Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor

Objective. To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1α 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 α 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 α1(II) and α1(IX), aggrecan, biglycan and link protein and collagen types α1(I) and α1(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-1α had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1α acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1α-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1α 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-1α 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-1α in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.