title: RANKL pretreatment plays an important role in the differentiation of pit-forming osteoclasts induced by TNF-α on murine bone marrow macrophages.
abstract: BACKGROUND
  Osteoclasts differentiated from bone marrow macrophages (BMMs) induced by TNF-α alone do not have resorbing activity. When BMMs are stimulated with receptor activator of NF-κB ligand (RANKL) before TNF-α stimulation, pit-forming osteoclasts are differentiated. However, the details of the effect of RANKL pretreatment on the pit-forming osteoclast differentiation by TNF-α have not been established. The aim of this study is to examine the condition of RANKL pretreatment for differentiation of pit-forming osteoclasts induced by TNF-α. Murine BMMs were stimulated with various concentrations of RANKL for 24h in the presence of M-CSF, then the medium was changed and TNF-α was added. Osteoclasts and pits formation were examined. Osteoprotegerin (OPG), decoy receptor of RANKL, was added to the culture to examine the necessity of co-existing RANKL with TNF-α on the formation of pit-forming osteoclasts. To investigate the influence of RANKL of sufficient concentration as pretreatment for pit-forming osteoclast formation by TNF-α, dose- and time-dependent changes of osteoclast formation were checked.
  
  
  RESULTS
  The pit formation by osteoclasts in response to TNF-α required 10ng/mL RANKL pretreatment. Stimulation with this concentration of RANKL led to the differentiation of mature osteoclasts in the 72h culture. The pit formation was not inhibited by the OPG.
  
  
  CONCLUSIONS
  These results suggested that the concentration of RANKL pretreatment, which also alone can differentiate BMMs into osteoclasts, may be important in the differentiation of pit-forming osteoclasts by TNF-α. In addition, the effects of TNF-α after RANKL treatment might be independent of RANKL.
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Yamashita
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. Electronic address: ukai@nagasaki-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ukai
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Yoshinaga
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Takamori
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Noguchi
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Atsutoshi
authors.initials: A
authors.lastname: Yoshimura
authors.affiliation: Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hara
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D053244:Osteoprotegerin / Q000494:pharmacology; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D053244:Osteoprotegerin; D053246:Receptor Activator of Nuclear Factor-kappa B; C506030:TNFRSF11B protein, human; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor
keywords: Osteoclast; Receptor activator of NF-κB ligand; Resorbing activity; Tumour necrosis factor-alpha