The Unfolded Protein Response in Human Airway and Alveolar Epithelial Cells

描述(由申请人提供)： 吸烟所致的慢性阻塞性肺疾病(COPD)以肺泡上皮细胞死亡、呼吸道化生和纤维化为特征。我们发现，非COPD的慢性吸烟者的肺对内质网应激表现出一种代偿性反应，称为未折叠蛋白反应(UPR)，并且在培养的呼吸道上皮细胞中，与UPR相关的转录因子ATF-4和Nrf2调节抗氧化基因的表达。这些数据增加了UPR保护肺细胞免受氧化应激和COPD发展的可能性。然而，当内质网应激足够严重，蛋白质动态平衡无法恢复时，UPR通过上调促凋亡转录因子CHOP的表达而导致细胞凋亡。这些观察结果提出了另一种可能性，即UPR可能通过诱导细胞凋亡而参与肺气肿的发病。我们之前的研究是在全肺裂解液和培养的呼吸道上皮细胞中进行的。我们还没有研究特定类型的肺细胞对香烟烟雾暴露的原位反应。因此，这个为期两年的项目的具体目标是表征香烟烟雾诱导的参与COPD发病机制的肺细胞，即呼吸道上皮细胞中的UPR。我们将在COPD受试者的呼吸道上皮细胞中检验香烟烟雾对UPR反应受损的假说。UPR标志性蛋白(即GRP78、钙网蛋白、钙粘蛋白和蛋白二硫键异构酶)、氧化防御(即Nrf2和ATF4)和凋亡(即CHOP)的表达将在COPD患者和非COPD患者的呼吸道上皮细胞中进行比较。激光捕获显微镜技术应用于HOPE固定、膨胀的肺组织切片，将从没有COPD的前吸烟者(n=10)和患有COPD的前吸烟者的黄金阶段II(n=10)和IV(n=10)中获取细胞。更好地了解CS诱导UPR的细胞学基础将有助于我们对COPD发病机制的理解。公共卫生相关性：吸烟是慢性阻塞性肺疾病(COPD)的危险因素，COPD是一种以炎症和肺细胞死亡为特征的疾病。然而，该病的发病机制仍不完全清楚，目前的治疗方法也不够充分。我们最近的工作表明，吸烟在肺内诱导的一种新的生化途径可以起到保护作用。这种反应及其生物学后果将在已知与COPD有关的细胞类型中进行研究。这些结果有望提高我们对吸烟相关肺部疾病的认识，并有助于开发新的更好的治疗方法。(摘要结束)

DESCRIPTION (provided by applicant): Cigarette smoking induced chronic obstructive pulmonary disease (COPD) is characterized by alveolar epithelial cell death and airway metaplasia and fibrosis. We have shown that the lungs of chronic cigarette smokers without COPD manifest a compensatory response to endoplasmic reticulum stress termed the unfolded protein response (UPR) and that the UPR-related transcription factors, ATF-4 and Nrf2, proteins which regulate anti-oxidant gene expression, are up-regulated by CS in cultured airway epithelial cells. These data raise the possibility that the UPR protects lung cells against oxidant stress and the development of COPD. However, when ER stress is sufficiently severe and protein homeostasis cannot be restored, the UPR causes apoptosis by up-regulating expression of the pro-apoptotic transcription factor, CHOP. These latter observations raise the alternative possibility that the UPR may contribute to the pathogenesis of emphysema by inducing cell apoptosis. Our previous studies were performed in whole lung lysates and cultured airway epithelial cells. We have not studied the in situ response of specific lung cell types to cigarette smoke exposure. Accordingly, the specific Aim of this 2 year project is to characterize the UPR induced by cigarette smoke in a lung cell involved in the pathogenesis of COPD, i.e., airway epithelial cells. We will test the hypotheses that UPR response to cigarette smoke is impaired in the airway epithelial cells of subjects with COPD. Expression of the UPR hallmark proteins (i.e., GRP78, calreticulin, calnexin and protein disulfide isomerase), oxidant defense (i.e., Nrf2 and ATF4) and apoptosis (i.e., CHOP) will be compared in airway epithelial cells from subjects with and without COPD. Cells will be harvested from ex-- smokers without COPD (n=10) and ex-smokers with COPD in GOLD stages II (n=10) and IV (n=10) using the technique of laser capture microscopy applied to HOPE fixed, inflated lung tissue sections. A better understanding of the cellular basis of the UPR induced by CS should enhance our understanding of the pathogenesis of COPD. PUBLIC HEALTH RELEVANCE: Cigarette smoking is a risk factor for chronic obstructive pulmonary disease (COPD), a disease characterized by inflammation, and lung cell death. However, the pathogenesis of this disease remains incompletely understood and current therapies are inadequate. Our recent work shows that a novel biochemical pathway induced in the lung by cigarette smoking can be protective. This response and its biological consequences will be studied in cell type known to be involved with COPD. The results obtained are expected to improve our understanding of cigarette smoke-related lung disease and contribute to the development of new and better treatments. (End of Abstract)