LMTK2 AND TGF BETA SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

摘要 囊性纤维化（CF）是白种人中最常见的隐性疾病，是由 编码囊性纤维化跨膜传导调节因子（CFTR）的基因， 氯离子通道90%的CF患者携带至少一个拷贝的CF 508等位基因。最近的研究 已经表明，两个小分子，折叠校正剂VX-809和增效剂VX-770， 组合并部分恢复人支气管上皮（HBE）细胞中的CFNF 508-CFTR离子通道功能。 然而，这些化合物对于具有IF 508突变的患者仅略微有效。一个关键限制 大多数CF患者产生高水平的转化生长因子（TGF）-β1。我们已发表的作品 结果显示，临床相关水平的TGF-β1抑制HBE细胞中TGF-508-CFTR的转录， 调节剂的上游，以阻断BNF 508-CFTR的拯救。高TGF-β1水平也是CF患者的主要因素， 炎症、上皮-间充质转化（EMT）和纤维化。TGF-β1启动信号转导 通过刺激组成型活性TGF-β1受体（TβR）-II与TβR-I相互作用并使其磷酸化， 质膜相比之下，蛋白磷酸酶1（PP 1）通过以下方式保护TβR-I免受组成性激活 非刺激细胞中的TβR-II。目前尚不清楚TGF-β1如何阻断PP 1与TβR-I相互作用， 信号我们在HBE细胞中的初步工作表明，由狐猴酪氨酸激酶组织的支架 在基底外侧质膜上的LMTK 2通过使TGF-β1的催化亚基失活而有利于TGF-β1信号传导。 PP 1（PP 1C），从而允许TβR-I的激活和信号转导。此外，我们的数据表明， 激活PP 1C阻断HBE细胞中的TGF-β1信号传导。我们的中心假设是TGF-β1稳定了 LMTK 2支架激活导致炎症、纤维化和TNF 508转录抑制的信号传导。 HBE细胞中的CFTR。在这样做时，LMTK 2允许TGF-β1拮抗TGF-β 508-CFTR蛋白的小分子拯救。 分子，和生物学的结果。因此，靶向LMTK 2支架代表了一种新的治疗策略 对于CF控制TGF-β1信号传导，减轻炎症和纤维化，并促进在 HBE细胞。在目的1中，我们将研究TGF-β1对TβR-I，LMTK 2， 和PP 1C。在目标2中，我们将测试TGF-β1是否在LMTK 2的表达水平募集和/或激活LMTK 2。 HBE细胞的基底侧质膜。在目标3中，我们将阐明TGF-β1信号转导是否可以被 通过阻断HBE细胞中PP 1C的LMTK 2失活而减弱。我们将使用最先进的研究工具。 由于TGF-β1信号传导是细胞类型和细胞环境依赖性的，我们将使用表达TGF-β 508的HBE细胞。 CFTR表现出许多与体内CF气道疾病相关的特征，是理想的 用于临床前实验的模型。我们预计，我们的研究将导致新的治疗靶向 在大多数CF患者中存在由高TGF-β1水平触发的过度TGF-β1信号传导，以保护气道 因此，它可以使小分子保持完整性，并允许小分子恢复CF 3F 508-CFTR功能。

ABSTRACT Cystic Fibrosis (CF), the most common recessive disease among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-activated chloride ion channel. Ninety percent of CF patients carry at least one copy of the ∆F508 allele. Recent studies have shown that two small molecules, the folding corrector VX-809 and the potentiator VX-770, act in combination and partially restore ∆F508-CFTR ion channel function in human bronchial epithelial (HBE) cells. However, these compounds are only marginally effective for patients with the ∆F508 mutation. A key limitation is that most CF patients produce high levels of Transforming Growth Factor (TGF)-β1. Our published work showed that clinically relevant levels of TGF-β1 repress ∆F508-CFTR transcription in HBE cells, acting upstream of modulators to block rescue of ∆F508-CFTR. High TGF-β1 levels also prime CF patients for inflammation, epithelial-mesenchymal transformation (EMT), and fibrosis. TGF-β1 initiates signal transduction by stimulating the constitutively active TGF-β1 receptor (TβR)-II to interact with and phosphorylate TβR-I at the plasma membrane. By contrast, Protein Phosphatase 1 (PP1) protects TβR-I from constitutive activation by TβR-II in non-stimulated cells. It is unknown how TGF-β1 blocks PP1 interaction with TβR-I to activate signaling. Our preliminary work in HBE cells indicates that the scaffold organized by Lemur Tyrosine Kinase (LMTK2) at the basolateral plasma membrane favors TGF-β1 signaling by inactivating the catalytic subunit of PP1 (PP1C), thus allowing activation of TβR-I and signal transduction. Moreover, our data indicate that activating PP1C blocks TGF-β1 signaling in HBE cells. Our central hypothesis is that TGF-β1 stabilizes the LMTK2 scaffold to activate signaling leading to inflammation, fibrosis, and transcriptional repression of ∆F508- CFTR in HBE cells. In so doing, LMTK2 allows TGF-β1 to antagonize ∆F508-CFTR protein rescue by small molecules, and worsens outcomes. Targeting the LMTK2 scaffold thus represents a novel therapeutic strategy for CF to control TGF-β1 signaling, attenuate inflammation and fibrosis, and facilitate rescue of ∆F508-CFTR in HBE cells. In Aim 1 we will examine TGF-β1 effects on the protein-protein interactions between TβR-I, LMTK2, and PP1C in HBE cells. In Aim 2, we will test whether TGF-β1 recruits and/or activates LMTK2 at the basolateral plasma membrane in HBE cells. In Aim 3, we will elucidate whether TGF-β1 signaling can be attenuated by blocking LMTK2 inactivation of PP1C in HBE cells. We will use state-of-the-art research tools. Because TGF-β1 signaling is cell-type and cell-context dependent we will use HBE cells expressing ∆F508- CFTR, which exhibit many of the characteristics associated with CF airway disease in vivo and are an ideal model for pre-clinical experimentation. We anticipate that our studies will lead to novel therapy targeting excessive TGF-β1 signaling triggered by high TGF-β1 levels present in most CF patients, to preserve airway integrity, and to allow small molecules to restore the ∆F508-CFTR function.