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）：气道分泌的形成凝胶的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，粘蛋白分泌过多导致大多数气道疾病中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稳态是至关重要的。气道杯状细胞粘蛋白分泌主要由P2 Y2嘌呤受体下游的信号传导途径控制，尽管另外的G蛋白偶联受体和受体酪氨酸激酶也可能起重要作用。气道液体层中的核苷酸对P2 Y2受体的激活导致第二信使二酰基甘油（DAG）和钙（Ca 2+）的产生，其一起导致稳健的粘蛋白分泌。值得注意的是，肺中分泌的大多数粘蛋白在基线时通过其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外泌机制的组分是结合DAG和Ca 2+的Munc 13引发蛋白，以及结合磷脂和Ca 2+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Munc 13蛋白之一Munc 13 -2的缺失导致小鼠中基线和刺激的粘蛋白分泌的部分缺陷：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分泌的实验工具。Munc 13 -4，表达的另一种亚型，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在基线分泌中不起作用。相反，Syt-2的缺乏导致刺激分泌的深刻缺陷，但我们推断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的相互作用可能解释这些自相矛盾的结果。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Munc 13和Syt蛋白上游的信号传导途径控制的，并且特定的Munc 13/Syt对划分基线和激动剂刺激的粘蛋白分泌。将使用小鼠模型来检验这一假设，并确定气道粘蛋白分泌调节的精确机制。在特定目标1中，我们将确定Munc 13 -2和Munc 13 -4在体内和体外气道粘蛋白分泌中的作用，并确定它们是如何被调节的，以及它们如何分别对基线和激动剂刺激的通路起作用。对于特定目标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差异地促进基线和激动剂刺激的途径，并确定Syt和Munc 13亚型之间是否存在显著的功能相互作用。 公共卫生相关性：粘蛋白分泌过多，导致肺气道中粘液的过度产生，实质上增加了几乎所有气道疾病的患者发病率和死亡率，常见疾病（慢性支气管炎和哮喘）和孤儿疾病（囊性纤维化和原发性纤毛运动障碍）都是如此，而不管起始环境和/或遗传损伤如何。因此，重要的是了解粘蛋白分泌的分子途径以及调节其分泌的分子途径，以实现有效的治疗设计和治疗。本项目将使用基因敲除和过表达小鼠模型来揭示参与调节粘蛋白分泌的两种蛋白质的作用，即负责启动分泌装置的Munc 13和作为最终细胞内钙传感器和分泌触发器的synaptotagmin。