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.

Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver and kidney slices.

Benzo(a)pyrene (BaP) and related aromatic hydrocarbons are suspected carcinogens; however, the molecular basis underlying tumorigenesis remains unclear. To identify acute molecular targets of BaP within the liver and kidney, precision-cut slices harvested from naive, adult female Sprague-Dawley rats were challenged with BaP (0.3-30 microM) for 0.5 to 24 h. BaP did not elicit cytotoxicity, as assessed by intracellular K+ and ATP content and histological evaluation over the 24-h period. To determine if molecular signaling pathways were maintained in precision-cut slices, induction of the aryl hydrocarbon receptor (AhR) pathway was assessed following BaP challenge. Induction of cytochrome P450IA1 (P450IA1) mRNA and protein expression was observed in both liver and kidney slices. c-fos and c-Ha-ras gene expression was enhanced in liver, but not kidney, slices by BaP. c-jun mRNA levels were decreased in liver and kidney slices, although the effect was earlier (0.5 h) in liver slices compared to kidney slices. BaP increased the DNA binding of nuclear proteins to the AP-1 consensus recognition element in liver, but decreased DNA binding in kidney slices. In contrast, DNA binding of NF-kappa B was not affected by BaP in either liver or kidney slices. These results suggest that acute BaP challenge is associated with altered expression of several growth-related genes and AP-1 signaling and establish precision-cut slices as a useful in vitro system to investigate the molecular basis of BaP-induced tumorigenesis, including organ-specific differences.

RNA polymerase II transcription complex assembly in nuclear extracts.

In vitro transcription systems based on nuclear extracts of eukaryotic cells continue to be valuable experimental systems for assessing function of promoter sequences and defining new activities involved in transcription complex assembly and activity, but many aspects of such systems have not been experimentally examined. Here, transcription complex assembly on the promoter from the long terminal repeat of mouse mammary tumor virus was assessed in vitro with a transcription system derived from nuclear extracts of cultured HeLa cells. The extent of preinitiation complex assembly on the promoter was limited by the availability of template, even though only a small fraction of the template present in the assays participated in transcription. These results support a model for transcription complex assembly in which template DNA has two alternative fates, one leading to assembly of a functional transcription complex, and another that leads to irreversible template inactivation. The observed kinetics of assembly reflects loss of template by both pathways and is dominated by a relatively rapid rate of template inactivation. Supplementing nuclear extracts with purified TATA binding protein increased the extent as well as the apparent rate of assembly. Both effects can be explained by a TATA binding protein-dependent increase in the rate of assembly that leads to altered partitioning of template between competing pathways.

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

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 microM), 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.