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.

摘要 在项目2中，我们将从力学上研究新发现的内皮细胞表达的作用 泛素E3连接酶CHFR(带有叉头和环指结构域的检查点)对肺屏障的调节作用 血管内皮细胞的完整性和先天免疫功能。VE-钙粘附素(VE-cad)在 内皮细胞黏附连接(AJs)是限制血浆通透性的“把关人” 大分子和吞噬中性粒细胞(PMN)流入组织。然而，关键机制是 在AJ引发VE-cad表达的丧失仍然难以捉摸。我们的支持数据显示：1)CHFR 通过K48连接的多泛素(PolyUb)链介导VE-cad泛素化导致VE-钙粘附素 降解，2)体内人肺内皮细胞或小鼠肺内皮细胞CHFR基因缺失 VE-钙粘附素的泛素化和降解；3)EC特异性缺失小鼠的ChfR(ChfR∆EC)也显著 减少强大的内皮屏障破坏介质血管生成素-2(Ang-2)的产生；4)CHFR 此外，在内皮细胞中，通过K48连接的PolyUb泛素化AKT1，减少了AKT1的表达，导致FoxO1 5)EC特异性缺失FOXO_1(FOXO_1∆EC)可阻止 CHFR和Ang-2的表达及VE-钙粘蛋白屏障的破坏；6)EC特异性的Chfr缺失 小鼠还增强了迁移的中性粒细胞吞噬和消除铜绿假单胞菌的能力。 基于这些令人兴奋的数据，在目标1中，我们将检验CHFR在肺内皮细胞中的表达， 在TLR4信号的下游，导致继发于泛素化的AJ的VE-钙粘蛋白表达的丧失 Ve-钙粘附素通过K48连接的多泛素链。在目标2中，我们将检验TLR4诱导的假设 CHFR表达增加FoxO1介导的Ang-2直接损伤肺内皮细胞屏障 在AKT1泛素化之后。在目标3中，我们将检验CHFR介导的VE- AJs中钙粘附素诱导中性粒细胞跨内皮细胞迁移是一种重要的宿主防御机制 调节移行的中性粒细胞的细菌清除能力。这些研究将使用一系列 生化、分子、活体实时活体成像和核心中可用的功能分析 CHFR如何通过泛素化依赖途径介导VE-钙粘蛋白和AKT1的降解 及其对内皮屏障完整性和肺内皮细胞天然免疫功能的影响。我们 将使用多种EC限制的基因敲除鼠标模型来实现上述目标。长期的 这些研究的目的是确定和开发针对ARDS的新的治疗方法 CHFR表达的操纵。此外，项目2将直接与其他项目整合，并 有助于了解肺内皮细胞的先天免疫功能及其在炎症机制中的作用 肺损伤。