A CCAAT/enhancer-binding protein site within antioxidant/electrophile response element along with CREB-binding protein participate in the negative regulation of rat GST-Ya gene in vascular smooth muscle cells.

Abstract

Studies were conducted to evaluate the negative regulatory function of rat (r)GST-Ya antioxidant/electrophile response element (ARE/EpRE) in vascular smooth muscle cells (vSMCs). We report that CCAAT/enhancer-binding protein (C/EBP)-beta interacts with ARE/EpRE in the rGST-Ya promoter and that aryl hydrocarbon receptor (AhR) is present within the protein complex binding to the C/EBP site. Overexpression of C/EBP-beta or C/EBP-alpha repressed, whereas AhR enhanced, 1.6CAT reporter activity in cells treated with benzo(a)pyrene (BaP). Overexpression of CREB-binding protein (CBP) nullified repression of rGST-Ya transcription. Human adenovirus E1A protein abrogated cotransactivation by CBP but an E1A mutant did not. Overexpression of C/EBPs abrogated stimulation of 1.6CAT by CBP or AhR alone, or in combination, regardless of BaP treatment. Similar profiles were observed using an AhRECAT construct. The C/EBP site within the ARE/EpRE inhibited chemical inducibility of the AhRE. The pattern of mouse GST-Ya regulation by BaP was similar to that of rGST-Ya. We conclude that multiple mechanisms mediate negative regulation of GST-Ya gene in vSMCs, most significant of which are that C/EBP-beta inhibits AhRE or ARE/EpRE inducibility of GST-Ya, limiting CBP levels compromise gene induction, functional interference exists between AhRE and ARE/EpRE, and AhR alone, or in combination with C/EBP-beta, functions as a repressor of the ARE/EpRE.