Asbestos and silica are fibrogenic minerals associated with the development of inflammation and type II epithelial cell injury and hyperplasia. Previous work suggests that these minerals elaborate active oxygen species (AOS) and induce the expression of antioxidant enzymes, most notably manganese-containing superoxide dismutase (MnSOD), in epithelial cells of the respiratory tract in vitro and after inhalation. Using a hamster tracheal epithelial (HTE) cell line, we have shown that responses to asbestos and chemical generating systems of AOS are biphasic - oxidants at high concentrations are toxic whereas these agents elicit cell proliferation at lower concentrations. The goal of this proposal is to determine the mechanisms of cell signalling by crocidolite and chrysotile asbestos, cristobalite silica and H202 which result in cell proliferation. Our work thus far shows that crocidolite asbestos causes activation of protein kinase C (PKC), increased levels of inositol phosphates, increased expression of c-jun/c-fos mRNA, and persistent induction of AP-1 transcription factors. Our objectives are to compare the ability of asbestos, silica and H202 at proliferative concentrations to activate cell signalling pathways as measured by changes in Ca++ uptake and/or intracellular Ca++ stores, diacylglycerol concentrations, PKC activity, and the hydrolysis of phosphoinositides in HTE cells and in primary isolates of rat trachea] epithelial cells (RTE) and rat lung fibroblasts (RLF). Activation of cell signalling pathways that lead to cell proliferation will be documented by measuring steady-state levels of c-jun and c-fos mRNA and activator protein-1 (AP1) transcription factor DNA binding activity. To assess whether extracellular antioxidants modulate the ability of fibrogenic minerals or H202 to induce cell proliferation, cell signalling events will be re-examined in cell cultures supplemented with MnSOD, catalase or scavengers of hydroxyl radical. To determine if cell proliferation by these agents is influenced by the overexpression of antioxidant enzymes in target cells, cell signalling events, induction of c-fos/c-jun, colony forming efficiency (CFE), and the incorporation of 5-bromodeoxyridine (BrdU) will be measured in stable transfected HTE cell lines that express elevated levels of MnSOD or catalase. These studies will reveal whether fibrogenic agents and AOS induce cell proliferation through common molecular mechanisms, and may lead to new approaches for the prevention or alleviation of oxidant-induced cell injury and/or proliferation in the lung.
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