Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance. The rate-limiting enzyme in the de novo synthesis of glutathione is gamma-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing catalytic and regulatory functions, respectively. Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) resulted in a time- and dose-dependent increase in the steady-state mRNA levels for both subunits. In order to identify sequences mediating the constitutive and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5'-flanking region (-3802 to +465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells. Constitutive expression was maximally directed by sequences between -202 and +22 as well as by elements between -3802 to -2752. The former sequence contains a consensus TATA box. Increased luciferase expression following exposure to 10 microM beta-NF was only detected in cells transfected with a reporter vector containing the full-length -3802:+465 fragment. Hence, elements directing constitutive and induced expression of the GCS heavy subunit are present in the distal portion of the 5'-flanking region, between positions -3802 and -2752. Sequence analysis revealed the presence of several putative consensus response elements in this region, including two potential antioxidant response elements (ARE3 and ARE4), separated by 34 base pairs. When cloned into the thymidine kinase-luciferase vector, pT81-luciferase, and transfected into HepG2 cells, both ARE3 and ARE4 increased basal luciferase expression approximately 20-fold. When cloned in tandem in their native arrangement the increase in luciferase activity was in excess of 100-fold, suggesting a strong interaction between the two sequences. Luciferase expression was elevated in beta-NF-treated cells transfected with the ARE4-tk-luciferase vector and all DNA fragments containing ARE4. In contrast, ARE3 did not direct increased luciferase expression in response to beta-NF nor did it significantly modify the magnitude of induction directed by ARE4. The influence of the ARE4 oligonucleotide on constitutive and induced expression was eliminated by introduction of a single base mutation, converting the core ARE sequence in ARE4 from 5'-GTGACTCAGCG-3' to 5'-GGGACTCAGCG-3'. When introduced into the full-length -3802:+465 segment, the same single base mutation also eliminated both functions. Collectively the data indicate that the constitutive and beta-NF-induced expression of the human GCS heavy subunit gene is mediated by a distal ARE sequence containing an embedded tetradecanoylphorbol-13-acetate-responsive element.
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PMID:Constitutive and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase heavy subunit gene is regulated by a distal antioxidant response element/TRE sequence. 905 46

Several regulatory elements, including AP-1 and NF-kappa B, are present in the 5'flanking region of the human glutamatex-cysteine ligase (EC 6.3.2.2, gamma-glutamyl-cysteine synthetase) catalytic subunit (GLCLC) gene. In this study, we investigated the role of redox-sensitive transcription factors in the regulation of GLCLC gene expression in LLC-PK1 cells that were exposed to the antioxidant butylated hydroxytoluene (BHT). Exposure of LLC-PK1 cells to 100 microM BHT induced expression of transcription factor AP-1, as demonstrated by an electrophoretic mobility shift assay. Peak AP-1 induction occurred after 3 h of incubation with BHT, BHT increased luciferase gene expression in cells that were transfected with a luciferase reporter vector containing an AP-1 element upstream of a SV40 promoter. Northern analysis showed that transcription of GLCLC gene in cells after incubation with BHT was increased 30% compared with control cells. Cellular glutathione concentrations were also significantly increased in cells exposed to BHT. In contrast, exposure of LLC-PK1 cells to 100 microM BHT did not alter expression of the transcription factor NF-kappa B. These results show that induction of transcription factor AP-1 by BHT is involved in transactivation of GLCLC gene expression.
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PMID:Up-regulation of glutamate-cysteine ligase gene expression by butylated hydroxytoluene is mediated by transcription factor AP-1. 953 46

