Molecular Pathways Regulating Airway Goblet Cell Mucin Secretion

调节气道杯状细胞粘蛋白分泌的分子途径

• 批准号: 8049606
• 负责人: C. William Davis
• 金额: $ 49.97万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049606
• 关键词: Agonist; Asthma; Breathing; Calcium; Cells; Chronic Bronchitis; Complex; Cystic Fibrosis; Cytoplasmic Granules; Data; Defect; Disease; Epitopes; Event; G-Protein-Coupled Receptors; Gel; Generations; Genetic; Goblet Cells; Homeostasis; Human; In Vitro; Inflammatory; Knock-out; Knockout Mice; Knowledge; Liquid substance; Lung; Mediating; Membrane Fusion; Molecular; Morbidity - disease rate; Mucins; Mucous body substance; Mus; Nucleotides; Obstruction; Orphan; P2Y2 receptor; Particulate; Pathway interactions; Patients; Phospholipids; Play; Primary Ciliary Dyskinesias; Production; Protein Binding; Protein Isoforms; Proteins; Purinoceptor; Receptor Protein-Tyrosine Kinases; Regulation; Role; SNAP receptor; Second Messenger Systems; Secretory Cell; Signal Pathway; Signal Transduction; Specific qualifier value; Testing; Therapeutic; Transgenic Organisms; gain of function; in vivo; injured airway; interest; knockout gene; mortality; mouse model; overexpression; pathogen; public health relevance; response; second messenger; sensor; synaptotagmin; synaptotagmin II; synaptotagmin VII; therapy design; tool

DESCRIPTION (provided by applicant): The secreted gel-forming mucins of the airway provide important protective functions through the clearance of inhaled pathogens and particulates; however, mucin hypersecretion causes airflow obstruction and airway injury in most diseases of the airways. Therefore, tight control of mucin production and secretion is critical for airway homeostasis. Airway goblet cell mucin secretion is principally controlled by signaling pathways downstream of P2Y2 purinoceptors, though additional G-protein coupled receptors and receptor tyrosine kinases may also play significant roles. Activation of P2Y2 receptors by nucleotides in the airway liquid layer leads to generation of the second messengers diacylglyerol (DAG) and calcium (Ca2+) that together result in robust mucin secretion. Significantly, the majority of mucins secreted in a lung are released, continuously, at baseline, by pathways whose regulation is unknown. Components of the cellular exocytic machinery known to respond to agonist-generated second messengers are the Munc13 priming proteins that bind DAG and Ca2+, and the SNARE-triggering Synaptotagmin (Syt) proteins that bind phospholipids and Ca2+. We have found that the absence of one of the two Munc13 proteins expressed in the airways, Munc13-2, results in partial defects in baseline and stimulated mucin secretion in mice: this mouse model therefore offers the first experimental tool useful in unraveling regulated baseline mucin secretion. Munc13-4, the other isoform expressed, presumably regulates agonist stimulated mucin secretion; however, our preliminary data indicate it may play no role in baseline secretion. In contrast, the absence of Syt-2 results in a profound defect in stimulated secretion, but we deduce a paradoxical increase in baseline mucin secretion. Additional Syt isoforms are expressed in mouse and human mucous cells whose interactions with Syt-2 might explain these paradoxical results. We hypothesize that baseline and agonist stimulated mucin secretion is controlled by signaling pathways upstream of Munc13 and Syt proteins in airway mucin secreting cells, and that specific Munc13/Syt pairs partition baseline and agonist stimulated mucin secretions. Mouse models will be used to test this hypothesis and to determine the precise mechanisms of regulation of airway mucin secretion. In Specific Aim 1, we will determine the roles of Munc13-2 and Munc13-4 in airway mucin secretion in vivo and in vitro, and identify how they are regulated and how they contribute individually to baseline and agonist stimulated pathways. For Specific Aim 2, we will determine the roles of Syt-2 and Syt-7 in airway mucin secretion in vivo and in vitro, identify whether and how they are regulated to contribute differentially to baseline and agonist stimulated pathways, and determine whether there are significant functional interactions between the Syt and Munc13 isoforms of interest. PUBLIC HEALTH RELEVANCE: Mucin hypersecretion, resulting in a overproduction of mucus in the airways of the lungs, substantially increases patient morbidity and mortality in literally all the airways diseases, common ones (chronic bronchitis and asthma) and orphan ones (cystic fibrosis and primary ciliary dyskinesia) alike, irrespective of the initiating environmental and/or genetic insult. Consequently, it is important to understand the molecular pathways by which mucins are secreted and by which their secretion is regulated, to enable effective therapeutic design and treatment. This project will use gene knockout and overexpression mouse models to reveal the roles of two kinds of proteins involved in regulating mucin secretion, Munc13, which is responsible for priming the secretory apparatus, and synaptotagmin, which acts as the final intracellular calcium sensor and trigger of secretion.

