SILICA-INDUCED NUCLEAR FACTOR- k B ACTIVATION: INVOLVEMENT OF REACTIVE OXYGEN SPECIES AND PROTEIN TYROSINE KINASE ACTIVATION

Abstract

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.

Silica particles cause NADPH oxidase–independent ROS generation and transient phagolysosomal leakage

“…Silica-induced ROS generation results in up-regulation of NF-κB and production of proinflammatory cytokines ( Rojanasakul et al. , 1999 ; Kang et al. , 2000 ).…”

“…Superoxide is converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase or by spontaneous dismutation, and H 2 O 2 is neutralized by peroxidases or catalase ( Kalyanaraman, 2013 ). An increase in cytoplasmic and mitochondrial reactive oxygen species (ROS) occurs upon silica exposure ( Kang et al. , 2000 ; Fazzi et al.…”

“…, 2014 ). This increase in ROS can result in NF-κβ activation ( Kang et al. , 2000 ) and up-regulation of expression of proinflammatory cytokines that contribute to fibrosis ( Rojanasakul et al.…”

Reprogramming of Tumor-Associated Macrophages with Anticancer Therapies: Radiotherapy versus Chemo- and Immunotherapies

“…It was shown that H 2 O 2 enhanced p65 NFκB DNA-binding and promoted p65 NFκB phosphorylation. This NFκB activation is followed by its translocation into the nucleus in macrophage and monocytic cell lines, and then induces M1 polarization (133). In parallel, it was shown that H 2 O 2 altered protein–protein interaction of p50–p50 NFκB, therefore reducing p50–p50 NFκB activation, supporting M1 activation (134).…”

Advances in the Use of N-Acetylcysteine in Chronic Respiratory Diseases

“…For instance, the NAC pretreatment of silica-exposed murine macrophage cell line 264.7 lowered the TNFα mRNA and protein levels and the mRNA levels of MIP-2, MIP-1α, MIP-1β, and MCP-1 [ 186 ]. Two other studies demonstrated that the silica exposure-induced overproduction of ROS is associated with the degradation of the NF-κB inhibitor IκB-α and activation of the NF-κB pathway because a pretreatment with NAC decreased a silica-induced accumulation of ROS and inhibited the suppression of IκB-α [ 179 ], or reduced the activation of NF-κB [ 187 ]. In alveolar macrophages isolated from silica-instilled rats [ 188 ] or exposed in vitro to silica, the silica-induced elevation in intracellular ROS was associated with a decrease in intracellular GSH and cysteine and a sustained presence of apoptotic alveolar macrophages.…”