Stage-specific Control of Connective Tissue Growth Factor (CTGF/CCN2) Expression in Chondrocytes by Sox9 and β-Catenin

CCN2/connective tissue growth factor is highly expressed in hypertrophic chondrocytes and is required for chondrogenesis. However, the transcriptional mechanisms controlling its expression in cartilage are largely unknown. The activity of the Ccn2 promoter was, therefore, investigated in osteochondro-progenitor cells and hypertrophic chondrocytes to ascertain these mechanisms. Sox9 and T-cell factor (TCF)·lymphoid enhancer factor (LEF) factors contain HMG domains and bind to related consensus sites. TCF·LEF factors are normally repressive but when bound to DNA in a complex with β-catenin become activators of gene expression. In silico analysis of the Ccn2 proximal promoter identified multiple consensus TCF·LEF elements, one of which was also a consensus binding site for Sox9. Using luciferase reporter constructs, the TCF·LEF·Sox9 site was found to be involved in stage-specific expression of Ccn2. Luciferase, electrophoretic mobility shift assay (EMSA), and ChIP analysis revealed that Sox9 represses Ccn2 expression by binding to the consensus TCF·LEF·Sox9 site. On the other hand, the same assays showed that in hypertrophic chondrocytes, TCF·LEF·β-catenin complexes occupy the consensus TCF·LEF·Sox9 site and activate Ccn2 expression. Furthermore, transgenic mice in which lacZ expression is driven under the control of the proximal Ccn2 promoter revealed that the proximal Ccn2 promoter responded to Wnt signaling in cartilage. Hence, we propose that differential occupancy of the TCF·LEF·Sox9 site by Sox9 versus β-catenin restricts high levels of Ccn2 expression to hypertrophic chondrocytes.