Lymphadenopathy is observed in knockin mouse lacking the SHP-2 MAPK pathway.

Differential regulation of MAP kinase activation by a novel splice variant of human MAP kinase phosphatase-2.

MAP kinase phosphatase-2 (MKP-2) is a member of the family of dual specificity phosphatases that functions to inactivate the ERK and JNK MAP kinase signalling pathways. Here, we identify a novel human MKP-2 variant (MKP-2-S) lacking the MAP kinase binding site but retaining the phosphatase catalytic domain. Endogenous MKP-2-S transcripts and proteins were found in PC3 prostate and MDA-MB-231 breast cancer cells and also human prostate biopsies. Cellular transfection of MKP-2-S gave rise to a nuclear protein of 33kDa which displayed phosphatase activity comparable to the formerly described long form of MKP-2 (MKP-2-L). Due to its lack of a kinase interacting motif (KIM), MKP-2-S did not bind to JNK or ERK; MKP-2-L bound ERK and to a lesser extent JNK. Protein turnover of adenoviral expressed MKP-2-S was accelerated relative to MKP-2-L, with a greater susceptibility to proteosomal-mediated degradation. MKP-2-S retained its ability to deactivate JNK in a similar manner as MKP-2-L and was an effective inhibitor of LPS-stimulated COX-2 induction. However, unlike MKP-2-L, MKP-2-S was unable to reverse serum-induced ERK activation or significantly inhibit endothelial cell proliferation. These findings reveal the occurrence of a novel splice variant of MKP-2 which is unable to bind ERK and may be significant in the dysregulation of MAP kinase activity in certain disease states, particularly in breast and prostate cancers.

Control of CNS Cell-Fate Decisions by SHP-2 and Its Dysregulation in Noonan Syndrome

Within the developing mammalian CNS, growth factors direct multipotent precursors to generate neurons versus glia, a process that if perturbed might lead to neural dysfunction. In this regard, genetic mutations resulting in constitutive activation of the protein tyrosine phosphatase SHP-2 cause Noonan Syndrome (NS), which is associated with learning disabilities and mental retardation. Here, we demonstrate that genetic knockdown of SHP-2 in cultured cortical precursors or in the embryonic cortex inhibited basal neurogenesis and caused enhanced and precocious astrocyte formation. Conversely, expression of an NS SHP-2 mutant promoted neurogenesis and inhibited astrogenesis. Neural cell-fate decisions were similarly perturbed in a mouse knockin model that phenocopies human NS. Thus, SHP-2 instructs precursors to make neurons and not astrocytes during the neurogenic period, and perturbations in the relative ratios of these two cell types upon constitutive SHP-2 activation may contribute to the cognitive impairments in NS patients.

Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

Germline mutations in the RET tyrosine kinase gene are responsible for the development of multiple endocrine neoplasia 2A and 2B (MEN2A and MEN2B). However, knowledge of the fundamental principles that determine the mutant RET-mediated signaling remains elusive. Here, we report increased expression of mitogen-activated protein kinase phosphatase-2 (MKP-2) in carcinomas developed in transgenic mice carrying RET with the MEN2A mutation (RET-MEN2A). The expression of MKP-2 was not only induced by RET-MEN2A or RET-MEN2B mutant proteins but also by the activation of endogenous RET by its ligand, glial cell line-derived neurotrophic factor (GDNF). MKP-2 expression was also evident in the MKK-f cell line, which was established from a mammary tumor developed in a RET-MEN2A transgenic mouse. Inhibition of MKP-2 attenuated the in vitro and in vivo proliferation of MKK-f cells, which was mediated by the suppression of cyclin B1 expression. Furthermore, we found that MKP-2 is highly expressed in medullary thyroid carcinomas derived from MEN2A patients. These findings suggest that the increased expression of MKP-2 may play a crucial role in oncogenic signaling downstream of mutant RET, leading to deregulation of cell cycle.

Crypt stem cells as the cells-of-origin of intestinal cancer

Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli (APC). As in most cancers, the cell of origin has remained elusive. In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells. Here we show that deletion of Apc in these stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma. These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks. The distribution of Lgr5+ cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30weeks, large adenomas are very rare in these mice. We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.

Snail coordinately regulates downstream pathways to control multiple aspects of mammalian neural precursor development.

The Snail transcription factor plays a key role in regulating diverse developmental processes but is not thought to play a role in mammalian neural precursors. Here, we have examined radial glial precursor cells of the embryonic murine cortex and demonstrate that Snail regulates their survival, self-renewal, and differentiation into intermediate progenitors and neurons via two distinct and separable target pathways. First, Snail promotes cell survival by antagonizing a p53-dependent death pathway because coincident p53 knockdown rescues survival deficits caused by Snail knockdown. Second, we show that the cell cycle phosphatase Cdc25b is regulated by Snail in radial precursors and that Cdc25b coexpression is sufficient to rescue the decreased radial precursor proliferation and differentiation observed upon Snail knockdown. Thus, Snail acts via p53 and Cdc25b to coordinately regulate multiple aspects of mammalian embryonic neural precursor biology.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8-positive solid lymphomas: a tissue-based variant of primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8, is consistently identified in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Here we report four cases of KSHV-bearing solid lymphomas that occurred in AIDS patients (cases 1 to 3) and in a human immunodeficiency virus (HIV)-seronegative person (case 4). The patients presented extranodal masses in the abdomen (cases 1, 3, and 4) or skin (case 2), and nodal involvement, together with Kaposi's sarcoma (case 3). The gastrointestinal tract was involved in two patients (cases 1 and 3). The patients did not develop a lymphomatous effusion. KSHV was detected in the tumor cells of all cases by immunohistochemistry and by polymerase chain reaction. Epstein-Barr virus was detected in two of the HIV-related cases. All KSHV-positive solid lymphomas exhibited PEL-like cell morphology. To investigate the relationship of these disorders to PEL and to other AIDS-associated diffuse large cell lymphomas, KSHV-positive solid lymphomas were tested for the expression of a set of genes that were previously shown by gene profiling analysis to define PEL tumor cells. The results showed that expression of this set of genes in KSHV-positive lymphomas is similar to that of PEL but distinct from KSHV-negative AIDS-associated diffuse large cell lymphomas. Because pathobiological features of KSHV-positive solid lymphomas closely mimic those of PEL, our results suggest that KSHV-positive solid lymphomas should be considered as a tissue-based variant of classical PEL, irrespective of HIV status.

Integrin-linked kinase expression is elevated in human cardiac hypertrophy and induces hypertrophy in transgenic mice.

BACKGROUND Although numerous signaling pathways are known to be activated in experimental cardiac hypertrophy, the molecular basis of the hypertrophic response inherent in human heart diseases remains largely unknown. Integrin-linked kinase (ILK) is a multifunctional protein kinase that physically links beta-integrins with the actin cytoskeleton, suggesting a potential mechanoreceptor role. METHODS AND RESULTS Here, we show a marked increase in ILK protein levels in hypertrophic ventricles of patients with congenital and acquired outflow tract obstruction. This increase in ILK was associated with activation of the Rho family guanine triphosphatases, Rac1 and Cdc42, and known hypertrophic signaling kinases, including extracellular signal-related kinases (ERK1/2) and p70 S6 kinase. Transgenic mice with cardiac-specific expression of a constitutively active ILK (ILK(S343D)) or wild-type ILK (ILK(WT)) exhibited a compensated ventricular hypertrophic phenotype and displayed an activation profile of guanine triphosphatases and downstream protein kinases concordant with that seen in human hypertrophy. In contrast, transgenic mice with cardiomyocyte-restricted expression of a kinase-inactive ILK (ILK(R211A)) were unable to mount a compensatory hypertrophic response to angiotensin II in vivo. CONCLUSIONS Taken together, these results identify ILK-regulated signaling as a broadly adaptive hypertrophic response mechanism relevant to a wide range of clinical heart disease.

Integration of Smad and MAPK pathways: a link and a linker revisited.

