Abstract

Background:Excessive abdominal fat deposition is a major challenge in the chicken farming industry, making it essential to elucidate the molecular mechanisms underlying chicken adipogenesis. Nuclear Respiratory Factor 1 (NRF1) has been reported to suppress chicken adipogenesis by downregulating peroxisome proliferator-activated receptor gamma (PPARγ) expression. Protein Phosphatase 1 Catalytic Subunit Gamma (PPP1CC) is a multifunctional phosphatase involved in various biological processes; however, its role in chicken adipogenesis remains unclear.Objective:This study aimed to investigate the functional role and underlying mechanism of PPP1CC in chicken preadipocyte differentiation.Methods:Co-immunoprecipitation (Co-IP) and immunofluorescence assays were performed to determine the interaction between PPP1CC and NRF1 in DF1 cells. Bioinformatic analysis predicted potential NRF1 dephosphorylation sites targeted by PPP1CC, based on which NRF1 mutants mimicking dephosphorylation were constructed. Phos-tag SDS-PAGE combined with Western blot analysis were used to verify PPP1CC-mediated dephosphorylation of wild-type NRF1. Dual-luciferase reporter assays were used to evaluate the effect of PPP1CC-mediated dephosphorylation on NRF1-regulatedPPARγP1 promoter transcriptional activity. ChIP-qPCR was employed to assess the occupancy of NRF1 to thePPARγP1 promoter upon PPP1CC overexpression. The effect of PPP1CC overexpression was assessed on preadipocyte differentiation using Oil Red O staining and marker gene expression analysis.Results:PPP1CC interacted with NRF1 in both the cytoplasm and nucleus of DF1 cells. Overexpression of PPP1CC significantly promoted NRF1 dephosphorylation during oleic acid-induced preadipocyte differentiation and increased endogenous NRF1 expression. Moreover, dual-luciferase assays showed that while PPP1CC strengthened the inhibitory effect of wild-type NRF1 onPPARγP1 promoter transcriptional activity, it exerted no additional suppression on the alr