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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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β-catenin may control chondrocyte hypertrophy non-cell autonomously.Tamoxifen was injected into pregnant females at 13.5 dpc. Embryos were harvested at 17.5 dpc and sections of the proximal tibia were analyzed by Safranin O staining, in situ hybridization with the indicated probes and immnohistochemistry. (A) Upregulated Wnt/β-catenin signaling was indicated by ectopic Lef1 expression and loss of Safranin O staining and Col2a1 expression (arrow). Pictures of higher magnification of the boxed area are shown as insets. Col10a1 expression was not ectopically detected in chondrocytes that had lost Col2a1 expression. (B) Increased p-Smad1/5/8 staining (red) was observed in chondrocyte patches of Catnbex3/+; Col2a1-CreER mouse embryos. Such increase in p-Smad1/5/8 staining was in the area with increased β-catenin staining (green). But p-Smad1/5/8 and β-catenin staining did not colocalize in many chondrocytes. Some cells with increased p-Smad1/5/8 staining (arrows) did not show increased β-catenin staining. (C) Distinct genetic interactions between Wnt/β-catenin and PTHrP signaling in regulating chondrocyte hypertrophy and maturation. The sequential process of chondrocyte hypertrophy and maturation in developing wild type and indicated mutant long bone cartilage were shown in the diagram in the upper panel. Hypertrophic chondrocytes are enlarged and undergo final maturation before they are replaced by the trabecular bone. Initiation of chondrocyte hypertrophy and final maturation are two separate processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling controls chondrocyte hypertrophy by inhibiting PTHrP signaling activity. However, Wnt/β-catenin signaling promoted final maturation of hypertrophic chondrocyte independently of PTHrP signaling.
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pone-0006067-g004: β-catenin may control chondrocyte hypertrophy non-cell autonomously.Tamoxifen was injected into pregnant females at 13.5 dpc. Embryos were harvested at 17.5 dpc and sections of the proximal tibia were analyzed by Safranin O staining, in situ hybridization with the indicated probes and immnohistochemistry. (A) Upregulated Wnt/β-catenin signaling was indicated by ectopic Lef1 expression and loss of Safranin O staining and Col2a1 expression (arrow). Pictures of higher magnification of the boxed area are shown as insets. Col10a1 expression was not ectopically detected in chondrocytes that had lost Col2a1 expression. (B) Increased p-Smad1/5/8 staining (red) was observed in chondrocyte patches of Catnbex3/+; Col2a1-CreER mouse embryos. Such increase in p-Smad1/5/8 staining was in the area with increased β-catenin staining (green). But p-Smad1/5/8 and β-catenin staining did not colocalize in many chondrocytes. Some cells with increased p-Smad1/5/8 staining (arrows) did not show increased β-catenin staining. (C) Distinct genetic interactions between Wnt/β-catenin and PTHrP signaling in regulating chondrocyte hypertrophy and maturation. The sequential process of chondrocyte hypertrophy and maturation in developing wild type and indicated mutant long bone cartilage were shown in the diagram in the upper panel. Hypertrophic chondrocytes are enlarged and undergo final maturation before they are replaced by the trabecular bone. Initiation of chondrocyte hypertrophy and final maturation are two separate processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling controls chondrocyte hypertrophy by inhibiting PTHrP signaling activity. However, Wnt/β-catenin signaling promoted final maturation of hypertrophic chondrocyte independently of PTHrP signaling.

