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

这个修改后的响应性应用程序将机械地调查新发现的 血管内皮细胞表达泛素E3连接酶CHFR(具有叉头和环指结构域的检查点)，调节血管内皮细胞的屏障完整性和天然免疫功能。表达于内皮细胞黏附连接(AJs)的Ve-钙粘附素(Ve-cadherin，Ve-cadherin)起着“把关人”的作用，限制血浆大分子的外溢和吞噬细胞的中性粒细胞(PMN)流入组织。然而，触发AJs VE-cad表达缺失的关键生化机制仍不清楚。我们的支持数据显示：1)CHFR通过K48连接的多泛素(PolyUb)链介导的泛素化诱导VE-钙粘附素 2)体内人肺内皮细胞(ECs)或小鼠内皮细胞CHFR的基因缺失阻止VE-钙粘蛋白的泛素化和降解；3)Chfr在(ChfrDEC)小鼠中的特异性缺失也减少了与减轻肺水肿有关的强大的内皮屏障干扰介质血管生成素-2(Ang-2)的产生；4)CHFR通过K48连接的PolyUb在ECs中另外泛素化AKT1，从而减少AKT1的表达，导致FoxO1核转位和激活增加；5)ECs特异性缺失FoxO1(FoxO1DEC)，阻止CHFR和Ang-2的表达，以及VEcadherin屏障的破坏；6)EC特异性的Chfr基因在小鼠体内的缺失也增强了中性粒细胞吞噬和清除铜绿假单胞菌的能力。基于这些令人兴奋的支持数据，在目标1中，我们将检验TLR4信号下游的肺内皮细胞CHFR表达通过K48连接的多泛素链泛素化VE-cad导致AJs VE-cad表达缺失的假设。在目标2中，我们将检验TLR4诱导的CHFR表达增加FoxO1介导的Ang-2产生以损伤 通过K48连接的多泛素链使AKT1泛素化，FoxO1负性调节因子降解后的内皮屏障。在目标3中，我们将验证CHFR介导的VE-cadherin在AJs的缺失诱导PMN跨内皮细胞迁移的假说，这是调节迁移的PMN清除细菌能力的重要宿主防御机制。这些研究将使用严格的生化、分子、活体实时成像和功能分析来确定CHFR如何通过泛素化依赖的途径介导VE-钙粘蛋白和AKT1的降解及其对内皮屏障完整性和内皮天然免疫功能的影响。我们将使用我们建立的EC限制性基因敲除小鼠模型(CHFR􀀀EC和FoxO1􀀀EC)来实现上述目的。这些研究的目的是通过操纵CHFR来识别和开发针对ARDS的新的治疗方法。

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.