Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells

Glutathione (GSH) is an important physiological antioxidant in lung epithel ial 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 synthet ase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gam ma-GCS-HS) mRNA at 24 h, Treatment with TNF-alpha also increased chloramphe nicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation o f the putative proximal AP-1-binding site (-269 to -263 base pairs), abolis hed TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predom inantly formed by dimers of c-Jun. Dexamethasone (3 mu M) produced a signif icant 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 m RNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-a lpha 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). Th ese data have implications for the oxidant/antioxidant balance in inflammat ory lung diseases.