Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.

Cisplatin is a first-line chemotherapeutic agent for ovarian cancer that acts by promoting DNA cross links and adduct. However drug resistance and considerable side effects including reproductive toxicity remain a significant challenge. PTEN is well known as a tumor suppressor function which plays a fundamental role in the regulation of the cell cycle, apoptosis and development of cancer. At the same time PTEN has been revealed to be critically important for the maintenance of the primordial follicle pool. In this study, we investigated the role of PTEN/Akt/FOXO3 pathway in cisplatin-induced primordial follicle depletion. Cisplatin induced ovarian failure mouse model was used to evaluate how this pathway involves. In vitro maturation was used for oocyte rescue after cisplatin damage. We found that cisplatin treatment decreased PTEN levels, leading to a subsequent increase in the phosphorylation of key molecules in the pathway. The activation of the PTEN/Akt/FOXO3 pathway cascade increased cytoplasmic translocation of FOXO3a in cisplatin-treated follicles, which in turn increased the pool size of growing follicles, and rapidly depleted the number of dormant follicles. Once activated, the follicles were more prone to apoptosis, and their cumulus cells showed a loss of luteinizing hormone (LH) receptor expression, which leads to failure during final maturation and ovulation. In vitro maturation to rescue oocytes in a cisplatin-treated mouse model resulted in successful maturation and fertilization. This study is the first to show the involvement of the PTEN/Akt/FOXO3 pathway in premature ovarian failure after cisplatin treatment and the possibility of rescue through in vitro maturation.

Some mutations in FOXL2 result in premature ovarian failure accompanied by blepharophimosis, ptosis, epicanthus inversus syndrome type I disease, and FOXL2-null mice exhibit developmental defects in granulosa cells. Recently, FOXL2 c.402C4G, a new somatic mutation that leads to a p.C134W change, was found in the majority of adult-type ovarian granulosa cell tumors (GCTs). In this study, we investigated the possible mechanisms by which the C134W mutation contributes to the development of GCTs. Wild-type (WT) and mutant FOXL2 displayed differential apoptotic activities. Specifically, WT FOXL2 induced significant granulosa cell death, but the mutant exhibited minimal cell death. The FOXL2-induced apoptotic response was greatly dependent on caspase 8, BID and BAK because the depletion of any of these three proteins inhibited FOXL2 from eliciting the full apoptotic response. Activation of caspase 8 and subsequent increased production of truncated BID, and oligomerization of BAK, and release of cytochrome c were all associated with the apoptosis induced by WT FOXL2 expression. In contrast, the mutant FOXL2 was unable to elicit the full array of apoptotic signaling responses. In addition, we found differential TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) upregulation between the WT and the mutant, and the silencing of TNF-R1 or Fas and the blockage of the death signaling mediated by TNF-R1 or Fas using TNF-Fc or Fas-Fc, respectively, resulted in significant attenuations of FOXL2-induced apoptosis. Moreover, granulosa cells that expressed either WT FOXL2 or mutant exhibited distinct cell death sensitivities on activation of death receptors and deprivation of serum. Thus, the differential activities of FOXL2 and its mutant may partially account for the pathophysiology of GCT development.