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形成的关键。死亡受体Fas在上皮细胞凋亡和随后的肺纤维化发展中起着重要作用。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）的发病机制中很重要，这表明氧化还原和内质网扰动、Fas、上皮细胞凋亡和纤维形成之间存在因果关系。在本基金的当前资助周期中，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）后得到增强。s -谷胱甘肽化是抗氧化剂分子谷胱甘肽与反应蛋白半胱氨酸（PSSG）的结合。我们最近发现，在肺上皮细胞中，并非所有的Fas都在表面表达，而是一种未完全加工成配体结合形式的Fas潜伏池定位于内质网。在FasL对表面Fas的刺激下，蛋白二硫异构酶ERp57诱导内质网内Fas的快速氧化加工。我们推测在这一过程中产生的过氧化氢（H2O2）负责s -谷胱甘肽酰化，该反应由谷胱甘肽s -转移酶P催化

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.