. However, secretion assays and Western blot analysis, following expression of c-myc-tagged XopD within its native Xcv chromosomic environment, allowed the detection of a unique band of a molecular mass close to 100 kDa [47]. Importantly, this protein was undetectable in culture supernatants of T3SS mutant bacteria cultured in secretion medium, demonstrating that this protein is secreted in a T3SS-dependent manner. In contrast, the XopD protein sequence annotated in the public databases predicts a protein of 545 amino acids with an expected molecular mass of 61 kDa. Noël and co-workers could not detect a (consensus) PIP box in the xopD promoter, but rather a putative hrp box, which is found in all hrpL-dependent promoters in Pseudomonas syringae and Erwinia spp [47]–[49]. Later inspection of the xopD promoter region identified the presence of a PIP box (ATCGC-N15-TTCGT), bound by HrpX, and a −10 putative sequence (TAAATT), which are situated 747 and 690 bp upstream the annotated start, respectively (Figure 1A) [35]. In agreement with these observations, the HrpX-dependent expression of XopD has been confirmed experimentally [35], [47].
10.1371/journal.pone.0015773.g001Figure 1
In silico analysis of the xopD locus.
(A) The xopD locus in Xcv 85-10 is shown (the genome interval for the Xcv 85-10 shown sequence and the xopD gene are respectively: 486209–489200 and 486544–488826). Open arrows indicate ORFs and filled arrows show promoter elements, such as the PIP box and the −10 sequence. The position of XopD translation start annotated in the public databases is indicated by an asterisk. (B) Schematic representation of XopD functional domains in Xcv 85-10, in comparison to its known protein homologues. The N-terminal extension, essential V and L residues in the DBD, tandemly repeated EAR motifs, conserved catalytic residues in the cysteine protease domain, and NLS motif are shown. (C) Sequence alignment of XopD from Xcv 85-10, hypothetical protein from Xcc B100 (YP_001902662), virulence protein from Xcc 8004 (AAY48282) or Xcc ATCC 33913 (AAM42168), peptidase C48 SUMO from Acidovorax avenae (EFA39722) and XopD from Xcc 147. The figure shows the longest ORF possible for all proteins. For XopD from Xcv, the first shown M residue corresponds to a UUG codon, not conserved among Xcc B100 and A. avenae coding sequences. The M residue previously annotated as the starting amino acid of the XopD protein is indicated by a red arrowhead. Putative translation starts situated in frame and upstream the previously annotated starting M are also indicated as follows: translation starts conserved among Xcv 85-10, Xcc B100 and Aea are indicated with a full red dot, otherwise, they appear indicated by an empty red dot. Yellow box: putative N-terminal extension; green box: DNA-binding domain (V and L residues in the helix-loop-helix domain essential for maximal DNA-binding [46] are indicated by an empty green dot); red boxes: tandemly-repeated EAR motifs [L/FDNLL/F(X)P] [44]; black box: SUMO protease domain (H, D and C catalytic core residues are indicated by a black empty dot); blue box: NLS. Gray and black highlighting of amino acids indicates, respectively, 70–80% and 90–100% of similarity.
In the view of these conflicting data, we set out to identify the starting amino acid of XopD and thereby determine the sequence of the XopD protein that is produced and secreted from Xcv. Our work confirms that the XopD protein sequence previously annotated in the databases is incomplete. We demonstrate that the functional protein expressed by Xcv presents a previously overlooked N-terminal extension of 215 amino acids that is essential for its T3SS-dependent secretion and virulence-associated functions in the plant cell.
Results
Sequence analysis of the xopD locus
In bacteria, the most frequently used codon for the initiation of translation is the triplet AUG, although GUG and, occasionally, UUG may also be used. The meaning of the GUG, or the rarely used UUG, codon depends on their context. When present within a gene, they provide valine (V), or leucine (L), as specified by the genetic code. However, present as first codons, they mediate the incorporation of methionine (M). Close inspection of the Xcv 85-10 genomic sequence neighbouring the annotated xopD gene shows that upstream of the annotated starting codon AUG, there are 6 AUG, 1 GUG and 5 UUG additional codons that are in frame and may potentially be used to start translation of the protein (Figure 1C). In addition, since HrpX-dependent expression of XopD was previously demonstrated, the presence of a PIP box and a −10 sequence upstream of the first putative starting codon suggested that translation of XopD may start immediately downstream these regulatory elements (Figure 1A).
BlastP analysis using the Xcv 85-10 XopD sequence starting at the first possible translation start (UUG, encoding or not a M residue depending on whether it is or not the translation start) identified three major hits corresponding to (i) a hypothetical, not yet characterized, protein from the strain B100 of Xantomonas campestris pathovar campestris (Xcc) [50], (ii) a protein from Acidovorax avenae, annotated as peptidase C48 SUMO/Sentrin/Ub11, and (iii) two virulence proteins that correspond to a shorter version of XopD deleted from its DBD, from the Xcc strains 8004 [51] and ATCC 33913 [52] (Figure 1B). In addition, sequencing of xopD using genomic DNA from the Xcc 147 strain [53] showed that this XopD protein is very similar to the one present in Xcc strains 8004 and ATCC 39913 (Figure 1B,C). It is noteworthy that the Met residue previously annotated as XopD starting amino acid is not conserved in Xcc B100 (Figure 1C; red arrow), suggesting that this may not be a starting amino acid for XopD. In contrast, the putative N-terminal extension of XopD is well conserved in both A. avenae and Xcc B100, indicating that it may be important for XopD function (Figure 1C).
T3SS-dependent secretion of an 85 kDa XopD protein from Xcv
To allow detection of the XopD protein that is produced by Xcv from its native promoter, Xcv genomic DNA was tagged with an HA epitope at the 3′end of xopD coding sequence using a suicide vector. XopD protein expression was analyzed in strains 85*, which allows constitutive expression of all hrp genes [54], and 85* ΔhrcV, which carries a deletion in a conserved component of the T3SS [55]. Western blot analysis of bacterial total protein extracts with an anti-HA antibody did not allow detection of any band at ∼61 kDa, which is the predicted molecular mass of the annotated XopD protein (Figure 2). In contrast, a unique band of ∼85 kDa was detected (Figure 2, lane 1), strongly suggesting that (i) there is a unique start for XopD translation and that (ii) the protein that is produced by Xcv presents a significant extension at the N-terminus of the annotated protein. No HA-tagged protein could be detected in the untransformed strain 85* (Figure 2, lane 2).
10.1371/journal.pone.0015773.g002Figure 2 Expression analysis of XopD.
Strains 85* (XopD-HA) (1), 85* (2) and 85* ΔhrcV (XopD-HA) (3) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants (200-fold concentrated) were analyzed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of XopD, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.
hrp-dependent secretion of XopD was next tested after incubation of bacteria in secretion medium. A single band of ∼85 kDa was detected in culture supernatants of the 85* strain (Figure 2, lane 1). XopD was not detectable in culture supernatants of T3SS mutant 85* ΔhrcV (Figure 2, lane 3), reflecting the presence of a functional T3SS secretion signal in the ∼85 kDa form of XopD. No protein could be detected in the supernatant fractions with an antibody against the intracellular chaperone GroEL, demonstrating that detection of proteins in the supernatant was not due to bacterial lysis (Figure 2).
Since the expected molecular mass for the longest possible ORF (UUG codon; Figure 1C) is 86 kDa, this analysis shows that the molecular mass of the XopD protein produced and secreted by Xcv 85* in a T3S-dependent manner is consistent with the protein starting at one of its very first putative translation starts.
Identification of a 215 amino acid N-terminal extension in XopD using mass spectrometry
A mass spectrometry analysis was thus conducted to determine the starting amino acid of the XopD protein produced by Xcv. HA-tagged XopD was immunopurified from Xcv 85* cultures and subjected to electrophoresis. After Coomassie staining of the gel, a ∼85 kDa band corresponding to XopD was excised, digested with trypsin and analyzed by mass spectrometry (Nano LC/ESI MS/MS). Trypsin cleaves at the C-terminal side of arginine (R) and lysine (K) residues, which are well distributed throughout the XopD sequence. In total, 28 peptides corresponding to XopD were detected (Figure 3A; Table 1), which represents a total XopD sequence coverage of 44%. The N-terminal region of the protein was particularly well covered (58% coverage for the newly identified N-terminal extension). MS/MS fragmentations for all peptides are provided in Figure S1.
10.1371/journal.pone.0015773.g003Figure 3 Analysis of the XopD protein sequence by mass spectrometry.
XopD216-760 protein sequence is shadowed. All possible translation starts situated in frame and upstream the annotated M216 are underlined. Peptides identified by Nano LC/ESI MS/MS analysis following trypsin (A) or V8 protease (B) digestion of the purified XopD protein are shown in bold. The 30 amino acid low complexity KAE-rich region is indicated in red.
10.1371/journal.pone.0015773.t001Table 1 Sequences and individual MASCOT scores of XopD peptides identified by Nano LC/ESI MS/MS analysis after trypsin digestion.
Peptide sequencea
,
b
MASCOT score
IFNFDYK 35
EMTEAADDYR 69
ENHGAGYNMHPLLESLPR 38
RNPTQVHADGSVHQMR 60
ILELISAYGDGK 107
GIPELQR 24
QMLQELNEDQR 68
DQVIHQIIR 50
RIEYCADPEYR 34
EVALSR 25
ITLSQR 29
AKAEAEAEAK 21
INEIMEYIPR 77
YEALEK 22
GDGSFGPGLPGILR 52
YMTPDQK 20
LYLASER 28
KLALAAPK 53
TLHQKPNLLLEISSK 26
LGEAVISGASQGIQTAIR 117
AWPAQPEASSSTFDDLESLDYR 90
IDVDNLPSPQDVADPELPPVR 80
ATSWLLDGHLR 64
AYTDDLAR 49
NKDAVAAYHYDSMAQK 24
VLDGTFDYAGGR 77
DLTDIEPDR 22
LAQAEQAPAESSIR 82
a The first 13 peptides are present in the XopD N-terminal extension.
b The starting M residue for the XopD protein annotated in the public databases in underlined in the INEIMEYIPR peptide.
Detection of a peptide (INEIMEYIPR; Figure 3; Table 1) encompassing the annotated starting M216 residue demonstrates that the XopD sequence annotated in the public databases is not complete. Furthermore, the first detected peptide is IFNFDYK, showing that the two possible start residues for XopD are the first and the second M shown in Figures 1C and 3.
To determine the starting amino acid of XopD, we next used the V8 protease, which cleaves at the C-terminal side of glutamic (E) and aspartic acid (D) residues, before conducting the mass spectrometry analysis. In total, 15 peptides were detected, which represents a XopD sequence coverage of 21% (Figure 3B; Table 2; Figure S2). Importantly, detection of the peptide MDRIFNFD demonstrates that the XopD protein that is produced by Xcv starts at the second predicted M of the longest possible ORF (XopD1-760, Figure 1C and 3). This represents an N-terminal extension of 215 amino acids compared to the annotated XopD protein (XopD216-760) and conservation of this sequence in both Xcc B100 and A. avenae (Figure 1C) suggests that this region may be important for XopD function(s).
10.1371/journal.pone.0015773.t002Table 2 Sequences and individual MASCOT scores of XopD peptides identified by Nano LC/ESI MS/MS analysis after digestion with the V8 protease.
Peptide sequencea
,b
MASCOT score
MDRIFNFD 25
YKKYREMTE 20
NHGAGYNMHPLLE 25
LQRSFPSFAAFLMD 41
YCADPEYRE 28
VALSRLE 37
SDCSGKITLSQRTLD 49
YIPRYE 22
LTRLGE 30
SLDYRQNYGYRE 22
LPPVRATSWLLD 26
AVAAYHYD 30
SMAQKDPQQRYLAD 23
MAAYHLGLDYQQTHE 29
VLAHRVLD 39
a The first 8 peptides are present in the XopD N-terminal extension.
XopD1-760 is able to form dimers in E. coli
To gain insight into the putative function of XopD N-terminal extension, XopD amino acid sequence (full length XopD1-760) was analyzed for its biochemical properties. Interestingly, we found a region rich in lysine, alanine and glutamic residues (KAE-rich; residues 168–202) with high propensity to form coiled-coil structures (Figure 4A). The presence of this type of coiled-coil structures generally indicates that the protein may interact with other amino acid chains of the same polypeptide to form oligomers or other polypeptides to form complexes. Further analysis predicted that this coiled-coil, KAE-rich region is likely to be involved in dimer formation, rather than trimer or other oligomeric structures (Figure 4A).
10.1371/journal.pone.0015773.g004Figure 4 XopD1-760, but not XopD216-760, is able to dimerize.
(A) Multicoil (http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi) prediction on XopD1-760 sequence. XopD amino acid sequence (amino acids 1 to 760) was analyzed for its propensity to form coiled-coil structures. A region comprising amino acid residues 168–202 showed high probability to form such coiled-coil structures. Furthermore, this coiled-coil region is likely to be involved in dimer formation (blue line), rather than trimer (red line) or other oligomeric structures. The (abcdefg) positions of the hepta-repeat in the coiled-coil region are indicated above the amino acid sequence. Above it, the relative positions of the hepta-repeat in the opposite chain within the putative dimer are indicated in blue. (B) Gel filtration analysis of HA-tagged XopD expressed in Xcv, and XopD1-760 and XopD216-760 expressed in E. coli as 6xHis-tagged fusions. Protein extracts were subjected to gel filtration chromatography on a Superdex S-200 column. 0.4 ml fractions were collected and aliquots were analyzed by Western blot. Fraction numbers of the elution profile re indicated by the numbers between the gels. The molecular mass estimated for each fraction (in kDa) is given at the top.
In the view of these observations, we first tested the oligomerization state of HA-tagged XopD produced by Xcv 85*. Size exclusion chromatography of bacterial total protein extracts, followed by Western blot analysis of the collected fractions, showed that the peak of elution of HA-tagged XopD corresponds to an estimated molecular mass of approximately 86 kDa, which coincides with the predicted molecular mass of an HA-tagged XopD monomer. This result (i) suggests that, despite the presence of a predicted coiled-coil structure, XopD is not able to dimerize in Xcv 85* and (ii) is consistent with the idea that, before translocation into the plant cell, T3Es are unfolded or associated to T3S chaperones to facilitate their passage through the secretion apparatus [56], [57].
The oligomerization state of XopD1-760 and XopD216-760 was next studied following overexpression of His-tagged protein versions in E. coli cells. The expected molecular mass for His-tagged XopD1-760 and XopD216-760 monomers is 89 and 65 kDa, respectively. After protein chromatography, aliquots from collected fractions were analyzed for the presence of XopD by immunoblot analysis. The estimated molecular mass of XopD216-760 ranged between 46 and 70 kDa whereas XopD1-760 elution ranged between 128 and 190 kDa (Figure 4B). These results strongly suggest that, consistent with the presence of a predicted coiled-coil structure, XopD1-760, but not XopD216-760, is able to dimerize in E. coli.
Subcellular localization and functions of XopD1-760 in N. benthamiana
The N-terminal domain of XopD216-760 was previously shown to be required for targeting the effector to subnuclear foci [43]. To determine the effect of the newly identified N-terminal extension of XopD on its subcellular localization in planta, XopD1-760 was fused to the Yellow Fluorescent Protein venus (YFPv) and transiently expressed, under the control of the 35S promoter, in Nicotiana benthamiana leaf epidermal cells. As XopD216-760, XopD1-760 was also localized in subnuclear foci (Figure 5A). Both proteins were detected by Western blot using an anti-GFP antibody (Figure 5B).
10.1371/journal.pone.0015773.g005Figure 5 Characterization of XopD1-760 expression and function in N. benthamiana.
(A) Confocal images of epidermal cells of N. benthamiana leaves expressing YFPv-tagged XopD216-760 and XopD1-760 36 hours after agroinfiltration. Bright field images are shown on the right. Bars = 15 µm. (B) Western blot analysis using an anti-GFP antibody shows expression of YFPv-tagged XopD216-760 and XopD1-760 constructs, 36 hours after agroinfiltration. Ponceau S staining of the membrane illustrates equal loading. (C) Cell death development 4 (upper panel) and 7 (lower panel) days after agroinfiltration of YFPv-tagged XopD216-760 (left) and XopD1-760 (right) constructs. (D) Cell death was quantified by measuring electrolyte leakage in N. benthamiana leaves expressing YFPv-tagged XopD216-760 (open circles) and XopD1-760 (filled circles) at the indicated time points after agroinfiltration. Mean and SEM values are calculated from 3 independent experiments (8 replicates/experiment). The statistical significance in mean conductivity values obtained with leaves expressing XopD216-760 or XopD1-760 was assessed by using a Student's t test (P value <10−5). (E) SUMO-conjugates detected by Western blot analysis using an anti-HA antibody 36 hours after agroinfiltration of the indicated constructs. Expression of XopD proteins was revealed using an anti-GFP antibody. Ponceau S staining of the membrane illustrates equal loading.
Agrobacterium-mediated transient expression of XopD216-760 in N. benthamiana leaves results in tissue necrosis by 4 to 7 days after agroinfiltration, likely reflecting cell death due to XopD accumulation and cytotoxicity (Figure 5C; [46]). Interestingly, up to 5 days after agroinfiltration, N. benthamiana leaves expressing XopD1-760 showed a significant delay in the development of the necrotic phenotype observed after transient expression of XopD216-760 (Figure 5C), despite similar levels of protein accumulation (Figure 5B). These results were confirmed by ion leakage measurements in leaf disk assays, which showed significantly lower conductivity values in leaves expressing YFP-tagged XopD1-760, compared to leaves that expressed XopD216-760 (Figure 5D). These observations indicate that the N-terminal extension of XopD may negatively regulate the previously described cytotoxic effect induced by XopD216-760 expression. From 7 days after agroinfiltration, a similar cell death phenotype was observed with both YFP-tagged XopD216-760 and XopD1-760 (Figure 5C).
Finally, the SUMO protease activity of YFP-tagged XopD1-760 was investigated after co-expression with an HA-tagged LeSUMO construct [43] in N. benthamiana leaves. Western blot analysis of HA-SUMO conjugates showed that, as in the case of XopD216-760, expression of XopD1-760 led to significant reduction in the detection of SUMO-modified proteins (Figure 5E). As previously described, XopD216-760-C470A, mutated in the conserved Cys residue in XopD catalytic core, was not able to hydrolyze the SUMO substrates [43] (Figure 5E). These data demonstrate that, similar to XopD216-760, XopD1-760 displays SUMO protease activity in planta.
Analysis of XopD1-760-mediated virulence functions in planta
We next investigated whether the newly identified N-terminal protein extension in XopD may have an effect on its function in planta. First, a previous report showed that Agrobacterium-mediated transient expression of XopD216-760 in N. benthamiana prevents the induction of the expression of the PR1 promoter (PR1p) fused to the GUS reporter gene after salicylic acid (SA) treatment [46]. Consistent with previous results, SA treatment induced PR1p transcriptional activation whereas, in the presence of XopD216-760, PR1p activation was significantly reduced (Figure 6A). Interestingly, co-expression of XopD1-760 in these assays led to a stronger repression of PR1p transcriptional activation, suggesting that the N-terminal extension of XopD is necessary to modulate XopD function in the host (Figure 6A).
10.1371/journal.pone.0015773.g006Figure 6
In planta analysis of XopD1-760-mediated virulence functions.
(A) Transactivation of the PR1 promoter after SA treatment in transient assays in N. benthamiana. Leaves were inoculated with A. tumefaciens carrying a 35S:PR1p-GUS fusion either alone (lanes 1, 2) or together with HA-tagged XopD216-760 (lane 3) or XopD1-760 (lane 4). 18 hours after agroinfiltration, leaves were mock-treated (white bar) or treated with 2 mM SA (grey bars). Fluorimetric GUS assays in leaf discs were performed 12 hours later. Mean values and SEM values were calculated from the results of four independent experiments, with four replicates per experiment. Statistical differences according to a Student's t test P value <0.05 are indicated by letters. MU, methylumbelliferone. (B) Western blot analysis showing expression of HA-tagged XopD216-760 and XopD1-760. Ponceau S staining illustrates equal loading. (C) Susceptible Pearson tomato leaves were inoculated with Xcv 85* or Xcv 85* ΔxopD, expressing an HA-tagged GUS control, Xcv 85* ΔxopD expressing HA-tagged XopD216-760 or Xcv 85* ΔxopD expressing HA-tagged XopD1-760. Inoculation was performed with bacterial suspensions of 1×105 cfu/ml. Representative symptoms observed 10 dpi are shown. Similar phenotypes were observed in four independent experiments. (D) Strains Xcv 85* expressing a GUS control (1) and 85* ΔxopD expressing either a GUS control (2), XopD216-760 (3) or XopD1-760 (4) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants concentrated (200-fold concentrated) were analysed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of GUS, XopD216-760 and XopD1-760, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.
Second, XopD was previously reported to delay the onset of symptom development in susceptible tomato leaves after inoculation with Xcv
[46]. In order to assess the role of XopD1-760 and XopD216-760 in Xcv symptom development in tomato, we first engineered a XopD null mutant in Xcv strain 85*. The sequence encoding the entire XopD ORF (XopD1-760) was deleted by homologous recombination to generate an Xcv 85* ΔxopD mutant strain. In agreement with previously published data, tomato leaves of the susceptible cultivar Pearson inoculated with Xcv ΔxopD developed cell death by 10 days post inoculation (dpi) whereas leaves inoculated with Xcv were relatively healthy at the same time point (Figure 6C). Interestingly, the mutant strain Xcv 85* ΔxopD expressing wild-type HA-tagged XopD1-760 from a constitutive lac promoter in a broad host range plasmid was complemented for symptom development, whereas transformation of the same strain with an equivalent construct to express XopD216-760 did not allow complementation and inoculated leaves became necrotic (Figure 6C). These data were confirmed by inoculation of susceptible tomato plants of the Moneymaker cultivar, which showed identical results (data not shown). Western blot analysis with an anti-HA antibody showed expression of both XopD216-760 and XopD1-760 in bacterial total protein extracts (Figure 6D). XopD216-760 lacks the first 215 amino acids of XopD, which contain the T3S-dependent secretion signal. As a result, XopD1-760, but not XopD216-760, could be detected in culture supernatants from bacteria incubated in secretion medium (Figure 6D; lanes 3,4). As expected, the HA-tagged GUS control protein expressed by Xcv 85* or Xcv 85* ΔXopD was detected in total bacterial extracts but not in culture supernatants (Figure 6D; lanes 1,2). These results demonstrate that XopD1-760 contains all necessary elements for functional N-terminal secretion and virulence function in planta. Together, our data (i) are consistent with XopD being a 760 amino acid protein and (ii) stress the biological significance of the newly identified N-terminal extension of XopD.
Discussion
Prediction of the translation start in bacterial proteins is particularly difficult, rendering systematic annotation of effector proteins a challenging task. Here, we took a mass spectrometry approach to determine the protein sequence of XopD following immunopurification from Xcv. After trypsin digestion, 13 peptides were identified upstream the formerly annotated starting M residue, confirming that the XopD protein sequence annotated in the public databases is not complete. Moreover, trypsin digestion of purified XopD led to detection of the peptide IFNFDYK (starting after R3 in Figure 3), showing that XopD may start either at the first (UUG codon) or the second (AUG codon) M residue shown in Figures 1C and 3. Detection of the peptide MDRIFNFD, after V8 protease treatment, confirmed that the newly identified N-terminal domain present in XopD produced and secreted by Xcv comprises 215 amino acids and that XopD is thus a 760 amino acid protein. This finding is consistent with the observation that initiation of translation of 84% of the predicted Xcv ORFs is predicted to start at an AUG codon, whereas only 4% of the predicted Xcv ORFs are predicted to start at a UUG triplet. In agreement with this assumption, out of a total of 28 inspected Xcv T3Es effectors, none is predicted to be translated starting from a UUG codon. Interestingly, the first predicted UUG codon in XopD from Xcv is not present in the XopD coding regions from either Xcc B100 or A. avenae, whereas the AUG codon, which is used to initiate XopD translation, is well conserved. Finally, inoculation of tomato susceptible plants with Xcv expressing XopD1-760 provided further confirmation that XopD from Xcv is a functional 760 amino acid secreted protein (see below).
In the case of T3Es, the existence of the T3S N-terminal secretion and translocation signals could theoretically help prediction of their starting amino acid. However, secretion signals are not conserved at the amino acid level, even among related or conserved effectors [36], [38] and, in some cases, they are highly tolerant of substitutions [58], [59]. Moreover, in many cases, the presence of several in frame putative initiation codons makes it difficult to determine the starting amino acid for a given effector. Several programs have been developed for prediction of T3S signals in secreted proteins from Gram-negative bacteria [60]–[62]. In the case of XopD, two different recent algorithms [60], [61] assigned the highest probability of secretion to XopD51-760, which, as shown in this study, does not correspond to the XopD protein secreted by Xcv, whereas the assigned probability of secretion for XopD1-760 was very low. This is in agreement with previous reports estimating that algorithm-based gene prediction may lead to up to 40% of wrongly assigned start codons [63]. Indeed, prediction of T3Es secretion is not straightforward, since real translation starts of T3Es have only occasionally been determined experimentally. In a few cases, manual changes of predicted translational start positions have been reported, particularly in the case of myristoylated T3Es, for which the presence of the conserved myristoylation motif facilitates determination of the translation start [64].
Several reasons may explain why the newly identified N-terminal extension of XopD was previously overlooked. First, XopD N-terminal extension presents a low coding probability using standard codon usage matrices. Indeed, the G+C content in this genomic region is lower (47%) than in the rest of the xopD gene (54%) and much lower than in the rest of the Xcv genome (65%), suggesting its acquisition from a different organism with a different codon usage. For instance, the ACUR0 matrix (alternative codon usage regions [65]), developed for systematic annotation of Ralstonia T3Es, assigns higher coding probability to this region, although, using this matrix, M41 is predicted to be XopD starting amino acid. Indeed, when analyzed for base composition, most ACURs differ significantly from the average 67% G+C content found in the entire genome, with variations ranging from 50–70% G+C content. Furthermore, ACURs are often associated with mobile genetic elements, suggesting that ACURs may have been acquired through horizontal transfer [65]. Interestingly, xopD acquisition by horizontal transfer during evolution was previously proposed [47].
T3SS-dependent translocation of XopD into plant cells was previously demonstrated using a translational C-terminal fusion with the calmodulin-dependent adenylate cyclase domain (Cya) of Bordetella pertussis cyclocysin [66]. In these assays, 815 bp corresponding to what was previously annotated as the xopD promoter region and the annotated XopD coding region (XopD216-760) were fused to the CyA sequence and detection of the CyA activity in pepper leaves reflected XopD translocation [43]. However, considering our present findings, the construct used for the CyA assays comprised the N-terminal extension identified here (XopD1-760) preceded by a promoter xopD region of only 171 bp. Interestingly, this small promoter region appears to contain all necessary elements to allow XopD expression and wrong annotation of XopD was thus not suspected. Likewise, previously reported complementation studies of a Xcv strain deleted from XopD were performed with a construct that contained the xopD promoter and the complete XopD coding sequence [46]. Although the authors did not describe the length of the xopD promoter sequence used for complementation of the Xcv ΔxopD mutant strain, it must have contained at least the N-terminal extension described in the present work and a promoter region that is long enough to allow complementation.
The present study demonstrates that the newly identified N-terminal extension in XopD is essential for its secretion and promotes XopD virulence function(s) in planta. First, XopD N-terminal domain appears to negatively regulate XopD-induced cytotoxicity. In addition, XopD1-760 is more efficient than XopD216-760 in repressing transcription of PR1 after SA treatment, further confirming the presence of important regulatory elements in the N-terminal stretch of XopD. Finally, inoculation of tomato plants showed that XopD216-760 is not secreted from Xcv due to the lack of its N-terminal T3S-dependent secretion signal and is thus not able to complement an Xcv ΔxopD mutant strain. In contrast, XopD1-760 is detected in supernatants from bacterial cultures and complements the Xcv ΔxopD mutant. This observation is consistent with the fact that translation of XopD is initiated at the first AUG codon (XopD1-760) and that all necessary elements for functional T3S-dependent secretion and in planta translocation are thus present in XopD1-760.
XopD was formerly described as a modular protein comprising a DNA-binding domain, two transcriptional repression motifs of the EAR type and a SUMO-protease domain [46]. Here, we describe a previously non-identified protein domain in XopD. Although determining the biological function of this protein domain is clearly beyond the scope of this study, our findings provide intriguing clues to the putative role of XopD N-terminal extension. For example, the fact that XopD KAE-rich domain presents a high probability to form coiled-coil structures, together with gel filtration analysis of XopD1-760 expressed in E. coli, strongly suggest that the KAE-rich domain is indeed involved in the formation of XopD dimers. Interestingly, detection of XopD dimers was not possible in Xcv, perhaps indicating that XopD is unfolded or associated to T3S chaperones prior to its injection in the plant cell. This idea is consistent with previous reports indicating that efficient effector translocation requires the assistance of specialized chaperones that promote the stability and/or secretion of their corresponding interaction partners, keeping them in a partially unfolded and, thus, secretion-competent conformation and guiding them to the secretion apparatus [56], [57]. In protein dimers, cooperativity between the two proteins that form the dimer may increase the binding affinity for DNA [67]. Dimerization can also enhance the specificity of DNA binding by doubling the length of the DNA region bound by the protein dimer [67]. XopD216-760 was previously described as a transcriptional repressor of plant target genes that displays non-specific DNA binding activity [46]. Therefore, our finding that the XopD N-terminal extension is involved in dimer formation opens new perspectives regarding the study of XopD specificity of DNA binding as well as the search of its host targets.
BLAST analysis of the KAE-rich region with a high propensity to form coiled-coil dimers (residues 177-202; Figure 4A) identified a number of hits corresponding to protein families with a high degree of amino acid sequence homology. Particularly interesting was the homology of this region of XopD with the TolA protein family. TolA are membrane proteins involved in colicin uptake [68]. Interestingly, these proteins also contain a region rich in K, A, E residues that has been shown to form long α-helical structures [69]. The high degree of similarity observed after sequence alignment of this KAE-rich region of TolA with XopD177-202 amino acid residues (Figure S3A) suggests that this region of XopD may adopt a similar structural conformation.
Further analysis of the sequence upstream of the KAE-rich region in XopD allowed us to identify a sequence (residues 165–175) with homology to the MarR (Multiple antibiotic resistance) family of proteins (Figure S3B). This family of transcriptional regulators is named after E. coli MarR, a repressor of genes that activate multiple antibiotic resistance and oxidative stress regulons [70]. MarR homologs are homodimers that bind sequence-specific palindromic or pseudopalindromic DNA via a winged HTH (helix-turn-helix) motif. The crystal structures of several members of the MarR family show that the winged HTH DNA-binding core is flanked by helices involved in protein dimerization [71], [72]. Interestingly, the XopD sequence containing residues 165–175 shows homology with the DNA recognition helix H3 (α4), which confers specificity of DNA binding to MarR family members [73]. It is noteworthy that XopD165-175 sequence, as its homologous sequence in MarR proteins, is located next to a domain (KAE-rich) involved in protein dimerization that may adopt a α-helical structure, based on its homology with the TolA protein family. The DNA recognition helix α4 in MarR proteins contains conserved arginine (R) residues that are also present in XopD (Figure S3B). Mutations in this recognition helix and, in particular, in conserved R73 and R77 residues in E. coli MarR, equivalent to R170 and R174 in XopD, abolish MarR DNA binding and repressor activities in whole cells and in vitro
[73]. More precisely, DNA-binding specificity mediated by the DNA recognition helix α4 in MarR proteins is determined by the specific contact(s) between residue R73 and the operator. Since XopD216-760 displays non-specific DNA binding activity [46], it is enticing to suggest that amino acids 165–175 of XopD1-760 may confer specificity for DNA recognition. Further studies are required to confirm the putative structural and functional similarities between this region of XopD and E. coli MarR proteins. Future work will determine whether this newly identified region in XopD and/or the KAE-rich domain, related to dimer formation, determine the specificity of the DNA binding activity displayed by the XopD protein.
Together, our data stress the difficulties associated to correct annotation of T3Es and open new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.
Materials and Methods
Bacterial strains and plasmids
Xcv 85* [54] and 85* ΔhrcV, strains carrying the hrpG* mutation which confers constitutive hrp gene expression [54] were cultivated overnight at 28°C in MOKA rich medium [74] or in secretion medium (MA) [75]. Plasmids were introduced into E. coli by electroporation and into Xcv by triparental mating using pRK2073 as helper plasmid [76], [77]. Oligonucleotide primers used for PCR amplification will be provided upon request.
Unless otherwise indicated, plasmids used in this study were constructed by Gateway technology (GW; Invitrogen) following the instructions of the manufacturer. PCR products flanked by the attB sites were recombined into the pDONR207 vector (Invitrogen) via a BP reaction to create the corresponding entry clones with attL sites. Inserts cloned into the entry clones were subsequently recombined into the destination vectors via an LR reaction to create the expression constructs.
XopD1-760 and XopD216-760 were amplified from Xcv 85-10 genomic DNA. XopD216-760 carrying a mutation in the conserved catalytic Cys residue was amplified from a pGEX-XopD216-760-C470A vector, kindly provided by Mary Beth Mudgett (Standford University, USA) [43]. 6xHis-, HA-, and YFPv-tagged constructs were generated by recombination of the corresponding entry vectors with pTH19 [78], pBin19-35S-GW-3HA, or pBin19-35S-GW-YFPv destination vectors, respectively (YFPv for YFPvenus, an enhanced form of the YFP; [79]–[80]). For complementation of the Xcv 85* ΔxopD mutant strain, XopD1-760 and XopD216-760 were recombined into a pLAFR6-GW-3HA vector (kind gift of Laurent Deslandes, LIPM, Castanet-Tolosan, France) that allows expression of HA-tagged XopD proteins under the control of a constitutive lac promoter.
For GUS reporter assays in N. benthamiana, a 1-kb fragment of the PR1 promoter (PR1p) was amplified from Arabidopsis Col-0 genomic DNA. The PCR product was cloned into the entry vector pDONR207 and subsequently recombined into the pKGWFS7 destination vector [81], resulting in a plant expression vector that contains a transcriptional fusion between the PR1p and the GUS reporter gene.
Epitope tagging of XopD and secretion assays
A 307 bp fragment containing the C-terminal end of XopD fused to an HA epitope was amplified by PCR. The amplified fragment was digested with BamHI and XbaI and cloned into the suicide plasmid pVO155 [82]. This construct was introduced into the Xcv 85* and Xcv 85* ΔhcrV strains. Secretion experiments were performed as described previously [75] and XopD was detected by Western blot analysis.
Expression and gel separation of HA-tagged XopD
500 ml of bacteria Xcv strain 85* expressing HA-tagged XopD were cultivated overnight at 28°C in MOKA rich medium. The bacterial pellet was washed, resuspended in protein extraction buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol (v/v), 1 mM DTT, 1 mM PMSF] and lysed using a French Press. Total protein extracts were ultracentrifuged at 100,000 g for 30 min at 4°C and the supernatant was subjected to immunoprecipitation using anti-HA affinity matrix (clone 3F10; Roche). Immunoprecipitated proteins were separated on a NuPage 4–12% Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. The proteins were stained using a commercial solution (PageBlue, Fermentas) and the band corresponding to XopD was excised from the gel for subsequent analysis by mass spectrometry.
Determination of XopD starting amino acid by mass spectrometry
The gel slice containing the XopD protein was digested by incubating with trypsin (Promega, Madison, WI, USA) or V8 protease (Roche) and the resulting peptides were extracted following established protocols [83]. The trypsin digest was then reconstituted in 18 µl 5% acetonitrile, 0.05% trifluoroacetic acid. 5 µL were analysed by nanoLC-MS/MS using an Ultimate 3000 system (Dionex, Amsterdam, The Netherlands) coupled to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The peptide mixture was loaded on a C18 precolumn (300 µm ID x 15 cm PepMap C18, Dionex) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5 to 50% gradient of solvent B during 80 min at 300 nl/min flow rate. Data were acquired with Xcalibur (LTQ Orbitrap Software version 2.2, Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode and was externally calibrated. Survey MS scans were acquired in the Orbitrap on the 300–2000 m/z range with the resolution set to a value of 60,000 at m/z 400. Up to 5 most intense multiply charged ions (2+, 3+ or 4+) per scan were CID fragmented in the linear ion trap. A dynamic exclusion window was applied within 60 sec. All tandem mass spectra were collected using normalized collision energy of 35%, an isolation window of 4 m/z, and 1 µscan. Other instrumental parameters included maximum injection times and automatic gain control targets of 250 ms and 500,000 ions for the FTMS, and 100 ms and10, 000 ions for LTQ MS/MS, respectively.
Data were analyzed using Xcalibur software (version 2.0.6, Thermo Fisher Scientific) and MS/MS centroid peak lists were generated using the extract_msn.exe executable (Thermo Fisher Scientific) integrated into the Mascot Daemon software (Mascot version 2.2.03, Matrix Sciences). Dynamic exclusion was employed within 60 seconds to prevent repetitive selection of the same peptide. The following parameters were set to create peak lists: parent ions in the mass range 400–4,500, no grouping of MS/MS scans, and threshold at 1,000. The data were searched against the protein database of Xanthomonas campestris pv vesicatoria 85-10 (NCBI) containing 4411 sequences to which the longest possible ORF for XopD was added (Figure 1C). Mass tolerances in MS and MS/MS were set to 5 ppm and 0.8 Da, respectively, and the instrument setting was specified as “ESI Trap”. Trypsin (specificity set for cleavage after K or R) and V8 (specificity set for cleavage after D or E) were designated as proteases, and one missing cleavage was allowed. Oxidation of methionine was searched as variable modification and carbamidomethylation of cysteine was set as fixed modification. All fragmentation spectra of peptides were manually checked as shown in Figure S1 and S2.
Protein expression in N. benthamiana
Agrobacterium-mediated transient expression in N. benthamiana leaves was performed as described [84].
Fluorescence Microscopy
YFPv fluorescence in N. benthamiana leaves was analyzed with a confocal laser scanning microscope (TCS SP2-SE; Leica) using a x63 water immersion objective lens (numerical aperture 1.20; PL APO). YFP fluorescence was excited with the 514 nm line ray of the argon laser and detected in the range between 520 and 575 nm. Images were acquired in the sequential mode (20 Z plains per stack of images; 0.5 µm per Z plain) using Leica LCS software (version 2.61).
XopD expression in E. coli
Expression vectors containing 6xHis-tagged XopD1-760 and XopD216-760 were transformed in E. coli Rosetta cells. For expression of recombinant proteins, cells were grown in Luria-Bertani (LB) medium at 28°C to OD600 = 0.6 to 0.8 and then induced with 0.2 mM isopropylthio-β-galactoside (Roche) for 4 h at 28°C. Cells were lysed in PBS, pH 8.0 and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) using a French Press.
Gel Filtration Analysis
Gel filtration was performed using a fast protein liquid chromatography system (Pharmacia) with HR 10/30 Superdex S-200 high-resolution columns (Pharmacia). Prior to chromatography, protein extracts were ultracentrifuged to remove any aggregates. All gel filtration assays were performed at 4°C with pre-filtered protein extracts. Column equilibration and chromatography were performed in PBS buffer. Fractions were collected every 0.4 ml and analyzed by Western blot.
Protein Gel Blot Analysis
Proteins were separated on NuPage 4%–12% Bis-Tris gels (Invitrogen) following the manufacturer's instructions and transferred onto Protran BA85 nitrocellulose membranes (Schleicher & Schuell) by wet electroblotting (Mini-Protean II system; Bio-Rad). For detection of HA-, YFPv- and His-tagged proteins, blots were respectively incubated with rat monoclonal anti-HA [clone EF10 (Roche); 1∶5,000] and mouse monoclonal anti-His6-peroxidase [clone-His-2 (Roche); 1∶50,000] antibodies, linked to horseradish peroxidase. Anti-GroEL rabbit polyclonal (Stressgen Biotechnologies Corporation) and mouse monoclonal anti-GFP IgG1 K [clones 7.1 and13.1 (Roche)] were used at 1∶10,000. Proteins were visualized using the Immobilon kit (Millipore) under standard conditions.
Fluorimetric GUS Assays
For GUS reporter assays, the indicated constructs were transiently expressed in N. benthamiana leaves using Agrobacterium. Leaves were sprayed with 2 mM salicylic acid (SA; SIGMA-Aldritch) 18 hours after agroinfiltration. 12 hours later, leaf discs were collected, frozen in liquid nitrogen and stored at −80°C until processing. GUS activity was measured using the substrate 4-methylumbelliferyl-β-D-glucuronide as described previously [80]. After protein extraction, 1 µg of total protein was used in replicates to measure enzymatic GUS activity of individual samples.
Construction of a Xcv 85* ΔxopD deletion mutant strain
An Xcv 85* xopD deletion mutant strain was constructed by using the sacB system [85]. Briefly, 830 bp upstream and 850 bp downstream regions of full-length xopD were amplified by PCR using Xcv 85-10 gDNA as template. PCR products were subsequently cloned in a GoldenGate-compatible pK18 plasmid (L. Noël, unpublished). GoldenGate is a cloning method based on the use of Type II restriction enzymes, BsaI in our study [86]. This plasmid was then introduced into Xcv 85* by triparental mating and deletion of xopD was verified by PCR.
For complementation of the Xcv 85* ΔxopD mutant strain, pLAFR6 vectors carrying either an HA-tagged GUS control (kind gift of Laurent Deslandes, LIPM, Castanet-Tolosan, France), XopD216-760 or XopD1-760 were introduced into Xcv 85* ΔxopD by triparental mating.
Inoculation of susceptible tomato cultivars
Whole leaves of Solanum lycopersicum cv Moneymaker or cv Pearson were inoculated with a 1×105 cfu/mL suspension of bacteria in 10 mM MgCl2 using a needleless syringe. Leaves of the same age on the same branch were used for each experimental test. Plants were kept under 16 h light/day at 28°C. Symptoms were analyzed 10 days after plant inoculation.
Quantification of cell death using electrolyte leakage
For electrolyte leakage measurements, 8 N. benthamiana leaf discs (6 mm diameter) were harvested 24 hours after agroinfiltration, washed and incubated at room temperature in 10 ml of distilled water before measuring conductivity.
Supporting Information
Figure S1
MS/MS spectra of peptides issued from tryptic hydrolysis of the XopD protein. Peptides were analyzed by LC-MS/MS using a capillary LC system coupled directly to an LTQ-Orbitrap mass spectrometer. Each MS/MS spectrum is a collection of ions produced by collision-induced dissociation of the intact peptide in the linear ion trap. The predominant b and y product ion peaks are labeled accordingly with the subscripts denoting their position in the identified peptide and 2+ or 3+ indicating doubly or triply protonated ions respectively. y and b ions that were detected on the graph are shown in bold.
(DOC)
Click here for additional data file.
Figure S2
MS/MS spectra of peptides issued from V8 hydrolysis of the XopD protein. Peptides were analyzed by LC-MS/MS using a capillary LC system coupled directly to an LTQ-Orbitrap mass spectrometer. Each MS/MS spectrum is a collection of ions produced by collision-induced dissociation of the intact peptide in the linear ion trap. The predominant b and y product ion peaks are labeled accordingly with the subscripts denoting their position in the identified peptide and 2+ or 3+ indicating doubly or triply protonated ions respectively. y and b ions that were detected on the graph are shown in bold.
(DOC)
Click here for additional data file.
Figure S3
Bioinformatics analysis of the N-terminal region of XopD. (A) Alignment of amino acid residues 177-202 of XopD with its homologous regions of the following proteins from the TolA family: TolA from Aeromonas salmonicida (A4SJ34), Erwinia chrysantemi (Q937K4), Escherichia coli (P19934), Haemophilus influenzae (P44678), Pseudomonas aeruginosa (P50600), Salmonella typhi (Q8Z8C1), Shewanella oneidensis (Q8EDJ7), Shigella dysenteriae (Q32II2), Tolumonas auensis (C4LBM3), Vibrio harveyi (A6AM98). (B) Alignment of amino acid residues 168-177 of XopD with its homologous regions of the following proteins from the MarR family: MarR from Escherichia coli (P27245), Enterobacter cloacae (Q9F4W7), Salmonella typhimurium (P0A2T4), Shigella flexneri (Q0T4J9), Citrobacter youngae (D4B9P6), Tolumonas auensis (C4LFI7), Bordetella petrii (A9ITE7), Burkholderia pseudomallei (Q63Z16), Klebsiella pneumoniae (C8T9Z4), Pseudomonas aeruginosa (B7V3Q1), Pseudomonas fluorescens (Q4K9R4), Pseudomonas putida (BIJ789), Pseudomonas syringae (Q48FY2). Positions of the conserved Arg residues responsible for DNA binding in MarR family members are indicated by an asterisk. Percentage of sequence identity is represented by colour intensity of blue boxes.
(DOC)
Click here for additional data file.
We are grateful to Mary Beth Mudgett for the kind gift of the Agrobacterium strain expressing HA-LeSUMO as well as the pGEX-XopD-C470A vector, and to Laurent Deslandes for the pLAFR6 vectors. We also thank Stéphane Genin and Laurent Deslandes for helpful discussions and critical reading of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: J.C. was funded by a grant from the French Ministry of National Education and Research. Mass Spectrometry work was supported by grants from the Fondation pour la Recherche Médicale (FRM; ‘Grands Equipements’), the Toulouse Midi-Pyrénées Génopole, the Midi-Pyrénées Regional Council (grant CR07003760) and the GIS-IBISA (Infrastructure en Biologie Santé et Agronomie). This work was supported by a French ANR-Jeunes Chercheurs grant (ANR JC08_324792) to S.R. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2120674510-PONE-RA-21122R210.1371/journal.pone.0014460Research ArticleGastroenterology and Hepatology/HepatologyOncology/Gastrointestinal CancersSurgery/Surgical OncologySurgery/TransplantationIdentification of Histone Deacetylase 3 as a Biomarker for Tumor Recurrence Following Liver Transplantation in HBV-Associated Hepatocellular Carcinoma HDAC3 in HCCWu Li-Ming Yang Zhe Zhou Lin Zhang Feng Xie Hai-Yang Feng Xiao-Wen Wu Jian Zheng Shu-Sen
*
Key Lab of Combined Multi-Organ Transplantation, Key Lab of Organ Transplantation, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, Ministry of Public Health, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Hoheisel Jörg EditorDeutsches Krebsforschungszentrum, Germany* E-mail: [email protected] and designed the experiments: LMW ZY SSZ. Performed the experiments: LMW ZY FZ XWF. Analyzed the data: LMW ZY LZ. Contributed reagents/materials/analysis tools: LMW ZY LZ FZ HYX JW. Wrote the paper: LMW ZY LZ JW SSZ.
2010 29 12 2010 5 12 e1446015 7 2010 23 11 2010 Wu et al.2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Recent studies have shown that high expression levels of class I histone deacetylases (HDACs) correlate with malignant phenotype and poor prognosis in some human tumors. However, the expression patterns and prognostic role of class I HDAC isoforms in hepatocellular carcinoma (HCC) remain unclear.
Methodology/Principal Findings
The expression patterns and clinical significance of class I HDAC isoforms were assessed by immunohistochemistry in a cohort of 43 hepatitis B virus-associated HCC patients treated with liver transplantation. In addition, the effects of HDAC inhibition on HCC cell behavior were investigated by knockdown of the HDAC isoform with short interfering RNA. Class I HDACs were highly expressed in a subset of HCCs with positivity for HDAC1 in 51.2%, HDAC2 in 48.8%, and HDAC3 in 32.6% of cases. The expression levels of HDAC isoforms were significantly associated with the proliferation index of HCC. Kaplan-Meier curves showed that a high expression level of HDAC2 or HDAC3 implicated significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed HDAC3 overexpression was an unfavorable independent prognostic factor (P = 0.002; HR 3.907). In vitro, inhibition of HDAC2 and HDAC3, but not HDAC1, suppressed proliferation and the invasiveness of liver cancer cells.
Conclusions/Significance
Our findings demonstrate that HDAC3 plays a significant role in regulating tumor cell proliferation and invasion, and it could be served as a candidate biomarker for predicting the recurrence of hepatitis B virus-associated HCC following liver transplantation and a potential therapeutic target.
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Introduction
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in liver. It ranks fifth in incidence and fourth in mortality rate in overall tumors [1], [2]. China is one of the highest prevalent areas of HCC, mainly because chronic hepatitis B carriers account for more than 10% of the Chinese population [3]. The prognosis of patients with HCC is generally poor, even after surgery or chemotherapy. Liver transplantation (LT) offers a potential curative option for patients with small HCC. However, frequent recurrence or metastasis after transplantation remains the main obstacle for long-term survival [4]. Therefore, elucidating the molecular mechanism of HCC recurrence is vital for the development of more effective therapeutic strategies.
Evidence has shown that modifications of acetylation play an important role in tumor progression and metastasis [5], [6]. Histone deacetylases (HDACs) are known to be one of the major enzymes that change the nucleosomal conformation of tumor cells via post-translational deacetylation of the core histones. Therefore, aberrant activation of HDACs leads to transcriptional repression of diverse genes mainly involved in the regulation of behavior of tumor cells such as proliferation, differentiation, angiogenesis and invasion, as well as migration and metastasis [7], [8].
So far, eighteen HDAC isoforms, grouped into four classes, have been described in humans [9]. Among them, the best characterized and probably biologically most relevant HDACs were class I isoforms HDAC1, HDAC2 and HDAC3. The expression patterns of HDAC1, HDAC2 and HDAC3 have been evaluated in different types of cancers, including gastric cancer [10], colorectal cancer [11], prostate cancer [12], breast cancer [13], renal cell cancer [14], and ovarian and endometrial carcinomas [15]. Elevated expression of class I HDAC has been shown to be an unfavorable independent prognostic factor in some of these tumor entities [16]. However, little is known on the expression pattern and biologic function of a single HDAC isoform in HCC, especially in HCC treated with LT.
Recently, a new group of chemotherapeutics called histone deacetylase inhibitors (HDIs) have emerged. HDIs, such as valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) target the HDAC enzyme family. VPA and SAHA, inhibiting class I and class II HDACs, causes growth arrest, differentiation and/or apoptosis of tumor cells, and considered to be potential substances for the treatment of malignant solid human tumors in the near future [17], [18]. However, the contribution of specific HDAC isoforms to the tumor progression, invasion and metastasis is still unclear.
In the present study, we investigated the expression patterns of HDAC1, HDAC2 and HDAC3 in HCC patients following LT and analyzed their relationship to the clinical phenotype, using a clinically well-characterized cohort of HCC patients treated with LT. Meanwhile, the functions of specific class I HDAC isoforms in liver cancer cells were also characterized.
Methods
Ethics statement
The study protocol was approved by the Institutional Review Board of Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health. Informed written consent was obtained according to the Declaration of Helsinki.
Study population and tissue samples
Forty-three HCC patients treated with LT during 2003 and 2005 in our hospital (First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China) were enrolled in this study. The eligibility criteria for the patients studied are as follows: (a) HCC diagnosed either before or after transplantation (as an incidental finding), which was confirmed by histopathological examination; (b) the patients were Han Chinese; (c) complete clinical and laboratory data such as portal vein tumor thrombi (PVTT), preoperative alpha-fetoprotein(AFP) level, histopathologic grading, tumor size, and tumor number were available before operation and during follow-up; (d) all patients were HBV-positive (HBsAg+) and none of them were hepatitis C virus (HCV)-positive; (e) none of the patients received preoperative adjuvant antineoplastic therapy; and (f) absence of de novo HCC nodules occurring in the transplanted liver. Follow-up data were obtained after LT for all 43 patients. The follow-up course and diagnostic criteria of recurrence has been described previously [19]. The distribution of the clinicopathologic data in the study cohort is given in Supplementary Table S1.
Cell lines
Liver cancer cell lines SMMC-7721 and HepG2, as well as the metastasis-capable human HCC cell lines MHCC97L (intermediate metastatic capability) and HCCLM3 (the most metastatic capability) were purchased from American Type Culture Collection (Manassas, VA) and Shanghai Institute of Cell Biology (Shanghai, China).All of the cell lines were maintained in the recommended culture conditions and incubated at 37°C in a humidified environment containing 5% CO2.
Immunohistochemistry
Tissue sections of 4-µm thickness were stained with monoclonal mouse HDAC1 antibody (1∶80; Abcam, Cambridge, UK), monoclonal mouse HDAC2 antibody (1∶5000; Abcam), monoclonal rabbit HDAC3 antibody (1∶80; Abcam) and monoclonal mouse Ki-67 antibody (1∶100; Zhongshan, Beijing, China). The immunohistochemistry procedure has been described previously [20]. Nuclear staining of HDAC1, HDAC2 and HDAC3 was scored using a semi-quantitative immunoreactivity scoring (IRS) system [11]. The immunostained sections were independently evaluated by two pathologists who were blind to the clinical data. Cases with an IRS from 0 to 6 were defined as HDAC negative, while cases with an IRS higher than 6 were defined as HDAC positive. The Ki-67 index was determined by counting Ki-67-positive nuclei per 100 tumor cells in a representative tumor section.
HDIs treatment and RNA interference
Cells were seeded into plates with a density of 5×104/mL, cultured for 24 hours, then treated with SAHA (Alexis Biochemicals, San Diego, CA) and VPA (Sigma, St.Louis, MO) at a concentration of 2.5 µM and 2 mM, respectively. After evaluating the effects of chemical HDI, we analyzed the specific function of Class I HDAC isoforms in liver cancer cells. Select validated short interfering RNA (siRNA) duplexes (Ambion, Austin, TX) were used to detect RNA interference-mediated down-regulation of HDAC1, 2, 3, and a nonsilencing siRNA was used as negative control. After 24 hours, cells were transfected with 33 nM siRNA using Lipofectamine2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The efficacy of transfection was checked by Reverse Transcription Polymerase Chain Reaction (RT-PCR) after 48 hours of incubation.
Cell viability assay and cell cycle analysis
Following treatment with HDIs or siRNA for 72 hours, cell viability was detected using a cell counting kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Cells were then incubated at 37°C for another 2 hours and measured at 450 nm and 630 nm. The experiment was repeated three times.
To detect the cell cycle alterations, cells were treated with HDAC1, 2, 3 siRNA, VPA and SAHA as described above. Cells were then harvested and stained with DNA PREP kit (Beckman Coulter, Fullerton, CA). The percentage of cells in sub G1, G0/G1, S, and G2/M phase was quantified using flow cytometry analysis according to the manufacturer's instructions (CYTOMICS FC 500, Beckman Coulter). The extent of apoptosis was assessed by the proportion of cells giving fluorescence in the hypodiploid sub G1 peak of the cell cycle. Analysis of cell cycle data was done with MULTICYCLE analysis software. All experiments were done in triplicates.
Cell invasion assay
Cell invasion analysis was performed using a Transwell (Millipore, Billerica, MA) based method. Seventy-two hours after RNA interference, 200 µL of 0.8×105 cells were applied to the upper compartment of the matrigel (BD Bioscience, San Jose, CA) coated filters, while the lower compartment was filled with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. After 48 hours, migrated cells on the bottom surface were fixed with methanol and stained with 0.1% Crystal Violet. The invading cells were examined, counted, and photographed using digital microscopy (×200). Four fields were counted per filter in each group.
Semiquantitative reverse-transcription polymerase chain reaction
Total RNA was extracted from all the cell lines using TRIZOL (Invitrogen, Carlsbad, CA). Two micrograms of total RNA was used for cDNA synthesis in each reaction. PCR were performed for HDAC1, HDAC2, HDAC3, and β-actin. The primers are listed on Supplementary Table S2. Relative gel band intensities of the products were measured by Kodak digital science image system (Kodak, Rochester, NY).
Statistical analysis
The association between Class I HDAC expression and clinicopathological variables was assessed by using either the Pearson's chi-square test or two sided Fisher's exact test, as appropriate. The correlation of Class I HDAC expression and tumor recurrence was evaluated according to Kaplan-Meier estimates. Cox regression was applied to assess variables significantly associated to recurrence in the univariate analysis. The detailed statistical tests used in the study are given in Results, and in the Tables and Figure legends. Statistical analysis was performed with SPSS 15.0 and GraphPad Prism 5.0. All tests were two tailed and P<0.05 was considered statistically significant.
Results
Expression patterns of Class I HDAC isoforms in HCC
High expression levels of all three HDAC isoforms (HDAC1, HDAC2 and HDAC3) in HCC nuclear were observed. In 43 cases, 22 (51.2%), 21 (48.8%) and 14 (32.6%) cases were positive for HDAC1, HDAC2 and HDAC3, respectively (Supplementary Table S1; Figure 1A). In some cases, additional cytoplasmic positivity was observed in a minority of tumor cells. Furthermore, all of the three HDAC isoforms were detectable in the nuclei of the bile duct. Stromal cells of HCC also displayed weak to moderate nuclear positivity for all of the three HDAC isoforms, which might be due to positive staining in fibroblasts and infiltrating inflammatory cells.
10.1371/journal.pone.0014460.g001Figure 1 The role of class I HDAC expression in HCC patients receiving liver transplantation.
(A) Representative HDAC1-positive (a,b), HDAC2-positive(c,d) and HDAC3-positive(e,f) samples are shown at 100× (a,c,e) and 400× magnification (b,d,f). HDAC negative (IRS from 0 to 6) and HDAC positive (IRS higher than 6). (B) Kaplan-Meier curves estimates the recurrence-free survival rates according to expression patterns of HDAC1, 2, 3, and combined HDAC expression in the cohort.
Correlation of Class I HDAC isoforms expression with clinicopathologic variables and cell proliferation
The association of the expression levels of HDAC isoform with clinicopathologic variables were further analyzed. Resultantly, the expression level of HDAC1 and HDAC2 was only associated with Ki-67 index (HDAC1, P = 0.021; HDAC2, P = 0.044), while HDAC3 was associated with Ki-67 index (P = 0.001) and tumor size (P = 0.005) (Supplementary Table S1). In addition, when the patients were grouped according to their overall class I HDAC expression pattern (all three isoforms negative versus one or two isoforms positive versus all three isoforms positive), positive expression of all three isoforms was associated with enhanced Ki-67 index as a surrogate marker of proliferation (P<0.001, one way ANOVA) and tumor size >5 cm (P = 0.033, χ2 test for trends; Supplementary Table S1).
Correlation of Class I HDAC isoforms expression and recurrence
To determine whether changes in class I HDAC expression were relevant to the recurrence of HCC patients treated with LT, univariate and multivariate survival analysis were performed. Patients with a high expression level of HDAC2 or HDAC3 were prone to earlier recurrence of HCC according to Kaplan-Meier estimates (Table 1, Figure 1B). Furthermore, we observed that the difference was pronounced when the HDAC all positive expression group was compared with HDAC all negative expression group (P = 0.015; Figure 1B). Univariate analysis also revealed that the clinicopathological variables could provide significant predictive values for recurrence including PVTT (P = 0.024), preoperative AFP level (P = 0.004), tumor size (P = 0.004), and tumor number (P = 0.017) (Table 1). These results were consistent with those previous reports in terms of clinical histological features [19], [21], which suggests that the selected samples in this study reflect the characteristics of LT patients in the Chinese population. Multivariate analysis further revealed that HDAC3 expression level was a novel independent factor (P = 0.002; hazard ratio 3.907) for predicting recurrence-free survival (Table 2).
10.1371/journal.pone.0014460.t001Table 1 Influence of HDAC isoform expression and clinicopathologic variables on HCC recurrence.
Variables Grading Recur P*
Negative Positive
Age(Years) ≤50 9 13 0.278
>50 12 9
Gender Female 1 1 0.788
Male 20 21
PVTT Negative 16 10 0.024
Positive 5 12
Preoperative AFP level (ng/ml) ≤400 15 8 0.004
>400 6 14
Histopathologic grading Well+moderate 17 16 0.884
Poor 4 6
Tumor size (cm) ≤5 12 4 0.004
>5 9 18
Tumor number Single 12 5 0.017
Multiple 9 17
HDAC1 Negative 13 8 0.228
Positive 8 14
HDAC2 Negative 16 6 0.010
Positive 5 16
HDAC3 Negative 19 10 0.001
Positive 2 12
HDAC groups All negative 10 5 0.015
Partially positive 10 6
All positive 1 11
*Log-rank test.
10.1371/journal.pone.0014460.t002Table 2 Multivariate Cox regression analysis of variables related to tumor recurrence at univariate analysis.
Variable HR(95% CI) P
AFP
<400 ng/ml 1.000
>400 ng/ml 3.520(1.441–8.598) 0.006
HDAC3
Negative 1.000
Positive 3.907(1.623–9.403) 0.002
Abbreviations: HR, risk ratio; 95% CI, 95% confidence interval.
Inhibition of cell proliferation and cell cycle alterations by treatment with VPA and SAHA
In vitro, we tested the effects of chemical HDI on liver cancer cells (SMMC-7721, HepG2, MHCC97L and HCCLM3). Treatment of liver cancer cells with SAHA (maximum dose 5 µM) and VPA (maximum dose 4 mM) both revealed a significant dose-dependent reduction in cell number after 72 hours (Figure 2A).
10.1371/journal.pone.0014460.g002Figure 2 Antiproliferative effects of chemical HDAC inhibitors and selective HDAC1, 2, 3 silencing in HCC cells.
(A) Treatment with either VPA or SAHA led to a significant dose-dependent reduction of HCC cell numbers after 72 h. (B) Effective silencing of HDAC1, 2, 3 mRNA in HepG2 after siRNA treatment for 48 hours. (C) Selective knockdown of HDAC3 and HDAC2 led to reduction of HepG2 cell numbers after 72 hours (**P<0.01, Student t test).
In addition, treatment of liver cancer cell lines with VPA (2 mM, 48 or 72 hours) resulted in an accumulation of cells in G0-G1 phase of the cell cycle. In contrast, treatment with SAHA (2.5 µM, 48 or 72 hours) led to an accumulation of cells in the G2-M phase (Figure 3A; Supplementary Table S3). Meanwhile, treatment with SAHA, and to a lesser extent of VPA, led to a significant induction of apoptosis of liver cancer cells in a time-dependent manner.
10.1371/journal.pone.0014460.g003Figure 3 Apoptosis-inducing and cellcycle alteration effects of HDIs and selective HDAC siRNA silencing in HCC.
(A) Flow cytometric analysis of apoptosis and cell cycle in 4 HCC cell lines after the treatment with the DMSO vehicle or the indicated concentrations of VPA and SAHA for 24, 48, or 72 hours. (B) Apoptosis and cellcycle alterations in HepG2 after selective silencing of HDAC1, 2, 3 for 48 hours.
Inhibition of cell proliferation and cell cycle alterations by specific silencing Class I HDAC isoforms
To further understand the function of Class I HDAC isoforms in liver cancer cells, specific class I HDAC isoforms were knockdowned by siRNA in HepG2 cell. The data showed that treatment with selective siRNA led to a specific reduction of mRNA expression of the HDAC isoforms (Figure 2B). In addition, selective knockdown of HDAC1, HDAC2 and HDAC3 resulted in a reduction of 5.3%, 19.7% and 29.7% in cell number, respectively. However, only the difference for HDAC2 and HDAC3 was statistically significant (P<0.05; Figure 2C).
Similarly, knockdown of HDAC2 and HDAC3 in HepG2 resulted in an accumulation of cells in G2-M and a reduction of cells in the S phase (Figure 3B; Supplementary Table S3), while specific knockdown of HDAC1 showed no obvious effect on the cell cycle after 48 hours. No significant induction of apoptosis in HepG2 was observed after treatment with isoform-specific siRNA in the present study (Figure 3B).
Inhibition of invasion of HCC cells by specific silencing Class I HDAC isoforms
As shown in Figure 4C, the inhibitory efficiency of siRNAs for gene transcription was significant in high-metastasic potential HCCLM3 cells. To determine whether knockdown of specific HDAC isoform had a crucial role in cell invasion, we performed an in vitro cell invasion assay. The result showed that the average number of invaded cells transfected with HDAC2 or HDAC3 siRNA significantly decreased when compared to those with negative control siRNA (Figure 4A, B). This indicates that the invasive potential of HCCLM3 cells was suppressed after transfection of HDAC2 and HDAC3 siRNA. In line with the hypothesis that HDAC2 and HDAC3 may be important contributors to the invasion of tumor cells, the expression levels of HDAC2 and HDAC3 influenced the metastatic behavior of the HCCLM3 cell line.
10.1371/journal.pone.0014460.g004Figure 4 Alteration of class I HDAC isoform levels in HCCLM3 cells changes its invasiveness in vitro.
(A) Selective knockdown of HDAC3 and HDAC2 led to reduced invasiveness of HCCLM3 (**P<0.01, Student t test). (B) Representative images of invasiveness of HCCLM3 cells transfected with negative siRNA (a) or siRNA against HDAC1(b), HDAC2(c), and HDAC3 (d). The transwell invasion assay showed that HCCLM3 cells transfected with siRNA against HDAC2,3 displayed a markedly decreased invasiveness behavior, as indicated by a significant decrease in the average number of cells invaded through the matrigel in comparison with the control siRNA. (C) Effective silencing of HDAC1, 2, 3 mRNA in HCCLM3 after siRNA treatment for 48 hours.
Discussion
In the present study, class I HDAC isoforms (HDAC1, HDAC2, and HDAC3) were highly expressed in a panel of HCC cases. High expression levels of HDAC2 and HDAC3 were associated with significantly reduced recurrence-free survival, with HDAC3 being an independent prognostic factor in this cohort. In addition, respective silencing of HDAC2 and HDAC3 suppressed proliferation and the invasiveness of HCC cell lines in vitro. To our knowledge, this is the first detailed systematic investigation of the expression pattern and the role of these three proteins in HCC.
Increasing numbers of studies suggest that expression of class I HDACs is associated with clinicopathological parameters or tumor prognosis in several types of cancer [10]–[12]. In a study of prostate cancer, Weichert et al [12] demonstrated that high expression levels of class I HDACs correlated with higher proliferative fractions (measured by Ki-67). Similar results were also observed in the present study. Expression of HDAC1 (P = 0.021), HDAC2(P = 0.044) and HDAC3 (P = 0.001) were all associated with the Ki-67 index of liver cancer cells (Supplementary Table S1). These data suggest that high HDAC activity leads to enhanced tumor cell activity. Based on these findings, we speculated that the expression level of class I HDACs might be associated with tumor recurrence in LT patients for HCC. We found that expression of HDAC3 was an independent factor influencing the risk of recurrence in HCC patients following LT, which is in line with the in vitro results of proliferation, cell cycle, and invasion (Figure 2, Figure 4). These data suggest HDAC3 expression may serve as a novel candidate prognosticator for HCC treated with LT, although the finding ought to be verified in a larger prospective study.
On the other hand, the functional studies of liver cell lines with siRNAs targeting class I HDACs showed no obvious difference between the knockdown of HDAC2 or HADC3 expression in the amount of viable cells, as well as in the amount of invasive cells. Some recent studies also have demonstrated that high HDAC2 expression is associated with shortened relapse-free survival time or overall survival time in different types of cancer [10], [12]. However, in the present study, only HDAC3, not HDAC2 showed an independent specific role in predicting recurrence of HCC following liver transplantation. This discrepancy might be due to the small sample size, which is similar to most of the cohorts previously investigated.
In addition, elevated HDAC1 expression showed no influence on the risk of recurrent HCC after LT in the present study, which is contrast to the previous study for 47 Japanese patients with surgically resected HCC [22]. Possible explanations for the discrepancy in these results include difference in the studied cohorts, including variation in the genetic and etiology backgrounds of the patients. Despite the important role of HDAC3 in tumor recurrence and its predictive implications, this study should be viewed as hypothesis generating, to be followed by larger prospective and multiethnic studies to confirm our findings.
Previous studies in vitro have suggested that chemical HDI (VPA and SAHA) could reduce cell proliferation, induce cell cycle arrest and apoptosis of several liver cancer cell lines [23]–[25]. Obviously, our results confirmed these effects in a range of liver cancer cells. However, current data on the cellular effects of a specific HDAC isoform knockdown in liver cancer is unclear. To clarify whether the effects of VPA and SAHA inhibition could be contributed to an inhibitory effect on one specific class I HDAC isoform, we investigated the effect of HDAC1, HDAC2, or HDAC3 on the proliferation, cell cycle, apoptosis and invasion of HCC cells by means of RNA silencing. Consistent with the above mentioned results in vivo (Supplementary Table S1), we found that silencing of HDAC2 or HDAC3 significantly reduced cell numbers and induced cell cycle arrest in HepG2 cells, suggesting that selected class I HDAC isoforms may play an important role in regulating tumor cell proliferation. Supporting our results, a previously published study demonstrated similar effects in colon cancer cell lines [26]. In addition, similar to the results of Spurling et al [27] in SW480 cells, the invasive capacity of HCCLM3 cells was significantly reduced after HDAC2 or HDAC3 knockdown, indicating that these two isoforms are involved in the invasion and metastasis processes of HCC cells. In the preset study, we chose HCCLM3 as the representative HCC cell line to analyze the invasiveness influence of HDACs, therefore, the observed results and mechanisms might be limited to this cell line. We can get more detailed data by evaluating additional metastatic potential cell lines, such as MHCC97L and MHCC97H. Taken together, these findings suggest that HDAC2 and HDAC3, especially HDAC3, may be important regulators for the proliferation and invasion of HCC cells, which at least partly explained the mechanism of tumor recurrence in vivo, and could be possible targets for suppressing tumor viability.
Our findings confirm the cellular biological basis that Class I HDACs exert a tumor-promoter effect in HCC through the induction of cell proliferation, invasion and metastasis. HCC cell proliferation and invasion involves in multiple steps and required alterations of a variety of tumor related genes, such as CDKN1A, E-cadherin, T-cadherin, DACT3, and MMP-2 [26], [28]–[30]. To comprehensively understand the exact molecular mechanism of the class I HDACs regulation, gene expression microarray analysis involving tumor proliferation and metastasis pathways, should be conducted.
The HBV contribution for the current findings should be highlighted in the present study. Recently, the induction of HDAC1 by HBV X protein has been reported [31], [32], and could act as a confounding factor. In the present study, the studied population almost unavoidably consisted of patients with hepatitis B virus-associated HCC because of the special situation in China. The analysis of HDACs expression in liver tissue of HCC patients with other etiological backgrounds might be very useful to ascertain the real predictive value of HDAC3 for HCC recurrence.
In summary, our results demonstrated the important role of class I HDACs, especially HDAC3, in liver cancer biology. Patients showing elevated HDAC3 expression had a significant negative prognostic impact in terms of recurrence-free survival, which indicates the potential use of this molecular marker to predict patient risk of recurrence after LT. More importantly, the prognostic impact of HDACs, together with our observation of the proposed interactions of HDAC isoforms with tumor cell proliferation and invasion, strongly suggests targeting class I HDACs might be an effective therapeutic strategy for HCC patients after LT.
Supporting Information
Table S1 Overall expression of class I HDAC isoforms in HCC treated with LT as well as the distribution of clinicopathologic data in this study cohort.
(0.10 MB DOC)
Click here for additional data file.
Table S2 Primers sequences for RT-PCR analysis of related genes described in this paper.
(0.03 MB DOC)
Click here for additional data file.
Table S3 Distribution of HepG2 cells in different phases of cell cycle after treatment with VPA, SAHA and HDAC1, HDAC2 as well as HDAC3 siRNA after 48 hours. Experiments were performed in triplicates.
(0.03 MB DOC)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was supported by National Basic Research Program of China (973 program) (No. 2009CB522400, http://www.most.gov.cn/kjjh/), National Natural Science Foundation of China (No. 30972946, http://www.nsfc.gov.cn/) and Science and Technology Fund for Excellent Young Talents of Medical and Health of Zhejiang Province (2009QN007,http://www.zjwst.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21249209PONE-D-10-0088410.1371/journal.pone.0016011OverviewBiologyEcologyEcological EnvironmentsMarine EnvironmentsEcosystemsEcosystem FunctioningConservation ScienceMarine EcologyPopulation BiologyPopulation DynamicsPopulation EcologyPopulation ModelingFisheries and Marine Animal Populations: Learning from the Long
Term Fisheries and Marine Animal
PopulationsStarkey David J.
1
*
Smith Tim D.
2
Barnard Michaela
1
1
Department of History, University of Hull,
Hull, United Kingdom
2
History of Marine Animal Populations, Redding,
California, United States of America
Fenton Brock EditorUniversity of Western Ontario, Canada* E-mail: [email protected] and designed the experiments: DJS TDS MB. Analyzed the data: DJS
TDS MB. Wrote the paper: DJS TDS MB.
2011 7 1 2011 6 1 e160119 8 2010 3 12 2010 Starkey et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Coding Early Naturalists' Accounts into Long-Term Fish Community Changes in the Adriatic Sea (1800–2000)
==== Body
The articles that comprise the HMAP Collection are products of the History of Marine
Animal Populations (HMAP) project. This is an international, multidisciplinary
initiative, the overarching aim of which is to improve knowledge and understanding
of the long-term interaction of humankind with the marine environment. HMAP
endeavours to attain this goal in three principal ways. First, a concerted effort is
being made to embed the approaches and methods developed by HMAP into the
institutional fabric of the universities that are hosting the project. This connects
closely with the second strand of the scheme, which is designed to develop the
parallel disciplines of historical marine ecology and marine environmental history
through the sponsorship of graduate studentships, workshops, summer schools,
conferences and the dissemination of research outputs. The third activity is the
co-ordination of a research programme embracing the efforts of over 100 scientists
in 18 countries working in teams tasked with investigating 12 regionally-specific,
two thematic and two global taxon-specific case studies [www.hmapcoml.org/projects]. HMAP has progressed fruitfully in
all three respects since its inception in 2000. It has established centres at the
universities of New Hampshire (USA), Roskilde (Denmark), Hull (UK), Murdoch
(Australia) and Trinity College Dublin (Ireland), where faculty members – some
of whom might be cast as ‘HMAP graduates’ - are responsible for leading
the project and cultivating its distinctive approach to marine environmental issues.
Here, and at numerous other educational institutions, the curricula have been
enriched by the introduction of programs of study that focus on the marine
dimensions of historical ecology and environmental history. Such learning and
teaching work is informed by research undertaken under the aegis of HMAP, which by
2009 had generated over 200 printed and online works [www.hmapcoml.org/publications], as well as a substantial
web-based data store [1], an online atlas of fisheries in the case study areas
[2] and an
image gallery [3].
The articles in the HMAP Collection add to that output. Some were generated by
scientists funded as part of the HMAP research effort, while others had their
genesis in papers presented at ‘Oceans Past II: Multidisciplinary Perspectives
on the History and Future of Marine Animal Populations’, an international
conference convened by HMAP and hosted by the Aquatic Ecosystem Research Laboratory
at the University of British Columbia in May 2009. The Collection testifies to the
vitality of the HMAP approach to the dynamic interaction of humankind and the marine
environment. This overview explains how that approach evolved, identifies the
research issues that lie at its heart, and outlines some of the contributions to
knowledge and understanding that HMAP research has yielded.
Introduction
The need for a long-term perspective
The History of Marine Animal Populations project was conceived in the late 1990s.
It swiftly hatched into the historical component of the primarily science-based
Census of Marine Life (CoML) program, which aims to assess and explain the
diversity, distribution, and abundance of life in the oceans - past, present and
future [4]. That
a historical dimension was integral to the initial CoML research agenda
indicates that the census-makers recognised how ‘a survey of contemporary
marine life would have much more value if compared with historical
information’ [5]. This recognition implied that significant explanatory
insight could ensue from measuring the present state of ocean life against
conditions in a ‘lost past’ that might to some extent be recovered
through historical research into the efforts of humans to extract fish from the
seas over the long term. In turn, such research promised to reveal the degree to
which marine life now, and in the future, has been ‘influenced and
cultured by the courage, ingenuity and folly of human endeavour’ in the
past [5].
The integration of history into the science-driven CoML reflected developments in
four disciplines - archaeology, history, biology and ecology - during the final
third of the twentieth century. In essence, specialisation in these fields of
enquiry led to the emergence of a number of sub-disciplines that converged in
the 1990s to offer a multidisciplinary, or holistic, perspective on the
long-term interaction of human and marine life. Since the 1960s, for instance,
developments in archaeological science have given rise to paleoecology,
archaeoichthyology and paleozoology. In one example of the fruits of these
sub-disciplines, the preservation of fish scales in anaerobic bottom sediments
off the coast of California enabled scientists to reconstruct 1600 years of
pelagic abundances [6]–[8]. Paleozoologists likewise
bridged the cultural divides of history and ecology during the 1990s by
analysing fish remains from archaeological sites to understand better the
diversity, distribution and abundance of species [9], [10]. Fish bones were also used
to test the accuracy of climate models, some of which predict that air and sea
temperatures will rise by approximately 3°C during the next 70–100
years. In order to understand some of the processes by which such global warming
might affect marine fish species near Denmark, researchers investigated fish
fauna deriving from one of the warmest prehistoric periods (the warm Atlantic
period: ca. 7000–3900 BC). A total of 108,000 fish bones were identified,
including those from species such as anchovy and black sea bream, which normally
live in warmer, more southerly waters, like those of the Mediterranean. When
temperatures cooled after the warm period ended, most of these species
disappeared from the archaeological record, suggesting that local abundances
declined [11]. However, since the early 1990s, many of these warm-water
species have reappeared in waters around Denmark as temperatures have risen,
suggesting that archaeological information can identify which species may become
common if global warming occurs.
Specialist sub-disciplines have evolved within the history discipline since the
1960s. Environmental history was one of these off-shoots, sprouting in the USA
during the 1970s and growing ever since, a pattern evident, albeit a little
later, in Europe, Asia, Australia and, despite institutional problems, South
America and Africa. However, the focus of the pioneering American environmental
historians was strongly on human agency and perception, with ecological factors
rarely afforded an explanatory role. Moreover, the sub-discipline developed out
of a strongly narrative and qualitative approach to history that had little
rapport with the quantitative approach of ecologists. The focus was very much on
frontier cultures of the prairies, bushlands, savannahs and steppes, while the
oceans were largely disregarded [12]. On the other hand,
maritime historians - another emerging specialist group – generally
adopted an economic and social approach, and were so preoccupied with naval and
shipping themes that they paid little heed to environmental issues. There were a
few fisheries historians, but they often found their subject of marginal
interest to mainstream historians and were rather fuzzy about the ecological
facets of fishing, knowing they could not be neglected, but making little effort
to understand them. Published overviews of fisheries were rare and generally
adopted a national, regional or port perspective, while environmental
considerations were incidental at best [13]. It was not until 1995
that the North Atlantic Fisheries History Association was established, but even
then few of its outputs dealt with the impact of harvesting on the seas [14]–[17]. Signs
of change were nevertheless evident as the twentieth century drew to a close. In
1995, scholars from the natural and social sciences, as well as the humanities,
participated in a conference convened at the Memorial University of Newfoundland
with the aim of assessing the scale, impact and management of fishing effort in
the North Atlantic region since c.1500 [18]. Three years later, Holm
and Starkey reported the results of ‘Fishing Matters’, a workshop
held in Denmark that brought together historians, social scientists, biologists,
oceanographers and fisheries managers to examine multidisciplinary approaches to
understanding the past and current scale and character of the fisheries [19]. On the
shores of the North Pacific, Pauly, Pitcher and Presiloshot organised a meeting
in 1998 aimed at mathematically reconstructing the state of the Strait of
Georgia, off Vancouver Island. Participants were drawn from various backgrounds,
their challenge being ‘to provide a vision for rebuilding the
Strait's once abundant resources’ [19].
The interest of natural scientists in the fisheries dates back to the late
nineteenth century, when governments began to address the question of
fluctuations in catches, largely because the social and economic costs of this
unpredictability were high. Two schools of thought emerged. Some scientists
explained the often large fluctuations and long-term declines from a single
species perspective. This simplicity allowed them to provide more concrete
advice to fisheries managers. Other scientists, however, saw the problem as
involving multiple interacting species, an ecological perspective that had its
origins in a terrestrial setting, and was now applied to the sea [20]. These
two perspectives initially developed concurrently, but by the 1920s they had
separated into two distinct disciplines - fisheries biology and marine ecology
[21],
[22].
Whereas fisheries biologists increasingly focused on self-regulating population
models that could be used as a basis for quantitative advice to fisheries
managers, marine ecologists concentrated on biodiversity, food-webs and
biological processes. For the fisheries biologists, the ultimate question was
how to predict sustainable catch levels, but with any other human impacts left
out. In contrast, the ultimate question for the marine ecologists was not what
is in the sea for us, the humans, but how do we understand nature on its own,
again with the humans left out. Nevertheless, both disciplines continued to
share the assumption that equilibrium or steady-state models were sufficient
because changes were expected to be followed by more or less rapid transitions
to new equilibrium conditions. The history of fisheries and ecosystems could
safely be ignored as the dynamics and new equilibrium states were determined
primarily by current conditions. By the 1950s, however, it was becoming clear
that ecosystems rarely remain steady for long, as ‘fluctuations lie in the
very essence of the ecosystems and of every one of the … populations’
[23].
Fisheries biologists and marine ecologists, who had hitherto perceived little or
no need to consider the history of the systems they were studying, were now
challenged with the reality that equilibrium baseline conditions were difficult
to define and that in any event present conditions were often strongly affected
by earlier events.
Fisheries biologists began to recognize the need for a more historical
perspective on some fisheries. Papers summarizing long-term landings were
therefore published, especially for fisheries where management depended on the
relationships between numbers of spawners and resulting number of recruits [24] or on
estimates of maximum sustainable yield [25]. Long time series of
landings for most fisheries, however, became unnecessary when management came
increasingly to depend on virtual population analyses, as these calculations
were only sensitive to the age structure of recent landings [22]. Another
sign of this growing historical awareness was the recognition that some formerly
important fishes no longer occurred in harvestable numbers, as Goode and Collins
had demonstrated in the 1880s for Atlantic halibut in New England waters [26]. This was
especially the case for whaling, where several populations of whales had nearly
been exterminated in the nineteenth century. Because of the extreme levels of
depletion of whales that had occurred, and because of the long lifespan of large
whales, the effects of whaling even 100 years previously were important to an
understanding of the current state of whale populations. Accordingly, by the
mid-1980s, scientists were combing through nineteenth-century whaling logbooks
for evidence of the scale and distribution of past catches [27].
A number of ecologists also made the historical turn. In 1995, Daniel Pauly
observed that most equilibrium or steady-state models are based on a given
dataset, often established by scientists within the last generation [28]. But what
happens to the equilibrium models if older data are introduced? We cannot know
from recent information the extent of the losses that have already happened. In
a seminal study of the Caribbean ecosystem, J.C.B. Jackson made the scathing
remark that the child assumes that the world as s/he sees it first is the
natural condition of the world – and ecologists have often assumed that
the natural or original condition is equal to the first scientific description
of a phenomenon. His critique was set within an empirical study of the trade in
Caribbean turtles that deployed evidence from eighteenth-century British
colonial trade statistics to ascertain that hundreds of thousands of turtles
were killed annually. This persuaded Jackson that the ecosystem of the Caribbean
would have looked very different to the scenario that conservation biologists
had supposed on the basis of information relating to the last couple of decades
[29]. The
lesson to ecologists of Jackson's historical analysis of Caribbean coral
reefs was that textbook descriptions of reef ecosystems were limited by the fact
that systematic descriptions by modern biologists only began in the 1950s.
Jackson put the case squarely to the ecologists: they needed to turn to
historical sources and rediscover the world. This argument was pressed home in
2001, when Jackson and his colleagues asserted that ‘more specific
paleoecological, archaeological, and historical data should be obtained to
refine the histories of specific ecosystems and as a tool for management’
[30].
Once more the scope of previous ecological and marine biological investigations
was criticized on the grounds that ‘most ecological research is based on
local field studies lasting only a few years and conducted sometime after the
1950s without longer term historical perspective’ [30].
With the long-term perspective finding favour in the marine sciences, and
ecological approaches assuming more importance in the analyses of archaeologists
and historians, a prospectus for a History of Marine Animal Populations project
was issued in 1999 [19]. This received the backing of the Sloan Foundation
and of the promoters of the CoML initiative, and a workshop was convened to
scope the HMAP project.
HMAP: parameters and key research questions
The initial phase of HMAP was characterized by discussions about the nature and
scope of what was appropriate and possible historically and scientifically that
would contribute to the present and future foci of the Census of Marine Life.
These deliberations culminated in a workshop held in Denmark in February 2000,
at which researchers from various disciplines involved in CoML sought to define
and encourage what was quickly seen to be a necessary and needed expansion of
both environmental history and historical ecology, as well as an important
component of the Census. The meeting yielded an analytical framework, a set of
hypotheses and an edited collection of papers [31].
It was agreed that the topical, spatial and temporal parameters of HMAP should be
broad: that is, the project should investigate the impact of human activity on
marine animal populations in the world's seas and oceans over the last
2,000 years. But four provisos were identified. First, much of what can be known
about the history of marine animal populations relates to the ‘human
edges’ of the ocean, the near shore and coastal waters where humans most
directly interacted with the sea in the past – as a source of food, a
means of transportation, a theatre of war and a recreational zone. Accordingly,
most historical records concern such activities in this realm. Second, the
research should focus on the human activity that has had the greatest impact on
marine ecosystems over historic time, the commercial fisheries conducted on the
human edges and (for certain species) in mid-oceanic waters. Third, in
generating knowledge about the impact and significance of the commercial
fisheries, it is inevitable that analyses will be skewed towards the extraction
of large animals, notably whales, large fish (such as cod and bluefin tuna), and
marketable smaller species (like herring), where the sheer size and/or
commercial value of the organisms encouraged captors to create and maintain
archival material. Fourth, moving from the unknown to the known history of the
oceans requires that the approaches and methods of archaeology, history, biology
and ecology be deployed in a truly multidisciplinary way.
A preliminary set of hypotheses was also formulated at the 2000 workshop
(http://hmapcoml.org/documents/Hypotheses.pdf). From this, four
broad research questions were devised:
How have the diversity, distribution, and abundance of marine animal
populations altered over the last 2,000 years?
Which factors have forced or influenced changes in the diversity,
distribution, and abundance of marine animal populations?
What has been the anthropogenic and biological significance of changes in
marine animal populations?
By what processes have marine ecosystems interacted with human
societies?
The workshop recognised that a range of practical factors would necessarily
restrict the research to sub-projects selected according to their intellectual
rigour, viability in terms of primary sources, cost and personnel, and fit with
the project's key research questions. Henceforth, the HMAP research effort
progressed on a case study basis, with a core of initial studies augmented by
later investigations that extended knowledge and understanding of past ocean
life over time and space. The HMAP Collection indicates that this process of
knowledge accretion continues unabated.
Results and Discussion
The HMAP Collection in an HMAP research context
The HMAP project has generated a sizeable volume of research products. Yet the
fact that over 60% of the papers presented to the Oceans Past II
Conference in May 2009 were delivered by researchers who had not previously been
engaged in the project suggests that the HMAP approach is being adopted by a
growing number of scientists and historians. The composition of the conference
programme also implied that new subjects are being investigated, while the
initial HMAP studies continue to stimulate research. The HMAP Collection
confirms these impressions, for it comprises papers that in different ways
extend the scope of HMAP, and papers that build upon established HMAP themes and
approaches.
Two papers in the HMAP Collection focus on the Northern Adriatic Sea. In one,
Shimrit Perkol-Finkel and Laura Airoldi push the frontiers of HMAP research into
the sub-tidal zone in their study of habitat resilience in algal forests of the
Adriatic coastline [32]. As they point out, habitat loss is often caused by
gradual, long-term changes that impair the ability of natural ecosystems to
absorb and recover from natural and human influences. Deploying a combination of
historical data, and quantitative in situ observations of
natural recruitment patterns, the authors argue that recent contractions in
forest areas along the urbanized coasts of the north Adriatic Sea were triggered
by accelerating cumulative impacts of natural- and human-induced habitat
instability, exacerbated by an increase in the occurrence and severity of
storms. Having examined the prospects for restoring such diminished habitats,
Perkol-Finkel and Airoldi emphasize that better protection of natural habitats
is required, as the restoration of pre-degradation environmental conditions, if
possible, is often not cost-effective. In the second Adriatic paper, Tomaso
Fortibuoni, Simone Libralato, Saša Raicevich, Otello Giovanardi and
Cosimo Solidoro assess the utility of qualitative evidence generated by
naturalists in the nineteenth and twentieth centuries. Deploying an innovative
methodology that facilitates the transformation of the descriptive accounts of
early naturalists into semi-quantitative information, the authors reconstruct
and quantitatively analyze a 200-year-long time series of fish community
structure indicators in the Northern Adriatic. Their findings chime with various
other HMAP studies in identifying long-term changes in fish community structure,
notably in this case the decline of Chondrichthyes, especially sharks, large
demersals, such as hake and angler fish, and large-sized and late-maturing
species like dusky grouper and brill [33].
The development of the Makassan sea cucumber (trepang) fishery,
as traced by Kathleen Schwerdtner Máñez and Sebastian C. A. Ferse
in the HMAP Collection, not only adds to HMAP's Asian case studies but also
offers an example of the commercial pressures that have driven the majority of
the world's fisheries over the centuries [34].
Trepang extraction enabled the people of Makassar,
Indonesia, to profit from the export of a commodity that was in great demand in
China. While this rendered them vulnerable to market fluctuations, the fishing
effort was shaped by patron-client relationships and marked by the ‘roving
bandit’, or bonanza, syndrome, whereby fishers exploit stocks in a
locality until they are depleted, when they move to another area. The fragility
of marine ecosystems in the face of seemingly rapacious human fishing activity
is also the central theme of another HMAP Collection paper, Ruth H Thurstan and
Callum Roberts's analysis of the ‘ecological meltdown’ that has
occurred in the Firth of Clyde, Scotland, since the introduction of more
intensive fishing methods in the mid-nineteenth century [35]. Previously an area that
supported productive herring, cod, haddock, turbot and flounder fisheries, the
impact of trawling reduced fish stocks to such a degree that the activity was
prohibited in 1889. This remained so until 1962, from which point the resumption
of trawling, and subsequently the deployment of ring-nets and fish finders,
caused the depletion of various species, leading the authors to conclude that
‘this once diverse and highly productive environment will only be restored
if closures or other protected areas are re-introduced’.
Although these studies relate to various marine species in different parts of the
world, their findings not only address HMAP's key research issues, but also
complement and reinforce the results of the project's initial case studies.
For instance, a series of investigations into the commercial fisheries of the
North Sea has revealed much about the chronology, scale and impact of the
extraction of marine life from these waters over the last thousand years.
Archaeological appraisals of dozens of medieval settlements showed that the
period c.950–1050 saw a major rise in fish consumption around the North
Sea [36].
Osteological analysis of fish bones, through their stable isotope signatures,
indicates that early medieval sites are dominated by the remains of freshwater
and migratory species such as eel and salmon, while later settlements reveal
that the consumption of marine species such as herring, cod, hake, saithe and
ling was widespread. In particular, evidence of traded cod – known as
‘stockfish’ - identified in urban areas in Norway, England, Belgium,
Germany, Denmark, Sweden, Poland and Estonia, and dated at or before the
mid-eleventh century, is abundant. The evidence also indicates that sea-going
vessels were widely deployed by the thirteenth century to catch deep-sea fish
such as ling. It is therefore apparent that substantial commercial fisheries had
emerged by the eleventh century. In turn, these were a function of major
economic and technological developments, as well as changes in consumption
patterns that were to form the basis of dietary preferences – embracing
religious practices of fasting and abstinence of red meat in favour of fish at
certain weekdays and through the 40 days of Lent - which lasted into the
seventeenth century.
With regard to the scale of activity, HMAP's North Sea researchers have
instilled a long-term perspective into the literature. Following in the wake of
Hutchings and Myers's pioneering reconstruction of catches of Atlantic cod
off Newfoundland and Labrador from 1508 to 1992 [37], the first estimate of
total removals of a species from the North Sea was developed for the
sixteenth-century Danish inshore fisheries for herring in Scania and Bohuslen.
Annual catches regularly reached a level of 35,000 tonnes [38]. By the late sixteenth
century, the Dutch had taken the lead in Northern European herring fisheries
with sea-going buysen harvesting the rich shoals off the coasts
of Scotland and the Orkneys. They landed catches of 60,000–75,000 tonnes
every year in the first quarter of the seventeenth century, when total removals
(including English, Scottish and Norwegian landings) amounted to upwards of
100,000 tonnes. Catches declined to about half of that level by 1700, and only
increased to about 200,000 tonnes in the late eighteenth century when the
Swedish and Scottish fisheries developed apace. By 1870, total removals reached
a level of 300,000 tonnes, which equals the Total Allowable Catch for North Sea
herring in 2007, as recommended by the International Council for the Exploration
of the Seas. This evidence demonstrates how fishermen in the age before steam
and trawl were able to remove large quantities of biomass from the sea. While
early modern catches seem to have been at a sustainable level, there are
indications that removals at much lower levels than those recommended by modern
standards had an effect on abundance. This can be deduced by standardizing the
catching capacity of North Sea herring fishing vessels across the technological
divide from sail to motor-powered vessels. Even by a conservative estimate,
analysis of catch-per-unit-effort indicates that stock abundance was ten times
higher in the 1600s than in the 1950s, and already by the 1800s, well before
steam was introduced, it had dropped to 50–60 percent of the level of the
seventeenth century. Accordingly, the impact of early modern removals of herring
was much greater than historians and ecologists had previously realised [39].
A similar finding emerged from an HMAP study of the extraction of ling and cod
from the North Sea. These species are classed as ‘top predators’. In
ecosystem theory, top predators play a controlling and balancing role for the
abundance of other species further down the food chain, and large numbers of top
predators are a sure sign of healthy biodiversity. Human hunting tends to focus
on top predators as the big fish are of the highest commercial value. At the
same time, removing the largest specimens weakens the ecosystem, for mature fish
are highly important for the reproduction of the population as their eggs are
healthier and more plentiful than the spawn of younger and smaller specimens. As
the fish continues to grow through its entire life, a decline in the length of
specimens caught is a clear indication that fishing is changing the age
structure and viability of the stock. Historical analysis demonstrated that
while the average length of North Sea ling in the mid to late nineteenth century
was about 1.5 metres, it had decreased to about 1.2 metres by the First World
War, and ling caught today is less than 1 metre on average. A century ago, cod
landed from the North Sea was usually 1-1½ metres long, while today it is
only about 50 cm. This means that while cod used to live to an age of eight or
ten years, in the early twenty-first century it is caught at less than three
years of age. As cod only spawns at the age of three years, the fisheries are
clearly removing cod at a critical stage [40].
An important aspect of any long-term perspective is the measurement of change
over time. This features prominently in the contribution of Tyler D Eddy,
Jonathan P.A. Gardner and Alejandro Pérez-Matus to the HMAP Collection,
which focuses on the role of baselines in the management of the lobster fishery
of the Juan Fernández Archipelago, Chile [41]. Having constructed baselines
of lobster abundance throughout the human history of the archipelago, the
authors examine the capacity of strategies such as marine reserves, effort
reduction and the stewardship of catches to utilize this primary economic
resource in a sustainable manner. Their findings indicate that stewardship
coupled with a 30% area closure through the erection of a marine reserve
would enable the stock to recover to a level midway between historic maxima and
the contemporary minimum. In other words, historical evidence is being used to
inform management targets and tools with the objective of rebuilding the lobster
population to a user-determined size, while also providing ecosystem and
biodiversity protection. This approach chimes well with the HMAP investigation
into the Scotian Shelf cod fisheries, using evidence derived from the detailed
log books that the skippers of fishing vessels were obliged to deposit with
customs officials in order to claim a government bounty on catches. Relating to
the period 1852–1866, thousands of these logbooks have been digitised and
analysed to reveal that in the 1850s the adult cod biomass was in the order of
1.26 million tonnes. Remarkably, in the 1990s the comparable estimate was 50,000
tonnes. In terms of extractions, the fishermen consistently removed 200,000
tonnes of live fish per year through the 1850s. For example, in 8.5 months
during 1855, the handlines used by fishermen in 43 schooners from Beverly,
Massachusetts, caught just over 8,000 tonnes of cod on the Scotian Shelf,
whereas in 15 months during 1999–2000 a total of just 7,200 tonnes of cod
was extracted from the same waters by the entire Canadian mechanized fishing
fleet, a return that fell short of the Total Allowable Catch by 11 percent [42]. This
long-term comparison points to a profound change in productivity on the Scotian
Shelf over the past 150 years, and a measurable reduction in abundance that is
even starker than the declines of lobster in Juan Fernández, and cod and
ling in the North Sea.
Like their counterparts in the Indonesian trepang fishery, New
Englanders were aware that their prey was diminishing in extent and responded by
moving to fishing grounds further offshore. By the late 1850s, many schooners
were undertaking longer voyages to the Gulf of St Lawrence and the Grand Banks,
where stocks were perceived to be larger. Another response, which was also
evident contemporaneously in the Firth of Clyde, was the application of new
technology. French fishermen introduced tub trawls to the Scotian Shelf fishery,
and soon the Americans no longer used the traditional handlines with 2–4
hooks per man, but longlines of upwards of 400–500 hooks per crewman.
While the catchment area of one boat increased immensely, and catches went up in
the short run, in a matter of a few years the fish stock was showing clear
depletion signals, with smaller individuals being caught and the
catch-per-unit-effort of fishermen declining [43]. In essence, these New
England fishermen were interacting with the marine environment in ways that were
not only evident in earlier historical settings, but have also become very
familiar since the 1850s: faced with environmental changes, which their activity
had precipitated, they extended the spatial scope, and enhanced the intensity,
of their fishing effort.
Spatial range is also a key theme of the research undertaken as part of the HMAP
World Whaling case study by Tim Smith, Randall Reeves, Elizabeth Josephson and
Judith Lund. This team has analyzed data relating to American offshore whaling
voyages to describe the historical distribution of five key groups of species
that were targeted by nineteenth-century whalers: sperm, right, humpback, gray
and bowhead whales. The data are presented in the form of world maps, showing
where the whalers went and where they encountered these species of whales [44], and will be
described in detail in a paper planned for the HMAP Collection. Comparing these
distributions with what is known of the present-day distribution of these
species identifies areas where whales do not appear to inhabit their historical
ranges, suggesting that comparisons of past and current ranges should be given
greater consideration in the management of whales today. This work builds upon a
body of research outputs generated by the team. In the early stages of the
project, they attempted to identify all of the world's whale fisheries,
from aboriginal harpoon fisheries with origins in antiquity to shore-based
commercial fisheries, and finally to high seas commercial fisheries beginning
before the nineteenth century and continuing today [45]. Noting that the
twentieth-century catches of the great whales were relatively well known [46], the team
focused most of their effort on the nineteenth century, when the main offshore
whaling nations were the United States, Britain and France, with the American
fishery being far and away the largest and best documented. The team assembled
summary data on all recorded American offshore whaling voyages [47], which
totalled more than 15,000, primarily in the eighteenth and nineteenth centuries.
They also extracted data from a sample of voyage logbooks kept by American
whalers, and rescued earlier data that had been extracted from some of these
logbooks in a study in the 1850s and another in the 1930s. These data provide
detailed spatial information on where whalers went and on the numbers of whales
taken on each sampled voyage.
The team used these offshore whaling voyage data, as well as other archival
sources from shore-based whaling operations, to develop more detailed
descriptions for several areas of the magnitude of whaling, and its impact on
for four key nineteenth-century target whales: right, sperm, gray and humpback.
Right whales were the earliest target species, beginning at least by 1050 AD and
continuing in the North Atlantic in some 33 fisheries for over 1000 years. Right
whales were pursued in all the world's oceans, both by shore-based and
offshore whalers. Total removals of right whales in the western North Atlantic
were estimated to have been at least 5,000 animals [48], far more than the current
population of several hundred animals, while roughly 25,000 right whales were
taken from New Zealand waters in the 1800s [49]. In the North Pacific, the
team demonstrated that rapid spatial shifts in right whaling occurred over the
decade of the 1840s and resulted in a swift decline in the rate at which whalers
encountered right whales [50]. The idea that the right whales were at one time
distributed broadly across the North Pacific was shown to be wrong, with the
highest encounter rates occurring east of 160 deg W and west of 170 deg E [51].
The other major target of nineteenth-century whalers were sperm whales. The team
drew on published and archival sources to identify more that 60 sperm whaling
grounds [52], and also addressed an apparent inconsistency between
order of magnitude declines in nineteenth-century sperm whale encounter rates
[53],
[54] and the
current status of this species globally, estimated at 70% of pre-whaling
abundance [55]. This latter study was based on estimates of current
global abundance and estimated catches in the eighteenth, nineteenth and
twentieth centuries of over one million animals. The team suggested that this
inconsistency is in fact most apparent in the North Pacific, and hypothesized
that the region north of 40 deg N latitude may have been a refuge for sperm
whales during the nineteenth century, a refuge that was breached in the
twentieth century [56]. While it is apparent that sperm whale abundance
declined in the first half of the eighteenth century in at least the North
Pacific, it is less clear if this was a major contributor to the eventual
decline of American whaling. The team demonstrated the limitations of global
analyses of sperm whaling, and used regional analyses to show that the effect of
whaling on sperm whales may have differed substantially between the Pacific and
the Atlantic [57]. Over the first half of the nineteenth century, rates
of encounter of sperm whales did not decline in the Atlantic as they had in the
Pacific, and by the second half of the nineteenth century American whalers had
retreated from the Pacific back into the Atlantic. Determining the causes of the
decline of American whaling is complicated by the complex spatial changes in
whales and whaling.
Humpback whales have been sought in the North Atlantic since the seventeenth
century, with at least one fishery continuing to the present, albeit at very low
levels. Both shore and offshore whalers pursued this species on its two North
Atlantic breeding grounds and in all of its many feeding grounds. The team
examined all archival information that it could find and developed estimates of
total removals of around 30,000 animals [58]. The number of humpback
whales alive before whaling (pre-whaling abundance) was estimated using
population models based on estimates of removal and present-day abundance to be
between 21,000 and 24,000 whales [59], [60]. This number is an order of magnitude lower than
estimates of average long-term abundance estimated from genetic variability
[61]. The
cause of this discrepancy is under investigation.
