Abstract
Necroptosis, a form of regulated necrotic cell death, is mediated by receptor interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL). However, the mechanism by which necroptosis promotes inflammation is still unclear. Here we report that the expression of cytokines is robustly upregulated in a cell-autonomous manner during necroptosis induced by tumor necrosis factor alpha (TNFα). We demonstrate that TNFα-induced necroptosis leads to two waves of cytokine production. The first wave, more transient and weaker than the second, is in response to TNFα alone; whereas the second wave depends upon the necroptotic signaling. We show that necroptosis promotes the transcription of TNFα-target genes in a cell-intrinsic manner. The activation of both NF-κB and p38 by the necroptotic machinery, RIPK1, RIPK3, and MLKL, is involved in mediating the robust induction of cytokine expression in the second wave. In contrast, necroptosis induced by direct oligomerization of MLKL promotes cytokine production at much lower levels than that of necroptosis induced with TNFα. Thus, we conclude that TNFα-induced necroptosis signaling events mediated by RIPK1 and RIPK3 activation, in addition to the MLKL oligomerization, promotes the expression of cytokines involving multiple intracellular signaling mechanisms including NF-κB pathway and p38. These findings reveal that the necroptotic cell death machinery mounts an immune response by promoting cell-autonomous production of cytokines. Our study provides insights into the mechanism by which necroptosis promotes inflammation in human diseases.
Highlights
Necroptosis is a regulated form of necrotic cell death that can be activated when cells are stimulated by the proinflammatory cytokine tumor necrosis factor alpha (TNFα) under apoptosis-deficient conditions[1,2]
Through a comparative transcriptome analysis of TNFα-induced necroptosis in the presence or absence of the RIPK1 kinase inhibitor Nec-1s, we discovered that necroptosis exerts a profound effect on transcription, activating the expression of a large number of genes
We showed that cytokine production mediated by mixed lineage kinase domain-like protein (MLKL) oligomerization alone was substantially lower than that induced by TSZ
Summary
Necroptosis is a regulated form of necrotic cell death that can be activated when cells are stimulated by the proinflammatory cytokine tumor necrosis factor alpha (TNFα) under apoptosis-deficient conditions[1,2]. D HT-29 cells were treated with TSZ for the indicated periods of time. The cell viability was determined by CellTiter-Glo. e HT-29 cells were treated with TSZ for the indicated periods of time. The cell lysates and culture media were collected separately, and analyzed by western blotting with indicated antibodies. H MEFs were treated for the indicated periods of time with TSZ. The expression levels of Cxcl[2] and Csf[2] were determined by qPCR. The cell viability was determined by CellTiter-Glo. i MEFs were treated as indicated. Cxcl[2] and Csf[2] mRNA levels were measured by qPCR after 4 h of treatment. The cell viability was determined by CellTiter-Glo after 13 h of treatment. Gene expression determined by qPCR was shown as fold induction compared with untreated cells in all figures.
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