The expression of inducible nitric oxide synthase in human retinal pigment epithelial cells under stimulation of proinflammatory cytokine tumor necrosis factor-α

Abstract

PurposeTo elucidate the effects of tumor necrosis factor (TNF)-α on the expression of inducible nitric oxide synthase (iNOS) in retinal pigment epithelial cells in vitro. MethodsARPE-19 cell lines were cultured with TNF-α stimulation, and then treated with proteasome inhibitors (MG132 or lactacystin) for 30 minutes. The expression of iNOS was determined by RT-PCR and Western blot. The expression of nitric oxide (NO) was determined by an enzyme-linked immunosorbent assay. The interaction of nuclear factor kappa-B (NF-κB) activation and iNOS induction was assessed by electrophoretic mobility shift assay. ResultsThe expression of iNOS in ARPE-19 was induced by TNF-α in a dose-dependent manner. Upregulation of iNOS resulted in increased production of NO. iNOS induced by TNF-α could be inhibited by MG-132 and lactacystin. Supershift assay revealed that NF-κB activation was responsible for iNOS induction. ConclusionTNF-α could induce iNOS expression and NO production in RPE cells, at least in part, via an NF-κB signal pathway.