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控的缺陷导致许多人类疾病，包括肺疾病。最近的证据表明，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，我们对这一过程所涉及的机制的理解是有限的。不了解这些机制直接限制了预防和治疗工作的有效性。本研究的总体目的是为了解fas介导的特定肺细胞凋亡及其调控的分子事件的机制提供科学依据。我们的初步研究结果表明，虽然Fas可以触发肺细胞凋亡，但Fas的表达水平及其被Fas配体（FasL）激活与Fas介导的细胞死亡易感性无关，这表明凋亡信号通路的调节因子一定存在。在这个项目中，我们将寻求确定控制fas介导的肺细胞死亡的关键调节因子并阐明其机制。该项目将专门测试以下假设：Fas介导的细胞凋亡和相关肺部病理的易感性可能由细胞fase抑制蛋白（c-FLIP）的表达水平决定，以及某些肺毒性药物对该蛋白的改变可以通过激活caspase-8和下游caspase级联使细胞对Fas介导的细胞死亡敏感。我们将利用各种分子生物学和生化技术确定c-FLIP的功能作用，并确定原代肺细胞中的死亡信号通路。我们还将验证通过翻译后修饰下调c- FLIP是通过caspase-8和NF-(B)信号通路控制fas介导的细胞死亡和存活的关键调控事件的假设。由于我们的初步研究结果表明活性氧（ROS）和泛素-蛋白酶体依赖途径在c-FLIP降解和Fas信号传导中的关键作用，我们将阐明潜在的机制，并确定特定的ROS及其细胞来源。此外，我们将利用位点定向诱变和基因缺失试验确定c-FLIP上针对该分子降解的特定泛素化位点。预计这些研究将为细胞死亡调控和相关肺部疾病的机制提供有价值的新信息，这将对风险评估和治疗干预具有重要意义。

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.