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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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Wnt antagonists inhibit BMP‐9‐induced osteogenic differentiation of MSCs and mouse embryonic fibroblasts (MEFs). (A) Effective transduction of C3H10T1/2 cells by adenoviral vectors expressing the Wnt signalling inhibitors DKK1, FrzB, sLRP‐5 and sLRP‐6. Left part: Subconfluent C3H10T1/2 cells were infected with a comparable titre of the four adenoviruses. At 36 hrs, infected cells were examined under a fluorescence microscope for GFP (Dkk1 and FrzB) or RFP (sLRP‐5 and sLRP‐6) expression. Right part: Subconfluent C3H10T1/2 cells were infected with the four Wnt inhibitor adenoviruses for 24 hrs, and stimulated with Wnt3A conditioned medium for another 12 hrs. Cells were fixed and subjected to immunofluorescence staining using an anti‐β‐catenin antibody. AdGFP infection and control IgG were used as controls. (B) Functional verification of adenoviral vectors expressing Wnt inhibitors. C3H10T1/2 cells were transfected with the Tcf4/LEF1 reporter TOP‐Luc and infected with increasing amounts of AdDkk1, AdFrzB, AdR‐sLRP‐5 and AdR‐sLRP‐6, along with AdWnt3A. At 36 hrs, cells were collected for luciferase assays. Data are present as mean ± S.D. (C) Effect of the Wnt inhibitors on BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. C3H10T1/2 cells were con‐infected with AdBMP‐9 and AdGFP, AdDkk1, AdFrzB, AdR‐sLRP‐5 or AdR‐sLRP‐6. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. (D) and (E) Effect of Dkk1 and FrzB on BMP‐9‐induced ALP activity in MEFs. Primary MEFs were co‐infected with AdBMP‐9 and AdGFP, or increasing titres of AdDkk1 or AdFrzB. ALP activities were quantitatively assessed at day 7 (Dkk1‐D7 and FrzB‐D7) and day 10 (Dkk1‐D10 and FrzB‐D10). Data are present as mean ± S.D. Histochemical staining of ALP activity (E) was done at 10 days after infection.
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f2: Wnt antagonists inhibit BMP‐9‐induced osteogenic differentiation of MSCs and mouse embryonic fibroblasts (MEFs). (A) Effective transduction of C3H10T1/2 cells by adenoviral vectors expressing the Wnt signalling inhibitors DKK1, FrzB, sLRP‐5 and sLRP‐6. Left part: Subconfluent C3H10T1/2 cells were infected with a comparable titre of the four adenoviruses. At 36 hrs, infected cells were examined under a fluorescence microscope for GFP (Dkk1 and FrzB) or RFP (sLRP‐5 and sLRP‐6) expression. Right part: Subconfluent C3H10T1/2 cells were infected with the four Wnt inhibitor adenoviruses for 24 hrs, and stimulated with Wnt3A conditioned medium for another 12 hrs. Cells were fixed and subjected to immunofluorescence staining using an anti‐β‐catenin antibody. AdGFP infection and control IgG were used as controls. (B) Functional verification of adenoviral vectors expressing Wnt inhibitors. C3H10T1/2 cells were transfected with the Tcf4/LEF1 reporter TOP‐Luc and infected with increasing amounts of AdDkk1, AdFrzB, AdR‐sLRP‐5 and AdR‐sLRP‐6, along with AdWnt3A. At 36 hrs, cells were collected for luciferase assays. Data are present as mean ± S.D. (C) Effect of the Wnt inhibitors on BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. C3H10T1/2 cells were con‐infected with AdBMP‐9 and AdGFP, AdDkk1, AdFrzB, AdR‐sLRP‐5 or AdR‐sLRP‐6. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. (D) and (E) Effect of Dkk1 and FrzB on BMP‐9‐induced ALP activity in MEFs. Primary MEFs were co‐infected with AdBMP‐9 and AdGFP, or increasing titres of AdDkk1 or AdFrzB. ALP activities were quantitatively assessed at day 7 (Dkk1‐D7 and FrzB‐D7) and day 10 (Dkk1‐D10 and FrzB‐D10). Data are present as mean ± S.D. Histochemical staining of ALP activity (E) was done at 10 days after infection.

Mentions: We sought to determine the effects of Wnt signalling inhibitors on BMP‐9‐induced osteogenic differentiation. To effectively introduce Wnt antagonists FrzB and DKK1 into MSCs, we previously constructed recombinant adenoviral vectors expressing FrzB or DKK1, along with the expression of a GFP marker [6, 16, 17]. We also constructed two adenoviral vectors that express the extracellular domains of human LRP‐5 and LRP‐6, namely sLRP‐5 and sLRP‐6, respectively, along with the expression of a RFP marker. The four adenoviral vectors were shown to effectively transduce C3H10T1/2 progenitor cells (Fig. 2A, left panel), and resulted in an apparent decrease of Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells (Fig. 2A, right panel). Furthermore, overexpression of the four inhibitors was shown to inhibit the Wnt3A‐induced β‐catenin/Tcf4 reporter activity in a dose‐dependent manner (Fig. 2B). These results demonstrated that adenovirus‐mediated expression of DKK1, FrzB, sLRP‐5 or sLRP‐6 effectively inhibits canonical Wnt/β‐catenin activity.


