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

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