Extinct in the North Atlantic in the 1600s, gray whales became a target of
whaling in the North Pacific, being pursued for several centuries in both their
western and eastern North Pacific populations. In the eastern North Pacific,
gray whaling was long conducted aboriginally on well-defined migration and
feeding grounds. In the 1850s, commercial whalers from several nations began to
focus on this species in its calving and breeding grounds in Baja California and
in the North Pacific feeding grounds. American shore-based whaling also
developed along the California coast. Although the total removals of eastern
North Pacific (or California) gray whales had previously been estimated, those
estimates were inconsistent with sightings-based estimates of current and recent
abundance, and as a result pre-whaling abundance has been poorly understood. The
World Whaling team revisited the estimates of nineteenth-century gray whaling in
the eastern North Pacific to determine if the source of this inconsistency was
biases in estimates of removals [62], [63]. Deploying substantially different methods than used
previously, their new estimates were remarkably similar to the earlier
estimates. Additional information is becoming available in the form of genetic
based estimates of long-term average abundance for both the eastern and western
North Pacific populations taken together [64] and improved estimates of
present day abundance [65]. Work is continuing to determine pre-whaling
abundance.
The HMAP Baltic case study has shed much light on the historical development of
fisheries in the region. Over the long term, little pressure was exerted by
commercial fishing on fish stocks in the inner parts of the Baltic. During the
late seventeenth century, for example, removals of fish biomass from the Gulf of
Riga were at least 200 times less than the level they reached in the late
twentieth century. In the earlier period, moreover, the bulk of the fishing
effort was expended in the rivers. Migratory fish species, such as sturgeon,
Atlantic salmon, brown trout, whitefish, vimba bream, smelt, eel and lamprey
were the most important commercial fish in the area, because they were abundant,
had high commercial value and were easily available. Over time, however, fishing
activity moved downstream and into the sea. Due to intensive fishing,
populations of many migratory species, especially sturgeon and Atlantic salmon,
contracted considerably and they became less commercially significant, while
marine fish, especially Baltic herring, increased in importance during the
nineteenth century [66]. In a paper to be published in this HMAP Collection,
Brian MacKenzie, Margit Eero and Henn Ojaveer focus on the relationship between
predators and prey to project whether the cod stock can recover if seals also
recover [67]. In so doing, they refer to the experience of earlier
centuries, for which the bulk of the evidence indicates that the two species
were present in some abundance. But this does not necessarily mean that the same
situation will occur in the future, for other variables, most notably fishing
pressure and climatic conditions, may well have changed. Drawing a schematic of
the upper trophic levels of the Baltic foodweb in different centuries, and
applying population modelling techniques, the authors provide research-informed
advice for resource management agencies.
This follows an established pattern, for the HMAP Baltic team has utilized
long-term perspectives to help solve other environmental issues. For instance,
in the absence of historical records before 1966, fishery managers asked if the
record high cod stock in the Baltic Sea in the late 1970s and early 1980s was a
unique occurrence, or whether it was likely to happen at regular intervals. The
question was unequivocally answered through the recovery of historical data back
to 1925, which showed that abundant cod stocks corresponded to a favourable
combination of four key drivers: incursions of saline water to the brackish
Baltic and hydrographic conditions that facilitated successful reproduction; low
marine mammal predation; a highly productive environment fuelled by nutrient
loading; and reduced fishing pressure. Such a conjunction of factors did not
take place at any other point in the twentieth century. While the cod biomass
was restricted from 1920 to 1950 by an abundance of marine mammals and low
ecosystem productivity, in the 1950s and 1960s stock levels were depressed by
high fishing pressure, and hydrographic conditions were rarely conducive to good
reproduction rates throughout the twentieth century, especially after 1985. The
late 1970s and early 1980s were therefore extraordinary in that a combination of
positive factors interacted to produce a large cod stock, a conclusion that will
perhaps inform the policies and targets of fisheries managers [68].
A further notable finding of HMAP Baltic research relates to the resilience of
fish to broad changes in temperature and other meteorological variables.
Archaeological evidence of fish fauna in the Atlantic warm period (c.7000-3900
BC) infers that there were many fish species in the waters around Denmark which
are now be found in warmer waters. However, cod was very abundant in the Stone
Age, even though temperatures were 2–4 degrees warmer those of the late
twentieth century, suggesting that significant cod populations can be maintained
in the Baltic region even if temperatures rise due to global warming, provided
that fishing mortalities are reduced. Climatic variables also influenced the
abundance and distribution of other species. For instance, during the Little Ice
Age of the late seventeenth century, cold-water marine fish (herring, flounder
and eelpout) sustained important fisheries in the Baltic, while the fishing
season for the major pelagic fish species was much later in the year, compared
to the much warmer conditions of the present day. Similarly, the magnitude and
composition of catches of herring and other coastal fish (e.g. perch and ide)
near Estonia in the mid and late nineteenth century, when fishing effort and
methods were constant, were chiefly governed by climatic fluctuations [69]. The
influence of another natural factor, the quality of the water, has also been
highlighted by historical analysis of the hydrographic event that ensued when
the North Sea breached the fragile coast of the Limfjord in 1825. In this
instance, the saltwater intrusion destroyed the habitat for freshwater whitefish
and created conditions that favoured saltwater species such as plaice – an
environmental shock that drastically altered the character of the human fishing
effort in this area [70].
A short view on the long-term perspective
A decade ago, the editors of the first collection of HMAP research papers
anticipated that the findings of the project's initial case studies would
make ‘a major contribution to knowledge and understanding of the complex,
delicate and important relationship between human societies and the marine
environment’ [31]. We can now report that the multidisciplinary
approach fostered by HMAP has generated a substantial corpus of work that offers
long-term perspectives on changes in stock abundance, the ecological impact of
large-scale human harvesting and the role of marine resource utilization in the
development of human societies. Such a view, in turn, broadens and deepens
knowledge of the contemporary condition of the marine environment and provides
the time series and ecological insight required to assess the future
sustainability of marine animal populations. We are confident that the papers in
this Collection – those currently available (as of December 2010) and
those to be added subsequently - will augment and enhance the HMAP contribution
to historical marine ecology and marine environmental history.
We acknowledge the central role of the Alfred P. Sloan Foundation in initiating and
supporting the Census of Marine Life, and in providing the core funding for the
History of Marine Animal Populations project. Jesse Ausubel's vision and
encouragement regarding the significance of the past to a Census of ocean life in
the present is much appreciated, and we gratefully acknowledge the contributions of
those who have been part of HMAP over this past decade. Our thanks are also due to
the HMAP Steering Group, which has laboured to keep us organised and on track, to
the other members of the publication group that co-ordinated the HMAP Collection,
Alison MacDiarmid, Bo Poulson and Matthew McKenzie, and to Professor Jeffrey
Hutchings of Dalhousie University and an anonymous reviewer for their helpful
comments on an earlier draft of this paper.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Alfred E Sloan Foundation Census of Marine Life Grant (www.coml.org). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
==== Refs
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J Med LifeJ Med LifeJMedLifeJournal of Medicine and Life1844-122X1844-3117Carol Davila University Press Romania 20302193JMedLife-03-19Original ArticlePrognostic factors in retroperitoneal fibrosis
Sinescu I *Surcel C *Mirvald C *Chibelean C *Gîngu C *Avram D *Hîrza M *Manu M *Lazar R *Savu C **Udrea A **** Center of Urological Surgery, Dialysis and Renal
Transplantation, Fundeni Clinical Institute, BucharestRomania** Department of Intensive Care and Anesthesiology, Fundeni Clinical Institute, BucharestRomania*** Polytechnic University, BucharestRomaniaCorrespondence to::C. Surcel, MD, Clinical Institute of Uronephrology, Dialisys and
Renal Transplantation Fundeni, 258 Fundeni Blvd., 022328, Bucharest, Romania,
[email protected] 2 2010 25 2 2010 3 1 19 25 07 10 2009 27 1 2010 ©Carol Davila University Press
2010This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work
is properly cited.The aim of this study is to evaluate effective prognostic factors in the evolution of patients
with retroperitoneal fibrosis and to establish the validity of fractal analysis in determining the disease
severity in these patients.
Material and Methods: This study included 19 patients (M/F: 5/14) treated for idiopathic
retroperitoneal fibrosis and bilateral obstructive renal failure between Jan 2004–Dec 2008. Patients
were identified retrospectively, searching for patients diagnosed with IRF, after retroperitoneal biopsy or,
in most cases the diagnosis rested on radiological findings, especially CT, with identification of
a retroperitoneal mass, the absence of other demonstrable renal or ureteric disease or any other pathology
that could explain the findings. CT was very useful in describing the retroperitoneal mass around the aorta
and inferior vena cava, the extent of the lesion and for monitoring the response to surgical treatment during
the follow–up. The data were evaluated about medical history, physical examination findings,
laboratory tests (serum urea and creatinine, blood sugar, sodium, potassium, bicarbonate levels, serum pH,
uric acid, haematocrit, white blood cell count), imaging methods (renal ultrasound, abdominal CT–scan,
MRI). At admission all patients had active disease with obstructive renal failure and underwent bilateral
ureteric stenting in order to normalize the BUN levels. After normalizing of BUN levels, ureterolysis and
omental wrapping was performed. Postoperatively, ureteric stents were removed after 1 month and remission of
renal disfunction was obtained in approximately 5 months (range 2–10 months). All patients were followed
for at least 1 year. Patients were regularly checked every 3 months.
Results: Of the 19 patients, there were 5 men and 14 women. The median age at diagnosis of RF was 50
years (range 42–64 years). The most frequent presenting symptoms were back or abdominal pain,
weakness, weight loss, oligoanuria, arterial hypertension and mild fever. The duration of symptoms before
diagnosis ranged from 6 to 18 months. At presentation all patients had active disease, presenting renal
dysfunction with a median serum creatinine of 5.18 mg/dl (range 1–15.4 mg/dl). Most of the patients
had moderate bilateral hydronephrosis (2nd degree hydronephrosis). In our study, all patients had
excellent prognosis, with full recovery of renal function in 78% of cases (15 patients). The
fractal dimension of the fibrosis mass contour correlates with level of renal function impairment. Even more,
the fractal dimension seems to slightly variate between CT evaluations (1.30±0.1), suggesting a
non aggressive pattern of extension of the fibrotic mass characteristic for benign lesions.
Conclusions: The imaging parameters did not predict the disease severity, except the increase in
fractal dimension of fibrosis surface area. Efficacy of bilateral ureteric stenting in improving renal function
is limited in most of the cases. Dispite the level of renal function impairment at admission, full recovery can
be achieved after bilateral ureteric stenting/nephrostomy and ureterolisis.
idiopathic retroperitoneal fibrosisureterolysisfractalobstructive renal failure
==== Body
Introduction
Retroperitoneal fibrosis (RPF) was first described in 1905 by the French urologist Albaran, but it became
fully recognized in 1948, with the classic publication by Ormond [1
]. Although its true incidence is unknown, estimates range from one case per 200,000 to 500,000
individuals per year. [2] It occurs predominantly in men in their fifth
and sixth decades of life, with a 3:1 male/female ratio, and no ethnic predisposition.[3] Retroperitoneal fibrosis is generally idiopathic, but can also be secondary to the use of certain drugs, malignant diseases, infections and surgery. The idiopathic form of the disease accounts for more than two thirds of cases and it is characterized by a white, woody and fibrous plaque that covers the retroperitoneal structures, i.e. the great vessels, ureters and the psoas muscle. It is usually centralized at the level of the fourth and fifth lumbar vertebrae and spread down to the pelvis; rarely, it extends into the roots of the mesentery, scrotum or continues above the diaphragm as fibrous mediastinitis. [1–3]
The clinical presentation of IRF is usually insidious with vague constitutional symptoms and generally low back pain that may be severe and non–responsive to anti–inflammatory drugs. The pathogenesis is still poorly elucidated, but recent evidence supports the hypothesis that the disease may be the result of an inflammatory state triggered by autoimmune responses [8–10]. Parum et al. [8], considering the high correlation of IRF with atheromatous peri–aortitis, considers that the disease may be due to an immune reaction to some components of atherosclerotic plaques such as low–density lipoprotein (LDL) and ceroid.
The introduction of medical therapy, mainly based on corticosteroids, has greatly improved patients' outcome,[5,6] and the availability of imaging techniques, such as computer tomography (CT) and magnetic resonance imaging (MRI), has provided non–invasive and reliable methods of diagnosis and follow–up. [7]
Both surgical and medical managements have been used in IRF. There are two main approaches; the first consists of surgical relief of the obstruction by ureterolysis (open or laparoscopic) with or without omental wrapping of the ureter, followed or not by corticosteroid therapy [6]. The second consists of relieving the obstruction by placing ureteric stent(s), followed by corticosteroid therapy alone or together with azathioprine or tamoxifen [6]. However, there are no prospective randomized trials to compare the two alternatives.
The aim of this study is to evaluate effective prognostic factors in the evolution of patients with retroperitoneal fibrosis and to establish the validity of fractal analysis in determining the disease severity in these patients.
Material and methods
This study included 19 patients (M/F: 5/14) treated for idiopathic retroperitoneal fibrosis and bilateral obstructive renal failure between Jan 2004-Dec 2008. Patients were identified retrospectively, by searching for the ones diagnosed with IRF, after retroperitoneal biopsy or, in most cases the diagnosis resting on radiological findings, especially CT, with the identification of a retroperitoneal mass, the absence of other demonstrable renal or ureteric disease or any other pathology that could explain the findings. CT was very useful in describing the retroperitoneal mass around the aorta and inferior vena cava, the extent of the lesion and for monitoring the response to surgical treatment during the follow–up. The data about medical history, physical examination findings, laboratory tests (serum urea and creatinine, blood sugar, sodium, potassium, bicarbonate levels, serum pH, uric acid, haematocrit, white blood cell count), imaging methods (renal echography, abdominal CT–scan, MRI) were evaluated.
All patients were followed for at least 1 year. Patients were regularly checked at every 3 months. At each control, patients were submitted to clinical examination and to the following laboratory tests: serum creatinine, complete blood cell count and urine analysis. Renal ultrasound and computed tomography (CT) were performed every 6 months until the achievement of remission. After remission, the same investigations were repeated every year.
At admission, all patients had active disease with obstructive renal failure and underwent bilateral ureteric stenting in order to normalize the BUN levels. CT scan was performed on a helical Siemens Emotions 2007 with 16 slices preoperatory in all cases and images were processed in the Department of Radiology of ‘Fundeni’ Clinical Institute. Parameters assessed on helical CT were: level of secondary hydronephrosis, fibrosis width in the transureteric frontal section, interureteric distance at L4 intervertebral discus, maximal cranio–caudal length in frontal section of fibrosis area and the fractal dimension of fibrosis mass. According to the level of renal function impairment we used native or contrast enhanced images.
At six months, contrast CT was performed at patients with normal renal function. Maximal cranio-caudal length was calculated in frontal sections and was considered as the maximal vertical extension of the fibrotic mass. Because the ICV and the abdominal aorta are not visible on frontal reconstructive section native CT scan, we considered the ureters as landmarks and the fibrotic mass occupying the space between the 2 ureters with a lateral extension beyond the line that crosses the ureters. The ureteric distance in native CT was calculated with the help of the ureteric stents. The width of the fibrotic mass was considered the line between the lateral extensions of the fibrotic mass at L4 intervertebral discus in a longitudinal section.[Fig 1]
Fig 1 Multiplanar frontal reconstruction in native CT scan after bilateral ureteric stenting (transureteric section)
Fractal analysis
In the last 10 years fractal analysis become a powerful tool for analyzing form, pattern and growth of biological systems and subsystems at microscopic and macroscopic scale [18, 19]. In our case, the fractal dimension can give information on the irregularity of the contour of the fibrosis masses. This kind of details are best captured by using the box counting method which provides us with a measure–fractal dimension (Fd) of the non/ smoothness of the contour. The fractal dimension is calculated by using the ‘box–counting’ algorithm because, in comparison to other methods, it offers two major advantages: it is easy to implement and can be applied on no matter how complex images.
Method description
The ‘box–counting’ algorithm [20] assumes the determination of the fractal dimension in accordance with the dependence of the object contour upon the used measure scale factor. It consists of successive image coverage with grids of squares of sides 2, 4, 8. It is important to constantly count the number of squares that contain parts of the contour. The points of coordinates (log(N(s)), log(1/s)), where s is the common side of the coverage squares, and N(s) the number of squares that contain the information, will be positioned approximately in a line and its slope will be the fractal dimension in ‘box–counting’ sense.
Thus, the ‘box–counting’ fractal dimension, derived from the Hausdorff coverage dimension [16, 17] is given by the following approximation:[Fig1]
Fig1 Hausdorff coverage dimension
where N(s) is the number of squares that contain information (parts of the extracted contour) and s–side length of the squares in the coverage grid.
It is expected, that for a smaller s, the above approximation should be better,[Fig2]
Fig2 Better approximation of Hausdorff
If this limit exists, it is called the ‘box–counting’ dimension of the measured object. In practice, this limit converges slowly, that is why the following expression is used:[Fig3]
Fig3 Better approximation of Hausdorff with limit coverages
This is the equation of a straight line of slope Fd, the ‘log–log’ curve described by the points of (log(N(s), log(1/s)) for different values of the cube's side's. Through linear regression (least squares method), the slope of the line that approximates the points' distribution is determined; this is the fractal dimension.
For an example of how the algorithm is used, we will consider the image of a region of the fibrosis mass for which we want to determine the fractal dimension of the contour–Fig 1.1. Traditionally, in order to determine the contour, the pixels over certain luminosity are being neglected Fig 1.2.a); this implies choosing a good threshold in order to capture the exact object of interest. When applying this procedure to medical images like CTs and MRIs, the errors can be larger than the extracted information itself. That is why we implemented a different method for contour extraction. The contour is captured at a series of different levels of luminosity Fig 1.2.b)–this method leading to better performances and being a novel approach towards macroscopic medical images processing. The different levels of luminosity are chosen within the range of shades of the analyzed object.
Fig 1.1 The initial image
Fig 1.2.a Extracted contour classical method
Fig 1.2.b Extracted contour proposed method
Next, we will apply the ‘box–counting’ algorithm, described above, for different
scale values s, using a software tool– MorfoFractal developed along with the contour extraction tool.
We have used images that contain those parts of the fibrosis mass that are not obstructed by nearby organs, so that we can obtain information on the free evolution of the mass. We took the CT from each patient, which contains as many such fibrosis regions and calculated the global fractal dimension.
Results
Clinical characteristics at presentation
Out of the 19 patients, 5 were men and 14 women. The median age at diagnosis of RF was 50 years old (range 42–64 years old). The most frequent presenting symptoms were back or abdominal pain, weakness, weight loss, oligoanuria, arterial hypertension and mild fever (Table 1). The duration of symptoms before diagnosis ranged from 6 to 18 months. At presentation all patients had active disease, presenting renal dysfunction with a median serum creatinine of 5,18 mg/dl (range 1–15.4 mg/dl). Five of them had a rapidly progressive renal failure with a serum creatinine (8.2mg/dl) and were oliguric or anuric at presentation. The median haematocrit of the patients group was 33% (range 29–40%).
Table 1 Clinical and laboratory characteristics at presentation of patients
Sex M/F 5/14
Age (year) 50 (42–64)
Creatinine (mg/dl) 5,18 (1–15.4)
Haematocrit (%) 33 (29–40)
Loin pain 15
Weight loss/weakness 10
Oliguria/Anuria 5
Fever 3
The diagnosis of IRF was made by CT in all patients and confirmed with a histological evaluation of the fibrotic mass. The biopsy of the mass was obtained during ureterolysis. At admission, all patients had active disease with obstructive renal failure and underwent bilateral ureteric stenting in order to normalize the BUN levels. After the normalizing of BUN levels, ureterolysis and omental wrapping was performed. Postoperatively, ureteric stents were removed after 1mth and remission of renal dysfunction was obtained in approximately 5 months (range 2–10 months).
We used bilateral ureteric stenting for all patients in order not only to normalize the BUN levels, but also to establish the landmarks on the native CT and to facilitate intraoperative dissection of the ureters. Five of our patients needed unilateral percutaneous nephrosthomy due to the inefficiency of bilateral stenting.[Table 2]
Table 2 CT characteristics and serum creatinine values during follow–up
UHN level Fibrosis width Interureteric distance Cranio–caudal length Fractal dimension Serum creatinine
Preop 1 mth 6 mth Preop 1 mth 6 mth Preop 1 mth 6 mth Preop 1 mth 6 mth Preop 1 mth 6 mth Preop 1 mth 6 mth
1 2 2 0 3.88 0 – 7.5 12.8 11 13.6 8.6 – 1.29 – – 2.4 1.1 0.7
2 2 1 1 6.22 5.4 – 6.2 12.4 12 16.2 10 4.3 1.28 1.23 – 2.3 1.2 1.3
3 3 3 1 5.82 0 – 5.7 11.4 11 10.2 4.5 – 1.43 – – 4.2 1.6 1.3
4 3 2 1 6.47 5.4 3.4 5.2 14.2 14 12.6 8.6 6.1 1.42 1.32 1.28 4.5 1.5 1.5
5 2 1 1 3.15 0 – 8.2 13.2 13 14.3 10 8.2 1.39 – – 4.5 1.4 1.3
6 2 2 1 7.27 7.2 – 7.2 10.4 9.5 10.6 5.6 – 1.38 1.36 – 4.3 2 1.1
7 2 1 0 5.53 3.1 – 8 8.2 8.6 7.8 4.4 – 1.36 1.31 – 3.8 1.1 1
8 1 1 2 6.31 5.8 – 6.3 7.4 7.2 10.4 14 13 1.25 1.15 – 1 1.2 1.6
9 1 1 1 7.34 0 – 7.3 – 8.4 5.4 5.6 5.4 1.28 – – 1.1 – 1.6
10 4 2 1 5.6 5.5 – 4.3 7.5 7.5 12.6 8.5 – 1.6 1.41 – 15.4 3.7 1.7
11 4 1 1 5.6 4.2 3.1 5.6 7.2 7.4 5.4 – – 1.49 1.45 1.36 6.8 2.1 1.4
12 3 2 1 6.3 3.4 3.4 6.5 10.4 10 5.6 5.6 4.8 1.55 1.49 1.39 10.6 1.7 1.1
13 2 1 0 6.7 0 – 5.8 7.6 7.2 6.3 – – 1.46 – – 5.6 2.1 0.7
14 2 1 0 8.2 4.3 – 5.6 8.5 8.2 10.5 4.8 – 1.37 1.28 – 3.1 0.8 0.7
15 2 1 0 6.3 6.4 3.1 6.3 6.3 6.3 7.8 – – 1.3 1.29 1.22 3.1 0.9 1
16 3 0 0 6.4 0 3.1 6.3 – 8.4 10.4 – 6.4 1.38 – 1.1 3.6 0.9 1
17 2 1 0 5.7 4.5 – 5.7 8.4 – 5.1 – – 1.53 – 1.32 6.9 0.9 0.7
18 3 1 1 8.4 4.7 4.4 7.2 – 8.8 13.1 – 8.4 1.58 – – 10 1.1 0.9
19 4 2 1 7.4 5.8 – 5.8 10.2 – 7.4 7.4 – 1.47 1.44 – 5.4 1.1 1.2
Median 2 1 1 6.24 5.1 3.4 6.35 9.8 9.4 9.56 9.8 6.8 1.41 1.33 1.06 5.18 3 1.1
Table 3 Median UHN, Median ureteric distance, Median fibrosis width, Fractal dimension, Serum creatinine
Median UHN Median ureteric distance Median fibrosis width Fractal dimension Serum creatinine
Preop 2 6.35 6.24 1.41 5.18
Preop 2 6.35 6.24 1.41 5.18
1 month 1 9.75 5.05 1.33 2.76
6 months 1 9.56 3.41 1.27 1.14
Most of the patients had moderate bilateral hydronephrosis (2nd degree hydronephrosis). In our study, all patients had excellent prognosis, with full recovery of renal function in 15 patients (78%).
The fractal dimension of the fibrosis mass correlates with the level of renal function impairment suggested by the creatinine level. Generally, in the case of preoperatory analyzed CTs, if the creatinine value is high, the fractal dimension is higher too, but the rate of increase in the case of Fd is lower as it can be deduced from Fig 3. This fact leads to our statement that the more aggressive the fibrosis (high Fd), the higher the level of renal function impairment.
The fractal dimension varies suggestively between CT evaluations in time (1.30 ±0.2), suggesting a non–aggressive pattern of extension of the fibrotic mass characteristic for benign lesions. In fact, the regression of the affected tissue width and structure complexity (Fd) can be clearly observed.
After the surgical intervention, it can be observed that the value of the fractal dimension of the mass is decreasing with approximately the same rate as the fibrosis width. The decrease in Fd results from the fact that the fibrosis contour is becoming smoother along with area diminution. Generally, the creatinine levels are dropping too, but much faster, mainly due to the presence of ureteric stents/nephrostomy.
Fig 3 Creatinine vs. Fractal dimension
Interureteric distance and fibrosis width at admission do not correlate with the serum creatinine value (Fig 4, Fig 5) and the recovery of the renal function, these confirming previous studies on RF.
Fig 4 Serum creatinine levels vs. hydronephrosis degree at admission.
Fig 5 Serum creatinine levels vs. fibrosis width at admission
Discussion
Malignancy was excluded based on clinical, laboratory and radiological grounds. CT scan did not show any
direct sign of malignancy or indirect signs such as cranial location of the mass, anterior displacement of
the aorta, lateral displacement of the ureters and/or bone destruction [13
]
All the patients had ureterolysis and omental wrapping. The advantages of surgery are the relief of obstruction with a recovery of renal function in about 70% of cases [11] and the possibility of taking samples of the invading mass to rule out lymphomas or metastatic cancer. However, obstruction may recur in about 22% of responders [12]. Moreover, surgery does not relieve the systemic manifestations of the disease that affect the majority of patients, symptoms that can be managed by the use of cortisone or immunosuppressive agents. Generally, however, ureterolysis remains the mainstay in treatment of this disease. The ureter is dissected free from the plaque, and in order to prevent it from being caught again by the fibrotic process laterally or intraperitoneally. An alternative procedure is to wrap the ureter with omentum to provide an effective barrier against re–entrapment by the fibrosis [15]. Postoperative CT in cases in which an omental wrap has been used shows a low–attenuation halo surrounding the opaque ureter [15].
Patients who have had ureterolysis commonly have a lateral bowing of the mid–portion of the ureter(s) [15]. Long–term follow–up with CT usually shows a progressive decrease in the size of the plaque, especially in patients treated with corticosteroids. However, the majority of patients will have a small residual mass that can persist for months to years [14].[Fig 6]
Fig 6 Postoperatory CT scans at 3 and 6 months. In figure A, low–attenuation halo is observed around the right ureter. Figure B, full remission of fibrotic mass at 6 months after surgery
Neither sufficient information is available on the long–term outcome of patients with RF, nor is there any study that compares the efficacy of different therapeutic options. In our study, most of our patients had excellent prognosis, with full recovery of renal function in 15 patients (78%). Four of our patients remained with one functional kidney, 2 required permanent ureteric stent and 2 nephrostomy with a mild but stable renal insufficiency (serum creatinine level >1.5 mg/dl).
Conclusions
The imaging parameters did not predict the disease severity, except for the increase in fractal dimension of fibrosis surface area. Efficacy of bilateral ureteric stenting in improving renal function is limited in most of the cases. Despite the level of renal function impairment at admission, full recovery can be achieved after bilateral ureteric stenting/nephrostomy and ureterolisis. However, as renal insufficiency may persist and local reactivations or other possible complications of the disease may occur, patients with IRF should be regularly monitored by CT scan at 6 months and should receive a prompt therapeutical intervention in order to treat the recurrences of the disease.
==== Refs
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10 Moroni G Del Papa N Moronetti LM Increased levels of circulating endothelial cells in chronic periaortitis as a marker of active disease
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12 Baker LRI Mallison WJW Gregory MC Idiopathic retroperitoneal fibrosis. A retrospective analysis of 60 cases Br J Urol 1988 60 497 503 3427331
13 Vivas I Nicolas AL Retroperitoneal fibrosis: typical and atypical manifestations Br J Radiol 2000 73 214 222 10884739
14 Amis ES Newhouse JH Essentials of uroradiology 1991 Boston Little, Brown
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18 Landini G Schroth G Fractals in Biology and Medicine 1998 Losa. Birkhauser Verlag 268 284
19 Dobrescu R Vailescu C Interdisciplinary Applications of Fractal and Chaos Theory 2004 Bucuresti Academia Romana
20 Crişan DA Popescu–Mina C Udrea A Image processing for biologic models using fractal techniques Proceedings of the 6th EUROSIM Congress on Modeling and Simulation 2007 | 20302193 | PMC3019032 | CC BY | 2021-01-04 21:16:12 | yes | J Med Life. 2010 Feb 15; 3(1):19-25 |
==== Front
PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 2124917810-PLPA-RA-2950R310.1371/journal.ppat.1001257Research ArticleNeurosciencePathology/NeuropathologyAerosols Transmit Prions to Immunocompetent and Immunodeficient Mice Prion Transmission by AerosolsHaybaeck Johannes
1
¤a
Heikenwalder Mathias
1
¤b
Klevenz Britta
2
Schwarz Petra
1
Margalith Ilan
1
Bridel Claire
1
Mertz Kirsten
1
3
Zirdum Elizabeta
2
Petsch Benjamin
2
Fuchs Thomas J.
4
Stitz Lothar
2
*
Aguzzi Adriano
1
*
1
Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland
2
Institute of Immunology, Friedrich-Loeffler-Institut, Tübingen, Germany
3
Department of Pathology, Clinical Pathology, University Hospital Zurich, Zurich, Switzerland
4
Department of Computer Science, Machine Learning Laboratory, ETH Zurich, Zurich, Switzerland
Westaway David EditorUniversity of Alberta, Canada* E-mail: [email protected] (AA); [email protected] (LS)¤a: Current address: Institute of Pathology, Medical University Graz, Graz, Austria
¤b: Current address: Institute of Virology, Technical University München/Helmholtz Zentrum München, Munich, Germany
Conceived and designed the experiments: JH MH BK PS IM CB KM EZ BP TJF LS AA. Performed the experiments: JH MH BK PS IM CB KM EZ BP TJF LS AA. Analyzed the data: JH MH BK PS IM CB KM EZ BP TJF LS AA. Contributed reagents/materials/analysis tools: JH MH BK PS IM CB KM EZ BP TJF LS AA. Wrote the paper: JH MH BK PS IM CB EZ BP TJF LS AA.
1 2011 13 1 2011 7 1 e100125722 3 2010 13 12 2010 Haybaeck et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.
Author Summary
Prions, which are the cause of fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs), can be experimentally or naturally transmitted via prion-contaminated food, blood, milk, saliva, feces and urine. Here we demonstrate that prions can be transmitted through aerosols in mice. This also occurs in the absence of immune cells as demonstrated by experiments with mice lacking B-, T-, follicular dendritic cells (FDCs), lymphotoxin signaling or with complement-deficient mice. Therefore, a functionally intact immune system is not strictly needed for aerogenic prion infection. These results suggest that current biosafety guidelines applied in diagnostic and scientific laboratories ought to include prion aerosols as a potential vector for prion infection.
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Introduction
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders that affect humans and various mammals including cattle, sheep, deer, and elk. TSEs are characterized by the conversion of the cellular prion protein (PrPC) into a misfolded isoform termed PrPSc
[1]. PrPSc aggregation is associated with gliosis, spongiosis, and neurodegeneration [2] which invariably leads to death. Prion diseases have been long known to be transmissible [3], and prion transmission can occur after oral, corneal, intraperitoneal (i.p.), intravenous (i.v.), intranasal (i.n.), intramuscular (i.m.), intralingual, transdermal and intracerebral (i.c.) application, the most efficient being i.c. inoculation [4], [5], [6], [7], [8], [9], [10], [11], [12]. Several biological fluids and excreta (e.g. saliva, milk, urine, blood, placenta, feces) contain significant levels of prion infectivity [13], [14], [15], [16], [17], and horizontal transmission is believed to be critical for the natural spread of TSEs [18], [19], [20], [21], [22], [23]. Free-ranging animals may absorb infectious prion particles through feeding or drinking [24], [25], and tongue wounds may represent entry sites for prions [26].
PrPSc has also been found in the olfactory epithelium of sCJD patients [27], [28]. Prion colonization of the nasal epithelium occurs in various species and with various prion strains [11], [12], [29], [30], [31], [32], [33], [34], [35], [36], [37]. In the HY-TME prion model, intranasal application is 10–100 times more efficient than oral uptake [29] and, as in many other experimental paradigms [38], [39], [40], [41], [42], [43], [44], the lymphoreticular system (LRS) is the earliest site of PrPSc deposition. A publication demonstrated transmission of chronic wasting disease (CWD) in cervidized mice via aerosols and upon intranasal inoculation [45], yet two studies reported diametrically differing results on the role of the olfactory epithelium or the LRS in prion pathogenesis upon intranasal prion inoculation [11], [12], perhaps because of the different prion strains and animal models used. These controversies indicate that the mechanisms of intranasal and aerosolic prion infection are not fully understood. Furthermore, intranasal administration is physically very different from aerial prion transmission, as the airway penetration of prion-laden droplets may be radically different in these two modes of administration.
Here we tested the cellular and molecular characteristics of prion propagation after aerosol exposure and after intranasal instillation. We found both inoculation routes to be largely independent of the immune system, even though we used a strongly lymphotropic prion strain. Aerosols proved to be efficient vectors of prion transmission in mice, with transmissibility being mostly determined by the exposure period, the expression level of PrPC, and the prion titer.
Results
Prion transmission via aerosols
Prion aerosols were produced by a nebulizing device with brain homogenates at concentrations of 0.1–20% (henceforth always indicating weight/volume percentages) derived from terminally scrapie-sick or healthy mice, and immitted into an inhalation chamber. As per the manufacturer's specifications, aerosolized particles had a maximal diameter of <10 µm, and approximately 60% were <2.5 µm [46].
Groups of mice overexpressing PrPC (tga20; n = 4–7) were exposed to prion aerosols derived from infectious or healthy brain homogenates (henceforth IBH and HBH) at various concentrations (0.1, 2.5, 5, 10 and 20%) for 10 min (Fig. 1A, Table 1). All tga20 mice exposed to aerosols derived from IBH (concentration: ≥2.5%) succumbed to scrapie with an attack rate of 100%. The incubation time negatively correlated with the IBH concentration (2.5%: n = 4, 165±54 dpi; 5%: n = 4, 131±7 dpi; 10%: n = 5, 161±27 dpi; 20%: n = 6, 133±8 dpi; p = 0.062, standard linear regression on standard ANOVA; Fig. 1A and F, Table 1, Table S1A).
10.1371/journal.ppat.1001257.g001Figure 1 Prion transmission through aerosols.
(A) tga20 mice were exposed to aerosols generated from 0.1%, 2.5%, 5%, 10% or 20% prion-infected mouse brain homogenates (IBH) for 10 min. (B) Groups of tga20 and (C) CD1 mice were exposed for 1, 5 or 10 min to aerosols generated from a 20% IBH. Experiments were performed twice (different colors). (D) C57BL/6, (E) 129SvxC57BL/6, and Prnp
o/o mice were exposed for 10 min to aerosols generated from 20% IBH. Kaplan-Meier curves describe the percentage of survival after particular time points post exposure to prion aerosols (y-axis represents percentage of living mice; x-axis demonstrates survival time in days post inoculation). Different colors and symbols describe the various experimental groups. (F) Jittered scatter plot of survival time against concentration of prion aerosols generated out of IBH with added linear regression fit (p = 0.0622). (G) Jittered scatter plot of survival time against exposure time for tga20 mice with added linear regression fit. The negative correlation between survival time and exposure time is significant (p<0.001***). (H) Consecutive paraffin sections of the right hippocampus of Prnp
o/o, tga20, CD1 and C57BL/6 mice stained with HE (for spongiosis, gliosis, neuronal cell loss), SAF84 (PrPSc deposits), GFAP (astrogliosis) and Iba-1 (microglia). All animals had been exposed to aerosols generated from 20% IBH for 10 min. Scale bars: 100µm.
10.1371/journal.ppat.1001257.t001Table 1 Survival times of different mouse strains exposed to prion aerosols for various periods.
Genotype inoculum concentr. exposure (min) n attack rate Incubation time (dpi)
tga20
0.1% 10 4 0/4 >300 >300 >300 >300
tga20
2.5% 10 4 4/4 120 121 208 219
tga20
5% 10 4 4/4 122 129 133 138
tga20
10% 10 5 5/5 124 133 161 167 191
tga20
20% 10 6 6/6 120 120 133 133 133 140
CD1 20% 1 3 0/3 >300 >300 >300
CD1* 20% 1 4 2/4 202 202 >300 >300
tga20
20% 1 3 3/3 134 174 189
tga20*
20% 1 3 3/3 203 204 205
CD1 20% 5 4 4/4 182 202 202 209
CD1* 20% 5 3 3/3 202 202 202
tga20
20% 5 4 4/4 136 170 170 174
tga20*
20% 5 4 4/4 127 134 136 136
CD1 20% 10 4 4/4 202 202 202 202
CD1* 20% 10 4 4/4 202 202 211 235
tga20
20% 10 4 4/4 134 138 142 142
tga20*
20% 10 4 4/4 132 133 134 134
C57BL/6 20% 10 4 4/4 164 182 188 188
129SvxC57BL/6 20% 10 5 5/5 155 180 182 184 197
Prnp
o/o
20% 10 3 0/3 >300 >300 >300
newborn tga20
20% 10 3 3/3 157 189 189
newborn CD1 20% 10 3 3/3 211 211 211
Upper panel: survival times of tga20 mice after 10-min exposure to aerosols generated from various concentrations of IBH. Lower panel: survival times of various mouse strains after exposure for 1, 5, or 10 min to infectious aerosols. Selected inoculations were repeated sequentially (asterisks) in order to estimate the reproducibility of these results.
tga20 mice exposed to aerosolized 0.1% IBH did not develop clinical scrapie within the observational period (n = 4; experiment terminated after 300 dpi), yet displayed brain PrPSc indicative of subclinical prion infection (Fig. 1A and 2A). In contrast, control tga20 mice (n = 4) exposed to aerosolized HBH did not develop any recognizable disease even when kept for ≥300 dpi, and their brains did not exhibit any PrPSc in histoblots and Western blots (data not shown).
10.1371/journal.ppat.1001257.g002Figure 2 PrPSc deposition in brains of mice infected with prion aerosols and profiling of NSE-PrP mice.
(A) Western blot analysis of brain homogenates (10%) from terminal or subclinical tga20 mice exposed to aerosols from 20% or 0.1% IBH for 10 min. PK+ or −: with or without proteinase K digest; kDa: Kilo Dalton. (B–C): Western blot analyses of brain homogenates from tga20 (B) or CD1 (C) mice exposed to prion aerosols from 20% IBH. (D) Histoblot analysis of brains from mice exposed to prion aerosols. Brains of tga20 mice challenged with aerosolized 10% (middle panel) or 20% (right panel) IBH showed deposits of PrPSc in the cortex and mesencephalon. Because the brain of a Prnp
o/o mouse showed no signal (left panel), we deduce that the signal in the middle and right panels represents local prion replication. (E) Histopathological lesion severity score analysis of 5 brain regions depicted as radar plots [51] (astrogliosis, spongiform change and PrPSc deposition) derived from tga20, CD1, C57BL/6 and 129SvxC57BL/6 mice exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. (F) Histopathological lesion severity score of 5 brain regions shown as radar blot (astrogliosis, spongiform change and PrPSc deposition) of i.c. prion inoculated tga20, CD1, C57BL/6 and 129SvxC57BL/6 mice. (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. (G) Survival curve and (H) lesion severity scores of NSE-PrP mice exposed to a 20% aerosolized IBH for 10 min. (I) Histological and immunohistochemical characterization of scrapie-affected hippocampi of NSE-PrP mice after exposure to aerosolized 20% IBH. Stain legend as in Fig. 1H. Scale bar: 100µm.
In the above experiments, and in all experiments described in the remainder of this study, all PrP-expressing (tga20 and WT) mice diagnosed as terminally scrapie-sick were tested by Western blot analysis and by histology: all were invariably found to contain PrPSc in their brains (Fig. 2) and to display all typical histopathological features of scrapie including spongiosis, PrP deposition and astrogliosis (Fig. 1H).
Correlation of exposure time to prion aerosols and incubation period
We then sought to determine the minimal exposure time that would allow prion transmission via aerosols (Fig. 1B, Table 1). tga20 mice were exposed to aerosolized IBH (20%) for various durations (1, 5 or 10 min) in two independent experiments. Surprisingly, an exposure time of only 1 min was found to be sufficient to induce a 100% scrapie attack rate. Longer exposures to prion-containing aerosols strongly correlated with shortened incubation periods (Fig. 1B and G, Table 1, Table S1A and B).
In order to test the universality of the above results, we examined whether aerosols can transmit prions to various mouse strains (CD1, C57BL/6; 129SvxC57BL/6) expressing wild-type (wt) levels of PrPC. CD1 mice were exposed to aerosolized 20% IBH in two independent experiments (Fig. 1C, Table 1). After 5 or 10-min exposures, all CD1 mice succumbed to scrapie whereas shorter exposure (1 min) led to attack rates of 0–50% [1 min exposure (first experiment): scrapie in 0/3 mice; 1 min exposure (second experiment): 2/4 mice died of scrapie at 202±0 dpi; 5 min (first experiment): n = 4, attack rate 100%, 202±12 dpi; 5 min (second experiment): n = 3, attack rate 100%, 202±0 dpi; 10 min (first experiment): n = 4, attack rate 100%, 202±0 dpi; 10 min (second experiment): n = 4, attack rate 100%, 206±16 dpi]. In CD1 mice exposed to prion-containing aerosols for longer intervals, we detected a trend towards shortened incubation times which did not attain statistical significance (Table S1A and S1B).
We also investigated whether C57BL/6 or 129SvxC57BL/6 mice would succumb to scrapie upon exposure to prion aerosols (Fig. 1D and E, Table 1). A 10 min exposure time with a 20% IBH led to an attack rate of 100% (C57BL/6: 10 min: n = 4; 185±11 dpi; 129SvxC57BL/6: n = 5; 10 min: 182±15 dpi). Control Prnp
o/o mice (129SvxC57BL/6 background; n = 3) were resistant to aerosolized prions (20%, 10 min) as expected (Fig. 1E and H, Table 1).
Incubation time and attack rate depends on PrPC expression levels
When tga20 mice were challenged for 10 min, variations in the concentration of aerosolized IBH had a barely significant influence on survival times (p = 0.062; Fig. 1F), whereas variations in the duration of exposure of tga20 mice affected their life expectancy significantly (p<0.001; Fig. 1G). Furthermore tga20 mice, which express 6–9 fold more PrPC in the central nervous system (CNS) than wt mice [46], [47], [48], succumbed significantly earlier to scrapie upon prion aerosol exposure for 10 min (20%) (tga20 mice: 134±4 dpi; CD1 mice: 202±12 dpi, p<0.0001; C57BL/6 mice: 185±11 dpi, p = 0.003; 129SvxC57BL/6 mice: 182±15 dpi, p = 0.003; Fig. 1B–E, Fig. S1, Table S1A and S1C). Incubation time was prolonged and transmission was less efficient in CD1 mice than in tga20 mice after a 1 min exposure to prion aerosols (20%). The variability of incubation times between CD1 mice was low (1st vs. 2nd experiment with 5-min exposure: p = 0.62, 1st vs. 2nd experiment with 10-min exposure: p = 0.27; Fig. 1C, Table 1). This suggests that 1 min exposure of CD1 mice to prion aerosols (20%) suffices for uptake of ≤1LD50 infectious units. This finding underscores the importance of PrPC expression levels not only for the incubation time but also for susceptibility to infection and neuroinvasion upon exposure to aerosols. Histoblot analyses confirmed deposition of PrPSc in brains of tga20 mice exposed to prion aerosols derived from 10% or 20% IBH, whereas no PrPSc was found in brains of Prnp
o/o mice exposed to prion aerosols (Fig. 2D).
We then performed a semiquantitative analysis of the histopathological lesions in the CNS. The following brain regions were evaluated according to a standardized severity score (astrogliosis, spongiform change and PrPSc deposition; [49]): hippocampus, cerebellum, olfactory bulb, frontal white matter, and temporal white matter. Scores were compared to those of mice inoculated i.c. with RML (Fig. 2E and F). Lesion profiles of terminally scrapie-sick mice (tga20, CD1, C57BL/6 and 129SvxC57BL/6) infected i.c. or through aerosols were similar irrespectively of genetic background or PrPC expression levels (Fig. 2E and F), with CD1 and 129SvxC57BL/6 hippocampi and cerebella displaying only mild histological and immunohistochemical features of scrapie regardless of the route of inoculation.
We attempted to trace PrPSc in the nasopharynx, the nasal cavity or various brain regions early after prion aerosol infection (1–6 hrs post exposure) and at various time points after intranasal inoculations (6, 12, 24, 72, 144 hrs, 140 dpi, and terminally) with various methods including Western blot, histoblot and protein misfolding analyses. However, none of these analyses detected PrPSc shortly after exposure to prion aerosols (6–72 hrs post prion aerosol exposure) whereas at 140 dpi or terminal stage PrPSc was detected by all of these methods (Fig. S2; data not shown).
PrPC expression on neurons allows prion neuroinvasion upon infection with prion aerosols
We then investigated whether PrPC expression in neurons would suffice to induce scrapie after exposure to prions through aerosols. NSE-PrP transgenic mice selectively express PrPC in neurons and if bred on a Prnp
o/o background (Prnp
o/o/NSE-PrP) display CNS-restricted PrP expression levels similar to wt mice [50].
Prnp
o/o/NSE-PrP (henceforth referred to as NSE-PrP) mice were exposed to prion aerosols (20% homogenate; 10 min). All NSE-PrP mice succumbed to terminal scrapie (216±8 dpi; n = 4; Fig. 1E, 2G, Table 1), although incubation times were significantly longer than those of wt 129SvxC57BL/6 mice (180±15 dpi; n = 5; p = 0.004). Histology and immunohistochemistry confirmed scrapie in NSE-PrP brains (Fig. 2H and I). Histopathological lesion severity score analysis (see above) revealed a lesion profile roughly similar to that of control 129SvxC57BL/6 mice (Fig. 2E, H). More severe lesions were observed in NSE-PrP cerebella whereas olfactory bulbs were less affected.
Real time PCR analysis revealed 2–4 transgene copies per Prnp allele in Prnp
o/o
/NSE-PrP mice.
A detailed quantitative analysis of PrPC expression levels at various sites of the CNS was performed by comparing the signals obtained by blotting various amounts of protein from NSE-PrP, wt and tga20 tissues (Fig. S3). A value of 100 was arbitrarily assigned to expression of PrPC in wt tissues; olfactory epithelia of tga20 and NSE-PrP mice expressed ≥350 and ∼30, respectively (Fig. S3A). In olfactory bulbs, tga20 and NSE-PrP mice expressed ≥150 and 30, respectively (Fig. S3B). In brain hemispheres tga20 and NSE-PrP mice expressed >250 and >150, respectively (Fig. S3C). Therefore, NSE-PrP mice expressed somewhat more PrPC than wt mice in brain hemispheres, but somewhat less in olfactory bulbs and olfactory epithelia.
Aerosolic prion infection is independent of the immune system
In many paradigms of extracerebral prion infection, efficient neuroinvasion relies on the anatomical and physiological integrity of several immune system components [40], [42], [43], [44]. To determine whether this is true for aerosolic prion challenges, we exposed immunodeficient mouse strains to prion aerosols. This series of experiments included JH
−/− mice, which selectively lack B-cells, and γCRag2
−/− mice which are devoid of mature B-, T- and NK-cells (Fig. 3A). Upon exposure to prion aerosoIs (20% IBH; exposure time 10 min) both JH
−/− and γCRag2
−/− mice succumbed to scrapie with a 100% attack rate (JH
−/−: n = 6, 181±21 dpi; γCRag2
−/−: n = 11, 185±41 dpi, p = 0.65). The incubation times were not significantly different to those of C57BL/6 wt mice exposed to prion aerosols (JH
−/− mice: p = 0.9; γCRag2
−/− mice: p = 0.7).
10.1371/journal.ppat.1001257.g003Figure 3 Prion transmission through aerosols in immunocompromised mice.
Survival curves, lesion severity score analysis (radar plots), and representative histopathological micrographs of mice with genetically or pharmacologically impaired components of the immune system (JH
−/−, γCRag2
−/−
A),129Sv mice treated with LTβR-Ig or with muIgG (B), and LTβR
−/−, and CD40
−/− mice (C). All mice were exposed for 10 min to aerosolized 20% IBH. Stain code: HE (spongiosis, gliosis, neuronal cell loss), SAF84 (PrPSc deposits), GFAP (astrogliosis) and Iba-1 (microglial activation) as in Fig. 1H. Scale bars: 100µm.
Histological and immunohistochemical analyses confirmed scrapie in all clinically diagnosed mice. Lesion severity score analyses (Fig. 3A and 3E) showed that JH
−/− and γCRag2
−/− mice had lower profile scores in cerebella and higher scores in hippocampi and frontal white matter than C57BL/6 mice. Slightly higher scores in temporal white mater areas and the thalamus could be detected in JH
−/− and γCRag2
/−/− mice, whereas γCRag2
−/− mice showed lower scores in olfactory bulbs. Consistently with several previous reports, γCRag2
−/− mice (n = 4) did not succumb to scrapie after i.p. prion inoculation (100µl RML6 0.1% 6 log LD50) even when exposed to a prion titer that was twice higher than that used for intranasal inoculations (data not shown).
Depending on the exposure time and the IBH concentration, tga20 mice developed splenic PrPSc deposits. In contrast, none of the scrapie-sick JH−/−, LTβR−/− and γCRag2
−/− mice displayed any splenic PrPSc on Western blots and/or histoblots (Fig. S4A–D) despite copious brain PrPSc.
Aerosol infection is independent of follicular dendritic cells
Follicular dendritic cells (FDCs) are essential for prion replication within secondary lymphoid organs and for neuroinvasion after i.p. or oral prion challenge [42], [44], [51]. Lymphotoxin beta receptor-Ig fusion protein (LTβR-Ig) treatment in C57BL/6 mice causes dedifferentiation of mature FDCs, resulting in reduced peripheral prion replication and neuroinvasion upon extraneural (e.g. intraperitoneal or oral) prion inoculation [52], [53]. We therefore investigated whether FDCs are required for prion replication after challenge with prion aerosols. C57BL/6 mice were treated with LTβR-Ig or nonspecific pooled murine IgG (muIgG) before and after prion challenge (−7, 0, and +7 days) (Fig. 3B). The effects of the LTβR-Ig treatment were monitored by Mfg-E8+/FDC-M1+ staining for networks of mature FDCs in lymphoid tissue. This analysis revealed a complete loss of Mfg-E8+/FDC-M1+ networks at the day of prion exposure and at 14 dpi (data not shown).
LTβR-Ig treatment and dedifferentiation of FDCs did not alter incubation times upon aerosol prion infection (LTβR-Ig: n = 3, attack rate 100%, 184±0 dpi; muIgG: n = 3, attack rate 100%, 184±0 dpi) (Fig. 3B, Table 2). The diagnosis of terminal scrapie was confirmed by histological and immunohistochemical analyses in all clinically affected mice (Fig. 3B; data not shown). Histopathological lesion severity scoring revealed that LTβR-Ig treated C57BL/6 mice displayed a higher score in all regions investigated than untreated C57BL/6 mice upon challenge with prion aerosols (20% IBH; 10 min) (Fig. 2E and 3B). We found slightly less severe scores in the olfactory bulbs of C57BL/6 mice treated with muIgG than in untreated C57BL/6 mice upon challenge with prion aerosols (Fig. 2E and 3B), and a slightly higher score in the temporal white matter (exposure to 20% aerosol for 10 min; Fig. 2E and 3B).
10.1371/journal.ppat.1001257.t002Table 2 Survival of mouse strains exposed to prion aerosols (upper panel) or intranasal administered prions (lower panel).
Genotype n Attack rate Survival (dpi)
Aerosol (20% IBH)
NSE-Prp 4 4/4 211 211 213 227
JH−/−
6 6/6 140 184 184 188 190 198
γCRag2−/−
11 11/11 121 134 141 165 169 187 224 224 224 224 227
C57BL/6 treated with LTbR-Ig 3 3/3 184 184 184
C57BL/6 treated with muIgG 3 3/3 184 184 184
LTβR−/−
4 4/4 272 272 272 272
CD40−/−
3 3/3 220 292 315
C3−/− C4−/−
3 3/3 363 363 420
intranasal inoculation
Prnp
o/o
8 0/8 >300 >300 >300 >300 >300 >300 >300 >300
C57BL/6 8 8/8 219 219 253 263 292 292 292 296
129SvxC57BL/6 5 5/5 187 213 213 235 235
Balbc 6 6/6 112 225 225 225 225 242
tga20
10 10/10 118 125 126 133 152 165 186 187 202 203
NSE-Prp 6 6/6 201 230 255 267 383 411
Rag1−/−
9 9/9 198 198 198 200 203 203 204 210 214
γCRag2−/−
16 16/16 224 224 224 229 241 242 242 242 244
244 256 258 264 265 265 319
C1qa−/−
4 4/4 256 284 291 319
CD21−/−
10 10/10 212 212 214 216 226 235 236 263 269 270
CXCR5−/−
5 5/5 190 245 363 363 403
C57BL/6 treated with LTβR-Ig 8 8/8 219 222 407 413 475 638 717 717
C57BL/6 treated with muIgG 9 9/9 176 242 242 242 255 255 263 271 272
LTβR−/−
6 6/6 223 252 263 314 346 348
TNFR1−/−
3 3/3 213 213 214
LTα−/−
6 6/6 233 234 238 255 262 283
C57BL/6 HBH 4 0/4 >300 >300 >300 >300
Balb/c HBH 4 0/4 >300 >300 >300 >300
Rag1−/− HBH 4 0/4 >300 >300 >300 >300
γCRag2−/− HBH 4 0/4 >300 >300 >300 >300
Prion aerosol infection of mice lacking LTβR or CD40L
LTβR signaling is essential for proper development of secondary lymphoid organs and for maintenance of lymphoid microarchitecture, and was recently shown to play an important role in prion replication within ectopic lymphoid follicles and granulomas [40], [41], [44]. To investigate the role of this pathway in aerogenic prion infections, LTβR
−/− mice were exposed to prion aerosols (20% IBH; 10 min exposure time). All LTβR
−/− mice succumbed to scrapie (LTβR
−/−: n = 4, 272±0 dpi) and displayed PrP deposits in their brains (Fig. 3C). Histological severity scoring of aerosol-exposed mice revealed higher scores in LTβR
−/− hippocampi and lower scores in cerebellum, olfactory bulb, frontal and temporal white matter than in C57BL/6 controls (exposure: 20%; 10 min; Fig. 2E and 3C).
We then investigated the role of CD40 receptor in prion aerosol infection. CD40
−/− mice fail to develop germinal centers and memory B-cell responses, yet CD40L
−/− mice show unaltered incubation times upon i.p. prion challenge [54]. Similarly to the other immunocompromised mouse models investigated, CD40
−/− mice developed terminal scrapie upon infection with prion aerosols with an attack rate of 100% (n = 3, 276±50 dpi). Lesion severity analyses of CD40
−/− mice revealed a slightly higher score in the cerebellum and the temporal white matter than in C57BL/6 mice (Fig. 2E and 3C). Therefore, LTβR and CD40 signaling are dispensable for aerosolic prion infection.
Components of the complement system are dispensable for aerosolic prion infection
Certain components of the complement system (e.g. C3; C1qa) play an important role in early prion uptake, peripheral prion replication and neuroinvasion after peripheral prion challenge [43], [55], [56]. We have tested whether this is true also for exposure to prion aerosols. Mice lacking both complement components C3 and C4 (C3C4
−/−) were exposed for 10 min to 20% aerosolized IBH. All C3C4
−/− mice succumbed to scrapie (n = 3, 382±33 dpi; Fig. 4A). Histopathological evaluation of all scrapie affected mice revealed astrogliosis, spongiform changes and PrP-deposition in the CNS (Fig. 4A).
10.1371/journal.ppat.1001257.g004Figure 4 Prion transmission through aerosols in complement-deficient and newborn mice.
(A) C3C4
−/− mice and (B) newborn tga20 and CD1 mice were exposed for 10 min to a 20% aerosolized IBH. Survival curves (right panels) as well as histological and immunohistochemical characterization of hippocampi indicate that all prion-exposed mice developed scrapie efficiently. Scale bars: 100µm.
No protection of newborn mice against prion aerosols
The data reported above argued in favor of direct neuroinvasion via PrPC-expressing neurons upon aerosol administration. However, a possible alternative mechanism of transmission may be via the ocular route, namely via cornea, retina, and optic nerve [57], [58]. In order to test this possibility, newborn (<24 hours-old) tga20 and CD1 mice, whose eyelids were still closed, were exposed for 10 min to prion aerosols generated from a 20% IBH. All mice succumbed to scrapie and showed PrP deposits in brains (tga20 mice: n = 3, 173±23 dpi; CD1 mice: n = 3, 211±0 dpi) (Fig. 4B). Newborn tga20 mice succumbed to scrapie slightly later (p = 0.0043) than adult tga20 mice, whereas no differences were observed between newborn and adult CD1 mice exposed for 10 min to prion aerosols generated from a 20% IBH (p = 0.392).
The brains of all animals contained PK-resistant material, as evaluated by Western blot analysis (data not shown). In addition, untreated littermates or other sentinels which were reared or housed together with aerosol-treated mice immediately following exposure to aerosols showed neither signs of scrapie nor PrPSc in brains, even after 482 dpi. This suggests that prion transmission was the consequence of direct exposure of the CNS to prion aerosols rather than the result of transmission via other routes like ingestion from fur by grooming or exposure to prion-contaminated feces or urine.
Lack of PrPSc in secondary lymphoid organs of immunocompromised, scrapie-sick mice after infection with prion aerosols
We further investigated additional mice for the occurrence of PrPSc in secondary lymphoid organs upon exposure to prion aerosols. PK-resistant material was searched for in spleens, bronchial lymph nodes (bln) and mesenteric lymph nodes (mln) at terminal stage of disease. C57BL/6, 129SvxC57BL/6, muIgG treated C57BL/6 mice, newborn tga20 mice as well as newborn CD1 mice contained PrPSc in the LRS, whereas LTβR−/− mice and C57BL/6 mice treated with LTβR-Ig lacked PrPSc deposits in spleens (Fig. S4A–F; Table 3).
10.1371/journal.ppat.1001257.t003Table 3 PrPSc deposition in spleens of mice challenged with a range of aerosolized prion concentrations and exposure times.
Genotype Splenic PrPSc in individual mice
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
Prnp
o/o
−
−
−
Newborn CD1 (20%; 10 min exp.)
+
+
+
CD1 (20%; 1 min exp.)
+
+
+
+
−
Nd Nd
CD1 (20%; 5 min exp.)
+
+
−
−
+
Nd Nd
CD1 (20%; 10 min exp.)
+
+
+
+
+
Nd Nd Nd
C57BL/6 (20%; 10 min exp.)
+
+
+
+
129SvxC57BL/6 (20%; 10 min exp.)
+
+
+
+
+
Newborn tga20 (20%; 10 min exp.)
+
+
+
tga20 (0.1%; 10 min exp.)
−
−
−
−
tga20 (2.5%; 10 min exp.)
+
+
+
+
tga20 (5%; 10 min exp.)
+
+
+
+
tga20 (10%; 10 min exp.)
+
+
+
+
Nd
tga20 (20%; 1 min exp.)
+
+
+
+
−
−
tga20 (20%; 5 min exp.)
+
+
+
+
+
+
Nd Nd
tga20 (20%; 10 min exp.)
+
+
+
+
+
+
+
+
tga20 (20%; 10 min exp.)
+
+
+
+
+
+
+
JH−/−
−
−
−
−
γCRag2−/−
−
−
−
−
−
−
−
−
−
−
−
C57BL/6 treated with LTβR-Ig
−
−
−
C57BL/6 treated with mu-IgG
+
+
Nd
LTβR−/−
−
−
−
−
PrPSc was assessed on Western blots and histoblots of spleens of Prnp
o/o, newborn CD1, adult CD1, 129SvxC57BL/6, newborn tga20, adult tga20, JH−/−, γCRag2−/−, C57BL/6 mice treated with LTβR-Ig or muIgG and LTβR−/− mice. +: PrPSc detectable in spleen; −: PrPSc undetectable; Nd: not determined; exp.: exposure time (minutes).
The efficiency of intranasal prion inoculation depends on the level of PrPC expression
To dissect aerosol-mediated from non-aerosolic contributions to prion exposure, we directly applied a prion suspension (RML 6.0, 0.1%, 40µl, corresponding to 4×105 LD50 scrapie prions) to the nasal mucosa of various mouse lines (Fig. S5). Since mice breathe exclusively through their nostrils [59], [60] we reasoned that this procedure would simulate aerosolic transmission with sufficient faithfulness although the mechanisms of prion uptake could still differ between aerosolic and intranasal administration [11].
tga20 (n = 10), 129SvxC57BL/6 (n = 5), C57BL/6 (n = 8) and Prnp
o/o mice (n = 8) were challenged intranasally with prions (Fig. S5). To test the possibility that the inoculation procedure itself might impact the life expectancy of mice, C57BL/6 mice (n = 4) were inoculated intranasally with healthy brain homogenate (HBH) for control (Fig. S5E). None of the animals that had been inoculated with HBH displayed a shortened life span, nor did they develop any clinical signs of disease - even when kept for ≥500 dpi. In contrast, after intranasal prion inoculation all C57BL/6, 129SvxC57BL/6 and tga20 mice succumbed to scrapie with an attack rate of 100% (Fig. S5A–C), whereas Prnp
o/o mice were resistant to intranasal prions (Fig. S5D). After intranasal inoculation, tga20 mice (n = 10, 160±28 dpi) displayed a shorter incubation time (Fig. S5C) than 129SvxC57BL/6 (n = 5, 217±20 dpi) or C57BL/6 mice (n = 8, 266±33 dpi; Fig. S5A and S5B). Further, histological and immunohistochemical analyses for spongiosis, astrogliosis and PrP deposition pattern confirmed terminal scrapie (Fig. S5J). A histopathological lesion severity score analysis revealed similar lesion profiles as detected after exposure to prion aerosols (Fig. S5K). However, in the olfactory bulb of tga20 and 129SvxC57BL/6 mice the score was lower upon intranasal administration than in the aerosol paradigm (Fig. 2E).
Finally, we tested whether prion transmission via the intranasal route would be enabled by selective PrPC expression on neurons. For that, we inoculated NSE-PrP mice. All intranasally challenged NSE-PrP mice (n = 6, 291±86 dpi) succumbed to scrapie. The incubation time until terminal disease did not differ significantly from that of 129SvxC57BL/6 control mice (n = 5, 217±20 dpi; p = 0.0868).
Intranasal prion transmission in the absence of a functional immune system
Next, we sought to determine which components (if any) of the immune system are required for neuroinvasion upon intranasal infection with prions. To address this question, Rag1
−/− and γCRag2
−/− mice were intranasally inoculated with prions (inoculum RML 6.0, 0.1%, 40µl, equivalent to 4×105 LD50 scrapie prions). Remarkably, all intranasally prion-inoculated Rag1
−/− (n = 9, 203±6 dpi) (Fig. 5A and H) and γCRag2
−/− mice (n = 16, 243±24 dpi) (Fig. 5D and G) succumbed to scrapie, providing evidence for a LRS-independent mechanism of prion neuroinvasion upon intranasal administration. Incubation times in Rag1
−/− were significantly different to those of intranasally challenged control mice (C57BL/6; attack rate 100%; n = 8, 266±33 dpi; p = 0.0009) whereas γCRag2
−/− mice were not different from those of intranasally challenged control mice (Balb/c: attack rate 100%, n = 6, 209±48 dpi, p = 0.099) (Fig. 5B and Fig. 5D).
10.1371/journal.ppat.1001257.g005Figure 5 Prion transmission by intranasal instillation.
(A) Rag1
−/− mice intranasally inoculated with RML6 0.1%, (B) C57BL/6 mice that have been intranasally inoculated with 3×105 LD50 prions. (C) Rag1
−/− mice i.c. inoculated with 3×105 LD50, (D) γCRag2
−/− mice intranasally inoculated with 4×105 LD50 or (E) Balb/c mice intranasally inoculated with 4×105 LD50 scrapie prions are shown. Survival curves (A–D) and respective Western blots (F–G) are indicative of efficient prion neuroinvasion. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Brain homogenates derived from a terminally scrapie-sick and a healthy C57BL/6 mouse served as positive and negative controls (s: sick; h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. (H and I) Histopathological lesion severity score described as radar blot (astrogliosis, spongiform change and PrPSc deposition) in 5 brain regions of both mouse lines exposed to prion aerosols. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter.
After intranasal prion administration, PrPSc was present in the CNS of Rag1
−/− or γCRag2
−/− mice. WB analysis corroborated terminal scrapie (Fig. 5G and H). Histopathological lesion severity scoring revealed a distinct lesion profile characterized by a high score in the temporal white matter and the thalamus in case of Rag1
−/− mice. In case of γCRag2
−/− mice the cerebellum, the olfactory bulb and the frontal white matter displayed lower scores (Fig. 5I and J). In contrast to the CNS spleens of the affected animals did not contain PK-resistant material in terminally sick Rag1
−/− and γCRag2
−/− mice (Fig. S6E).
For control, Rag1−/− as well as γCRag2
−/− mice were intranasally inoculated with HBH to test the possibility that intranasal inoculation itself impacts their life expectancy. None of the mice inoculated with HBH died spontaneously or developed scrapie up to ≥300 dpi (n = 4 each; Fig. S6A, C–D). Further, Balb/c mice and C57BL/6 mice (n = 4 each) inoculated intranasally with HBH (Fig. S5E and S6D) did not develop any disease for ≥300 dpi.
As a positive control, Rag1
−/− mice were i.c. inoculated with 3×105 LD50 scrapie prions. This led to terminal scrapie disease after approximately 130 days and an attack rate of 100% (n = 3, 131±8 dpi) (Fig. 5B and data not shown).
As additional negative controls, Rag1
−/− and γCRag2
−/− mice were i.p. inoculated with prions (100 µl RML 0.1%, 1×106 LD50). Although more infectious prions (approximately 2 fold more) were applied when compared to the intranasal route, i.p. prion inoculation did not suffice to induce scrapie in Rag1
−/− and γCRag2
−/− mice (attack rate: 0%, n = 4 for each group, experiment terminated after 400 dpi).
Relevance of the complement system for prion pathogenesis after intranasal challenge
The complement component C1qa is involved in facilitating the binding of PrPSc to complement receptors on FDCs [56]. Accordingly, C1qa−/− mice are resistant to prion infection upon low-dose peripheral inoculation. CD21
−/− mice are devoid of the complement receptor 1, display a normal lymphoid microarchitecture and show a reduction in germinal center size. The incubation time in CD21
−/− mice is greatly increased upon peripheral prion inoculation via the i.p. route [56].
To determine whether the complement system is involved in prion infection through aerosols, C1qa−/− and CD21
−/− mice were intranasally inoculated with prions. C1qa−/− mice and CD21
−/− mice succumbed to scrapie with an attack rate of 100% (C1qa−/− mice: n = 4, 288±26 dpi; CD21
−/− mice: n = 10, 235±24 dpi) (Figs. 6A–C), with CD21
−/− mice succumbing to scrapie slightly earlier when compared to C1qa−/− mice. However, survival times did not differ significantly from C57BL/6 control mice (n = 8, 266±33 dpi; C1qa−/− mice: p = 0.24; CD21
−/− mice: p = 0.05) (Fig. S5A and S5B). Western blot analysis of one terminally scrapie-sick C1qa−/− mouse revealed one PrPSc positive spleen (1/4) (Fig. S6F). Two terminally scrapie-sick CD21
−/− mice showed PK resistance in their spleens (2/10) (Fig. S6G). These results indicate that the complement components C1qa and CD21 are not essential for prion propagation upon intranasal application.
10.1371/journal.ppat.1001257.g006Figure 6 Intranasal prion transmission in immnunodeficient mice.
All mice were intranasally inoculated with 3×105 LD50 prions. (A) C1qa−/− mice intranasally inoculated and (B) CD21
−/− mice intranasally inoculated are shown. Survival curves illustrate survival after intranasal prion challenge. Respective Western blots of C1qa
−/− mice intranasally inoculated (C, left panel) and of CD21
−/− mice intranasally inoculated (C, right panel) are shown. Survival curves of CXCR5
−/− mice intranasally inoculated are shown (D). Respective Western blots of CXCR5
−/− mice intranasally inoculated. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends are as in Fig. 5.
CXCR5 deficiency does not shorten prion incubation time upon intranasal infection
CXCR5 controls the positioning of B-cells in lymphoid follicles, and the FDCs of CXCR5-deficient mice are in close proximity to nerve terminals, leading to a reduced incubation time after i.p. prion inoculation [39], [61]. Here we explored the impact of CXCR5 deficiency onto intranasal prion inoculation. CXCR5
−/− mice exhibited attack rates of 100%, and incubation times did not differ significantly from those of C57BL/6 mice (n = 5, 313±91 dpi; p = 0.32) (Fig. 6D). 3 out of 5 terminally scrapie-sick CXCR5
−/− mice revealed PK resistant material in their spleens (3/5), as detected by Western blot analysis (Fig. S6H).
Prion infection is independent of LTβR and TNFR1 signaling
Pharmacological inhibition of LTβR signaling strongly reduces peripheral prion replication and reduces or prevents prion neuroinvasion upon i.p. prion challenge [42], [44], [53]. To determine whether inhibition of LTβR signaling would affect prion transmission through the nasal cavity, we treated C57BL/6 mice with 100µg LTβR-Ig and for control with 100µg muIgG/mouse/week pre- and post-prion challenge (−7 days, 0 days, +7 days; 14 days). LTβR-Ig-treated mice were then inoculated intranasally with prions. 100% of the intranasally challenged mice died due to terminal scrapie (C57BL/6 mice treated with LTβR-Ig: n = 8, 476±200 dpi; Fig. 7A). MuIgG treated mice served as controls and showed an insignificantly shortened incubation time (attack rate: 100%, n = 9, 246±29 dpi) (Fig. 7B and C; C57BL/6 LTβR-Ig treated vs. muIgG treated mice: p = 0.014; C57BL/6 untreated vs. LTβR-Ig treated C57BL/6 mice: p = 0.021; C57BL/6 untreated vs. C57BL/6 muIgG treated mice: p = 0.22).
10.1371/journal.ppat.1001257.g007Figure 7 Intranasal prion transmission is independent of lymphotoxin signaling.
C57BL/6 mice treated with LTβR-Ig (A) or control muIgG (B), and mice lacking various components of the LT/TNF system (D–F, as indicated) were intranasally inoculated with 4×105 LD50 scrapie prions. Survival curves (A, B, D, E and G) and respective Western blots (C, F and H) indicate efficient prion infection and neuroinvasion. One animal that died early after intranasal inoculation (40 dpi) is reported as intercurrent death (i.d.) for reasons other than scrapie. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Controls and legends used are as in Fig. 1H.
We additionally challenged LTβR
−/−, TNFR1
−/− and LTα
−/− mice intranasally with RML prions (Fig. 7D–H). Under these conditions all LTβR
−/−, TNFR1
−/−
and LTα
−/− mice developed terminal scrapie (LTβR
−/− mice: n = 6, 291±52 dpi; TNFR1
−/− mice: n = 3, 213±1 dpi; LTα
−/− mice: n = 6, 251±20 dpi) (Fig. 7D–H). Terminal scrapie was confirmed by immunohistochemistry, histoblot and WB analysis (Fig. 7F and H, data not shown). Spleens of intranasally inoculated LTβR
−/− and TNFR1
−/− mice displayed no PK resistant material (LTβR
−/− mice: 0/6; TNFR1
−/− mice: 0/3). In LTα
−/− mice 1 out of 6 spleens contained PrPSc, while splenic PrPSc deposits of PK-resistant material were abundantly found in terminally scrapie-sick tga20 mice (tga20 mice: 2/10)(Fig. S6I–L).
Discussion
Although aerial transmission is common for many bacteria and viruses, it has not been thoroughly investigated for prion aerosols [11], [12], [29], [30], [31], [32], [33], [34], [62] and prions are not generally considered to be airborne pathogens. Yet olfactory nerves have been discussed as a possible entry site for prions [11], and indeed contact-mediated prion exposure of nostrils can efficiently infect various species. We therefore set out to investigate the possible hazards of prion infection deriving from exposure to prion aerosols. Our results establish that aerosolized prion-containing brain homogenates that aerosols are efficacious prion vectors.
Incubation time and attack rate after exposure to prion aerosols depended primarily on the exposure time, the PrPC expression level of recipients and, to a lesser degree, the prion titer of the materials used to generate prion aerosols in a standardized inhalation chamber. The paramount role of the exposure time suggests that the rate of transepithelial ingress of prion through the airways may be limiting even when prions are offered in relatively low concentrations. Conversely, the total prion uptake capacity by the respiratory system was never rate-limiting, because the incubation time of scrapie decreased progressively with higher concentrations and longer exposure times, and because we were unable to establish a response plateau. The latter phenomenon may be explained by the large alveolar surface potentially available for prion uptake.
Since it occurred in wt mice of disparate genetic backgrounds (C57BL/6; CD1; 129SvxC57BL/6), aerosolic infection may represent a universal phenomenon untied to the genetic peculiarities of any specific mouse strain (Fig. S7 features a representative panel of histological features in CD1 mice). However, in CD1 mice the rapidity of progression to clinical disease did not correlate with the exposure time at a given concentration of IBH used for generating prion aerosols, suggesting the existence of genetic factors modulating the saturation of aerogenic prion intake.
The passage of infectivity from the peritoneum to the brain requires a non-hematopoietic conduit that expresses PrPC
[63]. We therefore sought to determine whether such a conduit would be required for transfer of infectivity from the aerosols to the brains of recipients. Using NSE-PrP transgenic mice, we found that neuron-selective expression of PrPC sufficed to confer susceptibility of mice to prion infection by aerosols and intranasal application. Hence PrPC expression in non-neural tissues is not required for aerosolic or intranasal neuroinvasion.
Following peripheral exposure, many TSE agents accumulate and replicate in host lymphoid tissues, including spleen, lymph nodes, Peyers' patches, and tonsils [59], [64], [65], [66], [67], [68], [69] in B-cell and lymphotoxin-dependent process [70], [71]. After peripheral replication in the LRS, prions gain access to the CNS primarily via peripheral nerves [23]; the innervation of secondary lymphoid organs and the distance between FDCs and splenic nerve endings is rate-limiting step for neuroinvasion [38]
[39].
In contrast to the above, aerosolic and intranasal exposure led to prion infection in the absence of B-, T-, NK-cells and mature FDCs. Although a trend towards a slight delay in incubation time was detected in certain immunodeficient mice (e.g. LTβR−/− and C3C4−/−) and after LTβR-Ig treatment, these differences were not statistically significant, and all other immunodeficient (JH
−/−, Rag1
−/− and γCRag2
−/−) as well as complement-deficient (e.g. C3C4 and CD21) mice were susceptible to aerosolic and intranasal prion infection similarly to control mice. We conclude that transmission into the CNS upon aerosolic prion inoculation requires neither a functional adaptive immune system nor microanatomically intact germinal centers with mature FDCs. Further, the interference with LT signaling, be it by LTβR-Ig treatment or through ablation of the LTβR, indicates that the anatomical and functional intactness of lymphoid organs is dispensable for prion neuroinvasion, brain prion replication, and clinical scrapie.
Since genetic removal of the main cellular components of the LRS (e.g. by intercrosses with mice lacking T-, B-cells or NK-cells in, JH−/− or γCRag2−/− mice) as well as genetic (LTα−/−; LTβR−/−) or pharmacological (LTβR-Ig) depletion of follicular dendritic cells - the main cell responsible for prion replication in secondary lymphoid organs - did not change the course of disease upon infection with prion aerosols, we conclude that the above data demonstrate that the LRS is dispensable for prion infection through the aerogenic route. We therefore propose that airborne prions follow a pathway of direct prion neuroinvasion along olfactory neurons which extend to the surface of the olfactory epithelium. The infectibility of newborn mice supports this hypothesis, since these mice lacked a fully mature immune system at the time of prion exposure.
Our results contradict previous studies [12] claiming a role for the immune system in neuroinvasion upon intranasal prion infection, but are consistent with recent work [11] showing that prion neuroinvasion from the tongue and the nasal cavity can occur in the absence of a prion-infected LRS. Transmission of CWD to “cervidized” transgenic mice via aerosols and upon intranasal administration has also been shown [45].
Both LTβR
−/− and LTα
−/− mice lack Peyer's patches and lymph nodes as well as an intact NALT which may influence prion replication competence [11], [12], [29], [30], [31], [32], [33], [34], [63]. Furthermore, these mice display chronic interstitial pneumonia. Consistently with a role for LTβR-signaling in peripheral prion infection, these mice do not replicate intraperitoneally administered prions. On the other hand, TNFR1
−/− mice lack Peyer's patches, show an aberrant splenic microarchitecture, an abnormal NALT, but have intact lymph nodes where prion replication can occur efficiently [72]. However, prion replication efficacy in spleen is almost completely abrogated [73] and TNFR1
−/− mice die due to scrapie after a prolonged incubation time when peripherally challenged with prions.
In the present study, all LTα−/− mice succumbed to scrapie upon intranasal infection, whereas some LTα−/− mice acquired prion infection following nasal cavity exposure in a previous study [11]. The requirement for the LRS in intranasal prion infection may depend on the particular prion strain being tested and on the size of the administered inoculum. When present in sufficiently high titers, prions may be able to directly enter the nervous system via the nasal mucosa and olfactory nerve terminals (Fig. 8). However, at limiting doses, aerial prion infection may be potentiated by an LRS-dependent preamplification step (Fig. 8), e.g. in the bronchial lymph nodes (BLNs), the nose, the gut-associated lymphoid tissue (NALT; GALT), or the spleen. In this study, the particle size generated by the nebulizer ensured that the entire respiratory tract was flooded by the aerosol so that the prion-containing aerosolized brain homogenate would reach the alveolar surface of the lung. There, prions may also colonize airway-associated lymphoid tissues and gain access to the CNS (Fig. 8).
10.1371/journal.ppat.1001257.g008Figure 8 Model of the possible pathways of aerogenic prion transmission.
(left) Prion aerosols entering the nasal cavity (1) may directly migrate through the nasal epithelium towards olfactory nerve terminals (2). Subsequently, prions reach olfactory bulb neurons and colonize the limbic system and other regions of the brain (3). Prions may be taken up by the eyes from where they could be transported via the visual system (e.g. optic nerves) to the CNS. O: olfactory system; V: visual system. Alternatively (right) prions may be taken up by immune cells residing in (1) the nasal cavity, the lung, or (2) the gastrointestinal tract, from where they may be transferred to lymphoreticular system (LRS) components such as bronchial lymph nodes (BALT), nasal associated lymphoid tissue (NALT), gastrointestinal lymphoid tissue (GALT), mesenteric lymph nodes, or spleen for further amplification. Subsequently, prions traffic towards peripheral nerve terminals (PNS), from where they invade the central nervous system (CNS). SC: spinal cord. Arrows indicate possible migration directions of prions once they have invaded the spinal cord.
Infection through conjunctival or corneal structures was not required, since newborn mice succumbed to scrapie with an incidence of 100% despite having closed eyelids. While newborn tga20, but not CD-1, mice experienced slightly prolonged incubation times when compared to adult (6–8 week-old) mice of the same genotype, the anatomical structures of the nasopharynx (e.g. olfactory epithelium and olfactory nerves) are not similarly developed at postnatal day one when compared to adulthood, potentially leading to a less efficient prion uptake upon aerosol exposure (e.g. via olfactory nerves). Although unlikely, it can not be excluded that infection through conjunctival or corneal structures might contribute to a more efficient prion infection upon aerosol exposure. Be as it may, all newborn mice of either genotypes succumbed to terminal scrapie upon aerosol prion infection despite their lack of fully developed lymphoid organs, thereby bolstering our conclusion that the immune system is dispensable for prion transmission through aerosols.
In summary, our results establish aerosols as a surprisingly efficient modality of prion transmission. This novel pathway of prion transmission is not only conceptually relevant for the field of prion research, but also highlights a hitherto unappreciated risk factor for laboratory personnel and personnel of the meat processing industry. In the light of these findings, it may be appropriate to revise current prion-related biosafety guidelines and health standards in diagnostic and scientific laboratories being potentially confronted with prion infected materials. While we did not investigate whether production of prion aerosols in nature suffices to cause horizontal prion transmission, the finding of prions in biological fluids such as saliva, urine and blood suggests that it may be worth testing this possibility in future studies.
Material and Methods
Ethics statement
Animals were maintained under specific pathogen-free conditions and experiments were approved and conform to the guidelines of the Swiss Animal Protection Law, Veterinary office, Canton Zurich. Mouse experiments were performed under licenses 40/2002 and 30/2005 according to the regulations of the Veterinary office of the Canton Zurich and in accordance with the regulations of the Veterinary office Tübingen.
Aerosols
Exposure of mice to aerosols was performed in inhalation chambers containing a nebulizer device (Art.No. 73-1963, Pari GmbH, Munich, Germany) run with a pressure of 1.5 bar generating 100% particles below 10 µm with 60% of the particles below 2.5 µm and 52% below 1.2 µm. Such particle sizes are considered to be able to reach upper and lower airways [74]. Prion infected material used throughout this study was RML6 strain obtained from the brains of diseased CD1 mice in its 6th passage (RML6). Mice were exposed to aerosolized prion infected brain homogenates for one, five or ten minutes.
Intracerebral prion inoculation of mice
tga20 mice serving as indicator mice were inoculated i.c. with brain tissue homogenate using 30 µl volumes (RML6 0.1%, 3×105 LD50 scrapie prions). The animals were checked on a daily basis and were sacrificed when showing defined neurological signs such as severe gait disorders.
Intranasal prion application in mice
Mice were anesthetized with Ketamine/Xylazin hydrochloride anaesthesia. 10 µl of RML6 (0.1%) were intranasally inoculated in each nostril and on the nasal epithelium by using a 10 µl pipette. The mice were held horizontally during inoculation process and for 1 minute following the inoculation. The whole procedure was repeated after a break of 20 minutes, reaching a final volume of 40 µl of RML6, 0.1% (4×105 LD50 scrapie prions).
Intraperitoneal prion application in mice
Mice were anesthetized with Ketamine/Xylazin hydrochloride anaesthesia. 100 µl of RML6 (0.1%) 1×106 LD50 scrapie prions were i.p. inoculated into Rag1
−/− and γCRag2
−/− mice.
Western blotting
Tissue homogenates were prepared in sterile 0.32 M sucrose using a Fast Prep FP120 (Savant, Holbrook, NY, USA) or a Precellys 24 (Bertin Technologies). For detection of PrPSc 15µl of a brain homogenate were digested with Proteinase K (30 µg/ml) and incubated for 30 min at 37°C. For detection of PrPC no digestion was performed. Proteins were separated by SDS-PAGE and transferred to a PVDF (Immobilon-P, Millipore, Bedford, Mass., USA) or nitrocellulose membrane (Schleicher & Söhne). Prion proteins were detected by enhanced chemiluminescence (Western blotting reagent, Santa Cruz Biotechnology, Heidelberg, Germany) or ECL (from PerbioScience, Lausanne, CH), using mouse monoclonal anti-PrP antibody POM-1 and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG1 antibody (Zymed).
Histoblot analysis
Histoblots were performed as described previously [73]. Frozen brains that were cut into 12 µm-thick slices were mounted on nitrocellulose membranes. Total PrP, as well as PrPSc after digestion with 50 or 100 µg/ml proteinase K for 4 hrs at 37°C, were detected with the anti-prion POM1 antibody (1∶10000, NBT/BCIP, Roche Diagnostics).
Histological analysis
Formalin-fixed tissues were treated with concentrated formic acid for 60 min to inactivate prion infectivity. Paraffin sections (2µm) and frozen sections (5 or 10µm) of brains were stained with hematoxylin/eosin. Antibodies GFAP (1∶300; DAKO, Carpinteris, CA) for astrocytes were applied and visualized using standard methods. Iba-1 (1∶1000; Wako Chemicals GmbH, Germany) was used for highlighting activated microglial cells. Postfixation in formalin was performed for ∼8 hrs and tissues were embedded in paraffin. After deparaffinisation, for PrP staining sections were incubated for 6 min in 98% formic acid and washed in distilled water for 30 min. Sections were heated to 100°C in a steamer in citrate buffer (pH 6.0) for 3 min, and allowed to cool down to room temperature. Sections were incubated in Ventana buffer and stains were performed on a NEXEX immunohistochemistry robot (Ventana instruments, Switzerland) using an IVIEW DAB Detection Kit (Ventana). After incubation with protease 1 (Ventana) for 16 min, sections were incubated with anti-PrP SAF-84 (SPI bio, A03208, 1∶200) for 32 min. Sections were counterstained with hematoxylin.
Lesion profiling
We selected 5 anatomic brain regions from all investigated or at least 3 mice per experimental group. We evaluated spongiosis on a scale of 0–4 (not detectable, mild, moderate, severe and status spongiosus). Gliosis and PrP immunological reactivity was scored on a 0–3 scale (not detectable, mild, moderate, severe). A sum of the three scores resulted in the value obtained for the lesion profile for the individual animal. The ‘radar plots’ depict the scores for spongiform changes, gliosis and PrP deposition. Numbers correspond to the following brain regions: (1) hippocampus, (2) cerebellum, (3) olfactory bulb, (4) frontal white matter, (5) temporal white matter. Investigators blinded to animal identification performed histological analyses.
Misfolded Protein Assay (MPA)
Misfolded Protein Assay (MPA) was performed as described previously [75]. The assay, which was performed on a 96-well plate is divided into two parts: the PSR1 Capture and an ELISA. For the PSR1 Capture the set up of each reaction was as following: 3µL of PSR1 beads (buffer removed) and 100µl of 1× TBSTT were spiked with brain homogenate, incubated at 37°C for 1hr with shaking at 750rpm, the beads were washed on the plate washer (ELX405 Biotek) 8 times with residual 50 µl/well TBST. Then 75µl/well of denaturing buffer was added. This was incubated at RT for 10min with shaking at 750rpm. Subsequently 30µl/well of neutralizing buffer were added. An additional incubation at RT for 5min with shaking at 750rpm followed. The beads were pulled down with a magnet. The ELISA was performed as follows: 150µL/well of the sample was transferred to an ELISA plate which was coated with POM19. An incubation step at 37°C for 1hr with shaking at 300rpm followed. That was washed 6 times with wash buffer. POM2-AP conjugate had to be diluted to 0.01µg/mL in conjugate diluent. 150µL/well of diluted conjugate was added. Incubation at 37°C for 1hr without shaking followed. Washing 6 times with wash buffer was followed by preparation of enhanced substrate by adding 910µL of enhancer to 10mL of substrate (Lumiphos plus, Lumigen). 150µL/well of enhanced substrate was added. Incubation at 37°C for 30min was followed by reading by luminometer (Luminoskan Ascent) at default PMT, filter scale = 1.
Real-time RT-PCR for quantification of the transgene copy number in Prnp
o/o/NSE-PrP mice
Real-time PCR was performed on purified genomic DNA from mouse tails on a 7900 HT Fast Real-Time PCR System (AB). Data were generated and analyzed using SDS 2.3 and RQ manager 1.2 software. The following primers were used: Forward primer annealing in mouse Prnp gene intron 1: 5′ - GGT TTG ATG ATT TGC ATA TTA G - 3′. Reverse primer annealing in mouse Prnp gene exon 2: 5′ - GGA AGG CAG AAT GCT TCA GC - 3′. The PCR product is approximately 200 bps in length. For control, the mouse Lymphotoxin alpha gene was analyzed. The following primers were used: Forward primer annealing to the Exon 1 of the mouse Lymphotoxin alpha gene: 5′ - CCT GGT GAC CCT GTT GTT GG - 3′. Reverse primer annealing to the mouse Lymphotoxin alpha gene Intron 1: 5′ - GTG GGC AGA AGC ACA GCC - 3′. The PCR product is approximately 160 bps in lenght. Real time PCR analysis revealed 2–4 transgene copies per Prnp allele in Prnp
o/o
/NSE-PrP mice.
Statistical evaluation
Results are expressed as the mean+standard error of the mean (SEM) or standard deviation (SD) as indicated. Statistical significance between experimental groups was assessed using an unpaired two-sample Student's t-Test (Excel) and two-sample Welch t-Test for distributions with unequal variance (R). For survival analyses, Kaplan-Meier-survival curves were generated using SPSS or R software, statistical significance was assessed by performing log rank tests (R). Linear regression fits and analyses of variance (ANOVA) were conducted in R (www.r-project.org).
Supporting Information
Figure S1 Analysis of variance (ANOVA) for various genotypes regarding incubation times.
Dot plot of survival times for tga20, CD1, C57BL/6 and 129SvxC57BL/6 mice after 10 min exposure to IBH. The difference between genotypes is significant (p<0.001).
(0.14 MB TIF)
Click here for additional data file.
Figure S2 Prion transmission through aerosols or upon intranasal challenge cannot be directly followed by Western blot and PMA.
(A–D) Western blot analysis for PrPSc of the compartments olfactory epithelium (OE), olfactory bulb (OB), one brain hemisphere or the cerebellum at 60 days post aerosolic infection in different mouse strains (C57BL/6, CXCR5−/−, CD21−/−, Prnp
o/o, CD1, LTα−/−, NSE-PrP, TNFR1−/− mice). (+) and without (−) previous proteinase K (PK) treatment as indicated. Molecular weights (kDa) are indicated on the right side of the blots. kDa: Kilo Dalton. β-Actin served as a loading control. (E) Analysis of brain tissue homogenates from intranasally inoculated mice by the “misfolded protein assay” (MPA) reveals positive signals as indicators of PrpSc deposition in the control specimen (red) and negative signal in most cases investigated (blue, below wt level). The brain of a wt mouse was used as control (black). Y-axis: RLU: Relative (Chemi) Luminescence Units, X-axis: mouse number, blue column represents a 1∶10 dilution of a 10% brain homogenate. Data for uninfected brain controls are not shown here, but did not display a positive signal. (F) Analysis of olfactory bulb tissue homogenates from intranasally inoculated mice by the “misfolded protein assay” (MPA) reveals positive signals as indicators of PrpSc deposition in the control specimen (red) and in diseased mice (positive signals). Negative signals in most other cases investigated (blue bars under wt control level). The brain of a wt mouse was used as control (black). Y-axis: RLU: Relative (Chemi) Luminescence Units, X-axis: mouse number, blue column represents a 1∶10 dilution of a 10% brain homogenate. Data for uninfected brain controls are not shown here, but did not display a positive signal.
(0.68 MB TIF)
Click here for additional data file.
Figure S3 Detailed quantitative analysis of PrPC expression in the olfactory epithelium and the CNS.
(A) Quantification of PrPC expression levels by Western blotting in NSE-PrP, wt and tga20 mice. In the olfactory epithelium tga20 mice ≥3.5 fold higher PrPC expression compared to wt mice and approximately 11 fold higher PrPC expression compared to NSE-PrP mice. (B) In the olfactory bulb of tga20 mice ≥1.5 fold higher PrPC expression compared to wt mice and more than 3.5 fold higher PrPC expression compared to NSE-PrP mice. (C) In brain hemispheres of tga20 mice more than 2.5 fold higher PrPC expression compared to wt mice, and more than 1.5 fold higher PrPC expression compared to NSE-PrP mice. Molecular weights (kDa) are indicated on the right side of the blots. kDa: Kilo Dalton. β-Actin served as a loading control.
(1.48 MB TIF)
Click here for additional data file.
Figure S4 Splenic involvement after prion transmission through aerosols and involvement of the spleen.
(A) tga20 mice, (B) JH−/− mice, (C) LTβR−/− mice and (D) γCRag2
−/− mice in part show splenic deposits of PK resistant material, evaluated by Western blot analysis. (E) Histoblot analysis of spleens of C57BL/6, of Prnp
o/o and of Rag1
−/− mice. C57BL/6 mice reveal PK resistant deposits while Prnp
o/o and Rag1
−/− mice lack such deposits. (F) Western blot analysis of a representative tga20 spleen, mesenteric lymph node (mln) and bronchial lymph node (bln) lacking PK-resistant material. (+) and (−) with or without PK treatment. POM1 was used as primary antibody.
(2.81 MB TIF)
Click here for additional data file.
Figure S5 Prion transmission via the intranasal route.
Survival curves of (A) C57BL/6, (B) 129SvxC57BL/6 (C) tga20 and (D) Prnp
o/o mice that have been intranasally inoculated with RML6 0.1%. (E) Survival curve of C57BL/6 mice that have been intranasally inoculated with HBH. Western blots of brains of (F) C57BL/6 mice and of (G) tga20 mice that have been intranasally inoculated with 4×105 LD50 scrapie prions. Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Homogenate derived from a terminally scrapie-sick mouse served as positive control (s: sick), and healthy C57BL/6 mouse brain homogenate as negative control (h: healthy), respectively. Molecular weights (kDa) are indicated on the left side of the blots. (H) Survival curves of NSE-PrP mice intranasally inoculated with prions are shown (left panel). Respective Western blots of NSE-PrP mice intranasally inoculated with prions are shown (right panel). Brain homogenates were analyzed with (+) and without (−) previous proteinase K (PK) treatment as indicated. Homogenate derived from a terminally scrapie-sick mouse served as positive control (s: sick), and healthy C57BL/6 mouse tissue as negative control (h: healthy), respectively and i.d. indicates intercurrent death of animal. Molecular weights (kDa) are indicated on the left side of the blots. (I) Histoblot analysis of prion infected mouse brains. Left panel: shows healthy brain of a Prnp
o/o mouse as negative control, other panels demonstrate PrpSc deposits in brains of tga20 mice. Scale bars are indicated. (J) Histological and immunohistochemical characterization of scrapie affected mouse brains. Brain sections of Prnp
o/o, tga20, 129SvxC57BL/6 and C57BL/6 mice as evaluated by HE (for spongiosis, gliosis, neuronal cell loss), SAF84 (for PrPSc deposits), GFAP (for astrogliosis) and Iba-1 (for microglial activation). Scale bars: 100µm. (K) Histopathological lesion severity score of 5 brain regions described as radar blot (astrogliosis, spongiform change and PrPSc deposition) of intranasally prion inoculated tga20, C57BL/6 and 129SvxC57BL/6 mice.
(1.04 MB TIF)
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Figure S6 Prion transmission via the intranasal route, controls and involvement of the spleen.
(A) Rag1
−/− mice intranasally inoculated with HBH (40 µl) or (B) C57BL/6 intranasally inoculated with 4×105 LD50 scrapie prions. (C) γCRag2
−/− mice intranasally inoculated with HBH (40 µl) or (D) Balb/c mice intranasally inoculated with HBH are shown. Kaplan-Meier curves describe the percentage of survival after particular time points post intranasal prion inoculation (y-axis represents percentage of living animals; x-axis demonstrates survival time in days). Respective Western blots of (E) spleens and bln of terminal Rag1
−/− mice, (F) of spleens of terminal C1qa
−/− mice, (G) spleens of terminal CD21−/− mice, (H) spleens of terminal CXCR5−/− mice, (I) spleens of terminal LTβR−/− mice, (J) spleens of terminal TNFR1−/− mice, (K) spleens of terminal LTα−/− and LTβR−/− mice and (L) spleens of terminal tga20 mice.
(1.41 MB TIF)
Click here for additional data file.
Figure S7 Histological, immunohistochemical and immunoblot confirmation of prion disease upon aerosol infection.
(A, B) Histological and immunohistochemical characterization of scrapie affected CD1 mouse brains. (A) Representative olfactory bulbs (HE and Neurofilament stains) are shown. scale bars indicated (B) The cerebellum (upper row), the midbrain (second row from top), the frontal cortex (third row from top) and the olfactory bulb (lower row) display spongiosis, astrogliosis, microglial activation and PrpSc deposits upon prion infection via aerosols (HE, GFAP, Iba-1 and SAF-84 staining). Scale bars: 50 γm.
(8.63 MB TIF)
Click here for additional data file.
Table S1
Survival times of mouse strains exposed to prion aerosols for various periods.
(A) Analysis of variance for plates in Fig. 1F–G and Fig. S1. The time of exposure to aerosolized infectious brain homogenates, but not their concentration, significantly correlated with survival time. (B) Linear regression fits for survival time against exposure time in tga20 (Fig. 1G) and CD1 (Fig. S1) mice. Incubation times correlated negatively with PrP expression level. (C) Pair wise tests for differing mean survival time for tga20, CD1, C57BL/6 and 129SvxC57BL/6 mice after 10 minutes exposure to prion aerosols (Fig. S1), identifying Prnp gene copy number as the strongest independent variable. P<0.001: ***, P<0.01: **, P<0.05: *, P<0.1.
(0.06 MB DOC)
Click here for additional data file.
The authors thank Silke Gaedt and Rita Moos for technical assistance, Petra Reinhold for advice on aerosol treatment, Dr. Jeffrey Browning for providing LTβR-Ig, and Drs. Tobias Junt and Tracy O'Connor for discussions.
The authors have declared that no competing interests exist.
This work was supported in part by EU grants ANTEPRION and PRIORITY (LS), and the TSE-Forschungsprogramm des Landes Baden-Wuerttemberg, Germany (LS). This work was also supported by grants from the UK Department of Environment, Food and Rural Affairs (AA), the EU grants LUPAS and PRIORITY (AA), the Novartis Research Foundation (AA), and an Advanced Grant of the European Research Council to AA. MH was supported by the Foundation for Research at the Medical Faculty, the Prof. Dr. Max-Cloetta foundation and the Bonizzi-Theler Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21249152PONE-D-10-0188210.1371/journal.pone.0016068Research ArticleMedicineOncologyBasic Cancer ResearchMetastasisCancer TreatmentChemotherapy and Drug TreatmentCancers and NeoplasmsLung and Intrathoracic TumorsUp-Regulation of Sonic Hedgehog Contributes to TGF-β1-Induced Epithelial to Mesenchymal Transition in NSCLC Cells TGF-β1 Up-Regulate Sonic Hedgehog ProteinMaitah Ma'in Y.
1
Ali Shadan
2
Ahmad Aamir
1
Gadgeel Shirish
2
Sarkar Fazlul H.
1
*
1
Department of Pathology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America
2
Division of Hematology/Oncology, Department of Internal Medicine, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America
Agoulnik Irina EditorFlorida International University, United States of America* E-mail: [email protected] and designed the experiments: MYM FHS. Performed the experiments: MYM. Analyzed the data: MYM FHS SA AA SG. Contributed reagents/materials/analysis tools: FHS. Wrote the paper: MYM FHS SA.
2011 13 1 2011 6 1 e1606814 9 2010 5 12 2010 Maitah et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Lung cancer, especially non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths in the United States. The aggressiveness of NSCLC has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT). The acquisition of EMT phenotype induced by TGF-β1in several cancer cells has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in NSCLC remains unknown.
Methodology/Principal Findings
In this study we found for the first time that the induction of EMT by chronic exposure of A549 NSCLC cells to TGF-β1 (A549-M cells) led to the up-regulation of sonic hedgehog (Shh) both at the mRNA and protein levels causing activation of hedgehog signaling. These results were also reproduced in another NSCLC cell line (H2030). Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion. The aggressiveness of these cells was attenuated by the treatment of A549-M cells with pharmacological inhibitors of Hh signaling in addition to Shh knock-down by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin. In addition, knock-down of Shh by siRNA significantly attenuated EMT induction by TGF-β1.
Conclusions/Significance
Our results show for the first time the transcriptional up-regulation of Shh by TGF-β1, which is mechanistically associated with TGF-β1 induced EMT phenotype and aggressive behavior of NSCLC cells. Thus the inhibitors of Shh signaling could be useful for the reversal of EMT phenotype, which would inhibit the metastatic potential of NSCLC cells and also make these tumors more sensitive to conventional therapeutics.
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Introduction
An estimated 1.35 million individuals were diagnosed with lung cancer worldwide in 2009. Lung cancer is the most common cause of cancer related mortality in the United States, with more than 160,000 deaths per year and 85% of all lung cancers are non-small cell lung cancer (NSCLC) [1]. Greater than 70% of NSCLC patients, at present, shows metastases to the regional lymph nodes or to distant sites [2]. While, systemic therapy plays a major role in the management of most NSCLC patients, the benefits of systemic therapy are modest. The median survival of NSCLC patients with distant metastases ranges from 9-12 months, with median progression free survival (PFS) of only 3.5 to 5.5 months. Therefore, there is an urgent need to develop novel therapies based on newer understanding of the molecular mechanisms and pathways that participate in lung carcinogenesis for better and improved treatment of patients diagnosed with NSCLC.
Emerging evidence suggests that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype could be induced by Transforming Growth Factor-β (TGF-β) especially TGF-β1 among other factors, resulting in tumor invasiveness, and these EMT-type cells have been classified as cancer stem-like cells in recent studies [3]. The importance of EMT process have been established in embryonic development [4]. Lately, EMT has also been found to play a critical role in tumor invasion, metastatic dissemination and the acquisition of resistance to conventional therapies [5]–[12]. Moreover, EMT phenotype in cancers has been associated with poor clinical outcome in multiple cancer types including NSCLC, yet the molecular mechanisms underlying the induction of EMT by TGF-β1 remain ill-defined especially for NSCLC [13]–[17].
