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 5

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

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Integrin-mediated activation of Src is defective in Sh3bp2–/– osteoclast...
(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.