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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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Silencing of β‐catenin expression inhibit BMP‐9‐induced ectopic bone formation in vivo. (A) Knockdown of β‐catenin into C3H10T1/2 cells inhibits BMP‐9‐induced ectopic bone formation. C3H10T1/2 cells were co‐infected with AdBMP‐9 and varying titres of AdR‐simBC, or AdRFP for 15 hrs, and the co‐infection efficiency was examined under a fluorescence microscope. The infected cells were collected and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed and bony masses were retrieved. Representative gross images are shown (parts e to h). (B) BMP‐9‐induced bone formation in mouse embryonic fibroblasts (MEFs) is also inhibited by silencing β‐catenin expression. A similar set of experiments as described in (A) was carried out in MEFs. (C) MicroCT analysis. The retrieved masses from (A) were further subjected to microCT scanning. Representative reconstructed 3‐dimmensional images are shown. (D) Knockdown of β‐catenin reduces the volume of BMP‐9‐induced bone formation. Bone volume (mm3) was calculated based on microCT scanning data. The average bone volume of each group is shown. (E) The retrieved samples were decalcified and subjected to haematoxylin and eosin staining (parts a to d), Masson’s Trichrome staining (parts e to h) and Alcian Blue staining (parts i to l). Representative images are shown. For the Trichrome stain, decalcified ossified matrix stained dark red, whereas osteoid or cartilage matrix stained blue. For the Alcian Blue stain, cartilage stained blue. OB, osteoblast; OC, osteoid or cartilage‐like matrix; OM, ossified or mineralized matrix. Magnification, 150×.
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f6: Silencing of β‐catenin expression inhibit BMP‐9‐induced ectopic bone formation in vivo. (A) Knockdown of β‐catenin into C3H10T1/2 cells inhibits BMP‐9‐induced ectopic bone formation. C3H10T1/2 cells were co‐infected with AdBMP‐9 and varying titres of AdR‐simBC, or AdRFP for 15 hrs, and the co‐infection efficiency was examined under a fluorescence microscope. The infected cells were collected and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed and bony masses were retrieved. Representative gross images are shown (parts e to h). (B) BMP‐9‐induced bone formation in mouse embryonic fibroblasts (MEFs) is also inhibited by silencing β‐catenin expression. A similar set of experiments as described in (A) was carried out in MEFs. (C) MicroCT analysis. The retrieved masses from (A) were further subjected to microCT scanning. Representative reconstructed 3‐dimmensional images are shown. (D) Knockdown of β‐catenin reduces the volume of BMP‐9‐induced bone formation. Bone volume (mm3) was calculated based on microCT scanning data. The average bone volume of each group is shown. (E) The retrieved samples were decalcified and subjected to haematoxylin and eosin staining (parts a to d), Masson’s Trichrome staining (parts e to h) and Alcian Blue staining (parts i to l). Representative images are shown. For the Trichrome stain, decalcified ossified matrix stained dark red, whereas osteoid or cartilage matrix stained blue. For the Alcian Blue stain, cartilage stained blue. OB, osteoblast; OC, osteoid or cartilage‐like matrix; OM, ossified or mineralized matrix. Magnification, 150×.

Mentions: The above in vitro data demonstrated that canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. We sought to confirm these findings in vivo via stem cell implantation experiments. To knockdown β‐catenin expression in MSCs, we co‐infected C3H10T1/2 cells with AdBMP‐9 and different titres of AdR‐simBC (or AdRFP as controls) for 15 hrs (Fig. 6A, top row). The infected cells were collected and injected subcutaneously into athymic mice. At 5 weeks after injection, animals were killed, and the injection sites were retrieved. The gross appearance of the retrieved samples indicated that silencing β‐catenin expression inhibited the formation of ectopic bony masses in a dose‐dependent fashion (Fig. 6A, bottom row). A similar, if not more marked, inhibitory effect on bone formation was also observed in the implantation experiments using the MEF cells transduced with AdBMP‐9 and AdR‐simBC (Fig. 6B). When the ectopic bone formation was quantitatively analysed using microCT (Fig. 6C), silencing β‐catenin expression (at the high dose of AdR‐simBC) inhibited bone formation by 80% in C3H10T1/2 cells and 94% in MEFs, respectively (Fig. 6D). Further, consistent with our in vitro results, FrzB effectively inhibited BMP‐9‐induced ectopic bone formation in the MEF cells (Supporting Fig. 2C and D).


