Polarity gene discs large homolog 1 regulates the generation of memory T cells.

Mammalian ortholog of Drosophila cell polarity protein, Dlg1, plays a critical role in neural synapse formation, epithelial cell homeostasis, and urogenital development. More recently, it has been proposed that Dlg1 may also be involved in the regulation of T-cell proliferation, migration, and Ag-receptor signaling. However, a requirement for Dlg1 in development and function of T lineage cells remains to be established. In this study, we investigated a role for Dlg1 during T-cell development and function using a combination of conditional Dlg1 KO and two different Cre expression systems where Dlg1 deficiency is restricted to the T-cell lineage only, or all hematopoietic cells. Here, using three different TCR models, we show that Dlg1 is not required during development and selection of thymocytes bearing functionally rearranged TCR transgenes. Moreover, Dlg1 is dispensable in the activation and proliferative expansion of Ag-specific TCR-transgenic CD4(+) and CD8(+) T cells in vitro and in vivo. Surprisingly, however, we show that Dlg1 is required for normal generation of memory T cells during endogenous response to cognate Ag. Thus, Dlg1 is not required for the thymocyte selection or the activation of primary T cells, however it is involved in the generation of memory T cells.

ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling.

Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

Vav3 regulates osteoclast function and bone mass.

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

Differential requirements for Vav proteins in DAP10- and ITAM-mediated NK cell cytotoxicity.

Natural killer (NK) cells express multiple activating receptors that initiate signaling cascades through DAP10- or immunoreceptor tyrosine-based activation motif-containing adapters, including DAP12 and FcRgamma. Among downstream signaling mediators, the guanine nucleotide exchange factor Vav1 carries out a key role in activation. However, whether Vav1 regulates only some or all NK cell-activating pathways is matter of debate. It is also possible that two other Vav family molecules, Vav2 and Vav3, are involved in NK cell activation. Here, we examine the relative contribution of each of these exchange factors to NK cell-mediated cytotoxicity using mice lacking one, two, or all three Vav proteins. We found that Vav1 deficiency is sufficient to disrupt DAP10-mediated cytotoxicity, whereas lack of Vav2 and Vav3 profoundly impairs FcRgamma- and DAP12-mediated cytotoxicity. Our results provide evidence that these three Vav proteins function specifically in distinct pathways that trigger NK cell cytotoxicity.

Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells.

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late "transitional" stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)-induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho-guanine nucleotide exchange factors.

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice.

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells.

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.

Serum microRNA expression profiling in patients with multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease that is pathologically characterized by α­synuclein positive glial cytoplasmic inclusions in oligodendrocytes. The clinical diagnosis of MSA is often challenging as there are no established biomarkers and diagnoses are now based on clinical findings alone. At present, the etiology and pathogenesis of MSA are unclear. It has been reported that dysregulation of microRNA (miRNA/miR) serves an important role in neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The miRNA profile of patients with MSA remains to be established. The present study investigated the serum miRNA expression level of 10 patients with MSA, using microarray chips including 668 miRNAs. It was identified that 50 miRNAs were significantly upregulated and 17 miRNAs were significantly downregulated in the serum of the patients with MSA. The most upregulated miRNA was miR­16, which may induce the accumulation of α­synuclein. The target genes of some miRNAs upregulated in MSA (including miR­17, 20a, 24, 25, 30d and 451) were associated with autophagy­associated molecules. The present study concluded that the expression pattern of miRNAs may be a clinical biomarker for MSA and targeting these miRNAs may provide a novel treatment for MSA.

Cytoskeletal remodeling in lymphocyte activation.

The formerly distinct fields of lymphocyte signal transduction and cytoskeletal remodeling have recently become linked, as proteins involved in transducing signals downstream of lymphocyte antigen receptors have also been implicated in actin cytoskeleton remodeling, microtubule dynamics and regulation of cell polarity. These discoveries have fuelled interest in understanding both the role of the actin cytoskeleton as an integral component of lymphocyte activation and the interplay between lymphoid cell-cell contact sites (immunological synapse), retractile pole structures (uropod, distal pole complex), and Rho-family GTPases (Rac, Rho, Cdc42), their upstream activators (Dbl-family guanine nucleotide exchange factors) and their downstream effectors (WASp, Arp2/3, ADAP). To understand how these complex regulatory networks are wired, a new breed of computational biologists uses mathematical language to reproduce and simulate signaling circuits 'in silico'.

Effects of galectin-9 on apoptosis, cell cycle and autophagy in human esophageal adenocarcinoma cells.

The incidence of esophageal adenocarcinoma (EAC) is rapidly increasing in western countries. The overall mortality of this disease remains high with a 5-year survival rate of less than 20%, despite remarkable advances in the care of patients with EAC. Galectin-9 (Gal-9) is a tandem-repeat type galectin that exerts anti-proliferative effects on various cancer cell types. The aim of the present study was to evaluate the effects of Gal-9 on human EAC cells and to assess the expression of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. Gal-9 suppressed the proliferation of the EAC cell lines OE19, OE33, SK-GT4, and OACM 5.1C. Additionally, Gal-9 treatment induced apoptosis and increased the expression levels of caspase-cleaved cytokeratin 18, activated caspase-3 and activated caspase-9. However, it did not promote cell cycle arrest by reducing cell cycle-related protein levels. Furthermore, Gal-9 increased the level of the angiogenesis-related protein interleukin-8 (IL-8) and markedly altered miRNA expression. Based on these findings, Gal-9 may be of clinical use for the treatment of EAC.

