The peptide hormone ACTH can stimulate lipolysis and suppress leptin expression in murine adipocytes. These effects are mediated via the Melanocortin 2 Receptor (MC2-R), which is expressed when 3T3-L1 cells are induced to differentiate into adipocytes. In this study we have investigated the transcriptional activation of the MC2-R gene during adipogenesis.
5' deletion analysis of the murine MC2-R promoter revealed the presence of a transcriptional enhancer element between positions minus112 and minus53 relative to the transcription start site. A putative PPARgamma response element (PPRE) was identified within this 59bp region of the promoter at position minus95 and was the subject of further investigation. PPARgamma2 is a key regulator of adipogenesis and binds to its PPRE as a heterodimer with the Retinoid X Receptor alpha (RXRalpha).
When PPARgamma2 or RXRalpha were individually expressed, together with the minus112 reporter in preadipocytes, no effect was observed. However, co-expression of these two factors resulted in a 10-fold induction of reporter activity. This response to PPARgamma2/RXRalpha co-expression mapped to the putative PPRE at minus 95 and further deletion of the promoter past this site abolished the observed effect.
Radiolabelled double stranded oligonucleotide probes corresponding to the minus95 PPRE were prepared and used in EMSA. Binding was demonstrated in adipocyte, but not in preadipocyte nuclear extracts. When antibodies to PPARgamma and RXRalpha were preincubated with adipocyte nuclear extract, both were capable of supershifting the protein/DNA complex thereby demonstrating the ability of these proteins to bind to the minus95 PPRE. Finally, mutagenesis of the PPRE was performed that abolished binding in EMSA. When this mutation was introduced into the minus112 reporter, the response to PPARgamma2/RXRalpha co-transfection was reduced to 20percent that of the wild type reporter.
Taken together, these results demonstrate a role for PPARgamma/RXRalpha in the transcriptional up-regulation of the MC2-R during adipogenesis.