OVERLAPPING ANTIOXIDANT RESPONSE ELEMENT AND PMA RESPONSE ELEMENT SEQUENCES MEDIATE BASAL AND BETA-NAPHTHOFLAVONE-INDUCED EXPRESSION OF THEHUMAN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE CATALYTIC SUBUNIT GENE

gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in t he ne novo synthesis of GSH, is a heterodimer, consisting of a catalyt ic (GCS(h)) and a regulatory subunit (GCS(1)). We previously demonstra ted that the constitutive and beta-naphthoflavone (beta-NF)-induced ex pression of the GCS(h) gene is mediated by a distal antioxidant respon se element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 2 72, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCA GCG-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 GCS(h) gene was determined by mutational analyses. The internal activator protein-1 (A P-1)-binding sequence mediated constitutive expression of promoter/rep orter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins f rom nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particu larly 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 co nstructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cel ls. 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 t his element within a GCS(h) promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that AR E3 does not influence the constitutive or beta-NF-induced expression o f the GCS(h) gene.