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BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling.

Tang N, Song WX, Luo J, Luo X, Chen J, Sharff KA, Bi Y, He BC, Huang JY, Zhu GH, Su YX, Jiang W, Tang M, He Y, Wang Y, Chen L, Zuo GW, Shen J, Pan X, Reid RR, Luu HH, Haydon RC, He TC - J. Cell. Mol. Med. (2008)

Bottom Line: Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs).Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers.Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix.

View Article: PubMed Central - PubMed

Affiliation: The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China.

ABSTRACT
Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.

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Overexpression of a stabilized β‐catenin enhances BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. (A) Qualitative assessment of β‐catenin‐mediated augmentation of BMP‐9‐induced ALP activity. C3H10T1/2 cells were infected with varying titres of Adβ‐Cat* and/or AdBMP‐9. ALP activities were histochemically stained at 10 days after infection. (B) Quantitative assessment of β‐catenin‐mediated enhancement of BMP‐9‐induced ALP activity. C3H10T1/2 cells were co‐infected with a fixed titre of AdBMP‐9 and varying titres of Adβ‐Cat*. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D.
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f3: Overexpression of a stabilized β‐catenin enhances BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. (A) Qualitative assessment of β‐catenin‐mediated augmentation of BMP‐9‐induced ALP activity. C3H10T1/2 cells were infected with varying titres of Adβ‐Cat* and/or AdBMP‐9. ALP activities were histochemically stained at 10 days after infection. (B) Quantitative assessment of β‐catenin‐mediated enhancement of BMP‐9‐induced ALP activity. C3H10T1/2 cells were co‐infected with a fixed titre of AdBMP‐9 and varying titres of Adβ‐Cat*. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D.

Mentions: β‐Catenin is considered as the essential signalling mediator of the canonical Wnt pathway [27, 34, 40, 62, 64]. We examined whether overexpression of the stabilized β‐catenin exerted any effects on BMP‐9‐induced osteogenic differentiation. We previously constructed a recombinant adenoviral vector that expresses a stabilized form (S33Y) of human β‐catenin [17, 61, 65]. Overexpression of the stabilized β‐catenin alone in C3H10T1/2 resulted in a weak but detectable increase in ALP activity (Fig. 3A, top row). However, BMP‐9‐induced ALP activity was drastically enhanced by the overexpression of the stabilized β‐catenin (Fig. 3A, bottom row). A quantitative analysis of BMP‐9‐induced ALP activity further confirmed that β‐catenin can potentiate BMP‐9‐induced osteogenic differentiation of MSCs in a dose‐dependent manner (Fig. 3B).


BMP-9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signalling.

Tang N, Song WX, Luo J, Luo X, Chen J, Sharff KA, Bi Y, He BC, Huang JY, Zhu GH, Su YX, Jiang W, Tang M, He Y, Wang Y, Chen L, Zuo GW, Shen J, Pan X, Reid RR, Luu HH, Haydon RC, He TC - J. Cell. Mol. Med. (2008)

Overexpression of a stabilized β‐catenin enhances BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. (A) Qualitative assessment of β‐catenin‐mediated augmentation of BMP‐9‐induced ALP activity. C3H10T1/2 cells were infected with varying titres of Adβ‐Cat* and/or AdBMP‐9. ALP activities were histochemically stained at 10 days after infection. (B) Quantitative assessment of β‐catenin‐mediated enhancement of BMP‐9‐induced ALP activity. C3H10T1/2 cells were co‐infected with a fixed titre of AdBMP‐9 and varying titres of Adβ‐Cat*. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940786&req=5

f3: Overexpression of a stabilized β‐catenin enhances BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. (A) Qualitative assessment of β‐catenin‐mediated augmentation of BMP‐9‐induced ALP activity. C3H10T1/2 cells were infected with varying titres of Adβ‐Cat* and/or AdBMP‐9. ALP activities were histochemically stained at 10 days after infection. (B) Quantitative assessment of β‐catenin‐mediated enhancement of BMP‐9‐induced ALP activity. C3H10T1/2 cells were co‐infected with a fixed titre of AdBMP‐9 and varying titres of Adβ‐Cat*. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D.
Mentions: β‐Catenin is considered as the essential signalling mediator of the canonical Wnt pathway [27, 34, 40, 62, 64]. We examined whether overexpression of the stabilized β‐catenin exerted any effects on BMP‐9‐induced osteogenic differentiation. We previously constructed a recombinant adenoviral vector that expresses a stabilized form (S33Y) of human β‐catenin [17, 61, 65]. Overexpression of the stabilized β‐catenin alone in C3H10T1/2 resulted in a weak but detectable increase in ALP activity (Fig. 3A, top row). However, BMP‐9‐induced ALP activity was drastically enhanced by the overexpression of the stabilized β‐catenin (Fig. 3A, bottom row). A quantitative analysis of BMP‐9‐induced ALP activity further confirmed that β‐catenin can potentiate BMP‐9‐induced osteogenic differentiation of MSCs in a dose‐dependent manner (Fig. 3B).

Bottom Line: Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs).Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers.Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix.

View Article: PubMed Central - PubMed

Affiliation: The Second Affiliated Hospital and the Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, China.

ABSTRACT
Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.

Show MeSH
Related in: MedlinePlus