extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Comparison of proteomic profiles of serum, plasma, and modified media supplements used for cell culture and expansion

Background The culture and expansion of human cells for clinical use requires the presence of human serum or plasma in culture media. Although these supplements have been extensively characterized in their chemical composition, only recently it has been possible to provide by high throughput protein analysis, a comprehensive profile of the soluble factors contributing to cell survival. This study analyzed and compared the presence of 100 proteins including chemokines, cytokines and soluble factors in six different types of media supplements: serum, plasma, recalcified plasma, heat inactivated serum, heat inactivated plasma and heat inactivated recalcified plasma. Methods Serum, plasma, recalcified plasma, and heat inactivated supplements were prepared from ten healthy subjects. The levels of 100 soluble factors were measured in each sample using a multiplexed ELISA assay and compared by Eisen hierarchical clustering analysis. Results A comparison of serum and plasma levels of soluble factors found that 2 were greater in plasma but 18 factors were greater in serum including 11 chemokines. The levels of only four factors differed between recalcified plasma and plasma. Heat inactivation had the greatest effect on soluble factors. Supervised Eisen hierarchical clustering indicated that the differences between heat inactivated supplements and those that were not were greater than the differences within these two groups. The levels of 36 factors differed between heat inactivated plasma and plasma. Thirty one of these factors had a lower concentration in heat inactivated plasma including 12 chemokines, 4 growth factors, 4 matrix metalloproteases, and 3 adhesion molecules. Heat inactivated decalcified plasma is often used in place of heat inactivated serum and the levels of 19 soluble factors differed between these two supplements. Conclusion Our report provides a comprehensive protein profile of serum, plasma recalcified plasma, and heat inactivated supplements. This profile represents a qualitative and quantitative database that can aid in the selection of the appropriate blood derived supplement for human cell cultures with special requirements.

Background
There is a growing interest in the in vitro generation of large numbers and varieties of cell types for clinical applications including dendritic cells (DCs) to improve the effectiveness of immune therapies [1][2][3][4], cytotoxic T cell therapy of cancer [5], EBV induced lymphoma [6], and CMV infection [7,8], and to expand hematopoietic stem cells to provide greater quantities of transplantable cells [9,10]. The culture and expansion of cells usually involves the addition of cytokines and/or growth factors. In addition, serum or plasma is often added to the culture media [1,3,[6][7][8].
Although fetal calf serum has often been the standard media supplement for in vitro research studies, fetal calf serum is not suitable for the production of cells for clinical use. The use of bovine serum exposes patients to the potential risk of transmission of pathogens such as Creutzfeldt-Jakob disease (CJD) [11] and the development of a humoral or cellular response to bovine proteins [4]. In spite of these issues, some clinical protocols use fetal calf serum [3,6], but often human serum is used in place of fetal calf serum [1,7,8]. In some cases human plasma [1] or no media supplement is used [2,4,9,10].
While human serum is likely the best substitute for fetal calf serum, human plasma is more readily available than serum. Plasma is regularly produced by blood centers following good manufacturing practices from donors screened and tested for transfusion transmitted diseases. The plasma is stored frozen and is used for transfusion. In contrast, serum is not used for transfusion and is not routinely manufactured by blood centers.
The use of human plasma rather than serum in cell culture, however, presents some problems. Citrate, a calcium chelator, is the standard plasma anticoagulant used in the process of plasma collection. When plasma is added to culture media which usually contains calcium, fibrin or clots may form. This can be prevented by adding thrombin and calcium to plasma to induce clot formation and defibrination. Recalcified plasma is used in place of fetal calf serum in some clinical protocols.
When serum or plasma is used as a culture media supplement, it is often modified by heat inactivation which involves heating at 56°C for 1 hour to inactivate complement components and prevent the occurrence of complement mediated lysis in cell cultures [1]. However, heating has a large spectrum of denaturing effects and hence affects other serum and plasma factors.
Although serum, plasma, recalcified plasma, and heat inactivated serum and plasma are used to supplement culture media little is known about the differences in the composition of these supplements. The goal of this study was to determine if differences in the levels of soluble factors among these media supplements might contribute to the differences in their efficacy. Results from this study may assist in determining which plasma or plasmaderived components would be more appropriate for human cell cultures with special requirements.

Study design
A total of 450 mL of whole blood was collected from each of 10 healthy research donors. The blood was collected in two aliquots: one of 375 mL and the other 125 mL. The larger aliquot was collected in CP2D (citrate-phosphate-2dextrose) anticoagulant and was used to prepare plasma. One third of plasma prepared from the CP2D blood was processed to make recalcified plasma, one third to make heat inactivated plasma, and one third was not further processed. The 125 mL aliquot of blood was collected in a bag without anticoagulant and was used to make serum. One and one-half mL aliquots from all components were frozen at -80°C. Aliquots from each of the 10 subjects were tested for 100 soluble factors using a multiplex ELISA. In the first part of the study serum, plasma, recalified plasma and heat inactived plasma were tested. In the second part of the study samples of serum, plasma, and recalcified plasma stored at -80°C were thawed, heat inactivated and tested.

Blood collection
Whole blood was collected using a specially assembled set of blood collection bags consisting of a dry bag from a Pall collection bag system (Leukotrap WB system, CP2D/AS-3 double blood bag unit, Pall Corporation, East Hills, NY 11548, USA) and Baxter, 150 mL transfer bag (Baxter Health Corporation, Fenwal division, Deerfield, IL 60015, USA). The blood collection bags were assembled using a sterile connecting device (SCD 312, Terumo Medical Corp, Elkon, MD). The seven bag set included a phlebotomy needle tubing which was connected to a Y anastomoses tubing with one side connected to a collecting bag labeled (A), which contained 55 mL of CP2D as anticoagulant. Bag A was connected to a dry satellite bag labeled B, which in turn was connected to three 150 mL transfer bags labeled C, D, and E. The second end of Y blood withdrawal tubing was connected to a dry CLX satellite bag (Pall) labeled F, which was connected to second dry bag labeled G.
After 375 mL of whole blood was collected into the first primary bag (A), the tubing leading to bag (F) was then opened and 125 ml of blood was collected into this bag (F) which was placed on ice during phlebotomy. Bags A and F were separated at the end of the collection and the blood in bag A was used to prepare plasma and plasma products, while blood in bag F was used to prepare serum.

Plasma and serum preparation and storage
To prepare plasma the whole blood in bag A was centrifuged at 4000 rpm for 6.5 minutes (RC3C, Sorval Instruments, Newton, CT). Plasma was expressed into bag B which was then divided equally between the three satellite bags (C, D, and E) with a final volume of 60 to 75 mL in each bag. Plasma bag E was not processed further.
To prepare recalcified plasma calcium chloride (CaCl 2 ) and thrombin were added to bag C and the plasma was incubated at 37°C for one hour and then stored at 4°C overnight. The bag was then centrifuged at 4000 rpm for 6.5 minutes at room temperature and supernatant, recalcified plasma was expressed into a satellite bag.
Heat inactivated plasma was prepared by incubating plasma in bag D at 56°C for one hour. The bag was then centrifuged at 4000 rpm for 6.5 minutes and the expressed supernatant, heat inactivated plasma, was transferred into satellite bag.
To prepare serum, the whole blood in bag F was stored on ice for one hour. It was then centrifuged at 4000 rpm for 6.5 minutes and the supernatant was transferred into bag G.
Immediately after the preparation of each component was complete, 1.5 mL aliquots were placed into cryovials (1.8 mL Nunc) and snap frozen on dry ice and ethanol. After freezing, all samples were then transferred to -80°C for storage.

Analysis of plasma and serum proteins
The levels of 100 soluble factors were assessed on an ELISA-based platform (Pierce Search Light Proteome Arrays, Boston, MA) consisting of multiplexed assays that measured up to 16 proteins per well in standard 96 well plates [12]. The arrays were produced by spotting 2 × 2, 3 × 3, or 4 × 4 patterns of different monoclonal antibodies into each well of a 96-well plate. Following a typical sandwich ELISA procedure, signal was generated using a chemiluminescent substrate. The light produced at each spot in the array was captured by imaging the entire plate with a commercially available cooled CCD camera. The data was reduced using image analysis software that calcu-

Statistical analysis
Paired two tailed t-test of natural log transformed data was used to compare the plasma and serum soluble factor levels. Differences were considered significant at a cut off pvalue of ≤ 0.01. Relatedness of cytokine expression patterns in different types of plasma and serum was tested by applying unsupervised Eisen's hierarchical clustering methods [13] to the data set encompassing the soluble factors across all samples. Unsupervised clustering involved the sorting of both the soluble factors and the samples. In contrast, for unsupervised clustering the fac-tors were sorted, but not the samples. Twenty of the 100 factors were excluded from the analysis since their levels were under the assay's limits of detection . Those factors  were: TGF-β, IL-1β, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-15, IL
The most prominent dissimilarity among the 40 samples was a cluster of 15 factors whose levels were markedly lower in the 10 heat inactivated plasma than in the other samples ( Figure 2). These factors were fibrinogen, MIP-Unsupervised Hierarchical clustering (Eisen Kendall's) of 80 soluble factor levels among a total of 40 samples including 10 serum (S), 10 plasma (P), 10 recalcified plasma (CP), and 10 heat inactivated plasma (P-HI) samples prepared from whole blood from 10 healthy subjects (01 through 10) These results show that plasma and recalcified plasma are very similar in regards to the factors analyzed. However, serum and plasma are quite different as previously reported by our group [14] and heat inactivation affected the levels of several factors.

