Motojima M, Ando T, Yoshioka T
Biomedical Research Laboratories, Kureha Chemical Industry Co., 3-26-2 Hyakunin-cho, Shinjuku-ku, Tokyo 169-8503, Japan.
Biochem J. 2000 Jul 15;349(Pt 2):435-41. doi: 10.1042/0264-6021:3490435.
Angiotensin II (Ang II) up-regulates plasminogen-activator inhibitor type-1 (PAI-1) expression in mesangial cells to enhance extracellular matrix formation. The proximal promoter region (bp -87 to -45) of the human PAI-1 gene contains several potent binding sites for transcription factors [two phorbol-ester-response-element (TRE)-like sequences; D-box (-82 to -76) and P-box (-61 to 54), and one Sp1 binding site-like sequence, Sp1-box 1 (-72 to -67)]. We studied this region to determine the transcription factor(s) that mediates Ang-II-induced transcriptional activation of the PAI-1 gene. Various double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to various sequences in the proximal promoter region were transfected to mesangial cells to examine the effects on Ang-II-induced PAI-1 mRNA expression. Transfection with the full-length decoy (bp -87 to -45, D-P-ODN) markedly attenuated Ang-II-induced PAI-1 mRNA expression by up to 70%. Transfection with D-ODN (-87 to -71) and P-ODN (-66 to -45), which correspond to each of the two TRE-like sequences, did not attenuate the expression. Gel-shift assays using nuclear extracts prepared from Ang-II-treated mesangial cells and D-P-ODN showed three specific complexes. The major complex was supershifted by anti-Sp1 antibody. The methylation-interference experiment demonstrated that human recombinant Sp1 bound to the so-called GT box (TGGGTGGGGCT, -78 to -69), which contains the Sp1-box 1. The complex that migrated with anti-Sp1 antibody was enhanced in the cells treated with Ang II. Further, D-Sp1-ODN (-85 to -63) containing the GT box attenuated up-regulation of PAI-1 mRNA expression induced by Ang II to a level (68+/-9% inhibition) comparable to D-P-ODN, whereas ODN with four mutations in the GT box had no effect. Our findings suggest that binding of Sp1 or an Sp1-like transcription factor to the GT box in the PAI-1 promoter up-regulates PAI-1 gene transcription in mesangial cells stimulated with Ang II. This transcription-factor binding site may be targeted to control Ang-II-dependent extracellular matrix formation by mesangial cells.
血管紧张素II（Ang II）上调系膜细胞中纤溶酶原激活物抑制剂1型（PAI-1）的表达，以增强细胞外基质的形成。人PAI-1基因的近端启动子区域（bp -87至-45）包含几个转录因子的有效结合位点[两个佛波酯反应元件（TRE）样序列；D框（-82至-76）和P框（-61至-54），以及一个Sp1结合位点样序列，Sp1框1（-72至-67）]。我们研究了该区域，以确定介导Ang-II诱导的PAI-1基因转录激活的转录因子。将与近端启动子区域中各种序列相对应的各种双链诱饵寡脱氧核苷酸（ODN）转染到系膜细胞中，以检查对Ang-II诱导的PAI-1 mRNA表达的影响。用全长诱饵（bp -87至-45，D-P-ODN）转染可使Ang-II诱导的PAI-1 mRNA表达明显减弱，最多可减弱70%。用与两个TRE样序列中的每一个相对应的D-ODN（-87至-71）和P-ODN（-66至-45）转染并没有减弱该表达。使用从Ang-II处理的系膜细胞制备的核提取物和D-P-ODN进行凝胶迁移分析显示出三种特异性复合物。主要复合物被抗Sp1抗体超迁移。甲基化干扰实验表明，人重组Sp1与所谓的GT框（TGGGTGGGGCT，-78至-69）结合，该框包含Sp1框1。在用Ang II处理的细胞中，与抗Sp1抗体一起迁移的复合物增强。此外，含有GT框的D-Sp1-ODN（-85至-63）将Ang II诱导的PAI-1 mRNA表达上调减弱到与D-P-ODN相当的水平（68±9%抑制），而在GT框中有四个突变的ODN则没有效果。我们的研究结果表明，Sp1或类似Sp1的转录因子与PAI-1启动子中的GT框结合，可上调Ang II刺激的系膜细胞中PAI-1基因的转录。这个转录因子结合位点可能是控制系膜细胞中Ang-II依赖性细胞外基质形成的靶点。
