Regulation of human COL2A1 gene expression in chondrocytes. Identification of C-Krox-responsive elements and modulation by phenotype alteration.

To identify control motifs involved in human type II collagen gene transcription in both differentiated and dedifferentiated rabbit articular chondrocytes, transient transfection experiments were performed. A 715-base pair (bp) region of the first intron (+2127/+2842), including a 153-bp sequence so far uncharacterized (+2689/+2842), was found to mediate enhancer activity. In dedifferentiated chondrocytes, this enhancer activity was shown to be less effective than in primary cultures but still present. We then demonstrated that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in subcultured cells, it inhibited the gene activity via a 266-bp promoter. Multicopies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inhibited transcription in dedifferentiated chondrocytes. Additionally, we showed that C-Krox binds to several cis sequences that mediate its transcriptional effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-kappaB were increased. This was not associated with variations of mRNA levels, suggesting that post-transcriptional regulatory mechanisms could be involved in C-Krox expression. These results suggest that C-Krox plays a major role in type II collagen expression and the chondrocyte phenotype modulation.