Scleraxis and NFATc regulate the expression of the pro-α1(I) collagen gene in tendon fibroblasts
Scleraxis and NFATc regulate the expression of the pro-α1(I) collagen gene in tendon fibroblasts
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DOI：
10.1074/jbc.m610113200
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发表时间：
2007-06-15
影响因子：
4.8
通讯作者：
Rossert, Jerome
中科院分区：
文献类型：
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作者：
Lejard, Veronique;Brideau, Gaelle;Rossert, Jerome
单独的顺式作用元件的组合作用控制I型胶原基因的细胞特异性表达。特别是，我们已经表明，位于-3.2和-2.3 kb之间的两个短的元件，并命名为TSE 1和TSE 2是需要在肌腱成纤维细胞中表达的小鼠COL 1a 1基因。在这项研究中，我们分析了与TSE 1和TSE 2结合的反式作用因子。凝胶位移实验表明，scleraxis（SCX），这是一个基本的螺旋-环-螺旋转录因子，在肌腱成纤维细胞中选择性表达，结合TSE 2，优先作为SCX/E47异二聚体。在转染实验中，SCX和E47的过表达强烈增强了报告构建体的活性，所述报告构建体包含克隆在COL 1a 1最小启动子上游的四个TSE 2拷贝或COL 1a 1近端启动子的3.2 kb片段。TSE 1的分析表明，它包含一个NFATc转录因子的共识结合位点。这使我们表明，NFATc 4基因在发育中的小鼠肢体的肌腱和TT-D 6细胞中表达，TT-D 6细胞是一种具有肌腱成纤维细胞特征的细胞系。在凝胶迁移试验中，TSE 1结合TT-D 6细胞核提取物中存在的NFATc蛋白。在转染实验中，NFATc的过表达反式激活了一个报告构建体，该报告构建体含有克隆在COL 1a 1最小启动子上游的四个TSE 1拷贝。相反，在TT-D 6细胞中抑制NFATc蛋白的核转位强烈抑制COL 1a 1基因的表达。综上所述，这些结果表明，SCX和NFATc 4合作，以激活特异性肌腱成纤维细胞中的COL 1a 1基因。
The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis ( SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts.