Abstract
Src-null mice have higher bone mass because of decreased bone resorption and increased bone formation, whereas Abl-null mice are osteopenic, because of decreased bone formation. Compound I, a potent inhibitor of Src in an isolated enzyme assay (IC50 0.55 nM) and a Src-dependent cell growth assay, with lower activity on equivalent Abl-based assays, potently, but biphasically, accelerated differentiation of human mesenchymal stem cells to an osteoblast phenotype (1–10 nM). Compound I (≥0.1 nM) also activated osteoblasts and induced bone formation in isolated neonatal mouse calvariae. Compound I required higher concentrations (100 nM) to inhibit differentiation and activity of osteoclasts. Transcriptional profiling (TxP) of calvaria treated with 1 μM compound I revealed down-regulation of osteoclastic genes and up-regulation of matrix genes and genes associated with the osteoblast phenotype, confirming compound I's dual effects on bone resorption and formation. In addition, calvarial TxP implicated calcitonin-related polypeptide, β (β-CGRP) as a potential mediator of compound I's osteogenic effect. In vivo, compound I (1 mg/kg s.c.) increased vertebral trabecular bone volume 21% (microcomputed tomography) in intact female mice. Increased trabecular volume was also detected histologically in a separate bone, the femur, particularly in the secondary spongiosa (100% increase), which underwent a 171% increase in bone formation rate, a 73% increase in mineralizing surface, and a 59% increase in mineral apposition rate. Similar effects were observed in ovariectomized mice with established osteopenia. We conclude that the Src inhibitor compound I is osteogenic, presumably because of its potent stimulation of osteoblast differentiation and activation, possibly mediated by β-CGRP.
Footnotes
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- RANK
- receptor activator of nuclear factor κ-B
- rhRANKL
- recombinant human RANK ligand
- DMSO
- dimethyl sulfoxide
- BSA
- bovine serum albumin
- CT
- computed tomography
- μCT
- microCT
- rhM-CSF
- recombinant human macrophage colony-stimulating factor
- hMSC
- human mesenchymal stem cells
- 4-MUP
- 4-methylumbelliferyl phosphate
- OVX
- ovariectomized
- TRAP
- tartrate-resistant acid phosphatase
- ANOVA
- analysis of variance
- PTH
- parathyroid hormone
- β-CGRP
- calcitonin-related polypeptide, β
- SFK
- Src family kinase
- IL
- interleukin
- GSEA
- gene set enrichment analysis
- ES
- enrichment score
- OPG
- osteoprotegerin
- OCP
- osteoclast precursor
- BV
- bone volume
- TV
- total volume
- BS
- bone surface
- Tb.Th
- trabecular thickness
- Tb.N
- trabecular number
- Tb.Sp
- trabecular separation
- Conn.Den
- connectivity density
- GPCR
- G protein-coupled receptor.
- Received July 5, 2011.
- Accepted December 6, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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