Silica-induced nuclear factor-kappaB activation: involvement of reactive oxygen species and protein tyrosine kinase activation.
Nuclear factor-kappaB (NF-kappaB) is a multiprotein complex that may regulate a variety of inflammatory cytokines involved in the initiation and progression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kappaB in a mouse peritoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activation. In vitro exposure of mouse macrophages to silica (100 microg/ml) resulted in a twofold increase in ROS production, measured as the generation of chemiluminescence (CL), and caused activation of NF-kappaB. Silica-induced CL was inhibited 100% by superoxide dismutase (SOD) and 75% by catalase, while NF-kappaB activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, alpha-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine). Further evidence for the involvement of ROS in NF-kappaB activation is that 1 mM H2O2 enhanced NF-kappaB/DNA binding and that this activation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kappaB activation in silica-treated cells. Genistein and AG-494 also reduced NF-kappaB activation in H2O2-treated cells. Results confirm that tyrosine phosphorylation of several cellular proteins (approximate molecular mass of 39, 58-70, and 103 kD) was increased in silica-exposed macrophages and that genistein inhibited this silica-induced phosphorylation. In contrast, inhibitors of protein kinase A or C, such as H89, staurosporin, calphostin C, and H7, had no marked inhibitory effect on silica-induced NF-kappaB activation. The results suggest that ROS may play a role in silica-induced NF-kappaB activation in macrophages and that phosphorylation events mediated by tyrosine kinase may be involved in this activation.
Kang
J L
JL
Department of Physiology, College of Medicine, Ewha Medical Research Center, Ewha Womans University, Seoul, Korea. jihee@mm.ewha.ac.kr
Go
Y H
YH
Hur
K C
KC
Castranova
V
V
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D017382:Reactive Oxygen Species; D012822:Silicon Dioxide / Q000633:toxicity*; D014443:Tyrosine / Q000378:metabolism
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014443:Tyrosine; D012822:Silicon Dioxide; D004247:DNA; D011505:Protein-Tyrosine Kinases