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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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Synergistic effect of canonical Wnt and BMP‐9 on osteogenic differentiation of mesenchymal stem cells (MSCs). (A) Early osteogenic marker alkaline phosphatase (ALP) is induced by Wnt3A or BMP‐9. MSC line C3H10T1/2 cells were infected with a comparable titre of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was detected histochemically at the indicated time‐points. (B) and (C) Time‐course comparison between Wnt3A and BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with a comparable titre (MOI = 30 pfu/cell) of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was quantitatively assessed using a colorimetric assay (see ‘Materials and methods’) at the indicated time‐points. The dotted lines indicate the same level of relative ALP activity. (D) and (E) Synergistic effects between Wnt3A and BMP‐9 on osteogenic differentiation in MSCs. C3H10T1/2 cells were infected with AdBMP‐9 or AdGFP (MOI = 10 pfu/cell) and various concentrations of Wnt3A conditioned medium (D), or infected with AdWnt3A or AdGFP (MOI = 10 pfu/cell) and various concentrations of BMP‐9 conditioned medium (E). ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. All ALP assays were carried out in triplicate, and confirmed in at least two independent experiments.
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f1: Synergistic effect of canonical Wnt and BMP‐9 on osteogenic differentiation of mesenchymal stem cells (MSCs). (A) Early osteogenic marker alkaline phosphatase (ALP) is induced by Wnt3A or BMP‐9. MSC line C3H10T1/2 cells were infected with a comparable titre of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was detected histochemically at the indicated time‐points. (B) and (C) Time‐course comparison between Wnt3A and BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with a comparable titre (MOI = 30 pfu/cell) of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was quantitatively assessed using a colorimetric assay (see ‘Materials and methods’) at the indicated time‐points. The dotted lines indicate the same level of relative ALP activity. (D) and (E) Synergistic effects between Wnt3A and BMP‐9 on osteogenic differentiation in MSCs. C3H10T1/2 cells were infected with AdBMP‐9 or AdGFP (MOI = 10 pfu/cell) and various concentrations of Wnt3A conditioned medium (D), or infected with AdWnt3A or AdGFP (MOI = 10 pfu/cell) and various concentrations of BMP‐9 conditioned medium (E). ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. All ALP assays were carried out in triplicate, and confirmed in at least two independent experiments.

Mentions: Although the exact mechanisms remain to be fully delineated, both Wnt and BMP signalling pathways are shown to regulate the proliferation and differentiation processes of MSCs [7]. As shown in Fig. 1A, overexpression of either canonical Wnt3A or BMP‐9 was shown to effectively induce ALP activity in mesenchymal C3H10T1/2 cells. We previously demonstrated that, among the 14 types of BMPs, BMP‐9 is one of the most osteogenic BMPs, and yet is one of the least studied [5, 6, 12, 13, 27, 60]. Through microarray expression profiling, we demonstrated that Wnt3A and BMP‐9 regulate a set of mutual target genes, such as CTGF/CCN2, in MSCs [6, 16, 17]. Nonetheless, it is conceivable that a distinct set of downstream mediators is required for either Wnt3A or BMP‐9 induced osteogenic differentiation. As shown in Fig. 1B and C, a detailed time‐course experiment revealed that Wnt3A induced a stronger and earlier ALP activity than that of BMP‐9. At day 3, Wnt3A‐induced ALP activity is approximately threefold the ALP activity of BMP‐9. Wnt3A induced ALP activity peaked at day 6 (Fig. 1B), whereas BMP‐9‐induced ALP activity peaked at day 8 (Fig. 1C). Wnt3A was shown to enhance BMP‐9‐induced ALP activity (Fig. 1D), or vice versa (Fig. 1E), although the synergistic effects decreased significantly at late time‐points (i.e. day 10). These results suggest that there may be crosstalk between the two pathways.


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)

Synergistic effect of canonical Wnt and BMP‐9 on osteogenic differentiation of mesenchymal stem cells (MSCs). (A) Early osteogenic marker alkaline phosphatase (ALP) is induced by Wnt3A or BMP‐9. MSC line C3H10T1/2 cells were infected with a comparable titre of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was detected histochemically at the indicated time‐points. (B) and (C) Time‐course comparison between Wnt3A and BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with a comparable titre (MOI = 30 pfu/cell) of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was quantitatively assessed using a colorimetric assay (see ‘Materials and methods’) at the indicated time‐points. The dotted lines indicate the same level of relative ALP activity. (D) and (E) Synergistic effects between Wnt3A and BMP‐9 on osteogenic differentiation in MSCs. C3H10T1/2 cells were infected with AdBMP‐9 or AdGFP (MOI = 10 pfu/cell) and various concentrations of Wnt3A conditioned medium (D), or infected with AdWnt3A or AdGFP (MOI = 10 pfu/cell) and various concentrations of BMP‐9 conditioned medium (E). ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. All ALP assays were carried out in triplicate, and confirmed in at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

f1: Synergistic effect of canonical Wnt and BMP‐9 on osteogenic differentiation of mesenchymal stem cells (MSCs). (A) Early osteogenic marker alkaline phosphatase (ALP) is induced by Wnt3A or BMP‐9. MSC line C3H10T1/2 cells were infected with a comparable titre of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was detected histochemically at the indicated time‐points. (B) and (C) Time‐course comparison between Wnt3A and BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with a comparable titre (MOI = 30 pfu/cell) of AdWnt3A, AdBMP‐9 or AdGFP control (not shown). ALP activity was quantitatively assessed using a colorimetric assay (see ‘Materials and methods’) at the indicated time‐points. The dotted lines indicate the same level of relative ALP activity. (D) and (E) Synergistic effects between Wnt3A and BMP‐9 on osteogenic differentiation in MSCs. C3H10T1/2 cells were infected with AdBMP‐9 or AdGFP (MOI = 10 pfu/cell) and various concentrations of Wnt3A conditioned medium (D), or infected with AdWnt3A or AdGFP (MOI = 10 pfu/cell) and various concentrations of BMP‐9 conditioned medium (E). ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. All ALP assays were carried out in triplicate, and confirmed in at least two independent experiments.
Mentions: Although the exact mechanisms remain to be fully delineated, both Wnt and BMP signalling pathways are shown to regulate the proliferation and differentiation processes of MSCs [7]. As shown in Fig. 1A, overexpression of either canonical Wnt3A or BMP‐9 was shown to effectively induce ALP activity in mesenchymal C3H10T1/2 cells. We previously demonstrated that, among the 14 types of BMPs, BMP‐9 is one of the most osteogenic BMPs, and yet is one of the least studied [5, 6, 12, 13, 27, 60]. Through microarray expression profiling, we demonstrated that Wnt3A and BMP‐9 regulate a set of mutual target genes, such as CTGF/CCN2, in MSCs [6, 16, 17]. Nonetheless, it is conceivable that a distinct set of downstream mediators is required for either Wnt3A or BMP‐9 induced osteogenic differentiation. As shown in Fig. 1B and C, a detailed time‐course experiment revealed that Wnt3A induced a stronger and earlier ALP activity than that of BMP‐9. At day 3, Wnt3A‐induced ALP activity is approximately threefold the ALP activity of BMP‐9. Wnt3A induced ALP activity peaked at day 6 (Fig. 1B), whereas BMP‐9‐induced ALP activity peaked at day 8 (Fig. 1C). Wnt3A was shown to enhance BMP‐9‐induced ALP activity (Fig. 1D), or vice versa (Fig. 1E), although the synergistic effects decreased significantly at late time‐points (i.e. day 10). These results suggest that there may be crosstalk between the two pathways.

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