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

描述（由申请方提供）：气道炎性疾病，如哮喘、慢性阻塞性肺病（COPD）和囊性纤维化（CF）的特征在于粘液细胞化生和粘液过度产生。本申请通过研究气道炎症期间粘蛋白过度产生的新机制来解决这些疾病发病机制中的中心问题。人支气管上皮细胞（HBE）的炎症激活未折叠蛋白反应（UPR）。UPR激活与肌醇需要酶1（IRE 1）的刺激偶联，IRE 1是一种激活转录因子X-box结合蛋白1（XBP-1）的ER酶，XBP-1是许多细胞中蛋白质分泌所需的经典IRE 1依赖性途径。虽然最近的研究表明，IRE 1/XBP-1通路的激活是功能相关的气道上皮炎症介导的细胞因子分泌在体外和体内，IRE 1信号在气道炎症过程中的粘蛋白产生的作用还没有被调查。因为IRE 1也可以激活JNK和p38 MAP激酶以及NF-？B（非经典IRE 1途径），它们的激活可以诱导粘蛋白的转录，IRE 1也可能与粘蛋白的产生功能相关。IRE 1有两种亚型，1和2。我们发现IRE 1a是普遍表达的，但IRE 1 <$仅存在于肠道和呼吸道上皮细胞中。虽然在HBE中IRE 1 <$的表达比IRE 1a的表达高30倍以上，但IRE 1 <$在原代人肺泡II型细胞中不存在。此外，在HBE分化过程中发现IRE 1 <$2和MUC 5 B表达之间存在强相关性。在小鼠组织中，IRE 1？在肺实质中不存在，但其表达比在鼻咽、气管和支气管中的IRE 1a表达高6.5-8.5倍，鼻咽、气管和支气管组织表现出更高水平的粘液细胞。在野生型（wt）小鼠中，IRE 1？在Clara细胞和卵清蛋白（OVA）诱导的粘液细胞中表达，但在纤毛细胞中不表达，表明IRE 1？的特异性功能与粘蛋白产生有关。IRE 1-/-小鼠与野生型小鼠相比，OVA增加的气道上皮Muc 5 b和Muc 16的产生减弱。IRE 1 <$和参与粘蛋白产生或糖基化的基因之间的强相关性提供了IRE 1 <$在粘蛋白产生中的关键功能作用的进一步证据。我们的特定目标将检验以下主要假设：IRE 1是气道粘液细胞产生粘蛋白所必需的，它刺激粘蛋白转录和/或调节参与粘蛋白产生或糖基化的基因，其作用与IRE 1a作用不同，敲除IRE 1将减少与人类气道炎症性疾病相关的模型中的粘蛋白过度产生。研究将利用以粘液细胞化生和粘蛋白过度产生为特征的气道炎症的体外和体内模型。野生型和IRE 1-/-鼠气道上皮的原代培养物、表达不同水平IRE 1的HBE的原代培养物以及在气道上皮中表现出IRE 1缺失的Scnn 1b小鼠将用于测试IRE 1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，揭示了IRE 1作为哮喘，COPD和CF气道疾病的治疗靶点。 公共卫生相关性：粘蛋白过度产生是许多气道炎性疾病的特征。我们的研究将测试IRE 1的功能作用，IRE 1是一种参与内质网应激反应的蛋白质，在与气道炎症相关的粘蛋白产生中，并可能导致哮喘，COPD和CF患者的新疗法。

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.