Biomedical subjects

A C Wild

Publications and source records attributed to A C Wild.

4 recordsLinked to original sources

PubMed2000

Regulation of gamma-glutamylcysteine synthetase subunit gene expression: insights into transcriptional control of antioxidant defenses.

Gamma-glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5'-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-kappaB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery.

Animals↗

PubMed1999

Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2.

Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) or pyrrolidine dithiocarbamate (PDTC) resulted in the up-regulation of the gamma-glutamylcysteine synthetase catalytic (GCS(h)) and regulatory (GCS(l)) subunit genes. Increased expression was associated with an increase in the binding of Nrf2 to electrophile response elements (EpRE) in the promoters of these genes. Nrf2 overexpression increased the activity of GCS(h) and GCS(l) promoter/reporter transgenes. Overexpression of an MafK dominant negative mutant decreased Nrf2 binding to GCS EpRE sequences, inhibited the inducible expression of GCS(h) and GCS(l) promoter/reporter transgenes, and reduced endogenous GCS gene induction. beta-NF and PDTC exposure also increased steady-state levels of MafG mRNA. In addition to Nrf2, small Maf and JunD proteins were detected in GCS(h)EpRE-protein complexes and, to a lesser extent, in GCS(l)EpRE-protein complexes. The Nrf2-associated expression of GCS promoter/reporter transgenes was inhibited by overexpression of MafG. Inhibition of protein synthesis by cycloheximide partially decreased inducibility by PDTC or beta-NF and resulted in significant increases in GCS mRNA at late time points, when GCS mRNA levels are normally declining. We hypothesize that, in response to beta-NF and PDTC, the GCS subunit genes are transcriptionally up-regulated by Nrf2-basic leucine zipper complexes, containing either JunD or small Maf protein, depending on the particular GCS EpRE target sequence and the inducer. Following maximal induction, down-regulation of the two genes is mediated via a protein synthesis-dependent mechanism.

Base Sequence↗

PubMed1999

Pyrrolidine dithiocarbamate up-regulates the expression of the genes encoding the catalytic and regulatory subunits of gamma-glutamylcysteine synthetase and increases intracellular glutathione levels.

Time- and dose-dependent increases in the steady-state mRNA levels of the genes encoding the catalytic and regulatory subunits of the enzyme gamma-glutamylcysteine synthetase (GCS) were observed in HepG2 human hepatocarcinoma cells after exposure to pyrrolidine dithiocarbamate (PDTC). PDTC was demonstrated to manifest both antioxidant and pro-oxidant properties in HepG2 cells, as assessed by the decreased fluorescence of the redox-sensitive dye Dihydrorhodamine 123 and by the oxidation of glutathione respectively. Attempts to characterize the signalling pathway from PDTC exposure to increases in the expression of the GCS catalytic and regulatory subunit genes demonstrated that induction by PDTC could be partially blocked by treatment with the thiol agent N-acetylcysteine and by the copper chelator bathocuproine disulphonic acid. These findings suggested that the up-regulation of the two genes resulted from a PDTC-induced pro-oxidant signal, which was partially copper-dependent. In summary, these studies demonstrate that PDTC exposure elicits a cellular response in HepG2 cells, characterized by the induction of the genes encoding the two subunits of the enzyme GCS and increased de novo synthesis of the cellular protectant GSH.

Catalytic Domain↗

PubMed1998

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.

Antioxidants↗