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

描述（由申请人提供）：气道炎症性疾病，如哮喘、慢性阻塞性肺疾病（COPD）和囊性纤维化（CF），其特征是粘液细胞化生和粘液过量。本应用程序通过研究气道炎症期间粘液蛋白过量产生的新机制，解决了这些疾病发病机制中的一个核心问题。人支气管上皮炎症（HBE）可激活未折叠蛋白反应（UPR）。UPR激活与肌醇需要酶1 （IRE1）的刺激相结合，IRE1是一种内质网酶，可激活转录因子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IRE1依赖途径。尽管最近的研究表明，IRE1/XBP-1通路的激活在体外和体内与气道上皮炎症介导的细胞因子分泌功能相关，但尚未研究IRE1信号在气道炎症过程中粘蛋白产生中的作用。因为IRE1也可以激活JNK和p38 MAP激酶以及NF-？B（非规范IRE1途径），它们的激活可以诱导粘蛋白转录，IRE1也可能在功能上与粘蛋白的产生有关。IRE1有两种同工异构体，1和2。我们发现IRE1a是普遍表达的，但IRE1¿只存在于肠道和呼吸道上皮中。IRE1¿在HBE中的表达是IRE1a表达的30倍，而IRE1¿在人肺泡II型细胞中不存在。此外，在HBE分化过程中发现IRE1¿2与MUC5B表达有很强的相关性。在小鼠组织中，IRE1¿在肺实质中不存在，但其表达量是鼻咽部、气管和支气管中IRE1a表达量的6.5-8.5倍，而鼻咽部、气管和支气管的黏液细胞水平较高。在野生型（wt）小鼠中，IRE1¿在Clara细胞和卵清蛋白（OVA）诱导的黏液细胞中表达，但在纤毛细胞中不表达，这表明IRE1¿具有与黏液蛋白产生相关的特定功能。与wt相比，ova增加的IRE1¿-/-小鼠气道上皮Muc5b和Muc16的产生被减弱。IRE1¿与参与粘蛋白产生或糖基化的基因之间存在很强的相关性，进一步证明了IRE1¿在粘蛋白产生中的关键功能作用。我们的具体目标将验证主要假设，即IRE1¿是气道粘膜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，其作用不同于IRE1a的作用，敲除IRE1¿将减少与人类气道炎症疾病相关的模型中的粘蛋白过量产生。研究将利用以黏液细胞化生和黏液蛋白过量产生为特征的气道炎症的体外和体内模型。wt和IRE1¿-/-小鼠气道上皮原代培养，表达不同水平IRE1¿的HBE原代培养，以及在气道上皮中缺失IRE1¿的scn1b小鼠，将用于测试IRE1¿在粘蛋白产生中的作用。这些新研究揭示了IRE1¿作为哮喘、COPD和CF气道疾病的治疗靶点，可能具有很高的影响。

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.