Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos, and RasGRP1

Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.     

Cbl enforces Vav1 dependence and a restricted pathway of T cell development

Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl), an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1^-/-Cbl^-/- DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development.     

A novel pathway of human T cell activation via a 103 kD T cell activation antigen

A novel triggering signal for human proliferating and cytotoxic T lymphocytes defined by a 103 kD T cell-specific activation antigen (Tp103) is described. Tp103 is expressed on all proliferating normal T cells but is not present, or present only in low amounts, on resting peripheral blood T lymphocytes. Cross-linking of T cell and Fc receptor-positive accessory or target cells by an antibody against Tp103 leads to activation of the T cell. The proliferative response is due to an autocrine IL 2-dependent mechanism and can be inhibited by antibodies against the IL 2 receptor or by Cyclosporin A. Resting Tp103-positive T cells also respond to anti-Tp103. Although Tp103 is not linked to the antigen receptor/T3 complex, triggering via Tp103 can be inhibited by modulation of the T3 molecule. Thus, Tp103 defines a new antigen-independent pathway of T cell activation that can be regulated via other T cell surface structures.     

NR6A1 couples with cAMP response element binding protein and regulates vascular smooth muscle cell migration

Vascular smooth muscle cell (VSMC) migration is implicated in atherosclerosis and restenosis. Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is involved in regulating embryonic stem cell differentiation, reproduction, neuronal differentiation. Functional cooperation between cAMP response element modulator tau (CREMtau) and NR6A1 can direct gene expression in cells. cAMP response element binding protein (CREB) plays a key role in VSMC migration. In this study, we sought to determine whether CREB involved in NR6A1-modulated VSMC migration. VSMCs treated with platelet-derived growth factor-BB (PDGF-BB) displayed reduced mRNA and protein levels of NR6A1. Adenovirus-mediated expression of NR6A1 (Ad-NR6A1) could inhibit PDGF-BB- and serum-induced VSMC migration. The mRNA and protein expressions of secreted phosphoprotein 1 (SPP1) were down-regulated by NR6A1 overexpression. SPP1 promoter reporter activity was repressed by NR6A1. NR6A1 was found to physically couple with nuclear actin and the large subunit of RNA polymerase II. Furthermore, we showed that CREB interacted with NR6A1 in VSMCs. NR6A1 overexpression repressed cAMP response element (CRE) activity. ChIP assay revealed that NR6A1 bind to SPP1 promoter. Luciferase reporter assay showed that NR6A1 regulated SPP1 promoter activity via a putative CRE site. Adenovirus mediated local NR6A1 gene transfer attenuated stenosis after balloon-induced arterial injury in Sprague–Dawley rats. Taken together, this study provided experimental evidence that NR6A1 modulated SPP1 expression via its binding with CREB protein in VSMCs. We also revealed a NR6A1-CREB-SPP1 axis that serves as a regulatory mechanism for atherosclerosis and restenosis.     

Cholera toxin inhibits resting human T cell activation via a cAMP-independent pathway

The catalytic subunit of cholera toxin (CT) can chemically modify the alpha polypeptides of certain G-binding proteins and thus alter their function. In order to study the involvement of CT-sensitive G proteins in T cell activation, we have utilized CT in an in vitro system in which purified, resting human peripheral T cells are activated by anti-CD3 antibodies and rIL-2. Perturbation of the TCR/CD3 molecular complex by anti-CD3 antibodies causes changes in membrane phospholipids and induces a rise in cytoplasmic Ca2+. These events, however, are insufficient to allow progression into cellular proliferation and addition of IL-2 is required. Under these conditions, treatment of cells with a low concentration of CT (2 ng/ml) causes a significant inhibition of the anti-CD3-induced calcium event as well as the anti-CD3 plus IL-2-stimulated proliferation. Under our experimental conditions, inhibition of both proliferation and intracellular Ca2+ elevation by CT requires the involvement of the TCR/CD3 complex. This is supported by the observation that the toxin does not inhibit either the proliferation triggered by ionomycin and PMA or the Ca2+ influx induced by the ionophore. These data suggest that in TCR/CD3-mediated T cell activation CT acts at a point between TCR/CD3 perturbation and the generation of intracellular Ca2+. In view of the ability of CT to activate the alpha subunit of the G protein that stimulates adenyl cyclase (G alpha s), it is possible that the effect of CT on T cells is secondary to intracellular elevation of cAMP. However, measurement of cAMP levels both early after CT addition and at later time points, when proliferation is maximal, reveals lack of cyclic nucleotide accumulation. The presented data are consistent with the interpretation that the CT-mediated inhibition is caused by the modification of a G-binding protein that is either directly or indirectly associated with triggering of T cells via the TCR/CD3 molecular complex. The data also suggest that this protein is not G alpha s and it probably represents an as yet unidentified moiety or one of the several G proteins that have been recently described as regulators of phospholipase C activation.     

