Gap‐Junctional Communication Is Required for Mitotic Clonal Expansion during Adipogenesis

Abstract

Gap-junctional communication (GJC) plays critical roles in cell growth and differentiation. Several studies have demonstrated the involvement of GJC in myogenesis and osteogenesis; however, the role of GJC in adipogenesis has not been fully studied. Thus, we investigated the role of GJC in adipogenesis. 3T3-L1 preadipocytes were differentiated in the presence of gap junction inhibitor, 18-alpha-glycyrrhetinic acid (AGA), and accumulation of cytoplasmic triglycerides was measured. 3T3-L1 cells were transfected with 100 nM small interfering RNA duplexes targeting connexin (Cx) 43. The mRNA levels of CCAAT/enhancer-binding protein (C/EBP) alpha, peroxisome proliferator-activated receptor gamma, glucose transporter 4, C/EBPbeta, and Cx43 were measured by real-time polymerase chain reaction. The protein levels of C/EBPbeta were quantitated by Western blotting. The cell proliferation was measured by counting cell numbers, and DNA synthesis was measured by bromodeoxyuridine incorporation. AGA inhibited adipocyte differentiation dose-dependently. The lipid accumulation and the mRNA levels of C/EBPalpha, peroxisome proliferator-activated receptor gamma, and glucose transporter 4 were markedly reduced in AGA-treated adipocytes. The mRNA levels of C/EBPbeta did not decrease; however, C/EBPbeta [liver-enriched transcriptional activator protein (LAP)] expression and the C/EBPbeta (LAP)-to-C/EBP [liver-enriched transcriptional inhibitory protein (LIP)] ratio were reduced by AGA treatment. The increase in both cell number and DNA synthesis, which occurs during mitotic clonal expansion, was reduced by AGA in a dose-dependent fashion. The major component of gap junctions in 3T3-L1 cells was Cx43. Down-regulation of Cx43 using small interfering RNA reduced the expression of C/EBPbeta (LAP) and inhibited adipogenesis. Our data suggest that GJC plays some important roles in adipogenesis through inhibiting mitotic clonal expansion and modulating C/EBPbeta (LAP) expression.