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恢复被认为是纤维化的关键。死亡受体Fas在上皮细胞凋亡和随后的肺纤维化的发展中起重要作用。类似地，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发生之间存在因果关系。在该资助的当前资助周期中，我们发现Fas作为死亡受体的效力在其S-谷胱甘肽化（一种蛋白质氧化形式）后增强。S-谷胱甘肽化表示抗氧化剂分子谷胱甘肽与反应性蛋白半胱氨酸（PSSG）的缀合。我们最近发现，在肺上皮细胞中，并不是所有的Fas都表达在表面上，而是一个未完全加工成配体结合形式的潜在Fas库位于ER中。响应于FasL对表面Fas的刺激，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，在此过程中产生的过氧化氢（H2 O2）是负责S-谷胱甘肽化，在一个由谷胱甘肽S-转移酶P催化的反应中 （全面贸易优惠制）。这些发现阐明了Fas诱导的细胞凋亡的一个新的维度“ER中潜伏Fas的配体触发的氧化加工”作为调节细胞死亡强度的调节机制。ER内触发的确切氧化事件仍不清楚，ER内产生的H2 O2对上皮细胞凋亡的功能作用以及随后的纤维化发病机制尚不清楚。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含量的增加。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。在具体目标#1中，我们将确定ERp 57在氧化加工中的功能需求，以及随后肺上皮中Fas的S-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需求，以及肺纤维化的后续发展。在具体目标#3中，我们将评估Fas依赖性上皮细胞凋亡和随后的纤维化中ER中产生的H2 O2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，结合使用创新和临床相关策略对IPF和NSIP患者肺组织中的这些过程进行详细分析。鉴于Fas、ER应激和谷胱甘肽氧化还原扰动已独立地与纤维化的发病机制相关，完成拟议的实验可能会产生重大影响，并且旨在评估或靶向S-谷胱甘肽化的方法可能被证明是临床相关的诊断工具和潜在的治疗方法。

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.