title: Coculture of osteoclast precursors with rheumatoid synovial fibroblasts induces osteoclastogenesis via transforming growth factor beta-mediated down-regulation of osteoprotegerin.
abstract: OBJECTIVE
  The mechanisms of osteoclast maturation and the role of rheumatoid arthritis (RA) synovial fibroblasts in the control of osteoclastogenesis remain unclear. The purpose of this study was to determine the humoral factors that influence osteoclast differentiation resulting from mutual interactions between osteoclast progenitor cells and synovial fibroblasts.
  
  
  METHODS
  The cloned mouse macrophage cell line RAW 264.7 or isolated human CD14+ monocytes were cocultured with RA or osteoarthritis (OA) synovial fibroblasts in the presence of RANKL. Osteoclasts were visualized by staining for tartrate-resistant acid phosphatase (TRAP), and their functions were evaluated by bone resorption assay. Transforming growth factor beta (TGFbeta) and osteoprotegerin (OPG) levels were measured by enzyme-linked immunosorbent assay. Expression of pSmad2 and Smad7 was analyzed by Western blotting.
  
  
  RESULTS
  RANKL-mediated osteoclast formation was observed in cocultures of RAW cells with RA synovial cells, but not with OA synovial cells. This formation was inhibited by TGFbeta receptor kinase inhibitor or neutralizing TGFbeta antibody. Human CD14+ monocytes showed the same results with RAW 264.7, and bone resorption activity was consistent with osteoclast formation. RA synovial fibroblasts produced TGFbeta in response to cell-cell contact with RAW cells in a RANKL-dependent manner. TGFbeta reduced OPG production by RA synovial fibroblasts, but dose-dependently increased OPG secretion in OA synovial fibroblasts. TGFbeta decreased the expression of pSmad2 and increased the expression of Smad7 in RA synovial fibroblasts, but not OA synovial fibroblasts.
  
  
  CONCLUSIONS
  Suppression of OPG production by down-regulation of TGFbeta/Smad2 signaling may contribute to RANKL-mediated osteoclastogenesis from RA synovial fibroblasts.
authors.affiliation: Dokkyo Medical University School of Medicine, Mibu, Tochigi, Japan. hhase@dokkyomed.ac.jp
authors.forename: Hidenori
authors.initials: H
authors.lastname: Hase
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Kanno
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Kojima
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakurai
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Kobata
mesh_terms: D000818:Animals; D001172:Arthritis, Rheumatoid / Q000503:physiopathology*; D001862:Bone Resorption; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D019976:Cloning, Organism; D015536:Down-Regulation; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D051379:Mice; D009000:Monocytes / Q000502:physiology; D010003:Osteoarthritis / Q000503:physiopathology; D010010:Osteoclasts / Q000502:physiology*; D053244:Osteoprotegerin / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D051906:Smad7 Protein / Q000032:analysis; D013583:Synovial Membrane / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*
chemical_list: D053244:Osteoprotegerin; D053245:RANK Ligand; D018125:Receptors, Transforming Growth Factor beta; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta