TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.
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-alpha-induced osteoclastogenesis.


BACKGROUND
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-alpha-induced osteoclastogenesis.


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-alpha in the presence of M-CSF, and counted the number of TRACP(+) multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-kappaB 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.


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-alpha. 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-alpha-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP(+) multinucleate cells were formed, and TNF-alpha-mediated activation of JNK, NF-kappaB, 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-alpha-induced osteoclastogenesis.
Kanazawa
Kiyoshi
K
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Kudo
Akira
A
D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D001862:Bone Resorption; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005838:Genotype; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000196:embryology; D048670:MAP Kinase Kinase 4; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology; D047992:TNF Receptor-Associated Factor 2 / Q000502:physiology*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D009687:Nuclear Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D047992:TNF Receptor-Associated Factor 2; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D016173:Macrophage Colony-Stimulating Factor; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases