The signaling adapter protein DAP12 regulates multinucleation during osteoclast development

Introduction: Osteoclasts are bone-resorting 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 identified the DARs expressed. DAP12(-/-) osteoclasts developed in vitro with macrophage colony-stimulating 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 stimulation 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.