The Signaling Adapter Protein DAP12 Regulates Multinucleation during Osteoclast Development

Mary Beth Humphrey, Kouetsu Ogasawara, Wei Yao, Steven C. Spusta, Michael R. Daws, Nancy E Lane, Lewis L. Lanier, Mary C. Nakamura

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    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)224-234
    Number of pages11
    JournalJournal of Bone and Mineral Research
    Volume19
    Issue number2
    DOIs
    StatePublished - Feb 2004

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    Osteoclasts
    Proteins
    Reverse Transcriptase Polymerase Chain Reaction
    Osteogenesis
    Bone and Bones
    X-Ray Microtomography

    Keywords

    • Cellular differentiation
    • DAP12
    • Monocytes/macrophages
    • Osteoclasts
    • Rodent

    ASJC Scopus subject areas

    • Surgery

    Cite this

    The Signaling Adapter Protein DAP12 Regulates Multinucleation during Osteoclast Development. / Humphrey, Mary Beth; Ogasawara, Kouetsu; Yao, Wei; Spusta, Steven C.; Daws, Michael R.; Lane, Nancy E; Lanier, Lewis L.; Nakamura, Mary C.

    In: Journal of Bone and Mineral Research, Vol. 19, No. 2, 02.2004, p. 224-234.

    Research output: Contribution to journalArticle

    Humphrey, Mary Beth ; Ogasawara, Kouetsu ; Yao, Wei ; Spusta, Steven C. ; Daws, Michael R. ; Lane, Nancy E ; Lanier, Lewis L. ; Nakamura, Mary C. / The Signaling Adapter Protein DAP12 Regulates Multinucleation during Osteoclast Development. In: Journal of Bone and Mineral Research. 2004 ; Vol. 19, No. 2. pp. 224-234.
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    abstract = "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.",
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