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Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5

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Abstract

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin–transferrin–sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.

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Acknowledgments

The TOPEGFP reporter plasmid was kindly provided by Dr. M. Oshima (Cancer Research Institute, Kanazawa University, Japan). The pPyCAG-EGFP-IP plasmid vector was kindly provided by Dr. H. Niwa (RIKEN Center for Developmental Biology (CDB), Japan). This work was supported in part by a research grant from the dental research center, Nihon University School of Dentistry, a grant for collaborative research from the Graduate School of Dentistry, Nihon University, a research grant from Nihon University Multidisciplinary Research Grant for 2014, and a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (M.N., 25870785).

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Authors and Affiliations

  1. Department of Anatomy, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyodaku, Tokyo, 101-8310, Japan

    Masako Naito, Akiko Ohashi & Tomihisa Takahashi

  2. Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, 101-8310, Japan

    Masako Naito, Akiko Ohashi & Tomihisa Takahashi

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  1. Masako Naito
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  2. Akiko Ohashi
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  3. Tomihisa Takahashi
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Correspondence to Masako Naito.

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Naito, M., Ohashi, A. & Takahashi, T. Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5. Histochem Cell Biol 144, 261–272 (2015). https://doi.org/10.1007/s00418-015-1334-2

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  • DOI: https://doi.org/10.1007/s00418-015-1334-2

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