In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

Abstract

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-B, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1؊/؊ cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1 ؊/؊ cells treated with TNF.Although TRAF2 levels remained constant in TNF-treated wildtype cells, TNF stimulation of RIPK1 ؊/؊ cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-B2 to p52, a decrease in levels of cFLIP L , and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.In mouse embryonic fibroblasts (MEFs), 2 TNFR1 signaling is mediated by binding of the adaptor protein TRADD to its cytoplasmic domains. TRADD then recruits TRAF2 and RIPK1. TRAF2 binds to cIAP1 and cIAP2, which mediate ubiquitylation of RIPK1 (3-5). A series of ubiquitylation and phosphorylation events follow, leading to activation of TAK1, p65/RelA NF-B, and JNK (6 -8). When pro-survival signals mediated by NF-B are blocked, for example by deletion of genes for p65/ RelA, or addition of the translation inhibitor cycloheximide, ligation of TNFR1 results in recruitment of FADD, binding and activation of caspase-8, and apoptosis.Ligation-induced recruitment of RIPK1 to TNFR1 was thought to be necessary for activation of the IKK complex that phosphorylates IkB prior to its ubiquitylation by SCF BTrCP . In this way, RIPK1 was proposed to promote cell survival by allowing activation of the canonical NF-B. Consistent with this model, gene deletion of RIPK1 sensitizes cells to killing by TNF in combination with low doses of cycloheximide (2). However, we have recently shown that RIPK1 is not essential for activation of canonical p65/RelA NF-B in response to TNF, but plays a redundant role (1). This suggests that the survival effect of RIPK1 in response to TNF is mediated by some other mechanism.Like RIPK1 Ϫ/Ϫ cells, those deficient for TRAF2 are also more likely to die than wild-type MEFs when treated with low doses of cycloheximide and TNF. This sensitization was shown to be due to loss of function of the RING domain of TRAF2 (9). The fact that RIPK1 has been shown to interact directly with TRAF2 raised the possibility that they act in concert to inhibit cell death in response to TNF, but to date this has not been confirmed, and the mechanism has not been elucidated (10).We reasoned that the ability of TRAF2 and RIPK1 to promote survival of TNF-treated cells might be related. To test this hypothesis we compared TRAF2 Ϫ/Ϫ and RIPK1 Ϫ/Ϫ genedeleted cells. Here we show that RIPK1 prevents activatio...

RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

“…Of note, at the concentrations used, cell death occurred more rapidly with the TAK1 kinase inhibitor than with the SM. As negative control, we treated the MEFs with TNF in the presence of low concentration of CHX (TNF þ CHX) and, as previously reported, 13 observed that Ripk1 À / À MEFs were not protected but sensitized to this apoptotic trigger (Supplementary Figure S2). Interestingly, we found that inhibiting RIPK1 kinase activity by Nec-1-protected Ripk1 þ / þ MEFs to either TNF þ TAK1i or TNF þ SM as efficiently as the RIPK1 deficiency itself (Figures 1c and d, Supplementary Figures S1A and B).…”

“…5,11 Under these conditions, TNF-mediated death was shown not to depend on RIPK1. 12,13 cIAP1/2 are required for TNF-induced canonical NF-kB activation. 10,[14][15][16] Consequently, their depletion, obtained either genetically or by the use of Smac Mimetics (SM), also induces a switch to apoptosis.…”

TRAF2 is a biologically important necroptosis suppressor

“…In the absence of Z-VAD (TC), TRAF2 KO MEFs showed stronger TNFα-induced cell death and casapse-8 activation than did WT MEFs (Figures 1b and c), consistent with earlier evidence that TRAF2 suppresses cell-extrinsic apoptotic signaling. 34,40 TRAF2 siRNA knockdown in WT MEFs also enhanced TNFα-induced necroptosis (Figure 1d and Supplementary Figure 1C). Knockdown of RIPK1, RIPK3 or MLKL attenuated TCZ-induced death in TRAF2 KO MEFs, suggesting that TRAF2 regulates necroptosis via the (Figure 2b and Supplementary Figure 2A).…”

“…31,33,41,47 TRAF2 inhibits TNFα-induced apoptosis independent of its role in NF-κB activation. 40,48 TRAF2 also suppresses death-receptor-mediated apoptosis by promoting K48-ubiquitination and proteasomal degradation of activated caspase-8. 34 The present findings demonstrate that TRAF2 plays an additional role in curbing necroptotic TNFα signaling by acting both upstream and downstream of the necrosome.…”

IAPs: Guardians of RIPK1

“…While caspase-8-and FADD-deficient mice die at embryonic stage 10.5, they are rescued, at least in part, by co-deletion of RIPK1 and RIPK3. 52,83,84 Therefore, caspase-8 appears to be required to suppress caspase-independent necroptosis mediated by RIPK3.…”