Osteogenic Protein-1 Up-Regulation of the Collagen X Promoter Activity Is Mediated by a MEF-2-Like Sequence and Requires an Adjacent AP-1 Sequence

Bone morphogenetic proteins induce chondrogenesis and osteogenesis in vivo. To investigate molecular mechanisms involved in chondrocyte induction, we examined the effect of osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1 up-regulates collagen X mRNA levels and its promoter activity in a cell type- specific manner. Deletion analysis localizes the OP-1 response region to 33 bp (−310/−278), which confers OP-1 responsiveness to both the minimal homologous and heterologous Rous sarcoma virus promoter. Transforming growth factor-β2 or activin, which up-regulates the expression of a transforming growth factor-β-inducible p3TP-Lux construct, has little effect on collagen X mRNA and on this 33-bp region. Mutational analysis shows that both an AP-1 like sequence (−294/−285, TGAATCATCA) and an A/T-rich myocyte enhancer factor (MEF)-2 like sequence (−310/−298, TTT) in the 33-bp region are necessary for the OP-1 effect. Gel shift assays show interaction of distinct nuclear proteins from C5.18 cells with the AP-1-like and the MEF-2- like sequences. OP-1 rapidly induces nuclear protein interaction with the MEF-2-like sequence but not with the AP-1 like sequence. MEF-2-like binding activity induced by OP-1 is distinct from the MEF-2 family proteins present in C2C12 myoblasts, in which OP-1 does not induce collagen X mRNA or up-regulate its promoter activity. In conclusion, we identified a specific response region for OP-1 in the mouse collagen X promoter. Mutational and gel shift analyses suggest that OP-1 induces nuclear protein interaction with an A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in myoblasts, and up-regulates collagen X promoter activity, which also requires an AP-1 like sequence.