Comparison of serum and plasma
Previous studies have found that the levels of several factors differed between serum prepared from blood collected in BD vacutainer serum collection tubes and plasma prepared from blood collected in vacutainers with EDTA [14]. A comparison of the soluble factors in the 10 plasma samples prepared in this study from citrated blood collected in bags and the 10 serum samples collected in bags revealed that the levels of 20 factors differed between serum and plasma ( Table 1). The levels of fibrinogen and OPN were greater in plasma and levels of 18 factors were greater in serum. Among the other 18 factors whose levels were greater in serum than in plasma were 11 chemokines (MCP-2, MCP-3, MIP-1α, MIP-1β, MIP-3α, NAP-2, ITAC, lymphotactin, eotaxin 2, exodus 2, and RANTES) and 4 factors normally found in platelets NAP-2, RANTES, PDGF-BB, and PAI-1 total.

Comparison of factor levels in recalcified plasma, regular plasma and serum
To produce recalcified plasma thrombin and calcium were added to plasma. As expected, the levels of fibrinogen were lower in recalicified plasma than in plasma, (262 ± 38 microg/mL vs 4,810 ± 1,780 microg/L, p = 0.026). The levels of only 4 other factors differed between recalcified Factors that distinguish heat inactivated plasma from plasma, serum, and recalcified plasma Figure 2 Factors that distinguish heat inactivated plasma from plasma, serum, and recalcified plasma. Hierarchical clustering analysis was applied to 80 soluble factors measured in a total of 40 serum, plasma, and plasma derived components (as per Figure 1). The ten heat inactivated plasma components separated into a single group with one recalcified plasma component. A cluster of 15 soluble factors best distinguished the heat inactivated group from the other 29 components.
Heat inactivated plasma components serum plasma and recalcified plasma serum plasma and plasma (MDC, NAP-2, IL-16 and IFN-α) ( Table 2). The levels of MDC, IL-16, and IFN-α were greater in plasma and the level of NAP-2 was greater in recalcified plasma.
There were more differences between recalcified plasma and serum than between recalcified plasma and plasma. The levels of 19 factors differed between recalcified plasma and serum ( Table 3). Eighteen of the 19 factors were greater in serum and one of the factors was greater in recalcified plasma, OPN. Fourteen of the 19 factors that differed among plasma and serum also differed among recalcified plasma and serum (OPN, MIP-1β, MIP-3α, NAP-2, APO-A1, eotaxin 2, MCP-2, MCP-3, TIMP-1, Lymphotaxin, PDGF-BB, ITAC, PAI-1 total, and TNFR1). The 5 factors that differed among recalcified plasma and serum but not plasma and serum were MDC, MMP-2, L-selectin, APO-B100, and ENA-78. Values are mean ± one standard deviation.

Comparison of factor levels between freshly collected plasma and heat inactivated plasma
Heat inactivation was performed on a selected set of supplement samples right after collection to make a preliminary assessment of heat-induced protein levels variation. Plasma was selected as the candidate supplement for testing. A comparison of factor levels between heat inactivated plasma and plasma found that the levels of 36 factors changed with heat inactivation of plasma ( Table  4). The levels of 5 factors, MPO, MCP-1, eotaxin 2, MIP-1, PAI-1 total, and TNFR1, were greater in heat inactivated plasma ( Table 4). The 31 factors whose levels were greater in plasma than in heat inactivated plasma included several chemokines and growth factors.

Comparison of heat inactivated serum, plasma, and recalified plasma with untreated serum, plasma and recalcified plasma
Since heat inactivation changed the levels of many plasma factors, stored serum, plasma, and recalified plasma samples from the 10 subjects were heat inactivated and soluble factor levels were measured. A comparison of levels of soluble factors between all heat inactivated and untreated products using supervised hierarchical clustering found that, in general, there were more differences in protein lev-els between heat inactivated samples and those that were not heat inactivated than among samples that were heat inactivated or among samples that were not (Figure 3). Analysis using t tests found that the levels of 19 factors differed between heat inactivated serum and heat inactivated recalcified plasma (Table 5).

Discussion
This study reports the protein profile of 80 factors in serum, plasma, recalcified plasma and heat inactivated components. Significant differences in soluble factor profiles and concentrations were observed among these four supplements routinely utilized in clinical cell cultures. Serum and plasma showed very distinct profiles, recalcification of plasma had little affect on the levels of most soluble factors compared to non-recalcified plasma, and heat inactivation drastically changed plasma and serum protein content Serum differs from plasma and recalcified plasma in many ways. The levels of 18 of the 80 factors were greater in serum than in plasma. The increase in factors in serum was likely due to the stimulation of platelets by the coagulation cascade and the release of platelet factors NAP-2, RANTES, and PPGF-BB. The chemokine NAP-2 likely stimulates leukocytes leading to the production and release of cytokines, growth factors and other chemokines.
The levels of two factors were lower in serum than in plasma; fibrinogen and OPN. The lower levels of both factors are likely a direct effect of thrombin generation in serum. Thrombin catalyzes the conversion of fibrinogen to fibrin and OPN has a thrombin-cleavage site and it is degraded during the activation of the coagulation cascade [15]. The presence of thrombin-cleaved OPN in serum is likely to affect cells in culture since cleaved OPN promotes cell attachment and spreading to a greater extent than uncleaved OPN [15].
Since recalcified plasma has lower levels of fibrinogen than plasma, clots are less likely to form in media supplemented with recalcified plasma rather then plasma. However, the levels of almost all other factors are similar in plasma and recalcified plasma and the behavior of cells cultured in media plus recalcified plasma is likely be similar to those cultured in media plus plasma. Serum or plasma is often heat inactivated to prevent the activation of complement components during cell culture [1]. We found that heat inactivation affects the levels of many of the factors studied. The levels of almost one third, 31, of the plasma factors were reduced by heat inactivation. Heating denatures proteins and causes them to become insoluble and these results show that the effects of heat inactivation are very broad.
The levels of five factors increased with heat inactivation; MPO, eotaxin 2, MCP-1, PAI-1 and TNFR1. While plasma has marked reduced levels of cellular elements compared to whole blood, it does contain some platelets and leukocytes. The increase in the levels of these 5 factor levels following heat inactivation may be the result of heat-induced cell disruption and release of intracellular contents. MPO is an abundant neutrophil secondary granule protein, PAI-1 is found in platelets [16], TNFR1 is found on all leukocytes, eotaxin 2 in monocytes and macrophages [17] and MCP-1 in monocytes and T cells. Since the levels of many chemokines, growth factors and cell adhesion molecules changed as a result of heat inactivation, the behavior of cells in media supplemented with heat inactivated plasma is likely to be much different than the behavior of cells in media plus plasma.
Our laboratory often uses heat inactivated recalcified plasma rather then heat inactivated serum. Our multiplex protein analysis revealed several differences between the two types of supplements. Since the levels of many chemokines and cell adhesion molecules differed in heat inactivated serum and heat inactivated recalified plasma, the behavior of cells cultured in media plus heat inactivated serum may be different than from those cultured in media Supervised hierarchical clustering (Eisen Kendall's) of 80 soluble factors among 60 samples from 10 subjects Figure 3 Supervised hierarchical clustering (Eisen Kendall's) of 80 soluble factors among 60 samples from 10 subjects. The samples included 10 sera (S), 10 plasma (P), 10 recalified plasma (CP), 10 heat inactivated sera (S-HI), 10 heat inactivated plasma (P-HI), and 10 heat inactivated recalified plasma (CP-HI). The serum, plasma, and recalcified plasma were prepared, stored at -80°C and tested. The heat inactivated samples were prepared, stored at -80°C, thawed, heat inactivated, stored again at -80°C and tested. Red bar encompasses regular serum, plasma and recalcified plasma. Black bar heat inactivated samples.
plus heat inactivated recalcified plasma. These differences do not preclude the use of heat inactivated recalcified plasma, but when it is used in place of heat inactivated serum the cells must be carefully analyzed to ensure the final product has not changed.
Media without serum or plasma supplements have been used to culture a number of cell types including hematopoietic progenitor cells, dendritic, neutrophils, and leukemia cells [2,4,9,10,18,19]. However, when media with serum or plasma must be used, our report provides a useful characterization of serum/plasma components which can aid in selecting the most appropriate blood supplement for a particular type of cell culture or clinical hematological procedure. Additionally it can shed light on the soluble factors that are missing when investigators subject cultures to serum free media conditions Additional material


