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.
Highlights
This cell death modality operates independently of executioner caspases, but is negatively regulated by caspase-8 and is sensitized under conditions of caspase-8 inhibition or deficiency.[2]
The RIPK1–RIPK3– mixed lineage kinase domain like (MLKL)–phosphoglycerate mutase 5 (PGAM5)–Drp[1] axis operates during tumor necrosis factor (TNF)/zVAD-fmkinduced necroptosis in the presence of SMAC mimetics (SM) in human HT-29 cells and HeLa cells transfected with RIPK3.15 It is unknown whether this pathway is conserved among mammalian cells and to which extent it influences induced apoptosis
This TNF-induced apoptosis observed upon knockdown of RIPK3 or MLKL was reduced in the presence of the RIPK1 kinase inhibitor necrostatin-1 (Figure 2b), demonstrating its dependence on the kinase activity of RIPK1
Summary
This cell death modality operates independently of executioner caspases, but is negatively regulated by caspase-8 and is sensitized under conditions of caspase-8 inhibition or deficiency.[2]. The involvement of functional necroptosis in vivo greatly relies on the use of RIPK1 kinase inhibitors such as necrostatins[3,5] and the discovery of RIPK3 as a decisive pro-necroptotic kinase.[9,12,13] Members of the tumor necrosis factor (TNF) family are potent inducers of necroptosis. Induced necroptosis involves the formation of a necrosome complex consisting of the core components RIPK1, RIPK3 and mixed lineage kinase domain like (MLKL) that are negatively regulated by factors such as Fas associated death domain protein (FADD), caspase-8 and cellular FLICE inhibitory protein.[1,14] Despite the importance of necroptosis, its molecular components and the mechanisms of its regulation and execution remain elusive. MLKL was independently identified by two different groups, who showed that it is constitutively bound by a wild type but not by the kinase-dead RIPK3.15,16 During TNF-induced necroptosis, RIPK3 phosphorylates human MLKL at positions. Zhao et al.[16] identified MLKL by screening a short hairpin library
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