Cells develop by reading mixed signals. Nowhere is this clearer than in the highly dynamic processes that propel embryogenesis, when critical cell-fate decisions are made swiftly in response to well-orchestrated growthfactor combinations. Learning how diverse signaling pathways are integrated is therefore essential for understanding physiology. This requires the identification, in tangible molecular terms, of key nodes for pathway integration that operate in vivo. A report in this issue, on the integration of Smad and Ras/MAPK pathways during neural induction (Pera et al. 2003), provides timely insights into the relevance of one such node. Pera et al. (2003) report that FGF8 and IGF2—two growth factors that activate the Ras/MAPK pathway— favor neural differentiation and mesoderm dorsalization in Xenopus by inhibiting BMP (Bone Morphogenetic Protein) signaling. Mesoderm is formed from ectoderm in response to Nodal-related signals from the endoderm at the blastula stage and beyond (Fig. 1; for review, see De Robertis et al. 2000). BMP induces differentiation of ectoderm into epidermal cell fates at the expense of neural fates, and it ventralizes the mesoderm at the expense of dorsal fates (for review, see Weinstein and HemmatiBrivanlou 1999; De Robertis et al. 2000). Accordingly, neural differentiation and dorsal mesoderm formation are favored when BMP signaling is attenuated. Noggin, Chordin, Cerberus, and Follistatin, secreted by the Spemann organizer on the dorsal side at the gastrula stage, facilitate the formation of neural tissue by sequestering BMP (Weinstein and Hemmati-Brivanlou 1999; De Robertis et al. 2000). Experimentally blocking BMP signaling with a dominant-negative BMP receptor has a similar effect of promoting ectoderm neuralization (Weinstein and Hemmati-Brivanlou 1999). As it turns out, neural induction can also be achieved with FGF (fibroblast growth factor; Kengaku and Okamoto 1993; Lamb and Harland 1995; Hongo et al. 1999; Hardcastle et al. 2000; Streit et al. 2000; Wilson et al. 2000) and IGF (insulin-like growth factor; Pera et al. 2001; Richard-Parpaillon et al. 2002). Injection of transcripts encoding FGF8 or IFG2 into one animal-pole blastomere of a fourto eight-cell embryo results in an expanded neural plate at the injected side (Pera et al. 2003). Surprisingly, expression of a dominant-negative FGF receptor prevents neuralization of ectoderm explants by the BMP blocker Noggin (Launay et al. 1996). Likewise, the potent neuralizing effect of Chordin can be blocked by a dominant-negative FGF receptor or a morpholino oligonucleotide targeting the IGF receptor (Pera et al. 2003). Thus, the neuralizing effect of BMP inhibitors is somehow tied to FGF and IFG signaling. The question is, how? Because FGF8 and IFG2 activate MAPK, Pera et al. (2003) took heed from previous work showing that MAPK inhibits the BMP signal-transduction factor Smad1 (Kretzschmar et al. 1997a). Smad1 is directly phosphorylated by the BMP receptor, resulting in Smad1 activation (Kretzschmar et al. 1997b), and by MAPK in response to EGF, resulting in Smad1 inhibition (Kretzschmar et al. 1997a; Fig. 2). Smad transcription factors mediate gene responses to the entire TGF (Transforming Growth Factor) family, to which the BMPs belong (for review, see Massague 2000; Derynck and Zhang 2003). Smads 1, 5, and 8 act primarily downstream of BMP receptors and Smads 2 and 3 downstream of TGF , Activin and Nodal receptors. Smad proteins have two conserved globular domains—the MH1 and MH2 domains (Fig. 2). The MH1 domain is involved in DNA binding and the MH2 domain in binding to cytoplasmic retention factors, activated receptors, nucleoporins in the nuclear pore, and DNA-binding cofactors, coactivators, and corepressors in the nucleus (for review, see Shi and Massague 2003). Receptor-mediated phosphorylation occurs at the carboxy-terminal sequence SXS. This enables the nuclear accumulation of Smads and their association with the shared partner Smad4 to form transcriptional complexes that are interpreted by the cell as a function of the context (Massague 2000). Between the MH1 and MH2 domains lies a linker region of variable sequence and length. Attention was drawn to this region when it was found that EGF (epidermal growth factor), a classical activator of the Ras/ MAPK pathway, causes phosphorylation of the Smad1 linker at four MAPK sites (PXSP sequences; Kretzschmar et al. 1997a). This prevents the nuclear localization of Smad1 and inhibits BMP signaling. Mutation of these E-MAIL j-massague@ski.mskcc.org; FAX (212) 717-3298. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1167003.

Pancreatic tumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2.

Activating mutations within the K-ras gene occur in a high percentage of human pancreatic carcinomas. We reported previously that the presence of oncogenic, activated K-ras in human pancreatic carcinoma cell lines did not result in constitutive activation of the extracellular signal-regulated kinases (ERK1 and ERK2). In the present study, we further characterized the ERK signaling pathway in pancreatic tumor cell lines in order to determine whether the ERK pathway is subject to a compensatory downregulation. We found that the attenuation of serum-induced ERK activation was not due to a delay in the kinetics of ERK phosphorylation. Treatment with the tyrosine phosphatase inhibitor orthovanadate increased the level of ERK phosphorylation, implicating a vanadate-sensitive tyrosine phosphatase in the negative regulation of ERK. Furthermore, expression of a dual specificity phosphatase capable of inactivating ERK known as mitogen-activated protein (MAP) kinase phosphatase-2 (MKP-2) was elevated in most of the pancreatic tumor cell lines and correlated with the presence of active MAP kinase kinase (MEK). Taken together, these results suggest that pancreatic tumor cells expressing oncogenic K-ras compensate, in part, by upregulating the expression of MKP-2 to repress the ERK signaling pathway.

Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field

Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation.

Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli. In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-(13)C(2)]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-gamma or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1alpha activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for Hif-1alpha, the switch of PFK2 isoenzymes still occurs in LPS/IFN-gamma-activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1alpha-independent mechanisms.

NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.

Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses.

The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin–Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to βPIX. Rather, Scrib depletion disrupts E-cadherin–mediated cell–cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin–α-catenin fusion protein but not by E-cadherin–green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene.

The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.

Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

Purkinje cell compartments in the reeler mutant mouse as revealed by Zebrin II and 90-acetylated glycolipid antigen expression

The cerebellum is organized into a series of parasagittally aligned bands that may be revealed histologically in the adult mouse by largely complementary immunostaining of Purkinje cell sets with the monoclonal antibodies Zebrin II (ZII; antigen:aldolase C) and P-path (PP; antigen:90-acetyl glycolipids). We compared the normal staining pattern using these markers and an antibody to calbindin with that found in the reeler mutants (rl/rl), in which most Purkinje cell migration is halted beneath the cerebellar white matter. The results revealed that Purkinje cells in reeler mutants, despite their ectopic location in large subcortical masses, show a clear tendency to distribute into alternating zones that either stain for Zebrin II or for P-path, with variable transition zones of mixed labeling. However, the estimated number of zones was fewer than in the normal adult cortex: roughly 7–9 zones are revealed per side in the mutant compared with 14 major divisions in wild type mice. These results raise the possibility that neurons destined to express these markers are segregated during their migration and that the final phase of migration into the cortex might involve further splitting or interdigitation between cell sets expressing the two antigens.

Histone acetylation and DNA demethylation of T cells result in an anaplastic large cell lymphoma-like phenotype.

