Characterisation of γ‐glutamylcysteine synthethase‐heavy subunit promoter: a critical role for AP‐1

Rahman, Irfan; Smith, Christopher A.; Antonicelli, Frank; MacNee, William

doi:10.1016/s0014-5793(98)00410-4

Cited by 72 publications

(77 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Blocking AP-1 activity led to a fall in the basal mRNA level of all three genes and their constitutive promoter activities. This finding is similar to what has been reported also for the human GCL subunit promoters and GCLC mRNA level, where AP-1 activity is required for constitutive activity and basal expression (27)(28)(29). However, blocking AP-1 activity also blocked significantly the TBH-mediated increase in the promoter activity of the rat GCL subunits and GS as well as the mRNA levels of all three genes.…”

Section: Figsupporting

confidence: 90%

Role of AP-1 in the Coordinate Induction of Rat Glutamate-cysteine Ligase and Glutathione Synthetase bytert-Butylhydroquinone

Yang

Zeng

Lee

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

GSH synthesis occurs via two enzymatic steps catalyzed by glutamate-cysteine ligase (GCL, made up of two subunits) and GSH synthetase (GS). Recently, we described coordinate induction of GCL subunits and GS. To study GS transcriptional regulation, we have cloned and characterized a 2.2-kb 5-flanking region of the rat GS (GenBank TM accession number AF333982). One transcriptional start site is located at 51 nucleotides upstream of the translational start site. The rat GS promoter drove efficiently luciferase expression in H4IIE cells. Sequential deletion analysis revealed DNA regions that are involved in positive and negative regulation. One repressor identified was NF1. tert-Butylhydroquinone (TBH) exerted a dose-and time-dependent increase in the mRNA level and promoter activity of both GCL subunits and GS. TBH increased protein binding to several regions of the GS promoter, c-jun expression, and activator protein 1 (AP-1) binding activity to several of the putative AP-1-binding sites of the GS promoter. Blocking AP-1 binding with dominant-negative c-jun led to decreased basal expression and significantly blocked the TBH-induced increase in promoter activity and mRNA level of all three genes. In conclusion, AP-1 is required for basal expression of GCL and GS; while NF1 serves as a repressor of GS, increased AP-1 transactivation is the predominant mechanism for coordinate induction of GCL and GS expression by TBH.GSH is the main non-protein thiol in mammalian cells that participates in many critical cellular functions including antioxidant defense and cell growth (1-3). The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps: the formation of ␥-glutamylcysteine from glutamate and cysteine, and formation of GSH from ␥-glutamylcysteine and glycine. The first step of GSH biosynthesis is generally regarded as rate-limiting and catalyzed by glutamate-cysteine ligase (GCL, 1 also known as ␥-glutamylcysteine synthetase), whereas the second step is catalyzed by GSH synthetase (GS) (1). The GCL enzyme is composed of a catalytic (GCLC, M r ϳ73,000) and a modifier (GCLM, M r ϳ30,000) subunit that are encoded by different genes and dissociate under reducing conditions (4 -6). The catalytic subunit exhibits all of the catalytic activity of the isolated enzyme as well as feedback inhibition by GSH (6). The modifier subunit is enzymatically inactive but plays an important regulatory function by lowering the K m values of GCL for glutamate and raising the K i value for GSH (5, 7). Because GCL is a major determinant of the overall GSH synthesis capacity, regulation of GCL subunits has been a topic of extensive research (1). Changes in GCL activity can result from regulation at multiple levels affecting only the catalytic or modifier subunit or both. Both human and rat GCL promoters have been cloned (8 -12). Antioxidant response element (ARE, also known as electrophile-response element) and activator protein 1 (AP-1) are two cis-acting elements present in the promoter of both human GCL sub...

show abstract

Section: Figsupporting

confidence: 90%

Role of AP-1 in the Coordinate Induction of Rat Glutamate-cysteine Ligase and Glutathione Synthetase bytert-Butylhydroquinone

Yang

Zeng

Lee

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…We next elucidated the transcriptional regulatory mechanism by which TNF-␣ exerted its effect on the induction of ␥-GCS-HS. By using deletion and mutation studies, we have recently shown the critical role of a putative AP-1 site in oxidant-mediated regulation of the ␥-GCS-HS promoter (15). However, other investigators have suggested that an NF-Bbinding site in the ␥-GCS-HS promoter may be important in regulating ␥-GCS-HS gene expression (40,42).…”

Section: Discussionmentioning

confidence: 99%

“…A HindIII and SphI fragment (1138 bp) containing the promoter was isolated and subcloned into the polylinker of the promoterless plasmid pCAT Basic Vector (Promega). This construct was denoted pCBGCS and has been described previously (15,26,27).…”

Section: Methodsmentioning

confidence: 99%

“…These latter effects may be mediated by an interaction between the glucocorticoid-receptor complex and transcription factors such as NF-B and AP-1, which regulate transcription for inflammatory mediators such as cytokines (17,25). We have shown that AP-1 plays a critical role in enhancing ␥-GCS-HS mRNA expression, and therefore regulation of GSH synthesis (15,26,27). Thus corticosteroids may have an effect on intracellular GSH through a mechanism involving AP-1 (28).…”

mentioning

confidence: 90%

“…The heavy subunit possesses all of the catalytic activity (14). We have recently demonstrated that a putative AP-1 transcription factor-binding site is necessary for oxidant-mediated regulation of the catalytic ␥-GCS-HS gene promoter (15).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Molecular Mechanism of the Regulation of Glutathione Synthesis by Tumor Necrosis Factor-α and Dexamethasone in Human Alveolar Epithelial Cells

Rahman

Antonicelli

MacNee

1999

Journal of Biological Chemistry

Self Cite

122

View full text Add to dashboard Cite

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-␣ (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in ␥-glutamylcysteine synthetase (␥-GCS) activity and the expression of ␥-GCS heavy subunit (␥-GCS-HS) mRNA at 24 h. Treatment with TNF-␣ also increased chloramphenicol acetyltransferase (CAT) activity of a ␥-GCS-HS 5-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (؊269 to ؊263 base pairs), abolished TNF-␣-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-␣ increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 M) produced a significant decrease in the levels of GSH, decreased ␥-GCS activity and ␥-GCS-HS mRNA expression at 24 h. The increase in GSH levels, ␥-GCS-HS mRNA, ␥-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-␣ were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-␣ and dexamethasone modulate GSH levels and ␥-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.

show abstract

Anti‐inflammatory/Oxidative Stress Proteins and Peptides

Young

Mine

2010

Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals

View full text Add to dashboard Cite

Characterisation of γ‐glutamylcysteine synthethase‐heavy subunit promoter: a critical role for AP‐1

Cited by 72 publications

References 24 publications

Role of AP-1 in the Coordinate Induction of Rat Glutamate-cysteine Ligase and Glutathione Synthetase bytert-Butylhydroquinone

Role of AP-1 in the Coordinate Induction of Rat Glutamate-cysteine Ligase and Glutathione Synthetase bytert-Butylhydroquinone

Molecular Mechanism of the Regulation of Glutathione Synthesis by Tumor Necrosis Factor-α and Dexamethasone in Human Alveolar Epithelial Cells

Anti‐inflammatory/Oxidative Stress Proteins and Peptides

Contact Info

Product

Resources

About