Abstract
Adipogenesis has an important role in regulating energy homeostasis in mammals. 3T3-L1 preadipocytes have been widely used as an in vitro model for analyzing the molecular mechanism of adipogenesis. Previous reports indicated that the stage of contact inhibition (CI), through which the proliferating cells exit from the cell cycle, was required for granting preadipocyte the ability to differentiate. While this kind of the granting mechanism remains elusive. In the present study, we showed that DNA (cytosine-5) methyltransferase 3a (Dnmt3a) was upregulated at both the mRNA and protein level during the CI stage, and resulted in increasing promoter methylation of adipogenic genes. We further identified that the expression of Activator protein 2α (AP2α), a member of the transcription factor activator protein 2 (AP2) family, was highly correlated with the expression of Dnmt3a during the CI stage. In addition, we showed that AP2α transcriptionally upregulated Dnmt3a by directly binding to its proximal promoter region. Importantly, treatment of 3T3-L1 preadipocytes with AP2α-specific siRNAs inhibited the preadipocyte differentiation in a stage-dependent manner, supporting the conclusion that AP2α has an important role during the CI stage. Furthermore, overexpression of Dnmt3a partially rescued the impairment of adipogenesis induced by AP2α knockdown. Collectively, our findings reveal that AP2α is an essential regulator for granting preadipocyte the ability to differentiate through the upregulation of Dnmt3a expression during the CI stage.
Highlights
Adipogenesis has an important role in energy homeostasis of mammals
Some previous studies reported a few factors such as transcriptional factor 7-like 1, GATA-2 and GATA-3 that were involved in adipogenic competency during the contact inhibition (CI) stage,[12,13,14] the factors that function for regulation of adipogenesis in the CI stage are largely unknown
In 1998, Jiang et al.[22] found that the expression of Activator protein 2α (AP2α) was markedly elevated during the CI stage in preadipocytes and declined rapidly after MDI induction, and they further showed that AP2α was responsible for delaying C/EBPα expression to ensure the progression of mitotic clonal expansion in adipogenesis.[23,24,25]
Summary
Adipogenesis has an important role in energy homeostasis of mammals. Better understanding the mechanism underlying adipogenesis may provide novel therapeutic targets in the treatment of obesity and other metabolic diseases.1–4 3T3-L1 preadipocytes have been widely used as an in vitro model for studying the molecular mechanisms governing adipogenesis.5,6 3T3-L1 preadipocytes first undergo growth arrest by contact inhibition (CI), and differentiate into adipocytes upon exposure to a hormone cocktail including isobutylmethylxanthine, dexamethasone and insulin (MDI). Its homo- and heterodimers could transactivate target genes by binding to GC-rich consensus elements.[17] AP2α has an important role as the regulator during vertebrate development, cell growth and differentiation, carcinogenesis.[18,19,20,21] In 1998, Jiang et al.[22] found that the expression of AP2α was markedly elevated during the CI stage in preadipocytes and declined rapidly after MDI induction, and they further showed that AP2α was responsible for delaying C/EBPα expression to ensure the progression of mitotic clonal expansion in adipogenesis.[23,24,25] Recently, Received 14.8.16; revised 18.10.16; accepted 18.10.16; Edited by E Candi another study showed that epigenetic histone modifications underlay AP2α-mediated inhibition of C/EBPα expression.[26] the role of the elevation of AP2α during the CI stage in adipogenesis remains elusive. Our findings deliver an AP2α-mediated epigenetic programming for granting preadipocytes the ability to differentiate in the licensing stage
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