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. 2013 Oct;20(10):1381-92.
doi: 10.1038/cdd.2013.94. Epub 2013 Jul 26.

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

Y Dondelinger  1 , M A Aguileta, V Goossens, C Dubuisson, S Grootjans, E Dejardin, P Vandenabeele, M J M Bertrand
Affiliations

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

Y Dondelinger et al. Cell Death Differ. 2013 Oct.

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 TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-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 TNF-mediated 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-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-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.

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Figures

Figure 1
Figure 1
TAK1 kinase inhibition induces RIPK1 kinase-dependent apoptosis upon TNF stimulation. (a–h) Immortalized Ripk1+/+ and Ripk1−/− MEFs were pre-treated for 30 min with TAK1i or SM in the presence or absence of Nec-1 and subsequently stimulated with hTNF. The percentage of cell death (SytoxGreen fluorescence) (a–d) and caspase-3 activity (DEVD-AMC fluorescence) (e–h) was calculated in function of time using the Fluostar Omega fluorescence plate reader as indicated in the experimental procedures. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least three independent experiments. (i and j) Immortalized Ripk1+/+ and Ripk1−/− MEFs were pre-treated for 30 min with TAK1i (i) or SM (j) in the presence or absence of Nec-1 and subsequently stimulated with hTNF for 3 h. Cells were then lysed and immunoblotted as indicated
Figure 2
Figure 2
TAK1 and cIAP1/2 protect cells against TNF-mediated RIPK1-dependent apoptosis in a NF-κB-independent manner. (a–d) Control MEFs or MEFs overexpressing IκBαSR were pretreated with TAK1i (a and c) or SM (b and d) in the presence or absence of Nec-1 and subsequently stimulated with hTNF for 10 h. Cell death (a and b) and caspase-3 activity (c and d) was analyzed at the indicated time using the Fluostar Omega fluorometer. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least two independent experiments. (e–g) Control MEFs or MEFs overexpressing IκBαSR were pretreated with media (e), TAK1i (f) or SM (g) in the presence or absence of Nec-1 and subsequently stimulated with hTNF for, respectively, 4 h (e) or 2 h (f and g). Cells were then lysed and immunoblotted as indicated
Figure 3
Figure 3
TAK1 kinase inhibition induces complex IIb assembly without affecting RIPK1 ubiquitylation at complex I. (a and b) Immortalized Ripk1+/+ MEFs were pre-treated for 30 min with the indicated compounds in combination with TAK1i (a) or SM (b), followed by stimulation with hTNF for 2 h. Next, complex II was isolated by immunoprecipitation of caspase-8 and analyzed by immunoblotting. (c and d) Immortalized Ripk1+/+ MEFs were pre-treated for 30 min with TAK1i (c) or SM (d) and subsequently stimulated for 5 min with FLAG-hTNF. Complex I was immunoprecipitated and the ubiquitylation status of TNFR1-bound RIPK1 was analyzed by immunoblotting. The levels of cIAP1 in the lysates are also shown. The asterisk shows a non-specific band recognized by the anti-cIAP1 antibody. (e) Immortalized Ripk1+/+ MEFs were pretreated with TAK1i or SM and stimulated with either FLAG-hTNF for 5 min or hTNF for 2 h. Ubiquitylation status of RIP1 in the different complexes was evaluated by immunoblotting after immunoprecipitation of FLAG-hTNF for complex I or caspase-8 for complex II. (f) Immortalized Ripk1+/+ MEFs were pre-treated for 30 min with TAK1i or SM in the presence or absence of Nec-1 and subsequently stimulated with FLAG-hTNF for 5 min. Complex I was immunoprecipitated and recruitment of RIPK1 to TNFR1 was analyzed by immunoblotting
Figure 4
Figure 4
CYLD knockdown protects cells against RIPK1-dependent apoptosis induced by TNF and SM, but not by TNF and TAK1i. (a–e) Immortalized Ripk1+/+ MEFs were transfected with a control siRNA or siRNA-targeting CYLD. After 72 h, cells were pretreated with TAK1i (a and c) or SM (b and d) for 30 min followed by stimulation with hTNF. Cell death (a and b) and caspase-3 activity (c and d) were analyzed at the indicated time points using the Fluostar Omega fluorometer. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least two independent experiments. (e) Immortalized Ripk1+/+ MEFs were transfected with a control siRNA or siRNA-targeting CYLD. After 72 h, cells were pretreated with TAK1i or SM in the presence of zVAD for 30 min followed by the stimulation with hTNF for 2 h. Complex II assembly was analyzed by immunoblotting after immunoprecipitation of caspase-8
Figure 5
Figure 5
