DOK3 Modulates Bone Remodeling by Negatively Regulating Osteoclastogenesis and Positively Regulating Osteoblastogenesis

J Bone Miner Res. 2017 Nov;32(11):2207-2218. doi: 10.1002/jbmr.3205. Epub 2017 Aug 2.

Abstract

Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-κB ligand (RANKL) promotes osteoclast differentiation and function but requires costimulation of immunoreceptor tyrosine-based activation motif (ITAM)-coupled immunoreceptors. Triggering receptor expressed on myeloid cells-2 (TREM2) coupled to ITAM-adaptor protein DNAX activation protein 12kDA (DAP12) provides costimulation of intracellular calcium signaling during osteoclastogenesis. Previously, we found that downstream of kinase-3 (DOK3) physically associates with DAP12 to inhibit toll-like receptor (TLR)-induced inflammatory signaling in macrophages. However, whether and how DOK3 modulates DAP12-dependent osteoclastogenesis is unknown and the focus of this study. Bone microarchitecture and histology of sex- and age-matched wild-type (WT) and DOK3-deficient (DOK3-/- ) mice were evaluated. Male and female DOK3-/- mice have significantly reduced trabecular bone mass compared with WT mice with increased TRAP+ osteoclasts in vivo. In vitro, DOK3-/- bone marrow-derived macrophages (BMMs) have increased macrophage colony-stimulating factor (M-CSF)-induced proliferation and increased sensitivity to RANKL-induced osteoclastogenesis. Compared with WT, DOK3-/- osteoclasts are significantly larger with more nuclei and have increased resorptive capacity. Mechanistically, DOK3 limits osteoclastogenesis by inhibiting activation of Syk and ERK in response to RANKL and M-CSF. DOK3 is phosphorylated in a DAP12-dependent manner and associates with Grb2 and Cbl. Compared with DAP12-/- mice with high bone mass, DOK3- and DAP12- doubly deficient mice (DKO) have normalized bone mass, indicating that DOK3 also limits DAP12-independent osteoclastogenesis in vivo. In vitro osteoclasts derived from DKO mice are mononuclear with poor resorptive capacity similar to DAP12-/- osteoclasts. Histomorphometry reveals that DOK3-/- mice also have reduced osteoblast parameters. DOK3-/- osteoblasts have reduced in vitro osteoblastogenesis and increased osteoprotegerin (OPG) to RANKL expression ratio compared with WT osteoblasts. Co-culture of WT and DOK3-/- osteoblasts with pre-osteoclasts reveals a reduced capacity of DOK3-/- osteoblasts to support osteoclastogenesis. These data indicate that DOK3 regulates bone remodeling by negatively regulating M-CSF- and RANKL-mediated osteoclastogenesis and positively regulating osteoblastogenesis. © 2017 American Society for Bone and Mineral Research.

Keywords: DAP12; DOK3; M-CSF; RANKL; TREM2.

MeSH terms

  • Adaptor Proteins, Signal Transducing / deficiency
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism
  • Bone Remodeling* / drug effects
  • Bone Resorption / metabolism
  • Bone Resorption / pathology
  • Cell Differentiation / drug effects
  • Cell Proliferation / drug effects
  • Macrophage Colony-Stimulating Factor / pharmacology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Osteoblasts / drug effects
  • Osteoblasts / metabolism*
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism*
  • Osteogenesis* / drug effects
  • Osteoporosis / metabolism
  • Osteoporosis / pathology
  • Phosphorylation / drug effects
  • RANK Ligand / metabolism
  • RAW 264.7 Cells
  • Receptors, Immunologic / metabolism
  • Signal Transduction / drug effects

Substances

  • Adaptor Proteins, Signal Transducing
  • Dok3 protein, mouse
  • Membrane Glycoproteins
  • RANK Ligand
  • Receptors, Immunologic
  • Trem2 protein, mouse
  • Tyrobp protein, mouse
  • Macrophage Colony-Stimulating Factor