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CCN Proteins pp 219–237Cite as

Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1489))

Abstract

Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification; and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of 45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

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References

  1. Karaplis AC (2002) Embryonic development of bone and the molecular regulation of intramembranous and endochondral bone formation. In: Bilezikian JP, Raisz RG, Rodan GA (eds) Principles of bone biology, vol 1, 2nd edn. Academic, San Diego, CA, pp 33–58

    Chapter  Google Scholar 

  2. Pacifici M, Golden EB, Oshima O, Shapiro IM, Leboy PS, Adams SL (1990) Hypertrophic chondrocytes. The terminal stage of differentiation in the chondrogenic cell lineage? Ann N Y Acad Sci 599:45–57

    Article  CAS  PubMed  Google Scholar 

  3. Buckwalter JA, Mankin HJ (1998) Articular cartilage: tissue design and chondrocyte-matrix interactions. Instr Course Lect 47:477–486

    CAS  PubMed  Google Scholar 

  4. Hunziker EB (1994) Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Tech 28:505–519

    Article  CAS  PubMed  Google Scholar 

  5. Cancedda R, Descalzi Cancedda F, Castagnola P (1995) Chondrocyte differentiation. Int Rev Cytol 159:265–358

    Article  CAS  PubMed  Google Scholar 

  6. Maes C, Stockmans I, Moermans K, Van Looveren R, Smets N, Carmeliet P, Bouillon R, Carmeliet G (2004) Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival. J Clin Invest 113:188–199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Takigawa M, Takano T, Suzuki F (1981) Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture. J Cell Physiol 106:259–268

    Article  CAS  PubMed  Google Scholar 

  8. Takigawa M, Enomoto M, Shirai E, Nishii Y, Suzuki F (1988) Differential effects of 1α, 25-dihydroxychlecalciferol and 24R, 25-dihydroxycholecalciferol on the proliferation and the differentiated phenotype of rabbit costal chondrocytes in culture. Endocrinology 122:831–839

    Article  CAS  PubMed  Google Scholar 

  9. Trippel ST (1992) Role of insulin-like growth factors in the regulation of chondrocytes. In: Adolphe M (ed) Biological regulation of the chondrocytes. CRC Press, Boca Raton, pp 161–190

    Google Scholar 

  10. Kato Y, Iwamoto M, Koike T, Suzuki F, Takano Y (1990) Terminal differentiation and cacification in rabbit chondrocyte cultures grown in centrifuge tubes: Regulation by transforming growth factor-β and serum factors. Proc Natl Acad Sci U S A 85:9552–9556

    Article  Google Scholar 

  11. Nakanishi T, Kimura Y, Tamura T, Ichikawa H, Yamaai Y, Sugimoto T, Takigawa M (1997) Cloning of a mRNA preferentially expressed in chondrocytes by differential display-PCR from a human chondrocytic cell line that is identical with connective tissue growth factor (CTGF) mRNA. Biochem Biophys Res Commun 234(1):206–210

    Article  CAS  PubMed  Google Scholar 

  12. Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K, Takigawa M (2000) Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141:264–273

    CAS  PubMed  Google Scholar 

  13. Nishida T, Kubota S, Nakanishi T, Kuboki T, Yosimichi G, Kondo S, Takigawa M (2002) CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes. J Cell Physiol 192:55–63

    Article  CAS  PubMed  Google Scholar 

  14. Fujisawa T, Hattori T, One M, Uehara J, Kubota S, Kuboki T, Takigawa M (2008) CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes. Osteoarthritis Cartilage 16:787–795

    Article  CAS  PubMed  Google Scholar 

  15. Nishida T, Kawaki H, Baxter RM, Deyoung RA, Takigawa M, Lyons KM (2007) CCN2 (connective tissue growth factor) is essential for extracellular matrix production and integrin signaling in chondrocytes. J Cell Commun Signal 1:45–58

    Article  PubMed  PubMed Central  Google Scholar 

  16. Tamura T, Nakanishi T, Kimura Y, Hattori T, Sasaki K, Norimatsu H, Takahashi K, Takigawa M (1996) Nitric oxide mediates interleukin-1-induced matrix degradation and basic fibroblast growth factor release in cultured rabbit articular chondrocytes: a possible mechanism of pathological neovascularization in arthritis. Endocrinology 137:3729–3737

    CAS  PubMed  Google Scholar 

  17. Nishida T, Kubota S, Aoyama E, Janune D, Maeda A, Takigawa M (2011) Effect of CCN2 on FGF2-induced proliferation and MMP9 and MMP13 productions by chondrocytes. Endocrinology 152(11):4232–4241

