title: Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells.
abstract: Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Anatomy, Histology, Forensic Medicine and Orthopedics-Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy. alessio.dalessio@uniroma1.it
authors.forename: Alessio
authors.initials: A
authors.lastname: D'Alessio
authors.forename: Bianca
authors.initials: B
authors.lastname: Esposito
authors.forename: Claudia
authors.initials: C
authors.lastname: Giampietri
authors.forename: Elio
authors.initials: E
authors.lastname: Ziparo
authors.forename: Jordan S
authors.initials: JS
authors.lastname: Pober
authors.forename: Antonio
authors.initials: A
authors.lastname: Filippini
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism*; D002458:Cell Fractionation; D002784:Cholesterol / Q000172:deficiency; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000502:physiology*; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics; D011969:Receptors, Histamine H1 / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D015398:Signal Transduction; D012995:Solubility; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D034741:RNA, Small Interfering; D011969:Receptors, Histamine H1; D047888:Receptors, Tumor Necrosis Factor, Type I; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D006632:Histamine; D002784:Cholesterol; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human