A Simple and Reliable Assay of Proteoglycan Synthesis by Cultured Chondrocytes

  • Frédéric De Ceuninck
  • Audrey Caliez
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 100)

Abstract

A simple and reliable method to measure proteoglycan synthesis by chondrocytes in culture is described. Confluent chondrocytes in 24-well plates are labeled for 24–72 h with 35SO4 2− in the presence of stimulating agents. At the end of treatment, the secretion medium containing radiolabeled neosynthesized secreted proteoglycans (SP) is harvested, and cell-associated proteoglycans (CAP) are extracted with guanidine hydrochloride for 48 h. Aliquots of medium and cell extracts are distributed on Whatman paper and left to dry. SP and CAP are trapped by precipitation with the cationic detergent cetyl pyridinium chloride (CPC), whereas nonincorporated 35SO4 2− remains in solution. After drying, each spot corresponding to one well in the plate is cut, and its radioactivity is measured. Counts are proportional to the amount of neosynthesized proteoglycans. In a representative experiment using rabbit chondrocytes, total proteoglycan synthesis (SP plus CAP) was increased 3.5-fold after the addition of 1.34 nM insulin-like growth factor-1 (IGF-1) compared with nonstimulated cells. Further addition of a fourfold molar ratio of IGF binding protein-3 completely abolished this effect. This method can be used to measure proteoglycan synthesis by chondrocytes from many species, including human osteoarthritic chondrocytes, as well as guinea pig, rabbit, and rat chondrocytes.

Key Words

Chondrocyte proteoglycan synthesis glycosaminoglycan anabolism osteoarthritis assay cell culture cetyl pyridinium chloride precipitation, guanidine hydrochloride 

References

  1. 1.
    Kiani, C., Chen, L., Wu, Y. J., and Yang, B. B. (2002) Structure and function of aggrecan. Cell Res. 12, 19–32.CrossRefPubMedGoogle Scholar
  2. 2.
    Grover, J. and Roughley, P. J. (1993) Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan. Biochem. J. 291, 361–367.PubMedGoogle Scholar
  3. 3.
    Rosenberg, L. C., Choi, H. U., Tang, L. H., et al. (1985) Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages. J. Biol. Chem. 260, 6304–6313.PubMedGoogle Scholar
  4. 4.
    Heinegard, D., Larsson, T., Sommarin, Y., Franzen, A., Paulsson, M., and Hedbom, E. (1986) Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues. J. Biol. Chem. 261, 13,866–13,872.PubMedGoogle Scholar
  5. 5.
    Grover, J., Chen, X. N., Korenberg, J. R., and Roughley, P. J. (1995) The human lumican gene. Organization, chromosomal location, and expression in articular cartilage. J. Biol. Chem. 270, 21,942–21,949.CrossRefPubMedGoogle Scholar
  6. 6.
    SundarRaj, N., Fite, D., Ledbetter, S., Chakravarti, S., and Hassell, J. R. (1995) Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J. Cell Sci. 108, 2663–2672.PubMedGoogle Scholar
  7. 7.
    Costell, M., Gustaffson, E., Aszodi, A., et al. (1999) Perlecan maintains the integrity of cartilage and some basement membranes. J. Cell Biol. 147, 1109–1122.CrossRefPubMedGoogle Scholar
  8. 8.
    Grover, J. and Roughley, P., J. (1995) Expression of cell-surface proteoglycan mRNA by human articular chondrocytes. Biochem. J. 309, 963–968.PubMedGoogle Scholar
  9. 9.
    Barre, P. E., Redini, F., Boumediene, K., Vielpeau, C., and Pujol, J. P. (2000) Semiquantitative reverse transcription-polymerase chain reaction analysis of syndecan-1 and-4 messages in cartilage and cultured chondrocytes from osteoarthritic joints. Osteoarthritis Cartilage 8, 34–43.CrossRefPubMedGoogle Scholar
  10. 10.
    Pfander, D., Swoboda, B., and Kirsch, T. (2001) Expression of early and late differentiation markers (proliferating cell nuclear antigen, syndecan-3, annexin VI, and alkaline phosphatase) by human osteoarthritic chondrocytes. Am. J. Pathol. 159, 1777–1783.CrossRefPubMedGoogle Scholar
  11. 11.
    Hua, Q., Knudson, C. D., and Knudson, W. (1993) Internalization of hyaluronan by chondrocytes occurs via receptor-mediated endocytosis. J. Cell Sci. 106, 365–375.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Frédéric De Ceuninck
    • 1
  • Audrey Caliez
    • 1
  1. 1.Division de RhumatologieInstitut de Recherches ServierSuresnesFrance

Personalised recommendations