title: Evidence for cadherin-11 cleavage in the synovium and partial characterization of its mechanism.
abstract: BACKGROUND
  Engagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.
  
  
  METHODS
  Cadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.
  
  
  RESULTS
  Soluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.
  
  
  CONCLUSIONS
  Cadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard School of Medicine, Smith Research Building, 5th floor, 1 Jimmy Fund Way, Boston, MA, 02115, USA. enoss@partners.org.
authors.forename: Erika H
authors.initials: EH
authors.lastname: Noss
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard School of Medicine, Smith Research Building, 5th floor, 1 Jimmy Fund Way, Boston, MA, 02115, USA. gwatts@rics.bwh.harvard.edu.
authors.forename: Gerald F M
authors.initials: GF
authors.lastname: Watts
authors.affiliation: Exosomics Siena S.p.A., Strada del Petriccio e Belriguardo, 35, 53100, Siena, Italy. dzocco@exosomics.eu.
authors.forename: Davide
authors.initials: D
authors.lastname: Zocco
authors.affiliation: Harvard School of Dental Medicine, Department of Developmental Biology, REB 505, 190 Longwood Avenue, Boston, MA, 02115, USA. tracy_keller@hms.harvard.edu.
authors.forename: Tracy L
authors.initials: TL
authors.lastname: Keller
authors.affiliation: Harvard School of Dental Medicine, Department of Developmental Biology, REB 505, 190 Longwood Avenue, Boston, MA, 02115, USA. mwhitman@hms.harvard.edu.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Whitman
authors.affiliation: Hospital for Special Surgery, 535 east 70th Street, New York, NY, 10021, USA. BlobelC@hss.edu.
authors.forename: Carl P
authors.initials: CP
authors.lastname: Blobel
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard School of Medicine, Smith Research Building, 5th floor, 1 Jimmy Fund Way, Boston, MA, 02115, USA. david_marvin.lee@roche.com.|F. Hoffman-La Roche Ltd, Grenzacherstrasse 124, Building 69/Room 206, 4070, Basel, Switzerland. david_marvin.lee@roche.com.
authors.forename: David M
authors.initials: DM
authors.lastname: Lee
authors.affiliation: Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard School of Medicine, Smith Research Building, 5th floor, 1 Jimmy Fund Way, Boston, MA, 02115, USA. mbrenner@research.bwh.harvard.edu.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Brenner
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010003:Osteoarthritis / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D034741:RNA, Small Interfering; D013582:Synovial Fluid / Q000737:chemistry / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism*; D014162:Transfection
chemical_list: D015820:Cadherins; D010446:Peptide Fragments; D034741:RNA, Small Interfering; C086960:osteoblast cadherin