Regulation of Fas-Mediated Lung Cell Apoptosis

描述(由申请人提供)：细胞凋亡或程序性细胞死亡调节的缺陷导致许多人类疾病，包括肺部的疾病。近年来的研究表明，Fas(CD95)介导的细胞凋亡在多种肺部疾病的发病机制中起重要作用。然而，我们对这一进程所涉及的机制的了解有限。不了解这种机制直接限制了预防和治疗努力的有效性。本研究的总体目标是为从机制上理解Fas介导的细胞凋亡的分子事件及其在特定肺细胞中的调控提供科学基础。我们的初步研究结果表明，虽然Fas可以触发肺细胞的凋亡，但Fas的表达水平及其被Fas配体(FasL)激活与Fas介导的细胞死亡的易感性无关，这表明必须存在凋亡信号通路的调节因子。在这个项目中，我们将试图找出控制Fas介导的肺细胞死亡的关键调控因子，并阐明其机制。该项目将专门测试假设，即对Fas介导的细胞凋亡和相关的肺部病理的易感性可能由细胞FLICE抑制蛋白(c-FLIP)的表达水平决定，以及某些肺毒性药物改变该蛋白可以通过激活caspase-8及其下游的caspase级联而使细胞对Fas介导的细胞死亡敏感。我们将利用不同的分子生物学和生化技术来确定c-FLIP的功能作用，并在原代肺细胞中鉴定死亡信号通路。我们还将检验这样的假设，即通过翻译后修饰下调c-flip是一个关键的调控事件，通过caspase-8和NF-B信号通路控制Fas介导的细胞死亡和存活。由于我们的初步发现表明，活性氧物种(ROS)和泛素-蛋白酶体依赖途径在c-FLIP降解和Fas信号转导中起着关键作用，我们将阐明其潜在的机制，并确定参与其中的特定ROS及其细胞来源。此外，我们将使用定点突变和基因缺失分析来确定c-Flip上针对该分子降解的特定泛素化位点。预计这些拟议的研究将为细胞死亡调控机制和相关的肺疾病提供有价值的新信息，这将在风险评估和治疗干预中发挥重要作用。

DESCRIPTION (provided by applicant): Defects in apoptosis or programmed cell death regulation contribute to many human diseases, including those of the lung. Recent evidence indicates that Fas(CD95)-mediated apoptosis plays an important role in the pathogenesis of several pulmonary diseases. However our understanding of the mechanisms involved in the process is limited. Failure to understand such mechanisms directly limits the effectiveness of prevention and therapeutic efforts. The overall objective of this study is to provide a scientific basis for a mechanistic understanding of the molecular events involved in Fas-mediated apoptosis and its regulation in specific lung cells. Our preliminary findings indicate that while Fas can trigger apoptosis of lung cells, the expression level of Fas and its activation by Fas ligand (FasL) do not correlate with the susceptibility to Fas-mediated cell death, indicating that regulators of the apoptosis-signaling pathway must exist. In this project we will seek to identify key regulators controlling Fas-mediated cell death of lung cells and elucidate their mechanisms. The project will specifically test the hypotheses that susceptibility to Fas-mediated apoptosis and associated lung pathologies may be determined by the expression level of cellular FLICE-inhibitory protein (c-FLIP) and that alterations of this protein by certain pneumotoxic agents can sensitize cells to Fas- mediated cell death via an activation of caspase-8 and downstream caspase cascade. We will determine the functional role of c-FLIP and identify the death signaling pathways in primary lung cells using various molecular biology and biochemical techniques. We will also test the hypothesis that downregulation of c- FLIP through post-translational modifications is a critical regulatory event controlling Fas-mediated cell death and survival via caspase-8 and NF-(B signaling pathway. Because our preliminary findings indicate critical roles of reactive oxygen species (ROS) and ubiquitin-proteasome dependent pathway in c-FLIP degradation and Fas signaling, we will elucidate the underlying mechanisms and identify specific ROS involved and their cellular sources. Furthermore, we will determine specific ubiquitination sites on c-FLIP that target this molecule for degradation using site-directed mutagenesis and gene deletion assays. It is expect that the proposed studies will provide valuable new information on the mechanisms of cell death regulation and associated lung disorders which will be important in risk assessment and therapeutic intervention.