title: High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase.
abstract: OBJECTIVE
  To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells.
  
  
  METHODS
  Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP).
  
  
  RESULTS
  HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells.
  
  
  CONCLUSIONS
  These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.
authors.affiliation: Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, Japan. Electronic address: arikichi@kyu-dent.ac.jp.
authors.forename: W
authors.initials: W
authors.lastname: Ariyoshi
authors.affiliation: Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, Japan. Electronic address: t-oki@kyu-dent.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Okinaga
authors.affiliation: Department of Anatomy and Cell Biology, The Brody School of Medicine, East Carolina University, Greenville, NC, USA. Electronic address: knudsonc@ecu.edu.
authors.forename: C B
authors.initials: CB
authors.lastname: Knudson
authors.affiliation: Department of Anatomy and Cell Biology, The Brody School of Medicine, East Carolina University, Greenville, NC, USA. Electronic address: knudsonw@ecu.edu.
authors.forename: W
authors.initials: W
authors.lastname: Knudson
authors.affiliation: Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, Japan. Electronic address: tatsujin@kyu-dent.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Nishihara
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D018960:Hyaluronan Receptors / Q000276:immunology / Q000378:metabolism; D006820:Hyaluronic Acid / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D053245:RANK Ligand / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054460:rho-Associated Kinases / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors; D053245:RANK Ligand; D012333:RNA, Messenger; C505689:Tnfsf11 protein, mouse; D006820:Hyaluronic Acid; D054460:rho-Associated Kinases
keywords: CD44; Hyaluronic acid; Osteoclast; Receptor activator of NF-κB ligand; RhoA