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WNK and TGF-beta in Endothelial Migration

WNK 和 TGF-β 在内皮迁移中的作用

基本信息

批准号：
9918969
负责人：
MELANIE H. COBB
金额：
$ 40.5万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918969
关键词：
Atherosclerosis Binding Biochemical Biological Assay Blood Vessels Cardiovascular system Cell physiology Cell-Cell Adhesion Cells Cellular Structures Cessation of life Characteristics Co-Immunoprecipitations Complex Development Disease Embryo Endothelial Cells Endothelium Epithelial Cells Event Extravasation Fibrosis Functional disorder Future Gene Expression Genes Genetic Transcription Goals Homeostasis Impairment Inflammation Knock-out Knockout Mice Ligation Malignant Neoplasms Mediating Mesenchymal Molecular Pathologic Pathologic Neovascularization Pathway interactions Phenocopy Phenotype Phosphotransferases Physiology Pregnancy Process Protein Kinase Proteins Reporter Research Signal Pathway Signal Transduction Signaling Protein Testing Tight Junctions Tissues Transforming Growth Factor beta Ubiquitination angiogenesis base cell behavior cell motility experience experimental study kinase inhibitor migration occludin prevent protein function receptor response slug therapeutic target transcription factor vascular bed wound healing

项目摘要

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.

抽象的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 TGFβ 信号传导对内皮可塑性和稳态的影响。血管生成失败内皮特异性 WNK1 缺失小鼠导致胚胎死亡。我们发现两个重要的过程 TGFβ 和 WNK1 协调调节的内皮可塑性：细胞向迁移细胞的转化内皮-间质转化完全时发生的表型和紧密连接破坏。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 WNK1 调节的信号通路如何交叉控制内皮细胞行为和特征。 WNK 选择性激酶抑制剂、蛋白质消耗、基因编辑、邻近连接、基因表达和其他生化手段将用于基于细胞和组织的测定，以发现 WNK1 介导内皮细胞结构动态重组的机制与 TGFβ 协同作用的正常和病理生理性血管生成。在第一个具体目标中，我们将确定调节 TGFβ 和 WNK1 表达的信号通路之间的相互作用间充质转录因子 Slug (Snai2) 和随后诱导内皮细胞运动。我们假设 WNK1 通过作用于 TGFβ 调节来激活 Slug 表达和迁移 SMAD。 Slug 通过诱导抑制蛋白质来促进内皮细胞迁移和重塑细胞间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 WNK1 如何促进 TGFβ 诱导的紧密连接分解。响应 TGFβ， WNK1 底物激酶 OSR1 与紧密连接蛋白以及其他信号蛋白和 TGFβ 结合受体破坏紧密连接。抑制 WNK1 激酶活性可防止免疫共沉淀 OSR1 与 occludin 结合并防止内皮细胞中 TGFβ 诱导的紧密连接分解。我们假设 WNK1 是 TGFβ 诱导的紧密连接分解所必需的，通过其作用底物激酶 OSR1。我们将分析 WNK1/OSR1 如何参与 TGFβ 启动的紧密连接分解以发现需要他们合作的步骤。我们将识别组件 OSR1-occludin 复合物以及干扰 OSR1 功能对紧密连接破坏的影响。将使用救援策略来确定重要事件。我们的结果将定义范围 TGFβ 和 WNK1 信号传导机制之间的合作并揭示治疗机会疾病中 WNK1 通路的靶向。我们的研究结果将有助于更好地理解正常和病理性血管生成。