Induction of activator protein-1 through reactive oxygen species by crystalline silica in JB6 cells

We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H(2)O(2) in the mechanism of silica-induced AP-1 activation. Sodium formate (an ( small middle dot)OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H(2)O(2), but not O(2), may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H(2)O(2), play a significant role in silica-induced AP-1 activation.     

Silica induces cell cycle changes through PI-3K/AP-1 pathway in human embryo lung fibroblast cells

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein-1 (AP-1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant-negative mutants of phosphatidylinositol-3 kinase (Δp85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica-induced cell cycle alteration, AP-1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol-3 kinase (PI-3K)/AP-1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI-3K, Akt, or p70S6K could inhibit silica-induced overexpression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) and decreased expression of E2F-4. Taken together, silica could induce cell cycle changes through PI-3K/ AP-1 pathway in HELFs.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.