The Signaling Adapter Protein DAP12 Regulates Multinucleation during Osteoclast Development

Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12-associated receptors expressed by osteoclasts and examine function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts. Introduction: Osteoclasts are bone-resorbing cells derived from hematopoietic precursors in the myeloid lineage. In other myeloid cell types, the signaling adapter protein DAP12 transmits activating signals on ligation of a DAP12-associated receptor (DAR). The aim of this study was to clarify/the role of DAP12 signaling during osteoclast development. Materials and Methods: Osteoclasts from DAP12 ^-/- or control mice were analyzed in vitro for morphology, function, and for osteoclast markers. DARs were identified in osteoclast cultures through reverse transcriptase-polymerase chain reaction (RT-PCR). Bone density of DAP12^-/- and control mice were analyzed by microcomputed tomography. DAP12^-/- osteoclasts were retrovirally reconstituted with DAP12. RAW264.7 cells were transfected with FLAG-tagged DAP12 or TREM2 and stimulated by anti-FLAG antibody during in vitro osteoclastogenesis. Results: C57BL/6 DAP12-deficient mice have higher bone mass than C57BL/6 wildtype controls. We verified the presence of DAP12 in pre-osteoclasts and osteoclasts derived from C57BL/6 or the pre-osteoclast line RAW 264.7 and idenfified the DARs expressed. DAP12^-/- osteoclasts developed in vitro with macrophage colony-sfimulating factor (M-CSF) and RANKL formed only intensely TRACP ⁺ mononuclear cells and failed to generate multinuclear osteoclasts. These mononuclear cells are functional osteoclast-like cells because, by RT-PCR, they express other osteoclast markers and generate resorption pits on dentine slices, although quantitative assessment of bone resorption shows decreased resorption by DAP12^-/- osteoclasts compared with C57BL/6 osteoclasts. Restoration of DAP12 expression by retroviral transduction of DAP12 ^-/- osteoclast precursors rescued in vitro osteoclast multinucleation, Direct sfimulation of DAP12 expressed in RAW264.7 during in vitro osteoclastogenesis led to a marked increase in the number of TRACP ⁺ multinucleated osteoclast-like cells formed. Conclusion: Our studies indicate that stimulation of the DAP12 adapter protein plays a significant role in formation of multinuclear osteoclasts and that DAP12 and DARs likely participate in the regulation of bony remodeling.