γ-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 β-naphthoflavone (β-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 β-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 β-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 β-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 β-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from β-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 β-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or β-NF-induced expression of the GCSh gene.
Constitutive and β-Naphthoflavone-induced Expression of the Human γ-Glutamylcysteine Synthetase Heavy Subunit Gene Is Regulated by a Distal Antioxidant Response Element/TRE Sequence
