Downregulation of CLU mediates CART-induced apoptosis and suppressed proliferation in bovine granulosa cells†.

This study aimed to elucidate the molecular mechanisms by which cocaine- and amphetamine-regulated transcript peptide (CART) regulates granulosa cell (GC) fate in Bos taurus follicles. Bovine GCs were used as an in vitro model, and tandem mass tag-based quantitative proteomics was performed to identify differentially expressed proteins (DEPs) following CART treatment. CART significantly downregulated clusterin (CLU), the most altered DEP (P < 0.05). Functional studies demonstrated that CLU knockdown, mimicking CART action, significantly promoted GC apoptosis, as evidenced by increased Annexin V and PI fluorescence signals (all P < 0.05). CLU knockdown also significantly suppressed proliferation, with cell viability reduced by approximately 19-20% compared to controls in the CCK-8 assay (P < 0.05). CLU knockdown decreased the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL, P < 0.05) and cell-cycle regulators cyclin D2 (CCND2, P < 0.05), cyclin-dependent kinase 1 (CDK1, P < 0.05), and proliferating cell nuclear antigen (PCNA, P < 0.05), while elevating pro-apoptotic effectors BCL2-associated X (BAX, P < 0.05) and cleaved caspase-3 (CASP3, P < 0.05). However, CART co-treatment did not further exacerbate these effects (P > 0.05), indicating that CLU is a key effector molecule mediating CART's pro-apoptotic action. CART's pro-apoptotic and anti-proliferative effects on GCs likely also involve other downstream targets in the altered proteome. These results demonstrate that CART reprograms the GC proteome and that downregulation of CLU is a pivotal event driving this shift toward a pro-apoptotic and anti-proliferative state, providing insights into the protein-centric mechanisms by which CART influences GC fate.