Immune reactive cytokines, such as interferon (IFN)-γ, have multiple effects in glomerulonephritis. Superoxide anions (O2-), which are associated with the progression of glomerulonephritis, are mainly generated by nicotinamide adenine dinucleotide phosphate (reduced form) NAD(P)H oxidases. We determined the effects of IFN-γ on O2- production, phosphorylation of signal transducer and activator of transcription (STAT)-1α, and the mRNA and protein expressions of p22phox and Nox1, components of NAD(P)H oxidases, in human mesangial cells (HMCs). Significant increases in O2- production with IFN-γ were completely abolished by the flavin-containing enzyme inhibitor, diphenyleneiodonium (10 μmol/l), and the Janus-activated kinase (JAK)2 inhibitor, AG490 (100 μmol/l). Phosphorylated STAT-1α was detected after 5 min of IFN-γ stimulation using Western blot analysis, and binding to the gamma-activating site was observed from 30 min to 4 h, thereafter by electrophoretic mobility shift assay (EMSA). Super-shift analysis in EMSA revealed that the main transcription factor was STAT-1α. IFN-γ significantly increased the expression of p22phox mRNA and protein, although expression was inhibited by AG490. These data suggest that IFN-γ stimulates O2- production in HMCs via the JAK-STAT pathway and NAD(P)H oxidase.
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