Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells

The purpose of this study was to elucidate the signalling pathways involved in the cytokine-activated inducible nitric oxide synthase (NOS) response in a human kidney epithelial cell line, A498. Unstimulated cells did not express NOS. Exposure of A498 cells to a cytokine mixture consisting of interferon gamma, interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) increased nitrite production, NOS mRNA and protein expression. Pharmacological inhibition of tyrosine kinases, including janus kinase (JAY-2), and protein kinase C (PKC) inhibited cytokine-mediated nitrite production and NOS protein expression. The involvement of mitogen-activated protein kinases (MAPKs) was investigated. Inhibition of p38 MAPK, but not of an upstream activator of extracellular signal-regulated kinase (ERK), caused a decrease in NOS expression and nitrite production in response to cytokines. Electrophoretic mobility shift assay of nuclear extract from cytokine-stimulated cells demonstrated a pronounced binding to a nuclear factor kappaB (NF-kappaB) sequence present in the human iNOS promoter. Furthermore, the NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC) decreased cytokine-activated NOS protein expression and nitrite production. The present study has demonstrated that cytokine-stimulated NOS expression in human kidney epithelial cells involves activation of tyrosine kinases, including JAK2, PKC, p38 MAPK and NF-kappaB. (C) 2003 Elsevier Science B.V. All rights reserved.