Bone Research Laboratory, Texas Scottish Rite Hospital for Children, Dallas, Texas 75390, USA.
J Biol Chem. 2011 Sep 23;286(38):32995-3002. doi: 10.1074/jbc.M111.244236. Epub 2011 Aug 2.
Osterix (Osx) is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Satb2 is critical for osteoblast differentiation as a special AT-rich binding transcription factor. It is not known how Satb2 is transcriptionally regulated during bone formation. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. These results suggest that Osx controls Satb2 expression. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. 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. 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. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. This reveals a new additional link of the transcriptional regulation mechanism that Osx controls bone formation.
osterix (osx) 是一种成骨细胞特异性转录因子，对于成骨细胞分化和骨形成是必不可少的。osx 敲除小鼠完全缺乏骨骼。satb2 作为一种特殊的富含 at 的结合转录因子，对于成骨细胞分化至关重要。目前尚不清楚 satb2 在骨形成过程中是如何被转录调控的。在这项研究中，实时定量 rt-pcr 结果表明 satb2 在 osx 缺失的颅骨中表达下调。在使用四环素（tet）-off 系统的稳定 c2c12 间充质细胞中，过表达 osx 可刺激 satb2 的表达。此外，osx 的 siRNA 抑制导致成骨细胞中 satb2 的表达受到抑制。这些结果表明 osx 控制 satb2 的表达。瞬时转染实验表明，osx 以剂量依赖的方式激活 1kb satb2 启动子报告基因活性。为了确定 satb2 启动子对 osx 激活有反应的区域，构建了一系列 satb2 构建体的缺失突变体，并将最小区域缩小到 satb2 启动子的近端 130bp。进一步的点突变研究发现，两个 gc 丰富区的突变破坏了 osx 对 satb2 130bp 启动子的激活，表明这些 gc 丰富的结合位点负责 osx 对 satb2 的激活。凝胶阻滞实验表明，osx 直接与 satb2 启动子序列结合。chip 实验表明，内源性 osx 与成骨细胞中天然的 satb2 启动子结合。重要的是，satb2 siRNA 显著抑制了 osx 诱导的成骨细胞标记基因的表达。总之，我们的研究结果表明，osx 是骨形成过程中 satb2 的上游调节因子。这揭示了 osx 控制骨形成的转录调控机制的一个新的附加联系。
