Epithelial JNK-TGFb1 Signaling Axis in Airway Remodeling

描述(由申请人提供)：纤维化的发展代表了气道重塑的一个关键参数，可能是在不同细胞类型中操作的许多信号级联反应的结果。促纤维化细胞因子转化生长因子-1在纤维化的发生发展中起着重要作用，而转化生长因子-1诱导的信号转导结果高度依赖于细胞环境以及与其他信号通路的协同作用。来自我们实验室的激动人心的新的初步研究发现，c-jun-N末端激酶(JNK)在增强转化生长因子-1的促纤维化作用中发挥了关键作用，这与呼吸道上皮细胞(EMT)的间质转化有关。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-/-)的小鼠不能诱导间充质基因表达，也不能对卵蛋白攻击、转化生长因子-1或博来霉素治疗产生纤维化反应；3)从JNK1-/-小鼠分化的原代气管上皮细胞对转化生长因子-1诱导的间质基因和蛋白的表达不敏感；以及4)转化生长因子-1诱导的上皮向间质的转变需要JNK1。我们的数据表明，呼吸道上皮细胞来源的JNK依赖的信号在协调呼吸道纤维化中起着关键作用。这一设想的假设是，在小鼠气道重塑模型中，JNK1的激活是通过促进上皮细胞向间充质转化(EMT)而发生上皮下纤维化所必需的。我们推测JNK1通过受体Smads的协同连接蛋白磷酸化和c-jun的磷酸化增强了转化生长因子-1信号，导致间充质基因表达增强，这对转化生长因子-1诱导的纤维化至关重要。在特定的目标1中，我们将确定在转化生长因子-1诱导的原代培养的气管上皮细胞向间充质转化过程中对JNK1的需求。在特定的目标#2中，我们将确定小鼠呼吸道上皮细胞间充质基因表达的增加和上皮下纤维化的发展是否需要JNK1-转化生长因子信号轴。特定目的#3建议阐明JNK1依赖的c-jun或rSMADs连接区的磷酸化在增强转化生长因子-1诱导的信号转导中的相对重要性，而特定目的#4将揭示JNK1激活、c-jun和Smad3连接物磷酸化以及随后的c-jun和Smad4依赖的转录反应在OVA或TGF-1诱导的呼吸道纤维化中是否起关键作用。我们的研究结果基于细胞培养和转基因小鼠模型，将提供更好的理解导致呼吸道胶原沉积(疤痕)的分子信号，以及上皮细胞在这一过程中的相关性。项目简介：现在众所周知，排列在呼吸道(呼吸管)内的上皮细胞在防御吸入物质方面发挥着关键作用，并且对维持正常的肺功能非常重要。然而，我们的实验室已经发现，上皮细胞在促进肺组织僵硬方面发挥了负面作用，从而降低了肺功能。我们已经确定了一些关键的生化信号，它们促进了这一可能的负面事件。项目简介：这项拨款提案有四个具体目标，旨在测试这些生化信号在肺僵硬中的重要性，并将使用来自小鼠呼吸道的原代细胞培养，人类上皮细胞系，以及构建转基因小鼠。该项目的完成将为肺僵硬的过程提供新的见解，并可能导致限制肺僵硬的新疗法的开发，并有可能逆转这一过程。

DESCRIPTION (provided by applicant): The development of fibrosis represents a critical parameter of airway remodeling, and likely is the outcome of numerous signaling cascades operative in different cell types. The pro-fibrotic cytokine, transforming growth factor beta-1 (TGF-1) plays a cardinal role in the development of fibrosis, and the outcome of TGF-1 induced signaling is highly dependent upon the cellular environment, and the cooperation with other signaling pathways. Exciting new preliminary studies from our laboratory have identified a critical role for c-Jun-N- terminal-kinase (JNK) in augmenting the pro-fibrotic effects of TGF-1, in association with the causation of a mesenchymal transition of airway epithelial cells (EMT). 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 challenge or TGF-1, or bleomycin administration; 3) Primary tracheal epithelial cells derived from JNK1-/- mice are refractory to TGF-1-induced expression of mesenchymal genes and proteins; and 4) TGF-1 induced epithelial-to mesenchymal transition requires JNK1. Our data, suggest a critical role of airway epithelium-derived JNK- dependent signals in orchestrating airways fibrosis. The hypothesis of this proposal is that activation of JNK1 in the airway epithelium is required for the development of subepithelial fibrosis in murine models of airway remodeling by promoting epithelial to mesenchymal transition (EMT). We hypothesize that JNK1 enhances TGF-1 signaling, via coordinated linker phosphorylation of receptor SMADs, and phosphorylation of c-Jun, resulting in enhanced expression of mesenchymal genes, critical to TGF-1-induced fibrosis. In Specific Aim #1, we will determine the requirement for JNK1 in TGF-1-induced epithelial to mesenchymal transition in primary cultures of tracheal epithelial cells. In Specific Aim #2, we will determine whether increases in mesenchymal gene expression in airway epithelium, and development of subepithelial fibrosis in mice require a JNK1-TGF- signaling axis. Specific Aim #3 proposes to elucidate the relative importance of JNK1- dependent phosphorylation of c-Jun or rSMADs linker regions in augmenting TGF-1 induced signaling, whereas Specific Aim #4 will unravel whether JNK1 activation, c-Jun and SMAD3 linker phosphorylation, and subsequent c-Jun and SMAD4 dependent transcriptional responses within the airway epithelium are critical in the orchestration of OVA or TGF-1-induced airways fibrosis. The outcome of our study, which is based on cell culture and genetically altered mouse models, will provide a better understanding of the molecular signals that lead to collagen deposition (scarring) of airways, and the relevance of epithelial cells in this process. Project Narrative: Epithelial cells that line the airways (breathing tubes) are now well known to play a critical role in the defense against inhaled materials, and are important in maintaining normal lung function. However, our laboratories have identified that epithelial cells play a negative role in promoting stiffening of the lung tissue, thereby decreasing lung function. We have identified some critical biochemical signals that promote this possibly negative event. Project Narrative: This grant proposal has four specific aims to test the importance of these biochemical signals in lung stiffening, and will use primary cell cultures from mouse airways, a human epithelial cell line, and the construction of genetically altered mice. Completion of this project will provide new insights into the process of lung stiffening, and may lead to the development of new therapeutics to limit lung stiffening, and also potentially reverse this process.