title: Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis.
abstract: BACKGROUND
  The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.
  
  
  METHODS
  RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.
  
  
  RESULTS
  AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.
  
  
  CONCLUSIONS
  AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.
authors.affiliation: Department of Orthopaedic Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
authors.forename: De-chun
authors.initials: DC
authors.lastname: Geng
authors.forename: Yao-zeng
authors.initials: YZ
authors.lastname: Xu
authors.forename: Hui-lin
authors.initials: HL
authors.lastname: Yang
authors.forename: Guang-ming
authors.initials: GM
authors.lastname: Zhu
authors.forename: Xian-bin
authors.initials: XB
authors.lastname: Wang
authors.forename: Xue-song
authors.initials: XS
authors.lastname: Zhu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology*; D043885:Receptor, Cannabinoid, CB2 / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D007211:Indoles; D053245:RANK Ligand; D043885:Receptor, Cannabinoid, CB2; C094023:iodopravadoline