Reactive Oxygen Species Facilitate Adipocyte Differentiation by Accelerating Mitotic Clonal Expansion

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-beta (C/EBP beta) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBP beta is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBP beta is sequentially phosphorylated by MAPK and glycogen synthase kinase-3 beta, inducing C/EBP beta DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBP beta is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBP beta DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H2O2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor gamma. Interestingly, cell cycle progression (S to G2/M phase) was markedly enhanced by H2O2, whereas antioxidants caused an S phase arrest during the MCE. H2O2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBP beta, which is a hallmark for C/EBP beta binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBP beta. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBP beta DNA binding activity, along with the dual phosphorylation of C/EBP beta, is required for the MCE and terminal differentiation of adipocytes.