IL13-responsive genes in goblet cell metaplasia in asthma

描述(由申请人提供)：哮喘患者呼吸道上皮中杯状细胞(杯状细胞增生，GCH)数量的增加是哮喘患者因过敏原和环境毒素而产生有害的粘蛋白糖蛋白的主要原因。导致GCH的机制尚未阐明。该项目的重点是在白介素13诱导的过敏性哮喘小鼠模型中，通过激活Stat6信号转导通路，识别指导杯状细胞(杯状细胞化生，GCM)出现的基因和途径。在这个探索性/发展研究项目中要检验的总体假设是，IMS诱导的Stat6的激活激活了主要基因，例如带有Stat6顺式序列的转录因子，并且主要基因产物随后调节调节杯状细胞分化的开关基因。我们最近对暴露于IL13或PBS后的小鼠气管进行了时间表达谱分析，并鉴定了四个在IL13暴露后早期(1h)上调的候选主基因和几个候选开关基因，它们在中间时间(3.5h)显示出表达的变化，并在随后的时间(6h)恢复到基线水平。在目标1中，将对四个候选主控基因进行评估，以确定一个或多个是STAT6的主要靶标。候选主控基因将通过染色质免疫沉淀分析(ChIP)评估与IL4R的共定位以及与Stat6的结合，并在体外对小鼠气管上皮(MTE)细胞进行功能评估。在目标2中，将使用共聚焦显微镜评估候选Switch基因产物与主基因的细胞和亚细胞共定位。将评估主要基因产物与Switch基因启动子中的顺式元件的结合。在siRNA或将开关基因转导入MTE细胞后，将监测候选开关基因功能诱导GCM的能力。这项研究应该首次确定哺乳动物呼吸道系统中杯状细胞分化的途径。这将有助于开发药物来控制哮喘和其他慢性呼吸道疾病患者的GCH和粘液过度产生。

DESCRIPTION (provided by applicant): An increased number of goblet cells (goblet cell hyperplasia, GCH) in the airway epithelium is largely responsible for the deleterious overproduction of mucin glycoproteins in response to allergens and environmental toxins in patients with asthma. The mechanisms that result in GCH have not been elucidated. This project focuses on identifying genes and pathways that direct the appearance of goblet cells (goblet cell metaplasia, GCM) in the interleukin (IL)13-induced murine model of allergic asthma, which is mediated by activation of the Stat6 signal transduction pathway. The overall hypotheses to be tested in this Exploratory/Developmental Research project is that the IMS-induced activation of Stat6 activates master genes, e.g. transcription factors with Stat6 cis-sequences, and that master gene products subsequently regulate switch genes that mediate goblet cell differentiation. We have recently performed temporal expression array profiling of murine trachea following exposure to IL13 or PBS and identified four candidate master genes upregulated early (1 h) following IL13 exposure and several candidate switch genes, which exhibit altered expression at an intermediate time (3.5 h) and return to base line levels at a later time (6 h). In Aim 1, four candidate master genes will be evaluated to determine whether one or more are primary targets of Stat6. Candidate master genes will be evaluated for co-localization with IL4R and for binding to Stat6 by chromatin immunoprecipitation assay (ChIP) and functionally evaluated in vitro in murine tracheal epithelial (MTE) cells. In Aim 2, candidate switch gene products will be evaluated for cellular and sub- cellular co-localization with master genes using confocal microscopy. Binding of master gene products to cis-elements in switch gene promoters will be evaluated. The ability of candidate switch genes to functionally induce GCM will be monitored following siRNA or transduction of switch genes into MTE cells. This study should define for the first time a pathway for goblet cell differentiation in a mammalian airway system. This will contribute to the development of pharmacological agents to control GCH and mucus overproduction in patients with asthma and possibly other chronic airway diseases