Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity

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)-gamma 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-gamma, respectively. To elucidate the molecular mechanisms underlying IFN-gamma-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-gamma-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-gamma-induced PS exposure in C8KO MEFs. In human colorectal adenocarcinoma-derived HT29 cells, PS exposure and necroptosis were similarly induced by treatment with IFN-gamma in the presence of Smac mimetics and z-VAD-fmk. The removal of IFN-gamma 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-gamma for a significant interval of time before the induction of necroptosis by membrane rupture.