Programmed necrosis - a new mechanism of steroidogenic luteal cell death and elimination during luteolysis in cows

Takuo Hojo,Karolina Lukasik,Kiyoshi Okuda,Marta J Siemieniuch,Dariusz J Skarzynski,Agnieszka W Jonczyk,Katarzyna K Piotrowska-Tomala

doi:10.1038/srep38211

Abstract

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

Highlights

Ratio of Bcl-2, which protects against cell death, and Bax, a proapoptotic protein, determines cell fate[15]
Real-time PCR analysis showed that expression of RIPK1 and RIPK3 mRNA in bovine corpus luteum (CL) tissues was present throughout the luteal stages of the cycle and was estrous cycle dependent
There were no reports indicating that caspase-independent cell death can occur in the bovine CL during the course of luteolysis

Summary

Introduction

Ratio of Bcl-2, which protects against cell death, and Bax, a proapoptotic protein, determines cell fate[15]. Receptor-interacting protein kinase (RIPK) 1 and RIPK3 are known to play roles as sensors of cellular stress[22] and are essential kinases mediating the programmed necrosis pathway[21,23,24,25] Death ligands such as TNF and Fas ligand induce apoptosis and necrosis in a number of tissues[26,27]. To test the hypothesis that necroptosis serves as a necessary and/or accessory mechanism for CL cell death during luteolysis in the cow, we investigated: (1) the expression of RIPK1 and RIPK3 in bovine CL tissues throughout the estrous cycle and during PGF-induced luteolysis in vivo, (2) the local regulatory mechanism of RIPKs expression in luteal cells, and (3) the participation of RIPKs in LSC death mechanisms by using an in vitro cell culture system with necrostatin-1 (Nec-1), an allosteric inhibitor of RIPK1 activity

Methods

Results

Conclusion