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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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BMP‐9‐induced recruitment of both Runx2 and β‐catenin to osteocalcin promoter. (A) Schematic features of the genomic arrangement of the mouse osteocalcin promoter. The boxed regions represent exons and the filled boxes represent the coding region. The filled circles represent putative Runx2 binding sites, while the open circle represents the putative LEF1/Tcf4 binding site. CP‐1 and CP‐2 are the PCR primer pairs used for chromatin immunoprecipitation (ChIP) analysis. (B) Efficient transduction of MSCs by AdBMP‐9, AdβCat* and AdGFP for ChIP assay. Subconfluent C3H10T1/2 cells were infected with AdBMP‐9, AdβCat* or AdGFP. Expression of GFP was examined at 36 hrs under a fluorescence microscope, immediately prior to ChIP analysis. (C) Both BMP‐9 and β‐catenin induce recruitment of Runx2 to OC promoter. The infected cells were cross‐linked, sonicated and subjected to ChIP using a Runx2 antibody (Santa Cruz Biotechnology). The precipitated DNA‐protein complexes were de‐crosslinked and processed for PCR analyses using both pairs of primers listed in Fig. 7A. (D) BMP‐9 induces recruitment of β‐catenin to OC promoter. A similar ChIP procedure was carried out as described above, except that a β‐catenin antibody was used for immunoprecipitation. (E) Equal amount of DNA‐protein complex input was used for each ChIP sample. The arrows indicate the expected PCR products, while the asterisks denote the primer dimmers of PCR reactions. These results are representative of three independent experiments.
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f7: BMP‐9‐induced recruitment of both Runx2 and β‐catenin to osteocalcin promoter. (A) Schematic features of the genomic arrangement of the mouse osteocalcin promoter. The boxed regions represent exons and the filled boxes represent the coding region. The filled circles represent putative Runx2 binding sites, while the open circle represents the putative LEF1/Tcf4 binding site. CP‐1 and CP‐2 are the PCR primer pairs used for chromatin immunoprecipitation (ChIP) analysis. (B) Efficient transduction of MSCs by AdBMP‐9, AdβCat* and AdGFP for ChIP assay. Subconfluent C3H10T1/2 cells were infected with AdBMP‐9, AdβCat* or AdGFP. Expression of GFP was examined at 36 hrs under a fluorescence microscope, immediately prior to ChIP analysis. (C) Both BMP‐9 and β‐catenin induce recruitment of Runx2 to OC promoter. The infected cells were cross‐linked, sonicated and subjected to ChIP using a Runx2 antibody (Santa Cruz Biotechnology). The precipitated DNA‐protein complexes were de‐crosslinked and processed for PCR analyses using both pairs of primers listed in Fig. 7A. (D) BMP‐9 induces recruitment of β‐catenin to OC promoter. A similar ChIP procedure was carried out as described above, except that a β‐catenin antibody was used for immunoprecipitation. (E) Equal amount of DNA‐protein complex input was used for each ChIP sample. The arrows indicate the expected PCR products, while the asterisks denote the primer dimmers of PCR reactions. These results are representative of three independent experiments.

Mentions: It has been reported that there is crosstalk between Wnt and TGF‐β/BMP signalling pathways [7, 68, 69, 70]. We have demonstrated that β‐catenin acts synergistically with BMP‐9 or Runx2 on regulating osteocalcin promoter (Fig. 5A and B). It has been reported that Runx2 may physically interact with β‐catenin and Tcf/Lef1 complex [71, 72]. We asked whether BMP‐9 induced the recruitment of Runx2 and β‐catenin to the osteocalcin promoter. As illustrated in Fig. 7A, mouse osteocalcin promoter region contains at least four putative Runx2 binding sites and one putative LEF1/Tcf4 binding site, most of which are located within the proximal 1 kb region of the promoter. To assess the in vivo promoter binding activity, we carried out ChIP assays using the C3H10T1/2 cells infected with AdBMP‐9, Adβ‐catenin* or AdGFP (Fig. 7B). The presence of osteocalcin promoter fragments in the immunoprecipitated complexes was determined by semi‐quantitative PCR analysis using two pairs of primers (Fig. 7A). As expected, BMP‐9 stimulation induced the Runx2 binding to osteocalcin promoter (Fig. 7C, first lane), while the basal Runx2 binding to osteocalcin promoter was undetectable (Fig. 7C, third lane). Interestingly, expression of the stabilized β‐catenin also induced the Runx2 binding to osteocalcin promoter (Fig. 7C, second lane), suggesting that β‐catenin may facilitate the recruitment of Runx2 to osteocalcin promoter. Reciprocal immunoprecipitation using an anti‐β‐catenin antibody further demonstrated that BMP‐9 stimulation induced the recruitment of β‐catenin/Tcf complex to osteocalcin promoter (Fig. 7D). These results were reproducible in at least three independent experiments, and the initial input for each ChIP assay was comparable (Fig. 7E). These results suggest that β‐catenin may coordinate with Runx2 in BMP‐9‐regulated osteogenic signalling in pre‐osteoblast progenitors.


