Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes through antioxidant response elements (AREs). Our previous study showed that sulfur amino acid deprivation (SAAD) produces peroxides and induces rat glutathione S-transferase A2 (rGSTA2) through NF-E2-related factor 2 (Nrf2)/ARE activation via the pathway of phosphatidylinositol 3-kinase (PI3-kinase). The current study was designed to investigate the role of peroxynitrite in Nrf2/ARE activation and rGSTA2 induction. L-Arginine deficiency or N(G)-nitro-L-arginine methyl ester (L-NAME) reduced peroxide production induced by SAAD in H4IIE cells. Northern and Western blot analyses revealed that the levels of rGSTA2 mRNA and protein were significantly increased 24h after incubation of the cells in SAAD medium, which was inhibited by L-arginine deficiency or L-NAME. Subcellular fractionation and gel shift analyses revealed that SAAD increased the level of nuclear Nrf2 and activated ARE, which were also blocked by L-arginine deficiency or L-NAME. Whereas the exogenous NO donor S-nitroso-N-acetyl-penicillamine (SNAP) alone failed to significantly induce rGSTA2, SNAP enhanced SAAD-inducible rGSTA2 expression, verifying the notion that peroxynitrite derived from NO contributes to rGSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, increased the rGSTA2 mRNA and protein levels in a dose-dependent manner. SIN-1 increased the level of nuclear Nrf2 and activated Nrf2/ARE, which was supershifted by anti-Nrf2 and anti-Maf antibodies. SIN-1 increased the activity of PI3-kinase, as monitored by phosphorylation of Akt. SIN-1-inducible rGSTA2 expression was inhibited by PI3-kinase inhibitors. These results provide evidence that peroxynitrite plays an essential role in nuclear translocation of Nrf2 and ARE activation through the pathway of PI3-kinase and that nitric oxide synthase is involved in the induction of rGSTA2.