Since the acquisition of an EMT phenotype has emerged as an important mediator of cancer progression, cancer metastases and resistance to both chemotherapy and targeted drugs such as EGFR inhibitors, thus further mechanistic studies to ascertain the role of TGF-β1- induced EMT are warranted. The clinical relevance of EMT and drug insensitivity comes from recent studies showing an association between epithelial markers and sensitivity to erlotinib in NSCLC cell lines [17]–[19], suggesting that EMT-type cells are resistant to erlotinib, however the role of signaling molecules in mediating the induction of EMT by TGF-β1 is lacking. Among the various molecular pathways, the Hedgehog (Hh) signaling pathway has emerged as an important mediator of carcinogenesis and cancer metastases [20], [21]. Studies have shown that the Hh signaling pathway, a pathway normally active in human embryogenesis and tissue repair, is also active in many cancers including NSCLC [22]–[25]. Hh inhibitors are now being tested in preclinical and clinical settings based on findings that the inhibition of Hh signaling could inhibit cell growth, invasion and metastasis of cancer cells [26]–[28]. The Hh signaling pathway is comprised of the ligand sonic, indian, and desert hedghog (Shh, Ihh, Dhh, respectivly), and the cell surface molecules Patched (PTCH) and Smoothened (SMO). In the absence of an Hh ligand, PTCH causes suppression of SMO [29], [30]; however, upon ligand binding to PTCH, SMO protein translocates into the primary cilium, and leads to the activation of transcription factor GLI1, which then translocates to the nucleus, leading to the expression of Hh target genes [29], [30]. GLI1-mediated expression of genes is involved in cell growth and differentiation [29], and thus the activation of Hh signaling is believed to play an important role in tumor cell invasion and metastasis.
Based on the above findings and the lack of mechanistic studies in establishing the role of TGF-β1-induced activation of Hh signaling with respect to the acquisition of EMT and tumor cell aggressiveness, we used NSCLC cells as a preclinical model for the current study. Here we show for the first time that chronic exposure of A549 cells (NSCLC cells) to TGF-β1 led to the acquisition of EMT phenotype with concomitant up-regulation of sonic hedgehog (Shh) both at the mRNA and at the protein levels, which is consistent with findings in another NSCLC cell line (H2030). The up-regulation of sonic hedgehog was consistent with increased cell motility, invasion, and tumor cell aggressiveness. In addition, we found that this process could be attenuated by Shh siRNA as well as by chemical inhibitors of Hh signaling such as cyclopamine and GDC-0449. Moreover, we found that the inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin, suggesting that the acquisition of EMT phenotype by TGF-β1 in NSCLC cells is mechanistically mediated by the activation of Shh signaling because the knock-down of Shh by Shh specific siRNA attenuated TGF-β1-induced EMT phenotype.
Results
Induction of epithelial-to-mesenchymal transition (EMT) in A549 NSCLC cells by chronic exposure to TGF-β1
It has been reported that A549 cells undergoes EMT phenotypic changes upon exposure to TGF-β1 [13], [14]. This was seen as trans-differentiation, especially because the exposure was done for a short period of time (48–72 hours). In an attempt to recapitulate the in vivo situation where cells are chronically exposed to TGF-β1 in the tumor microenvironment, we exposed A549 cells to TGF-β1 up to three weeks. After 21 days of exposure to TGF-β1, A549 cells morphology was found to be completely changed to a mesenchymal phenotype (we termed this cells as A549-M cells), with an elongated and disseminated appearance (Fig. 1A). To confirm the mesenchymal phenotype, we assessed the expression of molecular markers of EMT such as ZEB1 mRNA, which has been reported earlier to serve as a mesenchymal marker [31], [32], and we found that ZEB1 was up-regulated, while the expression of E-cadherin mRNA, an epithelial marker, was down-regulated (Fig. 1B). Fibronectin protein, a mesenchymal marker [17], was also found to be highly up-regulated in A549-M cells (Fig. 1C).
10.1371/journal.pone.0016068.g001Figure 1 Induction of epithelial to mesenchymal transition (EMT) in A549 cells by chronic exposure to TGF-β1:
TGF-β1 was added to A549 cells in culture media and maintained for 21 days with changing medium every third day with freshly added TGF-β1. A) Phase contrast objective microscopic pictures at 10× magnification. A549 cells morphology changed to mesenchymal phenotype (A549-M cells). Cell shape appears elongated and non-polarized. B) qRT-PCR of A549 and A549-M cells. A549-M cells showed a lower E-cadherin “epithelial marker,” and a higher ZEB1 “EMT marker”, at the mRNA levels. Delta-delta-CT was calculated, considering GAPDH as internal control and A549 parental as reference control. C) Western blot analysis where A549-M cells showed up-regulation of fibronectin “mesenchymal” marker compared to A549 parental cells.
A549-M cells showed significant increase in cell migration and invasive characteristics compared to the A549 parental cells
Previous studies have shown that tumor cells with EMT phenotype are more motile [14], [17], [32], [33]. In order to further characterize A549-M cells, we performed a wound healing assay which showed increased cell migration of A549-M cells compared to parental cells (Fig. 2A). Moreover, we also found that A549-M cells are more invasive as documented by increased invasion as documented by matrigel-coated chamber assay (Fig. 2B), and A549-M cells also acquired more tumorigenic phenotype as documented by increased clonogenic growth (Fig. 2C).
10.1371/journal.pone.0016068.g002Figure 2 A549-M cells shows significant increase in migration, invasive, and tumorigenic characteristics compared to A549 parental cells:
TGF-β1-induced EMT phenotypic cells (A549-M cells) were generated as discussed under “Materials and Methods” section. A: showed wound healing assay results with its quantitative analysis. A549-M cells showed much higher motility compared to A549 parental cells. B and C showing the results of matrigel-coated membrane, and colony formation assays, respectively with its quantitative analysis. Significant increase was observed in the invasion and clonogenicity of A549-M cells compared to parental A549 cells. (* = p<0.05).
A549-M cells showed up-regulation of sonic hedgehog mRNA, and protein expression
In order to assess the mechanism by which chronic TGF-β1 treatment induced EMT and tumor cell aggressiveness, we focused our investigation on Hh signaling because it has been implicated in EMT induction, metastasis and invasion [20]–[24], [26], [34], [34]–[37]. Interestingly, we found a dramatic increase in the expression of Hh pathway ligand Shh both at the mRNA and protein levels in A549-M cells whereas the parental A549 cells showed undetectable levels of Shh mRNA (Fig. 3A and Fig. 3B), which is consistent with published data showing that A549 parental cells contains undetectable levels of Shh expression [35]. In order to further confirm our findings documenting up-regulation of Shh by TGF-β1 treatment, and the induction of EMT in A549 NSCLC cell lines, we treated another NSCLC cell line (H2030 cells) with TGF-β1 for two weeks, and we found a significant increase in the expression of Shh mRNA, which was consistent with the induction of EMT marker ZEB1 and down-regulation of epithelial marker E-cadherin (Fig. 4A). These results suggest that TGF-β1 induced EMT is mediated by the transcriptional activation of Shh, which is the first such report in the literature.
10.1371/journal.pone.0016068.g003Figure 3 A549-M cells showed up-regulation of sonic hedgehog (Shh) and GLI expression both at the mRNA and protein levels:
A and B showing qRT-PCR and Western blot results, respectively for the expression of Shh whereas C and D represent the expression status of GLI at the mRNA and protein levels, respectively in A549-M cells compared to parental A549 cells. E represent Western blot data of GLI1 expression in NIH-3T3 cell after culturing with A549-M-derived conditioned media showing higher levels of GLI1 expression. (* = p<0.05).
10.1371/journal.pone.0016068.g004Figure 4 Shh up-regulation is concomitant with TGF-β1-induced EMT in NSCLC cell lines.
The up-regulation of Shh contributes to the EMT induction through TGF-β1. (A) H2030 cell line was treated with TGF-β1 (5 ng/ml) for two weeks, and the media was changed every three days. The qRT-PCR data showed induced expression of EMT marker ZEB1 mRNA, and reduced expression of epithelial marker E-cadherin mRNA, which was consistent with up-regulation of Shh mRNA similar to those observed in A549 cells exposed to TGF-β1. (B, C and D) A549 cells was transfected with Shh siRNA (A549-siShh) or scrambled siRNA (A549-si-ve) for 24 hrs prior to treatment with TGF-β1 (5 ng/ml) for 48 hrs, then the cells where collected for assays or re-transfected for the second time with siRNA or scrambled siRNA for 24 hrs (total 6days after siShh transfection) prior to the second time treatment with TGF-β1 (5 ng/ml) for another 48 hrs (total 5days of TGF-β1 treatment). (B) Upper panel shows transfection efficiency, and lower panel shows cellular morphology following treatments. A549-siShh maintained epithelial morphology after treatment with TGF-β1 at both time points as shown in left and right panels, respectively. (C) qRT-PCR expression of Shh mRNA showing significant down-regulation following Shh siRNA transfection (D) qRT-PCR expression of ZEB1 and E-cadherin mRNA. A549-si-ve cells showed down-regulation of epithelial marker, E-cadherin consistent with significant induction in the expression of ZEB1 as expected whereas TGF-β1 failed to show any effect on these markers in A549-siShh cells.
Interestingly, GLI1 levels in both A549-M and parental cells were higher compared to normal human bronchial epithelial cells (NHBE cells) although the expression of GLI1 was much more increased in A549-M cells (Fig. 3C and Fig. 3D). The high levels of Hh target gene GLI1 in both A549-M and A549 cells was observed despite the undetectable levels of Shh in A549 parental cells, which suggests that the expression of GLI1 could be ligand-independent in the parental A549 cells. Moreover, since Shh protein expression in A549-M cells appears to induce the GLI1 expression by an autocrine process, we further investigated this possibility using NIH-3T3 cells cultured with condition-media of A549-M cells. NIH-3T3 cells express Hh signaling receptor and transcription factor GLI1, and our data confirmed showing increased Hh signaling consistent with increased expression of GLI1 (Fig. 3E) in NIH-3T3 cells cultured with A549-M condition media. These results clearly show that A549-M cells secrete active Shh which can then activate Hh signaling in NIH 3T3 cells, resulting in the activation of GLI1. We further investigated the possibility whether Shh up-regulation directly mediates EMT induction by TGF-β1 or not. We found that knock-down of Shh by siRNA significantly attenuated TGF-β1 induced EMT, which was confirmed morphologically and molecularly as presented below. A549 cells transfected with Shh siRNA 24 hrs prior to treatment with TGF-β1 for 48 hrs (A549-siShh) maintained epithelial morphology, while scrambled siRNA (A549-si-ve) showed transformation to mesenchymal morphology (Fig. 4B left panel). Likewise, A549-si-ve showed more EMT induction compared to A549-siShh following re-transfection with Shh siRNA and treatment with TGF-β1 [Total six days of Shh siRNA transfection and five days after TGF-β1 treatment; details under figure legend (Fig. 4B right panel)]. The Shh siRNA trasfection resulted in significant knock-down of Shh expression as shown by qRT-PCR (Fig. 4C). We found significant attenuation in the induction of EMT by TGF-β1 treatment in A549 cells with Shh knock-down (A549-siShh cells) as confirmed by qRT-PCR. A549-si-ve cells showed down-regulation of epithelial marker, E-cadherin consistent with significant induction in the expression of ZEB1 as expected (Fig. 4D, 48 hrs TGF-β1) whereas TGF-β1 failed to show effect on these markers in A549-siShh cells. Moreover, we found further attenuation in EMT induction following second round of Shh siRNA transfection and TGF-β1 treatment. A549-si-ve cells showed a significant increase in ZEB1 expression consistent with significant down-regulation of E-cadherin (Fig. 4D, 5 days TGF-β1) whereas TGF-β1 failed to show effect on these markers in A549-siShh cells. These results demonstrated for the first time that Shh up-regulation by TGF-β1 is mechanistically linked with TGF-β1 induced EMT in NSCLC cells.
Up-regulation of Shh in A549-M contributes to EMT-induced tumor cell migration and metastatic characteristics
Next, we investigated the role of increased expression of Shh in aggressive behavior such as migratory and metastatic potential of A549-M cells. We treated A549-M cells with Shh inhibitors such as cyclopamine and GDC-0449 and assessed their migration and invasion characteristics. Both cyclopamine and GDC-0449 significantly reduced cell migration and invasive capacity of A549-M cells (Fig. 5A–C and Fig. S1). Moreover, the treatment of A549-M cells by either cyclopamine (data not shown) or GDC-0449 showed partial reversal, where we observed incomplete attenuation of EMT phenotype, as documented by the reduced expression of fibronectin and ZEB1, and increased expression of epithelial marker E-cadherin (Fig. 5D and E).
10.1371/journal.pone.0016068.g005Figure 5 Up-regulation of Shh in A549-M cells contributes to increased tumor cells migration and metastatic characteristics:
A549-M cells were treated with Shh inhibitors such as Cyclopamine (2 µM) and GDC-0449 (20 nM) and assayed for wound healing (A), invasion (B) and clonogenic growth (C), and performed quantitative analysis showing attenuation of invasion by the treatment with Shh inhibitors. Western blot of A549-M cells before and after treatment with GDC-0449 (20 nM) for the expression of fibronectin (D). qRT-PCR for the expression of E-cadherin and ZEB1 mRNA in A549-M cells after treatment with GDC-0449 (20 nM) showing reversal of EMT phenotype compared to untreated A549-M cells (E). (* = p<0.05).
In order to further confirm the role of the induced expression of Shh by TGF-β1 in A549-M cells and its mechanistic association with increased cell migration, invasion and tumorigenesis, we knock-down the expression of Shh protein in A549-M cells by Shh-specific siRNA, and further assessed the transfection efficiency, which showed robust transfection efficiency (Fig. 6A). The knock-down of Shh protein in A549-M cells showed significant reduction in cell migration, invasion, and tumorigenic characteristics (Fig. 6B–D). The data clearly suggests the reversal of EMT morphology by the knock-down of Shh protein in A549-M cells (Fig. 6B inset). These data further confirmed that the inhibition in cell migration, invasion, and tumorigenic potential of A549-M cells is mechanistically mediated through the inhibition of Shh-mediated autocrine signaling.
10.1371/journal.pone.0016068.g006Figure 6 Reduction in A549-M cells motility, invasiveness, and tumorigenesis by specific knock-down of Shh using Shh-specific siRNA:
A549-M cells were transfected with Shh-specific siRNA (A): Transfection efficiency as assessed by GFP. The effect of knock-down of Shh was assessed by cell motility (wound healing) (B), invasion (C) and clonogenic growth (D) and further quantitated as detailed under “Materials and Methods” section, showing significant inhibition by Shh specific siRNA. (* = p<0.05).
Down-regulation of Shh autocrine signaling in additional NSCLC cell lines led to the reduction in tumor cell migration, invasion, and tumorigenic characteristics
In order to further investigate whether the inhibition of Shh autocrine signaling leads to the reduction in cell migration, invasion, and tumorigenesis in other NSCLC cell lines that expresses Shh, we chose H1299 and H1650 cell lines, both of which were derived from lung metastasis of NSCLC patients. Both cell lines have been shown to be resistant to chemotherapy and targeted therapy (e.g. Erlotinib) [17], [38], [39]. Our results confirmed that both the cell lines expressed Shh as documented by qRT-PCR and Western blot analysis (Fig. 7A). Treatment of H1650 cells with Shh inhibitors GDC-0449 showed decreased cell migration, invasion and tumorigenic characteristics (Fig. 7B–C and Fig. S1), which clearly provide strong experimental support in favor of the role Shh in EMT phenotype. It is important to note that the treatment of H1650 cells with GDC-0449 led to the partial reversal of the EMT phenotype as documented by the reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. 7D and E), which is consistent with the data in A549-M cells as presented under Fig. 5D and E. Moreover, these results are also consistent with the knock-down of Shh by Shh-specific siRNA in H1650 cells with robust transfection efficiency (Fig. 8A), and resulting in a significant reduction in cell migration and invasion (Fig. 8B and Fig. 8C). In addition, the treatment of H1299 cells with GDC-0449 or cyclopamine led to a significant reduction in invasion and tumorigenic behavior as assessed by clonogenic growth (Fig. S2A-B and Fig. S1). The treatment of H1299 cells with GDC-0449 also led to the partial reversal of the EMT phenotype as documented by reduced expression of fibronectin and ZEB1, and the increased expression of E-cadherin (Fig. S2C-D).
10.1371/journal.pone.0016068.g007Figure 7 Down-regulation of Shh autocrine signaling in NSCLC cell lines led to the reduction in tumor cell migration, invasion, and tumorigenesis:
A; both H1650 and H1299 cells expresses high levels of Shh mRNA compared to NHBE cells, and both cell lines have high Shh protein expression. B and C shows reduction in cell-invasion and the colony-forming ability of H1650 cells following treatment with Shh inhibitors such as GDC-0449 (20 nM). (D) Western blot of H1650 cells before and after treatment with GDC-0449 (20 nM) for the expression of fibronectin. (E) qRT-PCR for the expression of E-cadherin and ZEB1 mRNA in H1650 cells after treatment with GDC-0449 (20 nM) showing reversal of EMT compared to untreated H1650 cells. (* = p<0.05).
10.1371/journal.pone.0016068.g008Figure 8 Down-regulation of Shh signaling in NSCLC cells lines (H1650 cells) leads to reduced cell motility and invasion.
(A): Transfection efficiency was assessed by GFP. (B) Matrigel-Coated membrane assay where cells were labeled with DiIC12 fluorescent dye. (C) Matrigel-Coated membrane assay where cells were labeled with immune-staining kit (Quik staining kit). (B and C right panel) also show quantitative data analysis. (* = p<0.05).
Discussion
Previous studies have shown that the treatment of NSCLC cells (A549 cells) with TGF-β1 could induce EMT phenotype [13], [14], [16], a process that was originally reported to be involved in embryogenesis and gastrulation [4], [9], [40]. The induction of EMT in cancer cells confers these cells with the ability to become more motile and invasive with increased tumorigenic potential [4], [9], [11], [17], [18], [32], [33], [41]. Furthermore, the EMT phenotype appears to be involved in resistance to therapeutic agents. Thus, reversal of EMT by novel approaches may provide a tool by which one could enhance the effects of conventional therapeutic agents.
In this study, NSCLC cell lines (A549 and H2030) underwent EMT phenotypic changes (A549-M and H2030-M cells) after chronic exposure to TGF-β1, which was consistent with decreased expression of epithelial marker concomitant with increased expression of mesenchymal markers (Fig. 1A–C; Fig. 4A). In order to further characterize these cells, we assessed the ability of A549-M cells compared to A549 parental cells for cell migration, invasion and tumorigenic potential. Our data showed increased ability of A549-M cells for cell migration, invasion and tumorigenic potential compared to parental A549 cells (Fig. 2A–C). Interestingly, we also found that A549-M and H2030 cells showed high expression of Shh both at the mRNA and protein levels compared to parental cells (undetectable levels of Shh expression) (Fig. 3A–B; Fig. 4A). The up-regulation of Shh expression in A549-M cells is the first of its kind, which was also consistent with increased expression of GLI1 transcription factor, a downstream target gene of Hh signaling pathway (Fig. 3C–D) although the basal level of GLI1 expression was found to be high in the parental A549 cells. These results suggest that Hh signaling could be very active through non-canonical pathway (ligand-independence) in these cells. Our novel finding is very interesting not only because it connects two very important molecules of the developmental pathway such as TGF-β1 and Shh [42], [43] to tumor aggressiveness, but it is also consistent with published reports showing the role of EMT in tumor aggressiveness and metastasis [22], [25], [26], [31], [44]–[46]. However, no studies have shown the direct up-regulation of Hh ligand Shh mRNA and protein by TGF-β1 as documented in our current report although Shh has been reported to activate TGF-β family signaling through the ALK5-Smad 3 pathway in gastric cancer cells [21]. Moreover, it has been reported that TGF-β1 can induce GLI2 activation through Smad3 in pancreatic adenocarcinoma cell lines [47], and these published results suggest that there may exist a feed-back loop connecting TGF-β1 with Shh activation. Our finding also suggest that Hh signaling pathway reactivation in cancer epithelial cells within the tumor microenvironment could lead to the acquisition of aggressive phenotype of cancer cells within a tumor.
Although the mechanisms by which TGF-β1 can induce Hh ligand expression needs further investigation, our data clearly suggest that the activation of Shh signaling by TGF-β1 leads to increased tumor cell migration, invasion and tumorigenic potential of A549-M cells as documented by our mechanistic experiments using knock-down approach and by using chemical inhibitors of Shh signaling (Fig. 5A-C). Our results also suggest that the maintenance of EMT phenotype in A549-M cells may be related to the sustained activation of Hh. These results are also consistent with two other NSCLC cell lines that were derived from patients metastasis (H1650, H1299), and these two cell lines showed high basal levels of Shh expression, suggesting that lung metastatic cells have the ability to undergo EMT consistent with higher expression of Shh in vivo. Interestingly, the inhibition of TGF-β1-induced Shh signaling by pharmacological inhibitors or by siRNA decreased the ability of A549-M cells to migrate, invade and forming colony, and these results are consistent with previous reports showing that the activation of Shh signaling could increase invasion and metastasis [21], [23]–[26], [37], [44], [48].
We have also shown that the conditioned medium from A549-M cells has the ability to activate Shh downstream signaling in NIH 3T3 cells, which suggests that TGF-β1-induced EMT is mediated by the activation of Shh through both autocrine, paracrine or juxtacrine mechanisms although further mechanistic studies are warranted. Our results further showed the importance of Shh in EMT phenomenon, wherein inhibition of Shh signaling by GDC-0449 was able to down-regulate mesenchymal markers such as ZEB1 and fibronectin, which was consistent with up-regulation of epithelial marker such as E-cadherin (Fig. 5D and E and Fig. 7D and E). These results suggest that the attenuation of Shh signaling could reverse the EMT phenotype to mesenchymal-to-epithelial transition (MET) as shown in Fig. 6B (inset) where cells look more annular after Shh siRNA transfection, resulting in decreased cell migration, invasion and tumorigenic potential, which is consistent with the suggestion made by Feldmann et. al. [26]. More importantly, our data show for the first time that TGF-β1 induced EMT is mediated through up-regulation of Shh because knock-down of Shh by Shh specific siRNA significantly attenuated EMT induction by TGF-β1 treatment (Fig. 4B, C and D).
Clinically, NSCLC tumor tissues show higher levels of GLI1 expression compared to NSCLC cell lines [44], suggesting that the EMT phenotype with activated Shh signaling may be context-dependent such as what can be found in the tumor microenvironment where the tumor cells are chronically exposed to many factors including TGF-β1. This contention is partly supported by our data using four different NSCLC cell lines with epithelial vs. mesenchymal phenotype, and also suggested by a recently published report showing that chronic exposure to TGF-β1 in the tumor microenvironment may lead to the acquisition of EMT phenotype, which further leads to increased cell motility and invasiveness, resulting in tumor metastasis [46].
Based on existing evidence in the literature and our current data, we propose a model where epithelial tumor cells could be chronically exposed to TGF-β1 excreted by either stromal cells, immune cells or the tumor cells within the tumor microenvironment, resulting in the up-regulation of Shh both at the mRNA and at the protein levels and consequently causes activation of Hh signaling and the acquisition of EMT phenotype, which is responsible for tumor cell aggressiveness and metastasis (Fig. 9). Therefore, the inhibition of Shh signaling could be a useful approach for reducing tumor aggressiveness in NSCLC, and as such, the reversal of EMT could also be useful for re-sensitization of drug-resistant NSCLC to conventional therapeutics, which would likely contribute to the improved survival of patients who rightfully deserve better treatment outcomes.
10.1371/journal.pone.0016068.g009Figure 9 Schematic diagram showing activation of TGF-β receptor by TGF-β1which leads to the up-regulation of Shh expression.
The secreted Shh protein then activates Hh signaling pathway by inhibition of Patched (smoothened suppressor), which will repress smoothened, resulting in the activation of GLI1 and its translocation to the nucleus. GLI1 as a Hh transcription factor then could activate Hh target genes, which leads to the acquisition of EMT phenotype, and contributing to increased invasion, metastasis and drug resistance.
Materials and Methods
Cell Lines
The human lung adenocarcinoma cell lines, A549, H2030, H1299, H1650, and mouse fibroblast NIH-3T3 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained according to the American Type Culture Collection's instructions. The normal lung epithelial cell line (NHBE cells) was purchased from Lonza. NHBE cells where maintained and cultured according to Lonza's instructions. All the cell lines have been tested and authenticated using the Karmanos Cancer Center, Wayne State University's core facility (Applied Genomics Technology Center at Wayne State University) on March 13, 2009, and these authenticated cells were frozen for subsequent use. The method used for testing was short tandem repeat profiling using the PowerPlex 16 System from Promega. A549 cells were treated with TGF-β1 (5 ng/ml) for 21 days before experiments were conducted. Cells were treated with GDC-0449 (20 nM) or Cyclopamine (2 µM) for 72 hours, before conducting assays.
Reagents and antibodies
Anti-Shh N-terminal peptide antibody and recombinant human TGF-β1 protein was purchased from R&D Systems (Minneapolis, MN). Cyclopamine was purchased from Sigma (San Louis, MO) and diluted in dimethyl sulfoxide (control vehicle). GDC-0449 (20 nM) was obtained from Genentech. Rabbit anti-GLI1 was purchased from Abcam. Rabbit anti-fibronectin was obtained from Santa Cruz biotechnology (CA, USA). Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Affinity BioReagents (Golden, CO.). Mouse anti-β-actin was obtained from Sigma (St. Louis, MO). β-tubulin rabbit mAb was obtained from cell signaling (Danvers, MA).
Cell proliferation assay
Cells were treated with TGF-β1 for 21 days, Hh inhibitor for three-72 hour treatments, or knock-down with siRNA specific for Shh (si-Shh) for 48 hours. Prior to treatment, cells were seeded at 5×103 cells per 100 µl of culture medium per well in 96-well plates. The number of viable cells was assessed in triplicate wells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) according to the manufacturer's instructions. All assays were done in triplicates, and each experiment was repeated, at least, three times independently. Data reported here is one representative experiment.
Wound healing assay
Cells were treated with TGF-β1 for 21 days, Hh inhibitor for three-72 hour treatments, or knock-down with siRNA specific for Shh (si-Shh) for 48 hours. Prior to treatment, cells were seeded at 1×106 cells per well in a 6-well plates. Upon >90% confluence, cells were scraped across the cell monolayer using a plastic 200 µl tip. Photomicrographs were taken with Phase contrast objective microscope 4× magnification, at zero time point and after 24 hours. The measured ratio of the remaining wound area relative to the initial wound area was Quantified and reported. Quantification of the wound area using the NIH Image-J program was performed, and the results are expressed as the percentage of wound area change. Experiment was repeated at least three times, independently. Data reported here is one representative experiment.
Matrigel invasion assay
Cells were treated with TGF-β1 for 21 days, Hh inhibitor for three-72 hour treatments, or knock-down with siRNA specific for Shh (si-Shh) for 48 hours. Following seeding cells at 5×104 cells/well, invading cells at the bottom of the membrane and media in the lower chamber were detected by pre-labeled with DiIC12 (3) Fluorescent Dye or by post-staining using immune-staining Diff-Quick™ staining kit after removal of noninvasive cells. Cells were seeded in the upper chamber of a 24-insert with serum-free medium. Upper chambers coated with Matrigel (fluoro-block insert and MATRIGEL™ Invasion Chamber; BD Biosciences, USA). Lower chamber contained 10% FBS plus regular media. After 24 h of incubation, invading cells were examined by using a fluorescence microscope and photographed. The transfection efficiency was photographed at 10X, whereas invading cells was photographed at 4× magnification. TECAN Ultra imaging system was used to measure the fluorescence of invading cells. Immune-stained cells were also counted under phase contrast objective microscope (10× magnification). The experiment was repeated at least three times independently. Data reported here is one representative experiment.
Small interfering RNA (siRNA) transfection
Small interfering RNA (siRNA) specific for Shh (SHH Stealth RNAi™ siRNA) was purchased from Invitrogen. As a non-specific control siRNA, scrambled siRNA duplex was used which was also purchased from Invitrogen. Transfection was done using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) following the manufacturer's instruction. Experiment was repeated at least, three times independently. Data reported here is one representative experiment.
Western blot analysis
Whole-cell protein extraction was conducted using RIPA buffer [50 mM Tris, 150 mM NaCl, 1%TritonX-100, 0.1% sodiumdodecyl sulfate and 1% Nadeoxycholate (pH 7.4)] supplemented with protease inhibitors (1 mMphenylmethylsulfonyl fluoride, 10 µg/ml peptasin A, 10 µg/ml aprotinin and 5 µg/ml leupeptin). Protein concentrations were then measured using Bio-Rad protein assay kits (Bio-Rad, Hercules, CA). Next, the protein lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto nitrocellulose membranes (HybondTM-P; Amersham Biosciences, Piscataway, NJ), blocked with phosphate-buffered saline (PBS) containing 0.2% Tween 20 and 5% non-fat dry milk, incubated with primary antibody and then with horseradish peroxidase-labeled secondary antibody and developed using chemiluminiscent detection system, and the signals were then detected using X-ray film. Experiment was repeated at least three times independently. Data reported here is one representative experiment.
Clonogenic assay
Cells were treated with TGF-β1 for 21 days, Hh inhibitor for three-72 hour treatments, or knock-down with siRNA specific for Shh (si-Shh) for 48 hours. Prior to treatment, cells were plated at a density of 1×103 cells in 100-mm Petri dishes. Then the cells were incubated for 10–14 days at 37°C in a 5% CO2/5% O2/90% N2 incubator. Next, colonies were stained with 2% crystal violet and quantified using NIH Image-J software. The experiment was repeated at least three times independently. Data reported here is one representative experiment.
Quantitative Real-time PCR
Quantitative real-time RT-PCR analysis was conducted; 1 µg of total RNA from each sample was subjected to reverse transcription using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the manufacturer's protocol. Real-time PCR reactions were then carried out in a total volume of 25 µL reaction mixture (2 µL cDNA, 12.5 µL of 2 µL SYBR Green PCR Master Mix from Applied Biosystems, 1.5 µL of each 5 µmol/L forward and reverse primers, and 7.5 µL distilled H2O) using a SmartCycler II (Cepheid). The PCR program was started by 10 min at 95°C before 40 thermal cycles, each at 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method. Data was normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in each sample. GLI1 primers have been previously described [25]. Shh primers (Shh-forward: GTGGCCGAGAAGACCCTA, Shh-reverse: CAAAGCGTTCAACTTGTCCTTA. GAPDH, ZEB1, and E-cadherin primers were previously described [49]. Experiment was repeated at least, three times independently. Data reported here is one representative experiment.
Statistical analysis
The two-tailed v2 test was performed to determine the significance of the difference among the covariates. P values less than 0.05 were considered statistically significant. The SPSS software program (version 13.0, SPSS, Chicago, IL) was used for such analyses.
Supporting Information
Figure S1 Shh signaling inhibition decreases tumorigenic potential of NSCLC cells. (A) clonogenic growth assay of three parental NSCLC cell lines compared to A549-M cells before and after treatment with Hh inhibitor GDC-0449 (20 nM), and (B) represent quantitative data analysis of the data presented in panel-A. (* = p<0.05).
(TIFF)
Click here for additional data file.
Figure S2 Inactivation of Shh by cyclopamine and GDC-0449 (20 nM) led to the reduction in tumor cell invasion (A), and tumorigenic potential (B) of H1299 NSCLC cell line. Right panel shows quantitative analysis. (C) Western blot of H1299 cells before and after treatment with GDC-0449 (20 nM) for the expression of fibronectin. (D) qRT-PCR expression of E-cadherin and ZEB1 mRNA of H1299 cells after treatment with GDC-0449 (20 nM) showing reversal of EMT compared to untreated H1299 cells. (* = p<0.05).
(TIFF)
Click here for additional data file.
We would like to convey our special thanks to Genentech who kindly provided the GDC-0449 for our experiments.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors have no support or funding to report.
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BMC BiotechnolBMC Biotechnology1472-6750BioMed Central 1472-6750-11-22121099010.1186/1472-6750-11-2Research ArticleHeterologous expression, purification and characterization of nitrilase from Aspergillus niger K10 Kaplan Ondřej [email protected]ška Karel [email protected]íhal Ondřej [email protected] Rüdiger [email protected] Natallia [email protected]ěk Ondřej [email protected] Daniel [email protected] Oldřich [email protected] Anna [email protected]Šveda Ondřej [email protected]á Alicja B [email protected]ágelová Anna [email protected]ámová Kristýna [email protected] Maria [email protected] Jürgen [email protected]šková Jarmila [email protected]álek Jan [email protected] Michael [email protected]řen Vladimír [email protected]ínková Ludmila [email protected] Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague, Czech Republic2 Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-128 40 Prague, Czech Republic3 Centre of Biocatalysis and Biotransformation, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, 373 33 Nové Hrady, Czech Republic4 Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, Via Campo di Pile - Zona industriale di Pile, I-67100 L'Aquila, Italy5 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského náměstí 2, CZ-162 06 Prague, Czech Republic2011 6 1 2011 11 2 2 23 2 2010 6 1 2011 Copyright ©2011 Kaplan et al; licensee BioMed Central Ltd.2011Kaplan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.
Results
A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.
Conclusions
The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.
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Background
Nitrilases enable hydrolysis of nitriles to be performed under mild conditions and often in a stereo- or regioselective manner. These enzymes have thus great potential in organic synthesis but drawbacks such as instability, low activity or low selectivity lessen their practical use [1,2]. These limits may be overcome by searching for new nitrilases or improving known ones. Recently, the former approach has often made use of database mining [1-6].
According to GenBank search, not only bacteria, which have been intensively exploited as a source of nitrilases since the 1980s, but also filamentous fungi harbour a large number of nitrilase genes [7]. Apart from the teleomorph/anamorph pair Gibberella/Fusarium, the Aspergillus genus is a rich source of these enzymes, which exhibit low homology to bacterial nitrilases and thus may differ in their catalytic properties.
We have recently purified and characterized the first nitrilase in the Aspergillus genus, namely from the Aspergillus niger K10 strain [8], which was selected by nitrilase activity screening in filamentous fungi. In this study, the gene encoding this enzyme was amplified, cloned and sequenced and the protein deduced from gene sequencing was found to be highly homologous with a number of putative nitrilases in Aspergillus and Penicillium.
The natively expressed enzyme exhibited high specific activities towards (hetero) aromatic nitriles and was fairly stable under operational conditions for its use in nitrile hydrolysis [9]. Here, to potentiate its industrial utility, we expressed this enzyme in Escherichia coli. Heterologous expression has not been reported for any fungal nitrilases, as far as we know. On the other hand, a number of nitrilases from bacteria [1-4,6,10-13], and from the plant Arabidopsis thaliana [14,15] have been expressed in E. coli, as well as several cyanide hydratases from fungi [16].
Recombinant E. coli cells harbouring the A. niger gene produced the active enzyme (Nit-ANigRec). However, this enzyme differed in its catalytic properties from the wild-type enzyme that was purified from A. niger K10 (Nit-ANigWT). The quaternary structures of Nit-ANigRec and Nit-ANigWT were also different. Nitrilases and cyanide hydratases are proteins with unique structural properties, being able to exist in a number of different homooligomeric species - dimers, short homo-oligomeric spirals and extended helices [12,17-19]. The occurrence of these structural types in Nit-ANigRec and Nit-ANigWT was compared, indicating lower tendency of the former enzyme to form long helices. The homogeneity of this enzyme was enhanced by maturing (formation of species differing in molecular weight) during storage of the refolded enzyme, followed by size exclusion chromatography. The resulting protein appeared to be useful for analytical centrifugation and electron microscopy studies. It is also promising for nitrilase crystallization, which is thought to be impaired in enzymes forming the aforementioned helices [12].
A deeper insight into three-dimensional structures of nitrilases is impaired by missing crystal structures. The previous models of nitrilases from Pseudomonas fluorescens [1] and Rhodococcus rhodochrous [12,20] were therefore generated by exploiting their homology with crystallized members of the nitrilase superfamily. Here we have used an analogous approach to construct the first model of a fungal nitrilase, which is distantly related to the above bacterial enzymes.
Results
Determining the Aspergillus niger K10 nitrilase sequence
Previously, the determination of the N-terminal amino acid sequence of Nit-ANigWT suggested a high similarity of this enzyme to a group of highly conserved putative nitrilases (with ≥90% amino acid identity) from the Aspergillus genus (Additional file 1). This enabled us to design degenerate primers, which were based on the N-terminal and a conserved internal sequence of two putative Aspergillus fumigatus nitrilases. Combining the sequence data obtained from amplifications using both nitrilase-specific primers and from 5'-RACE and 3'-RACE amplifications provided a complete sequence of the nitrilase gene (GenBank:ABX75546).
The amino acid sequence deduced from this nitrilase gene confirmed that Nit-ANigWT was highly similar to putative nitrilases from Aspergillus (A. clavatus, A. fumigatus, A. flavus, A. nidulans, A. oryzae, A. terreus) and Neosartorya fischeri (teleomorph of Aspergillus fischerianus). While our study of Nit-ANigWT sequencing was in progress, a sequence of a nitrilase-coding gene (GenBank:XP_001389844) from another A. niger strain (CBS 513.88) was deposited in the database. The amino acid sequence of this hypothetical protein was 99% identical to that of the enzyme being studied by us. Later, another very similar nitrilase (with 89% amino acid identity) (GenBank:XP_002562104) was sequenced in Penicillium chrysogenum. However, neither of these two nitrilases has been studied at the protein level.
Nitrilase expression, purification and refolding
The expression of the enzyme was achieved with the pOK101 and pOK102 vectors and 7 out of the 9 E. coli strains tested (see Methods), the BL21(DE3) and BL21-CodonPlus(DE3)-RIL strains being exceptions. The absence of nitrilase activity in these strains may have been caused by low transformation efficiency, endonuclease activity or limited translation due to codon bias. In all other strains, the nitrilase was expressed after IPTG addition and formed about one half of the soluble cellular proteins, as determined by SDS-PAGE (not shown). However, the nitrilase activities of the recombinant strains were not as high. Total activities of BL21 or Rosetta-gami strains were between 65 and 230 U L-1, while those of Arctic Express strains were on the average lower (34-150 U L-1). These values were at most approx. twofold higher compared to those achieved in the native producer A. niger K10 (ca. 100 U L-1) [8]. However, the time required for maximum activity yield in E. coli was only ca. 20 h, which was approx. three times less than in A. niger. Of the above E. coli strains, the highest activity of approx. 230 U L-1 was obtained in BL21-Gold(DE3) strain carrying the pOK101 plasmid (pET-30(+) containing the nit gene). Further increase in total activity to approx. 500 U L-1 was brought about by variation of cultivation parameters (IPTG concentration, induction time, temperature; data not shown) and by co-expression of pTf16 plasmid (coding for the trigger factor). However, the latter accounted for only about 10% increase in the total activity. This strain designated E. coli BL21-Gold(DE3)(pOK101/pTf16) has been used throughout further work. Nit-ANigRec was purified from this culture to near homogeneity with an approx. 2-fold increase in specific activity and 35% yield (Table 1). As expected from the nitrilase activity of the whole cells, the specific activity of the purified enzyme for benzonitrile - 0.60 U mg-1 - was significantly (two orders of magnitude) lower than that of Nit-ANigWT - 91.6 U mg-1 [8].
Table 1 Purification of recombinant nitrilase from Aspergillus niger K10
Step Total protein,
mg Specific activity,
U mg -1 Total activity,
U Yield,
% Purification,
fold
Cell-free extract 444.4 0.29 130.5 100 -
Q-Sepharose 159.5 0.39 61.5 43.2 1.34
Sephacryl S-200 76.7 0.60 46.1 35.1 2.07
Enzyme activity was assayed with 25 mM benzonitrile (see Methods for details).
As the above results suggested the possibility of incorrect protein folding, the protein was fully denatured in 6 M guanidine-HCl and 2 M TCEP (tris-carboxyethylphosphine), and refolded in vitro. However, the best refolding conditions (see Methods) selected by screening the commercial iFOLD 1 system merely led to the recovery of the initial activity and not to its improvement.
According to SDS-PAGE analysis (not shown), the apparent molecular weight of the subunit of the purified enzyme (42.7 kDa) was higher than that of Nit-ANigWT - 38.5 kDa [8]. This indicated that the latter protein underwent a post-translational modification in its native environment. In order to clarify the molecular nature of this difference, we performed N-terminal sequencing and peptide mass mapping with both Nit-ANigRec and Nit-ANigWT (Figure 1). The N-terminal sequence of both enzymes was identical, indicating that the processing occurred most probably at the C-terminus of the enzyme. Indeed, peptide mass mapping using both trypsin and Asp-N in gel digestion revealed that the C-terminal tryptic peptide Val327 - Asn356 was present in Nit-ANigRec but absent in Nit-ANigWT. More specifically, the Asp-N generated peptide Asp298-Val313 detected in the recombinant protein was shortened to Asp298-Arg310 in the native enzyme. These results provide evidence that Nit-ANigWT was shortened by 46 amino acid residues at the C-terminus, and is composed of Met1 - Arg310 of amino acid sequence coded by the corresponding nitrilase gene.
Figure 1 Summary of the sequence analysis of heterologously expressed nitrilase vs. nitrilase isolated from the native organism (Nit-ANigRec and Nit-ANigWT respectively). Sequence analysis was performed by automated Edman degradation of nitrilase blotted onto PVDF membrane (underlined) in combination with peptide mass mapping using MALDI TOF mass spectrometry of peptides extracted after in gel digestion with trypsin (bold) or Asp-N protease (italics). The position of C-terminal truncation by 46 amino acids in the native preparation is indicated by an asterisk.
Preparation of homogeneous enzyme for structural studies
Electron microscopy study of Nit-ANigRec showed heterogeneous population of particles of different shapes from nearly isometric ones in size of about 14 nm to elongated ones reaching over 30 nm in length. Additionally, smaller particles of different shapes and some bigger clusters were also observed (Additional file 2). However, long filamentous structures typical for Nit-ANigWT [17] were not observed in this sample. Despite the limited ability of Nit-ANigRec to form the aforementioned filamentous structures, the purified enzyme was not suitable for structural studies or protein crystallography. The refolded protein (see above) also still exhibited some molecular heterogeneity as revealed by electron microscopy (Additional file 3). Though the gel-filtration chromatography fractionated the refolded enzyme as a single major peak (Mw ≅ 600 kDa), peak fronting and the appearance of a minor peak preceding the major one suggested that a small part of the enzyme aggregated into higher-molecular weight species (Figure 2A). After a 1-month storage of the major-peak protein fraction at 4°C, this aggregation occurred again, as well as a notable shift of the molecular mass of the enzyme towards lower values (about 500 kDa; Figure 2B). After removing the aggregated form, the rest of the enzyme remained rather stable, since after 10 more days of storage under the same conditions, fewer aggregates could be observed, without any further change in enzyme size (Figure 2C). Nevertheless, even after such "maturation" in enzyme quaternary structure, the enzyme was composed of a rather heterogeneous mixture of molecular forms, which were separated by gel filtration and examined by electron microscopy (Figure 2D through 2G). In the early eluting fractions the enzyme occurred in the form of short tubes and was rather heterogeneous, whereas the late eluting fractions contained the enzyme in more homogeneous forms (cf. Figure 2Dvs. 2G).
Figure 2 Effect of aging on the quaternary structure of recombinant refolded nitrilase. Gel filtration analysis on Superose 6B of freshly refolded enzyme (A), and enzyme stored for 30 and 40 days at 4°C (B and C, respectively). Fractions were collected from the last separation, and analyzed by electron microscopy using material eluted between 26 and 27 min (D), 30 and 31 min (E), 33 and 34 min (F), and 37-38 min (G). The homogeneous round-shaped particles observed in the latter fractions (G) were analyzed in an analytical ultracentrifuge using sedimentation velocity (H) and sedimentation equilibrium (I) experiments as detailed in Methods. Fitted data with residual plots showing goodness of fit are shown together with calculated continuous size distribution c(s) of sedimenting species.
Data obtained from sedimentation velocity analysis of the latter fractions (Figure 2H) suggested a rather broad mass distribution of sedimenting species with values of apparent sedimentation coefficients ranging between 10 and 30S, the majority (approx. 70%) of particles falling between 12 and 22S, in 95% confidence level. Integration of size distribution for the main particle fraction yielded a weight average sedimentation coefficient s* = 16.8 ± 2.4S (s20,w = 17.8 ± 2.0S) and a frictional coefficient ratio f/f0 = 1.42 corresponding to a moderately elongated particle. Global analysis of sedimentation equilibrium data (Figure 2I) resulted in weight average particle mass of 564 ± 5 kDa in 95% confidence level, with almost no observable tendency to aggregate in the time course of the experiment (as judged from the residual plot of fit analysis). Based on the value of sedimentation coefficient, frictional coefficient ratio and observed particle mass, the size and shape of the majority of particles was estimated as 20 × 10 ± 5 nm and this correlates with electron microscopy (Figure 2G). Taking into account the theoretical molar mass of nitrilase monomer is 40 kDa, we can conclude that majority of observed nitrilase oligomers was composed of 14 ± 2 nitrilase subunits, as deduced from a combination of data from SDS electrophoresis, gel filtration, electron microscopy and analytical ultracentrifugation, although higher oligomers were still present in significant amount.
In agreement with the results obtained by other techniques, DLS (dynamic light scattering) measurements (Table 2) confirmed the gradual decrease in size in the above protein fractions starting with 22.8 nm oligomers (Mw > 1 MDa) down to particles having a diameter of about 14.8 nm (Mw ≅ 370 ± 50 kDa). All fractions analysed by DLS (Table 2) are polydisperse with a polydispersity index (PdI) in the range 0.23-0.30 with equal data quality. Fractions with the lowest PdI correspond to the second half of the gel filtration peak. The smallest particle size and the highest homogeneity make these fractions most suitable for further analyses including prospective protein crystallization attempts.
Table 2 Measurement of size and heterogeneity of recombinant refolded nitrilase in fractions separated by gel filtration (see Figure 2) using dynamic light scattering (DLS)
Elution time, min Diameter, nm Mw, kDa PdI
32 22.8 1000 0.30
34 19.7 710 0.24
36 17.0 500 0.29
38 14.5 350 0.23
40 14.8 370 0.23
PdI = polydispersity index
Homology modelling and molecular dynamics
A BLAST search identified five proteins with relevant known structures: the NitFhit protein from Caenorhabditis elegans (pdb-code 1EMS) [21]; hypothetical protein Ph0642 from Pyrococcus horikoshii (1J31) [22]; Nit3 protein from Saccharomyces cerevisiae (a member of branch 10 of the nitrilase superfamily, pdb-code 1F89) [23]; the pyrimidine degrading enzyme from Drosophila melanogaster (2VHH) [24] and mouse nitrilase-2 (2W1V) [25] with corresponding identities of 22, 23, 20, 21 and 22%. Although the percentage of identity is at the lower threshold for homology modelling, 3D alignment with the SHEBA plug-in in YASARA showed a high conservation of secondary structure elements among the selected templates, thus supporting the attempt to at least obtain a useful low-resolution homology model. The C-terminal part (residues 316-356) was modelled based on the crystal structure of kinesin from Rattus norvegicus (2KIN, 29% of identity) [26] using residues 136-183 as a template, as this part has been lost in hydrolases. Figure 3 shows a structure-based multiple sequence alignment of nitrilase from Aspergillus niger with selected templates and sequences of previously published homology models of nitrilases from R. rhodochrous J1 [12,20] (identity 38%) and Pseudomonas fluorescens [1] (26%). Secondary structure is given as assigned by Procheck [27]. Figure 4A shows a view of the enzyme with the catalytic domain on the left and the active site in the domain center. The three long loops at the entrance of the active site are interesting features of the modelled structure. Loops including the residues that correspond to 236-252 and 55-64 between β2 and α2 and 236-252 between β10-β12 (coloured magenta in Figure 4A) in the primary sequence were found in just one template structure, 2VHH, but these residues were not resolved in the crystal structure [24]. A loop corresponding to 196-207 between β8 and α6 (yellow in Figure 4A) was not found in any template structure (Additional file 4). The nitrilase from Rhodococcus rhodochrous J1 [12] presents similar residues at the corresponding primary sequence positions, and similar external loops in its homology model. The nitrilase from Pseudomonas fluorescens lacks the insertion at the position similar to 196-207 but it has one additional loop between β14 and α7.
Figure 3 Multiple sequence alignment. (A) Multiple sequence alignment of A. niger K10 with template structures 1EMS [21], 1J31 [22], 1F89 [23], 2VHH [24] and 2W1V [25] (letters in upper case) and sequences of homologous nitrilases (letters in lower case) from Rhodococcus rhodochrous J1 (RrJ1) [12] and from Pseudomonas fluorescens (Pf-5) [1]. Clustal W scheme is used for marking similar residues. Amino acids from catalytic triad are strongly conserved; they are marked by red arrow and enclosed in blue rectangles. Secondary structures for template 1EMS and A. niger K 10 nitrilase model are shown above and under the aligned sequences, respectively, with numeration as in [12]. (B) Sequence alignment of A. niger K 10 with 2KIN [26] was used for modelling the C-terminal part. Secondary structure as assigned by Procheck [27] is shown for A. niger K 10 and for 2KIN above and under the aligned sequences, respectively.
Figure 4 Homology model of nitrilase (A) and active site amino acids (B) with docked benzonitrile. Loops formed by residues 55-64 and 236-252 are coloured magenta and loop formed by residues 196-207 is yellow. The catalytic domain is on the left side. Active site amino acids with docked benzonitrile (magenta) after 2 ns of molecular dynamics simulation (B). Hydrogen atoms are omitted. The catalytic triad is represented by Glu 48, Lys 130 and Cys 165. The only hydrogen bond (yellow dotted line) is created by hydrogen atom of Lys 130 and nitrogen of benzonitrile.
A docking attempt in AutoDock, using benzonitrile as the substrate, found a position in the centre of the enzyme with the lowest binding energy, and thus the highest affinity. This position involved the predicted triad of active residues (Figure 4B; Additional file 4), demonstrating the basic correctness of the modelled structure.
The geometrical parameters of spiral structures obtained from electron microscopy were used to draft a plausible multimeric arrangement. The electron micrographs corresponded in size and general shape to helical segments made up of dimers (Figure 5A and 5C). Hereby, taking into account the size and shape of the monomeric model, approximately 16 subunits would be organized in a spiral or helical arrangement and 8 dimers would form one helical turn that could be extended in both directions (Figure 5B and 5D). Similar loops are found at the C-surface in the helical-like form of the nitrilase from R. rhodochrous [12]. 14-16 subunits in the multimeric structure can be assumed for the aliphatic nitrilase from R. rhodochrous K22 [28] which has an identity of 42% with the nitrilase from A. niger K10.
Figure 5 Nitrilase multimer. (A) and (C) panels are images from electron microscope, (B) and (D) - overlay of top view and side view, respectively, of constructed multimer and image from electron microscope.
Comparison of reaction optima, substrate specificity, selectivity and stability of the heterologously expressed nitrilase and the nitrilase isolated from the native organism
The optimal reaction conditions of Nit-ANigRec and Nit-ANigWT were different. Nit-ANigRec exhibited a lower temperature optimum (38 vs. 45°C) when assayed after 10-min reaction time. Its activity decreased to 55 and 4% at 45 and 50°C, respectively, while Nit-ANigWT retained significant activity up to 55°C. The pH-range of Nit-ANigRec (ca. pH 5.5-9.5) was slightly shifted towards lower values compared to that of Nit-ANigWT (ca. pH 6-10).
Nit-ANigRec, incubated for 1 h at 40, 45 and 50°C, exhibited a residual activity > 80, 36 and 1.3%, respectively. Its stability was thus lower than that of Nit-ANigWT, which still exhibited 59, 24 and 6% of the maximum activity after 1-h incubation at 45, 50 and 55°C, respectively. The effects of various additives on Nit-ANigRec activity were similar to those reported for the Nit-ANigWT [8]. p-Hydroxymercuribenzoate, Hg2+, Ag+ and Al3+ ions completely abolished the activity of both preparations.
The relative activities of Nit-ANigWT decreased in the order 4-cyanopyridine > benzonitrile > 3-chlorobenzonitrile > 4-chlorobenzonitrile > phenylacetonitrile > 3-cyanopyridine > 2-cyanopyridine >> 2-phenylpropionitrile. In contrast, the best substrate of Nit-ANigRec was 2-cyanopyridine, followed by 3-cyanopyridine and 3-chlorobenzonitrile (Table 3). Thus 2-cyanopyridine was the only substrate for which Nit-ANigRec exhibited a similar or higher activity (9 U mg-1 of protein at 38°C after 10-min reaction time or 26 U mg-1 of protein 45°C after 1-min reaction time) compared to Nit-ANigWT (7 U mg-1 of protein at 45°C after 10-min reaction time). Other substrates were transformed at very low rates or not transformed at all. In Nit-ANigRec, the production of amide by-product was most significant with 2-cyanopyridine (23% amide in total product). Nevertheless, this was much less than with Nit-ANigWT, which gave a product consisting of up to 88% picolinamide. From the other substrates tested, the recombinant nitrilase produced only a maximum of 5% amide in the total product, in contrast to the enzyme isolated from the native organism producing high amounts of amides also from 4-chlorobenzonitrile and 4-cyanopyridine (Table 3).
Table 3 Substrate specificity and chemoselectivity of purified nitrilase isolated from A. niger K10 (Nit-ANigWT) and heterologously expressed nitrilase (Nit-ANigRec)
Substrate Relative activity, % Amide, molar % of total product
Nit-ANigWT Nit-ANigRec Nit-ANigWT Nit-ANigRec
Benzonitrile 27 4.9 9 0
2-Chlorobenzonitrile 0 0 - -
3-Chlorobenzonitrile 10 3.7 3 5
4-Chlorobenzonitrile 8.4 0.2 80 0
2-Cyanopyridine 2.4 100 88 23
3-Cyanopyridine 4.6 12.9 6 2
4-Cyanopyridine 100 0.8 36 0
Phenylacetonitrile 4.9 0.2 0 0
2-Phenylpropionitrile traces 0 0 0
Enzyme activity was assayed as described in Methods. The specific activities of Nit-ANigWT and Nit-ANigRec for their best substrates 4-cyanopyridine (306 U mg-1 at 45°C) and 2-cyanopyridine (9.0 U mg-1 at 38°C), respectively, were taken as 100%. Data represent the mean of four independent measurements with relative standard deviation values <5%.
All the compounds tested as potential stabilizers of the nitrilase (sugars, sugar alcohols, albumin, glycine) improved the Nit-ANigRec stability to a significant extent during either incubation at 38°C or repeated freezing/thawing cycles (Table 4). Without any stabilizer, the enzyme retained about 36 and 60% of its initial activity, respectively, but full activity was preserved in the presence of 1% glycine as the most powerful stabilizer. The combined action of freezing/thawing (20 cycles) and 3-h incubation at 45°C decreased nitrilase activity by >90% (to 0.057 U mg-1 protein). However, the same treatment in the presence of glycine, D-sorbitol, xylitol or glucose (5% each) allowed a 4-5-fold higher enzyme activity recovery (data not shown). A mixture of glycine and ammonium sulfate proved to be most efficient, enhancing the final activity by nearly a factor of 14 compared to the control without stabilizer.
Table 4 Effect of potential stabilizers on nitrilase activity
Compound (concentration) Residual activity, %
Method A Method B
None 36.3 60
Glycine (1%, w/v) 100 100
Sucrose (10%, w/v) 56.1 64.8
D-Glucose (10%, w/v) 73.7 92.7
Trehalose (10%, w/v) 48.5 89.7
D-Sorbitol (10%, w/v) 59.1 93.9
Xylitol (10%, w/v) 32.7 85.4
D-myo-inositol (10%, w/v) 57.3 97.0
D-Glycerol (10%, w/v) 54.4 86.6
Bovine serum albumin (0.1%, w/v) 56.7 87.0
Bovine serum albumin (1%, w/v) 64.9 80.6
Potential stabilizers were pre-incubated with the enzyme (0.28 mg of protein mL-1) for 180 min (method A) or added to enzyme solutions of the same protein concentration, which were then frozen and thawed 10 times (method B). The enzyme activity was assayed as described in Methods. The specific activity in the presence of 0.1% (w/v) glycine (0.70 and 0.67 U mg-1 protein in method A and B, respectively) was used as the reference value. Data represent the mean of four independent measurements with relative standard deviation values <5%.
Discussion
A large number of putative nitrilase and cyanide hydratase sequences are contained within the whole genomic sequences of fungi. As far as we know, none of the sequenced fungal nitrilases which were predicted to act on organic nitriles have been characterized, contrary to the situation with the fungal cyanide hydratases. Likewise, no sequence data have been available for the characterized nitrilases from Fusarium solani IMI196840 [29] and Fusarium oxysporum f. sp. melonis [30]. Only recently have partial amino acid sequences been identified in the nitrilases from Fusarium solani O1 [17] and Fusarium solani IMI196840 [31], the latter enzyme being probably different from that previously characterized in the same strain [29].
The putative nitrilases of the Aspergillus genus can be roughly divided into two groups, which share a relatively low degree of amino acid identity (30-40%) [7]. One of these groups is closely related to cyanide hydratases (with ca. 60-85% amino acid identity) and the A. niger K10 nitrilase was shown to be a member of this group. The high tendency of this enzyme to form amides from nitriles [8] is in accordance with its evolutionary relationship to cyanide hydratases, the reaction product of which is formamide [16,32].
The heterologous expression of the enzyme in E. coli BL21-Gold(DE3)(pOK101/pTf16) led to a notable increase in enzyme productivity (25.8 U L-1 h-1) under optimized conditions, which was fifteen times higher than in the native producer (approx. 1.7 U L-1 h-1). The potential to synthesize the active enzyme may be even higher in the heterologous producer as indicated by the high ratio of nitrilase to other cellular proteins. However, the output of Nit-ANitRec production was lessened by the low specific activity of the enzyme (at least when using benzonitrile as substrate). When the productivity of Nit-ANigRec and Nit-ANigWT was compared using the preferred substrate of the former enzyme, 2-cyanopyridine, that of the heterologous host was three orders of magnitude higher than that of the native producer.
In comparison with Nit-ANigWT, Nit-ANigRec produced a lower percentage of amide in total product from all substrates tested. With 2-cyanopyridine, the major products of the reaction were different, that is picolinic acid (77% of total product) and picolinamide (>80% of total product) with the Nit-ANigRec and Nit-ANigWT, respectively. Picolinic acid is an intermediate in the production of pharmaceuticals such as local anaesthetics. Nitrilases with satisfactory activities towards 2-cyanopyridine have rarely been reported. The best activity for this compound (approx. 1 U mg-1 protein) was reported in the thermostable nitrilase from Bacillus pallidus Dac521 [33]. This was much less than the activity determined for Nit-ANigRec (9 U mg-1 protein at 38°C).
Nit-ANigRec was less stable than the Nit-ANigWT but this drawback could be overcome by using some low-molecular-weight compounds or bovine serum albumin. These compounds, known collectively as osmolytes, have been recognized as efficient agents in protein stabilization [34]. Of the compounds tested, glycine (1%) was most efficient for the A. niger K10 nitrilase. Glycine and related compounds (sarcosine, betaine) were described as powerful agents able to protect proteins against thermal unfolding [34,35].
Nitrilases forming spiral structures differ from their nonspiral-forming homologs by two insertions of between 12 and 14 amino acids, and a C-terminal extension of up to 35 amino acids [36]. Recently, detailed structural reconstructions using electron microscopy and molecular modelling reported that the formation of spiral helices in the natively produced nitrilases may be related to the removal of 39 C-terminal amino acids from the wild-type protein [12]. This post-translational modification was postulated to be due to autocatalytic activity of this enzyme. The approx. 4-kDa difference in molecular weights of Nit-ANigRec and Nit-ANigWT suggested that a similar-sized peptide was cleaved in the latter enzyme. This assumption was verified by mass spectroscopic analysis, indicating missing cleavage of 46 amino acid residues at the C-terminus of Nit-ANigRec. The R. rhodochrous nitrilase consisting of full-length subunits was unable to form filamentous structures, which were reported for the post-translationally modified enzyme [12], and also for cyanide hydratase [19,37] and cyanide dihydratase [38]. In accordance with these observations, Nit-ANigWT was to a large extent composed of tube-like structures [17], while Nit-ANigRec exhibited a limited tendency to this arrangement.
The reason for the differences in catalytic properties (substrate specificity, reaction optima, amide formation, stability) between Nit-ANigRec and Nit-ANigWT is not clear but most probably it can be ascribed to differences in the post-translational processing of the two forms of the enzyme, and its subsequent effects on the folding, subunit interaction, and oligomerization of the enzyme. A recent mutational analysis revealed a number of effects caused by deletions or mutations in the C-terminal portion of arylacetonitrilase from Pseudomonas fluorescens EBC191 [39]. In this enzyme, the C-terminal deletions of up to 32 amino acids did not cause any differences in the catalytical properties. However, longer deletions of 47 to 67 amino acids resulted in reduction of enzymatic activity, increased formation of amide, and in changes in the enantiomeric selectivity. The effects caused by C-terminal deletions could be reversed by the addition of the corresponding sequences from another nitrilase [39]. It appears difficult to determine what is the relation of the above changes to those caused by 46 amino acid difference observed in the fungal nitrilase described here, and this issue certainly deserves detailed investigations in the future. It remains also unclear if missing post-translational modification is the primary event leading to partial enzyme misfolding, or if this misfolding negatively affected the autocatalytic cleavage of the enzyme.
The changes in catalytic behaviour could be also caused by differences in quaternary structure between Nit-ANigRec and Nit-ANigWT. Similarly, a small increase in activity was associated with fibre formation in cyanide dihydratase in Bacillus pumilus [36].
Attempts to express the enzyme in a eukaryotic host (Yarrowia lipolytica; D. Brady et al., personal communication) did not bring about any positive effect on the enzyme activity, which was barely detectable in the yeast cells. The effect of chaperone co-expression in E. coli was not very efficient in the heterologous expression of this enzyme either, though previous experiments suggested the importance of chaperones for the correct folding of proteins of the nitrilase superfamily. Chaperones were co-purified with nitrilases from Bacillus pallidus [33], Pseudomonas fluorescens [40] and A. niger [8] and also played an important role in folding D-carbamoylase [41]. In vitro re-folding of the enzyme from A. niger was also tested as a potential tool to improve its specific activity but did not prove successful. It appears that re-folding is not necessary for heterologous production of fungal cyanide hydratases or nitrilases as a number of them were produced as fully functional enzymes in E. coli [e.g., [12,16,19]].
As far as we know, little or nothing has been reported on the differences between heterologously expressed nitrilases and nitrilases isolated from the native organisms. This is probably because a number of known nitrilases have been purified and characterized either purely from the heterologous host or purely from the native producer. The enzyme with the highest homology to A. niger nitrilase, cyanide hydratase from A. nidulans (with 86% amino acid identity), was only examined with a single substrate (HCN) [16] and not compared with the purified enzyme from the wild-type producer as far as we know. Even if both enzyme forms were available in bacterial nitrilases [e.g., [12,42]], the enzymatic properties have rarely been compared under the same conditions. Therefore, potential differences between the nitrilases isolated from the native organisms and the heterologously expressed nitrilases may have gone unnoticed. Differences in biochemical properties of different nitrilase species may reflect partial misfolding of individual subunits, different post-translational modifications, but the diversity of the enzymes in terms of structural variants (dimers, short spirals, filaments) may be also important in this respect.
Conclusions
In conclusion, heterologous expression of a fungal nitrilase operating on organic nitriles was achieved in this study for the first time. The enzyme differed from that produced by the wild-type strain A. niger K10 in subunit molecular weight due to a missing post-translational modification (46 C-terminal amino acid cleavage). Nevertheless, it may still be useful for some biocatalytic applications, and even gained some advantages over the enzyme isolated from the native organism, such as a higher hydrolytic activity for 2-cyanopyridine. Purification of the enzyme from the heterologous host was straightforward, enabling tens of mg of the purified protein to be obtained for structural and activity studies. The refolded enzyme underwent changes in its oligomeric structure during storage and was finally fractionated to give a structurally almost homogeneous protein potentially useful for crystallographic nitrilase studies, which have so far been largely impaired by specific quaternary arrangement of these enzymes.
Methods
DNA manipulations
Total RNA was isolated from A. niger K10 using RNeasy Plant Mini Kit (Qiagen) and used to synthesize cDNA using SuperScript II Reverse Transcriptase (Invitrogen) and anchored oligo d(T)23 VN primer (NEB). Partial cDNA of the nitrilase gene was amplified using the degenerate forward primer NITR_NTERMFW01, 5'-AAY GCI GAR CCI GGI TGG TTY GA-3', derived from the N-terminal fragment sequence NAEPGWFD of Aspergillus fumigatus nitrilases [GenBank:EDP55254, XP_756085] and degenerate reverse primer NITR_INT2RE01, 5'-CAT RTA RTG ICC ICC RAA RTC IGC-3', derived from the internal peptide fragment sequence ADFGGHYM from the same A. fumigatus nitrilases. The PCR product (about 0.9 kb) obtained after 35 cycles of PCR on a Mastercycler personal cycler (Eppendorf) with proofreading DNA Polymerase Pfu Turbo (Stratagene) was cloned into the pBluescript SK+ vector (Stratagene) and sequenced using automated DNA sequencer (ABI PRISM 3130xl) according to the manufacturer's protocols.
To obtain 5' and 3' cDNA end sequences, a BD SMART RACE cDNA Amplification Kit (Clontech) was employed. 5'-RACE and 3'-RACE amplifications were performed using 30 cycles of PCR with BD Advantage 2 DNA Polymerase (Clontech). A common universal primer (UPM) was used for the amplifications of cDNA ends together with two distinct gene-specific primers: 5'-RACE reverse primer, NITR_RACE_FW01, 5'-CCG CGT CGG CCA CCT CAA CTG CTG GGA G-3', and 3'-RACE forward primer, NITR_RACE_RE01, 5'-CGT GGA CCT GCT CGC CCA AAG AGG CTG C-3', respectively. The resulting 5'-RACE and 3'-RACE PCR products (approx. 0.6 and 0.7 kb, respectively) were cloned and sequenced as described above.
In order to amplify the DNA fragment coding for the nitrilase sequence, forward primer 5'-GCC ATA TGG CAC CMG TCT TRA AGA AGT ACA A-3', (M = A or C; R = A or G), and reverse primer, 5'-GCA AGC TTT TAC TAG TTC TCC GAA TCC ACG GT-3' were used. The corresponding PCR product obtained after 30 cycles of PCR with Taq-DNA polymerase (Clontech) was ligated into the cloning vector pCR 2.1 TOPO (Invitrogen), cloned in One Shot® TOP10 E. coli Competent Cells (Invitrogen) and sequenced as described above.
Database searches were performed using the BLASTX and BLASTP programme [43,44]. Alignment of amino acid sequences was performed using ClustalW software [45].
For expression, the NdeI-HindIII fragment was ligated into the corresponding sites of the vectors pET-30a(+) (Novagen) and pRSET B (Invitrogen). The resulting vectors pOK101 and pOK102 were transformed into selected strains of E. coli (see below). Alternatively, E. coli was transformed with plasmid pOK101 and plasmid pTf16 (Takara) containing the tig gene which encodes the trigger factor.
Microbial cultures and plasmids
A. niger K10 was grown as described previously [8]. E. coli strains were grown in LB broth at 28°C (strains BL21(DE3), BL21-Gold(DE3), BL21-Gold(DE3)pLys, BL21-CodonPlus(DE3)-RIPL, BL21-CodonPlus(DE3)-RIL (Stratagene), Rosetta-gami 2 (DE3) (Novagen)) or 14°C (strains Arctic Express(DE3), Arctic Express(DE3)-RP and Arctic Express(DE3)-RIL (Stratagene)). Cultures of strains harbouring plasmid pOK101, pOK102 or both plasmids pOK101 and pTf16 (Takara) were performed with kanamycin (50 μg mL-1), ampicillin (150 μg mL-1) or ampicillin (150 μg mL-1) and chloramphenicol (20 μg.mL-1), respectively, in addition to selected antibiotics, with resistance to them encoded in the host chromosome, i.e. chloramphenicol (35 μg mL-1; strain BL21-Gold(DE3)pLys), tetracycline (12.5 μg mL-1; strain BL21-Gold (DE3), both chloramphenicol and tetracycline (strains BL21-CodonPlus(DE3)-RIPL and BL21-CodonPlus(DE3)-RIL, Rosetta-gami 2(DE3)), or gentamycin (20 μg.mL-1; Arctic Express strains). The expression of nitrilase was monitored after induction with IPTG (1 mM) by nitrilase activity assay using whole cells (see below) and by 12% SDS-PAGE [46] followed by Coomassie staining.
Enzyme purification
Nit-ANigRec was purified from the culture of E. coli BL21-Gold(DE3)(pOK101/pTf16). The culture was grown under the following optimized conditions: arabinose (2 g L-1) and IPTG (0.5 mM) addition to cultures with OD610 of 0.6 and 1.1, respectively, and cultivation temperature shift from 37 to 26°C after induction with IPTG. The cells were harvested at OD610 ≈ 8.7 (16 h after IPTG addition) and disrupted by sonication. After removing cell debris by centrifugation (13,000× g, 4°C, 30 min), the supernatant proteins were eluted through a Hi-Prep 16/10 Q FF column (Amersham Biosciences), with a linear gradient of NaCl (0.15-1 M) in Tris/HCl buffer (50 mM, pH 7.6). Active fractions were pooled, concentrated using an Amicon Ultra-4 unit (cut-off 10 kDa; Milipore) and injected into a Hi-Prep 16/60 Sephacryl S-200 column. The proteins, eluted with Tris/HCl buffer (50 mM, pH 7.6, 150 mM NaCl), were pooled, concentrated, analyzed by SDS-PAGE as described above and stored at -80°C.
The refolded enzyme was purified in two steps consisting in Q-Sepharose HP chromatography and gel filtration on Superose 6 Prep Grade. The sample was injected into a Q-Sepharose HP column (1.6 × 11.5 cm) pre-equilibrated with Tris/HCl buffer (50 mM, pH 7.5; 29 mM NaCl, 1 mM NaN3). Proteins were eluted with a linear gradient of NaCl (0.029-1 M) in Tris/HCl buffer (50 mM, pH 7.5; 29 mM NaCl, 1 mM NaN3), concentrated and injected into a Superose 6 Prep Grade column (1 × 25 cm). Proteins were eluted with Tris/HCl buffer (50 mM, pH 7.5; 150 mM NaCl). Those with a molecular weight of approx. 600 kDa (major peak) were collected and stored for one month at 4°C. Then the gel filtration was repeated, the fractions (eluted as the major peak of approx. 500 kDa) pooled, concentrated and stored at 4°C for a further 10 days. Gel filtration was repeated with this sample and the active fractions analyzed separately for enzyme activity, by analytical centrifugation and by electron microscopy.
Nit-ANigWT was purified as described previously [8].
Protein concentration was determined according to Bradford [47] using bovine serum albumin as the standard.
Refolding
Refolding conditions were screened using iFOLD Protein Refolding System 1 (Novagen). The purified enzyme (1 mg; 0.1 mL of Tris/HCl buffer, pH 7.6) was mixed with 1 mL of 6 M guanidine hydrochloride in phosphate-buffered saline (PBS) consisting of 10 mM sodium phosphate buffer (pH 7.4; 150 mM NaCl) supplemented with 1 mM of tris(2-carboxyethyl)phosphine hydrochloride and 0.03% N-lauryl sarcosine. Each of the refolding agent mixtures available in iFOLD Protein Refolding System 1 was transferred into a well of a Greiner BioOne UV Star microplate and 0.010 mL of enzyme solution added into each well. After a 20-h incubation of the microplate with periodic shaking, optical density at 340 nm was determined using a Safire microplate reader (TECAN). Samples with the lowest level of precipitation were assayed for nitrilase activity.
The refolding conditions selected via the above screening were used on a larger scale (1-10 mg protein). The purified enzyme (1-10 mg in Tris/HCl buffer, pH 7.5) was denatured as described above in a total volume of 10 ml. The sample was sonicated and mixed with 90 ml of 50 mM Tris buffer, pH 7.5, containing 100 mM NaCl, 20% glycerol, 12.5 mM methyl-β-D-cyclodextrin and 1 mM NaCl (buffer C12 according to iFOLD Protein Refolding System 1; approx. 10 mL mg-1 protein). Thereafter, the refolding mixture was dialyzed against 2 L of 50 mM Tris/HCl buffer (pH 7.5, 29 mM NaCl, 1 mM NaN3). After 4 h at 4°C, the dialysis buffer was changed for 2 L of fresh buffer, and dialysis was left to proceed overnight. The refolded nitrilase was recovered using Q-Sepharose and Superose 6 chromatographies as described above.
Mass spectrometry analysis
Peptide mass fingerprinting of fragments obtained by tryptic or Asp-N digestion of Nit-ANigRec and Nit-AnigWT was as decribed previously [8].
Determination of N-terminal amino acid sequence
The N-terminal sequences of Nit-ANigRec and Nit-ANigWT were analyzed as described previously [8].
Enzyme assays
The nitrilase activity was determined with 25 mM benzonitrile as described previously [8] with slight modification. If not stated otherwise, reaction temperature was 30 or 38°C with recombinant cells or Nit-ANigRec, respectively, instead of 45°C with Nit-AnigWT. Substrate specificity was determined using 25 mM of various nitriles as substrates under the above conditions. The substrates and reaction products were analyzed by HPLC.
Analytical HPLC
Benzonitrile, its analogues, phenylacetonitrile, 2-phenylpropionitrile and the corresponding reaction products (acids, amides) were analyzed using a Chromolith Flash RP-18 (Merck; 25 mm × 4.6 mm) in a mobile phase consisting of acetonitrile : water : H3PO4, 200 : 799 : 1 (flow-rate 2 mL.min-1; 35°C). Heterocyclic nitriles and their products were analyzed as described previously [9].
Analytical ultracentrifugation
Sedimentation velocity and sedimentation equilibrium experiments were performed using a ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) using an An50Ti rotor, and dual absorbance and laser interference optics. Before the experiment, 0.5 mL samples of nitrilase diluted to 0.4 mg.mL-1 were dialyzed for 20 h against 2 L of 50 mM Tris-HCl pH 7.5 with 150 mM NaCl and 1 mM NaN3, and the dialysis buffer was used as a reference and sample dilution buffer. The sedimentation velocity experiment was conducted at 15,000 rpm and 20°C using an epon double-sector cell (Beckman Coulter). Sample (400 μL) and dialysate (430 μL) were loaded into the sample and reference cells, respectively. Based on buffer composition and nitrilase amino acid sequence using the program SEDNTERP [48], buffer density and nitrilase partial specific volume were estimated to be 1.00585 g.mL-1 and 0.7331 mL.g-1, respectively. Absorbance scans were performed at 280 nm with 5 min intervals using a spacing of 0.003 cm in continuous scan mode and were analyzed with the program SEDFIT [49,50]. A continuous size-distribution for non-interacting discrete species model was calculated and the sedimentation coefficient value determined by integration. Sedimentation equilibrium ultracentrifugation runs were performed with nitrilase concentrations of 0.4, 0.2 and 0.1 mg.mL-1 and at speeds of 3,000, 3,500, 4,000, 4,500, 5,000, 5,500 and 6,000 rpm in a six-channel epon cell for 16 h at 20°C with 110 and 130 μL of sample and reference, respectively. Absorbance data was collected at 280 nm by averaging 20 scans with radial increments of 0.001 cm in step scan mode. The sedimentation equilibrium experiments were globally analyzed with the program SEDPHAT version 6.21 [50,51]. The size and shape of the sedimenting species was predicted using the Teller method in the program SEDNTERP.
Dynamic light scattering
The particle size distribution of the nitrilase solution from individual fractions obtained by gel filtration was assessed using the dynamic light scattering method (Malvern Instruments, ZEN3600) in a low volume glass cuvette (45 μL) at 18°C with an appropriate enzyme concentration in 50 mM TRIS, 150 mM NaCl, 1 mM NaN3, pH 7.5, sample volume 30 μL. The particle diameter values corresponding to the maxima of peaks in the mass distribution are reported. Molecular weight estimations were made using an empirical mass vs. size calibration curve of the instrument software (Dispersion Technology Software 5.03, Malvern Instrument).
Electron microscopy
Protein complexes were negatively stained (2% uranyl acetate) on glow discharge activated carbon coated grids [52]. Samples were viewed under a Philips CM100 electron microscope at 80 kV. Digital images were recorded using MegaView II slow scan camera at primary magnification of 64,000× resulting in pixel size of 0.98 nm.
Homology modelling and molecular dynamics
The search for homologous structures was done with BLAST [43,44]. Structures with the highest identity were extracted from the Protein Data Bank and used as templates for modelling. A structure-based multiple sequence alignment was done with the T-Coffee server [53] and manually corrected on the basis of consensus secondary structure prediction [54]. Three-dimensional models consisting of all non-hydrogen atoms were constructed with the package Modeller 9.1 [55] and validated with ProSA [56].
YASARA [57] was used for visualization, molecular dynamics simulation (MD) and building the multimeric structure. MD was run in water with Yamber2 force field, with a periodic boundary, in an NPT ensemble (temperature set at 298 K, constant pressure and constant number of particles in the cell). Substrate docking of substrate was done using AutoDock 4.0 [58].
Authors' contributions
LM, KB and OK designed research, LM and KB wrote the major parts of the paper. OK, OP, ABV, AR and OŠ carried out the molecular genetic studies. RE and NK designed the homology model. Structural studies were carried out by KB, DK and OV (re-folding, analytical ultracentrifugation), OB (electron microscopy), JD and JD (dynamic light scattering). OK and AM carried out biochemical characterization. JF analyzed and interpreted the gene sequence. VK, MC, KS and MK critically revised the manuscript draft. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Alignment of DNA sequences of fungal nitrilase and cyanide hydratase genes (pdf file).
Click here for file
Additional file 2
Electron micrograph of Nit-ANigRec before re-folding (tif file).
Click here for file
Additional file 3
Electron micrograph of Nit-ANigRec after re-folding (tif file).
Click here for file
Additional file 4
PDB-file of the refined homology model of nitrilase subunit, prepared in YASARA.
Click here for file
Acknowledgements
This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (grant number IAA500200708), Ministry of Education of the Czech Republic (grant numbers LC06010, OC09046, and MSM_21620808), Czech Science Foundation (305/09/H008 and 310/09/1407), COST/ESF CM0701 (short-term scientific mission grants no. COST-STSM-CM0701-4765 and COST-STSM-CM0701-4766 to A. Malandra) and institutional research concept AV0Z50200510 (Institute of Microbiology). We thank Dr. D. Brady (CSIR Johannesburg, South Africa) and his team for examining the feasibility of A. niger K10 nitrilase expression in Yarrowia lipolytica.
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RetrovirologyRetrovirology1742-4690BioMed Central 1742-4690-8-22121101810.1186/1742-4690-8-2ResearchThe cellular source for APOBEC3G's incorporation into HIV-1 Ma Jing [email protected] Xiaoyu [email protected] Jian [email protected] Quan [email protected] Zhenlong [email protected] Pingping [email protected] Jinming [email protected] Fei [email protected] Xuefu [email protected] Liyan [email protected] Lixun [email protected] Jiandong [email protected] Shan [email protected] Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing, PR China2 State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Science, Beijing, PR China3 Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada4 Microbiology & Immunology, McGill University, Montreal, Quebec, Canada2011 6 1 2011 8 2 2 6 9 2010 6 1 2011 Copyright ©2011 Ma et al; licensee BioMed Central Ltd.2011Ma et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<url>http://creativecommons.org/licenses/by/2.0</url>), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.
Results
Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.
Conclusions
Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.
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Background
Human APOBEC3G (hA3G) has been identified as one of anti-HIV-1 host factors [1]. hA3G belongs to an APOBEC superfamily containing at least 11 members, which share a cytidine deaminase motif (a conserved His-X-Glu and Cys-X-X-Cys zinc coordination motif) [2]. The APOBEC superfamily in humans includes APOBEC1, APOBEC2, APOBEC3A-H (hA3A-H), APOBEC4 and activation-induced cytidine deaminase (AID). The virus counters hA3G's anti-viral activity through the viral protein Vif (virion infectivity factor), which interacts with cytoplasmic hA3G as a part of Vif-Cul5-SCF complex, resulting in the ubiquitination and degradation of hA3G [3,4].
Viral encapsidation of hA3G is an essential step for its antiviral activity. Only if hA3G is encapsidated into the virions, can it exert its antiviral activity on the replication of progeny virons in the infectious target cells. This encapsidation of hA3G is facilitated by HIV-1 Gag. The nucleocapsid (NC) domain of Gag mediates the interaction of Gag with hA3G [5-9]. Although the Gag/hA3G interaction has been investigated extensively [10-12], the cellular source of viral hA3G remains unclear. It was found that hA3G in the HIV-1 virion was not reduced as much as the cellular hA3G in the presence of Vif. Furthermore, our previous work has also shown that the removal of the C-terminal region of hA3G results in a significant decrease in its cellular concentration without a corresponding decrease in its incorporation into viral particles [6]. These observations suggest that viruses may recruit hA3G from a particular intracellular pool, and the decrease in total cellular hA3G does not reflect any change occurring in this pool which acts as cellular source of viral hA3G.
The main cytoplasmic form of hA3G in H9 and 293T cells has been reported to be an enzymatically inactive, high-molecular-mass (HMM) ribonucleoprotein complex [13]. RNase treatment converts this complex to an enzymatically active, low-molecular-mass (LMM) form [13]. Biochemical studies have demonstrated the HMM hA3G complex associates with several cellular RNA binding proteins, as well as certain mRNAs and small non-coding RNAs [14-16]. hA3G has been shown to dynamically associate with various RNPs including ribosomes, miRNA-induced silencing complexes, RoRNPs, processing bodies, stress granules, and Staufen granules [14,16].
Recent work suggests that HIV-1 recruits hA3G from the cellular pool of newly-synthesized enzymes prior to its assembly into the HMM RNA-protein complexes, because of the appearance of viral hA3G shortly after its synthesis [17]. In favor of this hypothesis, most components of the HMM hA3G complex have not been found in virions containing hA3G. In addition, Khan et al. reported that encapsidation of hA3G into HIV-1 virions involves lipid raft association and does not correlate with hA3G oligomerization [18]. Nevertheless, another group showed that hA3G mutants failing to form the HMM complex were poorly incorporated into the HIV-1 particle, suggesting that the HMM hA3G may act as the cellular source for virion encapsidation [19].
The purposes of this study are to better characterize cellular distribution of hA3G, and provide insight into the cellular source for hA3G encapsidation into HIV-1. Our work herein shows that the majority of newly-synthesized hA3G interacts with lipid rafts, and acts as both the precursor of mature HMM hA3G complex and the cellular source of hA3G in HIV-1.
Results
The subcellular distribution of hA3G in P100 and S100 fractions
We first analyzed the cytoplasmic distribution of hA3G, using a subcellular fractionation assay. H9 cells, a human T-cell line expressing endogenous hA3G, were lysed by dounce homogenization in hypotonic TE buffer in the presence of RNase inhibitor and protease inhibitor. Similarly, 293T cells that do not express endogenous hA3G were transfected with a plasmid coding for HA (hemagglutinin) tagged hA3G, and then lysed 48 hours post-transfection. Following centrifugation of the cell homogenate at low speed to remove nuclei and unbroken cells, the resultant supernatant (S1) was ultra-centrifuged at 100,000 × g, resulting in pellet (P100) and supernatant (S100). Western blots of the P100 and S100 fractions were probed with either anti-hA3G or anti-HA for the samples derived from H9 cells or 293T cells, respectively (Figure 1A). Approximate 85% of total endogenous hA3G in H9 cells presented in the P100 (lanes 1 to 3), and a similar pattern was also obtained from hA3G transiently expressing in 293T cells (lane 4 to 6). Next, we analyzed the S1, P100 and S100 fractions prepared from 293T cells expressing hA3G, using a 4-35% discontinuous Opti-prep velocity gradient. Nine fractions were collected from the top to the bottom of the gradient, and then subjected to Western blot. In these gradients, hA3G in the S1 was found in both LMM fractions (including fractions 3 and 4) and HMM fractions (including fractions 7 and 8), as shown in the top panel of Figure 1B. hA3G in the P100 was solely detected in fractions 7 and 8 (middle panel, Figure 1B), and co-sediments with the HMM form of hA3G found in the S1, while hA3G in the S100 was only found in fractions 3 and 4 (bottom panel, Figure 1B). These results suggest that the majority of hA3G in the P100 and S100 fractions represented the HMM and LMM forms of hA3G respectively.
Figure 1 The cellular distribution of hA3G in P100 and S100 fractions. H9 cells and 293T cells expressing HA tagged hA3G were lysed in hypotonic TE buffer, and the resultant S1 was ultra-centrifuged, resulting in the P100 and the S100 fractions. The S1, P100 and S100 fractions prepared from 293T cells were analyzed by using a 4-35% discontinuous Opti-prep velocity gradient, as described in Methods. A. Western blots of the S1, P100, and S100 fractions were probed with either anti-hA3G (left) or anti-HA (right) for the samples derived from H9 cells or 293T cells, respectively. B. Nine fractions were collected from the top to the bottom of the gradient, then subjected to Western blot probed with anti-HA. The fraction numbers increase from the top to the bottom of the gradient.
Steady state hA3G in the cytoplasm appears in three different forms
hA3G has been shown to localize to lipid rafts, which are specialized membrane domains enriched in certain lipids, cholesterol and a specific set of proteins [5]. We reasoned that some of the HMM form of hA3G might result from association of soluble hA3G with lipid rafts. To examine this, P100 was further analyzed by floatation assay. After ultra-centrifugation at 100,000 × g overnight in sucrose gradient, all the collected fractions were subjected to Western Blot probed with anti-Caveolin-1 (lipid raft marker), anti-membrane transferrin receptor (TFR, a cytoplasm membrane bound protein) and anti-HA. As shown in Figure 2A, total HMM hA3G was fractionated into raft (lane 3 and 4) and non-raft (lane 7 to 9) fractions. Approximately 30% of the HMM form of hA3G associated with lipid rafts. Following treatment with mild nonionic detergent octyl glucoside, both the raft and non-raft fractions from HMM hA3G were subjected to the velocity gradient analysis. It shows that raft-associated hA3G was found to shift from the HMM fraction to the LMM fractions, while non-raft hA3G presented in fraction 8 at the bottom of the gradient and represented the HMM complex reported previously (Figure 2B). These data clearly demonstrate that a proportion (approximately 30%) of pelletable HMM hA3G is detergent-sensitive, which represents a LMM form of hA3G associated with lipid rafts, and the majority of pelletable hA3G appears to be mature HMM complexes.
Figure 2 Steady state hA3G in the cytoplasm appears in three different forms. A. 293T cells expressing HA tagged hA3G were lysed in hypotonic TE buffer. As described in Methods, P100 was prepared and treated with or without nonionic detergent octyl glucoside as indicated, then resolved by the sucrose floatation assay into the raft and non-raft proteins. Each fraction was analyzed by Western blot for the presence of hA3G, Caveolin-1 and TFR. B. The raft and non-raft fractions of hA3G were collected and treated with octyl glucoside, then resolved in the Opti-prep velocity gradient. Western blots of each fraction were probed with anti-HA. C. 293T cells expressing HA tagged hA3G were lysed in hypotonic TE buffer, and the S1 fractions were either untreated (lane 1 and 2) or treated with 0.5% Triton X-100 at 4°C (lane 3 and 4) and 37°C (lane 5 and 6), respectively. Following the ultra-centrifugation of the S1 fraction, Western blots of the P100 and S100 fractions were then probed with antibody specific for HA (top), TFR (middle), and caveolin-1 (bottom). S and P represent the S100 and P100 fractions, respectively. D. Fractions that respectively contain the soluble LMM (lane 1), RA LMM (lane 2) and HMM (lane 3), were subjected to immunoprecipitation with anti-HA, followed by Western blots of the immunoprecipitates probed with anti-HA, anti-RHA, and anti-Staufen, respectively.
A similar result was obtained by using cellular fractionation. We treated the S1 fraction containing hA3G with 0.5% Triton X-100 (TX-100) at 37°C, which will also resulted in solubilization of lipid rafts [20,21]. Following the ultra-centrifugation of the S1 fraction, Western blots of the P100 and S100 fractions were then probed with antibody specific for HA and caveolin-1 respectively. As shown in Figure 2C, the detergent treatment resulted in total release of caveolin-1 from the P100 fraction to the S100 fraction. Simultaneously, approximately 30% of pelletable HMM hA3G (lane 6) was reduced with a corresponding increase in the soluble LMM hA3G (lane 5). In contrast, incubation of the S1 fraction with TX-100 at a low temperature (4°C), a condition that only resolves cytoplasm membrane but not lipid rafts, did not affect the distribution of either hA3G or caveolin-1. A similar result was also obtained when H9 cells were used in the same experiment as described above (data not shown).
Together these data indicate that steady state hA3G in the cytoplasm appears as three different forms: LMM hA3G (or soluble hA3G), raft-associated LMM hA3G (RA LMM hA3G) and HMM hA3G complex. Consistent with the conclusion, a co-immunoprecipitation analysis shows that Staufen and RNA helicase A (RHA), two components found in the HMM complex [14], only associated with the HMM hA3G, but not with the LMM or RA LMM hA3G (Figure 2D).
The RA LMM hA3G acts as the precursor of the HMM hA3G complex
In an attempt to make a dynamic analysis of these three forms of hA3G, 293T cells expressing HA-tagged hA3G were labeled with [35S]methionine-[35S]cysteine for 10 min at 36 h posttransfection, followed by a chase period with cold methionine-cysteine. Aliquots of the cells were taken during the chase up to 3 hours, and then lysed hypotonically as previously described. The resultant S1 supernatant was firstly fractionated into S100 and P100 fractions. The P100 fractions were further treated with TX-100 at 37°C, and then separated by 100,000 × g centrifugation into supernatant and pellet. These fractions, which respectively contain the LMM, RA LMM, and HMM hA3G, were subjected to immunoprecipitation with anti-HA, followed by analysis of the distribution of radioactive hA3G using one-dimensional (1-D) PAGE (Figure 3A). The relative amount of radio-labeled hA3G in each fraction was determined by autoradiography and presented graphically in Figure 3B. The total amount of hA3G in each fraction was set as 100%. Results show that radio-labeled hA3G was present in the LMM and RA LMM, but not HMM fractions at 0-min of chase, i.e., after a 10-min pulse. The LMM hA3G decreased rapidly over the first 30 minutes of chase and remained stably thereafter, then reduced gradually after 2 hours post-radiolabel. In contrast, the amount of RA LMM hA3G increased during the first 30 minutes, and underwent a significant decrease thereafter. The radio-labeled hA3G in the HMM fraction increased gradually during the early time of chase, reached a peak by 1 to 2 hours and then declined. During the first 30 min chase period, the decrease of hA3G in LMM and the simultaneous increase of hA3G in RA LMM probably reflect a rapid movement of newly-synthesized hA3G to the lipid rafts. The distinct dynamics of newly-synthesized hA3G in RA LMM and HMM indicate that the RA LMM hA3G found here is not a breakdown product of the HMM hA3G complex, rather it is a distinct LMM form of hA3G. After 30 minutes chase, the amount of radio-labeled hA3G in the HMM complex increased significantly accompanied by a great reduction in newly-synthesized hA3G in RA LMM and the amount of soluble LMM hA3G remained stable. It is worthy to note that total hA3G was only reduced approximately 30% over 3 hours of chase, consistent with previous reports [22,23]. Although some LMM hA3G were degraded during the chase, the majority of LMM undergoing a significant decrease was most likely converted into the HMM form. This suggests that the RA LMM hA3G, instead of the LMM form, acts as the precursor of the HMM hA3G complex. All this reflects a dynamic course among LMM, RA LMM and HMM forms of hA3G, including a rapid movement of newly-synthesized hA3G from the LMM form to the lipid rafts, which serve as a precursor to the HMM hA3G complex.
Figure 3 The RA LMM hA3G acts as the precursor of the HMM complex. 293T cells expressing hA3G were radiolabeled and chased. Aliquots of the cells were collected, and then lysed hypotonically, as described in Methods. A. Total cell lysate and three fractions containing LMM, RA LMM and HMM form of hA3G were subjected to immunoprecipitation with anti-HA, respectively, followed by analysis of the distribution of radioactive hA3G using one-dimensional (1-D) PAGE. B. The relative amount of radio-labeled hA3G in each fraction was determined by autoradiography and presented graphically. The bar graphs represent the means of results of experiments performed at least three times, and the error bars represent standard deviations.
The correlation between the cellular distribution and viral incorporation of hA3G
Attempting to identify the cellular source of viral hA3G, we first determined if a correlation existed between the cellular distribution and viral incorporation of hA3G. We investigated the effect of a set of truncated mutations, which were described previously [6] and shown graphically in Figure 4A, upon the overall distribution of hA3G among the LMM form, the RA LMM form and the mature form of HMM hA3G complex, as described in Figure 2. The amounts of hA3G in these three forms were determined by Western blot (Figure 4B) and graphically shown in Figure 4C. It shows that hA3G missing amino acids 1-156 resulted in its failure to assemble into either the RA LMM or the mature form of the HMM complex. It is worth noting that the majority of two C-terminal deletions of hA3G resided in the RA LMM, i.e., approximately 70% of Δ157-384 formed the RA LMM and only 25% assembled into the mature HMM complex. This data suggest that the removal of the C-terminus of hA3G may impair its ability to convert the RA LMM into the mature HMM hA3G complex.
Figure 4 The correlation between the cellular distribution and viral incorporation of hA3G. A. Wild type and mutant hA3G. The filled rectangles represent the two zinc coordination units. The numbers represent the amino acid positions. B. 293T cells were co-transfected with hGag and either wild-type or mutated forms of hA3G, and the S1 fractions of the cell lysates were subjected to ultra-centrifugation and octyl glucoside treatment, resulting in the LMM, RA LMM and HMM forms of hA3G. The amounts of hA3G in the three forms were determined by Western blot. C. The cellular distributions of wild type and mutant hA3G are graphically shown. D. Western blot of cell lysates were probed with anti-HA. E. Western blot of virus like particle lysates probed with anti-HA. F. The relative amounts of mutated hA3G in the cell or viral lysates were normalized to wild-type hA3G, and then a ratio of viral to cellular hA3G was determined and used to measure its ability to be packaged into virions. The bar graphs in panel C and F represent the means of results of experiments performed at least three times, and the error bars represent standard deviations.
Next, we co-transfected 293T cells with plasmids coding for hGag and either wild-type or mutations of hA3G described above. The expression and viral incorporation of the hA3G variants were assessed by Western blots of cell and virion lysates, respectively (Figure 4D and 4E). Consistent with our previous report [6], Western blot analysis shows that hA3G missing amino acids 1-156 exhibited reduced incorporation into Gag VLPs, while the removal of the C-terminal portion of hA3G resulted in more efficient viral incorporation compared to wild type hA3G. The relative amounts of mutated hA3G in the cell or viral lysates were normalized to wild-type hA3G, and then a ratio of viral to cellular hA3G was determined and used to measure its ability to be packaged into virions (Figure 4F). A comparison of Figure 4F and Figure 4C indicates that the amount of hA3G residing in the RA LMM directly correlates with its ability to be incorporated into HIV-1. A similar quantitative change in the amounts of hA3G in the RA LMM and the virions provides further supporting evidence that the RA LMM represents the cellular source of viral hA3G.
The ability of hA3G to bind to Gag is insufficient for its incorporation into HIV-1
Several amino acid residues (i.e., W127) within the N-terminal part of the linker region play an important role in mediating the hA3G/Gag interaction. hA3G missing in this region will not likely bind to Gag, thus abolishing its incorporation into the virions [6,18,24,25]. While another mutation hA3G, Y124A, has been reported to possess the ability to bind to Gag but not to be packaged into virions [24]. To better define the role of the RA LMM in viral incorporation of hA3G, we further investigated the cellular distribution of hA3G Y124A. As shown in Figure 5A, hA3G Y124A was expressed at a similar level as wild type hA3G (left panel), whereas viral incorporation of hA3G Y124A was reduced by 3-4 folds (central panel), which is consistent with a previous report [24]. By co-immunoprecipitation analysis, similar amounts of hA3G and hA3G Y124A were detected in anti-p24 immunoprecipitates from the cell lysates (right panel), indicating that this mutant is able to interact with HIV-1 Gag as efficiently as wild-type hA3G. In contrast, viral inefficient encapsidation of W127A was mainly attributable to the loss of its interaction with HIV-1 Gag (Figure 5B). So, we further determined the distribution of wild type hA3G and hA3G Y124A among the LMM form, the RA LMM form and the mature form of HMM hA3G complex. In Figure 5C showed that the Y124A mutation, similar to the N-terminal hA3G truncations, caused a significant reduction in the RA LMM and HMM complex. This data suggest that, in addition to the ability to bind to HIV-1 Gag, the cellular distribution of hA3G is also critical for its viral incorporation.
Figure 5 The ability of hA3G to bind Gag is insufficient for its incorporation into HIV-1. 293T cells were co-transfected with plasmids coding for hGag and either hA3G Y124A (A) and W127A (B). Left panel: Western blot of cell lysates were probed with either anti-HA (top) or anti-β-actin (bottom). Middle panel: Western blot of virion lysates were probed with either anti-HA (top) or anti-p24 (bottom). Right panel: Western blots of rabbit anti-p24 immunoprecipitates were probed with either anti-HA (top) or mouse anti-p24 (bottom). C. Relative amounts of hA3G Y124A in the LMM, RA LMM and HMM hA3G complex were determined by Western blot and graphically presented. The bar graphs represent the means of results of experiments performed at least three times, and the error bars represent standard deviations.
Discussion
The purposes of this study are to better characterize cellular distribution of hA3G and to provide insight into the cellular source for hA3G encapsidation into HIV-1. In this work, we found that the majority of either endogenous hA3G in H9 cells, or hA3G transiently expressed in 293T cells, resided in the P100 fraction and were solely detected in the HMM fraction in a Opti-prep velocity gradient. Approximately 15% of the total hA3G appeared in the cytoplasm as a soluble form that was found in the S100 fraction after ultracentrifugation and in the LMM fraction of the velocity gradient. Using the criteria of sensitivity to the nonionic detergent octyl glucoside, we determined that the pelletable hA3G consisted of two distinct forms: RA LMM hA3G which were associated with lipid rafts and hA3G in the mature HMM complex. The HMM complex contains both Staufen and RNA helixase A, which is consistent with previous characterization of the mature HMM hA3G complex [14]. The results of a pulse-chase radiolabeling experiment revealed that the RA LMM hA3G represents the majority of newly-synthesized hA3G that associates with membrane lipid raft domains, and serves as the precursor of the HMM hA3G complex.
Although LMM hA3G can be converted to HMM complex when CD4 T cells are activated with various mitogens and cytokines [26,27], the mechanism by which hA3G is regulated to assemble into different complexes is largely unknown. Our work herein suggests two essential steps during the assembly of the HMM complex: 1) formation of the RA LMM precursor at lipid rafts and 2) conversion of this precursor into the mature HMM complex. For the first step, mutagenesis studies of hA3G revealed that the removal of amino acids 105 to 156, the linker region of hA3G, inhibited its localization at lipid raft domains to assemble the RA LMM and subsequent mature form of the HMM complex, thus resulting in a predominantly soluble LMM form of hA3G. Investigation of hA3G Y124A provides further evidence supporting the importance of the linker region for the assembly of the RA LMM and the distribution of hA3G. These data together suggest that hA3G amino acids 105-156 are required for its localization at lipid rafts where hA3G assemble into the RA LMM, the precursor of the HMM complex. A recent work has identified a novel cytoplasmic retention signal (CRS) within the linker region of hA3G [28]. The CRS residing within amino acids 113-128 is necessary and sufficient to retain hA3G in the cytoplasm. We reason that the CRS may be involved in the lipid rafts localization of hA3G and thereby restricts hA3G to the cytoplasm. Further studies are still needed to fine-map the motif within hA3G required for its cytoplasmic retention and lipid rafts localization, and to determine if a correlation exists between these two parameters.
It is worthy to note that all of the hA3G C-terminal deletions tested become pelletable and no soluble LMM form was detected. One explanation for this observation is a rapid conversion of the mutants from LMM to HMM. Alternatively, other groups and we have reported that N-terminal fragments of hA3G are inherently unstable [6,29], it is thus possible that the LMM form of these mutants has been degraded rapidly and association with lipid raft may increase their stability. In addition, significant accumulation of the RA LMM form of mutants suggests that the assembly of the mature HMM hA3G complex may involve sequences further down stream of hA3G 105-156; the removal of the C-terminus of hA3G may therefore impair its ability to convert the RA LMM into the HMM complex.
Previous works have shown that reduced cellular expression of the hA3G C-terminal truncations did not result in a corresponding decrease in its viral incorporation [6]. This suggests that viruses may recruit hA3G from a particular intracellular pool, i.e., the cellular distribution of hA3G may strongly influence its viral incorporation. Indeed, the fact that hA3G Y124A is able to bind to Gag in vitro, but fails to be packaged into virions, suggests that some other properties of this protein, such as specific cellular localization, are also required for the interaction of these two molecules occurring in vivo. Khan et al. reported that inability of the mutant to be packaged may result from its failure to associate with lipid rafts [10]. Studies of viral incorporation of truncated hA3G show that its ability to be packaged into virions directly correlates with its concentration in the RA LMM hA3G complex (Figure 4), suggesting that the RA LMM acts as a cellular source for hA3G virion encapsidation. In agreement with this hypothesis, hA3G Y124A that is deficient in its ability to form the RA LMM and HMM complexes (the precursor and mature forms), is unable to be packaged into virions, even though it is able to interact with Gag as efficiently as wild type hA3G (Figure 5). Furthermore, the movement of newly-synthesized hA3G to lipid raft domains to rapidly form the RA LMM is consistent with a previous finding that hA3G is incorporated into the virion shortly after its synthesis in cytoplasm [17]. These data together indicate that the RA LMM hA3G complex acts as the cellular source for its virion encapsidation.
Since HIV-1 Gag concentrates in the multivesicular bodies (MVB)/late endosomal compartments enriched in lipid rafts during virion assembly, one explanation for the role of the RA LMM hA3G in the incorporation is the localization in lipid rafts, where both Gag and hA3G concentrate, thereby interacting with each other. In agreement with the hypothesis, hA3G has been shown to associate with intracellular membrane rafts, and more specifically, late endosomal vesicles [5].
