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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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β‐Catenin also plays an important role in BMP‐9‐induced late stage of osteogenic differentiation. (A) Synergistic effect between β‐catenin and BMP‐9 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and infected with varying titres of Adβ‐Cat*, in the presence or absence of BMP‐9 conditioned medium. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (B) Synergistic effect between β‐catenin and Runx2 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and co‐infected with varying titres of Adβ‐Cat* and/or AdRunx2. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (C) Knockdown of β‐catenin inhibits BMP‐9‐induced activation of osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc and infected with varying titres of AdR‐simBC in the presence of BMP‐9 conditioned medium. Cells were collected for luciferase at the indicated time‐points. (D) Knockdown of β‐catenin inhibits BMP‐9‐induced expression of late osteogenic markers. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdR‐simBC or AdGFP for 10 days. Total RNA was isolated for RT‐PCR and qPCR analysis using primers specific for mouse osteocalcin and osteopontin. Experiments were done in triplicate. (E) Silencing of β‐catenin and FrzB overexpression inhibit BMP‐9‐induced mineralization. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdFrzB, AdR‐simBC, or AdGFP. At 14 and 21 days after infection, cells were fixed and subjected to Alizarin Red S staining. Representative images are shown (magnification, 40×).
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f5: β‐Catenin also plays an important role in BMP‐9‐induced late stage of osteogenic differentiation. (A) Synergistic effect between β‐catenin and BMP‐9 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and infected with varying titres of Adβ‐Cat*, in the presence or absence of BMP‐9 conditioned medium. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (B) Synergistic effect between β‐catenin and Runx2 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and co‐infected with varying titres of Adβ‐Cat* and/or AdRunx2. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (C) Knockdown of β‐catenin inhibits BMP‐9‐induced activation of osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc and infected with varying titres of AdR‐simBC in the presence of BMP‐9 conditioned medium. Cells were collected for luciferase at the indicated time‐points. (D) Knockdown of β‐catenin inhibits BMP‐9‐induced expression of late osteogenic markers. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdR‐simBC or AdGFP for 10 days. Total RNA was isolated for RT‐PCR and qPCR analysis using primers specific for mouse osteocalcin and osteopontin. Experiments were done in triplicate. (E) Silencing of β‐catenin and FrzB overexpression inhibit BMP‐9‐induced mineralization. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdFrzB, AdR‐simBC, or AdGFP. At 14 and 21 days after infection, cells were fixed and subjected to Alizarin Red S staining. Representative images are shown (magnification, 40×).

Mentions: Although ALP is a well‐established early osteogenic marker, it is hardly an accurate predictor of the late stage of osteogenic differentiation and bone formation [6, 7, 12, 13]. Thus, we sought to determine whether β‐catenin played any role in the BMP‐9‐induced late stage of osteogenic differentiation. Osteocalcin and osteopontin are well‐characterized markers of late osteogenesis [6, 7, 12, 13]. Using a commonly used Runx2‐regulated osteocalcin promoter reporter [63], we found that overexpression of the stabilized β‐catenin enhanced BMP‐9‐induced reporter activity (Fig. 5A). Osteocalcin is a known target of the osteogenic regulator Runx2 [66, 67], and overexpression of the stabilized β‐catenin acted synergistically on Runx2‐induced osteocalcin reporter activity (Fig. 5B). Conversely, silencing β‐catenin expression resulted in a drastic decrease in BMP‐9‐stimulated osteocalcin promoter activity (Fig. 5C). Quantitative PCR further confirmed that knockdown of the β‐catenin expression inhibited BMP‐9‐induced expression of the late osteogenic markers osteocalcin and osteopontin in C3H10T1/2 cells (Fig. 5D). Accordingly, either overexpression of FrzB or knockdown of β‐catenin expression was shown to effectively inhibit BMP‐9‐induced expression of osteocalcin and osteopontin in MEF cells (Supporting Fig. S2A). Furthermore, we found that either overexpression of FrzB or knockdown of β‐catenin expression effectively inhibited the BMP‐9‐induced in vitro mineralization in C3H10T1/2 cells (Fig. 5E) and in MEF cells (Supporting Fig. S2B). These findings suggest that canonical Wnt/β‐catenin signalling may play an important role in regulating both early and late stages of BMP‐9‐induced osteogenic differentiation.