extfieldsofstudy: Biology Medicine

text:
Cathepsin D, a Lysosomal Protease, Regulates ABCA1-mediated Lipid Efflux*

To identify genes involved in the regulation of plasma high density lipoprotein (HDL) cholesterol (HDL-C) levels, patients with low HDL-C and age- and sex-matched controls (normal HDL-C) were extensively characterized. Comparative transcriptome analysis was carried out in cholesterol-loaded monocyte-derived macrophages from low HDL subjects segregated into groups with or without cholesterol efflux defects or ABCA1 mutations. Clusters of differentially regulated genes were evident in the low HDL groups as compared with controls. Of particular note, expression of cathepsin D (CTSD), a lysosomal proteinase, was reduced by ∼50% in monocyte-derived macrophages of low HDL-C subjects, most significantly those with cholesterol efflux defects but without mutations in ABCA1 (p < 0.01). These results were verified by reverse transcription-PCR and replicated in a second cohort. We show here that blocking the activity or expression of CTSD, by pepstatin or CTSD small interfering RNA, respectively, reduced ABCA1 expression and protein abundance in both macrophages and CHO cells and apolipoprotein A-I-mediated lipid efflux by more than 70%. Conversely, expression of CTSD increased both ABCA1 mRNA expression and cellular ABCA1 protein. Consistent with its role in the proteolytic processing of prosaposin, inactivation of CTSD function resulted in the accumulation of glycosphingo-lipid and free cholesterol in late endosomes/lysosomes, a phenotype similar to NPC1 deficiency. Inhibition of CTSD also caused retention of ABCA1 in lysosomal compartments, reducing its trafficking to the plasma membrane. These studies demonstrate a novel and potentially important role for CTSD in intracellular cholesterol trafficking and ABCA1-mediated efflux. Therefore, decreased CTSD expression may contribute to low plasma HDL-C levels.

High density lipoprotein cholesterol (HDL-C) 3 deficiency is a major risk factor for cardiovascular diseases and has a complex metabolic and genetic etiology. Overproduction and/or impaired clearance of triglyceride-rich lipoproteins results in triglyceride enrichment of HDL and accelerated apoA-I catabolism and may account for low HDL-C in patients with other features of the metabolic syndrome. Low HDL can also result from mutations in a number of genes regulating HDL production or catabolism, including apolipoprotein A-I (1-4), lecithin:cholesterol acyltransferase (5-7), phospholipid transfer protein (8 -10), and ATP-binding cassette protein A1 (11)(12)(13)(14)(15)(16)(17)(18)(19).
Defective cellular phospholipid and cholesterol efflux is also a common contributory phenotype in familial low HDL syndromes, and in several of these kindreds, affected subjects are heterozygous carriers of ABCA1 mutations (17,20,21). These genetic studies demonstrate the key role of ABCA1 in cholesterol efflux to apoA-I, but as we have reported previously (22), sequence variants in ABCA1 account for a small proportion of hypoalphalipoproteinemia. In further studies in a cohort of low HDL and control subjects previously characterized (Ref. 22 and Sarov-Blat et al. 4 and Kiss et al., 5 submitted manuscripts), we have undertaken to identify other genes and gene products that contribute to lipid efflux and low HDL. High density oligonucleotide microarrays were used to analyze gene expression in cholesterol-loaded monocyte-derived macrophages (MDM) prepared from these subjects. Importantly, we demonstrate here that expression of cathepsin D (CTSD), a lysosomal proteinase (23), is reduced by 50% in the MDM of low HDL-C subjects, significantly in those with cholesterol efflux defects without mutations in ABCA1, compared with control subjects. CTSD is involved in intracellular catabolism in lysosomal compartments but also functions in hormone and antigen processing (24 -26). CTSD has been implicated in mitogenic activity in certain cancers (27,28), which may be related to the secretion of its pro-form (24), lysosomal membrane permeabilization, and apoptosis (29). In addition, genetic variants in CTSD have been identified as a risk factor for Alzheimer disease (30) and are the causative gene of neuronal ceroid lipofuscinosis (31,32). CTSD has also been previously shown to bind to and promote the maturation of prosaposin (pro-SAP) into saposins (A, B, C, and D), which are required for the hydrolysis of certain sphingolipids (33,34). We further demonstrate that CTSD thereby regulates intracellular sphingolipid metabolism, cholesterol trafficking, and ABCA1-mediated efflux. These findings are relevant to our understanding of the cellular etiology of certain low HDL states, with specific reference to the regulation of lysosomal enzymes involved in the intracellular cholesterol trafficking and therefore apoA-I/ ABCA1-mediated cellular cholesterol efflux.
Selection of Low HDL-C Subjects and Controls-Patients referred to the Lipid Clinic of the University of Ottawa Heart Institute were selected based on the following criteria: Caucasian, HDL-C Ͻ5th percentile for age and sex; triglyceride Ͻ95th percentile (most Ͻ75th percentile); and LDL cholesterol Ͻ75th percentile, measured while not on treatment with lipidmodifying medications. Exclusion criteria included clinical conditions or medications causative of low HDL-C, including all forms of diabetes. There was no other selection bias, since most subjects were recruited in chronologic order of referral. The control group was selected among healthy and normolipemic volunteers of the same ethnic background recruited from the Ottawa region. The study was approved by the University of Ottawa Heart Institute Human Research Ethics Committee, and written informed consent was obtained from all participants.
RNA Isolation in Human Subjects-Macrophages were homogenized with Qiashredder, and total mRNA was isolated using the RNeasy mRNA isolation kit (according to the manufacturer's instructions).
GeneChip Affymetrix Microarray-Gene expression profiles were analyzed by Affymetrix GeneChips, which cover 22,215 annotated genes. RNA quality and quantity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and RiboGreen RNA quantization reagent (Molecular Probes, Inc., Eugene, OR), respectively, in addition to measuring OD at 260 and 280 nm and 260/280 ratios. All samples possessed 18 and 28 S rRNA bands with no sign of RNA degradation. Affymetrix human U133A GeneChips (Affymetrix Inc., Santa Clara, CA) were used. Briefly, double-stranded cDNA was synthesized from each total RNA sample via oligo-T7-mediated reverse transcription. Biotin-labeled cRNA was synthesized using an in vitro transcription reaction with the Enzo BioArray high yield RNA transcript labeling kit (Enzo Diagnostics, Inc., Farmingdale, NY). Subsequently, the purified, biotin-labeled cRNA was fragmented into 50 -150-nucleotide fragments. Fragmented, biotinlabeled cRNA (15 mg), together with a known spike (according to Affymetrix recommendations) was hybridized to a U133A Gene-Chip for 16 -18 h at 45°C. An Affymetrix GeneChip fluidics station 450 was used to perform standard washing and streptavidin/ phycoerythrin staining. The chips were scanned using an Affymetrix GeneChip scanner 3000.
TaqMan Quantitative Real Time PCR-Total RNA (1 g) was converted to cDNA using a high capacity cDNA archive kit (Applied Biosystems), and the equivalent of 10 ng/well was arrayed into high density 384-well plates using a Biomek FX robot (Beckman Coulter), allowing expression profile generation for one gene at a time. Quantitative RT-PCR was carried out using a 7900HT Sequence Detector System (Applied Biosystems) in a 10-l reaction volume. TaqMan Universal PCR FIGURE 1. Cluster of the cathepsin family genes whose transcripts were decreased in low HDL groups (group A, low HDL, low efflux, no ABCA1 mutation; group B, low HDL, low efflux, ABCA1 mutation; group C, low HDL, normal efflux compared with control group D). Monocyte-derived macrophages isolated from patients with low HDL and age-and sex-matched controls were loaded with Ac-LDL, and global gene expression profiles were analyzed on duplicate GeneChip DNA-microarrays. A, hierarchical cluster dendrogram from TIGR MeV program for both genes and subjects. The cathepsin gene family was expressed similarly within low HDL groups and between control subjects. The red color represents up-regulated genes, whereas the green indicates down-regulated genes. The CTSD gene was the most differentially expressed member of the cathepsin genes and was significantly down-regulated in groups A and B compared with control (p Ͻ 0.01 and p Ͻ 0.016, respectively). B, plot of the CTSD probe set relative expression in microarray for individual subjects and average CTSD mRNA expression value for each group.
Master Mix 2X (Applied Biosystems) and universal PCR conditions recommended by the vendor were followed.
SYBR Green Real Time PCR-mRNA was isolated from mammalian cells using Trizol reagent (Invitrogen, Burlington, Canada). CTSD mRNA, ABCA1, and GAPDH mRNA were quantified using the Stratagene SYBR Green kit on a Lightcycler real time PCR machine according to the manufacturer's recommendations.
Cell Culture-Human blood monocytes were differentiated into macrophagic cells according to Cohen et al. (22). The monocytes were differentiated into macrophages over the course of 12 days in growth media, and the resulting cells were used in the efflux assay or for mRNA isolation. All macrophages were grown in RPMI medium supplemented with 10% pooled human serum, whereas J774 and CHO cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). All cells were grown at 37°C in 5% CO 2 and incubated with penicillin/streptomycin. Bone marrow-derived macrophages were flushed from mouse femurs. Macrophages were generated by incubating fetal liver cells (2 ϫ 10 6 cells/ml) or bone marrow cells (10 6 cells/ml) with DMEM, 10% FBS complemented with 15% L929 conditioned medium for 7 days. Three days prior to the isolation of peritoneal macrophages, mice were injected with 2 ml of 3% thioglycollate intraperitoneally. The animals were then euthanized with CO 2 , the peritoneal cavity was washed by an 8-ml injection of saline, and the cells were washed three times by centrifugation at 1000 ϫ g for 10 min with DMEM. The cells are then plated in 6-well plates containing DMEM, 10% FBS overnight.