A characteristic feature of anaplastic large cell lymphoma is the significant repression of the T-cell expression program despite its T-cell origin. The reasons for this down-regulation of T-cell phenotype are still unknown. To elucidate whether epigenetic mechanisms are responsible for the loss of the T-cell phenotype, we treated anaplastic large cell lymphoma and T-cell lymphoma/leukemia cell lines (n=4, each) with epigenetic modifiers to evoke DNA demethylation and histone acetylation. Global gene expression data from treated and untreated cell lines were generated and selected, and differentially expressed genes were evaluated by real-time reverse transcriptase polymerase chain reaction and western blot analysis. Additionally, histone H3 lysine 27 trimethylation was analyzed by chromatin immunoprecipitation. Combined DNA demethylation and histone acetylation of anaplastic large cell lymphoma cells was not able to reconstitute their T-cell phenotype. Instead, the same treatment induced in T cells: (i) an up-regulation of anaplastic large cell lymphoma-characteristic genes (e.g. ID2, LGALS1, c-JUN), and (ii) an almost complete extinction of their T-cell phenotype including CD3, LCK and ZAP70. In addition, suppressive trimethylation of histone H3 lysine 27 of important T-cell transcription factor genes (GATA3, LEF1, TCF1) was present in anaplastic large cell lymphoma cells, which is in line with their absence in primary tumor specimens as demonstrated by immunohistochemistry. Our data suggest that epigenetically activated suppressors (e.g. ID2) contribute to the down-regulation of the T-cell expression program in anaplastic large cell lymphoma, which is maintained by trimethylation of histone H3 lysine 27.

The Nuclear Effector of Wnt-Signaling, Tcf1, Functions as a T-Cell–Specific Tumor Suppressor for Development of Lymphomas

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

A genetic screen identifies an LKB1–MARK signalling axis controlling the Hippo–YAP pathway

The Hippo–YAP pathway is an emerging signalling cascade involved in the regulation of stem cell activity and organ size. To identify components of this pathway, we performed an RNAi-based kinome screen in human cells. Our screen identified several kinases not previously associated with Hippo signalling that control multiple cellular processes. One of the hits, LKB1, is a common tumour suppressor whose mechanism of action is only partially understood. We demonstrate that LKB1 acts through its substrates of the microtubule affinity-regulating kinase family to regulate the localization of the polarity determinant Scribble and the activity of the core Hippo kinases. Our data also indicate that YAP is functionally important for the tumour suppressive effects of LKB1. Our results identify a signalling axis that links YAP activation with LKB1 mutations, and have implications for the treatment of LKB1-mutant human malignancies. In addition, our findings provide insight into upstream signals of the Hippo–YAP signalling cascade.

Chronic lymphocytic leukemia with t(14;19)(q32;q13) is characterized by atypical morphologic and immunophenotypic features and distinctive genetic features.

The t(14;19)(q32;q13) involving the IGH@ and BCL3 loci is an infrequent cytogenetic abnormality detected in B-cell malignancies. We describe the clinicopathologic, cytogenetic, and molecular genetic characteristics of 14 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with t(14;19)(q32;q13). All patients (10 men and 4 women) had lymphocytosis; 10 had lymphadenopathy. Blood and bone marrow lymphocytes were predominantly small, but cytologically and immunophenotypically atypical. In all cases, t(14;19) was found in the neoplastic stem line; it was the sole abnormality in 4. Ten cases showed additional cytogenetic abnormalities, including trisomy 12 in 9 and complex karyotypes in 7. Fluorescence in situ hybridization demonstrated IGH@/BCL3 fusion gene in all cases. In all cases, the IGHV genes were unmutated, but only 7 expressed ZAP70. Seven cases preferentially used IGHV4-39. Our results indicate that t(14;19)(q32;q13) identifies a subset of CLL/SLL with distinctive clinicopathologic and genetic features. Furthermore, t(14;19) may represent an early, possibly primary, genetic event.

Integrins promote cytokinesis through the RSK signaling axis.

Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2−/− mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.

Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORβ

BACKGROUND Colorectal cancer remains one of the most common malignant tumors worldwide. Colorectal cancer initiating cells (CCICs) are a small subpopulation responsible for malignant behaviors of colorectal cancer. Aberrant activation of the Wnt pathways regulates the self-renewal of CCIC. However, the underlying mechanism(s) remain poorly understood. METHODS Via retroviral library screening, we identified Nuclear Receptor-Interacting Protein 2 (NRIP2) as a novel interactor of the Wnt pathway from enriched colorectal cancer colosphere cells. The expression levels of NRIP2 and retinoic acid-related orphan receptor β (RORβ) were further examined by FISH, qRT-PCR, IHC and Western blot. NRIP2 overexpressed and knockdown colorectal cancer cells were produced to study the role of NRIP2 in Wnt pathway. We also verified the binding between NRIP2 and RORβ and investigated the effect of RORβ on CCICs both in vitro and in vivo. Genechip-scanning speculated downstream target HBP1. Western blot, ChIP and luciferase reporter were carried to investigate the interaction between NRIP2, RORβ, and HBP1. RESULTS NRIP2 was significantly up-regulated in CCICs from both cell lines and primary colorectal cancer tissues. Reinforced expression of NRIP2 increased Wnt activity, while silencing of NRIP2 attenuated Wnt activity. The transcription factor RORβ was a key target through which NRIP2 regulated Wnt pathway activity. RORβ was a transcriptional enhancer of inhibitor HBP1 of the Wnt pathway. NRIP2 prevented RORβ to bind with downstream HBP1 promoter regions and reduced the transcription of HBP1. This, in turn, attenuated the HBP1-dependent inhibition of TCF4-mediated transcription. CONCLUSIONS NRIP2 is a novel interactor of the Wnt pathway in colorectal cancer initiating cells. interactions between NRIP2, RORβ, and HBP1 mediate a new mechanism for CCIC self-renewal via the Wnt activity.

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract, Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical beta-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3(-/-) mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3(-/-) and LtapLp/+ mutants, Dvl3(+/-);LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.

Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

Imaging the expression of transfected genes in vivo.

Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of "marker gene" and "marker substrate" in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK+ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2'-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK+ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[14C]FIAU accumulation was shown to be high in RG2TK+ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumulated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.

Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis

Tumor angiogenesis is an essential process for supplying rapidly growing malignant tissues with essential nutrients and oxygen. An angiogenic switch allows tumor cells to survive and grow, and provides them access to vasculature resulting in metastatic disease. Monocyte-derived macrophages recruited and reprogrammed by tumor cells serve as a major source of angiogenic factors boosting the angiogenic switch. Tumor endothelium releases angiopoietin-2 and further facilitates recruitment of TIE2 receptor expressing monocytes (TEM) into tumor sites. Tumor-associated macrophages (TAM) sense hypoxia in avascular areas of tumors, and react by production of angiogenic factors such as VEGFA. VEGFA stimulates chemotaxis of endothelial cells (EC) and macrophages. In some tumors, TAM appeared to be a major source of MMP9. Elevated expression of MMP9 by TAM mediates extracellular matrix (ECM) degradation and the release of bioactive VEGFA. Other angiogenic factors released by TAM include basic fibroblast growth factor (bFGF), thymidine phosphorylase (TP), urokinase-type plasminogen activator (uPA), and adrenomedullin (ADM). The same factors used by macrophages for the induction of angiogenesis [like vascular endothelial growth factor A (VEGF-A) and MMP9] support lymphangiogenesis. TAM can express LYVE-1, one of the established markers of lymphatic endothelium. TAM support tumor lymphangiogenesis not only by secretion of pro-lymphangiogenic factors but also by trans-differentiation into lymphatic EC. New pro-angiogenic factor YKL-40 belongs to a family of mammalian chitinase-like proteins (CLP) that act as cytokines or growth factors. Human CLP family comprises YKL-40, YKL-39, and SI-CLP. Production of all three CLP in macrophages is antagonistically regulated by cytokines. It was recently established that YKL-40 induces angiogenesis in vitro and in animal tumor models. YKL-40-neutralizing monoclonal antibody blocks tumor angiogenesis and progression. The role of YKL-39 and SI-CLP in tumor angiogenesis and lymphangiogenesis remains to be investigated.

Thymic expression of the golli-myelin basic protein gene in the SJL/J mouse

The T cell antigen-specific repertoire is thought to be shaped by thymic expression of self molecules. Since a myelin basic protein (MBP)-like gene (golli-MBP) has been reported to be expressed by cells of the immune system, the present study was undertaken to determine whether the golli-MBP gene was expressed in the mouse thymus and, if so, to characterize transcripts of this gene in this organ. Using exon-specific primers for MBP and golli-MBP, cDNA from thymus and other tissues was amplified, and the amplified products analyzed by Southern blotting with exon-specific oligonucleotide probes. The amplified products were subcloned, and the inserts characterized by DNA sequencing. The thymic transcripts were found to contain golli-MBP exons 1, 2, 3, 5A, 5B, 5C, 6, 7, 8, and 11.

Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages.

Infectious pathogens can selectively stimulate activation or suppression of T cells to facilitate their survival within humans. In this study we demonstrate that the trematode parasite Schistosoma mansoni has evolved with two distinct mechanisms to suppress T cell activation. During the initial 4- to 12-wk acute stages of a worm infection both CD4(+) and CD8(+) T cells are anergized. In contrast, infection with male and female worms induced T cell anergy at 4 wk, which was replaced after egg laying by T cell suppression via a known NO-dependent mechanism, that was detected for up to 40 wk after infection. Worm-induced anergy was mediated by splenic F4/80(+) macrophages (Mphi) via an IL-4-, IL-13-, IL-10-, TGF-beta-, and NO-independent, but cell contact-dependent, mechanism. F4/80(+) Mphi isolated from worm-infected mice were shown to induce anergy of naive T cells in vitro. Furthermore, naive Mphi exposed to live worms in vitro also induced anergy in naive T cells. Flow cytometry on in vivo and in vitro worm-modulated Mphi revealed that of the family of B7 costimulatory molecules, only programmed death ligand 1 (PD-L1) was selectively up-regulated. The addition of inhibitory mAb against PD-L1, but not PD-L2, to worm-modulated Mphi completely blocked the ability of these cells to anergize T cells. These data highlight a novel mechanism through which S. mansoni worms have usurped the natural function of PD-L1 to reduce T cell activation during early acute stages of infection before the subsequent emergence of egg-induced T cell suppression in the chronic stages of infection.

(99m)Tc-HisoDGR as a Potential SPECT Probe for Orthotopic Glioma Detection via Targeting of Integrin α5β1.

Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5β1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported. Developing α5β1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvβ3/αvβ5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5β1-specific probe (99m)Tc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5β1 in vivo, the mouse models of α5β1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of (99m)Tc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that (99m)Tc-HisoDGR represented a powerful molecular probe for integrin α5β1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5β1-targeted drugs, and assessing and monitoring the response to integrin α5β1-targeted drugs in clinical trials.

Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution

DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome

Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000–15,000 births. Affected females develop normally for 6–18 months, but then lose voluntary movements, including speech and hand skills. Most RTT patients are heterozygous for mutations in the X-linked gene MECP2 (refs. 3–12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis. Here we generate mice lacking Mecp2 using Cre-loxP technology. Both Mecp2-null mice and mice in which Mecp2 was deleted in brain showed severe neurological symptoms at approximately six weeks of age. Compensation for absence of MeCP2 in other tissues by MeCP1 (refs. 19,20) was not apparent in genetic or biochemical tests. After several months, heterozygous female mice also showed behavioral symptoms. The overlapping delay before symptom onset in humans and mice, despite their profoundly different rates of development, raises the possibility that stability of brain function, not brain development per se, is compromised by the absence of MeCP2.

Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node–residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node–residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.

Conversion of vascular endothelial cells into multipotent stem-like cells

Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor–dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell–like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.

Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity.

V domain-containing Ig suppressor of T-cell activation (VISTA) is a negative checkpoint regulator that suppresses T cell-mediated immune responses. Previous studies using a VISTA-neutralizing monoclonal antibody show that VISTA blockade enhances T-cell activation. The current study describes a comprehensive characterization of mice in which the gene for VISTA has been deleted. Despite the apparent normal hematopoietic development in young mice, VISTA genetic deficiency leads to a gradual accumulation of spontaneously activated T cells, accompanied by the production of a spectrum of inflammatory cytokines and chemokines. Enhanced T-cell responsiveness was also observed upon immunization with neoantigen. Despite the presence of multiorgan chronic inflammation, aged VISTA-deficient mice did not develop systemic or organ-specific autoimmune disease. Interbreeding of the VISTA-deficient mice with 2D2 T-cell receptor transgenic mice, which are predisposed to the development of experimental autoimmune encephalomyelitis, drastically enhanced disease incidence and intensity. Disease development is correlated with the increase in the activation of encephalitogenic T cells in the periphery and enhanced infiltration into the CNS. Taken together, our data suggest that VISTA is a negative checkpoint regulator whose loss of function lowers the threshold for T-cell activation, allowing for an enhanced proinflammatory phenotype and an increase in the frequency and intensity of autoimmunity under susceptible conditions.

The novel immunosuppressive enzyme IL4I1 is expressed by neoplastic cells of several B-cell lymphomas and by tumor-associated macrophages

We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

CD28 and ITK signals regulate autoreactive T cell trafficking

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

Foxp2 and Foxp1 cooperatively regulate lung and esophagus development.

The airways of the lung develop through a reiterative process of branching morphogenesis that gives rise to the intricate and extensive surface area required for postnatal respiration. The forkhead transcription factors Foxp2 and Foxp1 are expressed in multiple foregut-derived tissues including the lung and intestine. In this report, we show that loss of Foxp2 in mouse leads to defective postnatal lung alveolarization, contributing to postnatal lethality. Using in vitro and in vivo assays, we show that T1alpha, a lung alveolar epithelial type 1 cell-restricted gene crucial for lung development and function, is a direct target of Foxp2 and Foxp1. Remarkably, loss of a single Foxp1 allele in addition to complete loss of Foxp2 results in increased severity of morphological defects in mutant lungs and leads to perinatal loss of all Foxp2(-/-);Foxp1(+/-) mice. Expression of N-myc and Hop, crucial regulators of lung development, is compromised in Foxp2(-/-);Foxp1(+/-) mutants. In addition to the defects in lung development, esophageal muscle development is disrupted in Foxp2(-/-);Foxp1(+/-) embryos, a tissue where Foxp2 and Foxp1 are co-expressed. These data identify Foxp2 and Foxp1 as crucial regulators of lung and esophageal development, underscoring the necessity of these transcription factors in the development of anterior foregut-derived tissues and demonstrating functional cooperativity between members of the Foxp1/2/4 family in tissues where they are co-expressed.

Thrombopoietin-induced Polyploidization of Bone Marrow Megakaryocytes Is Due to a Unique Regulatory Mechanism in Late Mitosis

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

A novel Ku70 function in colorectal homeostasis separate from nonhomologous end joining

Ku70, a known nonhomologous end-joining (NHEJ) factor, also functions in tumor suppression, although this molecular mechanism remains uncharacterized. Previously, we showed that mice deficient for DNA ligase IV (Lig4), another key NHEJ factor, succumbed to aggressive lymphoma in the absence of tumor suppressor p53. However, the tumor phenotype is abrogated by the introduction of a hypomorphic mutant p53R172P, which impaired p53-mediated apoptosis but not cell-cycle arrest. However, Lig4−/−p53R172P mice succumbed to severe diabetes. To further elucidate the role of NHEJ and p53-mediated apoptosis in vivo, we bred Ku70−/− p53R172P mice. Unexpectedly, these mice were free of diabetes, although 80% of the mutant mice had abnormally enlarged colons with pronounced inflammation. Remarkably, most of these mutant mice progressed to dysplasia, adenoma and adenocarcinoma; this is in contrast to the Lig4−/−p53R172P phenotype, strongly suggesting an NHEJ-independent function of Ku70. Significantly, our analyses of Ku70−/−p53R172P colonic epithelial cells show nuclear stabilization of β-catenin accompanied by higher expression of cyclin D1 and c-Myc in affected colon sections than in control samples. This is not due to the p53 mutation, as Ku70−/− mice share this phenotype. Our results not only unravel a novel function of Ku70 essential for colon homeostasis, but also establish an excellent in vivo model in which to study how chronic inflammation and abnormal cellular proliferation underlie tumorigenesis and tumor progression in the colon.

