Role of the ER stress transcription factor XBP1S in the development of idiopathic pulmonary fibrosis

特发性肺纤维化(IPF)是一种病因不明的进展期和终末期肺部疾病。 没有解药。基因变化很可能会增加一个人患IPF的风险，然后暴露在某些 环境因素和/或衰老是疾病发病的诱因。MUC5B启动子变异体rs35705950 ∼存在于50%的特发性肺纤维化患者中，被认为是最强的已知风险因素(遗传和 否则)用于IPF的发展。该突变导致MUC5B基因和蛋白的过度表达。 外周肺内可见远端呼吸道上皮细胞和蜂窝状囊肿。这些观察结果提出了一个问题 为什么远端呼吸道MUC5B过度表达与IPF相关？最近，我们实验室鉴定出 内质网应激转录因子XBP1s首次在衬里的上皮细胞中高表达 IPF肺远端呼吸道和蜂窝囊通过与其直接结合激活MUC5B基因表达 推动者。此外，XBP1s对MUC5B启动子变异体进行差异调控。这些数据连接激活 启动子变异依赖模型中内质网应激并过度表达MUC5B可能损害粘液纤毛 远端呼吸道干细胞的清除和功能增加了肺疾病的易感性 纤维化症。我们假设XBP1s的激活诱导了MUC5B在远端呼吸道的表达， 促进肺纤维化。为了验证这一中心假设，我们提出了以下目标：1)XBP1s- 远端呼吸道上皮细胞介导的MUC5B分泌增加了发生 活体肺纤维化。我们将通过暴露XBP1特定的呼吸道上皮来评估XBP1的作用 博莱霉素致小鼠呼吸道上皮损伤的过度表达或缺失2)XBP1s介导的MUC5B 分泌物损害远端呼吸道干细胞修复损伤后外周肺上皮的功能。我们会 利用小鼠呼吸道上皮细胞-谱系示踪系统和体外二维和三维分化实验评价 XBP1s和粘蛋白在损伤后维持呼吸道干细胞动态平衡中的作用。3) XBP1s的激活和MUC5B启动子变异的存在会导致粘液分泌异常， 粘液纤毛清除和肌成纤维细胞分化激活受损。我们将分析 DAE分泌粘液的生化、生物物理特性和电生理 MUC5B启动子突变rs35705950在内质网应激基础和激活后。我们还将测试 表达XBP1s的DAE携带MUC5B启动子变异和/或直接致上皮损伤 促进肌成纤维细胞分化。完成本申请中提出的目标将提供新的 内质网应激介导DAE过度分泌粘蛋白促进肺纤维化的机制。 这些机制研究将可能确定用于早期诊断和治疗靶点的新生物标志物。 (例如，抑制XBP1的激活是解决MUC5B高分泌的一种有前途的方法，即使对于 那些携带MUC5B启动子变体的人)在远端肺组织中发挥作用，以防止或逆转纤维化疾病的进展。

Idiopathic pulmonary fibrosis (IPF) is a progressive and end-stage lung disease of unknown etiology and no cure. It is likely that genetic changes increase a person's risk of developing IPF, and then exposure to certain environmental factors and/or aging trigger the onset of the disease. The MUC5B promoter variant rs35705950 is present in ∼50% of individuals with IPF and is recognized as the strongest known risk factor (genetic and otherwise) for the development of IPF. This variant leads to overexpression of MUC5B mRNA and protein in both distal airway epithelia and honeycomb cysts in the peripheral lung. These observations raise the question of why excessive MUC5B expression in distal airways is associated with IPF? Recently, our laboratory identified for the first time that the ER stress transcription factor XBP1S is highly expressed in the epithelium lining the distal airways and honeycomb cysts of IPF lung, activates MUC5B gene expression by direct binding to its promoter. Further, XBP1S differentially regulating the MUC5B promoter variant. These data connected activation of ER stress with excessive MUC5B expression in a promoter variant-dependent model likely impairs mucociliary clearance and function of distal airway stem cells that increase susceptibility of development of pulmonary fibrosis. We hypothesize that activation of XBP1S induces MUC5B expression in the distal airways that promotes pulmonary fibrosis. To test this central hypothesis, we propose the following aims: 1) XBP1S- mediated MUC5B secretion in distal airway epithelium enhances susceptibility to development of pulmonary fibrosis in vivo. We will assess the role of XBP1S by exposing Xbp1 airway epithelium-specific overexpression or deletion mice to bleomycin induced respiratory epithelial injury. 2) XBP1S-mediated MUC5B secretion impairs distal airway stem cell function to repair peripheral lung epithelia after injury. We will utilize mouse airway epithelium-lineage tracing system and in vitro 2-D and 3-D differentiation assays to evaluate the role of XBP1S and mucin in maintenance of the airway stem cell homeostasis in response to injury. 3) Activation of XBP1S and presence of MUC5B promoter variant cause abnormal mucus secretion, impaired mucociliary clearance and activation of myofibroblast differentiation. We will analyze the biochemical and biophysical properties of the secreted mucus and electrophysiology of the DAE carrying the MUC5B promoter variant rs35705950 at baseline and after activation of ER stress. We will also test whether XBP1S-expressing DAE carrying the MUC5B promoter variant and/or hypoxia-induced epithelial injury directly promotes myofibroblast differentiation. Completing the aims proposed in this application will provide novel mechanisms underlying ER stress-mediated excessive mucin secretion by DAE promotes pulmonary fibrosis. These mechanistic studies will likely identify novel biomarkers for early diagnosis as well as therapeutic targets (e.g., suppression of XBP1S activation as a promising approach to resolve MUC5B hypersecretion, even for those carrying the MUC5B promoter variant) in the distal lung to prevent or reverse fibrotic disease progression.