H]Cholesterol and [ 3 H]Phospholipid Efflux-Macrophages and
CHO cells were labeled with LDL or Ac-LDL (50 g/ml) equilibrated with [ 3 H]cholesterol (5 Ci/ml) in 1% FBS medium for 24 h. The next day, the cells were washed twice with phosphate-buffered saline (PBS) and equilibrated overnight in serum-free medium. ApoA-I cholesterol efflux was monitored by counting the radioactivity in the medium after a 24-h incubation of 10 g/ml lipid-free ApoA-I, 0.1% BSA. Cholesterol efflux was expressed as the percentage of [ 3 H]cholesterol in the medium over total cellular [ 3 H]cholesterol. For phospholipid efflux, the same protocol was used as for cholesterol efflux, except that the 3 H-labeled phospholipids bound to immunoprecipitated apoA-I were counted, using a rabbit polyclonal anti-human apoA-I antibody. Since some CTSD is secreted by cells and could degrade protein in the medium, we analyzed by SDS-PAGE immunoprecipitated apoA-I from the medium after the efflux assay. No apoA-I degradation was observed (data not shown). Lipid Analysis-Free cholesterol and cholesteryl esters were extracted and separated by TLC on Sil-G TLC plates (EMD Chemicals, Darmstadt, Germany) using the solvent system hexane/diethylether/acetic acid (105:45:1.5) (35). Lipid bands were visualized with iodine vapors, isolated, and analyzed by ␤-scintillation counting.
siRNA Transfection and Expression Vectors-Cathepsin D siRNA obtained from Ambion (Austin, TX) was transfected into macrophage J774 cells using the TransIT-TKO transfection reagent (Mirus, Madison, WI). Briefly, 100 nM siRNA and 4 l of the transfection reagent were both mixed with 100 l of serum-free DMEM for 5 min. The solutions were then incubated at room temperature for 20 min. This mixture was applied to the DMEM, 10% FBS medium in each well of a 12-well plate (for cholesterol efflux) or 6-well plate (for RT-PCR and Western blotting) and incubated for 36 h. J774 or CHO cells were transiently transfected with human cathepsin D vector using Lipofectamine 2000 according to the manufacturer's recommendations.
Cell Surface Biotinylation and Western Blotting-Biotinylation was performed as described earlier (36) with slight modification. J774 or CHO cells were treated with or without pepstatin for 36 h, and surface proteins were biotinylated with 500 g/ml sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Pierce) for 30 min at 4°C on a platform rotator. Cells were then washed with ice-cold quench buffer (1 M Tris-HCl (pH 7.5)) and once with ice-cold phosphate-buffered saline. The cells were swollen on ice for 10 min and then homogenized with 20 strokes in a tight fitting Dounce homogenizer. After centrifugation at 2000 ϫ g, at 4°C, for 10 min to remove unbroken cells and nuclei, the supernatant was recentrifuged at 100,000 ϫ g, at 4°C, for 60 min. The resulting supernatant was discarded, and the final membrane pellet was resuspended in 250 l of immunoprecipitation buffer; 150 g of protein was added to 30 l of streptavidin-Sepharose (Amersham Biosciences) and incubated overnight on a platform mixer at 4°C. The gel was pelleted and washed five times with immunoprecipitation buffer. The Western blotting technique was performed as described before (36). Briefly, cells were washed twice with ice-cold PBS and scraped into 0.5 ml of radioimmune precipitation buffer and homogenized with 20 strokes in a tight fitting Dounce homogenizer. After centrifugation at 2000 ϫ g, at 4°C, for 10 min to remove unbroken cells and nuclei, the protein concentration was determined by a Lowry assay. The same amounts of proteins were electrophoresed on a SDS-polyacrylamide gel and transferred to nitrocellulose at 125 V for 4 h. The nitrocellulose membranes were then blocked with 5% milk in Tris-base-buffered solution and incubated overnight with CTSD, HSP60, ABCA1, or prosaposin antibodies. Horseradish peroxidase-linked secondary antibodies (Amersham Biosciences) were used, and bands were detected by chemiluminescence (Supersignal substrate; Pierce).
Fluorescence Microscopy-Cells were seeded on 35-mm dishes with attached glass coverslips (Fisher) and transfected with ABCAI-GFP. The coverslips were then mounted in a temperature-controlled chamber (37°C) in regular growth medium supplemented with 20 mM HEPES (pH 7.4) and visualized with an Olympus ϫ100 oil immersion objective, numerical aperture 1.4, on an Olympus IX80 laser-scanning confocal microscope operated by FV1000 software version 1.4a. The GFP was excited with a 515-nm laser, and the BODIPY-lactosylceramide was excited with the 488-nm line of a multiple-line argon laser, and the Lysotracker red (DND-99; Molecular Probes) was excited with the 543-nm line of a helium/neon green laser. Alternatively, for analysis of subcellular distribution of cholesterol, cells were seeded on glass coverslips similar to those above and incubated with regular growth medium with 10% FBS. 24 h later, cells were washed two times quickly with PBS and incubated with 200 l of 3.3% paraformaldehyde for 10 min. The paraformaldehyde was removed, and the cells were rinsed once with PBS and then quenched for 30 min with 50 mM NH 4 Cl. The coverslips were then visualized with an Olympus ϫ100 oil immersion objective, numerical aperture 1.35, on an Olympus 1X70 inverted microscope operated by TILLvisION software version 4.0 (TILL Photonics, Heidelberg, Germany). The protocol for recording images documenting filipin staining consisted of exciting the fluorophore with a Polychrome IV monochronometer at 380 nm with 1000-ms exposure time, utilizing a Fura filter cube (Chroma). Images were merged as RGB files using the TILLvisION software. The images were exported from the microscope software and processed with Adobe Pho- Statistical Analysis-The Resolver error model was used to build expression intensities. To determine significant differentially expressed genes within the groups, the TIGR MeV hierarchical cluster software (gift from the Canadian Bioinformatics workshop) was used for both genes and subjects. A p value of Ͻ0.05 was considered significant. The similarity metrics were based on Pearson correlation.
For TaqMan analysis, copy numbers of a given mRNA detected in each sample were calculated after normalization to the average log of the expression of three housekeeping genes (cyclophilin, glyceraldehyde-3-phosphate dehydrogenase, and B-actin). A one-way analysis of variance test, followed by Dunnett's test for multiple comparisons, was run separately for each gene to determine significant differences between groups.

