Occupational and environmental exposure to asbestos results in chronic pulmonary fibrosis and malignancies. Proliferative disorders caused by fibers require activated endothelial cells to promote tissue remodeling. However, the mechanisms for initiating responses in these cells exposed to asbestos are unclear. There are no known receptors for asbestos in any cell type; although the charged structure of these fibers may allow them to be ligands at receptors for matrix proteins, such as vitronectin. uPAR, the receptor for the protease, urokinase (uPA), is also a high affinity receptor for vitronectin (14). They have demonstrated that preventing the insertion of glycophosphatidylinositol-anchored (GPI) receptors, such as uPAR, into the membrane or inhibiting tyrosine kinases blocks morphological change in response to asbestos. Further, they observe tyrosine kinase-dependent nuclear translocation of nuclear factor kappaB (NF-kB) in endothelial cells responding to asbestos. Ceramic fibers of similar shape to asbestos, but different charge structure, do not elicit these responses. These preliminary data support the hypothesis that asbestos activates cells by binding to GPI receptors, which initiates tyrosine kinase dependent signal cascades. Further, they theorize that these cascades regulate the cell morphology, gene expression, and pericellular protease activity required for an invasive endothelial cell phenotype. The proposed studies will examine the role of uPAR in mediating the cellular effects of asbestos and will demonstrate how interaction with uPAR, or similar GPI receptors, initiates phenotypic change. Low passage, cultured porcine or human endothelial cells will be used to investigate the stated hypothesis. The role of uPAR, or other GPI receptors, will be demonstrated by using specific antibodies or soluble receptors to prevent asbestos from interacting with cell. Cell morphology and uPA proteolytic activity of control and asbestos-exposed cells will be endpoints to demonstrate functional changes relevant to an invasive phenotype. Kinases activated in response to asbestos will be identified. The hypothesis that asbestos activates these kinases by acting on a receptor or through formation of reactive oxygen species will be investigated. The localization of these kinases to glycolipid-rich membrane domains in resting cells and focal adhesions in asbestos exposed cells will be examined to link receptor occupancy and morphological change. Activity of signaling enzymes and the phosphorylation of IkB, the inhibitory subunit of NF-kB will be used to characterize the pathway for asbestos-induced translocation of NF-kB. Selective tyrosine kinase inhibitors. Transfections of mutant kinase genes, and cells isolated from kinase deficient mice will be used to establish that these kinases are required for responses to asbestos. These studies use novel approaches to define the pathobiology of asbestos. Mechanistic information gained will improve accurate assessment of the risks of fibrogenic fibers and will improve understanding of vascular responses to environmental toxins.
