Pulmonary epithelial and pleural mesothelial cells are primary targets of inhaled environmental particulates including asbestos fibers and particulate matter (PM). Exposure of these cell types to asbestos or PM leads to increases in expression of the early response prot(o)oncogenes, c-jun and c-fos, which are linked to the development of apoptosis and/or proliferation in other cell types. Recently, we have shown that asbestos fibers, in contrast to a number of nonpathogenic particles, stimulate the Extracellular Signal-Related Kinase (ERK) cascade, i.e. Mitogen-Activated Protein Kinase (MAPK) , through a mechanism involving autophosphorylation of the Epidermal Growth Factor Receptor (EGFR). In this proposal, we will test the hypothesis (#1) that activation of the EGFR by asbestos fibers modulates the ERK cell signalling pathway and the development of apoptosis in rat pleural mesothelial (RPM) and rat alveolar type II epithelial (RLE) cells. A second hypothesis to be tested is that EGFR expression plays a role in the development of epithelial cell apoptosis, a possible repair mechanism in removal of proliferating type II epithelial cells, and pulmonary fibrosis after inhalation of asbestos. To verify these hypotheses, phosphorylation of EGFR, activation of ERKs vs. c-Jun amino terminal kinases (JNKs), apoptosis and proliferation will be examined in RPM and RLE in vitro after addition of chrysotile or crocidolite asbestos in the presence and absence of selective inhibitors of EGFR and in transfection experiments using dominant negative EGFR mutant constructs. Transgenic mice overexpressing dominant negative mutant EGFR in vivo using lung epithelial-cell specific promoters also will be exposed by inhalation to asbestos and characterized, using quantitative markers for cell proliferation, apoptosis, and fibrosis, to determine if the severity and extent of these outcomes are altered significantly in comparison to transgene-negative litter mates. Results will indicate if EGFR receptor activation and the MAPK signalling cascade are intrinsic to epithelial cell injury and lung disease by inhaled xenobiotics and lead to the development of therapeutic strategies in man.
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