Platelet-derived growth factor-BB inhibits rat alpha 1D-adrenergic receptor gene expression in vascular smooth muscle cells by inducing AP-2-like protein binding to alpha 1D proximal promoter region

We have previously found that, in addition to mediating contraction of vasc ular smooth muscle, activation of alpha 1D-adrenergic receptors (AR) induce s smooth muscle cell (SMC) hypertrophy. Despite their importance, little is known about how alpha 1D-AR expression is regulated. Recently, we demonstr ated that platelet-derived growth factor (PDGF)-b receptor stimulation, but not various other growth factors, inhibits transcription of alpha 1D-, but not alpha 1A- or alpha 1B-ARs, resulting in reduced norepinephrine-mediate d SMC growth. To investigate this inhibitory mechanism, herein we cloned an d characterized 1.6 kb of the 5'-flanking region of the rat alpha 1D-AR gen e. Reporter gene transfection assays in rat aorta and vena cava SMCs showed that this 5'-flanking region, which lacks a TATA-box, possesses strong pro moter activity. Two transcription initiation sites and their flanking promo tor regions were identified, wherein the proximal promotor mediated PDGF-BB inhibition of transcription. Gel mobility shift assays suggested that Sp1 binds constitutively at two consensus sites within the 2399 base pair (bp)/ 2349-bp region of the proximal promotor. This constitutive binding was unaf fected by PDGF-BB. In contrast, a flanking motif (2384 bp/2349 bp), possess ing putative Sp1/activator protein-2 (AP-2) overlapping binding sites and l ocated upstream of the proximal transcription initiation site, was required for PDGF-BB inhibition of alpha 1D transcription. PDGF-BB increased AP-2 b inding to the distal AP-2 site in this region in the context of SMCs. Furth ermore, overexpression of AP-2 protein, by transgene transfection, dose-dep endently inhibited alpha 1D-AR activity driven by this motif. Thus, PDGF-BB may increase AP-2 binding within the proximal promoter to cause down-regul ation of alpha 1D-AR expression in SMCs when PDGF is elevated, such as in t he postnatal growing vascular wall and in vascular hypertrophic diseases.