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. 2010 Sep 1;123(Pt 17):2955-63.
doi: 10.1242/jcs.069872.

Cytoskeletal dysfunction dominates in DAP12-deficient osteoclasts

Wei Zou  1 , Tingting Zhu, Clarissa S Craft, Thomas J Broekelmann, Robert P Mecham, Steven L Teitelbaum
Affiliations

Cytoskeletal dysfunction dominates in DAP12-deficient osteoclasts

Wei Zou et al. J Cell Sci. .

Abstract

Despite evidence that DAP12 regulates osteoclasts, mice lacking the ITAM-bearing protein exhibit only mild osteopetrosis. Alternatively, Dap12(-/-) mice, also lacking FcRgamma, are severely osteopetrotic, suggesting that FcRgamma compensates for DAP12 deficiency in the bone-resorbing polykaryons. Controversy exists, however, as to whether these co-stimulatory molecules regulate differentiation of osteoclasts or the capacity of the mature cell to degrade bone. We find that Dap12(-/-) osteoclasts differentiate normally when generated on osteoblasts but have a dysfunctional cytoskeleton, impairing their ability to transmigrate through the osteoblast layer and resorb bone. To determine whether the FcRgamma co-receptor, OSCAR mediates osteoclast function in the absence of DAP12, we overexpressed OSCAR fused to FLAG (OSCAR-FLAG), in Dap12(-/-) osteoclasts. OSCAR-FLAG partially rescues the abnormal cytoskeleton of Dap12(-/-) osteoclasts grown on bone, but not those grown on osteoblasts. Thus, cytoskeletal dysfunction, and not arrested differentiation, is the dominant consequence of DAP12 deficiency in osteoclasts. The failure of osteoblasts to normalize Dap12(-/-) osteoclasts indicates that functionally relevant quantities of OSCAR ligand do not reside in bone-forming cells.

