title: TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function.
abstract: Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.
  
  
  BACKGROUND
  TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption.
  
  
  METHODS
  We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts.
  
  
  RESULTS
  Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF.
  
  
  CONCLUSIONS
  Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.
authors.affiliation: Department of Medicine, VA Medical Center and University of California, San Francisco, California 94121, USA. nbh@itsa.ucsf.edu
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Humphrey
authors.forename: Michael R
authors.initials: MR
authors.lastname: Daws
authors.forename: Steve C
authors.initials: SC
authors.lastname: Spusta
authors.forename: Eréne C
authors.initials: EC
authors.lastname: Niemi
authors.forename: James A
authors.initials: JA
authors.lastname: Torchia
authors.forename: Lewis L
authors.initials: LL
authors.lastname: Lanier
authors.forename: William E
authors.initials: WE
authors.lastname: Seaman
authors.forename: Mary C
authors.initials: MC
authors.lastname: Nakamura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001862:Bone Resorption / Q000235:genetics; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D002633:Chemotaxis / Q000235:genetics; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D053245:RANK Ligand; D034622:RNA Interference; D053246:Receptor Activator of Nuclear Factor-kappa B; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011971:Receptors, Immunologic; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C498716:Trem2 protein, mouse; C484963:Tyrobp protein, mouse; D016173:Macrophage Colony-Stimulating Factor