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)

β‐Catenin also plays an important role in BMP‐9‐induced late stage of osteogenic differentiation. (A) Synergistic effect between β‐catenin and BMP‐9 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and infected with varying titres of Adβ‐Cat*, in the presence or absence of BMP‐9 conditioned medium. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (B) Synergistic effect between β‐catenin and Runx2 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and co‐infected with varying titres of Adβ‐Cat* and/or AdRunx2. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (C) Knockdown of β‐catenin inhibits BMP‐9‐induced activation of osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc and infected with varying titres of AdR‐simBC in the presence of BMP‐9 conditioned medium. Cells were collected for luciferase at the indicated time‐points. (D) Knockdown of β‐catenin inhibits BMP‐9‐induced expression of late osteogenic markers. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdR‐simBC or AdGFP for 10 days. Total RNA was isolated for RT‐PCR and qPCR analysis using primers specific for mouse osteocalcin and osteopontin. Experiments were done in triplicate. (E) Silencing of β‐catenin and FrzB overexpression inhibit BMP‐9‐induced mineralization. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdFrzB, AdR‐simBC, or AdGFP. At 14 and 21 days after infection, cells were fixed and subjected to Alizarin Red S staining. Representative images are shown (magnification, 40×).
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f5: β‐Catenin also plays an important role in BMP‐9‐induced late stage of osteogenic differentiation. (A) Synergistic effect between β‐catenin and BMP‐9 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and infected with varying titres of Adβ‐Cat*, in the presence or absence of BMP‐9 conditioned medium. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (B) Synergistic effect between β‐catenin and Runx2 on osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc reporter and co‐infected with varying titres of Adβ‐Cat* and/or AdRunx2. At 48 hrs, cells were collected for luciferase assay. Data are present as mean ± S.D. (C) Knockdown of β‐catenin inhibits BMP‐9‐induced activation of osteocalcin promoter reporter. C3H10T1/2 cells were transfected with p6OSE2‐Luc and infected with varying titres of AdR‐simBC in the presence of BMP‐9 conditioned medium. Cells were collected for luciferase at the indicated time‐points. (D) Knockdown of β‐catenin inhibits BMP‐9‐induced expression of late osteogenic markers. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdR‐simBC or AdGFP for 10 days. Total RNA was isolated for RT‐PCR and qPCR analysis using primers specific for mouse osteocalcin and osteopontin. Experiments were done in triplicate. (E) Silencing of β‐catenin and FrzB overexpression inhibit BMP‐9‐induced mineralization. C3H10T1/2 cells were co‐infected with AdBMP‐9 and AdFrzB, AdR‐simBC, or AdGFP. At 14 and 21 days after infection, cells were fixed and subjected to Alizarin Red S staining. Representative images are shown (magnification, 40×).
Mentions: Although ALP is a well‐established early osteogenic marker, it is hardly an accurate predictor of the late stage of osteogenic differentiation and bone formation [6, 7, 12, 13]. Thus, we sought to determine whether β‐catenin played any role in the BMP‐9‐induced late stage of osteogenic differentiation. Osteocalcin and osteopontin are well‐characterized markers of late osteogenesis [6, 7, 12, 13]. Using a commonly used Runx2‐regulated osteocalcin promoter reporter [63], we found that overexpression of the stabilized β‐catenin enhanced BMP‐9‐induced reporter activity (Fig. 5A). Osteocalcin is a known target of the osteogenic regulator Runx2 [66, 67], and overexpression of the stabilized β‐catenin acted synergistically on Runx2‐induced osteocalcin reporter activity (Fig. 5B). Conversely, silencing β‐catenin expression resulted in a drastic decrease in BMP‐9‐stimulated osteocalcin promoter activity (Fig. 5C). Quantitative PCR further confirmed that knockdown of the β‐catenin expression inhibited BMP‐9‐induced expression of the late osteogenic markers osteocalcin and osteopontin in C3H10T1/2 cells (Fig. 5D). Accordingly, either overexpression of FrzB or knockdown of β‐catenin expression was shown to effectively inhibit BMP‐9‐induced expression of osteocalcin and osteopontin in MEF cells (Supporting Fig. S2A). Furthermore, we found that either overexpression of FrzB or knockdown of β‐catenin expression effectively inhibited the BMP‐9‐induced in vitro mineralization in C3H10T1/2 cells (Fig. 5E) and in MEF cells (Supporting Fig. S2B). These findings suggest that canonical Wnt/β‐catenin signalling may play an important role in regulating both early and late stages of BMP‐9‐induced osteogenic differentiation.

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