Glucocorticoid-induced TNF receptor, a costimulatory receptor on naive and activated T cells, uses TNF receptor-associated factor 2 in a novel fashion as an inhibitor of NF-kappa B activation

Glucocorticoid-induced TNFR (GITR) has been implicated as an essential regulator of immune responses to self tissues and pathogens. We have recently shown that GITR-induced cellular events promote survival of naive T cells, but are insufficient to protect against activation-induced cell death. However, the molecular mechanisms of GITR-induced signal transduction that influence physiologic and pathologic immune responses are not well understood. TNFR-associated factors (TRAFs) are pivotal adapter proteins involved in signal transduction pathways of TNFR-related proteins. Yeast two-hybrid assays and studies in HEK293 cells and primary lymphocytes indicated interactions between TRAF2 and GITR mediated by acidic residues in the cytoplasmic domain of the receptor. GITR-induced activation of NF-kappaB is blocked by A20, an NF-kappaB-inducible protein that interacts with TRAFs and functions in a negative feedback mechanism downstream of other TNFRs. Interestingly, in contrast with its effects on signaling triggered by other TNFRs, our functional studies revealed that TRAF2 plays a novel inhibitory role in GITR-triggered NF-kappaB activation.     

Tumor necrosis factor receptor (TNFR)-associated factor 5 is a critical intermediate of costimulatory signaling pathways triggered by glucocorticoid-induced TNFR in T cells

Tumor necrosis factor receptor (TNFR) family members such as glucocorticoid-induced TNFR (GITR) control T cell activation, differentiation, and effector functions. Importantly, GITR functions as a pivotal regulator of physiologic and pathologic immune responses by abrogating the suppressive effects of T regulatory cells and costimulating T effector cells. However, the molecular mechanisms underlying GITR-triggered signal transduction pathways remain unclear. Interestingly, GITR-induced stimulation of TNFR-associated factor (TRAF) 5-deficient T cells resulted in decreased activation of nuclear factor kappaB as well as the mitogen-activated protein kinases p38 and extracellular signal-regulated protein kinase, whereas activation of c-Jun N-terminal kinase was less affected. Consistent with impaired signaling, costimulatory effects of GITR were diminished in TRAF5-/- T cells. In sum, our studies indicate that TRAF5 plays a crucial role in GITR-induced signaling pathways that augment T cell activation.     

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.     

Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways

Tumor necrosis factor (TNF)-alpha-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-alpha-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-alpha engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-kappaB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-beta-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-alpha-induced activation of NF-kappaB and MAPKs was unaffected by methyl-beta-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-alpha-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-alpha-induced activation of RhoA.     

Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis

The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4(+)CD25(+) T regulatory (T(R)) cells and regulates their suppressive phenotype. Accordingly, we show that anti-GITR mAb treatment of SJL mice with proteolipid protein 139-151-induced experimental autoimmune encephalomyelitis significantly exacerbated clinical disease severity and CNS inflammation, and induced elevated levels of Ag-specific T cell proliferation and cytokine production. Interestingly, prior depletion of T(R) cells failed to result in exacerbated experimental autoimmune encephalomyelitis suggesting alternative targets for the anti-GITR mAb treatment. Importantly, naive CD4(+)CD25(-) T cells up-regulated GITR expression in an activation-dependent manner and anti-GITR mAb treatment enhanced the level of CD4(+) T cell activation, proliferation, and cytokine production in the absence of T(R) cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates T(R) cells and increases CD4(+)CD25(-) T cell effector function, thus enhancing T cell immunity.     

Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.     

