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) 1alpha 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-1alpha 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 alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(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-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha 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-1alpha 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-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.
Kolettas
E
E
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Muir
H I
HI
Barrett
J C
JC
Hardingham
T E
TE
D053674:Aggrecans; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D001374:Azacitidine / Q000494:pharmacology; D058578:Biglycan; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000276:immunology; D019902:Chondrocytes / Q000166:cytology* / Q000276:immunology*; D024043:Collagen Type II / Q000235:genetics; D024081:Collagen Type IX / Q000235:genetics; D006224:Cricetinae; D016326:Extracellular Matrix Proteins; D005333:Fetus / Q000166:cytology; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D006609:High Mobility Group Proteins / Q000235:genetics* / Q000276:immunology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology*; D037181:Lectins, C-Type; D008647:Mesocricetus; D010641:Phenotype; D011506:Proteins / Q000235:genetics; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000276:immunology; D000099249:Proteoglycan Link Protein
D053674:Aggrecans; D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine; D058578:Biglycan; D024043:Collagen Type II; D024081:Collagen Type IX; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D007334:Insulin-Like Growth Factor I; D007375:Interleukin-1; D037181:Lectins, C-Type; D011506:Proteins; D011509:Proteoglycans; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D000099249:Proteoglycan Link Protein