RESULTS
Cathepsin D Gene Expression Is Decreased in Monocyte-derived Macrophages of Low HDL Subjects-Subjects were divided into four age-and sex-matched groups based on the presence or absence of low HDL-C, cholesterol efflux defect, and functional mutations in ABCA1 as follows: group A, low HDL, low efflux, no ABCA1 mutation; group B, low HDL, low efflux, ABCA1 mutation; group C, low HDL, normal efflux; group D, normal HDL, normal efflux. The control subjects (group D) had normal plasma levels of triglycerides and LDL cholesterol, similar to low HDL subjects and, by definition, higher HDL-C and hence higher total cholesterol concentrations. The three low HDL groups demonstrated very similar lipid values, and all groups were closely matched for age, sex, and body mass index (data not shown). 4 MDM from low HDL and control subjects were loaded with AcLDL; mRNA was isolated from the cells of each subject and analyzed using duplicate Affymetrix U133A oligonucleotide arrays (see "Experimental Procedures"). Comparative gene expression analysis revealed the differential regulation of several pathways reported elsewhere. 4 In particular, a cluster of cathepsin genes, which are major aspartic proteases of endolysosomes, was found to be down-regulated in MDM of low HDL-C groups compared with control ( Fig. 1A; red represents higher relative expression, and green represents lower relative expression for each gene comparing all of the subjects for that gene). CTSD was significantly decreased in efflux-defective subjects compared with control (p Ͻ 0.01) (Fig. 1B). These differences were confirmed by real time PCR (group A, p Ͻ 0.01; group B, p Ͻ 0.016), and both microarray results and real time PCR values were also recapitulated in a second cohort for group A (RT-PCR series 2, p Ͻ 0.05) but not for group B, for which no additional subject was available. The decrease in CTSD observed in the low HDL group with normal efflux (group C) was not significant in either microarray series 1 (Fig.  1, A and B) or series 2 or in RT-PCR assays. Similarly, ABCA1 expression was not significantly changed in groups A and C (data not shown). These observations led to the hypothesis that reduced expression of CTSD may play a role in plasma HDL levels.
CTSD Activity Modulates ABCA1-mediated Lipid Efflux-CTSD, an aspartic protease involved in the proteolytic degradation of proteins in endosome-lysosome compartments, binds ceramide with high affinity (37). The interaction of ceramide with CTSD results in autocatalytic proteolysis of the 52-kDa prepro-CTSD to form the enzymatically active 48/32-kDa isoforms of CTSD (37). Pepstatin is also a potent inhibitor of CTSD, which binds CTSD and inhibits its autocatalytic activity and generates less active forms of CTSD (38) (supplemental Fig. 1).
To investigate the effect of CTSD modulators on lipid efflux, J774 macrophages were radioactively labeled by incubation with LDL (100 g/ml) equilibrated with [   treated with ceramide, an activator of CTSD (37), or pepstatin A, a specific high affinity inhibitor of CTSD (38), for 24 h in DMEM containing 0.2% BSA, prior to the efflux assay. Ceramide increased the apoA-I-mediated efflux of cholesterol ( Fig. 2A) and phospholipid (Fig. 2B) in a dose-dependent manner by more than 80%. In contrast, inhibition of CTSD by pepstatin A resulted in a dose-dependent decrease in the efflux of both cholesterol (Fig. 2C) and phospholipid (Fig. 2D). Treatment with pepstatin A also impaired cholesteryl ester mobilization (Fig. 2E) and slightly decreased cellular free cholesterol (Fig. 2F). These experiments clearly show that the modulation of CTSD activity affects apoA-I-mediated efflux of cholesterol and phospholipid.
We next investigated whether the impaired apoA-I-mediated efflux by pepstatin A was associated with decreased ABCA1 protein. These studies demonstrated that treatment with pepstatin A (10 g/ml) significantly reduced ABCA1 protein in J774 macrophages (Fig. 3A) and to an even greater extent in CHO cells (Fig. 3B), where it was also accompanied by decreased cholesterol efflux (Fig. 3C). Concurrently, cell surface ABCA1 was also reduced upon treatment with pepstatin A (data not shown). These results are consistent with the hypothesis that suppression of apoA-I-mediated lipid efflux by pepstatin A is the result of decreased cellular ABCA1 protein. J774 macrophages were treated with or without a stimulator of cholesterol efflux, cAMP, for 24 h in the presence or absence of pepstatin A, and cholesterol and phospholipid effluxes were determined. In both conditions, pepstatin A significantly decreased apoA-I-dependent efflux of cholesterol (Fig. 4A) and phospholipid (Fig. 4B) in J774 cells. Pepstatin A similarly reduced cholesterol efflux from bone marrow-derived-macrophages (Fig. 4C) and in LDL or Ac-LDL treated peritoneal macrophages (Fig. 4D). These results indicate that CTSD promotes lipid efflux through the ABCA1-dependent pathway in primary macrophages and in immortalized J774 cells. Determination of cholesterol efflux and ABCA1 protein level in J774 cells at three doses of pepstatin demonstrated a highly significant correlation of the two parameters (data not shown).

Modulation of the Expression Level of CTSD Regulates ApoA-Imediated Lipid
Efflux-For a more direct assay of the effect of CTSD functional deficiency on apoA-Imediated lipid efflux, J774 cells were treated with siRNA CTSD or dsRNA CTSD for 36 h. The addition of CTSD-specific siRNA significantly reduced cholesterol efflux in a dose-dependent manner (Fig. 5A) and phospholipid efflux (Fig. 5B). At 100 nM, CTSD siRNA decreased cholesterol efflux by ϳ80% and phospholipid efflux by about 50%. In contrast, the scrambled dsRNA had no effect on these parameters.
Western blot analysis of CTSD and ABCA1 after siRNA treatment demonstrated decreased expression of CTSD with siRNA but not dsRNA (Fig. 5D) and parallel changes in ABCA1 protein mass (Fig. 5C). These results further support the notion that CTSD regulates ABCA1 levels and cholesterol efflux.
To explore the potential role of CTSD overexpression on lipid efflux and ABCA1 protein, CHO or J774 cells were transfected with a human full-length CTSD plasmid for 24 h, lipid efflux was assayed, and ABCA1 protein level was determined. Consistent with the siRNA results, transfection of human CTSD in CHO cells increased apoA-I-mediated lipid efflux (Fig. 6, A and B) and the cellular level of ABCA1 protein (Fig.  6C). Having shown that CTSD increases both ABCA1 protein and cholesterol efflux, we next determined whether CTSD exerted its effect directly on ABCA1 gene transcription. Macrophages were treated with either pepstatin A or by overexpression of human CTSD for 24 h, and ABCA1 mRNA levels were measured by RT-PCR. As expected, ABCA1 expression was significantly increased (Ͼ50%) by CTSD overexpression and was significantly decreased (Ͻ50%) by pepstatin treatment (Fig. 6D). Overall, these findings demonstrate that CTSD induces cholesterol efflux at least in part by regulating ABCA1 transcription. Because a small proportion of CTSD is secreted (25), we evaluated the possibility that overexpressed CTSD present in the medium could degrade the acceptors for cholesterol efflux. After efflux assay, medium apoA-I was immunoprecipitated and analyzed by Western blot, but there was no evidence of apoA-I fragmentation (data not shown). This may reflect the fact that CTSD is mostly active at low pH.
CTSD Is Involved in the Maturation of Saposins-Prosaposin (pro-SAP) is a 73-kDa precursor of four protein cofactors, saposins A, B, C, and D, which are required for the enzymatic hydrolysis of glycosphingolipids by specific lysosomal hydrolases (33,34). The proteolytic degradation of pro-SAP and the maturation to different saposins is dependent on CTSD (39). To examine whether CTSD regulates the proteolysis of pro-SAP in our model, we transiently overexpressed CTSD in CHO cells and quantified the expression of CTSD, pro-SAP, and the control protein HSP60 by Western blotting. As shown in Fig. 7, prosaposin was hydrolyzed into proteolytic products of ϳ10 kDa upon overexpression of human CTSD. Interestingly, we also found that overexpression of CTSD increased the level of both pro-SAP and saposins, and these increases were proportional to the level of CTSD (Fig. 7, top). These data verify that CTSD functions in the proteolytic processing of prosaposin. Interestingly, prosaposin mRNA expression was found to be decreased in the microarrays for series 1 and 2 for low HDL subjects compared with control subjects (p Ͻ 0.001 and p Ͻ 0.03, respectively).

Inhibition of CTSD Causes the Intracellular
Accumulation of Cholesterol and Sphingolipids-Since saposins are sphingolipid-binding proteins involved in the degradation of glycosphingolipids, we hypothesized that dysregulation of the expression of pro-SAP or its co-activator CTSD could impair glycosphingolipid (GSL) metabolism and cholesterol traffic. Indeed, the cellular transport and storage of cholesterol is linked to GSL metabolism (40 -43), and the accumulation of GSL in human macrophage foam cells and fibroblasts causes the trapping of cholesterol in endosome/lysosome compartments (41,43). Thus, in CHO cells, we assessed the effect of pepstatin treatment on the cellular localization of GSL using BODIPY-lactosylceramide and free cholesterol accumulation using filipin staining. We demonstrated perinuclear fluorescence of BODIPY-lactosylceramide (BODIPY Lac-cer) in control cells (Fig. 8A), whereas, in cells treated with pepstatin A, there was predominantly bright punctate endosome/lysosome fluorescence (Fig. 8B) similar to the cellular phenotype of Niemann-Pick type C disease (NPC) (44). Extensive studies have also shown the accumulation of GSL and cholesterol in late endosomes/lysosomes in NPC cells (45). To test whether the addition of exogenous ceramide would also enhance cholesterol efflux in NPC1-deficient cells, we isolated bone marrow-derived macrophages from control and NPC1-deficient mice and measured apoA-I-mediated cholesterol efflux. The addition of ceramide (20 M) or cAMP increased cholesterol efflux in control cells (2-fold) but not in the NPC1-deficient cells (Fig.  8C). As expected, pepstatin also tended to cause enlarged punctate fluorescence of filipin-stained free cholesterol in endosome/lysosome-like structures, whereas in control cells cholesterol distribution was diffuse throughout the cell (data not shown). Taken together, these results demonstrate that inactivation of CTSD function results in the accumulation of GSL and free cholesterol in late endosomes/lysosomes.
Inhibition of CTSD Causes the Mistargeting of ABCA1 Protein to Late Endosomes/Lysosomes-ABCA1 is normally localized at the cell surface and in the late endosomal com-  DECEMBER 29, 2006 • VOLUME 281 • NUMBER 52 partment (46). To test whether inhibition of CTSD by pepstatin affected ABCA1 localization, we followed the distribution of ABCA1 in CHO cells by confocal microscopy using GFP-ABCA1 and Lysotracker red as a lysosomal marker. In these experiments, cycloheximide treatment was omitted to avoid interference with the degradation of lysosomal enzymes. Under these conditions, we observed no co-localization of ABCA1 with Lysotracker red (Fig. 9A) in control cells, but in cells treated with pepstatin, ABCA1 was found to colocalize extensively with lysosomes, as indicated by the yellow punctate fluorescence (Fig. 9B). These results show that inhibition of CTSD activity causes trapping of ABCA1 in a lysosomal compartment, impairing its trafficking to the plasma membrane.

