IRE1beta: A Novel Pathway for Regulation of Airway Epithelial Mucin Production

描述(申请人提供)：呼吸道炎症性疾病，如哮喘、慢性阻塞性肺疾病(COPD)和囊性纤维化(CF)，以粘液细胞化生和粘液过度生产为特征。这项应用通过研究一种导致呼吸道炎症期间粘蛋白过度产生的新机制，解决了这些疾病发病机制中的一个中心问题。人支气管上皮(HBE)炎症可激活未折叠蛋白反应(UPR)。UPR的激活与肌醇需要酶1(IRE1)的刺激相耦合，IRE1是一种ER酶，激活转录因子X-box结合蛋白1(XBP-1)，这是许多细胞中蛋白质分泌所需的典型的依赖IRE1的途径。尽管最近的研究表明，IRE1/XBP-1通路的激活在体外和体内都与呼吸道上皮炎症介导的细胞因子的分泌有关，但IRE1信号在呼吸道炎症过程中粘蛋白产生中的作用尚未被研究。由于IRE1还可以激活JNK和p38 MAP激酶和非典型性IRE1通路，并且它们的激活可以诱导粘蛋白转录，因此IRE1可能也与粘蛋白的产生有关。IRE1以1和2两种亚型存在。我们发现，IRE1a广泛表达，但只在肠道和呼吸道上皮细胞中存在。在HBE中，IRE1？的表达是IRE1a的30倍，而在原代培养的人肺泡II型细胞中不表达IRE1？此外，在HBE分化过程中，IRE1？2和MUC5B的表达有很强的相关性。在小鼠组织中，IRE1a在肺实质中不存在，但其表达水平是在鼻咽、气管和支气管组织中的6.5-8.5倍，这些组织表现出较高水平的粘液细胞。在野生型(Wt)小鼠中，IRE1在Clara细胞和卵清蛋白(OVA)诱导的粘液细胞中表达，但在纤毛细胞中不表达，提示IRE1？与粘蛋白的产生有关。卵子增加的呼吸道上皮细胞MUC5B和Muc16的产生在IRE1？-/-vs.wt小鼠中被钝化。IRE1与参与粘蛋白产生或糖基化的基因之间的强烈相关性进一步证明了IRE1在粘蛋白产生中的关键功能作用。我们的特定目标将检验最初的假设，即IRE1是呼吸道粘液细胞产生粘蛋白所必需的，它刺激粘蛋白转录和/或调节与粘蛋白产生或糖基化相关的基因，其作用与IRE1a作用不同，在与人类呼吸道炎症性疾病相关的模型中，敲除IRE1将减少粘蛋白的过度产生。研究将利用以粘液细胞化生和粘蛋白过度生产为特征的体外和体内呼吸道炎症模型。WT和IRE1-/-小鼠的原代培养物，表达不同水平IRE1的HBE原代培养物，以及在呼吸道上皮细胞中表现出IRE1的缺失的Scnn1b小鼠将被用来测试IRE1在粘蛋白产生中的作用。这些新的研究可能会产生很大的影响，揭示IRE1？作为哮喘、COPD和CF呼吸道疾病的治疗靶点。 公共卫生相关性：粘蛋白过度生产是许多呼吸道炎症性疾病的特征。我们的研究将测试参与内质网应激反应的IRE1？在与呼吸道炎症相关的粘蛋白产生中的功能作用，并可能为哮喘、COPD和CF患者带来新的治疗方法。

DESCRIPTION (provided by applicant): Airway inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are characterized by mucous cell metaplasia and mucus overproduction. This application addresses a central issue in the pathogenesis of these diseases by investigating a novel mechanism responsible for the overproduction of mucins during airway inflammation. Inflammation of human bronchial epithelia (HBE) activates the unfolded protein response (UPR). The UPR activation couples to stimulation of the inositol requiring enzyme 1 (IRE1), an ER enzyme that activates the transcription factor X-box binding protein 1 (XBP-1), the canonical IRE1-dependent pathway required for protein secretion in many cells. Although recent studies have shown that activation of the IRE1/XBP-1 pathway is functionally relevant for airway epithelial inflammation-mediated cytokine secretion in vitro and in vivo, the role of IRE1 signaling in mucin production during airway inflammation has not been investigated. Because IRE1 can also activate JNK and p38 MAP kinases and NF-?B (non-canonical IRE1 pathways), and their activation can induce mucin transcription, IRE1 might also be functionally relevant for mucin production. IRE1 exists in two isoforms, 1 and 2. We found that IRE1a was ubiquitously expressed, but IRE1¿ was present only in gut and respiratory epithelia. While IRE1¿ expression was > 30 fold higher than IRE1a expression in HBE, IRE1¿ was absent in primary human alveolar type II cells. Moreover, a strong correlation between IRE1¿2 and MUC5B expression was found during HBE differentiation. In mouse tissues, IRE1¿ was absent in lung parenchyma but its expression was 6.5-8.5 fold higher than IRE1a expression in nasopharynx, trachea, and bronchus, tissues that exhibit higher levels of mucous cells. In wild-type (wt) mice, IRE1¿ was expressed in Clara cells and ovalbumin (OVA)-induced mucous cells, but was absent in ciliated cells, suggesting a specific function for IRE1¿ related to mucin production. OVA-increased airway epithelial Muc5b and Muc16 production was blunted in IRE1¿-/- vs. wt mice. Further evidence for a key functional role of IRE1¿ in mucin production was given by a strong correlation between IRE1¿ and genes involved in mucin production or glycosylation. Our Specific Aims will test the primary hypothesis that IRE1¿ is required for mucin production by airway mucous cells, it stimulates mucin transcription and/or regulates genes involved in mucin production or glycosylation, its action is distinct from IRE1a action, and knocking out IRE1¿ will reduce mucin overproduction in models relevant for human airways inflammatory disease. Studies will utilize in vitro and in vivo models of airway inflammation characterized by mucous cell metaplasia and mucin overproduction. Primary cultures of wt and IRE1¿-/- murine airway epithelia, primary cultures of HBE expressing different levels of IRE1¿, and the Scnn1b mouse exhibiting deletion of IRE1¿ in airway epithelia will be used to test the role of IRE1¿ in mucin production. These novel studies may have a high impact by revealing IRE1¿ as a therapeutic target for asthma, COPD, and CF airways disease. PUBLIC HEALTH RELEVANCE: Mucin overproduction characterizes many airway inflammatory diseases. Our studies will test the functional role of IRE1¿, a protein involved in endoplasmic reticulum stress responses, in mucin production associated with airway inflammation and may lead to new therapies for asthma, COPD and CF patients.