Transcriptional regulation of a rat liver glutathione S-transferase Ya subunit gene. Analysis of the antioxidant response element and its activation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Abstract

Using transfection and gel retardation assays, we have characterized further the antioxidant response element (ARE) found in the 5'-flanking region of the rat glutathione S-transferase Ya subunit gene. The ARE core sequence (5'-GTGACAAAGC-3') is sufficient for transcriptional activation of the Ya subunit gene by metabolizable planar aromatic compounds, phenolic antioxidants, and hydrogen peroxide. When the ARE sequence is ligated to a chloramphenicol acetyltransferase reporter gene and transfected into HepG2 cells, chloramphenicol acetyltransferase activity is modestly inducible by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Since the ARE is responsive to TPA and shows some sequence similarity to an AP-1-binding site (Jun/Fos recognition motif), we have explored whether members of the Jun/Fos family of transcription factors might bind to the ARE. Using in vitro synthesized Jun and Fos, binding to the ARE could not be detected, whereas Jun/Fos binding to a classical AP-1-binding site, a TPA response element (TRE) from the human collagenase gene, could be demonstrated by gel retardation assays. If the 2 A nucleotides underlined in the ARE core sequence (5'-GTGACAAAGC-3') are changed to TC, the ARE sequence (ARE-TRE) becomes a high-affinity AP-1-binding site and retains xenobiotic inducibility. Removal of the -GC- dinucleotide at the 3'-end of the ARE or the ARE-TRE eliminates xenobiotic inducibility. However, the ARE-TRE construct without the -GC- dinucleotide is still a high-affinity AP-1 site and responsive to TPA. Taken together, our data suggest that the ARE is not a high-affinity binding site for the Jun/Fos heterodimer. Functionally, however, an AP-1-binding site can resemble an ARE in its response to various xenobiotics if a 3'-GC- dinucleotide is present.