Melatonin induces γ-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells

In the present study, we show that melatonin induces the expression of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of γ-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 α (RZR/RORα). ECV304 cells transiently transfected with a plasmid containing the γ-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the γ-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORα 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 γ-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.