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Rheumatology 2001; 40: 1146-1156
© 2001 British Society for Rheumatology
Original Papers |
Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK and
1 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, PO Box 12233, Research Triangle Park, NC 27709, USA
Objective. To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1
on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4+.2).
Methods. Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4+.2 cells cultured in the presence or absence of serum and IL-1 or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4+.2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W+.1, cultured under the different conditions and treatments.
Results. Early passage DES4+.2 cells expressed chondrocyte-specific molecules such as collagen types 1(II) and 1(IX), aggrecan, biglycan and link protein and collagen types 1(I) and 1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4+.2 and 10W+.1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1 had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1 acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1 correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments.
Conclusions. The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1 on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1 in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.
KEY WORDS: Chondrocytes, Gene expression, Cytokines, Apoptosis, Sox9.
Correspondence to: E. Kolettas, Cell and Molecular Physiology Unit, Laboratory of Physiology, University of Ioannina Medical School, 45110 Ioannina, Greece.
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