PI3-K/AKT regulation of NF-kappaB signaling events in suppression of TNF-induced apoptosis

We found that in MCF-7 breast carcinoma cells, PI3K and Akt suppressed a dose-dependent induction of apoptosis by tumor necrosis factor alpha (TNF). PI3K and Akt stimulated NF-kappaB activation in a dose-dependent manner, suggesting a common link between these two pathways. TNF has been shown to activate both an apoptotic cascade, as well as a cell survival signal through NF-kappaB. PI3K and AKT cell survival signaling were correlated with increased TNF-stimulated NF-kappaB activity in MCF-7 cells. We demonstrate that while both TNFR1 and NIK are partially involved in Akt-induced NF-kappaB stimulation, a dominant negative IkappaBalpha completely blocked Akt-NF-kappaB cross-talk. PI3K-Akt signaling activated NF-kappaB through both TNFR signaling-dependent and -independent mechanisms, potentially representing a mechanism by which Akt functions to suppress apoptosis in cancer.     

TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cell survival through RAF activation of NF-kappaB

Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-kappaB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-kappaB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

Tumor necrosis factor receptor 2 (TNFR2) signaling is negatively regulated by a novel, carboxyl-terminal TNFR-associated factor 2 (TRAF2)-binding site

Tumor necrosis factor (TNF) superfamily receptors typically induce both NF-kappaB and JNK activation by recruiting the TRAF2 signal transduction protein to their cytoplasmic domain. The type 2 TNF receptor (TNFR2), however, is a poor activator of these signaling pathways despite its high TRAF2 binding capability. This apparent paradox is resolved here by the demonstration that TNFR2 carries a novel carboxyl-terminal TRAF2-binding site (T2bs-C) that prevents the delivery of activation signals from its conventional TRAF2-binding site (T2bs-N). T2bs-C does not conform to canonical TRAF2 binding motifs and appears to bind TRAF2 indirectly via an as yet unidentified intermediary. Specific inactivation of T2bs-N by site-directed mutagenesis eliminated most of the TRAF2 recruited to the TNFR2 cytoplasmic domain but had no effect on ligand-dependent activation of the NF-kappaB or JNK pathways. By contrast, inactivation of T2bs-C had little effect on the amount of TRAF2 recruited but greatly enhanced ligand-dependent NF-kappaB and JNK activation. In wild-type TNFR2 therefore, T2bs-C acts in a dominant fashion to attenuate signaling by the intrinsically more active T2bs-N but not by preventing TRAF2 recruitment. This unique uncoupling of TRAF2 recruitment and signaling at T2bs-N may be important in the modulation by TNFR2 of signaling through coexpressed TNFR1.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Glucocorticoid-induced TNFR-related protein lowers the threshold of CD28 costimulation in CD8+ T cells

CD28 is well characterized as a costimulatory molecule in T cell activation. Recent evidences indicate that TNFR superfamily members, including glucocorticoid-induced TNFR-related protein (GITR), act as costimulatory molecules. In this study, the relationship between GITR and CD28 has been investigated in murine CD8(+) T cells. When suboptimal doses of anti-CD3 Ab were used, the absence of GITR lowered CD28-induced activation in these cells whereas the lack of CD28 did not affect the response of CD8(+) T cells to GITR costimulus. In fact, costimulation of CD28 in anti-CD3-activated GITR(-/-) CD8(+) T cells resulted in an impaired increase of proliferation, impaired protection from apoptosis, and an impaired rise of activation molecules such as IL-2R, IL-2, and IFN-gamma. Most notably, CD28-costimulated GITR(-/-) CD8(+) T cells revealed lower NF-kappaB activation. As a consequence, up-regulation of Bcl-x(L), one of the major target proteins of CD28-dependent NF-kappaB activation, was defective in costimulated GITR(-/-) CD8(+) T cells. What contributed to the response to CD28 ligation in CD8(+) T cells was the early up-regulation of GITR ligand on the same cells, the effect of which was blocked by the addition of a recombinant GITR-Fc protein. Our results indicate that GITR influences CD8(+) T cell response to CD28 costimulation, lowering the threshold of CD8(+) T cell activation.     

CD8 T cell-intrinsic GITR is required for T cell clonal expansion and mouse survival following severe influenza infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-x(L). In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphocyte survival, they clearly have nonredundant functions in protection from severe infection.