LMTK2 AND TGF BETA SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

摘要 囊性纤维化(CF)是高加索人中最常见的隐性疾病，由 CAMP激活的囊性纤维化跨膜电导调节因子(CFTR)编码基因 氯离子通道。90%的CF患者至少携带一份∆F508等位基因。最新研究 已经证明，两个小分子，折叠校正器VX-809和增强剂VX-770，作用于 结合并部分恢复人支气管上皮细胞的∆F508-cftr离子通道功能。 然而，这些化合物对∆F508突变的患者只有轻微的效果。一个关键限制 大多数CF患者产生高水平的转化生长因子-β-1。我们发表的工作 研究表明，临床相关水平的转化生长因子-β1抑制HBE细胞中∆F508-CFTR的转录，起作用 调制器上游，以阻止救援∆F508-cftr。高水平的转化生长因子-β1也为CF患者提供了 炎症、上皮间质转化(EMT)和纤维化。转化生长因子-β1启动信号转导 通过刺激固有活性的转化生长因子-β-1受体(T-βR)-II与T-βR-I相互作用并在 质膜。相比之下，蛋白磷酸酶1(PP1)通过以下途径保护TβR-I免受结构性激活 TβR-II在非刺激细胞中的表达。目前尚不清楚转化生长因子-β1是如何阻断Pp1与T-βR-I相互作用而激活的 发信号。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 (LMTK2)在基侧质膜通过失活转化生长因子-β1的催化亚单位来促进信号转导 Pp1(PP1C)，从而激活TβR-I和信号转导。此外，我们的数据表明， 激活PP1C可阻断HBE细胞中转化生长因子-β-1信号转导。我们的中心假设是转化生长因子-β1稳定了 LMTK2支架激活信号导致炎症、纤维化和∆F508转录抑制- HBE细胞中CFTR的表达。在这样做的过程中，LMTK2允许转化生长因子-β1通过小分子拮抗∆F508-cftr蛋白拯救 分子，并使结果更糟。因此，靶向LMTK2支架代表了一种新的治疗策略 益气活血方控制转化生长因子-β-1信号转导，减轻炎症和纤维化，促进∆F508-cftr的挽救。 HBe细胞。在目标1中，我们将研究转化生长因子-β1对T-βR-1、LMTK2、 和HBE细胞中的PP1C。在目标2中，我们将测试转化生长因子-β1是否在 HBE细胞的基侧质膜。在目标3中，我们将阐明转化生长因子-β1信号通路是否可以 通过阻断LMTK2失活HBE细胞中的PP1C而减弱。我们将使用最先进的研究工具。 由于转化生长因子-β1信号是细胞类型和细胞上下文相关的，我们将使用表达∆F508的HBE细胞- CFTR在体内表现出与CF呼吸道疾病相关的许多特征，是一种理想的 临床前实验的模型。我们预计，我们的研究将导致新的靶向治疗。 由高水平的转化生长因子-β1触发的过度转化生长因子-β1信号在大多数CF患者中存在，以保护气道 完整性，并允许小分子恢复∆F508-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.