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)

BMP‐9‐induced recruitment of both Runx2 and β‐catenin to osteocalcin promoter. (A) Schematic features of the genomic arrangement of the mouse osteocalcin promoter. The boxed regions represent exons and the filled boxes represent the coding region. The filled circles represent putative Runx2 binding sites, while the open circle represents the putative LEF1/Tcf4 binding site. CP‐1 and CP‐2 are the PCR primer pairs used for chromatin immunoprecipitation (ChIP) analysis. (B) Efficient transduction of MSCs by AdBMP‐9, AdβCat* and AdGFP for ChIP assay. Subconfluent C3H10T1/2 cells were infected with AdBMP‐9, AdβCat* or AdGFP. Expression of GFP was examined at 36 hrs under a fluorescence microscope, immediately prior to ChIP analysis. (C) Both BMP‐9 and β‐catenin induce recruitment of Runx2 to OC promoter. The infected cells were cross‐linked, sonicated and subjected to ChIP using a Runx2 antibody (Santa Cruz Biotechnology). The precipitated DNA‐protein complexes were de‐crosslinked and processed for PCR analyses using both pairs of primers listed in Fig. 7A. (D) BMP‐9 induces recruitment of β‐catenin to OC promoter. A similar ChIP procedure was carried out as described above, except that a β‐catenin antibody was used for immunoprecipitation. (E) Equal amount of DNA‐protein complex input was used for each ChIP sample. The arrows indicate the expected PCR products, while the asterisks denote the primer dimmers of PCR reactions. These results are representative of three independent experiments.
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Related In: Results  -  Collection

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f7: BMP‐9‐induced recruitment of both Runx2 and β‐catenin to osteocalcin promoter. (A) Schematic features of the genomic arrangement of the mouse osteocalcin promoter. The boxed regions represent exons and the filled boxes represent the coding region. The filled circles represent putative Runx2 binding sites, while the open circle represents the putative LEF1/Tcf4 binding site. CP‐1 and CP‐2 are the PCR primer pairs used for chromatin immunoprecipitation (ChIP) analysis. (B) Efficient transduction of MSCs by AdBMP‐9, AdβCat* and AdGFP for ChIP assay. Subconfluent C3H10T1/2 cells were infected with AdBMP‐9, AdβCat* or AdGFP. Expression of GFP was examined at 36 hrs under a fluorescence microscope, immediately prior to ChIP analysis. (C) Both BMP‐9 and β‐catenin induce recruitment of Runx2 to OC promoter. The infected cells were cross‐linked, sonicated and subjected to ChIP using a Runx2 antibody (Santa Cruz Biotechnology). The precipitated DNA‐protein complexes were de‐crosslinked and processed for PCR analyses using both pairs of primers listed in Fig. 7A. (D) BMP‐9 induces recruitment of β‐catenin to OC promoter. A similar ChIP procedure was carried out as described above, except that a β‐catenin antibody was used for immunoprecipitation. (E) Equal amount of DNA‐protein complex input was used for each ChIP sample. The arrows indicate the expected PCR products, while the asterisks denote the primer dimmers of PCR reactions. These results are representative of three independent experiments.
Mentions: It has been reported that there is crosstalk between Wnt and TGF‐β/BMP signalling pathways [7, 68, 69, 70]. We have demonstrated that β‐catenin acts synergistically with BMP‐9 or Runx2 on regulating osteocalcin promoter (Fig. 5A and B). It has been reported that Runx2 may physically interact with β‐catenin and Tcf/Lef1 complex [71, 72]. We asked whether BMP‐9 induced the recruitment of Runx2 and β‐catenin to the osteocalcin promoter. As illustrated in Fig. 7A, mouse osteocalcin promoter region contains at least four putative Runx2 binding sites and one putative LEF1/Tcf4 binding site, most of which are located within the proximal 1 kb region of the promoter. To assess the in vivo promoter binding activity, we carried out ChIP assays using the C3H10T1/2 cells infected with AdBMP‐9, Adβ‐catenin* or AdGFP (Fig. 7B). The presence of osteocalcin promoter fragments in the immunoprecipitated complexes was determined by semi‐quantitative PCR analysis using two pairs of primers (Fig. 7A). As expected, BMP‐9 stimulation induced the Runx2 binding to osteocalcin promoter (Fig. 7C, first lane), while the basal Runx2 binding to osteocalcin promoter was undetectable (Fig. 7C, third lane). Interestingly, expression of the stabilized β‐catenin also induced the Runx2 binding to osteocalcin promoter (Fig. 7C, second lane), suggesting that β‐catenin may facilitate the recruitment of Runx2 to osteocalcin promoter. Reciprocal immunoprecipitation using an anti‐β‐catenin antibody further demonstrated that BMP‐9 stimulation induced the recruitment of β‐catenin/Tcf complex to osteocalcin promoter (Fig. 7D). These results were reproducible in at least three independent experiments, and the initial input for each ChIP assay was comparable (Fig. 7E). These results suggest that β‐catenin may coordinate with Runx2 in BMP‐9‐regulated osteogenic signalling in pre‐osteoblast progenitors.

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