title: Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells.
abstract: 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-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-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-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-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.
authors.affiliation: Rayne Laboratory, Respiratory Medicine Unit, Department of Medicine (RIE), University of Edinburgh, Medical School, Edinburgh Eh8 9AG, United Kingdom. IR@srv1.med.ed.ac.uk
authors.forename: I
authors.initials: I
authors.lastname: Rahman
authors.forename: F
authors.initials: F
authors.lastname: Antonicelli
authors.forename: W
authors.initials: W
authors.lastname: MacNee
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005978:Glutathione / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*