Thesis or Dissertation RANKL pretreatment plays an important role in the differentiation of pit-forming osteoclasts induced by TNF-α on murine bone marrow macrophages

山下, 恭德

60 ( 9 )  , pp.1273 - 1282 , 2015-08-05 , Elsevier Ltd.
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 24 h 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 10 ng/mL RANKL pretreatment. Stimulation with this concentration of RANKL led to the differentiation of mature osteoclasts in the 72 h culture. The pit formation was not inhibited by the OPG. Conclusion 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.

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