DISCUSSION
These studies were designed to identify and evaluate novel candidate genes relevant to HDL metabolism and the etiology of low HDL syndromes. By focusing on genes differentially expressed in a group of subjects with low HDL, we identified a cluster of cathepsin genes. Notably, mRNA expression of CTSD, the major aspartic protease of endolysosomes (25), was decreased in the low HDL groups as compared with control subjects and significantly in groups with low efflux (p Ͻ 0.01) (Fig. 1). These results were confirmed in a second cohort that included a group with low HDL and efflux defect but no mutation in ABCA1, whereas the group with low HDL and normal efflux did not present with decreased CTSD. Although the decreased CTSD could not be verified in a second group with low HDL and efflux with ABCA1 mutations (for lack of subjects), the initial observation (Fig. 1B) suggests the possibility of an independent decrease in CTSD that compounds the ABCA1 defect. Alternatively, the decreased ABCA1 activity may decrease CTSD through a feedback loop.
Based on these observations, we hypothesized that CTSD activity might affect cellular cholesterol transport or efflux. CTSD binding of ceramide affects its autocatalytic activity (37), and in cells with acid sphingomyelinase deficiency, low endogenous ceramide (the product of the digestion of sphingomyelin) is associated with decreased CTSD activity (37). We showed that exogenous ceramide increases cholesterol and phospholipid efflux by increasing the autocatalytic activity of CTSD (supplemental Fig. 1). In agreement with our results, Witting et al. (47) have shown that the addition of ceramide increases cholesterol efflux and cell surface ABCA1. Alternatively, ceramide may also affect the cholesterol efflux through the feedback activation of acid sphingomyelinase (48).
CTSD has been shown to bind and stimulate the proteolytic processing of proSAP (39). The proSAP knock-out mice (49) and tissues from individuals with proSAP deficiency (50,51) are characterized by GSL accumulation (40 -43). Recently, our laboratory showed that the inhibition of prosaposin delivery to late endosomes by LRP1 and its adaptor, GULP, causes the accumulation of GSL and free cholesterol in late endosomes, resulting in down-regulation of ABCA1 and decreased cholesterol efflux (52). Consistent with these observations, we demonstrate that the inhibition of CTSD is also accompanied by the accumula-  tion of both BODIPY-lactosylceramide and free cholesterol in structures resembling endosome/lysosome compartments reminiscent of the cellular phenotype of NPC disease (44).
The identification of CTSD as an activator of pro-SAP has provided important insight into the mechanism by which CTSD modulates the metabolism of cellular cholesterol and GSL. Pro-SAP is the precursor of four protein cofactors, saposins A, B, C, and D, that are required for the enzymatic hydrolysis of sphingolipids by specific lysosomal hydrolases. Saposins have important functions in the degradation of ceramide (saposin D), glucocerebroside (saposin C), sulfatide (saposin B), and glycosylceramide (saposin C) (53). Recently, saposin B was also shown to bind and transfer phospholipids from one membrane to another (54), a process that can occur in lysosome/late endosome compartment and is specific for either phosphatidylcholine or phosphatidylinositol. Here, we show that overexpression of CTSD increases cellular protein levels of both pro-SAP and saposins, which control sphingolipid transport and the cholesterol efflux pathway. Sphingolipidoses are lysosomal storage diseases. One of the most common, Gaucher's disease, is caused by deficiency of glucocerebrosidase, a lysosomal acid ␤-glucosidase, which results in the accumulation of glucosylceramide in lysosomes. Notably, affected subjects have decreased plasma concentrations of HDL cholesterol and apoA-I (55,56). Niemann-Pick, type A or B, is caused by altered function of acid sphingomyelinase, which metabolizes the sphingomyelin to ceramide. The saposins A, C, and D are responsible for the hydrolytic activation of this pathway (57,58). Individuals with this disease also exhibit low HDL-cholesterol levels (59), as do patients with NPC disease (60). Furthermore, it has been reported that macrophages from acid sphingomyelinase knock-out mice accumulate sphingomyelin and exhibit defective cholesterol trafficking and efflux (61). Fibroblasts treated with lactosylceramide or with an inhibitor of glucocerebrosidase also accumulate glycosphingolipids, which results in inhibition of cholesterol efflux by the apoA-I/ ABCA1 pathway (62). Thus, disruption of one of the pathways FIGURE 9. Pepstatin causes mistargeting of ABCA1 protein that colocalizes with late endosomes/lysosomes. CHO cells were grown in 10% FBS and transfected with ABCA1-GFP for 24 h in the absence or presence of 10 g/ml pepstatin (panels A and B, respectively). The cells were then incubated with Lysotracker red, a lysosomal marker, for 10 min at room temperature and visualized by confocal microscopy. Confocal images of lysosomes are displayed in red, and GFP-ABCA1 is shown in green. In the presence of pepstatin ABCA1 colocalizes with Lysotracker in a lysosomal compartment. of sphingolipid metabolism can cause GSL and free cholesterol accumulation and alter cellular lipid homeostasis and HDL concentrations (Fig. 10). In this study, the low HDL subjects with efflux defects have low CTSD expression, suggesting a correlation between CTSD expression and HDL levels. This low HDL phenotype could also be additive with decreased expression of prosaposin (50). ABCA1 expression levels are not significantly decreased in group A (data not shown), suggesting that decreased CTSD may also affect efflux by dysregulation of ABCA1 trafficking rather than expression.
The expression and regulation of cathepsins are complex. They can act as transcriptional activators (63), and there can be cross-talks between cathepsins, where for example cathepsin L is involved in CTSD processing and regulation (64). The CTSD gene is controlled by a mixed promoter that contains features of both housekeeping and regulated genes (65)(66)(67)(68). It is regulated by estrogens (66,69) and by retinoids (65,70). Here, a decrease in CTSD results in decreases in saposins, which translate in glycosphingolipid accumulation in late endosomes that in turn blocks cholesterol transport, preventing regulation of cellular cholesterol and decreasing the efflux pathway. Thus, the regulation of CTSD in monocyte-macrophages by retinoic acid (65) is compatible with the general contribution of liver X receptors and retinoid X receptors to the control of cholesterol homeostasis and inflammation (71).
In conclusion, these studies show for the first time that CTSD regulates intracellular transport of phospholipid and cholesterol and ABCA1-mediated lipid efflux. The addition of pepstatin A, an inhibitor of CTSD, impaired apoA-I-mediated efflux in CHO cells and in macrophage cell lines. Specific siRNA oligonucleotides had a similar effect, causing a marked reduction in cholesterol and phospholipid efflux. Conversely, transfection of murine macrophages or CHO cells with a CTSD cDNA expression plasmid led to a moderate but significant increase in apoA-I-mediated lipid efflux. In addition, both mRNA and protein expression of ABCA1 was induced by CTSD and inhibited by pepstatin A. Taken together, these findings suggest that decreased CTSD expression can contribute to low HDL. These findings are relevant to our understanding of the regulation by lysosomal enzymes of intracellular cholesterol trafficking and efflux.


text:
A C-terminal Lysine That Controls Human P2X4 Receptor Desensitization*

Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X4 receptors exhibit intermediate desensitization when compared with P2X1 and P2X3 (much faster) and P2X2 and P2X7 (slower). We recorded membrane currents in HEK293 cells transfected to express the human P2X4 receptor. The decline in current during a 4-s application of ATP (100 μm) was about 30%; this was not different during whole-cell or perforated patch recording. Alanine-scanning mutagenesis of the intracellular C terminus identified two positions with much accelerated desensitization kinetics (Lys373: 92% and Tyr374: 74%). At position 373, substitution of Arg or Cys also strongly accelerated desensitization: however, in the case of K373C the wild-type phenotype was fully restored by adding ethylammonium methanethiosulfonate. At position 374, phenylalanine could replace tyrosine. These results indicate that wild-type desensitization properties requires an aromatic moiety at position 374 and an amino rather than a guanidino group at position 373. These residues lie between previously identified motifs involved in membrane trafficking (YXXXK and YXXGL) and implicates the C-terminal also in rearrangements leading to channel closing during the presence of agonist.