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)

Wnt antagonists inhibit BMP‐9‐induced osteogenic differentiation of MSCs and mouse embryonic fibroblasts (MEFs). (A) Effective transduction of C3H10T1/2 cells by adenoviral vectors expressing the Wnt signalling inhibitors DKK1, FrzB, sLRP‐5 and sLRP‐6. Left part: Subconfluent C3H10T1/2 cells were infected with a comparable titre of the four adenoviruses. At 36 hrs, infected cells were examined under a fluorescence microscope for GFP (Dkk1 and FrzB) or RFP (sLRP‐5 and sLRP‐6) expression. Right part: Subconfluent C3H10T1/2 cells were infected with the four Wnt inhibitor adenoviruses for 24 hrs, and stimulated with Wnt3A conditioned medium for another 12 hrs. Cells were fixed and subjected to immunofluorescence staining using an anti‐β‐catenin antibody. AdGFP infection and control IgG were used as controls. (B) Functional verification of adenoviral vectors expressing Wnt inhibitors. C3H10T1/2 cells were transfected with the Tcf4/LEF1 reporter TOP‐Luc and infected with increasing amounts of AdDkk1, AdFrzB, AdR‐sLRP‐5 and AdR‐sLRP‐6, along with AdWnt3A. At 36 hrs, cells were collected for luciferase assays. Data are present as mean ± S.D. (C) Effect of the Wnt inhibitors on BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. C3H10T1/2 cells were con‐infected with AdBMP‐9 and AdGFP, AdDkk1, AdFrzB, AdR‐sLRP‐5 or AdR‐sLRP‐6. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. (D) and (E) Effect of Dkk1 and FrzB on BMP‐9‐induced ALP activity in MEFs. Primary MEFs were co‐infected with AdBMP‐9 and AdGFP, or increasing titres of AdDkk1 or AdFrzB. ALP activities were quantitatively assessed at day 7 (Dkk1‐D7 and FrzB‐D7) and day 10 (Dkk1‐D10 and FrzB‐D10). Data are present as mean ± S.D. Histochemical staining of ALP activity (E) was done at 10 days after infection.
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f2: Wnt antagonists inhibit BMP‐9‐induced osteogenic differentiation of MSCs and mouse embryonic fibroblasts (MEFs). (A) Effective transduction of C3H10T1/2 cells by adenoviral vectors expressing the Wnt signalling inhibitors DKK1, FrzB, sLRP‐5 and sLRP‐6. Left part: Subconfluent C3H10T1/2 cells were infected with a comparable titre of the four adenoviruses. At 36 hrs, infected cells were examined under a fluorescence microscope for GFP (Dkk1 and FrzB) or RFP (sLRP‐5 and sLRP‐6) expression. Right part: Subconfluent C3H10T1/2 cells were infected with the four Wnt inhibitor adenoviruses for 24 hrs, and stimulated with Wnt3A conditioned medium for another 12 hrs. Cells were fixed and subjected to immunofluorescence staining using an anti‐β‐catenin antibody. AdGFP infection and control IgG were used as controls. (B) Functional verification of adenoviral vectors expressing Wnt inhibitors. C3H10T1/2 cells were transfected with the Tcf4/LEF1 reporter TOP‐Luc and infected with increasing amounts of AdDkk1, AdFrzB, AdR‐sLRP‐5 and AdR‐sLRP‐6, along with AdWnt3A. At 36 hrs, cells were collected for luciferase assays. Data are present as mean ± S.D. (C) Effect of the Wnt inhibitors on BMP‐9‐induced alkaline phosphatase (ALP) activity in MSCs. C3H10T1/2 cells were con‐infected with AdBMP‐9 and AdGFP, AdDkk1, AdFrzB, AdR‐sLRP‐5 or AdR‐sLRP‐6. ALP activities were quantitatively assessed at the indicated time‐points. Data are present as mean ± S.D. (D) and (E) Effect of Dkk1 and FrzB on BMP‐9‐induced ALP activity in MEFs. Primary MEFs were co‐infected with AdBMP‐9 and AdGFP, or increasing titres of AdDkk1 or AdFrzB. ALP activities were quantitatively assessed at day 7 (Dkk1‐D7 and FrzB‐D7) and day 10 (Dkk1‐D10 and FrzB‐D10). Data are present as mean ± S.D. Histochemical staining of ALP activity (E) was done at 10 days after infection.
Mentions: We sought to determine the effects of Wnt signalling inhibitors on BMP‐9‐induced osteogenic differentiation. To effectively introduce Wnt antagonists FrzB and DKK1 into MSCs, we previously constructed recombinant adenoviral vectors expressing FrzB or DKK1, along with the expression of a GFP marker [6, 16, 17]. We also constructed two adenoviral vectors that express the extracellular domains of human LRP‐5 and LRP‐6, namely sLRP‐5 and sLRP‐6, respectively, along with the expression of a RFP marker. The four adenoviral vectors were shown to effectively transduce C3H10T1/2 progenitor cells (Fig. 2A, left panel), and resulted in an apparent decrease of Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells (Fig. 2A, right panel). Furthermore, overexpression of the four inhibitors was shown to inhibit the Wnt3A‐induced β‐catenin/Tcf4 reporter activity in a dose‐dependent manner (Fig. 2B). These results demonstrated that adenovirus‐mediated expression of DKK1, FrzB, sLRP‐5 or sLRP‐6 effectively inhibits canonical Wnt/β‐catenin activity.

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