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

这个修改后的响应式应用程序将机械地调查新发现的 内皮细胞表达的泛素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先天免疫功能。内皮粘附连接处表达的VE-钙粘蛋白作为“看门人”发挥作用，限制血浆大分子外渗和吞噬性中性粒细胞（PMN）流入组织。然而，触发AJs VE-cad表达丧失的关键生化机制仍然难以捉摸。我们的支持性数据显示：1）CHFR通过K48连接的多聚泛素（poly-Ub）链介导的泛素化诱导VE-钙粘蛋白 2）在人肺内皮细胞（EC）或小鼠EC中CHFR的基因缺失阻止了VE-钙粘蛋白的泛素化和降解; 3）在（ChfrDEC）小鼠中EC特异性Chfr缺失也减少了与肺水肿减少相关联的强效内皮屏障破坏介质血管生成素-2（Ang-2）的产生; 4）CHFR通过K48连接的多聚Ub在EC中另外泛素化AKT 1，这降低AKT 1表达并导致FoxO 1核转位和活化增加; 5）FoxO 1的EC特异性缺失（FoxO 1DEC）抑制了CHFR和Ang-2的表达，破坏了VECadherin屏障;（6）EC特异性缺失Chfr也增强了PMNs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性数据，在目的1中，我们将检验以下假设：肺EC中CHFR的表达，TLR 4信号传导的下游，通过K48连接的多聚泛素链使VE-cad泛素化，导致AJs处VE-cad表达丧失。在目的2中，我们将检验TLR 4诱导的CHFR表达增加FoxO 1介导的Ang-2生成以损伤细胞的假设。 通过K48-连接的多聚泛素链，通过AKT 1的泛素化，FoxO 1负调节因子降解后，血管内皮屏障受损。在目标3中，我们将测试的假设，CHFR介导的损失VE-钙粘蛋白在AJs诱导跨内皮迁移的中性粒细胞，是一个重要的宿主防御机制，调节细菌清除能力的迁移的中性粒细胞。这些研究将采用严格的生物化学、分子、体内实时活体成像和功能测定来确定CHFR如何通过泛素化依赖性途径介导VE-钙粘蛋白和AKT 1的降解及其对内皮屏障完整性和内皮先天免疫功能的影响。我们将使用我们建立的EC限制性基因敲除小鼠模型（Chfr E3EC和FoxO 1 E3 EC）来实现上述目标。这些研究的目的是通过操纵CHFR来识别和开发针对ARDS的新治疗方法。

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.