Redox-based Fas Signaling in Allergic Airway Disease

描述(申请人提供)：死亡受体Fas在细胞凋亡的调控中起着关键作用。Fas途径的激活导致肺上皮细胞的凋亡，而Fas诱导的这些细胞的凋亡足以触发随后的纤维化发展。在这笔赠款的当前资助期间，我们做出了令人兴奋的新观察，即Fas死亡途径是通过谷氧还蛋白/S-谷胱甘肽氧化还原对来调节氧化还原的。当连接Fas时，硫醇修复酶谷氧还蛋白-1(Grx1)通过caspase8/3迅速降解，从而导致Fas半胱氨酸294位的S谷胱甘肽基化增加。S-Fas的谷胱甘肽基化促进其在脂筏中的募集，并增强与FasL的结合，caspase的激活和细胞凋亡，提供一个前馈扩增环来增强细胞凋亡信号的强度。缺乏Grx1的原代肺上皮细胞或成纤维细胞表达更多的Fas的S谷胱甘肽基化，并增强caspase活性和FasL诱导的细胞凋亡。相反，Grx1的过表达降低了Fas的S谷胱甘肽基化，并抑制了FasL诱导的半胱氨酸酶的激活和凋亡。这些令人兴奋的新观察提出了一个有趣的可能性，即谷氧还蛋白/S-谷胱甘肽氧化还原系统也通过控制肺上皮细胞的凋亡程度来影响肺内胶原沉积的程度。目前的假说是，Caspase依赖的Grx1降解导致的S谷胱甘肽的升高在上皮细胞的凋亡和随后的肺纤维化的发生中起了一定的作用，而Fas的S谷胱甘肽基化是这一过程中的一个关键靶点。我们还推测，通过逆转S谷胱甘肽基化的增加，肺上皮细胞中Grx1的表达增加，从而减少了上皮细胞的凋亡，并抑制了随后的纤维化形成。在特定的目标1中，我们将阐明S-Fas谷胱甘肽基化的分子基础，以及其增强Fas促凋亡功能的机制。在特定目的2中，我们将确定Fas的S-谷胱甘肽和S-谷胱甘肽在纤维化模型小鼠和纤维化肺疾病患者中的模式是否增加。在具体目标3中，我们将确定系统性或呼吸道上皮中缺乏Grx1的小鼠是否增加了发生肺纤维化的易感性，这与Fas和caspase的S谷胱甘肽基化增加有关。我们将解决Fas在增强肝纤维化易感性方面的需求。在特定的目标4中，我们将讨论在肺上皮细胞中转基因表达Grx1是否对纤维化的发展具有保护作用。完成这项更新申请的特定目标将提供有关最近发现的氧化还原对谷氧还蛋白/S谷胱甘肽基化在肺纤维化发展中的作用的重要新信息。公共卫生相关性：呼吸道(呼吸管)内的上皮细胞在抵御吸入物质方面发挥着重要作用，对维持正常的肺功能也很重要。然而，我们的实验室已经发现，上皮细胞在促进肺组织僵硬方面发挥了负面作用，从而降低了肺功能。我们已经确定了一些关键的氧化信号，这些信号通过导致上皮细胞死亡来促进这一可能的负面事件，并阻止正常的修复过程。这项拨款提案有四个具体目标，旨在测试这些生化信号在肺僵硬中的重要性，并将使用来自小鼠呼吸道、细胞系的原代细胞培养，以及构建转基因小鼠。该项目的完成将为肺僵硬的过程提供新的见解，并可能导致限制肺僵硬的新疗法的开发，并有可能逆转这一过程。

DESCRIPTION (provided by applicant): The death receptor Fas plays a critical role in the regulation of apoptosis. Activation of the Fas pathway causes apoptosis of lung epithelial cells, and Fas-induced apoptosis in these cells is sufficient to trigger the subsequent development of fibrosis. During the current funding period of this grant, we made the exciting novel observation that the Fas death pathway is redox regulated via the glutaredoxin/S-glutathionylation redox couple. Upon ligation of Fas, the thiol repair enzyme, glutaredoxin-1 (Grx1) is rapidly degraded via caspase 8/3, which causes increases in S-glutathionylation of Fas at Cysteine 294. S-glutathionylation of Fas promotes its recruitment into lipid rafts, and enhances binding of FasL, caspase activation and apoptosis, providing a feed forward amplification loop to enhance the strength of the apoptotic signal. Primary lung epithelial cells or fibroblasts that lack Grx1 display more S-glutathionylation of Fas and have enhanced caspase activity and FasL-induced apoptosis. Conversely, overexpression of Grx1 decreases S-glutathionylation of Fas and dampens FasL-induced caspase activation and apoptosis. These exciting new observations raise the intriguing possibility that the glutaredoxin/S-glutathionylation redox system also impact the extent of collagen deposition in the lung by controlling the extent of apoptosis in lung epithelial cells. The hypothesis of the current proposal is that increases in S-glutathionylation that occur as a result of caspase-dependent degradation of Grx1 play a causal role in apoptosis of epithelial cells and the subsequent development of pulmonary fibrosis, and that S-glutathionylation of Fas is a critical target in this process. We also speculate that augmentation of Grx1 in lung epithelium by reversing the increases in S-glutathionylation attenuates epithelial apoptosis, and dampens subsequent fibrogenesis. In Specific Aim 1, we will elucidate the molecular basis of S-glutathionylation of Fas, and the mechanism whereby it strengthens the pro-apoptotic function of Fas. In Specific Aim 2, we will determine whether patterns of S-glutathionylation and S-glutathionylation of Fas are increased in mouse models of fibrosis and patients with fibrotic lung disease. In Specific Aim 3, we will determine whether mice that lack Grx1 systemically or in airway epithelium have an enhanced susceptibility to the development of pulmonary fibrosis, in association with increases in S-glutathionylation of Fas and caspase activation. We will address the requirement of Fas in the enhanced susceptibility to fibrogenesis. In Specific Aim 4, we will address whether transgenic expression of Grx1 in lung epithelial cells confers protection against the development of fibrosis. Completion of the specific aims of this renewal application will provide important new information about the role of a recently discovered redox couple, glutaredoxin/S-glutathionylation in the development of pulmonary fibrosis. PUBLIC HEALTH RELEVANCE: Epithelial cells that line the airways (breathing tubes) play an important role in the defense against inhaled materials, and are important in maintaining normal lung function. However, our laboratories have identified that epithelial cells play a negative role in promoting stiffening of the lung tissue, thereby decreasing lung function. We have identified some critical oxidative signals that promote this possibly negative event, by causing death of the epithelial cells, and prevent the normal repair process. This grant proposal has four specific aims to test the importance of these biochemical signals in lung stiffening, and will use both primary cell cultures from mouse airways, cell lines, and the construction of genetically altered mice. Completion of this project will provide new insights into the process of lung stiffening, and may lead to the development of new therapeutics to limit lung stiffening, and also potentially reverse this process.