Silicosis remains a serious inflammatory lung disease for which no successful treatment is available. During the previous cycle of this grant we showed that patients with silicosis have poor response to lung transplantation. We also showed that silica promotes activation of p42 MAPK/ERK and induces phosphorylation of TNFR1, a process that modulates silica-induced cytotoxicity in macrophages. RAW 264.7 macrophages experience ERK-mediated phosphorylation of TNFR1 and are protected from apoptosis. In contrast, IC21 macrophages do not phosphorylate TNFR1, and experience enhanced apoptosis in response to silica. Silica induction of macrophage apoptosis is associated with mitochondrial dysfunction and the oxidation of cardiolipin (CL) that inversely correlate with TNFR1 phosphorylation. The mechanisms responsible for the silica-induced oxidation of CL are unknown but may involve the peroxidase activity of cytochrome c (cyt c). The mechanism(s) by which phosphorylated TNFR1 protects macrophages from silica-induced apoptosis and its effect on silicosis is unknown and constitutes the question addressed in this renewal application. We have assembled a team of researchers with expertise in TNF1 and lipid biology to study the importance of TNFR1 phosphorylation on silica-induced lung injury. Specifically, we will study the role of the intracellular TNFR1 in silicosis and its effects on caspase activation and mitochondrial damage. We hypothesize that following exposure of macrophages to silica TNFR1 undergoes phosphorylation and inhibits the formation of the pro-apoptotic signaling complex thus preventing apoptosis. Our central hypothesis is that phosphorylated TNFR1 inhibits the peroxidase activity of cyt C and limits the silica-induced oxidation of CL thus protecting macrophages from silica-induced apoptosis and ameliorating silicosis in mice. To test this hypothesis we propose the following specific aims: 1) To determine the mechanism(s) by which phosphorylated TNFR1 protects RAW 264.7 macrophages from silica-induced apoptosis. 2) To determine whether phosphorylated TNFR1 modulates the peroxidase activity of cyt c and reduces CL oxidation during silica-induced macrophage apoptosis. 3) To determine the consequences of in vivo expression of phosphorylated TNFR1 on silica-induced lung injury in mice. We will isolate macrophages from the bone marrow of C57BL/6 mice, to stably transfect them with viral constructs encoding for TNFR1 mutants to prevent or mimic TNFR1 phosphorylation. Subsequently, stably transfected macrophages will be transferred to silica-exposed mice to evaluate the effect of these cells on the development of silica induced lung injury in mice. PROJECT NARRATIVE: The term silicosis refers to the fibronodular lung disease caused by long-term inhalation of free silica. In the United States, more than 2 million people are exposed to silica every year and recent surveys indicate that a high percentage of these individuals are exposed to levels of silica above the permissible exposure limits. Subsequently, there were an estimated 3,600-7,300 newly recognized silicosis cases in the US from 1987 to 1996. Although preventive measures have decreased mortality attributable to silica the reality is that a large number of silica-exposed patients are still dying as a result of this disease. CDC surveys indicate that nine states, including Pennsylvania, report high rates of silica- induced mortality, more than 3 deaths/million people/year. These reports also point out that Western Pennsylvania exhibits a much higher mortality due to silicosis when compared to the rest of the country and clusters of counties (where mining is an important activity) around the city of Pittsburgh are identifiable that contribute disproportionably (> 14.8 deaths/million/year) to this mortality. Emphasizing the importance of these reports is our experience (described in our preliminary data) at the University of Pittsburgh with lung transplantation in patients afflicted with silicosis. We identified 12 patients that received lung transplantation for this disease from 1986-2004 . The outcome of these patients was contrasted with that of a group of 79 patients that received lung transplantation for IPF, a disease for which lung transplantation confers survival benefit. Our data indicate that patients with silicosis have poor survival rates (2.4 years) and experienced earlier rejection of their lung grafts (0.9 years) following transplantation than patients with IPF (5.34 and 2.4 years for survival and graft rejection respectively). Consequently, these data indicate that silicosis still is a serious and frequently lethal lung disease for which no successful treatment is available. ? ? ?
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