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. 2004 Apr 20;101(16):6158-63.
doi: 10.1073/pnas.0401602101. Epub 2004 Apr 8.

VSports手机版 - The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase

Attila Mócsai  1 , Mary Beth Humphrey, Jessica A G Van Ziffle, Yongmei Hu, Andrew Burghardt, Steven C Spusta, Sharmila Majumdar, Lewis L Lanier, Clifford A Lowell, Mary C Nakamura
Affiliations

The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase

"V体育官网" Attila Mócsai et al. Proc Natl Acad Sci U S A. .

Abstract

Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor gamma-chain (FcRgamma), are severely osteopetrotic. DAP12(-/-)FcRgamma(-/-) bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk(-/-) progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk(-/-) osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12(-/-) FcRgamma(-/-) cells VSports手机版. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-kappaB ligand cytokines, DAP12 and FcRgamma have overlapping roles in supporting osteoclast development in osteoblast-osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling. .

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Figures

Fig. 1.
Fig. 1.
Osteopetrosis in mice lacking DAP12 and FcRγ.(A) Three-dimensional reconstitution of micro-CT scans of proximal tibia and 3D trabecular (Tb.) quantitative parameters (mean ± SEM) of bone structure. Significant differences from wild-type are shown (*, P < 0.05; **, P < 0.001). (B) Hematoxylineosin staining of decalcified sections of the primary spongiosa of proximal tibias. BV/TV, relative bone volume. TRI-SMI, 3D reconstruction image SMI.
Fig. 2.
Fig. 2.
Lack of Syk phosphorylation in DAP12-/-FcRγ-/- osteoclast-like cells. (A) OCLs stained with anti-Syk and phalloidin. (B) Expression of Syk in in vitro OCLs (OC) and macrophages (MΦ) compared with the macrophage marker CD11b and actin by immunoblotting. (C) Precipitation of whole-cell lysates with GST–Syk fusion protein containing the SH2 domains of Syk or DAP12 antiserum probed with anti-phosphotyrosine antibody. Whole-cell lysates show expression of DAP12, FcRγ, and actin. (D) Immunoprecipitation (IP) with anti-Syk followed by phosphotyrosine (PY) immunoblot. (E) Immunoblot of whole-cell lysate for Y519/520 phosphorylated Syk, total Syk, or actin.
Fig. 3.
Fig. 3.
DAP12/FcRγ and Syk are required for in vitro generation of osteoclast-like cells. (A) TRAP-stained OCLs generated in RANKL and 10 (“low”) or 100 ng/ml (“high”) M-CSF. TRAP+ multinucleated cells (MNC = 3 or more nuclei per cell) enumerated as mean ± SEM of 3 wells (2 cm2 per well). (B) RT-PCR analysis of OCLs cultured in RANKL and 10 ng/ml M-CSF. 1, GAPDH; 2, calcitonin receptor; 3, cathepsin K; 4, integrin β3; 5, OSCAR; 6, RANK. DAP12-/-, DAP12-/-FcRγ-/-, and syk-/- groups were statistically different (P < 0.001) from wild type in both conditions.
Fig. 4.
Fig. 4.
DAP12/FcRγ and Syk are required for functional resorption of mineralized matrix. (A) OCLs generated in RANKL and M-CSF (10 or 100 ng/ml) on an artificial calcium phosphate substrate. The percentage of the resorption of substrate (dark areas) was quantified by dark-field microscopy and expressed as the mean ± SEM of three samples. (B) Bone resorption by OCLs on dentine slices with RANKL and 10 ng/ml M-CSF, visualized by toluidine blue staining and light microscopy. Resorption in the DAP12-/-, DAP12-/-FcRγ-/-, and syk-/- groups was statistically different (P < 0.001) from wild type in all conditions.
Fig. 5.
Fig. 5.
SH2 domains of Syk and an intact DAP12 ITAM are required for in vitro osteoclast differentiation and function. TRAP+ MNC (A) and the percent resorption (B) of calcium phosphate substrate by syk-/- precursors infected with retrovirus encoding vector alone, wild-type syk, or a SH2-dead mutant (R194A) of syk at indicated M-CSF concentrations. In both conditions, TRAP+ MNC number and percent resorption in the SykWT groups was statistically different (P < 0.01) from vector or SykSH2-Dead, with no difference between vector and SykSH2-Dead groups (P > 0.05). (C) Anti-Syk blot of whole-cell ligands from retrovirally transduced or wild-type OCLs. TRAP+ MNC (D) and percent resorption (E) from DAP12-/-FcRγ-/- precursors infected with virus-encoding vector alone, wild-type DAP12, or ITAM tyrosine mutants (Y65, Y76, or both) of DAP12 cultured in 10 ng/ml M-CSF. (F) Expression of FLAG epitope on cells retrovirally transduced with FLAG-tagged DAP12 or FLAG-tagged DAP12 mutants.
Fig. 6.
Fig. 6.
Coculture partially restores in vitro osteoclast formation in DAP12-/- cells but not DAP12-/-FcRγ-/- or syk-/- cells. OB from wild-type mice were used to stimulate osteoclast precursors from wild-type, DAP12-/-, FcRγ-/-, DAP12-/-FcRγ-/-, or syk-/- mice. The number of TRAP+ MNC (A) and the percent resorption (B) on calcium phosphate substrate is shown.
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