Molecular Pathways Regulating Airway Goblet Cell Mucin Secretion

描述(申请人提供)：呼吸道的分泌性凝胶形成粘蛋白通过清除吸入的病原体和颗粒提供重要的保护功能；然而，在大多数呼吸道疾病中，粘蛋白过度分泌会导致气流阻塞和呼吸道损伤。因此，严格控制粘蛋白的产生和分泌对于维持呼吸道内环境的稳定至关重要。虽然额外的G蛋白偶联受体和受体酪氨酸激酶也可能发挥重要作用，但气道杯状细胞粘蛋白的分泌主要受P2Y2嘌呤受体下游的信号通路控制。由呼吸道液体层中的核苷酸激活的P2Y2受体导致第二信使二酰甘油(DAG)和钙(Ca~(2+))的产生，共同导致粘蛋白的旺盛分泌。值得注意的是，肺中分泌的大多数粘蛋白在基线水平上由调节未知的途径持续释放。已知的对激动剂产生的第二信使做出反应的细胞胞外机制的组件是与DAG和钙离子结合的Munc13启动蛋白，以及结合磷脂和钙离子的圈套触发突触素(SYT)蛋白。我们发现，在呼吸道表达的两种Munc13蛋白中的一种Munc13-2的缺失会导致小鼠基线和刺激粘蛋白分泌的部分缺陷：因此，这个小鼠模型提供了第一个可用于解开调节的基线粘蛋白分泌的实验工具。Munc13-4，另一个表达的异构体，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，它可能在基线分泌中没有作用。相反，缺乏SYT-2会导致刺激分泌的严重缺陷，但我们推导出基线粘蛋白分泌的矛盾增加。其他SYT亚型在小鼠和人的粘液细胞中表达，它们与SYT-2的相互作用可能解释了这些矛盾的结果。我们假设基础和激动剂刺激的粘蛋白分泌是由呼吸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对分割基础和激动剂刺激的粘蛋白分泌。小鼠模型将被用来检验这一假说，并确定调节呼吸道粘蛋白分泌的精确机制。在特定的目标1中，我们将确定Munc13-2和Munc13-4在体内和体外呼吸道粘蛋白分泌中的作用，并确定它们是如何调节的，以及它们如何分别对基线和激动剂刺激的途径做出贡献。对于特定的目标2，我们将确定SYT-2和SYT-7在体内和体外呼吸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的通路做出不同的贡献，并确定SYT和Munc13亚型之间是否存在显著的功能相互作用。 公共卫生相关性：粘蛋白高分泌导致肺部呼吸道粘液过度产生，大大增加了所有呼吸道疾病、常见疾病(慢性支气管炎和哮喘)和孤儿(囊性纤维化和原发纤毛运动障碍)的患者发病率和死亡率，无论最初的环境和/或遗传损害如何。因此，重要的是要了解粘蛋白分泌和调节的分子途径，以便能够进行有效的治疗设计和治疗。该项目将使用基因敲除和过度表达的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，synaptopagmin负责最终的细胞内钙传感器和分泌触发。

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.