3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions
Noam Levaot, … , Ernst Reichenberger, Robert Rottapel
Noam Levaot, … , Ernst Reichenberger, Robert Rottapel
Published July 18, 2011
Citation Information: J Clin Invest. 2011;121(8):3244-3257. https://doi.org/10.1172/JCI45843.
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Research Article Bone biology

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.

Authors

Noam Levaot, Paul D. Simoncic, Ioannis D. Dimitriou, Andrew Scotter, Jose La Rose, Adeline H.M. Ng, Thomas L. Willett, Chiachien J. Wang, Salima Janmohamed, Marc Grynpas, Ernst Reichenberger, Robert Rottapel

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Figure 7

3BP2 is required for Abl activation in osteoblasts.

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3BP2 is required for Abl activation in osteoblasts. (A) Abl and 3BP2 wer...
(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.