Mentions: As PTHrP signaling controls chondrocyte hypertrophy cell autonomously [23], we tested whether the Wnt/β-catenin signaling pathway also acts cell autonomously. We expressed an activated form of β-catenin in subsets of chondrocytes using a mouse line with the exon 3 of β-catenin floxed (CatnbEx3) [24]. This mouse line was crossed with a tamoxifen (TM) inducible Cre line, Col2a1-CreER [25]. As the tamoxifen induced Cre activity is not robust in all cells, the activated form of β-catenin is only expressed in a subset of chondrocytes in the CatnbEx3/+; Col2a1-CreER embryos following TM injection. Since Lef1 is a transcription target of Wnt/β-catenin signaling [16], in these subset of chondrocytes, Lef1 expression was upregulated (Fig. 4A). In addition, bone formation was increased in the TM injected CatnbEx3/+; Col2a1-CreER embryos indicated by von Kossa staining (Fig. 4A), suggesting that Wnt/ β-catenin signaling in chondrocytes may also promote bone formation in the perichondrium non-cell autonomously. Interestingly, Safranin O staining and Col2a1 expression was significantly downregulated in the chondrocytes that expressed the activated form of β-catenin (Fig. 4A). However, these cells did not ectopically express Col10a1, a marker for hypertrophic chondrocytes (Fig. 4A). This is in sharp contrast to the normal process of chondrocyte hypertrophy in which Col2a1 expression is turned off, while Col10a1 expression is switched on. This is also drastically different from the Pthr1−/− clones in cartilage [23], in which loss of PTHrP signaling led to cell autonomous expression of Col10a1. In addition, while PTHrP signaling does not affect the determination of chondrocyte lineage [26], [27], activated Wnt signaling in the developing cartilage can change chondrocyte identity to the ones that resemble cells found in the joint interzone or joint [28]. These data suggest that activated β-catenin signaling acts cell autonomously to control chondrocyte identity, but non-cell autonomously in regulating chondrocyte hypertrophy. Among the secreted signaling molecules that can regulate chondrocyte hypertrophy, Growth differentiation factor 5 (Gdf5) and Bmp2 expression are upregulated by Wnt/β-catenin signaling [28]–[30]. To address the potential role of these genes, we examined Bmp signaling activities in our mutant mice (Fig. 4B). In and around chondrocytes that express activated β-catenin, we observed stronger phosphorylation of Smad1, 5 and 8, indicating increased Bmp signaling [31]. As Bmp/Gdf5 signaling has been shown to promote chondrocyte hypertrophy [32]–[34], Gdf5 and Bmp2 are likely to participate in mediating the non-cell autonomous function of Wnt/β-catenin signaling in 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)

β-catenin may control chondrocyte hypertrophy non-cell autonomously.Tamoxifen was injected into pregnant females at 13.5 dpc. Embryos were harvested at 17.5 dpc and sections of the proximal tibia were analyzed by Safranin O staining, in situ hybridization with the indicated probes and immnohistochemistry. (A) Upregulated Wnt/β-catenin signaling was indicated by ectopic Lef1 expression and loss of Safranin O staining and Col2a1 expression (arrow). Pictures of higher magnification of the boxed area are shown as insets. Col10a1 expression was not ectopically detected in chondrocytes that had lost Col2a1 expression. (B) Increased p-Smad1/5/8 staining (red) was observed in chondrocyte patches of Catnbex3/+; Col2a1-CreER mouse embryos. Such increase in p-Smad1/5/8 staining was in the area with increased β-catenin staining (green). But p-Smad1/5/8 and β-catenin staining did not colocalize in many chondrocytes. Some cells with increased p-Smad1/5/8 staining (arrows) did not show increased β-catenin staining. (C) Distinct genetic interactions between Wnt/β-catenin and PTHrP signaling in regulating chondrocyte hypertrophy and maturation. The sequential process of chondrocyte hypertrophy and maturation in developing wild type and indicated mutant long bone cartilage were shown in the diagram in the upper panel. Hypertrophic chondrocytes are enlarged and undergo final maturation before they are replaced by the trabecular bone. Initiation of chondrocyte hypertrophy and final maturation are two separate processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling controls chondrocyte hypertrophy by inhibiting PTHrP signaling activity. However, Wnt/β-catenin signaling promoted final maturation of hypertrophic chondrocyte independently of PTHrP signaling.
