TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members

Abstract

TNF receptor 1 signaling induces NF-κB activation and necroptosis in L929 cells. We previously reported that cellular inhibitor of apoptosis protein-mediated receptor-interacting protein 1 (RIP1) ubiquitination acts as a cytoprotective mechanism, whereas knockdown of cylindromatosis, a RIP1-deubiquitinating enzyme, protects against tumor necrosis factor (TNF)-induced necroptosis. We report here that RIP1 is a crucial mediator of canonical NF-κB activation in L929 cells, therefore questioning the relative cytoprotective contribution of RIP1 ubiquitination versus canonical NF-κB activation. We found that attenuated NF-κB activation has no impact on TNF-induced necroptosis. However, we identified A20 and linear ubiquitin chain assembly complex as negative regulators of necroptosis. Unexpectedly, and in contrast to RIP3, we also found that knockdown of RIP1 did not block TNF cytotoxicity. Cell death typing revealed that RIP1-depleted cells switch from necroptotic to apoptotic death, indicating that RIP1 can also suppress apoptosis in L929 cells. Inversely, we observed that Fas-associated protein via a death domain, cellular FLICE inhibitory protein and caspase-8, which are all involved in the initiation of apoptosis, counteract necroptosis induction. Finally, we also report RIP1-independent but RIP3-mediated necroptosis in the context of TNF signaling in particular conditions.

Figure 1

Knockdown of HOIL-1, HOIP or A20 sensitizes to TNF-induced necroptosis, whereas depletion of TRADD or NEMO does not affect the cell viability. (a) Upon knockdown of RIP1, L929 cells were stimulated with TNF for the indicated times. IκBα degradation and RIP1 knockdown efficiency was analyzed using western blotting. Asterisk indicates an nonspecific band. Flow cytometric analysis of the percentage of Sytox-positive cells after TNF stimulation for 4 h (reaching around 50% cell death in control setup) of L929 cells in which TRADD or NEMO (b), HOIL-1 or HOIP (d), or A20 was knocked down (f). Error bars indicate S.D.s of triplicates of three independent experiments. Cell survival analysis using a MTT assay after 20 h of TNF stimulation of L929 cells in which TRADD or NEMO (c), HOIL-1 or HOIP (e), or A20 was knocked down (g). Error bars indicate S.Ds of triplicates. Knockdown efficiency of the indicated proteins was determined by western blotting or RT-PCR. ^*P<0.05 (Mann–Whitney U-test)

Figure 2

FADD, caspase-8 or c-FLIP knockdown sensitizes to TNF-induced necroptosis. (a) Flow cytometric analysis of the percentage of Sytox-positive cells after 3 h of TNF stimulation (reaching around 25% cell death in control setup) and cell survival analysis using a MTT assay after 20 h of TNF stimulation of L929 cells in which FADD was knocked down. (b) Flow cytometric analysis of the percentage of Sytox-positive cells after 3 h of TNF stimulation of L929 cells in which caspase-8 or c-FLIP was knocked down. Error bars indicate S.D.s of triplicates of at least two independent experiments. (c) Cell survival analysis using a MTT assay after 20 h of TNF stimulation of L929 cells in which caspase-8 or c-FLIP were knocked down. Error bars indicate S.Ds of triplicates and data are representative of at least three independent experiments. Knockdown efficiency of the indicated proteins was determined by western blotting or RT-PCR

Figure 3

RIP1 knockdown shifts TNF-induced TRADD-independent necroptosis to TRADD-dependent apoptosis. (a) Flow cytometric analysis of the percentage of Sytox-positive cells at the indicated time points after TNF stimulation of L929 cells in which RIP1 is knocked down. Knockdown efficiency was determined by western blotting. (b) Western blot analysis of caspase-3 processing at the indicated time points after TNF stimulation of L929 cells in which RIP1 is knocked down. (c) Flow cytometric analysis of the percentage of caspase-3 activity and PI-positive cells at the indicated time points after TNF stimulation of L929pCasper3-BG cells in which RIP1 or RIP1+TRADD are knocked down. Knockdown efficiency of the indicated proteins was determined by western blotting. (d) Cells were stained with Hoechst 33242 (blue) and PI (red) for live-cell imaging and monitored for 8 h. Overlay snapshots of the fluorescent and transmitted light images at the indicated time points after TNF stimulation of L929 cells in which RIP1 is knocked down. Error bars indicate S.Ds of triplicates and data are representative of at least three independent experiments.

Figure 4

RIP3-dependent necroptosis when RIP1 and caspase-8 or TRADD are knocked down. (a) Flow cytometric analysis of the percentage of caspase-3 activity and PI-positive cells at the indicated time points after TNF stimulation of L929pCasper3-BG (FRET-based apoptosis sensor) cells in which RIP1, RIP1+caspase-8, or RIP1+caspase-8+RIP3 are knocked down. (b) Flow cytometric analysis of the percentage of caspase-3 activity and PI-positive cells at the indicated time points after TNF stimulation of L929pCasper3-BG cells in which RIP1, RIP1+TRADD and RIP1+TRADD+RIP3 are knocked down. Error bars indicate S.Ds of triplicates and data are representative of at least two independent experiments. Knockdown efficiency of the indicated proteins was determined by western blotting

Figure 5

Summary of the RNAi study of TNF-induced necroptosis in L929 cells. At TNFR1 complex II, the apoptotic proteins FADD, caspase-8 and c-FLIP suppress TNF-induced necroptosis. RIP1 knockdown shifts TNF-induced TRADD-independent necroptosis to TRADD-dependent apoptosis. The combined knockdown of RIP1 and caspase-8 blocks apoptosis but does not affect the extent of cell death. This RIP1-independent necroptotic cell death can be blocked by deleting RIP3 as well.