Fas-Induced Apoptosis and Inflammatory Lung Injury

描述(申请人提供)：Fas/Fas配体(FasL)死亡信号通路已被证明在肺部炎症和纤维化的发展中发挥重要作用，但我们对涉及的机制了解有限。特别是，控制Fas表达的转录机制和参与肺中毒后Fas激活的信号事件在很大程度上尚不清楚。本研究的总体目标是为从机制上理解Fas诱导的细胞凋亡和肺部病理过程中涉及的分子事件提供科学基础，并为风险评估和开发有效的治疗方法提供有用的信息。首先，我们将研究博莱霉素和FasL处理后特定肺细胞中Fas表达的转录机制。我们将利用凝胶电泳法、基因缺失分析和定点突变等方法，鉴定Fas基因启动子上与Fas激活相关的核转录因子和调控元件。其次，我们将确定导致转录因子动员和诱导特定肺细胞凋亡的早期分子事件。具体地说，这一目标将检验博莱霉素和FasL刺激产生的特定活性氧物种(ROS)在介导Fas激活和随后诱导细胞凋亡中发挥重要作用的假说。我们还将研究细胞内关键抗氧化剂和抗凋亡蛋白Bcl-2在这一过程中的可能作用。电子自旋共振将被用来鉴定涉及的特定ROS，免疫印迹和基因转染研究将被用来阐明Bcl-2的作用。最后，由于我们的初步研究表明，FasL治疗在体外和体内都能引起肿瘤坏死因子的上调，而且这种分子在肺部疾病和细胞凋亡中起着至关重要的作用，因此我们将测试FasL诱导的肺毒性是否依赖于肿瘤坏死因子的激活。同样，将使用Fas和肿瘤坏死因子基因敲除小鼠来研究Fas信号在肿瘤坏死因子诱导的毒性中的可能作用。我们期望这些研究将为Fas诱导细胞凋亡的分子机制及其在肺部发病机制中的作用提供有价值的新信息。

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.