Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-kappaB in RAW 264.7 cells.
The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low concentrations of sodium nitroprusside (SNP), a well-known nitric oxide (NO) donor, was investigated. The peak of intracellular GSH was reached at 6 h after exposure of the cells to SNP (0.1-0.5 mM), and this was preceded by the induction of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-alpha-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-kappaB, significantly suppressed the SNP-induced elevation of GSH protein and gamma-GCS mRNA, while curcumin, an inhibitor of AP-1, was less effective. Electrophoretic mobility shift assay (EMSA) showed that SNP exposure markedly increased the DNA binding of NF-kappaB, but not that of AP-1. Deletion or mutagenesis of the NF-kappaB site in the gamma-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and gamma-GCS mRNA. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low concentrations of SNP occurs through the operation of the de novo GSH pathway, and is mediated by transcriptional up-regulation of the gamma-GCS gene, predominantly at the NF-kappaB binding site in its promoter.
Kurozumi
Risa
R
The Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. j3102702@ed.noda.tus.ac.jp
Kojima
Shuji
S
D000818:Animals; D003474:Curcumin / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation
D016328:NF-kappa B; D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D009599:Nitroprusside; D009569:Nitric Oxide; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003474:Curcumin