Critical roles of a cyclic AMP responsive element and an E-box in regulation of mouse renin gene expression

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin expressing cells, express high levels of renin mRN A from their endogenous Ren-1(c) gene. We have previously identified a 242- base pair enhancer (coordinates -2866 to -2625 relative to the CAP site) up stream of the mouse Ren-1(c) gene. This enhancer, in combination with the p roximal promoter (-117 to +6), activates transcription nearly 2 orders of m agnitude in an orientation independent fashion. To further delimit sequence s necessary for transcriptional activation, renin promoter-luciferase repor ter gene constructs containing selected regions of the Ren-1(c) enhancer we re analyzed after transfection into As4.1 cells. These results demonstrate that several regions are required for full enhancer activity. Sequences fro m -2699 to -2672, which are critical for the enhancer activity, contain a c yclic AMP responsive element (CRE) and an E-box. Electrophoretic mobility s hift assays demonstrated that transcription factors CREB/CREM and USF1/USF2 in As4.1 cell nuclear extracts bind to oligonucleotides containing the Ren -1(c) CRE and E-box, respectively. These two elements are capable of synerg istically activating transcription from the Ren-1(c) promoter. Moreover, mu tation of either the CRE or E-box results in almost complete loss of enhanc er activity, suggesting the critical roles these two elements play in regul ating mouse Ren-1(c) gene expression. Although the Ren-1(c) gene contains a CRE, its expression is not induced by cAMP in As4.1 cells. This appears to reflect constitutive activation of protein kinase A in As4.1 cells since t reatment with the protein kinase A inhibitor, H-89, caused a significant re duction in Ren-1(c) gene expression and this reduction is mediated through the CRE at -2699 to -2688.