Regulation of Fas-Mediated Lung Cell Apoptosis

Fas 介导的肺细胞凋亡的调节

• 批准号: 7100360
• 负责人: Yon Rojanasakul
• 金额: $ 36.63万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7100360
• 关键词: CD95 molecule; apoptosis; cell differentiation; cysteine endopeptidases; free radical oxygen; gene induction /repression; laboratory rat; lung; nuclear factor kappa beta; oxidative stress; p53 gene /protein; posttranslational modifications; site directed mutagenesis; ubiquitin

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.

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发挥着重要作用。然而，我们对该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。本研究的总体目标是为理解 Fas 介导的细胞凋亡及其在特定肺细胞中的调节所涉及的分子事件的机制提供科学基础。我们的初步研究结果表明，虽然 Fas 可以引发肺细胞凋亡，但 Fas 的表达水平及其由 Fas 配体 (FasL) 的激活与 Fas 介导的细胞死亡的易感性并不相关，这表明细胞凋亡信号通路的调节剂必须存在。在这个项目中，我们将寻求确定控制 Fas 介导的肺细胞细胞死亡的关键调节因子，并阐明其机制。该项目将具体测试以下假设：Fas 介导的细胞凋亡和相关肺部病理的易感性可能是由细胞 FLICE 抑制蛋白 (c-FLIP) 的表达水平决定的，并且某些肺毒性药物对该蛋白的改变可以使细胞对Fas 通过激活 caspase-8 和下游 caspase 级联介导细胞死亡。我们将使用各种分子生物学和生化技术确定 c-FLIP 的功能作用并识别原代肺细胞中的死亡信号通路。我们还将检验以下假设：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。因为我们的初步研究结果表明 c-FLIP 的关键作用通过研究 c-FLIP 降解和 Fas 信号传导中的活性氧 (ROS) 和泛素蛋白酶体依赖性途径，我们将阐明其潜在机制并确定所涉及的特定 ROS 及其细胞来源。此外，我们将确定特定的 ROS 来源。 c-FLIP 上的泛素化位点通过定点诱变和基因缺失分析靶向该分子进行降解，预计所提出的研究将提供有关细胞死亡调节和相关肺部疾病机制的有价值的新信息。风险评估和治疗干预。