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Related In: Results  -  Collection

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pone-0006067-g004: β-catenin may control chondrocyte hypertrophy non-cell autonomously.Tamoxifen was injected into pregnant females at 13.5 dpc. Embryos were harvested at 17.5 dpc and sections of the proximal tibia were analyzed by Safranin O staining, in situ hybridization with the indicated probes and immnohistochemistry. (A) Upregulated Wnt/β-catenin signaling was indicated by ectopic Lef1 expression and loss of Safranin O staining and Col2a1 expression (arrow). Pictures of higher magnification of the boxed area are shown as insets. Col10a1 expression was not ectopically detected in chondrocytes that had lost Col2a1 expression. (B) Increased p-Smad1/5/8 staining (red) was observed in chondrocyte patches of Catnbex3/+; Col2a1-CreER mouse embryos. Such increase in p-Smad1/5/8 staining was in the area with increased β-catenin staining (green). But p-Smad1/5/8 and β-catenin staining did not colocalize in many chondrocytes. Some cells with increased p-Smad1/5/8 staining (arrows) did not show increased β-catenin staining. (C) Distinct genetic interactions between Wnt/β-catenin and PTHrP signaling in regulating chondrocyte hypertrophy and maturation. The sequential process of chondrocyte hypertrophy and maturation in developing wild type and indicated mutant long bone cartilage were shown in the diagram in the upper panel. Hypertrophic chondrocytes are enlarged and undergo final maturation before they are replaced by the trabecular bone. Initiation of chondrocyte hypertrophy and final maturation are two separate processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling controls chondrocyte hypertrophy by inhibiting PTHrP signaling activity. However, Wnt/β-catenin signaling promoted final maturation of hypertrophic chondrocyte independently of PTHrP signaling.
Mentions: As PTHrP signaling controls chondrocyte hypertrophy cell autonomously [23], we tested whether the Wnt/β-catenin signaling pathway also acts cell autonomously. We expressed an activated form of β-catenin in subsets of chondrocytes using a mouse line with the exon 3 of β-catenin floxed (CatnbEx3) [24]. This mouse line was crossed with a tamoxifen (TM) inducible Cre line, Col2a1-CreER [25]. As the tamoxifen induced Cre activity is not robust in all cells, the activated form of β-catenin is only expressed in a subset of chondrocytes in the CatnbEx3/+; Col2a1-CreER embryos following TM injection. Since Lef1 is a transcription target of Wnt/β-catenin signaling [16], in these subset of chondrocytes, Lef1 expression was upregulated (Fig. 4A). In addition, bone formation was increased in the TM injected CatnbEx3/+; Col2a1-CreER embryos indicated by von Kossa staining (Fig. 4A), suggesting that Wnt/ β-catenin signaling in chondrocytes may also promote bone formation in the perichondrium non-cell autonomously. Interestingly, Safranin O staining and Col2a1 expression was significantly downregulated in the chondrocytes that expressed the activated form of β-catenin (Fig. 4A). However, these cells did not ectopically express Col10a1, a marker for hypertrophic chondrocytes (Fig. 4A). This is in sharp contrast to the normal process of chondrocyte hypertrophy in which Col2a1 expression is turned off, while Col10a1 expression is switched on. This is also drastically different from the Pthr1−/− clones in cartilage [23], in which loss of PTHrP signaling led to cell autonomous expression of Col10a1. In addition, while PTHrP signaling does not affect the determination of chondrocyte lineage [26], [27], activated Wnt signaling in the developing cartilage can change chondrocyte identity to the ones that resemble cells found in the joint interzone or joint [28]. These data suggest that activated β-catenin signaling acts cell autonomously to control chondrocyte identity, but non-cell autonomously in regulating chondrocyte hypertrophy. Among the secreted signaling molecules that can regulate chondrocyte hypertrophy, Growth differentiation factor 5 (Gdf5) and Bmp2 expression are upregulated by Wnt/β-catenin signaling [28]–[30]. To address the potential role of these genes, we examined Bmp signaling activities in our mutant mice (Fig. 4B). In and around chondrocytes that express activated β-catenin, we observed stronger phosphorylation of Smad1, 5 and 8, indicating increased Bmp signaling [31]. As Bmp/Gdf5 signaling has been shown to promote chondrocyte hypertrophy [32]–[34], Gdf5 and Bmp2 are likely to participate in mediating the non-cell autonomous function of Wnt/β-catenin signaling in 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