Unbiased genome-wide screen to identify genes regulating mucous cell hyperplasia

描述(由申请人提供)： 研究挑战领域：翻译研究15，挑战主题04-HL-102以开发综合策略来阐明肺部疾病的机制摘要/摘要项目概要：本挑战研究项目将进行1)小鼠基因组范围的shRNA文库筛选，以确定诱导或抑制杯状细胞分化和粘液高分泌的基因和相关基因网络，以及2)广泛的小分子化合物文库筛选，以确定与急、慢性肺部疾病相关的调节粘液细胞活性的化合物和过程。小鼠和人类的呼吸上皮细胞(RECs)将被改造成指示由SPDEF(SAM指向结构域ETS样因子)控制的遗传网络活动的基因，我们最近发现的SPDEF基因是肺内粘液细胞编程的“主调节器”。REC将被改造为表达SPDEF和/或FOXA3(使用慢病毒载体)和两个不同的SPDEF靶基因表达盒(农业2-荧光素酶和Muc5A/C-renilla)。将进行机器人高通量筛选，以利用小鼠shRNA文库和大型、特征良好的化学库筛选人支气管上皮细胞(HBECs)和小鼠肺上皮(MLE)细胞，以识别调控SPDEF定位、水平和靶基因表达(活性)的基因。抑制SPDEF活性和调节SPDEF活性的基因和分子将被识别和表征。利用QRT-PCR、免疫组织化学和原位杂交法，将在小鼠模型(体内)和REC细胞(体外)组织中评估候选基因的表达位置及其与并发粘液细胞增殖和粘液过度生成的肺部疾病过程的关系。将对调节杯状细胞活动的遗传和化学信号通路的性质进行建模，并优先选择候选基因和分子进行进一步研究。该项目利用了最近发现的SPDEF在与杯状细胞增生和粘液过度生成相关的肺部疾病的发病机制中的重要作用。CCHMC最近获得的接近全基因组的Lenti-shRNA文库的可访问性，以及与辛辛那提大学和CCHMC合作建立的基因组研究所(GRI)现有的出色的高通量筛选设施，使这一大规模项目成为可能。该项目将提供对杯状细胞增生、粘液细胞过度生成的发病机制的见解，创造用于研究影响呼吸道的肺部疾病的试剂，并识别治疗常见、慢性和急性肺部疾病的治疗感兴趣的基因和分子，包括慢性阻塞性肺疾病、慢性阻塞性肺病和哮喘。这项建议回应了广泛的挑战领域15“翻译科学”和挑战主题04-HL-102，并基于我们最近确定的SPDEF在控制决定杯状细胞增殖和粘液过度生产的基因网络中所起的关键作用。杯状细胞增生和粘液细胞分泌和功能异常伴随并参与了常见肺部疾病的发病，包括哮喘、囊性纤维化(CF)和慢性阻塞性肺疾病(COPD)。同样，粘液过多与急性和慢性感染和炎症有关，导致慢性上呼吸道疾病，包括慢性鼻窦炎、过敏性鼻炎、中耳疾病以及导致全世界呼吸道发病率和死亡率的肺部疾病。发现和利用调控杯状细胞增殖和粘液高分泌的分子，将有助于了解肺部疾病的发病机制，提高常见呼吸系统疾病的诊断和治疗水平。目前的应用是基于我们的初步数据和假设：杯状细胞的分化和粘液的过度产生是由控制SPDEF活性的信号和转录网络调节呼吸道的粘液细胞活性所介导的。其应用主要集中在对SAM点结构域ETS样因子(SPDEF)表达和功能调控有用的基因和小分子的鉴定上。杯状细胞增生和粘液细胞过度生成与常见的慢性和急性呼吸系统疾病的发病、发病率和死亡率有关，包括慢性阻塞性肺疾病(COPD)、囊性纤维化(CF)和哮喘，这些肺部疾病共同影响着全球数百万人。目前，人们对杯状细胞分化相关的基因、遗传网络和确切的病理后果知之甚少，也缺乏预测易感性和改善预后的标志物和潜在的治疗靶点。该项目将确定调控杯状细胞分化和粘液高分泌的基因和分子途径，以提供对慢性肺部疾病的诊断和预后洞察，以及它对先天免疫和通常与这些疾病相关的继发感染的影响。该项目将确定潜在的治疗目标和分子，为这些慢性肺部疾病的未来治疗提供基础，这些慢性肺部疾病共同构成了世界范围内的一个主要健康问题。

DESCRIPTION (provided by applicant): Research Challenge Area: Translational Studies 15 , Challenge Topic 04-HL-102 to Develop Integrated Strategies to Elucidate Mechanisms of Lung Disease Summary/Abstract Project Synopsis: This Challenge Research project will undertake 1) a mouse genome-wide shRNA library screen to identify genes and associated gene networks inducing or inhibiting goblet cell differentiation and mucous hypersecretion, and 2) an extensive small molecule compound library screen to identify compounds and processes regulating mucous cell activity relevant to both acute and chronic lung diseases. Mouse and human Respiratory Epithelial Cells (RECs) will be engineered with genes indicating the activity of a genetic network controlled by Spdef (Sam pointed domain Ets-like factor), a gene we recently showed is a "master regulator" of mucous cell programming in the lung. RECs will be engineered to express SPDEF and/or FOXA3 (using lentiviral vectors) and two distinct SPDEF target gene expression cassettes (Agr2-luciferase and Muc5A/C-renilla). A robotic, high-throughput screen will be undertaken to utilize a mouse shRNA library and a large, well-characterized chemical library screen human bronchial epithelial cells (HBECs) and mouse lung epithelial (MLE) cells to identify genes regulating SPDEF localization, levels, and target gene expression (activity). Genes and molecules inhibiting and those modulating SPDEF activity induces SPDEF nuclear localization and stability will be identified and characterized. The sites of expression of candidate genes and their association with the pulmonary disease processes complicated by mucous cell hyperplasia and mucous hyperproduction will be assessed in tissue from mouse models (in vivo) and in REC cells (in vitro), utilizing QRT-PCR, immunohistochemistry, and in situ hybridization. The nature of the genetic and chemical signaling pathways regulating goblet cell activity, will be