RIPK3 deficiency affects TNFR1-mediated RIPK1-dependent apoptosis. (a–d) Immortalized Ripk3+/+ and Ripk3−/− MEFs were pre-treated for 30 min with TAK1i (a and c) or SM (b and d) and subsequently stimulated with hTNF. Cell death (a and b) and caspase-3 activity (c and d) were analyzed at the indicated time points using the Fluostar Omega fluorometer. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least three independent experiments. (e and f) Immortalized Ripk3+/+ and Ripk3−/− MEFs were pre-treated for 30 min with SM (e) or TAK1i (f), followed by stimulation with hTNF for the indicated periods of time. Cells were then lysed and immunoblotted as indicated. (g) Primary Ripk3+/+ and Ripk3−/− MEFs were pre-treated for 30 min with biotinylated-IETD-fmk and SM and subsequently stimulated with hTNF for 4 h. Cells were then lysed and caspase-8 bound to biotinylated-IETD-fmk was immunoprecipitated. The levels of pro-caspase-8 in the lysates and of cleaved-caspase-8 in the immunoprecipitate were then revealed by immunoblotting. (h–l) Immortalized Ripk1+/+ MEFs were lentivirally transduced with either a control miRNA or a miRNA-targeting RIPK1 or RIPK3. (h) Repression of RIPK1 or RIPK3 protein levels was revealed by immunoblotting. (i–l) miRNA-transduced Ripk1+/+ MEFs were pre-treated for 30 min with TAK1i (i and k) or SM (j and l) and subsequently stimulated with hTNF. Cell death (i and j) or caspase-3 activity (k and l) was analyzed at the indicated time points using the Fluostar Omega fluorometer. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least two independent experiments
Figure 6
Figure 6
The kinase activity of RIPK3 and its intact RHIM domain are dispensable for TNFR1-mediated RIPK1-dependent apoptosis. (a–d) Immortalized Ripk3−/− MEFs were lentivirally reconstituted with an empty control vector (CTRL), wild-type Ripk3 (WT), kinase-dead Ripk3 (KD) or RHIM mutated Ripk3 (RHIM*). (a–d) The reconstituted Ripk3−/− MEFs were pre-treated for 30 min with SM (a) or TAK1i (b) and subsequently stimulated with hTNF. Caspase-3 activity (a and b) and cell death (d) were analyzed at the indicated time points using the Fluostar Omega fluorometer. Error bars indicate the standard deviation from triplicate samples. The results are representative of at least three independent experiments. (c) RIPK3 expression levels in Ripk3+/+ and reconstituted Ripk3−/− MEFs were compared by immunoblotting
Figure 7
Figure 7
ROS scavenging inhibits TNFR1-mediated RIPK1-dependent apoptosis. (a) Immortalized Ripk1+/+ MEFs were pre-treated for 30 min with TAK1i in the presence of Nec-1, BHA or NAC and subsequently stimulated with hTNF for 4 h. Cell death was analyzed using the Fluostar Omega fluorescence plate reader. Error bars indicate standard deviation from triplicate samples. Experiments are representative of at least three independent experiments. (b–d) Immortalized Ripk1+/+ MEFs were pre-treated for 30 min with TAK1i (b and c) or SM (d) in the presence or absence of BHA (b and d) or NAC (c). Cells were then stimulated by hTNF for the indicated time and cell lysates were analyzed by immunoblotting. (e) Immortalized Ripk1+/+ MEFs were pre-treated with the indicated compounds for 30 min and then stimulated with hTNF for 2 h. TNFR1 complex IIb was isolated by caspase-8 immunoprecipitation and analyzed by immunoblotting. (f) Immortalized Ripk1+/+ MEFs were pre-treated or not with BHA for 30 min before stimulation with FLAG-hTNF for 5 min. TNFR1 complex I was analyzed by immunoprecipitation using FLAG beads followed by immunoblotting. (g) Immortalized Ripk3+/+ and Ripk3−/− MEFs were left unstimulated or pre-treated for 30 min with TAK1i in the presence or absence of zVAD-fmk and subsequently stimulated with hTNF for 3 h. DHR-123 was added for the last 30 min of incubation at 37 °C. Cellular ROS in PI-negative cells was monitored using the BD LSRII flow cytometer. The results are normalized to the percentage of DHR123+/PI- cells detected in unstimulated Ripk3+/+ MEFs. Error bars indicate standard deviation from triplicate samples. The results are representative of two independent experiments. (h) Immortalized Ripk3+/+ and Ripk3−/− MEFs were pre-treated with TAK1i in the presence or absence of zVAD-fmk, followed by stimulation with hTNF for 2 h. TNFR1 complex IIb assembly was analyzed by immunoprecipitation of caspase-8
Figure 8
Figure 8
Model for the two cell death checkpoints in TNF-induced apoptosis. TNF stimulation leads to the assembly of the plasma membrane TNFR1 complex I. Within this complex, RIPK1 is rapidly conjugated with Ub chains. These Ub chains act as scaffolds for the recruitment and activation of the TAB2/3-TAK1 complex and the IKK complex, which subsequently leads to the activation of the canonical NF-κB signaling pathways that drive transcription of genes that prevent cell death — the first cell death checkpoint (1). Accordingly, when the NF-κB response is inhibited, either genetically or by the use of the general translation inhibitor cycloheximide (CHX), TNFR1 ligation switches from a pro-survival to a RIPK1-independent pro-apoptotic response by assembly of the cytoplasmic TNFR1 complex IIa. In addition to its role in NF-κB activation, TAK1 regulates a second cell death checkpoint at the level of complex I that prevents RIPK1 from integrating the death complex (2). Indeed, when TAK1 activity in complex I is affected, either indirectly by avoiding TAK1 recruitment through the use of Smac Mimetics (SM) — which affects RIPK1 ubiquitylation — or directly by inhibiting its kinase activity (TAK1i), TNF stimulation induces RIPK1 kinase-dependent apoptosis by assembly of the cytosolic death complex IIb. Because CYLD acts upstream of TAK1 recruitment, its repression does not protect cells from apoptosis induced by TAK1 kinase inhibition. RIPK3 contributes to full caspase-8 activation by acting downstream of complex IIb, in a kinase and intact RHIM-independent manner. ROS regulates TNF-induced RIPK1-dependent caspase activation by acting upstream of complex IIb assembly, and possibly also downstream of complex IIb via RIPK3
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