Bone morphogenetic protein-2 (BMP-2) signaling to the Col2alpha1 gene in chondroblasts requires the homeobox gene Dlx-2.

To understand the role of Dlx genes in the process of chondrogenesis, we studied the expression of Dlx-2 and Dlx-5 mRNAs in a mouse clonal chondroblast cell line, TMC23. We also examined the involvement of Dlx2 in the bone morphogenetic protein-2 (BMP-2) signaling to the type II collagen gene, Col2alpha1, in this cell line. In this report, we show that the TMC23 cells express Dlx-2 and Dlx-5 mRNAs, and the levels can be upregulated by recombinant BMP-2 at an early stage of chondroblast differentiation. Addition of rBMP-2 dramatically increased type II collagen expression at both the mRNA and the protein level. Also, rBMP-2 increased transcription of Col2alpha1, as shown by stimulation of a chondrocyte-specific Col2alpha1 enhancer. The mechanism involves Dlx-2, as the stimulatory effect of rBMP-2 on the Col2alpha enhancer was blocked by an antisense oligonucleotide against Dlx-2 mRNA. The rBMP-2 signaling to the Col2alpha1 enhancer was also blocked by a dominant-negative Smad1 expression vector. These data demonstrate that Dlx-2 is a downstream target of the BMP-2 signaling pathway in chondroblasts. Therefore, we propose a model in which rBMP-2 stimulates Dlx-2 expression, which then serves as a necessary transcription factor for Col2alpha1 gene expression through a chondrocyte-specific enhancer fragment.