APE1/REF-1 AFFECTS ADIPOCYTE DIFFERENTIATION BY REGULATING ADIPOGENIC TRANSCRIPTION FACTORS IN 3T3-L1 CELLS

Abstract

Objective: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. Design and method: To determine the changes in APE1/Ref-1 expression during the adipogenic differen-tiation of preadipocytes, we investigated APE1/Ref-1 expression on days 0, 2, 4, 6, and 8 of 3T3-L1 cell differentiation (Figure 1A) To evaluate the effect of APE1/Ref-1 on MDI-induced adipocyte differentiation, 3T3-L1 cells were transfected with adenovirus expressing APE1/Ref-1. Having established that overexpression of APE1/Ref-1 suppressed adipocyte differ-entiation, the effect of gene silencing of APE1/Ref-1 during MDI-induced adipocyte differ-entiation was evaluated in 3T3-L1 cells with transfection of APE1/Ref-1 siRNA. Finally, the effect of E3330, a redox inhibitor of APE1/Ref-1, on adipocyte differentiation was evaluated in 3T3-L1 cells. Results: In this study, we investigated the effect of APE1/Ref-1 on the regulation of adi-pocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression sig-nificantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-alpha and peroxisome proliferator-activated receptor gamma, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-alpha, PPAR-gamma, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-alpha, PPAR-gamma, and aP2 during adipocyte differentiation. Conclusions: In conclusion, our study demonstrated that endogenous APE1/Ref-1 is downregu-lated during adipocyte differentiation, whereas overexpression of APE1/Ref-1 inhibits ad-ipocyte differentiation; in contrast, silencing APE1/Ref-1 and redox inhibition using E3330 promotes adipocyte differentiation through the modulation of expression of adipogenic transcriptional factors. Overall, these results suggest that the redox function of APE1/Ref-1 exerts an anti-adipogenic function by inhibiting adipocyte differentiation.