Galectin-9 suppresses the proliferation of gastric cancer cells in vitro.

Gastric cancer is the second-leading cause of cancer-related mortality worldwide, and the prognosis of advanced gastric cancer remains poor. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has recently been demonstrated to exert anti-proliferative effects on various types of cancer cells. The aim of our present study was to evaluate the effects of Gal-9 on human gastric cancer cells and the expression levels of microRNAs (miRNAs) associated with the antitumor effects of Gal-9 in vitro. In our initial experiments, Gal-9 suppressed the proliferation of gastric cancer cell lines in vitro. Our data further revealed that Gal-9 increased caspase-cleaved keratin 18 (CCK18) levels in gastric cancer cells. Additionally, Gal-9 reduced the phosphorylation of vascular endothelial growth factor receptor-3 (VEGFR-3) and insulin-like growth factor-1 receptor (IGF-1R). Furthermore, miRNA expression levels were markedly altered with Gal-9 treatment in vitro. In conclusion, Gal-9 suppressed the proliferation of human gastric cancer cells by inducing apoptosis. These findings suggest that Gal-9 could be a potential therapeutic target in the treatment of gastric cancer.

The Vav family: at the crossroads of signaling pathways.

The Vav family of Rho-guanine nucleotide exchange factors (GEFs) is thought to control a diverse array of signaling pathways emanating from antigen receptors in lymphocytes, although the exact mechanism by which Vav exerts its function is only beginning to emerge. Vav proteins are modular and contain the Dbl-homology domain, typical of all known Rho-GEFs, in addition to several other structural domains characteristic of proteins involved in signal transduction. Recently, our laboratory generated mice congenitally lacking all three Vav isoforms, providing genetic evidence that the Vav family is critical and nonredundant in T- and B-lymphocyte development and function and is essential in the formation of the adaptive immune system. These experiments also demonstrated that Vav proteins are indispensable for both T-cell receptor- and B-cell receptor-induced Ca++ fluxes. However, detailed analyses of Vav-deficient mice revealed unexpected complexity of Vav involvement in cellular activation. Notably, we observed lineage-specific Vav regulation of mitogen-activated protein kinase signaling, in which Vav was required in T-cells, but not in B-cells. Moreover, the three Vav proteins appear to function specifically in distinct signaling pathways emanating from activating receptors of natural killer cells that trigger natural cytotoxicity.

Novel mechanism of tumor suppression by polarity gene discs large 1 (DLG1) revealed in a murine model of pediatric B-ALL.

Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel "pre-leukemic" stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers.

VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans.

BACKGROUND: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. CONCLUSIONS/SIGNIFICANCE: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.

Vav proteins control MyD88-dependent oxidative burst.

The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.

An ITAM-signaling pathway controls cross-presentation of particulate but not soluble antigens in dendritic cells.

Dendritic cells (DC) possess a unique capacity for presenting exogenous antigen on major histocompatibility class I, a process that is referred to as cross-presentation, which serves a critical role in microbial and tumor immunity. During cross-presentation, antigens derived from pathogen-infected or tumor cells are internalized and processed by DCs for presentation to cytotoxic T lymphocytes (CTLs). We demonstrate that a signaling pathway initiated by the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors DAP12 and FcRgamma utilizes the Vav family of Rho guanine nucleotide exchange factors (GEFs) for processing and cross-presentation of particulate, but not soluble, antigens by DCs. Notably, this novel pathway is crucial for processing and presentation of particulate antigens, such as those associated with Listeria monocytogenes bacteria, yet it is not required for antigen uptake. Mechanistically, we provide evidence that in DCs, Vav GEFs are essential to link ITAM-dependent receptors with the activation of the NOX2 complex and production of reactive oxygen species (ROS), which regulate phagosomal pH and processing of particulate antigens for cross-presentation. Importantly, we show that genetic disruption of the DAP12/FcRgamma-Vav pathway leads to antigen presentation defects that are more profound than in DCs lacking NOX2, suggesting that ITAM signaling also controls cross-presentation in a ROS-independent manner.

Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics.

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.

Essential role for Vav1 in activation, but not development, of gammadelta T cells.

Vav1 is a guanine nucleotide exchange factor essential in the development and function of alphabeta lineage T cells. Here we report that in contrast to profound effects on pre-TCR- or alphabeta TCR-dependent events in thymocyte development, Vav1 deficiency has no detectable effect on the development of gammadelta T cells. Strikingly, however, these gammadelta T cells are markedly deficient in signaling through the gammadelta TCR, as evidenced by a lack of proliferation and cytokine production in response to stimulation with anti-gammadelta TCR antibodies. We propose that Vav1 has a unique and non-redundant role in the initiation of signaling downstream of the gammadelta TCR in lymphocytes.

Vav3 is regulated during the cell cycle and effects cell division.

Vav3 is a member of the family of guanine nucleotide exchange factors implicated in the regulation of Rho GTPases. Although the exact in vivo function of Vav3 is unknown, evidence from several studies indicates a role distinct from Vav2 or Vav1. Here we report that the expression of Vav3 is regulated during the cell cycle. Strikingly, Vav3 was transiently up-regulated in HeLa cells during mitosis, whereas enforced expression of Vav3 perturbed cytokinesis and led to the appearance of multinucleated cells. These effects of Vav3 were RhoA-dependent, required phosphorylation of the regulatory tyrosine 173, but were not enhanced by N-terminal truncations. Thus, this report establishes that expression of Vav3 is strictly regulated in a cell cycle-dependent manner and implicates Vav3 in the control of cytokinesis.