Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-κB in RAW 264.7 cells

Abstract

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 γ-glutamylcysteine synthetase (γ-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-α-Tosyl- l-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-κB, significantly suppressed the SNP-induced elevation of GSH protein and γ-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-κB, but not that of AP-1. Deletion or mutagenesis of the NF-κB site in the γ-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and γ-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 γ-GCS gene, predominantly at the NF-κB binding site in its promoter.