Abstract
Previous studies in this laboratory have demonstrated that transcriptional deregulation of c-Ha-ras expression is associated with the induction and maintenance of proliferative vascular smooth muscle cell (SMC) phenotypes by benzo[a]pyrene (BaP). We examined previously undescribed cis-acting elements within the proximal 5′ regulatory region of c-Ha-ras (−550 to +220) for their ability to influence BaP-induced transcription in murine SMCs. BaP-inducible DNA binding activity was demonstrated at a site located −30 relative to the major start site cluster at +1 that exhibits extensive homology to a consensus aryl hydrocarbon response element (AHRE), as well as a site located at −543 that contains a consensus electrophile response element (EpRE). In vitro cross-linking studies revealed the specific interaction of 104- and 96-kDa proteins with the putative AHRE and of an 80-kDa protein with the EpRE. The use of monoclonal antibodies to the aryl hydrocarbon receptor transcription factor in competition electrophoretic mobility shift assays indicated this protein is specifically induced by BaP to interact at the AHRE within thec-Ha-ras 5′ regulatory region. Transient transfection with an Ha-ras promoter construct containing the putative AHRE but lacking the EpRE linked to the chloramphenicol acetyl transferase reporter gene, followed by challenge with BaP (0.3, 3.0, and 30 μm), revealed transcriptional activation that was not statistically significant. However, insertion of an oligonucleotide composed of the EpRE immediately upstream of basal sequences at −330 was associated with strong activation of transcription by BaP. These data indicate that c-Ha-ras gene expression is modulated by BaP via a complex mechanism that likely involves interactions among multiple regulatory elements. We conclude that c-Ha-ras expression is regulated by BaP at the transcriptional level, a response that may constitute an epigenetic basis of atherogenesis.
Footnotes
- Received February 24, 1997.
- Accepted August 27, 1997.
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Send reprint requests to: Dr. Kenneth S. Ramos, Dept. of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843-4466. E-mail:kramos{at}cvm.tamu.edu
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This work was supported in part by National Institute of Environmental Health Sciences Grants ES04849 (K.S.R.) and ES05728 (C.M.B.).
- The American Society for Pharmacology and Experimental Therapeutics
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