Runt-related transcription factor 2 (Runx2) and Osterix (Osx) are the master transcription factors in bone formation. Nonetheless, genes acting downstream of both Runx2 and Osx have yet to be fully characterized. Here, we investigate the downstream targets of both Runx2 and Osx in osteoblasts.
DNA microarray analysis was conducted on calvarial RNA from wild-type, Runx2 heterozygous, Osx heterozygous, and Runx2/Osx double heterozygous embryos. Expression and transcriptional responses of the selected target gene were analyzed in MC3T3-E1 osteoblastic cells.
The expression of unique cartilage matrix-associated protein (Ucma) was decreased in Runx2/Osx double heterozygous embryos. In contrast, Ucma expression was increased in osteoblasts overexpressing both Runx2 and Osx. Ucma expression was initiated mid-way through osteoblast differentiation and continued throughout the differentiation process. Transcriptional activity of the Ucma promoter was increased upon transfection of the cells with both Runx2 and Osx. Runx2-and Osx-mediated activation of the Ucma promoter was directly regulated by Runx2-and/or Sp1-binding sites within its promoter. During osteoblast differentiation, the formation of mineralized nodules in Ucma-overexpressing stable clones occurred earlier and was more enhanced than that in the mock-transfected control. Mineralized nodule formation was strongly augmented in the cells cultured in a medium containing secretory Ucma proteins.
Ucma is a novel downstream gene regulated by both Runx2 and Osx and it stimulates osteoblast differentiation and nodule formation.