Translational Regulation in Bronchial Epithelial Cells

描述(由申请人提供)：本提案的目的是阐明参与炎症反应的支气管上皮细胞中参与炎症反应的mRNAs的转录后机制控制翻译和亚细胞定位。呼吸道上皮细胞直接暴露在环境中，是抵御空气中颗粒物、过敏原和感染性物质的第一道防线。越来越多的证据表明，支气管上皮细胞不仅在维持呼吸道的理化稳态方面发挥着关键的、多方面的作用，而且在呼吸道疾病的发病机制中也发挥着重要的作用。在过敏性呼吸道炎症过程中，上皮细胞既是介质产生的来源，也是重塑过程的靶点。然而，对于炎症反应过程中这些细胞中介质、效应和重塑基因表达的转录后调节，以及当呼吸道炎症变得持续时，例如哮喘时，这种调节可能如何改变，人们知之甚少。我们使用在液-气界面培养的原代正常或哮喘人支气管上皮(HBE)细胞、人支气管上皮BEAS-2B细胞和过敏性呼吸道炎症的活体小鼠模型进行了广泛的初步研究。这些结果表明，哮喘患者的支气管上皮细胞经历了miRNA轮廓/功能和P小体的协调改变，P小体是用于存储和/或降解翻译抑制的mRNAs的细胞质结构域。这些变化导致蛋白质合成的过度活跃状态，有助于维持炎症介质的过度产生，从而使哮喘炎症的慢性本质永久化。在拟议的研究中，我们将：1)确定miR-26和miR-16丰度的降低是否有助于在哮喘原代HBE细胞中观察到持续的IL-6水平升高；2)确定在哮喘原代HBE细胞中显著下调的一组miRNAs在控制支气管上皮细胞翻译机制活动中的作用；以及3)确定P小体减少是否是激活的支气管上皮细胞的标志，以及P小体组装和拆卸的改变如何影响支气管上皮细胞中的炎症反应。哮喘患者的呼吸道上皮细胞基本上是异常的，但这种异常的分子和生化基础在很大程度上仍不清楚。我们将使用最先进的方法来获得关键的洞察力，以了解炎性介质产生的翻译调节，以及哮喘时这种调节可能改变的病理机制。拟议的研究涉及一个尚未探索但令人兴奋的呼吸道炎症研究的新领域，并可能将新的概念和方法引入呼吸道炎症领域。这一结果有可能确定新的靶点，以加快开发与哮喘相关的慢性呼吸道炎症的新治疗方法。

DESCRIPTION (provided by applicant): The objective of this proposal is to elucidate the post-transcriptional mechanisms controlling translation and subcellular localization of mRNAs involved in inflammatory responses in bronchial epithelial cells. Epithelial cells of the bronchial airways are directly exposed to the environment and are the first line of defense against airborne particulate matter, allergens and infectious agents. Mounting evidence suggests that bronchial epithelial cells play a pivotal, multifaceted role not only in the maintenance of physico-chemical homoeostasis of the airways but also in the pathogenesis of airway diseases. During allergic airway inflammation, the epithelium is both a source of mediator production as well as a target of remodeling processes. Yet, little is known about post-transcriptional regulation of mediator, effector and remodeling gene expression in these cells during the inflammatory response, and about how this regulation may be altered when airway inflammation becomes persistent, such as in asthma. We have carried out extensive preliminary studies using primary normal or asthmatic human bronchial epithelial (HBE) cells cultured at the liquid-air interface, human bronchial epithelial BEAS-2B cells, and an in vivo mouse model of allergic airway inflammation. The results suggest that bronchial epithelial cells from asthmatics undergo a coordinated alteration of miRNA profile/function and of P- bodies, cytoplasmic domains for storage and/or degradation of translationally repressed mRNAs. These changes lead to a hyperactive state of the protein synthesis that contributes to sustaining excess production of inflammatory mediators and thus perpetuating the chronic nature of asthmatic inflammation. In the proposed study we will: 1) Determine whether a reduction in miR-26 and miR-16 abundance contributes to the persistent, elevated level of IL-6 observed in asthmatic primary HBE cells; 2) Define the role of a group of miRNAs that are significantly down-regulated in asthmatic primary HBE cells in controlling the activity of translation machinery in bronchial epithelial cells; and 3) Determine whether a reduction in P-bodies is a hallmark of activated bronchial epithelial cells, and how alteration of P-body assembly and disassembly influences the inflammatory response in bronchial epithelial cells. The airway epithelium is fundamentally abnormal in asthma, but the molecular and biochemical bases for this abnormality remains largely undefined. We will employ state-of-the-art approaches to gain critical insights into translational regulation of inflammatory mediator production and the pathological mechanism by which this regulation may be altered in bronchial epithelial cells in asthma. The proposed studies involve an unexplored but exciting new area of research in airway inflammation and are likely to introduce novel concepts and methodologies to the field of airway inflammation. The results have a potential for identifying new targets to speed development of new therapeutic approaches to the chronic airway inflammation associated with asthma.