Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Feb 1;94(2):403-18.
doi: 10.1002/jcb.20253.

Beta-catenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation

Gabriel Mbalaviele  1 , Sharmin Sheikh, Joseph P Stains, Valerie S Salazar, Su-Li Cheng, Di Chen, Roberto Civitelli
Affiliations

Beta-catenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation

Gabriel Mbalaviele et al. J Cell Biochem. .

Abstract

Mutations of critical components of the Wnt pathway profoundly affect skeletal development and maintenance, probably via modulation of beta-catenin signaling. We tested the hypothesis that beta-catenin is involved in mesenchymal lineage allocation to osteogenic cells using a beta-catenin mutant with constitutive transcriptional activity (DeltaN151). Although this stable beta-catenin had no effects by itself on osteogenic differentiation of multipotent embryonic cell lines, it synergized with bone morphogenetic protein-2 (BMP-2) resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression, and matrix mineralization. Likewise, DeltaN151 and BMP-2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, DeltaN151 prevented adipogenic differentiation from pre-adipocytic or uncommitted mesenchymal cells in vitro. Intriguingly, the synergism with BMP-2 on gene transcription occurred without altering expression of Cbfa1/Runx2, suggesting actions independent or downstream of this osteoblast-specific transcription factor. Thus, beta-catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP-2, in part via Tcf/Lef dependent mechanisms. In vivo, this synergism leads to increased new bone formation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Stable β-catenin synergizes with BMP-2 in inducing osteogenesis of C3H10T1/2 cells without affecting cell proliferation. Western analysis of whole cell lysates of C3H10T1/2 cells transduced with a retroviral vector carrying either ΔN151 or LacZ, showing a strong reactive band corresponding to the truncated, ΔN151 β-catenin mutant selectively in virally transduced cells (A, inset). This band matches the specific band detected in the lysate from the packaging cell line, 293GPG cells transfected with a ΔN151 expression construct. Cells transduced with either LacZ or ΔN151 retroviral vectors were cultured for 7 days (A) or 14 days (B) in the absence or in the presence of different concentrations of BMP-2, and alkaline phosphatase activity was determined as a marker of osteogenic commitment. Retrovirally transduced cells were plated onto 96-well dishes at low density and cultured for 48 h before labeling with 5-bromo-2′-deoxy-uridine (BrdU) as an index of cell proliferation (C). *P <0.05 vs. 0; #P <0.05 vs. LacZ (t test for unpaired samples).
Fig. 2
Fig. 2
Stable β-catenin and BMP-2 synergize to induce matrix mineralization by C3H10T1/2 cells. Cells transduced with retroviral vectors carrying either LacZ or ΔN151 were grown to confluence in 24-well dishes and cultured for 14 days in the presence or in the absence of 100 ng/ml BMP-2, and then stained with either alizarin red or Von Kossa as markers of mineralized matrix, as indicated. Note the alizarin red stain covering about 50% of the wells in cultures transduced with ΔN151 treated with BMP-2 (J), with calcified “nodules” visible in the micrographs (K, L). While minimal calcification occurred in LacZ-transduced cells treated with BMP-2 (D, E, F), no alizarin red or Von Kossa stain was detected in either LacZ (A, B, C) or ΔN151 (G, H, I) transduced cultures in the absence of BMP-2. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 3
Fig. 3
Stable β-catenin and BMP-2 synergize to induce osteogenic commitment by C2C12 cells. Cells transduced with retroviral vectors carrying either LacZ or ΔN151 were cultured for 7 days in the presence or in the absence of 100 ng/ml BMP-2 to induce osteogenic commitment that was monitored by development of alkaline phosphatase activity (A). Shown in the inset is a Western blot using an anti-β-catenin antibody in whole cell lysates of LcaZ or ΔN151 transduced cell, demonstrating efficient expression of the truncated β-catenin mutant. C2C12 cells transduced with either LacZ or ΔN151 retroviral vectors were grown to confluence in 24-well dishes and cultured for 14 days in the presence or in the absence of 100 ng/ml BMP-2, as indicated, and then stained with alizarin red. Note the extensive alizarin red stain covering most of the well surface in cultures transduced with ΔN151 and treated with BMP-2 (H), whereas substantially less calcification is present in control, LacZ-transduced cells treated with BMP-2 (D). Also note intense, diffuse red stain in the micrographs, and many dome shaped nodules with a dark red stained center (E, I). Extremely faint alizarin red stain was detected in either LacZ (B) or ΔN151 (F) in the absence of BMP-2. Microscopic examination reveals no extracellular staining and a spindle, elongated cell shape in confluent areas (C, I). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 4
Fig. 4
