The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay - PubMed (original) (raw)
The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay
Seda Çöl Arslan et al. PLoS One. 2011.
Abstract
Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. TAK1 impairs ROS-mediated cell death triggered by catalytically active RIP1.
(A) Left panel TAK1 KO MEFs transfected with a control siRNA (siC) or with RIP1 targeting siRNAs were stimulated with TNFα and cell survival was analyzed by crystal violet staining (CV). The bar graphs depict mean values ± SEM %. Right panel Cell lysates were analyzed by western blotting (WB) with α-RIP1 and α-NEMO antibodies to control the efficiency of RIP1 down-regulation. (B) TAK1 KO MEFs were transfected as in (A) and stimulated with TNFα. Intracellular ROS accumulation was analyzed by flow cytometry. (C) TAK1 KO MEFs were treated with DMSO or Necrostatin-1 (Nec-1) for 1 hour and then stimulated with TNFα. Butylated hydroxyanisole (BHA) was added together with TNFα. Cell survival was analyzed by CV. The bar graphs depict mean values ± SEM %. (D) TAK1 KO MEFs were treated with DMSO, Nec-1 or BHA as in (C) and stimulated with TNFα for indicated time points. ROS accumulation was analyzed as in (B).
Figure 2. TNFα-induced caspase activity in TAK1 deficient cells is not essential for ROS-mediated cytotoxicity.
(A) TAK1 KO and WT MEFs were stimulated with TNFα as indicated and analyzed by WB with the indicated antibodies to detect IκBα degradation and the cleavage of RIP1, caspase-8 (p18) and caspase-3 (p15). (B) TAK1 KO MEFs were pre-treated with DMSO or zVAD for 1 hour, stimulated with TNFα and analyzed by WB for cleaved caspase-8, caspase-3 and IKKβ. (C) TAK1 KO MEFs were pre-treated as in (B) and stimulated with TNFα. Cell survival was analyzed by CV. The bar graphs depict mean values ± SEM %. (D) TAK1 KO MEFs were pre-treated as in (B) and stimulated with TNFα. ROS accumulation was evaluated by flow cytometry. (E) TAK1 KO MEFs were pre-treated with DMSO or Nec-1 for 1 hour, stimulated with TNFα and analyzed as in (B). BHA was added together with TNFα. (*) denotes the cleaved fragments of caspase-3 (p15) and RIP1.
Figure 3. TAK1 prevents TNFα-induced necrosome formation and stabilizes polyubiquitinated RIP1 in complex-I.
(A) TAK1 KO MEFs were stimulated with TNFα and lysed for immunoprecipitation with an α-FADD antibody to isolate the necrosome complex. Co-immunoprecipitated proteins were analyzed by WB with the indicated antibodies. (B) TAK1 KO MEFs were pre-treated with DMSO, Nec-1 or zVAD for 60 minutes and then stimulated with TNFα. Cell lysates were analyzed by WB as in (A). (C) WT and TAK1 KO MEFs were stimulated with Flag-TNFα for indicated time points. TNFα-bound TNF-RI and additional complex-I members were immunoprecipitated by α-Flag M2 affinity beads and analyzed by WB. Ubi-RIP1 depicts complex-I-associated polyubiquitinated RIP1. (*) denotes the p43 fragment of caspase-8 and cFLIPL. n.s, non-specific band.
Figure 4. TAK1 blocks TNFα-induced necrosis independently of its kinase activity.
(A) TAK1 KO MEFs transfected with a WT or a kinase-dead mutant (K63W) construct of TAK1 along with untransfected and empty-vector (EV) transfected cells were stimulated with TNFα and analyzed by WB for IκBα degradation as well as TAK1 and NEMO expression. (B) Cells were stimulated with Flag-TNFα and Ubi-RIP1 interaction with complex-I was analyzed by immunoprecipitating Flag-TNFα-bound TNF-RI and associated factors by α-Flag M2 affinity beads as in Fig. 3C. (C) Indicated cells were stimulated with TNFα and necrosome formation was investigated by immunoprecipitating FADD as in Fig. 3A and 3B. (D) Cells were stimulated with TNFα and cell survival was analyzed by CV. The bar graphs depict mean values ± SEM %. (E) Cells were stimulated with TNFα and caspase activation was assessed by WB analysis. (F) Cells were treated with TNFα and intracellular ROS accumulation was analyzed by flow cytometry. (*) denotes the p43 fragment of caspase-8 and cFLIPL. (#) denotes cleaved caspase-3 (p15). n.s., non-specific band.
References
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