Silica-induced nuclear factor-kappa B activation: Involvement of reactive oxygen species and protein tyrosine kinase activation

Nuclear factor-kappa B (NF-kappa B) is a multiprotein complex that may regu late a variety of inflammatory cytokines involved in the initiation and pro gression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kappa B in a mouse per itoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activa tion. In vitro exposure of mouse macrophages to silica (100 mu g/ml) result ed in a twofold increase in ROS production, measured as the generation of c hemiluminescence (CL), and caused activation of NF-KB. Silica-induced CL wa s inhibited 100% by super oxide dismutase (SOD) and 75% by catalase, white NF-kappa B activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, cr-tocopherol, pyrrolidine dithiocarbamate, or N-ace tylcysteine). Further evidence for the involvement of ROS in NF-kappa B act ivation is that 1 mM H2O2 enhanced NF-kappa B/DNA binding and that this act ivation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kappa B a ctivation in silica-treated cells. Genistein and AG-494 also reduced NF-kap pa B activation in H2O2-treated cells. Results con-firm that tyrosine phosp horylation of several cellular proteins (approximate molecular mass of 39, 58-70, and 703 kD) was increased in silica-exposed macrophages and that gen istein inhibited this silica-induced phosphorylation. In contrast, inhibito rs of protein kinase A or C, such as H89, staurosporin, calphostin C, and H 7, had no marked inhibitory effect on silica-induced NF-kappa B activation. The results suggest that ROS may play a role in silica-induced NF-kappa B activation in macrophages and that phosphorylation events mediated by tyros ine kinase may be involved in this activation.