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)-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.

Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis.

Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL). Disruption of Caspase-8 expression causes embryonic lethality in mice, which is rescued by depletion of either Ripk3 or Mlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown of Caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripk1 and Ripk3; however, the enhancement of RA-induced cell differentiation is independent of Mlkl and necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3. In Caspase-8-deficient as well as Caspase-8- and Mlkl-deficient mouse embryos, the expression of RA-specific target genes was obviously enhanced. Thus, Caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo.

Molecular Mechanism of Apoptosis by Amyloid ß-Protein Fibrils Formed on Neuronal Cells.

Aggregational states of amyloid ß-protein (Aß) are critical for its neurotoxicity, although they are not well-characterized, particularly after binding to the cell membranes. This is one reason why the mechanisms of Aß neurotoxicity are controversial and elusive. In this study, the effects of toxic Aß-(1-42) fibrils formed in the membrane on cellular processes were investigated using human neuroblastoma SH-SY5Y cells. Consistent with previous observations, fibrillar Aßs formed on the membranes induced activation of caspase-3, the effector caspase for apoptosis. Knockdown analyses of the initiator caspases, caspase-8 and caspase-9, indicated that the apoptosis was induced via activation of caspase-8, followed by activation of caspase-9 and caspase-3. We also found that inflammation signaling pathways including Toll-like receptors and inflammasomes NOD-, LRR-, and pyrin domain-containing protein 3 are involved in the initiation of apoptosis by the Aß fibrils. These inflammation-related molecules are promising targets for the prevention of apoptotic cell death induced by Aß.

FLASH/casp8ap2 is indispensable for early embryogenesis but dispensable for proliferation and differentiation of ES cells.

FLICE/caspase-8-associated huge protein (FLASH)/casp8ap2 is involved in various cellular functions, such as cell cycle progression, transcriptional regulation, the regulation of apoptosis, and the regulation of histone gene expression. The down-regulated expression of FLASH has been shown to inhibit cell cycle progression in the S phase in many kinds of mice and human cell lines and the inhibition of cell cycle progression may be attributed to the suppressed expression of replication-dependent histone genes. We here demonstrated that the induced knockout of FLASH never affected cell cycle progression in ES cells, in which the expression of core histone genes was decreased to levels similar to those in human KB cells sensitive to the knockdown of FLASH. In addition, the FLASH conditional knockout ES cells could differentiate normally into not only mesodermal and endodermal cells, but also trophoblasts. In order to investigate the function of FLASH in early embryogenesis in vivo, we also examined a FLASH mutant mouse, in which FLASH mutant allele did not express FLASH mRNA in embryos and most adult organs, except for the testis. FLASH mutant embryos died between E3.5 and E8.5. Furthermore, the in vitro cultivation of FLASH mutant embryos generated by in vitro fertilization showed embryonic lethality at the pre-implantation stage by inhibiting the hatching of embryos and their adherence to substrates. Taken together, these results indicate that FLASH plays an important role in early embryogenesis, but is not essential for either the proliferation or differentiation of ES cells.