Fas-Induced Apoptosis and Inflammatory Lung Injury

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DESCRIPTION (provided by applicant): The Fas/Fas ligand (FasL) death-signaling pathway has been shown to play an important role in the development of pulmonary inflammation and fibrosis, but our understanding of the mechanisms involved is limited. In particular, the transcriptional machinery controlling Fas expression and the signaling events involved in Fas activation following pneumotoxic exposure are largely unknown. The overall objective of this study is to provide a scientific basis for a mechanistic understanding of the molecular events involved in Fas induced apoptosis and lung pathologies and to provide information that will be useful in risk assessment and in the development of effective therapeutic approaches for the diseases. First, we will investigate the transcriptional machinery controlling Fas expression in specific lung cells following bleomycin and FasL treatment. We will identify specific nuclear transcription factors and control elements on the Fas gene promoter that are involved in Fas activation using electrophoretic gel shift assay, gene deletion assay, and site-directed mutagenesis. Second, we will determine the early molecular events leading to transcription factor mobilization and induction of apoptosis in specific lung cells. Specifically, this aim will test the hypothesis that specific reactive oxygen species (ROS) generated during bleomycin and FasL stimulation play an essential role in mediating Fas activation and subsequent induction of apoptosis. We will also investigate the possible role of Bcl-2, a key intracellular antioxidant, and anti-apoptotic protein, in this process. Electron spin resonance will be used to identify specific ROS involved, and immunoblot and gene transfection studies will be performed to elucidate the role of Bcl-2. Lastly, because our preliminary studies have shown that FasL treatment causes an in vitro and in vivo upregulation of TNF, and because this molecule has been shown to play a crucial role in pulmonary disorders and apoptosis, we will therefore test whether FasL-induced pulmonary toxicity is dependent on TNF activation. Likewise, the possible role of Fas signaling in TNF-induced toxicity will be investigated using Fas and TNF gene knockout mice. We expect that the proposed studies will provide valuable new information on the molecular mechanisms of Fas induced apoptosis and its role in lung pathogenesis.