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Negative regulation of rat GST-Ya gene via Antioxidant/Electrophile response element is directed by a C/EBP-like site.

The present studies were conducted to evaluate functional interactions between aryl hydrocarbon and antioxidant/electrophile response elements (AhRE and ARE/EpRE, respectively) in transcriptional regulation of the rat (r)GST-Ya gene. Transient transfection of an AhRECAT reporter construct into vascular smooth muscle cells (vSMCs) or HepG2 cells showed that benzo(a)pyrene (BaP) (0.3-30 microM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0. 1-10 nM), but not hydrogen peroxide (H(2)O(2)) (100-400 microM), increased gene transcription. ARE/EpRE did not mediate gene inducibility by any of the chemicals in vSMCs but increased transcription in HepG2 cells treated with BaP or H(2)O(2), but not TCDD. Gene inducibility in response to all chemicals was repressed in both cell types transfected with a 1.6CAT full-length promoter construct containing the AhRE and ARE/EpRE in genomic context. Site-directed mutagenesis of 1.6CAT showed that a CCAAT/enhancer-binding protein (C/EBP)-like site within the ARE/EpRE directed negative regulation of the rGST-Ya gene in vSMCs and HepG2 cells. These results show that ARE/EpRE in rGST-Ya does not function as a positive cis-acting regulatory element in all cell types, and that in the context of the full-length rGST-Ya promoter a C/EBP-like site directs negative regulation of the gene by BaP and related chemicals.