Overlapping antioxidant response element and PMA responseelement sequences mediate basal and β-naphthoflavone-induced expression of the human γ-glutamylcysteine synthetase catalytic subunit gene

Abstract

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.

γ-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells

“…Because the GC box was not required for AP-1 binding but was critical for the induction of gene expression via EpRE [52,53], the motif responsible for both basal and HNE-inducible promoter activity appeared to be EpRE rather than the embedded AP-1-binding sequence. This is consistent with a previous report on the roles of the GC box in the EpRE of modulatory subunit of glutamate cysteine ligase (GCLm) [54], in which a GC box mutation abrogated β-naphthoflavone-induced EpRE function and had reduced binding efficiency. Studies on the role of the GC box in constitutive gene expression, however, are controversial.…”

“…Studies on the role of the GC box in constitutive gene expression, however, are controversial. Similarly to what was observed here with rat GGT promoter 5, deletion of the GC box abrogated EpRE-mediated basal expression of the rat GSH S-transferase Ya [55] and cystine/glutamate exchange transporter [56]; however, deletion of the GC box had no effect on the constitutive expression of hGCLm and mice heme oxygenase-1 (HO-1) [54,57].…”

H2O2-dependent Activation of GCLC-ARE4 Reporter Occurs by Mitogen-activated Protein Kinase Pathways without Oxidation of Cellular Glutathione or Thioredoxin-1

“…1A, 2-fold increases at 3 milliunits/ml glucose oxidase) in control cells. To explore H 2 O 2 signaling, we used luciferase under the regulation of ARE4, the antioxidant response element from GCLC, which is known to respond to ROS (28,29 Fig. 1A; however, these cells showed no differences in the level of GSSG and GSH compared with control cells (Cont1) (Fig.…”

“…The NF-E2-related factor, Nrf2, can bind to and activate ARE by complexing with AP-1-related proteins such as c-Jun, Jun-B, Jun-D, or c-Fos, while small Maf proteins (MafG and MafK) inhibit gene expression by complexing with Nrf2 (24 -27). Con-stitutive and ␤-naphthoflavone-induced expression of human GCLC gene is mediated by a distal ARE sequence, ARE4, in which an embedded AP-1 site is well conserved (28).…”

Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid

“…The basal and inducible expression of these GCL substituents seem to be mediated by means of the antioxidant response element (ARE) (13)(14)(15). The ARE is a cis-acting enhancer sequence that transcriptionally regulates Phase II detoxification enzymes, which are critical for maintaining cellular redox status and protecting against oxidative damage (16).…”