cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Abstract

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.

Figure 1

BV6 treatment sensitizes cells to necrosis induced by TNF, but not to necrosis induced by anti-Fas, poly(I:C), or H₂O₂. (a) L929 cells were treated with 1 μM BV6 for the indicated durations. Cells were lysed and cIAP1/2 and XIAP were immunoblotted. (b) L929 cells were pre-treated with IFNβ, zVAD-fmk, or medium in the presence of BV6, and then stimulated with the mentioned triggers for the indicated times. Cell death (% Sytox positivity) was analyzed by flow cytometry. Data are representative of three independent experiments. Error bars represent standard deviation. (c) L929 cells were pre-treated with BV6 and stimulated with a serial dilution of TNF. After 20 h, cell viability was determined by an MTT assay. Error bars represent standard deviations of triplicates. (d) L929 cells were pre-treated with BV6 and stimulated with TNF. Cell lysates were made, and processed caspase-3 was checked by western blot. Stimulation with anti-Fas was included as a positive control for apoptosis. Cell death was determined by flow cytometry. (e) FADD−/− Jurkat cells were pre-treated with BV6 and stimulated with TNF for 24 h. Cell death (PI positivity) was analyzed by flow cytometry. Levels of cIAP1, cIAP2 and XIAP were checked on western blot. ^*P<0.05; ^**P<0.01 (Mann–Whitney U-test)

Figure 2

Loss of cIAP1 sensitizes L929 cells to TNF-induced necrosis. (a) Protein levels of cIAP1, cIAP2, and XIAP were repressed by using RNAi. Knockdown efficiency was checked by western blot. Detection of cIAP2 is below detection limit. (b) mRNA levels of cIAP2 were tested using RT-PCR. cDNA from macrophages was included as a positive control. (c) L929 cells with repressed levels of cIAP1, cIAP2, or XIAP were stimulated with TNF (10 000 IU/ml) for 2, 4, or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Results are representative of at least three independent experiments. Error bars indicate standard deviation. ^***P<0.001 (Mann–Whitney U-test)

Figure 3

cIAP1 repression induces RIP1 kinase activity. (a) L929 cells were pre-treated with 10 μM Nec-1 and 1 μM BV6 for 2 h and stimulated with TNF for 4 h. (b) RIP3 levels in L929 were reduced by using RNAi. After 72 h, cells were pre-treated with BV6 and stimulated with TNF for 6 h. Results are representative of at least three independent experiments. Error bars indicate standard deviations. ^*P<0.05; ^**P<0.01 (Mann–Whitney U-test). (c) L929 cells were pre-treated with BV6 in the presence or absence of Nec-1. Next, cells were stimulated with TNF and lysed. ERK activation (phosphorylated ERK) was checked by western blot. (d) cIAP1 and RIP1 levels were repressed using RNAi in L929 cells. Next, ERK phosphorylation was detected after TNF stimulation. Knockdown efficiency was checked by western blot. (e) L929 cells were pre-treated with BV6 and stimulated with TNF. (f) After TNF stimulation, RIP1 was immunoprecipitated and samples were treated with λ-phosphatase. (g) L929 cells were pre-treated with BV6 and Nec-1, followed by TNF stimulation. RIP1 protein levels were visualized using western blot

Figure 4

cIAP1 and TAK1 regulate the formation of the necrosome complex. (a) TAK1 levels in L929 were repressed by using RNAi. After 72 h, cells were pre-treated with Nec-1 and triggered with TNF. (b) L929 cells were pre-treated with Nec-1 (10 μM) and TAK1 kinase inhibitor 5Z-7 (1 μM) for 1 h, and then stimulated with TNF (10 000 IU/ml) for 2 h. (c) CYLD levels were reduced in L929 cells as described above, and then stimulated with TNF for 4 or 6 h. Cell death (% Sytox positivity) was analyzed by flow cytometry. Knockdown efficiency was checked by western blot. Results are representative of at least three independent experiments. Error bars indicate standard deviations. ^*P<0.05; ^***P<0.001 (Mann–Whitney U-test). (d and e) L929 cells were pre-treated with BV6 or 5Z-7, followed by TNF stimulation. FADD was immunoprecipitated and immunoblotted

Figure 5

cIAP1 and TAK1 depletion augments RIP1/RIP3-dependent ROS generation induced by TNF. (a) L929 cells were pre-treated with BV6 and Nec-1, and then stimulated with TNF (10 000 IU/ml). (b) RIP3 levels in L929 cells were reduced for 72 h using RNAi, and then treated with BV6 and TNF. Protein levels of (c) cIAP1 or XIAP and (d) TAK1 or CYLD were reduced by using RNAi, followed by TNF stimulation. (e) L929 cells were pre-treated with BV6 and 100 μM BHA. (f) p22phox (encoded by the Cyba gene) and Ndufb8 levels were repressed using RNAi, followed by BV6 treatment and TNF stimulation. Knockdown efficiency was checked by western blot or RT-PCR. Cell death (% Sytox positivity) and ROS generation (ΔMFI (DHR123)) were analyzed simultaneously by flow cytometry. Results are representative of at least two independent experiments. Error bars indicate standard deviations