Cooperative Regulation of Chondrocyte Differentiation by CCN2 and CCN3 Shown by a Comprehensive Analysis of the CCN Family Proteins in Cartilage

Authors

  • Harumi Kawaki,

    1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Satoshi Kubota,

    1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Akiko Suzuki,

    1. Division of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Noureddine Lazar,

    1. Laboratoire d'Oncologie Virale et Moléculaire, UFR de Biochimie, Paris, France
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  • Tomohiro Yamada,

    1. Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Tatsushi Matsumura,

    1. Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Toshihiro Ohgawara,

    1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
    2. Department of Oral and Maxillofacial Surgery and Biopathological Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
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  • Takeyasu Maeda,

    1. Division of Oral Anatomy, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
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  • Bernard Perbal,

    1. Laboratoire d'Oncologie Virale et Moléculaire, UFR de Biochimie, Paris, France
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  • Karen M Lyons,

    1. Department of Orthopedic Surgery, UCLA School of Medicine, Los Angeles, CA, USA
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  • Masaharu Takigawa

    Corresponding author
    1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan
    • Address reprint requests to: Masaharu Takigawa, DDS, PhD, Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8525, Japan
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  • The authors state that they have no conflicts of interest.

Abstract

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, we conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins on chondrocyte proliferation, differentiation, and the expression of chondrocyte-associated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes in vitro, whereas Ccn3 expression drastically increased. In contrast, the addition of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes, whereas decreasing the Ccn3 expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

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