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，我们将从机制上研究新发现的内皮细胞表达的作用 泛素 E3 连接酶 CHFR（叉头和无名指结构域检查点）在调节肺屏障中的作用 血管内皮细胞的完整性和先天免疫功能。 VE-钙粘蛋白 (VE-cad) 表达于 内皮粘附连接（AJ）充当限制血浆渗透性的“看门人” 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 AJ 处 VE-cad 表达缺失的机制仍然难以捉摸。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-cad 泛素化，产生 VE-cadherin 降解，2) 人肺内皮细胞 (EC) 或小鼠 EC 体内 CHFR 基因缺失被阻止 VE-钙粘蛋白的泛素化和降解； 3) 小鼠中 Chfr 的 EC 特异性缺失 (ChfrΔEC) 也显着 减少强效内皮屏障破坏介质血管生成素-2 (Ang-2) 的产生； 4）CHFR 另外，EC 中还通过 K48 连接的聚泛素泛素化 AKT1，从而减少 AKT1 表达并导致 FoxO1 核转位和激活； 5) 小鼠中 EC 特异性删除 FoxO1 (FoxO1ΔEC) 可防止 CHFR 和 Ang-2 的表达以及 VE-钙粘蛋白屏障的破坏； 6) EC 特有的 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，我们将检验以下假设：肺 EC 中 CHFR 的表达， TLR4 信号下游，导致 AJ 处 VE-钙粘蛋白表达丧失，继发于泛素化 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 TLR4 诱导的假设 CHFR 表达增加 FoxO1 介导的 Ang-2 生成，直接损伤肺内皮屏障 AKT1 泛素化后。在目标 3 中，我们将检验 CHFR 介导的 VE- 丢失的假设 AJ 处的钙粘蛋白诱导 PMN 跨内皮迁移，因此是一种重要的宿主防御机制 调节迁移的 PMN 的细菌消除能力。这些研究将采用一系列 核心中提供生化、分子、体内实时活体成像和功能测定来定义 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 及其对内皮屏障完整性和肺内皮先天免疫功能的影响。我们 将使用多种EC限制性敲除小鼠模型来完成上述目标。长期来看 这些研究的目的是通过以下方式确定和开发针对 ARDS 的新治疗方法 CHFR 表达的操纵。此外，项目 2 将直接与其他项目集成， 有助于了解肺内皮的先天免疫功能及其在炎症机制中的作用 肺损伤。