The adaptor SAP controls NK cell activation by regulating the enzymes Vav-1 and SHIP-1 and by enhancing conjugates with target cells.

The adaptor SAP, mutated in X-linked lymphoproliferative disease, has critical roles in multiple immune cell types. Among these, SAP is essential for the ability of natural killer (NK) cells to eliminate abnormal hematopoietic cells. Herein, we elucidated the molecular and cellular bases of this activity. SAP enhanced NK cell responsiveness by a dual molecular mechanism. It coupled SLAM family receptors to the kinase Fyn, which triggered the exchange factor Vav-1 and augmented NK cell activation. SAP also prevented the inhibitory function of SLAM family receptors. This effect was Fyn independent and correlated with uncoupling of SLAM family receptors from the lipid phosphatase SHIP-1. Both mechanisms cooperated to enable conjugate formation with target cells and to stimulate cytotoxicity and cytokine secretion by NK cells. These data showed that SAP secures NK cell activation by a dichotomous molecular mechanism, which is required for conjugate formation. These findings may have implications for the role of SAP in other immune cell types.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

TNF-stimulated MAP kinase activation mediated by a Rho family GTPase signaling pathway.

The biological response to tumor necrosis factor (TNF) involves activation of MAP kinases. Here we report a mechanism of MAP kinase activation by TNF that is mediated by the Rho GTPase family members Rac/Cdc42. This signaling pathway requires Src-dependent activation of the guanosine nucleotide exchange factor Vav, activation of Rac/Cdc42, and the engagement of the Rac/Cdc42 interaction site (CRIB motif) on mixed-lineage protein kinases (MLKs). We show that this pathway is essential for full MAP kinase activation during the response to TNF. Moreover, this MLK pathway contributes to inflammation in vivo.

DLG1 influences distal ureter maturation via a non-epithelial cell autonomous mechanism involving reduced retinoic acid signaling, Ret expression, and apoptosis.

The absence of Discs-large 1 (DLG1), the mouse ortholog of the Drosophila discs-large tumor suppressor, results in congenital hydronephrosis characterized by urinary tract abnormalities, reduced ureteric bud branching, and delayed disconnection of the ureter from the common nephric duct (CND). To define the specific cellular requirements for Dlg1 expression during urogenital development, we used a floxed Dlg1 allele and Pax2-Cre, Pax3-Cre, Six2-Cre, and HoxB7-Cre transgenes to generate cell type-restricted Dlg1 mutants. In addition, we used Ret(GFP) knockin and retinoic acid response element-lacZ transgenic mice to determine the effects of Dlg1 mutation on the respective morphogenetic signaling pathways. Mutation of Dlg1 in urothelium and collecting ducts (via HoxB7-Cre or Pax2-Cre) and in nephron precursors (via Pax2-Cre and Six2-Cre) resulted in no apparent abnormalities in ureteric bud branching or in distal ureter maturation, and no hydronephrosis. Mutation in nephrons, ureteric smooth muscle, and mesenchyme surrounding the lower urinary tract (via the Pax3-Cre transgene) resulted in congenital hydronephrosis accompanied by reduced branching, abnormal distal ureter maturation and insertion, and smooth muscle orientation defects, phenotypes very similar to those in Dlg1 null mice. Dlg1 null mice showed reduced Ret expression and apoptosis during ureter maturation and evidence of reduced retinoic acid signaling in the kidney. Taken together, these results suggest that Dlg1 expression in ureter and CND-associated mesenchymal cells is essential for ensuring distal ureter maturation by facilitating retinoic acid signaling, Ret expression, and apoptosis of the urothelium.

Cutting edge: cell-autonomous control of IL-7 response revealed in a novel stage of precursor B cells.

During early stages of B-lineage differentiation in bone marrow, signals emanating from IL-7R and pre-BCR are thought to synergistically induce proliferative expansion of progenitor cells. Paradoxically, loss of pre-BCR-signaling components is associated with leukemia in both mice and humans. Exactly how progenitor B cells perform the task of balancing proliferative burst dependent on IL-7 with the termination of IL-7 signals and the initiation of L chain gene rearrangement remains to be elucidated. In this article, we provide genetic and functional evidence that the cessation of the IL-7 response of pre-B cells is controlled via a cell-autonomous mechanism that operates at a discrete developmental transition inside Fraction C' (large pre-BII) marked by transient expression of c-Myc. Our data indicate that pre-BCR cooperates with IL-7R in expanding the pre-B cell pool, but it is also critical to control the differentiation program shutting off the c-Myc gene in large pre-B cells.

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.

Scaffolding proteins DLG1 and CASK cooperate to maintain the nephron progenitor population during kidney development.