描述（由申请人提供）：气道的分泌凝胶形成的粘液通过清除吸入的病原体和颗粒物提供了重要的保护功能；然而，粘蛋白过度分泌会导致大多数气道疾病的气流阻塞和气道损伤。因此，严格控制粘蛋白的产生和分泌对于气道体内平衡至关重要。气道杯状细胞粘蛋白分泌主要由P2Y2 purinoceptors下游的信号通路控制，尽管其他G蛋白偶联受体和受体酪氨酸激酶也可能起重要作用。气道液层中核苷酸对P2Y2受体的激活导致第二核糖基甘油烯醇（DAG）和钙（Ca2+）的产生，从而导致强大的粘蛋白分泌。值得注意的是，肺中分泌的大多数粘蛋白在基线时连续释放，其调节未知的途径。已知响应激动剂生成的第二个使者的细胞外胞索机械的成分是结合DAG和Ca2+的Munc13启动蛋白，以及结合磷脂磷脂和Ca2+的SNARE触发突触蛋白（SYT）蛋白。我们发现，在气道中表达的两种Munc13蛋白之一Munc13-2的缺失导致小鼠基线和刺激粘蛋白分泌的部分缺陷：因此，该小鼠模型提供了第一个实验工具，可用于解开调节的基线粘蛋白粘蛋白分泌。 Munc13-4，另一种表达的同工型可能调节激动剂刺激粘蛋白分泌。但是，我们的初步数据表明它在基线分泌中可能没有任何作用。相反，缺乏SYT-2会导致刺激分泌的严重缺陷，但我们推断出基线粘蛋白分泌的矛盾增加。其他SYT同工型在小鼠和人类粘液细胞中表达，它们与SYT-2的相互作用可能解释了这些矛盾的结果。我们假设基线和激动剂刺激了粘蛋白的分泌，这是通过气道粘蛋白分泌细胞上游的信号通路和SYT蛋白上游控制的，而特定的Munc13/Syt Pairs Pairs Parairs Partition Basinear和激动剂刺激了粘蛋白分泌。小鼠模型将用于检验该假设，并确定气道粘蛋白分泌调节的确切机制。在特定的目标1中，我们将确定Munc13-2和Munc13-4在体内和体外的气道粘蛋白分泌中的作用，并确定它们如何受到调节以及它们如何对基线和激动剂刺激途径单独贡献。对于特定目标2，我们将确定SYT-2和SYT-7在体内和体外的气道粘蛋白分泌中的作用，确定它们是否以及如何调节它们是否对基线和激动剂刺激的途径有所不同，并确定SYT和MUNC13 ISOFORM之间是否存在显着的功能相互作用。 公共卫生相关性：粘蛋白的过度分泌，导致肺气道中的粘液过度生产，从而大大增加了所有气道疾病的患者发病率和死亡率，常见的疾病（慢性支气管炎和哮喘）和孤儿（孤立的环境）和原发性的环境和基因般的环境和基因般的环境和基因构成。因此，重要的是要了解粘蛋白分泌的分子途径并通过其分泌的分泌，以实现有效的治疗设计和治疗。该项目将使用基因敲除和过表达小鼠模型来揭示两种蛋白质在调节粘蛋白分泌中涉及的蛋白质的作用，即负责启动分泌仪的Munc13和SynaptoTagmin，它们是最终的细胞内钙传感器和分泌物的触发器。