Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors

Abstract

During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata.

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

“…SOX9 is a member of the sex-determining region Y-type high mobility group box protein family and has been shown to be required for chondrocyte-specific gene expression and chondrogenesis (48)(49)(50). We also observed the involvement of the SOX factors in the dedifferentiation process (51). Interestingly, recent investigations have established a connection between SOX9 and the protein CD-RAP.…”

αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

“…SOX9 is a transcription factor that regulates the expression of a series of cartilage matrix genes during chondrogenesis (49). With the progression of dedifferentiation, the expression level and the transcription activity of SOX9 decline (50,51), while the activity of SOX9 may be negatively regulated by ERK signaling (47). We evaluated the expression level of SOX9 in monolayer-cultured chondrocytes and found that it did not change significantly in the early phase of dedifferentiation investigated in the present study.…”

Regulation of Human COL9A1 Gene Expression

“…Electrophoretic mobility-shift assays (EMSAs) were carried out as previously described with minor modifications (25). Briefly, binding reactions consisted of 12.5 mM Hepes, pH 7.9, 50 -100 mM NaCl, 5% glycerol, 2 mg/ml bovine serum albumin, 2 g poly(dG-dC), 0.1 mM EDTA, 0.1 mM dithiothreitol, 1 ng of 32 P-end labeled double-stranded oligonucleotide probe, and 10 -15 g of nuclear protein.…”