Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 - A novel target of Runx2
Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 - A novel target of Runx2
复制标题
C2C12 肌源性祖细胞的成骨分化需要 Fos 相关抗原 Fra-1 - Runx2 的新靶标
DOI：
10.1016/j.bbrc.2012.11.033
复制
发表时间：
2013-01-04
影响因子：
3.1
通讯作者：
Hong, An
中科院分区：
文献类型：
--
作者：
Yu, Shouhe;Geng, Qianqian;Hong, An
骨形成是需要间充质干细胞分化成成骨细胞的发育过程。已确定Runx 2紧密调节成骨细胞分化和骨形成。Fos相关抗原Fra-1是骨形成的必需因子。目前的证据并不支持Fra-1和Runx 2在骨生成中的关系。在此，我们探讨了Runx 2在C2 C12肌源性祖细胞成骨分化过程中调节Fra-1表达的可能性。在Tet-on稳定的C2 C12细胞系（C2 C12/Runx 2（Dox）亚系）中，Runx 2活化后，Fra-1的表达被快速诱导。瞬时转染实验表明Runx 2激活Fra-1启动子-报告基因活性，提示Fra-1可能是Runx 2的直接靶点。为了确定由Runx 2激活的Fra-1启动子的最小区域，制备了一系列Fra-1启动子缺失构建体。通过瞬时转染实验，我们确定了最小区域为近端342 bp（-84至+258）。在该区域内预测了两个潜在的Runx 2结合位点（在位置+139和+208处）。与野生型相比，Runx 2基序+208位的突变显著降低了Fra-1启动子活性，而Runx 2基序+139位的突变没有影响。电泳迁移率变动分析（EMSA）证实在+208位存在一个非典型Runx 2结合元件，染色质免疫沉淀（ChIP）分析揭示Runx 2通过该位点与天然Fra-1启动子结合。最后，Fra-1的强制表达导致碱性磷酸酶（ALP）的上调，这是早期成骨细胞分化的标志物。总之，这些结果表明，Fra-1是C2 C12肌原祖细胞成骨分化过程中Runx 2的直接靶点。(C)2012 Elsevier Inc. All rights reserved.
Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2(Dox) sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (-84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells. (C) 2012 Elsevier Inc. All rights reserved.