DLG1 (discs-large homolog 1) and CASK (calcium/calmodulin-dependent serine protein kinase) interact at membrane-cytoskeleton interfaces and function as scaffolding proteins that link signaling molecules, receptors, and other scaffolding proteins at intercellular and synaptic junctions. Dlg1-null mice exhibit hydronephrosis, hydroureter, and occasionally hypoplastic kidneys, whereas Cask-null mice do not. To investigate whether DLG1 and CASK cooperate in the developing urogenital system, we generated mice deficient in both DLG1 and CASK either 1) globally, 2) in metanephric mesenchyme, or 3) in nephron progenitors. With each approach, Dlg1;Cask double-knockout (DKO) kidneys were severely hypoplastic and dysplastic and demonstrated rapid, premature depletion of nephron progenitors/stem cells. Several cellular and molecular defects were observed in the DKO kidneys, including reduced proliferation and increased apoptosis of cells in the nephrogenic zone and a progressive decrease in the number of cells expressing SIX2, a transcription factor essential for maintaining nephron progenitors. Fgf8 expression was reduced in early-stage DKO metanephric mesenchyme, accompanied by reduced levels of components of the Ras pathway, which is activated by fibroblast growth factor (FGF) signaling. Moreover, Dlg1(+/-);Cask(-/-) (het/null) kidneys were moderately hypoplastic and demonstrated impaired aggregation of SIX2-positive cells around the ureteric bud tips. Nephron progenitor-specific het/null mice survived with small kidneys but developed glomerulocystic kidney disease and renal failure. Taken together, these results suggest that DLG1 and CASK play critical cooperative roles in maintaining the nephron progenitor population, potentially via a mechanism involving effects on FGF signaling.

The role of SAP97 in synaptic glutamate receptor dynamics.

Proteins of the PSD-95-like membrane-associated guanylate kinase (PSD-MAGUK) family are vital for trafficking AMPA receptors (AMPARs) to synapses, a process necessary for both basal synaptic transmission and forms of synaptic plasticity. Synapse-associated protein 97 (SAP97) exhibits protein interactions, such as direct interaction with the GluA1 AMPAR subunit, and subcellular localization (synaptic, perisynaptic, and dendritic) unique within this protein family. Due in part to the lethality of the germline knockout of SAP97, this protein's role in synaptic transmission and plasticity is poorly understood. We found that overexpression of SAP97 during early development traffics AMPARs and NMDA receptors (NMDARs) to synapses, and that SAP97 rescues the deficits in AMPAR currents normally seen in PSD-93/-95 double-knockout neurons. Mature neurons that have experienced the overexpression of SAP97 throughout development exhibit enhanced AMPAR and NMDAR currents, as well as faster NMDAR current decay kinetics. In loss-of-function experiments using conditional SAP97 gene deletion, we recorded no deficits in glutamatergic transmission or long-term potentiation. These results support the hypothesis that SAP97 is part of the machinery that traffics glutamate receptors and compensates for other PSD-MAGUKs in knockout mouse models. However, due to functional redundancy, other PSD-MAGUKs can presumably compensate when SAP97 is conditionally deleted during development.

Vav family Rho guanine nucleotide exchange factors regulate CD36-mediated macrophage foam cell formation.

Lipid-laden macrophages or "foam cells" are the primary components of the fatty streak, the earliest atherosclerotic lesion. Although Vav family guanine nucleotide exchange factors impact processes highly relevant to atherogenesis and are involved in pathways common to scavenger receptor CD36 signaling, their role in CD36-dependent macrophage foam cell formation remains unknown. The goal of the present study was to determine the contribution of Vav proteins to CD36-dependent foam cell formation and to identify the mechanisms by which Vavs participate in the process. We found that CD36 contributes to activation of Vav-1, -2, and -3 in aortae from hyperlipidemic mice and that oxidatively modified LDL (oxLDL) induces activation of macrophage Vav in vitro in a CD36 and Src family kinase-dependent manner. CD36-dependent uptake of oxLDL in vitro and foam cell formation in vitro and in vivo was significantly reduced in Vav null macrophages. These studies for the first time link CD36 and Vavs in a signaling pathway required for macrophage foam cell formation.

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.

Requirement for cyclin D3 in lymphocyte development and T cell leukemias.

The D-type cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. Cyclin D3 gene is rearranged and the protein is overexpressed in several human lymphoid malignancies. In order to determine the function of cyclin D3 in development and oncogenesis, we generated and analyzed cyclin D3-deficient mice. We found that cyclin D3(-/-) animals fail to undergo normal expansion of immature T lymphocytes and show greatly reduced susceptibility to T cell malignancies triggered by specific oncogenic pathways. The requirement for cyclin D3 also operates in human malignancies, as knock-down of cyclin D3 inhibited proliferation of acute lymphoblastic leukemias deriving from immature T lymphocytes. These studies point to cyclin D3 as a potential target for therapeutic intervention in specific human malignancies.

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.

Restrictions limiting the generation of DNA double strand breaks during chromosomal V(D)J recombination.

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRbeta locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

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.

Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLCgamma2 signaling axis in mice.

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.

DLGH1 is a negative regulator of T-lymphocyte proliferation.

Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1(-/-) T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.

Discs large (Dlg1) complexes in lymphocyte activation.

T cell antigen recognition involves the formation of a structured interface between antigen-presenting and T cells that facilitates the specific transmission of activating and desensitizing stimuli. The molecular machinery that organizes the signaling molecules and controls their disposition in response to activation remains poorly understood. We show here that in T cells Discs large (Dlg1), a PDZ domain-containing protein, is recruited upon activation to cortical actin and forms complexes with early participants in T cell activation. Transient overexpression of Dlg1 attenuates basal and Vav1-induced NFAT reporter activation. Reduction of Dlg1 expression by RNA interference enhances both CD3- and superantigen-mediated NFAT activation. Attenuation of antigen receptor signaling appears to be a complex, highly orchestrated event that involves the mutual segregation of important elements of the early signaling complex.

Vav GEFs are required for beta2 integrin-dependent functions of neutrophils.

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.

Essential role of Vav family guanine nucleotide exchange factors in EphA receptor-mediated angiogenesis.

Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2-/- Vav3-/- mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.