Overlapping antioxidant response element and PMA response element sequences mediate basal and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase catalytic subunit gene.
gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and beta-naphthoflavone (beta-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCAGCG-3') containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and beta-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and beta-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or beta-NF-induced expression of the GCSh gene.
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Department of Human Oncology, University of Wisconsin Medical School, 600 Highland Avenue, K4/316 CSC, Madison, WI 53792, USA.
Gipp
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Mulcahy
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D000975:Antioxidants / Q000494:pharmacology*; D001665:Binding Sites / Q000502:physiology; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005807:Genes, Overlapping; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000201:enzymology; D009687:Nuclear Proteins / Q000032:analysis; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D019324:beta-Naphthoflavone / Q000494:pharmacology*
D000975:Antioxidants; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D019324:beta-Naphthoflavone; D005721:Glutamate-Cysteine Ligase; D013755:Tetradecanoylphorbol Acetate