Transcriptional Regulation of Vascular Endothelial Growth Factor (VEGF) by Osteoblast-specific Transcription Factor Osterix (Osx) in Osteoblasts
Transcriptional Regulation of Vascular Endothelial Growth Factor (VEGF) by Osteoblast-specific Transcription Factor Osterix (Osx) in Osteoblasts
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DOI：
10.1074/jbc.m111.288472
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发表时间：
2012-01-13
影响因子：
4.8
通讯作者：
Zhang, Chi
中科院分区：
文献类型：
--
作者：
Tang, Wanjin;Yang, Fan;Zhang, Chi
Osterix(OSX)是一种成骨细胞特异性转录因子，是骨形成和成骨细胞分化所必需的。骨形成的关键步骤是用带血管的骨取代无血管软骨模板。成骨和血管生成相互联系，共享一些重要的调控因素。血管内皮生长因子(VEGF)参与血管生成和成骨。在成骨细胞中，对血管内皮生长因子表达的转录调控还不是很清楚。在本研究中，实时定量RT-PCR结果显示，在OSX缺失的颅骨细胞中，VEGF的表达下调，成骨细胞标志物骨钙素的表达缺失。使用Tet-Off系统在稳定的C2C12间充质细胞中过表达OSX导致骨钙素和血管内皮生长因子的表达上调。SiRNA抑制OSX可抑制成骨细胞中血管内皮生长因子的表达。这些结果提示OSX对血管内皮细胞生长因子的表达具有调控作用。转染实验表明，OSX激活了血管内皮生长因子启动子活性。检测了一系列的血管内皮生长因子启动子缺失突变体，并将最小的OSX响应区定义为血管内皮生长因子启动子近端140个碱基的区域。另外的点突变被用来鉴定负责OSX激活血管内皮生长因子启动子的两个GC富集区。凝胶漂移实验表明，OSX与血管内皮生长因子启动子序列直接结合。染色质免疫沉淀分析表明，在原代成骨细胞中，内源性OSX与天然的血管内皮生长因子启动子有关。此外，免疫组织化学染色显示OSX条件性基因敲除小鼠胫骨中的VEGF蛋白水平降低。我们提供了OSX控制血管内皮生长因子表达的第一个证据，表明OSX在协调成骨和血管生成方面具有潜在的作用。
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 downregulated 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.