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J. Biol. Chem., Vol. 275, Issue 35, 27421-27438, September 1, 2000

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Regulation of Human COL2A1 Gene Expression in Chondrocytes
IDENTIFICATION OF C-Krox-RESPONSIVE ELEMENTS AND MODULATION BY PHENOTYPE ALTERATION^*

Chafik Ghayor^§, Jean-François Herrouin^§¶, Christos Chadjichristos, Leena Ala-Kokko, Masaharu Takigawa^**, Jean-Pierre Pujol, and Philippe Galéra

From the  Laboratoire de Biochimie du Tissu Conjonctif, Centre Hospitalier Universitaire de Caen, Faculté de Médecine, Avenue de la Côte de Nacre, 14032, Caen Cedex, France, the  Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, 90220 Oulu, Finland, and the ^** Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Shikata-cho, Okayama 700, Japan

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-B 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.

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