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

In the present study, we show that melatonin induces the expression of gamm a-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glut athione (GSH) synthesis, in ECV304 human vascular endothelial cells. One mi cromolar melatonin induced the expression of gamma-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoreti c mobility shift assay showed that melatonin stimulates the DNA-binding act ivity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor alpha (RZR/ROR alpha). ECV304 cells trans iently transfected with a plasmid containing the gamma-GCS promoter-lucifer ase construct showed increased luciferase activity when treated with melato nin. The melatonin-dependent luciferase activity was found in the gamma-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 wa s repressed in the promoter containing RZR/ROR alpha site. In addition, cel l cycle analysis showed that melatonin increases the number of cells in the G(0)/G(1) phase; however, treatment of the cells with buthionine sulfoximi ne, a specific inhibitor of gamma-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. (C) 1999 Elsevie r Science Inc.