Conclusions
This work thus provides the first evidence for the existence of RA LMM hA3G complex and leads toward a better understanding of the regulation of hA3G regarding its antiviral and cellular functions. The potential implications of this work for the development of anti-HIV therapeutics include either enhanced viral incorporation of hA3G by accumulation of the RA LMM complex, or increased accumulation of the LMM complex by blocking its localization at lipid rafts, which may produce a Vif-resistant post-entry inhibition on HIV-1 replication found in resting T cells.
Methods
Plasmid construction
The hGag plasmid, which encodes the HIV-1 Gag sequence, produces mRNA whose codons have been optimized for mammalian codon usage, and was a kind gift from G Nabel, NIH [30]. The construction of wild-type and mutant forms of hA3G has been previously described [6]. hA3G Y124A was constructed using a site-directed mutagenesis kit (Stratagene) and confirmed by sequencing.
Cell, transfection and virus purification
The culture and transfection of HEK-293T cells with these plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, California), and the isolation of virions 48 h posttransfection from the cell supernatant, were done as previously described [6,31]. Unless stated otherwise, 293T cells were transfected with 1 μg of hGag and 1 μg of plasmid coding for wild type or mutant forms of hA3G. The total amount of plasmid DNA used for transfection was kept constant in controls by replacing plasmid coding for hA3G with the empty vector, pcDNA3.1.
Protein Analysis
Cellular and viral proteins were extracted with RIPA buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% NP40, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 100 mg/ml PMSF). Equal amounts of protein (determined by a Bio-Rad assay) were analyzed by SDS PAGE (10% acrylamide), followed by blotting onto nitrocellulose membranes (Amersham Pharmacia). Western blots were probed with the following antibodies that are specifically reactive with: HIV-1 capsid (Zepto Metrocs Inc.), HA, TFR (Invitrogen) and caveolin-1 (Santa Cruz Biotechnology Inc.), β-actin (Sigma), RNA helicase (a gift from Chen Liang [32]), Staufen (a gift from Andrew Mouland [33]). Detection of proteins was performed by enhanced chemiluminescence (NEN Life Sciences Products), using as secondary antibodies anti-mouse and anti-rabbit, both obtained from Amersham Life Sciences. Bands in Western blots were quantitated using the ImageJ 1.35 s automated digitizing system (NIH).
Subcellular fractionation
293T cells were lysed 48 h post-transfection at 4°C in hypotonic medium; lysis was done by Dounce homogenization in 1.0 ml of hypotonic TE buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.01% ß-mercaptoethanol) supplemented with protease inhibitor cocktail (Complete; Boehringer Mannheim) and RNase inhibitors (Ambion). The cell homogenate was then centrifuged at 1,500 × g for 30 minutes to remove nuclei and unbroken cells. The supernatant (S1) was then centrifuged for 1 h at 100,000 × g in an SW55Ti rotor (Beckman, Columbia, Md.) at 4°C, resulting in the supernatant (S100) and the pellet (P100). To resolve cytoplasmic membrane or lipid rafts, the S100 and the P100 suspended in 1 ml of hypotonic TE buffer was incubated with 0.5% Triton X-100 at 4°C or 37°C for 15 minutes, respectively.
Resolution of hA3G into the LMM and HMM forms was performed, using a 4-35% discontinuous Opti-prep velocity gradient. This gradient was prepared in advance by layering 0.5 ml of 35%, 0.5 ml of 30%, 0.5 ml of 25%, 1.5 ml of 20%, 0.5 ml of 15%, 0.5 ml of 10%, and 0.5ml of 4% Opti-prep from bottom to top. 0.5 ml of the S1, S100 or the P100 re-suspended in hypotonic TE buffer was layered on top of the gradient, and then centrifuged at 100,000 × g in a Beckman SW55Ti rotor for 1 h at 4°C. Nine fractions (0.5 ml) were collected and diluted with an equal volume of 2× TNT, and each fraction was subjected to Western blot or immunoprecipitation analysis.
Memberane floatation assay (raft association)
293T cells expressing HA tagged hA3G were lysed and fractionated into S100 and P100 as described above. The pellet P100 was resuspended in TNE buffer (100 mM Tris-HCl, 600 mM NaCl, 16 mM EDTA, supplemented with protease inhibitor cocktail and RNase inhibitors) containing either 0.5% Triton X-100 or 0.5% nonionic detergent n-octyl-β-D-glucopyranoside (octyl glucoside). Following mixed with 85.5% sucrose (w/v) in TNE lysis buffer to obtain 73% sucrose (w/v), samples were placed at the bottom of ultracentrifuge tubes, and overlaid with 65% (w/v) sucrose and 10% sucrose (w/v) in TNE lysis buffer. Then samples were centrifuged at 4°C in a SW55 rotor for 16 hours at 35,000 rpm to obtain raft and non-raft associated fractions [18,34]. Nine equal fractions were collected from the top, followed by analysis of Western blot.
Immunoprecipitation assay
293T cells from 100 mm plates were collected 48 hours post transfection, and lysed in 500 μl TNT buffer (20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1% Triton X-100). Insoluble material was pelleted at 1800 × g for 30 minutes. Equal amounts of protein were incubated with 5 μl HA (or p24)-specific antibody for 16 hours at 4°C, followed by the incubation with protein A-Sepharose (Pharmacia) for two hours. The immunoprecipitate was then washed three times with TNT buffer and twice with phosphate-buffered saline (PBS). After the final supernatant was removed, 30 μl of 2X sample buffer (120 mM Tris HCl, pH 6.8, 20% glycerol, 4% SDS, 2% β-mercaptoethanol, and 0.02% bromphenol blue) was added, and the precipitate was then boiled for 5 minutes. After microcentrifugation, the resulting supernatant was analyzed using Western blots, as previously described [35].
Pulse-chase radiolabeling experiments
Transfected 293T cells were labeled 36 h posttransfection with 400 μCi of [35S]methionine-[35S]cysteine for 15 minutes and then chased for various lengths of time in Dulbecco modified Eagle medium containing 10% fetal bovine serum and 100 μM cysteine and methionine. After being washed, the cells were lysed hypotonically by Dounce homogenization in 1 ml of hypotonic TE buffer at 4°C, and the cell lysates were centrifuged at 1,500 × g for 30 min to remove nuclei and unbroken cells. The resulting S1 supernatant (1 ml) was fractionated into S100 and P100 fractions by centrifuging for 1 h at 100,000 × g in SW55Ti rotor at 4°C. The P100 fractions were further treated with octyl glucoside, and then separated by 100,000 × g centrifugation into a supernatant and pellet which contain RA LMM and HMM hA3G, respectively. The immunoprecipitation with anti-HA was performed, followed by analysis of the distribution of radioactive hA3G using one-dimensional (1-D) PAGE and autoradiography.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
First three authors contributed equally to this work. JM, XYL and JX have made equal contributions to conception and design, acquisition of data, and analysis and interpretation of data. JM was involved in drafting the manuscript. QZ, ZLL and PPJ carried out the mutation construction. FG, LYY and XFY participated in the design of the study and performed the statistical analysis. LXZ and JDJ were involved in revising the manuscript and helping to draft the manuscript. SC supervised the project and commented on the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported in part by Nature Science Foundation of China 30973569 (S.C.), The National S&T Major Special Project on Major New Drug Innovation 2009ZX09103-138 (S.C.) and 2009ZX09303-005 (X.F.Y. and C.S.), and the Canadian Institutes for Health Research grant (S. C.).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21283593PONE-D-10-0104710.1371/journal.pone.0016256Research ArticleAgricultureAgricultural BiotechnologyGenetically Modified OrganismsCropsBiologyBiochemistryProteinsNutrient and Storage ProteinsBiotechnologyGenetic EngineeringGenetically Modified FoodsPlant BiotechnologyGenetically Modified OrganismsTransgenic PlantsPlant SciencePlant BiotechnologyTransgenic PlantsPlantsTransgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein Protein Biofortification of Cassava RootsAbhary Mohammad
1
Siritunga Dimuth
2
Stevens Gene
3
Taylor Nigel J.
1
Fauquet Claude M.
1
*
1
International Laboratory for Tropical Agricultural Biotechnology, Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America
2
Department of Biology, University of Puerto Rico-Mayaguez, Mayaguez, Puerto Rico
3
University of Missouri-Delta Center, Portageville, Missouri, United States of America
Newbigin Edward EditorUniversity of Melbourne, Australia* E-mail: [email protected] and designed the experiments: CMF MA NJT. Performed the experiments: MA DS GS. Analyzed the data: MA CMF DS NJT. Wrote the paper: MA NJT CMF. Generated the greenhouse and field experiments: DS GS. Produced the transgenic cassava plants: NJT.
2011 25 1 2011 6 1 e1625620 8 2010 20 12 2010 Abhary et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181–194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.
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Introduction
Cassava (Manihot esculenta) is cultivated throughout the tropics and is a major staple food crop across tropical sub-Saharan Africa [1]. In a previous study [2], we established that the starchy roots of cassava contain no storage proteins, possess levels of total protein in the range 0.7 to 2.5% dry weight (dw) (compared to 7 to 14% in cereals [3]) and are almost completely devoid of sulfur-containing amino acids [4]–[6]. Insufficient protein intake is a major causal factor of protein energy malnutrition (PEM), which is estimated to affect one in four of the world's children [7]. With the lowest protein∶energy ratio (P∶E) of any staple food, resource-poor populations that rely on cassava as their major source of calories are at high risk of PEM [8] and developing conditions such as Kwashiorkor [9] and related pathological disorders [10].
Few reports exist of successful transgenic modification of starchy storage organs to accumulate storage proteins. Chakraborty et al. (2000) [11] achieved a 40% increase in the protein content of potato tubers to reach 10 to 14% dw, by accumulation of the seed albumin protein from Amaranthus. The protein content of sweet potato tubers was enhanced two- to threefold to obtain 12% dw by expression of the artificial storage protein ASP1 [12] but this occurred in a non-specific manner, with similar levels of artificial protein also accumulated in the transgenic shoot tissues. When the same ASP1 gene was expressed in cassava under the control of the CaMV 35S promoter [13], no increase in protein content was achieved in the storage root organs.
In the present study, cassava was genetically modified using the patatin promoter [14] to direct transgenic expression of zeolin to the tuberous roots. Zeolin is a fusion product between phaseolin, the major storage protein in common beans (Phaseolus vulgaris), and a truncated gamma-zein protein from maize (Zea mays), which directs the fused polypeptide to form stable protein bodies within the ER [15]. Previous reports have shown expression of zeolin to result in significant accumulation of this storage protein in leaf tissues of tobacco and alfalfa [16]. We report here production of transgenic cassava plants expressing zeolin in which storage roots accumulated up to 12.5% dw protein, a greater than fourfold increase compared to controls with no associated accumulation of the protein product in leaf tissues. Analysis of transgenic plants grown under greenhouse and field conditions have confirmed that this trait is stable when plants are propagated vegetatively, it does not impact plant development and it was correlated with a significant reduction in the cyanogen content of both leaf and root tissues.
Results
Production and analysis of transgenic plants
Cassava plants were genetically transformed with pILTAB601 and pILTAB600, in which zeolin is expressed under the control of the CaMV 35S and patatin promoters respectively. A total of 210 in vitro regenerated plantlets were screened, from which a subset was indentified possessing one to two copies of the zeolin transgene (Figure S1). Eleven low copy transgenic lines, seven driven by the patatin promoter and four by the 35S promoter expressing zeolin at the RNA level, were transferred to soil in pots and grown in the greenhouse to produce storage roots.
Total protein was extracted from peeled storage roots at 8 weeks after planting and every 30 days thereafter, and levels determined by Bradford assay. All seven transgenic lines in which the patatin promoter drove zeolin expression, had accumulated 9.75% to 10.60% dw protein within their storage roots by six months after planting, a 3.0- to 3.5-fold increase compared to controls (Figure 1A). Total protein content accumulated at slightly different rates between the transgenic lines (Figure 1B), but was not correlated with RNA expression levels (Figure S1C). When total protein extract was loaded on an SDS gel, strong bands corresponding to the size of zeolin were found at significantly higher levels in transgenic storage root tissues than in leaves (Figure 1C). Conversely, plants in which zeolin expression was driven by the 35S promoter showed no significant increase in protein content of storage roots (Figure 1A) as determined by Bradford assay. In these plants presence of zeolin was observed in leaf extracts but barely detectable in storage root tissues (Figure 1C) when expressed under this constitutive promoter. Western blotting with antibodies specific to the phaseolin component confirmed presence of zeolin in the respective tissues (Figure 1D).
10.1371/journal.pone.0016256.g001Figure 1 Protein accumulation in leaves and peeled storage roots of transgenic cassava expressing zeolin under control of the patatin and 35S promoters.
A) Protein content in storage roots as quantified by Bradford assay after 7 months of growth in pots in the greenhouse. Non-transgenic cassava cv. 60444 (dark green bar) and transgenic plant line with empty gene vector only (pCambia2300) (light green bar) show total protein content of less than 3% dw, while all seven transgenic lines expressing zeolin under control of the patatin promoter (blue bars) accumulate total protein at 9.5% to 10.3% dw. Storage roots from plants expressing zeolin under control of the 35S promoter (orange bars) do not accumulate protein at levels above controls. B) Accumulation of total protein in peeled storage roots of pot grown transgenic plants expressing zeolin under control of the patatin promoter with time, as assessed by Bradford assay. Total protein content of non-transgenic cassava cv. 60444 (green bar) shows no increase over a 7 month cultivation period in the greenhouse, while the three transgenic events studied accumulated protein at slightly differing rates, but all reached approximately 10% dw by the end of the 7 month period. C) SDS-PAGE of crude protein extracted from leaves and roots of in vitro transgenic cassava lines expressing the zeolin transgene. Forty micrograms of protein extract were loaded as follows: lane 1: protein ladder; lanes 2 and 3: non-transgenic cassava cv. 60444 leaves and roots respectively; lanes 4 and 5: leaves and roots of transgenic cassava line transformed with pILTAB601 in which 35S drives expression of zeolin; and lanes 6 and 7: leaves and roots transgenic cassava line transformed with pILTAB600 in which the patatin promoter drives expression of zeolin. Presence of distinct band at 65 kDa in transgenic plants, but not in the control, indicates accumulation of zeolin in leaves of plants transgenic for 35S-zeolin and roots of plants transgenic for patatin-zeolin, but not vice versa. D) Western blot analysis of leaves (L) and peeled storage roots (R) harvested from greenhouse soil beds at 7 months of age. Total protein was isolated, 100 µg loaded in each lane and detected with specific anti-zein antibodies. Presence of zeolin protein was detected in all tissues, but accumulated at 3.5 times greater amounts in roots when expressed under the control of patatin versus the 35S promoter with the reverse pattern seen in leaves. E) Immuno-printing of 50–100 µm thick sections of cassava storage roots from transgenic line pILTAB600-25 in which the patatin promoter drives zeolin. Plants were grown in pots in the greenhouse and harvested from one to sevens months of age with non-transgenic cv. 60444 used as a control (top right panel). Presence of zeolin is clearly seen in roots of all ages in the transgenic, but not the control root sections, with significant accumulation in the core (xylem parenchyma) as well as the outer peel layer. Bars = 1 cm.
Immunoprinting of cassava storage roots further indicated that the transgenically expressed zeolin accumulated within both peel and xylem parenchyma storage tissues of tuberous roots (Figure 1E), while immunofluorescence studies revealed sub-cellular localization of zeolin within novel, globular protein bodies distributed within xylem parenchyma cells of 10-week-old storage roots (Figure S2). These structures were isolated by Ficoll gradient allowing identification of spherical bodies, 5 to 7 µm in diameter under the scanning electron microscope (Figure S2). No such structures were observed in non-transgenic controls and have never previously been reported within tissues of this species.
Accumulation of storage protein and performance of fully-grown cassava plants
Three transgenic lines in which zeolin was driven by the patatin promoter were planted in soil beds in a greenhouse to facilitate production of mature plants. Total protein content within peeled storage roots harvested from these plants at 7 months of age reached 10 to 11% dw in all three transgenic lines tested (Figure 2A). Plants of the same lines, plus one in which zeolin was under control of the 35S promoter, were also established in the field at the University of Puerto Rico (Mayaguez) and assessed for agronomic characteristics and protein content of the storage roots at 11 months of age. No morphological differences were observed between transgenic and non-transgenic plants and no significant differences were apparent for average shoot or root yields, harvest index or dry matter and starch content of tuberous roots harvested from transgenic and control plants (Figure 2C) (Figure S3). Total protein content of field grown, peeled storage roots in which zeolin was driven by the patatin promoter reached 11.9 to 12.5% dw, a 4.1- to 4.3-fold increase compared to non-transgenic controls. As observed in greenhouse grown plants (Figure 1A), no increase in total protein content was detected in roots in which zeolin was under control of the 35S promoter (Figure 2A).
10.1371/journal.pone.0016256.g002Figure 2 Characterization of fully-grown zeolin expressing plants.
A) Total protein content of peeled storage roots harvested from transgenic cassava plants expressing zeolin grown in greenhouse soil beds and in the field. Non-transgenic plant 60444 (green bar); plant line transgenic for empty gene vector control (pCambia2300) (light green bar); plants transgenic for patatin promoter-zeolin (pILTAB600; blue bars); plants transgenic for 35S promoter-zeolin (pILTAB601; orange bar) n = 7. Assessment of total protein content by Bradford assay of storage roots shows similar results from both locations with non-transgenic, empty vector control and 35S driven zeolin varying from 2.9 to 3.1% dw, and all three events transgenic for patatin driven zeolin accumulating 3 to 4 times this amount of total protein. Total protein content from roots of field-grown plants was higher in all three lines compared to greenhouse-grown, with maximum levels of total protein reaching 12.3% dw in line pILTAB600-25. B) Evaluation of important agronomic traits of zeolin expressing cassava plants grown for 11 months in the field in Puerto Rico. Non-transgenic cv. 60444 plant line (green bar), plant line transgenic for empty gene vector control (pCambia2300) (light green bar), plants transgenic for patatin promoter driven zeolin transgenic lines (pILTAB600) (blue bars); plants transgenic for 35S promoter driven zeolin (orange bar) n = 12. Transgenic events showed no significant differences compared to controls across traits studied.
Western blot analysis was performed on flour extracted from 7-month-old field grown, peeled storage roots and leaf tissues. As observed from greenhouse grown plants, zeolin was found to be present in both organ types but in significantly higher amounts in transgenic storage roots compared to leaves of the same plant when patatin was the promoter employed to drive transgene expression. Use of the 35S promoter produced the opposite effect, with little accumulation in the roots but significant presence of transgenically derived protein in the leaves (Figure 1C).
Amino acid profile and forms of accumulated zeolin
Peeled storage roots were collected from 7-month-old plants of the patatin driven zeolin-expressing line pILTAB600-25 grown in greenhouse soil beds and analyzed to determine amino acid composition. Levels of total (free+bound) amino acids increased in transgenic roots (from 3.67% to 11.87% dw) to reach 3.5 to 4.0 times that of non-transgenic controls (Figure 3A), amounts closely corresponding to the total increase in protein content. Conversely, levels of free amino acids were found to be reduced approximately threefold (from 0.62% to 0.22% dw) in these tissues compared to wild type controls. Unlike non-transgenic plants, the most abundant free amino acids detected in transgenic cassava were Aspartate and Glutamate (Figure 3A), both of which are known to be forms of nitrogen transport in cassava [17].
10.1371/journal.pone.0016256.g003Figure 3 Analysis of amino acid and cyanogenic content in cassava tissues transgenic for zeolin.
A) Total amino acid profile of storage roots of non-transgenic cassava cv. 60444 (green bars) and transgenic line pILTAB600-25 (blue bars) in which zeolin is driven by the patatin promoter. Increase in amounts of individual amino acids in transgenic root tissue varied from a four- to ninefold increase compared to the non-transgenic control in a manner that reflects the expect amino acid profile for zeolin. B, C) Levels of cyanogens present in cassava leaf and root tissues of seven-month-old, pot grown plants. All transgenic lines showed a reduction in cyanogen content within leaves and storage roots compared to the non-transgenic control and was apparent in transgenic events in which zeolin was under control of either the patatin (pILTAB600, blue bars) or 35S (pILTAB601, orange bars) promoters. A maximum reduction of 55% was observed in roots of pILTAB600-25, the transgenic event that accumulated the highest levels of total protein within its storage roots.
Compositional analysis of total amino acids revealed levels of individual amino acids reflecting that of zeolin, such that all essential amino acids (except tryptophan, which is absent in zeolin) were enhanced 4- to 9-fold in transgenic cassava roots (Figure 3A). Importantly, the sulfur-containing amino acids cysteine and methionine, known to be very low in cassava [4], were elevated 9- and 4.5-fold respectively in zeolin-expressing roots as compared to non-transgenic control tissues (Figure 3A). Considering that total protein content increased from ∼3% to 11% dw in this transgenic line (Figure 2A), six amino acids (Asp, Glu, Val, Met, Leu and Phe) showed a 100% match with the calculation for this level of zeolin, while Thr, Ala, Cys and Lys increased by more than 120% (Figure 3A). This data indicates that the modified total amino acid profile can be largely accounted for by accumulation of zeolin, but also in a minor way by the presence of other cassava proteins. While micro-Kjeldahl nitrogen results showed that total nitrogen content increased from 4.3% to 12.1% (2.8×) in the roots of transgenic plants grown in soil beds, Bradford assay measurements showed that the protein content was elevated from 3.0% to 11.3% (3.8×) in the roots of the same transgenic plants.
Further studies were carried out on the nature of the accumulated zeolin and associated proteins. Western blot analysis was performed on crude protein extracted from peeled storage roots of greenhouse grown, transgenic cassava. Four distinct forms of zeolin were observed (Figure S2), and respective levels of abundance determined by band intensity. Approximately 6% of the total zeolin was found present as a monomer, 76% as a trimer, 14% as a trimer bound to BiP chaperone, and 4% in a higher molecular weight, glycosylated form [16], [18]. When protein bodies were isolated without a reducing agent and loaded onto a native polyacrylamide gel, band patterning showed a higher molecular weight than expected for zeolin (Figure S2), once more suggesting that other proteins were associated with the transgenically expressed product. This high molecular weight band was eluted, sonicated and loaded onto a SDS-PAGE denaturing gel, allowing detection of proteins at different sizes, ranging from 15–90 kDa (Figure S2). The 90 kDa band was digested and confirmed by MS-ID to consist of zeolin protein, BiP chaperone and ER resident ATPases, while smaller bands contained precursor proteins, including rubisco precursors, ATPases precursors and ER membrane proteins (Table SI). To determine whether zeolin was associated with folding chaperones, an ATP-affinity assay was performed on crude proteins extracted from transgenic storage roots. Results showed that zeolin was present tightly linked to BiP chaperone in the transgenic cassava root tissues (Figure S2).
Impact of transgenically accumulated protein on cyanogenic glycoside content
Cassava is well known as a crop species that generates and stores cyanogenic glycosides within its tissues [17], [19], [20]. Greenhouse grown plants were analyzed to determine if accumulating transgenically derived storage protein would impact the cyanogenic glycoside content of such plants. Analysis of zeolin expressing plants using an Orion CN-sensitive electrode, revealed a reduction in cyanogenic content in both leaves and storage root tissues of up to 55% compared to non-transgenic control plants (Figure 3B). Cyanogen reduction was observed in all transgenic lines and occurred when both patatin and 35S promoters were used to drive zeolin expression.
Discussion
Although an excellent source of carbohydrate, storage roots of cultivated cassava contain only 2–3% dw protein [21]. Modifying this large storage organ to become a sink for protein through conventional methods is significantly constrained by lack of naturally occurring germplasm with high protein content and the complexity of cassava breeding programs [22]. Transgenic approaches are therefore an attractive method for directing accumulation of nutritionally balanced, or industrially valuable protein to these large storage organs. The present study was initiated to demonstrate capacity to transgenically modify cassava to accumulate storage protein of known amino acid profile within the tuberous roots. While the zeolin product described here is not intended for release to African farmers, the results described represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein [2], to one capable of supplying both calories and nutritionally balanced proteins at dietary significant quantities to those dependent on the crop.
Zeolin expression in cassava under control of the patatin promoter caused de novo formation of protein bodies in storage root cells in a manner never previously reported within tissues of this species (Figure S2B), and resulted in an increase in total protein content of approximately fourfold to reach 12.5% dw (Figure 2A). Detailed analysis of these protein bodies confirmed that they consisted of different forms of zeolin and carried the nutritionally valuable, amino acid profile of the predicted storage protein, in this case phaseolin (Figure S2C, D, E).
When commencing this work there was concern that modification of cassava to accumulate significant levels of storage protein in the starch-rich storage roots would not be stable and/or would lead to disrupted physiology and altered phenotype of the transgenic plants. Greenhouse and field studies revealed this not to be the case, with similar levels of protein accumulation recorded across more than three years of testing in three different locations (Figure S3A, C, E). In addition, agronomic characteristics of the transgenic plants determined during confined field trials, showed no significant changes compared to control plants (Figure 2B). A significant pleiotropic effect of zeolin expression was seen, however, as a correlated reduction in free amino acid levels and cyanogen content in transgenic plants (Figure 2B, C). Cyanogenic levels, considered by some to be a food safety concern in this crop [10], were reduced by up to 55% in both leaf and storage root tissues and occurred when both the 35S and patatin promoters were used to drive zeolin expression.
Earlier work to enhance the nutritional quality of cassava storage roots [13], expressed the storage protein ASP1 [23] under control of the 35S constitutive promoter, but the resulting plants did not accumulate protein within their tuberous roots [24]. Likewise, in the present study, when the constitutive 35S promoter was used to drive expression of the transgene (Figure 2C, D), total protein content of root storage organs was never significantly elevated above that of the non-transgenic controls (Figures 1A & 2A). This finding indicates that the use of a suitable promoter is required to achieve accumulation of protein products in cassava storage roots. Cassava storage roots are not tubers but modified roots, which have evolved to store starch within proliferated xylem parenchyma. Considered sink specific with high activity in root tissues [14], the Class II patatin promoter used in this study was found to drive expression of zeolin RNA in leaves at levels approximately 50% of that detected in cassava storage root tissues (Figure S1C). Accumulation of the zeolin protein when derived under the control of the patatin promoter was, however, almost totally restricted to the storage roots, and remained at very low levels in leaf tissues (Figure 1C & 1D). We hypothesize, therefore, that the observed promoter-dependent, organ specific accumulation of zeolin may result, at least in part, from source/sink partitioning of nitrogen resources between the leaves and storage roots. For example, reduced levels of cyanogen of up to 55% within leaf tissues, when zeolin was expressed by either the 35S or patatin promoter, supports previous evidence that a flow of cyanogens exists between leaves and storage roots in cassava [17], [19], [20]. Furthermore, micro-Kjeldahl results showed that the total nitrogen increased by three folds in the roots of the transgenic plants and the total amino acid analysis showed that glutamate and aspartate were abundant in the storage roots of zeolin expressing plants. The difference between micro-Kjeldahl (3×) and Bradford assay measurements of the total protein content (4×) is due to the non-proteinaceous nitrogen content in cassava. Studies are ongoing to determine if this non-proteinaceous nitrogen pool, which is unique among the major crop species, is being utilized to support synthesis of storage protein in the tuberous roots of transgenic cassava plants, and if growth under conditions of differing soil nitrogen availability can affect levels of storage protein accumulation.
Cassava has the lowest protein∶energy ratio (P∶E) of any staple food, with protein content ranging from 1 to 3% dw. Thus, a two-year-old child consuming 50% of his/her dietary energy as wild type cassava will receive about 5 g dietary protein, equivalent to 35% of their daily protein requirement. The same child consuming the same amount of modified cassava accumulating storage protein at levels achieved in the present study would obtain approximately 18 g of dietary protein, or more than 100% of their daily requirement. This illustrates that genetic modification of cassava could be a potentially important component of delivering enhanced nutrition to at-risk populations in the tropics.
Accumulation of storage protein in transgenic cassava to the levels achieved in this study was not limited by a capacity to synthesize particular amino acids, which were elevated between 4.5 and 11 times compared to that of wild type, and in a manner that reflected the amino acid profile of zeolin (Figure 3B). Cassava storage roots provide a large harvest index with reported yields of up to 70 tons/ha [4], translating to a potential of 2.9 tons/ha protein product at the 12.5% dw levels achieved here. Demonstrated capacity to modify this large organ into a novel sink for nitrogen indicates that cassava likely possesses significant potential to produce and accumulate a range of proteins with nutritional, industrial or pharmaceutical value in a manner that deserves further investigation.
Materials and Methods
Plasmid construction
The chimeric zeolin gene was amplified from its original vector, using primers ZaF:5′AGATCTATGATGGAGCAAGGGTTCC3′ and ZeR:5′AGGTACCCTGTTTGTTGATCAGCTTC3′, with BglII and KpnI restriction sites designed within the primer sequence. The PCR product was cloned into pGEM-T vector (Promega), sequenced, the inserted fragment digested with BglII and KpnI and cloned downstream of the CaMV 35S or the Class II patatin promoter. Cassettes comprising promoter-zeolin-tNOS were cloned into the pCambia2300 binary vector (http://www.cambia.org/daisy/bioforge_legacy/3724.html), to generate pILTAB600 for patatin promoter and pILTAB601 for 35S promoter versions respectively.
Plant transformation and selection of transgenic lines
Agrobacterium tumefaciens strain LBA4404 harboring pILTAB600 and pILTAB601, and pCambia2300 as a control were used to genetically transform cassava friable embryogenic callus of the West African cultivar 60444 [25]. Transformed cells were recovered, and plants regenerated on selection media, propagated and sampled for DNA, RNA and protein analysis. DNA was extracted from leaves of in vitro plants according to the Dellaporta method [26] and used for PCR and Southern blot analysis. In the latter, 10 µg of DNA were digested with BglII or BglII+KpnI, loaded on a 0.8% w/v agarose gel, transferred onto N+ Hybond membrane (GE Healthcare) and hybridized with a non-radioactive probe for the zeolin gene, prepared using the AlkPhos kit (GE Healthcare). Extraction of RNA from transgenic events was performed from in vitro plants and from 7-month-old plants grown in the greenhouse at DDPSC using Trizol reagent according to the manufacturer's instructions. First strand cDNA was generated using the Superscript II system (Invitrogen) from 1 µg of RNA template and subsequent PCR performed using 1 µg of cDNA and zeolin primers ZeF and ZeR. Northern blots were performed by loading 10 µg of total RNA onto 1% w/v formaldehyde agarose gel, transfered to N+ Hybond membrane and hybridized using a AlkPhos non-radioactive DNA probe generated from the zeolin sequence. RNA levels were determined using ImagQuant software to measure band intensity.
Greenhouse growth conditions
Transgenic cassava events with low (1 to 2) copy number, high RNA expression and high protein content were propagated and transferred to soil in 7 cm pots grown in the greenhouse, and evaluated for protein accumulation. Supplemental lighting was provided for 14 hours daily with 1000 W metal halide fixtures. Artificial lights provided an average photosynthetically active radiation (PAR) of 250 µmol m−2 s1. Air temperature was maintained at 28°C during the day and 25°C at night. Fertilizer was applied at 200 ppm N three times per week using Jack's 15-16-17 Peat Lite and once a week with Jack's 15-5-15 Cal-Mag.
Vegetative clones of transgenic cassava were also evaluated by planting directly into soil beds in a greenhouse. Plants were established at DDPSC in 7 cm pots and transferred when 25 cm tall to the University of Missouri Delta Research Station, Portageville, MO. The soil consisted of Tiptonville sandy loam soil (fine-loamy, mixed, thermic Typic Argiudolls), determined to contain 60 kg NO3-N ha−1, 184 kg Bray-1 P ha−1, and 351 kg ammonium acetate extractable K ha−1. Soil was mechanically tilled and cassava plants transplanted into the earth floor at 1 m spacing. Supplemental lighting was provided for 16 hours with Sun System III 1000 W metal halide lights, providing an average PAR of 1175 µmol m−2 s−1. Air temperature was thermostatically maintained at 25 to 30°C. No fertilizer was applied during this experiment.
Confined field trials
Transgenic cassava was grown for 11 months at the Isabella Agriculture Research Station of the University of Puerto Rico, Mayaguez in Northeast Puerto Rico, in a randomized block design with 3 reps and 8 plants/line/rep. A uniform distance of 1.5 m spacing was maintained between each plant and the entire plot surrounded by a row of wildtype 60444 plants. All steps to confine and contain the transgenic material were observed as regulated by USDA APHIS. Yield data comprising the number of roots above and below ground mass were measured per plant at harvest, 11 months after planting. Dry matter content and starch content were calculated by computing specific gravity X per plant root, where X = Wwater/(Wair−Wwater). Wair is normal weight of 3 to 5 kg of roots while Wwater is the weight in water of those same roots. Dry matter percentage and starch percentage is then equal to 158.3x−142 and 112.1x−106.4, respectively [27]. Harvest index was calculated by assessing the proportion of root fresh weight as a ratio of the total fresh weight of the plant [28].
Immuno-fluorescence and immuno-printing
Sections of peeled cassava storage roots 50 to 100 µm thick were obtained from 10-week-old plants grown in DDPSC greenhouses in 7 cm pots. Sections were fixed in 4% para-formaldehyde and washed with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Fixed cells were treated with cell wall lysing enzyme Drisalase (Sigma) for 15 min, washed five times with TBST, incubated with anti-phaseolin antibodies for one hour, washed five times with TBST and incubated again with Alexa-Fluor 488 fluorescent secondary antibodies (Invitrogen) for another hour. Cells were washed five times with TBST buffer and spread on glass slides for visualization under the confocal microscope (Nikon Eclipse E-800 C1) with 100 ms exposure.
For immunoprinting, the same storage roots were washed with sterile water, cut into 1 to 3 mm thick sections, squashed onto nitrocellulose membrane and allowed to dry at room temperature. The membrane was blocked with 5% v/v skimmed milk and hybridized with anti-phaseolin antibodies, washed five times with TBST buffer and incubated with HRP secondary antibodies for an hour at room temperature. The signal was developed after washing with TBST buffer and exposing the membrane to X-ray film.
Micro-Kjeldahl and amino acid analysis
The nitrogen content and amino acid composition of transgenic line pILTAB600-25 was compared with non-transgenic roots collected from 7-month-old soil bed grown plants. Harvested cassava storage roots were peeled, sliced into pieces 1 cm thick, lyophilized and ground into fine powder using a coffee grinder. The amino acid composition of flour produced from transgenic and non-transgenic cassava was analyzed by the Dept. of Chemistry, University of Missouri, Columbia, MO by Chemical analysis, AOAC Official Method 982.30 E (a,b,c), chp. 45.3.05, 2006.
Protein extractions and Western blot analysis
Storage root samples were cut into 1 cm cubes, dried using a freeze-dryer and crushed into powder with a pestle and mortar. Total protein was extracted from dry samples collected at different growth stages according to Mainieri et al
[15], and quantified by Bradford assay. A 100 mg tissue sample was placed in a 2 ml tube with a ceramic bead, 1 ml of protein extraction buffer and homogenized using FastPrep machine (MP Biomedicals). The homogenate was filtered through layers of cheesecloth, protein precipitated with TCA and quantified. Equal protein concentration were loaded on a 12% w/v polyacrylamide gel and blotted onto a nitrocellulose membrane for Western blot analysis. Anti-phaseolin primary antibodies were used after blocking the membrane with 5% skimmed milk in TTBS buffer, and HRP secondary anti-bodies used for subsequent quantification after film development using ImageQuant software (GE Healthcare).
Mass-spectrometry identification (MS-ID)
Eluted protein bands from the native (without a reducing agent or SDS) and denaturing PAGE were digested and subjected to nano-LC-ESI-MS/MS analysis. Nano-LC was performed by LC Packings Ultimate system (San Francisco, CA) equipped with a Dionex C18 PepMap100 column (75 µm i.d.) (Sunnyvale, CA) flowing at 180 nL/min. Peptides (5 µl injections) were resolved on a gradient that started at 95% solvent A (5% acetonitrile, 0.1% formic acid) and 5% solvent B (95% acetonitrile, 0.075% formic acid in MilliQ water) for 3 min, then increasing from 5 to 25% B over 5 min, from 25 to 60% B over the next 32 min, and from 60 to 95% B over the final 5 min. Mass spectrometric analysis was performed on an ABI QSTAR XL (Applied Biosystems/MDS Sciex) hybrid QTOF MS/MS mass spectrometer equipped with a nanoelectrospray source (Protana XYZ manipulator). Positive mode nanoelectrospray was generated from fused-silica PicoTip emitters with a 10 µm aperture (New Objective) at 2.5 kV. TOF mass and product ion spectra were acquired using information dependent data acquisition (IDA) in Analyst QS v1.1 with mass ranges for TOF MS and MS/MS at m/z 300–2000 and 70–2000, respectively. Every second, a TOF MS precursor ion spectrum was accumulated, followed by three product ion spectra, each for 3 s. Switching from TOF MS to MS/MS was triggered by the mass range of peptides (m/z 300–2000), precursor charge state [2]–[4] and ion intensity (>50 counts). The DP, DP2, and FP settings were 60, 10, and 230, respectively, and rolling collision energy was used.
Determination of ATP-affinity bound proteins
Five grams of three-month-old transgenic, peeled cassava storage roots harvested from transgenic line pILTAB600-25, grown in 7 cm pots in DDPSC greenhouses, were gently homogenized in TBS buffer containing proteinase inhibitor cocktail (Sigma) and filtered using four layers of cheesecloth. Cell lysate was passed through a 22 µm filter, loaded on 5, 10, 15, and 20% Ficoll gradient and centrifuged for 3 hr at 15 000 rpm in 4°C. Precipitated proteins were collected and incubated with ATP-Sephasrose (Jena-Bioscience) to capture ATP bound proteins following manufacturer instructions. ATP bound proteins were loaded on native and denaturing acrylamide gels and protein bands eluted and identified using MS-ID. The same precipitate recovered from Ficoll was washed with TBS buffer, blotted on nitrocellulose membrane and hybridized with anti-phaseolin and secondary Alexa-fluor 488 antibodies for evaluation under the fluorescent and scanning electron microscope (Hitachi TM-1000 tabletop).
Determination of cyanogenic content
Cassava leaf and root samples were collected from plants at six months of age growing in 7 cm pots in the greenhouse and 100 mg fresh tissue ground in a closed tube containing 1 ml TBS buffer using a FastPrep machine (MP Biomedicals). Tubes were incubated for 10 min at room temperature then placed in 50 ml closed tubes containing 9 ml of 5M NaOH. Content of cyanogenic compounds was measured using an Orion CN-sensitive electrode (Thermo-Scientific) after preparing a standard curve of KCN. The electrode sensitivity was evaluated by measuring the HCN captured in 5M NaOH from β-glucosidase activity on known concentrations of pure linamarin purchased from (Sigma) and processed in the same way.
Supporting Information
Figure S1
Transgenic nature and expression of zeolin in cassava.
a) PCR amplification of DNA from a subset of the 210 in vitro transgenic plants produced, using primer set ZeF and ZeR specific for zeolin. Non-transgenic cv. 60444 was used as a negative control and pILTAB600 plasmid as a positive control, followed by transgenic cassava transformed with genetic constructs pILTAB601 and pILTAB600. b) Southern blot of DNA extracted from in vitro leaf tissue of transgenic cassava plants. Lanes 3–11: plants transformed with pILTAB600 and lanes 12–19 plants transformed with pILTAB600. Non-transgenic cassava cv.60444 was used as a negative control and plasmid pILTAB600 as positive control. Upper panel shows DNA digested with BglII to confirm integration and copy number of the zeolin transgene, lower panel shows DNA digested with BglII and KpnI to confirm intact nature of integrated sequence. c) Determination of zeolin RNA expression in transgenic tissues. Northern blotting was performed on 10 µg total RNA extracted from in vitro and seven month old greenhouse grown leaves and roots of plants transgenic for zeolin under control of the 35S (pITAB601) and patatin (pILTAB600) promoters. Expression levels were equivalent for both constructs in leaves and roots (L and R respectively) from in vitro grown plants, but significantly different in plants grown in the greenhouse. In the latter, the 35S promoter drove 3–4 times higher expression of zeolin in the leaves than in the roots, while patatin drove zeolin RNA expression at significantly higher levels in the roots than in leaf tissue. Transgenic tobacco leaff and root tissues were used as a positive control.
(TIF)
Click here for additional data file.
Figure S2
Characterization of zeolin accumulation in roots of transgenic cassava. a) left panel; Western blot analysis of 50 µg protein extracted without reducing agent from transgenic cassava storage roots from six different plants (clones) of transgenic line pILTAB600-25, using antibody against phaseolin. Four different forms of zeolin protein were visualized in accordance with Bellucci et al., 13, with smallest band size representing newly synthesized zeolin polypeptide found as a monomer, zeolin trimers then zeolin trimers bound to the folding chaperone BiP. Right panel; re-probing with BiP specific antibodies confirmed binding of zeolin to BiP chaperones in cassava root tissue. c) Localization of zeolin accumulation as protein bodies within the xylem parenchyma of cassava storage roots. (top left panel) non-transgenic cassava storage root cells. (top right panel) cells of transgenic storage roots in which zeolin is labeled with fluorescent secondary antibody showing zeolin accumulating as discrete bodies. (lower panel) scanning electron microscopy of purified zeolin protein bodies isolated from transgenic cassava storage roots of plant line p600-25. Zeolin protein bodies range in size from 3 to 7 µm in diameter. c) Native PAGE of purified protein bodies isolated from storage root tissue of thee pILTAB600-25 plant lines showing signal at higher MW size than expected for zeolin (65 kDa). d) SDS-PAGE gel of the high MW bands from c after elution and sonication. All bands were eluted and analyzed by MS-ID, confirming upper band as zeolin with BiP chaperone and smaller protein bands (arrowed) as rubisco precursors e) ATP affinity assay for total protein extracted from transgenic cassava storage roots of pILTAB600-25. Bands were eluted and analyzed by MS-ID to identify zeolin associated with ATP chaperones, ATPase precursors and ER membrane proteins.
(TIF)
Click here for additional data file.
Figure S3
Transgenic cassava plants of cv. 60444 expressing the chimeric storage protein zeolin.
a) Transgenic plants producing storage roots (b) growing in pots in the greenhouse at DDPSC c) Transgenic plants expressing transgenic storage protein growing in a soil bed in the greenhouse. Plants reach 3.5 m in height and produce up to 10 storage roots per (d) plant within six months. e) Transgenic plants expressing zeolin growing in confined field trial at the University of Puerto Rico, Mayaguez. Plants in the field showed no significant morphological differences to the non-transgenic controls and produced mature storage roots within 10–11 months.
(TIF)
Click here for additional data file.
Table S1
Protein identity of eluted and digested protein bands from the SDS-PAGE (Figure S2D and S2E). All bands from Figure S2D and S2E were eluted, digested, and analyzed by MS-ID as detailed in Materials and Methods. The table provides: GI Accession number for the blast result, Protein Identity, Source of the Protein, Score Identity to the aligned protein from the source organism, and Peptide Coverage percentage of the sequenced amino acids in the digested proteins compared to the source organism proteins.
(DOC)
Click here for additional data file.
The authors thank Dr. A. Vitale for providing the zeolin gene tobacco seeds and phaseolin antibody, Drs. R. H. Berg, L. Hicks and E. Herman (DDPSC) for assistance with microscopy, mass spectrometry and BiP antibody respectively, and Dr. M. Manary for advice on pediatric nutrition. We extend gratitude to C. Trembley and D. Burkhart (DDPSC) for excellent care of greenhouse grown plants.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The Bill and Melinda Gates Foundation funded this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21347313PONE-D-10-0390810.1371/journal.pone.0016776Research ArticleBiologyMicrobiologyVirologyViral classificationRNA virusesMedicineInfectious DiseasesViral DiseasesVeterinary ScienceAnimal TypesLaboratory AnimalsVeterinary DiseasesVeterinary VirologyVeterinary MicrobiologyExperimental Infection of Mice with Avian Paramyxovirus Serotypes 1 to 9 Experimental Infection of Mice with APMV 1 to 9Khattar Sunil K.
1
Kumar Sachin
1
Xiao Sa
1
Collins Peter L.
2
Samal Siba K.
1
*
1
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
2
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland, United States of America
Qiu Jianming EditorUniversity of Kansas Medical Center, United States of America* E-mail: [email protected] and designed the experiments: SKK PLC SKS. Performed the experiments: SKK SK SX. Analyzed the data: SKK PLC SKS. Contributed reagents/materials/analysis tools: SKK SKS. Wrote the paper: SKK PLC SKS.
2011 10 2 2011 6 2 e1677622 10 2010 29 12 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.2011This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.The nine serotypes of avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds worldwide. APMV-1, also called Newcastle disease virus, was shown to be attenuated in non-avian species and is being developed as a potential vector for human vaccines. In the present study, we extended this evaluation to the other eight serotypes by evaluating infection in BALB/c mice. Mice were inoculated intranasally with a prototype strain of each of the nine serotypes and monitored for clinical disease, gross pathology, histopathology, virus replication and viral antigen distribution, and seroconversion. On the basis of multiple criteria, each of the APMV serotypes except serotype 5 was found to replicate in mice. Five of the serotypes produced clinical disease and significant weight loss in the following order of severity: 1, 2>6, 9>7. However, disease was short-lived. The other serotypes produced no evident clinical disease. Replication of all of the APMVs except APMV-5 in the nasal turbinates and lungs was confirmed by the recovery of infectious virus and by substantial expression of viral antigen in the epithelial lining detected by immunohistochemistry. Trace levels of infectious APMV-4 and -9 were detected in the brain of some animals; otherwise, no virus was detected in the brain, small intestine, kidney, or spleen. Histologically, infection with the APMVs resulted in lung lesions consistent with broncho-interstitial pneumonia of varying severity that were completely resolved at 14 days post infection. All of the mice infected with the APMVs except APMV-5 produced serotype-specific HI serum antibodies, confirming a lack of replication of APMV-5. Taken together, these results demonstrate that all APMV serotypes except APMV-5 are capable of replicating in mice with minimal disease and pathology.
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Introduction
The family Paramyxoviridae is large and diverse and includes viruses that have been isolated from a wide variety of mammalian and avian species around the world [1]. Some members of the family are responsible for major human and animal diseases, while others cause inapparent infections. The viruses belonging to this family are pleomorphic and enveloped and contain a non-segmented, negative-sense, single-stranded RNA genome of 13–19 kb. On the basis of virus structure, genome organization and sequence relatedness, the family Paramyxoviridae is divided in to two subfamilies: Paramyxovirinae and Pneumovirinae
[1]. The subfamily Paramyxovirinae is divided into five genera: Respirovirus (including Sendai virus and human parainfluenza virus types 1 and 3), Rubulavirus (including simian virus type 5, mumps virus, and human parainfluenza virus types 2 and 4), Morbillivirus (including measles and canine distemper viruses), Henipavirus (comprising Hendra and Nipah viruses), and Avulavirus (comprising the avian paramyxoviruses [APMVs]). Subfamily Pneumovirinae contains two genera, Pneumovirus (including human respiratory syncytial virus and bovine respiratory syncytial virus) and Metapneumovirus (comprising human metapneumovirus and the avian metapneumoviruses) [1], [2].
All the paramyxoviruses isolated from avian species are classified into genus Avulavirus, representing the APMVs, and genus Metapneumovirus, representing the avian metapneumoviruses. The APMVs have been divided into nine serotypes (APMV 1 to 9) based on hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays [3]. Recently, APMVs were isolated from Rockhopper Penguins in the Falkland Islands. Serological and genome characterizations suggested that these viruses probably represent a new serotype (APMV-10) [4]. APMV-1, which includes all strains of NDV, has been extensively characterized because virulent NDV strains are important causes of disease in chickens. Complete genome sequences have been determined for a number of NDV strains, and there is extensive information on NDV molecular biology and pathogenesis [5]–[15]. Comparatively little is known about the other eight APMV serotypes. As an initial step towards their characterization, complete genome sequences of APMV serotypes 2 to 9 were recently determined [16]–[26].
APMV-1 (NDV) strains are divided into three pathotypes: highly virulent (velogenic) strains that cause severe respiratory and neurologic diseases in chickens; moderately virulent (mesogenic) strains that cause milder disease, and nonpathogenic (lentogenic) strains that cause inapparent infection. In contrast, much less is known about the biological characteristics and pathogenicity of APMV-2 to -9. APMV-2 has been associated with severe respiratory disease and drop in egg production in turkeys [27], [28]. APMV-3 has been associated with encephalitis and high mortality in caged birds, respiratory diseases in turkeys and stunted growth in young chickens [29], [30]. APMV-5 causes disease in budgerigars that is characterized by depression, dyspnoea, diarrhea and high mortality [31]. APMV-6 and -7 cause mild respiratory disease in turkeys and are associated with a drop in egg production [32]–[34]. APMV-4, -8, and -9, isolated from ducks, waterfowl, and other wild birds did not produce any clinical signs of viral infection in chickens [35]–[39]. Recently, we performed experimental infection of 1-day-old chicks and 4-week-old chickens and turkeys with APMV-2 strains, Yucaipa and Bangor, and documented viral infection in the gastrointestinal and respiratory tracts [40]. We also performed experimental infection of 2-week-old chickens and turkeys with APMV-3 strains, Netherlands and Wisconsin, and documented viral infection in the brain, lungs, spleens, trachea, pancreas and kidney [41].
In the last 10 years, reverse genetic techniques have made it possible to engineer NDV as a potential vaccine vector for delivery of a number of foreign antigens not only in avian hosts but also in murine and nonhuman primate models [42]–[50]. In addition, NDV has been evaluated as a promising broad spectrum oncolytic agent in a mouse model [51]–[54]. Safety of different strains of NDV, due to attenuation by host range restriction, has been documented in these animal models [55]. In the future, we plan to use reverse genetic techniques to evaluate other APMV serotypes (serotype 2 to 9) as vaccine vectors and oncolytic agents. However, small laboratory animal models will be needed to evaluate their replication, immunogenicity and safety. In the current study, we sought to evaluate mice as a small animal model for infection with APMV 2 to 9. We inoculated BALB/c mice intranasally with APMV serotypes 1 to 9 and monitored clinical signs and viral replication and tropism in a number of possible target organs. We further studied the serological responses to APMV infection in these animals. The results showed that BALB/c mice are susceptible to infection with all of the APMV serotypes except serotype 5. Viral replication occurred mostly in the upper and lower respiratory tracts. The data generated using this animal model will be helpful in elucidating mechanisms of immunity and pathogenesis and evaluating candidate recombinant vaccines.
Materials and Methods
Cells and viruses
Chicken embryo fibroblast (DF1) and African green monkey kidney (Vero) cell lines, obtained from the American Type Culture Collection (ATCC, Manassas, VA), were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. The prototype strains of all nine APMV serotypes used in this study were: APMV-1 strain LaSota/46, APMV-2 strain chicken/Yucaipa/California/56, APMV-3 strain parakeet/Netherland/449/75, APMV-4 strain duck/Hongkong/D3/75, APMV-5 strain budgerigar/Kunitachi/74, APMV-6 strain duck/HongKong/18/199/77, APMV-7 strain dove/Tennessee/4/75, APMV-8 strain goose/Delaware/1053/76, and APMV-9 strain duck/New York/22/78. All of the strains except for serotype 5 were obtained from National Veterinary Services Laboratory, Ames, Iowa. APMV-5 strain budgerigar/Kunitachi/74 was kindly provided by Dr. Ian Brown, the Veterinary Laboratories Agency, Weybridge, Surrey, UK. All of the APMV serotypes except serotype 5 were grown in the allantoic cavity of 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs. APMV serotype 5 was grown in Vero cells. The allantoic fluids from infected eggs were collected 96 h post-inoculation and virus titers were determined by hemagglutination (HA) assay with 0.5% chicken RBC. In the case of APMV-5, the titer was determined by plaque assay on Vero cells. The APMV-5 samples were inoculated in triplicate onto 24-well plates of Vero cells at 80% confluency, incubated for 1 h, washed with PBS, overlaid with 0.8% methylcellulose, and observed for plaque production until 7 days post inoculation (dpi). The cells were fixed with methanol and stained with 1% crystal violet. Values for each tissue sample were based on average plaque count from three wells.
Preparation of hyperimmune antiserum against the viral nucleocapsid (N) protein of each serotype of APMV
Each APMV serotype strain described above was purified on a sucrose gradient and the virion proteins were separated on a 10% SDS-polyacrylamide gel and negatively stained using E-Zinc TM reversible stain kit (Pierce, Rockford, IL). The N protein specific band was excised from the gel and destained with Tris-glycine buffer pH 8. The excised gel band was minced in a clean pestle and mixed with elution buffer (50 mM Tris-HCl buffer pH 8, 150 mM NaCl, 0.5 mM EDTA, 5 mM DTT and 0.1% SDS) and transferred to the upper chamber of a Nanosep centrifugal device (Pall Life Sciences, Ann Arbor, MI, USA). After centrifugation two times, the eluted protein in the supernatant was quantified and 0.2 mg of protein was mixed with complete Freund's adjuvant and injected subcutaneously into a rabbit. After two weeks, a booster immunization was given with 0.2 mg of protein mixed with incomplete Freund's adjuvant and 2 weeks later the hyperimmune sera were collected. The sera were tested by Western blot and were found to recognize specifically the N protein of the respective APMV serotypes (data not shown).
Experimental infection of Mice
BALB/c mice aged 4 weeks, of either gender, were obtained from Charles River Laboratories, Wilmington, Massachusetts. Mice were housed in microisolator cages in our Bio Safety Level-2 facility and provided water and food ad libitum. Mice were housed in groups of 6 for inoculation of each APMV serotype. Mice were anaesthetized by isofluorane and inoculated intranasally with 50 µl of freshly harvested allantoic fluid containing 27 HA units of each APMV serotype, except for serotype 5 which contained 3×103 PFU/ml of cell culture harvested virus. A group of 6 mice was mock infected with normal allantoic fluid. Mice were weighed and examined two times daily for clinical signs, change in activity and behavior. Three mice from each group were euthanized at 3 day post inoculation (dpi) and other three at 14 dpi by CO2 asphyxiation. Necropsies were performed immediately and tissues from lung, nasal turbinate, brain, small intestine, kidney and spleen were collected. One half of each tissue sample was immediately processed for virus detection and quantification and the other half was stored in 10% neutral buffered formalin for histopathology and immunohistochemistry experiments. On 14 dpi, blood was collected from each mouse just before euthanasia and sera were prepared for analysis. All the animal research was conducted according to the guidelines approved by Institutional Animal Care and Use Committee of the University of Maryland. The protocol number is R-09-47 [Immunogenicity and protection efficacy of recombinant NDV expressing foreign antigens in mammalian models (mice and ferrets)].
Virus detection and quantification
Tissues collected from different organs were homogenized in 1 ml of ice-cold DMEM. Tissue homogenates were centrifuged at 4°C for 10 min at 1000× g and supernatants were collected. For virus detection, 100 µl of clarified homogenate from each tissue was injected into allantoic cavities of five 9-day-old embryonated SPF chicken eggs. Eggs were incubated at 37°C for 4 days. Allantoic fluid was collected from each egg and the presence of virus was detected by hemagglutination (HA) test. For quantification of virus, clarified homogenate from each tissue was diluted in serial 10-fold dilutions in DMEM. The dilutions were inoculated in triplicate onto DF1 cell monolayers in 96-well plates and incubated at 37°C for 1 h. The wells were overlaid with 0.8% methylcellulose in DMEM supplemented with 10% normal allantoic fluid and 2% FBS and incubated at 37°C in a CO2 incubator for 4 days. The monolayers were washed with PBS, permeabilized with 100% cold methanol, and incubated with a 1∶300 dilution of primary N-specific antibody in PBS produced as described above. After three washes with PBS, plates were incubated with a 1∶1000 dilution of secondary antibody (Alexa Fluor 488 conjugated goat anti rabbit immunoglobulin G antibodies). After a further wash cycle, plates were viewed under a fluorescent microscope and fluorescent foci were counted.
Histopathology and Immunohistochemistry
Tissues from infected and control mice were examined by histopathology and immunohistochemistry. Paraffin embedded sections of all the tissues were prepared at Histoserve, Inc. (Maryland, USA). The 5 micron tissue sections were stained with hematoxylin and eosin for histopathology. The severity of inflammation in lungs and nasal turbinate of each APMV infected mouse was assessed based on the extent of fibrin, edema, and mixed inflammatory cells (neutrophils, macrophages, lymphocytes, plasma cells), necrotic cellular debris, and hemorrhage in alveolar spaces and septae.
Immunohistochemical staining was performed on lung and nasal turbinate tissues to detect viral N protein using the following protocol. Briefly, tissue sections were deparaffinized in 2 changes of xylene (5 mins each) and hydrated by incubation in 2 changes each of 100% (3 mins each), 95% (1 min each), and 80% (1 min each) ethanol followed by washing in distilled water. Slides were heated for 40 mins in a staining dish containing sodium citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) until the temperature reached 95–100°C, followed by cooling to room temperature. Sections were washed two times in PBS-Tween 20 for 2 min each, blocked with 2% normal goat serum in PBS for 30 mins, and incubated with 1∶200 dilution of primary antibody (hyperimmune sera raised against N protein of a particular APMV serotype) in PBS for 1 h at room temperature. The sections were washed three times with PBS-Tween 20 for 2 min each and incubated with 1∶1000 dilution of secondary antibody (Alexa Fluor 488 conjugated goat anti rabbit immunoglobulin G antibodies) in PBS for 1 h. After a further wash cycle, the sections were mounted with glycerol and viewed under fluorescent microscope.
Serological analysis
The antibody levels in serum samples collected from mice infected with APMVs on 14 dpi, were evaluated by hemagglutination inhibition (HI) assay except in the case of APMV-5 [56]. In our standard HI assay, the normal mice sera produced nonspecific HI. Therefore a modified HI assay was used. Briefly, 25 µl of mice serum was first treated with 50 µl of receptor destroying enzyme II (catalog number YCC 340–122; Accurate Chemical and Scientific, Westbury NY) at a 1∶3 ratio (vol/vol) at 37°C overnight. Then 25 µl of 5% sodium citrate was added and incubation was continued at 56°C for 30 min. Each serum sample was allowed to cool to room temperature and 100 µl of packed chicken RBCs were added. After incubation at 4°C for 30 min, samples were centrifuged at 1000× g for 10 min. Supernatants were used for HI assay. For the HI assay, twofold serial dilutions of treated sera (50 µl) were prepared, and each dilution was combined with 4 HA units of a particular APMV serotype. Following 1 h of incubation, 50 µl of 1% chicken RBC was added and incubated for 30 min at room temperature, and hemagglutination was scored. In the case of APMV-5, antibody titers were measured by a plaque reduction assay. Briefly, the sera were heat inactivated at 56°C for 30 mins. Ten-fold dilutions of sera were made and mixed with a constant amount of APMV-5 (3×103 PFU), and incubated at room temperature for 1 h in a shaker. The antigen antibody mixtures were analyzed by plaque assay as described above. The serum titer that reduced plaque numbers by 70% was the end point titer.
Results
Clinical disease and gross pathology
Four-week-old BALB/c mice in groups of six were inoculated by the intranasal route with 27 HA units of APMV serotypes 1 to 9 except in the case of serotype 5, for which 3×103 PFU/ml of virus were inoculated. Three animals from each group were sacrificed 3 dpi, and the remaining three were sacrificed 14 dpi. None of the mice infected with APMV serotypes 3, 4, 5 and 8 displayed any overt clinical signs and loss of weight (Fig. 1). At 1 dpi, the mice infected with APMV serotype 1 and 2 had a pronounced decrease in their physical activity, a tendency to huddle and very ruffled fur compared to control mice. The mice infected with APMV serotypes 6, 7 and 9 also presented these clinical signs, which were less marked than those of the mice infected with serotypes 1 and 2. Mice infected with serotypes 1 and 2 exhibited more weight loss than mice infected with serotypes 6, 7 and 9. The loss in weight was observed until day 4 in mice infected with serotypes 1 and 2, until day 3 in mice infected with serotypes 6 and 9, and until day 2 in mice infected with serotype 7. Further, the weight gain remained poor in mice infected with serotypes 1 and 2 after 4 dpi. None of the mice infected with serotypes 1 to 9 died of disease. When the animals were sacrificed 3 dpi and 14 dpi, the following tissues were collected: lung, nasal turbinate, brain, spleen, kidney and small intestine. In all cases, gross examination revealed normal tissue morphology with no noticeable gross lesions.
10.1371/journal.pone.0016776.g001Figure 1 Weight loss in mice infected with APMV serotypes 1 to 9.
Mice in groups of 6 were inoculated with allantoic fluid containing 27 HA units of each APMV serotype except serotype 5, which contained 3×103 PFU/ml of cell culture harvested virus. The control group was inoculated with normal allantoic fluid. The mice were weighed daily and weight lost was calculated as a percent of the weight on day 0. Data depict the mean ± SD from 3 mice per group.
Virus isolation and titration in tissue samples
To determine the sites of APMV replication, one half of each sample of lung, nasal turbinate, brain, spleen, kidney and small intestine tissue collected 3 dpi and 14 dpi was homogenized and clarified supernatants were prepared. For each of the serotypes except APMV-5 (which does not grow in the allantoic cavity of eggs), aliquots were inoculated into the allantoic cavity of embryonated 9-day-old chicken eggs, and 4 dpi the allantoic fluid was collected and assayed for virus infection by HA test (Table 1). For all of the serotypes of APMV except serotype 5, virus was detected from the lungs and nasal turbinates at 3 dpi. APMV serotypes 4 and 9 also were detected in the brain in two mice each at 3 dpi. Virus was not detected in any other tissues isolated 3 dpi, or in any tissue isolated 14 dpi.
10.1371/journal.pone.0016776.t001Table 1 Virus isolation from the indicated tissue harvested from mice 3 dpi with APMV serotypes 1 to 9*.
APMV Serotype Lung Nasal turbinate Spleen Kidney Small intestine Brain
1
+++ +++ - - - -
2
+++ +++ - - - -
3
+++ +++ - - - -
4
+++ +++ - - - ++
5
ND ND ND ND ND ND
6
+++ +++ - - - -
7
+++ +++ - - - -
8
+++ +++ - - - -
9
+++ +++ - - - ++
* Mice in groups of 3 were inoculated with 50 ìl of allantoic fluid containing 27 HA units of each APMV serotype except serotype 5, which contained 3×103 PFU/ml of cell culture harvested virus. The control group was inoculated with normal allantoic fluid. Tissues were harvested 3 dpi and homogenized, and clarified supernatant fluid was inoculated into 9-day-old embryonated eggs and tested for virus 4 days later by HA assay.
+ = each + indicates isolation of virus from one mice.
- = no virus was isolated.
ND = not detected.
For quantification of the replication of each APMV, homogenates of tissue samples that were positive for virus isolation in eggs were used to generate 10-fold dilution series that were inoculated onto chicken DF1 cells and incubated under methylcellulose overlay. The tissue homogenates from APMV-5-infected mice were analyzed in parallel. Immunofluorescence staining for the N protein 4 dpi revealed foci with strong positive intracellular staining in the case of samples from the lungs and nasal turbinates for each of the APMV serotypes except APMV-5, for which no virus was detected (Fig. 2). The mean virus titers from lungs of the mice infected with APMV serotypes 1, 2, 3, 4, 6, 7, 8 and 9 were 2×102, 3×102, 7×101, 2×102, 2×102, 3×101, 5×102 and 9×102, respectively (Table 2). The mean virus titers from nasal turbinates of the mice infected with APMV serotypes 1, 2, 3, 4, 6, 7, 8 and 9 were 3×101, 6×, 2×101, 1×102, 3×102, 1×101, 2×102 and 8×102, respectively. These results indicated the titer of APMV serotype 9 was higher than other serotypes in the lung and nasal turbinates, suggesting that it was the most permissive APMV for replication in mice. The brain tissue samples of APMV serotypes 4 and 9 infected mice that were positive for infection of embryonated eggs were negative in cell culture. No virus was detected in any sample from day 14, indicating viral clearance. The APMV-5 samples also were inoculated onto African green monkey Vero cells, where no virus could be detected from any of the tissue harvested from mice 3 dpi or 14 dpi by immunofluorescence staining. This indicated that APMV-5, alone among the APMV serotypes, was not recoverable from infected mice.
10.1371/journal.pone.0016776.g002Figure 2 Immunofluorescence visualization of foci formed in DF-1 cells infected with each of the 9 APMV serotypes.
Mice were infected with each of the APMV serotypes and lungs were harvested 3 dpi and homogenized, and the clarified supernatants were inoculated onto DF-1 cells that were incubated 4 days under methycellulose prior to immunostaining with polyclonal antiserum specific to the N protein of the respective serotype. Viruses: APMV-1 (panel B), APMV-2 (panel C), APMV-3 (panel D), APMV-4 (panel E), APMV-5 (panel F), APMV-6 (panel G), APMV-7 (panel H), APMV-8 (panel I), APMV-9 (panel J), and mock-infected (panel A).
10.1371/journal.pone.0016776.t002Table 2 Virus titers in the indicated tissue harvested from mice 3 dpi with APMV serotypes 1 to 9, expressed as mean fluorescent foci/gram of tissue.
APMV serotype Lung Nasal Turbinate Brain
1
2×102
3×101
-
2
3×102
6×101
-
3
7×101
2×101
-
4
2×102
1×102
-
5
- - -
6
2×102
3×102
-
7
3×101
1×101
-
8
5×102
2×102
-
9
9×102
8×102
-
All the values are the mean of 3 mice per group.
*Titers were determined by serial dilution of clarified tissue homogenates from the experiment in Table 1 onto DF1 cells, which were overlaid with methylcellulose and immunostained 4 days later with N protein-specific antisera to detect viral foci.
- = no virus was isolated.
Immunonohistochemistry
The remaining half of each of the tissue samples isolated on 3 dpi and 14 dpi from the mice infected with the APMV serotypes was fixed and embedded in paraffin, and tissue sections were prepared. The tissue sections representing all of the collected tissue samples from the virus-infected and mock-infected animals from 3 dpi and 14 dpi were deparaffinized and immunostained using polyclonal antiserum specific to the N protein of the corresponding APMV serotype. Large amounts of APMV-specific N antigen was detected at 3 dpi by immunofluorescence staining of lungs and nasal turbinate tissue samples of mice infected with all the serotypes of APMV except serotype 5, which was negative (Fig. 3a and 3b). In the lungs of mice infected with each of the APMV serotypes except serotype 5, the viral antigen was localized mainly on the respiratory epithelium lining small and medium bronchi. In nasal turbinates of mice infected with each of the APMV serotypes except serotype 5, the virus specific immunoflourescence was observed on nasal epithelium lining the turbinate bone. No viral N antigen was detected in tissue samples from brain, spleen, kidney and small intestine of mice 3 dpi or 14 dpi with any the APMV serotypes, or from the lungs and nasal turbinates 14 dpi.
10.1371/journal.pone.0016776.g003Figure 3 Immunohistochemistry of sections of lungs (3a) and nasal turbinates (3b) harvested from mice 3 dpi with each of the 9 APMV serotypes.
Mice were mock-infected (panel A) or infected with APMV-1 (panel B), APMV-2 (panel C), APMV-3 (panel D), APMV-4 (panel E), APMV-5 (panel F), APMV-6 (panel G), APMV-7 (panel H), APMV-8 (panel I), and APMV-9 (panel J). Immunofluoresence was performed with polyclonal antiserum specific to the respective serotype N protein (magnification, ×400). In sections of the lungs from mice infected with different APMVs, immunofluorescence was evident around the bronchial epithelium (Fig. 3a). In sections of the nasal turbinates from mice infected with different APMVs, immunofluorescence was evident around the bronchial epithelium, at the apical surface of the ciliated epithelial cells and in the cytoplasm (Fig. 3b). Bronchioles are shown by arrow.
Histopathology
Tissue sections also were stained with hematoxylin and eosin and examined for histopathology. Lesions were observed in the lungs at 3 dpi in all the mice infected with each of the APMV serotypes except serotype 5, for which no histopathology was evident. Lesions were more severe in the alveoli and interstitial tissue, and were less severe in the airways. Severe multifocal to coalescing acute necrotizing bronchointerstitial pneumonia was noticed in lung tissues infected with APMV-1 and APMV-2, while mild to subacute bronchointerstitial pneumonia was observed in the other APMVs infected mice (Fig. 4). There was involvement of bronchiolar epithelium in some of the tissue samples. In some of the lung samples, alveolar hemorrhage and fibrin, which may be necrotizing, was observed. In addition, syncytia involving type II pneumocytes was evident for each of the APMVs except for APMV-4, APMV-5, and APMV-8 (Fig. 4 marked arrow). Microscopic lesions were not found in the lungs of any mouse at 14 dpi. In addition, lesions were not found in any other tissues on either day.
10.1371/journal.pone.0016776.g004Figure 4 Histopathology in sections of lungs harvested from mice 3 dpi with each of the APMV serotypes.
Mice were mock-infected (panel A) or infected with APMV-1 (panel B), APMV-2 (panel C), APMV-3 (panel D), APMV-4 (panel E), APMV-5 (panel F), APMV-6 (panel G), APMV-7 (panel H), APMV-8 (panel I), and APMV-9 9panel J). Sections were stained with hematoxylin and eosin (magnification, ×400). In mock-infected mice, the bronchiole is lined by a single layer of epithelial cells, alveoli are filled with air (although partially collapsed) and lined by flattened type I pneumocytes and there is mild acute alveolar hemorrhage likely secondary to CO2 euthanasia. APMV-5 infected mice had no detectable lesions and were indistinguishable from mock-infected mice. In APMV-1 and -2 infected mice, severe subacute diffuse necrotizing bronchointerstitial pneumonia was observed (panels B and C). Further, alveolar spaces and septae are filled with hemorrhage, fibrin, edema, and mixed inflammatory cells (neutrophils, macrophages, lymphocytes, plasma cells), as well as necrotic cellular debris (panels B and C). In APMV-1 infected mice, alveolar spaces/interstitium are more severely affected than are the airways (panel B). Further, bronchiolar epithelial cells are hypertrophied with karyomegally and occasional individual cell necrosis/apoptosis was also observed (panel B). In APMV-7 and -9 infected mice, there is type II pneumocyte hypertrophy as well as endothelial cell hypertrophy (black arrows in panels H and J). In APMV-8 infected mice, there are neutrophils in expanded alveolar septae (black arrow in panel I). However, mild bronchointerstitial pneumonia was observed in mice infected with some APMVs with different degrees of bronchiole damage (panels D, E and G to J).
Seroconversion
The sera of the mice infected with the different serotypes of APMV were collected at 14 dpi. Sera from mice infected with each of the APMV serotypes except serotype 5 were analyzed for virus-specific antibodies by a modified HI assay using chicken erythrocytes. The HI titers of the pre-infection and control mice were 2 or less. An HI titer of greater than 8 was considered positive. Every infected mouse seroconverted, indicating that viral replication had occurred (Table 3). The mean serum HI titers of mice infected with APMV serotypes 1, 2, 3, 4, 6, 7, 8 and 9 were 1∶64, 1∶64, 1∶64, 1∶16, 1∶16, 1∶64, 1∶64, 1∶64, respectively. In the case of APMV-5, which does not hemagglutinate, antibody titers were measured by a plaque reduction assay. However, none of the mice infected with APMV-5 developed detectable neutralizing serum antibodies.
10.1371/journal.pone.0016776.t003Table 3 Serum antibody responses against APMV serotypes 1–9 in infected micea.
APMV serotype HI antibody titerb
1
1∶64
2
1∶64
3
1∶64
4
1∶16
5
-
6
1∶16
7
1∶64
8
1∶64
9
1∶64
All the values are averages from three independent experiments.
a Mice in groups of 3 were inoculated as in Table 1. Serum samples were collected before inoculation and 14 dpi.
b The hemagglutination inhibition (HI) titer is expressed as the reciprocal of the highest serum dilution causing complete inhibition of four HA units of NDV.
- = not detected.
Discussion
APMVs are frequently isolated from wide variety of avian species around the world and have been grouped into nine serotypes based on antigenic relatedness involving the HN protein. Among the nine serotypes, APMV-1 (NDV) is the most studied member due to its importance as a major pathogen of poultry. APMV serotypes 2 to 9 are frequently isolated from both domestic and wild birds, but have been largely uncharacterized until recently. APMV-1 has been shown to infect not only avian species but also non-avian species, although its replication is restricted in non-avian hosts. APMV-1 is being developed as promising viral vaccine vector for delivery of a number of foreign antigens of animal and human pathogens. Further, APMV-1 has been developed as safe and effective oncolytic agent both in mouse models and also in human clinical trials [51]–[53], [57]–[59]. Therefore, it is of interest to also evaluate APMV serotypes 2 to 9 as potential vaccine vectors and oncolytic agents. However, the ability of APMV serotypes 2 to 9 to replicate in mammalian hosts was unknown.
The goal of this study was to evaluate intranasal infection by representative APMVs in mice to assess pathogenesis in a non-avian host and to identify a small laboratory animal model for further studies. Mice were evaluated for permissiveness to infection by APMV serotypes 1 to 9, clinical disease, magnitude and location of replication in the respiratory tract, dissemination to other tissues, disease, histopathology, and induction of antibodies. Inbred mice have been commonly used to study replication and pathogenesis of various viruses, to screen attenuation phenotypes of live virus vaccines, and to evaluate immune responses and protective efficacy elicited by virus vaccine candidates. This animal model is desirable for a number of reasons (i) Inbred mice represent a genetically homogenous model for which many reagents are available (ii) The degree of replication in the respiratory tract and other organs is readily measured (iii) Pathogenesis is readily monitored (iv) Innate, cellular, humoral and mucosal immune responses can be readily measured and (v) The mouse has the same body temperature as humans, and is a convenient mammalian host.
In this study, we have evaluated the replication and pathogenicity of APMV serotypes 2 to 9 in mice by a natural (intranasal) route of infection. Mice infected with different serotypes of APMV showed relatively mild or no clinical signs. Mice infected with APMV serotypes 1, 2, 6, 7 and 9 exhibited loss of weight that was more marked with serotypes 1 and 2. However, these mice recovered fully by 7th day of infection and showed no further clinical signs. The order of severity of clinical disease and weight loss was: 1, 2>6, 9>7. The other serotypes did not induce clinical disease. Mice infected with any of the APMV serotypes exhibited no gross pathological lesions in any of the organs.
Each of the APMV serotypes replicated to low-to-moderate titers in the lungs and nasal turbinates on 3 dpi except for serotype 5, for which infectious virus could not be recovered from any tissue at any time point. In the case of APMV serotypes 4 and 9, the brain tissue samples from two of the three infected mice in each group were positive for infectious virus 3 dpi when assayed in embryonated eggs, but were negative for virus recovery in cell culture. This suggests that the viral titers in the brain samples were very low. Infectious virus was not detected in any other tissue samples for any serotype. These findings indicate that replication by the APMV serotypes is largely restricted to the upper and lower respiratory tracts. In our study virus could not be detected in spleen in contrast to previous findings [60]. These discrepancies could be due to the differences in route of inoculation in two studies. Consistent with this, mild histopathological lesions were observed 3 dpi in the respiratory tract for all of the serotypes except APMV-5, which was negative. Lesions were not observed in any other tissues. Also, immunohistochemical staining detected viral N antigen in the lungs and nasal turbinates 3 dpi, but not in any other tissue. An interesting finding was the presence of large amounts of viral antigens at the epithelial cell linings, suggesting that these viruses have tropism towards the superficial layer of epithelial cells. On 14 dpi, virus was not detected in any of the tissues for any of the APMV serotypes, either by virus isolation or immunostaining. In addition, no histopathologic lesions remained. Thus, the infections had completely resolved by 14 dpi.
The different APMV serotypes induced a spectrum of disease severity. At one extreme, mice infected with serotypes 1 and 2 exhibited ruffled fur, huddling, decreased physical activity, and substantial weight loss. At the other extreme, serotypes 3, 4, 5, and 8 did not exhibit any disease signs apart from marginal weight loss in some instances. Serotypes 6, 7, and 9 were intermediate in disease signs. The clinical disease that was observed was short-lived. Increased disease was not reflected by substantial increases in the titer of infectious virus recovered from the lungs and nasal turbinates. For example, APMV-1 and -2 induced the most clinical disease, but the viral titers observed with APMV-1 and -2 were similar to those with APMV-4, -6, -8, and -9 for which disease was either less (APMV-6 and -9) or not apparent (APMV-4 and -8). Similarly, the amount of antigen observed in the epithelial linings of the lungs and nasal turbinates was extensive for all of the viruses except APMV-5, and thus usually was not accompanied by severe disease. These findings are favorable with regard to the possible use of these viruses as vaccine vectors, since extensive antigen expression with minimal disease and minimal-to-moderate virus replication are desired characteristics. Serological analysis demonstrated a humoral response in the mice inoculated with different serotypes of APMVs except serotype 5, a further indication that APMV-5 did not replicate significantly. These results show that, with the exception of serotype 5, the APMVs are capable of infecting mice by the intranasal route with extensive antigen expression and minimal disease.
Previously, pathogenicity of APMV-3 strain Netherlands in 9-day-old embryonated chicken eggs, 1-day and 2-week-old chickens and turkeys was examined [41]. The mean death time (MDT) in 9-day-old embryonated chicken eggs was 112 h and intracerebral pathogeneicity index (ICPI) in 1-day-old chicks was 0.39. It caused mild respiratory disease in 1-day-old chickens and turkeys after inoculation through oculonasal route. In 2-week-old chicken and turkeys, it did not cause any disease. All the birds were seroconverted and the virus was detected in brain, lungs, alimentary tract, spleen, trachea, pancreas and kidney. Subbiah et al. [40] studied pathogenicity of APMV-2 strain Yucaipa in 9-day-old embryonated chicken eggs, 1-day and 4-week-old chickens and turkeys. The MDT in 9-day-old embryonated chicken eggs was more than 168 h and ICPI in 1-day-old chicks was zero. After oculonasal inoculation, it did not cause any disease in 4-week-old chickens and turkeys, although all the birds were seroconverted. The virus was detected in respiratory and alimentary tracts. In another study, experimental infection of 1-day-old chicks with APMV serotypes 2, 4 and 6 resulted in viral infection and virus was recovered from infected birds' trachea, lungs, gut, and pancreas [61]. Similar to the findings in chickens, we were able to detect the virus in respiratory tract of mice but we were not able to isolate infectious virus or detect viral antigen for any of the APMV serotypes in the spleen, kidney and small intestines of the mice, suggesting that the tropism of these APMVs is more restricted in mice, presumably reflecting reduced replication and host-range restriction in this non-avian species. However, the observation that trace amounts of APMV-4 and -9 were detected in the brain will need to be further evaluated.
Our results identified BALB/c mice as a small animal model that supported replication in the respiratory tract of all of the APMVs except for APMV-5, which failed to infect detectably. Except for APMV-5, each APMV induced a substantial humoral immune response. The replication of APMVs in mice produced a spectrum of mild, short-lived disease that was restricted to the respiratory tract. These results suggest that, like APMV-1, the other APMV serotypes are candidates for evaluation in non-human primates as potential vaccine vectors attenuated by host rang restriction. In conclusion, this study is the first comparative report on the replication and pathogenicity of prototype strains of all nine APMV serotypes in mice. Our results lay the foundation for a good laboratory animal model for testing the replication and pathogenicity of APMV strains.
We thank Philip Martin, Pathologist (Center for Advanced Preclinical Research, SAIC/NCI-Frederick, Bld 539, 225 b Frederick, Maryland 21702) for analysis of histopathological sections. We also thank Daniel Rockemann, Flavia Dias and all our laboratory members for their excellent technical assistance and help.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This research was supported by NIAID contract no. N01A060009 (85% support) and NIAID, NIH Intramural Research Program (15% support). The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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==== Front
Br J Cancer
Br J Cancer
British Journal of Cancer
0007-0920
1532-1827
Nature Publishing Group
6605972
10.1038/sj.bjc.6605972
21102589
Translational Therapeutics
Pre-treatment levels of circulating free IGF-1 identify NSCLC patients who derive clinical benefit from figitumumab
Free IGF-1 predicts sensitivity to figitumumab
Gualberto A 12*
Hixon M L 2
Karp D D 3
Li D 1
Green S 1
Dolled-Filhart M 4
Paz-Ares L G 5
Novello S 6
Blakely J 7
Langer C J 8
Pollak M N 9
1 The Department of Clinical Development and Medical Affairs, Pfizer Oncology, New London, CT 06320, USA
2 Department of Pathology and Laboratory Medicine, Warren Alpert Medical School at Brown University, Providence, RI 02903, USA
3 Department of Thoracic Oncology, MD Anderson Cancer Center, Houston, TX 77030, USA
4 HistoRx Inc., New Haven, CT 06511, USA
5 Department of Medical Oncology, Virgen del Rocio University Hospital, Seville 41013, Spain
6 Department of Medical Oncology, University of Turin, Orbassano, Turin 10043, Italy
7 Department of Medical Oncology, West Clinic, Memphis, TN 38120, USA
8 Hematology/Oncology Division, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA
9 Department of Medical Oncology, McGill University, Montreal, Quebec H3T 1E2, Canada
* E-mail: [email protected] or [email protected] Current address: Millennium: The Takeda Oncology Company, 35 Landsdowne Street, Cambridge, MA 02139, USA
04 01 2011
23 11 2010
104 1 6874
26 07 2010
04 10 2010
05 10 2010
Copyright © 2011 Cancer Research UK
2011
Cancer Research UK
https://creativecommons.org/licenses/by/4.0/ This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.
Background:
Phase III trials of the anti-insulin-like growth factor type 1 receptor (IGF-IR) antibody figitumumab (F) in unselected non-small-cell lung cancer (NSCLC) patients were recently discontinued owing to futility. Here, we investigated a role of free IGF-1 (fIGF-1) as a potential predictive biomarker of clinical benefit from F treatment.
Materials and method:
Pre-treatment circulating levels of fIGF-1 were tested in 110 advanced NSCLC patients enrolled in a phase II study of paclitaxel and carboplatin given alone (PC) or in combination with F at doses of 10 or 20 mg kg−1 (PCF10, PCF20).
Results:
Cox proportional hazards model interactions were between 2.5 and 3.5 for fIGF-1 criteria in the 0.5–0.9 ng ml−1 range. Patients above each criterion had a substantial improvement in progression-free survival on PCF20 related to PC alone. Free IGF-1 correlated inversely with IGF binding protein 1 (IGFBP-1, ρ=−0.295, P=0.005), and the pre-treatment ratio of insulin to IGFBP-1 was also predictive of F clinical benefit. In addition, fIGF-1 levels correlated with tumour vimentin expression (ρ=0.594, P=0.021) and inversely with E-cadherin (ρ=–0.389, P=0.152), suggesting a role for fIGF-1 in tumour de-differentiation.
Conclusion:
Free IGF-1 may contribute to the identification of a subset of NSCLC patients who benefit from F therapy.
IGF-IR
IGF-1
figitumumab
NSCLC
==== Body
pmcThe insulin growth factor (IGF) system is comprised of the IGF ligands (IGF-1 and IGF-2), the IGF binding proteins (IGFBPs 1–7) that regulate ligand bioactivity, the cell surface receptors insulin-like growth factor type 1 receptor (IGF-IR) and IGF-2R, the adaptor proteins insulin receptor substrate (IRS)-1 and -2 and downstream signalling pathways (Pollak, 2008). Signalling through the IGF-IR plays important roles in normal growth and development as well as in the initiation and progression of neoplasia (Chitnis et al, 2008). There is considerable current interest in targeting the IGF-1 receptor as a therapeutic strategy in oncology, with more than a dozen drug candidates undergoing clinical evaluation (Gualberto and Pollak, 2009). In NSCLC, the IGF-IR has been shown to be frequently expressed in tumour tissue as well as to mediate the proliferation of lung cancer cell lines (Favoni et al, 1994). Also, high IGF-1 levels have been associated with higher incidence and aggressiveness of NSCLC (Spitz et al, 2002). These data suggest that targeting the IGF-IR could be a viable approach for the treatment of NSCLC.
Figitumumab (F) is a selective inhibitor of the IGF-IR that has been well tolerated in initial studies (Gualberto, 2010). Figitumumab enhances the tumour growth inhibition of chemotherapy and targeted agents in pre-clinical models (Cohen et al, 2005). A recently completed phase II study concluded that F increases the response rate and progression-free survival (PFS) benefit of paclitaxel and carboplatin as first-line treatment of patients with advanced NSCLC (Karp et al, 2009). However, pivotal trials of this agent in NSCLC were recently discontinued owing to futility. These results stress the need to identify patient subpopulations that may preferentially benefit from F therapy. This paper summarises a series of preliminary ancillary studies conducted to characterise plasma markers that could identify a subset of patients who derive benefit from the addition of F to standard NSCLC therapy.
Materials and methods
Patients
Study 1002 was a multiple-centre, open-label, randomised phase II trial that investigated the efficacy of the combination of F with paclitaxel (P) and carboplatin (C) as treatment for patients with chemotherapy-naïve stage IIIB or IV NSCLC (Karp et al, 2009). Briefly, patients had histologically or cytologically confirmed NSCLC not amenable to curative treatment. Eligible patients had at least one unidimensionally measurable lesion according to the Response Evaluation Criteria in Solid Tumours and Eastern Cooperative Oncology Group performance status of 0/1. Eligible patients were randomised 2:1 (PCF arm : PC arm) to receive P at 200 mg m−2 intravenously (i.v.) over 3 h and C with area under the plasma concentration–time curve of 6, i.v. over 15–60 min every 3 weeks with or without F at doses of 10 or 20 mg kg−1 in two sequential cohorts (PCF10, PCF20). The protocol was conducted in accordance with Good Clinical Practice guidelines and was approved by each participating institutional ethics review boards. All patients signed written informed consent before enrolment.
Laboratory assessments
Plasma samples were collected from fasted patients before trial treatments. Levels of total IGF-1, free IGF-1 (fIGF-1), IGF-2, IGFBP-1, IGFBP-2, IGFBP-3, insulin and cotinine were determined at a central laboratory (Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA), using the ELISA method. Antibodies and reagents were from Beckman-Coulter Diagnostic System Laboratories (Webster, TX, USA) as follows: total IGF-1 (DSL 10-2800), fIGF-1 (DSL 10-9410), IGF-2 (DSL 10-2600), IGFBP-1 (DSL 10-7810), IGFBP-2 (DLS 10-7100), IGFBP-3 (DSL 10-6600) and insulin (DSL 10-1610). Cotinine was determined using a kit (CO096A) from Calbiotech (Spring Valley, CA, USA). Glucose and creatinine were determined at the local clinical study sites using standard techniques. E-cadherin and vimentin were quantified using fluorescent immunohistochemistry (F-IHC) and an automated analysis system. Briefly, tumour tissues were deparaffinised, hydrated in water and antigen retrieval performed using standard techniques. Incubation with primary antibodies was conducted for 1 h at room temperature. Primary antibodies were mouse anti-E-cadherin (M3612, clone NCH38, 2 μg ml−1; Dako, Carpinteria, CA, USA) and mouse anti-vimentin (MS-129, 0.07 μg ml−1; Thermo Scientific, Rockford, IL, USA). Each primary antibody was included in a cocktail with rabbit anti-pan-cytokeratin (Z0622, 1:200; Dako) for the identification of epithelial regions and non-nuclear regions. Image capture, review, validation and scoring were carried out using a PM2000 epi-fluorescence microscopy system and AQUAnalysis software (HistoRx, New Haven, CT, USA).
Statistical analysis
Progression-free survival curves were constructed using the Kaplan–Meier method, and differences analysed by the log-rank test using the MedCalc software (Mariakerke, Belgium). Receiver-operating characteristic (ROC) analysis, area under the curve estimates and significance tests were accomplished using the built-in functions of MedCalc. Cox proportional hazards regression analysis was used to determine biomarker interactions. Biomarker data were analysed using ANOVA and correlations determined using Pearson's correlation tests.
Results
Pre-treatment plasma fIGF-1 levels identify NSCLC patients who may benefit from the addition of F to standard chemotherapy.
A total of 156 patients were randomised 2 : 1 to receive PC with or without F at doses of 10 or 20 mg kg−1 (PC, PCF10, PCF20). Plasma samples were obtained on cycle 1 day 1 before dosing from 110 of these patients. Patients were 68% men and had a median age of 64 years (range: 36–85). Forty-nine percent of them had tumours of adenocarcinoma histology and 80% were stage IV. Thirty-five patients received PC, 40 PCF10 and 35 PCF20. The overall efficacy results of this trial have been reported previously (Karp et al, 2009). Patients receiving the PCF20 regimen had a clinical benefit in terms of PFS of approximately 6 weeks over PC alone, while no clinical benefit over that of PC alone was observed with PCF10 (3.53, 3.60, 5.0 months median PFS for PC, PCF10 and PCF20, respectively, P=0.015). Median concentrations of baseline total IGF-1, free IGF-I, total IGF-2, insulin, and IGFBP-1, -2, -3 were, respectively, 219.3, 0.6, 578.1, 23.6, 15.1, 2.3 and 3627.8 ng ml−1. Potential biomarker associations with patient demographics were investigated. Higher baseline levels of fIGF-1 were observed in female patients (0.78 ng ml−1, 0.22–1.38 95% CI, N=36) than in male patients (0.52 ng ml−1, 0.07–1.50 95% CI, N=74, P=0.005), and in patients with tumours of adenocarcinoma histology (0.76 ng ml−1, 0.13–1.68 95% CI, N=53) than in those with other histologies (0.55 ng ml−1, 0.06–1.4 95% CI, P=0.03). No other differences were identified.
Progression-free survival was estimated in the subset of patients providing biomarker samples using the Kaplan–Meier method. Median PFS was similar to that in the overall trial population: 2.97, 3.63 and 5.6 months, respectively, for the PC, PCF10 and PCF20 cohorts (P=0.002). The ability of pre-treatment biomarker tests to identify patients who experienced prolonged clinical benefit on PCF treatment, for example, PFS longer than 6 months, was investigated using ROC analysis. Receiver operating characteristic curves plot the true positive rate (sensitivity) in function of the false positive rate (100-specificity, if expressed as percentage) at different biomarker cutoff points, and the 95% CI of the value for the area under the ROC curve (AUC) can be employed to test for predictive value (ROC curve AUC must be >0.5 for the test to be potentially useful) (Zweig and Campbell, 1993). Receiver operating characteristic plots were also employed to screen for markers able to identify patients with reduced clinical benefit, for example, patients with PFS shorter than 3 months. Table 1 summarises the AUC values and significance of the ROC analyses for the baseline analytes of patients receiving PCF20. As expected, the AUC 95% CI were wide owing to the small sample size (N=35); however, it was observed that high pre-treatment levels of fIGF-1 (at least 0.54 ng ml−1) in patients receiving PCF20 were associated (P=0.007) with PFS >6 months, whereas low pre-treatment fIGF-1 levels were associated (P=0.026) with PFS <3 months. When patients receiving PCF10 and PCF20 were analysed together (N=75), significant AUCs (P⩽0.05) were also identified. Figure 1 shows the ROC plots for all patients treated with F (10, 20 mg kg−1) using PFS benefit beyond 3, 4, 5 and 6 months as end points. Although our sample size was insufficient to investigate differences in AUC, the results represented in the figure were suggestive that fIGF-1 is a better predictor of long-term clinical benefit of F combination therapy. In contrast, pre-treatment fIGF-1 levels were not predictive of PFS in patients receiving PC only (not shown), suggesting that this parameter was not a general prognostic marker for the outcome of chemotherapy. Of interest, high pre-treatment levels of IGFBP-1 were associated (P=0.04) with PFS <3 months (Table 1).
The potential interaction between pre-treatment fIGF-1 criteria and the PFS benefit derived from F treatment was then investigated using the Cox proportional hazards models. Free IGF-1 cutoff criteria from 0.1 to 0.9 ng ml−1 were examined. Useful criteria were found in the range of 0.5–0.9 ng ml−1. For PCF20 vs PC, the estimated treatment–biomarker interaction terms from Cox proportional hazards models were between 2.5 and 3.5 for fIGF-1 criteria in the 0.5–0.9 ng ml−1 range, with one-sided P-values of 0.11 (0.5 ng ml−1 criterion), 0.09 (0.6 ng ml−1 criterion), 0.030 (0.7 ng ml−1 criterion), 0.033 (0.8 ng ml−1 criterion) and 0.026 (0.9 ng ml−1 criterion) adjusted for multiple testing. Hazard ratios for patients with fIGF-1 values above or below the 0.5–0.9 ng ml−1 criteria are shown in Figure 2A. Patients above each criterion had a substantial observed improvement in PFS on the PCF20 arm, while only a modest effect was observed below the criterion. For example, the PFS hazard ratio (PCF20/PC) was 4.2 for patients with fIGF-1 above 0.8 ng ml−1, but only 1.9 for patients with fIGF-1 equal or below 0.8 ng ml−1. A significant biomarker/treatment interaction for PCF10 was only observed at the fIGF-1 >0.8 ng ml−1 (P=0.027) and >0.9 ng ml−1 (P=0.04) criteria. Figure 3A–C shows specific examples of Kaplan–Meier curves for all patients in the biomarker cohort (Figure 3A; N=110), those with a baseline fIGF-1 level equal or lower than 0.7 ng ml−1 (Figure 3B; N=64) and those with a baseline fIGF-1 level higher than 0.7 ng ml−1 (Figure 3B; N=46). Median PFS for patients who had a baseline fIGF-1 level above 0.7 ng ml−1 were 2.63 (PC), 3.97 (PCF10) and 6.53 months (PCF20), respectively (P=0.0007). In contrast, no apparent differences in median PFS between the treatment cohorts were observed in those who had a baseline fIGF-1 level equal or below 0.7 ng ml−1. Additional analysis of PFS by fIGF-1 quartiles further indicated that clinical benefit of F combination therapy increased with baseline fIGF-1 levels. Tumours in patients with plasma fIGF-1 levels at the highest quartile (4th quartile, fIGF-1 ⩾1 ng ml−1) derived particular benefit from the addition of F to standard chemotherapy (Figure 4).
Effect of the insulin to IGFBP-1 ratio
The findings described above prompted us to investigate the mechanisms underlying the heterogeneity of fIGF-1 levels among NSCLC patients. It is well known that the bioactivity of IGF-1 and IGF-2 is modulated by 6, potentially 7, binding proteins (IGFBPs), of which IGFBP-1 and -3 are the best characterised (Pollak, 2008). Using the Pearson’s parametric correlation test, a significant inverse correlation was identified between pre-treatment fIGF-1 and IGFBP-1 levels (ρ=−0.295, P=0.005), but no significant association was observed between fIGF-1 and IGFBP-2 (P=0.37) or IGFBP-3 (P=0.9). Receiver operating characteristic plots were conducted to investigate potential associations between PFS rate and the pre-treatment ratios of IGF-1, IGF-2 and insulin to IGFBP-1, -2 and -3. A high baseline ratio of insulin (μU ml−1) to IGFBP-1 (ng ml−1) was found to be predictive (P=0.05–0.1) of the PFS rate of patients receiving PCF20 at multiple time points in the 3–8 months post-treatment period. No other significant associations were identified.
Insulin/IGFBP-1 ratio cutoffs criteria of 0.6–1 were then examined using Cox proportional hazards models. For PCF20, all cutoff criteria appeared to be potentially useful, with one-sided P-values for treatment–biomarker interaction of 0.07 (insulin/IGFBP-1 ratio of 0.5), 0.06 (ratio of 0.6), 0.02 (ratio of 0.7), 0.02 (ratio of 0.8), 0.03 (ratio of 0.9), 0.01 (ratio of 1) and 0.08 (ratio of 1.1), respectively, adjusted for multiple testing (Figure 2B). For PCF10, cutoff criteria of interest were 0.9 (P=0.04) and 1 (P=0.05). Kaplan–Meier estimates of median PFS in patients enrolled in Study 1002 who had a baseline insulin/IGFBP-1 ratio above 0.8 were 2.83 (PC), 3.86 (PC10) and 5.60 months (PC20), respectively (P=0.016; Figure 2D–F).
Other potential predictors
Other potential predictors of F activity were investigated. These included creatinine and creatine clearance, body mass index, fasting glucose to insulin ratio, the homeostatic model assessment of insulin sensitivity (HOMA) and the quantitative insulin sensitivity check index (QUICKI). None of these parameters reached significance in both the ROC and the Cox biomarker interaction analyses. A potential interaction with smoking was investigated using patient reported smoking habits and measuring pre-treatment cotinine levels, a serum metabolite of nicotine. Cotinine was detected in 0 of 13 patients who declared to have never smoke, eight of 61 patients who declared to be ex-smokers and 12 of 17 smokers. No effect of cotinine levels or smoking status on the PFS of patients receiving F was identified.
High plasma fIGF-1 is associated with high vimentin and low E-cadherin expression in NSCLC
We have previously shown that the IGF-IR and other molecules associated to the IGF-IR pathway (e.g. IGF-2R, IRS-1, -2) are overexpressed in NSCLC tumours undergoing epithelial-to-mesenchymal transition (EMT) (Gualberto et al, 2009). Epithelial-to-mesenchymal transition is a key feature of tumour infiltration and metastasis that is characterised at a molecular level by the expression of mesenchymal markers, such as vimentin, and the downregulation of epithelial differentiation markers, such as E-cadherin (Kalluri and Weinberg, 2009). We hypothesised that high circulating levels of fIGF-1 could be associated with high IGF bioactivity in the tumour microenvironment, and this could favour EMT. As a first step investigating the relationship between fIGF-1 in plasma and tumour EMT, E-cadherin and vimentin levels were quantified using F-IHC in tumour biopsies of 45 patients enrolled in Study 1002. The relationship between circulating fIGF-1 and tissue marker expression (F-IHC AQUA scores) was investigated using Pearson's correlation analysis. Free IGF-1 correlated directly with tumour vimentin (ρ=0.594; P=0.021), and inversely with E-cadherin expression (ρ=−0.389; P=0.152) and the tumour E-cadherin/vimentin ratio (ρ=−524, P=0.007). Of note, tumours in patients who had high levels of circulating fIGF-1 had barely detectable levels of E-cadherin (Figure 5), suggesting high degree of tissue de-differentiation.
Discussion
Many targeted therapies are active only for a subset of patients, and the characterisation of predictive biomarkers to identify those patients who are likely to benefit has been a key aspect of drug development. Examples include predicting benefit from trastuzumab therapy in breast cancer by assessing HER2/neu amplification (Sauter et al, 2009) and from cetuximab in colorectal cancer by assessing K-ras mutations (Siena et al, 2009). To date, predictive biomarkers for targeted therapies in NSCLC have been defined largely in the context of agents that target the EGF receptor family (Shepherd and Tsao, 2010), although recent data suggest a predictive value of EML4-ALK gene fusion for the clinical benefit derived from ALK inhibition (Koivunen et al, 2008). We undertook the current investigation to identify potential biomarkers that would allow for the prospective selection of patients who could benefit from the addition of F to standard chemotherapy of NSCLC.
Higher pre-treatment fIGF-1 levels were found to be predictive of the clinical benefit derived from the addition of F to chemotherapy in NSCLC patients. Although these results require confirmation in larger studies, it is of interest that consistent with our results, preliminary data from a recently discontinued phase III of PCF20 vs PC in patients with non-adenocarcinoma NSCLC revealed a biomarker/treatment interaction for baseline levels of fIGF-1. Median overall survival times were 10.2 and 7.0 months, respectively, for patients with a baseline fIGF1 >1 ng ml−1 receiving PCF20 and PC alone (Jassem et al, 2010). Analysis of this phase III study continues and final results will be discussed elsewhere.
In view of the considerable interindividual heterogeneity in levels of IGFs and their binding proteins, we hypothesise that plasma hormone levels might have particular utility in personalised therapy. This is a departure from the more common paradigm of emphasising molecular characteristics of the tumour in searching for predictive biomarkers. The reasoning underlying this hypothesis is that tumours that develop in a patient with high IGF bioactivity are more likely to become dependent on (or even ‘addicted’ to) IGF-1 receptor signalling, and therefore may be more sensitive to F therapy. Our results suggest that pre-treatment levels of fIGF-1 are predictive of the clinical benefit of F therapy in NSCLC, independent of any tumour characteristics. The possibility that more precise identification of patients who may benefit from IGF-IR targeting could be achieved by the use of algorithms that combines patient hormone levels and tumour characteristics remains open, but could not be explored in this study owing to small sample size. We observed however that tumours of patients with elevated circulating fIGF-1 expressed higher levels of vimentin and lower levels of E-cadherin, suggesting EMT. We speculate that this tumour characteristic is secondary to the hormonal environment. It has been shown previously that IGFs can induce neo-expression of mesenchymal markers and E-cadherin downregulation (reviewed by Julien-Grille et al (2005)). Insulin growth factors have also been shown to enhance the phosphorylation of β-catenin, causing its dissociation from membrane E-cadherin and translocation to the cytoplasm/nucleus (Playford et al, 2000; Morali et al, 2001). These interactions are thought to facilitate the coupling of IGF-IR activation with migration, invasiveness and metastasis.
High insulin to IGFBP-1 ratio was also predictive of the clinical benefit derived from F therapy, and an inverse correlation was observed between fIGF-1 and IGFBP-1. A role for IGFBP-1 in the control of IGF-1 bioactivity has been described previously (Bereket et al, 1996; Attia et al, 1999). Of note, over 90% of serum IGF-1 circulates in a complex with IGFBP-3 and another glycoprotein, acid-labile subunit (ALS). This complex is large (150 kDa), unable to transverse the endothelial barrier and has a long half-life, acting as a serum reservoir of IGF-1 (Kelley et al, 1996). In contrast, IGF-1 bound to IGFBP-1 does not form complexes with ALS, is able to cross the endothelial barrier and is, consequently, more likely to play a role in the regulation of IGF-1 bioactivity at extravascular tissues (Lee et al, 1993).
