title: Vascular smooth muscle cell-specific regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1) transcription by Sp1 is mediated via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.
abstract: Smooth muscle cells (SMC) play a central role in common vascular pathologies such as atherosclerosis and restenosis. Understanding the molecular regulation of SMC proliferation at a transcriptional level may provide important clues for the targeted control of vascular hyperplasia. We recently reported the capacity of the transcription factor Sp1 to down-regulate p21(WAF1/Cip1) production thereby reducing p21(WAF1/Cip1)-cyclin D1-Cdk4 complex formation and inhibiting vascular SMC proliferation (Kavurma and Khachigian [2003] J. Biol. Chem. 278, 32537-32543). We have now localized the Sp1-response elements in the p21(WAF1/Cip1) promoter responsible for p21(WAF1/Cip1) repression in WKY12-22 SMCs. The proximal region of the p21(WAF1/Cip1) promoter contains five distinct Sp1-binding elements that we have termed A, B, C, D, and E. Electrophoretic mobility shift analysis revealed that SMC nuclear Sp1 interacts with all five Sp1-binding sites, and each of these sites is critical for Sp1 repression of the p21(WAF1/Cip1) promoter, since mutation in any one element ablates repression, and in some cases results in activation. In contrast, only elements C, D, and E are bound by Sp1 in endothelial cells. Sp1 overexpression activates the p21(WAF1/Cip1) promoter in this cell type. Furthermore, mutation in any of these five elements is not sufficient to prevent activation of the p21(WAF1/Cip1) promoter by Sp1 in endothelial cells. Surprisingly, double mutations of elements C and E facilitates superactivation by Sp1 in both cell types, whereas triple mutations of C, D, and E inactivate the promoter. These findings demonstrate cell type-specific regulation of p21(WAF1/Cip1) transcription by Sp1 via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.
authors.affiliation: The Centre for Vascular Research, Department of Pathology, The University of New South Wales, Australia.
authors.forename: Mary M
authors.initials: MM
authors.lastname: Kavurma
authors.forename: Levon M
authors.initials: LM
authors.lastname: Khachigian
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C493958:Cdkn1a protein, rat; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D011494:Protein Kinases; C065999:Sp1 kinase