Profiles of antioxidant/electrophile response element (ARE/EpRE) nuclear protein binding and c-Ha-ras transactivation in vascular smooth muscle cells treated with oxidative metabolites of benzo[a]pyrene.
Activation of nuclear protein binding to the antioxidant/electrophile response element (ARE/EpRE) by benzo[a]pyrene (BaP) in vascular smooth muscle cells (vSMCs) is associated with transcriptional deregulation of c-Ha-ras. This response may be mediated by oxidative intermediates of BaP generated during the course of cellular metabolism. To test this hypothesis, the profile of ARE/EpRE protein binding and transactivation elicited by BaP was compared with that of 3-hydroxy BaP (3-OH BaP) (0.03 to 3.0 microM), BaP 7,8-dihydrodiol (BaP 7,8-diol) (0.03 to 3.0 microM), BaP 3,6-quinone (BaP 3,6-Q) (0.0003 to 3.0 microM), and H(2)O(2) (25 to 100 microM). Specific protein binding to the consensus c-Ha-ras ARE/EpRE was observed in vSMCs treated with all BaP metabolites at concentrations considerably lower than those required for the parent compound. H(2)O(2), a by-product of BaP 3,6-Q redox cycling, also increased binding to the ARE/EpRE. Treatment of vSMCs with oxidative BaP metabolites or H(2)O(2) transactivated the c-Ha-ras promoter in all instances, but the response was consistently half of the maximal induction elicited by BaP. Similar proteins cross-linked specifically to the consensus c-Ha-ras ARE/EpRE sequence in cells treated with BaP or its oxidative intermediates. The protein binding profile in the c-Ha-ras promoter was similar to that in the NADPH:quinone reductase gene (NQO(1)) and the glutathione S-transferase Ya gene (GSTYa) promoters, but the relative abundance of individual complexes was promoter-specific. We conclude that oxidative intermediates of BaP mediate activation of nuclear protein binding to ARE/EpRE and contribute to transcriptional de-regulation of c-Ha-ras in vSMCs.
Miller
K P
KP
Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA.
Chen
Y H
YH
Hastings
V L
VL
Bral
C M
CM
Ramos
K S
KS
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011485:Protein Binding; D015533:Transcriptional Activation / Q000187:drug effects*
D000975:Antioxidants; D009687:Nuclear Proteins; D001564:Benzo(a)pyrene