Oxidant signaling for airway remodeling and inflammation

描述（由申请人提供）： 哮喘是一种慢性气道疾病，其特征在于气流阻塞、炎症、高反应性和重塑。气道重塑部分是由于平滑肌细胞生长增加，并且气道平滑肌细胞的免疫调节作用最近已被认识到。活性氧已被证明是气道平滑肌细胞中的信号介质。然而，活性氧如何参与细胞信号事件的机制在很大程度上仍然不确定。缺乏这方面的知识会妨碍开发旨在预防和/或治疗哮喘的新治疗策略。我的长期目标是了解气道平滑肌细胞中活性氧信号的机制。本申请的目的是评估哮喘介质诱导的膜联蛋白A1（脂皮质素）羰基化的机制和功能。中心假设是由哮喘介质（包括血小板衍生生长因子（PDGF）和内皮素-1）产生的活性氧促进膜联蛋白A1（调节炎症和细胞生长）的羰基化作为信号转导机制。这一假说是基于平滑肌细胞中强有力的初步数据而提出的，这些数据表明：（i）PDGF和内皮素-1，哮喘的重要介质，促进蛋白质羰基化;（ii）质谱鉴定膜联蛋白A1，（抗炎、抗增殖和促凋亡分子）是一种羰基化的蛋白质;（iii）膜联蛋白A1与促进平滑肌细胞增殖和存活以及炎症反应的蛋白质相互作用;（iv）膜联蛋白A1的过表达减少平滑肌细胞数目。（v）膜联蛋白A1的羰基化之后是蛋白酶体依赖性降解，和（vi）铁螯合抑制人气道平滑肌细胞中PDGF诱导的bcl-2和白细胞介素-13 mRNA表达的上调。此外，在体内用过敏原处理小鼠以诱导哮喘促进膜联蛋白A1的降解。这项研究的基本原理是，一旦获得了调节气道平滑肌细胞细胞生长和炎症反应的信号通路的知识，它将导致可用于预防和/或治疗哮喘的新策略。本申请的目的将通过追求三个具体目标来实现：1）通过人气道平滑肌细胞中的细胞信号传导介质确定膜联蛋白A1羰基化的机制，2）确定蛋白质羰基化在膜联蛋白A1降解机制中的作用，以及3）定义膜联蛋白A1在气道平滑肌细胞生长和炎症反应调节中的作用。这项工作具有创新性，因为它将研究活性氧信号传导的新机制，并研究膜联蛋白A1在气道平滑肌调节细胞生长和炎症反应中的新作用。我的预期是，包括PDGF和内皮素-1在内的哮喘介质促进金属催化的膜联蛋白A1羰基化，通过蛋白酶体机制特异性影响细胞生长、凋亡和炎症信号传导。这些结果将是重要的，因为它们有望为哮喘的预防和治疗干预提供新的药物。此外，这些结果将从根本上推进肺细胞生物学领域。(End摘要）

DESCRIPTION (provided by applicant): Asthma is a chronic disorder of the airways that is characterized by airflow obstruction, inflammation, hyperreactivity and remodeling. Airway remodeling is, in part, due to increased smooth muscle cell growth, and immunomodulatory roles of airway smooth muscle cells have recently been recognized. Reactive oxygen species have been shown to serve as signaling mediators in airway smooth muscle cells. Mechanisms of how reactive oxygen species are involved in cell signaling events, however, remain largely undefined. Lack of such knowledge interferes with the development of new therapeutic strategies that are designed to prevent and/or treat asthma. My long-range goal is to understand the mechanism of reactive oxygen species signaling in airway smooth muscle cells. The objective of this application is to evaluate the mechanism and functions of annexin A1 (lipocortin) carbonylation induced by mediators of asthma. The central hypothesis is that reactive oxygen species produced by mediators of asthma including platelet-derived growth factor (PDGF) and endothelin-1 promote carbonylation of annexin A1 (which regulates inflammation and cell growth) as a signal transduction mechanism. The hypothesis has been formulated on the basis of strong preliminary data in smooth muscle cells which suggest that: (i) PDGF and endothelin-1, important mediators of asthma, promote protein carbonylation; (ii) Mass spectrometry identified that annexin A1 (an anti-inflammatory, anti-proliferative and pro-apoptotic molecule), is one protein that is carbonylated; (iii) Annexin A1 interacts with proteins which promote proliferation and survival of smooth muscle cells and inflammatory responses; (iv) Overexpression of annexin A1 reduces smooth muscle cell number, (v) Carbonylation of annexin A1 is followed by proteasome- dependent degradation, and (vi) Iron chelation inhibits PDGF-induced upregulation of bcl-2 and interleukin-13 mRNA expression in human airway smooth muscle cells. Further, in vivo treatment of mice with allergens to induce asthma promoted degradation of annexin A1. The rationale for the proposed research is that, once knowledge of signaling pathways that regulate cell growth and inflammatory responses in airway smooth muscle cells has been obtained, it will lead to new strategies that can be used to prevent and/or treat asthma. The objective of the application will be accomplished by pursuing three specific aims: 1) Identify the mechanism of annexin A1 carbonylation by cell signaling mediators in human airway smooth muscle cells, 2) Determine the role of protein carbonylation in the mechanism of annexin A1 degradation, and 3) Define the roles of annexin A1 in the regulation of airway smooth muscle cell growth and inflammatory responses. The proposed work is innovative because it will investigate a novel mechanism for reactive oxygen species signaling and study the novel roles of annexin A1 in airway smooth muscle regulation for cell growth and inflammatory responses. It is my expectation that mediators of asthma including PDGF and endothelin-1 promote metal-catalyzed annexin A1 carbonylation that specifically influences cell growth, apoptotic and inflammatory signaling via mechanisms involving proteasomes. These results will be significant because they are expected to provide new agents for preventative and therapeutic interventions for asthma. In addition, the results will fundamentally advance the field of lung cell biology. (End of Abstract)