Insulin growth factor binding protein-1 levels and its IGF-1 binding capacity are tightly regulated by insulin, with IGFBP-1, as a result, acting as a bridging molecule between the insulin and IGF systems (Sakai et al, 2001; Borai et al, 2007). Thus, our data suggest that insulin may affect the risk/benefit of anti-IGF-IR therapy by regulating fIGF-1 levels. This is not in contradiction with other potential effects of insulin on cancer therapy; for example, those mediated by insulin receptors on tumour cells (Gualberto and Pollak, 2009). A role for IGFBP-1 in cancer has not been extensively studied, but it is known that low IGFBP-1 levels are associated with poor prognosis in at least one tumour type, colorectal cancer (Wolpin et al, 2009). Further research on IGFBP-1 functions could contribute to a better understanding of the effects of carbohydrate metabolism on cancer outcome. Overall, our data are consistent with previous reports associating low IGF-1 bioactivity with longer overall, disease-free and event-free survival in NSCLC (Chang et al, 2002; Han et al, 2006). In principle, the utilisation of circulating factors as predictive biomarkers would appear to be more convenient than measurements requiring fresh or frozen neoplastic tissue. However, it should be noted that current assay methodologies, particularly those measuring IGF-1 bioactivity, are controversial and imperfect (Frystyk, 2007). For example, in some applications, measurement of fIGF-1 offers no additional information beyond that provided by total IGF-1 (Juul et al, 1997), a measurement which is much more widely used than fIGF-1, but that is itself challenging (Brugts et al, 2008a). This issue is further complicated by the relatively small fraction of total IGF-1 bound to IGFBP-1 (Attia et al, 1999). In our hands, the fIGF-1 assay had an intra-assay imprecision of approximately 10% at the criteria of interest (0.5–0.9 ng ml−1); however, differences were observed across reagent lots. Thus, further analytical validation would be desirable before the prospective use of this assay in randomised studies. Structural studies have recently shown that the binding residues for IGFBP-1 and -3 on IGF-1 are overlapping but distinct (Dubaquié and Lowman 1999; Dubaquié et al, 2001). These differences could be exploited to develop new assays for the quantification of the fIGF-1 fraction specifically released from IGFBP-1. Development of such reagents could be important for a more personalised use of anti-IGF-IR therapy.
This exploratory study was not sufficiently powered for subset safety analysis. No major differences in the frequency or severity of adverse events between subgroups of patients with low vs high baseline fIGF-1 were apparent (not shown). Low levels of IGF-1 bioactivity have been associated with increased risk of cardiovascular mortality, whereas fasting IGFBP-1 levels were associated with more favourable cardiovascular risk profiles (Janssen et al, 1998; Brugts et al, 2008b). Of note, IGF-1 bioactivity changes during the progression of the metabolic syndrome, increasing in parallel to HOMA-IR and hyperinsulinaemia, but decreasing drastically when patients reach frank diabetes (Brugts et al, 2010). Insulin growth factor-1 bioactivity is also limited at low total IGF-1 levels (Brugts et al, 2010). Thus, treatment with anti-IGF-IR therapy in patients with low levels of total IGF-1 and/or glucose intolerance should be approached with caution. The ongoing analysis of the safety profile of larger F studies may contribute to a better understanding of the potential role of fIGF-1 in the assessment of the risk/benefit of anti-IGF-IR therapy.
In conclusion, our data provide preliminary evidence that fIGF-1 is a predictive biomarker of the clinical benefit, in terms of PFS, of F therapy in NSCLC. Confirmation of these findings in larger studies is needed. We also recognise the need to optimise assay methods and to further study the interactions between serum markers and tumour characteristics in NSCLC and other cancer types in which IGF-IR targeting is currently being investigated.
We thank our patients for their participation in this trial. This work was supported in part by NIH PHS Grants ES015704 (to MLH), and by Pfizer Inc.
Figure 1 Receiver-operating characteristic curves for fIGF-1 as a predictive maker for PFS benefit at 3–6 months in PCF-treated patients (10–20 mg kg−1 F).
Figure 2 (A) Hazard ratio of Study 1002 patients receiving treatment with PCF (10 or 20 mg kg−1) vs PC alone according to baseline fIGF-1 levels. (B) A hazard ratio of patients receiving treatment with PCF (10 or 20 mg kg−1) vs PC alone according to baseline insulin to IGFBP-1 ratio.
Figure 3 Kaplan–Meier plots of PFS in Study 1002 patients. (A) All patients with available baseline fIGF-1 level data (N=110). (B and C) Patients with baseline fIGF-1 ⩽0.7 (B, N=64) or >0.7 ng ml−1 (C, N=46). (D) All patients with available baseline insulin/IGFBP-1 ratio data (N=82). (E and F) Patients with baseline insulin/IGFBP-1 ratio ⩽0.7 (E, N=34) or >0.7 ng ml−1 (F, N=48).
Figure 4 Progression-free survival of PC, PCF10 and PCF20 patients by fIGF-1 quartile. The central box represents the values from the lower to upper quartile (25–75 percentile). In the box plots, the middle line represents the median. A line extends from the minimum to the maximum value, excluding ‘outside’ values that are displayed as separate points. An outside value is defined as a value that is smaller than the lower quartile minus 1.5 times the interquartile range, or larger than the upper quartile plus 1.5 times the interquartile range (inner fences). These values are plotted with a square marker.
Figure 5 Cyanine-5 fluorescence images representative of vimentin and E-cadherin expression in tumours from Study 1002 patients. Insets show cytokeratin (green) and DAPI (blue) fluorescence sample stainings.
Table 1 ROC for baseline IGF-IR-related serum analytes in patients receiving PCF20
PFS at 3 months PFS at 6 months
Analyte ROC AUC 95% CI P-value ROC AUC 95% CI P-value
Total IGF-1 0.368–0.758 0.566 0.476–0.846 0.140
fIGF-1 0.536–0.852 0.026 0.661–0.933 0.007
IGF-2 0.430–0.811 0.290 0.460–0.834 0.215
Insulin 0.387–0.769 0.457 0.464–0.832 0.213
IGFBP-1 0.531–0.880 0.040 0.405–0.784 0.401
IGFBP-2 0.498–0.862 0.086 0.361–0.752 0.650
IGFBP-3 0.306–0.652 0.842 0.478–0.809 0.178
Abbreviations: AUC=area under the ROC curve; CI=confidence interval; IGF=insulin-like growth factor; fIGF-1=free insulin-like growth factor 1; IGFBP=insulin-like growth factor binding protein; IGF-IR=insulin-like growth factor type 1 receptor; PCF=paclitaxel and carboplatin in combination with figitumumab; PFS=progression-free survival; ROC=receiver operating characteristics.
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Int J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 10.3390/ijms12010385ijms-12-00385ArticleRNA Interference Targeting Slug Increases Cholangiocarcinoma Cell Sensitivity to Cisplatin via Upregulating PUMA Zhang Kejun 1†Chen Dong 1*†Wang Xingang 1Zhang Shaoyan 2Wang Jigang 3Gao Yuan 4Yan Bomin 11 General Surgery of the Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shan Dong Province 266003, China; E-Mails: [email protected] (K.Z); [email protected] (X.W.); [email protected] (B.Y.)2 Laboratory of the Affiliated Hospital of Medical College, Qingdao University, Qingdao, Shan Dong Province 266003, China; E-Mail: [email protected] Pathology, The Affiliated Hospital of Medical College, QingDao University, Qingdao, Shan Dong Province 266003, China; E-Mail: [email protected] Molecular Biology, The Affiliated Hospital of Medical College, QingDao University, Qingdao, Shan Dong Province 266003, China; E-Mail: [email protected]*Author to whom correspondence should be addressed; E-Mail: [email protected].† These authors contributed equally to this work.
2011 14 1 2011 12 1 385 400 9 12 2010 6 1 2011 7 1 2011 © 2011 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.2011This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug’s ability to silence expression suppresses the growth of cholangiocarcinoma cells and/or sensitizes cholangiocarcinoma cells to chemotherapeutic agents through induction of apoptosis. We targeted the Slug gene using siRNA (Slug siRNA) via full Slug cDNA plasmid (Slug cDNA) transfection of cholangiocarcinoma cells. Slug siRNA, cisplatin, or Slug siRNA in combination with cisplatin, were used to treat cholangiocarcinoma cells in vitro. Western blot was used to detect the expression of Slug, PUMA, and E-cadherin protein. TUNEL, Annexin V Staining, and cell cycle analysis were used to detect apoptosis. A nude mice subcutaneous xenograft model of QBC939 cells was used to assess the effect of Slug silencing and/or cisplatin on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug and PUMA. TUNEL was used to detect apoptosis in vivo. The results showed that PUMA and E-cadherin expression in cholangiocarcinoma cells is Slug dependent. We demonstrated that Slug silencing and cisplatin both promote apoptosis by upregulation of PUMA, not by upregulation of E-cadherin. Slug silencing significantly sensitized cholangiocarcinoma cells to cisplatin through upregulation of PUMA. Finally, we showed that Slug silencing suppressed the growth of QBC939 xenograft tumors and sensitized the tumor cells to cisplatin through PUMA upregulation and induction of apoptosis. Our findings indicate that Slug is an important modulator of the therapeutic response of cholangiocarcinoma cells and is potentially useful as a sensitizer in cholangiocarcinoma therapy. One of the mechanisms is the regulation of PUMA by Slug.
cisplatinchemotherapyslugPUMAE-cadherin
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1. Introduction
Despite the aggressive multidisciplinary cancer therapies that have been used clinically, the prognosis of cholangiocarcinoma patients is extremely poor due to the low resection rate and the tolerance of the cancer to chemotherapy and radiotherapy [1,2]. For this reason, it is important to find new methods to enhance the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents. Gene therapy for cholangiocarcinoma is potentially a promising approach. The identification of molecules involved in the regulation and induction of apoptosis in cholangiocarcinoma has recently been reported and has generated new biological treatments aimed at enhancing chemotherapy-induced cell apoptosis [3–5].
The human Slug gene belongs to the highly conserved Slug/Snail family of transcription repressors, master regulators of neural crest cell specification and melanocyte migration during development in vertebrates [6–10]. Previous studies have demonstrated that the overexpression of Slug can be found in many kinds of cancer [11–14]. Moreover, Slug activates multiple signal intermediates, such as E-cadherin, which are key factors that influence the events of tumor invasion and metastasis [15,16]. In addition, Slug promotes survival and hinders cell death by directly suppressing PUMA, a key BH3-only antagonist of the anti-apoptotic Bcl-2 protein [17,18]. A previous study has demonstrated that PUMA is an important modulator of the therapeutic response of lung cancer cells and is potentially useful as a sensitizer in lung cancer therapy [19]. There have been a few studies devoted to the role of Slug in the chemoresistance of cancer cells to anti-cancer agents. A recent report indicated the possibility that Slug enhances chemoresistance of malignant mesothelioma cells to doxorubicin, paclitaxel, and vincristine [20]. It has been reported by Kurrey NK that Slug plays a critical role in the ability of a cancer cell to acquire stem cell characteristics to resist radiotherapy or chemotherapy-mediated cellular stress [21]. Roberta et al. [22] has reported that Slug down-regulation facilitates the apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo, and that Slug silencing enhances the efficacy of cisplatin and fotemustine in the treatment of melanoma [23].
However, to the best of our knowledge, the roles and possible mechanisms of Slug in the chemoresistance of cholangiocarcinoma cells to cisplatin have not been previously reported. The aim of this study is to investigate and define the ability and mechanism of Slug silencing to increase the susceptibility of cholangiocarcinoma cell lines to the currently used cytotoxic drug cisplatin.
2. Materials and Methods
2.1. Cell Culture
The human cholangiocarcinoma cell lines QBC939, RBE, ICC-9810 and FRH 0201 were purchased from ATCC and conserved in the central laboratory of the first affiliated Hospital of Suzhou University, China. Cells were grown in HAMs F12 medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 humidified atmosphere.
2.2. siRNA and cDNA Transfection
The siRNA targeting Slug (Slug siRNA, sc-38393) and mock siRNA (mock, sc-37007), the siRNA targeting PUMA (PUMA siRNA, sc-37153) and mock siRNA (mock, sc-33007) was obtained from Santa Cruz Biotechnology. Mock siRNA is a non-targeting 0–25nt siRNA designed to serve as a negative control. In a six well tissue culture plate, we seeded 2 × 105 cholangiocarcinoma cells per well in 2 mL antibiotic-free normal growth medium supplemented with FBS. We then incubated the QBC939 cells at 37 °C in a CO2 incubator until the cells were 60–80% confluent for 24 hours. The following solutions were prepared: Solution A: For each transfection, 2–8 μL of siRNA duplex (i.e., 0.25–1 μg siRNA) was diluted into 100 μL siRNA transfection medium: sc-36868 (Santa Cruz). Solution B: For each transfection, 6 μL of siRNA transfection reagent: sc-29528 (Santa Cruz, CA, USA) was diluted into 100 μL siRNA transfection medium: sc-36868(Santa Cruz, CA, USA). The siRNA duplex solution was then added (Solution A) directly to the diluted transfection reagent (Solution B) using a pipette and mixed gently by pipetting the solution up and down, then incubated for 30 minutes at room temperature. We then washed the cells once with 2 mL of siRNA transfection medium: sc-36868. For each transfection, we added 0.8 mL siRNA transfection medium to each tube containing the siRNA transfection reagent mixture (Solution A + Solution B) and then mixed gently and overlayed the mixture onto the washed cells. The cells were then incubated for 6 hours at 37 °C in a CO2 incubator. A volume of 1 mL of normal growth medium containing 2 times the normal serum and antibiotics concentration (2 × normal growth medium) was then added without removing the transfection mixture, and the cells were subsequently incubated for an additional 24 hours, at which time the medium was aspirated and we proceeded immediately to the next step. We performed Western blot on the cells 0–72 h after the addition of fresh medium in the step above. Mock siRNA containing a scrambled sequence that would not lead to the specific degradation of cellular Slug mRNA was transfected as above. The plasmid vector with the full coding region of human Slug (pcDNA3-Slug cDNA, Slug cDNA in brief) and the mock vector (pcDNA3-EGFP, mock cDNA in brief) were created in our laboratory (16). Transfection of Slug cDNA was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. In brief, 1 μg Slug cDNA was mixed with 3 μg Lipofectamine 2000 at a final concentration of 2 μg Slug cDNA/mL dissolved in OPTI-MEM I (Invitrogen), and the resulting complex was added to the cells and the FRH 0201cells were incubated with the complex for 4 h. FRH 0201 cells were washed with PBS and further incubated with the culture medium for specified time periods ranging from 0–72 h. Stably expressed Slug siRNA (mock) clones were selected by using medium containing G418 (500 μg/mL) for 28 days. Cells were routinely maintained in selection media containing 200 μg/mL of G418-sulfate to avoid overgrowth of nontransfected cells.
2.3. Western Blot
We collected approximately 2.0 × 107 cells (at different conditions) by low-speed centrifugation (e.g., 200 × g) at room temperature for 5 minutes and carefully removed the culture medium. Next, we washed the pellet with PBS at room temperature, and again collected cells by low-speed centrifugation and carefully removed the supernatant. We added 1.0 mL of ice cold RIPA buffer with freshly added Protease Inhibitors and gently resuspended the cells in RIPA buffer with a pipette and incubated the cells on ice for 30 minutes. We then further disrupted and homogenized the cells by hydrodynamic shearing (21-gauge needle), dounce homogenization (Optional: Add 10 μL of 10 mg/mL PMSF stock), and then incubated the cells for 30 minutes on ice. Next, samples were transfered to microcentrifuge tube(s) and centrifuged at 10,000 × g for 10 minutes at 4 °C. The supernatant fluid represented the total cell lysate and the supernatant was transferred to a new microfuge tube and represented the whole cell lysate. Total protein was measured in the extract by the Bradford assay. Primary antibodies were as follows: Anti-Slug (1:200 dilution), Anti–PUMA (1:400 dilution), and Anti-ß-actin (1:500 dilution), all from Santa Cruz Biotechnology. The membrane was probed using horseradish peroxidase–conjugated immunoglobulins (DAKO) as described previously (24). Western blot images were captured using an Epi Chemi II Darkroom and Sensicam imager with Labworks 4 software (UVP).
2.4. Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling Assay
Cells in different groups were cultured on chamber slides for 24 h. Apoptosis of the cells was evaluated on the basis of the TUNEL assay using the Dead End Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. All assays were performed in quadruplicate.
2.5. Cell Cycle Analysis
The cells in different groups and time points were washed twice with PBS and fixed with 70% ethanol/PBS. They were then treated with 0. 5 mg/mL RNase (Sigma) in PBS with 0.1% saponin, and incubated at 37 °C for 30 min before staining with 20 μg/mL PI for 30 min at 4 °C. The cells (1 × 106) were then analyzed for DNA content using a FACSCalibur flow cytometer equipped with CellQuest software (Becton Dickinson Immunocytometry Systems).
2.6. Annexin V Staining
The Annexin V assays were performed according to the manufacture’s protocol (PharMingen). Briefly, the cultured cells were collected, washed with binding buffer, and incubated in 200 μL of a binding buffer containing 5 μL of Annexin-V-FITC. The nuclei were counterstained with PI. The percentage of apoptotic cells was determined using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).
2.7. Xenograft Tumors
Immunodeficient female mice, 4 to 6 weeks old, were purchased from the Shanghai Animal Center. Autoclaved cages containing food and water were changed once a week. Mouse body weight was measured every 3 to 4 days. On the day of tumor cell inoculation, tumor cells at 70% to 80% confluence were trypsinized and resuspended in fetal bovine serum-free culture medium. Xenograft tumors were established by subcutaneous injection of 5 × 106 BQC939 cells (stable transfected with Slug siRNA or mock) into the flanks of 4 to 6 week old female Nude mice (n = 6 per group). In the combination model, cisplatin (3 mg/kg/d) was injected intraperitoneally into the mice on days 6–8. Mock transfected BQC939 cells were injected into separate tumors in the same animals. Tumor growth was monitored every other day with calipers for 28 days to calculate tumor volume according to the formula [length × width2]/2.
2.8. Immunohistochemistry in Xenograft Tumors
Tumor samples fixed in 10% neutral buffered formalin were embedded in paraffin using automatic embedding equipment, after which 5 μm sections were prepared. Immunohistochemical analyses for Slug and PUMA were performed on paraffin-embedded sections of mice treated with Slug siRNA or mock siRNA according to the manufacturer’s instruction.
2.9. TUNEL Staining in Vivo
Paraffin-embedded tumor sections were used to identify apoptotic cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining following the vendor’s protocol. A section was incubated with nuclease to generate DNA strand breaks for positive control. Endogenous peroxidase activity was quenched by 5% H2O2 (in methanol, v/v) and sections were incubated with terminal deoxynucleotidyl transferase labeling buffer followed with terminal deoxynucleotidyl transferase enzyme and biotinylated nucleotides (for negative control, labeling buffer was used instead of terminal deoxynucleotidyl transferase enzyme). Sections were incubated with streptavidin-conjugated horseradish peroxidase followed with 3,3′-diaminobenzidine solution (Sigma) and counterstained with diluted hematoxylin. Apoptosis was evaluated by counting the TUNEL-positive cells together with the total number of cells at 5 randomly selected fields at ×400 magnification in each tumor; the data are presented as percent TUNEL-positive cells.
2.10. Statistics
All statistical analyses were performed using SPSS13.0 software. The results were presented as mean ± SD of three separate assays. Differences between various groups were assessed using the ANOVA or Dunnett t-test. A P value of <0.05 was considered to indicate statistical significance.
3. Results
3.1. Slug Regulates PUMA and E-Cadherin Expression in Cholangiocarcinoma Cells
Slug expression was examined in a panel of four cholangiocarcinoma cell lines QBC939, RBE, ICC-9810, and FRH0201 by Western blot. The results indicated that the cell line FRH 0201 exhibited the lowest expression level of Slug and that QBC939 exhibited the highest expression level of Slug (Figure 1A). In this regard, the cell lines FRH 0201 and QBC939 were chosen for the studies.
The cell line FRH0201 was transiently transfected with either full length human Slug cDNA vector or the mock vector for 48 h to increase the expression of Slug. The cell line QBC939 was transiently transfected with Slug siRNA for 48 h to knock down Slug. In Slug siRNA-transfected QBC939 cells, Slug expression was barely detectable compared with parental cells (Figure 1B), whereas Slug cDNA-transfected FRH 0201 cells expressed a higher level of Slug compared with parental cells (Figure 1C).
Slug is a suppressor of PUMA transcription (18). To evaluate whether Slug plays a role in the regulation of PUMA in cholangiocarcinoma cell lines, we analyzed the expression of PUMA in Slug siRNA-transfected QBC939 cells and Slug cDNA-transfected FRH 0201 cells by Western blot. In Slug siRNA-transfected QBC939 cells, PUMA was up-regulated compared with parental cells (Figure 1B). By contrast, in Slug cDNA-transfected FRH 0201, no PUMA expression was seen compared with parental cells (Figure 1C).
It has been recently reported that the Slug transcription factor directly represses E-cadherin expression in many epithelial cancers associated with epithelial-mesenchymal transitions. The reverse correlation of Slug and E-cadherin expression has been noted in many malignant cells. We found in our study that Slug siRNA-transfected QBC939 cells showed a remarkble upregulation of E-cadherin protein compared with parental cells (Figure 1B) and that a remarkable downregulation of E-cadherin protein was shown in Slug cDNA-transfected FRH 0201 cells compared with parental cells (Figure 1C). These observations provided direct evidence that Slug regulated E-cadherin and PUMA expression in human cholangiocarcinoma cells.
3.2. Slug Inhibition by siRNA Promotes Apoptosis in Cholangiocarcinoma Cells
It has been previously demonstrated that Slug is involved in the control of apoptosis [17]. We next hypothesized that Slug silencing may have proapoptotic effects on cholangiocarcinoma cells. To investigate this, cell apoptosis was detected in QBC93 cells transiently transfected with Slug siRNA or mock for 48h by TUNEL analysis. It showed that the apoptosis rate in Slug siRNA-transfected QBC939 cells was significantly increased compared with the control or mock-transfected QBC939 cells (*P < 0.05, Figure 2).
To explore whether Slug inhibition by RNA interference promotes apoptosis by upregulating PUMA, and not by upregulating E-cadherin, we examined the effects of Z-DEVD-CHO, a caspase-3 inhibitor, on the internucleosomal degradation of DNA. It showed that the treatment of QBC939 cells with the inhibitor combined with transfection of Slug siRNA for 48 h resulted in significantly decreased cell apoptosis in contrast to only Slug siRNA treated groups (Figure 2, *P < 0.05).
3.3. Cisplatin promotes Cholangiocarcinoma Cells Apoptosis in Vitro
This study demonstrated that cisplatin induced apoptosis in human cholangiocarcinoma cell lines QBC939 and FRH0201 in a concentration- and time-dependent manner. As shown in Figure 3A and Figure B, treating the cholangiocarcinoma cells with 0.1 μg/mL cisplatin for 72 h caused only a slight increase in the proportion of apoptotic cells in the two cell lines. However, increasing the cisplatin concentration (20 μg/mL) for 72 h resulted in a sharp increase in the proportion of apoptotic cells, suggesting that cell death occurred in a dose-dependent manner (compared to control, *P < 0.05, **P < 0.01, ***P < 0.001).
When the cholangiocarcinoma cells were exposed to 20 μg/mL cisplatin, the proportion of apoptotic cells increased in a time-dependent manner. Almost 70% (FRH 0201), over 40% (QBC939) of the other cell population underwent apoptosis after 72 h, compared with ≤5% of the control (BPS-treated) cells (Figure 3A,B).
Similar results were obtained when the apoptosis was monitored by cell cycle analysis. Exposure to 20 μg/mL cisplatin for 12–24 h had no remarkable effect on the cell cycle distribution of the cholangiocarcinoma cells. However, the cisplatin treatment for 48–72 h resulted in a progressive increase in the sub-G1 cell fraction. (Figure 3C, D) (contrast to control, *P < 0.05, **P < 0.01, ***P < 0.001).
3.4. Cisplatin Cytotoxicity Is Associated with PUMA Induction
The effects of cisplatin on the intracellular level of PUMA were analyzed to additionally examine the relationship between cell death and PUMA in the QBC939 and FRH 0201 cholangiocarcinoma cells. The cells were treated with various cisplatin concentrations for 72 h, or the cells were treated with 20 μg/mL cisplatin concentrations for various lengths of time. The PUMA and E-cadherin extracted from the cells were subjected to Western blot analysis. A representative result of QBC939 and FRH 0201 is shown in Figure 4A–D. The amount of PUMA was increased in a concentration-dependent and time-dependent manner, reaching a maximum level at 20 μg/mL of cisplatin or 72 h treatment.
The results demonstrated that cisplatin did not obviously promote or reduce E-cadherin expression (Figure 4A–D). These observations provided direct evidence that cisplatin-induced apoptosis was not E-cadherin- dependent.
To determine whether the induction of cell death in cisplatin-treated cholangiocarcinoma cells was due, in part, to regulation of PUMA expression, QBC939 and FRH 0201 cells were exposed to a cisplatin concentration of 20 μg/mL for 6 h, after which time the cisplatin-treated cells were transfected PUMA siRNA for 72 h to knock down PUMA. This study demonstrated that induction of PUMA was not shown in any of the PUMA siRNA treated sample populations (Figure 4E,F). Cisplatin combined with PUMA siRNA did not induce obvious apoptosis in QBC939 and FRH 0201 cells (Figure 4G,H, *P < 0.01). By contrast, mock-transfected cells exhibited PUMA levels or an apoptosis level similar to that found in cells treated only with cisplatin (Figure 4G,H).
3.5. Slug Silencing and Cisplatin Treatment Act in Concert to Induce Apoptosis in Cholangiocarcinoma Cells
Given the activity of Slug in cell survival through regulation of proapoptotic factor PUMA, and the fact that cisplatin promotes apoptosis by upregulating PUMA, we assessed the apoptosis susceptibility of Slug siRNA-transfected QBC939 cells in the presence of 5 μg/mL cisplatin for 48 h. At the end of each treatment, cells were fixed and stained for TUNEL analysis. siRNA-transfected QBC939 cells combined with 5 μg/mL cisplatin showed a significantly increased apoptosis rate compared with siRNA-transfection only or cisplatin treatment alone (*P < 0.05, **P < 0.01) (Figure 5).
3.6. Slug Silencing Suppresses Cholangiocarcinoma Tumor Growth and Sensitizes Cholangiocarcinoma Xenografts to Cisplatin in Vivo
To determine whether Slug silencing confers antitumor activity in vivo, established 6 × 106 QBC939 cells (stable transfection with Slug siRNA or mock siRNA) were injected into the flanks of 4 to 6 week old female Nude mice (n = 6 per group). Mock siRNA had no effect on tumor growth compared to the control alone, with tumors doubling in volume in 28 days compared to Slug siRNA treatment groups (**P < 0.01; Figure 6A).
Furthermore, we analyzed the tumor sections from control, mock siRNA, and Slug siRNA groups for Slug and PUMA using immunohistochemistry. We observed significant expression levels of Slug in control and mock-treated tumor sections (Figure 6B). However, expression levels were drastically reduced in the tumor sections of mice treated with Slug siRNA. Furthermore, we observed drastically increased PUMA in Slug siRNA tumor sections (Figure 6B).
Analyzing tissue sections from tumors revealed that Slug siRNA, but not mock, significantly increased apoptosis in the tumors as assessed by TUNEL staining (Figure 6C). These data show that Slug silencing effectively inhibits the growth of Cholangiocarcinoma tumors through apoptosis induction.
Slug silencing was found to promote apoptosis by cisplatin in vitro. We wanted to determine whether such effects can be obtained in the QBC939 xenograft tumor model. To investigate the potential additive or synergistic effects, we used cisplatin (3 mg/kg/d for 4 days) in these experiments. Cisplatin treatment alone resulted in 20% growth inhibition compared with mock or PBS control, respectively (*P < 0.05, Figure 6A). In contrast, Slug siRNA combined with cisplatin resulted in >60% growth suppression (***P < 0.001, Figure 6A), indicating synergism. Analysis of tumor sections revealed that the Slug siRNA and cisplatin combination resulted in a significant increase in apoptosis compared with Slug siRNA or cisplatin alone (Figure 6C). These results suggest that Slug silencing enhances the therapeutic response of QBC939 tumors to cisplatin through apoptosis induction.
4. Discussion
Resistance of cholangiocarcinoma to chemotherapy is a major problem in cancer treatment [27–29]. Currently, it is generally acknowledged that chemotherapeutic agents exert their cytotoxic effects through the activation of apoptosis and promotion of apoptosis can increase the chemosensitivity of cancer cells [30,31]. For this reason, inhibited expression of some specific genes that inhibit apoptotic cell death can cause malignant cells to be relatively sensitive to the cytotoxic effect of chemotherapeutic agents.
The appearance of novel strategies for cancer treatment is based on the selective downregulation of specific targets involved in neoplastic progression. Slug seems to be a relevant target for such therapeutic intervention. Slug is detectable in many types of cancer, and its presence has been associated with poor prognosis in many malignant tumors [32–35]. Not only is Slug an inducer of epithelial-mesenchymal transition (EMT) and cell movement, it is also considered to be a factor favoring cell survival [16]. Previous studies have demonstrated that Slug overexpression exhibits a radioprotective function in TK6 cells demonstrating that it has potential as a candidate for gene therapeutic radioprotection of normal tissues [36]. Mancini et al. [37] has demonstrated that Slug overexpression contributes to apoptosis resistance in leukemic progenitors. Manuela Mancini reported that Slug overexpression was involved in prolonged survival and imatinib(IM)resistance of Chronic myelogenous leukemia(CML) progenitors [37]. In this report, we demonstrated for the first time that Slug silencing could efficiently promote apoptosis in cholangiocarcinoma cells. Annexin-V/PI and TUNEL staining revealed that apoptotic cell death was abundant in cells with slug repression, but almost completely absent in cells transfected with mock-siRNA. These data demonstrate that the Slug silencing gene facilitates apoptosis.
Slug, a highly conserved zinc finger transcriptional repressor, has been reported to antagonize apoptosis of hematopoietic progenitor cells by repressing PUMA transactivation [18]. In another study, it was demonstrated that suppression of the Slug gene facilitates apoptosis of fibroblast-like synoviocytes (FLS) by increasing PUMA transactivation [38].
Our current findings show that when Slug was knocked down in the QBC939 cells, the PUMA and E-cadherin proteins were upregulated in the cells with the Slug supression. When caspase-3, a downstream gene induced by upregulation of PUMA, was blocked, the cell apoptosis caused by Slug silencing was reduced. These data demonstrate that Slug silencing facilitates apoptosis by PUMA upregulation, and not by E-cadherin upregulation.
Recent studies have demonstrated that Slug silencing increases sensitivity to apoptosis induced by cisplatin, fotemustine, imatinib mesylate, etoposide, or doxorubicin [39,40]. Our study demonstrated that Slug silencing markedly enhances cisplatin-induced apoptosis in cholangiocarcinoma cells in vivo and in vitro, and that Slug overexpression could contribute to impaired apoptosis (data not shown). The mechanism by which Slug silencing induced an increase in cisplatin-induced apoptosis involves the upregulation of PUMA; moreover, Slug suppression increases the capacity of cisplatin to block the cell cycle and induce cell death and such a phenomenon is also accompanied by the upregulation of PUMA.
Previous studies have demonstrated that cisplatin-induced apoptosis in human bladder cancer cells and renal tubular cells was dependant upon PUMA [41,42]. However, Carly St has reported that cisplatin is a MAPK pathway dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects [43]. The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. In the present study, we found that cisplatin caused significant induction of apoptosis of human cholangiocarcinoma cells in a time and concentration dependent manner and that this was associated with arrest of the cell cycle in G0-G1. We also found that cisplatin cytotoxicity is associated with PUMA induction, and when PUMA was suppressed the apoptosis induced by cisplatin was blocked. This suggests that an apoptosis-inducing mechanism triggered by cisplatin operates via PUMA in the cholangiocarcinoma cells. Another study, however, has found that PUMA induction by chemotherapeutic agents is abrogated in most human non-small-cell carcinoma(HNSCC)cell lines, and that cisplatin does not induce any increase in PUMA expression [44].
To investigate whether the combination of Slug knockdown and cisplatin treatment can synergistically inhibit tumor growth and increase cholangiocarcinoma cell sensitivity to cisplatin, we used a well-established Xenograft tumor model in severe combined immunodeficient mice. Slug silencing or cisplatin alone inhibited Xenograft tumor growth, and the effects of both did reach statistical significance. This result is in contrast with the findings of the in vitro assays. When Slug-silenced cells were treated with cisplatin, significant growth suppression and apoptosis was demonstrated.
Our results suggest that this combination of cisplatin and Slug suppression may be useful for chemoprevention and/or therapy of cholangiocarcinoma and possibly other types of cancer. Indeed, in recent unpublished studies we found that this combination exerts a synergistic inhibition of the growth of cholangiocarcinoma cells. These findings suggest the combination of cisplatin with Slug suppression might be an effective regimen for the chemoprevention and/or chemotherapy of various types of human malignancies.
Acknowledgements
This work was supported by a grant from the Natural Science Foundation of Hainan Province (No. 809043) and the Department of Education Science Foundation of HaiNan Province, China (No. hjkj-2010-34).
Figure 1 Slug regulation-induced processing of Slug and PUMA. (A) The protein fractions of Slug in QBC939, RBE, ICC-9810, and FRH 0201 cells were subjected to Western blot analysis. FRH 0201 exhibited the lowest expression level of Slug and QBC939 exhibited the highest expression level of Slug. (B) The protein fractions of Slug, E-cadherin, and PUMA in QBC939 cells after transfection with Slug siRNA for 48 h were subjected to Western blot analysis. Slug expression was barely detectable compared with parental cells (**P < 0.01), and a remarkble upregulation of E-cadherin and PUMA protein was shown compared with parental cells (*P < 0.05). (C) The protein fractions of Slug, E-cadherin, and PUMA in the FRH 0201 cell line after transfection with Slug cDNA for 48 h were subjected to Western blot analysis. Remarkable upregulation of Slug protein was shown compared with parental cells (**P < 0.01), and remarkable downregulation of PUMA and E-cadherin proteins was shown compared with parental cells (*P < 0.05).
Figure 2 TUNEL staining was performed for QBC939 cells transiently transfected with Slug siRNA or mock transfected for 48hs. Green nuclear staining indicates apoptotic cells. The percentage of TUNEL-positive cells was quantified. Columns and bars represent the mean and standard deviation of three independent determinations, respectively. Significant differences between the controls (mock) and the Slug siRNA groups are indicated by *P < 0.05.
Figure 3 Cisplatin-induced apoptosis in QBC939 and FRH 0201 cells. A, C, QBC939 and FRH0201 cells (1 × 106/mL) were exposed to the designated concentrations of cisplatin for the indicated times, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. The data are expressed as a mean value of the percentage of apoptotic cells from three independent experiments performed in duplicate (*P < 0.05, **P < 0.01, ***P < 0.001). B, D, At the indicated time points after treatment with 20 μg/mL cisplatin, the cells were harvested and fixed in 70% ethanol. After staining with PI, the apoptotic DNA content was analyzed by flow cytometry. The number of apoptotic cells in the sub-G1 fraction is expressed as a percentage of the total number of cells (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 4 Induction of apoptosis by cisplatin is independent of PUMA mechanism. (A, B) The QBC939 and FRH 0201 cells were exposed to various cisplatin concentrations for 72 h. The PUMA and E-cadherin protein expression levels in the cell lysate were examined by Western blot analysis using anti-PUMA (E-cadherin) antibody, and the antibodies against ß-actin which served as an internal control. (C, D) The QBC939 and FRH 0201 cells were exposed to 20 μg/mL cisplatin for the indicated times. The PUMA and E-cadherin protein expression level was measured by Western blot. (E, F) QBC939 and FRH0201 cells (1 × 106/mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, PUMA protein expression level was measured by Western blot. (G, H) QBC939 and FRH0201 cells (1 × 106/mL) were exposed to 20 μg/mL cisplatin combined with PUMA siRNA for 72 h, after which time the percentage of apoptotic cells was determined by flow cytometric analysis, as described in “Materials and Methods”. Cisplatin combined with PUMA siRNA did not induce obvious apoptosis in QBC939 and FRH 0201 cells (*P < 0.01).
Figure 5 TUNEL staining was performed to assess the potential cooperation of Slug silencing and cisplatin treatment in inducing apoptosis in QBC939 cells. Green nuclear staining indicates apoptotic cells. The percentage of TUNEL-positive cells was quantified. Columns and bars represent mean and standard deviation of three independent determinations, respectively. Significant differences between the controls (mock) and the Slug siRNA groups are indicated by *P < 0.05, **P < 0.01.
Figure 6 Slug silencing sensitizes cholangiocarcinoma xenograft tumors to cisplatin. A, growth bar of QBC939 tumors (n = 6 per group) subjected to Slug siRNA, mock, and PBS and/or combined with cisplatin treatment for 28 days. B, Immunohistochemistry analysis for Slug and PUMA in different groups. C, TUNEL-positive cells in three groups were detected in the control, cisplatin, or Slug siRNA plus cisplatin treatments. Columns and bars represent the mean of three independent determinations and S.D., respectively. *P < 0.05, **P < 0.01, ***P < 0.01.
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J Lab PhysiciansJLPJournal of Laboratory Physicians0974-27270974-7826Medknow Publications India 21346899JLP-2-7010.4103/0974-2727.72152Original ArticleAn Approach to Uropathogenic Escherichia Coli in Urinary Tract Infections Ranjan K Prabhat Ranjan Neelima Chakraborty Arindam 1Arora D R 2Department of Microbiology, Pt. B D Sharma Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India1 Department of Microbiology, Kasturba Medical College, Mangalore, Karnataka, India2 Department of Microbiology, Maharaja Agrasen Medical College, Agroha, Hisar, Haryana, IndiaAddress for correspondence: Dr. K. Prabhat Ranjan, E-mail: [email protected] 2010 2 2 70 73 © Journal of Laboratory Physicians2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Purpose:
To study the occurrence and characterization of Uropathogenic Escherichia Coli (UPEC) in cases with urinary tract infections.
Materials and Methods:
A total of 220 symptomatic cases from urinary tract infections and 50 stool samples from apparently healthy individuals were included. The colonies identified as Escherichia Coli were screened for virulence factors, that is, hemolysin, Mannose Resistant and Mannose Sensitive Hemagglutination (MRHA, MSHA), Cell surface hydrophobicity, and Serum resistance.
Results:
Among the 220 cases 91 (41.36%) were hemolytic, 68 (30.90%) showed MRHA, 58 (26.36%) were cell surface hydrophobicity positive, and 72 (32.72%) were serum-resistant. In 50 controls, three (6%) were hemolytic, six (12%) showed MRHA, nine (18%) showed cell surface hydrophobicity, and 12 (24%) were serum-resistant. The difference between cases and controls for hemolysis and MRHA were significant (P<0.001 and P<0.01, respectively). A total of 14 atypical E. coli were isolated from the urine and all showed the presence of one or the other virulence markers. Out of the 18 mucoid E.coli isolated, 10 were serum-resistant.
Conclusions:
The present study revealed that out of 220 urinary isolates, 151 could be labeled as UPEC.
Escherichia Colihemolysinhemagglutionationserum resistancesurface hydrophobicityvirulence factor
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INTRODUCTION
Urinary tract infection (UTI) is one of the most important causes of morbidity and mortality. E.coli is the most frequent urinary pathogen isolated from 50 – 90% of all uncomplicated urinary tract infections.[1] E.coli present in the gastrointestinal tract as commensals provide the pool for initiation of UTI. It has been traditionally described that certain serotypes of E.coli are consistently associated with uropathogenicity and are designated as Uropathogenic E.coli (UPEC). These isolates express chromosomally encoded virulence markers. These markers of UPEC are expressed with different frequencies in different disease states, ranging from asymptomatic bacteriuria to chronic pyelonephritis.
In the late 1970s, it was recognized for the first time that E.coli strains causing urinary tract infections typically agglutinate human erythrocytes, despite the presence of Mannose,[2] and this was mediated mainly by fimbriae. Subsequently an array of virulence factors were proposed as virulence markers for uropathogenic isolates of E.coli. It is now recognized that there are a subset of fecal E.coli having the above-mentioned factors that can colonize the periurethral area, enter the urinary tract, and cause symptomatic disease. These are currently defined as UPEC.[3]
However, most of these studies have been carried out on patient isolates and no studies have been carried out on commensal / gut isolates. In addition, the relative importance of these bacterial factors has not been validated. In the present study an attempt has been made to answer these two questions with respect to four proposed virulence factors namely hemolysin, hemagglutination, cell surface hydrophobicity, and serum resistance.
MATERIALS AND METHODS
Samples
The study was conducted in the Department of Microbiology, Maharaja Agrasen Medical College. Urine samples from a total of 220 E. coli from all age groups of symptomatic cases of UTI, belonging to the Departments of Urology, Nephrology, Medicine, and Pediatrics were studied for the detection of virulence markers. A total of 50 fecal isolates from apparently healthy individuals who had come for a routine health checkup were screened for E. coli and studied further for virulence markers and included as controls. Urine samples were processed as per standard protocol.[4] Lactose fermenting colonies on MacConkey’s agar (MA) showing significant bacteriuria (105 CFU/ml) were processed and identified as E. coli by standard biochemical tests.[5] Standard uropathogenic E. coli serotypes O4 and O6 and E.coli ATCC 25922 were used as controls for detection of the virulence markers. The additional features noticed in terms of colony morphology were, whether they were mucoid / non-mucoid and the biochemical reactions, whether they showed hemolysis on sheep blood agar after incubation overnight, and whether they were typical or atypical isolates. An isolate was considered as typical if it was a lactose fermenter and aerogenic and atypical if it was a non-lactose fermenter and anaerogenic. E. coli thus obtained from cases with significant counts (105 CFU/ml) were screened for virulence markers.
Detection of virulence factors
Hemolysin production
The plate hemolysis test was done for the detection of α-hemolysis produced by the E. coli.[6] The bacteria were inoculated into 5% sheep blood agar and incubated overnight at 35°C. Hemolysin production was detected by the presence of a zone of complete lysis of the erythrocytes around the colony and clearing of the medium.
Hemagglutination
E. coli grown on Mac Conkey agar plates were inoculated into 5 ml of phosphate buffered saline pH 7.4 (PBS) and incubated for five days at 37°C, to obtain fimbriae E. coli. The pellicle that formed on the surface was noted and subcultured onto a colonization factor antigen (CFA) agar; 0.5 ml of fresh group A-positive blood was obtained from the blood bank and added to an equal amount of Alsever’s solution. This was washed thrice and 3% erythrocyte suspension was prepared with PBS. The colonies of E. coli growth on CFA were emulsified on a VDRL slide in PBS, to form a milky white suspension. To this an equal volume of 3% suspension of erythrocytes was added and gently mixed with a wooden applicator. The slide was rotated manually for three to five minutes, observed for hemagglutination macroscopically, within 10 minutes. To determine the mannose-resistant hemagglutination, colonies from the CFA were emulsified on a slide in PBS. A drop of 2.5% of mannose was added. To this mixture, an equal volume of 3% suspension of erythrocytes was added and gently mixed with a wooden applicator. The slide was rotated for three to five minutes and observed for hemagglutination (HA). Hemagglutination was designated as a mannose-resistant hemagglutination (MRHA) if the HA was observed with and without mannose to the same degree, and mannose sensitive hemagglutination (MSHA) if HA was inhibited in the presence of mannose.[6] The following controls were used: E. coli ATCC 25922 for MSHA. UPEC serotypes 06 and 011 for MRHA.[5]
Cell surface hydrophobicity
The cell surface hydrophobicity of E. coli was determined by the salt aggregation test (SAT).[67] One loopful (10 µL) of bacterial suspension made in phosphate buffer was mixed with an equal volume of ammonium sulfate solution of different molarity, that is, from 0.3125 m through 5.0 m, on a glass slide and observed for one minute, while rotating. The highest dilution of ammonium sulfate solution, giving a visible clumping of bacteria, was scored as the salt aggregation test (SAT) value. Strains showing aggregation in 0.002 m phosphate buffer alone (pH 6.8) were considered to be auto aggregative. E. coli strains that had a SAT value ≤ 1.25 m were considered hydrophobic.[67]
Serum resistance
Serum resistance was studied using a fresh culture of the isolates.[67] Overnight cultures of E. coli, grown at 37°C on blood agar, were harvested and the cells were suspended in Hank’s balanced salt solution (HBSS). The bacterial suspension (0.05 mL) was incubated with serum (0.05 mL) at 37°C for 180 minutes. Ten microliters of samples were withdrawn and spread on blood agar plates, which were then incubated at 37°C for 18 hours and the viable count was determined. Resistance of bacteria to serum bactericidal activity was expressed as the percentage of bacteria surviving after 180 minutes of incubation with serum, in relation to the original count. Bacteria were termed serum sensitive, if the viable count dropped to 1% of the initial value, and resistant if >90% of the organisms survived after 180 minutes.
Statistical Analysis
The chi square test was used to compare the occurrence of virulence markers in both the cases and controls. P value less than 0.05 was considered significant.
RESULTS
A total of 220 E. coli were isolated from the urine samples, from UTI. Out of these, 191 samples had significant bacteriuria with counts 105 CFU/mL and 29 had probably significant bacteriuria with counts between 104 and 105 CFU/mL.
Phenotypic and biochemical characteristics of E. coli
A total of 18 E. coli isolates were mucoid lactose fermenting colonies. All controls were non-mucoid lactose fermenters. Out of 220 isolates 14 were non-lactose fermenters and anaerogenic and considered as atypical E. coli.
Virulence markers of Uropathogenic E. coli obtained from cases and controls
The performance of the standard uropathogenic E. coli (UPEC) isolates was satisfactory in all the four assays. Occurrence of UPEC with virulence markers in different combinations among cases and controls.
Hemolysin
Among the 220 cases 91 (41.36%) were hemolytic. Among 50 controls, three (6%) were hemolytic. The difference between the cases and controls, for hemolysin production, was highly significant (P<0.001).
Hemagglutination
A total of 68 (30.90%) among 220 cases and six (12%) among 50 controls showed mannose-resistant hemagglutination. There was a significant (P<0.01) difference in MRHA between cases and controls.
Cell surface hydrophobicity
A total of 58 (26.36%) among 220 cases and nine (18%) among 50 controls were cell-surface hydrophobic. There was no significant difference (P>0.05) between the cases and controls, for cell surface hydrophobicity.
Serum resistance
Among 220 cases, 72 (32.72%) E. coli were serum-resistant and among 50 controls, 12 (24%) E. coli were serum-resistant. There was no significant difference (P>0.05) in the cases and controls for serum resistance. The results of virulence markers among E. coli isolates from cases and controls.
Atypical / Mucoid E. coli and virulence
Of the 18 mucoid lactose fermenters, 10 were serum-resistant and three were hemolytic, two were MRHA, and one was MSHA. Among the 14 non-lactose fermenters, all had one or more virulence markers.
DISCUSSION
Considering the high degree of morbidity and mortality of UTIs, the subject of uropathogenic E. coli was receiving increasing attention. Cell morphology and molecular biology studies have revealed that uropathogenic E.coli express several surface structures and secrete protein molecules, some of them cytotoxic and peculiar to the strains of E. coli, causing UTI.[1] Hence, it is important to identify UPEC from non-UPEC isolates in the urinary samples. Results in this study showed that most of the urinary isolates from cases had more than one virulence marker. In this case-control study we conclude that UPEC strains are definitely associated with the aetio-pathogenesis of UTI. The occurrence of multiple virulence factors in the UPEC strains further strengthens the concept of the association of UPEC with urinary pathogenicity. It was interesting to note that UPEC with multiple virulence factors were significantly more in the cases than in the controls.
Phenotypic and biochemical characterization revealed interesting findings. A total of 18 urinary isolates were mucoid and all these were obtained from the cases only. No mucoid strains were isolated from the control group. Furthermore, 10 of these 18 were serum-resistant. The mucoid strains were capsulated[8] and the capsule conferred serum and phagocyte resistance to some E. coli strains.[3] This property was attributed to the content of sialic acid, which reduced the ability of the bacterial surface to activate and complement by an alternative pathway. However, eight mucoid strains were susceptible to the serum indicating that non-capsular factors had a role in serum resistance. Thus, the conventional phenotypic marker, such as the capsule, was also an important virulence factor for the UPEC. In the present study, 14 (6.36%) of the 220 isolates were atypical E. coli. Altered phenotypes could be due to an altered genetic makeup. Bhat et al.[9] studied 210 E. coli strains isolated from the urine and found 26 (12.4%) to be atypical. In their study of the 26 atypical urinary isolates, 12% were hemolytic and three had hydrophobicity (< 0.156) and were MRHA positive and all the atypical E.coli had one or the other virulence marker, indicating that an atypical phenotype probably contributed to their virulence. The cytolytic protein toxin secreted by most hemolytic E.coli strains was α-hemolysin. E.coli also produced cell-associated lysin on blood agar plates and hemolysin caused a clear zone of lysis.[10] In the present study, although the nature of hemolysin was not further characterized it could be considered as a cytotoxic necrotizing factor (hemolysin). The difference between cases and controls for the production of hemolysin was highly significant (P<0.001). This was similar to the Johnson et al.[2] study, where hemolysin was produced by 38% of the urinary isolates and 12% of the fecal isolates. Hemagglutination was mediated by the fimbriae.[11] MRHA could be mediated by P fimbriae. Thus, the MRHA positive strains could be considered as UPEC, most likely having P fimbriae.[2] In the present study there was a significant difference in MRHA between cases and controls (P<0.01). This was similar to a study by Johnson[2] et al, where 58% of the urinary isolates and 19% of the fecal isolates showed MRHA. The expression of type 1 fimbriae was indicated by MSHA. MSHA were more in the fecal strains than in urinary isolates, in our study. More work is required to assess the role of MSHA in pathogenecity. The role of the cell-surface hydrophobicity in mediating bacterial adherence to mammalian cells was conceived by Mudd and Mudd.[12] Crystalline surface layers: the ‘S’ layer present on both gram negative and gram positive organisms play a role in this.[13] Hydrophobicity is a recently described novel virulence mechanism by E. coli. In the present study, although there was no significant difference in cell-surface hydrophobicity between the cases and controls (P>0.05), more isolates from the cases were hydrophobic. Taylor[14] reviewed that bacteria were killed by normal human serum through the lytic activity of an alternative complement system. Bacterial resistance to killing by serum, results from the individual or combined effects of the capsular polysaccharide, O polysaccharide, and surface proteins.[15] Although more E. coli isolates from the cases were serum-resistant compared to the controls, the difference was not statistically significant (P>0.05). The O group designation reveals little about a strain. The apparent virulence associated with certain groups may be mediated through other virulence factors like P fimbriae, hemolysin, and serum resistance, which is commonly associated with UTI-associated strains.[2]
In this study, 151 out of 220 isolates had one or more virulence factors. When a comparison was done between the urinary and fecal E.coli isolates, hemolysin production, presence of capsule, and the capacity to cause MRHA, emerged as important virulence factors. The hemolysin, especially α-hemolysin, also known as cytotoxic necrotizing factor, is strongly proinflammatory leading to secretion of IL-6 and chemotaxins, which sets pace for the pathogenesis of renal disease. The capacity to cause MRHA is due to various adhesions mainly P fimbriae, P-associated fimbriae, and FIC fimbriae, seen in pyelonephritis cases. These adhere to the fibronectin on the uroepithelial cells contributing to the persistence.
We believe that the methods of detection of the above-mentioned virulence markers are reasonably easy and screening them in a clinical microbiology laboratory is a worthwhile exercise.
Source of Support: Nil
Conflict of Interest: None declared.
==== Refs
REFERENCES
1 Steadman R Topley N Brumfitt W Jeremy MT The virulence of Escherichia coli in urinary tract. Urinary tract infections Hamilton Miller. Chapter 3 1998 th ed London Chapman and Hall Publication 37 41
2 Johnson JR Virulence factors in Escherichia Coli urinary tract infection Clin Microbiol Rev 1991 4 81 28
3 Warren JW Schrier RW Gottschalk CW Host parasite interactions and host defence mechanisms Diseases of the kidney 1997 6th ed London Little Brown 873 94
4 Central Laboratory Standards Institute (CLSI) Performance standards for antimicrobial disc susceptibility tests, Approved standards 10th ed CLSI document
5 Forbes BA Sahm DF Alice S Weissfeld Infections of the urinary tract in Bailey and Scott’s. Diagnostic Microbiology 2002 St. Louis USA Missouri Mosby 927 38
6 Siegfried L Kmetova M Puzova H Molokacova M Filka J Virulence associated factors in Escherichia Coli strains isolated from children with urinary tract infections J Med Microbiol 1994 41 127 32 7913974
7 Raksha R Srinivasa H Macaden RS Occurrence and characterization of uropathogenic Escherichia Coli in urinary tract infections Indian J Med Microbiol 2003 21 102 7 17642991
8 Altwegg Bockemuhl J Collier L Balows A Escherichia and Shigella Topley and Wilsons Microbiology and Microbial infections. Systematic bacteriology 1998 9th ed London Edward Arnold 940 3
9 Bhat GK Bhat GM Atypical Escherichia Coli in urinary tract infections Trop Doctor 1995 25 127
10 Smith HW The hemolysins of Escherichia Coli J Pathol Bacteriol 1963 85 197 211 13989441
11 Duguid JP Clegg S Wilson ML The fimbrial and non fimbrial haemagglutinins of Escherichia Coli J Med Microbiol 1979 12 213 27 379341
12 Mudd S Mudd EB The penetration of bacteria through capillary spaces IV. A kinetic mechanism in interfaces J Exp Med 1924 40 633 45 19868947
13 Sleytr B Messner P Crystalline surface layers of bacteria Ann Rev Microbiol 1983 37 311 39 6416145
14 Taylor PW Bactericidal and bacteriolytic activity of serum against gram negative bacteria Microbiol Rev 1983 47 46 83 6343827
15 Montenegro MA Bitter-Suermann D Timmis JK Agüero ME Cabello FC Sanyal SC Serum resistance and pathogenicity related factors in clinical isolates of Escherichia Coli and other gram negative bacteria J Gen Microbiol 1985 131 1511 21 3900279 | 21346899 | PMC3040084 | CC BY | 2021-01-04 19:39:28 | yes | J Lab Physicians. 2010 Jul-Dec; 2(2):70-73 |
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J Lab PhysiciansJLPJournal of Laboratory Physicians0974-27270974-7826Medknow Publications India 21346900JLP-2-7410.4103/0974-2727.72153Original ArticlePrevalence of Pseudomonas aeruginosa in Post-operative Wound Infection in a Referral Hospital in Haryana, India Ranjan K Prabhat Ranjan Neelima Bansal Satish K 1Arora D R Department of Microbiology, Maharaja Agrasen Medical College and Hospital, Agroha (Hisar),Haryana, India1 Department of Surgery, Maharaja Agrasen Medical College and Hospital, Agroha (Hisar),Haryana, IndiaAddress for correspondence: Dr. K Prabhat Ranjan, E-mail: [email protected] 2010 2 2 74 77 © Journal of Laboratory Physicians2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background:
The objective of our study was to determine the prevalence of Pseudomonas aeruginosa in the isolates of postoperative wound and its susceptibility pattern to commonly used antibiotics.
Materials and Methods:
During a 2-year period, specimens were received as postoperative wound swabs in Microbiology Laboratory, Maharaja Agrasen Medical College, Agroha (Hisar), Haryana, India.
Result:
Of the 300 bacterial isolates, 89 (29.6%) were P. aeruginosa, followed by Escherichia coli (61, 20.3%), Klebsiella spp. (50, 16.6%), Staphylococcus aureus (43, 14.3%), Proteus spp. (19, 6.3%), Acinetobacter spp. (9, 3.0%), and Citrobacter freundii (2, 0.6%). There was no growth in 27 (9.0%) specimens.
Conclusion:
P. aeruginosa isolation was higher in male patients and most common in the age group of 21-40 years. The susceptibility pattern showed the organism to be most commonly susceptible to imipenem, followed by meropenem, cefoperazone/sulbactam, ticarcillin/clavulanate, and amikacin.
Pseudomonas aeruginosapostoperative woundprevalencenosocomialantibiotic
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INTRODUCTION
Postoperative wound infection or surgical site infection is an important cause of health care associated infections among surgical patients. Patients who develop wound infections have longer hospital stays, more expensive hospitalizations, and increased mortality.[1] The development of wound infections depends on the integrity and protective functions of the skin.[2]
Pseudomonas aeruginosa is a leading cause of health care associated infections, ranking second among gram-negative pathogens as reported by the United States national nosocomial infection surveillance system. P. aeruginosa contributes substantially to wound-related morbidity and mortality worldwide. The organism enters into the blood, causing sepsis that may spread to the skin and leads to ecthyma gangrenosum, a black necrotic lesion.[3] It produces several substances that are thought to enhance the colonization and infection of host tissue.[4] These substances together with a variety of virulence factors, including lipopolysaccharides (LPSs), exotoxin A, leukocidin, extracellular slime, proteases, phospholipase, and several other enzymes, make P. aeruginosa the most clinically significant pathogen among non-fermenting bacteria. P. aeruginosa has the capacity to carry plasmids containing genes that regulate antimicrobial resistance, and this feature has led to the appearance of some strains that are resistant to normally reliable antibiotics.[5] Out of these, there are multiple reasons for postoperative wound infections, which have been validated and documented as risk factors.
A risk factor is any recognized contribution to an increase in postoperative wound infection.[6] The virulence and invasive capability of the organisms have been reported to influence the risk of infection, but the physiological state of the tissue in the wound and immunological integrity of the host seem to be of equal importance in determining whether infection occurs or not.[7] Primary infections are usually more serious, appearing within 5–7 days of surgery. These infections are mostly related to endogenous flora and some other environmental sources in the operating theater. The deep-seated sepsis developing within 30 days after a surgery and before the wound has been dressed reflect a theater infection.[8] Some of the studies support the concept that a reduction in postoperative wound infection is directly related to increased education and awareness of its causes, and its prevention is greatly aided by critically evaluated infection control practice.[9]
The prevalence of primary wound infection is correlated to the bacteriological cleanliness of the operation. Clean operation (<2%) does not involve opening a viscous or cutting across mucus membranes. In contaminated operations (20%), a viscous normally containing bacteria or a membrane normally colonized with bacteria is incised, while in clean–contaminated operations (<10%), a viscous or membrane which is usually sterile, is incised.[10] Health care associated infections tend to be more superficial and frequently follow the dressing of wounds in the ward. Similarly, skin infections such as boils or abscesses developing at sites other than the operation site indicate that the infection was acquired in the ward.[7] Wound infection after contaminated operations is usually caused by the bacteria normally residing in the opened viscous or on the incised mucus membrane, i.e. the bacteria belong to the patient’s own normal flora, or have gained entry while the patient is in a hospital.[11] These include operations which are carried out through a field already contaminated by bacteria such as abscesses and colon operations.[10] Bacteriological studies have shown that postoperative wound infection is universal and that the bacterial types present vary with geographic location, bacteria residing on the skin, clothing at the site of wound, time between wound and examination.[12] Facultative anaerobic gram-negative bacilli, Streptococci and Staphylococci remain in the colon, regardless of the type of preparation. The bowel and postoperative infection in colon and rectal surgery without systemic intraoperative prophylaxis can be as high as 50%.
In the recent years, the growing incidence of P. aeruginosa has been of particular interest. The incidence of P. aeruginosa in postoperative wound infection is becoming more serious in developing countries because of lack of general hygienic measures, mass production of low quality antiseptic and medicinal solutions for treatment, and difficulties in proper definition of the responsibilities among the hospital staff.[13] The hospital-acquired nature of infections with P. aeruginosa has been noted and while some patients suffer endogenous infections, the vast majority is acquired from exogenous sources. So, the objective of our study was to determine the prevalence of P. aeruginosa in the isolates of postoperative wounds in our hospital and its antimicrobial susceptibility pattern.
MATERIALS AND METHODS
The study was conducted in the bacteriology laboratory, Department of Microbiology, Maharaja Agrasen Medical College and Hospital, Agroha (Hisar), India. All the specimens received from patients hospitalized from April 2007 to March 2009 were processed for isolation and identification of bacterial pathogens, according to the standard microbiological techniques.[14]
Clinical specimens
Postoperative wound swabs were collected aseptically with two sterile cotton wool swabs for each sample from different wards in the hospital. One swab was for Gram stain and the other one was for culture.
Culture media and biochemical tests
The following media were used and tests were conducted in this study: blood agar, MacConkey agar, chocolate agar, nutrient agar, mannitol salt agar, Simmon citrate agar, peptone water, indole production test, motility test, methyl red test, voges proskauer test, catalase, coagulase, urease, and oxidase tests. All the above media and reagents were obtained from HiMedia, Mumbai, India. The media were prepared according to the manufacturers’ instructions.
All wound swabs collected for bacteriology investigations during the study period were treated according to the established methods of treating wound swabs. Gram stain preparations were made from one swab and culture are processed from another swab.
The plates were incubated at 37°C for 18–24 hours in an incubator. The plates were read the following day but extended to 48 hours if there was no bacterial growth within 24 hours. Isolated colonies were subjected to Gram staining and biochemical tests for identification.
Identification was carried out according to the standard biochemical tests.[14]
Antibiotic susceptibility testing
Antimicrobial susceptibility test were carried out on isolated and identified colonies of P. aeruginosa using commercially prepared antibiotic disk (HiMedia) on Mueller Hinton agar plates by the disk diffusion method, according to the Central Laboratory Standards Institute (CLSI) guidelines.[15] Antibiotic testing was not done of other bacterial isolates in this study since our focus was on the prevalence of P. aeruginosa. The standard strain of P. aeruginosa (ATCC 27853) was used as a control. Antibiotics used in our study were piperacillin (100 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), meropenem (10 µg), ampicillin/sulbactam (10/10 µg), piperacillin/tazobactam (100/10 µg), ticarcillin/clavulanate (75/10 µg), cefoperazone/sulbactam (75/10 µg), gentamicin (10µg), tobramicin (10 µg), amikacin (30 µg), and ciprofloxacin (5 µg).
RESULTS
A total of 300 specimens were obtained from postoperative wounds, including superficial and deep-seated infections of all patients hospitalized at surgical, pediatrics, orthopedic, obstetrics, and gynecology wards.
Isolation
The most common isolated organism from postoperative wounds was P. aeruginosa (89 isolates, 29.6%), followed by Escherichia coli (61 isolates, 20.3%), Klebsiella spp. (50 isolates, 16.6%), Staphylococcus aureus (43 isolates, 14.3%), Proteus spp. (19 isolates, 6.3%), Acinetobacter spp. (9 isolates, 3.0%), and Citrobacter freundii (2 isolates, 0.6%). There was no growth in 27 (9.0%) samples. The abscess drainage was the most common type of postoperative wound (17.90%), followed by the surgery of diabetic foot (12.82%), cesarean section (11.72%), and open knee surgical wound (11.35%) [Table 1].
Table 1 The number of wound swabs in relation to the type of surgery
Type of surgery Number of specimens Percentage
Abscess drainage 49 17.90
Diabetic foot 35 12.82
Cesarean section 32 11.72
Open knee wound 31 11.35
Liver abscess 23 8.42
Herniorrhaphy 17 6.22
Abdominal abscess 16 5.80
Nail removal 12 4.39
Perianal fistu 11 4.02
Septoplasty 10 3.66
Mastoidectomy 10 3.66
Neck abscess 7 2.56
Skin grafting 6 2.19
Lipoma excision 5 1.83
Bone excision 5 1.83
Thyroidectomy 4 1.46
The frequency of P. aeruginosa isolation in relation to age is shown in Table 2. The most frequent isolation of the P. aeruginosa was noted in the age group of 21–40 years (48.6%), followed by those in the age group of 41–60 years (40.6%), 0–20 years (5.0%), and >60 years (4.0%). We found the relationship between postoperative wound infections and sex. The prevalence rate was higher in male (58%) patients compared with females (42%).
Table 2 The frequency of P. aeruginosa isolation in relation to the age group
Age group (years) Number of specimens Number of isolation Percentage
0–20 15 8 5.0
21–40 146 53 48.6
41–60 122 44 40.6
>60 17 7 4.0
Susceptibility
P. aeruginosa was most commonly susceptible to imipenem (76.9%), followed by meropenem (70.4%), cefoperazone/sulbactam (62.1%), ticarcillin/clavulanate (60.7%), and amikacin (53%) [Table 3].
Table 3 The susceptibility pattern of P. aeruginosa isolated in postoperative wounds
Antibacterial Percentage of susceptibility
Imipenem 76.9
Meropenem 70.4
Cefoperazone/sullbactam 62.1
Ticarcillin/clavulanate 60.7
Amikacin 53
Piperacillin/tazobactam 45.8
Ciprofloxacin 36
Ceftazidime 35.8
Tobramicin 30.5
Gentamicin 29.1
Cefepime 25.2
Piperacillin 13.6
Ampicillin/sulbactam 12
DISCUSSION
A surgical wound infection is a postoperative complication that brings about embarrassment to the surgeon, considerable financial burden, undue discomfort to the patient, and sometimes death. Our study shows that P. aeruginosa was most prevalent (29.6%) among all the pathogens isolated from the surgical wound. Our results were consistent with similar studies carried out by Anupurba and colleagues which showed P. aeruginosa was isolated in 32% of isolates.[16] Oguntibegri and Nwobu, in their study, concluded it to be 33.3%[17] and Hani and colleagues found a prevalence rate of 27.78%.[18] Stephen and colleagues, in a similar study, reported a frequency of P. aeruginosa isolation rate of 18.8%.[19] We therefore report it as a significant finding which is in agreement with that obtained in other hospitals. The frequency of P. aeruginosa isolation was found to be maximal in patients who underwent cesarean section in the study by Oguntibeju and Nwobu[19] and in those with surgical wound infections and undergoing cesarean section in the study by Hani and colleagues.[20] In our study, it was most commonly isolated in procedures involving drainage of abscesses and diabetic foot operations, followed by cesarean section operations.
When factors such as age and sex of the patient were considered, we found the occurrence of P. aeruginosa to be higher in males and in patients in the age group 21–40 years. Stephen and colleagues found that P. aeruginosa was more commonly isolated from patients in the age group 21–30 years.[21] We found the prevalence rate to be higher in male (58%) patients compared to females (42%). Jamshaid and colleagues also reported that P. aeruginosa infections were more common in males, and Stephen and colleagues also reported in their study that male patients had higher isolation rates.[21]
The maximal susceptibility of P. aeruginosa isolates was against imipenem (76.9%) and meropenem (70.4%). Navaneeth and colleagues, in their study, noted 88% susceptibility against each of imipenem and meropenem, among P. aeruginosa isolates.[22] Bonfiglio and colleagues, in their study, summarized that meropenem was the most active compound against P. aeruginosa isolates, followed by amikacin.[23] Although we found carbapenems to be the most successful drugs in vitro against P. aeruginosa, there is a likelihood of resistance to even these as seen in studies carried out on multidrug-resistant phenotype of P. aeruginosa.[24] Resistance to carbapenems is most likely to occur by the interplay of excess β-lactamase production, impermeability via a loss of porin protein Opr D, together with the up-regulation of multidrug efflux systems, primarily MexA MexB Opr M.[25] Our study shows that there is an increased rate of incidence of P. aeruginosa in postoperative wound infections. The most common causative agent of postoperative infections was P. aeruginosa, followed by E. coli, Klebsiella spp., S. aureus, Proteus spp., and Acinetobacter calcoaceticus. Other less common causes were Streptococcus pyogenes, Enterococcus faecalis and C. freundii26. This is in agreement with survey studies carried out in various hospitals. The infection appears to be common in hospitals with relaxed hygienic measures and is dependent on age, sex and even duration of stay in the hospital. The primary reason for this increase in postoperative infection rate with prolonged preoperative hospitalization may be the colonization of patients with hospital-acquired resistant microorganisms.
Source of Support: Nil
Conflict of Interest: None declared.
==== Refs
REFERENCES
1 Kirkland KB Briggs JP Trivette SL Wilkinson WE Sexton DJ The impact of surgical-site infections in the 1990s: attributable mortality, excess length of hospitalization, and extra costs Infect Control Hosp Epidemiol 1999 20 725 30 10580621
2 Calvin M Cutaneous wound repair Wounds 1998 10 12 32
3 Khan JA Iqbal Z Rahman SU Farzana K Khan A Report: prevalence and resistance pattern of Pseudomonas aeruginosa against various antibiotics Pak J Pharm Sci 2008 21 311 5 18614431
4 Bodey GP Bolivar R Fainstein V Jadeja L Infections caused by Pseudomonas aeruginosa Rev Infect Dis 1983 5 279 313 6405475
5 Livermore DM Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa : our worst nightmare? Clin Infect Dis 2002 34 634 40 11823954
6 Mousa H Aerobic, anaerobic and fungal burn wound infections J Hosp Infect 1997 37 317 23 9457609
7 National Nosocomial Infections Surveillance (NNIS) System. NNIS report, data summary from January 1992 to June 2002, issued August 2002 Am J Infect Control 2002 30 458 75 12461510
8 Leigh DA Emmanuel FX Sedgwick J Dean R Post-operative urinary tract infection and wound infection in women undergoing caesarean section: A comparison of two study periods in 1985 and 1987 J Hosp Infect 1990 15 107 16 1969432
9 Leigh DA Emmanuel FX Sedgwick J Dean R Post-operative urinary tract infection and wound infection in women undergoing caesarean section: A comparison of two study periods in 1985 and 1987 J Hosp Infect 1990 15 107 16 1969432
10 Russell RC Williams NS Bulstrode CJ Bailey and Love’s Short Practice of Surgery 2000 23rd ed USA Oxford Press 87 98
11 Andenaes K Lingaas E Amland PF Giercksky KE Abyholm F Preoperative bacterial colonization and its influence on post operative wound infection in plastic surgery J Hosp Infect 1996 34 291 9 8971618
12 Trilla A Epidemiology of nosocomial infections in adult intensive care units Intensive Care Med 1994 20 1 4 8163751
13 Bertrand XM Thouverez C Patry P Balvay Talon D Pseudomonas aeruginosa: antibiotic susceptibility and genotypic characterization of strains isolated in the intensive care unit Clin Microbiol Infect 2002 7 706 8 11843917
14 Forbes BA Sahm DF Weissfeld AS Forbes BA Sahm DF Weissfeld AS Pseudomonas, Burkholderia, and similar organisms Bailey and Scott’s Diagnostic Microbiology 2002 11th ed Louis Mosby Inc 448 61
15 Central Laboratory Standards Institute (CLSI) Central Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disc susceptibility tests, Approved standards Vol 29 CLSI document M02-A10, No1
16 Anupurba S Bhattacharjee A Garg A Sen MR Antimicrobial susceptibility of Pseudomonas aeruginosa from wound infections Indian J Dermatol 2006 51 286 8
17 Oguntibeju OO Nwobu RAU Occurrence of Pseudomonas aeruginosa in post-operative wound infection Pak J Med Sci 2004 20 187 92
18 Masaadeh HA Jaran AS Incident of Pseudomonas aeruginosa in post-operative wound infection Am J Infect Dis 2009 5 1 6
19 Oguntibeju OO Nwobu RAU Occurrence of Pseudomonas aeruginosa in post-operative wound infection Pak J Med Sci 2004 20 187 92
20 Masaadeh HA Jaran AS Incident of Pseudomonas aeruginosa in post-operative wound infection Am J Infect Dis 2009 5 1 6
21 Siguan SS Ang BS Pala IM Baclig RM Aerobic Surgical Infection: surveillance on microbiological etiology and antimicrobial sensitivity pattern of commonly used antibiotics Phil J Microbiol Infect Dis 1990 19 27 33
22 Navaneeth BV Sridaran D Sahay D Belwadi MR A preliminary study on metallo-beta-lactamase producing Pseudomonas aeruginosa in hospitalized patients Indian J Med Res 2002 116 264 7 12807154
23 Bonfiglio G Carciotto V Russo G Stefani S Schito GC Debbia E Antibiotic resistance in Pseudomonas aeruginosa: an Italian survey J Antimicrob Chemother 1998 41 307 10 9533479
24 Goossens H Susceptibility of multi-drug-resistant Pseudomonas aeruginosa in intensive care units: results from the European MYSTIC study group Clin Microbiol Infect 2003 9 980 3 14616692
25 Kohler T Michea-Hamzehpour M Epp SF Pechere JC Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems Antimicrob Agents Chemother 1999 43 424 7 9925552
26 Lewis CM Zervos MJ Clinical manifestations of Enterococcal infection Eur Clin Microbial Infec Dis 1990 9 111 7 | 21346900 | PMC3040092 | CC BY | 2021-01-04 19:39:29 | yes | J Lab Physicians. 2010 Jul-Dec; 2(2):74-77 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21408027PONE-D-10-0653510.1371/journal.pone.0017850Research ArticleBiologyMolecular Cell BiologyMedicineDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryOncologyBasic Cancer ResearchCancer PreventionCancer TreatmentAnti-Tumor Activity of a Novel Compound-CDF Is Mediated by Regulating miR-21, miR-200, and PTEN in Pancreatic Cancer Targeted Killing of CSCs by CDFBao Bin
1
Ali Shadan
2
Kong Dejuan
1
Sarkar Sanila H.
1
Wang Zhiwei
1
Banerjee Sanjeev
1
Aboukameel Amro
2
Padhye Subhash
3
Philip Philip A.
2
Sarkar Fazlul H.
1
*
1
Department of Pathology, Wayne State University, Detroit, Michigan, United States of America
2
Division of Hematology/Oncology Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America
3
Dr. D. Y. Patil University, Pimpri, Pune, India
Zhang Lin EditorUniversity of Pennsylvania, United States of America* E-mail: [email protected] and designed the experiments: BB SA DK SHS ZW SB AA SP PAP FHS. Performed the experiments: BB SA DK SHS ZW AA. Analyzed the data: BB SA ZW AA FHS. Contributed reagents/materials/analysis tools: PAP SP FHS. Wrote the paper: BB SA FHS.
2011 9 3 2011 6 3 e1785013 12 2010 10 2 2011 Bao et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The existence of cancer stem cells (CSCs) or cancer stem-like cells in a tumor mass is believed to be responsible for tumor recurrence because of their intrinsic and extrinsic drug-resistance characteristics. Therefore, targeted killing of CSCs would be a newer strategy for the prevention of tumor recurrence and/or treatment by overcoming drug-resistance. We have developed a novel synthetic compound-CDF, which showed greater bioavailability in animal tissues such as pancreas, and also induced cell growth inhibition and apoptosis, which was mediated by inactivation of NF-κB, COX-2, and VEGF in pancreatic cancer (PC) cells.
Methodology/Principal Findings
In the current study we showed, for the first time, that CDF could significantly inhibit the sphere-forming ability (pancreatospheres) of PC cells consistent with increased disintegration of pancreatospheres, which was associated with attenuation of CSC markers (CD44 and EpCAM), especially in gemcitabine-resistant (MIAPaCa-2) PC cells containing high proportion of CSCs consistent with increased miR-21 and decreased miR-200. In a xenograft mouse model of human PC, CDF treatment significantly inhibited tumor growth, which was associated with decreased NF-κB DNA binding activity, COX-2, and miR-21 expression, and increased PTEN and miR-200 expression in tumor remnants.
Conclusions/Significance
These results strongly suggest that the anti-tumor activity of CDF is associated with inhibition of CSC function via down-regulation of CSC-associated signaling pathways. Therefore, CDF could be useful for the prevention of tumor recurrence and/or treatment of PC with better treatment outcome in the future.
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Introduction
Pancreatic cancer (PC) is one of the most lethal malignant diseases with worst prognosis, which is ranked as the fourth leading cause of cancer-related deaths in the United States [1]. Over the past two decades, numerous efforts have been made in improving treatment and survival PC patients but the outcome has been disappointing. This disappointing outcome is due to many factors among which de novo resistance (intrinsic) and acquired (extrinsic) resistance to conventional therapeutics (chemotherapy and radiation therapy) including gemcitabine alone or in-combination with other cytotoxic or targeted agents. Emerging evidence suggest that the resistance could in fact be due to the enriched existence of tumor initiating cells, also classified as cancer stem-like cells (CSC) in a tumor mass [2]–[6]. The CSCs have the capacity of self-renewal and the potential to regenerate into all types of differentiated cells giving rise to heterogeneous tumor cell populations in a tumor mass, which contributes to tumor aggressiveness [2]–[6]. Thus, the failure to eliminate these special cells is considered to be one of the underlying causes of poor treatment outcome with conventional therapeutics, suggesting that newer and novel therapeutic strategies must be developed for the targeted killing of drug resistant CSCs in order to eradicate the risk of tumor recurrence for improving the survival of patients diagnosed with PC.
In search of novel yet non-toxic agents, attention has been focused on natural agents for several years. One such agent is curcumin (diferuloylmethane), which is derived from the plant Curcuma longa (Linn) grown in tropical Southeast Asia [7]–[9]. Curcumin has been shown to inhibit the growth of a variety of tumor cells; however, the poor bioavailability of curcumin limits its application in the clinic. Recently, we have developed a novel synthetic analogue of curcumin, 3,4-difluoro-benzo-curcumin [we named it as Difluorinated-Curcumin or in short CDF [10], [11]], which showed greater bioavailability in pancreatic tissues, and also inhibited cell growth, DNA-binding activity of NF-κB, Akt, COX-2, and the production of PGE2 and VEGF, and caused induction of miR-200 and inactivation of miR-21 in PC cells [12]. Since miR-200 is associated with the acquisition of epithelial to mesenchymal transition (EMT), which is also believed to be associated with CSCs or cancer stem-like cells, here we investigated the effects of CDF on CSC function.
Here we report, for the first time, that CDF could inactivate many functions of CSCs including self-renewal capacity as demonstrated by the inhibition of sphere-forming (pancreatospheres) ability of drug-resistant PC cells, which was consistent with inactivation of CSC biomarkers such as CD44 and EpCAM. We also showed anti-tumor activity of CDF alone and in-combination with gemcitabine, which was consistent with inactivation of miR-21, and consequently increased expression of PTEN, attenuation of the DNA binding activity of NF-κB inhibition in the expression of COX-2, and activation in the expression of miR-200 in tumor remnants of a xenograft mouse model of human PC, all of which provide convincing in vivo activity of CDF which is consistent with in vitro findings.
Results
AsPc-1 and MIAPaCa-2 cell lines and their clones were chosen for this study because of their relatively resistant nature. The CSC characteristics of these cell lines using stem cell markers' Lin28B and Nanog by RT-PCR, and EpCAM and CD44 by western blot showed an increase in expression level in the PC-GTR cell lines compared to their parental cell lines (Figure 1). Hence we chose these to test our hypothesis that CDF is more effective than curcumin even in resistant cell lines and also their resistant clones–GTR.
10.1371/journal.pone.0017850.g001Figure 1 Comparative expression of Lin28B (A) and Nanog (B) mRNA by qRT-PCR showed increased expression in resistant cell lines compared to parental cell lines, supporting the CSC characteristics of these cell lines.
The characteristics of CSCs were further confirmed by the protein expression of EpCAM and CD44 (C).
CDF strongly prevents clonogenicity and invasion of PC cells compared to gemcitabine and curcumin
We selected the concentration of 20 nmol/L of gemcitabine and 4 µmol/L of curcumin or CDF to conduct clonogenic assay following our previous publication [12]. The results demonstrated that there was a significant reduction in clonogenicity of AsPC-1 and MIAPaCa-2 cells treated with curcumin and CDF, but not with gemcitabine (Figure 2A). However, CDF treatment had a much greater and significant reduction in colony formation compared to curcumin. AsPC-1-GTR and MIAPaCa-2-GTR cells had an 80% reduction of clonogenicity with CDF treatment, whereas, only 20–30% reduction of clonogenicity was observed with gemcitabine or curcumin treatment (Figure 2A). Overall, CDF treatment showed a significant reduction in clonogenicity of human PC cells, suggesting the superiority of CDF.
10.1371/journal.pone.0017850.g002Figure 2 CDF and Curcumin decreased clonogenicity and invasion in AsPC-1, AsPC1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR cells. Clonogenic assay (A) Invasion assay (B).
Fluorescence of the invaded cells was read using ULTRA Multifunctional microplate reader (TECAN) at excitation/emission wavelengths of 530/590 nm. Basal level of ABCG2 expression showing relatively higher expression in drug resistant cell lines (C).
CDF or curcumin treatment decreased PC cell migration and invasion. The results showed that 4 µmol/L of curcumin had minimal inhibition of invasion whereas similar concentration of CDF showed significant inhibition of invasion (Figure 2B). The basal level of ABCG2 expression was found in parental cell lines (de novo drug resistant cells); however, the level of expression of ABCG2 was further increased in drug resistant (acquired drug resistant) cell lines (Figure 2C).
CDF inhibited viability of human PC cells more than curcumin and gemcitabine as evaluated by MTT assay
Initially MTT assay was conducted to examine the effect of different concentrations of gemcitabine (1 to 50 nmol/L), and curcumin or CDF (2–6 µmol/L) on cell survival after 72h of treatment (data not shown). Subsequently, 4 µmol/L of CDF or curcumin, and 20 nmol/L of gemcitabine were used to treat individually as well as in combination with gemcitabine for 72h. The results showed that CDF treatment in combination with gemcitabine caused a remarkable reduction of cell survival in all four cell lines compared to curcumin and gemcitabine combination treatment (Figure 3). Furthermore, analysis of drug combination treatment showed that the combination index after treatment with CDF in combination with gemcitabine was less than 1.00 (Figure 3), suggesting the synergistic effect of CDF combination. In contrast, the combination index with curcumin and gemcitabine was more than 1.00 (Figure 3), showing non-synergistic effect. Overall, these results suggest that CDF caused a much more significant reduction of cell survival in PC cells, compared to gemcitabine/curcumin alone or their combinations compared to CDF and gemcitabine combination.
10.1371/journal.pone.0017850.g003Figure 3 CDF and its combination with gemcitabine inhibited cell viability.
MTT assay was conducted in all four cell lines after 72h of treatment with CDF, curcumin, or its combination with gemcitabine. Untreated control has been assigned a value of 100%. The p value shown represents comparisons between single agent and their combinations by using paired t-test. The combination Index (CI) <1 for CDF and Gemcitabine combination indicates synergism.
CDF remarkably increased pancreatosphere disintegration of PC cells
To examine the effect of treatments on the sphere forming ability of PC cells (pancreatosphere) and disintegration of pancreatospheres, we conducted sphere disintegration assay for 10 days to generate the formation of pancreatospheres, followed by 5 days of drug treatment. The results show that there was a remarkable increase of sphere disintegration by curcumin and CDF treatment, not by gemcitabine treatment (Figure 4). However, the greatest effect on disintegration was observed in response to the CDF treatment (Figure 4), once again suggesting that CDF is much more superior in inhibiting the functions of cancer stem-like cells.
10.1371/journal.pone.0017850.g004Figure 4 CDF remarkably increased disintegration of pancreatospheres in AsPC-1, AsPC1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR cells.
P values were calculated by the paired t test.
CDF inhibited pancreatospheres formation in PC cells
To examine the effect of drugs on CSC self-renewal capacity in PC cells, we conducted sphere formation assay for 1 week and four weeks (Figure 5A and B). The results indicated that CDF in combination with gemcitabine completely eliminated pancreatospheres formation after four weeks of treatment compared to gemcitabine and curcumin combination in PC cells even in gemcitabine-resistant PC cells, suggesting that CDF may cause the pancreatospheres more sensitive to gemcitabine than that of curcumin treatment, and could be useful for targeted killing of CSCs. Figure 5C shows the effect of different concentration of gemcitabine and CDF on 2nd passage of pancreatospheres in pre-treated primary pancreatospheres of AsPC-1 cells. CDF treatment remarkably inhibited 2nd passage of pancreatospheres in a dose-dependent manner. Furthermore, CDF-pre-treated cells exhibited a greater effect than non-CDF-pre-treated cells.
10.1371/journal.pone.0017850.g005Figure 5 CDF decreased the formation of pancreatospheres in AsPC-1 and AsPC-1-GTR cells 1 week treatment (A); 4 weeks treatment (B); AsPC-1 and CDF-pre-treated AsPC-1 cells treated with gemcitabine and CDF (C).
A significant reduction in pancreatospheres was observed in cells treated with CDF shown by asterisk
CDF decreased CD44 and EpCAM expression in pancreatospheres of PC cells
We examined the effect of drugs on CSC biomarkers, CD44 and EpCAM in pancreatospheres of AsPC-1 and AsPC-1-GTR cells by confocal microscopy (Figure 6). The results indicate that CDF decreased CD44 and EpCAM expression in pancreatospheres, suggesting the inhibitory effect of CDF on pancreatosphere formation may be associated with the inhibition of CD44 and EpCAM expression.
10.1371/journal.pone.0017850.g006Figure 6 CDF treatment decreased the expression of CD44 and EpCAM, the known markers of CSCs.
Expression in pancreatospheres of AsPC-1 and AsPC-1-GTR cells was assessed by confocal microscopy (Magnification X250).
CDF in combination with Gemcitabine inhibited Pancreatic Tumor Growth in vivo much more than curcumin combination
We have used a subcutaneous xenograft tumor model where the tumor was induced by MIAPaCa-2 cells in CB17-SCID mice. CDF treatment in combination with gemcitabine significantly inhibited tumor growth in MIAPaCa-2 tumors much more than curcumin and gemcitabine combination (Figure 7A) as well as compared to either untreated controls or those treated with a single drug. The mice did not show any weight loss during the treatment period (30 days), suggesting that these treatment had no major adverse effects on animals.
10.1371/journal.pone.0017850.g007Figure 7 CDF exhibited anti-tumor activity in MIAPaCa-2 cells induced tumors in a xenograft mouse model, which was consistent with inhibition of NF-κB DNA binding, COX-2, miR-21, and caused re-expression of miR200 in tumor remnants.
Anti-tumor activity and changes in tumor weight from each group of animals (A). The arrow indicates starting day of the treatment. NF-κB DNA binding activity of tumor tissues; and NF-κB competition control study with unlabeled NF-κB oligonucleotide (B). Western blots analysis of COX-2, PTEN and β-actin expression in tumor remnants (C); miR-21, miR-200b and miR-200c expression in tumor remnants as measured by real-time RT-PCR (D). P values were calculated by the paired t test.
CDF with Gemcitabine significantly decreased NF-κB Activation in vivo
NF-κB activation was determined in the CDF or curcumin, and/or gemcitabine treated tumor remnants derived from MIAPaCa-2 cells induced tumors as shown above. CDF and curcumin as single agent down-regulated NF-κB activation whereas gemcitabine activated NF-κB level, which was abrogated in combination treatment with CDF. The combination treatment of CDF with gemcitabine showed a significant decrease in NF-κB level compared to curcumin and gemcitabine treatment (Figure 7B), suggesting that the inactivation of NF-κB could be one of the molecular mechanisms by which CDF elicits its anti-tumor activity against PC tumors.
CDF effects on protein expression in vivo
The COX-2, PTEN, and β-actin expression was determined by Western blot. A significant down-regulation in the expression of COX-2 was observed in both the combination, but the effect was more pronounced in CDF combination group. The expression of phosphatase and tension homolog (PTEN), a tumor suppressor gene was found to be decreased in MIAPaCa-2 cells; however, the expression of PTEN was up-regulated when treated with CDF (Figure 7C). These results suggest that CDF is much more effective than curcumin. Since PTEN is a known target of miR-21, which has been reported to be up-regulated in PC [13]–[15], we assessed the expression levels of miR-21 in tumor remnants as shown below.
Modulation in the expression of miR-21 and miR-200 family in vivo
We determined the expression levels of miR-21, miR-200b and miR-200c in MIAPaCa-2 tumors by real time RT-PCR. Over-expression of miR-21 was observed in MIAPaCa-2 tumors whereas we found a significant reduction in the expression of miR-21 in tumors treated with either CDF alone or in combination with CDF and gemcitabine (Figure 7D). We further determined the expression levels of miRNA-200b and miR-200c in tumor tissues which are known regulators of EMT and found to be significantly low in MIAPaCa-2 cells (Figure 7D). In contrast, we found that the CDF treatment with or without gemcitabine combination showed increased expression of both miR-200b, and miR-200c, but the effect with curcumin or its combination was minimal, suggesting the superiority of CDF in suppressing the expression of miR-21, resulting in the re-expression of PTEN, and re-expression of miR-200 which could be responsible for the reversal of EMT phenotype in cells treated with CDF. Overall, these results suggest that the phenotypic characteristics of MIAPaCa-2 tumors are consistent with enriched population of CSCs and EMT characteristics, and these drug resistant cells could be killed either by CDF alone or in combination with gemcitabine.
Pancreatospheres enhanced tumor growth in vivo
Under traditional experimental conditions, we normally inject one million cells for assessing tumor growth; however, for investigating the greater potential of tumor growth by pancreatospheres, we injected only 5,000 cells in mice as a proof-of-concept study. The tumor weight was remarkably increased as the days progressed (Figure 8A). The level of miR-21 was increased between tumors implanted with one million of parental cells compared to pancreatospheres (Figure 8B). The animal was euthanized after 30 days because of tumor burden, and gross tumors are shown in Figure 8C indicating larger tumors as well as loco-regional lymph node metastasis whereas tumors derived from parental cells did not show any metastasis over a period of 30 days. The tumor-derived cells showed significant inhibition of pancreatospheres when treated with CDF (Figure 8D). Overall, these results suggest that CSCs (pancreatospheres) can be grown in mice and CDF could be useful for the killing of these drug resistant cells (Figure. 8).
10.1371/journal.pone.0017850.g008Figure 8 Tumor growth pattern of pancreatospheres derived from MIAPaCa-2 cells.
(A). 5,000 pancreatospheres were inoculated in mice using 1∶1 matrigel, progressive tumor growth over a period of 30 days. Moderate increase in the expression of miR-21 as measured by real-time RT-PCR was observed in tumors derived from pancreatospheres compared to tumors derived from parental cells by injecting one million cells and tumor was assessed over the same period of time (B). Photographs showing tumor growth, arrow points to tumor and asterisk (*) refers to loco-regional lymph node metastasis whereas we did not find any metastasis when one million parental cells were injected (C). Tumor cells harvested from the tumors derived from pancreatospheres were treated with CDF showed significant inhibition in the formation of pancreatospheres (D).
Discussion
In this study, we have demonstrated that a synthetic analogue of curcumin, CDF, is significantly more effective compared to curcumin in the killing of gemcitabine-resistant pancreatic cancer (PC) cells that consists high proportion of cells with cancer stem cells (CSCs) or cancer stem-like cells characteristics. The inhibition of cell growth could in part be due to better cellular uptake, retention and reduced metabolic inactivation of CDF by PC cells, which is consistent with our published findings on cellular and animal pharmacokinetics data [10], [11]. Our previous reports indicate that CDF inhibits NF-κB and COX-2 activity in PC cells in vitro
[12]. Here we confirm these observations in vivo using a mouse xenograft model. Thus, the killing of gemcitabine-resistant PC cells by CDF is associated with inactivation of NF-κB and COX-2 signaling pathway which is very important because these pathways are known to contributes to drug-resistance of PC cells to chemotherapeutic agents [16]–[18].
CSCs comprises only a very small proportion of cells in a tumor mass and posses the ability to self-renew and give rise to differentiated tumor cells [3]–[5], [19]. The CSC theory has fundamental clinical implications especially because CSC has been identified in many malignant tumor tissues including pancreatic cancers and considered to be highly resistant to chemo-radiation therapy than differentiated daughter cells [3]–[5], [20]; however, CSCs isolated from human tumors are usually insufficient for further mechanistic studies. The existence of CSCs provides an explanation for the clinical observation that tumor regression alone may not correlate with patient survival [21] because of tumor recurrence, which is in part due to the presence of CSCs. Therefore, targeting self-renewal pathways and the killing of CSCs might provide more specific approach for eliminating cells that are the root cause of tumor recurrence. A potential challenge in this regard is the development of therapies that selectively affect CSCs while sparing normal stem cells that may rely on similar mechanisms for self-renewal. In this study, we have demonstrated that CDF not only inhibit cell growth of PC cells, but also inhibit CSC self-renewal capacity as assessed by sphere formation (pancreatospheres) assays. Therefore, CDF could have a greater potential to inhibit cancer growth as documented by our xenograft mouse model of gemcitabine resistant PC cells, which appears to be mediated via inhibition of CSC self-renewal capacity.
Emerging evidence suggests the role of microRNA (miRNA) in many biological processes [22]–[25]. Among many miRNAs, miR-21, commonly considered as an oncogene, is over-expressed in many solid tumors including PC and has been reported to be associated with tumor progression, poor survival and drug resistance [13], [14], [26], [27]. In our previous report, we have demonstrated that the expression of miR-21 is up-regulated in gemcitabine-resistant PC cells and that its expression can be significantly down-regulated by CDF treatment in vitro
[12]. The increased expression of miR-21 is known to be associated with inactivation of PTEN, a know tumor suppressor gene, resulting in activation of PI3K/Akt/mTOR signaling pathway, leading to aggressive tumor growth [15], [28]. In this study, we confirmed that CDF treatment could results in the down-regulation of miR-21, resulting in the up-regulation of PTEN in vivo, suggesting that the anti-tumor activity of CDF is associated with up-regulation of PTEN resulting from the inactivation of miR-21 expression.
In contrast to miR-21, miR-200 family is known as tumor suppressor and they are usually down-regulated in some tumors including PC and the loss of expression of miR-200 family contribute to the acquisition of EMT phenotype and drug resistance. Down-regulation of miR-200 by siRNA technique has been shown to be associated with EMT phenotype while re-expression of miR-200 can result in the reversal of EMT phenotype [29], [30]. In our previous publication [12], we demonstrated that CDF treatment could re-express miR-200 in PC cells. Here we showed, for the first time, that CDF can up-regulate miR-200b and miR-200c in tumor remnants in vivo, consistent with significantly greater inhibition of tumor growth in the xenograft mouse model when CDF was used in combination with gemcitabine. These results suggest that the anti-tumor activity of CDF is mediated via re-expression of miR-200 which may potentially results in the reversal of EMT phenotype and could also lead to overcome drug resistance in PC.
In conclusion, CDF showed much more pronounced growth inhibitory effect, inhibited CSC self-renewal consistent with inactivation of CSC biomarkers (CD44 and EpCAM) in PC cells especially in gemcitabine-resistant PC cells compared to curcumin. In xenograft mouse model of human PC tumors induced by MIAPaCa-2 cells, CDF exhibits anti-tumor activity by regulating COX-2, PTEN, miR-21, miR-200, and NF-κB in vivo. These results strongly suggest that CDF could be a novel agent for the treatment of PC in general but gemcitabine-resistant PC in particular by attenuating the behavior of CSCs.
Materials and Methods
Ethics Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Any animal found unhealthy or sick were promptly euthanized. The protocol was approved by the Committee on the Ethics of Animal Experiments of Wayne State University institutional Users Animal Care Committee (Permit Number: A-10-03-08).
Cell Culture, Drugs and Reagents
Human pancreatic cancer cell lines AsPC-1, and MIAPaCa-2 were purchased from ATCC (Manassas, VA). These two cell lines AsPC-1 and MIAPaCa-2 were exposed to 200 nmol/L of gemcitabine and 5 µmol/L of tarceva (erlotinib) every other week for about 6 months to create gemcitabine and tarceva resistant (GTR) cell lines, named as AsPC-1-GTR and MIAPaCa-2-GTR, respectively. As a result, AsPC-1, AsPC-1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR were chosen for this study based on their differential sensitivities to gemcitabine. All the cell lines have been authenticated (Applied Genomics Technology Center at Wayne State University) on March 13, 2009 and these authenticated cells were frozen for subsequent use. The method used for testing was short tandem repeat profiling using the PowerPlex 16 System from Promega. Gemcitabine and curcumin were purchased from Eli Lilly (Indianapolis, IN) and Sigma-Aldrich (St. Louis, MO), respectively. CDF was synthesized as described in our earlier publication [10], [11]. Gemcitabine was dissolved in water, whereas CDF and curcumin were dissolved in DMSO with a final concentration of 0.1% DMSO in medium.
Antibodies
Antibodies against ABCG2, and PTEN were purchased from Santa Cruz (Santa Cruz, CA). Antibody to COX-2 and β-actin was acquired from Cayman Chemicals (Ann Arbor, MI), and Sigma Chemicals (St. Louis, MO).
Clonogenic assay
Clonogenic assay was conducted to examine the effect of drugs on cell growth in PC cells, as described previously [12]. 5×104 cells were plated in a six-well plate. After 72h of exposure to 20 nmol/L of gemcitabine, 4 µmol/L of CDF or curcumin, the cells were trypsinized, and 1,000 single viable cells were plated in 100-mm Petri dishes. The cells were then incubated for 10 to 12 days at 37°C in a 5% CO2/5% O2/90% N2 incubator. Colonies were stained with 2% crystal violet and counted.
Invasion assay
The invasive activity of cells was tested by using BD BioCoat Tumor Invasion Assay System (BD Biosciences, Bedford, MA) according to the manufacturer's protocol as described previously [31]. Briefly 5×104 cells were seeded with serum free medium supplemented with curcumin or CDF into the upper chamber and bottom wells were filled with complete medium in the system. The, fluorescence was read using Microplate Reader (TECAN) at 530/590 nm and were photographed.
Cell survival assay
MTT assay was conducted using AsPC-1, AsPC-1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR as described previously [12] after 72 h of treatment. Combination index and Isobologram for combination treatment were also calculated and plotted using CalcuSyn software (Biosoft, Cambridge, United Kingdom) to determine synergy based on the method of Chou and Talalay [32].
Sphere formation/disintegration assay
Single cell suspensions of cells were plated on ultra low adherent wells of 6-well plate at 1,000 cells/well in sphere formation medium [33]. After 7 days, the spheres termed as “pancreatospheres” were collected by centrifugation and counted [33]. For sphere disintegration assay, 1,000 cells/well were incubated for 10 days, following 5 days of drug treatment, which examined the effect of drug treatment on disintegration of pancreatospheres as described previously [33]. The pancreatospheres were collected by centrifugation and counted under a microscope.
Confocal microscopy
Single cell suspensions of AsPC-1 and AsPC-1-GTR cells were plated using ultra low adherent wells of 6-well plate at 3,000 cells/well in sphere formation medium. After 7 days of treatment, the pancreatospheres were collected by centrifugation, washed with 1xPBS, and fixed with 3.7% parformaldehyde. CD44 and EpCAM antibodies were used for immunostaining assay, as described previously [29]. The CD44 or EpCAM-labeled pancreatospheres were photographed by confocal microscopy (Leica TCS SP5) using software LAS AF 1.2.0 Build 4316.
Protein extraction and Western blot analysis
Proteins were extracted from all four cell lines and also from animal tumor tissues as described previously [12]. Relative level of ABCG2 was evaluated for all four cell lines. The effects on COX-2, PTEN and β-actin expression were evaluated on tumor tissues by Western blot analysis. as described previously [12].
Animal Experiments
The animal protocol was approved by the Animal Investigation Committee, Wayne State University, Detroit, MI. Female CB17 SCID mice 4 wks old were purchased from Taconic Farms (Germantown, NY) and fed Lab Diet 5021 (Purina Mills, Inc., Richmond, IN). Small fragments of the MIAPaCa-2 xenograft were implanted subcutaneously and bilaterally into mice for the drug-efficacy trials. Once the mice developed palpable tumors, they were randomly selected into the following treatment groups (n = 5/group): (1) untreated control; (2) CDF (5 mg/mouse/day), intragastric once daily for 12 days; (3) curcumin (5 mg/mouse/day), intragastric once daily for 12 days; (4) gemcitabine (1 mg/mouse/day), intravenous every third day for a total of three doses; (5) CDF and gemcitabine using the doses indicated above; (6) curcumin and gemcitabine using the doses as indicated above. Tumor measurements and changes in weight were performed and tissue was stored at −70°C for RNA and protein extraction.
Electrophoretic Mobility Shift Assay (EMSA) assay for assessing the DNA binding activity of NF- κB
Nuclear proteins were prepared from tumors tissue using a Dounce homogenizer with 400 µl of ice cold lysis buffer extracted as described earlier [34]. EMSA was performed using the Odyssey Infrared Imaging System with NF-κB IRDye labeled oligonucleotide from LI-COR, Inc. (Lincoln, NE). Ten µg of the nuclear protein extract was used as described earlier [34]. The NF-κB competition control study was conducted using unlabeled NF-κB consensus oligonucleotide. The samples were loaded and run at 30 mA for 1 hour. The gel was scanned using Odyssey Infrared Imaging System (LI-COR, Inc.).
TaqMan miRNA Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
To determine the expression of miRNAs (miRNA-200b, miR-200c, and miR-21) in MIAPaCa-2 tumors, we used TaqMan MicroRNA Assay kit (Applied Biosystems) following manufacturer's protocol. 5 ng of total RNA was reverse transcribed and real-time PCR reactions were carried as described earlier [30], using Smart Cycler II (Cepheid). Data were analyzed using Ct method and were normalized by RNU6B expression.
Growth of CSC in xenograft model
5,000 pancreatospheres were isolated and implanted in mice with 1:1 matrigel. The growth rate was observed for a period of 30 days. RNA was extracted from the tumor tissue for subsequent molecular assays as presented under figure legend.
Statistical Analysis
Comparisons of treatment outcome were tested for statistical difference by the paired t tests. Statistical significance was assumed at a P value of <0.05.
Competing Interests: The authors have declared that no competing interests exist.
Funding: National Cancer Institute, NIH grants 5R01CA131151, 3R01CA131151-02S1, and 5R01CA132794 (F.H. Sarkar). We thank Puschelberg and Guido foundations for their generous financial contribution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21464949PONE-D-10-0279910.1371/journal.pone.0018076Research ArticleBiologyAnatomy and PhysiologyCardiovascular SystemCardiovascular AnatomyCell PhysiologyBiochemistryCytochemistryImmunocytochemistryProteinsGrowth FactorsMolecular Cell BiologyCell AdhesionCadherinsCellular TypesEndothelial CellsMedicineCardiovascularPeripheral Vascular DiseasesVascular BiologyPlacenta Growth Factor-1 Exerts Time-Dependent Stabilization of Adherens Junctions Following VEGF-Induced Vascular Permeability PlGF-1 Antagonizes VEGF-Induced PermeabilityCai Jun
1
Wu Lin
1
Qi Xiaoping
1
Shaw Lynn
2
Li Calzi Sergio
2
Caballero Sergio
2
Jiang Wen G.
