title: Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.
abstract: Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.
authors.affiliation: Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Japan.|Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Sakaue
authors.affiliation: Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Fujisaki
authors.affiliation: Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Japan.|Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Hironao
authors.initials: H
authors.lastname: Nakayama
authors.affiliation: Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Japan.|Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Maekawa
authors.affiliation: Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Hiyoshi
authors.affiliation: Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Kubota
authors.affiliation: Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Joh
authors.affiliation: Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Hironori
authors.initials: H
authors.lastname: Izutani
authors.affiliation: Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Japan.|Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Toon, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
mesh_terms: D015703:Antigens, CD / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015820:Cadherins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002450:Cell Communication / Q000187:drug effects; D044842:Cullin Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D000075687:NEDD8 Protein; D011500:Protein Synthesis Inhibitors; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D014452:Ubiquitins / Q000032:analysis / Q000502:physiology*; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D044842:Cullin Proteins; D003513:Cycloheximide; D003517:Cyclopentanes; D000075687:NEDD8 Protein; D011500:Protein Synthesis Inhibitors; D011743:Pyrimidines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014452:Ubiquitins; D000099152:Cadherin 5; C503645:CUL3 protein, human; C082860:NEDD8 protein, human; C539933:pevonedistat
keywords: Cullin 3; MLN4924; endothelial barrier function; neddylation; vascular endothelial-cadherin