Adipocyte differentiation is a complex process involving several signaling pathways. Molecular mechanisms regulating the very early stage of adipocyte differentiation is not fully appreciated yet. Several inducible genes at the early stage of preadipocyte differentiation have been identified, including the regulator of G protein signaling 2 (RGS2), a member of the RGS protein superfamily. This study aimed to clarify the precise induction profile of RGS2 and to determine the essential transcription element(s) regulating RGS2 expression in differentiating 3T3-L1 preadipocytes. RGS2 mRNA expression was elevated immediately at 1 h after differentiation initiation and it remained high until the late stage of differentiation. The putative promoter sequence (approximately 3,000 bp) of the mouse RGS2 gene was isolated and the RGS2 promoter activity was significantly upregulated 3 h after inducing differentiation. The primary signaling pathway leading to RGS2 transcriptional activation appeared to be cAMP-dependent. Sequential deletion and site-directed mutagenesis strategies demonstrate that the RGS2 promoter sequence truncated down to 78 bp in size retained full inducibility by the differentiation stimuli. Mutation of a Sp1 site within the 78 bp region significantly blocked promoter activity. In addition, high expression of Sp1 transcription factor was noted prior to and paralleling the differentiation process. Taken together, our data suggest that RGS2 transcription is immediately induced via a cAMP-dependent pathway after initiation of 3T3-L1 differentiation and the RGS2 mRNA level remains consistently high throughout the differentiation progression. A Sp1 site within RGS2 promoter appeared to be a crucial response element to regulate RGS2 transcription.
(c) 2008 Wiley-Liss, Inc.