Bone Research Laboratory, Texas Scottish Rite Hospital for Children, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
PLoS One. 2011;6(10):e26504. doi: 10.1371/journal.pone.0026504. Epub 2011 Oct 18.
Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.
osterix(osx)是一种成骨细胞特异性转录因子，对于间充质干细胞向成骨细胞分化是必需的。在 osx 敲除小鼠中，没有骨形成。维生素 d 受体(vdr)是核激素受体超家族的成员，调节靶基因转录，以确保适当的钙稳态和骨骼发育的控制。在这里，我们提供了几条证据表明，vdr 基因是 osx 在成骨细胞中转录调节的靶基因。例如，与野生型颅骨相比，来自 osx 缺失胚胎的颅骨中 vdr 表达明显减少。osx 的稳定过表达刺激 c2c12 间充质细胞中 vdr 的表达。通过 sirna 抑制 osx 表达导致 vdr 下调。相比之下，vdr 缺失小鼠的成骨细胞中 osx 水平保持不变。使用瞬时转染测定的机制方法表明，osx 以剂量依赖的方式直接激活 vdr 启动子的 1kb 片段。为了确定 vdr 启动子对 osx 有反应的区域，研究了一系列 vdr 启动子缺失突变体，并将 osx 反应最小区域精修到 vdr 启动子的近端 120bp。额外的点突变用于鉴定两个 gc 丰富的区域，这些区域负责 osx 激活 vdr 启动子。染色质免疫沉淀测定表明，内源性 osx 与体内原代成骨细胞中的天然 vdr 启动子相关。总之，这些数据强烈支持 osx 在 vdr 基因表达中的直接调节作用。它们进一步提供了新的见解，潜在的机制和途径，osx 在成骨细胞中控制和在成骨细胞分化过程中。
