Fewer bones are formed through intramembranous ossification, a process in which bones are formed directly from mesenchymal condensations. Most bones are formed through endochondral ossification, which needs the template of cartilage. This reveals a new additional link of the transcriptional regulation mechanism that Osx controls bone formation.īone formation involves two processes: endochondral and intramembranous ossification. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. Further point mutation studies found that two GC-rich region mutations disrupted the Satb2 130bp promoter activation by Osx, suggesting that these GC-rich binding sites were responsible for Satb2 activation by Osx. To define the region of Satb2 promoter responsive to Osx activation, a series of deletion mutants of Satb2 constructs were made, and the minimal region was narrowed down to the proximal 130 bp of the Satb2 promoter. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. These results suggest that Osx controls Satb2 expression. ![]() Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. ![]() In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. It is not known how Satb2 is transcriptionally regulated during bone formation. Satb2 is critical for osteoblast differentiation as a special AT-rich binding transcription factor. Osterix (Osx) is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation.
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