title: Shear stress is a positive regulator of thimet oligopeptidase (EC3.4.24.15) in vascular endothelial cells: consequences for MHC1 levels.
abstract: OBJECTIVE
  Thimet oligopeptidase (TOP, endopeptidase EC3.4.24.15) is a soluble metallopeptidase known to be expressed within the mammalian vasculature. We examine for the first time the relationship between TOP expression and laminar shear stress, a haemodynamic force associated with endothelium-mediated vascular homeostasis.
  
  
  RESULTS
  Human and bovine aortic endothelial cells were exposed to physiological levels of laminar shear (0-10 dynes/cm², 24-48 h) and monitored for TOP expression using promoter activity assay, qRT-PCR, western blotting, and immunocytochemistry. Using a luciferase reporter encoding the full-length rat TOP promoter, initial studies demonstrated shear-dependent promoter activation (~five-fold). TOP mRNA and protein were also consistently up-regulated by shear, events which could be completely prevented by pre-treatment of cells with either N-acetylcysteine, superoxide dismutase, or catalase, confirming ROS involvement. Consistent with this, targeted inhibition of NADPH oxidase (apocynin, NSC23766, NOX4 siRNA) had a similar blocking effect. Finally, in view of its pivotal role in cellular antigen presentation and major histocompatibility complex (MHC) class-1 regulation, we hypothesized that the shear-dependent induction of TOP may lower MHC1 expression. In this respect, we observed that recombinant TOP over-expression in static HAECs dose-dependently depleted MHC1 (>60%), while siRNA-mediated blockade of TOP induction in sheared HAECs led to substantially elevated MHC1 (~66%).
  
  
  CONCLUSIONS
  Our findings demonstrate that laminar shear positively regulates endothelial TOP expression. Moreover, a role for ROS production by NADPH oxidase is indicated. Finally, our studies suggest that shear-dependent TOP induction down-regulates MHC1 levels, pointing to a role for TOP in the flow-mediated regulation of endothelial immunogenicity.
authors.affiliation: School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Anthony F
authors.initials: AF
authors.lastname: Guinan
authors.forename: Keith D
authors.initials: KD
authors.lastname: Rochfort
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fitzpatrick
authors.forename: Tony G
authors.initials: TG
authors.lastname: Walsh
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Pierotti
authors.forename: Susan
authors.initials: S
authors.lastname: Phelan
authors.forename: Ronan P
authors.initials: RP
authors.lastname: Murphy
authors.forename: Philip M
authors.initials: PM
authors.lastname: Cummins
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000276:immunology* / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D033081:Shear Strength; D013314:Stress, Mechanical
chemical_list: D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase
keywords: Endothelium; MHC1; NADPH oxidase; Shear stress; Thimet oligopeptidase