Divergent reprogramming routes lead to alternative stem-cell states

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

A systemic granulomatous response to Schistosoma mansoni eggs alters responsiveness of bone-marrow-derived macrophages to Toll-like receptor agonists.

Macrophages play a pivotal role in innate and acquired immune responses to Schistosoma mansoni. Classical (M1) or alternative (M2) activation states of these cells further delineate their roles in tissue damage through innate immunity or fibrotic remodeling, respectively. In the present study, we addressed the following question: Does systemic Th2-type cytokine polarization evoked by S. mansoni affect macrophage differentiation and activation? To this end, we analyzed bone marrow-derived macrophages from mice with S. mansoni egg-induced pulmonary granulomas and unchallenged (or naïve) mice to determine their activation state and their response to specific TLR agonists, including S. mansoni egg antigens. Unlike naïve macrophages, macrophages from Th2-polarized mice constitutively expressed significantly higher "found in inflammatory zone-1" (FIZZ1) and ST2 (M2 markers) and significantly lower NO synthase 2, CCL3, MIP-2, TNF-alpha, and IL-12 (M1 markers). Also, compared with naïve macrophages, Th2-polarized macrophages exhibited enhanced responses to the presence of specific TLR agonists, which consistently induced significantly higher levels of gene and protein levels for M2 and M1 markers in these cells. Together, these data show that signals received by bone marrow precursors during S. mansoni egg-induced granuloma responses dynamically alter the development of macrophages and enhance the TLR responsiveness of these cells, which may ultimately have a significant effect on the pulmonary granulomatous response.

CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling via transactivation of GSK-3β and Axin2 expression

Caudal-related homeobox transcription factor 2 (CDX2), an intestine-specific nuclear transcription factor, has been strongly implicated in the tumourigenesis of various human cancers. However, the functional role of CDX2 in the development and progression of colorectal cancer (CRC) is not well known. In this study, CDX2 knockdown in colon cancer cells promoted cell proliferation in vitro, accelerated tumor formation in vivo, and induced a cell cycle transition from G0/G1 to S phase, whereas CDX2 overexpression inhibited cell proliferation. TOP/FOP-Flash reporter assay showed that CDX2 knockdown or CDX2 overexpression significantly increased or decreased Wnt signaling activity. Western blot assay showed that downstream targets of Wnt signaling, including β-catenin, cyclin D1 and c-myc, were up-regulated or down-regulated in CDX2-knockdown or CDX2-overexpressing colon cancer cells. In addition, suppression of Wnt signaling by XAV-939 led to a marked suppression of the cell proliferation enhanced by CDX2 knockdown, whereas activation of this signaling by CHIR-99021 significantly enhanced the cell proliferation inhibited by CDX2 overexpression. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that CDX2 transcriptionally activates glycogen synthase kinase-3β (GSK-3β) and axis inhibition protein 2 (Axin2) expression by directly binding to the promoter of GSK-3β and the upstream enhancer of Axin2. In conclusion, these results indicated that CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling.

Chemokine-like receptor 1 (CMKLR1) and chemokine (C-C motif) receptor-like 2 (CCRL2); two multifunctional receptors with unusual properties.

Chemokine-like receptor 1 (CMKLR1), also known as ChemR23, and chemokine (C-C motif) receptor-like 2 (CCRL2) are 7-transmembrane receptors that were cloned in the late 1990s based on their homology to known G-protein-coupled receptors. They were previously orphan receptors without any known biological roles; however, recent studies identified ligands for these receptors and their functions have begun to be unveiled. The plasma protein-derived chemoattractant chemerin is a ligand for CMKLR1 and activation of CMKLR1 with chemerin induces the migration of macrophages and dendritic cells (DCs) in vitro, suggesting a proinflammatory role. However, in vivo studies using CMKLR-deficient mice suggest an anti-inflammatory role for this receptor, possibly due to the recruitment of tolerogenic plasmacytoid DCs. Chemerin/CMKLR1 interaction also promotes adipogenesis and angiogenesis. The anti-inflammatory lipid mediator, resolving E1, is another CMKLR1 ligand and it inhibits leukocyte infiltration and proinflammatory gene expression. These divergent results suggest that CMKLR1 is a multifunctional receptor. The chemokine CCL5 and CCL19 are reported to bind to CCRL2. Like Duffy antigen for chemokine receptor (DARC), D6 and CCX-CKR, CCRL2 does not signal, but it constitutively recycles, potentially reducing local concentration of CCL5 and CCL19 and subsequent immune responses. Surprisingly, chemerin, a ligand for CMKLR1, is a ligand for CCRL2. CCRL2 binds chemerin and increases local chemerin concentration to efficiently present it to CMKLR1 on nearby cells, providing a link between CCRL2 and CMKLR1. Although these findings suggest an anti-inflammatory role, a recent study using CCRL2-deficient mice indicates a proinflammatory role; thus, CCRL2 may also be multifunctional. Further studies using CMKLR1- or CCRL2-deficient mice are needed to further define the role of these receptors in immune responses and other cellular processes.

Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks.

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.

Adult c-Kit(+) progenitor cells are necessary for maintenance and regeneration of olfactory neurons.

The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.

Hedgehog signaling regulates brain tumor-initiating cell proliferation and portends shorter survival for patients with PTEN-coexpressing glioblastomas.

The identification of brain tumor stem-like cells (BTSCs) has implicated a role of biological self-renewal mechanisms in clinical brain tumor initiation and propagation. The molecular mechanisms underlying the tumor-forming capacity of BTSCs, however, remain unknown. Here, we have generated molecular signatures of glioblastoma multiforme (GBM) using gene expression profiles of BTSCs and have identified both Sonic Hedgehog (SHH) signaling-dependent and -independent BTSCs and their respective glioblastoma surgical specimens. BTSC proliferation could be abrogated in a pathway-dependent fashion in vitro and in an intracranial tumor model in athymic mice. Both SHH-dependent and -independent brain tumor growth required phosphoinositide 3-kinase-mammalian target of rapamycin signaling. In human GBMs, the levels of SHH and PTCH1 expression were significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. In addition, we show that hyperactive SHH-GLI signaling in PTEN-coexpressing human GBM is associated with reduced survival time. Thus, distinct proliferation signaling dependence may underpin glioblastoma propagation by BTSCs. Modeling these BTSC proliferation mechanisms may provide a rationale for individualized glioblastoma treatment.

Neural crest cell lineage segregation in the mouse neural tube.

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.

Identification and characterization of a novel monocyte/macrophage differentiation-dependent gene that is responsive to lipopolysaccharide, ceramide, and lysophosphatidylcholine.

A novel differentiation-dependent cDNA (DIF-2) has been isolated from human mononuclear phagocytes by differential display. The full-length cDNA was cloned and sequenced. DIF-2 consists of 156 amino acids and has a predicted isoelectric point of 8.84. The mRNA is expressed in freshly isolated monocytes and is downregulated significantly when monocytes are subjected to differentiation. A similar differentiation-dependent downregulation is observed in normal hepatocytes compared to undifferentiated HepG2 cells. The mRNA expression in monocytes is sensitive to lipopolysaccharide and ceramide which both strongly increase DIF-2 transcription, while lysophosphatidylcholine results in a weaker upregulation of DIF-2 expression. A DIF-2 homologous gene has been previously isolated from mouse fibroblasts and was shown to be a serum growth factor-inducible immediate early gene. Our results indicate that DIF-2 represents a gene which is regulated in differentiation processes and strongly responsive to lipopolysaccharide, ceramide and lysophosphatidylcholine.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2.

UNLABELLED Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein we examined the expression profiles of long noncoding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling, and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had a poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling, and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2. CONCLUSION Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling, and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects on the treatment of HCC.

Loss of lamin B1 results in prolongation of S phase and decondensation of chromosome territories.

Nuclear lamin B1 (LMNB1) constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of LMNB1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion. In addition, we observed telomere attrition and an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrated that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that LMNB1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S phase due to activation of Chk1. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing.

