MK3 Gene Upregulates Granulosa Cell Apoptosis Through the TNF/P38 MAPK Pathway in Chicken

In poultry production, the laying rate is a critical economic trait, as high egg production significantly enhances profitability. Mitogen-activated protein kinase-activated protein kinase 3 (<i>MK3</i>) is a member of the mitogen-activated protein kinase (MAPK) family, which plays an important role in follicular development. Our previous RNA-seq analysis revealed that <i>MK3</i> expression was significantly altered in the ovaries of laying hens exposed to normal versus light-deprivation conditions. Based on previous RNA-seq analysis of chicken ovaries, this study focused on the <i>MK3</i> gene to explore its role in regulating apoptosis of follicular granulosa cells in laying hens. The results demonstrated that <i>MK3</i> overexpression induced granulosa cell apoptosis by modulating the expression of key proliferation- and apoptosis-related genes, including <i>FAS</i>, <i>Caspase3</i>, <i>BCL2</i>, and <i>C-myc</i>. These findings were further validated using specific siRNA-mediated knockdown of <i>MK3</i>. Flow cytometry, CCK-8, and EdU assays consistently showed that <i>MK3</i> facilitated apoptosis and inhibited granulosa cell proliferation. Additionally, dual-luciferase reporter assays revealed that the transcription factor <i>WT1</i> bound to the <i>MK3</i> promoter and enhanced its transcriptional activity. Mechanistically, <i>MK3</i> regulated granulosa cell apoptosis through the TNF/P38 MAPK pathway. This conclusion was corroborated by treatment with the P38 inhibitor GS-444217 and specific siRNA targeting components of the pathway. In summary, <i>MK3</i> promotes granulosa cell apoptosis in the follicles of laying hens, is transcriptionally regulated by <i>WT1</i>, and exerts its pro-apoptotic effects via the TNF/P38 MAPK pathway.