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The Wnt/beta-catenin pathway interacts differentially with PTHrP signaling to control chondrocyte hypertrophy and final maturation.

Guo X, Mak KK, Taketo MM, Yang Y - PLoS ONE (2009)

Bottom Line: Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling.Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression.In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics Section, National Human Genome Research Institute, Bethesda, MD, USA.

ABSTRACT
Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through beta-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling. Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/beta-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.

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Analysis of chondrocyte hypertrophy in β-catenin and Smo mutant embryos.(A) Skeletal preparation of 18.5 dpc embryos. Mineralization (stained by Alizarin red) in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was more advanced than that in the Catnbc/c; Col2a1-Cre mutant embryo, but less advanced than that in the Smoc/c; Col2a1-Cre mutant embryo. (B) Sections of the developing distal tibia at 15.5 dpc were examined by in situ hybridization with PTHrP probes. PTHrP expression was slightly reduced only in the periarticular chondrocytes in Catnbc/c; Col2a1-Cre mutant embryos compared to that in wild type embryos. Catnbc/c; Smoc/c; Col2a1-Cre and Smoc/c; Col2a1-Cre embryos have similar expression of PTHrP. (C) Safranin O staining and Col10a1 expression in the developing humerus at 15.5 dpc. Non-hypertrophic chondrocyte region (white line) was expanded in the Catnbc/c; Col2a1-Cre mutant, but reduced in the Smoc/c; Col2a1-Cre mutant embryo, compared that in the wild type control. Chondrocyte hypertrophy in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was delayed than that in the Smoc/c; Col2a1-Cre mutant embryo, but accelerated than that in the Catnbc/c; Col2a1-Cre mutant embryo.
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pone-0006067-g002: Analysis of chondrocyte hypertrophy in β-catenin and Smo mutant embryos.(A) Skeletal preparation of 18.5 dpc embryos. Mineralization (stained by Alizarin red) in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was more advanced than that in the Catnbc/c; Col2a1-Cre mutant embryo, but less advanced than that in the Smoc/c; Col2a1-Cre mutant embryo. (B) Sections of the developing distal tibia at 15.5 dpc were examined by in situ hybridization with PTHrP probes. PTHrP expression was slightly reduced only in the periarticular chondrocytes in Catnbc/c; Col2a1-Cre mutant embryos compared to that in wild type embryos. Catnbc/c; Smoc/c; Col2a1-Cre and Smoc/c; Col2a1-Cre embryos have similar expression of PTHrP. (C) Safranin O staining and Col10a1 expression in the developing humerus at 15.5 dpc. Non-hypertrophic chondrocyte region (white line) was expanded in the Catnbc/c; Col2a1-Cre mutant, but reduced in the Smoc/c; Col2a1-Cre mutant embryo, compared that in the wild type control. Chondrocyte hypertrophy in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was delayed than that in the Smoc/c; Col2a1-Cre mutant embryo, but accelerated than that in the Catnbc/c; Col2a1-Cre mutant embryo.

Mentions: The above data suggest that active PTHrP signaling is required for Wnt/β-catenin signaling to regulate chondrocyte hypertrophy, but PTHrP signaling may not require β-catenin to regulate chondrocyte hypertrophy. This predicts that in the absence of β-catenin, PTHrP signaling levels can still determine the rate of chondrocyte hypertrophy. To test this, we need to partially reduce PTHrP signaling by reducing PTHrP expression. In this case, β-catenin removal should still enhance the residual PTHrP signaling, which will lead to a delay in chondrocyte hypertrophy. To achieve these genetic manipulations, we partially reduced PTHrP expression by partially reducing Ihh signaling as Ihh controls PTHrP expression. Ihh signaling was reduced by removing Smoothened (Smo), which encodes the signaling receptor for Ihh, in the cartilage in the Smoc/c; Col2a1-Cre embryo [17] and Fig. 2A). PTHrP expression was diminished in the cartilage, but still detectable in the joint region in the Smoc/c; Col2a1-Cre embryo (Fig. 2B). Chondrocyte hypertrophy was accelerated in the Smoc/c; Col2a1-Cre embryo, as indicated by both Safranin O staining and analysis of Col2a1 and Col10a1 expression (Fig. 2C). When β-catenin was also removed in the Smoc/c; Catnbc/c; Col2a1-Cre double mutant embryo, chondrocyte hypertrophy was delayed compared to that in the Smoc/c; Col2a1-Cre embryos (Fig. 2C), despite PTHrP expression levels being similarly reduced in both (Fig. 2B). In addition, we observed previously that when PTHrP expression was similarly upregulated in Ptch1c/−; Col2a1-Cre and Ptch1c/−; Catnbc/−; Col2a1-Cre embryos, there was a further delay of chondrocyte hypertrophy when β-catenin is removed [12]. Thus, in the absence of β-catenin, PTHrP signaling still inhibits chondrocyte hypertrophy in a dose-dependent manner, whereas in the absence of PTHrP signaling, β-catenin has no effect on chondrocyte hypertrophy.


The Wnt/beta-catenin pathway interacts differentially with PTHrP signaling to control chondrocyte hypertrophy and final maturation.

Guo X, Mak KK, Taketo MM, Yang Y - PLoS ONE (2009)

Analysis of chondrocyte hypertrophy in β-catenin and Smo mutant embryos.(A) Skeletal preparation of 18.5 dpc embryos. Mineralization (stained by Alizarin red) in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was more advanced than that in the Catnbc/c; Col2a1-Cre mutant embryo, but less advanced than that in the Smoc/c; Col2a1-Cre mutant embryo. (B) Sections of the developing distal tibia at 15.5 dpc were examined by in situ hybridization with PTHrP probes. PTHrP expression was slightly reduced only in the periarticular chondrocytes in Catnbc/c; Col2a1-Cre mutant embryos compared to that in wild type embryos. Catnbc/c; Smoc/c; Col2a1-Cre and Smoc/c; Col2a1-Cre embryos have similar expression of PTHrP. (C) Safranin O staining and Col10a1 expression in the developing humerus at 15.5 dpc. Non-hypertrophic chondrocyte region (white line) was expanded in the Catnbc/c; Col2a1-Cre mutant, but reduced in the Smoc/c; Col2a1-Cre mutant embryo, compared that in the wild type control. Chondrocyte hypertrophy in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was delayed than that in the Smoc/c; Col2a1-Cre mutant embryo, but accelerated than that in the Catnbc/c; Col2a1-Cre mutant embryo.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2698152&req=5

pone-0006067-g002: Analysis of chondrocyte hypertrophy in β-catenin and Smo mutant embryos.(A) Skeletal preparation of 18.5 dpc embryos. Mineralization (stained by Alizarin red) in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was more advanced than that in the Catnbc/c; Col2a1-Cre mutant embryo, but less advanced than that in the Smoc/c; Col2a1-Cre mutant embryo. (B) Sections of the developing distal tibia at 15.5 dpc were examined by in situ hybridization with PTHrP probes. PTHrP expression was slightly reduced only in the periarticular chondrocytes in Catnbc/c; Col2a1-Cre mutant embryos compared to that in wild type embryos. Catnbc/c; Smoc/c; Col2a1-Cre and Smoc/c; Col2a1-Cre embryos have similar expression of PTHrP. (C) Safranin O staining and Col10a1 expression in the developing humerus at 15.5 dpc. Non-hypertrophic chondrocyte region (white line) was expanded in the Catnbc/c; Col2a1-Cre mutant, but reduced in the Smoc/c; Col2a1-Cre mutant embryo, compared that in the wild type control. Chondrocyte hypertrophy in the Catnbc/c; Smoc/c; Col2a1-Cre double mutant embryo was delayed than that in the Smoc/c; Col2a1-Cre mutant embryo, but accelerated than that in the Catnbc/c; Col2a1-Cre mutant embryo.
Mentions: The above data suggest that active PTHrP signaling is required for Wnt/β-catenin signaling to regulate chondrocyte hypertrophy, but PTHrP signaling may not require β-catenin to regulate chondrocyte hypertrophy. This predicts that in the absence of β-catenin, PTHrP signaling levels can still determine the rate of chondrocyte hypertrophy. To test this, we need to partially reduce PTHrP signaling by reducing PTHrP expression. In this case, β-catenin removal should still enhance the residual PTHrP signaling, which will lead to a delay in chondrocyte hypertrophy. To achieve these genetic manipulations, we partially reduced PTHrP expression by partially reducing Ihh signaling as Ihh controls PTHrP expression. Ihh signaling was reduced by removing Smoothened (Smo), which encodes the signaling receptor for Ihh, in the cartilage in the Smoc/c; Col2a1-Cre embryo [17] and Fig. 2A). PTHrP expression was diminished in the cartilage, but still detectable in the joint region in the Smoc/c; Col2a1-Cre embryo (Fig. 2B). Chondrocyte hypertrophy was accelerated in the Smoc/c; Col2a1-Cre embryo, as indicated by both Safranin O staining and analysis of Col2a1 and Col10a1 expression (Fig. 2C). When β-catenin was also removed in the Smoc/c; Catnbc/c; Col2a1-Cre double mutant embryo, chondrocyte hypertrophy was delayed compared to that in the Smoc/c; Col2a1-Cre embryos (Fig. 2C), despite PTHrP expression levels being similarly reduced in both (Fig. 2B). In addition, we observed previously that when PTHrP expression was similarly upregulated in Ptch1c/−; Col2a1-Cre and Ptch1c/−; Catnbc/−; Col2a1-Cre embryos, there was a further delay of chondrocyte hypertrophy when β-catenin is removed [12]. Thus, in the absence of β-catenin, PTHrP signaling still inhibits chondrocyte hypertrophy in a dose-dependent manner, whereas in the absence of PTHrP signaling, β-catenin has no effect on chondrocyte hypertrophy.

Bottom Line: Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling.Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression.In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics Section, National Human Genome Research Institute, Bethesda, MD, USA.

ABSTRACT
Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through beta-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/beta-catenin and PTHrP signaling. Wnt/beta-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/beta-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/beta-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.

Show MeSH
Related in: MedlinePlus