Nicotine-induced fibronectin expression in lung injury and repair

描述（由申请人提供）： 摘要：烟碱乙酰胆碱受体（nAChR）是由神经元和非神经元细胞表达的能够进行信号转导的五聚体通道。虽然许多关于nAChR功能的已知信息与神经系统有关，但关于它们在非神经系统中的作用的信息正在迅速出现。nAChR在肺中表达，并且在非人灵长类动物中进行的有趣的工作表明，尼古丁激活nAChR导致肺结构/功能异常。我们探讨了nAChR激活导致基质表达的机制，发现这种作用是通过称为17 nAChR的nAChR的特定亚组介导的。在肺成纤维细胞中激活这些受体后，我们观察到MEK-1/Erk通路的诱导和转录因子CREB的磷酸化。反过来，CREB刺激纤连蛋白的表达，一种与肺损伤和修复有关的基质糖蛋白。这些事件被17种nAChR拮抗剂阻断，并且不存在于17种nAChR KO原代肺成纤维细胞中。此外，我们发现nAChR激活通过MEK-1/ERK依赖性途径刺激肺成纤维细胞增殖。在用尼古丁长期刺激的小鼠中进行了类似的观察，但在发育和成年17 nAChR KO动物中没有观察到。最近，我们记录了暴露于香烟烟雾提取物的成纤维细胞中纤连蛋白的增殖和表达的增加，并观察到暴露于烟草烟雾的小鼠肺中纤连蛋白蛋白沉积的增加，证实了该过程与烟草相关疾病的相关性。我们还发现，在长期暴露于尼古丁的情况下，博莱霉素诱导的肺损伤后，肺修复机制似乎受损。这些数据揭示了非神经元nAChR在控制肺组织重塑中的新作用。基于上述，我们假设肺中的17 nAChR活化通过刺激成纤维细胞的增殖和它们通过MEK-1/ERK依赖性信号过度表达基质组分如纤连蛋白来促进组织重塑。在基线时，这导致基质组成的细微变化，而不会引起明显的后果，如纤维化。相反，在肺损伤的情况下，这导致过度的组织重塑，促进低效的修复和纤维化。这将在具体目标中得到检验，这些目标旨在：1）检查17个nAChR在肺成纤维细胞的增殖、肌成纤维细胞转分化和基质表达中的作用，以及MEK-1/Erk在野生型与17个nAChR KO肺成纤维细胞中介导这些事件中的作用，2）在两种肺损伤模型中使用野生型和17 nAChR KO小鼠检查17 nAChR在肺损伤和修复中的作用（博莱霉素模型和异源气管移植模型）; 3）检测17 nAChR持续激活在损伤后肺修复中的作用。 公共卫生相关性： 项目叙述本项目将探讨损伤后肺组织重塑的细胞机制。组织重塑是以结缔组织成分（也称为细胞外基质）的表达、沉积和降解的改变为特征的过程。这对现役战斗人员很重要，但对退伍军人也很重要，因为在这一人群中常见的许多肺部疾病（例如，COPD，肺纤维化），与组织重塑的改变有关。有趣的是，烟草及其成分与许多这些疾病有关，因此，人们认为它们促进了组织重塑的改变。但是，负责这些事件的机制尚不清楚。该项目将通过关注烟碱受体以及这些受体的激活如何促进肺细胞外基质相对组成的改变来探索这些过程中所涉及的机制。

DESCRIPTION (provided by applicant): ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are pentameric channels expressed by neuronal and non-neuronal cells that are capable of signal transduction. Although much of what is known about the function of nAChRs relates to the nervous system, information about their roles in non-neuronal systems is rapidly emerging. nAChRs are expressed in lung and interesting work performed in non-human primates suggests that nAChR activation by nicotine leads to pulmonary structural/functional abnormalities. We explored the mechanisms by which nAChR activation leads to matrix expression and found that this effect is mediated through a specific subgroup of nAChRs termed 17 nAChRs. Upon activation of these receptors in lung fibroblasts, we observed induction of the MEK-1/Erk pathway and phosphorylation of the transcription factor CREB. In turn, CREB stimulated the expression of fibronectin, a matrix glycoprotein implicated in lung injury and repair. These events were blocked by 17 nAChR antagonists and were not present in 17 nAChR KO primary lung fibroblasts. In addition, we found that nAChR activation stimulates lung fibroblast proliferation through MEK-1/Erk-dependent pathways. Similar observations were made in mice chronically stimulated with nicotine, but not in developing and adult 17 nAChR KO animals. More recently, we documented an increase in the proliferation and expression of fibronectin in fibroblasts exposed to cigarette smoke extract, and observed an increase in fibronectin protein deposition in the lungs of mice exposed to tobacco smoke confirming the relevance of this process to tobacco-related disease. We also found that lung repair mechanisms appeared to be impaired after bleomycin-induced lung injury in the setting of chronic exposure to nicotine. These data unveil a novel role for non-neuronal nAChRs in control of lung tissue remodeling. Based on the above, we hypothesize that 17 nAChR activation in lung promotes tissue remodeling by stimulating the proliferation of fibroblasts and their overexpression of matrix components like fibronectin through MEK-1/Erk-dependent signals. At baseline, this leads to subtle alterations in matrix composition without causing overt consequences such as fibrosis. In contrast, in the setting of lung injury, this leads to exaggerated tissue remodeling that promotes inefficient repair and fibrosis. This will be tested in specific aims designed to: 1) Examine the role of 17 nAChRs in the proliferation, myofibroblast transdifferentiation, and matrix expression of lung fibroblasts, and the role of MEK-1/Erk in mediating these events in wildtype vs. 17 nAChR KO lung fibroblasts, 2) Examine the role of 17 nAChRs in lung injury and repair using wildtype and 17 nAChR KO mice in two models of lung injury (bleomycin model and the model of heterologous tracheal transplantation), and 3) Examine the effect of persistent 17 nAChR activation in lung repair after injury. PUBLIC HEALTH RELEVANCE: Project Narrative This project will explore the cellular mechanisms involved in tissue remodeling of the lung after injury. Tissue remodeling is a process characterized by alterations in the expression, deposition, and degradation of connective tissue components (also called extracellular matrices). This is important to active combatants but also to veterans in view that many pulmonary disorders typically seen in this population (e.g., COPD, lung fibrosis) that are associated with alterations in tissue remodeling. Interestingly, tobacco and its components have been implicated in many of these disorders and, therefore, it is thought that they promote alterations in tissue remodeling. But, the mechanisms responsible for these events are unknown. This project will explore the mechanisms involved in these processes by focusing on nicotinic receptors and how activation of these receptors promotes alterations in the relative composition of the lung extracellular matrix.