Novel E3 Ubiquitin Ligase CHFR Regulates Endothelial Barrier Integrity and Innate Immune Function

新型 E3 泛素连接酶 CHFR 调节内皮屏障完整性和先天免疫功能

基本信息

批准号：
10297258
负责人：
CHINNASWAMY TIRUPPATHI
金额：
$ 38.38万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10297258
关键词：
AKT1 gene Acute Address Adherens Junction Adult Respiratory Distress Syndrome Angiopoietin-2 Biochemical Biological Assay Blood Vessels Cell surface Cells Coupled Data Down-Regulation Edema Endothelial Cells Endothelium Event Extravasation FOXO1A gene Feasibility Studies Gatekeeping Generations Genetic Genetic Transcription Head Host Defense Host Defense Mechanism Human Inflammatory Injury Innate Immune Response Knock-out Knockout Mice Life Link Lung Mediating Mediator of activation protein Molecular Mus Nodal Nuclear Translocation P-Cadherin Pathogenicity Pathway interactions Permeability Phagocytes Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Plasma Polyubiquitin Process Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa pneumonia Pulmonary Edema Ring Finger Domain Role Signal Transduction TIE-2 Receptor TLR4 gene Testing Therapeutic Time Tissues Ubiquitin Ubiquitination Up-Regulation Vascular Endothelial Cell bactericide base cadherin 5 defense response in vivo innate immune function intravital imaging lung injury macromolecule migration mouse model neutrophil new therapeutic target novel novel therapeutic intervention pathogen prevent protein degradation pulmonary function respiratory response tissue injury transcription factor ubiquitin-protein ligase vascular inflammation vascular injury

项目摘要

This revised and responsive application will investigate mechanistically how newly discovered endothelial cell expressed ubiquitin E3 ligase CHFR (checkpoint with fork-head and ring finger domain) regulates the barrier integrity and the innate immune function of vascular endothelial cells. VE-cadherin expressed at endothelial adherens junctions (AJs) functions as a “gatekeeper” to restrict extravasation of plasma macromolecules and influx of phagocytic neutrophils (PMNs) into tissue. However, the key biochemical mechanisms triggering the loss of VE-cad expression at AJs have remained elusive. Our Supporting Data show: 1) CHFR-mediated ubiquitylation through K48-linked polyubiquitin (poly-Ub) chains induced VE-cadherin degradation, 2) genetic deletion of CHFR in human lung endothelial cells (ECs) or mouse ECs in vivo prevented ubiquitylation and degradation of VE-cadherin; 3) EC-specific deletion of Chfr in (ChfrDEC) mice also reduced the generation of the potent endothelial barrier-disrupting mediator angiopoietin-2 (Ang-2) which was coupled to reduction in pulmonary edema; 4) CHFR additionally ubiquitylates AKT1 via K48-linked poly-Ub in ECs, which reduced AKT1 expression and led to increased FoxO1 nuclear translocation and activation; 5) ECspecific deletion of FoxO1 (FoxO1DEC) in mice prevented expression of CHFR and Ang-2, and disruption of VEcadherin barrier; and 6) EC-specific deletion of Chfr in mice also enhanced the ability of PMNs to phagocytose and eliminate Pseudomonas aeruginosa. Based on these exciting Supporting Data, in Aim 1, we will test the hypothesis that expression of CHFR in lung ECs, downstream of TLR4 signaling, causes the loss of VE-cad expression at AJs by ubiquitylation of VE-cad through K48-linked polyubiquitin chains. In Aim 2, we will test the hypothesis that TLR4-induced CHFR expression increases FoxO1-mediated Ang-2 generation to injure the endothelial barrier subsequent to the degradation of the FoxO1 negative regulator through the ubiquitylation of AKT1 via K48-linked polyubiquitin chains. In Aim 3, we will test the hypothesis that CHFR-mediated loss of VE-cadherin at AJs induces transendothelial migration of PMNs and is an essential host-defense mechanism regulating bacterial elimination capacity of transmigrated PMNs. These studies will employ rigorous biochemical, molecular, in vivo real-time intravital imaging, and functional assays to define how CHFR mediates the degradation of VE-cadherin and AKT1 through the ubiquitylation-dependent pathway and its consequences on endothelial barrier integrity and innate immune function of the endothelium. We will use ECrestricted knockout (Chfr􀀀EC and FoxO1􀀀EC) mouse models generated by us to accomplish the above aims. The intent of these studies is to identify and develop novel therapeutic approaches targeting ARDS via the manipulation of CHFR.

经过修订且响应迅速的应用将从机械上调查新发现的方式内皮细胞表达泛素E3连接酶CHFR（带叉头和环形域的检查点）调节血管内皮细胞的屏障完整性和先天免疫功能。在内皮粘附连接（AJS）上表达的VE-钙粘蛋白是限制血浆大分子的渗出以及吞噬性嗜中性粒细胞（PMN）的影响的“守门人”。但是，触发AJS上VE-CAD表达丧失的关键生化机制仍然难以捉摸。我们的支持数据显示：1）CHFR介导的泛素化通过K48连接的聚偶像素（poly-UB）链诱导的VE-钙粘着蛋白降解，2）CHFR在人肺内皮细胞（ECS）或小鼠EC中的遗传缺失可防止VE-钙粘着蛋白的泛素化和降解； 3）（CHFRDEC）小鼠中CHFR的EC特异性缺失还减少了有效的内皮屏障 - 干扰介体Angiopietin-2（Ang-2）的产生，该介体与肺水肿的减少相连； 4）CHFR还通过EC中的K48连接的聚-UB泛素化Akt1，从而降低了Akt1的表达并导致FOXO1核易位和激活增加； 5）小鼠中FOXO1（FOXO1DEC）的Ecspific缺失阻止了CHFR和ANG-2的表达，并破坏了维卡德蛋白屏障； 6）CHFR在小鼠中的EC特异性缺失还增强了PMN对吞噬剂和消除铜绿假单胞菌的能力。基于这些令人兴奋的支持数据，在AIM 1中，我们将测试以下假设：TLR4信号下游CHFR在TLR4信号传导下游的表达会导致通过通过K48链接的多泛素链的VE-CAD泛素化VE-CAD通过VE-CAD的泛素化而导致VE-CAD表达的丧失。在AIM 2中，我们将检验以下假设：TLR4诱导的CHFR表达会增加FOXO1介导的ANG-2生成以损害通过AKT1通过K48连接的多泛素链的泛素化降解后，内皮屏障在降解后进行了降解。在AIM 3中，我们将检验以下假设：CHFR介导的AJS上VE-钙黏着蛋白的丧失诱导PMN的跨内皮迁移，并且是调节翻译PMN细菌消除能力的必不可少的宿主防御机制。这些研究将采用严格的生化，分子，体内实时浸润成像和功能性测定，以定义CHFR如何通过依赖性依赖性依赖性途径及其对内皮障碍的障碍和天生的免疫功能的后果来介导VE-cadherin和akt1的降解。我们将使用我们为实现上述目的而生成的Ecrented敲除（CHFRMA EC和FOXO1MA EC）的鼠标模型。这些研究的目的是通过操纵CHFR来识别和开发针对ARD的新型热方法。