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. 2008 Mar;14(3):306-14.
doi: 10.1038/nm1716. Epub 2008 Feb 24.

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation

Matthew J Hilton  1 , Xiaolin Tu, Ximei Wu, Shuting Bai, Haibo Zhao, Tatsuya Kobayashi, Henry M Kronenberg, Steven L Teitelbaum, F Patrick Ross, Raphael Kopan, Fanxin Long
Affiliations

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation

Matthew J Hilton et al. Nat Med. 2008 Mar.

Abstract

Postnatal bone marrow houses mesenchymal progenitor cells that are osteoblast precursors. These cells have established therapeutic potential, but they are difficult to maintain and expand in vitro, presumably because little is known about the mechanisms controlling their fate decisions. To investigate the potential role of Notch signaling in osteoblastogenesis, we used conditional alleles to genetically remove components of the Notch signaling system during skeletal development. We found that disruption of Notch signaling in the limb skeletogenic mesenchyme markedly increased trabecular bone mass in adolescent mice. Notably, mesenchymal progenitors were undetectable in the bone marrow of mice with high bone mass. As a result, these mice developed severe osteopenia as they aged. Moreover, Notch signaling seemed to inhibit osteoblast differentiation through Hes or Hey proteins, which diminished Runx2 transcriptional activity via physical interaction. These results support a model wherein Notch signaling in bone marrow normally acts to maintain a pool of mesenchymal progenitors by suppressing osteoblast differentiation. Thus, mesenchymal progenitors may be expanded in vitro by activating the Notch pathway, whereas bone formation in vivo may be enhanced by transiently suppressing this pathway.

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Figures

Figure 1
Figure 1
Skeletal phenotype of PPS mice at 8 weeks of age. (a) X-ray radiographs of hindlimbs. Red double-headed arrows denote length of tibia. Green arrowheads point to trabecular bone region. (b) Medial, longitudinal section through 3-D reconstruction of the tibia by μCT. Double-headed arrow: expanded growth plate; asterisk: excessive bone; arrow: normal growth plate. (c) H&E staining of medial longitudinal sections through the tibia. (d-h) Higher magnification of boxed areas in c. 2°: secondary ossification center; NH: nonhypertrophic region; H: hypertrophic region; TB: trabecular bone; M: marrow.
Figure 2
Figure 2
Skeletal phenotype of PNN mice at 8 weeks of age. (a) X-ray radiographs of hindlimbs. Red double-headed arrows denote length of tibia. Green arrowheads point to trabecular bone region. (b) Medial longitudinal section through 3-D reconstruction of the tibia by μCT. Double-headed arrow: expanded growth plate; asterisk: excessive bone; arrow: normal growth plate. (c) H&E staining of medial longitudinal sections through the tibia. (d) Higher magnification of boxed areas in c from top to bottom shown in left-to-right order. 2°: secondary ossification center; NH: nonhypertrophic region; H: hypertrophic region; TB: trabecular bone; M: marrow. (e) TRAP staining on medial longitudinal sections through the tibia. Osteoclasts stain red. (f) Higher magnification of boxed areas in e, number of osteoclasts normalized to trabecular bone perimeter (No. OC/mm), and osteoclast surface normalized to bone surface (OC.S./B.S.). (g) Area micrographs from longitudinal sections of tibia after calcein double labeling, and percentage of calcein double-labeled bone surface over total bone surface (Dls/Tbs) in trabecular bone region. (h) Number of osteoblasts per trabecular bone area on sections. Cuboidal and flat osteoblasts were scored separately. *: p<0.05, n=3.
Figure 3
Figure 3
Molecular and histological analyses in PNN and wild type (WT) embryos. (a) H&E staining of medial longitudinal sections through the tibia of E18.5 embryos with various combinations of Notch1 (N1) and Notch2 (N2) alleles. Insets: higher magnification of boxed regions: M: marrow; arrowheads: occasional myeloid cells. Double-headed arrows: hypertrophic zone. (b) In situ hybridization on adjacent longitudinal sections through the tibia from E18.5 wild type (WT) versus PNN littermates. Double-headed arrows denote length of expression domain; yellow contours demarcate chondro-osseous junction; arrows indicate last low of hypertrophic chondrocytes. (c) H&E staining and in situ hybridization of adjacent medial longitudinal sections through the tibia of E14.5 littermates. Double-headed arrows: lengths of hypertrophic zones or expression domains. Yellow arrowheads denote major expression domains of Ihh.
Figure 4
Figure 4
Notch regulation of bone marrow mesenchymal progenitors. (a) Bone marrow CFU-F assays for wild type (WT) versus PNN mutant littermates. (b) AP staining after 8 days in osteogenic medium following CFU-F assays. (c) AP quantitative assay for high-density BMSCs cultures in osteogenic medium. (d) Real-time PCR of osteoblast markers in BMSCs from 15-week-old mice cultured for 10 days in regular medium. (e) Phase-contrast pictures of high-density BMSCs cultures in adipogenic medium. Note lipid droplets only in wild type cells. (f) CFU-F assays for wild type BMSCs with or without 1 μM DAPT. Arrow denotes a typical “type I” CFU-F. (g) AP staining immediately following CFU-F assays of wild type BMSCs in f. (h) Representative “type II” CFU-Fs from g. *: p<0.05, n=3.
Figure 5
Figure 5
Progressive bone loss in mature PNN mice. (a) 3-D reconstruction of metaphyseal trabecular bone in wild type (WT) versus PNN mice at different ages. (b) H&E staining of medial longitudinal sections through the tibia of 26-week-old mice. Arrows: bone trabeculae. Right panels: higher magnification of boxed regions in corresponding left panels. Double-lined arrows: chondrocytes. Vertical lines denote height of growth plate. Asterisk: area devoid of chondrocytes.
Figure 6
Figure 6
Mechanisms for Notch function in bone. (a) Western analyses for Notch1 and 2 in protein extracts from tibiae and femora of 8-week-old PNN versus wild type (WT) mice. Signal intensity normalized to α-tubulin. (b) Real-time PCR with RNA from tibiae of 8-week-old mice. (c) Real-time PCR with RNA from BMSCs of 15-week-old mice. (d-f) Luciferase reporter assays in CHO cells. *p < 0.01, ** p = 0.05, n=3, p values in f calculated against samples transfected with pCS2+MT empty vector and pCMV-Runx2 (black bar). (g) Co-immunoprecipitation of Hes1 or Hey1 with Runx2 in ST2 cells.
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References

    1. Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells. 2001;19:180–92. - PubMed
    1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–6. - PubMed
    1. Chiba S. Notch signaling in stem cell systems. Stem Cells. 2006;24:2437–47. - PubMed
    1. Schroeter EH, Kisslinger JA, Kopan R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature. 1998;393:382–6. - PubMed
    1. Honjo T. The shortest path from the surface to the nucleus: RBP-J kappa/Su(H) transcription factor. Genes Cells. 1996;1:1–9. - PubMed

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