Liu Y, Zhong X, Li W, Brattain M G, Banerji S S
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA.
J Biol Chem. 2000 Apr 21;275(16):12231-6. doi: 10.1074/jbc.275.16.12231.
Progression of MCF-7 cells from early passage (MCF-7E, <200 passage) to late passage (MCF-7L, >500 passage) correlates with a loss of sensitivity to exogenous TGFbeta1. We have previously shown that loss of TGFbeta sensitivity is due to decreased expression of the transforming growth factor receptor type II (TbetaRII) and is associated with increased tumorigenicity in nude mice. Reduced TbetaRII expression in MCF-7L cells is caused by decreased TbetaRII 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 TbetaRII transcription. Mutation of the Sp1 site resulted in decreased transcription of TbetaRII 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 Sp1 site from the TbetaRII 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 TbetaRII promoter construct in MCF-7L cells induced the expression from the promoter-CAT constructs and resulted in an increase of endogenous TbetaRII protein levels. These results demonstrate that the transcriptional repression of TbetaRII in MCF-7L cells is caused, in part, by lower Sp1 levels.
MCF-7细胞从早期传代（MCF-7E，传代次数<200）发展到晚期传代（MCF-7L，传代次数>500）与对外源性转化生长因子β1（TGFβ1）敏感性的丧失相关。我们之前已经表明，TGFβ敏感性的丧失是由于II型转化生长因子受体（TβRII）表达降低，并且与裸鼠中肿瘤发生能力增加有关。MCF-7L细胞中TβRII表达降低是由该细胞系中TβRII启动子活性降低所致。我们之前使用该启动子的5'缺失构建体进行的研究表明，MCF-7L细胞无法将最小启动子（-47至+2）转录至与MCF-7E细胞相同的水平。该启动子的这一区域在距离主要转录起始位点-25的位置含有一个Sp1元件。在本研究中，我们调查了Sp1在TβRII转录中的作用。Sp1位点的突变导致MCF-7E和MCF-7L细胞中TβRII转录降低，表明该位点在该启动子的转录中发挥作用。使用来自TβRII启动子的近端SpSp1位点进行的凝胶迁移试验表明，与MCF-7L细胞相比，从MCF-7E细胞分离的核蛋白与DNA形成的蛋白质复合物增强。超迁移分析确定这种结合活性为Sp1。对Sp1水平的蛋白质印迹分析表明，与MCF-7L细胞相比，MCF-7E细胞中Sp1蛋白增加，这与结合活性增加相一致。与MCF-7L细胞相比，MCF-7E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，这也证明了Sp1活性存在差异。在MCF-7L细胞中将Sp1表达载体与TβRII启动子构建体共转染可诱导启动子-CAT构建体的表达，并导致内源性TβRII蛋白水平增加。这些结果表明，MCF-7L细胞中TβRII的转录抑制部分是由较低的Sp1水平引起的。
