Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1
Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1
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DOI：
10.1074/jbc.271.5.2731
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发表时间：
1996-02-02
影响因子：
4.8
通讯作者：
Mackman, N
中科院分区：
文献类型：
--
作者：
Cui, MZ;Parry, GCN;Mackman, N
组织因子（TF）基因表达迅速诱导上皮细胞的佛波酯12-肉豆蔻酸酯13-乙酸酯和血清。我们已经证明，这种诱导是由一个新的血清反应区（SRR）（-111至+14 bp）内的人TF启动子介导的。在这项研究中，我们的特点是顺式作用的遗传元件内的SRR调节的基础和诱导表达的TF基因在HeLa细胞，凝胶迁移率变动分析使用寡核苷酸跨越整个SRR确定了三个12碱基对（bp）的基序内的亚区1，2和3，结合组成型表达的Sp1和诱导表达的EGR-1。通过与Sp1和EGR-1位点、突变和抗体超移位实验的竞争，分析与这些12-bp基序结合的蛋白质，表明它们各自含有重叠6 bp的不同EGR-1和Sp1位点。使用含有野生型TF启动子（-111至+14 bp）或在三个Sp1和/或EGR-1位点中含有突变的衍生物的质粒转染的HeLa细胞的功能研究检查了基础和诱导表达。Sp1位点介导基础启动子活性，并且Sp1和EGR-1位点都是由佛波醇12-肉豆蔻酸酯13乙酸酯或血清最大诱导TF启动子所需的。这些数据表明，TF基因在HeLa细胞中的表达受Sp1和EGR-1的调节。
Tissue factor (TF) gene expression is rapidly induced in epithelial cells by phorbol 12-myristate 13-acetate and serum. We have shown that this induction is mediated by a novel serum response region (SRR) (-111 to +14 bp) within the human TF promoter. In this study, we characterized cis-acting genetic elements within the SRR that regulated basal and inducible expression of the TF gene in HeLa cells, Gel mobility shift assays using oligonucleotides spanning the entire SRR identified three 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound constitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR-1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids containing the wild-type TF promoter (-111 to +14 bp) or derivatives containing mutations in the three Sp1 and/or EGR-1 sites examined basal and inducible expression, The Sp1 sites mediated basal promoter activity, and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13 acetate or serum. These data indicated that TF gene expression in HeLa cells was regulated by both Sp1 and EGR-1.