title: Platelet-derived growth factor-BB inhibits rat alpha1D-adrenergic receptor gene expression in vascular smooth muscle cells by inducing AP-2-like protein binding to alpha1D proximal promoter region.
abstract: We have previously found that, in addition to mediating contraction of vascular smooth muscle, activation of alpha1D-adrenergic receptors (AR) induces smooth muscle cell (SMC) hypertrophy. Despite their importance, little is known about how alpha1D-AR expression is regulated. Recently, we demonstrated that platelet-derived growth factor (PDGF)-beta receptor stimulation, but not various other growth factors, inhibits transcription of alpha1D-, but not alpha1A- or alpha1B-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 alpha1D-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 alpha1D 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 alpha1D-AR activity driven by this motif. Thus, PDGF-BB may increase AP-2 binding within the proximal promoter to cause down-regulation of alpha1D-AR expression in SMCs when PDGF is elevated, such as in the postnatal growing vascular wall and in vascular hypertrophic diseases.
authors.affiliation: Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7545, USA.
authors.forename: X
authors.initials: X
authors.lastname: Xin
authors.forename: N
authors.initials: N
authors.lastname: Yang
authors.forename: J E
authors.initials: JE
authors.lastname: Faber
mesh_terms: D000818:Animals; D000077214:Becaplermin; D019976:Cloning, Organism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C547721:Adra1d protein, rat; D004268:DNA-Binding Proteins; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000077214:Becaplermin