Vascular Calcification in Chronic Kidney Disease is Induced by Bone Morphogenetic Protein-2 via a Mechanism Involving the Wnt/β-Catenin Pathway

Background: Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo a phenotypic transformation into osteoblast-like cells, is one of the emergent risk factors for the accelerated atherosclerosis process characteristic of chronic kidney disease (CKD). Phosphate is an important regulator of VC. Methods: The expression of different smooth muscle cell or osteogenesis markers in response to high concentrations of phosphate or exogenous bone morphogenetic protein 2 (BMP-2) was examined by qRT-PCR and western blotting in rat VSMCs. Osteocalcin secretion was measured by radioimmunoassay. Differentiation and calcification of VSMCs were examined by alkaline phosphatase (ALP) activity assay and Alizarin staining. Short hairpin RNA-mediated silencing of β-catenin was performed to examine the involvement of Wnt/β-catenin signaling in VSMC calcification and osteoblastic differentiation induced by high phosphate or BMP-2. Apoptosis was determined by TUNEL assay and immunofluorescence imaging. Results: BMP-2 serum levels were significantly higher in CKD patients than in controls. High phosphate concentrations and BMP-2 induced VSMC apoptosis and upregulated the expression of β-catenin, Msx2, Runx2 and the phosphate cotransporter Pit1, whereas a BMP-2 neutralization antibody reversed these effects. Knockdown of β-catenin abolished the effect of high phosphate and BMP-2 on VSMC apoptosis and calcification. Conclusions: BMP-2 plays a crucial role in calcium deposition in VSMCs and VC in CKD patients via a mechanism involving the Wnt/β-catenin pathway.

Role of boundary conditions in an experimental model of epithelial wound healing.

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.

Human Cyclin a Is Required for Mitosis until Mid Prophase

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

Opposing Effects of PKCθ and WASp on Symmetry Breaking and Relocation of the Immunological Synapse

The immunological synapse (IS) is a junction between the T cell and antigen-presenting cell and is composed of supramolecular activation clusters (SMACs). No studies have been published on naive T cell IS dynamics. Here, we find that IS formation during antigen recognition comprises cycles of stable IS formation and autonomous naive T cell migration. The migration phase is driven by PKCtheta, which is localized to the F-actin-dependent peripheral (p)SMAC. PKCtheta(-/-) T cells formed hyperstable IS in vitro and in vivo and, like WT cells, displayed fast oscillations in the distal SMAC, but they showed reduced slow oscillations in pSMAC integrity. IS reformation is driven by the Wiscott Aldrich Syndrome protein (WASp). WASp(-/-) T cells displayed normal IS formation but were unable to reform IS after migration unless PKCtheta was inhibited. Thus, opposing effects of PKCtheta and WASp control IS stability through pSMAC symmetry breaking and reformation.

PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome.

Recently, mutations in genes involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor have been identified in a new subclass of congenital disorders of glycosylation (CDGs) with a distinct spectrum of clinical features. To date, mutations have been identified in six genes (PIGA, PIGL, PIGM, PIGN, PIGO, and PIGV) encoding proteins in the GPI-anchor-synthesis pathway in individuals with severe neurological features, including seizures, muscular hypotonia, and intellectual disability. We developed a diagnostic gene panel for targeting all known genes encoding proteins in the GPI-anchor-synthesis pathway to screen individuals matching these features, and we detected three missense mutations in PGAP2, c.46C>T, c.380T>C, and c.479C>T, in two unrelated individuals with hyperphosphatasia with mental retardation syndrome (HPMRS). The mutations cosegregated in the investigated families. PGAP2 is involved in fatty-acid GPI-anchor remodeling, which occurs in the Golgi apparatus and is required for stable association between GPI-anchored proteins and the cell-surface membrane rafts. Transfection of the altered protein constructs, p. Arg16Trp (NP_001243169.1), p. Leu127Ser, and p. Thr160Ile, into PGAP2-null cells showed only partial restoration of GPI-anchored marker proteins, CD55 and CD59, on the cell surface. In this work, we show that an impairment of GPI-anchor remodeling also causes HPMRS and conclude that targeted sequencing of the genes encoding proteins in the GPI-anchor-synthesis pathway is an effective diagnostic approach for this subclass of CDGs.

The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase

Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.

Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D:Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

Alphavbeta6-Fyn signaling promotes oral cancer progression.

We have previously shown that the integrin beta6 is neo-expressed in invasive oral squamous cell carcinoma (SCC) and is correlated with oral tumor progression. However, the mechanism by which the integrin beta6 promotes oral tumor progression is not well understood. The purpose of the present study was to determine whether integrin beta6 signaling activates Fyn and thus promotes oral squamous cell carcinoma progression. We analyzed the integrin beta6 signaling complex and investigated the function of these signaling molecules in oral SCC cells. We found that, upon ligation of the integrin beta6 with fibronectin, beta6 complexed with Fyn and activated it. The activation of Fyn recruited and activated focal adhesion kinase to this complex. This complex was necessary to activate Shc and to couple beta6 signaling to the Raf-ERK/MAPK pathway. This pathway transcriptionally activated the matrix metalloproteinase-3 gene and promoted oral SCC cell proliferation and experimental metastasis in vivo. These findings indicate that integrin beta6 signaling activates Fyn and thus promotes oral cancer progression.

Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins

Inhibition of αvβ3 or αvβ5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking β3 integrins or both β3 and β5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither β3 nor β5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of β3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the β3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in β3-null endothelial cells. These data indicate that αvβ3 and αvβ5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of αv-integrin antagonists in anti-angiogenic therapeutics.

Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity

Patients with asthma, a major public health problem, are at high risk for serious disease from influenza virus infection, but the pathogenic mechanisms by which influenza A causes airway disease and asthma are not fully known. We show here in a mouse model that influenza infection acutely induced airway hyper-reactivity (AHR), a cardinal feature of asthma, independently of T helper type 2 (TH2) cells and adaptive immunity. Instead, influenza infection induced AHR through a previously unknown pathway that required the interleukin 13 (IL-13)–IL-33 axis and cells of the non-T cell, non-B cell innate lymphoid type called 'natural helper cells'. Infection with influenza A virus, which activates the NLRP3 inflammasome, resulted in much more production of IL-33 by alveolar macrophages, which in turn activated natural helper cells producing substantial IL-13.

CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS.

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood-brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1alpha, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

Notch activation promotes cell proliferation and the formation of neural stem cell-like colonies in human glioma cells

Since Notch signaling plays a critical role in stem cells and oncogenesis, we hypothesized that Notch signaling might play roles in cancer stem cells and cancer cells with a stem cell phenotype. In this study, we accessed potential functions of the Notch pathway in the formation of cancer stem cells using human glioma. Using RT-PCR, we found that most human astrogliomas of different grades expressed moderate to high level of Notch receptors and ligands. mRNA of Hes5 but not Hes1, both of which are major downstream molecules of the Notch pathway, was also detected. In human glioma cell lines BT325, U251, SHG-44, and U87, mRNA encoding different types of Notch receptors were detected, but active form of Notch1 (NIC) was only detected in SHG-44 and U87 by Western blot. Interestingly, proliferation of these two glioma cell lines appeared faster than that of the other two lines in which NIC was not detected. We have over-expressed NIC of Notch1 in SHG-44 cells by constitutive transfection to evaluate the effects of Notch signaling on glioma cells. Our results showed that over-expression of NIC in SHG-44 cells promoted the growth and the colony-forming activity of SHG-44 cells. Interestingly, over-expression of NIC increased the formation neurosphere-like colonies in the presence of growth factors. These colonies expressed nestin, and could be induced to cells expressing neuron-, astrocyte-, or oligodendrocyte-specific markers, consistent with phenotypes of neural stem cells. These data suggest that Notch signaling promote the formation of cancer stem cell-like cells in human glioma.

Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation

The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.

Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

Shc coordinates signals from intercellular junctions and integrins to regulate flow-induced inflammation

Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell-cell and cell-matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor kappaB-dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell-cell and cell-matrix adhesions to elicit flow-induced inflammatory signaling.

ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.

AIMS The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. METHODS AND RESULTS ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. CONCLUSION Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Oncolytic adenovirus armed with IL-24 Inhibits the growth of breast cancer in vitro and in vivo

BACKGROUND Interleukin-24 (IL-24) is a cytokine that belongs to the IL-10 family. It can selectively induce cancer cell apoptosis which has been utilized as a cancer gene therapy strategy. METHODS A recombinant type five adenovirus containing IL-24 gene (designated CNHK600-IL24) was constructed, whose replication is activated only in tumor cells. The replication of CNHK600-IL24 in breast tumor cells and fibroblasts were assessed by TCID50 and MTT assay; the secretion of IL-24 was measured by ELISA and western blotting. The in vivo anti-tumor effect of CNHK600-IL24 was investigated in nude mice carrying orthotopic or metastatic breast tumor. RESULTS We observed that CNHK600-IL24 could replicate efficiently and resulted in high level IL-24 expression and massive cell death in human breast cancer cell MDA-MB-231 but not in normal fibroblast cell MRC-5. In addition, orthotopic breast tumor growth in the nude mice model was significantly suppressed when CNHK600-IL24 was administered. In the metastatic model generated by tail vein injection, CNHK600-IL24 virotherapy significantly improved survival compared with the same virus expressing EGFP (median survival CNHK600-IL24, 55 days vs. CNHK600-EGFP, 41 day, p < 0.05 Mantal-Cox test). A similar phenomenon was observed in the metastatic model achieved by left ventricular injection as suggested by in vivo luminescence imaging of tumor growth. CONCLUSION The oncolytic adenovirus armed with IL-24, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of breast cancer.

Subversion of Systemic Glucose Metabolism as a Mechanism to Support the Growth of Leukemia Cells.

From an organismal perspective, cancer cell populations can be considered analogous to parasites that compete with the host for essential systemic resources such as glucose. Here, we employed leukemia models and human leukemia samples to document a form of adaptive homeostasis, where malignant cells alter systemic physiology through impairment of both host insulin sensitivity and insulin secretion to provide tumors with increased glucose. Mechanistically, tumor cells induce high-level production of IGFBP1 from adipose tissue to mediate insulin sensitivity. Further, leukemia-induced gut dysbiosis, serotonin loss, and incretin inactivation combine to suppress insulin secretion. Importantly, attenuated disease progression and prolonged survival are achieved through disruption of the leukemia-induced adaptive homeostasis. Our studies provide a paradigm for systemic management of leukemic disease.

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction

Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets

Immune tolerance and activation depend on precise control over the number and function of immunosuppressive Foxp3(+) regulatory T (T reg) cells, and the importance of IL-2 in maintaining tolerance and preventing autoimmunity is clear. However, the homeostatic requirement for IL-2 among specific populations of peripheral T reg cells remains poorly understood. We show that IL-2 selectively maintains a population of quiescent CD44(lo)CD62L(hi) T reg cells that gain access to paracrine IL-2 produced in the T cell zones of secondary lymphoid tissues due to their expression of the chemokine receptor CCR7. In contrast, CD44(hi)CD62L(lo)CCR7(lo) T reg cells that populate nonlymphoid tissues do not access IL-2-prevalent regions in vivo and are insensitive to IL-2 blockade; instead, their maintenance depends on continued signaling through the co-stimulatory receptor ICOS (inducible co-stimulator). Thus, we define a fundamental homeostatic subdivision in T reg cell populations based on their localization and provide an integrated framework for understanding how T reg cell abundance and function are controlled by unique signals in different tissue environments.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells.

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Significant correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy

The aim of the present study was to investigate whether the gene expression levels of LKB1 and LGR5 correlated with clinical outcome in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy (CRT). Residual cancer cells were obtained from 52 patients with locally advanced rectal cancer treated with preoperative CRT. Total RNA was then isolated from formalin-fixed, paraffin-embedded specimens using microdissection. The expression levels of LKB1 and LGR5 genes were measured using real-time reverse-transcription polymerase chain reaction and by immunohistochemistry. In addition, in vitro studies were performed using colon cancer cell lines to study the serial changes of LKB1, LGR5 and PRKAA1 (AMPK) gene expression levels after irradiation. Our data demonstrate that specimens obtained from patients with poor pathological response and tumor recurrence had significantly higher gene expression levels of LKB1 and LGR5 than those without them (P < 0.05), and there was a significant positive correlation between LKB1 and LGR5 gene expression after CRT (Spearman’s ρ: 0.429, P = 0.0023). The patients with high expression levels of both LKB1 and LGR5 had a significantly lower recurrence-free survival compared with the other group (P = 0.0055, 95 % confidence interval: 1.39–11.08). Lastly, in vitro studies demonstrated a similar pattern of serial gene expression among LKB1, LGR5 and PRKAA1 after irradiation. Our results suggest that LKB1 and LGR5 expression may be implicated in resistance to CRT, therefore contributing to tumor relapse in patients with locally advanced rectal cancer treated with preoperative CRT.

Metabolic regulation by the mitochondrial phosphatase PTPMT1 is required for hematopoietic stem cell differentiation.

The regulation and coordination of mitochondrial metabolism with hematopoietic stem cell (HSC) self-renewal and differentiation is not fully understood. Here we report that depletion of PTPMT1, a PTEN-like mitochondrial phosphatase, in inducible or hematopoietic-cell-specific knockout mice resulted in hematopoietic failure due to changes in the cell cycle and a block in the differentiation of HSCs. Surprisingly, the HSC pool was increased by ∼40-fold in PTPMT1 knockout mice. Reintroduction of wild-type PTPMT1, but not catalytically deficient PTPMT1 or truncated PTPMT1 lacking mitochondrial localization, restored differentiation capabilities of PTPMT1 knockout HSCs. Further analyses demonstrated that PTPMT1 deficiency altered mitochondrial metabolism and that phosphatidylinositol phosphate substrates of PTPMT1 directly enhanced fatty-acid-induced activation of mitochondrial uncoupling protein 2. Intriguingly, depletion of PTPMT1 from myeloid, T lymphoid, or B lymphoid progenitors did not cause any defects in lineage-specific knockout mice. This study establishes a crucial role of PTPMT1 in the metabolic regulation of HSC function.

The antidiabetic effect of mesenchymal stem cells is unrelated to their transdifferentiation potential but to their capability to restore Th1/Th2 balance and to modify the pancreatic microenvironment.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.

Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase.

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Materials and Methods Figs. S1 to S15 References Supporting Online Materials

Endosomal Toll-like receptors (TLRs) 7 and 9 recognize viral pathogens and induce signals leading to the activation of nuclear factor κB (NF-κB)-dependent proinflammatory cytokines and interferon regulatory factor 7 (IRF7)-dependent type I interferons (IFNs). Recognition of viral nucleic acids by TLR9 requires its cleavage in the endolysosomal compartment. Here, we show that TLR9 signals leading to the activation of type I IFN, but not proinflammatory cytokine genes, require TLR9 trafficking from endosomes to a specialized lysosome-related organelle. Furthermore, we identify adapter protein-3 as the protein complex responsible for the trafficking of TLR9 to this subcellular compartment. Our results reveal an intracellular mechanism for bifurcation of TLR9 signals by selective receptor trafficking within the endosomal system.

Cdk1 is sufficient to drive the mammalian cell cycle

Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.

Identification of an autoantigen demonstrates a link between interstitial lung disease and a defect in central tolerance.

Interstitial lung disease (ILD) is a common manifestation of systemic autoimmunity characterized by progressive inflammation or scarring of the lungs. Patients who develop these complications can exhibit significantly impaired gas exchange that may result in hypoxemia, pulmonary hypertension, and even death. Unfortunately, little is understood about how these diseases arise, including the role of specific defects in immune tolerance. Another key question is whether autoimmune responses targeting the lung parenchyma are critical to ILD pathogenesis, including that of isolated idiopathic forms. We show that a specific defect in central tolerance brought about by mutations in the autoimmune regulator gene (Aire) leads to an autoreactive T cell response to a lung antigen named vomeromodulin and the development of ILD. We found that a human patient and mice with defects in Aire develop similar lung pathology, demonstrating that the AIRE-deficient model of autoimmunity is a suitable translational system in which to unravel fundamental mechanisms of ILD pathogenesis.

