Self-assembled RANK induces osteoclastogenesis ligand-independently.
By immunoprecipitation assay, we showed that mouse RANK was self-assembled through its cytoplasmic domain located at position 534-539, whose domain was different form TRAF binding domains. Moreover, overexpression experiments showed that oligomeric RANK, which was self-associated, induced osteoclastogenesis ligand-independently.


BACKGROUND
TNF receptor I or II (TNF-RI or TNF-RII) is thought to induce its own trimerization by ligand binding; however, recently TNF-RI or TNF-RII was shown to form a trimer through its extracellular domain without ligand binding. RANK, which plays an important role in osteoclast differentiation, is a member of the TNF receptor family. Here, we studied the self-assembly of mouse RANK.


METHODS
Self-assembly of mouse RANK was examined by immunoprecipitation assay using 293T cells that had been transfected with the full-length RANK (Full) fused to FLAG tag (Full-FLAG) and Full fused to HA tag (Full-HA) without soluble RANKL (sRANKL). To explore the binding site for self-assembly, FLAG-tagged RANK C-terminal deletion mutants, 461-, 511-, 533-, 539-, and 544-FLAG, were constructed, and immunoprecipitation was performed. To examine whether RANK overexpression induced osteoclastogenesis, osteoclast progenitors that were derived from wildtype bone marrow cells, in which RANK was overexpressed, were cultured with monocyte-macrophage colony-stimulating factor (M-CSF), and TRACP staining was performed. We examined whether overexpression of each five individual C-terminal mutants induced osteoclastogenesis in osteoclast progenitors. To study the involvement of TRAF6 in RANK-induced osteoclastogenesis, osteoclast progenitors, in which RANK was overexpressed, were cultured with M-CSF and TNF receptor-associated factor (TRAF)6 decoy peptides (T6DP) that inhibit the interaction of RANK with TRAF6.


CONCLUSIONS
Immunoprecipitation experiments showed that RANK was self-assembled without sRANKL. Among the five individual mutants, only 539- and 544-FLAG mutants were associated with Full-HA ligand-independently, suggesting that self-association of RANK was regulated by its cytoplasmic domain located at position 534-539. Overexpression of full-length RANK induced osteoclast differentiation, and this differentiation was suppressed by treatment with T6DP. Overexpression of RANK deletion mutants revealed that only 539- and 544-FLAG induced osteoclastogenesis. The five C-terminal mutants had the TRAF6 binding domain in their cytoplasmic regions, suggesting that ligand-independent osteoclastogenesis requires the receptor oligomerization of RANK.
Kanazawa
Kiyoshi
K
Department of Biological information, Tokyo Institute of Technology, Yokohama, Japan.
Kudo
Akira
A
D000135:Acid Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001862:Bone Resorption / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003804:Dentin / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006388:Hemagglutinins / Q000235:genetics / Q000276:immunology; D006801:Humans; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D041681:NIH 3T3 Cells; D009842:Oligopeptides; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D011485:Protein Binding; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D014162:Transfection
D002352:Carrier Proteins; D006388:Hemagglutinins; D007527:Isoenzymes; C492279:L-T6DP-1 peptide; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D009842:Oligopeptides; D010455:Peptides; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011993:Recombinant Fusion Proteins; D048029:TNF Receptor-Associated Factor 6; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C085706:FLAG peptide; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase