Eyries Mélanie, Agrapart Monique, Alonso Amalia, Soubrier Florent
Institut National de la Santé et de la Recherche Médicale, Unit 525, Faculté de Médecine Pitié-Sapétrière, Paris, France.
Circ Res. 2002 Nov 15;91(10):899-906. doi: 10.1161/01.res.0000042703.39845.b4.
Angiotensin-converting enzyme (ACE) is an enzyme that plays a major role in vasoactive peptide metabolism, and it has been implicated in various cardiovascular diseases. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, has been shown to increase ACE mRNA at the transcriptional level in human umbilical vein endothelial cells. We have investigated the transcriptional mechanism involved in protein kinase C induction of the ACE gene. Deletion and transfection analyses have revealed that two regions are required for PMA-inducible gene expression. The first is a G+C-rich region located in the proximal ACE promoter bearing overlapping consensus recognition sequences for stimulatory protein-1 (Sp1) and early growth response gene 1 (Egr-1). Electrophoretic mobility shift assay and supershift experiments have shown that Egr-1 is present in the specific nucleoprotein complex induced by PMA in human umbilical vein endothelial cells. The second region is located in the distal ACE promoter. DNase I footprinting analysis restricted this region to a 21-bp element containing a cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate-responsive element sequence. Electrophoretic mobility shift assays and supershift analyses have revealed that activating protein 1 (AP-1) is the transcription factor binding the cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate-responsive element located in the ACE promoter after PMA stimulation. Mutations of either Egr-1 or AP-1 binding sites partially abrogate ACE expression induced by PMA, whereas mutation of both sites totally abrogates PMA-induced ACE expression. Treatment of cells with PD98059, a mitogen-activated protein kinase kinase-1-specific inhibitor, inhibited PMA-induced ACE expression. Our results demonstrate that the two transcription factors, Egr-1 and AP-1, are involved in the PMA-induced ACE transcriptional activation in human endothelial cells via the activation of the extracellular signal-regulated kinase 1/2 signaling pathway.
血管紧张素转换酶（ACE）是一种在血管活性肽代谢中起主要作用的酶，它与多种心血管疾病有关。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），一种蛋白激酶C激活剂，已被证明可在人脐静脉内皮细胞中在转录水平上增加ACE mRNA。我们研究了蛋白激酶C诱导ACE基因的转录机制。缺失和转染分析表明，PMA诱导的基因表达需要两个区域。第一个是位于近端ACE启动子中的富含G + C的区域，带有刺激蛋白-1（Sp1）和早期生长反应基因1（Egr-1）的重叠共有识别序列。电泳迁移率变动分析和超迁移实验表明，Egr-1存在于PMA诱导的人脐静脉内皮细胞中的特异性核蛋白复合物中。第二个区域位于远端ACE启动子中。DNase I足迹分析将该区域限制在一个含有cAMP反应元件/12 - O - 十四烷酰佛波醇13 - 乙酸酯反应元件序列的21个碱基对的元件上。电泳迁移率变动分析和超迁移分析表明，激活蛋白1（AP-1）是PMA刺激后结合ACE启动子中cAMP反应元件/12 - O - 十四烷酰佛波醇13 - 乙酸酯反应元件的转录因子。Egr-1或AP-1结合位点的突变部分消除了PMA诱导的ACE表达，而两个位点的突变则完全消除了PMA诱导的ACE表达。用丝裂原活化蛋白激酶激酶-1特异性抑制剂PD98059处理细胞可抑制PMA诱导的ACE表达。我们的结果表明，两种转录因子Egr-1和AP-1通过细胞外信号调节激酶1/2信号通路的激活参与了人内皮细胞中PMA诱导的ACE转录激活。