G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

Alkylating agent melphalan augments the efficacy of adoptive immunotherapy using tumor-specific CD4+ T cells.

In recent years, the immune-potentiating effects of some widely used chemotherapeutic agents have been increasingly appreciated. This provides a rationale for combining conventional chemotherapy with immunotherapy strategies to achieve durable therapeutic benefits. Previous studies have implicated the immunomodulatory effects of melphalan, an alkylating agent commonly used to treat multiple myeloma, but the underlying mechanisms remain obscure. In the present study, we investigated the impact of melphalan on endogenous immune cells as well as adoptively transferred tumor-specific CD4(+) T cells in tumor-bearing mice. We showed that melphalan treatment resulted in a rapid burst of inflammatory cytokines and chemokines during the cellular recovery phase after melphalan-induced myelodepletion and leukodepletion. After melphalan treatment, tumor cells exhibited characteristics of immunogenic cell death, including membrane translocation of the endoplasmic reticulum-resident calreticulin and extracellular release of high-mobility group box 1. Additionally, there was enhanced tumor Ag uptake by dendritic cells in the tumor-draining lymph node. Consistent with these immunomodulatory effects, melphalan treatment of tumor-bearing mice led to the activation of the endogenous CD8(+) T cells and, more importantly, effectively drove the clonal expansion and effector differentiation of adoptively transferred tumor-specific CD4(+) T cells. Notably, the combination of melphalan and CD4(+) T cell adoptive cell therapy was more efficacious than either treatment alone in prolonging the survival of mice with advanced B cell lymphomas or colorectal tumors. These findings provide mechanistic insights into melphalan's immunostimulatory effects and demonstrate the therapeutic potential of combining melphalan with adoptive cell therapy utilizing antitumor CD4(+) T cells.

DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress

The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.

Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Role of TRPML and two-pore channels in endolysosomal cation homeostasis.

The transient receptor potential (TRP) channels TRPML1, TRPML2, and TRPML3 (also called mucolipins 1-3 or MCOLN1-3) are nonselective cation channels. Mutations in the Trpml1 gene cause mucolipidosis type IV in humans with clinical features including psychomotor retardation, corneal clouding, and retinal degeneration, whereas mutations in the Trpml3 gene cause deafness, circling behavior, and coat color dilution in mice. No disease-causing mutations are reported for the Trpml2 gene. Like TRPML channels, which are expressed in the endolysosomal pathway, two-pore channels (TPCs), namely TPC1, TPC2, and TPC3, are found in intracellular organelles, in particular in endosomes and lysosomes. Both TRPML channels and TPCs may function as calcium/cation release channels in endosomes, lysosomes, and lysosome-related organelles with TRPMLs being activated by phosphatidylinositol 3,5-bisphosphate and regulated by pH and TPCs being activated by nicotinic acid adenine dinucleotide phosphate in a calcium- and pH-dependent manner. They may also be involved in endolysosomal transport and fusion processes, e.g., as intracellular calcium sources. Currently, however, the exact physiological roles of TRPML channels and TPCs remain quite elusive, and whether TRPML channels are purely endolysosomal ion channels or whether they may also be functionally active at the plasma membrane in vivo remains to be determined.

Tissue-inherent fate of GPI revealed by GPI-anchored GFP transgenesis.

To clarify the fate of glycosylphosphatidylinositol (GPI) in mammals, we developed GPI-anchored enhanced green fluorescent protein (EGFP-GPI) and transgenic mice carrying this fusion construct. When it was introduced to culture cells, the EGFP-GPI protein was correctly sorted to plasma membranes and microsomes depending on GPI biosynthesis. Transgenic mice carrying EGFP-GPI were found to show a broad transgene expression. Histologically, a prominent polarized localization of EGFP-GPI protein was observed in various epithelia, the nervous system and liver and secreted from some exocrine glands, as well as non-polarized presence in non-epithelial tissues, demonstrating a tissue-inherent manner of GPI sorting.

SWI/SNF-Brg1 regulates self-renewal and occupies core pluripotency-related genes in embryonic stem cells.

The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.

Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia.

Gene expression profiling of 207 uniformly treated children with high-risk B-progenitor acute lymphoblastic leukemia revealed 29 of 207 cases (14%) with markedly elevated expression of CRLF2 (cytokine receptor-like factor 2). Each of the 29 cases harbored a genomic rearrangement of CRLF2: 18 of 29 (62%) had a translocation of the immunoglobulin heavy chain gene IGH@ on 14q32 to CRLF2 in the pseudoautosomal region 1 of Xp22.3/Yp11.3, whereas 10 (34%) cases had a 320-kb interstitial deletion centromeric of CRLF2, resulting in a P2RY8-CRLF2 fusion. One case had both IGH@-CRLF2 and P2RY8-CRLF2, and another had a novel CRLF2 rearrangement. Only 2 of 29 cases were Down syndrome. CRLF2 rearrangements were significantly associated with activating mutations of JAK1 or JAK2, deletion or mutation of IKZF1, and Hispanic/Latino ethnicity (Fisher exact test, P < .001 for each). Within this cohort, patients with CRLF2 rearrangements had extremely poor treatment outcomes compared with those without CRLF2 rearrangements (35.3% vs 71.3% relapse-free survival at 4 years; P < .001). Together, these observations suggest that activation of CRLF2 expression, mutation of JAK kinases, and alterations of IKZF1 cooperate to promote B-cell leukemogenesis and identify these pathways as important therapeutic targets in this disease.

Antiapoptotic BCL-2 is required for maintenance of a model leukemia.

Resistance to apoptosis, often achieved by the overexpression of antiapoptotic proteins, is common and perhaps required in the genesis of cancer. However, it remains uncertain whether apoptotic defects are essential for tumor maintenance. To test this, we generated mice expressing a conditional BCL-2 gene and constitutive c-myc that develop lymphoblastic leukemia. Eliminating BCL-2 yielded rapid loss of leukemic cells and significantly prolonged survival, formally validating BCL-2 as a rational target for cancer therapy. Loss of this single molecule resulted in cell death, despite or perhaps attributable to the presence of other oncogenic events. This suggests a generalizable model in which aberrations inherent to cancer generate tonic death signals that would otherwise kill the cell if not opposed by a requisite apoptotic defect(s).

A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Tetraspanin 7 (TSPAN7) expression is upregulated in multiple myeloma patients and inhibits myeloma tumour development in vivo.

BACKGROUND Increased expression of the tetraspanin TSPAN7 has been observed in a number of cancers; however, it is unclear how TSPAN7 plays a role in cancer progression. METHODS We investigated the expression of TSPAN7 in the haematological malignancy multiple myleoma (MM) and assessed the consequences of TSPAN7 expression in the adhesion, migration and growth of MM plasma cells (PC) in vitro and in bone marrow (BM) homing and tumour growth in vivo. Finally, we characterised the association of TSPAN7 with cell surface partner molecules in vitro. RESULTS TSPAN7 was found to be highly expressed at the RNA and protein level in CD138(+) MM PC from approximately 50% of MM patients. TSPAN7 overexpression in the murine myeloma cell line 5TGM1 significantly reduced tumour burden in 5TGM1/KaLwRij mice 4 weeks after intravenous adminstration of 5TGM1 cells. While TSPAN7 overexpression did not affect cell proliferation in vitro, TSPAN7 increased 5TGM1 cell adhesion to BM stromal cells and transendothelial migration. In addition, TSPAN7 was found to associate with the molecular chaperone calnexin on the cell surface. CONCLUSION These results suggest that elevated TSPAN7 may be associated with better outcomes for up to 50% of MM patients.

Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated herpesvirus.

K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.