The role of Sp1 in the differential expression of transforming growth factor-β receptor type II in human breast adenocarcinoma MCF-7 cells
The role of Sp1 in the differential expression of transforming growth factor-β receptor type II in human breast adenocarcinoma MCF-7 cells
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DOI：
10.1074/jbc.275.16.12231
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发表时间：
2000-04-21
影响因子：
4.8
通讯作者：
Banerji, SS
中科院分区：
文献类型：
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作者：
Liu, Y;Zhong, XH;Banerji, SS
MCF-7细胞从早期传代（MCF-7 E，500代）的进展与对外源性TGF β 1的敏感性丧失相关。我们先前已经表明，TGF β敏感性丧失是由于转化生长因子受体II型（T β RII）表达降低所致，并与裸鼠致瘤性增加相关。MCF-7 L细胞中T β RII表达降低是由于该细胞系中T β RII启动子活性降低所致。我们先前使用该启动子的5'缺失构建体的研究揭示，MCF-7 L细胞不能支持最小启动子（-47至+2）转录至与MCF-7 E细胞相同的水平。启动子的这个区域在距离主要转录起始位点的-25位含有Sp1元件。在这项研究中，我们研究了Sp1在T β RII转录中的作用。Sp1位点的突变导致MCF-7 E和MCF-7 L细胞中T β RII的转录降低，表明该位点在该启动子的转录中起作用。凝胶位移分析使用近端Spl位点从T β RII启动子显示增强的DNA：蛋白质复合物的形成与MCF-7 E细胞相比，MCF-7 L细胞分离的核蛋白。Western blot分析表明，MCF-7 E细胞中Sp1蛋白的表达水平高于MCF-7 L细胞，且Sp1蛋白的结合活性也高于MCF-7 L。与MCF-7 L细胞相比，MCF-7 E细胞中Sp1依赖性胰岛素样生长因子II启动子构建体的转录水平更高，也证明了Sp1的差异活性。在MCF-7 L细胞中共转染Sp1表达载体与T β RII启动子构建体诱导启动子-CAT构建体的表达，并导致内源性T β RII蛋白水平的增加。这些结果表明，MCF-7 L细胞中T β RII的转录抑制部分是由较低的Sp1水平引起的。
Progression of MCF-7 cells from early passage (MCF-7E, 500 passage) correlates with a loss of sensitivity to exogenous TGF beta 1, We have previously shown that loss of TGF beta sensitivity is due to decreased expression of the transforming growth factor receptor type II (T beta RII) and is associated with increased tumorigenicity in nude mice. Reduced T beta RII expression in MCF-7L cells is caused by decreased T beta RII promoter activity in this cell line. Our previous studies using 5' deletion constructs of this promoter revealed that MCF-7L cells were unable to support transcription of the minimal promoter (-47 to +2) to the same levels as the MCF-7E cells. This region of the promoter contains an Sp1 element at position -25 from the major transcription start site. In this study, we investigated the role of Sp1 in T beta RII transcription. Mutation of the Sp1 site resulted in decreased transcription of T beta RII in MCF-7E and MCF-7L cells, indicating that this site played a role in transcription of this promoter. Gel shift assays using the proximal Spl site from the T beta RII promoter showed enhanced DNA:protein complex formation with nuclear proteins isolated from MCF-7E cells compared with MCF-7L cells. Supershift analysis identified this binding activity as Sp1, Western blot analysis of Sp1 levels demonstrated that MCF-7E cells contain increased Sp1 protein compared with MCF-7L cells, paralleling the increased binding activity. Differential Sp1 activity was also demonstrated by higher levels of transcription of an Sp1-dependent insulin-like growth factor II promoter construct in MCF-7E cells compared with MCF-7L cells. Co-transfection of an Sp1 expression vector with a T beta RII promoter construct in MCF-7L cells induced the expression from the promoter-CAT constructs and resulted in an increase of endogenous T beta RII protein levels. These results demonstrate that the transcriptional repression of T beta RII in MCF-7L cells is caused, in part, by lower Sp1 levels.