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 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.

γ-Glutamylcysteine inhibits oxidative stress in human endothelial cells

“…Also, a putative binding site for NF-κB was found at − 4 227 bases in the GSS promoter, but none for PPARs. These findings are consistent with previous studies which documented the existence of NF-κB binding site in both GSS and GCS-HC promoters (Iwanaga et al 1998;Kurozumi and Kojima 2005;Lee et al 2005;Yang et al 2005). Overexpression with either NF-κB p50 or p65 increases promoter activities of GSS and GCS-modifier subunit (GCS-LC) .…”

“…al's study (Lee et al 2005). Similarly, there was one putative binding site for NF-κB at −2 537-base in the GCS-HS promoter region, but none for PPARs, consistent with previous studies (Iwanaga et al 1998;Kurozumi and Kojima 2005;Yang et al 2005). …”

NF‐κB pathway is involved in griseofulvin‐induced G2/M arrest and apoptosis in HL‐60 cells

“…Super-shift assay was performed as previously described [Kurozumi and Kojima, 2005] with some modification. Briefly, 2.5 ml of anti-p50 or anti-p65 antibodies (Santa Cruz Biotech, Santa Cruz, CA) were added to the sample for 60 min at 378C.…”

Gestation linked radical oxygen species fluxes and vitamins and trace mineral deficiencies in the ruminant

“…This is because radical formation is, in the first place, an essential component of cell metabolism, taking part in the catalysis of numerous enzymatic activities [2,11] and in the fine control of cell metabolism through the control local redox potential [11,12]. ROS can be induced, as second messenger, by cytokines, oncogenes, growth factors or hormones [11,[13][14][15][16]. ROS of external origin or resulting from some insult to the cells or from deviation of the usual metabolism of oxygen can mimic the effects of ROS induced as constitutive second messenger and can trigger activation of many transcription factors through the release of signaling proteins [11,17,18].…”

“…Suppression of ROS fluxes can suppress the expression of both Mn-and Cu/Zn-SOD [78]. Moreover, the coordination of the whole set of regulatory events and its relation to oxygen partial pressure can be altered by variation of the cellular contents of the • NO radical, because this compound inhibits HIF-1α stabilization under hypoxia [52] or its oxidation under normal oxygenation [91], or, again, triggers GSH synthesis to change the redox environment and the control of gene expression [15].…”