Src activation of NF-kappaB augments IL-1beta-induced nitric oxide production in mesangial cells.

NF-kappaB is a critical transcription factor that is involved in glomerulonephritis and inflammatory host responses and a critical transactivator of the inducible nitric oxide (NO) synthase gene in mesangial cells. The Src protein tyrosine kinases (SFK) are involved in several signaling pathways and have been proposed to mediate cytokine activation of NF-kappaB in a few cell types. However, the specific involvement of SFK in IL-1beta induction of NO production has not been clearly established. Accordingly, pharmacologic and molecular tools were used to clarify this issue in cultured murine mesangial cells. The SFK antagonist 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine (PP2) dramatically inhibited IL-1beta-mediated induction of endogenous NO production as measured by the Griess reaction, as well as the induction of NF-kappaB p50/p65 DNA-binding activity in gel shift assays and the activity of an NF-kappaB-responsive promoter-reporter construct transiently transfected into the cells. Immunoprecipitation and immunoblotting with anti-IkappaBalpha and anti-phosphotyrosine antibodies revealed that PP2 also inhibited IL-1beta-stimulated tyrosine phosphorylation of IkappaBalpha, a requisite step in NF-kappaB activation in this signaling cascade. In agreement with the pharmacologic inhibition studies, siRNA directed against c-Src specifically limited c-Src protein expression and inhibited IL-1beta-mediated induction of NF-kappaB DNA-binding activity, whereas control siRNA had no effect. Conversely, overexpression of constitutively active c-Src augmented basal and IL-1beta-mediated induction of NF-kappaB DNA-binding activity and NO production. Thus, SFK play a key role in IL-1beta-induced NO production in mesangial cells and do so via tyrosine phosphorylation of IkappaBalpha and consequent NF-kappaB activation.