Epithelial JNK-TGFb1 Signaling Axis in Airway Remodeling

描述（由申请人提供）：上皮下纤维化的发展代表了哮喘气道重塑的一个重要特征，上皮细胞在其中的关键作用正在显现。促纤维化细胞因子转化生长因子β-1（TGF-β 1）在纤维发生中起着重要作用。TGF-β 1信号传导的结果高度依赖于与其他信号传导途径的合作。我们最近发现c-Jun-N-terminal- kinase 1（JNK 1）在增强TGF-β 1的促纤维化作用中具有关键作用，与气道上皮细胞（EMT）的间充质转化有关。具体来说，我们已经证明：1）JNK主要在卵白蛋白致敏和激发小鼠的气道上皮中活化; 2）缺乏JNK 1（JNK 1-/-）的小鼠不能诱导间充质基因或对卵白蛋白，TGF-β 1或博来霉素产生纤维化反应; 3）TGF-β 1诱导的EMT需要JNK 1，通过连接结构域中SMAD 3的JNK 1依赖性磷酸化。这些数据表明，气道上皮来源的JNK 1依赖性信号在协调气道纤维化中发挥着关键作用。本文提出的假设是气道上皮中JNK 1的活化是通过促进上皮-间充质转化（EMT）而在屋尘螨诱导的气道疾病中发展上皮下纤维化所必需的。具体来说，我们假设JNK 1通过连接结构域中SMAD 3的磷酸化增强TGF-β 1信号传导，这增强了SMAD 3降低let-7 microRNA（miRNA）表达的能力。let-7 miRNA的减少反过来增强了高迁移率族A2（HMGA 2）基因的表达，HMGA 2基因是EMT的近端调节因子，导致上皮下胶原沉积。在具体目标1中，我们将确定连接结构域中SMAD 3的JNK 1依赖性磷酸化在抑制肺上皮细胞中let-7 microRNA（miRNA）以及随后增强高迁移率族A2（HMGA 2）基因中的重要性。我们还将分析let-7 miRNA和HMGA 2在哮喘患者支气管上皮细胞中的表达，并与对照组进行比较，确定哮喘和正常上皮细胞对TGF-β 1诱导的EMT的易感性，并阐明JNK和SMAD 3在其中的作用。在特定目标2中，我们将阐明气道上皮内JNK 1的激活是否在室内尘螨诱导的上皮向间充质转化、气道纤维化和体内呼吸力学改变的协调中至关重要，并利用在CRE重组酶介导的消融后全球缺乏JNK 1或特别是在细支气管上皮内的小鼠。在具体目标3中，我们将通过建立TetOP-FLAG-SMAD 3-EPSM小鼠和TetOP-FLAG-SMAD 3-野生型（WT）转基因小鼠（作为对照）来确定SMAD 3连接结构域磷酸化的重要性，并由此减少let-7 g miRNA在体内促进上皮向间充质转化、气道纤维化和呼吸力学改变。我们将评估let-7 g pre-miRNA的递送对HDM诱导的重塑的影响。

DESCRIPTION (provided by applicant): The development of subepithelial fibrosis represents an important feature of airway remodeling in asthma, and a critical role for epithelial cells herei is emerging. The pro-fibrotic cytokine, transforming growth factor beta-1 (TGF-¿1) plays a cardinal role in fibrogenesis. The outcome of TGF-¿1 signaling is highly dependent upon cooperation with other signaling pathways. We have recently identified a critical role for c-Jun-N-terminal- kinase 1 (JNK1) in augmenting the pro-fibrotic effects of TGF-¿1, in association with the causation of a mesenchymal transition of airway epithelial cells (EMT). Specifically, we have demonstrated that: 1) JNK is predominantly activated in airway epithelium from ovalbumin-sensitized and challenged mice; 2) mice lacking JNK1 (JNK1-/-) fail to induce mesenchymal genes or develop fibrosis in response to ovalbumin, TGF-¿1, or bleomycin; 3) TGF-¿1-induced EMT requires JNK1, through JNK1-dependent phosphorylation of SMAD3 in the linker domain. These data, suggest a critical role of airway epithelium-derived JNK1-dependent signals in orchestrating airways fibrosis. The hypothesis addressed herein is that activation of JNK1 in the airway epithelium is required for the development of subepithelial fibrosis in house dust mite- induced airways disease by promoting epithelial-mesenchymal transition (EMT). Specifically, we hypothesize that JNK1 enhances TGF-¿1 signaling via phosphorylation of SMAD3 in the linker domain, which enhances the competency of SMAD3 to decrease expression of let-7 microRNA (miRNA). Decreases in let-7 miRNA in turn enhances expression of the high mobility group A2 (HMGA2) gene, a proximal regulator of EMT, events that lead to subepithelial collagen deposition. In Specific Aim 1 we will determine the importance of JNK1-dependent phosphorylation of SMAD3 in the linker domain in repressing let-7 microRNA (miRNA) in lung epithelial cells, and subsequent enhancement of the high mobility group A2 (HMGA2) gene. We will also analyze expression of let-7 miRNA and HMGA2 in bronchial epithelial cells derived from asthmatics, compared to controls, and determine the susceptibility of asthmatic and normal epithelial cells to TGF-¿1-induced EMT, and elucidate the role of JNK and SMAD3 therein. In Specific Aim 2 we will elucidate whether activation of JNK1 within the airway epithelium is critical in the orchestration of house dust mite-induced epithelial to mesenchymal transition, airways fibrosis, and altered respiratory mechanics in vivo, and utilize mice that globally lack JNK1, or specifically within the bronchiolar epithelium, following and CRE-recombinase mediated ablation. In Specific Aim 3 we will determine the importance of SMAD3 linker domain phosphorylation and resultant decreases of let-7g miRNA in promoting epithelial to mesenchymal transition, airways fibrosis, and altered respiratory mechanics in vivo, via the creation of TetOP-FLAG-SMAD3-EPSM mice, which are refractory to phosphorylation by JNK in the linker domain, and TetOP-FLAG-SMAD3-wild type (WT) transgenic mice, as a control. We will assess the impact of delivery of let-7g pre-miRNA on HDM-induced remodeling.