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Figures

Fig. 1.
Fig. 1.
DAP12-deficient macrophages in co-culture yield few osteoclasts following removal of osteoblasts. (A) DAP12 and FcRγ expression by WT, Dap12−/− and DKO macrophages was determined by immunoblot. Actin serves as loading control. (B–D) WT, Dap12−/− and DKO macrophages were co-cultured with WT osteoblasts. After 7 days, osteoblasts were removed using 0.1% collagenase and residual cells stained for TRAP activity. (B) Representative wells. (C) Histological appearance of TRAP-stained cells (40×). (D) Quantification of osteoclast number. Error bars represent s.d.
Fig. 2.
Fig. 2.
DAP12, but not FcRγ rescues DKO osteoclastogenesis in co-culture. DKO macrophages were retrovirally transduced with DAP12, FcRγ or empty vector (A–D) or DAP12–FcRγ domain hybrids (E–H). The transduced cells were co-cultured with osteoblasts for 7 days. Osteoblasts were removed and cells stained for TRAP activity. (A) DAP12 and FcRγ expression by transduced DKO macrophages as determined by immunoblot. (B) Representative TRAP-stained wells following osteoblast removal. (C) Osteoclasts identified by TRAP staining following osteoblast removal (40×; inset 200×). (D) Quantification of osteoclast number in wells illustrated in B and C. (E) TRAP-stained wells of DKO osteoclasts transduced with DAP12, FcRγ, vector or domain hybrids of the two ITAM adaptors. F represents FcRγ and D, DAP12. First letter indicates extracellular, second letter transmembrane and third letter, cytoplasmic domain. (F) Quantification of osteoclast number in E. (G) TRAP-stained wells containing co-culture generated Dap12−/− osteoclasts transduced with WT DAP12 or its D52A or 2YF mutant. (H) Quantification of osteoclast number in G. Error bars represent s.d.
Fig. 3.
Fig. 3.
Cytoskeleton-deficient DAP12 osteoclasts form in normal numbers in co-culture. WT and Dap12−/− macrophages, transduced with GFP-actin, were co-cultured with osteoblasts for 7 days. Cells were examined before osteoblast removal. (A) The cells were stained for TRAP activity. WT, but not Dap12−/− osteoclasts, are spread (200×). (B) Quantification of osteoclast number in A. Error bars represent s.d. (C) Fluorescent microscopy reveals actin rings in WT co-cultured osteoclasts. By contrast, those lacking DAP12, in contact with osteoblasts fail to organize podosomes (dot-like structures) into rings (200×). (D) WT and Dap12−/− macrophages, transduced with GFP-actin, were cultured in M-CSF and RANKL, for 3 days, to generate pre-fusion osteoclasts. The cells were lifted and placed on a confluent layer of osteoblast-like ST-2 cells expressing RFP-m-Cherry, with continued presence of the osteoclastogenic cytokines. Two days later the cells were examined, in the Z-plane, by confocal microscopy. (*, podosome belt; 630×). (E) WT and Dap12−/− macrophages were co-cultured with osteoblasts, with or without RANKL (100 ng/ml) and/or M-CSF (50 ng/ml), for 7 days. The cells were stained for TRAP activity (200×).
Fig. 4.
Fig. 4.
OSCAR-FLAG activation induces fusion of Dap12−/− osteoclasts. (A) OSCAR-FLAG-transduced, cytokine-generated WT osteoclasts, were immunostained with anti-FLAG mAb (left panel). Arrowhead indicates localization of transduced protein in plasma membrane. Vector-transduced WT osteoclasts serve as negative control (right panel) (200×). (B) WT and Dap12−/− OSCAR-FLAG-transduced macrophages were cultured with RANKL and M-CSF for 3 days. OSCAR in FcRγ immunoprecipitates was determined by anti-FLAG immunoblot. IgG serves as negative control (TCL; total cell lysates). (C) OSCAR-FLAG-transduced Dap12−/− macrophages were cultured with RANKL and M-CSF for 4 days. Then cell were lifted with 0.02% EDTA and re-plated on FLAG-mAb-coated plates for 30 minutes. FcRγ immunoprecipitates were immunoblotted for phosphotyrosine (p-Y) and FcRγ. (D,E) WT, Dap12−/−, FcRγ, DKO and Syk−/− macrophages, transduced with OSCAR-FLAG, were cultured, with M-CSF and RANKL, in FLAG mAb- or IgG-coated wells. After 6 days, the cells were stained for TRAP activity (200× for D). (F) Dap12−/− macrophages, transduced WT OSCAR-FLAG, OSCARR231A-FLAG or vector, were cultured for 6 days, with M-CSF and RANKL, on anti-FLAG mAb. The cells were stained for TRAP activity (200×). (G) OSCAR-FLAG or empty vector was transduced into DAP12-deficient macrophages. The cells were cultured on anti-FLAG mAb- or IgG-coated wells containing RANKL and M-CSF. After 6 days, the cells were stained for TRAP activity.
Fig. 5.
Fig. 5.
OSCAR-FLAG activation does not affect osteoclast differentiation. (A) Dap12−/− macrophages transduced with OSCAR-FLAG or vector were cultured, for 4 days, with M-CSF and increasing amounts of RANKL (RL) in anti-FLAG mAb- or IgG-coated plates. NFATc1 and Src were determined by immunoblot. Actin serves as loading control. (B) OSCAR-FLAG-transduced Dap12−/− macrophages were cultured with RANKL and M-CSF for 0, 4 or 6 days in wells coated with anti-FLAG mAb or IgG. Osteoclast differentiation markers were determined by qPCR. (C) OSCAR-FLAG-transduced Dap12−/− macrophages, cultured on anti-FLAG mAb- or IgG-coated plates, were exposed to RANKL for 15 minutes or M-CSF for 5 minutes. MAP kinase activation was determined by immunoblot. (D) OSCAR-FLAG-transduced WT and Dap12−/− macrophages (KO) were cultured in M-CSF, without (Mφ) or with (OC) RANKL for 4 days on FLAG mAb- or IgG-coated plates. BrdU incorporation was assessed by ELISA. (E,F) OSCAR-FLAG-transduced Dap12−/− macrophages were cultured with RANKL and M-CSF for 4 days, on FLAG mAb- or IgG-coated plates. Then cells were deprived of either M-CSF (E) or RANKL (F) for indicated times. Apoptosis was determined by ELISA (*P<0.05; **P<0.01; error bars represent s.d.).
Fig. 6.
Fig. 6.
OSCAR-FLAG partially rescues Dap12−/− osteoclast function on bone. (A) OSCAR-FLAG-transduced Dap12−/− macrophages were cultured, for 6 days, with RANKL and M-CSF on FLAG-mAb- or IgG-coated plates. The actin cytoskeleton was visualized by FITC-phalloidin staining. Arrowheads indicate small circular actin structures appearing in OSCAR-FLAG-bearing Dap12−/− cells on anti-FLAG mAb (200×). (B) WT or OSCAR-FLAG- or vector-transduced Dap12−/− macrophages were cultured with RANKL and M-CSF on FLAG mAb- or IgG-coated bone. The actin cytoskeleton was visualized by FITC-phalloidin staining. (C) Quantification of area encompassed by actin rings in WT, and ring-like structures in OSCAR-FLAG-expressing Dap12−/− cells on bone coated with anti-FLAG mAb or IgG. Vector-transduced Dap12−/− osteoclasts contain no such actin structures. (D) Bone resorption pits generated by cells detailed in B (200×). (E) Quantification of bone resorption pit area generated by cells detailed in D. No resorption pits are excavated by vector-transduced Dap12−/− osteoclasts. (F) OSCAR-FLAG- or vector-transduced Dap12−/− macrophages were cultured, for 4 days, in M-CSF, without (Mφ) or with (OC) RANKL. Total OSCAR expression was determined by immunoblot. OSCAR-FLAG were also analyzed by anti-FLAG immunoblot. Numbers represent densitometrically determined OSCAR:actin ratio, normalized to that endogenous in macrophages. (G) OSCAR-FLAG- or vector-transduced Dap12−/− macrophages were cultured with osteoblasts for 7 day. The cells were stained for TRAP activity before (W OB) and following (W/O OB) removal of osteoblasts (200×). (H) OSCAR-FLAG or vector transduced Dap12−/− macrophages were cultured with RANKL and M-CSF for 4 days. Then cell were lifted and re-plated on anti-FLAG mAb- or IgG-coated plates for 1 hour. Vav3 tyrosine phosphorylation was determined by immunoprecipitation and immunoblot. Numbers represent densitometric analysis of phosphorylated tyrosine:Vav3 ratio normalized to vector transductant. *P<0.05; **P<0.01; error bars represent s.d.
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