# The Negative Role of Ankyrin-Repeat and SOCS-Box Protein 9 in PAR1 Expression and the MAPK Signaling Pathway in Bovine Granulosa Cells

## TL;DR

ASB9 reduces PAR1 expression and disrupts the MAPK pathway in bovine granulosa cells, leading to fewer cells through reduced growth and increased cell death.

## Contribution

This study reveals ASB9's negative role in PAR1 regulation, MAPK signaling, and granulosa cell survival in bovine reproduction.

## Key findings

- ASB9 downregulates PAR1 expression in bovine granulosa cells despite thrombin stimulation.
- ASB9 inhibits the MAPK signaling pathway, affecting cell function in granulosa cells.
- ASB9 reduces granulosa cell numbers by suppressing proliferation and promoting apoptosis.

## Abstract

Ankyrin-repeat and SOCS-box protein 9 (ASB9), a protein involved in regulating biological processes, is induced in bovine granulosa cells by luteinizing hormone (LH). Our study on how ASB9 functions in these cells found that ASB9 induction by LH appears to negatively regulate the expression of protease-activated receptor 1 (PAR1), a protein that normally increases with thrombin treatment. This happens even in the presence of thrombin. Furthermore, research indicates that ASB9 plays a negative role in the MAPK signaling pathway, which is important for cell function. This study also revealed that ASB9 negatively affects the number of granulosa cells by inhibiting their proliferation and increasing their apoptosis (programmed cell death). In summary, ASB9’s function in bovine granulosa cells is to downregulate PAR1, suppress the MAPK pathway, and reduce cell numbers by inhibiting proliferation and promoting apoptosis, suggesting that it plays a significant role in the ovulatory process.

Ankyrin-repeat and SOCS-box protein 9 (ASB9) is a member of the ASB family of proteins, which act as a substrate recognition component of E3 ubiquitin ligases and regulate various reproductive processes. ASB9 was previously identified as being induced in bovine granulosa cells (GCs) by LH/hCG, and its binding partners, including protease-activated receptor 1 (PAR1), were reported. The aim of this study was to decipher ASB9’s mechanisms of action in GCs and determine whether ASB9 induction by LH/hCG is necessary for the regulation of PAR1 and the signaling pathways involved in GC function and activity. Cultured GCs 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 GCs suggests PAR1-mediated control. ASB9 induction appeared to have a negative effect on the MAPK pathway, although thrombin treatment briefly (within an hour) blocked the negative effect of ASB9 on PAR1. Proliferation assays showed that ASB9 negatively regulated the GC number while increasing apoptosis. These data provide evidence of ASB9’s mode of action and its potent functional effects on PAR1 regulation, GC proliferation, and, potentially, the ovulatory process in bovine species.