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

Abstract

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNFinduced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-b-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNFmediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-jB (NF-jB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-jB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation. Tumor necrosis factor (TNF) is a pleiotropic cytokine that controls a variety of cellular responses, including proliferation, differentiation, inflammatory cytokine production, survival and death. 1 TNF signals by binding and activating two cell surface receptors, TNF receptor (TNFR) 1 and TNFR2, 2 but most of its biological activities have been associated with TNFR1. 3 In most cell types, TNFR1 activation does not induce cell death but instead leads to the transcriptional upregulation of genes encoding pro-survival and pro-inflammatory molecules. 4 This function is mediated by the assembly of a plasma membranebound multiprotein signaling complex, referred to as TNFR1 complex I, 5 that contains TNF receptor-associated death domain (TRADD), receptor-interacting protein kinase 1 (RIPK1, also know...

NIK promotes tissue destruction independently of the alternative NF-κB pathway through TNFR1/RIP1-induced apoptosis

“…We recently showed that the RIP1 kinase inhibitor necrostatin-1 (Nec-1) prevented SM/TNFα-induced caspase activation. 19 Similarly, we observed that Tweak/TNFα-or agonist LTβR /TNFα-induced caspase activation was equally inhibited in the presence of Nec-1 or Z-Val-Ala-DL-Aspfluoromethylketone (z-VAD-FMK) (Figures 2c and d). We also found that Tweak/TNFα-or agonist LTβR/TNFα-induced RIP1/caspase-8 assembly was fully abrogated in the presence of Nec-1 (Figures 2e and f).…”

“…These results are consistent with previous studies demonstrating a role for Nec-1 in the prevention of SM/TNFα-induced RIP1/caspase-8 assembly and apoptosis. 19,41 We further found that the DEVDase activity induced by Tweak/TNFα co-stimulation was strongly reduced in Bax/Bak − / − double KO MEFs when compared with Bax/Bak +/+ counterparts MEFs (Supplementary Figure S2e). Likewise, treatment of Bax/ Bak − / − MEFs with Tweak and TNFα resulted in a slight alteration of caspase-8 processing but in a complete inhibition of caspase-3 cleavage when compared with Bax/ Bak +/+ counterparts control MEFs, indicating that caspase-8 processing precedes the Bax/Bak-mediated activation of the executioner caspases (Supplementary Figure S2f).…”

“…17,18 The former assembles when cells are deficient in p65 or pretreated with cycloheximide (CHX), whereas the latter forms in the absence of c-IAP1/2, in TAK1 kinase-inhibited conditions or in NEMO-depleted cells. 19,20 In addition, under caspase-repressed conditions RIP1 associates with RIP3 to mediate necroptosis, a form of programmed necrosis that takes place during viral infection or TNF alpha (TNFα)-induced systemic inflammatory response syndrome (SIRS). [21][22][23][24][25] Synthetic small molecules mimicking Smac (Smac mimetics (SMs)) emerged as tools to induce c-IAP1/2 depletion and were consequently shown to sensitize particular cancer cell lines to TNFR1-mediated RIP1-dependent death.…”

TAK1 control of cell death

“…49 In line with this, combined inhibition of caspase and RIPK3 rescues TNFa-induced cell death in Tak1-deficient cells. 58,[77][78][79] These results suggest that, although the default pathway of cell death in Tak1-deficient cells is apoptosis, Tak1 deficiency engages an alternative RIPK1-RIPK3 necroptosis pathway if the default pathway is blocked. Interestingly, even though RIPK1 was believed to be an essential mediator of RIPK3 activation, necroptosis can still be engaged with Ripk1 deletion.…”

A siRNA screen reveals the prosurvival effect of protein kinase A activation in conditions of unresolved endoplasmic reticulum stress

“…To identify genes positively involved in the ER stress-induced cell death pathway, genes providing at least 30% protection upon repression were selected and defined as 'hits' (Figures 1b and c and Supplementary Tables 1 and 2). This led us to the identification of 22 genes (Figure 1c), out of which 7 were validated by kinetic cell death analysis measuring nuclear SytoxGreen fluorescence in death cells (Fluostar Omega fluorescence plate reader, BMG Labtech, Ortenberg, Germany), as previously described 19 (Figures 1d-j and Supplementary Figure 1). Of note, two of these seven genes, Pmaip1 (Noxa) and Bad, are reported mediators of ER stress-induced death, 20,21 confirming the validity of our screen.…”