Reactive Oxygen Species Facilitate Adipocyte Differentiation by Accelerating Mitotic Clonal Expansion

Abstract

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-␤ (C/EBP␤) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBP␤ is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBP␤ is sequentially phosphorylated by MAPK and glycogen synthase kinase-3␤, inducing C/EBP␤ DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBP␤ is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBP␤ DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H 2 O 2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor ␥. Interestingly, cell cycle progression (S to G 2 /M phase) was markedly enhanced by H 2 O 2 , whereas antioxidants caused an S phase arrest during the MCE. H 2 O 2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBP␤, which is a hallmark for C/EBP␤ binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBP␤. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBP␤ DNA binding activity, along with the dual phosphorylation of C/EBP␤, is required for the MCE and terminal differentiation of adipocytes.

G0/G1 switch gene 2 has a critical role in adipocyte differentiation

“…Labeled cells were analyzed using a FACS caliber flow cytometry system, and data were analyzed using the ModFit software (BD Biosciences) as described. 30 Cell labeling with BrdU and immunofluorescence analysis. Labeling with BrdU was done using the BrdU Labeling and Detection Kit I (Roche, Mannheim, Germany) according to the manufacturer's instructions.…”

c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma 2

“…Interestingly, both ATM and c-Abl were found to be activated not only by DNA damage induction but also by insulin treatment (20,34), suggesting a possible mechanism of c-Abl activation by adipogenic induction through ATM. It was also demonstrated that genotoxic drugs or reactive oxygen species can promote adipocyte differentiation of cultured mesenchymal progenitors of adipocytes (35,36). An intriguing possibility that should be further investigated is that controlled DNA damage is being used during normal adipogenesis to activate key regulators like c-Abl.…”

Dexras1 mediates glucocorticoid-associated adipogenesis and diet-induced obesity

“…The 3T3-L1 preadipocytes were cultured and differentiated into adipocytes as described previously (27). Briefly, 3T3-L1 preadipocytes were maintained in DMEM containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 8 μg/mL biotin, supplemented with 10% (vol/vol) heat-inactivated calf serum at 37°C, in an atmosphere of 90% air and 10% CO 2 .…”

“…Protein samples of equal amount were separated by SDS/ PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed using the following antibodies: polyclonal antibodies against PPARγ (Cell Signaling Technology) and C/EBPα (27); and mouse monoclonal antibodies against PPARγ, β-actin (Santa Cruz Biotechnology), and FLAG (Sigma).…”