b The p-RIPK3, RIPK3, and GAPDH protein levels in the indicated cells treated with or without IFN-γ for 36 h were measured by western blotting. a The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-γ, IFN-γ + zVAD, or IFN-β, and cell survival was determined by measuring the ATP levels. ZBP1 is required for IFN-induced necroptosis.
For d, the data shown are representative of three independent experiments A two-tailed Student’s t-test was applied to determine the indicated P values. The error bars indicate the mean ± s.e.m. The quantitative data shown are from n = 3 independent experiments. f The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-β, or IFN-β + zVAD and with CHX and ActD for 36 h, and cell survival was determined by measuring the ATP levels. e The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-γ, or IFN-γ + zVAD and with CHX (10 µg/ml) and ActD (1 µg/ml) for 36 h, and cell survival was determined by measuring the ATP levels. d WT and RIPK1 KO cells were treated with or without IFN-γ, nifuroxazide, or a JAK inhibitor for 36 h, and the indicated proteins were analyzed by western blotting. c qRT-PCR analysis was performed to determine the relative CXCL10 mRNA level in WT and RIPK1 KO L929 cells treated with or without IFN-γ, nifuroxazide, or a JAK inhibitor. b The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-β, or IFN-β + zVAD with or without nifuroxazide or a JAK inhibitor for 36 h, and cell survival was determined by measuring the ATP levels. a The indicated knockout L929 cell lines were treated with DMSO (ctrl), zVAD, IFN-γ, or IFN-γ + zVAD and nifuroxazide (50 µM) or a JAK inhibitor (1 µM) for 36 h, and cell survival was determined by measuring the ATP levels. IFN-β/γ-induced cell death is dependent on the JAK1-STAT1 signaling pathway and de novo protein synthesis. For d– f, the data shown are representative of the results of three independent experiments All the abovementioned error bars indicate the mean ± s.e.m. g The indicated knockout L929 cell lines were treated with IFN-γ and IFN-γ + zVAD for 36 h, and cell survival was determined by measuring the ATP levels. f The RIPK3, MLKL, and GAPDH levels in the indicated knockout L929 cell lines were measured by western blotting. e WT, FADD KO, RIPK1 KO, and caspase-8 KO L929 cells were treated with IFN-β for 36 h, and the indicated proteins were detected by western blotting.
d WT, FADD KO, RIPK1 KO, and caspase-8 KO L929 cells were treated with IFN-γ for the indicated time periods, and the indicated proteins were detected by western blotting. c WT and RIPK1 KO MEF cells were treated with DMSO (ctrl), zVAD (20 µM), IFN-γ, IFN-γ + zVAD, IFN-β, or IFN-β + zVAD, and cell survival was determined by measuring the ATP levels. b The indicated knockout L929 cell lines were treated with PBS (ctrl) or IFN-β (1000 U/ml) for 36 h, and cell survival was determined by measuring the ATP levels. Cell viability was determined by measuring the ATP levels. a The indicated knockout L929 cell lines were treated with PBS (ctrl) or IFN-γ (10 ng/ml) for 36 h.
IFN-β/γ induces necroptosis in RIPK1/FADD/caspase-8 deficient cells. ZBP1-mediated necroptosis in IFN-treated cells is likely physiologically relevant, as ZBP1 KO mice were significantly protected against acute systemic inflammatory response syndrome (SIRS) induced by TNF + IFN-γ. The antinecroptotic function of RIPK1, FADD, and caspase-8 in IFN-treated cells is most likely executed by caspase-8-mediated cleavage of RIPK3, since the inhibitory effect on necroptosis was eliminated when the caspase-8 cleavage site in RIPK3 was mutated. The N-terminal domain (ND) of ZBP1 is important for ZBP1-ZBP1 homointeraction, and its RHIM domain in the C-terminal region interacts with RIPK3 to initiate RIPK3-dependent necroptosis. We found that the pronecroptotic signal from IFN stimulation depends on new protein synthesis and identified ZBP1, an IFN-stimulated gene (ISG) product, as the de novo synthesized protein that triggers necroptosis in IFN-stimulated cells. We show that L929 fibroblast cells became susceptible to IFN-induced necroptosis when RIPK1, FADD, or Caspase-8 was genetically deleted, confirming the antinecroptotic role of these proteins in IFN signaling.
ZBP1 GENE BIOEDIT ACTIVATOR
Here, we report that the DNA-dependent activator of IFN regulatory factors (ZBP1, also known as DAI) is required for both type I (β) and type II (γ) IFN-induced necroptosis. IFN-induced necroptosis has been reported in cells deficient in receptor-interacting protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8, but the mechanism is largely unknown. Interferons (IFNs) play an important role in immunomodulatory and antiviral functions.
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