Enhancement of nuclear factor-kappa B activation and protein tyrosine phosphorylation by a tyrosine phosphatase inhibitor, pervanadate, involves reactive oxygen species in silica-stimulated macrophages

Reactive oxygen species (ROS) and phosphorylation events mediated by tyrosi ne kinase are involved in silica-induced nuclear factor-kappa B (NF-kappaB) activation. Protein tyrosine phosphatase (PTPase) acts to limit protein ty rosine phosphorylation. In the present study, we investigated the role of P TPase in NF-kappaB activation and tyrosine phosphorylation in silica-stimul ated macrophages, and the involvement of ROS in these responses. Treatment of mouse peritoneal macrophages (RAW264.7 cells) with a PTPase inhibitor, p ervanadate, markedly enhanced the DNA-binding activity of NF-kappaB in the presence or absence of silica. The stimulatory effect of pervanadate on NF- kappaB activation was also demonstrated in LPS-stimulated macrophages. A sp ecific inhibitor of protein tyrosine kinase (PTK;), genistein, prevented th e NF-kappaB activation induced by ptr vanadate in the presence of silica wh ile inhibitors of protein kinase A or C. such as staurosporine or H7, had n o inhibitory effect on NF-F;B activation. A variety of antioxidants, such a s catalase, superoxide dismutase, N-acetyl cysteine (NAC), and pyrrolidine dithiocarbamate, inhibited NF-kappaB activation induced by pervanadate in t he presence of silica. Furthermore, pervanadate markedly enhanced silica- o r LPS-induced protein tyrosine phosphorylation in cells. Treatment of macro phages with NAC abolished the increase in tyrosine phosphorylation in cells stimulated with the combination of pervanadate and either silica or LPS or with silica alone. The results suggest that PTPase may play a crucial role in the negative regulation of silica-signaling pathways leading to NF-kapp aB activation in macrophages. Furthermore. ROS appear to be involved in dow nstream signaling between PTPase inhibition and NF-kappaB activation. (C) 2 000 Elsevier Science Ireland Ltd. All rights reserved.