Abstract
Phosphatidylserine (PS), an anionic phospholipid enriched in the inner leaflet of the plasma membrane, is exposed to the outer leaflet during apoptosis. PS exposure was recently shown to be induced during tumor necrosis factor-induced necroptosis. We herein demonstrated that interferon (IFN)-γ induced necroptosis in Caspase-8-knockout mouse-derived embryonic fibroblasts (C8KO MEFs), as well as in WT MEFs co-treated with the pan-caspase inhibitor, z-VAD-fmk. PS exposure and necroptosis were significant after 6- and 24-h treatments with IFN-γ, respectively. To elucidate the molecular mechanisms underlying IFN-γ-induced PS exposure, we generated C8KO MEF-derived cell lines without the expression of RIPK3 (receptor-interacting protein kinase 3), an essential molecule in tumor necrosis factor-induced necroptosis, and IFN-γ-induced PS exposure and necrotic cell death were shown to be specifically inhibited by the loss of RIPK3 expression. Furthermore, the down-regulated expression of MLKL (mixed lineage kinase domain-like protein), a key molecule for inducing membrane rupture downstream of RIPK3 in necroptosis, abolished IFN-γ-induced PS exposure in C8KO MEFs. In human colorectal adenocarcinoma-derived HT29 cells, PS exposure and necroptosis were similarly induced by treatment with IFN-γ in the presence of Smac mimetics and z-VAD-fmk. The removal of IFN-γ from PS-exposing MEFs after a 6-h treatment completely inhibited necroptotic cell death but not the subsequent increase in the number of PS-exposing cells. Therefore, PS exposure mediated by RIPK3-activated MLKL oligomers was induced by a treatment with IFN-γ for a significant interval of time before the induction of necroptosis by membrane rupture.
Highlights
Phosphatidylserine (PS), an anionic phospholipid enriched in the inner leaflet of the plasma membrane, is exposed to the outer leaflet during apoptosis
Interferon (IFN), which constitutes a family of cytokines with antiviral, cell growth–suppressive, and immunomodulatory acting protein kinase; MLKL, mixed lineage kinase domain-like protein; IFN, interferon; mouse embryonic fibroblasts (MEFs), mouse embryonic fibroblast; IZ, IFN-␥ and z-VAD-fmk; MFG-E8, milk fat globule-EGF-factor 8; PI, propidium iodide; C8KO, caspase-8 knockout; iC8KO, immortalized C8KO; shRNA, short hairpin RNA; z, benzyloxycarbonyl; fmk, fluoromethyl ketone; TCZ, tumor necrosis factor (TNF)-␣, cycloheximide, and z-VAD-fmk; RHIM, RIP homotypic interaction motif; NSA, necrosulfonamide; FBS, fetal bovine serum; CHO, Chinese hamster ovary; qRTPCR, quantitative Reverse Transcription PCR
To investigate whether PS is exposed during the IFN-␥– induced necroptotic process, primary WT mouse embryonic fibroblasts (MEFs) with the Balb/c genetical background were treated with IFN-␥ and z-VAD-fmk (IZ). z-VAD-fmk is a pancaspase inhibitor that blocks the protease activities of various caspases, including caspase-8
Summary
Long-term PS exposure is induced before the induction of necroptosis in IFN-␥–treated MEFs. PS exposure and cell death were observed in IFN-␥–treated HT29 cells in the presence of Smac mimetics (Fig. S8A), they were not significantly inhibited by co-treatment with a human MLKL-specific inhibitor necrosulfonamide (NSA) [27] (data not shown). After a 24-h treatment with IFN-␥, only ϳ20% and less than 10% of parental iC8KO17e were shown to expose PS and die, respectively, and both PS exposure and cell death were inhibited by knockdown of RIPK3 expression using shRipk (Fig. S9C). IFN-␥ treatment mainly induces PS exposure dependently on RIPK3 but hardly induces necroptosis in iC8KO17e MEFs. The oligomerization of activated MLKL is essential for PS exposure and the execution of necroptotic cell death in MEFs. We investigated whether IFN-␥–induced PS-exposing MEFs with activated MLKL are fated to die by necroptosis or remain alive after the removal of IFN-␥. The present results suggest that caspase8 –inactivated MEFs treated with IFN-␥ for at least 6 h expose PS on their cell surface through the activation of MLKL after the removal of IFN-␥, do not die by MLKL-mediated necroptosis, and may be resuscitated to normal PS-negative cells
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