3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

Abstract

A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2-/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cell-intrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2-/- osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2-/- osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages.

Figures

Figure 1. Decreased bone density and strength in 12-week-old Sh3bp2–/– mice.

(A) H&E staining of tibia from 12-week-old Sh3bp2–/– and wild-type mice. (B) μCT reconstruction of the trabecular region below the distal femur growth plate (upper panel) and the cortical region of midshaft femurs (lower panel) of Sh3bp2–/– and wild-type mice. μCT-derived measurements of (C) BV/TV, (D) trabecular number (Tb.N), (E) trabecular thickness (Tb.Th), and (F) trabecular separation (Tb.Sp). n = 9; *P < 0.05. (G) μCT-derived measurements of the cortical bone volume fraction (BV/TV). n = 12; *P < 0.05. (H) Mechanical testing of vertebrae from wild-type and Sh3bp2–/– mice. n = 5; *P < 0.05. Data are presented as mean ± SEM.

Figure 2. Impaired bone resorption by Sh3bp2–/– osteoclasts.

Histomorphometric analysis of (A) OcS/BS, (B) N.Oc/BS, and (C) BV/TV in 12-week-old mice. n = 6; *P < 0.05. (D) Analysis of N.Nuclei/Oc in tibias of 12-week-old mice. Osteoclasts were sorted into groups with 1–2, 3–5, and 6+ N.Nuclei/Oc. n = 6. Quantitative PCR analysis of tnfsf11 (E) and tnfsf11b (F) transcript levels in the bones of 12-week-old wild-type and Sh3bp2–/– mice. n = 5. (G) ELISA measurement of basal serum CTX-I levels in 12-week-old wild-type and Sh3bp2–/– mice. n = 5; *P < 0.01. (H) TEM of vertebral sections from 4-week-old wild-type and Sh3bp2–/– mice showing representative osteoclasts exhibiting mature and attenuated ruffled borders from wild-type or Sh3bp2–/– mice, respectively. Scale bars: 2 μm. (I) Quantification of osteoclasts with mature ruffled borders. n = 50 osteoclasts from 3 wild-type and 3 Sh3bp2–/– mice (approximately 15 osteoclasts/mouse); P < 0.01. (J) Length measurements of 5-month-old wild-type or Sh3bp2–/– mice. The distance between the snout and anus is shown. n = 15 wild-type and 13 Sh3bp2–/– mice; P < 0.01. (K) μCT reconstruction of the femora of 16-week-old wild-type recipient mice, 8 weeks following the transfer of wild-type or Sh3bp2–/– bone marrow. Nonirradiated control is shown on the left. (L–N) μCT-derived measurements of (L) BV/TV, (M) Tb.Th, and (N) Tb.N. n = 8; *P < 0.05. (O and P) Histomorphometric analysis of (O) OcS/BS and (P) N.Oc/BS in the tibias of 16-week-old wild-type recipient mice, 8 weeks following the transfer of wild-type or Sh3bp2–/– bone marrow is shown. n = 4; *P < 0.05. Data are presented as mean ± SEM.

Figure 3. Impaired bone deposition in Sh3bp2–/– mice.

(A) Histomorphometric analysis of osteoblast surface to bone surface (ObS/BS). n = 6; *P < 0.05. (B) Serum PINP levels in wild-type and Sh3bp2–/– 12-week-old mice as measured by ELISA. n = 9; *P < 0.01. (C) Dynamic histomorphometric analysis of bones from 12-week-old wild-type and Sh3bp2–/– mice. Measurement of BFR, MAR, and MS/BS is shown. n = 5; *P < 0.01. (D) μCT reconstruction of the femurs of 16-week-old wild-type or Sh3bp2–/– mice, 8 weeks following the transfer of wild-type bone marrow. Nonirradiated control is shown on the left. μCT-derived measurements of (E) BV/TV, (F) Tb.N, and (G) Tb.Th. n = 8 mice; *P < 0.05. (H) Dynamic histomorphometric analysis of tibias from 16-week-old wild-type or Sh3bp2–/– mice, 8 weeks following the transfer of wild-type bone marrow. Measurement of BFR is shown. n = 3, *P < 0.05. Data are presented as mean ± SEM.

Figure 4. 3BP2 is required for osteoclast function but not differentiation.

(A) TRAP staining of osteoclasts derived from wild-type and Sh3bp2–/– BMMs cultured for 5 days. Scale bars: 100 μm. (B) Quantification of osteoclast area. n = 4; *P < 0.01. (C) Resorptive pits formation assay on dentin discs of wild-type or Sh3bp2–/– BMMs cultured for 7 days. (D) Comparison of wild-type and Sh3bp2–/– osteoclast resorption pit area. n = 3; *P < 0.01. (E) Quantification of TRAP-positive and multinucleated (>3 nuclei/cell) osteoclasts derived from wild-type and Sh3bp2–/– BMMs cultured for 3 days. (F) Western blot analysis of NFATc1 protein levels in wild-type and Sh3bp2–/– BMMs grown at different time intervals. (G) Semiquantitative PCR analysis of Calcr, Ctsk, Acp5, and Itgb3 expression in wild-type and Sh3bp2–/– osteoclasts grown for 5 days. Gapdh mRNA levels served as the normalization reference. (H) Phalloidin staining of wild-type and 3BP2-deficient osteoclasts cultured for 5 days. Scale bars: 100 μm (upper panels); 5 μm (lower panels). (I) The percentage of cell perimeter occupied by high-density podosome belts in H was quantified. *P < 0.01. Data are presented as mean ± SEM.

