Transcriptional regulation of the mouse steroidogenic acute regulatory protein gene by the cAMP response-element binding protein and steroidogenic factor 1

Abstract

Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5′-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMτ (but not CREMα and CREMβ) have qualitatively similar effects on StAR promoter activity in response to (Bu) 2 cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu) 2 cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMτ and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMτ and SF-1 results in enhanced activation of the StAR promoter.

The role of JUN in the regulation of PRKCC-mediated STAR expression and steroidogenesis in mouse Leydig cells

“…Nevertheless, an oligonucleotide probe encompassing the JUN motif can specifically bind MA-10 NE in a PRKCC-responsive manner as well as in vitro translated JUN, and that DNAprotein binding was affected by mutations in the JUN site, JUN Ab, and consensus JUN. It is noteworthy that the JUN motif in the STAR promoter is analogous to a CRE-like element that interacts with CRE-DNA-binding proteins (Manna et al 2002a(Manna et al , 2003a. Similar overlapping sequences have been shown to target the CCAAT/ enhancer-binding protein b (Silverman et al 1999(Silverman et al , 2006.…”

“…Luciferase activity in the cell lysates was determined by the Dual-luciferase reporter assay system (Promega), using a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA), as described previously (Manna et al 2002a, 2003a.…”

“…All western blotting experiments were repeated three to five times. EMSA experiments were carried out with MA-10 nuclear extracts (NE) and in vitro transcribed/translated JUN (Manna et al 2002a(Manna et al , 2003a(Manna et al , 2004 (Manna et al 2002a(Manna et al , 2004. Briefly, NE (12 mg) and in vitro translated JUN (2-5 mg) were incubated in 20 ml reaction buffer (10 mM HEPES, 1 mM EDTA, 4% Ficoll, 10 mM dithiothreitol, 1 mg poly dIdC, 40 mg/ml BSA, and 2 mM ZnSO 4 , pH 7 .…”

Crosstalk of CREB and Fos/Jun on a single cis-element: transcriptional repression of the steroidogenic acute regulatory protein gene

“…While the StAR gene promoter lacks a consensus CRE, several transacting factors within the 150 bp (relative to the transcription start site) cAMP-responsive region have been demonstrated to be instrumental in StAR gene expression (Manna et al 2003a, Clem et al 2005. Previously, we have identified three CRE-like elements (including an overlapping CRE2/AP-1 motif, TGACTGA, K81/K75), and the roles of CRE (CREB/CRE modulator (CREM)) and AP-1 (Fos/Jun) family members in transcription of the StAR gene have been demonstrated (Manna et al 2002b(Manna et al , 2003b(Manna et al , 2004. However, those studies did not provide information on the molecular events by which these families of proteins associate with the CRE2/AP-1 site to regulate the transcriptional machinery involved in StAR gene expression.…”

Natural Flavonoids in StAR Gene Expression and Testosterone Biosynthesis in Leydig Cell Aging

“…The cAMP-responsive element (CRE)-like sites were found in StAR promoter DNA sequences. The results from electrophoretic mobility shift assays (EMSA) demonstrated the binding of CREB to StAR promoter (Manna et al, 2002;Manna et al, 2003). PKA-phosphorylation is critical for the activity of CREB (Meyer & Habener, 1993;Montminy et al, 1986), because the phosphorylation of CREB is needed for the recruitment of its cofactor CBP (CREB binding protein) that is involved in the transcriptional activity of CREB (Chrivia et al, 1993;.…”