3
Vinores Stanley A.
4
Antonetti David
5
Ahmed Asif
6
Grant Maria B.
2
Boulton Michael E.
1
*
1
Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States of America
2
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, United States of America
3
Department of Surgery, School of Medicine, Cardiff University, Cardiff, United Kingdom
4
Ophthalmology, Johns Hopkins University, Wilmer Eye Institute, Baltimore, Maryland, United States of America
5
Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania, United States of America
6
BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom
Fadini Gian Paolo EditorUniversity of Padova Medical School, Italy* E-mail: [email protected] and designed the experiments: JC MEB MBG AA. Performed the experiments: JC LW LS SL SC XQ. Analyzed the data: SAV AA WGJ DA MBG MEB. Contributed reagents/materials/analysis tools: DA AA. Wrote the paper: JC MEB MBG DA.
2011 25 3 2011 6 3 e180764 10 2010 24 2 2011 Cai et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.
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Introduction
Increased vascular permeability is an inciting event in numerous human vascular pathologies such as ischemic stroke, diabetic complications, tumorogenesis and rheumatoid arthritis [1], [2], [3]. Intercellular junctions between endothelial cells control vascular permeability and integrity. This barrier function requires the expression and organization of VE-cadherin and claudin-5, which are essential components of adherens junctions (AJs) and tight junctions (TJs) respectively [1], [4] in the blood-brain and blood-retinal barriers. TJs and AJs may act as two resistors that act in series with the TJs more restrictive to ions and small molecules than the AJs [3], [5], [6]. As opposed to epithelial cells where the AJs and TJs can clearly be distinguished by ultrastructural analysis, in endothelial cells of the blood-brain and blood retinal barrier these junctional complexes are intermingled [1], [7]. Cells require AJ formation to build TJs [6], [8] and recent reports indicate that co-ordinated disruption of VE-cadherin intracellular interactions culminates in the restructuring of both AJs and TJs and the subsequent opening of endothelial cell-cell junctions [1], [4], [7]. Furthermore, VE-cadherin is involved in the formation of TJs, regulates claudin-5 expression and is required for VEGF-induced endothelial cell survival [1], [7], [9].
VEGF increases vascular permeability by inducing VE cadherin destabilization [7] and inducing the endocytosis of both the AJ protein VE-cadherin [10] and the TJ protein occluding [3], [11] through a phosphorylation-dependent signalling pathway. The VEGF family includes VEGF-A and placenta growth factor (PlGF) which can exist as homo- or heterodimers [12], [13]. PlGF, which has a 42% amino acid sequence identity with VEGFA [14], occurs in at least four isoforms, PlGF-1, PlGF-2, PlGF-3, PlGF-4 as a result of alternative splicing [14], [15]. PlGF-2 has high heparin binding affinity whereas neither PlGF-1, PlGF-3 nor PlGF-4 bind heparin. VEGF is considered to increase vascular permeability through VEGFR-2, however, the role of PlGF, which specifically binds VEGFR-1, has been more controversialwith both pro- and anti-angiogenic effects proposed [15]. This is likely to in part be due to the isoform of PlGF examined and the models used. Most studies have focussed on PlGF-2 since this is the only isoform present in the mouse [16], [17]. PlGF-2 deficiency protects mice against vascular permeability [18] while mice overexpressing PlGF-2 have enhanced VEGF-induced permeability [19], [20]. The latter is perhaps counterintuitive since VEGFR-1 is considered to be a potent negative regulator of VEGFR-2 activity [21], [22], [23]. Exogenous PlGF-2 has been reported to have either no direct or a direct effect on in vitro or in vivo permeability but, in general, most studies agree that PlGF-2 enhances VEGF-induced permeability [15], [24], [25], [26], [27], [28], [29]. The role of PlGF in angiogenesis is unclear given a) the variance in outcomes of PlGF-2 blockade on tumor angiogenesis [30], [31] and b) PlGF-1 antagonizes VEGF-induced angiogenesis in some models [27], [32], [33] but promotes angiogenesis in others [34].
Here, we present evidence demonstrating that PlGF-1, but not PlGF-2, can inhibit VEGF-induced endothelial cell permeability in the normal vasculature but only during a critical window approximately 6 hours after VEGF induction of permeability. We show PlGF-1 stabilizes both AJs and TJs in vitro using endothelial monolayers and in vivo using the retinal vasculature of mice. PlGF-1 stabilization of junctions occurs by a carefully orchestrated series of events including dephosphorylation of VE-cadherin, reduced cleavage of VE-cadherin, and increased VE-cadherin expression through transactivation of Sp1 and Sp3 within the VE-cadherin promoter. These studies strongly suggest that early detection is paramount to therapeutic success and that if therapeutic agents are to be administered in the identical manner as nature has carefully orchestrated then as much attention must be given to when a therapy is initiated and its dosing regimen as is typically given to identifying the actual therapeutic agent. Our results also provide validity to the intermittent intravitreal administration of anti-VEGF agents as an optimal therapeutic strategies rather than sustained release.
Results
PlGF-1, but not PlGF-2, exerts a temporal regulation of VEGF-induced permeability
Given the controversy regarding the effect of PlGF on vascular permeability we first asked whether there was a temporal dependence of the effect of PlGF on VEGF-induced vascular permeability and if this was isoform dependent. We have identified a critical window during which hPlGF-1 can inhibit VEGF-induced permeability (Fig. 1a). The addition of VEGF to cultured retinal microvascular endothelial cells caused a significant decrease in transendothelial resistance and an increase in the transendothelial flux of flourescent dextran, which was sustained over a 24 hour period (Fig. 1a, b). Furthermore this was dose-dependent with 200 ng/ml hPlGF-1 exerting the maximum inhibition of VEGF-induced permeability while 10 ng/ml hPlGF-1 only had a weak effect. Neither the simultaneous treatment of cultured retinal endothelial cells with hPlGF-1 and VEGF (Fig. 1a,b) nor 3 or 24 hour pre-treatment with hPlGF-1 followed by VEGF (Fig. 2) had any significant effect on VEGF-induced permeability. However, addition of hPlGF-1 6 hr post-treatment with VEGF resulted in a complete inhibition of VEGF-induced permeability (P<0.05) (Fig. 1a, b). By contrast, 24 hr post-treatment with hPlGF-1 had no significant effect on VEGF-induced permeability (Fig. 2). Neutralizing antibody to remove secreted VEGF caused a modest decrease in permeability and abolished any hPlGF-1-induced effect suggesting that even the constitutive secretion of endogenous VEGF is sufficient to affect barrier function (Fig. 2). By contrast, hPlGF-2 had no effect on in vitro barrier function either when applied alone or in combination with VEGF (Fig. 1c,d).
10.1371/journal.pone.0018076.g001Figure 1 PlGF-1, but not PlGF-2, exerts a temporal-dependent regulation of VEGFA-induced permeability.
(a) & (c) Temporal changes in transendothelial resistance across a microvascular endothelial monolayer grown on a Transwell insert with the five conditions including the unstimulated group, vehicle (saline), VEGFA alone, PlGF alone, simultaneous VEGFA + PlGF and PlGF 6 hours post VEGFA (n = 5 independent experiments). VEGFA was used at 100 ng/ml. Data for hPlGF-1 and hPlGF-2 is at 100 ng/ml with the exception of PlGF 6 hours post VEGFA in which results are shown for PlGF at 10 ng/ml (α), 100 ng/ml (β) and 200 nglml (γ). hPlGF-1 was used in (a) and hPlGF-2 in (c). (b) & (d) Show paracellular macromolecular permeability to 40 kDa Dextran-FITC using the conditions described above for (a) and (c) (n = 5 independent experiments). In the case of PlGF 6 hours or 24 hours post VEGF, Transwell inserts were transferred to new wells containing basal medium without fluorescent dextran. (e) Leakage of systemic FITC-labeled albumin into the retina of C57BL/6 mice receiving one of the following intravitreal injections: VEGF; hPlGF-1 or mPlGF-2; VEGF plus hPlGF-1 or mPlGF-2; 0.9% saline vehicle; VEGF followed by hPlGF-1 or mPlGF-2 6 or 24 hours later; VEGF followed by 0.9% saline 6 or 24 hours later. VEGF was given at a concentration of 60 ng/µl while hPlGF-1 and mPlGF-2 were injected at 10, 60 or 120 ng/µl. 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin and retinas were taken for analysis 2 hours later (n = 10-20 per group). (f) Representative confocal microscopy showing dilated vessels and leakage of FITC-labeled albumin in the retinas of mice receiving vehicle only (i), VEGF A (ii), simultaneous VEGF + hPlGF-1 (iii), VEGFA followed by hPlGF-1 6 hours later (iv), simultaneous VEGF + mPlGF-2 (v), or VEGFA followed by mPlGF-2 6 hours later (vi). As shown in (g), PlGF-1 reduced Leakage of systemic FITC-labeled albumin into the retina of C57BL/6 mice receiving intravitreal injection of VEGF A in a dose-dependent manner. Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Student's t test and ANOVA for multiple comparisons). Scale bar = 50 µM.
10.1371/journal.pone.0018076.g002Figure 2 The effect of PlGF-1 on VEGFA-induced permeability is highly dependent on the timing and order of exposure.
(a) Effect of VEGFA alone, hPlGF-1 pretreatment for 3 hr or 24 hr exposure to VEGF and hPlGF-1 24 hours post VEGF on temporal changes in transendothelial resistance across a microvascular endothelial monolayer grown on a transwell insert effected by (n = 4 independent experiments). VEGFA and hPlGF-1 were used at 100 ng/ml. (b) paracellular macromolecular permeability to 40 kDa Dextran-FITC using the conditions described in (a) (n = 4 independent experiments). (c) The effect of neutralization of endogenous VEGF on PlGF-induced transendothelial resistance (n = 4 independent experiments). VEGFA neutralizing antibody was used at 10 µg/ml. (d) Paracellular macromolecular permeability to 40 kDa Dextran-FITC using the conditions described in (c) (n = 4 independent experiments). Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Student's t test and ANOVA for multiple comparisons).
To confirm that the temporal effect of hPlGF-1 we repeated our studies in mice. Intravitreal injection of VEGF in C57Bl6 mice resulted in significant intraretinal leakage of systemically introduced fluorescent albumin. Similar to the culture data, neither pretreatment, simultaneous treatment, 24 hour post-treatment with hPlGF-1 nor treatment with mPlGF-2 resulted in any significant change in VEGF-induced permeability (Fig. 1e). However, intravitreal injection of hPlGF-1 6 hr post-treatment with VEGF resulted in a complete inhibition of VEGF-induced fluorescent albumin leakage into the retina. Confocal microscopy of flat mount retinal preparations showed intraretinal fluorescent albumin in greater than 90% of the retina in VEGF treated animals, confirming increased vascular leakage compared to vehicle only controls (Fig. 1f). By marked contrast, minimal vascular leakage of fluorescent albumin was observed in animals receiving intravitreal injection of hPlGF-1 6 hr post-treatment with VEGF while animals receiving pretreatment or simultaneous treatment with hPlGF-1 showed considerable vascular leakage (Fig. 1f). The ability of hPlGF-1 to block VEGF-induced permeability when injected 6 hr following VEGF was dose-dependent with 60 and 120 ng per eye almost completely blocking fluorescence leakage while 10 ng per eye had no significant effect.
VEGFR-1 is a negative regulator of VEGFR-2 but only after both receptors are activated
To determine whether endothelial permeability is regulated by specific VEGF receptors subtypes, VEGF-E, selective for VEGFR-2, was tested. VEGF-E did not induce detectable changes in endothelial permeability indicating that VEGFR2 is not the dominant receptor in regulating VEGF-induced permeability (Fig. 3a,b). This was supported by neutralization of VEGFR-2 which only reduced VEGF-A induced permeability by less than 30% at 1 and 12 hours post VEGF injection, suggesting the requirement for a second receptor. Blocking VEGFR-1 with a neutralizing antibody abolished the effects of VEGF on the changes in TER and permeability of the endothelial cell monolayer, confirming that VEGF must bind to both VEGFR-1 and R-2 to elicit a maximal increase in permeability in cultured cells (Fig 3a, b). Neutralizing antibodies both to VEGFR-1 and VEGFR-2 were able to block VEGF-induced vascular permeability in mice (Fig. 3c).
10.1371/journal.pone.0018076.g003Figure 3 VEGFR-1 is a critical regulator of VEGF-induced permeability.
(a) Temporal changes in transendothelial resistance across a microvascular endothelial monolayer grown on a transwell insert treated with VEGFA alone (100 ng/ml), VEGFE alone (100 ng/ml), and VEGFA (100 ng/ml) + a neutralizing antibody (2 µg/ml) to VEGFR-1 or VEGFR-2 (n = 4 independent experiments). (b) Paracellular macromolecular permeability to 40 kDa Dextran-FITC using the conditions described in (a) (n = 4 independent experiments). (c) The effect of neutralizing antibodies to VEGFR-1 (4 or 12 ng/eye) and VEGFR-2 (0.5 or 1.0 ng/eye) on VEGF-induced permeability in C57BL/6 mice. Neutralizing antibodies were given by intravitreal injection and VEGF (60 ng/eye) was injected 6 hours later. 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin and retinas were taken for analysis 2 hours later (n = 6 per group). Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Student's t test and ANOVA for multiple comparisons).
PlGF-1 stabilizes both AJs and TJs
Because the barrier function of the endothelial monolayer is closely associated with both TJs and AJs [1], [35], [36] we performed immunohistochemistry to determine the temporal and spatial changes in junctional complexes following VEGF and hPlGF-1 treatment both in vitro and in vivo. Endothelial monolayers demonstrated strong staining of the lateral membranes for both VE-cadherin, claudin-5 (Fig. 4) and ZO-1 (data not shown). When exposed to hPlGF-1 alone cells retained intact interendothelial cell-to-cell contact (Fig. 5) with an intense increase in interendothelial junctional VE-cadherin staining at 12 and 24 hours post treatment with no change in the TJ proteins (Fig. 4). In contrast, VEGF-treated cells showed a rapid loss of both tight and adherens junctional complex integrity over a 24 hour period. For VE-cadherin, a reduction of staining at the intercellular junctions with a more particulate staining profile was observed by 5-minute post VEGF treatment and the formation of numerous intercellular gaps was observed by 12 hours (Fig. 4). Changes in claudin-5 and ZO-1 staining were not observed until 1 hour post VEGF treatment consistent with reports that changes in TJs occur later than alterations of AJs [7], [36]. Addition of hPlGF-1 at 6 hours post VEGF treatment significantly restored VE-cadherin, claudin 5 and ZO-1 staining within 1 hour. Staining intensity for VE-cadherin increased further over 24 hours and loss of cell-cell contacts was prevented (Fig. 4).
10.1371/journal.pone.0018076.g004Figure 4 PlGF-1 stabilizes both AJs and TJs in vitro following VEGF-induced permeability.
Representative pictures of confluent cultures of microvascular endothelial cells treated with VEGFA alone or VEGFA followed by hPlGF-1 6 hours later triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI) and assessed at different times over 24 hours using confocal microscopy. VEGFA and hPlGF-1 were used at 100 ng/ml. Scale bar = 100 µm.
10.1371/journal.pone.0018076.g005Figure 5 PlGF-1 alone stabilizes AJs in microvascular endothelial cells.
Representative pictures of confluent cultures of microvascular endothelial cells treated with hPlGF-1 alone (100 ng/ml) stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI) and assessed at different times over 24 hours using confocal microscopy. Scale bar = 100 µm.
We next correlated the spatial relationship between AJ and TJ proteins expression and changes in paracellular vascular permeability of the mouse retinal vasculature (Fig. 6). A typical staining pattern of the vascular network that demarcates the lateral membranes of microvascular endothelial cells was observed for both VE-cadherin and claudin-5 in mouse eyes without injection, with intravitreal injection of the PBS vehicle or exposure to hPlGF-1 alone over a 48 hour period. Intravitreal injection of VEGF resulted in an almost complete loss of staining of the junctional network for greater than 90% of the retinal vessels indicative of loss of junctional complexes and this was confirmed by excessive leakage of fluorescent albumin into the retina (Fig. 6). By contrast, eyes which had received hPlGF-1 6 hrs after VEGF exhibited a pattern of VE-cadherin and claudin 5 staining similar to that seen for controls in over 75% of the retina although a few areas remained in which junctional complexes seemed to be less well formed. However, if hPlGF-1 was given at the same time as VEGF there was significant destabilization of the junctional proteins although the effect was not as great as when VEGF was given alone.
10.1371/journal.pone.0018076.g006Figure 6 PlGF-1 stabilizes both AJs and TJs in retinal vessels of mice following VEGF-induced vascular permeability.
The upper panel (a) shows representative confocal images of retinal vessels in flat mount preparations from control (no injection) C57BL/6 mice and animals receive a 1 µl intravitreal injection of: vehicle (PBS); VEGFA; hPlGF-1; simultaneous VEGFA + hPlGF-1; VEGFA followed by hPlGF-1 6 hours later. VEGF was given at a concentration of 60 ng/µl and hPlGF-1 at 60 ng/µl. 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin. Two hours later, animals were perfusion fixed with paraformaldehyde. Retinas were prepared as flat mounts and immunostained with VE-cadherin or claudin-5 (red) and FITC-conjugated agglutinin to visualize retinal vessels. (n = 10-20 per group). Scale bar = 50 µM. The lower panel (b)shows representative merged higher power images of retinal vessels stained for VE-cadherin or claudin-5 (red) and FITC-conjugated agglutinin (green). Scale bar = 10 µM.
PlGF-1 reverses VEGF-induced phosphorylation of VE-cadherin followed by claudin-5
VE-cadherin phosphorylation is believed to play a critical role in vascular permeability as VEGF induces phosphorylation of VE-cadherin in AJs and this parallels increases in cell permeability [1]. Putative phosphorylation sites on VE-cadherin include Y658, Y685, Y731 and S665. Exogenous VEGF results in tyrosine phosphorylation of VE-cadherin within 5 minutes, significantly before phosphorylation of claudin-5 occurs. Similarly, dephosphorylation of VE-cadherin was evident within 5-10 minutes in cells treated with hPlGF-1 at 6 hours post VEGF and this occurred significantly earlier than dephosphorylation of claudin-5 (Fig. 7a–d). VE-Cadherin phosphorylation appeared to be predominantly regulated at Y658 and Y731 (Fig. 7e). No change in VE-cadherin phosphorylation was observed when hPlGF-1 was given prior to, or in combination with, VEGF or when hPlGF-1 was administered alone. These observations show that VE-cadherin phosphorylation occurs before claudin 5 phosphorylation and is consistent with alterations in AJs preceding TJs. It has been proposed that endothelial AJs can regulate TJs by VE-cadherin-regulation of claudin-5 [7], [36]. Consistent with previous reports [37], [38], phosphorylation of claudin-5 at T207 is associated with increased permeability. Tyrosine phosphorylation of claudin-5 occurred within 15 minutes following VEGF treatment (Fig. 7). Dephosphorylation of claudin-5 was evident only in cells treated with hPlGF-1 at 6 hours post VEGF-induced permeability and not when hPlGF-1 was given prior to, or in combination with, VEGF. hPlGF-1 alone had no effect on claudin-5 phosphorylation status. Neutralization of VEGFR-2 significantly decreases VEGF-induced phosphorylation of both VE-cadherin and claudin-5 and hPlGF-1 has no effect. By contrast, neutralization of VEGFR-1 significantly increased VEGF-induced phosphorylation of VE-cadherin and claudin-5 and this was not influenced by hPlGF-1 Figure.
10.1371/journal.pone.0018076.g007Figure 7 PlGF-1 reverses VEGF-induced phosphorylation of VE-cadherin followed by claudin-5.
Representative immunoblots showing time-dependent phosphorylation of VE-cadherin (a) and claudin-5 (b) following treatment of endothelial cells with VEGFA alone, hPlGF-1 alone, simultaneous VEGFA + hPlGF-1 and hPlGF-1 6 hours post VEGF for periods up to 12 hours (n = 4 independent experiments). VEGFA and hPlGF-1 were used at 100 ng/ml. Membrane fractions were immunoprecipitated with PY 20 and Western blot undertaken for VE-cadherin and Claudin-5. Laser densitometry quantification of immunoblots showing the relative ratio of VE-cadherin (c) and claudin-5 (d) phosphorylation to the heavy chain of PY20 (n = 4 independent experiments). (e) Representative immunoblots and laser densitometry showing the effect of the treatments in (a) on the phosphorylation status of VE-cadherin Y658 and Y731 (n = 4 independent experiments). (1) = VEGFA; (2) = PlGF-1; (3) = PlGF-1 6 hr after VEGFA; (4) = simultaneous VEGFA + PlGF-1 and (5) = VEGF A 24 hr after PlGF-1. (f) & (g) The effect of neutralizing antibodies to VEGFR1 or VEGFR2 (2 µg/ml) on the phosphorylation of VE-cadherin and Claudin-5 treated as described in (a). Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Mann-Whitney U test).
PlGF-1 promotes expression of VE-cadherin but not TJ proteins
An increase in the levels of interendothelial junctional VE-cadherin can result from recruitment of either pre-existing VE-cadherin or newly synthesized molecules of VE-cadherin. To distinquish between these two possibilities, the VE-cadherin protein levels were quantified by Western blotting. Western blot analysis showed that VEGF only marginally affected VE-cadherin expression (Fig. 8a). In contrast, after 1 hour of hPlGF-1 treatment, the amount of VE-cadherin protein increased to approximately 50% compared to untreated cells. Extension of hPlGF-1 treatment time up to 24 hr sustained significant VE-cadherin protein levels in endothelial cells. Additionally, hPlGF-1 induced a marked time-dependent increase in VE-cadherin protein expression in VEGF-stimulated cells (Fig. 8a). To further explore the changes in VE-cadherin protein levels in the context of membrane-association, a cell-based ELISA assay was used to measure cell surface VE-cadherin. hPlGF-1 -treated monolayers showed a strong increase in the level of cell surface VE-cadherin by 1 hour (Fig. 8b). In contrast, VEGF induced a significant reduction of the level of cell surface VE-cadherin by 1 hour, while hPlGF-1 mediated a slight submaximal increase by 12 hours in VEGF-stimulated cells (Fig. 8b). RT-PCR revealed markedly increased expression of VE-cadherin after hPlGF-1 treatment with the levels of VE-cadherin mRNA increased 0.5 to 1 times and the most pronounced increase was observed at 1 hr (Fig. 8c). VEGF failed to induce any significant changes in VE-cadherin mRNA and as expected, hPlGF-1 significantly elevated (∼100%) the amount of VE-cadherin mRNA in the VEGF-stimulated cells. VEGF caused a reduction in occludin but this was not reversed by exogenous hPlGF-1 even when applied 6 hours after VEGF (Fig. 9). Neither VEGF nor hPlGF-1 caused detectable changes in protein or mRNA expression of claudin 5.
10.1371/journal.pone.0018076.g008Figure 8 PlGF-1 promotes expression of VE-cadherin and reduces cleavage of cell surface VE-cadherin and regulates VE-cadherin expression.
Confluent microvascular endothelial cultures were exposed to VEGFA; hPlGF-1; simultaneous VEGFA + hPlGF-1; VEGFA followed by hPlGF-1 6 hours later and assessed for total VE-cadherin expression by Western blot. VEGFA and hPlGF-1 were used at 100 ng/ml. (a) Top: representative immunoblots for VE-cadherin and, upon reblot, α-tubulin. Bottom: laser densitometry analysis demonstrating the relative ratio of VE-cadherin to the house keeping protein α-tubulin (n = 3 independent experiments). (b) The level of cell surface VE-cadherin on microvascular endothelial cells determined using a cell-based ELISA. Values were calculated as the percent relative to the unstimulated group (n = 4 independent experiments). (c) VE-cadherin mRNA levels quantified using QRT-PCR. Values are displayed as mean transcript copies normalized against GAPDH as the housekeeping gene (n = 3 independent experiments). Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Mann-Whitney U test).
10.1371/journal.pone.0018076.g009Figure 9 PlGF-1 does not regulate the expression of the TJ proteins claudin-5 or occludin.
(a) Confluent microvascular endothelial cultures were exposed to VEGFA followed by hPlGF-1 6 hours later for varying times and VE-cadherin and claudin-5 mRNA levels quantified using QRT-PCR. VEGFA and hPlGF-1 were used at 100 ng/ml. Values are displayed as mean transcript copies normalized against GAPDH as the housekeeping gene (n = 3). (b) Using the same experimental conditions, claudin-5 and occludin expression were assessed by Western blot. Left, representative immunoblots for claudin-5 and occludin and, upon reblot, α-tubulin. Bottom, laser densitometry analysis demonstrating the relative ratio of claudin-5 and occludin to the house keeping protein α-tubulin (n = 3). Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Mann-Whitney U test).
PlGF-1 regulates VE-cadherin expression through the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter
To identify whether VEGF and hPlGF-1 could directly regulate VE-cadherin gene expression through the trans-activating Sp1 and Sp3 interaction with the VE-cadherin gene promoter [39] we analyzed by electrophoretic mobility shift assay (EMSA) the interaction of a VE-cadherin promoter oligonucleotide probe (-70/-39), containing the identified GT box (-50/-44) as shown in Fig. 10a, with the nuclear proteins extracted from endothelial cells. In unstimulated cells, a single DNA-protein complex was observed (Fig. 10b). After VEGF, hPlGF-1 or combination treatment, an additional three DNA-protein binding bands appeared on the gel. The upper two bands (Sp1 and Sp3, respectively) were much closer to each other than the remaining slower migrating bands (Fig. 10b). PlGF induced considerable weak intensities of the second and third bands compared with the treatments of VEGF (Fig. 10b,c). In a supershift assay, anti-Sp3 antibody was able to almost completely shift the second, third and fourth bands, suggesting that the last two bands also represented Sp3, whereas anti-Sp1 antibody only partially shifted the first protein-DNA band (Fig. 10b). Interestingly, in the cells pre-exposed to VEGF, PlGF reduced significantly the intensity of the second band (Sp3) without detectable change in the intensities of the first, third and fourth bands (Fig. 10c).
10.1371/journal.pone.0018076.g010Figure 10 PlGF-1 regulates VE-cadherin expression through the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter.
(a) Nucleotide sequence of the -169/+20 region of the VE-cadherin gene. All numberings are related to the transcriptional start site (+1). The sequence belonging to the first exon is boldface. The sequence of oligonucleotide probe is underlined, and putative Sp1 (GT box) is boxed. (b) Representative EMSA analysis of microvascular endothelial nuclear proteins binding to Sp1 recognition sequences with the promoter of the VE-cadherin gene in response to vehicle (unstimulated), VEGFA alone, hPlGF-1 alone and hPlGF-1 6 hours following VEGF. VEGFA and hPlGF-1 were used at 100 ng/ml. VEGFA and PlGF alone or in combination. Supershift complexes were observed with anti-Sp1 and anti-Sp3 antibodies, respectively, indicating Sp1 and Sp3 binding to the GT box (n = 3). From the top to bottom (1) = first band; (2) = second band; (3) = third band and (4) = fourth band. (c) Quantitative analysis of the trans-activating Sp1 and Sp2 interaction with the VE-cadherin promoter. The fluorescent density of the bands were normalized to the fluorescent density of VE-cadherin promoter oligonucleotide probe. Data are represented as means ± s.e.m. *p<0.05, ** by p<0.01 (Mann-Whitney U test).
Discussion
The significance of our observations is that we show hPlGF-1 represents a potent endogenous antagonist of VEGFA-induced vascular permeability and that this is highly dependent on elevated VEGF and the timing of the subsequent hPlGF-1 exposure. This work highlights the potential importance of the precise timing of the initial administration of anti-VEGF therapies and equal attention to the time intervals of subsequent dosing. Our results suggest that repeated treatments with inhibitors of the VEGF signalling pathway may offer greater success than sustained inhibition in keeping with nature's strategy to maintain vascular health. The mechanisms for blood-retinal barrier breakdown are complex and the results of the present study provide evidence that multiple mechanisms are involved. It is noteworthy that the window of effectiveness for hPlGF-1 is 6 hours after VEGF treatment, which coincides with the peak of VEGF-induced vasopermeability [40], [41].
The surprising finding that hPlGF-1 can reverse VEGF-induced pathological vascular permeability, but only during a critical window of time helps explain some of the controversy surrounding the reported pro- and anti-angiogenic effects of PlGF [15], [30], [31]. Here we demonstrate that hPlGF-1 but not hPlGF-2, is a potent antipermeability factor but only for a few hours after VEGF-A exposure. Although pre-exposure to hPlGF-1 alone or simultaneous hPlGF-1/VEGFA treatment led to increased expression of VE-cadherin, this was not sufficient to prevent VEGF-induced permeability. Our data show that the cells need to be primed with VEGF before they can respond to hPlGF-1 inhibition and utilize VE-cadherin to stabilize the endothelial junctions. In pathological neovascularisation, PlGF expression occurs following elevated VEGF levels [42] leading to the notion that hPlGF-1 stabilizes fragile and dysfunctional new vessels [43]. Furthermore, previous in vitro and in vivo studies have relied on knockout or transgenic mice in which the changes in PlGF expression were sustained throughout life rather than, as we recreated, pathophysiological conditions which involve significant oscillations in growth factor levels.
Our second key finding is that vascular permeability is driven by VEGFR-1. This has significant implications for therapeutic intervention in vascular diseases. Given that VEGF-E did not stimulate vascular permeability and neutralization of VEGFR-1 abolished VEGF-A-induced permeability in cultured cells, we convincingly demonstrated that VEGF-induced permeability is directed primarily through VEGFR-1 with VEGFR-2 playing a supportive role. Interestingly, neutralization of either VEGFR-1 and VEGF-2 in the mouse retina blocked VEGF-induced retinal vessel leakage further supporting the interdependence of these two receptors in the regulation of vascular permeability. Given that VEGFA and hPlGF-1 are clearly both regulating vascular permeability via VEGFR-1, it would suggest that hPlGF-1 could inhibit vascular permeability by competing with VEGF-A for binding to VEGFR-1. However, on endothelial cells the Kd values of VEGF-A to VEGFR-1 and VEGFR-2 range from 9-26 and 100-770 pM, respectively [29], [44], [45], [46] while the binding affinity of PlGF for VEGFR-1 was shown to be 230 pM [29], [46]. An alternative explanation is that although both VEGF and PlGF bind to VEGFR-1, albeit at distinct sites, they may induce distinct biological responses through the phosphorylation of different tyrosine residues within the intracellular domain of VEGFR-1 [23].
It is unclear why PlGF-1 and PlGF-2 have opposing effects on VEGF-induced permeability since they both signal through VEGFR-1 (although PlGF-2 additionally binds to neuropilin-1 and -2) [15], [47]. Since most cells in the retina express VEGFR-1 and VEGFR-2 it is possible that PlGF-2 acts indirectly via non-vascular cells, explaining its lack of effect in vitro on pure endothelial cell cultures, while PlGF-1 acts directly on the vascular endothelium. Cao and colleagues have proposed that hPlGF-1 acts by heterodimerization with VEGFA, thus limiting its angiogenic potential while PlGF-2 acts as a homodimer and proangiogenic regulator [27], [32].
Our data show that hPlGF-1 regulates vascular permeability at the level of AJs and that changes in TJs are dependent on, and subsequent to, dephosphorylation of VE-cadherin. The importance of AJs in maintaining barrier function is derived from data that shows that genetic deletion of VE-cadherin, or inhibition of its adhesive function, results in increased permeability and disruption of endothelium integrity, whereas enhancing VE-cadherin-dependent adhesion can protect the integrity of endothelium [7], [9], [48]. The temporal changes in staining patterns for junctional proteins together with VE-cadherin phosphorylation always preceding phosphorylation of claudin 5 endorses the emerging view that endothelial AJs control TJ integrity [7], [36]. The observation that neutralization of VEGFR-2 blocked VEGF-induced phosphorylation of both VE-cadherin and claudin-5 while neutralization of VEGFR-1 significantly increased phosphorylation emphasises the negative regulatory role of VEGFR-1 in vascular permeability and that the ratio of VEGF receptors at the junctional complexes may determine the integrity of the junctional complexes.
Our data strongly predicts that targeting AJs rather than TJs will likely offer an alternative therapeutic option for reducing vascular permeability. Furthermore, our study demonstrates that VEGF has the capacity to disassemble endothelial junctions via reduced availability of VE-cadherin at the cell surface together with redistribution from cell-cell contacts, rather than by alteration of VE-cadherin expression. In contrast, hPlGF-1 directly increases VE-cadherin expression and enhances the density of VE-cadherin along the interendothelial junctions supporting our conclusion that hPlGF-1 plays a critical role in the maintenance and stabilization of vascular barrier function. However, the time-dependent effect of hPlGF-1 on VEGF-induced permeability is in agreement with the notion that interendothelial cellular junctions have the capacity to dissemble and assemble upon various stimulations and that the restoration of endothelial cell-to-cell contacts requires the synthesis of VE-cadherin [49]. hPlGF-1 may directly increase the expression of AJ proteins via the Sp1 family of transcription factors. The Sp1 family induces conformational change in the DNA structure to facilitate the recruitment of distal DNA-bound transcription factors and the assembly of the transcription initiation complex via protein-protein interaction [50], [51]. Recent studies have revealed that two members of the Sp1 family, Sp1 and Sp3, are known to be co-expressed in several tissue/cell types including endothelial cells [39], [52] and to interact with the identical consensus such as the GT box[50]. In addition, the GT box occupies a key position within the VE-cadherin promoter [51]. hPlGF-1 dramatically reduces the Sp3 binding level from Sp1/Sp3 complexes. In this sense, the relative levels of Sp1 and Sp3 in the Sp1/Sp3 complexes may be more crucial than the absolute amount of Sp1 and Sp3 in term of initiation of VE-cadherin gene transcription in endothelial cells. Based on the cis-activating functions of Sp1 and Sp3, we speculate that activation of VEGF receptors can unconditionally cause Sp1 nuclear translocation and binding to the VE-cadherin promoter and can induce Sp3 nuclear translocation which competes or blocks Sp1 binding to the promoter of VE-cadherin gene.
In conclusion, this work highlights the need for a more complete understanding of how temporal expression of pro- and anti-angiogenic agents function in vivo to regulate vascular permeability which will be essential in order to maximise the therapeutic potential of anti-angiogenic therapies and therapies that directly treat increased vascular permeability.
Materials and Methods
Materials
For in vitro studies, we used recombinant human hPlGF-1(R&D Systems, Minneapolis, MN, USA) and recombinant human hPlGF-2 (Cell Sciences, Canton, MA, USA). For in vivo studies, we used recombinant hPlGF-1 and mouse mPlGF-2 (R&D Systems, Minneapolis, MN, USA). Recombinant VEGF165 was purchased from R&D systems (R&D Systems, Minneapolis, MN, USA) and recombinant Orf Virus-HB-VEGF-E was obtained from (Cell Sciences, Canton, MA, USA). VEGFA neutralizing antibody was obtained from (R&D Systems, Minneapolis, MN, USA) and VEGFR-1 and VEGFR-2 neutralizing antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Microvascular endothelial cell culture
Retinal microvascular endothelial cells were isolated as previously described [22]. In brief, isolated bovine retinas in ice cold Eagle's minimal essential medium (MEM) with HEPES were homogenized by a Teflon-glass homogeniser and microvessels trapped on an 83 µm nylon mesh. Vessels were transferred into 2×MEM containing 500 µg/ml collagenase, 200 µg/ml pronase (BDH, UK) and 200 µg/ml DNase at 37°C for 20 min. The resultant vessel fragments were trapped on 53 µm mesh, washed with cold MEM and pelleted at 225 g for 10 min. The pellet was resuspended in microvascular endothelial cell basal medium (MCDB131) with growth supplement (Invitrogen, CA) at 37°C, 5% CO2 for 3 days. Purity was confirmed by Factor VIII and VE-cadherin staining. Cells were used between passage 1 and 3.
Growth factor treatment
Confluent endothelial cultures were rendered quiescent for 45 min in serum-free medium. Growth factors, including VEGF-A, VEGF-E, hPlGF-1 or hPlGF-2 (alone or in combination) were added at 100 ng/ml, unless stated otherwise, based on our previous studies [43], [53] and in the sequences indicated in the text for different time periods.
Neutralization of VEGF and VEGFRs in vitro
In some experiments the effect of endogenous VEGF on hPlGF-1 induced permeability was blocked by co-administration of a neutralizing antibody against VEGFA (10 µg/ml). To confirm the relative role of VEGFRs in VEGF and/or hPlGF-1 regulation on permeability a neutralizing antibody to either VEGFR-1 or VEGFR-2 (2 µg/ml) was added in combination with the growth factors as described previously [43].
TER Measurement
Endothelial cells were grown to confluence on porous polyester membrane inserts (6.5 mm diameter, 0.4 µm pore size; Transwell, Corning, Cambridge, MA). The upper and lower compartments contained 100 µl and 0.5 ml of media, respectively. For experimental treatments, various growth factors were added to the upper compartment. TER measurements were performed using an EVOM volt-ohmmeter connected to a 6.5-mm Endohm unit (World Precision Instruments, Sarasota, FL). At the indicated time intervals, resistance readings (Ω) were obtained from each insert and multiplied by the membrane area (Ω × cm2) as values of TER. The resistance value of an empty culture insert (no cells) was subtracted. Data were collected from triplicate inserts per treatment in each experiment.
Paracellular permeability assay
Endothelial cells were grown to confluence on porous polyester membrane inserts (6.5 mm diameter, 0.4 µm pore size; Transwell, Corning, Cambridge, MA). The growth medium in the upper chamber was replaced with 100 µl of growth medium containing a 1 mg/ml FITC-dextran 20 or 40 and the cells were equilibrated at 37°C for 15 min. Then different growth factors were added to the inserts and the insert was moved to a fresh lower well containing 0.5 ml of the growth medium for various periods of times. Samples from the lower chamber (50 µl) were taken in triplicate and placed in 96-well cluster plates for measuring fluorescent intensity using a fluorescent plate reader with excitation at 530 nm and emission at 590 nm.
In vivo retinal permeability measurements
All animal studies were performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Florida, and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eight-week-old C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice received the following intravitreal injections (1 µl) with a 32-gauge needle into one eye: VEGF; hPlGF-1 or mPlGF-2; VEGF plus hPlGF-1 or mPlGF-2; 0.9% saline vehicle; VEGF followed by hPlGF-1 or mPlGF-2 6 or 24 hours later; VEGF followed by 0.9% saline 6 or 24 hours later. VEGF was given at a concentration of 60 ng/µl while hPlGF-1 and mPlGF-2 were injected at 10, 60 or 120 ng/µl. Unstimulated control is the baseline fluorescence in untreated animals. Forty six hours after the first injection mice received tail vein injections of FITC-labeled albumin (0.5 mg in 50 µl vehicle). The mice were returned to their cages and their cages were placed on heating pads set to low to maintain normal body temperature. After 2 hours the mice were treated in two ways: A) For albumin leakage measurements animals were killed and the eye that received the intravitreal injection enucleated and the retinas removed and placed in PBS. The retina were rinsed in buffer and disrupted mechanically with a polytron homogenizer in 1 ml of buffer (50 mM ammonium acetate and 150 mM NaCl, pH 7.4), and cleared by centrifugation at 12,000 g for 15 min at 4°C. The supernatant fraction was transferred to a new tube, diluted. FITC-albumin was quantified against a standard curve of FITC-albumin using a FLUOstar Optima spectrofluorometer (BMG Labtechnologies) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. B) For histology and immunostaining the mice were perfused by cardiac puncture with 10 ml of 1% paraformaldehyde in citrate buffer (pH 4.2) which was pre-warmed to 37°C the eyes that received the intravitreal injection enucleated.
Neutralization of VEGF and VEGFRs in vivo
To confirm the role of VEGFR-1 and VEGFR-2 in VEGF-induced retinal microvascular permeability microvascular permeability neutralizing antibodies to VEGFR-1 (4 or 12 ng/eye) or VEGFR-2 (0.5 or 1.0 ng/eye) were given by intravitreal injection in C57BL/6 mice 6 hours prior to injection of VEGF (60 ng/eye). 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin and retinas were taken for analysis 2 hours later (n = 6 per group).
Immunocytochemical analysis
Endothelial cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Subsequently, the cells were washed with PBS, permeabilized with 0.1% triton X-100 in PBS for 5 min at room temperature and blocked with 10% normal goat serum in PBS at room temperature for 30 min. The cells were then incubated with goat polyclonal anti-VE-cadherin antibody (Santa Cruz Biotechnology) (1∶100), rabbit poly anti-claudin 5 (Cell Signalling, MA) and rabbit poly anti-ZO-1 (Santa Cruz Biotechnology, CA) in PBS containing 1% bovine serum albumin at room temperature for 1 h, and with the secondary antibody, Alexa Fluor 549-labeled anti-goat IgG (Molecular Probe) (1∶1000) for VE-cadherin and Alexa Fluor 488-labeled donkey anti-rabbit IgG (Invitrogen, CA) for claudin 5 and ZO-1 in 1% BSA in PBS at room temperature for 1 hour in dark. Then the cells were examined and photomicrographs were obtained using a DSU-Olympus IX81 confocal microscope. Flat mount retinas from the mouse studies were permeabilized with 0.2% Triton X-100 and non-specific binding was blocked by 10% normal goat serum in PBS for overnight at 4°C. The retinas were then transferred to a solution of primary antibody and incubated for 24 hours at 4°C. The primary antibodies were rabbit anti-VE-Cadherin (1∶100, Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-Claudin-5 (1∶3000, Abcam Inc., Cambridge, MA, USA). The retinas were transferred to the secondary antibody for 24 hours at 4°C after washing in PBS with 0.2% Triton X-100. The secondary antibody was Cy3 conjugated goat anti-rabbit IgG (1∶250). The retinas were then incubated 30 minutes at room temperature in 1∶500 FITC -conjugated agglutinin in 10 mM HEPES, 150 mM NaCl and 0.1% Tween 20. Retinas were flat mounted onto microscope slides and covered in aqueous VectaShield mounting medium (Vector Laboratories, Inc., Burlingame, CA, UAS) for observation by confocal microscopy. Digital confocal images were captured with an Olympus DSU-Olympus IX81 confocal microscope with identical photomultiplier tube gain settings. Maximum projections generated from z-section stacks of confocal images are processed identically in experimental and control retinas.
Western blotting analysis
VE-cadherin protein expression was assessed in the cell lysates through standard Western blotting analysis. Equal amounts of protein from each sample were resolved by 10% SDS polyacrylamide gel and transferred onto nitrocellulose membrane. The membranes were incubated with goat polyclonal anti-VE-cadherin, and rabbit polyclonal anti-Occludin, anti-claudin 5 and anti-ZO-1 antibodies (1∶250, Santa Cruz Biotechnology, CA, USA; Cell Signalling, Canton, MA, USA) at room temperature for 2 hr. α-tubulin acted as the loading control. The membranes were then washed with 5% milk/TBS containing 0.05% Tween-20 followed by HRP-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) (1∶4000) at room temperature for 1 hr. Following washing, the membranes were incubated with ECL (Santa Cruz Biotechnology, CA, USA) and exposed to Biomax MR film. Band intensity was determined by laser densitometry from a minimum of three separate experiments.
RT-PCR analysis of VE-cadherin, claudin 5, Occludin and ZO-1 expression
Total RNA was isolated from cells treated with growth factors by using TRIzol Reagent (Invitrogen), and then reversed transcribed using Reverse-iT™ (Abgene). Bovine VE-cadherin, claudin 5, Occludin and ZO-1 transcripts were amplified at 1.5 mM MgCl2 using the primer pairs (VE-cadherin, forward: 5′-CTAACAGCCCTTCCTTGCAG-3′, reverse: 5′- CTTTGAGTTGGACCCGTGAT -3′; Claudin 5, forward: 5′-TCGTCGCGCTGTTC GTGACC -3′, reverse: 5′-ATGGGCACGGTCGGGTCGTA-3′; Occludin, forward 5′-CCGGAAGATGAAATTCTCCA-3′, reverse 5′-CAGCTCCCATTAAGGTTCCA-3′; ZO-1, forward: 5′-CGCCTTTGGACAAAGAGAAG-3′, reverse 5′- TTTTAGGATCACCCGA CGAG-3′). As control for the amount of mRNA input we amplified bovine glyceraldehyde-phosphate-dehydrogenase (GAPDH) at 54.93°C annealing temperature, 1.5 mM MgCl2 concentration with forward primer 5′-GGGTCATCATCTCTGCACCT-3′ and reverse primer 5′-GGTCATAAGTCCCTCCACGA-3′. A total of 10 µl aliquots of amplified products were separated electrophoretically on a 1.5% agarose gel stained with ethidium bromide and illuminated with UV light and analyzed using NIH Image software.
Real-time quantitative PCR analysis
The CFX 96 Real Time PCR Detection System (BioRad, Hercules, CA) was used to quantify the mRNA level of VE-cadherin (copies/µl from internal control) in endothelial cells with bovine VE-cadherin primers (forward: 5′-CTAACAGCCCTTCCTTGCAG-3′; reverse: 5′- CTTTGAGTTGGACCCGTGAT -3′), the Amplifluor system (Intergen Inc, UK), real time-quantitive polymerase chain reaction (Q-PCR) master matrix (Abgene, Surrey, UK) and a universal probe (UniPrimerTM). Real-time conditions were 95°C for 15 min, followed by 65 cycles at 95°C for 15 s, 55°C for 60 s and 72°C for 20 s. The results of the test molecules were normalised against levels of β-actin. The level of the VEGF transcript from a given sample was automatically calculated by the software from an internal standard, a method previously described [54].
VE-cadherin cell surface ELISA
Confluent RMEC monolayers were rinsed with MECBM containing growth supplement and fixed with 4% paraformaldehyde in PBS for 20 min. After two washes with PBS containing 0.1%BSA, the cells were incubated with goat polyclonal anti-VE-cadherin antibody (Santa Cruz Biotechnology) (1∶200) for 2 hr. The cells then washed for three times with PBS containing 0.1% BSA and incubated with HRP-conjugated secondary antibody (1:1000). The monolayer was then rinsed four times with PBS containing 0.1%BSA, followed by one wash with PBS. For detection, equal parts of the substrate reagents hydrogen peroxide and 3,3′,5,5′-tetramethylment were added to each well. After colour development, 1 N HCl was added to stop the reaction. Absorbance was measured at 450 nm using ELISA (Quantikine®, R & D system) according to the manufacturer’s instruction.
Electrophoretic Mobility Shift Assay (EMSA)
Generation of the nuclear extract from cells was performed using nuclear extraction kit (Chemicon® International, Inc) according to the manufactory instruction. The protein concentration was determined by using the BCA protein assay kit (Perbio Science UK Ltd). The oligonucleotides (5′- CATCTGCCCTCATCTGGGAATGGGGTGAGGGG -3′ and 5′- CCCCTCACCCCATTCCCAGATGAGGGCTGATG -3′) were synthesized (Sigma-Aldrich Company Ltd). Four µg of nuclear extracts were prepared in a final volume of 20 µl containing 34 mM KCl, 5 mM MgCl2, 0.1 mM dithiothreitol, and 3 µg of poly(dI-dC). After 10 min on ice, the DNA probe was added, and the incubation was continued for 20 min at room temperature. The specific antibodies (1:20) were added to the mixture before the addition of the DNA probe and incubated 20 min on ice. Finally, the samples were added with 7 µl of a 20% (w/v) Ficoll solution, and analyzed on 5% non-denaturing polyacrylamide gels in 0.5× TBE. The gels were stained with fluorescence-based EMSA kit (Molecular Probes, Inc), which uses fluorescent dye for detection-SYBR® Green EMSA nucleic acid gel for DNA. Fluorescence intensity was determined by laser densitometry from a minimum of three separate experiments.
Statistical analysis
All experiments were repeated at least three times. The TER, paracellular permeability and VE-cadherin cell surface ELISA data at different time points were assessed using a Student's t test plus ANOVA for multiple comparisons. The Mann-Whitney test was used to determine statistical significance in the data of VE-cadherin expression both obtained using Western blotting analysis and Q-PCR. Results are expressed as mean±standard deviation. p<0.05 is considered statistically significant.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by National Institutes of Health (NIH) grants: EY018358 to MEB; EY007739, EY012601 and U01 HL087366 to MBG; EY017164 to SAV; EY012021 to DAA. Other funding included the Wellcome Trust, UK (MEB, AA), Research to Prevent Blindness (SAV), Medical Research Council (G0601295 and G0700288) and British Heart Foundation (RG/09/001/25940) to AA and the Juvenile Diabetes Research Foundation (DAA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J PregnancyJ PregnancyJPJournal of Pregnancy2090-27272090-2735Hindawi Publishing Corporation 2149074410.1155/2010/789748Research ArticleAntenatal Steroid Therapy for Fetal Lung Maturation and the Subsequent Risk of Childhood Asthma: A Longitudinal Analysis Pole Jason D.
1, 2, 3
*Mustard Cameron A.
1
To Teresa
1, 3
Beyene Joseph
1, 3, 4
Allen Alexander C.
5
1Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada M5T 3M72Pediatric Oncology Group of Ontario, Toronto, ON, Canada M5G 1V23Child Health Evaluative Sciences, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X84Department of Clinical Epidemiology & Biostatistics, McMaster University, Hamilton, ON, Canada L8N 3Z55Perinatal Epidemiology Research Unit, Departments of Obstetrics and Gynecology and Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada B3K 6R8*Jason D. Pole: [email protected] Editor: Sam Mesiano
2010 15 6 2010 2010 78974826 1 2010 6 4 2010 Copyright © 2010 Jason D. Pole et al.2010This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This study was designed to test the hypothesis that fetal exposure to corticosteroids in the antenatal period is an independent risk factor for the development of asthma in early childhood with little or no effect in later childhood. A population-based cohort study of all pregnant women who resided in Nova Scotia, Canada, and gave birth to a singleton fetus between 1989 and 1998
was undertaken. After a priori specified exclusions, 80,448 infants were available for analysis.
Using linked health care utilization records, incident asthma cases developed after 36 months of
age were identified. Extended Cox proportional hazards models were used to estimate hazard
ratios while controlling for confounders. Exposure to corticosteroids during pregnancy was
associated with a risk of asthma in childhood between 3–5 years of age: adjusted hazard ratio of
1.19 (95% confidence interval: 1.03, 1.39), with no association noted after 5 years of age:
adjusted hazard ratio for 5–7 years was 1.06 (95% confidence interval: 0.86, 1.30)
and for 8 or greater years was 0.74 (95% confidence interval: 0.54, 1.03). Antenatal steroid therapy appears to be an independent risk factor for the development of asthma between 3 and 5 years of age.
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1. Introduction
Research on the etiology and natural history of asthma has identified a web of predisposing factors (e.g., genetics, atopy), causal factors that may sensitize the airways (e.g., animal dander, dust mites, workplace allergens) and contributing factors (perinatal events such as mode of delivery, exposure to cigarette smoke during pregnancy, gestational age and childhood respiratory infections, air quality, socioeconomic status, and pollution) [1–6]. Conclusions that can be drawn from this research outline a myriad of exposures and complex interactions in the etiology of pediatric asthma [7–10].
Given the complex nature of pediatric asthma etiology, factors in the perinatal period that would predispose individuals to asthma are of particular interest [11, 12]. Corticosteroid (CS) therapy, administered during labour and delivery to accelerate fetal lung maturation, has not been fully examined as a potential risk factor for the development of asthma in humans [13]. CS therapy has been shown to alter the development of the fetal lung and has been linked, in animal studies, to changes in brain chemistry and subsequent hypertension later in life [14–28]. Complex time-dependent relationships between CS therapy and subsequent lung function in animals has been noted [29].
The use of CS therapy, in Canada, among preterm infants before 34 weeks' gestation has increased in the last 15 years from less than 25 percent to approximately 60 percent [30, 31]. This increase of CS therapy follows from randomized controlled trial evidence indicating increased infant survival among preterm infants exposed to antenatal CS therapy [32].
A limited number of previous studies have investigated lung function (not asthma) in childhood among those previously exposed to CS therapy [33–36]. The small sample sizes and convenience samples of these studies do not provide sufficient evidence for or against an association between CS therapy and asthma [13]. Therefore, a large population-based cohort study where confounding by indication can be controlled is warranted.
This study was designed to test the hypothesis that fetal exposure to corticosteroids in the antenatal period is an independent risk factor for the development of asthma in childhood. Further, the risk of asthma is hypothesized to be greater in the early childhood years and attenuated in later childhood.
2. Methods
A population-based cohort study of all pregnant women who resided in Nova Scotia, Canada and gave birth to a singleton fetus between January 1, 1989, and December 31, 1998, and lived to discharge was undertaken.
Data from The Maternal-Child Health Database (MCHD) were used. The MCHD is a longitudinal population-based database of all mothers and infants delivered while resident in Nova Scotia that is linked to health services utilization data. Four independently collected linked databases comprise the MCHD used in this study including data from the Nova Scotia Atlee Perinatal Database (NSAPD), the Nova Scotia physician visits Medical Services Insurance File database, the Nova Scotia Vital Statistics Database and the Nova Scotia portion of the Canadian Institute for Health Information hospital admissions database.
Pregnancies that resulted in the birth of more than one infant (twins, triplets, quadruplets) differ from singleton pregnancies both physiologically and obstetrically and therefore were not eligible for inclusion. Mothers who suffer from thyroid conditions (due to higher levels of hormones already present) and mothers experiencing active asthma during the gestational period (due to increased risk of obstetrical complications and the possibility of fetal exposure to steroids because of the pharmaceutical interventions used to manage the mothers asthma) were also excluded [37–39].
Previous work in Canada has established that health service administrative records can be used to identify and describe children with asthma [40–43]. The definition of asthma employed in this study was based on this previous work, determined by examining physician visits or hospitalizations where the primary diagnostic field was for asthma or asthma-like conditions (asthma: ICD-9-CM code 493, bronchitis: ICD-9-CM code 490, or bronchiolitis: ICD-9-CM code 466). Subjects who experienced at least two health care interactions in any 365 day period, starting at 156 weeks post birth (3 years), where the diagnosis was asthma or an asthma-like condition and at least one visit was not in the winter period (December to March inclusive), or experienced one hospitalization specifically for asthma (ICD-9-CM code 493) were considered to be asthmatic [40]. This definition captures all levels of asthma severity.
A second more conservative definition of asthma was developed to examine the potential that the primary definition would be too encompassing and therefore have a high level of misclassification. The conservative definition of asthma was similar to the primary definition except that only interactions where asthma (ICD-CM code 493) was the primary diagnostic field were considered.
The primary exposure of interest is antenatal CS therapy. Maternal systemic steroid therapy is coded in the NSAPD [44]. The first dose of the first course of betamethasone or dexamethasone coupled with the timing of the administration is recorded. For this study, infants were considered exposed to CS therapy if either of these drugs was administered regardless of timing.
Numerous potential risk factors for childhood asthma have been identified in the literature. Potential confounders considered in this analysis included the infant's sex, gestational age at birth, maternal age, one minute Apgar score, administration of surfactant, infant's birth weight, delivery by caesarian section, maternal smoking during the gestational period, socio-economic status (SES), hyaline membrane disease, bronchopulmonary dysplasia, maternal insulin dependant diabetes mellitus, gestational diabetes, number of siblings, and year of birth.
The infant's gestational age at birth, in completed weeks, was examined both continuously and as a dichotomous variable indicating full term/preterm, with full term being 37 or more completed weeks of gestation. Infants born at an early gestational age are at increased risk of poor lung function and preterm labour is often used as an indication for CS therapy [30, 45–47].
2.1. Confounding
Control for confounding by indication was achieved in this study given not all infants who are preterm or low birth weight were exposed to CS therapy and a portion of infants who are full-term or full birth weight will have been exposed. There is a portion of infants in each year of the cohort where the therapy is clinically indicated, but for various reasons not administered (e.g., where mothers presented to the hospital too late). Also, there is a portion of infants in each year of the cohort where the therapy is clinically indicated and administered, but for various reasons delivery was able to be delayed until the infant was delivered full term. Therefore, each year a proportion of preterm and low birth weight infants are unexposed and a portion of full-term infants are exposed to CS therapy allowing for differences in the development of childhood asthma to be examined.
Maternal age was examined continuously. Given maternal age has been shown to be related to preterm birth and related to obstetrical complications leading to the administration of CS, maternal age was only considered as a confounder in this analysis [31, 48]. The infants one minute Apgar score (examined in three categories, 0–3, 4–6, 7–10) has been shown to be an independent risk factor for childhood asthma [49]. Delivery by caesarean section was examined as a dichotomous variable where, regardless of other obstetrical interventions during delivery (e.g., the use of forceps prior to surgery), if the infant was ultimately born by caesarean section it was considered a caesarean section. Delivery by caesarean section is related to CS therapy and CS therapy has been shown to be associated with poor lung function in the infant [49, 50]. Maternal smoking during pregnancy was examined as a dichotomous variable. Cigarette exposure during the gestational period has been shown to be related to the development of childhood asthma and risk of obstetrical complications (including prematurity) that are related to the administration of CS [47, 51–56]. Differences in the complication rate and the potential for threatened preterm labour is increased when the mother is diagnosed with either type 1 diabetes mellitus or gestational diabetes.
Hyaline membrane disease is a respiratory condition in the infant that is often associated with preterm delivery and subsequent morbidity. CS therapy was primarily instituted to ameliorate this disease. However, the administration of surfactant to alleviate various respiratory difficulties in the infant after birth, such as hyaline membrane disease, has been shown to be related to both long term lung function and the use of CS therapy [57–60].
Due to the declining incidence of asthma over the time period under study, the year of birth was used to control for birth cohort effects (temporal effects).
A greater number of older siblings in a household has been shown to be associated with reduced risk of childhood asthma and forms part of the basis for the hygiene hypothesis [61–63]. Also related to the hygiene hypothesis is the socioeconomic status of the infants family [64, 65]. Mothers who are of low socioeconomic status are also more likely to have various pregnancy complications that include threatened preterm labour [66]. Therefore, two socioeconomic status measures were used in this study. Marital status at the time of delivery (married or common law versus single, widowed, divorced, or separated) and quintiles of neighbourhood income (based on the postal code of residence at the time of delivery).
2.2. Analysis
Extended Cox proportional hazards regression models were used to analyse the data [67]. Time zero was recorded as the date of birth. Duration of follow-up was measured in weeks. Subjects were censored the week they were identified as having asthma, died or were lost to follow-up. Given the outcome of asthma was not considered before 156 weeks the minimum amount of follow-up time required to be included in this analysis was therefore 156 weeks.
A Cox proportional hazards regression stratified by antenatal steroid therapy was utilized to assess the time dependant hazard comparing subjects that received and did not receive steroid therapy. A kernel smoothing method was utilized to remove the extreme variability in the time-specific hazard estimates [68, 69].
Given that one mother may give birth to more than one infant in the cohort an examination of robust variance estimates was undertaken using a sandwich estimator. The nonindependence altered the variance estimates by less than 5 percent so no consideration of this nonindependence was given in subsequent analysis.
To quantitatively assess if the hazard ratio for the association between exposure to antenatal corticosteroids and the subsequent development of asthma changes with time, an extended Cox proportional hazards regression with time-dependant covariates was used. The effect of steroid administration through time was described by three hazard ratios; one when time was between 156 and 260 weeks, one when time was between 261 and 364 weeks and one when time was greater than 364 weeks. These time periods were selected based on an examination of the smoothed figure derived from the stratified Cox model.
All potential confounders were examined with nested models with the time-dependant covariates for steroid administration. Each potential confounder's effect on the parameter estimates for antenatal steroid therapy (early, mid and late) was assessed by examining the percentage difference in the parameter estimates from a model with and without the potential confounder. Differences larger than 10 percent in any one of the parameter estimates was considered evidence of confounding [70]. Potential cofounders that did not demonstrate evidence of confounding were not retained in the final models.
Thus, the statistical model for the final analysis was as follows:
(1) h(t,X(t))=h0(t)exp [β1(steroid)g1(t)+β2(steroid)g2(t) +β3(steroid)g3(t)+βx],
where
(2) g1(t)={1if 156<t≤260 weeks,0if t<260 weeks,g2(t)={1if 261≤t≤364 weeks,0if t<261 or t>364 weeks, g3(t)={1,if t≥365 weeks,0,if t<365 weeks, βx={x other covariates.
All statistical analyses were performed using SAS version 8.2.
3. Results
There were a total of 113,145 births between January 1, 1989, and December 31, 1998, in Nova Scotia. Exclusions included 1,325 fetal, neonatal or infant deaths that occurred before 1 year of age, 2,408 infants who were delivered from a multiple gestation, 4,958 infants who were born to mothers experiencing active asthma during the gestational period and a further 89 infants were excluded because they were born to mothers experiencing endocrine abnormalities.
There were 104,365 infants eligible for record linkage. Birth records for 18,627 infants could not be successfully linked to the health care follow-up data due to errors or missing information with the provincial unique identifier. Due to deaths, migration, and other events an additional 3,484 infants did not have 156 or more weeks of follow-up time. A further 1,671 infants did not have either a birth weight or gestational age recorded (which is required for the analysis), 135 infants were greater than or less than three standard deviations from their mean sex-specific birth weight for gestational age and therefore were excluded. This left 80,448 infants for analysis.
Table 1 provides descriptive statistics for the study sample by asthma status. Both a row and column percentage is provided to assist in the interpretation of the relationships. The asthma incidence among birth cohorts peaked in 1989 and has been on a decline since. The number of siblings, income, marital status, mean birth weight, mean gestational age and mean maternal age at birth are similar between asthmatics and nonasthmatics. Infants born preterm had an elevated incidence of asthma over the follow-up period. As expected, children who developed asthma had higher prevalence of caesarean section, surfactant administration, maternal smoking, hyaline membrane disease and bronchopulmonary dysplasia compared to children who did not develop asthma. The median overall follow-up time was nearly 8.5 years (440 weeks). On average asthma developed in this cohort at approximately 5.5 years of age (287 weeks).
Table 2 provides the rates of antenatal steroid therapy administration and preterm birth by year of birth. The use of antenatal steroid therapy increased 3-fold over the 10-year time period of the study, from a rate of 7.5 in 1989 to 23.7 per 1,000 births in 1998. The preterm birth rate increased approximately 30 percent over the same period, from a rate of 40.5 in 1989 to 52.7 per 1,000 live births in 1998.
Figure 1 provides the smoothed adjusted hazard function over time, stratified by antenatal steroid therapy. Adjustments were made for the infant's sex, gestational age at birth and year of birth. Given the smoothing algorithm used a bandwidth of 75 weeks, the tales of the curves have not been estimated. The figure provides evidence that the hazard for developing asthma differs by antenatal steroid therapy dependant on time. The difference is greatest in the early period, diminishing over the middle period with little differences noted beyond 7 years (400 weeks).
Table 3 contains the estimated adjusted hazard ratios for the development of asthma for antenatal steroid therapy from 3 to 5, 6 to 7 and 8, or greater years of age along with the estimates for all other confounders in the model. Adjustments were made for infant's sex, gestational age at birth and an indicator for perterm birth, one minute Apgar score, administration of surfactant, infant's birth weight, delivery by caesarian section, maternal smoking during the gestational period, hyaline membrane disease, bronchopulmonary dysplasia, number of siblings and year of birth. Unadjusted hazard ratio estimates are also provided for the antenatal steroid exposure. The effect of antenatal steroid therapy on the development of asthma is seen to be larger in early childhood (HR = 1.19, 95% CI: 1.03, 1.39) with no effect noted in mid childhood (HR = 1.06, 95% CI: 0.86, 1.30) and potentially a protective effect in late childhood (HR = 0.74, 95% CI: 0.54, 1.03).
When considering the more conservative definition of asthma the prevalence of asthma dropped from 25.4% to 18.0%. The estimated adjusted hazard ratios for the development of asthma for antenatal steroid therapy from 3 to 5, 6 to 7 and 8 or greater years of age were HR = 1.37 (95% CI: 1.15, 1.62), HR = 1.17 (95% CI: 0.93, 1.49) and HR = 0.92 (95% CI: 0.67, 1.26), respectively. Adjustments were made for the same confounders as in the main model.
4. Discussion
Antenatal steroid therapy appears to be an independent risk factor for the development of childhood asthma after controlling for confounding. The risk appears to be time-dependent with the highest risk early in childhood and diminishing as the child ages. The reasons for this increase risk are not obvious but many animal studies have established latent effects of corticosteroid exposure in utero.
Although a mechanistic link between antenatal corticosteroid therapy and the onset of asthma in childhood has not been fully established [71], wide ranging effects of corticosteroid therapy have been demonstrated. The alteration in brain chemistry and hypothalamic-pituitary-adrenal (HPA) axis, the shift in immune function and other wide-ranging latent effects such as changes in kidney development and subsequent hypertension all provide biological plausibility for a link to childhood asthma.
Time-dependent complications associated with antenatal corticosteroid therapy have been demonstrated [15, 16]. Corticosteroids have been shown to affect HPA development in primates, sheep and to a limited extent in humans [17–20]. The HPA axis is important in regulating the physical growth and organ development of the neonate [18]. The timing of exposure to corticosteroids within a species, late in gestation, has been shown by Matthews to be critical with regard to the effect on the HPA axis [17, 21]. Published and unpublished work by Clifton in Australia indicates that corticosteroid exposure not only alters the HPA axis but also influences the Th1/Th2 ratio in favour of Th2 [72].
Strengths of this study are rooted in the large population cohort that were assembled and followed for extended lengths of time. The availability of potential confounders for control was extensive. Control for confounding by indication was also inherent in this study design providing considerable methodological strength.
The retrospective nature of this study design has a number of limitations. The reliability and validity of an asthma diagnosis in the MSI data is unknown. Errors in the primary reason for admission in the CIHI Hospital Discharge Data may occur when several competing causes of admission are present in any one individual. Errors of this sort, with both databases, would have a conservative effect on the estimate of association generated in this study.
When considering the more conservative definition of asthma the pattern of early childhood risk for the development of asthma after antenatal exposure to corticosteroids remains the same as with the main asthma definition. As expected with this definition the risk estimate, particularly in the early childhood period is greater. This sensitivity analysis lends credence to the hazard ratio estimates using the main asthma definition.
Limitations in the measure of exposure exist, in that there is no measure of the CS that crosses the placenta and is biologically available to each fetus. Information that pertains to multiple exposure to CS therapy and the actual amount of medication administered is not recorded in the NSAPD. With all data limitations outlined, bias would only result if there was a systematic difference between those exposed to CS therapy and those not exposed. There is no indication that physician billing or hospital discharge records would be different based on exposure.
Given that this study is focussed on mothers and infants from the province of Nova Scotia, Canada, any findings from this current study would need to be replicated in other populations to strengthen external validity. Although no factors specifically related to the biological effects of CS therapy are postulated to differ between this population and others, other factors related to childhood asthma may differ. The trend in asthma incidence by birth year for this population appears to be different than that of other populations and therefore replication of these findings in different populations is warranted.
Continued research into the perinatal factors in the etiology of childhood asthma is warranted. Information continues to surface that draws attention to the potential long term consequences of exposures in utero and subsequent disease risk. Cohort studies that examine the time-dependant effects are crucial in establishing periods of disease susceptibility. Although only a small, but significant, elevated risk for childhood asthma and antenatal steroid exposure was demonstrated in the current study, further research into the smallest possible dose required for the steroid to achieve the desired post-natal effect could be undertaken potentially limiting the long term consequences such as childhood asthma.
Acknowledgments
The authors acknowledge the operational funding from the Canadian Institutes of Health Research (CIHR) (Funding Reference no. MOP-77693) and the Allergy, Genes and Environment Network (AllerGen NCE Inc.) (Project no. FP-2005-1). Dr. J. D. Pole contributed to the conception of the study, study design, analysis, and interpretation of the data, editorial preparation, and review of the manuscript. Dr. J. D. Pole also had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs. C. A. Mustard, T. To, J. Beyene and A. C. Allen contributed to the study design, interpretation of the data, editorial preparation and review of the manuscript. All authors have no conflict of interest or financial interests related to this study. This work was funded by and operating grant from the Canadian Institutes of Health Research and the AllerGen Networks of Centres of Excellence.
Figure 1 Smoothed Hazard Function Estimates by Antenatal Steroid Therapy. Note: Adjusted for gender, gestational age and year of birth.
Table 1 Descriptive characteristics of the study sample by asthma status.
Asthma
No Yes Total
% %
Num Row Column Num Row Column Num %*
Total 59,975 74.6 100.0 20,473 25.4 100.0 80,448 100.0
Birth year
1989 3,823 65.1 6.4 2,051 34.9 10.0 5,874 7.3
1990 5,816 66.5 9.7 2,931 33.5 14.3 8,747 10.9
1991 5,671 67.2 9.5 2,773 32.8 13.5 8,444 10.5
1992 6,020 71.7 10.0 2,374 28.3 11.6 8,394 10.4
1993 6,135 74.0 10.2 2,152 26.0 10.5 8,287 10.3
1994 6,524 75.9 10.9 2,067 24.1 10.1 8,591 10.7
1995 6,503 77.4 10.8 1,896 22.6 9.3 8,399 10.4
1996 6,793 80.7 11.3 1,629 19.3 8.0 8,422 10.5
1997 6,465 81.8 10.8 1,440 18.2 7.0 7,905 9.8
1998 6,225 84.3 10.4 1,160 15.7 5.7 7,385 9.2
Antenatal steroid exposure 773 68.1 1.3 362 31.9 1.8 1,135 1.4
Number of siblings
0 25,916 72.2 43.2 9,972 27.8 48.7 35,888 44.6
1 21,753 75.1 36.3 7,199 24.9 35.2 28,952 36.0
2 8,691 78.1 14.5 2,435 21.9 11.9 11,126 13.8
3+ 3,608 80.7 6.0 864 19.3 4.2 4,472 5.6
Preterm birth (<37 weeks) 2,534 68.1 4.2 1,189 31.9 5.8 3,723 4.6
Caesarean section 10,873 71.9 18.1 4,253 28.1 20.8 15,126 18.8
Income
Quintile 1 12,693 73.6 21.5 4,547 26.4 22.7 17,240 21.4
Quintile 2 12,619 74.3 21.4 4,359 25.7 21.7 16,978 21.1
Quintile 3 11,368 75.2 19.3 3,749 24.8 18.7 15,117 18.8
Quintile 4 12,435 74.6 21.1 4,235 25.4 21.1 16,670 20.7
Quintile 5 9,873 75.7 16.7 3,174 24.3 15.8 13,047 16.2
Surfactant administration 139 59.9 0.2 93 40.1 0.5 232 0.3
1 Minute Apgar score
0–3 1,309 70.4 2.2 550 29.6 2.7 1,859 2.3
4–6 4,375 71.9 7.3 1,712 28.1 8.4 6,087 7.6
7–10 54,070 74.9 90.5 18,137 25.1 88.9 72,207 89.8
Maternal smoking 16,421 72.6 27.4 6,211 27.4 30.3 22,632 28.1
Married/Common law 42,671 74.6 71.1 14,566 25.4 71.1 57,237 71.1
Hyaline membrane disease 672 62.3 1.1 406 37.7 2.0 1,078 1.3
Bronchopulmonary dysplasia 97 54.2 0.2 82 45.8 0.4 179 0.2
Duration: weeks (mean, SD) 512.3 155.3
287.2 116.6
455.0 176.2
Birth weight: grams (mean, SD) 3,487.4 554.7
3,444.3 592.1
3,476.5 564.7
Gestational age: weeks (mean, SD) 39.5 1.7
39.3 1.9
39.4 1.7
Maternal age: years (mean, SD) 26.7 5.3
26.4 5.2
26.6 5.3
Note: All totals do not add to grand total given missing values for some variables.
*Percent of total cohort.
Table 2 Rates of antenatal steroid therapy administration and preterm birth by year of birth.
Total births Steroid administration
Preterm Full term Preterm birth
Num Num Rate Num Rate Num Rate
Year of birth
1989 5,874 26 109.24 18 3.19 238 40.52
1990 8,747 49 129.63 18 2.15 378 43.21
1991 8,444 58 164.31 28 3.46 353 41.80
1992 8,394 79 201.02 40 5.00 393 46.82
1993 8,287 69 195.47 36 4.54 353 42.60
1994 8,591 68 181.82 48 5.84 374 43.53
1995 8,399 78 186.16 34 4.26 419 49.89
1996 8,422 99 235.71 46 5.75 420 49.87
1997 7,905 106 261.08 60 8.00 406 51.36
1998 7,385 99 254.50 76 10.86 389 52.67
Total 80,448 731 196.35 404 5.27 3,723 46.28
Note: Rate is per 1,000 births.
Table 3 Association between antenatal steroid therapy exposure and childhood asthma.
Adjusted Unadjusted
HR 95% CI HR 95% CI
Antenatal steroid exposure
3–5 years 1.19 1.03–1.39 1.55 1.36–1.77
6–7 years 1.06 0.86–1.30 1.36 1.12–1.65
8+ years 0.74 0.54–1.03 0.98 0.72–1.34
Male gender 1.24 1.21–1.28 —
Preterm birth (<37 weeks) 1.03 0.95–1.12 —
Number of siblings
0 1.00 —
1 0.90 0.88–0.93 —
2 0.78 0.74–0.82 —
3+ 0.67 0.62–0.72 —
Bronchopulmonary Dysplasia 0.98 0.74–1.31 —
Hyaline Membrane Disease 1.14 1.01–1.30 —
Surfactant Administration 1.07 0.82–1.40 —
1 Minute Apgar Score
0–3 1.00 —
4–6 0.95 0.86–1.05 —
7–10 0.87 0.79–0.95 —
Caesarean section 1.11 1.07–1.15 —
Maternal smoking 1.12 1.09–1.16 —
Birth weight (per 500 grams) 1.00 0.98–1.01 —
Gestational age (weeks) 0.97 0.96–0.98 —
Birth year
1989 1.47 1.37–1.59
1990 1.45 1.35–1.56 —
1991 1.49 1.39–1.60 —
1992 1.28 1.19–1.37 —
1993 1.21 1.12–1.30 —
1994 1.14 1.06–1.23 —
1995 1.12 1.04–1.20 —
1996 0.99 0.92–1.07 —
1997 1.02 0.95–1.11 —
1998 1.00 —
Note: Adjustment for all other confounders in table.
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==== Front
PLoS BiolPLoS BiolplosplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 21483722PBIOLOGY-D-10-0137510.1371/journal.pbio.1001040Research ArticleBiologyA Chaperonin Subunit with Unique Structures Is Essential for Folding
of a Specific Substrate Function of Cpn60β4 on Folding of
NdhHPeng Lianwei
1
Fukao Yoichiro
2
Myouga Fumiyoshi
3
Motohashi Reiko
4
Shinozaki Kazuo
3
Shikanai Toshiharu
1
*
1 Department of Botany, Graduate School of
Science, Kyoto University, Sakyo-ku, Kyoto, Japan2 Plant Global Educational Project, Graduate
School of Biological Sciences, Nara Institute of Science and Technology,
Takayama, Ikoma, Nara, Japan3 Gene Discovery Research Group, RIKEN Plant
Science Center, Yokohama, Japan4 Faculty of Agriculture, University of
Shizuoka, Shizuoka, JapanHorwich Arthur L. Academic EditorYale School of Medicine/HHMI, United States of America* E-mail: [email protected] author(s) have made the following declarations about their contributions:
Conceived and designed the experiments: LP TS. Performed the experiments: LP
YF. Analyzed the data: LP YF . Contributed reagents/materials/analysis
tools: FM RM KS. Wrote the paper: LP TS.
4 2011 5 4 2011 5 4 2011 9 4 e100104020 12 2010 23 2 2011 Peng et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Type I chaperonins are large, double-ring complexes present in bacteria (GroEL),
mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating
the folding of newly synthesized, translocated, or stress-denatured proteins. In
Escherichia coli, GroEL comprises 14 identical subunits and
has been exquisitely optimized to fold its broad range of substrates. However,
multiple Cpn60 subunits with different expression profiles have evolved in
chloroplasts. Here, we show that, in Arabidopsis thaliana, the
minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with
Cpn60α1 and Cpn60β1–β3 and is specifically required for the
folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex
(NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4.
Furthermore, the unique C-terminus of Cpn60β4 is required for the full
activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH.
Our findings suggest that this unusual kind of subunit enables the Cpn60 complex
to assist the folding of some particular substrates, whereas other dominant
Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the
folding of other obligate substrates.
Author Summary
Chaperonins assist the folding of some nascent and denatured proteins to their
native, functional forms. Each chaperonin consists of a pair of protein
complexes resembling two stacked toroids; folding occurs inside the toroid
cavity. Chaperonins are ubiquitous in both bacteria and more complex nucleated
cells, as well as in the intracellular organelles that have evolved from
bacteria by endosymbiosis: mitochondria and, in plants, chloroplasts. They are
indispensable for cellular function. Many different chaperonin subunits have
evolved in various species of bacteria as well as in most mitochondria and
chloroplasts. The physiological and functional relevance of these multiple
chaperonin subunits is poorly understood, however. In this study, we have
characterized the minor chaperonin subunit Cpn60β4 from
Arabidopsis chloroplasts, which differs in structure from
other chloroplast chaperonins. When the Cpn60β4 gene is
defective, the plants fail to accumulate one protein complex in particular: the
chloroplast NADH dehydrogenase-like complex (NDH). We discovered that
Cpn60β4 forms a complex with other Cpn60 α and β
subunits and that this complex is essential for the folding of the NDH subunit
NdhH. Cpn60β4 has a unique protein “tail” that is required for
the efficient folding of NdhH. Our findings suggest that Cpn60β4 has evolved
with distinctive structural features that facilitate the folding of one specific
substrate and that this strategy is used by plants to satisfy their conflicting
requirements for chaperonins with both specialized and general functions.
==== Body
Introduction
Chaperonins are large double-ring assemblies that assist in the efficient folding of
substrate proteins (reviewed in [1]–[3]). Two types of chaperonins have been identified: type I in
bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), and type II in
archaea (thermosome) and eukaryotic (TRiC/CCT) cytosol (reviewed in [4]). Whereas a
type I chaperonin ring is composed of seven subunits, a type II chaperonin ring
consists of eight or nine subunits that are not identical but are homologous. Type I
chaperonin requires co-chaperonin GroES/Hsp10 for substrate encapsulation, whereas
type II chaperonin is independent of GroES/Hsp10 factors. Both types of chaperonins
utilize ATP as energy to drive a series of structural rearrangements that allow them
to capture, encapsulate, and release the substrate proteins (reviewed in [4]).
The GroEL/GroES complex from Escherichia coli (E.
coli) represents the type I chaperonins and its structure and function
have been studied extensively (reviewed in [4],[5]). GroEL consists of 14 identical
subunits of ∼57 kDa and these subunits form two heptameric rings stacked
back-to-back with a central cavity in each ring [6]. Each subunit contains three
domains. An equatorial domain comprises the ATP/ADP binding site and an apical
domain contains the hydrophobic surface toward the ring cavity for polypeptide
binding. The intermediate domain links the equatorial and apical domains [6],[7]. The
co-chaperonin GroES is a homoheptameric single-ring composed of 10 kDa subunits
[8]. GroES can
rapidly bind the substrate-captured GroEL ring (cis ring) in the
presence of ATP; hence, the GroEL/GroES complex provides an encapsulated cavity for
protein folding [9],[10]. Due to structural rearrangements in the apical and
intermediate domains, the cis cavity becomes enlarged and the
physical features of the cavity wall change. This process lasts about 10–15 s
and is accompanied by the hydrolysis of seven ATP molecules. After hydrolysis, ATP
and other non-native peptides bind to the GroEL in the trans ring,
triggering dissociation of the GroES from the opposite ring. The folded protein is
then released from the chaperonin complex (reviewed in [1]–[5]).
Proteome-wide analysis of chaperonin-dependent protein folding has shown that GroEL
interacts with about 250 different proteins and these substrates are categorized
into three classes [11]. The class I substrates are independent of GroEL/GroES,
whereas class II substrates are partially dependent on GroEL/GroES, and they can
utilize other chaperone systems, such as DnaK, for folding. A total of 84 proteins
are grouped into class III and they are potential obligate substrates of GroEL/GroES
in vivo [11]. More
recently, Fujiwara et al. [12] employed a more direct approach by testing the solubility
of class III substrates in GroE-depleted cells and found that only ∼60%
(49 out of 84) of the class III proteins are absolutely dependent on GroEL/GroES for
folding. Furthermore, an additional eight proteins that were not identified as class
III proteins were also found to be GroEL/GroES obligate substrates and the authors
defined these 57 proteins as class IV obligate substrates [12]. The majority of the class IV
proteins are involved in metabolic reactions. Bioinformatic analysis has shown that
nearly half of the class IV proteins contain TIM-barrel folds. In addition to these
TIM-barrel folds, FAD/NAD(P)-binding domains, PLP-dependent transferase-like folds,
and thiolase folds are also highly enriched in the group [12]. These data suggest that
GroEL/GroES has been optimized to facilitate the folding of a variety of substrates
during evolution.
The basic features of the mechanisms for GroEL/GroES-mediated folding of the
nonnative substrates have been demonstrated by a great number of functional and
structural studies. However, these studies have focused primarily on the model
chaperonin system that is composed of uniform subunits, such as GroEL from
E. coli. In contrast to E. coli, nearly
30% of bacterial genomes contain two or more chaperonin genes [13]. Furthermore,
almost all mitochondria and chloroplasts studied in higher plants possess multiple
chaperonin subunits [14]. There have been few reports focusing on the role played
by multiple chaperonin genes. In Sinorhizobium meliloti, one of the
five GroEL paralogs, GroEL1 was shown to be required for NodD protein folding [15]. However,
overexpression of another GroEL protein can suppress the defect of the
groEL1 mutant [15]. There are similar reports in other bacteria;
Bradyrhizobium japonicum possesses at least five highly
conserved groESL operons. Although nitrogen fixation activity was
reduced to approximately 5% of the wild-type (WT) level in the double mutant
defective in groEL3 and groEL4, overexpression of
two of the other groESL operons partially suppressed this phenotype
[16]. Of the
three chaperonin genes of Rhizobium leguminosarum, only
Cpn60.1 is essential for growth. Overexpression of the
Cpn60.3 gene in the cpn60.1 mutant sustains
bacterial growth, but the complemented strain is sensitive to high temperature,
suggesting that Cpn60.3 does not facilitate the folding of particular proteins
supporting the growth of bacteria at high temperature [17]. By contrast, the specificity of
GroEL1 function in Mycobacterium smegmatis seems to be absolute.
This chaperonin is not essential for growth but is required for mycolic acid
biosynthesis during mature biofilm formation [18]. GroEL1 may be specifically
involved in the folding of two proteins, KasA and SmEG4308, which are required for
mycolic acid synthesis, or in converting KasA between two isoforms [18]. These lines of
evidence suggest that the functions of the multiple chaperonin subunits are
specialized, although they have different degrees of specificity.
Chloroplast type I chaperonin complex (Cpn60) is similar in structure to GroEL and
also consists of two stacked heptameric rings [19],[20]. In contrast to GroEL, which
is composed of identical subunits, Cpn60 comprises two different subunit types,
Cpn60α and Cpn60β [21]–[23], and they are only approximately 50% identical to
each other [14]. In
vitro reconstitution studies of the chloroplast Cpn60 complex suggested a
stoichiometry of α7β7 in the Cpn60 complex [24], which is in accordance with
the observation that roughly equal amounts of α and β subunits are present
in chaperonin oligomers purified from spinach chloroplasts [25]. However, it is still unclear
how these subunits are organized within a complex. Furthermore, the
Arabidopsis thaliana genome contains two genes encoding
Cpn60α subunits and four genes encoding Cpn60β subunits [14], and they have
different expression profiles [26],[27]. Cpn60α1 (At2g28000), Cpn60β1 (At1g55490), and
Cpn60β2 (At3g13470) are the dominant Cpn60 subunits, whereas Cpn60α2
(At5g18820), Cpn60β3 (At5g56500), and Cpn60β4 (At1g26230) are present at
very low levels. Disruption of the Cpn60α1 gene results in
general defects in plastid function, leading to embryonic lethality [28], which
highlights the critical role of Cpn60α1 in maintaining plastid function. The
cpn60β1β2 double mutant also shows the lethal phenotype
[29],
suggesting that Cpn60α1 and Cpn60β1–β2 form a heterooligomer that
provides the housekeeping chaperonin function in chloroplasts by assisting the
folding of a wide range of proteins.
Multiple subunits also occur in type II chaperonin CCT and certain subunits are
responsible for the binding of specific substrates, such as actin and tubulin [30]. Recently, it
has been suggested that different subunits of CCT play unique roles in determining
substrate specificity [31]. However, few reports have focused on the function of the
multiple subunits in the chloroplast chaperonin system. In particular, whereas the
amino acid sequences of the Cpn60β1–β3 subunits share
90%–95% identity, Cpn60β4 is only 60% identical to
each of the other three Cpn60β subunits [14]. So far, there has been no
explanation why plants evolved this unusual kind of Cpn60β subunit. In this
study, we showed that Cpn60β4 is strictly and specifically required for the
folding of the NdhH protein, a subunit of the chloroplast NADH dehydrogenase-like
complex (NDH).
Results
The Arabidopsis crr27 Mutant Is Specifically Defective in
NDH Activity
NDH is a multi-subunit complex embedded in the thylakoid membrane and is involved
in chlororespiration and photosystem I (PSI) cyclic electron transport (Figure 1A) [32]. The
activity of NDH can be monitored as a post-illumination rise in chlorophyll
fluorescence (Figure 1B) due
to reduction of the plastoquinone (PQ) pool by the NDH complex in the dark [33]. Based on
this phenomenon, we isolated dozens of Arabidopsis mutants
specifically defective in NDH activity, which we referred to as
chlororespiratory reduction (crr) mutants.
Characterization of these mutants led to the identification of several NDH
subunits and a large body of proteins involved in the expression of subunit
genes and the assembly or stabilization of the NDH complex (reviewed in [34],[35]). Here, we
identify two mutants, cpn60α1 and crr27,
neither of which showed the increase in fluorescence after actinic light (AL)
illumination, indicating impaired NDH activity (Figure 1B). In contrast to
crr mutants, the cpn60α1 mutant
exhibited retarded growth and pale green-leaf phenotypes (Figure 1C). Map-based cloning identified a
single amino acid substitution (D335A) at a conserved position in
cpn60α1 (Figure 1D). Although the total levels of Cpn60α and Cpn60β
were increased in cpn60α1, possibly due to complementation
effects, the levels of many other chloroplast proteins, including NDH subunits,
were reduced to various extents (Figure S1), supporting the idea that Cpn60
has a diverse set of substrates. The reduction in NDH
(25%–50%) in cpn60α1 at least partly
explains the failure to detect NDH activity by chlorophyll fluorescence.
10.1371/journal.pbio.1001040.g001Figure 1 Characterization of cpn60α1 and
crr27 mutants.
(A) A schematic model of NDH function. The NDH complex mediates electron
transfer from the stromal reducing pool to plastoquinone (PQ). PQ
reduction in the dark depends on NDH activity and can be detected by the
transient rise of chlorophyll (Chl) fluorescence after illumination with
actinic light (AL). For simplicity, this model does not include the
information that NDH interacts with PSI. Cyt, cytochrome; PC,
plastocyanin; Fd, ferredoxin. (B) Determination of NDH activity using
Chl fluorescence analysis. The bottom curve indicates a typical trace of
Chl fluorescence in the WT plants. Leaves were exposed to AL for 5 min.
AL was turned off and the subsequent transient rise in fluorescence
ascribed to NDH activity was monitored using a PAM Chl fluorometer.
Insets are magnified traces from the boxed area.
crr27-1+Cpn60β4 and
crr27-1+Cpn60β4-HA represent
crr27-1 transformed by the WT genomic
Cpn60β4 and genomic
Cpn60β4 fused to the HA epitope-tag,
respectively. crr27-1+35S::Cpn60β4-HA and
crr27-1+35S::Cpn60β1-HA represent
crr27-1 transformed with Cpn60β4 and
Cpn60β1 cDNA, respectively, fused to the sequence encoding the
HA-tag expressed under the control of the CaMV 35S
promoter. Fluorescence levels were standardized to the maximum
fluorescence levels of closed PSII (Fm) by applying
saturating-light pulses (SP). ML, measuring light; Fo,
minimum fluorescence level of open PSII. (C) Visible phenotype of
mutants. Seedlings were cultured at 50 µmol photons
m−2 s−1 for 4 wk after
germination. (D and E) Mutations in cpn60α1 (D) and
three crr27 mutant alleles (E) are indicated. (F)
RT-PCR analysis of the Cpn60β4 transcript in WT and
crr27 mutants. ACT8 was used as a
control.
The crr27 mutants were isolated by screening Ds
transposon-tagged lines using PAM (pulse amplitude modulation) fluorometry [36],[37]. Unlike
cpn60α1, crr27 mutants did not exhibit
any visible phenotype besides impaired NDH activity (Figure 1B and 1C). The
Cpn60β4 gene was knocked out by Ds or
T-DNA insertions in three crr27 alleles, and reverse
transcription (RT)-PCR analysis did not detect any Cpn60β4
transcripts (Figure 1E and
1F). Full NDH activity was rescued by the introduction of the WT
Cpn60β4 gene into crr27-1 (Figure 1B). Two chlorophyll
fluorescence parameters, ETR (electron transport rate) and NPQ (nonphotochemical
quenching), indicate subtle defects in photosynthesis and are therefore often
used to characterize mutants with defective photosynthetic apparatus. ETR was
only slightly reduced and NPQ was not affected in crr27-1
(Figure
S2), which is consistent with the phenotypes of other
crr mutants with specific defects in NDH activity.
Accumulation of NDH Subcomplex Is Impaired in the Absence of
Cpn60β4
NDH interacts with at least two copies of PSI to form the NDH-PSI supercomplex
(Figure 2A) [35], which can be
separated by blue native (BN)-PAGE [38],[39]. To study the role of
Cpn60β4 in biogenesis of the NDH complex, thylakoid protein complexes from
WT and crr27 mutants were separated by BN-PAGE. No difference
was found in the major complex bands between WT and crr27
(Figures 2B and S3A).
However, band I, corresponding to the NDH-PSI supercomplex detected in WT, was
replaced by band II, corresponding to the subsupercomplex in
crr27, as in the NdhL-defective ndhl
(crr23) mutant (Figure
2B) [38]. Based on extensive genetic and biochemical
characterizations, we divided the NDH complex into four categories: membrane,
lumen, and A and B subcomplexes (Figure 2A) [39]. Previous mass analysis revealed that only subcomplex
A, which is composed of four plastid-encoded subunits (NdhH–NdhK) and four
nucleus-encoded subunits (NdhL–NdhO), was absent in band II (Figure 2A) [39]. Immunoblot
analysis confirmed that the levels of subcomplex A subunits NdhH and NdhL were
dramatically decreased in crr27, whereas subunits of the other
NDH subcomplexes, NDH18, NDF1, and FKBP16-2, were only slightly reduced (Figure 2C). Consistent with
the invisible growth phenotype of crr27, identical levels of D1
(PSII complex), PsaA (PSI complex), and cytochrome (Cyt) f (Cyt
b
6
f complex) were detected in
crr27 and WT (Figure 2C). In addition, no significant difference in stromal
protein levels was detected between WT and crr27-1 mutants by
clear native (CN)-PAGE and subsequent two-dimensional (2D)/SDS-PAGE (Figure
S3B). From these results, we conclude that the accumulation of NDH
subcomplex A was specifically impaired in the absence of Cpn60β4.
10.1371/journal.pbio.1001040.g002Figure 2 Accumulation of NDH subcomplex A was impaired in
crr27.
(A) A schematic model of the NDH-PSI supercomplex in chloroplasts. The
NDH complex is divided into four subcomplexes and interacts with at
least two copies of PSI to form the NDH-PSI supercomplex corresponding
to Band I, which can be detected by BN-PAGE (B). Eleven plastid-encoded
subunits are depicted by green letters; nucleus-encoded subunits are
depicted by red letters. Subcomplex A, surrounded by the red line, is
missing in the sub-NDH-PSI supercomplex corresponding to Band II, which
is detected in crr27 and ndhl mutants
(B). (B) BN-PAGE analysis of thylakoid protein complexes. After
electrophoresis, the gel was stained with CBB. The
complexes are identified in the original non-stained gel in Figure
S3A. (C) Immunoblot analysis of the thylakoid proteins from
various genetic backgrounds with the indicated antibodies. Thylakoid
proteins were loaded on an equal chlorophyll basis, and the series of
dilutions is indicated.
Heterooligomeric Chaperonin Complex Formation of Cpn60β4 with
Cpn60β1–β3 and Cpn60α1
Previous transcriptomic analysis [26] indicated that the expression level of the
Cpn60β
4 gene is lower than that of
other Cpn60β genes. Furthermore, the Cpn60β4 subunit
could not be visualized with Coomassie Brilliant Blue (CBB) staining in the 2D
CN/SDS-PAGE gel, whereas other Cpn60β subunits and Cpn60α1 were detected
[27],
suggesting that the stoichiometry of Cpn60β4 is extremely low compared to
the other Cpn60β subunits. To confirm the accumulation of Cpn60β4 in WT,
we separated total stromal protein complexes isolated from WT plants by CN-PAGE.
The protein band corresponding to the position of the Cpn60 complex was excised
from the gel (Figure S4A) and analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) analysis using the linear ion-trap triple quadrupole
(LTQ)-Orbitrap XL-HTC-PAL system, which provides high mass accuracy, high
resolution, and high sensitivity. The values of Mowse score, Protein match, and
emPAI (exponentially modified Protein Abundance Index) are commonly used to
estimate relative protein levels. LC-MS/MS analyses detected the Cpn60β4
protein, but its level was significantly lower than those of the other three
Cpn60β proteins (Figure S4B; Table S1).
Consistent with the apparent mutant phenotype (Figures 1 and 2), we confirmed the accumulation of
Cpn60β4 in WT (Figure S4C).
In contrast to cpn60α1, the crr27 mutation
did not affect total Cpn60α or Cpn60β levels (Figures 3A and S1A). To
study the role of Cpn60β4, a chimeric gene encoding an HA (influenza
hemagglutinin protein epitope) tag fused to the C-terminus of Cpn60β4 was
introduced into crr27-1. This transformation fully restored NDH
activity (Figure 1B),
indicating that the HA-tag did not affect the function of Cpn60β4. We also
overexpressed HA-tagged Cpn60β1 and Cpn60β4 in crr27-1
under control of the cauliflower mosaic virus (CaMV) 35S
promoter. NDH activity and NDH subcomplex A level were rescued only in the
35S::Cpn60β4-HA lines (Figures 1B and 2C), indicating that Cpn60β1 cannot
complement the function of Cpn60β4, even under the control of the same
promoter.
10.1371/journal.pbio.1001040.g003Figure 3 Analysis of the Cpn60β4 subunit.
(A) Cpn60β4 is localized to the chloroplast stroma. Freshly isolated
chloroplasts from various genotypes were separated into membrane and
stromal fractions. Immunoblot analysis was performed using the indicated
antibodies. RbcL and Cyt f were detected as loading and
fractionation controls. (B) Stromal protein complexes isolated from
crr27-1 complemented by Cpn60β4-HA were
separated by CN-PAGE, followed by 2-dimensional SDS-PAGE. The proteins
were immunodetected with specific antibodies. A short arrow indicates
the position of the Cpn60 complex. (C) Heterooligomeric complex
formation between Cpn60β4, Cpn60α1, and
Cpn60β1–β3. Chaperonin complex containing Cpn60β4 was
purified from the crr27-1 mutant plants expressing
HA-tagged Cpn60β4 using the µMACS HA isolation kit. After
elution, total proteins were separated by 7.5% SDS-PAGE and
stained with CBB. The signals were quantitatively analyzed with
Imagemaster software (Amersham Pharmacia Biotech). The stoichiometry of
Cpn60β4, Cpn60β1–β3, and Cpn60α1 in the specific
chaperonin complex containing Cpn60β4 was estimated to be 17:37:46
and 14:39:47 in the two independent purifications (E1 and E2),
respectively.
Cpn60β4 localized to the chloroplast stroma (Figure 3A) and co-migrated with other
Cpn60β and Cpn60α subunits in CN-PAGE (Figures 3B and S4),
suggesting that Cpn60β4 is an intrinsic subunit of the Cpn60 complex. To
examine this possibility, HA-tagged Cpn60β4 was enriched from the stromal
fraction isolated from crr27-1 plants complemented with
Cpn60β4-HA using the µMACS HA isolation kit (Miltenyi Biotec) under
previously established conditions [11]. Because the additional HA
tag in the C-terminus does not affect the function of Cpn60β4 (Figure 1B), their interacting
proteins might also be co-purified. Total isolated proteins were separated by
SDS-PAGE (Figure
S5A) and further analyzed by LC-MS/MS analysis. Both Cpn60α1 and
Cpn60β1–β4 subunits were detected in the purified sample and MS
analysis showed that they were the most abundant proteins (Tables 1 and S2),
implying that Cpn60β4 forms a specific heterooligomeric Cpn60 complex with
Cpn60α1 and other Cpn60β subunits.
10.1371/journal.pbio.1001040.t001Table 1 Summary of the chaperonin and NDH subunits detected in the
Cpn60β1 and Cpn60β4 IP fractions.
Protein Name Cpn60β1 IP Cpn60β4 IP emPAI Ratioa
Mowse Score Protein Match Coverage (%) Mowse Score Protein Match Coverage (%)
Cpn60α1 2,177 64 69 6,369 188 72 3.9
Cpn60β1 6,794 230 70 5,591 202 68 0.47
Cpn60β2 5,689 178 59 5,356 182 67 0.87
Cpn60β3 3,624 119 36 3,448 123 37 1.26
Cpn60β4 226b
13b
3b
3,348 156 74 101
NdhJ 57 2 15 — — — —
NdhH 44 2 4 614 24 53 43.4
a. emPAI Ratio means the ratio between emPAI score of proteins
isolated from Cpn60β4 IP and Cpn60β1 IP fraction.
b. The sequences of these 13 peptides are identical between
Cpn60β1 and Cpn60β4, suggesting that they are sequenced from
Cpn60β1. RT-PCR showed that crr27-1 is a null
mutant (Figure
1F).
The molecular masses of Cpn60β4-HA, Cpn60β1–β3, and Cpn60α1
are 64.4, 58.2, and 57.1 kDa, respectively, which enables us to distinguish them
in SDS-PAGE. The purified proteins were subjected to 7.5% SDS-PAGE and
three major bands with molecular masses of approximately 60 kDa were visualized
with CBB staining (Figure
3C). Based on the mobility, three bands should correspond to
Cpn60β4-HA, Cpn60β1–β3, and Cpn60α1 (Figure 3C). Quantitative estimation of these
signals showed that the level of Cpn60α1 is about 50% of all of the
chaperonin subunits, which is consistent with the proposal that the Cpn60
complex is composed in a α7β7 stoichiometry [24],[25]. However, the level of
Cpn60β4 in the chaperonin complex was lower (∼15%) (Figure 3C), implying that
approximately two molecules of Cpn60β4 are included in double rings, as well
as five molecules of other Cpn60β subunits. Based on the stoichiometry of
the Cpn60β4 subunit in total Cpn60 subunits [26],[27], the majority of the Cpn60
complex is unlikely to contain Cpn60β4, and we estimated its stoichiometry
in the specific complex including Cpn60β4.
NdhH Is Specifically Associated with Cpn60β4
Given that Cpn60β4 is an intrinsic subunit of the chaperonin complex (Figure 3) and that the NDH
subcomplex A was missing in the crr27 mutants (Figure 2), it is very likely
that the specific chaperonin complex containing Cpn60β4 is required for the
folding of at least one subunit of the NDH subcomplex A. If this is the case,
the interacting NDH subunit would be copurified with Cpn60β4-HA. To
determine differences in substrate specificity, protein complexes containing
Cpn60β1 were isolated using the 35S::Cpn60β1-HA lines.
Neither Cpn60 nor NDH subunits were detected in untransformed WT plants, which
were used as a negative control (Figure S5A; Table S2),
excluding the possibility of non-specific binding to the magnetic beads.
Cpn60α1 and Cpn60β1–β3 were co-purified with both Cpn60β1
and Cpn60β4 (Tables 1
and S2).
These results confirmed that Cpn60α1 and Cpn60β1–β4 form a
heterooligomeric complex in vivo.
The ratio between protein emPAI scores can be used to estimate the relative
amounts of protein in the different samples [40]. The emPAI ratios of
Cpn60α1 and Cpn60β1–β3 detected in Cpn60β4- and
Cpn60β1-purified samples were 3.9 and 0.47–1.26
(Cpn60β4/Cpn60β1), respectively, suggesting that comparable amounts of
Cpn60 complexes were used for MS analysis. This estimation was confirmed by the
similar intensity of the Cpn60 subunit bands detected by SDS-PAGE (Figure
S5A). Interestingly, 24 peptides of an NDH subunit, NdhH, were found in
the Cpn60β4-purified fraction. No other NDH subunits or NDH biogenesis
factors were found in any sample other than the NdhJ detected in the
Cpn60β1-purified extraction (Table 1). Although NdhH was also co-purified with Cpn60β1, only
two NdhH peptides were detected (Table 1 and Figure S5B) and the emPAI ratio of NdhH from
Cpn60β4- and Cpn60β1-purified samples was 43.4. Aside from the
nonspecific proteins detected in WT as well as chaperonin subunits detected in
Cpn60β4-IP sample, NdhH was the most abundant protein found in the
Cpn60β4-purified sample (Table S2). These results indicate that the
Cpn60 complex containing Cpn60β4 can specifically recognize unfolded NdhH.
NdhH was also detected in the Cpn60β1-purified sample (Table 1), suggesting that
Cpn60 complexes containing Cpn60β1 also can interact with NdhH. However,
when Cpn60β1-HA was introduced into crr27-1, the
transformation did not rescue NDH activity (Figures 1 and 2), suggesting that Cpn60 complexes lacking
Cpn60β4 cannot produce native NdhH even though they can bind to it. This
idea is consistent with the fact that crr27 accumulates the
band II subsupercomplex (Figure
2).
