Abstract
Atherosclerosis and restenosis are common vascular disorders that involve excess proliferation of smooth muscle cells (SMCs) in the artery wall. In this study we demonstrate the anti-mitogenic, pro-apoptotic role of the zinc finger transcription factor Sp1 in vascular SMCs and define the underlying molecular mechanism via its capacity to repress the expression of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 at the level of transcription, mRNA, and protein. SMC proliferation inducible by a dominant-negative mutant form of Sp1 was abrogated by antisense strategies targeting p21WAF1/Cip1. Conversely, antisense p21WAF1/Cip1 induced apoptosis in SMCs overexpressing dominant-negative-Sp1. p21WAF1/Cip1 overexpression alone stimulated proliferation and inhibited apoptosis. Sp1 down-regulated p21WAF1/Cip1 expression in SMCs. Sp1 blocked assembly of cyclin D1-Cdk4-p21WAF1/Cip1 complex formation whose integrity is critical for G1->S transition. Moreover, Rb phosphorylation, which lies immediately downstream of the cyclin D1-Cdk4-p21WAF1/Cip1 complex, was blocked either by Sp1 overexpression or antisense p21WAF1/Cip1. These findings, using complementary approaches, demonstrate the inverse relationship between Sp1 and p21WAF1/Cip1 in SMCs and the capacity of Sp1 to regulate SMC proliferation and apoptosis via its repression of p21WAF1/Cip1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis
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Arteries / metabolism
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Blotting, Western
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Bromodeoxyuridine / pharmacology
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CDC2-CDC28 Kinases*
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Cell Cycle
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Cell Division
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Cell Line
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Cells, Cultured
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Cyclin D1 / metabolism*
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / metabolism*
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Down-Regulation
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Endothelium, Vascular / cytology*
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Etoposide / pharmacology
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Genes, Dominant
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Mutation
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Myocytes, Smooth Muscle / pathology*
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Nucleic Acid Synthesis Inhibitors / pharmacology
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Oligonucleotides, Antisense / pharmacology
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Promoter Regions, Genetic
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Protein Binding
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins*
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RNA, Messenger / metabolism
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Rats
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Rats, Inbred WKY
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Retinoblastoma Protein / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Sp1 Transcription Factor / genetics
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Sp1 Transcription Factor / metabolism
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Sp1 Transcription Factor / physiology*
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Time Factors
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Transcription, Genetic*
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Zinc Fingers
Substances
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Cdkn1a protein, rat
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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Nucleic Acid Synthesis Inhibitors
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Oligonucleotides, Antisense
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Proto-Oncogene Proteins
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RNA, Messenger
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Retinoblastoma Protein
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Sp1 Transcription Factor
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Cyclin D1
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Etoposide
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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Cdk2 protein, rat
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Cdk4 protein, rat
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinases
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Bromodeoxyuridine