Runx2, a transcription factor known to be essential for osteoblast maturation and skeletogenesis, is also expressed in pre-cartilaginous mesenchymal condensations in the developing embryo. It is therefore necessary to understand the control and consequential regulatory activity of the Runx2 gene within the context of chondrogenic differentiation of a mesenchymal progenitor cell. We identify the homeodomain protein Nkx3.2 as a potent sequence-specific repressor of the Runx2 promoter that acts through a regulatory element 0.1 kb upstream from the site of transcriptional initiation. The biological significance of this repression is established by utilizing bone morphogenic protein 2 (BMP-2)-induced chondrogenic differentiation of pluripotent C3H10T1/2 cells as a model for the initial events of mesenchymal chondrogenesis. We demonstrate that induction of the chondrogenic phenotype and endogenous Nkx3.2 expression is accompanied by a repression of Runx2 gene activity. Bypassing Runx2 repression by adenoviral-mediated introduction of Runx2 into C3H10T1/2 cells can prevent the induction of chondrogenesis, but cannot reverse the chondrogenic phenotype once it has been initiated, as evidenced by Sox9 and type II collagen expression and extracellular matrix deposition. Our results demonstrate that Runx2 is a direct transcriptional target of Nkx3.2, and that repression of Runx2 at the onset of chondrogenesis is a prerequisite for the activation of a chondrocyte-specific program of gene expression. We postulate that Runx2 is a critical link in BMP-2-mediated initiation of mesenchymal chondrogenesis that results in activation of Sox9 at least in part through the Nkx3.2-dependent repression of Runx2.

Cartilage formation is an intricate process that requires temporal and spatial organization of regulatory factors in order for a mesenchymal progenitor cell to differentiate through the distinct stages of chondrogenesis. Gene function during this process has best been studied by analysis of in vivo cartilage formation in genetically altered mouse models. Mouse embryonic fibroblasts (MEFs) isolated from such mouse models have been widely used for the study of growth control and DNA damage response. Here, we address the potential of MEFs to undergo chondrogenic differentiation. We demonstrate for the first time that MEFs can enter and complete the program of chondrogenic differentiation ex vivo, from undifferentiated progenitor cells to mature, hypertrophic chondrocytes. We show that chondrogenic differentiation can be induced by cell-cell contact or BMP-2 treatment, while in combination, these conditions synergistically enhance chondrocyte differentiation resulting in the formation of 3-dimensional (3-D) cartilaginous tissue ex vivo. Temporal expression profiles of pro-chondrogenic transcription factors Bapx1 and Sox9 and cartilaginous extracellular matrix (ECM) proteins Collagen Type II and X (Coll II and Coll X) demonstrate that the in vivo progression of chondrocyte maturation is recapitulated in the MEF model system. Our findings establish the MEF as a powerful tool for the generation of cartilaginous tissue ex vivo and for the study of gene function during chondrogenesis.