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: 8217298
• 负责人: C. William Davis
• 金额: $ 49.6万
• 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/8217298
• 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）：气道分泌的凝胶形成粘蛋白通过清除吸入的病原体和颗粒物提供重要的保护功能；然而，在大多数气道疾病中，粘蛋白分泌过多会导致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稳态至关重要。气道杯状细胞粘蛋白分泌主要由 P2Y2 嘌呤受体下游信号通路控制，尽管其他 G 蛋白偶联受体和受体酪氨酸激酶也可能发挥重要作用。气道液层中的核苷酸激活 P2Y2 受体会导致第二信使二酰甘油 (DAG) 和钙 (Ca2+) 的产生，两者共同导致粘蛋白的强劲分泌。值得注意的是，肺部分泌的大多数粘蛋白在基线时通过调节未知的途径连续释放。已知响应激动剂产生的第二信使的细胞胞吐机制的组成部分是结合 DAG 和 Ca2+ 的 Munc13 启动蛋白，以及结合磷脂和 Ca2+ 的 SNARE 触发突触结合蛋白 (Syt) 蛋白。我们发现，气道中表达的两种 Munc13 蛋白 Munc13-2 中的一种缺失，会导致小鼠基线出现部分缺陷并刺激粘蛋白分泌：因此，该小鼠模型提供了第一个可用于解开受调节的基线粘蛋白的实验工具分泌。 Munc13-4，另一种表达的亚型，可能调节激动剂刺激的粘蛋白分泌；然而，我们的初步数据表明它可能对基线分泌没有作用。相比之下，Syt-2 的缺失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 亚型在小鼠和人类粘液细胞中表达，其与 Syt-2 的相互作用可能可以解释这些矛盾的结果。我们假设基线和激动剂刺激的粘蛋白分泌是由气道粘蛋白分泌细胞中 Munc13 和 Syt 蛋白上游的信号通路控制的，并且特定的 Munc13/Syt 对划分基线和激动剂刺激的粘蛋白分泌。小鼠模型将用于检验这一假设并确定气道粘蛋白分泌调节的精确机制。在具体目标 1 中，我们将确定 Munc13-2 和 Munc13-4 在体内和体外气道粘蛋白分泌中的作用，并确定它们的调节方式以及它们如何单独对基线和激动剂刺激途径做出贡献。对于具体目标 2，我们将确定 Syt-2 和 Syt-7 在体内和体外气道粘蛋白分泌中的作用，确定它们是否以及如何被调节以对基线和激动剂刺激途径做出不同的贡献，并确定是否存在感兴趣的 Syt 和 Munc13 亚型之间存在显着的功能相互作用。 公共健康相关性：粘蛋白分泌过多，导致肺部气道粘液过量产生，大大增加了几乎所有气道疾病、常见疾病（慢性支气管炎和哮喘）和罕见疾病（囊性纤维化和原发性纤毛病）的患者发病率和死亡率。运动障碍）类似，无论起始环境和/或遗传损伤如何。因此，了解粘蛋白分泌及其分泌调节的分子途径非常重要，以实现有效的治疗设计和治疗。该项目将利用基因敲除和过度表达小鼠模型来揭示两种参与调节粘蛋白分泌的蛋白质的作用：Munc13（负责启动分泌装置）和突触结合蛋白（作为最终的细胞内钙传感器和触发器）分泌。