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.
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.
Nguyen
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Department of Molecular Biology, Merck Frosst Center for Therapeutic Research, Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec, Canada.
Rushmore
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TH
Pickett
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CB
D000818:Animals; D000975:Antioxidants; D001483:Base Sequence; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D005982:Glutathione Transferase; D013755:Tetradecanoylphorbol Acetate