Phosphatidylinositide 3-kinase priming couples c-FLIP to T cell activation

Cellular FLICE (FADD-like interleukin-1-beta-converting enzyme)-inhibitory protein (c-FLIP) inhibits death receptor-induced apoptosis by binding to FADD (Fas-associated death domain protein) and pro-caspase-8. c-FLIP has also been shown to transmit activation signals and to enhance interleukin (IL)-2 production. However, c-FLIP-mediated T cell activation is difficult to detect in most cells. We found that in DO11.10 T cells, c-FLIP expression led to inhibition of IL-2 production, in contrast to the readily detectable c-FLIP-induced activation in Jurkat cells. A direct comparison revealed that distinct signal pathways were regulated by c-FLIP in Jurkat cells and DO11.10 cells. We investigated whether constitutively activated phosphatidylinositide 3-kinase (PI3K) in Jurkat cells stimulated c-FLIP. Inhibition of PI3K in Jurkat cells abrogated a c-FLIP-mediated increase in IL-2 production. In addition, c-FLIP coordinated with active PI3K for ERK activation. Furthermore, introduction of PTEN back into Jurkat cells eliminated the stimulatory effect of c-FLIP on IL-2 production and ERK activation. Our results suggest that priming with PI3K promotes the coupling of c-FLIP to T cell activation.     

Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.     

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

Role of the stress kinase pathway in signaling via the T cell costimulatory receptor 4-1BB.

4-1BB is a member of the TNFR superfamily expressed on activated CD4+ and CD8+ T cells. 4-1BB can costimulate IL-2 production by resting primary T cells independently of CD28 ligation. In this study, we report signaling events following 4-1BB receptor aggregation using an Ak-restricted costimulation-dependent T cell hybridoma, C8.A3. Aggregation of 4-1BB on the surface of C8.A3 cells induces TNFR-associated factor 2 recruitment, which in turn recruits and activates apoptosis signal-regulating kinase-1, leading to downstream activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). 4-1BB ligation also enhances anti-CD3-induced JNK/SAPK activation in primary T cells. Overexpression of a catalytically inactive form of apoptosis signal-regulating kinase-1 in C8.A3 T cells interferes with activation of the SAPK cascade and with IL-2 secretion, consistent with a critical role for JNK/SAPK activation in 4-1BB-dependent IL-2 production. Given the ability of both CD28 and 4-1BB to induce JNK/SAPK activation, we asked whether hyperosmotic shock, another inducer of this cascade, could function to provide a costimulatory signal to T cells. Osmotic shock of resting primary T cells in conjunction with anti-CD3 treatment was found to costimulate IL-2 production by the T cells, consistent with a pivotal role for JNK/SAPK in T cell costimulation.     

p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway

Clearance of fibrin through proteolytic degradation is a critical step of matrix remodeling that contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis, and pulmonary disease. However, the molecular mechanisms that regulate fibrin deposition are not known. Here, we report that the p75 neurotrophin receptor (p75^NTR), a TNF receptor superfamily member up-regulated after tissue injury, blocks fibrinolysis by down-regulating the serine protease, tissue plasminogen activator (tPA), and up-regulating plasminogen activator inhibitor-1 (PAI-1). We have discovered a new mechanism in which phosphodiesterase PDE4A4/5 interacts with p75^NTR to enhance cAMP degradation. The p75^NTR-dependent down-regulation of cAMP results in a decrease in extracellular proteolytic activity. This mechanism is supported in vivo in p75^NTR-deficient mice, which show increased proteolysis after sciatic nerve injury and lung fibrosis. Our results reveal a novel pathogenic mechanism by which p75^NTR regulates degradation of cAMP and perpetuates scar formation after injury.