    Article  CAS  PubMed  Google Scholar 

  18. Nishida T, Kubota S, Fukunaga T, Kondo S, Yosimichi G, Nakanishi T, Takano-Yamamoto T, Takigawa M (2003) CTGF/Hcs24, hypertrophic chondrocyte-specific gene product, interacts with perlecan in regulating the proliferation and differentiation of chondrocytes. J Cell Physiol 196:265–275

    Article  CAS  PubMed  Google Scholar 

  19. Nishida T, Nakanishi T, Asano M, Shimo T, Takigawa M (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J Cell Physiol 184:197–206

    Article  CAS  PubMed  Google Scholar 

  20. Maeda A, Nishida T, Aoyama E, Kubota S, Lyons KM, Kuboki T, Takigawa M (2009) CCN family 2/connective tissue growth factor modulates BMP signalling as a signal conductor, which action regulates the proliferation and differentiation of chondrocytes. J Biochem 145:207–216

    Article  CAS  PubMed  Google Scholar 

  21. Nishida T, Kubota S, Aoyama E, Takigawa M (2013) Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus. Osteoarthritis Cartilage 21:700–709

    Article  CAS  PubMed  Google Scholar 

  22. Kawata K, Eguchi T, Kubota S, Kawaki H, Oka M, Minagi S, Takigawa M (2006) Possible role of LRP1, a CCN2 receptor, in chondrocytes. Biochem Biophys Res Commun 345:552–559

    Article  CAS  PubMed  Google Scholar 

  23. Janune D, Kubota S, Nishida T, Kawaki H, Perbal B, Iida S, Takigawa M (2011) Novel effects of CCN3 that may direct the differentiation of chondrocytes. FEBS Lett 585:3033–3040

    Article  CAS  PubMed  Google Scholar 

  24. Kawaki H, Kubota S, Suzuki A, Lazar N, Yamada T, Matsumura T, Ohgawara T, Maeda T, Perbal B, Lyons KM, Takigawa M (2008) Cooperative regulation of chondrocyte differentiation by CCN2 and CCN3 shown by a comprehensive analysis of the CCN family proteins in cartilage. J Bone Miner Res 23:1751–1764

    Article  CAS  PubMed  Google Scholar 

  25. Tomita N, Hattori T, Ito S, Aoyama E, Yao M, Yamashiro T, Takigawa M (2013) Cartilage–specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/CTGF) stimulates insulin-like growth factor expression and bone growth. PLoS One 8, e59226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fujisawa T, Hattori T, Takahashi K, Kuboki T, Yamashita A, Takigawa M (1999) Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. J Biochem 125:966–975

    Article  CAS  PubMed  Google Scholar 

  27. Nishida T, Kondo S, Maeda A, Kubota S, Lyons KM, Takigawa M (2009) CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition. Bone 44:24–31

    Article  CAS  PubMed  Google Scholar 

  28. Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ (1995) Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP). J Biol Chem 270:9420–9428

    Article  CAS  PubMed  Google Scholar 

  29. Takigawa M, Tajima K, Pan H-O, Enomoto M, Kinoshita A, Suzuki F, Takano Y, Mori Y (1989) Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res 49:3996–4002

    CAS  PubMed  Google Scholar 

  30. Iwamoto-Enomoto M, Takigawa M (1992) Regulation of tumor-derived and immortalized chondrocytes. In: Adolphe M (ed) Biological regulation of the chondrocytes. CRC Press, Boca Raton, pp 321–338

    Google Scholar 

  31. Mukhopadhyay K, Lefebvre V, Zhou G, Garofalo S, Kimura JH, de Crombrugghe B (1995) Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-α1 (II) collagen gene. J Biol Chem 270:27711–27719

    Article  CAS  PubMed  Google Scholar 

  32. Farndale RW, Sayers CA, Barrett AJ (1982) A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect Tissue Res 9:247–248

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by grants from the programs Grants-in-Aid for Scientific Research (C) to T.N. (#JP26462810) and to S.K. (#JP25462886) and Scientific Research (B) to M.T. (#JP15H05014) from the Japan Society for the Promotion of Sciences, Japan.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Takashi Nishida D.D.S., Ph.D.

  2. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Satoshi Kubota

  3. Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Satoshi Kubota

  4. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Masaharu Takigawa

Authors
  1. Takashi Nishida D.D.S., Ph.D.
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  2. Satoshi Kubota
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  3. Masaharu Takigawa
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Corresponding authors

Correspondence to Takashi Nishida D.D.S., Ph.D. or Masaharu Takigawa .

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Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

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Nishida, T., Kubota, S., Takigawa, M. (2017). Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 1489. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6430-7_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6430-7_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6428-4

  • Online ISBN: 978-1-4939-6430-7

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