Assembly of NDH Subcomplex A Is Impaired in crr27
Although the functional NDH complex is localized to the thylakoids, three
assembly intermediate complexes including NdhH are present in the chloroplast
stroma (Figure
S6) [41]. Nuclear-encoded factors CRR6 and CRR7 may be
required for integration of these intermediates into thylakoids to form the
functional NDH complex. In crr27-1, the level of
stroma-localized NdhH was significantly reduced (Figure
S6A). Furthermore, 2D CN/SDS-PAGE and immunoblot studies showed that the
accumulation of the 500 kDa and 400 kDa intermediate complexes was impaired in
crr27-1 (Figure S6B), implying that only NdhH folded
by the Cpn60 complex including Cpn60β4 can be efficiently incorporated into
these two assembly intermediates and further into thylakoids. These results also
suggest that the folding of NdhH via the Cpn60 complex containing Cpn60β4
occurs at the initial step of NDH subcomplex A biogenesis.
The Unique C-Terminus of Cpn60β4 Is Required for Its Specific
Function
Based on a transcriptome database of Arabidopsis, ATTED-II [42], we found
that the Cpn60β4 gene, but not other Cpn60
genes, is co-expressed with genes encoding NDH subunits and NDH biogenesis
factors (Table
S3). This pattern is consistent with our findings that Cpn60β4 is
specifically required for the folding of NdhH. However, the question remains as
to why the other Cpn60β proteins cannot complement the function of
Cpn60β4.
The mycobacterial GroEL1 has a histidine-rich C-terminus that appears to be
critical for its specific function in association with proteins required for
bacterial biofilm formation [18]. Cpn60β4 also contains a C-terminal extension
that is not conserved in other Cpn60β proteins (Figures 4A and S7).
Although the C-terminus of Cpn60β4 is not conserved in plants, the region is
rich in positively charged residues (Figure 4A). To study whether the C-terminus
is important for Cpn60β4 function, HA-tagged Cpn60β4 lacking the
C-terminus or exchanged by the short C-terminal tail of Cpn60β1 was
expressed in crr27-1 (Figure 4B). 2D CN/SDS-PAGE immunoblot
analysis showed that the mutant versions of Cpn60β4 can be incorporated into
the Cpn60 complex (Figure
4C), excluding the possibility that the C-terminus of Cpn60β4 is
required for the stabilization or formation of the chaperonin complex. Although
the levels of mutant Cpn60β4 were approximately twice those of the WT
version of Cpn60β4 in the stroma, NdhH levels in thylakoids were reduced by
approximately one half in the Cpn60β4-HA lines (Figure 4B), resulting in the reduction of NDH
activity (Figure 4D). These
results indicate that the folding efficiency of NdhH was reduced in the absence
of the Cpn60β4-specific C-terminus.
10.1371/journal.pbio.1001040.g004Figure 4 C-terminal of Cpn60β4 is required for the Cpn60β4
function.
(A) C-terminal alignment of Cpn60 subunits. Cpn60β4 from
Arabidopsis thaliana (At), Arabidopsis
lyrata (Al), Oryza sativa (Os),
Zea mays (Zm), and Populus
trichocarpa (Pt) were aligned with the
Arabidopsis Cpn60β1–β3 and
Cpn60α1 and the E. coli GroEL. Positions of the
deletion and the C-terminal swapping are shown. The number of negatively
(−) and positively (+) charged residues in the region is
indicated. (B) Protein blot analysis of NdhH and Cpn60β4. RbcL and
Cyt f were used as loading controls. The series of
dilutions is indicated. (C) The C-terminus of Cpn60β4 is not
essential for the assembly of the chaperonin complex. Stromal protein
complexes isolated from crr27-1 complemented by
Cpn60β4 truncated in the C-terminus (Cpn60β4(-C)-HA) and from
crr27-1 transformed by Cpn60β4, in which the
C-terminus was exchanged for that of Cpn60β1
(Cpn60β4(+β1C)-HA), were separated by CN-PAGE, followed by
2-D SDS-PAGE. The proteins were immunodetected with specific antibodies.
A short arrow indicates the position of the Cpn60 complex. (D) NDH
activity monitored by Chl florescence as in Figure 1B.
crr27-1+Cpn60β4-HA,
crr27-1 transformed by WT genomic
Cpn60β4;
crr27-1+Cpn60β4(-C)-HA,
crr27-1 transformed by Cpn60β4 truncated in the
C-terminus; crr27-1+Cpn60β4(+β1C)-HA,
crr27-1 transformed by Cpn60β4, in which the
C-terminus was exchanged for that of Cpn60β1;
crr27-1+Cpn60β4(CM)-HA,
crr27-1 transformed by Cpn60β4 containing the
amino acid alterations in the wall of central cavity. All of the
proteins were fused to the HA-epitope tag on their C-termini. (E) The
net charge of each subunit wall exposed to the central cavity in the
cis ring. As shown in Figure
S7, the multiple charged residues, which are highly conserved
among Cpn60β4 but not in Cpn60β1–β3 subunits, were
altered in AtCpn60β4 to the corresponding amino acids in
AtCpn60β1, resulting in a net charge of −4
(AtCpn60β4(CM)). (F) Immunoblot analysis of NdhH and Cpn60β4
from crr27-1 expressing the AtCpn60β4(CM)-HA
protein. (G) Protein blot analysis of NdhH and Cpn60β4.
crr27-1+Cpn60β4(ΔC+CM)-HA,
crr27-1 transformed by a multiple mutant version of
Cpn60β4 containing the amino acid alterations in the wall of central
cavity (AtCpn60β4(CM)) and truncated in its C-terminus
(Cpn60β4(-C)). The proteins were fused to the HA-tag on their
C-termini. RbcL and Cyt f were used as loading controls
and the series of dilutions is indicated.
In the absence of the C-terminal tail, NdhH is still partially assembled (Figure 5B). We also
transformed crr27 with Cpn60β1 fused with the Cpn60β4
C-terminal tail, but NdhH folding activity was not complemented (unpublished
data). These results suggest that other features of Cpn60β4 are required for
its specific function. Protein sequence alignment revealed that the ATP/ADP and
Mg2+ binding sites are highly conserved between Cpn60β4
and the other three Cpn60β subunits (Figure S7), which is consistent with the fact
that Cpn60β4 is an intrinsic subunit of the Cpn60 complex (Figure 3). However, the
proposed substrate-binding residues are less conserved (Figure S7),
which may explain why Cpn60β4 has a high affinity specifically for NdhH.
Protein sequence alignment also showed that up to 31 amino acid residues are
highly conserved among the putative Cpn60β4 orthologs, but their properties
are different from the corresponding residues in other Cpn60β subunits
(Figures
S7 and S8). Three-dimensional (3-D) structure
analyses mapped these residues to the apical, intermediate, and equatorial
domains of the Cpn60β4 subunit (Figure S8).
10.1371/journal.pbio.1001040.g005Figure 5 Evolutionary analyses of the chloroplast Cpn60β4
subunits.
(A) A phylogenetic tree of the chloroplast chaperonin 60 and
cyanobacterial GroEL proteins. Chaperonin subunits from fully sequenced
genomes of angiosperms (Arabidopsis thaliana,
Arabidopsis lyrata, Populus
trichocarpa, Zea mays, and Oryza
sativa) and a bryophyte (Physcomitrella
patens) were retrieved from GenBank or Phytozome (http://www.phytozome.net/). The GroEL sequences from
Synechocystis SP. PCC 6803 and a Cpn60β
sequence from Marchantia polymorpha, which is used to
complement the crr27-1 mutant in (B), are also
included. The tree was generated using both Maximum-likelihood (ML) and
Bayesian methods. ML and Bayesian consensus trees were topologically
congruent and only ML topology is shown and drawn to scale. Numbers at
each node in the ML tree signify bootstrap/posterior probability values
(>50%) from ML and Bayesian methods, respectively. Support
values less than 50 are shown as hyphens (−). The sequence
alignments used to generate the ML and Bayesian tree are available in
Dataset S1. Proteins highlighted with yellow boxes were
investigated in this study. (B) Immunodetection of chloroplast proteins
from WT, crr27-1, crr27-1 complemented
by AtCpn60β4-HA, and three lines of crr27-1
complemented by MpCpn60β-HA (L1–L3).
Of the 31 conserved residues in Cpn60β4, several charged amino acids are
located inside the cavity (Figure S8). The negatively charged GroEL
cavity wall is required for rapid folding of some substrates [43]. In
E. coli, each GroEL subunit has 27 negatively and 21
positively charged amino acids exposed to the central cavity in the
cis-conformation, resulting in a net charge of −6
[44].
By analogy with E. coli GroEL, Cpn60α1 and Cpn60β1 have
net charges of −4 and −6, respectively. However, Cpn60β4 in
Arabidopsis has a more positive charge of 0, and this trend
is found for Cpn60β4 in other plants (charges ranging from −2 to
+2) (Figure 4E). To
investigate the significance of the positively charged cavity wall in the
folding of NdhH, the multiple charged residues, which are highly conserved in
Cpn60β4 but not in Cpn60β1–β3 subunits, were converted to the
corresponding amino acids of AtCpn60β1 and the mutant genes were introduced
into crr27-1 plants. The sites correspond to 5 out of 31 amino
acid residues indicated in Figure S8. Although the mutant Cpn60β4
has a net charge of −4 (Figure 4E), NdhH level and NDH activity were fully rescued in the
transformed plants (Figure 4D and
4F). We also transformed a version of Cpn60β4 with the amino acid
alterations on the wall of the central cavity and the deletion of the C-terminus
into crr27-1. In these lines, NdhH levels in thylakoids were
reduced to ∼50% of the Cpn60β4-HA lines (Figure 4G), similar to the results in
crr27-1 transformed by Cpn60β4 lacking its C-terminus
(Figure 4B). These
results suggest that the positive charge of the cavity wall is not crucial for
the folding of NdhH. The residues specifically conserved in the putative
Cpn60β4 orthologs are dispersed throughout the molecule except for five
positively charged sites facing the cavity wall (Figure S8),
and it is not feasible to determine the sites responsible for the specific
function by the site-directed mutagenesis. Proper folding of NdhH may require
both the drastic alteration in the sequence as well as the C-terminal
extension.
Evolution of the Cpn60β4 Subunit
Both chloroplast Cpn60 and NDH complexes are thought to have originated from
their cyanobacterial ancestors, GroEL2 [45] and NDH-1 [32]. NdhH is
highly conserved in Arabidopsis, Physcomitrella
patens (moss) and cyanobacteria (Figure S9).
NdhH is a 45.5 kDa protein with α+β domains (Figure S9)
[46], and
theoretically it can be fully encapsulated within the chaperonin cage. In
contrast to NdhH, the structure of Cpn60 is not conserved among organisms. To
clarify the evolution and ancestry of Cpn60β subunits in plants, we compared
the amino acid sequences of members in several fully sequenced genomes (Figure 5A). In addition to the
distinct clades of Cpn60α and Cpn60β, Cpn60β proteins were further
divided into two clades: putative AtCpn60β4 orthologs and other Cpn60β
genes (major Cpn60β). The orthologs of AtCpn60β1–β3 were found
in the closely related Arabidopsis lyrata, but two major
Cpn60β proteins of poplar (Populus trichocarpa) were
related only to AtCpn60β3. In contrast, the major Cpn60β subunits of
monocots form a different subclade, and maize (Zea mays) and
rice (Oryza sativa) each contain two major Cpn60β subunits
(Figure 5A). These facts
suggest that gene duplication of major Cpn60β subunit genes took place
independently both in monocots and eudicots. In contrast, a single copy of the
putative Cpn60β4 ortholog was detected in angiosperms (Figure 5A).
A total of three Cpn60β subunits were found in Physcomitrella
patens and they are related to major Cpn60β subunits in
angiosperms (Figure 5A).
Notably, no ortholog of Cpn60β4 was found in P. patens. The
phylogenetic tree indicates that the origin of Cpn60β4 can be traced to the
origin of land plants and that Cpn60β4 was lost in the descendent lineage of
bryophytes. Due to the low bootstrap support of the evolutionary relationships
between angiosperm and bryophyte major Cpn60β subunits (72/0.90 as shown in
Figure 5), it is also
likely that the Cpn60β4 orthologs were produced by a gene duplication event
that took place only in a common ancestor of angiosperms and underwent a rapid
rate of evolution to obtain the novel function. In any case, P.
patens should use a different mechanism to assist the folding of
NdhH, as it also contains the chloroplast NDH complex.
To study whether the Cpn60β subunits in bryophytes can facilitate the folding
of NdhH, we introduced the Cpn60β gene isolated from
liverwort (Marchantia polymorpha) into crr27-1
(Figure 5B). We
identified only one gene copy encoding Cpn60β in M.
polymorpha, possibly due to incomplete genome information.
Immunoblots detected a trace amount of NdhH in the thylakoids of three
transgenic lines, although the levels of MpCpn60β were comparable to those
of AtCpn60β4 in crr27-1 transformed by
AtCpn60β4-HA (Figure 5B), suggesting that MpCpn60β
partially rescues the phenotype of crr27-1 and that Cpn60β
in bryophytes retains its ability in assisting the folding of NdhH. As
MpCpn60β forms the heterologous Cpn60 complex with AtCpn60α, we could
not compare the efficiency of the NdhH folding with the complex including
AtCpn60β.
Discussion
Although multiple chaperonin genes are present in a significant proportion of
bacteria and eukaryotes, the function and biological significance of this kind of
divergent evolution have yet to be revealed [13]. Generally, it is thought that
the major subunits fulfill the housekeeping chaperonin function. The minor
chaperonin subunits may increase the general chaperoning ability by elevating the
chaperonin abundance in response to different environmental conditions. In this
study, we demonstrated that the highly divergent chloroplast chaperonin subunit
Cpn60β4 is specifically and strictly required for the folding of the NDH subunit
NdhH.
Although Cpn60β4 is highly divergent from the major Cpn60β subunits (Figure 5), it is an intrinsic
chaperonin subunit and forms a heterooligomeric Cpn60 complex with Cpn60α1 and
other Cpn60β subunits (Figure
3 and Table 1),
suggesting the involvement of this specific Cpn60 complex in assisting the folding
of some proteins. In line with this idea and the crr27 phenotype,
the NDH subunit NdhH was copurified with the heterooligomeric Cpn60 complex
including Cpn60β4 (Table
1). Although the Cpn60 complex lacking Cpn60β4 also interacts with NdhH
with less affinity (Tables 1
and S2), it
cannot produce native NdhH for further NDH complex assembly (Figures 1 and 2), implying that Cpn60β4 is required for
both high-affinity binding and folding of NdhH. In contrast with the observation in
R. meliloti, B. japonicum, and R.
leguminosarum
[15]–[17], in which the
function of the unusual chaperonins can be partially replaced by other chaperonins,
Cpn60β4 is absolutely required for the folding of NdhH. These data support the
proposal that multiple chaperonin subunits have evolved to assist the folding of
specific proteins, although the functional specialization is not absolute in some
cases.
What is the structural basis for the functional specialization of Cpn60β4? We
clarified that the unusual C-terminus of Cpn60β4 is required for the full
activity of the Cpn60 complex containing Cpn60β4 for folding NdhH. The
aforementioned GroEL1 protein in M. smegmatis also
has an unusual histidine-rich C-terminus, which was shown to be essential for the
specific function of GroEL1 [18]. These observations suggest that modification of the
C-terminus is necessary to facilitate the folding of specific targets. This idea is
consistent with the fact that the many bacterial genomes encode an additional
chaperonin with an unusual C-terminus [13].
The C-terminus of Cpn60β4 is not required for the formation of the specific
chaperonin complex containing Cpn60β4 (Figure 4C). Thus, it should have some special
functions in other steps during the folding of NdhH. The chaperonin complex
containing Cpn60β4 and its substrate NdhH can be purified via the HA tag fused
with the C-terminus of Cpn60β4 (Table 1 and Figure
3C). As the cis ring is capped by a co-chaperone, it is
likely that the C-terminal tail of Cpn60β4 extends from the
trans ring so that the chaperonin complex containing
Cpn60β4 can be trapped by microbeads coupled with HA antibody. Alternatively,
the chaperonin complex was purified by the C-terminal tail extruding from the
cis ring, which was not capped but already associated with the
substrate NdhH. The protruded C-terminal tail of Cpn60β4 might promote the high
affinity binding with NdhH, ensuring that the nonnative NdhH can be efficiently
captured by the Cpn60 complex containing Cpn60β4. In addition, enclosure of the
nonnative NdhH protein inside the cavity by a co-chaperonin will lead to the
encapsulation of the C-terminus. Consequently, the C-terminus might also contribute
to the specific function of Cpn60β4 in assisting the folding of NdhH in the
cavity. It has been reported that changing the length of the C-terminus of GroEL can
affect the folding speed of some substrates [43],[47]. Farr et al. provided further
evidence that the elongated C-terminus perturbed the ATPase activity, and the
disturbance of the rate of ATP hydrolysis resulted in the modification of the
folding rate of some substrates [48]. The physical properties of the C-terminus [49] such as the
length [43],[47], hydrophilicity
[50], and
hydrophobicity [43]
have been proposed to be critical for the substrate folding in the cavity. We also
found that the C-terminus of the Cpn60β4 is rich in positively charged residues
(Figure 4A). Thus, it is
likely that these unusual C-termini provide specific environments in the chaperonin
cavity and/or modify the chaperonin ATPase rate of the chaperonin complex.
The deletion of up to 27 residues of the C-terminal tail of GroEL does not affect the
growth of E. coli
[51],[52], suggesting
that the C-terminal motif does not play an essential role in assisting the folding
of substrates. It is also true that Cpn60β4 lacking its unusual C-terminal tail
still can partially assist the folding of NdhH (Figure 4B). Our results showed that the charged
residues exposed on the cavity wall do not play a critical role for the specific
function (Figure 4F). In
addition, we discovered many residues that are potentially important for the
specific function of Cpn60β4 (Figures S7 and S8). Among
them, several residues are mapped to the intermediate domain or near the ATP/ADP
binding site (Figure
S8). The E. coli GroEL with specific mutations in this
region can improve the folding activity for green fluorescent protein (GFP), most
likely through the adjustment of the ATPase activity [53]. Recently, the apical domain of
GroEL1 from Mycobacterium tuberculosis was shown to be sufficient
for binding the specific substrate KasA [54]. Notably, some residues are
specifically conserved in the apical domain of the putative Cpn60β4 orthologs
(Figure
S8). In addition, other conserved residues in AtCpn60β4 orthologs may
be required for the formation of the Cpn60 complex including Cpn60β4 with
certain stoichiometry or to provide certain physical features of the cavity wall.
With the exception of the C-terminal extension, we could not specify the residues
that are required for Cpn60β4 function. It is likely that some residues that are
specifically conserved in the putative Cpn60β4 orthologs cumulatively contribute
to their specific function, and this drastic evolution may have been necessary to
assist the folding of NdhH. However, it is puzzling that MpCpn60β, which is
related to the major AtCpn60β subunits, partly complemented the function of
Cpn60β4 and that bryophytes do not contain the Cpn60β4 orthlogs (Figure 5).
Specific mutations of GroEL can improve the folding activity of a specific protein
[53]. However,
mutated GroEL has a reduced ability to fold a variety of natural substrates,
suggesting a conflict between the specific ability of GroEL to fold particular
substrates and its general ability in assisting the folding of a wide range of
substrates [53]. By
combining all of the features acquired during the evolution of plants, Cpn60β4
allows the Cpn60 complex to assist the folding of the specific substrate, NdhH.
However, other Cpn60 subunits, especially the major Cpn60β proteins, may have
become optimized to support the efficient folding of other obligate substrates.
Through this kind of divergent evolution, the chaperonin system can resolve the
apparent conflict between specialization and generalization of its function.
Materials and Methods
Plant Material and Growth Conditions
Arabidopsis thaliana (ecotypes Col-0 and Nössen) plants
were grown in a growth chamber (50 µmol photons m−2
s−1, 16 h photoperiod, 23°C) for 3 to 4 wk.
cpn60α1 was mutagenized by ethyl methanesulfonate [55].
crr27-3 (ecotypes Nössen) was isolated from a
collection of Ds transposon insertion lines [36].
crr27-1 (SALK_136518, Col-0) and crr27-2
(SALK_064887, Col-0) were obtained from the ABRC Stock Center. For
complementation experiments, vectors were transferred into Agrobacterium
tumefaciens C58C by electroporation, and the bacteria were used to
transform crr27-1 by floral dipping.
Chlorophyll Fluorescence Analysis
Chlorophyll fluorescence was measured using a MINI-PAM (pulse amplitude
modulation) portable chlorophyll fluorometer (Walz, Effeltrich, Germany). The
transient increase in chlorophyll fluorescence after turning off AL was
monitored as previously described [33]. Leaves were exposed to
AL (50 µmol photons m−2 s−1) for 5 min.
AL was turned off and the subsequent transient rise in fluorescence ascribed to
NDH activity was monitored. Fluorescence levels were standardized to the maximum
fluorescence levels of closed PSII (Fm) by applying
saturating-light pulses (SP).
To investigate the light intensity dependence of two chlorophyll fluorescence
parameters, ETR and NPQ, measuring light (650 nm, 0.1 µmol photons
m−2 s−1) was used to excite the minimum
fluorescence at open PSII centers in the dark-adapted state
(Fo). A saturating pulse of white light (800
ms, 8,000 µmol photons m−2 s−1) was
applied to determine the maximum fluorescence at closed PSII centers in the dark
(Fm) or during light illumination
(Fm’). The steady-state fluorescence level
(Fs) was recorded during AL illumination (15–1,000
µmol photons m−2 s−1). These
photosynthetic parameters were recoded 2 min after the change of AL intensity.
ΦPSII was calculated as (Fm′ –
Fs)/Fm′. ETR and NPQ were calculated
as ΦPSII×photon flux density (µmol photons
m−2 s−1) and (Fm –
Fm′)/Fm′, respectively.
Thylakoid Membrane Isolation, BN-PAGE, CN-PAGE, and Immunoblot
Analysis
Freshly isolated chloroplasts were osmotically ruptured in buffer containing 20
mM HEPES–KOH (pH 7.6), 5 mM MgCl2, and 2.5 mM EDTA. Thylakoid
membranes were separated from the stromal fraction by centrifugation (17,000 g
for 10 min at 4°C). CN-PAGE, BN-PAGE, and 2-D CN/SDS-PAGE were performed
according to previous reports [27],[38]. For immunoblot analysis, thylakoid and stromal
proteins were loaded by equal chlorophyll and protein content, respectively. The
stromal protein contents were determined with a Bio-Rad Protein Assay Kit (cat.
No. 500-0006). Immunoblot signals were detected with an ECL plus Western
Blotting Detection Kit (GE Healthcare) and visualized with a Luminescent image
analyzer (LAS)-3000 (Fuji Film). Immunoblots were quantified by Imagemaster
software (Amersham Pharmacia Biotech).
RNA Isolation and RT-PCR Analysis
Total RNA was isolated from Arabidopsis leaves with an RNeasy
Plant Mini Kit (Qiagen) and treated with DNase I (Invitrogen). Total RNA (5
µg) was reverse transcribed using the SuperScript III First-Strand
Synthesis System (Invitrogen) in a total volume of 20 µl. The cDNA was
used in 35 cycles of PCR with the following primers: 5′-TGGCTCTGTCACCAAGAAGCTTCAG-3′ and
5′-GCTTTCTGGGTGAATCCGTTGGTAA-3′. RT-PCR
products were separated on agarose gels and detected by ethidium bromide
staining.
Affinity Chromatography of the Chaperonin-Substrate Complexes
Cpn60-substrate complexes were isolated from crr27-1 plants
expressing HA-tagged Cpn60β subunits with the µMACS HA isolation kit
under the conditions previously established [11], with a minor modification.
Freshly isolated chloroplasts were osmotically ruptured in lysis buffer (50 mM
Tris-HCl pH 8.0, 0.01% Tween 20, 10 mM MgCl2, 20 mM glucose,
20 U/ml hexokinase) plus protease inhibitors (Complete mini, Roche). Within 10 s
after lysis, ADP was added to a final concentration of 10 mM. Thylakoid
membranes were pelleted by centrifugation at 20,000 g for 10 min at 4°C and
the supernatants were transferred to new tubes. NaCl was added to the
supernatants to a final concentration of 150 mM and then mixed with 50 µl
anti-HA MicroBeads (Miltenyi Biotec.). After incubation for 2 h at 4°C, the
beads were transferred to columns placed in a magnetic field. Columns were
rinsed four times with 200 µl washing buffer I (50 mM Tris-HCl pH 8.0,
1% Triton, 0.5% Sodium deoxycholate, 150 mM NaCl, 5 mM ADP) and
twice with 200 µl washing buffer II (50 mM Tris-HCl pH 8.0, 1%
Triton, 150 mM NaCl, 5 mM ADP). After final washing with 200 µl washing
buffer III (25 mM Tris-HCl pH 7.5, 5 mM ADP), the chaperonin-substrate complex
was eluted with elution buffer (50 mM Tris-HCl pH 6.8, 50 mM DTT, 1% SDS,
1 mM EDTA, 0.005% bromophenol blue, 10% glycerol). Total protein
was separated on 12.5% SDS-PAGE gels (Perfect NT Gel, DRC) and stained
with CBB. SDS-PAGE lanes were cut into four slices and analyzed by LC-MS/MS
analyses.
Mass Spectrometric Analysis and Database Searching
Peptide Preparation and LC-MS/MS analyses were performed as previously described
[39]. The
excised bands were treated twice with 25 mM ammonium bicarbonate in 30%
(v/v) acetonitrile for 10 min and 100% (v/v) acetonitrile for 15 min, and
then dried in a vacuum concentrator. The dried gel pieces were treated with 0.01
mg/ml trypsin (sequence grade; Promega)/50 mM ammonium bicarbonate and incubated
at 37°C for 16 h. The digested peptides were recovered twice with 20
µl 5% (v/v) formic acid/50% (v/v) acetonitrile. The
extracted peptides were combined and then dried in a vacuum concentrator.
LC-MS/MS analyses were performed on an LTQ-Orbitrap XL-HTC-PAL system. MS/MS
spectra were compared by the MASCOT server (v. 2.2) against TAIR8 (The
Arabidopsis Information Resource) with the following search parameters: set-off
threshold at 0.05 in the ion-score cut-off; peptide tolerance, 10 ppm; MS/MS
tolerance, ±0.8 Da; peptide charge, 2+ or 3+; trypsin as enzyme
allowing up to one missed cleavage; carboxymethylation on cysteines as a fixed
modification; and oxidation on methionine as a variable modification.
Phylogenetic Analysis
Chaperonin protein sequences were first aligned using the CLUSTALX 1.83 program
[56].
The protein alignment was further refined manually and 534 conserved sites were
used for phylogenetic analysis. Phylogenetic trees were constructed by using
both the maximum likelihood (ML) and Bayesian methods to ensure the robustness
of our analysis. ML trees were constructed by using PHYML version 2.4 [57] with WAG
model selected via MODELTEST 3.06 [58], and support for each
branch was assessed using bootstrap analyses with 100 bootstrap replicates.
Bayesian trees were constructed using MrBayes software [59] with the WAG model.
Four chains of Markov chain Monte Carlo were run, sampling one tree every 1,000
generations for 1,000,000 generations, starting with a random tree. The first
50,000 generations were excluded as burn-in to ensure that the chains reached
stationary. The posterior probability was used to estimate nodal robustness.
Homology Modeling
The structure model of the Cpn60β4 protein was obtained by homology modeling
using the SWISS-MODEL server (http://swissmodel.expasy.org/) [60], and the crystal structure
of E. coli GroEL (PDB 1AON, Chain A) was used as a modeling
template. The 3-D predicted structure was visualized using the PyMol
software.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative
or GenBank/EMBL databases under the following accession numbers: At
(Arabidopsis thaliana) Cpn60α1 (At2g28000),
AtCpn60α2 (At5g18820), AtCpn60β1 (At1g55490), AtCpn60β2 (At3g13470),
AtCpn60β3 (At5g56500), AtCpn60β4 (At1g26230), AtNdhH (BAA84443), Al
(Arabidopsis lyrata) Cpn60α1 (481708), AlCpn60α2
(488768), AlCpn60β1 (474606), AlCpn60β3 (495739), AlCpn60β4
(890123), Sy (Synechocystis SP. PCC 6803) GroEL-1 (NP_440731),
SyGroEL-2 (NP_442170), SyNdhH (CAA43057), GroEL (Escherichia
coli) (NP_418567), and Pp (Physcomitrella patens)
NdhH (BAC85094).
Supporting Information
Figure S1 Protein blot analysis of chloroplast proteins. Equal amounts of total protein
extracted from the leaves of WT, cpn60α1, and
crr27-1 plants were separated by SDS-PAGE and
immunodetected by antibodies against stromal proteins Cpn60α,
Cpn60β, and RbcL (A) or thylakoid proteins NdhH, NDH18, NDF1, FKBP16-2,
PsaA, Cyt f, D1, and LHCII (B), which represent the
thylakoid protein complexes indicated.
(TIF)
Click here for additional data file.
Figure S2 In vivo analysis of electron transport activity. (A) Light-intensity
dependence of ETR. ETR is depicted relative to the maximum value of
ΦPSII × light intensity (μmol photons
m−2 s−1) in the WT (100%). (B)
Light-intensity dependence of NPQ of chlorophyll fluorescence. All values
represent the mean ± SD (n = 5)
in (A) and (B).
(TIF)
Click here for additional data file.
Figure S3 Analysis of thylakoid and stromal protein complexes by native PAGE. (A)
Freshly isolated thylakoid membranes from WT (ecotype Col-0 and
Nössen), crr27, and ndhl plants were
solubilized in 1%
n-dodecyl-β-d-maltoside (DM) at a chlorophyll
concentration of 1 µg μl−1, and the protein
sample was separated by 5%–12% BN-PAGE. The major
protein complexes were assigned based on a previous report [39]. Band I,
NDH-PSI supercomplex detected in WT; Band II, sub-NDH-PSI supercomplex
detected in the crr27 and ndhl mutants.
(B) Stromal protein complexes isolated from WT and crr27-1
plants were separated by CN-PAGE followed by 2-dimensional SDS-PAGE. After
electrophoresis, protein was stained with CBB. The positions of the
molecular markers are indicated above the gel. The Cpn60 complex was
assigned on the basis of a previous report [27].
(TIFF)
Click here for additional data file.
Figure S4 Detection of Cpn60β4 in WT plants. (A) CN-PAGE analysis of stromal
protein complexes isolated from WT plants. After electrophoresis, the CN-gel
was stained with CBB. The protein band corresponding to the position of the
Cpn60 complex (the boxed area indicated by an arrow) was excised from the
gel and analyzed by LC-MS/MS analysis. (B) Summary of the chaperonin
subunits detected in the excised band. (C) The peptides of Cpn60β4
detected in the excised band by MS analysis. Total proteins detected are
listed in Table S1.
(TIFF)
Click here for additional data file.
Figure S5 Immunoaffinity purification of Cpn60-substrate complexes. (A) Chaperonin
complexes containing Cpn60β1 and Cpn60β4 subunits were purified from
crr27-1 mutants expressing HA-tagged Cpn60β1 and
Cpn60β4, respectively. After elution, total proteins were separated by
SDS-PAGE and stained with CBB. Due to the low abundance of Cpn60β4, more
stromal proteins were used for the purification, leading to an increase in
non-specific binding of some proteins. Lanes were cut into four slices and
total protein was analyzed by LC-MS/MS analysis. CK, WT plants without any
transformation were used as a negative control. (B) NdhH peptides detected
from the chaperonin complexes containing Cpn60β1 (upper) and Cpn60β4
(bottom) are indicated in red bold.
(TIFF)
Click here for additional data file.
Figure S6 Analysis of stromal protein complexes isolated from wild-type and
crr27-1 plants. (A) Immunodetection of NdhH isolated
from the stroma of wild type (WT), crr27-1,
ndhl, and crr27-1 plants complemented
with Cpn60β4. Equal amounts of stromal protein (8 µg) were loaded
onto each well. WT thylakoid proteins corresponding to 2.5 µg
chlorophyll were also analyzed. RbcL was used as a loading control. An
asterisk represents non-specific signals. (B) Stromal protein complexes
isolated from WT and crr27-1 plants were separated by
CN-PAGE followed by 2-dimensional SDS-PAGE. The proteins were immunodetected
with specific antibodies against NdhH and RbcL. Vertical arrows indicate the
positions of three assembly intermediates, which include NdhH.
(TIF)
Click here for additional data file.
Figure S7 Protein alignment of the mature chaperonin proteins. The origin of all
chaperonin subunits is shown in Figure 5. GroEL from E. coli was also included
in the alignment. Residues involved in ATP/ADP and Mg2+
binding are marked with +. Red and blue sharps (#) represent the
residues for the substrate binding proposed by Fenton et al. [61] and
Buckle et al. [62], respectively. The charged residues exposed to
the central cavity in the cis ring are labeled with
asterisks. The residues marked with red asterisks were selected for
introducing the mutations as shown in Figure 4F. Amino acid residues that are
highly conserved in the putative Cpn60β4 orthologs and with different
properties from the corresponding residues in major Cpn60β subunits are
marked with solid red circles under the protein sequences. These residues
were also highlighted as spheres in the structure model of Cpn60β4 in
Figure
S8. The amino acid mutated in the cpn60α1
mutant is labeled with a triangle. Positions of the deletion and the
C-terminal swapping are shown.
(TIFF)
Click here for additional data file.
Figure S8 Predicted structure of the Arabidopsis Cpn60β4 subunit.
A 3-dimensional (3D) structural model of Arabidopsis
Cpn60β4 was generated with the Swiss Model alignment server. The
structure model of E. coli GroEL (PDB 1AON, Chain A) was
used as a modeling template. Predicted 3-D structure was displayed by using
the PyMOL software. The apical domain (Ap), intermediate domain (Int), and
equatorial domain (Eq) are shown in cyan, green, and blue, respectively.
Highly conserved ATP/ADP and Mg2+ binding sites are marked
with magenta spheres. Residues that are highly conserved in Cpn60β4
subunits but exhibit different properties with the corresponding residues in
other Cpn60β are highlighted with yellow and red spheres. Among them,
sites marked with red, blue, and cyan arrows represent residues near the ATP
binding site or in the intermediate domain, residues exposed to the central
cavity in the cis-conformation of the chaperonin complex,
and one residue involved in the substrate binding, respectively. Sites
marked with red spheres (indicated with green arrows) were selected for
introducing the mutations shown in Figure 4F.
(TIFF)
Click here for additional data file.
Figure S9 Structure and evolution of NdhH. (A) Ribbon models of the Complex I Subunit
Nqo4 (PDB 2fugV) from Thermus thermophilus. Nqo4
corresponds to NdhH in the chloroplast NDH complex. (B) A predicted
structure of the Arabidopsis NdhH. Homology models were
generated with the Swiss Model alignment server. The α-helix is shown in
red, β-sheet in blue, and other domains in yellow. (C) Protein alignment
of the NdhH subunits from Arabidopsis thaliana (AtNdhH),
Physcomitrella patens (PpNdhH), and
Synechocystis SP. PCC 6803 (SyNdhH).
(TIFF)
Click here for additional data file.
Table S1 Total protein detected in the excised band corresponding to the Cpn60 complex
in CN-gel.
(XLS)
Click here for additional data file.
Table S2 Total protein detected in the WT-IP, Cpn60β1-IP, and Cpn60β4-IP
fractions.
(XLS)
Click here for additional data file.
Table S3
r value between Cpn60β4 and
Cpn60α1 with NDH complex-related or chaperonin
subunit genes.
(DOC)
Click here for additional data file.
Dataset S1 Protein sequences used for alignment and phylogenetic tree construction.
(DOC)
Click here for additional data file.
We are grateful to M. Hayer-Hartl for her critical reading of this manuscript, to D.
Gao and M. Hasebe for their help and advice in the phylogenetic analyses, and to F.
Kessler for valuable discussion. We would also like to thank T. Kohchi for providing
the information regarding the genome of Marchantia polymorpha. We
also thank K. Tamura for help in purification of the chaperonin-substrate complexes,
A. Tahara and M. Okamura for their technical assistance, and T. Endo, H. Mi, A.
Makino, and M. Nakai for providing antibodies.
The authors have declared that no competing interests exist.
This work was supported by Grants 17GS0316, 22114509, 22247005 from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan and a grant from
the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for
Agricultural Innovation; GPN0008). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
Abbreviations
ALactinic light
BN-PAGEblue native PAGE
CN-PAGEclear native PAGE
crr
chlororespiratory reduction
Cytcytochrome
emPAIexponentially modified Protein Abundance Index
HAinfluenza hemagglutinin protein epitope
LC-MS/MSliquid chromatography-tandem mass spectrometry
NDHNADH dehydrogenase-like complex
PAMpulse amplitude modulation fluorometry
PQplastoquinone
PSIphotosystem I
PSIIphotosystem II
WTwild type
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21494603PONE-D-10-0477310.1371/journal.pone.0018434Research ArticleBiologyBiochemistryProteinsDNA-binding proteinsGeneticsGene expressionRNA interferenceMolecular cell biologyGene expressionDNA transcriptionRNA interferenceMedicineClinical ImmunologyImmunologic TechniquesImmunohistochemical AnalysisGastroenterology and HepatologyGastrointestinal CancersPancreasOncologyCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerIdentification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer Identification of RegIV as GLI1 Target GeneWang Feng
1
Xu Ling
2
Guo Chuanyong
2
Ke Aiwu
2
Hu Guoyong
2
Xu Xuanfu
2
Mo Wenhui
2
Yang Lijuan
1
Huang Yinshi
2
He Shanshan
2
Wang Xingpeng
1
2
*
1
Department of Gastroenterology, The First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
2
Department of Gastroenterology, The Tenth People's Hospital of Shanghai, Tongji University, Shanghai, People's Republic of China
Uversky Vladimir N. EditorUniversity of South Florida College of Medicine, United States of America* E-mail: [email protected] and designed the experiments: FW XPW. Performed the experiments: FW LX GYH XFX WHM LJY YSH. Analyzed the data: FW LX. Contributed reagents/materials/analysis tools: XPW CYG. Wrote the paper: FW AWK SSH.
2011 11 4 2011 6 4 e1843410 10 2010 4 3 2011 Wang et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background and Aims
GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.
Methods
GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA).
Results
The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells.
Conclusion
GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.
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Introduction
Pancreatic cancer (PC) is the fourth most common cancer-related cause of mortality in the Western world [1]–[3] and has a dismal prognosis despite considerable progress in management. The median survival of PC is less than 6 months; the 5-year survival rate is less than 5% [1], [2]. More than 80% present with unresectable disease; one-third have local disease while the remainder have distant metastases. Research over the last two decades has shown that PC is a genetic disease fundamentally, caused by inherited germline and acquired somatic mutations in cancer-associated genes. Tumor progression model for PC in which the pancreatic ductal epithelium progresses from normal to increased grades of pancreatic intraepithelial neoplasia (PanINs) to invasive cancer. Multiple alterations in genes that are important in PC progression have been identified, for example K-ras, INK4A, p53, and SMAD4/DPC4 [4], [5]. PC is characterized by near-universal mutations in K-ras and frequent deregulation of crucial embryonic signalling pathways, including the Hedgehog (HH) signaling pathway [6], [7]. A better understanding of the mechanisms underlying the development of PC might help to improve early diagnosis and potentially identify molecular therapeutic targets.
The hedgehog (HH) signaling pathway was first identified in the embryonic development of Drosophila [8] and has been shown to be crucial for growth and patterning in the pancreas during embryonic development. HH signaling regulates cell differentiation and organ formation during embryonic development , and is expressed in pancreatic epithelial cells [9], [10]. Constitutive activation of HH signaling is detected in a variety of human cancers, including pancreatic cancer [9]–[13]. Given its misexpression in both metastatic pancreatic cancer cell lines and in precursor lesions (PanIN) [14], HH signal activation may be involved in both early and late pancreatic tumorigenesis.
Of the three mammalian ligands in the HH family, Sonic (SHH), Desert (DHH), and Indian (IHH) Hedgehog [15], the former has been associated with both pancreatic organogenesis and pancreatic cancer. HH signals are transmitted and modified by two transmembrane proteins, patched (PTCH) and smoothened (SMO), and by downstream transcription factors that are members of the glioma-associated oncogene (GLI) family (GLI1, 2, and 3). GLI2 and GLI3 have transactivation and repressive domains, whereas GLI1 likely functions only as a transactivator and transcriptional target of the HH pathway itself [16]–[19]. The regenerating gene (Reg) family, a group of small secretory proteins, is involved in cell proliferation or differentiation in digestive organs [20], is upregulated in several gastrointestinal cancers, and functions as trophic or antiapoptotic factors [21]–[23]. RegIV, a member of the regenerating gene family, is involved in digestive tract malignancies, including the stomach [24], colorectum [25], [26], and pancreas [27], [28], as well as in benign diseases such as ulcerative colitis [29]. RegIV overexpression in tumor cells has been associated with cell growth, survival, adhesion, and resistance to apoptosis. Recently, RegIV overexpression was reported to be associated with the initiation and progression of pancreatic cancer, and was suggested as a promising tumor marker to screen early stage PC and target for adjuvant therapy in PC [28], [30].
The functions of GLI1 and RegIV appeared to be similar in our review of the literature; thus, we investigated the expression and correlation of GLI1 and RegIV in PC tissues and cell lines. We also explored the possible mechanism between GLI1 and RegIV, by using ChIP and EMSA assays.
Materials and Methods
Cell lines and tissues
Human pancreatic cancer cell lines, PANC-1, AsPC-1, BxPC-3, CaPan-2 and SW1990, were purchased from Chinese Academy of Sciences Committee Type Culture Collection cell bank. PANC-1 was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco BRL, USA), the other types of Cells were cultured in RPMI-1640 (Gibco BRL, USA), and all the mediums were supplemented with 10% FBS (Gibco BRL, USA), penicillin G (100 U/ml), streptomycin (100 ug/ml). Cells were incubated at 37°C with 5% CO2. Twelve pairs of PC and corresponding non-cancerous pancreas tissues were obtained from Shanghai Tenth People's Hospital with full written ethical consent. None of these patients had received chemotherapy or radiation therapy prior to cancer resection. Another 9 paired tissues slices was obtained from pathology department of Shanghai Tenth People's Hospital. The study was approved by the Ethical Committee of Tongji University School of Medicine and Life Sciences.
Short Hairpin RNA (shRNA) Design and Vector Production
Interfering sequences corresponding to distinct regions of GLI1mRNA, as well as negative control with no homology for human or mouse genes were designed by Shanghai GeneChem Biotech (Table 1). Three siRNA duplexes were screened for GLI1 knock-down by Western blot analysis in cotransfection experiments with GLI1 expression plasmid in HEK 293T cells. The most successful sequence and one non-silencing Luciferase sequence were designed into a shRNA oligonucleotide template consisting of sense, hairpin loop, antisense, and terminator sequences, all of which were flanked by restriction enzyme sites to facilitate directional sub-cloning. The resulting vectors encoded GFP under transcriptional control of the EF1 promoter and a H1 promoter upstream of cloning restriction sites (MluI and ClaI) to allow the introduction of oligonucleotides encoding shRNAs. Either shRNA against GLI1 or a nonsilencing-Luciferase shRNA was located under the H1 promoter (Figure 1). The correct insertion of the specific shRNA was further confirmed by direct DNA sequencing.
10.1371/journal.pone.0018434.g001Figure 1 Construction of the pLVTHM vector encoding anti-GLI1 shRNA.
For the GLI1-shRNA vector, two single strand DNAs encoding two linkers, the target sequences and a loop element, were synthesized. These were annealed to double stranded DNA, and ligated into the pLVTHM following MluI and CalI digestion. The short hairpin form of shGLI1 is expressed under the control of human H1 promoter. The vector also contains a human EF1-α promoter driving the GFP marker gene for tracking transduced cells. The vectors were generated by transient transfection of pRsv-REV, pMDlg-pRRE, pMD2G, and the shRNA encoding pLVTHM into 293T cells. Abbreviations: RRE, Rev response element; cPPT, central polypurine tract; EF1-α, human elongation factor 1-α promoter; H1, the human H1 promoter; GFP, green fluorescent protein; PRE, human hepatitis virus posttranscriptional regulatory element.
10.1371/journal.pone.0018434.t001Table 1 Sequences of primers used in this study for GLI1-shRNA constructs.
5' STEMP Loop STEMP
shGLI1-1
CGCGTCCCC
CTCCACAGGCATACAGGAT
TTCAAGAGA
ATCCTGTATGCCTGTGGAG
CGATTTCCAAAAA
CTCCACAGGCATACAGGAT
TCTCTTGAA
ATCCTGTATGCCTGTGGAG
shGLI1-2
CGCGTCCCC
CGTGAGCCTGAATCTGTGTAT
TTCAAGAGA
ATACACAGATTCAGGCTCACG
CGATTTCCAAAAA
CGTGAGCCTGAATCTGTGTAT
TCTCTTGAA
ATACACAGATTCAGGCTCACG
shGLI1-3
CGCGTCCCC
GCTCAGCTTGTGTGTAATTAT
TTCAAGAGA
ATAATTACACACAAGCTGAGC
CGATTTCCAAAAA
GCTCAGCTTGTGTGTAATTAT
TCTCTTGAA
ATAATTACACACAAGCTGAGC
scramble
CGCGTCCCC
GCCAGCGTTAACCAGACTA
TTCAAGAGA
TAGTCTGGTTAACGCTGGC
CGATTTCCAAAAA
GCCAGCGTTAACCAGACTA
TCTCTTGAA
TAGTCTGGTTAACGCTGGC
For production of the lentiviral vector, HEK 293T cells were cultured to 30–40% confluence by the following day. The next day, the medium was replaced with DMEM/10% FBS without antibiotics. Subsequently, 20 µg of shRNA plasmid DNA (nonsense shRNA or GLI1 targeting shRNA; GeneChem Biotech, Shanghai, China), 7.5 µg pMD2G, 10 µg pRsv-Rev, and 15 µg pMDLg-pRRE were mixed with sterile ddH2O to a final volume of 1800 µl, then mixed with 200 µl of 2.5 M CaCl2. The DNA mix was oxygenated and 2000 µl 2×PBS (pH 7.05) added in drops, and incubated at room temperature for 30 minutes. The transfection mixture was added to its respective plates and incubated overnight. The medium was replaced after 12 hours with DMEM supplemented with 10% FBS. After 48 hours, the conditioned medium containing shRNA lentivirus was collected and filtered through 0.45-µm pore size cellulose acetate filters, and stored on ice. The virus was concentrated by spinning at 70,000 G for 2 hours and resuspended with 500 µl PBS. The transduction unit (TU) titer was assessed on HEK 293T cells in the presence of polybrene 8 µg/mL (Sigma-Aldrich, St. Louis, MO, USA). Titers of 2–5×108 TU/ml were routinely achieved.
Overexpression-GLI1 Lentiviral Vector Construction
Human GLI1 cDNA was purchased from Open-Biosystem (USA). The complete cDNA sequence of GLI1 was generated by PCR using the forward primer, 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGTTCAACTCGATGACCCCAC-3′; and reverse primer, 5′-TCATCCTTGTAGTCGCTAGCGGCACTAGAGTTGAGGA-3′; then inserted into a pGC-FU-EGFP-3FLAG Vector (GeneChem Company, Shanghai, China). Transformants were analyzed by sequencing. The resultant 3320-bp fragment was confirmed by sequencing (Figure S1) and compared with the sequence of the GLI1 gene expression region in GenBank (NM_005269.2). To produce lentiviral stock, 293FT cells were cultured to 70–85% confluence the following day. The complete culture medium was removed. Cells were then exposed to 5 mL medium (Opti-MEM; Invitrogen) with complexes containing packaging helper construct (GeneChem Company, Shanghai, China), 20 µg expression plasmid DNA (pGC-FU-EGFP-3FLAG-GLI1), or control plasmid DNA (pGC-FU-EGFP-3FLAG) with 100 µl lipofectamine 2000 (Invitrogen, USA) in the presence of polybrene (8 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). After incubation for 24 hours, the infection medium was replaced with complete culture medium. Lentivirus-containing supernatants were harvested 72 hours after transfection. The supernatants were centrifuged to remove pellet debris and stored at −80°C. Titers of 2–5×107 TU/ml were routinely achieved.
Lentiviral Transfection
Cells (1×105) in a six-well plate were transfected with the lentiviral vector at a multiplicity of infection (MOI) = 5 (PANC-1) or 20 (BxPC-3) in the presence of 8 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 72 hours of transfection, the medium was replaced with 2 ml complete culture medium. 48 hours after transfection, GLI1 expression was established by real time-PCR and Western blot analysis.
Flow Cytometry
Cells were adjusted to 1×106 cells/100 µL and used for flow cytometry. A total of 10,000 events were analyzed to determine transfection efficiency using FACS Calibur (Becton Dickinson, USA) Cell-Quest software.
qRT-PCR
Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed with the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, CA, USA). GLI1 and RegIV mRNA expression was analyzed by qRT-PCR using SYBR Green Dye. β-actin was used as the housekeeping gene. Target gene expression was normalized to β-actin and analyzed by the 2−ΔΔCT formula. The primer sequences are as follows: GLI1, forward: TTCCTACCAGAGTCCCAAGT, reverse: CCCTATGTGAAGCCCTATTT, RegIV, forward: CGCTGAGATGAACCCCAAG, reverse: TGAGAGGGAAGTGGGAAGAG. β-actin, forward: AAGGGACTTCCTGTAACAATGCA, reverse: CTGGAACGGTGAAGGTGACA. All reactions were performed at least three times.
Western blot analysis
Cells were rinsed twice in PBS, then lysed for 2 hours in RIPA lysis buffer on ice and centrifuged at 12,000 rpm for 10 minutes at 4°C. Protein concentration was determined by the standard BCA method (BCA™ Protein Assay Kit, Pierce, USA). 50 µg of total protein was separated by SDS-PAGE using 6% or 12% polyacrylamide gel with Mini-PROTEAN Tetra Cell (Bio-Rad, USA). GLI1 and RegIV protein in gel was transferred to a 0.45-µm nitrocellulose membrane with Mini Transfer Cell and Trans-blot SD Semi-Dry Transfer Cell (Bio-Rad, USA) respectively.
The immunoreagents used for Western blot were rabbit monoclonal antibody against GLI1 (1∶200; Santa Cruz, USA) and goat polyclonal anti-RegIV antibody (1∶100; Santa Cruz, USA). Mouse polyclonal anti-β-actin antibody (1∶5000; Santa Cruz, USA) was used as loading control. The blots were developed by a standard enhanced chemiluminescence (ECL) method (Pierce, USA). All experiments were repeated several times and gave similar results.
Immunohistochemistry
Tumor sections were deparaffinized, rehydrated, and treated with 10 mM citrate buffer (pH 6.0) at 95°C to retrieve antigens. After quenching endogenous peroxidase activity with H2O2 and blocking with 10% normal horse serum, the sections were incubated sequentially with the primary antibodies goat anti-RegIV (1∶100; Santa Cruz, California, USA), rabbit anti-GLI1 (1∶200; Santa Cruz, California, USA), biotinylated secondary antibodies, and the ABC reagent (Gene Tech, Shanghai, China). The immunostaining was visualized with 3.3-diaminobenzidine (Gene Tech, Shanghai, China). The sections were then counterstained with hematoxylin. Negative controls were performed in each case by replacing the primary antibody with PBS.
Chromatin immunoprecipitation (CHIP)
We modified the previously reported protocol [31] for chromatin immunoprecipitation (CHIP). In brief, PANC-1 cells (3×107) were cross-linked with 1% formaldehyde. The fixation reaction was stopped by adding 10 ml Glycin (0.125 M), then chromatin was collected with 1 mL IP buffer containing protease inhibitor cocktails. Chromatin was sheared by using a sonicator with a 4 mm tip probe 3 times for 10 second pulses (60 W, 80 W, and 100 W, respectively, 90 s intervals) in an ice box. Crosslinking was reversed by adding 20 µL of 5 M NaCl overnight at 65°C. DNA was extracted using phenol/chloroform assay. 20 µL of DNA was electrophoresed on a 1.5% agarose gel and the rest was preserved at −20°C as INPUT DNA. Soluble chromatin was immunoprecipitated with 2 µg anti-GLI1 rabbit monoclonal antibody (Santa Cruz, California, USA). The 2 µg mouse IgG (Santa Cruz, California, USA) was added as a random control, RNA polymerase II as a positive control, and β-actin antibody as a negative control. DNA-protein immune complexes were eluted and reverse cross-linked, and DNA was extracted with phenol/chloroform and precipitated. The presence of the RegIV promoter domain containing GLI1 motifs in immunoprecipitated DNA was identified by PCR using the following primers: RegIV-A for site 1 (118 bp), forward: 5′-5-TGGTCCCTTCCAGACTTA-3-3′, reverse: 5′- TCCAGTATAGATGGCAAA -3′. RegIV-B for site 2 (131 bp), forward: 5′-CTAACCCTTTGCCATCTA -3′, reverse: 5′-GACCTGGACACTGAACCTTG-3′. RegIV-C for site 3 (70 bp), forward: 5′-CTATGCTGCTCACAAGGA-3′, reverse: 5′-GTGTTACATAACGGGTTT-3′. RegIV-D for site4 (70 bp), forward: 5′-TGTAACACACTCTGTTGATGTAAGC-3′, reverse: 5′- CTATTTGAGCTTCTCCCGCAG-3′. RegIV-E for sites 3 and 4 (226 bp), forward: 5′-CTCGGAAGGTTTCTAATC-3′, reverse: 5′- TTCAACATGCGTGAGTTT-3′. RegIV-F for sites 3 and 4 (481 bp), forward: 5′-CTATGCTGCTCACAAGGA-3′, reverse: 5′-AGACGGCTTCAGAATGTA-3′. RegIV-G for site 5 (315 bp), forward: 5′-TTCCTGAGGCAAGAAGAT-3′, reverse: 5′-CCAAGATTTAACCCAACA-3′. The PCR conditions for the RegIV promoter region were: denaturation 30 seconds at 94°C, annealing 30 s, elongation 1 minute at 72°C. Annealing temperatures for RegIV-A-G were 55°C, 56°C, 56°C, 47°C, 56°C, 56°C, and 52°C, respectively. The amplification of the RegIV promoter region was analyzed after 35 cycles. All experiments were repeated at least three times.
Electrophoretic Mobility Shift Assays (EMSA)
Nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, USA). EMSA and supershift EMSA with digoxin-labeled probes were performed using the DIG-Gel shift kit according to the manufacturer's instructions (Roche, Basel, Switzerland). The sequences of the oligonucleotides used were 5′-AGAACATGGATGATCATGTCA-3′ (binding motif underlined). Mutant oligonucleotides used were 5′-AGAACAAAAAATTTTATGTCA-3′. In the supershift study, 5 µg rabbit monoclonal antibody against GLI1 was incubated with 5 µg of nuclear extract on ice for 30 minutes before addition of the labeled probe, and further incubated on ice for 30 minutes. The entire 20 µl binding reaction was resolved on a 7% polyacrylamide gel and transferred to a positively charged nylon membrane (Bio-Rad, USA) in 0.5× Tris borate-EDTA buffer.
Statistical analysis
Quantitative data are expressed as the mean ± standard deviation (SD). Real-time PCR data was analyzed according to the differences of target gene expression by the paired t-test and were 2−ΔΔCT transformed before analysis. The relationship between GLI1 and RegIV expression was analyzed using Spearman. IHC data was analyzed using the Chi-squared test. A p-value of less than 0.05 was considered statistically significant.
Results
GLI1 and RegIV expression in pancreatic cancer tissues
To study GLI1 and RegIV expression in PC, qRT-PCR and IHC were used in 12 paired biopsy tissues. GLI1 expression was higher in 9 cases (9/12) compared with adjacent normal pancreatic tissues (p = 0.011; Figure 2); RegIV expression was higher in 9 cases (9/12) (p = 0.011; Figure 2). There was a positive correlation between GLI1 and RegIV in PC tissues (R = 0.795, p<0.0001; Figure 2). On IHC, we found RegIV to be expressed only in beta cells of normal endocrine pancreatic tissues, which confirmed Oue's report [37]. On IHC, GLI1 and RegIV expression were higher in most PC compared with normal tissues (15/21 versus 4/21, p = 0.001; 14/21 versus 5/21, p = 0.005; respectively; Figure 3). 15 of 21 PC cases had high expression of GLI1 protein, among which 11 cases expressed high levels of RegIV protein (p = 0.001; Figure 3).
10.1371/journal.pone.0018434.g002Figure 2 GLI1 and RegIV mRNA expression in PC tissues and adjacent normal tissues.
The expression of GLI1 RegIV mRNA in 12 pairs of PC tissues and adjacent normal tissues(A–B). DNA from the samples were collected from surgical biopsies, and relative GLI1 and RegIV mRNA expression were detected by qRT-PCR. Statistical correlation between the expression of GLI1 and RegIV mRNA in 12 pairs of PC tissues and adjacent normal tissues was analyzed by Spearman's test (C). All results were normalized to β-actin mRNA expression. The data are presented as the mean ± SD and were calculated by the paired t-test. Significantly different between two groups: *p<0.05. ** p<0.01. NS: not significant.
10.1371/journal.pone.0018434.g003Figure 3 Expression of GLI1 and RegIV proteins was analyzed by IHC in PC and adjacent normal tissues.
All samples were collected, formalin-fixed, paraffin-embedded, and detected by IHC. Representative pictures are shown. Positive staining of GLI1 was observed at the PDAC cell nucleus (A); however, adjacent normal tissues exhibited no or faint staining for GLI1 (B). In adjacent normal pancreatic tissues, only islet cells showed positive staining of RegIV (D), while the positive staining of RegIV was observed as well as goblet-like cytoplasm granules in PC tissues (C). All photomicrographs were obtained at ×200 magnification. Scale bars, 100 µm.
The correlation between GLI1 and RegIV
We tested GLI1 and RegIV expression in 5 PC cell lines by qPCR and Western blot. There was a positive correlation between the level of GLI1 and RegIV mRNA and protein (R = 0.958, p = 0.011 and R = 0.939, p = 0.018, respectively; Figure 4). GLI1 and RegIV were overexpressed in PC versus normal pancreatic cells.
10.1371/journal.pone.0018434.g004Figure 4 The expression of GLI1 and RegIV in 5 PC cell lines.
Expression of GLI1 and RegIV proteins in 5 pancreatic cancer cell lines as detected by Western blot analysis on cell extracts, using anti-GLI1 and anti-RegIV antibodies (A). β-actin was used as the loading control in all experiments. The results were quantified by determining the intensities of the bands compared with that of β-actin (B). Statistical correlation between expression of GLI1 and RegIV protein in 5 PC cell lines was analyzed by Pearson's test (C). Relative GLI1 and RegIV mRNA expression were examined by qRT-PCR (D). The expression of GLI1 and RegIV mRNA was normalized to β-actin mRNA expression. Statistical correlation between the expression of GLI1 and RegIV mRNA in 5 PC cell lines was analyzed by Pearson's test (E). All data are presented as the mean ± SD of three independent experiments.
RegIV expression changed with GLI1 expression in PANC-1 and BxPC-3
To further verify the relationship between GLI1 and RegIV in PC cells, we designed and constructed shRNA-GLI1 lentiviral vector, and transfected it into PANC-1, a PC cell line with the highest expression of GLI1 (Figure 4). 48 hours after transfection, efficiency of transfection was shown by flow cytometry (FCM) to be more than 95% (Figure S2); stable fluorescence could still be detected even after 20 passages (Figure S3).
Afterwards, qRT-PCR and Western blot were used to detect RegIV expression in GLI1-shRNA-PANC-1 cells. Cells without transfection were used as controls, while cells transfected with scramble shRNA were used as negative controls. RegIV mRNA decreased by 94.7±0.3% when GLI1 mRNA decreased by 82.1±3.2%. RegIV protein decreased by 84.1±0.5% when GLI1 protein decreased by 76.7±2.2% (Figure 5). This suggested that RegIV expression decreased when GLI1 was silenced by RNAi.
10.1371/journal.pone.0018434.g005Figure 5 RegIV expression changed with GLI1 in PC cells.
PANC-1 cells were transfected with GLI1-shRNA or GFP-shRNA, BxPC-3 cells were transfected with LV-GLI1-eGFP or LV-eGFP, then expression of GLI1 mRNA relative to that of β-actin mRNA was assessed by qRT-PCR (A, D). After transfection, expression of GLI1 proteins was analyzed by Western blot (C, F). The inset shows a substantial decrease in RegIV expression by real-time RT-PCT (B, E) and Western blot analysis (C, F). The results were normalized to that of β-actin expression. All data are presented as the mean ± SD of three independent experiments.
We further designed and constructed a lentivirus vector that expressed GLI1, and transfected it into BxPC-3, with the lowest GLI1 expression in the 5 cell lines (Figure 4), to determine whether RegIV expression changed along with GLI1. 48 hours after transfection, qRT-PCR and Western blot were used to detect RegIV in the LV-GLI1-BxPC-3 cells. Cells without transfection were used as controls, while cells transfected with empty lentivirus vector were used as negative controls. RegIV mRNA increased by 729.1±4.3% when GLI1 mRNA increased by 924.5±5.3%. RegIV protein increased by 339.0±3.7% when GLI1 protein increased by 362.1±3.5% (Figure 5). This implies that RegIV expression increased when GLI1 was overexpressed. Based on these results, we concluded that GLI1, a transcription factor, might regulate RegIV gene expression.
Identification of candidate Gli1 binding sites in the RegIV promoter
The positive correlation between GLI1 and RegIV in both PC tissue and cell lines prompted us to search the RegIV promoter for potential GLI1 binding sites to the DNA consensus sequence 5′-GACCACCCA-3′
[42]. Database analysis revealed four potential sites located upstream of the transcriptional start site (Figure 6). The homology of each GLI1 binding site to the canonical consensus sequence varied from 67% (sites 1, 2, 3, and 5) to 78% (site 4), which suggested that the RegIV gene promoter might bind to GLI1.
10.1371/journal.pone.0018434.g006Figure 6 Potential GLI1 binding sites on the RegIV promoter and homology to the GLI1 consensus sequence.
(A) Location of the potential GLI1 binding sites (numbers 1–5) in relation to the structure of the gene. P represents the transcriptional start site. The gray frame represents the variable splicing site. Blank frames represent exons. (B) Position of the binding sites on the promoter in relation to the P transcriptional start site and the sequence homology to the GLI1 consensus binding sequence, GACCACCCA.
Confirmation of GLI1 protein bound to promoter region of RegIV gene by CHIP
The sonicated chromatin solution assay showed that the total DNA fragment appeared smeared in the 100 bp to 1 kb range in the 80 W group (Figure S4). The result of DNA electrophoresis showed the predicted DNA band in INPUT, GLI1-Ab, and postive control groups using human RegIV primer-D-F, and not in the IgG and negative control groups (Figure 7). Only INPUT and the positive control showed the predicted band using human RegIV primer-A-C, G but not in GLI-Ab, IgG, and negative groups (data not shown). The results of sequence analysis showed that the sequences were the same as that of the RegIV gene promoter of site 4 (Figure S5, S6, S7). All data suggested that GLI1 was bound to the RegIV gene promoter of site 4 (GATCATCCA), and regulated RegIV in PC through the HH signaling pathway.
10.1371/journal.pone.0018434.g007Figure 7 Modulation of GLI1 binding on RegIV promoter was assessed by Chromatin immunoprecipitation (ChIP) assay.
The locations of RegIV primer-A-G in the promoter region of RegIV gene. The numbers on the schematic of the RegIV gene (numbers 1–5) correspond to the potential GLI1 binding sites. P represents the transcriptional start site. Lysates from PANC-1 cells were subjected to Chromatin immunoprecipitation by anti-GLI1 antibody. Human RegIV primer-A-G were used to amplify the RegIV promoter region containing the putative GLI1-binding site. Sonicated chromatin were used as INPUT DNA control. IgG, RNA polymerase II, and β-actin Ab were used as random controls, positive controls, and negative controls. (B) Only INPUT, positive control, and GLI1-Ab showed the predicted band in ethidium bromide-stained agarose gels using the CHIP-PCR products which were amplified by RegIV primer-D(i), RegIV primer-E(ii), and RegIV primer-F(iii). No detectable transcript was observed in amplified template from IgG or negative control and a positive control lane confirmed the expected fragment. Molecular weight standards (Marker) were used to estimate the size of the amplified bands.
Confirmation of GLI1 bound to the RegIV promoter by EMSA
As described above, the GLI1 binding site in the promoter region of the RegIV gene was confirmed with ChIP-PCR. We then used EMSA assays to directly address whether GLI1 binds RegIV in vivo. We synthesized specific oligonucleotides containing the GLI1 element present in the RegIV promoter in EMSA experiments with nuclear extracts from PANC-1 cell lines. As shown in Figure 8, incubation of PANC-1 cells extracts with the biotin-labeled GLI1-RegIV sequence produced a DNA-protein band shift. These DNA-protein complexes were specific to the GLI1 site by successful competition assays using different folds of excess unlabeled GLI1-RegIV and mutant labeled GLI1-RegIV oligonucleotides. To confirm the binding of GLI1 to the GLI1-RegIV sequence, these EMSA reactions were further incubated with anti-GLI1 antibody. As shown in Figure 8, the addition of this antibody resulted in a supershifted complex in addition to the DNA-protein band. These data demonstrated the presence of GLI1 in the nuclear protein complex that binds the GLI1 binding site of the RegIV promoter (−528∼−520).
10.1371/journal.pone.0018434.g008Figure 8 Analyses of the binding of GLI1 to the Reg IV promoter by Electrophoretic Mobility Shift Assays (EMSA).
EMSA was performed with nuclear extracts of PANC-1 cells (lanes 2 to 7) or without nuclear extracts (lane 1). The RegIV probe was generated by annealing single-stranded and end-labeled oligonucleotides containing the RegIV promoter region (nucleotides −528∼−520). Competition experiments were performed using 1-fold (lanes 3), 10-fold (lanes 4), and 100-fold (lanes 5) excess of unlabeled oligonucleotides, respectively (lanes 3 to 5) or 100-fold mutant labeled oligonucleotides (lane 6). For super-shift, anti-GLI1 antibody (lane 7) was incubated with nuclear extracts before being added to the reaction.
Discussion
In this study, we confirmed that GLI1 and RegIV were overexpressed in PC tissue and cell lines, confirmed by other reports [12], [20], [32]. We also demonstrated a significantly positive correlation between the expression of GLI1 and RegIV. RNA interference and overexpression experiments showed that RegIV expression changed with GLI1 expression in PC cell lines; this was confirmed by CHIP and EMSA. This is the first report that GLI1 can modulate RegIV expression by binding to the RegIV gene promoter, and that GLI1 is a RegIV transcriptional factor.
The HH signaling pathway, including transcription factor GLI1, is involved in the development of many kinds of cancers, including PC [12], [13], [32]–[35]; however, the mechanism has not been fully elucidated. Thus far, only a few downstream targets of GLI1 have been identified, including GLI1, PTCH, HHIP, CCND, Snail, Bcl-2, cyclin D2, FOX-F1, -L1, -M1, Follistatin, and N-Myc [36]. We demonstrated that the HH-GLI1 signaling pathway could regulate RegIV expression by a serie of experiments, including CHIP and EMSA. In our literature review, we learned that RegIV expression in different cell types was associated with regeneration, and cell growth, survival, adhesion, and resistance to apoptosis. RegIV is systematically overexpressed in stomach [24], colon [25], [26], and pancreas cancers [27], [28] and in diseases that predispose to colon cancer such as ulcerative colitis [29]. IHC analysis has confirmed RegIV expression in gastric, colorectal, and pancreatic carcinoma [27], [37], [38], and that RegIV has a potential role in diagnosing digestive tract neuroendocrine tumors [39]. RegIV gene amplification is an early event in pancreatic cancer development [30], and elevated RegIV was found in the sera of patients with PC [28]. PC-derived cells overexpressing RegIV protein grew more rapidly and were more resistant to gemcitabine treatment [30]. RegIV overexpression was thought to be associated with an unfavorable response to adjuvant chemoradiotherapy in patients with PC [40]. Other studies showed that RegIV was associated with a relatively favorable prognosis in patients with gallbladder carcinoma after surgical resection [41]. Thus, we concluded that the HH/GLI1/RegIV cascade may be an important pathway in PC development.
Chromatin immunoprecipitation (CHIP) is a reliable procedure used to determine whether a protein binds to or is localized to a specific DNA sequence in vivo. Through CHIP and promoter analysis, we identified a direct transcriptional target gene of GLI1, although the GLI1-binding element (GATCATCCA) showed a 2 nucleotide difference (underlined) from a previously identified 9-nucleotide GLI1-binding sequence (GACCACCCA) [42]. EMSA is one of the most sensitive methods for studyting protein-DNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA sequence. “Supershift assay” is a term used to unambiguously identify a protein present in the protein-nucleic acid complex. The EMSA and supershift assay also confirmed GLI1 to be bound on site 4 of the RegIV promoter motif. Those results suggested that GLI1 can bind to RegIV gene promoters on site 4 in vivo. Based on these results, we concluded that GLI1 transcriptionally regulates RegIV gene in PC cells.
Although the biological function of RegIV is poorly understood, it has been reported that RegIV may function as a growth and antiapoptotic factor in gastric, colon, and pancreatic cancers [27], [29], [43], [44]. The expression of RegIV may contribute to liver metastasis through induction of MMP7 by RegIV [44], and is a potent activator of the EGFR/Akt/AP-1 signaling pathway in colon cancer cells. It also increases the expression of Bcl-2, Bcl-xl, and survival proteins, all associated with the inhibition of apoptosis [44]. However, the role of RegIV in migration and invasion, and whether GLI1 contributes to proliferation, migration, and invasiveness through RegIV regulation in PC is still unclear. Whether RegIV is transcriptionally regulated by GLI1, thus imposing its effect on pancreatic carcinogenesis, the pathways responsible require further investigation. The coherence of different molecular events would be partly elucidated by revealing the mechanism of transcriptional regulation between GLI1 and RegIV, which may be determined by investigating the effects of GLI1 and RegIV on common signaling pathways such as the EGFR/Akt/AP1 cascade, as reported recently both in HH and RegIV. Our work may contribute to the body of research on pancreas carcinogenesis and provide insight into the correlated network of signaling pathways through the GLI1/RegIV axis.
In conclusion, the SHH-GLI1 signaling pathway regulates the transcription of RegIV gene in PC. This is the first report that demontrates GLI1 as a transcriptional factor that regulates RegIV expression in PC. Our work may help to elucidate the molecular mechanism of the SHH-GLI1 signaling pathway and promote earlier diagnosis and treatment of PC. The newly identified GLI1/RegIV axis provides a new insight into PC pathogenesis. Additional studies are required to determine whether the biological behavior of GLI1 in PC may be achieved by regulating RegIV.
Supporting Information
Figure S1
The result of sequence analysis of positive clone products in overexpression-GLI1 lentiviral vector construction. The resultant 3320-bp fragment was confirmed by sequencing which is same with the sequence of the GLI1 gene expression region in GenBank (NM_005269.2).
(TIF)
Click here for additional data file.
Figure S2
Transduction efficiency of PANC-1 cells by lentivirus vector were evaluated by FCM. Cells were transfected with GFP-vector. Transduction efficiency based on the fluorescent signalwas analyzed by FCM. (A) PANC-1 cells without transfection were used as the blank control; (B) Cells transfected with GFP-shRNA as random control; (C) Cells transfected with GLI1-shRNA as experiment group.
(TIF)
Click here for additional data file.
Figure S3
Transduction efficiency of PANC-1 cells by lentivirus vector, phase contrast and GFP expression under a fluorescent microscope. Transduction efficiency of PANC-1 cells by GLI1 silencing vector. PANC-1 cells were transfeced with the GLI1-shRNA vector. The corresponding phase-contrast image(left panel), the GFP fluorescence (middle panel) and the merged image (right panel) are shown at a magnification of ×200. GFP expression reveals high transduction efficiency, with more than 90% of cells being transduced.
(TIF)
Click here for additional data file.
Figure S4
Electropheretogram of sonicated chromatin solution in different conditions. Sonicated chromatin solution in different conditions were electrophoresed on 1.5% agarose gel containing ethidium bromied. DNA sizes appear smear at a range of 100 bp to 1 kb range in 80 W group.
(TIF)
Click here for additional data file.
Figure S5
The result of sequence analysis of CHIP products which amplified by RegIV primer-D. The result showed that the sequence amplified with RegIV primer-D is the same as that of RegIV gene promoter region containing GLI1-binding site 4.
(TIF)
Click here for additional data file.
Figure S6
The result of sequence analysis of CHIP products which amplified by RegIV primer-E. The result showed that the sequence amplified with RegIV primer-E is the same as that of RegIV gene promoter region containing GLI1-binding site 4.
(TIF)
Click here for additional data file.
Figure S7
The result of sequence analysis of CHIP products which amplified by RegIV primer-F. The result showed that the sequence amplified with RegIV primer-F is the same as that of RegIV gene promoter region containing GLI1-binding site 4.
(TIF)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported in part by National Natural Science Foundation of China (No. 81072065), Foundation for Shanghai Science and Technology Committee (No. 09JC1412200, No. 09410705400), and Doctoral Fund of Ministry of Education of China (No. 20090072110022). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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10 Hebrok M 2003 Hedgehog signaling in pancreas development. Mech Dev 120 45 57 12490295
11 Watkins DN Berman DM Burkholder SG Wang B Beachy PA 2003 Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422 313 317 12629553
12 Berman DM Karhadkar SS Maitra A Montes De Oca R Gerstenblith MR 2003 Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425 846 851 14520411
13 Thayer SP di Magliano MP Heiser PW Nielsen CM Roberts DJ 2003 Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425 851 856 14520413
14 Morton JP Mongeau ME Klimstra DS Morris JP Lee YC 2007 Sonic hedgehog acts at multiple stages during pancreatic tumorigenesis. Proc Natl Acad Sci U S A 104 5103 5108 17372229
15 Ingham PW McMahon AP 2001 Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15 3059 3087 11731473
16 Dai P Akimaru H Tanaka Y Maekawa T Nakafuku M 1999 Sonic Hedgehog-induced activation of the Gli1 promoter is mediated by GLI3. J Biol Chem 274 8143 8152 10075717
17 Lee J Platt KA Censullo P Ruiz i Altaba A 1997 Gli1 is a target of Sonic hedgehog that induces ventral neural tube development. Development 124 2537 2552 9216996
18 Wang B Fallon JF Beachy PA 2000 Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100 423 434 10693759
19 Aza-Blanc P Lin HY Ruiz i Altaba A Kornberg TB Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities. Development 2000;127 4293 4301
20 Zhang YW Ding LS Lai MD 2003 Reg gene family and human diseases. World J Gastroenterol 9 2635 2641 14669303
21 Bishnupuri KS Luo Q Sainathan SK Kikuchi K Sureban SM 2010 Reg IV regulates normal intestinal and colorectal cancer cell susceptibility to radiation-induced apoptosis. Gastroenterology 138 616 626 19900450
22 Sekikawa A Fukui H Fujii S Takeda J Nanakin A 2005 REG Ialpha protein may function as a trophic and/or anti-apoptotic factor in the development of gastric cancer. Gastroenterology 128 642 653 15765400
23 Kuniyasu H Oue N Sasahira T Yi L Moriwaka Y 2009 Reg IV enhances peritoneal metastasis in gastric carcinomas. Cell Prolif 42 110 121 19143768
24 Oue N Hamai Y Mitani Y Matsumura S Oshimo Y 2004 Gene expression profile of gastric carcinoma: identification of genes and tags potentially involved in invasion, metastasis, and carcinogenesis by serial analysis of gene expression. Cancer Res 64 2397 2405 15059891
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26 Zhang Y Lai M Lv B Gu X Wang H 2003 Overexpression of Reg IV in colorectal adenoma. Cancer Lett 200 69 76 14550954
27 Takehara A Eguchi H Ohigashi H Ishikawa O Kasugai T 2006 Novel tumor marker REG4 detected in serum of patients with resectable pancreatic cancer and feasibility for antibody therapy targeting REG4. Cancer Sci 97 1191 1197 16918991
28 Takayama R Nakagawa H Sawaki A Mizuno N Kawai H 2010 Serum tumor antigen REG4 as a diagnostic biomarker in pancreatic ductal adenocarcinoma. J Gastroenterol 45 52 59 19789838
29 Nanakin A Fukui H Fujii S Sekikawa A Kanda N 2007 Expression of the REG IV gene in ulcerative colitis. Lab Invest 87 304 314 17260007
30 Legoffic A Calvo E Cano C Folch-Puy E Barthet M 2009 The reg4 gene, amplified in the early stages of pancreatic cancer development, is a promising therapeutic target. PLoS One 4 e7495 19834624
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39 Li FY Ren XB Xu EP Huang Q Sheng HQ 2010 RegIV expression showing specificity to gastrointestinal tract and its potential role in diagnosing digestive tract neuroendocrine tumor. J Zhejiang Univ Sci B 11 258 66 20349522
40 Eguchi H Ishikawa O Ohigashi H Takahashi H Yano M 2009 Serum REG4 level is a predictive biomarker for the response to preoperative chemoradiotherapy in patients with pancreatic cancer. Pancreas 38 791 798 19546835
41 Tamura H Ohtsuka M Washiro M Kimura F Shimizu H 2009 Reg IV expression and clinicopathologic features of gallbladder carcinoma. Human Pathology 40 1686 1692 19716164
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Int J Prev MedIJPVMInternational Journal of Preventive Medicine2008-78022008-8213Medknow Publications India IJPVM-1-260Brief CommunicationUltrafast Mid-IR Laser Scalpel; Approaching to Scar-less Surgery Nik Saeid Amini M.Sc, M.D, Ph.D11 Developmental & stem cell Biology at Sickkids hospital University of Toronto, Toronto, Canada Email: [email protected] [email protected] 2010 1 4 260 263 29 9 2010 05 10 2010 © International Journal of Preventive Medicine2010This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Lasers have in principle the capability to cut at the level of a single cell, the fundamental limit to minimally invasive procedures and restructuring biological tissues. To date, this limit has not been achieved due to collateral damage on the macroscale that arises from thermal and shock wave induced collateral damage of surrounding tissue. Here, we report on a novel concept using a specifically designed Picosecond IR Laser (PIRL) that selectively energizes water molecules in the tissue to drive ablation or cutting process faster than thermal exchange of energy and shock wave propagation, without plasma formation or ionizing radiation effects. The targeted laser process imparts the least amount of energy in the remaining tissue without any of the deleterious photochemical or photothermal effects that accompanies other laser wavelengths and pulse parameters.
Skin wound healing is a regenerative process requiring the coordinated regulation of a variety of cell types and cell signalling pathways.1 This healing process is comprised of overlapping and linked phases : inflammation, proliferation (new tissue formation), and tissue remodeling.2 Coordination of these phases, together with cellular responses to tissue damage, shapes the outcome of healing tissue, resulting in a scar.2 During the proliferative phase of wound healing, mesenchymal (fibroblast-like) cells migrate into the healing wound, proliferate, and produce a disorganized matrix, providing the initial tensile strength to the wound, and regulating the size of the scar that will form.3
While most wounds heal with a scar that is acceptable to the patient, large scars cause conin surgical technique, siderable functional and cosmetic deformities, as well as psychological stress, and patient dissatisfaction. The biggest problem is the formation of scar tissue that impairs function, a problem in nearly all surgeries to some extent. Currently available approaches to optimize wound repair include refinements in surgical technique, nutritional supplementation, and the use of local wound care modalities.4 Despite these approaches, there has been little progress in the ability to regulate wound size.
The laser was first used as a surgical tool shortly after its invention as an alternative to mechanical surgical tools.5 In principle, lasers offer the prospect of performing surgery at the fundamental limit by exploiting the spatial phase coherence of laser radiation to focus sufficient intensity for ablation or cutting at the single cell level. Although lasers have emerged as a valuable surgical tool, conventional surgical lasers, having pulse durations longer than nanoseconds, impair the proliferative phase of healing due to thermally-induced cell damage in the surrounding tissue.6 Conventional medical lasers show benefits over mechanical surgical tools only in a very limited number of procedures.7
We recently reported on a novel laser source, the Picosecond IR Laser (PIRL), explicitly designed to exploit a newly discovered ablation mechanism in which the selective excitation of water’s vibrational modes couples directly to translation motions within tissue, the very motions involved in ablation, faster than any other material. By achieving superheating on picoseconds timescales, the nucleation sites for the ablative phase transition have nanometer (molecular) dimensions, avoiding cavitation and associated shock wave induced damage that has been one of the major stumbling blocks in using lasers for surgery. The strong acoustic attenuation at the 100 GHz frequency range further ensures that all the absorbed photon energy ablates tissue on time scales much faster than heat transfer can damage adjacent tissue of the targeted area. The pulse duration and heating rate is also specifically designed to avoid multiphoton ionization effects,8 that lead to highly reactive species known to be a major problem with other ionizing radiation sources. Using the PIRL system as a surgical tool and comparing it with a conventional laser and mechanical surgical tools, we performed a wound healing study on mice and compared the resultant ablative and tissue damaging characteristics, as well as the final impact on scar size.
To determine how the various modalities ablate tissue differently, the skin of the mouse subject was cut to a linear full thickness cut using the PIRL system, a commercial Er:YAG surgical laser (long pulse) at the exact same wavelength, or a conventional surgical scalpel. Transmission electron microscopy and scanning electron microscopy of the incised border revealed that the conventional laser damaged the skin border up to 800 μm away from the visible edge and the surgical scalpel caused dissociation of extracellular matrix fibres up to 400 μm further from the edge (Figure 1A–C). By comparison, cuts done with the PIRL system had sharp edges and minimal damage to adjacent tissue. The PIRL system generated a cutting gap of 8 μm, smaller than the diameter of a single skin fibroblast which was observed in the same skin sections. In contrast, the measured gap for scalpel incisions ranged from 40 to 120 micrometers and 650 μm for the conventional laser (data not shown). Wounds that were formed using the PIRL system had a higher number of viable skin cells immediately adjacent to the cut as compared to the other modalities (data not shown). Taken together, these results show that PIRL produces substantially less damage to the extracellular matrix and cells surrounding the wound, and ablates a much lower volume of tissue to execute the same function in comparison to a conventional laser and surgical scalpel.
Figure 1 Minimal tissue ablation with less damage of surrounding tissues by using the PIRL laser. Scanning electron microscopy (SEM) of skin at the cut borders. (A) The PIRL laser kept the collagen layer intact. (B) The conventional laser damaged (burned) skin and deformed the collagen fibres resulting in a damaged, irregular extracellular matrix surface. (C) The scalpel damaged the skin by shearing between the collagen fibres and exposing individual cells (Arrow shows an adiopocytes which is exposed in this image). (D-F) Representative histologic sections of healed skin of excisional 4mm circular full thickness wounds using the three methods at 9 days post wounding. (D) PIRL Laser (E) Conventional Laser (F) Skin Biopsy Punch. (G-I) Schematic of cutting modalities. (G) The well absorbed PIRL pulses cause superheating of water inside the tissue on the picosecond timescale, ejecting the tissue faster than energy can diffuse to the surroundings area. The remaining adjacent tissue shows minimal damage compared to the other two modalities. (H) Conventional surgical lasers cut by depositing heat until the tissue melts or burns away. The damage zone in this case, can reach up to 800 µm away from the ablated edge. (I) The mechanical scalpel cuts skin by producing shear forces which exceed the elastic limit of the tissue. This causes a border of damage around the incision which reaches as far as 400 µm from the borders of the incision.
In order to evaluate the amount of tissue damage and its effect on scar formation, we removed the same amount of tissue (excision of 4mm circular, full thickness, wounds) using the three methods and compared scar formation at different time points. Despite the same amount of tissue ablated by all modalities, the width of the scar formed by the PIRL system was half that of the wounds produced using either a conventional surgical laser or a scalpel at 9 days post-wounding (Figure 2 D–F). A similar trend was observed when incision of linear wounds were performed (data not shown). Moreover, there was a lower proliferation rate, as measured using KI-67 staining and aniline blue staining showed higher levels of collagen in the early stages of wounds produced using the PIRL system, suggesting that these wounds mature faster, and thus have a shorter proliferative phase.
These observations show that PIRL ablates the minimal amount of tissue and causes less damage to surrounding tissue (Figure 1G–I). Selective ablation process owes its efficacy to the ultrafast time scale of the ablation process. The process occurs on timescales comparable or faster than even collision induced energy redistribution between molecules within the excited zone. We have observed whole proteins, even weakly bound protein complexes driven into the gas phase as intact neutral species using mass spectroscopy.9 These molecules, especially the protein complexes, are extremely fragile and heretofore have never been observed in laser ablation without undergoing thermally driven fragmentation. This result shows that even at a molecular level there is minimal heat deposition into the constituent biological molecules. The key factor is the time scale under which the energy is preferentially partitioned within the excited water molecules that act as a propellant to drive the molecules into the gas phase and provide the cutting actions. The choice of pulse duration was made to be in this limit but not so short as to increase the peak power above the threshold for multiphoton ionization effects. In all cases, it is important to note that the forces remain far more localized than those involved in the use of mechanical tools, which need to exceed the shear elastic limit of the tissue in order to cut.
The use of PIRL can open up new surgical methods where scar tissue formation is particularly debilitating.10 This approach may have general applications in reducing hyperplastic scarring and also cosmetic application in the revision of existing hyperplastic scars. Moreover by decreasing the healing time, this new surgical modality may result in increased patient comfort and decreased risk of infections due to infection in surgery. The PIRL system is a new tool for scar prevention, promising outstanding results and improved surgical outcomes. As stated by Fitz Gibbon11 : “By your scars you will be judged”.
Conflict of interest statement: the author declares that he has no conflict of interest.
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REFERENCES
1 Ito M Yang Z Andl T Cui C Kim N Millar SE Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding Nature 2007 447 7142 316 20 17507982
2 Gurtner GC Werner S Barrandon Y Longaker MT Wound repair and regeneration Nature 2008 453 7193 314 21 18480812
3 Ashcroft GS Dodsworth J van Boxtel E Tarnuzzer RW Horan MA Schultz GS Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels Nat Med 1997 3 11 1209 15 9359694
4 Wadman M Scar prevention: the healing touch Nature 2005 436 7054 1079 80 16121148
5 Solon LR Aronson R Gould G Physiological implications of laser beams Science 1961 134 1506 8 13915009
6 Paltauf G Dyer PE Photomechanical processes and effects in ablation Chem Rev 2003 103 2 487 518 12580640
7 Sakimoto T Rosenblatt MI Azar DT Laser eye surgery for refractive errors Lancet 2006 367 9520 1432 47 16650653
8 Girard B Yu D Armstrong MR Wilson BC Clokie CM Miller RJ Effects of femtosecond laser irradiation on osseous tissues Lasers Surg Med 2007 39 3 273 85 17311312
9 Franjic K Talbot F Miller RJ Desorption by Impulsive Vibrational Excitation (DIVE) and postionization for the detection of biological molecules, using mass spectrometry (Submitted June 2010) PCCP 2010 Available from: URL:http://www.plosone.org/article/ (in press)
10 Sanders DL Reinisch L Wound healing and collagen thermal damage in 7.5-microsec pulsed CO(2) laser skin incisions Lasers Surg Med 2000 26 1 22 32 10637000
11 FitzGibbon GM The commandments of Gillies Br J Plast Surg 1968 21 3 226 39 4877604 | 21566783 | PMC3075523 | CC BY | 2021-01-04 19:24:20 | yes | Int J Prev Med. 2010 Fall; 1(4):260-263 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21541336PONE-D-10-0360410.1371/journal.pone.0019194Research ArticleBiologyAnatomy and PhysiologyPhysiological ProcessesAgingBiochemistryMetabolismMetabolic PathwaysOxygen MetabolismNucleic acidsDNADNA metabolismChemical BiologyModel OrganismsAnimal ModelsRatMolecular Cell BiologyCellular Stress ResponsesAge Related Changes in NAD+ Metabolism Oxidative Stress and
Sirt1 Activity in Wistar Rats Age Related Changes in NAD+
MetabolismBraidy Nady
1
Guillemin Gilles J.
1
2
Mansour Hussein
3
Chan-Ling Tailoi
3
Poljak Anne
4
Grant Ross
1
5
*
1
Department of Pharmacology, School of Medical Sciences, Faculty of
Medicine, University of New South Wales, Sydney, Australia
2
St Vincent's Centre for Applied Medical Research, Sydney,
Australia
3
Retinal and Developmental Neurobiology Lab, Discipline of Anatomy and
Histology, School of Medical Sciences, University of Sydney,
Australia
4
Bioanalytical Mass Spectrometry Facility, University of New South Wales,
Sydney, Australia
5
Australasian Research Institute, Sydney Adventist Hospital, Sydney,
Australia
Xu Aimin EditorUniversity of Hong Kong, China* E-mail: [email protected] and designed the experiments: NB RSG. Performed the experiments: NB
HM. Analyzed the data: NB GG RSG. Contributed reagents/materials/analysis
tools: GG TCL AP RSG. Wrote the paper: NB RSG.
2011 26 4 2011 6 4 e191946 10 2010 29 3 2011 Braidy et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key
regulator of metabolism, stress resistance and longevity. Apart from its role as
an important redox carrier, NAD+ also serves as the sole substrate for
NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an
important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins
which play an important role in a wide variety of processes, including
senescence, apoptosis, differentiation, and aging. We examined the effect of
aging on intracellular NAD+ metabolism in the whole heart, lung, liver and
kidney of female wistar rats. Our results are the first to show a significant
decline in intracellular NAD+ levels and NAD∶NADH ratio in all organs
by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These
changes in [NAD(H)] occurred in parallel with an increase in lipid
peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in
total antioxidant capacity in these organs. An age dependent increase in DNA
damage (phosphorylated H2AX) was also observed in these same organs. Decreased
Sirt1 activity and increased acetylated p53 were observed in organ tissues in
parallel with the drop in NAD+ and moderate over-expression of Sirt1
protein. Reduced mitochondrial activity of complex I–IV was also observed
in aging animals, impacting both redox status and ATP production. The strong
positive correlation observed between DNA damage associated NAD+ depletion
and Sirt1 activity suggests that adequate NAD+ concentrations may be an
important longevity assurance factor.
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Introduction
Multiple degenerative processes are implicated in natural senescence. As aging is
associated with progressive decline in organ function, elucidating the complex
pathways controlling the rate of aging is of significant clinical importance [1]. An important
mechanism contributing to aging is oxidative stress. The “free-radical theory
of aging”, initially proposed by Harman (1956) suggests that oxidative damage
occurs with advanced aging due to an imbalance between free radical and reactive
species (ROS) production, and cellular antioxidant defense mechanisms [2]. Elevated levels
of intracellular ROS through hydrogen peroxide treatment, or deficiency of ROS
scavenging enzymes such as superoxide dismutase (SOD1) knockdown, has been shown to
induce premature senescence and reduce cellular life span [3], [4], [5].
The mitochondria, represents the main producer of cellular ROS in the human body, and
approximately 1–2% of the oxygen molecules consumed during normal
respiration are converted into highly reactive superoxide anion, which is rapidly
dismutated to H2O2 by the superoxide dismutases [6]. Other pathways
and events able to produce ROS include peroxisomal metabolism, enzymatic synthesis
of nitric oxide, phagocytic leukocytes, heat, ultraviolet (UV) light, therapeutic
drugs, and ionizing radiation [7]. Intracellular ROS, due to their high reactivity, can
interact with a spectrum of biological molecules, leading to the oxidation of
several macromolecules, such as protein, lipids, and nucleic acids [8]. As a result,
vital functions, such as energy production, maintenance of plasma membrane
potential, and cellular ionic homeostasis may be impaired in the early stage of
oxidative stress [8]. Excessive oxidative insult may also stimulate secondary
events leading to cell death via an apoptotic mechanism [9].
A major factor associated with age-related diseases is the increase of oxidative DNA
damage [7]. It is
estimated that at least 5000 single-stranded DNA breaks occur during a single cell
cycle as a result of ROS production [10], [11]. Approximately 1% of these DNA breaks are
converted into double-stranded DNA breaks, primarily during DNA replication.
Accumulation of unrepaired DNA damage induced by ROS can lead to arrest or induction
of transcription, induction of signal transduction pathways, replication errors and
genomic instability [10], [11]. These molecular changes are observed in both cancer and
aging, and this supports the notion that chronic oxidative damage to DNA might
trigger cancer and promote aging [10], [11].
The removal of oxidative DNA damage through repair of DNA single strand breaks by DNA
base excision repair, is facilitated by Poly(ADP-ribose) polymerase-1 (PARP) [12], [13], [14]. PARP is an
abundant protein modifying nuclear enzyme involved in DNA repair. The enzymatic
activity of PARP is strongly activated in cells in response to treatment with ROS
such as H2O2
[15]. Activation of
PARP leads to the transfer of ADP-ribose moieties from NAD+, to the
target protein [16]. Since PARP uses NAD+ as the only
endogenous substrate for poly-ADP-ribosylation, PARP activity is dependent on the
amount of NAD+ available, and may act as a nuclear energy sensor.
Under physiological conditions, mild activation of PARP can regulate several
cellular processes, including DNA repair, cell cycle progression, cell survival,
chromatin remodeling, and genomic stability [13], [17]. However, overactivation of PARP
can repress genomic transcription and reduce cell survival. NAD+, in
addition to being a substrate for PARP, also serves as an important redox carrier to
power oxidative phosphorylation and ATP production [18]. Depletion of
NAD+ following PARP hyperactivation has been shown to deplete
intracellular ATP stores leading to the release of apoptosis-inducing factors (AIF)
and consequent cell death due to energy restriction [19]. PARP activation has been
implicated in the pathogenesis of hypertension, atherosclerosis, lung injury,
haemorrhagic shock, and diabetic cardiovascular and kidney complications [20], [21], [22], [23], [24]. In these
diseases, the oxidant-mediated endothelial cell injury is dependent on PARP
activation, and can be attenuated by pharmacological PARP inhibitors [25], [26]. Therefore,
tight regulation of PARP activity is crucial to prevent the development of several
age-related pathological disorders.
In addition to its role in PARP activity, another essential factor that is greatly
affected by changes in intracellular NAD+ levels is the class III
histone deacetylases known as sirtuins, or silent information regulator of gene
transcription [27].
Gene silencing by this family of enzymes has been correlated directly with longer
lifespan in yeast and worms [28]. In yeast, sir2 plays a critical role in transcriptional
silencing and maintenance of genomic stability [29], [30]. Sirt1 is the human homolog of
sir2 and appears to be involved in several physiological functions including the
control of gene expression, cell cycle regulation, apoptosis, DNA repair,
metabolism, and aging [31], [32], [33]. Sirt1 can deacetylate numerous proteins such as tumor
suppressor protein, p53, which modulates various genes that control damaged DNA
[34]. The
deacetylase activity of SIRT proteins is dependent on the intracellular
NAD+ content [27]. They catalyse a unique reaction that releases
nicotinamide, acetyl ADP-ribose (AADPR), and the deacetylated substrate [27]. Impaired SIRT1
activity due to PARP mediated NAD+ depletion allows increased
activity of several apoptotic effectors such as p53, therefore sensitising cells to
apoptosis. Adequate NAD+ levels are therefore critical to
maintaining Sirt1 activity which can delay apoptosis and provide vulnerable cells
with additional time to repair even after repeated exposure to oxidative stress
[35].
Though a number of studies have demonstrated elevated levels of oxidative stress
associated damage in aged tissue, to our knowledge, no study has yet reported on
changes in NAD+ levels during the aging process. In this study, we
have characterized and quantified changes in NAD+ metabolism in the
liver, heart, kidney and lung from female wistar rats aged from 3 to 24 months,
spanning life stages from young adulthood to old age [36]. We quantified the levels of
oxidative stress (in the form of protein carbonyls, lipid peroxidation, and
oxidative DNA damage), total antioxidant and NAD+ levels and PARP,
Sirt1 and mitochondrial activities in these tissues at different stages of life. Our
results suggest that oxidative stress induced NAD+ depletion could
play a significant role in the aging process, by compromising, energy production,
DNA repair and genomic surveillance.
Materials and Methods
Reagents and Chemicals
Phosphate buffer solution (PBS) was from Invitrogen (Melbourne, Australia).
Nicotinamide, bicine, β-nicotinamide adenine dinucleotide reduced form
(β-NADH), 3-[-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT), alcohol dehydrogenase (ADH), sodium pyruvate, TRIS,
γ-globulins, N-(1-naphthyl) ethylenediamine dihydrochloride, EGTA, EDTA,
tricarboxylic acid (TCA), Hepes, proteinase K, Percoll, mannitol, 1,-dithio
DL-threitol (DTT), KCN, decylubiquinone, succinate, antimycin, rotenone,
cytochrome c, and sodium borohydride were obtained from
Sigma-Aldrich (Castle-Hill, Australia). Phenazine methosulfate (PMS) was
obtained from ICN Biochemicals (Ohio, USA). Bradford reagent was obtained from
BioRad (Hercules, CA, USA). DAPI and polyclonal antibodies (pAb) for β-actin
and all chemicals used for Western blots (unless otherwise stated) were obtained
from Sigma-Aldrich (Castle-Hill, Australia). Polyclonal antibodies for detection
of (E)-4- Hydroxynonenal, Sirt1, phospho-H2AX-ser139 were obtained from Alexis
Biochemicals (San Diego, CA, USA). Monoclonal antibody for the detection of
Poly(ADP-ribose) was purchased from Alexis Biochemicals (San Diego, CA, USA).
Polyclonal antibody for the detection of acetylated p53 was obtained from Abcam
(Cambridge, UK). Monoclonal antibody to total p53 was obtained from Millipore
(Melbourne, Australia). Alexa 488- or Alexa 594-conjugated anti-mouse IgG or
anti-rabbit were purchased from Invitrogen. All commercial antibodies were used
at the concentrations recommended by the manufacturer.
Animals
Female wistar rats were used and experiments were designed to target the
following age groups: 3 months old (equivalent to a young human adult aged 20
years), 12 month old (equivalent to a middle-aged human aged 40 years) and 24
month old (equivalent to an aged human greater than 80 years) [37]. The
animals were individually housed in an environmentally controlled room under a
12∶12 hour light/dark cycle at 23°C and were given commercial rat chow
and water ad libitum. All experiments were performed according
to the Animal Ethics Committee of the University of Sydney. Anaesthesia was
induced with a mixture of O2, NO2, and 5%
halothane, and then maintained by an intraperitoneal injection of sodium
pentobarbitone (60 mg kg−1). Transcardial perfusion was
performed using 0.1 M DPBS, pH 7.4. Whole heart, lung, liver and kidney, were
removed, washed with DPBS and used immediately for a variety of biochemical and
histochemical procedures.
Assay of o- and m- tyrosine for the
Measurement of Protein Carbonyl Formation
The assay for o- and m- tyrosine was performed
as previously described [38]. Briefly, calibration standards of phenylalanine (313
pmol and 50 nmol) and o- and m- tyrosine (313
fmol to 50 pmol) were used for quantification. Labelled
[13C6]-phenylalanine was used as an internal
standard and was added to all samples and standards. The liver, heart, kidney
and lung were placed in tapered glass vial. The protein was then allowed to
precipitate (∼18 h, −20°C) following the addition of 10 volumes of
acetone/HCl (100∶1, v∶v). The precipitate was centrifuged (14,000 g,
30 min, ambient temperature) and the supernatant discarded. Internal standard
(10 µl, 1 mg/ml [13C6]-phenylalanine)
containing trace quantities of [13C6]-tyrosine,
was added to all samples and standards, and dried under reduced pressure (<1
Torr, 1–2 h). Afterwards, gas phase acid hydrolysis was performed at
150°C for 1 h. Samples and standards were then derivatised using a
three-step derivatisation approach, using heptafluorobutyric anhydride (HFBA),
pentafluorobenzylbromide (PFBBr) and N,O-bis(trimethylsilyl)-trifluoroacetamide
(BSTFA), vacuum dried for 10 min, and reconstituted in 50 µl ethyl
acetate. Each standard and sample (1 µl) was injected into the GC/MS using
pressure pulse splitless loading (6890N gas chromatograph interfaced to 5973
mass selective detector; Agilent technologies, Ryde, Australia), with methane as
the ECNI reagent gas. Chromatography was performed on a fused silica capillary
column (HP-5MS; 30 m×0.25 mm i.d.×0.25 µm, film thickness),
with helium carrier gas and using the following chromatographic parameters:
inlet temperature 250°C, transfer line temperature 280°C, programmed
oven temperature gradient with initial temperature of 150°C for 3 min, then
a temperature ramp of 30°C/min to 290 and a final time of 3 min at
290°C. Final o- and m- tyrosine levels
were expressed relative to phenylalanine levels.
Western Blots for the Detection of 4-Hydroxynonenal, Poly(ADP)-ribose, Sirt1,
and Total/Acetylated p53 Protein Expression
The whole liver, heart, kidney and lung were homogenised in RIPA lysis buffer
containing 50 mM Tris-HCl (pH 7.4); IGEPAL 1%; 0.25%
Na-deoxycholate; 1 mM EDTA, 150 mM NaCl; 1 µg/ml each of aprotinin,
leupeptin and pepstatin; 1 mM Na3VO4; and 1 mM NaF. After
1 h, the homogenate was obtained by centrifuging at 14,000 g, for 30 min at
4°C. Protein concentration was determined using the Bradford protein assay.
Equal amounts of protein extract (30 µg) were dissolved in Laemmli sample
buffer (Hercules, CA, USA), boiled for 5 min, electrophoresed on
8–12% (v/v) polyacrylamide SDS-PAGE gels (BioRad, Hercules, CA,
USA), and electrotransferred onto PVDF membranes (BioRad, Hercules, CA, USA).
Membranes were blocked with 5% non-fat milk dissolved in TBS for 1 h and
incubated with primary antibody overnight at 4°C. The primary antibodies
used are described in Table
1. After incubation with primary antibodies, membranes were washed in
TBS-Tween-20 and incubated with HRP conjugated secondary antibodies (Sigma,
Castle Hill, Australia) for 1 h at room temperature. After further washing in
TBS-Tween-20, the membranes were incubated with an ECL plus reagent (RPN2132,
Amersham) and protein bands were visualised on X-ray films. The bands were
quantified by ImageJ software, and normalised to β-actin, which served as an
internal control.
