Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-α 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- α (TNF-α) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-α (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in γ-glutamylcysteine synthetase (T- GCS) activity and the expression of γ-GCS heavy subunit (γ-GCS-HS) mRNA at 24 h. Treatment with TNF-α also increased chloramphenicol acetyltransferase (CAT) activity of a γ-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-α-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-α increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 μM) produced a significant decrease in the levels of GSH, decreased γ-GCS activity and γ-GCS-HS mRNA expression at 24 h. The increase in GSH levels, Γ-GCS-HS mRNA, Γ-GCS-HS promoter activity, and AP- 1 DNA binding produced by TNF-α were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-α and dexamethasone modulate GSH levels and γ-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.