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背后的分子机制。 WNK1与转化生长因子β信号转导对内皮细胞可塑性和动态平衡的影响血管生成失败 内皮特异性WNK1基因缺失的小鼠导致胚胎死亡。我们发现有两个过程对 转化生长因子β和WNK1协同调节的内皮可塑性：细胞向迁移性细胞的转化 随着内皮-间充质的完全转变而发生的表型和紧密连接破裂。 这些过程有助于正常的血管生理学和病理生理学。我们会调查的 转化生长因子β和WNK1调控的信号通路在控制内皮细胞行为和 特点。WNK选择性激酶抑制剂，蛋白耗尽，基因编辑，邻近连接，基因 表达和其他生化手段将被用于基于细胞和组织的分析，以发现 WNK1介导内皮细胞结构动态重组的机制 潜在的正常和病理生理血管生成与转化生长因子β同步。在第一个具体目标中，我们 将确定转化生长因子β和WNK1信号通路之间的相互作用，以调节 间充质转录因子slug(SNAI2)和随后对内皮细胞运动的诱导。我们 WNK1通过作用于β调节的转化生长因子激活Slug表达和迁移的假设 斯马德。鼻涕虫通过诱导抑制内皮细胞迁移和重塑的蛋白质来促进内皮细胞的迁移和重塑 细胞-细胞黏附和增强间充质细胞的迁移表型。在第二个具体目标中，我们将 确定WNK1如何促进转化生长因子β诱导的紧密连接解离。为回应转化生长因子β， WNK1底物激酶OSR1与其他信号蛋白和转化生长因子β结合于紧密连接蛋白 受体可以分解紧密的连接。抑制WNK1激酶活性可防止免疫共沉淀 用阻断素阻断OSR1，并阻止转化生长因子β诱导的内皮细胞紧密连接解离。我们 假设WNK1是转化生长因子β通过其作用诱导的紧密连接解离所必需的 底物激酶OSR1。我们将分析WNK1/OSR1如何参与转化生长因子β启动的紧密连接 分解以发现需要他们合作的步骤。我们将在中确定组件 OSR1-occludin复合体以及干扰OSR1功能对紧密连接击穿的影响。 关键事件将通过救援战略确定。我们的结果将定义 转化生长因子β和WNK1信号机制的合作及发现治疗机会 WNK1通路在疾病中的靶向性。我们的发现将使我们更好地理解正常和 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.