USP8 promotes ferroptosis-associated senescence-like phenotype in chicken ovarian follicular granulosa cells via ACSL4.

Abstract

Ferroptosis is an iron-dependent form of cell death triggered by intracellular lipid peroxidation. It has been well documented to participate in multiple mammalian ovarian diseases; however, its functional significance in avian reproduction remains largely unclear. Here, we explored the role of the deubiquitinating enzyme ubiquitin-specific protease 8 (USP8) in governing ferroptosis and subsequent cellular senescence in chicken follicular granulosa cells. Combined loss- and gain-of-function assays demonstrated that USP8 positively modulates granulosa cell ferroptosis. This regulatory effect was evidenced by altered cell viability, glutathione (GSH) depletion, increased malondialdehyde (MDA) and reactive oxygen species (ROS) levels, and differential expression of the ferroptosis-associated markers FSP1, SLC7A11, and NCOA4. Mechanistically, USP8 regulates ACSL4 ubiquitination and proteasomal stability, leading to increased ACSL4 protein abundance, which in turn enhances ACSL4-dependent ferroptosis in granulosa cells. Functional analyses further showed that USP8-promoted ferroptosis is associated with a senescence-like phenotype in granulosa cells, which is characterized by the upregulation of P16, P21, and P53, as well as prominent G1-phase cell cycle arrest. Notably, ACSL4 knockdown effectively reversed the ferroptosis and senescent phenotypes caused by USP8 dysregulation. Collectively, this study identifies a novel USP8-ACSL4-ferroptosis-senescence axis that may control granulosa cell fate and follicular atresia in chickens. Our findings suggest that USP8 and ACSL4 could be explored as potential targets for preserving granulosa cell function, though this remains to be validated in vivo.