Bone Research Laboratory, Texas Scottish Rite Hospital for Children, Dallas, Texas 75219, USA.
J Biol Chem. 2012 Jan 13;287(3):1671-8. doi: 10.1074/jbc.M111.288472. Epub 2011 Nov 22.
Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.
osterix(osx) 是一种成骨细胞特异性转录因子，对于骨形成和成骨细胞分化是必需的。骨形成的关键步骤是用血管化的骨替代无血管的软骨模板。成骨和血管生成是相互关联的，它们共享一些必要的调节因子。血管内皮生长因子(vascular endothelial growth factor, vegf) 参与血管生成和成骨。在成骨细胞中，vegf 表达的转录调控尚不清楚。在这项研究中，定量实时 rt-pcr 结果显示，vegf 表达在 osx 缺失的颅骨细胞中下调，骨钙素表达缺失。使用 tet-off 系统在稳定的 c2c12 间充质细胞中过表达 osx 导致骨钙素和 vegf 表达上调。osx 的 siRNA 抑制导致成骨细胞中 vegf 表达受到抑制。这些结果表明 osx 控制 vegf 表达。转染实验表明，osx 激活了 vegf 启动子活性。研究了一系列 vegf 启动子缺失突变体，并将 osx 反应的最小区域定义为 vegf 启动子的近端 140bp 区域。额外的点突变体用于鉴定两个 gc 丰富区，它们负责 osx 激活 vegf 启动子。凝胶移位实验表明 osx 直接结合 vegf 启动子序列。染色质免疫沉淀实验表明，内源性 osx 与原代成骨细胞中的天然 vegf 启动子结合。此外，免疫组织化学染色显示，osx 条件性敲除小鼠胫骨中 vegf 蛋白水平降低。我们首次证明 osx 控制 vegf 表达，提示 osx 在协调成骨和血管生成中可能发挥作用。
