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: 10706515
• 负责人: Asrar B. Malik
• 金额: $ 42.91万
• 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/10706515
• 关键词: AKT1 gene; Acute Respiratory Distress Syndrome; Address; Adherens Junction; Angiopoietin-2; Binding; Biochemical; Biological Assay; Blood Vessels; Cell Communication; Cell surface; Cells; Data; Down-Regulation; Edema; Endocytosis; Endothelial Cells; Endothelium; Event; FOXO1A gene; Failure; Gatekeeping; Generations; Genetic; Genetic Transcription; Host Defense; Host Defense Mechanism; Human; Individual; Inflammation; Inflammatory; Injury; Innate Immune Response; Innate Immune System; Knockout Mice; Life; Link; Lung; Lung immune response; Mediating; Mediator; Methods; Molecular; Mus; Natural Immunity; Nodal; Nuclear Translocation; Pathogenicity; Pathway interactions; Permeability; Phagocytes; Phagocytosis; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Plasma; Polyubiquitin; Process; Protac; Proteins; Pseudomonas aeruginosa; Pulmonary Edema; Respiratory Failure; Ring Finger Domain; Role; Secondary to; Signal Pathway; Signal Transduction; TIE-2 Receptor; TLR4 gene; Testing; Time; Tissues; Up-Regulation; Vascular Endothelial Cell; bactericide; 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; response; superresolution microscopy; transcription factor; transcriptomics; ubiquitin-protein ligase; vascular endothelial protein tyrosine phosphatase; 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，我们将从机制上研究新发现的内皮细胞表达的 泛素E3连接酶CHFR（checkpoint with fork-head and ring finger domain）对肺屏障的调节 完整性和血管内皮细胞的先天免疫功能。VE-钙粘蛋白（VE-cad）表达于 内皮粘附连接（AJs）作为“看门人”来限制血浆渗透性， 大分子和吞噬性中性粒细胞（PMN）流入组织。然而，关键机制 触发AJs处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连接的多聚泛素（poly-Ub）链介导VE-钙粘蛋白的泛素化，导致VE-钙粘蛋白 2）在体内防止人肺内皮细胞（EC）或小鼠EC中CHFR的遗传缺失 3）小鼠EC特异性Chfr缺失（Chfr缺失EC）也显著降低了VE-钙粘蛋白的泛素化和降解。 减少了有效的内皮屏障破坏介质血管生成素-2（Ang-2）的产生; 4）CHFR 此外，通过K48连接的多聚Ub在EC中泛素化AKT 1，这降低了AKT 1的表达，并导致FoxO 1 5）小鼠中EC特异性FoxO 1缺失（FoxO 1缺失EC）阻止了细胞核转位和激活。 CHFR和Ang-2的表达，以及VE-钙粘蛋白屏障的破坏;和6）在血管内皮细胞中， 小鼠还增强了移行的PMNs吞噬和清除铜绿假单胞菌的能力。 基于这些令人兴奋的数据，在目的1中，我们将检验CHFR在肺EC中的表达， TLR 4信号转导的下游，导致继发于泛素化的AJs处VE-钙粘蛋白表达的丧失。 VE-钙粘蛋白通过K48连接的多聚泛素链。在目标2中，我们将检验TLR 4诱导的细胞凋亡的假设。 CHFR表达增加FoxO 1介导的Ang-2生成直接损伤肺内皮屏障 在AKT 1的泛素化之后。在目标3中，我们将检验CHFR介导的VE-1丢失的假设。 AJs上的钙粘蛋白诱导中性粒细胞的跨内皮迁移，因此是一种重要的宿主防御机制 调节迁移的中性粒细胞的细菌清除能力。这些研究将采用 生物化学、分子、体内实时活体成像和功能测定， CHFR如何通过泛素化依赖性途径介导VE-钙粘蛋白和AKT 1的降解 及其对肺内皮的内皮屏障完整性和先天免疫功能的影响。我们 将使用多种EC限制性基因敲除小鼠模型来实现上述目标。长期 这些研究的目的是确定和开发针对ARDS的新治疗方法， CHFR表达的操纵。此外，项目2将直接与其他项目相结合， 有助于了解肺内皮的天然免疫功能及其在炎症机制中的作用 肺损伤