Glioblastoma is one of the most malignant of all neoplasms, and often shows resistance to chemotherapy and radiation therapy. Ionizing radiation activates transcriptional factors, such as nuclear factor kappa-B (NF-kappa B). Previously we found that glutathione (GSH) synthesis is induced by cytokines mediated by NF-kappa B (Urata et al. J. Biol. Chem., 1996). Here, we present direct evidence that NF-kappa B activated by ionizing radiation induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme of GSH synthesis, using T98G human glioblastoma cells. T98G cells have approximately 14-times the level of intracellular GSH of NB9 cells, radiation-sensitive neuroblastoma cells. In T98G cells, 30-Gy of ionizing radiation was required for the activation of NF-kappa B on an electrophoretic mobility shift assay and the induction of gamma-GCS mRNA on Northern blots and a nuclear run-on assay. However, when T98G cells were treated with buthionine sulfoximine, 3-Gy of ionizing radiation stimulated the DNA-binding activity of NF-kappa B and the expression of gamma-GCS. We constructed chimeric genes containing various regions of gamma-GCS promoter gene and the coding region for Luciferase. T98G cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with ionizing radiation. The luciferase activity stimulated by ionizing radiation was found in the gamma-GCS promoter containing the NF-kappa B binding site, whereas not in that containing its mutated site. These results suggest that GSH synthesis is upregulated by ionizing radiation mediated by NF-kappa B and a high concentration of GSH in T98G cells causes downregulation of the NF-kappa B-DNA binding activity in response to ionizing radiation. The irresponsiveness of the intracellular signal transduction cascade to irradiation may be a factor in the resistance of T98G cells to radiation therapy.
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PMID:Nuclear factor kappa B dependent induction of gamma glutamylcysteine synthetase by ionizing radiation in T98G human glioblastoma cells. 962 82

Impairment of endothelial cells by oxidized low density lipoprotein (OxLDL) is believed to be the first step in atherogenesis. It is also believed that oxidative stress/antioxidant imbalance is involved in the cell damage by OxLDL. However, little is known about the interaction between OxLDL and antioxidants. In this study, we show that treatment of human vascular endothelial cells with OxLDL caused a gradual increase of glutathione (gamma-glutamylcysteinyl glycine, GSH) levels in 24 h. OxLDL increased the intracellular levels of reactive oxygen species (ROS) and stimulated the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the GSH synthesis, the mitogen-activated protein kinase (MAPK) activity, and the AP-1-DNA binding activity. The luciferase activity of gamma-GCS promoter containing AP-1 site was activated by OxLDL. Collectively, OxLDL induces gamma-GCS expression mediated by AP-1 resulting in an increase of GSH levels. The MAPK activity stimulated by ROS may be involved in the activation of AP-1. The increase in GSH by OxLDL may afford cellular protection against OxLDL-induced oxidative stress.
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PMID:Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells. 1021 47

In the present study, we show that melatonin induces the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of gamma-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor alpha (RZR/RORalpha). ECV304 cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the gamma-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORalpha site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of gamma-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.
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PMID:Melatonin induces gamma-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells. 1051 88

Glutamate cysteine ligase (GCL; also referred to as gamma-glutamylcysteine synthetase, GCS) catalyzes the rate-limiting step of glutathione synthesis. The GCL holoenzyme is composed of a catalytic (GCLC; also called GCS(h)) and a modifier (GCLM; also called GCS(l)) subunit, each encoded by a unique gene. Wild-type and mutant promoter/luciferase reporter transgenes containing the promoter region of each GCL subunit gene were transfected into A549 (lung carcinoma), HEK 293 (transformed embryonic kidney), HepG2 (hepatocellular carcinoma), and RD (skeletal muscle rhabdomyosarcoma) cells to examine potential cell-type related differences in transcriptional regulation. In A549, HepG2, and RD cells, maximal basal expression of the GCLC transgene required the full-length (-3802 bp) promoter. Maximal expression in HEK 293 cells was uniquely directed by cis-elements contained within the -2752 to -1286 bp fragment of the promoter. No differences in GLCM promoter function were detected among these 4 cell lines. GCL subunit induction in each cell line by pyrrolidine dithiocarbamate (PDTC), phenethyl isothiocyanate (PEITC), and beta-naphthoflavone (beta-NF) was examined by RNAse protection assays. Although both genes were similarly induced in HepG2 cells by beta-NF, PDTC, and PEITC, neither was induced by beta-NF in A549, HEK 293, and RD cells. PDTC and PEITC induced GCLM to a much greater extent than GCLC in HEK 293 cells and failed to induce GCLC in RD cells. Neither subunit was induced by any of the agents in A549 cells. These studies indicate that the GCL subunit genes are independently regulated and display cell-type specific differences in both basal and inducible expression.
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PMID:Cell-type specific differences in glutamate cysteine ligase transcriptional regulation demonstrate independent subunit control. 1135 35

