Endocrine Abstracts

Runx2 and Osterix(Osx) have been known as the master transcription factors for bone formation. However, genes that act downstream of both Runx2 and Osx have not been fully studied. To investigate downstream target genes of Runx2 and Osx, DNA microarray was conducted in calvaria of WT, Runx2^ΔC/+, Osx^+/−, and Runx2^ΔC/+; Osx^+/− double heterozygous mice designated WT, Runx2^het, Osx^het, and Double^het respectively. Compared to WT, the expression of Ucma, an unique cartilage matrix-associated protein, was decreased in Runx2^het or Osx^het and it was more decreased in Double^het. In contrary, Ucma expression was increased in either Runx2 or Osx overexpressed osteoblasts and it was more increased in both Runx2 and Osx overexpressed osteoblasts. To examine Ucma expression during osteoblast differentiation, MC3T3-E1 osteoblastic cells were cultured for 30 days in the medium supplemented with ascorbic acid and β-glycerophosphate. Ucma expression exhibited in the middle of differentiation and continued during differentiation. To investigate whether Runx2 and Osx modulate the transcriptional activity of Ucma, Ucma promoter was co-transfected with Runx2 and/or Osx expression plasmid into MC3T3-E1 cells. The transcriptional activity of Ucma promoter was more increased when both Runx2 and Osx expression vectors were used. In Ucma promoter, two Sp1 and three Runx binding sites were existed. The Runx2- and Osx-mediated activations of Ucma promoter were directly regulated through Runx and/or Sp1 binding sites. The formation of mineralized nodules in Ucma-overexpressing stable clone was earlier and more increased than those of the mock control. Moreover, mineralized nodule formation was highly increased in MC3T3-E1 cells cultured in the medium including Ucma proteins secreted from Ucma-overexpressing cells. Collectively, this study suggests that Ucma is a novel downstream target gene regulated by both Runx2 and Osx and has a positive effect to enhance osteoblast differentiation and nodule formation.