Receptor desensitization can determine the time course of transmitter action and profoundly alter sensitivity to drugs. Among P2X receptors, ion currents through homomeric P2X 4 receptors exhibit intermediate desensitization when compared with P2X 1 and P2X 3 (much faster) and P2X 2 and P2X 7 (slower). We recorded membrane currents in HEK293 cells transfected to express the human P2X 4 receptor. The decline in current during a 4-s application of ATP (100 M) was about 30%; this was not different during whole-cell or perforated patch recording. Alanine-scanning mutagenesis of the intracellular C terminus identified two positions with much accelerated desensitization kinetics (Lys 373 : 92% and Tyr 374 : 74%). At position 373, substitution of Arg or Cys also strongly accelerated desensitization: however, in the case of K373C the wild-type phenotype was fully restored by adding ethylammonium methanethiosulfonate. At position 374, phenylalanine could replace tyrosine. These results indicate that wild-type desensitization properties requires an aromatic moiety at position 374 and an amino rather than a guanidino group at position 373. These residues lie between previously identified motifs involved in membrane trafficking (YXXXK and YXXGL) and implicates the C-terminal also in rearrangements leading to channel closing during the presence of agonist.
P2X receptors belong to a family of ATP-gated ion channels, seven subunits of which have been currently identified and cloned (P2X 1-7 ) (1). Functional P2X receptors probably form as trimers, incorporating the same or different subunits (2,3). The P2X subunits are considered to have two hydrophobic transmembrane domains with intracellular N and C termini and a large extracellular ectodomain containing conserved cysteines (1); the ectodomain also contains residues that appear to contribute to the binding site for ATP (4,5).
In the central nervous system, P2X receptors have been implicated in mediating ATP-dependent fast excitatory neurotransmission (6 -9). The P2X 4 subunit is highly abundant in the central nervous system, with expression in both spinal cord and brain, including dentate gyrus, CA1/ CA3 pyramidal, and cerebellar Purkinje cells (10 -12); they are particularly localized to glutamatergic synapses (12). Upon activation, P2X 4 can regulate cellular Ca 2ϩ levels via direct permeation and by the activation of voltage-dependent Ca 2ϩ channels. P2X receptor activity has therefore been proposed to be important in synaptic plasticity (13,14).
The time course of the effect of ATP at P2X receptors is strongly influenced by receptor desensitization, a feature common to most other ligand-gated ion channels. Desensitization manifests itself as a decline in current amplitude during agonist occupancy of the receptor, and in the case of P2X 4 , it can be modified pharmacologically (15). Recombinant P2X receptors display varying degrees of desensitization; membrane current recordings show that P2X 1 and P2X 3 undergo fast desensitization (tens of milliseconds), P2X 4 exhibits moderate desensitization (several seconds), and P2X 2 , P2X 5 , and P2X 7 show less desensitization (1,16,17). For various P2X receptors, desensitization can be regulated by cellular signaling events, which further suggests its importance in P2X receptor physiology. Several studies have demonstrated regulation of P2X desensitization by protein kinase activity, either via direct N-terminal phosphorylation (at a highly conserved protein kinase C site) or via phosphorylation of an associated protein (18,19). P2X 3 desensitization can be decreased by the calcineurin-mediated dephosphorylation of the N terminus (20).
Experiments using chimeric constructs of P2X 1 and P2X 2 subunits identified desensitization critical domains as the transmembrane segments and the intracellular juxtamembrane regions (up to 15 amino acids in length). Other domains within the C terminus of P2X 2 have been identified by functional analysis of splice variants (21,22). P2X receptors have very different C-terminals, and the regions important for P2X 4 subunits are not known. Given the likely importance of the P2X 4 receptor in central synaptic physiology, it seemed useful to understand further the molecular mechanisms that may contribute to P2X 4 desensitization.

EXPERIMENTAL PROCEDURES
Human P2X 4 subunit with a C-terminal EMYPME epitope tag subcloned into pcDNA3.1(ϩ) was used as the template for all mutagenesis reactions. Mutagenesis was achieved using Pfu turbo polymerase and QuikChange methodology (Stratagene). Truncated receptors were generated by the introduction of a premature stop codon by mutagenesis. HEK293 cells were grown in 10% fetal calf serum in Dulbecco's minimal essential medium at 37°C with 5% CO 2 in a humidified incubator. Cells were transiently transfected with plasmids encoding wild-type or mutant P2X receptor (1 g) and enhanced green fluorescent protein (0.1 g) using Lipofectamine 2000 (Invitrogen).
Whole-cell recordings were made from HEK293 cells at room temperature, 24 -48 h after transfection. The extracellular solution contained (mM): 145 NaCl, 2 KCl, 2 CaCl 2 , 1 MgCl 2 , 13 D-glucose, and 10 HEPES. The intracellular pipette solution contained (mM): 145 NaCl, 10 HEPES, and 10 EGTA. Solutions had pH 7.3 after titration with 5 M NaOH. Chemicals were purchased from Sigma (Poole, UK). Pipettes had resistances of 3-5 M⍀. For perforated patch-clamp, perforation was achieved by the addition of 120 g/ml amphotericin in the patch pipette solution. Perforation typically occurred 5-8 min after gigaseal formation. ATP was applied using an RSC 200 system (Biological Science Instruments, Grenoble, France). Ethyl ammonium methane thiosulfonate (MTSEA 2 : Toronto Research Chemicals) was prepared as a concentrated stock, stored at Ϫ20°C, and diluted in extracellular solution immediately prior to application. MTSEA was applied by superfusion at a rate of ϳ2 ml/min. For immunohistochemistry, transfected cells were fixed with 4% paraformaldehyde for 5 min followed by permeabilization with 0.1% Triton X-100. Cells were blocked for 30 min (phosphate-buffered saline, 0.5% bovine serum albumin) and incubated with primary antibody (rabbit anti-EE, 1:500; Bethyl Laboratories) in blocking solution for 1-2 h at room temperature. After washing, cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody (goat anti-rabbit, 1:200; Jackson ImmunoResearch) for 1-2 h at room temperature.
Numerical data are expressed as mean Ϯ S.E., and differences were tested by Student's t test and analysis of variance.

RESULTS
Decline in Response with Repeated ATP Application-We distinguish two forms of desensitization at the human P2X 4 receptor. The first is the decline in the peak current with repeated brief applications of ATP. Thus, in whole-cell recording, the peak current amplitude progressively declined when ATP (100 M, 2 s) was applied at 1-min intervals (Fig.  1a). This decline was well fit with a single exponential ( ϭ 93 Ϯ 6.2 s, n ϭ 25 cells), reaching a plateau level at ϳ20% of the initial peak amplitude. We observed no recovery from desensitization with repeated application when the application interval was increased to 5, 10, or 20 min (data not shown). P2X 4 currents recorded using the perforated patch configuration (amphotericin 120 g/ml) did not show decline with repeated applications ( Fig. 1, a and b). However, we were unable to influence the decline in response by a range of other perturbations. Ivermectin (3 M, 3 min) did not alter the kinetics of current decline ( ϭ 98 Ϯ 5.4 s; n ϭ 8, p Ͼ 0.05) (Fig. 1a), although it increased peak current amplitudes ϳ3-fold. The rate of current decline was unchanged in external solutions containing 0 mM calcium and also not different when the recording pipette contained ATP (1 mM), GTP (1 mM), staurosporine (100 nM), genistein (100 M), phosphatidylinositol 4,5-bisphosphate (50 M), or U73122 (10 M). P2X 4 ⌬Y378, which is truncated so as to lack the endocytic motif YEQGL (Fig. 2), showed properties not different from wild type receptors ( ϭ 91 Ϯ 5.2 s, n ϭ 12 cells, p Ͼ 0.05).
Decline in Response during ATP Application-The second form of desensitization is the decline in the membrane current during the continued application of ATP, and this is the subject of the remainder of this paper. Currents evoked by ATP (100 M) rose to their peak amplitude in 410 Ϯ 8.2 ms, and the average peak amplitude was 151 Ϯ 30 pA (n ϭ 25 cells). During a 5-s application of ATP (100 M), wild-type currents declined to a value that was 32 Ϯ 7.9% (n ϭ 25 cells) of their initial peak amplitude. These values were obtained in the whole-cell configuration, but the decline in current during the application was not different when recorded using the perforated patch configuration (27 Ϯ 5.2%, n ϭ 12 cells, p Ͼ 0.05) (Fig. 1c). However, the decline during the application was  it was also independent of holding potential (Ϫ60 mV versus ϩ60 mV).
Truncations and Alanine Scanning in the C Terminus-The C terminus of the human P2X 4 receptor extends from residue Leu 358 , at the end of the second transmembrane domain, to Gln 388 . A truncation that deleted the final eleven amino acids (⌬Y378) had properties not different from wild type P2X 4 ; it showed a similar desensitization during the application (Fig. 2) and a similar decline with repeated applications. Removal of the residues from and including Lys 373 or Lys 362 resulted in receptors that were non-functional (Fig. 2b).
These results indicate that maintained channel opening in the presence of ATP requires Lys 373 (or the equivalent side chain -S-S-(CH 2 ) 2 -NH 3 ϩ ). We asked therefore in how many subunits of a homotrimeric receptor was the lysine required? We co-expressed wild-type and P2X 4 [K373A] receptors in nominally equal amounts. Whole-cell recordings showed typical ATP-activated currents, but the decline during the application was intermediate between that seen for cells expressing either wild-type or P2X 4 [K373A] receptors alone (Fig. 4c). In fact, the current declined by 58 Ϯ 8.4% over 4 s (n ϭ 12 cells) during a 4-s application. This decline in the current was not different from that which would have been observed from an equal mixture of wild type and [K373A] substituted receptors. If one assumes that heterotrimer formation occurs readily between subunits containing alanine and those containing lysine and that only two (rather than four) phenotypes can occur, then this result indicates that a single alanine-containing subunit does not exert a dominant effect with respect to the desensitization kinetics (Fig. 4c).

