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：
9765942
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/9765942
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 Wound Healing 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

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的分子机制在内皮可塑性和稳态上带有TGFβ信号传导的WNK1。失败的血管生成内皮特异性的WNK1无效小鼠导致胚胎死亡。我们发现两个过程对于由TGFβ和WNK1协调调节的内皮可塑性：细胞转化为迁移与完整的内皮间质转变发生的表型和紧密分解。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相互控制内皮细胞行为和特征。 WNK选择性激酶抑制剂，蛋白质耗竭，基因编辑，接近结扎，基因表达和其他生化手段将用于基于细胞和组织的测定中，以发现 WNK1介导内皮细胞结构的动态重组的机制与TGFβ相连的正常和病理生理血管生成。在第一个特定目标中，我们将确定调节调节表达的TGFβ和WNK1信号通路之间的相互作用间充质转录因子sl（SNAI2）和随后诱导内皮细胞运动。我们假设WNK1通过对TGFβ调节的作用激活SLUG表达和迁移 Smads。 SLUG促进了通过诱导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的间充质表型。在第二个特定目标中，我们将确定WNK1如何促进TGFβ诱导的紧密连接拆卸。为了响应TGFβ， WNK1底物激酶OSR1与其他信号蛋白和TGFβ结合了紧密的连接蛋白受体分解紧密的连接处。抑制WNK1激酶活性可防止共免疫沉淀 OSR1带有occludin并防止内皮细胞中TGFβ诱导的紧密连接拆卸。我们假设WNK1是TGFβ诱导的紧密连接拆卸所必需的底物激酶OSR1。我们将分析WNK1/OSR1如何参与TGFβ引起的紧密连接分解以发现需要合作的步骤。我们将在 OSR1-OCCLUDIN复合物以及OSR1功能对紧密连接分解的影响。将使用救援策略建立基本事件。我们的结果将定义 TGFβ和WNK1信号传导机制和揭示治疗机会之间的合作靶向疾病中的WNK1途径。我们的发现将导致对正常和病理血管生成。