Vicinal dithiol-binding agent, phenylarsine oxide, inhibits inducible nitric-oxide synthase gene expression at a step of nuclear factor-kappaB DNA binding in hepatocytes

Abstract

Inflammatory cytokine interleukin 1beta induces inducible nitric-oxide synthase (iNOS) mRNA and its protein, which are followed by increasing the production of nitric oxide, in primary cultures of rat hepatocytes. Nuclear factor-kappaB (NF-kappaB), an important transcription factor for iNOS gene expression, is also activated and translocated to the nucleus. In the present study, we found that vicinal dithiol-binding agent, phenylarsine oxide (PAO), inhibited the induction of iNOS protein and mRNA as well as the release of nitrite (nitric oxide metabolite) into the culture medium. Simultaneous addition of a vicinal dithiol compound, 2, 3-dimercaptopropanol, with PAO completely abolished these inhibitions. PAO could not prevent either degradation of an inhibitory protein, IkappaB, of NF-kappaB or translocation of NF-kappaB to the nucleus. However, electrophoretic mobility shift assay demonstrated that PAO decreased the interaction between NF-kappaB and its binding consensus oligonucleotide. Transfection experiments with iNOS promoter-luciferase construct revealed that PAO inhibited NF-kappaB binding to DNA. These results indicate that PAO inhibits iNOS gene expression at a step of NF-kappaB binding to DNA by modifying its vicinal dithiol moiety, which may play a crucial role for the iNOS regulation in hepatocytes.