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)

Silencing of β‐catenin expression inhibit BMP‐9‐induced ectopic bone formation in vivo. (A) Knockdown of β‐catenin into C3H10T1/2 cells inhibits BMP‐9‐induced ectopic bone formation. C3H10T1/2 cells were co‐infected with AdBMP‐9 and varying titres of AdR‐simBC, or AdRFP for 15 hrs, and the co‐infection efficiency was examined under a fluorescence microscope. The infected cells were collected and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed and bony masses were retrieved. Representative gross images are shown (parts e to h). (B) BMP‐9‐induced bone formation in mouse embryonic fibroblasts (MEFs) is also inhibited by silencing β‐catenin expression. A similar set of experiments as described in (A) was carried out in MEFs. (C) MicroCT analysis. The retrieved masses from (A) were further subjected to microCT scanning. Representative reconstructed 3‐dimmensional images are shown. (D) Knockdown of β‐catenin reduces the volume of BMP‐9‐induced bone formation. Bone volume (mm3) was calculated based on microCT scanning data. The average bone volume of each group is shown. (E) The retrieved samples were decalcified and subjected to haematoxylin and eosin staining (parts a to d), Masson’s Trichrome staining (parts e to h) and Alcian Blue staining (parts i to l). Representative images are shown. For the Trichrome stain, decalcified ossified matrix stained dark red, whereas osteoid or cartilage matrix stained blue. For the Alcian Blue stain, cartilage stained blue. OB, osteoblast; OC, osteoid or cartilage‐like matrix; OM, ossified or mineralized matrix. Magnification, 150×.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940786&req=5

f6: Silencing of β‐catenin expression inhibit BMP‐9‐induced ectopic bone formation in vivo. (A) Knockdown of β‐catenin into C3H10T1/2 cells inhibits BMP‐9‐induced ectopic bone formation. C3H10T1/2 cells were co‐infected with AdBMP‐9 and varying titres of AdR‐simBC, or AdRFP for 15 hrs, and the co‐infection efficiency was examined under a fluorescence microscope. The infected cells were collected and subjected to subcutaneous injection into athymic mice. At 5 weeks after implantation, animals were killed and bony masses were retrieved. Representative gross images are shown (parts e to h). (B) BMP‐9‐induced bone formation in mouse embryonic fibroblasts (MEFs) is also inhibited by silencing β‐catenin expression. A similar set of experiments as described in (A) was carried out in MEFs. (C) MicroCT analysis. The retrieved masses from (A) were further subjected to microCT scanning. Representative reconstructed 3‐dimmensional images are shown. (D) Knockdown of β‐catenin reduces the volume of BMP‐9‐induced bone formation. Bone volume (mm3) was calculated based on microCT scanning data. The average bone volume of each group is shown. (E) The retrieved samples were decalcified and subjected to haematoxylin and eosin staining (parts a to d), Masson’s Trichrome staining (parts e to h) and Alcian Blue staining (parts i to l). Representative images are shown. For the Trichrome stain, decalcified ossified matrix stained dark red, whereas osteoid or cartilage matrix stained blue. For the Alcian Blue stain, cartilage stained blue. OB, osteoblast; OC, osteoid or cartilage‐like matrix; OM, ossified or mineralized matrix. Magnification, 150×.
Mentions: The above in vitro data demonstrated that canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. We sought to confirm these findings in vivo via stem cell implantation experiments. To knockdown β‐catenin expression in MSCs, we co‐infected C3H10T1/2 cells with AdBMP‐9 and different titres of AdR‐simBC (or AdRFP as controls) for 15 hrs (Fig. 6A, top row). The infected cells were collected and injected subcutaneously into athymic mice. At 5 weeks after injection, animals were killed, and the injection sites were retrieved. The gross appearance of the retrieved samples indicated that silencing β‐catenin expression inhibited the formation of ectopic bony masses in a dose‐dependent fashion (Fig. 6A, bottom row). A similar, if not more marked, inhibitory effect on bone formation was also observed in the implantation experiments using the MEF cells transduced with AdBMP‐9 and AdR‐simBC (Fig. 6B). When the ectopic bone formation was quantitatively analysed using microCT (Fig. 6C), silencing β‐catenin expression (at the high dose of AdR‐simBC) inhibited bone formation by 80% in C3H10T1/2 cells and 94% in MEFs, respectively (Fig. 6D). Further, consistent with our in vitro results, FrzB effectively inhibited BMP‐9‐induced ectopic bone formation in the MEF cells (Supporting Fig. 2C and D).

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