Stable β-catenin prevents adipogenic differentiation from pre-adipocytic 3T3-L1 and uncommitted C3H10T1/2 cells. Whole cell lysates were obtained from confluent 3T3-L1 cells transduced with retroviral vectors carrying either LacZ or ΔN151, and subjected to Western analysis using an anti-β-catenin antibody (A). Note the doublet band corresponding to the truncated β-catenin mutant only in the ΔN151 transduced cells. Cells grown to confluence in 24-well dishes were cultured for further 7 days in the absence or in the presence of an adipogenic cocktail, and then stained with Oil Red O as marker of adipogenic cells. Note the bright red stain covering the entire wells in 3T3-E1 cultures transduced with LacZ treated with the adipogenic cocktail (D). Almost all cells in these cultures contain red lipid droplets (E). Less intense but evident stain is present in the C3H10T1/2 well (K). Conversely, no lipid containing cells were detected in both cell lines, transduced with either LacZ (B, C, J), or ΔN151 (F, G, L) vectors in the absence of adipogenic cocktail. Cultures transduced with ΔN151 showed only a small number of Oil Red O positive cells scattered throughout culture (H, I, M). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 5
Fig. 5
Stable β-catenin does not trans-differentiate adipogenic committed cells. C3H10T1/2 cells were transduced with retroviral vectors carrying either LacZ or ΔN151, and incubated in the absence or in the presence of an adipogenic cocktail for 9 days. They were then exposed to 100 ng/ml BMP-2 or vehicle, as indicated for 14 days, and processed for alizarin red stain. Note the much stronger red stain in ΔN151 transduced and BMP-2 treated cells relative to control, LacZ transduced cells, and the relatively much lower intensity of the red stain in BMP-2 treated cells pre-exposed to the adipogenic cocktail in both cell transfectants. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 6
Fig. 6
Active β-catenin promotes bone formation in vivo. A solution containing LacZ or ΔN151 was injected subcutaneously over the parietal bone of the calvariae of 1 month-old mice daily for 5 days in the absence or presence of 10 μg/kg/day BMP-2, as indicated. Mice (n =5 per group) were sacrificed 21 days later, bones sections were stained, and thickness of the new bone (delimited by a subperiosteal cement line) was measured. Transduction of ΔN151 by itself did not induce detectable changes relative to LacZ transduction (A, C), but it markedly enhanced de novo bone formation stimulated by BMP-2 (B, D, E). *P <0.05 vs. control (Ct); #P <0.05 vs. LacZ+BMP-2 (t test for unpaired samples). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 7
Fig. 7
The osteogenic stimulation by β-catenin involves Tcf/Lef activity. C3H10T1/2 cells transduced with either LacZ or ΔN151 retroviral vectors were transfected with pcDNA3 or dominant negative XTcf-3 (DN XTcf3), grown to confluence, and co-transfected with the TOPFLASH and a renilla-luciferase reporter constructs (A). Promoter activities are expressed as firefly/renilla chemiluminescence ratio. Note that ΔN151 activates TOPFLASH, an effect that is inhibited by DN XTcf-3. Cells transduced with ΔN151 were transfected (48 h later) with either pcDNA3 or DN XTcf3, cultured for 14 days in the presence or in the absence of 100 ng/ml BMP-2, and stained for alizarin red (B). Note that alizarin red staining in cultures transduced with ΔN151 and treated with BMP-2 is partially inhibited by DN XTcf-3. *P <0.02 vs. LacZ (t-test for unpaired samples). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Fig. 8
Fig. 8
Active β-catenin and BMP-2 synergistically up-regulate the osteocalcin gene independent of Osterix and Cbfa1/Runx2. C3H10T1/2 cells transduced with retroviral vectors carrying either LacZ or ΔN151 were cultured for 7 days in the absence or presence of 100 ng/ml BMP-2, as indicated. Total RNA extracts were used for real-time PCR, as detailed under Materials and Methods, using specific primers to amplify osteocalcin (A). Data are expressed as the abundance of mRNA for each PCR product relative to that of GAPDH. Cells were transduced with either LacZ or DN151 retroviral vectors, grown to confluence, and transfected with either a rat osteocalcin promoter-luciferase reporter (OCN-Luc; B), TOPFLASH (C), or Smad binding element-luciferase construct (SBE-Luc; D), in the absence or presence of 100 ng/ml BMP-2 for 72 h. A renilla luciferase reporter construct was also co-transfected to control for transfection efficiency, and promoter activities are expressed as firefly/renilla chemiluminescence ratio. Note that ΔN151 synergizes with BMP-2 on the osteocalcin promoter, but the synergism does not occur on the TOPFLASH or the SBE-Luc elements. C3H10T1/2 cultured as just described were processed for real time PCR detection of Osterix mRNA (E), or for immunoblotting using an anti-Cbfa1 antibody (F). The membrane was washed and re-blotted using an anti-GAPDH antibody to control for loading. Note that while ΔN151 did not affect Osterix mRNA or Cbfa1/Runx2 protein expression. *P <0.05 vs. LacZ or ΔN151; #P <0.05 vs. LacZ and ΔN151 (t test for unpaired samples).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H, Gross G. Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol. 1993;12:871–880. - PubMed
    1. Bain G, Muller T, Wang X, Papkoff J. Activated β-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction. Biochem Biophys Res Commun. 2003;301:84–91. - PubMed
    1. Barolo S, Posakony JW. Three habits of highly effective signaling pathways: Principles of transcriptional control by developmental cell signaling. Genes Dev. 2002;16:1167–1181. - PubMed
    1. Barth AI, Pollack AL, Altschuler Y, Mostov KE, Nelson WJ. NH2-terminal deletion of β-catenin results in stable colocalization of mutant β-catenin with adenomatous polyposis coli protein and altered MDCK cell adhesion. J Cell Biol. 1997;136:693–706. - PMC - PubMed
    1. Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med. 2002;346:1513–1521. - PubMed

Publication types

MeSH terms

LinkOut - more resources