Role of interferon regulatory factor-1 and mitogen-activated protein kinase pathways in the induction of nitric oxide synthase-2 in retinal pigmented epithelial cells

Abstract

Bovine retinal pigmented epithelial cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Experiments were performed to investigate the involvement of interferon regulatory factor-1 (IRF-1) on NOS-2 induction and its regulation by NOS-2 inhibitors such as pyrrolidine dithiocarbanate (PDTC), an antioxidant, or protein kinase inhibitors. Analysis by transitory transfections showed that LPS, alone or with IFN-γ, stimulated activity of the murine NOS-2 promoter fragment linked upstream of luciferase and its suppression by PDTC and by the different protein kinase inhibitors, genistein (tyrosine kinase inhibitor), PD98059 (mitogen-actived protein (MAP) kinase kinase inhibitor), and SB 203580 (p38 MAP inhibitor). Using specific antibodies, we have confirmed that extracellular signal-regulated kinases and p38 MAP kinase were activated by LPS and IFN-γ in retinal pigmented epithelial cells. Analysis by reverse transcriptase-polymerase chain reaction, Western blot, and electrophoretic mobility shift assay demonstrated that IFN-γ alone or combined with LPS induced an accumulation of IRF-1 mRNA and protein and IRF-1 DNA binding. Transfections assays with the IRF-1 promoter showed an induction of this promoter with IFN-γ, potentiated by LPS. The decrease of LPS/IFN-γ-induced IRF-1 promoter activity, IRF-1 synthesis, and IRF-1 activation, by PDTC, genistein, PD98059, and SB 203580, could explained in part the inhibition of the NOS-2 induction by these compounds. Our results demonstrate that IRF-1 is necessary for NOS-2 induction by LPS and IFN-γ and that its synthesis requires the involvement of a redox-sensitive step, the activation of tyrosine kinases, and extracellular signal-regulated kinases 1/2 and p38 MAP kinases.