Vol. 56, Issue 6, 1152-1161, December 1999

Platelet-Derived Growth Factor-BB Inhibits Rat 1D-Adrenergic Receptor Gene Expression in Vascular Smooth Muscle Cells by Inducing AP-2-Like Protein Binding to 1D Proximal Promoter Region

Xiaohua Xin, Nengyu Yang, and James E. Faber

Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina

We have previously found that, in addition to mediating contraction of vascular smooth muscle, activation of 1D-adrenergic receptors (AR) induces smooth muscle cell (SMC) hypertrophy. Despite their importance, little is known about how 1D-AR expression is regulated. Recently, we demonstrated that platelet-derived growth factor (PDGF)- receptor stimulation, but not various other growth factors, inhibits transcription of 1D-, but not 1A- or 1B-ARs, resulting in reduced norepinephrine-mediated SMC growth. To investigate this inhibitory mechanism, herein we cloned and characterized 1.6 kb of the 5'-flanking region of the rat 1D-AR gene. Reporter gene transfection assays in rat aorta and vena cava SMCs showed that this 5'-flanking region, which lacks a TATA-box, possesses strong promoter activity. Two transcription initiation sites and their flanking promotor 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 399 base pair (bp)/349-bp region of the proximal promotor. This constitutive binding was unaffected by PDGF-BB. In contrast, a flanking motif (384 bp/349 bp), possessing putative Sp1/activator protein-2 (AP-2) overlapping binding sites and located upstream of the proximal transcription initiation site, was required for PDGF-BB inhibition of 1D transcription. PDGF-BB increased AP-2 binding to the distal AP-2 site in this region in the context of SMCs. Furthermore, overexpression of AP-2 protein, by transgene transfection, dose-dependently inhibited 1D-AR activity driven by this motif. Thus, PDGF-BB may increase AP-2 binding within the proximal promoter to cause down-regulation of 1D-AR expression in SMCs when PDGF is elevated, such as in the postnatal growing vascular wall and in vascular hypertrophic diseases.