CRE-like response element regulates expression of rat alpha 2D-adrenergic receptor gene in vascular smooth muscle

Abstract

Contractile and binding studies indicate that alpha 2-adrenergic receptors (ARs) are differentially expressed by vascular smooth muscle cells (SMCs) according to vascular segment (artery, arteriole, vein). In the present study, alpha 2D-AR mRNA was two- to threefold higher in vena cava than in aorta. To understand vascular regulation of alpha 2D-AR expression in these cells, we sequenced 2.8 kb of the 5' flanking region of the alpha 2D-AR gene. Notable features include two potential TATA boxes, an adenosine 3',5'-cyclic monophosphate response element (CRE)-like binding element, and an Sp1 element. Comparison of the rat and human genes revealed an overall homology of 74% over the 1.87-kb sequence 5' to the translation initiator methionine, including complete homology at the distal TATA, CRE-like, and Sp1 sites, alpha 2D-AR transcription starts from the guanine nucleotide 18 base pair downstream from the distal TATA box. Reporter gene constructs demonstrated strong alpha 2D-AR promoter activity, but with several differences in construct activity, in both rat aorta and vena cava SMCs. Analysis of an essential promoter fragment revealed two regions protected by aorta and vena cava SMC nuclear proteins. The core sequences of these protected regions are TGACGCTA and TATAA. The former CRE-like element conferred specific binding of both aorta and vena cava nuclear proteins. In addition, promoter activity was increased 300% by forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate, indicating that the CRE-like element may regulate alpha 2D-AR expression in vascular tissue.