E3 Ubiquitin Ligase CHFR Regulates Lung Endothelial Barrier Integrity and Innate Immunity through Control of VE-cadherin Expression

E3 泛素连接酶 CHFR 通过控制 VE-钙粘蛋白表达来调节肺内皮屏障完整性和先天免疫

• 批准号: 10494617
• 负责人: Asrar B. Malik
• 金额: $ 36.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10494617
• 关键词: AKT1 gene; Acute Respiratory Distress Syndrome; Address; Adherens Junction; Angiopoietin-2; Biochemical; Biological Assay; Blood Vessels; Cell surface; Cells; Data; Down-Regulation; Edema; Endothelial Cells; Endothelium; Event; FOXO1A gene; Failure; Gatekeeping; Generations; Genetic; Genetic Transcription; Head; Host Defense; Host Defense Mechanism; Human; Individual; Inflammatory; Injury; Innate Immune Response; Innate Immune System; Instruction; Knockout Mice; Life; Link; Lung; Lung immune response; Mediating; Mediator of activation protein; Methods; Microscopy; Molecular; Mus; Natural Immunity; Nodal; Nuclear Translocation; P-Cadherin; Pathogenicity; Pathway interactions; Permeability; Phagocytes; Phagocytosis; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Plasma; Polyubiquitin; Process; Protac; Proteins; Pseudomonas aeruginosa; Resolution; Ring Finger Domain; Role; Secondary to; Signal Pathway; Signal Transduction; TIE-2 Receptor; TLR4 gene; Testing; Time; Tissues; Ubiquitin; Up-Regulation; Vascular Endothelial Cell; bactericide; base; cadherin 5; cellular imaging; defense response; in vivo; innate immune function; intravital imaging; lung injury; macromolecule; migration; mouse model; neutrophil; new therapeutic target; novel; novel strategies; novel therapeutic intervention; pathogen; prevent; protein degradation; pulmonary function; respiratory; response; transcription factor; transcriptomics; ubiquitin-protein ligase; vascular injury

ABSTRACT In Project 2 we will investigate mechanistically the role of the newly discovered endothelial cell expressed ubiquitin E3 ligase CHFR (checkpoint with fork-head and ring finger domain) in regulating lung barrier integrity and the innate immune function of vascular endothelial cells. VE-cadherin (VE-cad) expressed at endothelial adherens junctions (AJs) functions as a “gatekeeper” to restrict the permeability of plasma macromolecules and influx of phagocytic neutrophils (PMNs) into tissue. However, the key mechanisms triggering the loss of VE-cad expression at AJs have remained elusive. Our Supporting Data show: 1) CHFR mediates ubiquitylation of VE-cad through K48-linked polyubiquitin (poly-Ub) chains resulting in VE-cadherin degradation, 2) genetic deletion of CHFR in human lung endothelial cells (EC) or mouse EC in vivo prevented ubiquitylation and degradation of VE-cadherin; 3) EC-specific deletion of Chfr in mice (Chfr∆EC) also significantly reduced the generation of the potent endothelial barrier-disrupting mediator angiopoietin-2 (Ang-2); 4) CHFR additionally ubiquitylated AKT1 via K48-linked poly-Ub in ECs, which reduced AKT1 expression and led to FoxO1 nuclear translocation and activation; 5) EC-specific deletion of FoxO1 (FoxO1∆EC) in mice prevented the expression of CHFR and Ang-2, and the disruption of VE-cadherin barrier; and 6) EC-specific deletion of Chfr in mice also enhanced the ability of transmigrated PMNs to phagocytose and eliminate Pseudomonas aeruginosa. Based on these exciting 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-cadherin expression at AJs secondary to ubiquitylation of VE-cadherin 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 directly the lung endothelial barrier subsequent to ubiquitylation of AKT1. In Aim 3, we will test the hypothesis that CHFR-mediated loss of VE- cadherin at AJs induces transendothelial migration of PMNs and is thus an essential host-defense mechanism regulating bacterial elimination capacity of the transmigrated PMNs. These studies will employ a repertoire of biochemical, molecular, in vivo real-time intravital imaging, and functional assays available in the Cores to define how CHFR mediates the degradation of VE-cadherin and AKT1 through the ubiquitylation-dependent pathways and its consequences on endothelial barrier integrity and innate immune function of the lung endothelium. We will use a variety of EC-restricted knockout mouse models to accomplish the above aims. The long-term objective of these studies is to identify and develop novel therapeutic approaches targeting ARDS via manipulation of CHFR expression. Furthermore Project 2 will be directly integrated with the other Projects and help to inform the innate immune function of the lung endothelium and its role in the mechanism of inflammatory lung injury.

抽象的 在项目2中，我们将机械地研究新发现的内皮细胞的作用 泛素E3连接酶CHFR（带叉 - 头和环形域的检查点）调节肺屏障 血管内皮细胞的完整性和先天免疫功能。 VE-cadherin（VE-CAD）表示 内皮粘附连接（AJS）充当“守门人”，以限制等离子体的渗透性 大分子和吞噬中性粒细胞（PMN）对组织的影响。但是，关键机制 触发AJ处VE-CAD表达的丢失仍然难以捉摸。我们的支持数据显示：1）CHFR 通过K48连接的多泛素（聚-UB）链中VE-CAD的泛素化，导致VE-钙粘着蛋白 降解，2）在人体肺内皮细胞（EC）或小鼠EC中遗传缺失在体内阻止 VE-辅助蛋白的泛素化和降解； 3）CHFR在小鼠（CHFRΔEC）中的EC特异性缺失也显着 减少了有效的内皮屏障的产生，破坏了介体血管生成素-2（ANG-2）； 4）CHFR 另外，通过EC中的K48连接的聚-UB对AKT1进行了泛素化，从而降低了Akt1的表达并导致FOXO1 核易位和激活； 5）小鼠中FOXO1（FOXO1ΔEC）的EC特异性缺失阻止了 CHFR和ANG-2的表达，以及VE-钙粘蛋白屏障的破坏； 6）CHFR的EC特定删除 小鼠还提高了翻译PMN为吞噬剂并消除铜绿假单胞菌的能力。 基于这些令人兴奋的数据，在AIM 1中，我们将检验以下假设：CHFR在肺ECS中的表达， TLR4信号传导的下游会导致aJS继发于泛素化的AJS表达的丧失 通过K48连接的多泛素链的VE-钙粘蛋白。在AIM 2中，我们将测试TLR4诱导的假设 CHFR表达增加了FOXO1介导的Ang-2生成，直接伤害肺部内皮屏障 在Akt1的泛素化之后。在AIM 3中，我们将检验以下假设：CHFR介导的VE-损失 AJS的钙粘蛋白诱导PMN的跨内皮迁移，因此是必不可少的宿主防御机制 调节翻译PMN的细菌消除能力。这些研究将采用 生化，分子，体内实时内加静脉内成像和核心中可用的功能测定 CHFR如何通过泛素化依赖性途径介导VE-钙粘蛋白和AKT1的降解 及其对肺部内皮的内皮屏障完整性和先天免疫功能的后果。我们 将使用各种EC限制的敲除鼠标模型来实现上述目标。长期 这些研究的目的是通过 操纵CHFR表达。此外，项目2将直接与其他项目集成 帮助告知肺部内皮的先天免疫功能及其在炎症机制中的作用 肺部受伤。