E3 Ubiquitin Ligase CHFR Regulates Lung Endothelial Barrier Integrity and Innate Immunity through Control of VE-cadherin Expression

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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.