10.1371/journal.pone.0019194.t001Table 1 List of primary antibodies used.
Antibody Type Antigen Dilution Source
(E)-4-Hydroxynonenal
Polyclonal Free HNE (E)-4-hydroxynonenal 1∶1000 Alexis Biochemicals
Phospho-H2AX-Serine139
Polyclonal Phosphorylated H2AX at Serine 139 1∶1000 Alexis Biochemicals
Poly(ADP-ribose) (10H)
Monoclonal Poly(ADP-ribose) 1∶400 Alexis Biochemicals
Sirt1
Polyclonal Sirt1 1∶2000 Alexis Biochemicals
Acetyl K386
Polyclonal Acetylated p53 at lysine 386 1∶1000 Abcam
p53 Oncogene
Monoclonal Total p53 oncogene 1∶1000 Millipore
Total Antioxidant Capacity Assay
The total antioxidant capacity in aging liver, heart, kidney and lung was
evaluated by Trolox equivalent antioxidant capacity assay using a standard
antioxidant assay kit (Cayman, Michigan, USA). Briefly, the tissue was
homogenised on ice in a buffer containing 5 mM potassium phosphate; 0.9%
NaCl; and 0.1% glucose, pH 7.4. The samples were centrifuged at 10,000 g
for 15 min at 4°C, and the supernatant was collected for the assay. Briefly,
10 µl of sample was added to 10 µl of Metmyoglobin and 150 µl
of Chromogen. The reaction was initiated by adding 40 µl of hydrogen
peroxide working solution. The samples were incubated on a shaker for 5 min at
room temperature and the absorbance was read at 405 nm using the Model 680XR
microplate reader (BioRad, Hercules). The absorbance values obtained for the
samples were compared with a standard curve obtained using Trolox
(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid), a permeable cell
derivative of vitamin E commonly employed as an antioxidant. The results were
expressed as mmol Trolox/L/mg protein.
DNA Damage Assay
Since H2AX phosphorylation at serine 139 correlates very closely with each
double-stranded DNA break, phosphor-H2AX is a sensitive marker for DNA damage
[39]. We
measured phosophorylation of H2AX at serine 139 using a DNA damage assay kit
(Active Motif, Carlsbad, California, USA). Briefly, the liver, heart, kidney and
lung was suspended on ice in a buffer containing 1 mM PMSF and pelleted at
10,000 g for 4 min at 4°C. Afterwards 200 µl of warm SDS lysis buffer
containing 1 µg/ml each of aprotinin, leupeptin and pepstatin and 1 mM
PMSF were then added and incubated for 10 min on ice. All samples were sonicated
to generate 100 - to 1000-bp DNA fragments. 5 µg/ml of
anti-phospho-H2AX-ser139 was added to the lysate with cold DPBS and allowed to
incubate overnight at 4°C. The primary antibody was removed by washing the
lysate 3 times with 200 µl DPBS. Alexis 488 goat anti-rabbit IgG secondary
antibody (1∶1000) was added to the lysate and incubated for 1 hour at room
temperature. The secondary antibody was removed by washing the lysate twice with
200 µl DPBS. The fluorescence was read using Fluostar Optima Fluorometer
(NY, USA). Filter excitation and emission was set at 485 nm and 520 wavelengths
respectively.
Isolation and Extraction of Nuclei for PARP and Sirt1 deacetylase Activity
Assays
Aliquots of homogenate from the liver, heart, kidney and lung (without protease
inhibitors) were spun through 4 ml of 30% sucrose solution containing 10
mM Tris HCl (pH 7.4); 10 mM NaCl; and 3 mM MgCl2 at 1,300 g for 10
min at 4°C. The remaining pellet was washed with cold 10 mM Tris-HCl (pH
7.4) containing 10 mM NaCl. The nuclei was later suspended in 50–100
µl extraction buffer containing 50 mM Hepes KOH (pH 7.4); 420 mM NaCl; 0.5
mM EDTA; 0.1 mM EGTA; and glycerol 10%, sonicated for 30 s, and allowed
to stand on ice for 30 min. After centrifugation at 10,000 g for 10 min, an
aliquot of the supernatant was used to determine protein concentration using the
Bradford protein assay.
PARP Activity Assay
PARP activity was measured in nuclear extracts from the liver, heart, kidney and
lung of young, middle-aged, and aged rats, using a new operational protocol
relying on the chemical quantification of NAD+ modified from
Putt et al. [40]. The final reaction mixture contained 10 mM
MgCl2, Triton X-100 (1%), and 20 µM
NAD+ in 50 mM Tris buffer, pH 8.1. The plate was then
incubated for 1 hour and the amount of NAD+ consumed was
measured by the NAD(H) microcycling assay using the Model 680XR microplate
reader (BioRad, Hercules). The results were expressed as NAD+
consumed/h/mg protein.
Sirt1 deacetylase Activity
Sirt1 deacetylase activity was evaluated in nuclear extracts from the liver,
heart, kidney and lung of young, middle-aged, and aged rats, using the Cyclex
SIRT1/Sir2 Deacetylase Flourometric Assay Kit (CycLex, Nagano, Japan). The final
reaction mixture (100 µl) contained 50 mM Tris-HCl (pH 8.8), 4 mM MgCl2,
0.5 mM DTT, 0.25 mA/ml Lysyl endopeptidase, 1 µM Trichostatin A, 200
µM NAD+, and 5 µl of nuclear sample. The samples
were mixed well and incubated for 10 min at room temperature and the
fluorescence intensity (ex. 340 nm, em. 460 nm) was measured every 30 s for a
total of 60 min immediately after the addition of fluorosubstrate peptide (20
µM final concentration) using Fluostar Optima Fluorometer (NY, USA) and
normalised by the protein content. The results are reported as relative
fluorescence/µg of protein (AU).
Measurement of Intracellular NAD+/NADH Levels
Total intracellular NAD (NADH+NAD+) concentration was
measured spectrophotometrically using the thiazolyl blue microcycling assay
established by Bernofsky and Swan (1973) adapted for 96 well plate format by
Grant and Kapoor (1998) [41], [42]. Briefly, each assay contained 100 mM bicine, pH 7.8;
0.42 mM MTT, and 1.66 mM PMS. For NAD+ measurement, 20 µl
of ADH in 0.15% ethanol was added to the reaction mixture. The amount of
NAD and NADH were measured as the change in absorbance at 590 nm at 37°C for
10 minutes with a Model 680XR microplate reader (BioRad, Hercules). The ratio of
NAD+/NADH was calculated based on results of
NAD+ and NADH concentrations.
Isolation of Mitochondria
All procedures were carried out at 0–4°C. Briefly, the liver, heart,
kidney and lung were excised, washed with DPBS, and treated with proteinase K (1
mg/ml) for 30 s, washed with buffer A (250 mM mannitol; 0.5 mM EGTA; 5 mM Hepes;
and fatty acid free bovine serum albumin 0.1%, pH 7.4 at 4°C). The
samples homogenised with buffer A using a Teflon pestle. The homogenate was
centrifuged twice at 600 g for 5 min at 4°C. The supernatants were mixed and
centrifuged at 10,300 g for 10 min at 4°C. The mitochondrial pellets were
suspended in 0.5 ml buffer A and transferred to ultracentrifuge tubes containing
1.4 ml buffer B (225 mM mannitol; 1 mM EGTA; 25 mM Hepes; and fatty acid free
bovine serum albumin (BSA) 0.1%, pH 7.4 at 4°C) and 0.6 ml Percoll.
The mixture was centrifuged at 105,000 g for 30 min at 4°C. The pure
mitochondrial fraction was purified by washing twice with buffer A at 10,300 g
for 10 min at 4°C to remove the Percoll and frozen to −80°C. An
aliquot was used to determine the mitochondrial protein content using the
Bradford protein assay.
Determination of Mitochondrial Complexes I, II, III, and IV
activities
To determine the complex I activity, submitochondrial fractions (0.6 mg/ml) were
incubated for 3 min in buffer solution containing 250 mM sucrose; 50 mM
potassium-phosphate; 1 mM KCN; 50 µM decylubiquinone; 0.8 µM
antimycin, pH 7.4. The reaction was started following the addition of NADH (100
µM). The activity of NADH CoQ oxidoreductase was measured as the rate of
oxidation of NADH at 340 nm using the Model 680XR microplate reader (BioRad,
Hercules). The activity of complex I was expressed as nmol oxidised NADH/min/mg
protein. The activity of complex II was measured in buffer solution containing
submitochondrial fractions (0.03 mg/ml); 100 mM potassium-phosphate; 20 mM
succinate; 0.8 µM antimycin; 50 µM rotenone; 2 µM KCN; 50
µM DCIP, pH 7.4. The reaction was initiated by the addition of 50 µM
decylubiquinone. The activity of succinate:DCIP oxireductase was measured as the
rate of reduction of 2, 6-DCIP at 600 nm with 520 nm as reference wavelength.
The activity of complex II was expressed in nmol reduced DCIP/min/mg protein.
The activity of complex III was measured in buffer solution containing
submitochondrial fractions (0.03 mg/ml); 35 mM potassium-phosphate; 5 mM
MgCl2; 2.5 mg/ml BSA; 50 mM rotenone; 1.8 mM KCN; 2 mM
decylubiquinone, pH 7.5. The reaction was initiated by the addition of 125
µM cytochrome c. The activity of ubiquinol:cytochrome
c reductase was measured as the rate of reduction of
cytochrome c at 550 nm with 580 nm as the reference wavelength.
The activity of complex III was expressed in nmol reduced cytochrome
c/min/mg protein. The activity of complex IV was measured
in a buffer solution containing submitochondrial fractions (0.1 mg/ml); 50 mM
potassium-phosphate, pH 6.8. The reaction was started by the addition of 75
µM cytochrome c previously reduced with sodium
borohydride and measuring the absorbance at 550 nm. The activity of cytochrome
c oxidase was expressed as nmol oxidised cytochrome
c/min/mg protein.
Immunoflourescence Staining for 4-Hydroxynonenal, Phospho-H2AX-Serine139,
Poly(ADP)-ribose, and Sirt1
Liver, heart, kidney and lung whole mounts were immersion-fixed with ice cold
70% ethanol for 20 mins followed by 4% paraformaldehyde (PFA), pH
7.4 for 10 min at 4°C. Immunostaining was established on brain whole mount.
Fixed tissue were permeabilised with 1% Triton-X 100 in DPBS for 30 min
at room temperature and non-specific binding was blocked with 1% BSA in
DPBS for 2 h before application of primary antibodies. All specific primary mAb
or pAb antibodies were diluted in 1% BSA in PBS and incubated overnight
at 4°C. Tissue whole mounts were washed three times with DPBS and incubated
for 1 h at 37°C with the appropriate labeled secondary antibodies (goat
anti-mouse IgG or goat anti-rabbit IgG coupled to Alexa 488 or Alexa 594).
Nuclear staining was performed using DAPI at 1 µg/ml for 5 min at room
temperature. The slides were washed 3 times in DPBS at room temperature and
coverslips were mounted on glass slides with Flouromount-G and were examined
with an Olympus BX60 fluorescence microscope fitted with a SensiCam digital
camera. Titration series and conjugate experimental controls were performed on
both primary and secondary antibodies to optimise for low background noise,
maximal stain sensitivity, avoidance of non-specific staining and
cross-reactivity.
Bradford Protein Assay for the Quantification of Total Protein
DNA damage, total antioxidant capacity, PARP and Sirt1 activities,
NAD+ concentration, and mitochondrial function were adjusted
for variations in protein number using the Bradford protein assay described by
Bradford [43].
Data Analysis
Results obtained are presented as the means ± the standard error of
measurement (SEM) of at least eight animals per age group analysed in duplicate.
One way analysis of variance (ANOVA) and post hoc Tukey's multiple
comparison tests were used to determine statistical significance between
treatment groups. Differences between treatment groups were considered
significant if p was less than 0.05 (p<0.05).
Results
Age-related Increase in Protein Carbonyl Formation
Hydroxyl radical attack on the amino acid phenylalanine generates
o-, m- and p- tyrosine
were detected using gas chromatography/mass spectrometry (GC/MS) [44], [45]. Figure 1 shows the age-related
changes in o- (Fig. 1A) and m- tyrosine (Fig. 1B) levels in the liver, heart, kidney
and lung. Our results show a significant increase in both o- and
m- tyrosine formation with age, with the highest rate of increase
occurring after 12 months of age in all organs (p<0.01).
10.1371/journal.pone.0019194.g001Figure 1 Increased formation of oxidatively modified proteins in the liver,
heart, kidney, and lung with age.
Increased levels of (A) o- tyrosine and (B)
m- tyrosine were reported in the brain after 12
months of age compared to 3 month old rats. All values are means
± S.E from tissue obtained from eight different rats for each age
group. Significance *p<0.01 compared to 3 month old
rats.
4-HNE as a Biomarker for Lipid Peroxidation in Aging
Peroxidative damage to cellular lipid constituents was determined by using 4-HNE
as a marker of lipid peroxidation. Increased 4-HNE protein expression using
western blotting was observed in the liver (p<0.01) (Fig. 2A), heart (p<0.01) (Fig. 2B), kidney (p<0.01)
(Fig. 2C) and lung
(p<0.01) (Fig. 2D),
consistent with an increase in protein carbonyl formation (Fig. 1).Using immunohistofluorochemistry, we
stained frozen sections for 4-HNE (Fig. 2E). Two age related changes are clearly visible: (1) There is
an increase in 4-HNE expression consistent with the previous western blot
studies (Fig. 2A–D).
(2) Aged tissue appears to demonstrate significantly lower cell volume,
indicative of extensive cell loss occurring during the aging process.
10.1371/journal.pone.0019194.g002Figure 2 Increased oxidative damage to lipids in selected brain regions with
age.
(A) Liver, (B) heart, (C) kidney, and (D) lung were analysed by Western
blotting using anti-4HNE antibody. The blots shown are representative
tracings of an experiment done eight times. Graphs are mean ± S.E
from tissue obtained from eight different rats for each age group. Each
bar of the quantification graph represents the corresponding band for
each age group. Significance *p<0.01 compared to 3
month old rats. (E) Immunodetection of 4-HNE in the liver, heart,
kidney, and lung from 3 month, 12 month and 24 month old rats. 4-HNE
(green) and DAPI (blue). Higher immunoreactivity for 4-HNE was observed
in 12 and 24 month old rats compared to 3-month old rats.
Total Antioxidant Capacity Declines with Aging
To investigate changes in endogenous antioxidant function age, we measured total
antioxidant capacity in-situ using the Trolox assay. We
observed a significant reduction in total antioxidant activity by 12 months of
age in all organs with the most rapid decline occurring between 12 and 24 months
(p<0.01) (Fig. 3).
10.1371/journal.pone.0019194.g003Figure 3 Total antioxidant capacity significantly declined in the rat liver,
heart, kidney, and lung with age.
All values are means ± S.E from tissue obtained from eight
different rats for each age group. Significance
*p<0.01 compared to 3 month old rats.
DNA Damage Increases with Aging
The aging process is also considered to cause changes in the DNA repair process
leading to increased vulnerability to DNA damage [7]. We assessed DNA damage by
measuring the phosphorylation of H2AX at Serine139 residue using a flourometric
assay. Increased DNA damage was observed in all aged organs assessed in this
study by 12 months of age with accelerated damage occurring between 12 and 24
months (Fig. 4) (p<0.01).
Immunohistofluorochemistry (Fig.
4C) confirmed the increase in phospho-H2AX-Serine139.
10.1371/journal.pone.0019194.g004Figure 4 Increased DNA damage was reported in the rat liver, heart, kidney,
and lung with age.
(A) DNA damage was determined by measuring the flourescence of
phosphorylated H2AX at Serine139 using the Fluostar Optima Fluorometer
(NY, USA). All values are means ± S.E from tissue obtained from
eight different rats for each age group. Significance
*p<0.01 compared to 3 month old rats. (B)
Immunodetection of phosphor-H2AX-Serine139 in the liver, heart, kidney,
and lung from 3 month, 12 month and 24 month old rats.
Phosphor-H2AX-Serine139 (green) and DAPI (blue). Higher immunoreactivity
for phosphor-H2AX-Serine139 was observed in 12 and 24 month old rats
compared to 3-month old rats.
PARP Activation & Poly(ADP-ribose) polymers
To demonstrate a direct link between DNA damage and poly-ADP-ribosylation, we
measured PARP activity in nuclei from the liver, heart, kidney and lung of aging
rats. Our results show a significant (p<0.01) up-regulation of PARP activity
in all organs by 12 months of age (Fig. 5A). This is consistent with increased formation of
poly(ADP-ribose) nuclear protein in the liver (Fig. 5B), heart (Fig. 5C), kidney (Fig. 5D),and lung (Fig. 5E), using western blot and
immunohistofluorochemistry (Fig.
5F).
10.1371/journal.pone.0019194.g005Figure 5 Increased Poly(ADP-ribose) activity in the brain with age.
(A) PARP activity was determined in aging tissue using a
spectrophotometric assay. All values are means ± S.E from tissue
obtained from eight different rats for each age group. Significance
*p<0.01 compared to 3 month old rats. (B) Western
blotting for poly(ADP-ribose) in (i) liver, (ii) heart, (iii) kidney,
and (iv) lung with aging using anti-Poly(ADP-ribose) (10H) antibody. The
blots shown are representative tracings of an experiment done eight
times. Graphs are mean ± S.E from tissue obtained from eight
different rats for each age group. Each bar of the quantification graph
represents the corresponding band for each age group. Significance
*p<0.01 compared to 3 month old rats. (C)
Immunodetection for poly(ADP-ribose) in the liver, heart, kidney and
lung from 3 month, 12 month and 24 month old rats. Poly(ADP-ribose)
(green) and DAPI (blue). Higher immunoreactivity for poly(ADP-ribose)
was observed in 12 and 24 month old rats compared to 3-month old
rats.
Intracellular NAD+(H) Levels
As NAD+ is the sole substrate for PARP we assessed the impact of
PARP activation on intracellular NAD+ content in the same aging
rat tissue. We found that increased PARP activity was associated with a
significant (p<0.01) reduction in the NAD+ content by 24
months of age in each of the organs investigated (Fig. 6A). This is in line with a significant
increase in NADH (Fig. 6B)
(p<0.01) and a significant decline in the NAD+∶NADH
ratio (Fig. 6C) (p<0.01).
Most of the decline in intracellular NAD+ content was observed
after 12 months of age, and appears to be more noteworthy in the heart.
10.1371/journal.pone.0019194.g006Figure 6 Increased PARP activation alters pyridine nucleotide metabolism with
aging.
(A) NAD+ content and (B) NADH content were determined in
the rat liver, heart, kidney and lung with aging using a
spectrophotometric assay. All values are means ± S.E from tissue
obtained from eight different rats for each age group. Significance
*p<0.01 compared to 3 month old rats. (C)
NAD+∶NADH ratio was determined as the total
NAD+ content divided by total NADH levels. All
values are means ± S.E from tissue obtained from eight different
rats for each age group. Significance *p<0.01 compared
to 3 month old rats.
Sirt1 deacetylase Activity (but not protein levels) Declines with
Aging
Sirt1 is localised in the nucleus [27]. Therefore, both Sirt1 protein level and activity
were measured in the liver, heart, kidney and lung of aging rats. Aging induced
a significant decline in Sirt1 activity in the liver (p<0.01), heart
(p<0.01), kidney (p<0.01), and lung (p<0.01) (Fig. 7A) consistent with decreased substrate
(NAD+) availability (Fig.
6). In contrast, we observed that aging significantly (p<0.01)
increased the amount of Sirt1 protein in the nucleus in all organs examined in
this study, using both western blotting (Fig. 7B–E) and
immunohistofluorochemistry (Fig.
7F).
10.1371/journal.pone.0019194.g007Figure 7 Reduced NAD+ levels contributes to reduced Sirt1
activity in the liver, heart, kidney and lung with aging.
(A) Reduced Sirt1 activity was observed in the rat liver, heart, kidney
and lung after 12 months of age using a flourometry. All values are
means ± S.E from tissue obtained from eight different rats for
each age group. Significance *p<0.01 compared to 3
month old rats. (B) Western blotting for Sirt1 in (i) liver, (ii) heart,
(iii) kidney, and (iv) lung with aging using anti-Sirt1 antibody. The
blots shown are representative tracings of an experiment done eight
times. Graphs are mean ± S.E brains from tissue obtained from
eight different rats for each age group. Each bar of the quantification
graph represents the corresponding band for each age group. Significance
*p<0.01 compared to 3 month old rats. (C)
Immunodetection for Sirt1 in the liver, heart, kidney and lung from 3
month, 12 month and 24 month old rats. Sirt1 (red) and DAPI (blue).
Higher immunoreactivity for Sirt1 was observed in 12 and 24 month old
rats compared to 3-month old rats.
Increased p53 Acetylation in Aging Brain
To confirm the effect of decreased NAD+ levels and Sirt1 function
on the acetylation status of p53, we measured both acetylated and total p53
protein levels using western blotting. As shown in Figure 8, we found a significant
age-dependent increase in acetylated p53 expression in the liver (p<0.01),
heart (p<0.01), kidney (p<0.01), and lung (p<0.01). However, no change
was observed in total p53 content between young and aged rats in all organs
investigated.
10.1371/journal.pone.0019194.g008Figure 8 Reduced Sirt1 activity induces p53 acetylation in the liver, heart,
kidney and lung with aging.
Acetylated p53 and total p53 levels were determined by Western blotting
in (A) liver, (B) heart, (C) kidney, and (D) lung with aging using
anti-acetylated p53 and anti-total p53 antibodies. The blots shown are
representative tracings of an experiment done eight times. Graphs are
mean ± S.E brains from tissue obtained from eight different rats
for each age group. Each bar of the quantification graph represents the
corresponding band for each age group. Significance
*p<0.01 compared to 3 month old rats.
Impaired Mitochondrial Respiratory Chain Activity with Aging
Although mitochondrial production of ROS has been shown to increase with
advancing age [46], changes in the mitochondrial redox status with age
in the heart, lung, liver and kidney has not, to our knowledge, been evaluated.
We measured the activities of the mitochondrial respiratory complexes as shown
in Figure 9A–D. The
activity of all complexes decreased after 12 months of age, and reached
statistical significance at 24 months in the liver (p<0.01), heart
(p<0.01), kidney (p<0.01) and lung (p<0.01). Fig. 8E shows the ATP levels determined in
all organs during the aging process. ATP levels significantly (p<0.01)
declined in the heart, lung, liver and kidney after 12 months of age. This is in
line with increased PARP activation and decline in cellular NAD+
content during the aging process.
10.1371/journal.pone.0019194.g009Figure 9 Oxidative stress-mediated reduction in mitochondrial respiratory
chain activity in the liver, heart, kidney and lung with age.
Reduced (A) complex I, (B) complex II, (C) complex III, and (D) complex
IV, activities at 24 months of age. All values are means ± S.E
from tissue obtained from eight different rats for each age group.
Significance *p<0.01 compared to 3 month old rats.
Discussion
There is growing consensus around the hypothesis that oxidatively damage proteins,
lipids, and nucleic acids play a key role in the aging process. Oxidative stress
occurs in several degenerative diseases including neurodegenerative disease (e.g.
Alzheimer's, Parkinson's), rheumatoid arthritis, atherosclerosis,
diabetes, and cardiovascular disease [47]. This study is the first to
investigate the impact of age associated changes in oxidative stress levels on both
intracellular NAD+ levels and activity of the
‘longevity’ enzyme Sirt1. This study is also the first to report changes
in o- and m- tryosine (specific markers of protein
oxidation) in aging heart, lung, liver and kidney.
In this report we provide consistent evidence of accumulating oxidative damage to
tissue proteins, lipids and nuclear DNA with age. Reactive oxygen species (ROS) are
byproducts of normal cellular physiology [48]. A delicate balance between ROS
generation and detoxification is maintained by several cellular antioxidant defense
systems [49],
[50]. These
comprise antioxidant enzymes such as SOD1, catalase, glutathione peroxidase; and
endogenous non-enzymatically acting compounds, such as vitamins A, C and E [9]. Nevertheless,
various endogenous and exogenous triggers may stimulate the overproduction and
accumulation of ROS leading to oxidative damage [9]. While oxidative stress increases
with aging, it is yet unclear at which age free-radicals may act to initiate the
senescence process. Young and aged rat models are relatively homogenous in their
gene expressions in the various organs. This may be in contrast to young adult and
aged human populations which may demonstrate much greater heterogeneity. Unlike aged
rodents, humans may diverge in their rates of aging, as they translate from middle
age to old age. In this study at middle age (i.e.12 months), female wistar rats
already exhibit higher levels of oxidative stress induced damage to proteins,
lipids, and DNA, with a lower total antioxidant efficiency than young adult animals
of 3 months of age. Our results suggest an age-dependent increase in oxidative
stress in these organs that appears to accelerate after middle age (i.e.12 months).
These data support the existence of a progressive disequilibrium between the
formation of ROS and the antioxidant protective mechanisms during the process of
normal aging [9] in
rats that may be applicable to the human population.
Apart from increased levels of protein oxidation and lipid peroxidation, DNA is also
susceptible to oxidative damage by ROS [7]. We assessed DNA damage by
measuring the phosphorylation of histone H2AX at Serine139 residue. H2AX
phosphorylation is considered a specific reporter of double-stranded DNA damage
[39]. The level
of DNA damage increased with age in all organs consistent with an increase in
protein carbonyls and lipid peroxidation. Experimental data from other laboratories
have shown a similar increase in DNA damage, (e.g. 8-oxoguanine) in the heart, lung,
liver and kidney of aging rats [51].
The DNA damage associated with oxidative stress is known to activate the
NAD+-dependent DNA repair enzyme, PARP, which assists in
combination with the enzymes of the base-excision-and-repair complex of the cell in
energy-consuming repair processes [12], [52], [53]. The vital role of PARP activation in several diseases
has been obtained from experimental studies using PARP inhibitors, PARP knock-down
mice, and clinical trials. Recent studies have shown the protective effects of PARP
inhibitors on ischaemic heart and liver, diabetic kidney disease, and
endotoxin-induced acute lung injury, arguably due to the preservation of
NAD+ levels [20], [21], [22], [23], [24]. PARP activation can also
promote apoptosis by stabilising p53, and mediating the translocation of
apoptosis-inducing factor from mitochondria to the nucleus [19], [54]. Several lines of
evidence reported here show that PARP activation increases in the aging heart, lung,
liver, and kidney (Fig. 5), and
depletes cellular NAD+ levels (Fig. 6). Theoretically this should lead to a
reduction in Sirt1 deacetylase activity and accumulation of acetylated p53.
Consistent with this prediction we observed that increased PARP activation (Fig. 5) was associated with
NAD+ depletion (Fig. 6) which was accompanied by a decrease in Sirt1 deacetylase
activity (Fig. 7).
It is well established that genotoxic stress can kill cells by depleting the
intracellular NAD+ pools due to extensive use of
NAD+ as a substrate for PARP [12], [13], [16], [19]. Results from the present study,
consistent with others, suggests that both PARP and Sirt1 compete for the same
limiting intracellular NAD+ pool [55]. One study showed that PARP
activation depleted intracellular NAD+ levels and reduced Sirt1
deacetylase activity in DNA damaged in cardiac myocytes [55]. Replenishing intracellular
NAD+ levels by increasing NAD+ levels was able
to restore cellular viability but only in the presence of functional Sirt1 [55]. As
NAD+ serves as a cofactor for key glycolytic and mitochondrial
enzymes, vulnerable cells subsequently lose their ability to carry out energy
dependent functions including the maintenance of cell wall integrity and DNA/RNA
synthesis and consequently die [56]. We demonstrated, for the first time, that aging in the
rat is associated with decreased tissue levels of NAD+. This may
lower the rate of cell survival in response to several endogenous and exogenous
stressors. As NAD+ is the only substrate for both PARP and Sirt1
activities, maintaining optimal NAD+ content is crucial to
counteract the age-related degeneration observed during the aging process [56], [57].
As a substrate for the sirtuin family of NAD+ dependent deacetylases,
known as silent information regulators of gene function [27]. This discovery suggests that
gene silencing by this system might be related to metabolic rate and hence to
NAD+ concentrations, whose levels therefore may be directly
proportional to a longer lifespan [28]. While several studies have demonstrated the protective
roles of sirtuin activators during aging [30], [34], [35], [58], [59], [60], [61], [62], [63], little is known regarding role
of Sirt1 in the aging heart, lung, liver, and kidney.
The present results show that aging increases the amount of available Sirt1 protein
and activities of Sirt1 in nuclear extracts in the heart, lung, liver, and kidney.
The activity per molecule of Sirt1 appears to become lower in these organs as the
animal ages, suggesting the presence of more inactive or less active molecules in
aged animals. These observations are in line with a previous study which reported a
decrease in Sirt1 activity but not Sirt1 protein expression in skeletal muscle of
aged rats [64].Two
possibilities, which are not mutually exclusive, may explain this observation: 1)
Oxidative damage may potentially inhibit Sirt1 activity in a similar way to several
other proteins [65]
and/or 2) It is also likely that the age-associated drop in NAD+
content due to increased demand by PARP in the DNA repair process limits the Sirt1
substrate NAD+ thereby limiting Sirt1 activity. A compensatory
mechanism which increases the production of the Sirt1 protein [64] would increase its relative
success when competing for the limited NAD+ pool.. Sirt1 deficiency
has been shown to accentuate apoptosis of renal medullary cells [66] and cardiac
mycocytes [55].
Over-expression of Sirt1 has been shown to promote the resistance of these cells to
oxidative insult, both in vitro and in vivo
[67]. The
protective function of Sirt1 has been reported in other organs such as the heart,
neurons, lung, liver [67], [68], [69], [70]. Given the pivotal role of Sirt1 as an anti-stress and
anti-aging protein, targeting Sirt1 by either Sirt1 activators or increasing its
substrate (i.e. NAD+) availability may prove therapeutically
beneficial for the prevention of age-related cardiovascular, respiratory, hepatic,
and renal dysfunction.
Because Sirt1 activity is affected by the NAD+∶NADH ratio [71], we determined
the NAD+ and NADH content in aging heart, lung, liver, and kidney.
Our results show a significant decline in the NAD+∶NADH ratio
with aging, consistent with a decline in NAD+ levels and an increase
in NADH levels. This indicates that age related metabolic changes may influence
NAD+ and NADH upstream of Sirt1 similar to what was observed in
yeast Sir2α and in the skeletal muscle [71].
To examine the downstream effect of Sirt1 deacetylase activity in cellular processes,
we measured the expression of both total and acetylated p53 in aging rat tissue. The
main tumour suppressor protein, p53 regulates the expression of several gene
products that either lead to cell cycle arrest in G1 phase and prevent DNA
replication immediately before the repair of damage, or cause cell death via an
apoptotic mechanism [72]. We observed a significant increase in acetylated p53
protein while no change was observed in total p53 protein in all organs was
apparent. Higher acetylation levels of p53 have been reported in cardiac myocytes
following treatment with the oxidant H2O2, probably resulting
from decreased NAD+ and impaired Sirt1 deacetylase activity [55]. A recent
report showed that microRNA 34a (miR-34a), a tumour suppressor gene inhibiting Sirt1
expression also increases acetylated p53 levels, thus stimulating transcriptional
targets of p53 responsible for promoting cell-cycle arrest, and apoptosis [73]. While p53
has been shown to be bind to PARP and be poly(ADP-ribosylated), an effect which
enhances their pro-apoptotic activity, Sirt1-dependent deacetylation inhibits their
apoptotic activity [55]. These results suggest that changes in intracellular
NAD+ levels may regulate the post-translational acetylation of
p53, thus providing further evidence of the association between NAD+
levels, oxidative DNA damage-mediated PARP activation, and reduced Sirt1 deacetylase
activity [55].
One factor which may sensitise cells to increased DNA damage is impaired
mitochondrial function [74]. The mitochondrial electron transport system can trigger
the formation of superoxide leading to increased production of
H2O2 by superoxide dismutase [49], [50]. Reduced electron flow through
the mitochondrial respiratory chain, particularly through the inhibition of complex
I or complex III, favours the enhanced production of superoxide and
H2O2
[75]. Together,
with the age-dependent increase in oxidative stress and decline in
NAD+ and ATP content, we found a tendency to the reduction in
the activity of the respiratory complexes with age in all organs. Sipos et al.
(2003) showed that mitochondrial formation of H2O2 due to
complex I inhibition is more clinically relevant than ROS production due to
inhibition of complex III and IV in situ
[76]. Using the
same experimental paradigm, Rodriguez et al. (2008) reported similar mitochondrial
oxidative failure in the diaphragm of senescent prone mice [49].
It has also been demonstrated that an increased NADH∶NAD+
ratio, favours ROS generation by the catalytic activity of α-Ketoglutarate
dehydrogenase (α KGDH) [77]. The dependence on ROS production by α KGDH on the
intracellular NADH content may play a crucial role in promoting the accumulation of
free radicals in aging. When the reoxidation of NADH is impaired due to reduced
complex I activity, the NADH∶NAD+ ratio increases, thus
reducing the activity of several NAD+ dependent dehydrogenases [77]. Moreover,
NADH is also able to promote the formation of H2O2 in the
presence of iron [78]. Given the observations in the present study, increased
ROS production in aging may be a consequence of impaired mitochondrial respiratory
function, and may, at least in part, be attributed to increased
NADH∶NAD+ ratio inducing free radical production via
αKGDH and Fenton chemistry.
Overall, our results show that oxidative stress increased with age, (appearing to
accelerate more rapidly after mid life), in association with reduced antioxidant
defence mechanisms, and mitochondrial dysfunction; data consistent with the
predictions of the free-radical theory of aging. The current study, however,
provides new insight into the potential mechanisms of aging involved in response to
oxidative damage, particularly focusing on NAD+ metabolism and the
roles of PARP and Sirt1 in the heart, lung, liver, and kidney (Fig. 10). Adequate NAD+
concentrations may therefore be an important longevity assurance factor.
10.1371/journal.pone.0019194.g010Figure 10 Schematic representation for the association between oxidative stress,
PARP-mediated and decline in NAD+ content, and
NAD+ dependent functions with aging.
Oxidative stress to DNA activates PARP leading to poly(ADP-ribosylation) of
proteins in a reaction which consumes NAD+. Depletion of
cellular NAD+ stores attenuates the activity of Sirt1
deacetylase leading to hyperacetylation of p53, and consequently tilting the
balance to cell death via an apoptotic mechanism.
Therefore, we hypothesize that strategies targeted toward maintaining adequate
NAD+ content during the aging process may prove a novel and
potentially effective mechanism for retarding oxidative stress mediated cell
degeneration, and age associated disorders.
The authors would like to thank the staff from the Bioanalytical Mass Spectrometry
Facility at the University of New South Wales for assistance with GC-MS
experiments.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by grants to Professor Tailoi Chan-Ling from the National
Health and Medical Research Council of Australia, and to Dr. Gilles J. Guillemin
and Professor Tailoi Chan-Ling from the Rebecca Cooper Medical Research
Foundation (Sydney, Australia). Nady Braidy is the recipient of an Australian
Postgraduate Award at the University of New South Wales. Hussein Mansour is the
recipient of a University of Sydney Medical Foundation/Bluesand Research
Scholarship. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21559417PONE-D-11-0078710.1371/journal.pone.0019338Research ArticleBiologyNeuroscienceCellular NeuroscienceNeurobiology of Disease and RegenerationMedicineMental HealthTherapiesPassive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease Passive Immunization in LBD ModelMasliah Eliezer
1
2
*
Rockenstein Edward
1
Mante Michael
1
Crews Leslie
2
Spencer Brian
1
Adame Anthony
1
Patrick Christina
1
Trejo Margarita
1
Ubhi Kiren
1
Rohn Troy T.
3
Mueller-Steiner Sarah
4
Seubert Peter
4
Barbour Robin
4
McConlogue Lisa
4
Buttini Manuel
4
Games Dora
4
Schenk Dale
4
1
Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America
2
Department of Pathology, University of California San Diego, La Jolla, California, United States of America
3
Department of Biology, Boise State University, Boise, Idaho, United States of America
4
ELAN Pharmaceuticals, South San Francisco, California, United States of America
McAlonan Grainne M. EditorThe University of Hong Kong, Hong Kong* E-mail: [email protected] work: ER MM. Immunohistochemistry: AA CP. Electron microscopy: EM MT. Antibody characterization: TTR PS RB LM MB AA CP. Conceived and designed the experiments: EM ER LM DG DS. Performed the experiments: EM ER MM LC BS AA CP MT SMS RB LM MB DG. Analyzed the data: EM ER LC KU DG DS. Contributed reagents/materials/analysis tools: EM SMS PS RB LM MB DG. Wrote the paper: EM LC KU DG DS.
2011 29 4 2011 6 4 e1933822 12 2010 28 3 2011 Masliah et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.
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Introduction
Neurodegenerative conditions with accumulation of α-synuclein (α-syn) are common causes of dementia and movement disorders in the aging population. Disorders where the clinical and pathological features of Alzheimer's Disease (AD) and Parkinson's Disease (PD) overlap are known as Lewy body disease (LBD) [1].
α-Syn is a natively unfolded protein [2] found at the presynaptic terminal [3] and may play a role in synaptic plasticity [4]. Abnormal α-syn accumulation in synaptic terminals and axons plays an important role in LBD [5], [6], [7], [8]. Recent work has suggested that α-syn oligomers rather than fibrils might be the neurotoxic species [9], [10].
While in rare familial cases mutations in α-syn might contribute to oligomerization [11], it is unclear what triggers α-syn aggregation in sporadic forms of LBD. Alterations in α-syn synthesis, aggregation or clearance have been proposed to impact the formation of toxic oligomers [12], [13], [14]. Therefore, strategies directed at promoting the clearance of oligomers may be of therapeutic value for LBD. Previous studies have used gene therapy targeting selective regions to increase α-syn clearance via autophagy or by reducing α-syn synthesis [12], [15].
However, neurodegenerative processes in LBD are more widespread than originally suspected [16] therefore there is a need for therapeutic approaches that target toxic α-syn in multiple neuronal populations simultaneously. For this reason we began to explore an immunotherapy approach for LBD and have previously shown that active immunization with recombinant α-syn ameliorates α-syn related synaptic pathology in a transgenic (tg) mouse model of PD [17]. Previous studies have shown that intracellular antibodies (intrabodies) can inhibit α-syn aggregation [18], [19] and that copolymer-1 immunotherapy reduces neurodegeneration in a PD model [20].
The mechanisms through which α-syn immunotherapy might work are unclear given that native α-syn is cytoplasmic. However, it is possible that antibodies may recognize abnormal α-syn accumulating in the neuronal plasma membrane [10], [17], [21], [22] or secreted forms of α-syn. In support of this possibility, studies have shown that oligomerized α-syn is secreted in vitro [23] and in vivo [24] via exocytosis, contributing to the propagation of the synucleinopathy. Moreover, α-syn is present in the cerebrospinal fluid of α-syn tg mice and in patients with LBD [25], [26].
This study examined whether passive immunization with an antibody against the C-terminus (CT) of α-syn (hereafter referred to as the 9E4 antibody) was able to recognize and clear a-syn aggregates in a-syn tg mice. We show that the 9E4 antibody crossed into the CNS and ameliorated behavioral deficits and neuropathological alterations in α-syn transgenic mice. In addition we show that 9E4 is able to reduce the accumulation of calpain-cleaved and oligomerized a-syn aggregates. These results imply that passive immunization against the CT of α-syn may be an important therapeutic alternative in patients with PD and DLB.
Materials and Methods
Transgenic mouse model and passive immunization
For this study mice over-expressing α-syn under the PDGF-β promoter (Line D) were utilized [27], [28]. This model was selected because mice from this line develop α-syn aggregates distributed through the temporal cortex and hippocampus similar to what has been described in LBD accompanied by behavioral deficits [29], [30].
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity and was chosen for the immunization study. A total of 40 α-syn tg mice (6 m/o, n = 20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control. An additional group of non-tg mice treated with the 9E4 antibody (n = 12) and the IgG1 control (n = 12) was included as control for behavioral and neuropathological studies. Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA). At the end of the studies, mice were tested for functional effects in the water maze. Brains and peripheral tissues were removed and divided sagittally.
For studies of antibody trafficking into the CNS the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. For this experiment non-tg (total n = 18) and α-syn tg mice (total n = 18) (6 m/o) were injected intravenously (IV) with the 9E4-FITC or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrificed 3, 14 and 30 days after injection (n = 3 per group). CSF from these mice was used to immunolabel cortical sections from antibody-naive animals.
As an additional control to monitor the passage of FITC-labeled antibodies across the blood-brain barrier, mice were injected with FITC tagged β-syn. The For this experiment non-tg (total n = 8) and α-syn tg mice (total n = 8) (6 m/o) were injected intravenously (IV) with the FITC-labeled β-syn or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg. Mice were sacrified at 14 days post-injection. CSF from these mice was also used to immunolabel slides from FITC tagged β-syn niave non-tg and α-syn tg mice.
Upon sacrifice, the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at −70°C for subsequent protein analysis. All experiments described were approved by the animal subjects committee at the University of California at San Diego (UCSD) and were performed according to NIH recommendations for animal use. UCSD is an Institutional Animal Care and Use Committee accredited institution and the UCSD Animal Subjects Committee approved the experimental protocol (S02221) followed in all studies according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines.
Preparation of antibodies for passive immunization
Previous active immunization experiments have suggested that the most effective antibodies were against CT-epitopes of α-syn [17], for this study a panel of antibodies were developed 1) clone 8A5 (α-syn epitope 125–140; IgG1); 2) clone 9E4 (α-syn epitope 118–126; IgG1) and 6H7 (α-syn epitope 1–4: IgG1) (Table 1). The 9E4 and 6H7 antibodies were prepared by immunizing with recombinant human α-syn, while 8A5 was generated with purified bovine α-syn. Epitope mapping was done on 15-mer peptides that had a 3 amino acid difference (i.e. 1–15, 3–18, etc, which gave an epitope resolution of +/−2 amino acids). For control experiments a non-immune IgG1 (clone 27-1) prepared under similar conditions was used. Initial immunoblot and immunohistochemical examination using these antibodies on untreated non-tg and α-syn tg mice demonstrated that the 9E4 displayed the most specificity for human α-syn therefore this antibody was chosen for the immunization study.
10.1371/journal.pone.0019338.t001Table 1 Antibodies used for this study.
Antibodyname Type of antibody Epitope/specificity Antibody Isotype ExperimentalUse
27-1 Monoclonal Control IgG1 Control for Immunization
6H7 Monoclonal NT 1-4 IgG1 Initial Characterization
8A5 Monoclonal CT 125-140 IgG1 InitialCharacterization
9E4 Monoclonal CT 118-126 IgG1 Passive Immunization
FL-α-syn Polyclonal FL Affinity purified ICC and IB
CC-α-syn Polyclonal CT 122-123 Affinity purified ICC and IB
CT = C-terminus; FL = full length; CC = Calpain-cleaved; ICC = immunocytochemistry; IB = Immunoblot.
Behavioral testing
Water maze
In patients with LBD, α-syn accumulates not only in subcortical nuclei but also in the temporal cortex and limbic system and accounts for cognitive deficits in these patients [31]. Similarly, in our PDGF-α-syn tg mice, protein accumulation occurs in the temporal cortex and hippocampus [28]. In this context and as previously described [29], in order to evaluate the functional effects of passive immunization treatment in mice, groups of non-tg and α-syn tg animals were tested in the water maze. For this purpose, a pool (diameter 180 cm) was filled with opaque water (24°C) and mice were first trained to locate a visible platform (days 1–3) and then a submerged hidden platform (days 4–7) in three daily trials 2–3 min apart. Mice that failed to find the hidden platform within 90 seconds were placed on it for 30 seconds. The same platform location was used for all sessions and all mice. The starting point at which each mouse was placed into the water was changed randomly between two alternative entry points located at a similar distance from the platform. In addition, on the final day of testing the platform was removed and the time spent by mice in the correct quadrant was measured (Probe test). The duration of the probe test was 40 secs. Time to reach the platform (escape latency) was recorded with a Noldus Instruments EthoVision video tracking system (San Diego Instruments, San Diego, CA) set to analyze two samples per second.
Pole Test
For the pole test, animals were placed head upward on top of a vertical wooden pole 50 cm long and 1 cm in diameter. When placed on the pole, animals orient themselves downward and descend the length of the pole. Groups of mice received training that consisted of five trials for each session. For testing, animals received five trials and the time taken to descend (T-total) was measured.
Rotarod
Mice were analyzed for 2 days in the Rotarod (San Diego Instruments, San Diego, CA), as previously described (Masliah et al., 2000). On the first day, mice were trained for five trials: the first one at 10 rpm, the second at 20 rpm, and the third to the fifth at 40 rpm. On the second day, mice were tested for seven trials at 40 rpm each. Mice were placed individually on the cylinder and the speed of rotation increased from 0 to 40 rpm over a period of 240 s. The length of time mice remained on the rod (fall latency) was recorded and used as a measure of motor function.
ELISA analysis of brain and plasma antibody concentrations
Antibody levels in the brain and plasma of immunized mice were determined as previously described [17]. Briefly, using 96-well microtiter plates coated with 0.4 µg per well of purified full-length α-syn. Samples were incubated overnight followed by goat anti-mouse IgG alkaline phosphatase-conjugated antibody (1∶7500, Promega, Madison, WI). The plate was read at wavelengths of 450 nm and 550 nm. Results were plotted on a semi-log graph with relative fluorescence units versus serum dilution. Antibody titer was defined as the dilution at which there was a 50% reduction from the maximal antibody binding.
Immunoblot analysis
Briefly, as previously described, brains were homogenized and divided into cytosolic and membrane fractions [12], [15]. For immunoblot analysis, 20 µg of total protein per lane was loaded into 4–12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For characterization of the antibodies samples from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and N-Terminal (NT) α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). To determine the effects of the immunotherapy in levels of α-syn blotted samples from treated α-syn tg were probed with antibodies against calpain-cleaved α-syn (CC α-syn) which recognizes a C-terminal fragment of α-syn [32], full length α-syn (FL α-syn rabbit polyclonal (1∶1000, Millipore, Temecula CA). For the analysis of synaptic proteins, monoclonal antibodies against Synapsin I (1∶1000, Millipore, Temecula, CA) and PSD95 (UC Davis/NIH Neuro-Monoclonal Antibody Facility, Davis, CA) were used. In order to determine the effects of the immunotherapy on levels of total tau and PHF-tau blotted samples from treated α-syn tg were probed with antibodies against total tau (1∶1000, Dako, Carpinteria, CA) and PHF-tau (1∶1000, UC Davis/NIH Neuro-Monoclonal Antibody Facility, Davis, CA). Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1∶5000, Santa Cruz Biotechnology, Santa Cruz, CA), visualization with enhanced chemiluminescence and analysis with a Versadoc XL imaging apparatus (BioRad, Hercules, CA). Analysis of β-actin (Sigma) levels was used as a loading control.
Immunocytochemical and neuropathological analyses
For characterization of the antibodies used for immunotherapy, vibratome sections from untreated non-tg and α-syn tg mice were incubated the with monoclonal antibodies against CT and NT-α-syn (9E4, 6H7 and 8A5, ELAN Pharmaceuticals). Analysis of α-syn accumulation for the immunotherapy experiment was performed in serially-sectioned, free-floating, blind-coded vibratome sections by incubating the sections overnight at 4°C with a polyclonal antibody against total α-syn (1∶500, affinity purified rabbit polyclonal, Millipore) [27] and with an antibody against the calpain-cleaved CT-α-syn [32], followed by secondary antibodies tagged with FITC or biotinylated goat anti-rabbit IgG1 (1∶100, Vector Laboratories, Inc., Burlingame, CA), Avidin D-HRP (1∶200, ABC Elite, Vector) and detection with the Tyramide Signal Amplification™-Direct (Red) system (1∶100, NEN Life Sciences, Boston, MA). In order to determine the effects of immunotherapy on levels of total tau and PHF-tau blotted samples from treated α-syn tg were probed with antibodies against total tau (1∶500, Dako, Carpinteria, CA) and PHF-tau (1∶500, UC Davis/NIH Neuro-Monoclonal Antibody Facility, Davis, CA). Antibodies against Zo-1 (1∶500, Millipore, Temecula, CA), Iba-1 (1∶1000, Wako, Richmond, VA) and GFAP (1∶1000, Millipore, Temecula, CA) were used to examine the effects of passive immunization with 9E4 on vasculature or glial cell activation respectively. All sections were processed simultaneously under the same conditions and experiments were performed in triplicate in order to assess the reproducibility of results.
Stereological analysis and quantification of neocortical and hippocampal intra-neuronal FL-αsyn and CC-αsyn immunoreactivity was conducted by the disector method using the Stereo-Investigator System (MBF Bioscience, Williston, VT) and the results were averaged and expressed as cell counts per 0.1 mm3. Neocortical and hippocampal FL-αsyn and CC-αsyn immunoreactive neuropil was assessed in digital images analyzed with the Image Quant software by selecting and area to exclude cell bodies, setting the threshold levels and expressing the data as pixel intensity (arbitary units).
Double immunolabeling and fluorescence co-labeling
To determine the co-localization between α-syn and lysosomal and autophagy markers double-labeling experiments were performed, as previously described [15]. For this purpose, vibratome sections were immunolabeled with the rabbit polyclonal antibodies against α-syn (Millipore, affinity purified polyclonal, 1∶500) or CC α-syn [27], [32] and LC3 (Abcam) or cathepsin-D (Dako, 1∶100). The α-syn immunoreactive structures were detected with the Tyramide Signal Amplification™-Direct (Red) system (1∶100, NEN Life Sciences, Boston, MA) while LC3 and cathepsin-D was detected with FITC tagged antibodies (Vector, 1∶75). Co-labeling experiments were performed with antibodies against cathepsin-D and LC3 detected with tyramide red in sections from mice that received IV injections with the 9E4-FITC or IgG1-FITC. All sections were processed simultaneously under the same conditions and experiments were performed in triplicate in order to assess the reproducibility of results. Sections were imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss) with an attached MRC1024 LSCM (laser scanning confocal microscope) system (BioRad) [27].
Electron microscopy and immunogold analysis
Briefly, vibratome sections were postfixed in 1% glutaraldehyde, treated with osmium tetraoxide, embedded in epon araldite and sectioned with the ultramicrotome (Leica, Germany). Grids were analyzed with a Zeiss OM 10 electron microscope as previously described [33]. For immunogold labeling, sections were mounted in nickel grids, etched and incubated with biotin-tagged antibodies against mouse IgG1 to detect the circulating antibodies utilized for immunization or with antibodies against α-syn followed by labeling with 10 nm Aurion ImmunoGold particles (1∶50, Electron Microscopy Sciences, Fort Washington, PA) with silver enhancement. A total of 125 cells were analyzed per condition. Cells were randomly acquired from 3 grids, and electron micrographs were obtained at a magnification of 25,000X.
For morphometric analysis of synapses from each section, a total of 20 electron micrographs were obtained at a final magnification of 12,000x. Electron micrographs were digitized and analyzed with the Quantimet 570C (Leica, Deerfield, IL) to determine the density of synapses per unit of volume.
Neuronal cell cultures and treatments
The rat neuroblastoma cell line B103 was used for in vitro experiments [34]. This model was selected because over expression of α-syn in these cells interferes with neuronal plasticity (reduced neurite outgrowth and adhesion) but does not result in overt cell death [35], [36]. This model mimics the early pathogenic process of PD where cell death is preceded by reduced neurite outgrowth and synaptic alterations. For all experiments, cells were plated in complete media (Dulbecco's Modified Eagle Medium [Invitrogen, Carlsbad, CA] supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA) and infected with LV expressing α-syn or controls at a multiplicity of infection (MOI) of 40. After infection, cells were incubated for 48 hr in a humidified 5% CO2 atmosphere at 37°C. All experiments were conducted in triplicate to ensure reproducibility. To investigate the effects of the antibody treatment on autophagy and α-syn, neuronal cells were grown as described above and were then plated onto poly L-lysine coated glass coverslips at a density of 5×104 cells. Five hours after plating, cells were infected with the LV-αsyn LV-control and incubated for 24 hours with 9E4 (3 µg/ml) or IgG1 control in the presence or absence of inhibitors of the autophagy pathway – 3-methyladenine (3-MA, 10 mM, Sigma) or inducers – rapamycin (200 nM, Sigma) as previously described [15]. All coverslips were also co-infected with an LV expressing LC3-GFP at an MOI of 40. Cultures were then washed 2X with serum-free DMEM and then fed either complete media or serum-free media for 12 hours before fixation with 4% PFA. Briefly as previously described [37], coverslips were treated with Prolong Gold anti-fading reagent with DAPI (Invitrogen) and imaged with the LSCM to determine the number of GFP-positive granular structures consistent with autophagolysosomes using semiautomatic image analysis system and the ImageQuant software. For each condition an average of 50 cells were analyzed.
Statistical analysis
All experiments were done blind-coded and in triplicate. Values in the figures are expressed as means ± SEM. To determine the statistical significance, values were compared using one-way analysis of variance (ANOVA) with post hoc Dunnett's test when comparing to the IgG1 control. Additional comparisons were done using Tukey-Kramer or Fisher post hoc tests. Repeated-measures two-way ANOVA was used to analyze the water maze and Rotarod data when comparing immunized mice to the non-tg or IgG1 treated controls. The differences were considered to be significant if p values were less than 0.05.
Results
Initial characterization of novel monoclonal α-syn antibodies and selection of antibody for passive immunotherapy in α-syn tg mice
The PDGFβ-α-syn tg mice were selected for the present study as they display accumulation of α-syn in cortical and sub-cortical regions and neuropathological and behavioral deficits consistent with LBD [12], [17], [27], [28], [29], [38]. In order to initially characterize the specificity of the mouse monoclonal antibodies and to select the one to be used for immunotherapy (Table 1) tissue sections and brains homogenates from non-tg and α-syn tg mice were examined by immunoblot and immunohistochemistry.
By immunoblot analysis (Figure S1A), the NT-α-syn antibody (6H7) identified the α-syn monomer at 14 kDa in both the α-syn tg mice and to a lesser degree in the non-tg mice. The antibodies against CT- αsyn (8A5, 9E4) antibodies specifically recognized the α-syn monomer at 14 kDa in the α-syn tg mice (Figure S1A). No immunoreactivity was observed with the IgG1 control (Figure S1A). The FL α-syn antibody recognized monomeric α-syn in the α-syn tg mice (Figure S1A). Consistent with a previous report [32] the antibody against the calpain-cleaved (CC) α-syn, which recognizes a C-terminally cleaved fragment of α-syn, produced a distinctive pattern detecting a native band at 14 kDa. No cross-reactivity was observed with these antibodies in the non-tg animals (Figure S1A).
Immunohistochemical analysis demonstrated that, compared to the non-immune IgG1, (Figure S1B, C) antibodies against the NT (6H7) (Figure S1D,E) and CT of α-syn (8A5) (Figure S1F, G), (9E4) (Figure S1H,I) strongly immunolabeled the neuropil and the intra-neuronal α-syn aggregates in the temporal cortex of the α-syn tg mice. In the non-tg mice, there was a mild immunoreactivity with the 8A5 antibody (Figure S1F), which was more prominent with the 6H7 antibody (Figure S1D). With the 9E4 antibody no immunoreactivity was detected in the non-tg mice (Figure S1H).
The patterns of immunostaining of the antibodies used for immunotherapy were compared to a polyclonal antibody against FL-α-syn (Figure S1J, K) and to the antibody against CC-α-syn, (Figure S1L,M). In the α-syn tg mice, both antibodies immunolabeled the intra-neuronal α-syn aggregates and the neuropil. The polyclonal antibody against FL α-syn immunolabeled the neuropil in the non-tg mice (Figure S1J), no immunoreactivity was detected with the antibody against calpain-cleaved α-syn in these mice (Figure S1L).
Collectively these results demonstrate that the 9E4 antibody directed against the CT of α-syn displayed the most specificity for human α-syn, therefore this antibody was chosen for the subsequent passive immunization study.
An antibody against CT-α-syn ameliorates motor and learning deficits and synaptic pathology in α-syn tg mice
Following the initial screening and subsequent selection of the 9E4 antibody, α-syn tg and non-tg mice were passively immunized with either 9E4 or the control IgG1. Antibody titers in the passively immunized α-syn tg and non-tg mice were analyzed by ELISA. On average titer levels were comparable between passively immunized α-syn tg and non-tg mice, though a greater variability was observed within the α-syn tg group (Figure 1A).
10.1371/journal.pone.0019338.g001Figure 1 Plasma antibody titers and effects of passive immunization on motor behavior in passively immunized α-syn tg mice.
(A) Antibody titers determined by ELISA in non-tg or α-syn tg mice immunized with the C-terminal antibody (9E4) or IgG1 controls. Horizontal lines represent the mean of the data, whilst the points represent the spread of the individuals in each group. To examine the effects of immunization with the 9E4 antibody on motor behavior in the α-syn tg, mice were tested in the rotarod and pole tests. (B) Pole test performance (time taken to traverse pole) by non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (C) Rotarod performance (time spent on rotating rod) by non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. Error bars represent mean ± SEM. When analyzing rotarod results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg group by repeated-measures two-way ANOVA and (#) indicates p<0.05, when comparing α-syn tg mice immunized with 9E4 to IgG1 immunized α-syn tg mice using repeated-measures two-way ANOVA. When analyzing pole test results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg group by one-way ANOVA with post hoc Dunnett's and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by one-way ANOVA with post hoc Dunnett's.
The effects of passive immunization on motor behavior in the α-syn tg mice was assessed using the rotarod and pole test. Results from the pole test demonstrated a motor impairment in the IgG1-treated α-syn tg mice compared to IgG1-treated non-tg mice, evidenced by the significantly longer time taken by the α-syn tg mice to traverse the pole as determined by one-way ANOVA (Figure 1B). Immunization with the 9E4 antibody significantly reduced the time taken by the α-syn tg mice to traverse the pole when compared to IgG1-treated α-syn tg mice (Figure 1B). The time taken to traverse the pole by the α-syn tg mice immunized with the 9E4 antibody did not significantly different from the time taken by the non-tg mice, as determined by one-way ANOVA (Figure 1B).
Statistical analysis of the Rotarod results using repeated-measures two-way ANOVA demonstrated that IgG1-treated α-syn tg mice spent significantly less time on the rotating rod in comparison to IgG1-treated non-tg mice, suggesting that the α-syn tg mice have deficits in motor coordination (Figure 1C). In contrast, α-syn tg mice immunized with the 9E4 antibody spent a significantly longer time on the rod when compared to IgG1-treated α-syn tg mice, as determined by repeated-measures two-way ANOVA (Figure 1C). The time spent on the rod by α-syn tg mice immunized with the 9E4 antibody did not differ from that of the non-tg controls.
In order to evaluate the effects of passive immunization with the CT α-syn antibody on memory and learning, following the 6-month immunization period, mice were tested in the water maze. During the initial training part of the test when the platform was visible (days 1-3), all groups performed at comparable levels, though a greater variability was observed in the IgG1-treated α-syn tg mice (Figure 2A, B, cued platform), as determined by repeated measures two-way ANOVA. At day 2 of the visible platform 9E4-treated α-syn tg appear to reach the platform at a faster rate compared to the other 3 group. However at the end of the visible period of training all 4 groups of mice performed similarly. Following the cued platform session, the mice underwent 4 days of testing during which the platform was submerged and hidden from view (days 4–7). On the first day of testing with the hidden platform all groups performed comparably, indicating that that were all able to swim and locate the platform. Over the next 3 days of testing the performance of the non-tg mice improved in terms of the distance of their swim path and the time taken to locate the platform, During the submerged platform segment of the test, the performance of the IgG1-treated α-syn tg mice did not improve to the same extent as that observed in the non-tg mice (Figure 2A, B, hidden platform). Upon statistical analysis of performance with the submerged platform (days 4–7) using repeated-measures two-way ANOVA a significant difference was observed between the IgG1-treated α-syn tg mice and non-tg controls, with the IgG1-treated α-syn mice taking a significantly longer path and longer time to locate the hidden platform in comparison to their non-tg littermates (Figure 2A, B, hidden platform). These results indicate that the α-syn tg mice have a deficit in the learning and memory skills associated with this task.
10.1371/journal.pone.0019338.g002Figure 2 Effects of passive immunization on behavioral performance in the water maze in passively immunized α-syn tg mice.
(A) Performance in the water maze (distance taken to locate platform) during training with the cued platform (days 1–3) and with the platform submerged (days 4–7) in non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. (B) Performance in the water maze (time to locate the platform) during training with the cued platform (days 1–3) and with the platform submerged (days 4–7) in non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. (C) Probe test performance (time spent in correct quadrant) (day 8) in non-tg mice or α-syn tg, mice immunized with IgG1 or 9E4. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (D) Representative images of the swim paths of animals from each group when the platform was visible (Day 3, trial 4, red box indicates the location of the platform), hidden (Day 7, trial 4, red box indicates the location of the platform which is now submerged below the opaque surface) or absent (Day 8, Probe Test). When analyzing the water maze results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg IgG1 group by repeated-measures two-way ANOVA and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by repeated-measures two-way ANOVA. When analyzing probe test results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg IgG1 group by one-way ANOVA with post hoc Dunnett's and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by one-way ANOVA with post hoc Dunnett's.
Analysis using repeated-measures two-way ANOVA demonstrated that mice immunized with the 9E4 antibody took a significantly shorter path and time to locate the hidden platform in comparison to IgG1-treated α-syn tg mice (Figure 2A, B, hidden platform), indicating that passive immunization with this antibodies was able to ameliorate the memory and learning deficit observed in the IgG1-treated α-syn tg mice. The time taken by the 9E4 immunized α-syn tg mice to find the submerged platform did not differ significantly from that of the non-tg mice as determined by repeated-measures two-way ANOVA. In the non-tg mice immunization with the 9E4 antibody or the IgG1 control had no deleterious effect upon their performance during the cued or hidden portions of the water maze test (Figure 2A, B).
Following the final day of testing with the submerged platform the mice underwent a Probe test. During this test the platform was removed completely and the time spent by the mice in the correct quadrant (that corresponding to the previous location of the platform) was measured. A longer time spent in the correct quadrant is indicative of a learning effect wherein the mice remember the previous location of the platform and spend an increased period of time looking for it, whilst a shorter time in this quadrant indicates a memory deficit. Statistical analysis using one-way ANOVA demonstrated a significant decrease in the amount of time spent in the correct quadrant by IgG1-treated α-syn tg mice in comparison to non-tg control mice (Figure 2C) suggestive of a memory deficit in these mice. In contrast, α-syn tg mice immunized with the 9E4 antibody spent longer in the target area when compared to IgG1-treated α-syn tg mice, as determined by one-way ANOVA (Figure 2C). The time spent in the correct quadrant by α-syn tg mice immunized with the 9E4 antibody did not differ from that of the non-tg controls. These results suggest that immunization with the 9E4 antibody was able to ameliorate the memory deficits observed in the IgG1-treated α-syn tg mice. No significant effects were observed in the probe test in non-tg mice treated with the 9E4 antibody or the IgG1 control (Figure 2C).
Representative images of the swim paths of mice from the different groups are shown in Figure 2D for day 3, trial 4 (platform visible, red box indicates location of platform) and day 7, trial 4 (platform hidden, red box indicates location of platform which now submerged in the opaque water). When the platform was visible all mice appeared to take a comparable path length to the platform (Figure 2A D- Day 3, trial 4, Platform visible). When the platform was submerged, IgG1-treated α-syn tg mice took a significantly longer path and a more convoluted path to find the platform (Figure 2A, D - Day 7, trial 4, Platform hidden). In contrast, 9E4-immunized α-syn tg mice were able to locate the hidden platform with a time comparable to that observed in the IgG1-treated non-tg controls and with a much more direct path than that observed with IgG1-treated α-syn tg mice (Figure 2A, D - Day 7, trial 4, Platform hidden). In the probe test portion of the water maze IgG1-treated α-syn tg mice did not spend a significant amount of time in the correct quadrant (Figure 2C, D - Day 8, Probe Test). In contrast, the α-syn tg mice that had been immunized with 9E4 spent much longer in the correct quadrant, comparable to the time observed in the IgG1-treated non-tg controls (Figure 2C, D - Day 8, Probe Test).
Since improvements in behavioral performance may be related to enhanced synaptic connectivity, synaptic structure was analyzed by electron microscopy. Ultrastructural analysis (Figure 3) showed that, compared to non-tg mice (Figure 3A), α-syn tg mice treated with IgG1 displayed a significant reduction in the number of postsynaptic densities (PSDs) and presynaptic terminal diameter (Figure 3C, E, F), as determined by one-way ANOVA. In contrast, immunization with the 9E4 antibody significantly increased the number of PSDs (Figure 3D, E) and the diameter of pre-synaptic terminals in the immunized α-syn mice (Figure 3D, F) in comparison to the IgG1-treated α-syn tg mice, as determined by one-way ANOVA. PSD number and pre-synaptic terminal diameter in the α-syn tg mice immunized with the 9E4 antibody did not significantly differ from that in the non-tg mice (Figure 3A, D, E, F).
10.1371/journal.pone.0019338.g003Figure 3 Analysis of the effects of passive immunization on synaptic structure and markers in α-syn tg animals.
The effect of immunization with the 9E4 antibody on synaptic markers was evaluated in the temporal cortex of non-tg and α-syn tg mice by electron microscopy and immunoblot analysis. Representative electron micrographs are from the temporal cortex layers 5–6 obtained at 15,000 X. (A) non-tg mice immunized with IgG1 control. (B) non-tg mice immunized with 9E4. (C) α-syn tg mice immunized with the IgG control. (D) α-syn tg mice immunized with the 9E4 antibody. (E, F) Image analysis of the numbers of post-synaptic densities (PSD) and mean presynaptic terminal diameters respectively. (G) Representative immunoblot for PSD95, a postsynaptic marker and Synapsin I, a presynaptic marker, in non-tg mice or α-syn tg mice immunized with the IgG control or the 9E4 antibody. (H, I) Analysis of the levels of PSD95 and Synapsin I immunoreactive bands respectively. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immunized α-syn tg mice to IgG1-immunized non-tg mice and (#) indicates p<0.05 when comparing α-syn tg mice immunized with 9E4 to IgG1 immunized α-syn tg mice using one-way ANOVA with post hoc Dunnett's.
Consistent with the ultrastructural examination, immunoblot analysis (Figure 3G) demonstrated that levels of PSD95 were significantly reduced in the IgG1 treated α-syn tg mice compared to non-tg mice, as determined by one-way ANOVA (Figure 3G, H). The mice immunized with the 9E4 antibody displayed significantly higher levels of PSD95 when compared to IgG1-treated α-syn tg mice, as determined by one-way ANOVA (Figure 3G, H). PSD levels in mice immunized with the 9E4 antibody were not significantly different from those in non-tg mice, as determined by one-way ANOVA (Figure 3G, H). Similarly, immunoblot analysis of Synapsin I, a presynaptic marker, demonstrated significantly reduced levels in the IgG1 treated α-syn mice in comparison to non-tg control mice (Figure 3G, I). Immunization with the CT-α-syn antibody 9E4 significantly increased synapsin levels in the immunized α-syn tg mice in comparison to the IgG1-treated α-syn tg mice (Figure 3G, I), as determined by one-way ANOVA. Synapsin levels in the 9E4-immunized α-syn tg mice did not differ significantly from those observed in the non-tg mice (Figure 3G, I).
In the non-tg mice immunization with the 9E4 antibody or IgG1 control had no deleterious effects upon PSDs, pre-synaptic terminals or on synapsin I levels (Figure 3A, B, E-I).
Passive immunotherapy with a CT α-syn antibody reduces the accumulation of calpain-cleaved α-syn aggregates
To investigate whether the behavioral and synaptic improvements in the immunized α-syn tg mice were associated with reduced accumulation of α-syn, immunochemical studies were performed with antibodies against FL and CC α-syn. CC-α-syn has been proposed to serve as a substrate for aggregation and antibodies against this epitope have been shown to identify abnormal α-syn aggregates that otherwise are not detected in control human brain or in wild-type mice [32]. With the polyclonal FL α-syn antibody there was no significant difference in the intra-neuronal inclusions in the temporal cortex in 9E4-treated α-syn tg mice in comparison to IgG1-treated α-syn tg mice (Figure 4C, D, I), however there was a small but significant reduction of α-syn immunoreactivity in the neocortical neuropil of the 9E4 treated mice in comparison to IgG1-treated α-syn tg mice (Figure 4C, D, J). In the hippocampus there were no significant differences in FL α-syn immunoreactivity in intra-neuronal inclusions or neuropil between the 9E4-treated α-syn tg mice and IgG1-treated α-syn tg mice control groups (Figure 4G, H, K, L)
10.1371/journal.pone.0019338.g004Figure 4 Comparative immunohistochemical analysis with antibodies against full length or calpain-cleaved a-syn in passively immunized a-syn tg mice.
To examine the effects of immunization on α-syn accumulation, immunohistochemical analysis using antibodies against FL-α-syn (layers 5–6) and CC-α-syn was conducted. Panels illustrate laser scanning confocal images of the temporal cortex and hippocampus (CA3) immunolabeled with antibodies against FL and CC α-syn immunoreactivity. (A, C) Temporal cortex of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (B, D) Temporal cortex of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (E, G) Hippocampus of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (F, H) Hippocampus of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (I) Image analysis of the numbers of neocortical α-syn immunoreactivity neurons with the FL α-syn antibody. (J) Analysis of the levels of α-syn immunoreactivity in the neuropil in the neocortex in sections labeled with the FL α-syn antibody. (K) Image analysis of the numbers of hippocampal α-syn immunoreactivity neurons with the FL α-syn antibody. (L) Analysis of the levels of α-syn immunoreactivity in the neuropil in the hippocampus in sections labeled with the FL α-syn antibody. (M, O) Temporal cortex of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (N, P) Temporal cortex of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (Q, S) Hippocampus of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (R, T) Hippocampus of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (U) Image analysis of the numbers of neocortical α-syn immunoreactivity neurons with the CC α-syn antibody. (V) Analysis of the levels of α-syn immunoreactivity in the neuropil in the neocortex in sections labeled with the CC α-syn antibody. (W) Image analysis of the numbers of hippocampal α-syn immunoreactivity neurons with the CC α-syn antibody. (X) Analysis of the levels of α-syn immunoreactivity in the neuropil in the hippocampus in sections labeled with the CC α-syn antibody. Scale bar = 30 µM. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immunized α-syn tg mice to IgG1-imunized non-tg mice by one-way ANOVA with post hoc Dunnett's. (#) Indicates p<0.05, when comparing α-syn tg mice immunized the 9E4 α-syn antibody to IgG1-treated α-syn tg mice by one-way ANOVA with post hoc Dunnett's.
In contrast, immunohistochemical analysis with the antibody against CC α-syn showed a significant reduction in the levels of immunoreactivity in the both the numbers of immunolabeled intra-neuronal aggregates and the neuropil in the temporal cortex and hippocampus in the α-syn tg mice treated with the 9E4 antibody when compared to α-syn tg mice treated with the IgG1 control (temporal cortex, Figure 4O, P, U, V), hippocampus, Figure 4S, T, W, X).
In the temporal cortex of α-syn tg mice immunized with the 9E4 antibody intra-neuronal α-syn immunoreactivity, as detected by the CC-α-syn antibody was reduced 43% when compared to α-syn tg mice treated with the IgG1 control. Similarly, neocortical α-syn immunoreactive neuropil in α-syn tg mice immunized with the 9E4 antibody displayed a 57% reduction in the accumulation of CC α-syn when compared to α-syn tg mice treated with the IgG1 control. In the hippocampus of α-syn tg mice immunized with the 9E4 antibody intra-neuronal α-syn immunoreactivity, as detected by the CC-α-syn antibody was reduced 90% when compared to α-syn tg mice treated with the IgG1 control.
Hippocampal α-syn immunoreactive neuropil in α-syn tg mice immunized with the 9E4 antibody displayed a 33% reduction in the accumulation of CC α-syn when compared to α-syn tg mice treated with the IgG1 control.
In the IgG1- or 9E4-treated non-tg mice the FL and CC-α-syn antibodies detected minimal levels of α-syn in neocortical or hippocampal neurons (Figure 4A, B, I, E, F, K, M, N, U, V, Q, R, W, X). However the FL α-syn antibody was able to detect low levels of α-syn in the neuropil of these regions in the non-tg mice (Figure 4A, J, E, L).
Consistent with immunohistochemical findings, immunoblot analysis with the polyclonal antibody against FL α-syn (Figure 5A) showed similar levels of α-syn monomer (Figure 5B) and oligomer species (Figure 5C) in the soluble fraction of 9E4-treated α-syn tg mice when compared IgG1-treated α-syn tg mice. Levels of α-syn oligomers in the insoluble fraction were significantly reduced in the α-syn tg mice immunized with the 9E4 antibody compared to IgG1-treated α-syn tg mice (Figure 5D, F), whilst levels of α-syn monomers were low and appeared unaffected by immunization (Figure 5D, E).
10.1371/journal.pone.0019338.g005Figure 5 Immunoblot analysis with antibodies against full length and calpain-cleaved a-syn in passively immunized α-syn tg mice.
To evaluate the effects of immunization on α-syn accumulation, immunoblot analysis using antibodies against FL-αsyn and CC-α-syn was conducted. (A) Representative immunoblot with anti-FL α-syn of the soluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (B, C) Analysis of the levels of the α-syn immunoreactive bands corresponding to the monomer and oligomers respectively, as detected by the FL α-syn antibody in the soluble fraction. (D) Representative immunoblot with anti-FL α-syn of the insoluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (E, F) Analysis of α-syn monomer or oligomer levels respectively, detected by the FL α-syn antibody in the insoluble fraction. (G) Representative immunoblot with anti-CC α-syn of the soluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (H, I) Analysis of α-syn monomer or oligomer levels respectively detected by the CC α-syn antibody in the soluble fraction. (J) Representative immunoblot with anti-CC α-syn of the insoluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (K, L) Analysis of α-syn monomer or oligomer levels respectively, detected by the CC α-syn antibody in the insoluble fraction. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immiunized α-syn tg mice with IgG1-immunized non-tg mice using one-way ANOVA with post hoc Dunnett's. (#) indicates p<0.05, when comparing α-syn tg mice immunized with 9E4 with IgG1 immunized α-syn tg mice using one-way ANOVA with post hoc Dunnett's.
Immunoblot analysis with the antibody against CC α-syn in the soluble fraction showed a significant decrease in α-syn monomers and oligomers in 9E4-treated α-syn tg mice, in comparison to the IgG1-treated α-syn tg mice (Figure 5G–I). In the insoluble fraction the CC α-syn antibody showed a reduction of approximately 70% in the levels of monomeric α-syn band (Figure 5J, K) and a 95% reduction in the levels of the bands corresponding to oligomers in the insoluble fraction in the 9E4-treated α-syn tg mice group in comparison to the IgG1-treated α-syn tg mice (Figure 5J, L). In the non-tg mice immunization with the 9E4 antibody had no effect upon levels of FL- or CC-α-syn (Figure 5).
Given the recent results from genome-wide association studies suggesting that tau may play an important role in α-synucleinopathies such as PD [39], [40] we examined the effect of passive immunization with 9E4 on levels of tau and PHF-tau in the α-syn tg mice (Figure S2). Immunohistochemical analysis of the frontal cortex using an antibody against tau did not show significantly different levels of total tau between IgG1-treated α-syn tg and non-tg mice (Figure S2A, C, E), in contrast, IgG1-treated α-syn tg mice had significantly higher levels of PHF-tau, a four-fold increase in comparison to IgG1-treated non-tg mice (Figure S2F, H, J). 9E4 immunization did not alter levels of total tau (Figure S2B, D, E,) or PHF-tau (Figure S2G, I, J) in either group. Immunoblot analysis of total and PHF-tau levels was consistent with the immunohistochemical results and demonstrated no effect of 9E4 immunization on levels of total or PHF-tau in α-syn tg or non-tg mice (Figure S2K–M).
As passive immunization has been suggested to perturb vasculature we performed immunohistochemical analysis with the endothelial cell marker Zo-1 to examine the effects of immunization with the 9E4 antibody. In IgG1-treated non-tg and α-syn tg mice Zo-1 immunoreactivity was observed in the neuropil in association with the microvasculature and immunization with 9E4 had no effect upon Zo-1 immunoreactivity in either group (Figure S3A–E).
Immunohistochemical analysis of glial cell reactivity surrounding the vasculature was performed using markers against microglial and astroglial activation (Iba-1 and GFAP, respectively). IgG1- and 9E4-treated non-tg mice showed similar patterns of Iba-1 immunoreactivity in the neuropil around the blood vessels (Figure S3F, G, J). There was a moderate increase in Iba-1 immunoreactive microglial cells in the IgG1-treated α-syn tg mice in comparison to the IgG1-treated non-tg mice (Figure S3F, H, J) and no difference in Iba-1 immunoreactivity was observed the IgG1- or 9E4-treated α-syn tg mice (Figure S3H–J).
In the non-tg mice scattered GFAP immunoreactive astroglial cells were observed in the neuropil surrounding blood vessels (Figure S3K), no differences in the levels of GFAP were observed between IgG1- and 9E4-treated non-tg mice (Figure S3K, L, O). In contrast, the IgG1-treated α-syn tg mice had a robust increase in GFAP immunoreactivity in comparison to the IgG1-treated non-tg mice (Figure S3K, M, O) and passive immunization with 9E4 was able to reduce GFAP immunoreactivity in the α-syn tg mice (Figure S3M–O) resulting in a normalization of astroglial cells around the blood vessels in these mice.
Collectively the results thus far demonstrate that the 9E4 antibody is specific for human α-syn, significantly ameliorates the motor and memory/learning deficits examined in the α-syn tg mice and is effective at reducing the accumulation of α-syn in α-syn tg mice. Additionally these beneficial effects of 9E4 did not perturb the microvasculature.
A monoclonal antibody against CT-α-syn traffics into the CNS and localizes to lysosomes
In order to evaluate the trafficking of 9E4 into the CNS, 9E4 and a control IgG1 were labeled with FITC and injected intravenously into non-tg and α-syn tg. At 3 days post injection low levels of the 9E4 antibody were detected in the brain, while high levels were detected in plasma (Figure 6A). At 14 and 30 days post injection higher levels were detected in the brain with decreasing levels in the plasma as detected by ELISA (Figure 6A). By immunohistochemistry the 9E4-FITC antibody was detected in association with neurons in the brains of α-syn tg mice at 30 days post injection (Figure 6B). The 9E4-FITC antibody was detected in association with granular structures in neurons distributed in the deeper layers of the temporal cortex and the CA1-2 region of the hippocampus only in the brains of α-syn tg mice (Figure 6C, D). Control experiments with a non-immune IgG1-FITC show only background labeling in α-syn tg mice (Figure 6E). In non-tg mice only low levels of 9E4-FITC labeling were detected in blood vessels (Figure 6F).
10.1371/journal.pone.0019338.g006Figure 6 Trafficking of the FITC-tagged α-syn 9E4 antibody in tg mice.
To investigate the distribution of the 9E4 antibody after passive immunization, the FITC tagged antibody was injected intravenously (IV) and analyzed by ELISA and confocal microscopy. (A) Antibody titers in the plasma and brain at 3, 14 and 30 days post-injection in mice immunized with the 9E4 antibody, determined by ELISA. (B) Image analysis of 9E4-FITC positive neurons in the α-syn tg mice at 3, 14 and 30 days post-injection. (C, D) Representative laser scanning confocal images of the signal in the FITC channel in the temporal cortex of α-syn tg mouse 30 days following intravenous IV injection with the FITC-tagged 9E4 antibody. Arrows highlight labeled intra-neuronal granular-like structures. (E) No signal is detected in the FITC channel in α-syn tg mouse 30 days following IV injection with the FITC-tagged IgG1 control antibody. (F) No signal in the FITC channel in non-tg mouse 30 days following IV injection with the FITC-tagged 9E4 antibody. (G) Confocal image of a section from an antibody-naive α-syn tg mouse immunolabeled with cerebrospinal fluid (CSF) from a mouse immunized with 9E4-FITC. (H) Confocal image of a section from non-tg mouse immunolabeled 9E4-FITC antibody. Scale bar (C, E–H) = 50 µM; (D) = 10 µM. N = 20 per group, 12 months of age. Error bars represent mean ± SEM.
To further confirm that the 9E4-FITC antibody crossed the blood-brain barrier (BBB) and circulated in the CNS, CSF from mice immunized with the IgG-FITC or 9E4-FITC antibodies was used to label sections from antibody-naive α-syn tg mice. These studies demonstrated that the CSF from mice immunized with the 9E4-FITC antibody immunolabeled synapses and neurons in the antibody-naive α-syn tg (Figure 6G), in contrast no labeling was observed with the CSF of mice treated with non-immune IgG1-FITC (Figure 6H).
As an additional control to assess the passage of a FITC-labeled protein into the brain, α-syn tg and non-tg mice were injected with FITC-labeled β-syn (Figure S4). No signal in the FITC channel was observed upon direct visualization of cortical sections from these mice (Figure S4A, B), as would be expected given that β-syn is a predominantly cytoplasmic protein and, unlike α-syn, has not been reported at the plasma membrane. However when CSF from mice injected with the FITC-labeled β-syn was used to immunolabel sections from naive α-syn tg and non-tg mice (those that had not been injected with the FITC-β-syn), a clear immunoreactivity was observed in the cortex of both non-tg and α-syn tg mice (Figure S4C and D).
To determine whether α-syn co-localizes to the structures decorated by 9E4-FITC, co-labeling experiments were performed. Laser scanning confocal microscopy in sections from α-syn tg mice showed that the granular structures within the neurons labeled with 9E4-FITC antibody co-localized with α-syn immunoreactivity (Figure 7A–C). These, intra-neuronal structures labeled by the 9E4-FITC displayed LC3 (Figure 7D–F) and cathepsin-D immunoreactivity (Figure 7G–I). To corroborate the localization of the 9E4 antibody to the lysosomes, immuno-electron microscopic analysis was performed with a gold-tagged anti-mouse antibody. Ultrastructural analysis confirmed the presence of immunogold particles in the lysosomes and autophagosomes in the brains of α-syn tg mice treated with 9E4 (Figure 7J, K). In contrast, no specific labeling of lysosomes or autophagosomes was detected in the brains of α-syn tg mice treated with the non-immune IgG1 (Figure 7L, M) or in the neurons non-tg mice treated with 9E4 (Figure 7N, O).
10.1371/journal.pone.0019338.g007Figure 7 Co-localization of the FITC-tagged α-syn 9E4 antibody with lysosomal and autophagosomal markers.
To analyze the sub-cellular distribution of the 9E4 antibody immunohistochemical and ultrastructural analysis was conducted in 9E4-FITC immunized α-syn tg mice. (A–C) Representative confocal image of a brain section from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against α-syn. Arrows indicate co-localization of the 9E4-FITC signal with α-syn in neuronal granular-like structures. (D–F) Confocal image from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against LC3. Arrows indicate co-localization of the 9E4-FITC with LC3 in neuronal autophagosome-like structures. (G–I) Confocal image from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against cathepsin D. Arrows indicate co-localization of the 9E4-FITC with cathepsin-D in neuronal lysosomal-like structuresLC3. (J, K) Representative electron micrographs of sections from an α-syn tg mouse immunized with the 9E4 antibody and immunolabeld with gold-tagged anti-mouse antibody. (L, M) Electron micrographs of sections from an α-syn tg mouse immunized with the control IgG1 antibody and immunolabeld with gold-tagged anti-mouse antibody. (N, O) Representative electron micrographs of sections from a non-tg mouse immunized with the 9E4 antibody and immunolabeld with gold-tagged anti-mouse antibody. No reactivity is observed in lysosomes or autophagosomes. Scale bar (A–I) = 10 µM; (J–O) magnification 25,000x.
Taken together, these studies suggest that the 9E4 antibody can cross the BBB and bind α-syn and it is possible that the resulting antibody-antigen complex may then be endocytosed and transferred into the lysosomal compartment for degradation.
Passive immunotherapy with a monoclonal antibody against CT-α-syn activates the autophagy pathway
Since passive immunotherapy in this system appears to promote clearance of α-syn via a lysosomal pathway we investigated whether treatment with the 9E4 antibody activated the autophagy pathway in the α-syn tg immunized mice. Compared to control experiments where α-syn tg mice treated with IgG1 displayed discrete LC3 and cathepsin-D immunoreactivity granules, in the α-syn tg mice treated with the 9E4 antibody there was a significant increase in the neuronal levels of LC3 (Figure 8A-C) and cathepsin-D immunoreactivity (Figure 8D–F) This was accompanied by a decrease in the levels of intra-neuronal and synaptic CC α-syn accumulation and the compartmentalization of α-syn to granular structures (Figure 8G-I). Double labeling experiments confirmed that in the α-syn tg mice treated with the 9E4 antibody α-syn colocalized with the lysosomal (cathepsin-D) (Figure 8J–L) and autophagy (LC3) (Figure 8M–O) markers. Additional immunohistochemical was performed to confirm the co-localization of α-syn with LC3 in the 9E4 treated α-syn tg mice, which was absent in the IgG1 controls (Figure S5A–I). Further electron microscopy demonstrated a significant increase in the levels of gold-labeled α-syn particles in the phagosomes of 9E4 immunized α-syn tg mice in comparison to IgG1 controls (Figure S5J–N). Consistent with the immunohistochemistry, immunoblot analysis showed levels of LC3 breakdown and Beclin-1 immunoreactivity were increased in α-syn tg mice treated with the 9E4 antibody compared to the non-immune IgG1 (Figure 9A, B). Other genes expressed during autophagy, such as Atg 7 and Atg 10, remained stable with the 9E4 treatment in the α-syn tg mice (Figure 9A, B).
10.1371/journal.pone.0019338.g008Figure 8 Immunocytochemical analysis of the effects of passive immunization with 9E4 in markers of lysosomes and autophagy in α-syn tg mice.
To examine the sub-cellular distribution of the 9E4 antibody immunohistochemical and ultrastructural analysis was conducted in 9E4 immunized α-syn tg mice. (A) LC3 immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (B) LC3 immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (C) Analysis of LC3 immunoreactivity in α-syn tg mice immunized with IgG1 or 9E4 antibody. (D) Cathepsin-D immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (E) Cathepsin-D immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (F) Analysis of cathepsin-D immunoreactivity in α-syn tg mice immunized with IgG1 or 9E4 antibody. (G) CC α-syn immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (H) CC α-syn immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (I) Analysis of % area of CC α-syn immunoreactive neuropil in α-syn tg mice immunized with IgG1 or 9E4 antibody. (J–L) Co-localization of α-syn and cathepsin-D immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (M–O) Co-localization of α-syn and LC3 immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. Scale bar (A, B) = 30 µM (D, E, G and H) = 20 µM, (J–O) = 10 µM. (*) Indicates p<0.05, when comparing IgG1 to 9E4 group by unpaired Student's t test. Error bars represent mean ± SEM.
10.1371/journal.pone.0019338.g009Figure 9 Immunoblot analysis of the effects of passive immunization with 9E4 in molecular components of the autophagy pathway in α-syn tg mice.
(A) Immunoblot analysis of mTor, Beclin 1, LC3, Atg 5, Atg 7, and Atg 10 protein immunoreactivity in α-syn tg mice that had been immunized with either the IgG1 control or 9E4 antibody. (B) Analysis of mTor, Beclin 1, LC3, Atg 5, Atg 7, and Atg 10 protein levels in α-syn tg mice that had been immunized with either the IgG1 control or 9E4 antibody. (*) Indicates p<0.05, when comparing IgG1 to 9E4 group by unpaired Student's t test. Error bars represent mean ± SEM.
To confirm that the 9E4 antibody promotes clearance of α-syn aggregates via autophagy, in vitro experiments were performed in a neuronal cell line (B103 rat neuroblastoma cells [34]) expressing Lenti-virus (LV) α-syn and the reporter gene LC3-GFP. Under basal conditions neuronal cells expressing a control LV and treated with IgG1 only displayed discrete LC3-GFP granules (Figure 10A). Neuronal cells overexpressing α-syn showed the presence of enlarged LC3-GFP granules that co-localized with a-syn (Figure 10B). Treatment of the neuronal cells infected with the LV- α-syn with the 9E4 antibody resulted in an increase number of normal appearing LC3-GFP granules with a considerable decrease in the accumulation of α-syn in the cytoplasm (Figure 10C). In these neuronal cells, granular α-syn deposits co-localized to LC3-GFP structures representing autophagosomes (Figure 10C). The effects of the 9E4 antibody at reducing α-syn and elevating LC3-GFP were enhanced by rapamycin (an inducer of autophagy) (Figure 10E, G and H) and were blocked by 3-MA (an inhibitor of autophagy) (Figure 10F, I and J).
10.1371/journal.pone.0019338.g010Figure 10 The effects of the 9E4 monoclonal antibody on promotion of α-syn clearance via autophagy in a neuronal cell model.
(A) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control and treated with the IgG1 control antibody. (B) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the IgG1 control antibody. (C) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the 9E4 antibody. (D) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control, treated with the IgG1 control antibody and rapamycin, an inducer of autophagy. (E) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin, an inducer of autophagy. (F) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA, an inhibitor of autophagy. (G) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (H) Analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (I) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. (J) Quantitative analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. Scale bar (A–F) = 10 µM (*) Indicates p<0.05 compared to LV-control infected and vehicle-treated cultures by one-way ANOVA with post-hoc Dunnett's test. (#) Indicates p<0.05 compared to LV-control infected and vehicle-treated cultures by one-way ANOVA with post-hoc Tukey-Kramer test. Error bars represent mean ± SEM.
Taken together, these results support the possibility that passive immunization with antibodies against the CT of α-syn promotes clearance of α-syn aggregates via autophagy.
Discussion
The present study is the first to demonstrate that passive immunization with an antibody directed at the CT of α-syn is able to reduce memory/learning deficits and promote clearance of cortical and hippocampal a-syn aggregates in tg mice expressing human α-syn under the PDGFβ promoter. This is consistent with a previous study utilizing active immunization where epitope mapping indicated that the best results in terms of reducing a-syn was observed with antibodies that preferentially recognize the CT of a-syn [17]. Though the reason for the enhanced activity of the antibodies against the CT of a-syn is not completely understood, recent studies have supported the possibility that the generation of neurotoxic a-syn aggregates involves CT cleavage [41] and interactions with the CT domain of a-syn [10], [42]. Therefore it is possible that antibody targeting of this region may reduce the generation of this toxic species.
Calcium dependent calpain activation cleaves a-syn at the CT between amino acids 121–123 [32]. The cleavage of a-syn at either the NT or CT end of a-syn could be detected in the brains of patients with PD and DLB using two site-directed calpain-cleavage antibodies [32]. Calpain can cleave a-syn in vitro, leading to its aggregation and adoption of a b-sheet conformation. Therefore, immunization with antibodies against the CT of a-syn might be protective either by blocking the CT cleavage of a-syn, recognizing and promoting the clearance of CT fragments and aggregates of a-syn or by blocking the interaction of CT fragments with FL a-syn. In support of this possibility the present study showed that passive immunotherapy reduced the accumulation and formation of CT fragments compared to FL a-syn. The antibody against the calpain-cleaved a-syn utilized for this study has been shown to recognize both the free a-syn fragments as well as those complexed in oligomers [32]. These antibodies are sensitive at recognizing sets of a-syn aggregates that appear relevant to the disease process, given that such immunoreactivity is not found in control cases or other neurodegenerative disorders [32], [43]. In the brains of patients with DLB as well as in the tg models, a-syn aggregates containing CT-fragments not only accumulate in the cell bodies but also in axons and nerve terminals. In the tg animals, immunotherapy reduced the accumulation of a-syn preferentially in the neuropil. This was associated with improved behavioral performance in the water maze and expression of the post-synaptic markers such as PSD95. This is consistent with recent studies supporting the view that neurotoxic a-syn accumulates preferentially in synapses and that a-syn interferes with synaptic function [44], [45]. Therefore promoting the clearance of a-syn aggregates could be beneficial and result in functional improvements.
Potential explanations for the preferential beneficial effects of antibodies against the CT of a-syn include the possibility the CT epitopes in a-syn might be more readily exposed facilitating recognition by the antibody. For example, molecular modeling and nuclear magnetic resonance studies suggest that while the NT of a-syn interacts with the membrane [22], the CT tail is more rigid [46], capable of penetrating the membrane and be freely exposed to the external membrane surface where it can be recognized by the antibodies [21], [22]. Given that under physiological conditions a-syn is primarily a cytosolic protein it is puzzling how the antibodies utilized for the passive immunization strategies described in this study might recognize and trigger the clearance of a-syn. In this regard it is worth noting that under pathological condition the aggregated a-syn tends to accumulate in the membrane [21], [47], [48], [49] and to be exposed to the extracellular compartment. Moreover, neurotoxic a-syn oligomers can be secreted via exosomes into the extracellular space [50], [51], [52] and can be detected in the CSF [26], [53]. This suggests that the antibodies might recognize a-syn aggregates lying in the cell surface that in turn might be internalized and cleared via the autophagy pathway. In support of this possibility, in the present study we showed that systemically administered FITC-tagged antibodies against a-syn traffic into the CNS and are internalized by a-syn containing neurons and identified by double labeling and immunogold in lysosomes and autophagosomes. Moreover, a-syn was detected in LC3-positive granular structures further supporting a role for the autophagy-mediated clearance of a-syn in the immunized animals. This is consistent with recent studies showing that activating the autophagy pathway with pharmacological [54], [55] or gene therapy approaches promote elimination of a-syn aggregates and ameliorates the deficits in tg mice [12], [15].
Previous work by our group has shown that active immunization with recombinant α-syn ameliorates α-syn related synaptic pathology in a tg mouse model of PD [17], however given the common immunological problems that have often been associated with active immunization we chose to pursue a passive immunization protocol for this study. Our results indicate that passive immunization is as effective as active for the sequestration and removal of α-syn aggregates. It is interesting to note that both the passive and active immunization approaches to α-syn eventually result in the recruitment of the autophagocytic pathway indicating that key mechanisms may be involved in the degradation of α-syn.
Passive immunization with antibodies against amyloid-beta (Ab has been extensively investigated as a potential treatment modality for AD [56], [57], [58], [59]. These studies have been bolstered by the fact that Ab is secreted and easily accessible to antibody recognition. However a number of recent studies have shown that similarly to a-syn, immunotherapy can reduce the accumulation of other membrane bound and intracellular protein aggregates such as tau [60], [61], PrP [62] and huntingtin [63].
In conclusion, we show that a monoclonal antibody against CT α-syn traffics into the CNS, recognizes α-syn aggregates in affected neurons and ameliorates behavioral and neuropathological alterations in α-syn tg mice. Taken together, the results from this study support the view that passive immunization with antibodies against the CT of a-syn might have therapeutic potential in the treatment of PD and DLB.
Supporting Information
Figure S1
Immunochemical characterization of the specificity of a-syn antibodies utilized for passive immunotherapy. Immunoblot and immunohistochemical analysis was conducted to characterize the different α-syn antibodies and to select the one that was subsequently used for passive immunotherapy. Western blot analysis was performed with soluble fraction from the temporal cortex. (A) Immunoblot analysis in non-tg and α-syn tg mice with IgG1 control and the mouse monoclonal antibodies against -α-syn- 6H7, 8A5, 9E4 and the polyclonal antibodies against full-length (FL) α-syn and Calpain cleaved (CC) α-syn. (B, C) Background levels of immunostaining with the control IgG1 in non-tg and α-syn tg, respectively. (D, E) Representative confocal images with the 6H7 antibody in non-tg and α-syn tg, displaying immunostaining in the neuropil and neurons in the temporal cortex respectively. (F, G) Confocal images with the 8A5 antibody in non-tg and α-syn tg, displaying immunostaining in the neuropil and neurons respectively. (H, I) Confocal images with the 9E4 antibody in non-tg showing no specific labeling and immunostaining in the neuropil and neurons in α-syn tg. (J, K) FL α-syn immunoreactivity in non-tg and α-syn tg, respectively. (L, M) CC α-syn immunoreactivity in non-tg and α-syn tg, respectively). N = 3 per group, 6 months of age. Scale bar (B-M) = 30μM.
(TIF)
Click here for additional data file.
Figure S2
Effects of passive immunization with a C-terminal α-syn antibody on tau. Levels of total and PHF-tau were examined by immunohistochemistry and immunoblot analysis to asses the effects of passive immunization with 9E4. (A, B) Representative brightfield images of total tau in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively. (C, D) Representative brightfield images of total tau in the frontal cortex of IgG1- and 9E4-treated α-syn tg mice, respectively. (E) Quantitative analysis of total tau levels in the frontal cortex of IgG1 and 9E4-treated non-tg and α-syn tg mice. (F, G) Representative brightfield images of PHF-tau in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively. (H, I) Representative brightfield images of PHF-tau in the frontal cortex of IgG1- and 9E4-treated α-syn tg mice, respectively. (J) Quantitative analysis of PHF-tau levels in the frontal cortex of IgG1 and 9E4-treated non-tg and α-syn tg mice. (K) Immunoblot of levels of total and PHF-tau in the frontal cortex of IgG1 and 9E4 treated non-tg and α-syn tg mice. (L) Analysis of total tau levels in the frontal cortex of IgG1 and 9E4-treated non-tg and α-syn tg mice as determined by immunoblot. (M) Analysis of PHF-tau levels in the frontal cortex of IgG1 and 9E4-treated non-tg and α-syn tg mice as determined by immunoblot. Scale bar = 40uM. Error bars represent mean ± SEM. (*) Indicates p<0.05, when comparing α-syn tg immunized with IgG1 or 9E4 to IgG1-treated non-tg mice by one-way ANOVA with post hoc Dunnett's.
(TIF)
Click here for additional data file.
Figure S3
Effects of passive immunization with a C-terminal α-syn antibody on vasculature or markers of glial cell reactivity. (A, B) Representative brightfield images of the endothelial cells marker Zo-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively (arrows indicate location of blood vessels). (C, D) Representative brightfield images of Zo-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated α-syn tg mice, respectively (arrows indicate location of blood vessels). (E) Analysis of % of Zo-1 immunoreactive neuropil in the frontal cortex of IgG1- and 9E4-treated non-tg and α-syn tg mice. (F, G) Representative brightfield images of the microglial marker Iba-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively. (G, H) Representative brightfield images of Iba-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated α-syn tg mice, respectively. (E) Analysis of Iba-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg and α-syn tg mice. (F, G) Representative brightfield images of the microglial marker Iba-1 immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively. (K, L) Representative brightfield images of the astroglial cell marker GFAP immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg mice, respectively (arrows indicate location of blood vessels). (M, N) Representative brightfield images of GFAP immunoreactivity in the frontal cortex of IgG1- and 9E4-treated α-syn tg mice, respectively (arrows indicate location of blood vessels). (E) Analysis of GFAP immunoreactivity in the frontal cortex of IgG1- and 9E4-treated non-tg and α-syn tg mice. Scale bar (A-D) = 80uM, (F-N) = 50μM. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immunized α-syn tg mice to IgG1-imunized non-tg mice by one-way ANOVA with post hoc Dunnett's. (#) Indicates p<0.05, when comparing α-syn tg mice immunized the 9E4 α-syn antibody to IgG1-treated α-syn tg mice by one-way ANOVA with post hoc Dunnett's.
(TIF)
Click here for additional data file.
Figure S4
FITC-labeled β-syn crosses the blood-brain barrier but does not bind neurons in α-syn tg mice. (A, B) Signal in the FITC channel upon direct visualization of temporal cortex sections from non-tg and α-syn tg mice injected with FITC-labeled β-syn, respectively. (C, D) Representative confocal images of the neocortex of non-tg and α-syn mice, respectively, immunolabeled with CSF from mice injected with FITC-labeled β-syn displaying immunostaining of the neuropil. Scale bar (A-I) = 10μM.
(TIF)
Click here for additional data file.
Figure S5
Further characterization of LC3 immunoreactivity and phagosome involvement of α-syn clearance in α-syn tg mice immunized with 9E4. (A) α-syn immunoreactivity in α-syn tg mouse immunized with the IgG1 control antibody. (B) LC3 immunoreactivity in α-syn tg mouse immunized with the IgG1 control antibody. (C) Co-localization of α-syn and LC3 immunoreactivity in α-syn tg mouse immunized with the IgG1 control antibody. (D, G) α-syn immunoreactivity in α-syn tg mouse immunized with the 9E4 antibody. (E, H) LC3 immunoreactivity in α-syn tg mouse immunized with the 9E4 antibody. (F, I) Co-localization of α-syn and LC3 immunoreactivity in α-syn tg mouse immunized with the 9E4 antibody. (J, K) Immuno-gold electron micrographs of phagosomes from α-syn tg mouse immunized with the IgG1 control antibody, very few gold particles were detected. (L, M) Immuno-gold electron microscopy images of phagosomes from α-syn tg mouse immunized with the 9E4 antibody, abundant gold particles were detected.(N) Quantitative analysis of gold particles in phagosomes from α-syn tg mice immunized with either IgG1 control or 9E4 antibody Scale bar (A-I) = 10μM (J-M) = 0.5μM Error bars represent mean ± SEM. (*) Indicates p<0.05, when comparing IgG1 to 9E4 group by one way ANOVA with post hoc Dunnet's.
(TIF)
Click here for additional data file.
Competing Interests: The authors have declared the following conflict of interest: Sarah Mueller-Steiner, Peter Seubert, Robin Barbour, Lisa McConlogue, Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Funding: This work was funded by National Institutes of Health (NIH) grants AG 11385, AG 18840, AG 022074 and NS 044233 and by ELAN Pharmaceuticals. NIH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sarah Mueller-Steiner, Peter Seubert, Robin Barbour, Lisa McConlogue, Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals, who, in collaboration with the group at UCSD, were intellectually involved in the conception and execution of the in vitro and in vivo passive immunization experiments.
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