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 gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of gamma-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 alpha (RZR/RORalpha). ECV304 cells transiently transfected with a plasmid containing the gamma-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the gamma-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORalpha 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 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.
Urata
Y
Y
Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Japan.
Honma
S
S
Goto
S
S
Todoroki
S
S
Iida
T
T
Cho
S
S
Honma
K
K
Kondo
T
T
D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D008550:Melatonin / Q000494:pharmacology*; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D018384:Oxidative Stress / Q000187:drug effects; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D018168:Receptors, Retinoic Acid; D015534:Trans-Activators; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014162:Transfection; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D012333:RNA, Messenger; C539738:RORA protein, human; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D018168:Receptors, Retinoic Acid; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D019328:Buthionine Sulfoximine; D020122:tert-Butylhydroperoxide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D008550:Melatonin