RRC ID 44723
Author Ariyoshi W, Okinaga T, Knudson CB, Knudson W, Nishihara T.
Title High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase.
Journal Osteoarthritis Cartilage
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.
Volume 22(1)
Pages 111-20
Published 2014-1-1
DOI 10.1016/j.joca.2013.10.013
PII S1063-4584(13)00986-2
PMID 24185105
MeSH Animals Antibodies, Monoclonal / immunology Cell Differentiation / drug effects Cell Differentiation / physiology Cells, Cultured Coculture Techniques Dose-Response Relationship, Drug Down-Regulation / drug effects Down-Regulation / physiology Enzyme Activation / drug effects Enzyme Activation / physiology Hyaluronan Receptors / immunology Hyaluronan Receptors / metabolism Hyaluronic Acid / administration & dosage Hyaluronic Acid / chemistry Hyaluronic Acid / metabolism Hyaluronic Acid / pharmacology* Mice Molecular Weight Monocytes / cytology Monocytes / drug effects Osteoclasts / cytology Osteoclasts / drug effects* RANK Ligand / antagonists & inhibitors* RANK Ligand / genetics RANK Ligand / metabolism RANK Ligand / physiology RNA, Messenger / genetics Stromal Cells / cytology Stromal Cells / drug effects Stromal Cells / metabolism rho-Associated Kinases / physiology*
IF 4.793
Times Cited 27
Human and Animal Cells RAW 264(RCB0535) ST2(RCB0224)