Redox-based Fas Signaling in Allergic Airway Disease

项目摘要 过度的上皮细胞凋亡和缺乏适当的上皮恢复被认为是纤维化的关键。 死亡受体Fas在上皮细胞凋亡中起着重要的作用，并且随后的细胞凋亡的发展也是一个重要的过程。 肺纤维化同样，氧化环境的变化，以及内质网（ER） 应激被认为在特发性肺纤维化（IPF）的发病机制中是重要的，这表明 氧化还原和ER扰动、Fas、上皮细胞凋亡和纤维化之间的因果关系。于本 我们发现，Fas作为死亡受体的效力在其 S-谷胱甘肽化，蛋白质氧化的一种形式。S-谷胱甘肽化代表抗氧化剂 分子谷胱甘肽转化为反应蛋白半胱氨酸（PSSG）。我们最近发现在肺上皮细胞中 不是所有的Fas都表达在表面上，而是未完全加工成配体的潜在Fas库 结合形式，位于ER中。响应于用FasL刺激表面Fas，蛋白质二硫键 异构酶ERp 57诱导内质网内Fas的快速氧化加工。我们推测氢 在此过程中产生的过氧化物（H2 O2）负责S-谷胱甘肽化， 由谷胱甘肽S-转移酶P（GSTP）催化。这些发现揭示了Fas的一个新维度- 诱导细胞凋亡的“配体触发的氧化处理的潜伏Fas在ER”作为一种调节机制， 调节细胞死亡的强度。在ER内触发的确切氧化事件仍然未知， ER内产生的H2 O2在上皮细胞凋亡中的功能作用，以及随后的 纤维化不清楚。本文要解决的中心假设是，ERp 57催化的Fas加工 导致ER中H2 O2含量增加。H2 O2的增加反过来又需要允许GST催化的 Fas的S-谷胱甘肽化，增加上皮细胞凋亡，从而导致肺纤维化。在特定 目的#1我们将确定ERp 57在氧化加工中的功能需求，以及随后的S- 肺上皮中Fas的谷胱甘肽化，以及由此产生的肺纤维化的发展。具体目标 #2旨在探索GSTP在Fas的S-谷胱甘肽化中的功能要求，以及随后的 肺纤维化的发展。在具体目标#3中，我们将评估H2 O2的功能重要性 在Fas依赖性上皮细胞凋亡和随后的纤维化中在ER中产生。我们将使用 补充细胞培养和小鼠转基因方法，再加上这些过程的详细分析 在IPF以及NSIP患者的肺组织中，使用创新和临床相关策略。 完成拟议的实验可能会产生实质性的影响，因为Fas，ER应激， 谷胱甘肽氧化还原紊乱与纤维化的发病机制独立相关， 旨在评估或靶向S-谷胱甘肽化的方法可能被证明具有临床相关性， 诊断工具和潜在的治疗方法。

PROJECT SUMMARY 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.