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Keywords:

  • TNF receptor-associated factor 2;
  • osteoclast differentiation;
  • TNF-α;
  • RANKL

Abstract

TRAF2-deficient mice show embryonic lethality, and we developed a new in vitro differentiation system to show the function of TRAF2 in osteoclastogenesis, in which osteoclast progenitors are derived from the fetal liver of TRAF2-deficient mice. Using this system, we showed that TRAF2 is required for TNF-α-induced osteoclastogenesis.

Introduction: TNF receptor-associated factor 2 (TRAF2) is a signal transducer for RANK and for two TNF receptor isotypes, TNFR1 and TNFR2. Because TRAF2-deficient mice show embryonic lethality, it has remained unclear whether TRAF2 is crucial in RANKL- or TNF-α-induced osteoclastogenesis.

Materials and Methods: Osteoclast progenitors derived from fetal liver were cultured in the presence of monocyte macrophage colony-stimulating factor (M-CSF), and flow cytometry for characterization of surface markers on these cells was performed. To examine the involvement of TRAF2 in osteoclast differentiation, we cultured osteoclast progenitors from TRAF2-deficient and wildtype mice with soluble RANKL or TNF-α in the presence of M-CSF, and counted the number of TRACP+ multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-κB activation in osteoclast progenitors was examined by Western blot analysis and electrophoretic mobility shift assay, respectively. Nuclear factor of activated T cells (NFATc1) expression and activation were analyzed by RT-PCR and immunofluorescence staining, respectively. To examine whether TRAF2 overexpression induced osteoclastogenesis, TRAF2 was overexpressed in osteoclast progenitors form wildtype bone marrow by retrovirus infection.

Results and Conclusions: Osteoclast progenitors from normal fetal liver, which were cultured with M-CSF, expressed surface molecules c-fms, Mac-1, and RANK, and could differentiate into TRACP+ multinucleate cells in the presence of soluble RANKL or TNF-α. RANKL-induced osteoclastogenesis gave a reduction of 20% in the progenitors from TRAF2-deficient mice compared with that of the cells from littermate wildtype mice, whereas TNF-α-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP+ multinucleate cells were formed, and TNF-α-mediated activation of JNK, NF-κB, and NFATc1 was defective. TRAF2 overexpression induced differentiation of osteoclast progenitors from wildtype mice into TRACP+ multinucleate cells. These results suggest that TRAF2 plays an important role in TNF-α-induced osteoclastogenesis.