Activation of nuclear protein binding to the antioxidant/electrophile response element in vascular smooth muscle...

This laboratory has previously shown that binding of nuclear proteins to the antioxidant/electrophile response element (ARE/EpRE) participates in deregulation of vascular gene expression by benzo(a)pyrene (BaP), a suspected atherogen. In the present study, oligonucleotides representing ARE/EpREs within the c-Ha-ras and glutathione-S-transferase (GST-Ya) promoters were employed to evaluate the role of flanking sequences in stabilizing protein:DNA interactions in BaP-treated vascular smooth muscle cells (vSMCs). We also wanted to define promoter-specific patterns of protein recognition to ARE/EpREs in this cell system. In electrophoretic mobility shift assays (EMSA), optimal protein binding to a human Ha-ras ARE/EpRE variant sequence fitted to match the extended mouse(m) GST-Ya ARE/EpRE core (5'-TMAnnRTGAYnnnGCR-3') was dependent on 5' nucleic acid sequence. Using immobilized DNA affinity chromatography (IDAC), we identified four nuclear proteins of M(r) 62, 60, 50, and 30 kDa that associated specifically with the mGSTYa ARE/EpRE. Photo crosslinking to a BrdU-substituted hHa-ras or mGST ARE/EpRE probe identified specific proteins of M(r) 80, 60, 55, 25, 23 kDa or 80, 60, 55, 27, 25, 23 kDa, respectively. Protein:DNA complexes detected using IDAC eluate overlapped with those observed in crude nuclear extracts. Chemical treatments known to modulate ARE/EpRE protein binding in vSMCs did not alter overall protein:DNA affinity and/or sequence recognition to either hHa-ras or mGST-Ya elements. We conclude that nucleotide sequences 5' to the core ARE/EpRE influence specific binding of nuclear proteins and that multiple proteins bind to ARE/EpREs in a promoter-specific manner in vSMCs.