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，并且上皮细胞的关键作用正在显现。促纤维化细胞因子转化生长因子β1在纤维化形成中起重要作用。转化生长因子-1信号转导的结果高度依赖于与其他信号通路的协同作用。我们最近发现c-jun-N-末端激酶1(JNK1)在增强转化生长因子-β1的促纤维化作用中起着关键作用，这与呼吸道上皮细胞(EMT)的间质转化有关。具体地说，我们已经证明：1)JNK主要在卵蛋白致敏和攻击的小鼠的呼吸道上皮细胞中激活；2)缺乏JNK1(JNK1-/-)的小鼠未能诱导间充质基因或对卵蛋白、转化生长因子β1或博莱霉素产生反应而形成纤维化；3)转化生长因子β1诱导的子宫内膜转化需要JNK1，通过JNK1依赖的连接域中SMAD3的磷酸化来实现。这些数据表明，呼吸道上皮细胞来源的JNK1依赖的信号在协调呼吸道纤维化中起着关键作用。本文提出的假设是，在尘螨引起的呼吸道疾病中，JNK1的激活是通过促进上皮-间充质转化(EMT)而导致的上皮下纤维化的发生所必需的。具体地说，我们假设JNK1通过在连接器域磷酸化Smad3来增强TGF-β1信号，从而增强Smad3降低let-7 microRNA(MiRNA)表达的能力。Let-7miRNA的减少反过来又增强了高迁移率组A2(HMGA2)基因的表达，HMGA2是EMT的近端调节因子，导致上皮下胶原沉积。在特定的目标1中，我们将确定JNK1依赖的连接器域Smad3的磷酸化在抑制肺上皮细胞let-7 microRNA(MiRNA)以及随后增强高迁移率组A2(HMGA2)基因方面的重要性。我们还将分析哮喘患者和对照组支气管上皮细胞中let-7miRNA和HMGA2的表达，并测定哮喘和正常支气管上皮细胞对转化生长因子β1诱导的EMT的敏感性，并阐明JNK和Smad3在其中的作用。在具体目标2中，我们将阐明JNK1在呼吸道上皮细胞内的激活是否在室内尘螨诱导的上皮细胞向间充质转化、呼吸道纤维化和体内呼吸力学改变的协调中起关键作用，并利用整体缺乏JNK1的小鼠，或在Cre-重组酶介导的消融后的细支气管上皮细胞内。在具体目标3中，我们将通过建立TetOP-FLAG-SMAD3-EPSM小鼠和TetOP-FLAG-SMAD3-EPSM转基因小鼠作为对照，确定Smad3连接器域的磷酸化和由此导致的let-7G miRNA的减少在促进上皮向间充质转化、呼吸道纤维化和体内呼吸力学改变方面的重要性。我们将评估let-7g Pre-miRNA的交付对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.