Redox-based Fas Signaling in Allergic Airway Disease

描述(由申请人提供)：过度的上皮细胞凋亡和缺乏适当的上皮修复被认为是纤维化形成的关键。死亡受体Fas在上皮细胞凋亡及随后的肺纤维化发展中起重要作用。同样，氧化环境的改变以及内质网(ER)应激在特发性肺纤维化(IPF)的发病机制中也被认为是重要的，这表明氧化还原和ER扰动、Fas、上皮细胞凋亡和纤维化之间存在因果联系。在这笔赠款的当前资金周期中，我们发现Fas作为死亡受体的效力在其S谷胱甘肽基化(一种蛋白质氧化形式)后得到增强。S-谷胱甘肽化代表了抗氧化剂分子谷胱甘肽与反应蛋白半胱氨酸的偶联。我们最近发现，在肺上皮细胞中，并不是所有的Fas都在表面表达，而是在内质网中定位了一个潜伏的Fas池，该池没有完全加工成配体结合的形式。作为对FasL刺激表面Fas的反应，蛋白质二硫键异构酶ERp57诱导内质网内Fas的快速氧化处理。我们推测，在这个过程中产生的过氧化氢(H_2O_2)是由谷胱甘肽S转移酶P催化的谷胱甘肽-谷胱甘肽基化反应。 (GSTP)。这些发现揭示了Fas诱导的细胞凋亡的一个新维度--“配体触发内质网中潜在Fas的氧化处理”，作为调节细胞死亡强度的调节机制。内质网中触发的确切氧化事件尚不清楚，内质网中产生的过氧化氢在上皮细胞凋亡和随后的纤维化发病机制中的功能作用也不清楚。这里要解决的中心假设是，ERp57催化的Fas处理导致内质网中过氧化氢含量增加。H_2O_2的增加反过来又需要允许GSTP催化的GSTP催化的Fas的谷胱甘肽基化，从而增强上皮细胞的凋亡，从而导致肺纤维化。在特定的目标#1中，我们将确定ERp57在氧化过程中的功能需求，以及随后肺上皮细胞Fas的S谷胱甘肽基化，以及由此导致的肺纤维化的发展。具体目的2旨在探讨谷胱甘肽转移酶在S体内的功能需求--Fas的谷胱甘肽基化，以及随后的肺纤维化的发展。在特定的目标#3中，我们将评估内质网中产生的过氧化氢在Fas依赖的上皮细胞凋亡和随后的纤维化形成中的功能重要性。我们将使用互补细胞培养和小鼠转基因方法，结合使用创新的和临床相关的策略，详细分析IPF患者和NSIP患者的肺组织中的这些过程。拟议实验的完成可能会产生实质性的影响，因为Fas、内质网应激和谷胱甘肽氧化还原扰动已经独立地与纤维化的发病机制联系在一起，而旨在评估或靶向S谷胱甘肽基化的方法可能被证明作为诊断工具和潜在的治疗方法在临床上具有相关性。

DESCRIPTION (provided by applicant): Excessive epithelial apoptosis and lack of proper epithelial restitution are believed to be critical to fibrogenesis. The death receptor Fas plays a cardinal role in epithelial apoptosis, and the subsequent development of pulmonary fibrosis. Similarly, changes in the oxidative environment, as well as endoplasmic reticulum (ER) stress are believed to be important in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suggesting a causal link between redox and ER perturbations, Fas, epithelial apoptosis, and fibrogenesis. During the current funding cycle of this grant we discovered that the potency of Fas as a death receptor is enhanced following its S-glutathionylation, a form of protein oxidation. S-glutathionylation represents the conjugation of the antioxidant molecule glutathione to reactive protein cysteines (PSSG). We recently unraveled that in lung epithelial cells not all Fas is expressed on the surface but that a latent pool of Fas which is not fully processed into the ligand binding form, is localized in the ER. In response to stimulation of surface Fas with FasL, the protein disulfide isomerase, ERp57, induces rapid oxidative processing of Fas within the ER. We speculate that hydrogen peroxide (H2O2) produced during this process is responsible for S-glutathionylation, in a reaction that is catalyzed by glutathione S-transferase P (GSTP). These discoveries illuminate a new dimension of Fas- induced apoptosis "ligand-triggered oxidative processing of latent Fas in the ER" as a regulatory mechanism to regulate the strength of cell death. The exact oxidative events triggered within the ER remain unknown, and the functional role of H2O2 generated within the ER for epithelial apoptosis, and subsequent pathogenesis of fibrosis are unclear. The central hypothesis to be addressed herein is that ERp57-catalyzed processing of Fas leads to increases of H2O2 content in the ER. Increases in H2O2 are in turn required to permit GSTP-catalyzed S-glutathionylation of Fas, augmenting epithelial apoptosis, thereby leading to pulmonary fibrosis. In Specific Aim #1 we will determine the functional requirement of ERp57 in oxidative processing, and subsequent S- glutathionylation of Fas in lung epithelium, and the resultant development of pulmonary fibrosis. Specific Aim #2 seeks to explore the functional requirement of GSTP in S-glutathionylation of Fas, and the subsequent development of pulmonary fibrosis. In Specific Aim #3 we will assess the functional importance of H2O2 generated in the ER in Fas-dependent epithelial apoptosis and subsequent fibrogenesis. We will use complementary cell culture and mouse transgenic approaches, coupled to detailed analysis of these processes in lung tissues from patients with IPF as well as NSIP, using innovative and clinically relevant strategies. Completion of proposed experiments is likely to exert a substantial impact given that Fas, ER stress, and glutathione redox perturbations have been independently linked to the pathogenesis of fibrosis, and methodologies aimed at assessing or targeting S-glutathionylation may prove to be clinically relevant as diagnostic tools and potential therapeutics.