Figure 5. Integrin-mediated activation of Src is defective in Sh3bp2–/– osteoclasts.

(A) FACS analysis of β3 integrin expression on the surface of wild-type or Sh3bp2–/– osteoclasts cultured for 3 days. Sh3bp2–/– (–/–), wild-type (+/+), and unstained (US). (B) Western blot analysis of active Src levels in wild-type or 3BP2-deficient preosteoclasts cultured for 3 days, lysed in suspension, or 1 hour after plating on osteopontin (Opn). Lysates were probed with anti-Src or anti–Src pY416 antibodies. (C) Western blot analysis of active Src levels in wild-type and Sh3bp2–/– osteoclasts cultured for 5 days. Lysates were probed with anti-Src and anti–Src pY416 antibodies. (D) Coimmunoprecipitation of endogenous complexes of 3BP2 with Src in Raw264.7 cells cultured for 5 days. C, IgG control. (E) Western blot analysis of activated Syk, Vav1, and Vav3 in wild-type and Sh3bp2–/– BMMs cultured for 5 days. Syk, Vav1, and Vav3 proteins were immunoprecipitated and probed for phosphotyrosine or with an anti–Vav3 pY173 antibody. (F) Rac1GTP was coprecipitated from lysates derived from wild-type or Sh3bp2–/– osteoclasts using immobilized recombinant Pak1-RBD protein and probed with an anti-Rac1 monoclonal antibody. (G) Sh3bp2–/– osteoclasts were infected by a retrovirus expressing either 3BP2 or Src Y527F. Empty vector was used as a control. Following infection, cells were cultured for 5 days and then stained for TRAP. (H) As in G, infected cells were plated on dentin and cultured for 10 days. Dentin chips were stained with Toluidine to reveal resorption pits. Original magnification, ×10 (G); ×5 (H). Data are presented as mean ± SEM.

Figure 6. Impaired osteoblast maturation in Sh3bp2–/– mice.

(A) In vitro osteoblast maturation assay. Bone marrow–derived stromal cells were cultured for 21 days and stained for ALP after 7, 14, or 21 days of culture. (B) Quantification of ALP activity after 14 days of culture. n = 9; *P < 0.05. (C) Collagen I deposition as determined by picric acid staining after 7, 14, and 21 days of culture. (D) Mineralized nodule formation as determined by von Kossa staining after 7, 14, and 21 days of culture. (E) FACS analysis of Sca-1+CD29+CD45–CD11b– skeletal stem cells from 12- to 13-week-old wild-type or Sh3bp2–/– mice. n = 7. (F) Quantification of CFU-F from wild-type and Sh3bp2–/– bone marrow–derived adherent cells cultured for 2 weeks. n = 12. (G) ALP staining of CFU-F cultured for 28 days. (H) Quantification of CFU-Ob from G. n = 6; *P < 0.05. (I) Immunofluorescence staining for Runx2 (red) or nuclei (DAPI). Original magnification, ×10. (J) Decreased mineralized nodules of Sh3bp2–/– calvarial-derived osteoblasts cultured for 21 days and stained with Alizarin red (K). Quantification of calvarial osteoblast mineralized nodules in I. n = 6; *P < 0.01. (L) Quantitative PCR of runx2, osterix, and osteocalcin transcript levels in calvarial osteoblasts cultured for 21 days. n = 3; *P < 0.05. Data are presented as mean ± SEM.

Figure 7. 3BP2 is required for Abl activation in osteoblasts.

(A) Abl and 3BP2 were immunoprecipitated and Western blotted, respectively, at various time points during differentiation of MC3T3 cells (n = 4). The blots were quantified by densitometry and relative values plotted. (B) Abl and 3BP2 were reciprocally coimmunoprecipitated from MC3T3 cells cultured for 14 days. Beads with nonrelevant polyclonal antibodies or isotype-matched monoclonal antibodies were used as controls. W.C.L., whole cell lysates; C, IgG control. (C) Lysates from MC3T3 infected with a 3BP2-expressing retroviral vector or an empty vector control were probed with an anti–Abl pY245 specific antibody. (D) Abl protein was immunoprecipitated from mineralizing wild-type or Sh3bp2–/– calvarial osteoblasts and probed with anti–Abl pY245 specific antibody. (E) Binding curve of the Abl SH3 domain to the FITC-conjugated 3BP2 diproline peptide, as measured by fluorescence polarization. 2 irrelevant 3BP2-derived peptides were used as negative controls. (F) The activity of recombinant SH3SH2 kinase Abl protein was measured in a continuous kinase assay in the presence of increasing concentrations of the 3BP2 diproline peptide or the Aprotinin-negative control peptide. The data in E and F are representative of 3 and 4 independent experiments, respectively. (G) Wild-type and Sh3bp2–/– calvarial-derived osteoblast progenitors were infected with the Abl-FKBP–expressing retroviral vector or an empty vector control and grown under osteogenic conditions in the presence of AP20187 for 28 days. Cells were then stained with alizarin red and quantified following elution (H), n = 18. Data are presented as mean ± SEM.