Receptor interacting protein kinases-dependent necroptosis as a new, potent mechanism for elimination of the endothelial cells during luteolysis in cow

Abstract

Necroptosis is an alternative form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent. In the present study, to clarify if necroptosis in luteal endothelial cells (LECs) participates and contributes for bovine luteolysis, we investigated RIPK1 and RIPK3 localization in luteal tissue and their expression in cultured LECs after treatment with selected immune factors - mediators of luteolytic action of prostaglandin F2α (PGF). In addition, effects of tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM), and/or nitric oxide donor - NONOate (100 μM) on viability and CASP3 activity in the cultured LECs were investigated. Furthermore, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on RIPKs and CASPs expression, were evaluated. Localization of RIPK1 and RIPK3 protein in the cultured LECs were determined. In cultured LECs, expression of RIPKs mRNA were up-regulated by TNF + IFNG at 12 h, and by PGF (1 μM) or NONOate at 24 h, respectively (P < 0.05). Although NONOate decreased cell viability, it prevented TNF + IFNG-stimulated CASP3 activity in cultured LECs. Nec-1 prevented TNF + IFNG-induced RIPK1 and CASP3 mRNA expression at 12 h and prevented RIPK3 mRNA expression. These findings suggest that RIPKs-dependent necroptosis which are induced by TNF + IFNG, PGF or NO could be potent mechanism responsible for LECs cell death and disappearance of luteal capillaries in regressing bovine CL.