CART affects the proliferation, apoptosis, and estradiol secretion of bovine granulosa cells by downregulating the expression of LRRC51

Abstract

This study aimed to elucidate the molecular mechanisms by which cocaine- and amphetamine-regulated transcript peptide (CART) influences granulosa cell (GC) proliferation, apoptosis, and estradiol (E₂) production in Bos taurus follicles. Bovine GCs were used as an in vitro model, and integrated transcriptomic and metabolomic analyses were performed to identify differentially expressed genes and metabolites following CART treatment. CART significantly reduced the mRNA abundance of leucine rich repeat containing 51 (LRRC51) and increased the concentration of 2-methoxyestrone, both of which are enriched in the steroid hormone biosynthesis pathway. These findings suggest that CART may impair follicular development by suppressing LRRC51 expression. Furthermore, methylation-specific PCR (MS-PCR) revealed that CART elevated the methylation level of the LRRC51 promoter region in GCs, providing a potential mechanism for the downregulation of LRRC51. Functional studies showed that LRRC51 knockdown significantly decreased E₂ concentration, increased 2-methoxyestrone levels, suppressed GC viability, and promoted apoptosis. Knockdown also markedly reduced steroidogenic acute regulatory protein (STAR), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and cyclin D2 (CCND2) protein abundance, while upregulating Bcl-2 interacting mediator (Bim) and caspase-3 (CASP3) protein expression. Conversely, LRRC51 overexpression enhanced E₂ secretion and GC viability. Importantly, CART treatment abrogated the stimulatory effects of LRRC51 overexpression on GC viability and E₂ accumulation, while amplifying the inhibitory effects of LRRC51 knockdown on cell viability and apoptosis. Collectively, these results demonstrate that CART downregulates LRRC51 expression via promoter methylation, thereby modulating GC viability, apoptosis, and E₂ synthesis and metabolism, and provide new insights into the molecular regulation of follicular maturation.