Role of Ankyrin-Repeat and SOCS-Box Protein 9 (ASB9) in PAR1 Inhibition and the MAPK Signaling Pathway in the Bovine Ovary

ASB9 is a member of the ASB family of proteins, which act as a substrate-recognition component of E3 ubiquitin ligases and regulate various biological processes. We previously identified ASB9 as induced in bovine granulosa cells (GC) by LH/hCG and reported ASB9 binding partners including protease-activated receptor 1 (PAR1). We aimed to study ASB9 mechanisms of action in GC and determine whether ASB9 induction by LH/hCG is necessary for the regulation of PAR1 and signaling pathways involved in GC function and activity. Cultured GC were treated with different doses of FSH, LH and thrombin. RT-qPCR analyses revealed that thrombin increased PAR1 expression, while FSH had no effect on PAR1. Treatment with LH significantly downregulated PAR1, even in the presence of thrombin, possibly via ASB9. The phosphorylation profile of MAPK3/1 in thrombin-treated GC suggest a PAR1-mediated control. ASB9 induction appeared to have a negative effect on MAPK pathway although thrombin treatment blocked briefly (within an hour) the negative effect of ASB9 on PAR1. Proliferation assays showed that ASB9 negatively regulates GC number, while increasing apoptosis. These data provide evidence for ASB9 mode of action and its potent functional effects on PAR1 regulation, GC proliferation and potentially the ovulatory process in bovine species.