Critical Roles of a Cyclic AMP Responsive Element and an E-box in Regulation of Mouse Renin Gene Expression

Abstract

Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin expressing cells, express high levels of renin mRNA 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) upstream of the mouseRen-1^c gene. This enhancer, in combination with the proximal promoter (−117 to +6), activates transcription nearly 2 orders of magnitude in an orientation independent fashion. To further delimit sequences necessary for transcriptional activation, renin promoter-luciferase reporter gene constructs containing selected regions of the Ren-1^c enhancer were analyzed after transfection into As4.1 cells. These results demonstrate that several regions are required for full enhancer activity. Sequences from −2699 to −2672, which are critical for the enhancer activity, contain a cyclic AMP responsive element (CRE) and an E-box. Electrophoretic mobility shift 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 synergistically activating transcription from the Ren-1^c promoter. Moreover, mutation of either the CRE or E-box results in almost complete loss of enhancer activity, suggesting the critical roles these two elements play in regulating mouseRen-1^c gene expression. Although theRen-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 treatment with the protein kinase A inhibitor, H-89, caused a significant reduction in Ren-1^c gene expression and this reduction is mediated through the CRE at −2699 to −2688.