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis, is made up of two subunits, a catalytic (heavy) subunit (GCLC) and a modifier (light) subunit (GCLM), which are differentially regulated. Increased hepatic GCLC expression occurs during rapid growth, oxidative stress and after ethanol treatment. To facilitate studies of GCLC transcriptional regulation, we have cloned and characterized a 1.8 kb 5'-flanking region of the rat GCLC (GenBank accession number AF218362). A consensus TATA box and one transcriptional start site are located at 302 and 197 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The rat GCLC promoter was able to efficiently drive luciferase expression in H4IIE cells. Sequential deletion analysis revealed that three DNA regions, -595 to -111, -1108 to -705 and -705 to -595, are involved in positive (the first two regions) and negative (the latter region) gene regulation. Specific protein binding to these regions was confirmed by DNase I footprinting and electrophoretic mobility-shift assays (EMSAs). Ethanol-fed livers exhibit increased protein binding to region -416 to -336 on DNase I footprinting analysis, which was found to be NF-kappaB and AP-1 on EMSA and supershift analysis. Acetaldehyde treatment of H4IIE cells led to a time- and dose-dependent increase in GCLC mRNA levels, binding of NF-kappaB and AP-1 to the GCLC promoter, and luciferase activity driven by the GCLC promoter fragment containing these binding sites.
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PMID:Cloning and characterization of the 5'-flanking region of the rat glutamate-cysteine ligase catalytic subunit. 1143 94

Glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione (GSH) synthesis, is made up of two subunits, a catalytic (GCLC) and a modifier (GCLM) subunit, which are differentially regulated. Increased GCLM expression occurs under certain oxidative stress conditions. To facilitate studies of GCLM transcriptional regulation, we have cloned and characterized a 1.86-kb 5'-flanking region of the rat GCLM (GenBank Accession No. AF311745). A TATA-like element and one transcriptional start sites are located at 364 and 93 nucleotides upstream of the translational start site, respectively. The promoter contains consensus binding sites for many transcription factors including activator protein 1 (AP-1), transcription factor 11 (TCF11), heat shock transcription factor (HSF), and nuclear factor kappa B (NFkappaB). The rat GCLM promoter was able to drive efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions, -649 to -154 and -1251 to -649, are involved in positive and negative gene regulation, respectively. Candidate transcription factors were identified by DNase I footprinting.
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PMID:Cloning and analysis of the rat glutamate-cysteine ligase modifier subunit promoter. 1554 Mar 69

Glutamate-cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 microM) induces the GCLM and GCLC mRNAs approximately 10- and approximately 2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3 kb; all exons, intron-exon junctions, and 4.7 kb of 5'-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5'-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5'-flanking deletions (4.7-0.5 kb), showed high basal activity but only modest ( approximately 2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5'-most transcription start-site. Site-directed mutagenesis of this -9 EPRE demonstrated minimal (30-40%) decreases in tBHQ induction and no effect on basal activity-suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at -9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5'- or 3'-ward of the 4.7-kb region studied.
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PMID:Glutamate-cysteine ligase modifier subunit: mouse Gclm gene structure and regulation by agents that cause oxidative stress. 1200 77

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.
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PMID:Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence. 1207 Jan 77


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