Necroptosis in primate luteolysis: a role for ceramide

Abstract

The corpus luteum (CL) is a transient endocrine organ, yet molecular mechanisms resulting in its demise are not well known. The presence of phosphorylated mixed lineage kinase domain-like pseudokinase pMLKL(T357/S358) in human and nonhuman primate CL samples (Macaca mulatta and Callithrix jacchus) implied that necroptosis of luteal cells may be involved. In M. mulatta CL, pMLKL positive staining became detectable only from the mid-late luteal phase onwards, pointing to necroptosis during regression of the CL. Cell death, including necroptosis, was previously observed in cultures of human luteal granulosa cells (GCs), an apt model for the study of the human CL. To explore mechanisms of necroptotic cell death in GCs during culture, we performed a proteomic analysis. The levels of 50 proteins were significantly altered after 5 days of culture. Interconnectivity analysis and immunocytochemistry implicated specifically the ceramide salvage pathway to be enhanced. M. mulatta CL transcriptome analysis indicated in vivo relevance. Perturbing endogenous ceramide generation by fumonisin B1 (FB1) and addition of soluble ceramide (C2-CER) yielded opposite actions on viability of GCs and therefore supported the significance of the ceramide pathway. Morphological changes indicated necrotic cell death in the C2-CER treated group. Studies with the pan caspase blocker zVAD-fmk or the necroptosis blocker necrosulfonamid (NSA) further supported that C2-CER induced necroptosis. Our data pinpoint necroptosis in a physiological process, namely CL regression. This raises the possibility that the primate CL could be rescued by pharmacological inhibition of necroptosis or by interaction with ceramide metabolism.

Human Luteinized Granulosa Cells—A Cellular Model for the Human Corpus Luteum

“…To examine to what extend IVF-derived cultured GCs resemble their in vivo -counterparts, and are an appropriate model for the study of the CL (21), we combined a number of techniques, e.g., live cell imaging, cell death measurements, immunocytochemistry, and mass spectrometry analysis. We specifically studied GCs cultured from day 2 up to day 5 after retrieval from patients.…”

“…Large luteal cells express CYP11A1, a protein known to be crucial for progesterone production. Mass spectrometry revealed that it is highly expressed in IVF-derived human GCs (21, 23). Also low density lipoprotein receptor (LDL-R), 3β-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein (StAR) were found by mass spectrometry in cultured GCs.…”

Regulation of Necroptosis by Phospholipids and Sphingolipids

“…Accumulation of endogenous ceramide was shown to promote necroptotic cell death in human luteal granulosa cells, implicating the possible involvement of ceramide synthases in necroptosis induction [53]. More recent studies indicated that upregulation of C 16 -ceramide triggered mitophagy-mediated necroptosis, suggesting important crosstalk between ceramide production and necroptotic cell death in lung structural cells [54].…”

Western Blotting and Flow Cytometry for Evaluating Necroptosis of Cells (Review)

“…Для оценки интенсивности процессов некроптоза метод вестерн-блоттинга применяется с целью определения некроптоз-ассоциированных белков и является на данный момент основной техникой, применяемой для идентификации некроптоза. В частности, киназа RIPK1, фосфорилированная по остатку серина в 166 позиции, киназа RIPK3, фосфорилированная по остатку серина в 232 положении, псевдокиназа MLKL, фосфорилированная по остатку треонина в 357 позиции и по остатку серина в 358 положении, используются в качестве маркеров некроптоза [2,11,18]. При фосфорилировании RIPK1, RIPK3 и MLKL активируются, что и наблюдается при некроптозе.…”