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 transcri ption in both differentiated and dedifferentiated rabbit articular chondroc ytes, transient transfection experiments were performed, A 715-base pair (b p) 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 l ess effective than in primary cultures but still present. We then demonstra ted that a zinc finger protein, C-Krox, activates COL2A1 gene transcription in differentiated chondrocytes through the enhancer region, whereas in sub cultured cells, it inhibited the gene activity via a 266-bp promoter. Multi copies of the C-Krox binding site were found to mediate transactivation in both primary cultures and passaged cells, whereas C-Krox overexpression inh ibited transcription in dedifferentiated chondrocytes. Additionally, we sho wed that C-Krox binds to several cis sequences that mediate its transcripti onal effects. During chondrocyte dedifferentiation, the protein levels and binding activity of C-Krox were reduced, whereas those of NF-KB were increa sed. This was not associated with variations of mRNA levels, suggesting tha t post-transcriptional regulatory mechanisms could be involved in C-Krox ex pression. These results suggest that C-Krox plays a major role in type II c ollagen expression and the chondrocyte phenotype modulation.