Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

Abstract

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

Figure 1

Transient knockdown of components along the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis in L929sahFas cells. Knockdown of RIPK1, RIPK3 or MLKL causes a switch from TNF-induced necroptosis to apoptosis. Seventy-two hours after knockdown of L929sahFas cells with the indicated siRNAs, cells were stimulated in triplicate with TNF (10 000 U/ml) or in duplicate with agonistic anti-Fas Ab (250 ng/ml). (a) The 6-h time point is shown±S.E.M (n=3). Cell death and caspase activity were determined as described in Materials and Methods. *P<0.05 and **P<0.01. (b) The 12-h time point is shown±S.E.M (n=3). Cell death and caspase activity were determined as described in Materials and Methods. *P<0.05. (c) Seventy-two hours after knockdown of murine RIPK1, RIPK3, Drp1 or Parkin, the knockdown efficiency of these targets was validated by western blotting, and (d) 72 h after knockdown of murine PGAM5, MLKL and Pink1, the knockdown efficiency of these targets was validated by RT-PCR because detection by antibody was unreliable

Figure 2

Knockdown of RIPK3 or MLKL results in delayed cell death that switches from TNF-induced necroptosis to a delayed, Nec-1 sensitive apoptosis. (a) Seventy-two hours after knockdown of L929sahFas cells with the indicated siRNAs, cells were stimulated with TNF (10 000 U/ml) or agonistic anti-Fas Ab (250 ng/ml) in the presence of 1 μg/ml HOECHST and 3 μg/ml propidium iodide (PI). Imaging was performed on a BDPathwayTM 855 (n=2). Chromatin condensation is indicated with arrows and plasma membrane blebbing with arrowheads. Scale bar indicates 20 μm. (b) Seventy-two hours after knockdown of L929sahFas cells with the indicated siRNAs, cells were pretreated with 10 μM necrostatin-1 (N1) for 1 h, followed by TNF stimulation (10 000 U/ml) in triplicate. Cell death and caspase activity were determined as described in Materials and Methods. The 12-h time point is shown±S.E.M (n=3)

Figure 3

Knockdown of Drp1 delays cell proliferation but does not reduce mitochondrial respiration. (a) Forty-eight hours after transient knockdown of L929 cells with the indicated siRNAs, cells were seeded at 10 000 cells in a 96-well plate (eight replicates), and after 24 h live cells (Hoechst+/PI-) were counted using automated imaging. Data are expressed as percentage of control±S.E.M (n=3). *P<0.05. (b) Forty-eight hours after reverse transfection of L929 cells with the indicated siRNAs, cells were seeded at 10 000 cells in a 96-well plate, and 24 h later the cells were incubated in fresh medium supplemented with SytoxGreen (1.6 μM) and Triton-X100 (0.1% w/v). Subsequently, maximal fluorescence of Sytoxgreen was determined by fluorometry. Data are expressed as percentage of control±S.E.M (n=3). *P<0.05. (c) Basal, maximal and minimal oxygen consumption rates (OCR) in L929 cells were analyzed as described in Materials and Methods. Briefly, 10 000 cells were seeded in a 96-well plate (eight replicates) for each knockdown. After 24 h, cellular oxygen consumption rates were determined. The data show the kinetics of four consecutive measurements of baseline respiration. Subsequently, the protonophore CCCP was added (12 μM) to each well to dissipate the mitochondrial membrane potential and thereby promote maximal electron transport and corresponding maximal oxygen consumption. Injection of the complex I inhibitor rotenone (Rot, 2.5 μM) and complex III inhibitor Antimycin-A (AM, 5 μM) in each well was followed by four consecutive measurements (at 5 min intervals) to determine the minimal mitochondrial respiration. Finally, the amount of viable cells (HOECHST+/PI- cells) in the microchamber of each well was determined by automated imaging. The data represent the oxygen consumption rate (pmol O₂ per minute) per 1000 cells±S.E.M (n=3)

Figure 4

The Drp1 inhibitor mdivi inhibits TNF-induced necroptosis and mitochondrial respiration. (a) L929sahFas cells were pretreated with mdivi (0–250 μM) for 1 h and respiration was analyzed as described in Materials and Methods. After four consecutive measurements of basal respiration, the protonophore CCCP was injected (12 μM) and maximal respiration was determined. Rotenone (2.5 μM) and Antimycin-A (5 μM) were then injected to determine the mitochondrial baseline respiration. A representative experiment is shown (n=2). (b) After pretreatment with mdivi (0–250 μM), L929sahFas cells were stimulated with TNF for 6 h, and cell death was determined by flow cytometry; it is expressed as percentage of SytoxGreen positive cells control±S.E.M (n=2). *P<0.05