Molecular Medicine YAP Controls Endothelial Activation and Vascular Inflammation Through TRAF6 Yang Lv, Kyungho Kim, Yue Sheng, Jaehyung Cho, Zhijian Qian, You-Yang Zhao, Gang Hu, Duojia Pan, Asrar B. Malik, Guochang Hu Rationale: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. Objective: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. Methods and Results: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell–specific deletion of YAP. Conclusions: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced Downloaded from http://ahajournals.org by on July 25, 2018 inflammation and organ failure. (Circ Res. 2018;123:43-56. DOI: 10.1161/CIRCRESAHA.118.313143.) Key Words: acute lung injury ◼ E-selectin ◼ endothelial cells ◼ neutrophils ◼ sepsis ◼ ubiquitination epsis is the leading cause of intensive care unit admissions, ultimately organ dysfunction.3 Therefore, maintenance or re- Swith high mortality and morbidity. In septic patients, wide- constitution of vascular endothelial homeostasis after a major spread vascular leakage and inflammation secondary to endo- systemic injury is a crucial determinant of outcome in sepsis.1,4 thelial activation and dysfunction is the primary mechanism of However, the underlying mechanisms limiting the severity of multiorgan failure.1 Endothelial cells are the key sentinel cells endothelial activation during sepsis remain to be identified. detecting microbial infection through the activation of pattern Meet the First Author, see p 3 recognition receptors to mount an inflammatory response. Endothelial activation during sepsis leads to the production of Activation of NF-κB (nuclear factor-κB) is critical for multiple proinflammatory cytokines and upregulation of adhe- regulating the expression of inflammatory genes and adhesion sion molecules E/P-selectins and ICAM (intercellular adhe- molecules.5 The NF-κB activation cascade is initiated by endo- sion molecule)-1 essential for polymorphonuclear neutrophil toxin lipopolysaccharide binding to TLR (Toll-like receptor)-4, (PMN) rolling, adhesion, and transendothelial PMN migra- which activates adaptor protein MyD88 (myeloid differen- tion.2 Sustained activation of endothelium results in massive tiation factor)-dependent and MyD88-independent signaling infiltration of PMNs, hyperinflammation, tissue damage, and pathways.6,7 MyD88 associates with IRAK (IL [interleukin]-1 Original received April 14, 2018; revision received May 16, 2018; accepted May 21, 2018. In April 2018, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.36 days. From the Department of Anesthesiology (Y.L., Guochang Hu) and Department of Pharmacology (K.K., J.C., A.B.M., Guochang Hu), University of Illinois College of Medicine, Chicago; Department of Pharmacology, Nanjing Medical University, Jiangsu, China (Y.L., Gang Hu); Department of Medicine and Cancer Research Center, The University of Illinois Hospital and Health Sciences System, Chicago (Y.S., Z.Q.); Program for Lung and Vascular Biology, Stanley Manne Children’s Research Institute, Ann & Robert H. Lurie Children’s Hospital of Chicago, IL (Y.-Y.Z.); Departments of Pediatrics and Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL (Y.-Y.Z.); and Howard Hughes Medical Institute and Department of Physiology, UT Southwestern Medical Center, Dallas, TX (D.P.). The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 118.313143/-/DC1. Correspondence to Guochang Hu, MD, PhD, Department of Anesthesiology, University of Illinois College of Medicine, 3200W UICH, MC 515, 1740 W Taylor St, Chicago, IL 60612-7239. E-mail [email protected] © 2018 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.118.313143 43 44 Circulation Research June 22, 2018 Novelty and Significance What Is Known? Systemic endothelial activation and dysfunction induce vascu- lar inflammation and disruption of microvascular barrier integ- • Microvascular endothelial activation and dysfunction are critical deter- rity leading to microvascular leak, tissue edema, shock, organ minants in the progression from sepsis to organ failure. • YAP (Yes-associated protein) transcriptionally promotes cell survival, failure, and ultimate death. However, the molecular mechanisms chemotaxis, differentiation, and proliferation. responsible for controlling endothelial activation remains un- known. Herein, we identified a crucial role of cytoplasmic YAP What New Information Does This Article Contribute? as a checkpoint modulator of endothelial activation and vascular inflammation. YAP suppressed inflammatory signaling by inter- • Endothelial-specific deletion of YAP exaggerates sepsis-induced acting with TRAF6 and prevented the activation of NF-κB by en- vascular inflammation, lung inflammatory injury, and cardiovascular hancing degradation of TRAF6 via K48 ubiquitination as well as dysfunction. suppressing K63-linked ubiquitination. Our results suggest that • YAP limits NF-κB (nuclear factor-κB) activation via modification of the endothelial YAP may be a novel therapeutic target for prevention signaling adaptor TRAF6 (tumor necrosis factor receptor-associated and treatment of sepsis-induced tissue inflammatory injury and factor 6). organ failure. TRAF6 leads to its degradation via the 26S proteasome, where- Nonstandard Abbreviations and Acronyms as K63-linked polyubiquitination is essential for activation of CKO conditional knockout TAK1 kinase and subsequent NF-κB signaling.13,14 CLP cecal ligation and puncture The Hippo signaling pathway consists of a serine kinase HA hemagglutinin cascade with associated regulatory and scaffolding proteins HEK human embryonic kidney that transcriptionally regulate the expression of genes control- HLMVECs human lung microvascular endothelial cells ling growth. The Ser/Thr kinase Hippo in Drosophila (Mst1 ICAM intercellular adhesion molecule [mammalian sterile 20-like kinase] and Mst2 in mammals) IκB inhibitor protein κB and Sav1 (salvador) form a complex that phosphorylates and activates LATS1/2 (large tumor suppressor) kinases, which IKK IκB kinase Downloaded from http://ahajournals.org by on July 25, 2018 in turn phosphorylate YAP (Yes-associated protein) and TAZ IL interleukin (transcriptional coactivator with PDZ-binding motif), the 2 IRAK IL-1 receptor-associated kinase downstream effectors of the Hippo pathway, causing them to MyD88 myeloid differentiation factor be retained in the cytoplasm.15–17 Dephosphorylation of YAP NF-κB nuclear factor-κB and TAZ induces their nuclear translocation and activates ex- PMN polymorphonuclear neutrophil pression of genes that promote cell proliferation and inhibit sICAM soluble ICAM apoptosis.18,19 YAP is a potent oncogene first identified by its siRNA small interfering RNA ability to associate with Yes and Src tyrosine kinases.20 As a sVCAM soluble vascular cell adhesion molecule transcriptional coactivator, YAP activates the expression of TAK transforming growth factor-β–activated kinase target genes important for cell survival, chemotaxis, differen- TAZ transcriptional coactivator with PDZ-binding motif tiation, and proliferation.21,22 Activation of YAP or loss of its TLR Toll-like receptor upstream negative regulators leads to striking overgrowth and TNF tumor necrosis factor tumor phenotypes.23 Although Hippo/YAP signaling is impor- TRAF6 TNF receptor-associated factor 6 tant in oncogenesis, little is known about how YAP signals WT wild-type inflammation. We previously showed that Gram-positive bac- YAP Yes-associated protein teria activated Hippo signaling through Toll-dependent path- way in Drosophila.24 It is unknown if Hippo/YAP signaling also regulates inflammatory response induced by sepsis. receptor-associated kinase),