DISCUSSION
Our results distinguish two modes of P2X 4 receptor desensitization. In our whole-cell experimental conditions, P2X 4 responses declined both during ATP applications lasting a few seconds and with applications repeated at 1-min intervals (Figs. 1a and 2a). With perforated patch recording to minimize dialysis of intracellular contents, the decline with repeated application ("run-downP") was completely prevented, whereas the decline during ATP applications was the same as in whole-cell recordings (Fig. 1). The prevention of P2X 4 rundown by per- forated patch recording is comparable with the work by Lewis and Evans (25), who showed that the run-down of a P2X 1 -like current in smooth muscle cells did not occur with amphotericin-permeabilized recordings. The phenomena of run-down has been observed for P2X 1 , P2X 3 , P2X 4 , and P2X 7 ; other P2X receptors give robust steady-state currents with repeated ATP applications. A likely explanation for P2X 4 run-down in whole-cell recording is that a diffusible cytosolic factor that tonically regulates the receptor is lost during intracellular dialysis. The identity of such a factor is not obvious, but from our experiments it seems not to be ATP, GTP, or phosphatidylinositol 4,5-bisphosphate. The mechanism underlying the decline in P2X 4 response with repeated ATP application is also not likely to involve AP2-mediated receptor internalization. This is because the truncation P2X 4 [⌬ Y378], and the point mutations Y378A and L382A, each of which lack the AP2-dependent endocytic motif YXXG⌽ responsible for receptor internalization in neurons (27), exhibited a decline in response with repeated ATP application that was not different from wild type receptors. These truncated receptors would presumably use the YXXV motif (beginning at Tyr 372 ) for their normal trafficking (24).
Ivermectin increases the currents evoked by ATP at P2X 4 receptors (26). A recent careful study by Priel and Silberberg (15) distinguished two effects: the first is an increase in the maximum current that is seen at concentration around 0.3 M, and the second is a slowing of deacti-vation that occurs at 10-fold higher concentrations. Both these effects were observed in our experiments, using 3 M ivermectin. However, in ivermectin there was no change in the decline of the peak currents observed with repeated application of ATP. Parenthetically, we also noted that the increase in current by ivermectin was observed in the truncated P2X 4 receptor P2X 4 [⌬Y378].
The C terminus of P2X 4 is short (30 residues) relative to that of other P2X receptors: 62% of these residues are either charged or aromatic (tyrosine) (Fig. 2a). By alanine scanning we identified two residues (Lys 373 and Tyr 374 ) as being important for the time course of desensitization during the ATP application. Both K373A and Y374A mutations resulted in receptors with much accelerated decline in response during ATP application as compared with wild-type receptor currents (Fig. 3a). These residues lie between two other functionally important regions for P2X receptors. These are the YXXXK motif that is found in all P2X family members (Tyr 367 to Lys 371 , human P2X 4 ) and the YEQGL sequence (Tyr 378 to Leu 382 ) found uniquely in P2X 4 subunits. The first of these contributes to receptor stabilization at the plasma membrane (23). The second has been shown to bind directly to the 2 subunit of the adaptor protein AP-2, where it occupies a site comparable to that seen for the more common endocytic motif YXX⌽ (24). This binding is key to clathrin-mediated receptor internalization undergone by P2X 4 receptors (27). These two residues that we have identified form the heart of the YKYV motif that is not normally used for clathrin-mediated endocytosis of P2X4 receptors (24,27).
We asked how critical was the requirement for tyrosine at position 374 and found that P2X 4 [Y374F] functioned essentially as the wild-type receptor with respect to the decline in current during the ATP application (Fig. 3a). This indicates that the requirement for aromaticity at this position for the channel to remain open during agonist occupancy. It further indicates that Tyr 374 is unlikely to be phosphorylated: for example, previous work (28) on P2X 7 receptors has indicated that repeated activation leads to receptor dephosphorylation and that this underlies the decline in the responses in that case.
On the other hand, arginine could not substitute for lysine at position 373 (Fig. 3a). This suggests that the critical moiety is not simply the positive charge but other features of the lysine side chain (such as the hydrocarbon chain). We tested this by expressing the P2X 4 [K373C] receptor and using MTSEA to add a side chain of similar length (-S-S-CH 2 -CH 2 -NH 3 ϩ ) to that of lysine (-CH 2 -CH 2 -CH 2 -CH 2 -NH 3 ϩ ).
This completely restored the wild-type phenotype. Control experiments showing that MTSEA did not modify the wild-type channel or the fast desensitizing mutant P2X 4 [K373A], and that the effect of MTSEA was prevented by intracellular cysteine (29 -31), strongly support the interpretation that MTSEA is acting by modification of the cysteine side chain at position 373. This indicates that lysine rather than arginine is required for structural reasons (e.g. the arginine side chain -CH 2 -CH 2 -CH 2 -NH-C(NH 2 ) ϭ NH 2 ϩ is too long to be accommodated) or perhaps that the lysine is subject to posttranslational modification. For example, post-translational modification of lysine has been shown to be important for the function of other ion channels (32). However, there are no consensus motifs for lysine modification apparent at position 373 and the relatively fast time course (5-10 min: Fig. 4b) with which MTSEA restored the wild type phenotype effect also argues against such an interpretation. The simplest explanation of why K373A mutation accelerates the decline in response during ATP application is that the critical positioning of this positive charge is required for channel opening to be maintained when ATP is bound. This implies that gating transitions involve not only conformational changes in the ectodomain and transmembrane hydrophobic regions but also the first part of the C terminus (33)(34)(35)(36). It is difficult to make further mechanistic interpretation, given that the residue at this position is lysine in one other P2X subunit (P2X 1 , which shows rapid desensitization) and negatively charged (Glu or Asp) in all others. In a previous study by Koshimizu et al. (22), the region 376 -381 (EDYEQG) was identified as being important in controlling P2X 4 desensitization during sustained applications of ATP. They measured the decline in intracellular calcium concentration over periods of several hundred seconds, so it is difficult to interpret those results with respect to the present studies in which ionic current was measured over a period of 5 s. Under our experimental conditions, these residues do not participate in receptor desensitization, as determined by alanine substitution or truncation. Previous work by Royle et al. (27) has shown that this region contains the first four amino acids of the motif (YEQGL) that interacts with the 2 subunit of the adaptor protein AP2 and is thus involved in receptor internalization. In general terms, this would be consistent with our interpretation that the decline in the current during the time frame of a few seconds that we have studied is unrelated to receptor internalization.
P2X receptors are generally considered to operate as trimers (1). Our final question was whether the rapid desensitization was observed when only one of the three subunits carried alanine at position 373 (i.e. P2X 4 [K373A]⅐P2X 4 ⅐P2X 4 ) (Fig. 4); in other words, did this mutation confer a dominant phenotype. The result of the co-expression of wild-type and mutant subunits in equal amounts (Fig. 4c) strongly indicates that accelerated desensitization is seen only in channels with two or more subunits carrying the alanine (i.e. P2X 4 [K373A]⅐P2X 4 [K373A]⅐P2X 4 ). This would imply that normal channel function can occur even though one of the three subunits is mutated to alanine at this position. We cannot exclude the alternative explanation for the results of the co-expression, which is that the lysine to alanine mutation prevents the formation of heteromeric channels, and that the currents observed flow through approximately equal parts of the homomeric wild type P2X 4 and mutant P2X 4 [K373A] channels. This seems unlikely given that the P2X 4 [K373A] has properties similar in many respects to wild type channels, which shows that alanines in this position (at least with three of them) do not themselves prevent formation of functional homomers.
In summary, we have identified Lys 373 and Tyr 374 in the C terminus of  the P2X 4 receptor as being key determinants of P2X 4 receptor desensitization on the time scale of seconds. This implicates the juxtamembrane C terminus in playing an important role in determining the duration of the physiological action of ATP at the P2X 4 receptors.