Interferon-γ enhances superoxide production in human mesangial cells via the JAK–STAT pathway

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