The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

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Abstract

To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (ca) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of β-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner.

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Materials and methods

Preparation of plasmids and adenoviruses. Plasmids encoding hemagglutinin (HA)-tagged constitutively active TCF/LEF-1 (caLEF-1) and HA-tagged dominant negative TCF/LEF-1 (dnLEF-1) were provided by Hecht [12]. The sequence encoding dnLEF-1 lacks amino acids 17–264 of murine LEF-1 and the sequence encoding caLEF-1 includes amino acids 695–781 of β-catenin fused to the C-terminus of dnLEF-1. Plasmids expressing mouse β-catenin were provided by Akiyama (University of Tokyo, Tokyo, Japan). Plasmids

Modulation of the canonical Wnt signaling pathway by adenoviral vectors

To modulate the canonical Wnt signaling pathway, we constructed adenoviral vectors expressing caLEF-1 (Ad-caLEF-1), dnLEF-1 (Ad-dnLEF-1) or siRNA for β-catenin (Ad-siβ-catenin). To confirm the function of Ad-caLEF-1 and Ad-dnLEF-1, we transduced an undifferentiated mesenchymal cell line C3H10T1/2 with each vector at different dosages. Western blot analysis revealed that both mutant proteins were well expressed in a dose-dependent fashion, with caLEF-1 and dnLEF-1 being detected at 35 and 24 kDa,

Discussion

In the current paper, we demonstrate that the canonical Wnt signaling pathway promotes chondrogenic differentiation in C3H10T1/2; that the pathway promotes hypertrophy in C3H10T1/2, ATDC5, and primary chondrocytes; that the pathway increases the Sox9 mRNA level in C3H10T1/2, ATDC5, and primary chondrocytes; and that, in the absence of Sox9, the canonical Wnt signaling pathway is not able to induce chondrogenic differentiation or hypertrophy.

Among different Wnt proteins, Wnt1, Wnt3a, Wnt4,

Acknowledgments

We thank Dr. A. Hecht for plasmids expressing caLEF-1 and dnLEF-1, Dr. T. Akiyama for a plasmid expressing β-catenin, Dr. B. deCrombrugghe for Sox9−/− ES cells, and Drs. M. Miyagishi and K. Taira for the U6icassette vector.

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    Funding source: Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (#16659400).

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