Vav1 GEF activity is required for T cell mediated allograft rejection.

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.

Vav1 couples the T cell receptor to cAMP response element activation via a PKC-dependent pathway.

The transcription factor cAMP-responsive element binding protein (CREB) is a regulator of the expression of several genes important for lymphocyte activation and proliferation. However, the proximal signaling events leading to activation of CREB in T cells upon antigen receptor stimulation remain unknown. Here we identify a role for Vav1 in the activation of the cAMP response element (CRE), the binding site for CREB. T cell receptor (TCR)/CD28 - induced costimulation of Jurkat T cells expressing Vav1 but not a GEF-deficient mutant showed increased CRE activation (7.2+/-2.4 fold over control), whereas Vav1 downregulation by siRNA reduced activation of CRE by 2.6+/-1.3 fold. Inhibition of PKC and MEK but not p38 could reduce Vav1-mediated CRE activation, suggesting that Vav1 transmits TCR and CD28 signals to activation of CRE via PKC and ERK signaling pathways. As a consequence, downregulation of Vav1 impaired the expression of several CRE-containing genes like cyclin D1, INFgamma and IL-2, whereas overexpression of Vav1 enhanced CRE-dependent gene expression. Furthermore, cAMP-induced CRE-dependent transcription and gene expression was also modulated by Vav1, but did not require activation of PKC and the GEF function of Vav1. Our data provide insights into the signal transduction events regulating CRE-mediated gene expression in T cells, which affects T cell development, proliferation and activation. We identify Vav1 as an essential component of TCR-induced CRE activation and gene expression, which underlines the central role for Vav1 as key player for TCR signal transduction and gene expression.

Looking beyond death: a morphogenetic role for the TNF signalling pathway.

Tumour necrosis factor alpha (TNFalpha) is a pro-inflammatory mediator with the capacity to induce apoptosis. An integral part of its apoptotic and inflammatory programmes is the control of cell shape through modulation of the cytoskeleton, but it is now becoming apparent that this morphogenetic function of TNF signalling is also employed outside inflammatory responses and is shared by the signalling pathways of other members of the TNF-receptor superfamily. Some proteins that are homologous to the components of the TNF signalling pathway, such as the adaptor TNF-receptor-associated factor 4 and the ectodysplasin A receptor (and its ligand and adaptors), have dedicated morphogenetic roles. The mechanism by which TNF signalling affects cell shape is not yet fully understood, but Rho-family GTPases have a central role. The fact that the components of the TNF signalling pathway are evolutionarily old suggests that an ancestral cassette from unicellular organisms has diversified its functions into partly overlapping morphogenetic, inflammatory and apoptotic roles in multicellular higher organisms.

PtdIns(4,5)P-restricted plasma membrane localization of FAN is involved in TNF-induced actin reorganization.

The WD-repeat protein factor associated with nSMase activity (FAN) is a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades. However, the precise functional involvement of FAN in specific cellular TNF responses remain unclear. Here, we report the involvement of FAN in TNF-induced actin reorganization and filopodia formation mediated by activation of Cdc42. The pleckstrin-homology (PH) domain of FAN specifically binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P), which targets FAN to the plasma membrane. Site-specific mutagenesis revealed that the ability of FAN to mediate filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding motif, or by the disruption of intramolecular interactions between the PH domain and the adjacent beige and Chediak-Higashi (BEACH) domain. Furthermore, FAN was shown to interact with the actin cytoskeleton in TNF-stimulated cells via direct filamentous actin (F-actin) binding. The results of this study suggest that PH-mediated plasma membrane targeting of FAN is critically involved in TNF-induced Cdc42 activation and cytoskeleton reorganization.

NF-kappaB-independent down-regulation of XIAP by bortezomib sensitizes HL B cells against cytotoxic drugs.

The proteasome inhibitor bortezomib has been shown to possess promising antitumor activity and significant efficacy against a variety of malignancies. Different studies demonstrated that bortezomib breaks the chemoresistance in different tumor cells basically by altering nuclear factor-kappaB (NF-kappaB) activity. NF-kappaB has been shown to be constitutively active in most primary Hodgkin-Reed-Sternberg (H-RS) cells in lymph node sections and in Hodgkin lymphoma (HL) cell lines and was suggested to be a central molecular switch in apoptosis resistance in HL. Here we report a bimodal effect of bortezomib in HL cells. Whereas high-dose bortezomib induced direct cytotoxicity that correlated with decreased NF-kappaB activity, low-dose bortezomib sensitized HL cells against a variety of cytotoxic drugs without altering NF-kappaB action. Strikingly, bortezomib induced marked XIAP down-regulation at the posttranslational level that was independent of the NF-kappaB status. Similarly, RNA interference (RNAi)-mediated XIAP down-regulation generated susceptibility to cytostatic agents. The results identify XIAP as an NF-kappaB-independent target of bortezomib action that controls the chemoresistant phenotype of HL cells.

Acid sphingomyelinase is indispensable for UV light-induced Bax conformational change at the mitochondrial membrane.

Ultraviolet light-induced apoptosis can be caused by DNA damage but also involves immediate-early cell death cascades characteristic of death receptor signaling. Here we show that the UV light-induced apoptotic signaling pathway is unique, targeting Bax activation at the mitochondrial membrane independent of caspase-8 or cathepsin D activity. Cells deficient in acid sphingomyelinase (ASMase) do not show UV light-induced Bax activation, cytochrome c release, or apoptosis. In ASMase-deficient cells, the apoptotic UV light response is restored by stable or transient expression of human ASMase. Bax conformational change in ASMase(-/-) cells is also caused by synthetic C(16)-ceramide acting on intact cells or isolated mitochondria. The results suggest that UV light-triggered ASMase activation is essentially required for Bax conformational change leading to mitochondrial release of pro-apoptotic factors like cytochrome c and Smac.