Autophagy regulates airway epithelial cell mucin secretion

 描述（由申请人提供）：气道疾病的意义：包括哮喘、慢性阻塞性肺病和囊性纤维化在内的气道疾病是发病率和死亡率的重要原因。慢性炎症性气道疾病的一个关键特征是粘蛋白的过度分泌和粘稠粘液引起的气道阻塞。气道上皮细胞如何适应气道分泌性粘蛋白的产生和分泌增加的这种应激尚不清楚。自噬作为一种调节分泌的新途径：自噬虽然在真核生物中高度保守，但以前被认为主要是在压力下调节营养循环。现在已经确定，自噬对于包括蛋白质分泌在内的多种反应是必不可少的。此外，在细胞应激期间的高蛋白质产生和分泌期间，自噬似乎特别需要。我们的数据刚刚发表在《自噬6》杂志上，表明在气道上皮细胞中，自噬是Th 2炎症期间MUC 5AC分泌所必需的。然而，自噬调节气道中粘蛋白分泌的机制尚不清楚。研究计划：在本申请中，我们提出了我们的初步数据，使用体外人类上皮细胞和 Th 2炎症的体内小鼠模型，证明自噬是MUC 5Ac分泌和活性氧（ROS）产生所需的。我们假设这些共有的表型实际上是相互关联的，并且自噬依赖的ROS可能是调节分泌的一种新机制。为了验证这一假设，首先，我们将使用原代人气道上皮细胞（hTEC），在IL-13存在和不存在的情况下，检查钙在自噬介导的基础和ATP激活的分泌性粘蛋白MUC 5AC和MUC 5 B分泌中的作用。第二，我们将确定是否自噬依赖性ROS调节MUC 5AC和/或MUC 5 B独立于粘蛋白生产使用hTEC。第三，我们将使用自噬缺陷小鼠Atg 16 l1 HM/HM在体内Th 2炎症模型中检查自噬、ROS和粘蛋白分泌之间的关系。这项研究应用将推进我们的知识气道生物学使用相关的人类疾病模型，以确定新的治疗策略的目标。这项工作将通过一个结构化的职业发展计划来推进，目标是成为一名独立的调查员。职业发展：作为一名医生-科学家，从经验丰富的研究人员那里获得强有力的指导至关重要。我的主要导师Joseph Sisson博士完全致力于支持我成为气道疾病独立研究者的职业目标。他提供了资源来建立我的实验室，保护我的研究时间与有限的临床职责，所以我已经开始在职业发展计划的背景下进行上述实验。该计划包括由经验丰富的调查人员对正式的导师委员会进行监督，以确保重要的职业里程碑得到满足，正式的课程工作，以及在内布拉斯加大学和世界各地开展新的合作。

 DESCRIPTION (provided by applicant): Significance of airway disease: Airway disease including asthma, chronic obstructive pulmonary disease, and cystic fibrosis are a significant cause of morbidity and mortality. A key feature of chronic inflammatory airway disease is hyper secretion of mucin proteins and airway obstruction by thick tenacious mucus. How the airway epithelial cell adapts to this stress of increased production and secretion of airway secretory mucins is not well understood. Autophagy as a novel pathway to regulate secretion: Autophagy, while highly conserved amongst eukaryotes, was previously thought to primarily regulate nutrient recycling in times of stress. It has now been established that autophagy is essential for multiple responses including protein secretion. Furthermore, autophagy appears to be particularly required during periods of high protein production and secretion during cellular stress. Our data just published in the Journal, Autophagy6, demonstrates that in the airway epithelia, autophagy is required for secretion of MUC5AC during Th2 inflammation. However, the mechanism of autophagy regulated mucin secretion in the airway is not known. Research Plan: In this application we present our preliminary data using in vitro human epithelial cells and in vivo mouse models of Th2 inflammation, demonstrating that autophagy is required for both MUC5Ac secretion and reactive oxygen species (ROS) generation. We hypothesize that these shared phenotypes are in fact connected and that autophagy-dependent ROS may be a novel mechanism that regulates secretion. To test this hypothesis, first, we will examine the role of calcium in autophagy-mediated basal and ATP-activated secretion of the secretory mucins, MUC5AC and MUC5B in the absence and presence of IL-13 using primary human airway epithelial cells (hTEC). Second, we will determine whether autophagy-dependent ROS regulates MUC5AC and or MUC5B independent of mucin production using hTEC. Third, we will examine the relationship among autophagy, ROS, and mucin secretion in an in vivo Th2 inflammation model using the autophagy deficient mice, Atg16l1HM/HM. This research application will advance our knowledge of airway biology using relevant models of human disease with the goal of identifying new therapeutic strategies. This work will advance through a structured program of career development with a goal of becoming an independent investigator. Career Development: As a physician-scientist, it is critically important to have strong mentorship from experienced investigators. My primary mentor, Dr. Joseph Sisson is fully committed to support my career goal of becoming an independent investigator in airway diseases. He has provided resources to establish my lab, protect my research time with limited clinical duties, so that I have begun work on experiments outlined above within the context of a career development plan. This plan includes oversight by experienced investigators on a formal mentorship committee to ensure that important career milestones are being met, formal course work, and development of new collaborations at the University of Nebraska and around the world.