Identification of benzo[a]pyrene-inducible cis-acting elements within c- Ha-ras transcriptional regulatory sequences

C. M. Bral, Kenneth Ramos

Research output: Contribution to journalArticle

43 Citations (Scopus)

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 the c-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.

Original languageEnglish (US)
Pages (from-to)974-982
Number of pages9
JournalMolecular Pharmacology
Volume52
Issue number6
StatePublished - Dec 1997
Externally publishedYes

Fingerprint

Antioxidant Response Elements
Benzo(a)pyrene
Response Elements
Hydrocarbons
Nucleic Acid Regulatory Sequences
Transcriptional Activation
Aryl Hydrocarbon Receptors
Proteins
ras Genes
Electrophoretic Mobility Shift Assay
Chloramphenicol
Transferases
Reporter Genes
Vascular Smooth Muscle
Epigenomics
Oligonucleotides
Smooth Muscle Myocytes
Transfection
Atherosclerosis
Transcription Factors

ASJC Scopus subject areas

  • Pharmacology

Cite this

Identification of benzo[a]pyrene-inducible cis-acting elements within c- Ha-ras transcriptional regulatory sequences. / Bral, C. M.; Ramos, Kenneth.

In: Molecular Pharmacology, Vol. 52, No. 6, 12.1997, p. 974-982.

Research output: Contribution to journalArticle

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