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J. Biol. Chem., Vol. 275, Issue 35, 27366-27376, September 1, 2000

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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^*

Yun-Houng Chen and Kenneth S. Ramos

From the Department of Physiology and Pharmacology, College of Veterinary Medicine, and Center for Environmental and Rural Health, Texas A & M University, College Station, Texas 77843 4466

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)- 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- or C/EBP- 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- 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-, functions as a repressor of the ARE/EpRE.

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