Khachigian L M, Williams A J, Collins T
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1995 Nov 17;270(46):27679-86. doi: 10.1074/jbc.270.46.27679.
The platelet-derived growth factor (PDGF) A-chain has been implicated in the initiation and progression of vascular occlusive lesions. The elements in the human PDGF-A promoter that mediate increased expression of the gene in vascular endothelial cells have not been identified. A potent inducer of PDGF-A expression in endothelial cells is phorbol 12-myristate 13-acetate (PMA). 5'-Deletion and transfection analysis revealed that a G+C-rich region in the proximal PDGF-A promoter is required for PMA-inducible gene expression. This region bears overlapping consensus recognition sequences for Sp1 and Egr-1. PMA induces Egr-1 mRNA expression within 1 h, whereas PDGF-A transcript levels increase after 2-4 h. Constitutive levels of Sp1 are not altered over 24 h. A specific nucleoprotein complex is formed when an oligonucleotide bearing the G+C-rich element is incubated with nuclear extracts from PMA-treated cells. The temporal appearance of this complex is consistent with the transient increase in Egr-1 transcripts. Antibodies to Egr-1 completely supershift the PMA-induced complex. Interestingly, increased nuclear levels of Egr-1 attenuate the ability of Sp1 to interact with the oligonucleotide, implicating competition between Egr-1 and Sp1 for the G+C-rich element. Binding studies with recombinant proteins demonstrate that Egr-1 can displace Sp1 from this region. Insertion of the G+C-rich element into a hybrid promoter-reporter construct confers PMA inducibility on the construct. Mutations that abolish Egr-1 binding also abrogate expression induced by PMA or overexpressed Egr-1. These findings demonstrate that PMA-induced Egr-1 displaces Sp1 from the G+C-rich element and activates expression driven by the PDGF-A proximal promoter in endothelial cells. The Sp1/Egr-1 displacement mechanism may be an important regulatory circuit in the control of inducible gene expression in vascular endothelial cells.
血小板衍生生长因子（PDGF）A链与血管闭塞性病变的发生和发展有关。尚未确定人类PDGF - A启动子中介导该基因在血管内皮细胞中表达增加的元件。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）是内皮细胞中PDGF - A表达的有效诱导剂。5' - 缺失和转染分析表明，近端PDGF - A启动子中富含G + C的区域是PMA诱导基因表达所必需的。该区域具有Sp1和Egr - 1重叠的共有识别序列。PMA在1小时内诱导Egr - 1 mRNA表达，而PDGF - A转录水平在2 - 4小时后增加。Sp1的组成型水平在24小时内未改变。当将带有富含G + C元件的寡核苷酸与PMA处理细胞的核提取物一起孵育时，会形成一种特异性核蛋白复合物。该复合物的瞬时出现与Egr - 1转录本的短暂增加一致。针对Egr - 1的抗体完全使PMA诱导的复合物发生超迁移。有趣的是，Egr - 1核水平的增加减弱了Sp1与寡核苷酸相互作用的能力，这意味着Egr - 1和Sp1之间对富含G + C元件的竞争。与重组蛋白的结合研究表明，Egr - 1可以从该区域取代Sp1。将富含G + C的元件插入杂交启动子 - 报告基因构建体中可赋予该构建体PMA诱导性。消除Egr - 1结合的突变也消除了PMA或过表达的Egr - 1诱导的表达。这些发现表明，PMA诱导的Egr - 1从富含G + C的元件上取代SpI，并激活内皮细胞中由PDGF - A近端启动子驱动的表达。Sp1 / Egr - 1取代机制可能是控制血管内皮细胞中诱导基因表达的重要调节途径。
