Redox-based Fas Signaling in Allergic Airway Disease

描述（由申请人提供）：死亡受体Fas在细胞凋亡的调节中起关键作用。Fas通路的激活导致肺上皮细胞的凋亡，并且这些细胞中Fas诱导的凋亡足以触发随后的纤维化发展。在本基金的当前资助期内，我们进行了令人兴奋的新观察，即Fas死亡途径是通过谷氧还蛋白/S-谷胱甘肽化氧化还原对进行氧化还原调节的。在连接Fas时，巯基修复酶谷氧还蛋白-1（Grx1）通过半胱天冬酶8/3迅速降解，这导致Fas在半胱氨酸294处的S-谷胱甘肽化增加。Fas的S-谷胱甘肽化促进其募集到脂筏中，并增强FasL的结合、半胱天冬酶活化和凋亡，提供前馈放大环以增强凋亡信号的强度。缺乏Grx1的原代肺上皮细胞或成纤维细胞显示更多的Fas S-谷胱甘肽化，并具有增强的caspase活性和FasL诱导的凋亡。相反，Grx1的过表达降低了Fas的S-谷胱甘肽化，并抑制了FasL诱导的caspase活化和凋亡。这些令人兴奋的新观察结果提出了一种有趣的可能性，即谷氧还蛋白/S-谷胱甘肽化氧化还原系统也通过控制肺上皮细胞凋亡的程度来影响肺中胶原沉积的程度。目前的建议的假设是，在S-谷胱甘肽的增加，发生的结果半胱天冬酶依赖性降解的Grx1发挥因果作用的上皮细胞凋亡和随后的肺纤维化的发展，和S-谷胱甘肽的Fas是一个关键的目标，在这个过程中。我们还推测，通过逆转S-谷胱甘肽化的增加，肺上皮中Grx1的增加减弱了上皮细胞凋亡，并抑制了随后的纤维化。在具体目标1中，我们将阐明Fas的S-谷胱甘肽化的分子基础，以及它增强Fas促凋亡功能的机制。在特定目标2中，我们将确定在纤维化小鼠模型和纤维化肺病患者中Fas的S-谷胱甘肽化和S-谷胱甘肽化模式是否增加。在特定目标3中，我们将确定全身或气道上皮缺乏Grx1的小鼠是否对肺纤维化的发展具有增强的易感性，与Fas的S-谷胱甘肽化和半胱天冬酶活化的增加相关。我们将讨论Fas在增强纤维化易感性中的作用。在具体目标4中，我们将讨论肺上皮细胞中Grx 1的转基因表达是否能保护肺纤维化的发展。完成这项更新申请的具体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：排列在气道（呼吸管）上的上皮细胞在防御吸入物质方面起着重要作用，并且在维持正常肺功能方面很重要。然而，我们的实验室已经确定，上皮细胞在促进肺组织硬化，从而降低肺功能方面起着负面作用。我们已经确定了一些关键的氧化信号，通过引起上皮细胞的死亡来促进这种可能的负面事件，并阻止正常的修复过程。这项拨款提案有四个具体目标，以测试这些生化信号在肺硬化中的重要性，并将使用来自小鼠气道的原代细胞培养物，细胞系和基因改造小鼠的构建。该项目的完成将为肺硬化过程提供新的见解，并可能导致新疗法的开发，以限制肺硬化，并可能逆转这一过程。

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.