Regulation of Fas-Mediated Lung Cell Apoptosis

细胞凋亡或程序性细胞死亡调控的缺陷导致许多人类疾病，包括 那些是肺的。最近的证据表明，Fas(CD95)介导的细胞凋亡在 几种肺部疾病的发病机制。然而，我们对所涉及的机制的理解 在这一过程中是有限的。对这种机制的不了解直接限制了 预防和治疗努力。这项研究的总体目标是为 Fas介导的细胞凋亡及其调控的分子机制研究进展 特定的肺细胞。我们的初步发现表明，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Tofas易感性无关。 介导细胞死亡，表明必须存在凋亡信号通路的调节器。在这个项目中 我们将努力寻找控制Fas介导的肺细胞死亡的关键调控因子，并阐明它们的 机械装置。该项目将专门测试对Fas介导的细胞凋亡易感性的假设 而相关的肺病理可能由细胞FLICE抑制基因的表达水平来决定 蛋白(c-FLIP)，该蛋白被某些肺毒性药物改变可使细胞对Fas- 通过激活caspase-8及其下游的caspase级联途径介导细胞死亡。我们将决定 C-FLIP的功能作用及在原代肺细胞中识别死亡信号通路的研究 分子生物学和生化技术。我们还将检验这样一种假设，即下调c- 翻转翻译后修饰是控制Fas介导的细胞死亡的关键调控事件 并通过caspase-8和NF-kB信号通路存活。因为我们的初步调查结果显示 活性氧簇(ROS)和泛素-蛋白酶体依赖途径在c-FLIP降解中的作用 和Fas信号，我们将阐明潜在的机制，并确定特定的ROS参与和他们的 蜂窝信号源。此外，我们将确定c-flip上的特定泛素化位点，以此为目标 利用定点突变和基因缺失分析进行降解的分子。预计， 拟议中的研究将提供关于细胞死亡调节和机制的宝贵新信息 相关的肺部疾病，这在风险评估和治疗干预中将是重要的。

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 toFas- 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-kB 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.