Gap-junctional communication is required for mitotic clonal expansion during adipogenesis

Abstract

Objective: 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. Research Methods and Procedures: 3T3-L1 preadipocytes were differentiated in the presence of gap junction inhibitor, 18-α- 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) α, peroxisome proliferator-activated receptor γ, glucose transporter 4, C/EBPβ, and C×43 were measured by real-time polymerase chain reaction. The protein levels of C/EBPβ were quantitated by Western blotting. The cell proliferation was measured by counting cell numbers, and DNA synthesis was measured by bromodeoxyuridine incorporation. Results: AGA inhibited adipocyte differentiation dose-dependently. The lipid accumulation and the mRNA levels of C/EBPa, peroxisome proliferator-activated receptor γ, and glucose transporter 4 were markedly reduced in AGA-treated adipocytes. The mRNA levels of C/EBPβ did not decrease; however, C/EBPβ [liver-enriched transcriptional activator protein (LAP)] expression and the C/EBPβ (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 C×43. Down-regulation of C×43 using small interfering RNA reduced the expression of C/EBPβ (LAP) and inhibited adipogenesis. Discussion: Our data suggest that GJC plays some important roles in adipogenesis through inhibiting mitotic clonal expansion and modulating C/EBPβ (LAP) expression. Copyright © 2007 NAASO.