ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2006) 11 P574

Prolonged expression of the ACTH receptor, MC2-R, during 3T3-L1 adipocyte differentiation is dependent upon switching promoter usage to an adipocyte-specific, C/EBP-driven, downstream promoter

LA Noon1, AJL Clark1, PJ O’Shaughnessy2 & PJ King1

1Queen Mary, University of London, London, United Kingdom; 2University of Glasgow, Glasgow, United Kingdom.

The peptide hormone ACTH stimulates lipolysis and suppresses leptin production in adipocytes via the G-protein coupled seven transmembrane receptor, MC2-R. We have shown previously that PPARγ2 is the primary factor responsible for transactivation of the already identified murine MC2-R promoter in the differentiating 3T3-L1 adipocyte cell line. In this study we show that despite the activity of this promoter being transient during differentiation, MC2-R mRNA remains elevated at later time points during adipogenesis. Analysis of MC2-R transcripts in terminally differentiated 3T3-L1 cells reveals that they initiate from a transcriptional start site in the first intron of the murine MC2-R, 1.4 kb downstream of the previously identified start site. Placing the genomic sequences in intron 1 from the end of exon 1 to the end of the novel first exon upstream of the luciferase gene in the promoterless vector pGL3 we showed that this region behaves as an adipocyte-specific promoter activated with late kinetics, ie it becomes active after the decline in activity of the 5′ promoter. Introduction of a 5′ deletion series of this novel promoter into differentiated adipocytes indicates that a C/EBP site, 87 bp upstream of the transcriptional initiation site, is necessary for activity. Mutation of this site inactivates the promoter in differentiated 3T3-L1 cells and EMSA and ChIP analyses reveal that this site is bound by C/EBP factors. Real time PCR analysis of mRNA initiating from the two start sites shows that there is a switch in promoter usage from the 5′ to the 3′ promoter around day 5, indicating the complex regulation of murine MC2-R during adipogenesis.

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