modeled and candidate genes and molecules prioritized for further studies. The project takes advantage of recent identification of the important role of SPDEF in the pathogenesis of lung diseases associated with goblet cell hyperplasia and mucous hyperproduction. The accessibility of a near genome-wide lenti-shRNA library recently acquired by CCHMC and the outstanding high-throughput screening facility present at the Genome Research Institute (GRI) that was founded in partnership with the University of Cincinnati and CCHMC makes this large scale project feasible. The project will provide insights into the pathogenesis of goblet cell hyperplasia, mucous cell hyperproduction, create reagents for the study of pulmonary diseases affecting the airways, and identify genes and molecules of therapeutic interest for the treatment of common, chronic and acute pulmonary disorders, including CF, COPD and asthma. This proposal is responsive to the broad challenge area 15 "Translational Science" and Challenge topic 04-HL-102, and is based on our recent identification of the critical role that SPDEF plays in controlling a network of genes that determines goblet cell hyperplasia and mucous hyperproduction. Goblet cell hyperplasia and abnormalities of mucous cell secretion and function accompany and contribute to the pathogenesis of common pulmonary diseases, including asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). Likewise, mucous hyperproduction is associated with acute and chronic infection and inflammation that contributes to chronic upper respiratory disorders, including chronic sinusitis, allergic rhinitis, middle ear disease, as well as pulmonary disease that contribute to respiratory morbidity and mortality world wide. The identification and utilization of molecules that regulate goblet cell hyperplasia and mucous hyperproduction will be useful in understanding the pathogenesis of lung disease, and improving the diagnosis and treatment of common respiratory disorders. The present application is based on our preliminary data and the hypothesis: that goblet cell differentiation and mucous hyperproduction are mediated by signaling and transcriptional networks controlling Spdef activity regulate mucous cell activity in the respiratory tract. The application focuses to the identification of both genes and small molecules useful in controlling the expression and function of Sam pointed domain Ets-like factor (SPDEF). Goblet cell hyperplasia and mucous cell hyperproduction are associated with and contribute to the pathogenesis, morbidity and mortality attributed to common chronic and acute respiratory disorders, including Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF), and asthma, together, lung diseases that affect millions of individuals world-wide. At present, there is little understanding of the genes, genetic networks, and precise pathological consequences related to goblet cell differentiation and a lack of markers and potential therapeutic targets to predict susceptibilities and improve outcomes, respectively. This project will identify genes and molecular pathways regulating goblet cell differentiation and mucous hyperproduction that will provide diagnostic and prognostic insight into chronic lung disease, and its impact on innate immunity and secondary infections commonly associated with these disorders. This project will identify potential therapeutic targets and molecules that will provide the basis for future therapies of these chronic lung diseases that, together, comprise a major health problem world-wide.