Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of β‐catenin inhibits BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs and mouse embryonic fibroblasts (MEFs). (A) Construction and verification of siRNAs targeting mouse β‐catenin expression, simBC. The three listed candidate siRNA sites were validated using our recently developed pSOS system and subcloned into an adenoviral shuttle vector. The resultant adenovirus AdR‐simBC, which also expresses RFP, was used to infect C3H10T1/2 cells. The infection efficiency was assessed under a fluorescence microscope. With immunofluorescence stain using an anti‐β‐catenin antibody, AdR‐simBC effectively knocked down Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells. BF, bright field. (B) AdR‐simBC effectively inhibits Tcf4/LEF1 reporter activity. C3H10T1/2 cells were transfected with TOP‐Luc reporter and infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. At 36 hrs, cells were collected for luciferase assay. (C) Silencing of β‐catenin inhibits Wnt3A‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (D) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (E) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MEFs. Primary MEFs were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. All data are present as mean ± S.D.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4940786&req=5

f4: Knockdown of β‐catenin inhibits BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs and mouse embryonic fibroblasts (MEFs). (A) Construction and verification of siRNAs targeting mouse β‐catenin expression, simBC. The three listed candidate siRNA sites were validated using our recently developed pSOS system and subcloned into an adenoviral shuttle vector. The resultant adenovirus AdR‐simBC, which also expresses RFP, was used to infect C3H10T1/2 cells. The infection efficiency was assessed under a fluorescence microscope. With immunofluorescence stain using an anti‐β‐catenin antibody, AdR‐simBC effectively knocked down Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells. BF, bright field. (B) AdR‐simBC effectively inhibits Tcf4/LEF1 reporter activity. C3H10T1/2 cells were transfected with TOP‐Luc reporter and infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. At 36 hrs, cells were collected for luciferase assay. (C) Silencing of β‐catenin inhibits Wnt3A‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (D) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (E) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MEFs. Primary MEFs were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. All data are present as mean ± S.D.

Mentions: We next sought to determine the importance of β‐catenin in BMP‐9‐induced osteogenic differentiation by silencing β‐catenin expression in MSCs. Using our recently developed pSOS system [56], we selected and validated three siRNA sites targeting the coding region of mouse β‐catenin (Fig. 4A). An adenovirus pool that expresses all three siRNA sites was then generated. The resultant AdR‐simBC, which also expresses RFP marker, was shown to effectively transduce C3H10T1/2 progenitor cells (Fig. 4A). AdR‐simBC was shown to inhibit Wnt3A conditioned medium‐induced nuclear accumulation of β‐catenin protein (Fig. 4A), as well as to inhibit Wnt3A‐induced β‐catenin/Tcf4 transcriptional activation in a dose‐dependent manner (Fig. 4B). These results demonstrated that silencing β‐catenin expression was achieved by adenovirus‐mediated expression of β‐catenin siRNAs.


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)

Knockdown of β‐catenin inhibits BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs and mouse embryonic fibroblasts (MEFs). (A) Construction and verification of siRNAs targeting mouse β‐catenin expression, simBC. The three listed candidate siRNA sites were validated using our recently developed pSOS system and subcloned into an adenoviral shuttle vector. The resultant adenovirus AdR‐simBC, which also expresses RFP, was used to infect C3H10T1/2 cells. The infection efficiency was assessed under a fluorescence microscope. With immunofluorescence stain using an anti‐β‐catenin antibody, AdR‐simBC effectively knocked down Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells. BF, bright field. (B) AdR‐simBC effectively inhibits Tcf4/LEF1 reporter activity. C3H10T1/2 cells were transfected with TOP‐Luc reporter and infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. At 36 hrs, cells were collected for luciferase assay. (C) Silencing of β‐catenin inhibits Wnt3A‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (D) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (E) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MEFs. Primary MEFs were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. All data are present as mean ± S.D.
© Copyright Policy
Related In: Results  -  Collection

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

f4: Knockdown of β‐catenin inhibits BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity in MSCs and mouse embryonic fibroblasts (MEFs). (A) Construction and verification of siRNAs targeting mouse β‐catenin expression, simBC. The three listed candidate siRNA sites were validated using our recently developed pSOS system and subcloned into an adenoviral shuttle vector. The resultant adenovirus AdR‐simBC, which also expresses RFP, was used to infect C3H10T1/2 cells. The infection efficiency was assessed under a fluorescence microscope. With immunofluorescence stain using an anti‐β‐catenin antibody, AdR‐simBC effectively knocked down Wnt3A‐induced β‐catenin accumulation in C3H10T1/2 cells. BF, bright field. (B) AdR‐simBC effectively inhibits Tcf4/LEF1 reporter activity. C3H10T1/2 cells were transfected with TOP‐Luc reporter and infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. At 36 hrs, cells were collected for luciferase assay. (C) Silencing of β‐catenin inhibits Wnt3A‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of Wnt3A conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (D) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MSCs. C3H10T1/2 cells were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. (E) Silencing of β‐catenin inhibits BMP‐9‐induced ALP activity in MEFs. Primary MEFs were infected with varying titres of AdR‐simBC, in the presence or absence of BMP‐9 conditioned medium. ALP activities were quantitatively assessed at the indicated time‐points. All data are present as mean ± S.D.
Mentions: We next sought to determine the importance of β‐catenin in BMP‐9‐induced osteogenic differentiation by silencing β‐catenin expression in MSCs. Using our recently developed pSOS system [56], we selected and validated three siRNA sites targeting the coding region of mouse β‐catenin (Fig. 4A). An adenovirus pool that expresses all three siRNA sites was then generated. The resultant AdR‐simBC, which also expresses RFP marker, was shown to effectively transduce C3H10T1/2 progenitor cells (Fig. 4A). AdR‐simBC was shown to inhibit Wnt3A conditioned medium‐induced nuclear accumulation of β‐catenin protein (Fig. 4A), as well as to inhibit Wnt3A‐induced β‐catenin/Tcf4 transcriptional activation in a dose‐dependent manner (Fig. 4B). These results demonstrated that silencing β‐catenin expression was achieved by adenovirus‐mediated expression of β‐catenin siRNAs.

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