Analysis of the mechanism of TNF receptor-mediated NF-kB activation.

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1. To investigate the functional role of TRAF5 in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-kB or JNK/SAPK by TNF, CD27, and CD40 was not abrogated in traf5^<-/-> mice.However, traf5^<-/-> B cells showed defects in proliferation and upregulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. CDZ7-mediated costimulatory signal was also impaired in traf5^<-/-> T cells. Collectively, these results demonstrated that TRAF5 is involved in CD40- and CD27- mediated signaling.2. To investigate the function of TRAF2 and TRAF5 further, we have generated TRAF2- and TRAF5-double deficient mice. TNF-induced nuclear translocation of NF-kB was severely impaired in murine embryonic fibroblasts (MEFs) derived from traf2^<-/-> traf5^<-/-> (DKO) mice, while IL-1-induced nuclear translocation of NF-kB was not impaired.Moreover, viability of MEFs from DKO mice, but not from traf2^<-/-> mice, dramatically reduced in the presence of TNF alone. Collectively, these results indicate that both TRAF2 and TRAF5 are implicated in TNF-induced NF-kB activation and also transmit anti-apoptotic signals.3. To investigate the molecular mechanism of increased sensitivity of DKO MEFs to TNF-induced cell death, we examined the expression level of various apoptosis-related genes. Among various genes investigated, induction of A1/Bf1-1, a member of the anti-apoptotic Bc1-2 family, was severely impaired in DKO MEFs compared to wild-type ones. However, stable transfection of A1/Bf1-1 into DKO MEFs only partially inhibited TNF-induced cell death. A molecule other than A1/Bf1-1 might be critically involved in protection against TNF-induced cell death.