WNK and TGF-beta in Endothelial Migration

摘要 这项研究的长期目标是确定相互作用的分子机制 WNK 1与TGFβ信号转导对内皮可塑性和稳态的影响。血管生成失败， 内皮特异性WNK 1缺失小鼠导致胚胎死亡。我们发现两个过程对于 TGFβ和WNK 1协同调节的内皮可塑性：细胞向迁移性细胞的转化 表型和紧密连接破坏，发生完全内皮-间充质转化。 这些过程有助于正常的血管生理学和病理生理学。我们将调查 TGFβ和WNK 1调节的信号通路如何在控制内皮细胞行为中交叉， 特色WNK选择性激酶抑制剂，蛋白质耗竭，基因编辑，邻近连接，基因 表达和其他生物化学手段将用于基于细胞和组织的测定，以发现 WNK 1介导内皮细胞结构动态重组的机制 与TGFβ串联的潜在正常和病理生理血管生成。在第一个具体目标中，我们 将决定TGFβ和WNK 1信号通路之间的相互作用，这些信号通路调节 间充质转录因子Slug（Snai 2）和随后的内皮细胞运动诱导。我们 假设WNK 1通过作用于TGFβ调节的细胞因子来激活Slug表达和迁移， SMAD。Slug通过诱导抑制内皮细胞增殖的蛋白质促进内皮细胞迁移和重塑， 细胞-细胞粘附并增强迁移间充质表型。在第二个具体目标中，我们将 确定WNK 1如何促进TGFβ诱导的紧密连接解体。在对TGFβ的反应中， WNK 1底物激酶OSR 1与紧密连接蛋白沿着与其他信号蛋白和TGFβ结合 受体来破坏紧密连接。抑制WNK 1激酶活性可防止免疫共沉淀 OSR 1与occludin的结合，并阻止TGFβ诱导的内皮细胞紧密连接解体。我们 假设WNK 1是TGFβ诱导的紧密连接解体所必需的， 底物激酶OSR 1。我们将分析WNK 1/OSR 1如何参与TGFβ启动的紧密连接 他们会发现需要他们合作的步骤。我们将确定组件， OSR 1-occludin复合物和干扰OSR 1功能对紧密连接破坏的影响。 将使用救援策略确定重要事件。我们的研究结果将确定 TGFβ和WNK 1信号传导机制之间的合作，并发现治疗的机会 在疾病中靶向WNK 1通路。我们的发现将有助于更好地了解正常和 病理性血管生成

Abstract The long term goal of this research is to determine the molecular mechanisms underlying the interactions of WNK1 with TGFβ signaling on endothelial plasticity and homeostasis. Failed angiogenesis in endothelial-specific WNK1 null mice led to embryonic death. We have found two processes important for endothelial plasticity that are coordinately regulated by TGFβ and WNK1: conversion of cells to a migratory phenotype and tight junction breakdown that occurs with complete endothelial-mesenchymal transition. These processes contribute to normal vascular physiology and to pathophysiology. We will investigate how signaling pathways regulated by TGFβ and WNK1 intersect in controlling endothelial cell behavior and characteristics. WNK-selective kinase inhibitors, protein depletion, gene editing, proximity ligation, gene expression and other biochemical means will be used in cell- and tissue-based assays to discover the mechanisms through which WNK1 mediates dynamic reorganization of endothelial cell structures underlying normal and pathophysiological angiogenesis in tandem with TGFβ. In the first specific aim, we will determine interactions between TGFβ and WNK1 signaling pathways that regulate expression of the mesenchymal transcription factor Slug (Snai2) and subsequent induction of endothelial cell motility. We hypothesize that WNK1 activates Slug expression and migration through actions on TGFβ-regulated SMADs. Slug promotes endothelial cell migration and remodeling by inducing proteins that repress cell-cell adhesion and enhance a migratory mesenchymal phenotype. In the second specific aim, we will determine how WNK1 promotes TGFβ-induced tight junction disassembly. In response to TGFβ, the WNK1 substrate kinase OSR1 binds to tight junction proteins along with other signaling proteins and TGFβ receptors to break down tight junctions. Inhibiting WNK1 kinase activity prevents co-immunoprecipitation of OSR1 with occludin and prevents TGFβ-induced tight junction disassembly in endothelial cells. We hypothesize that WNK1 is required for TGFβ-induced tight junction disassembly through actions of its substrate kinase OSR1. We will analyze how WNK1/OSR1 participate in TGFβ-initiated tight junction break down to discover the steps requiring their cooperation. We will identify components in OSR1-occludin complexes and the effects of interfering with OSR1 function on tight junction breakdown. Essential events will be established using rescue strategies. Our results will define the extent of cooperation between TGFβ and WNK1 signaling mechanisms and uncover opportunities for therapeutic targeting of the WNK1 pathway in disease. Our findings will lead to a better understanding of normal and pathological angiogenesis.