Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells
Sp1 sites mediate activation of the plasminogen activator inhibitor-1 promoter by glucose in vascular smooth muscle cells
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DOI：
10.1074/jbc.273.14.8225
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发表时间：
1998-04-03
影响因子：
4.8
通讯作者：
Vaughan, DE
中科院分区：
文献类型：
--
作者：
Chen, YQ;Su, M;Vaughan, DE
本研究旨在观察高血糖对体外培养的大鼠主动脉平滑肌细胞纤溶酶原激活物抑制剂-1（派-1）表达的直接影响，利用一系列含有派-1 5 '侧翼序列的荧光素酶报告基因构建物，在转染到RASM中的荧光素酶（从-6.4个碱基对（bp）到-42个碱基对（bp））中，我们发现葡萄糖（25 mM）一致地诱导荧光素酶活性增加4倍，其中响应定位于-85和-42 bp之间的序列。位于感兴趣区域的两个推定的Sp1结合位点的突变基本上消除了葡萄糖反应。电泳迁移率变动分析与放射性标记的寡核苷酸含有两个假定的Sp1结合位点从派-1启动子和RASM细胞核提取物显示，葡萄糖治疗显着改变的主要蛋白质-DNA复合物的流动性模式。超位移分析表明，转录因子Sp1存在于控制和高血糖条件下的复合物。这些结果表明，葡萄糖通过作用于派-1 5 '侧翼区-85和-42 bp之间的两个相邻的Sp1位点来调节RASM细胞中派-1基因的表达，并且从Sp1复合物中释放转录阻遏物可以解释RASM细胞中派-1基因在高糖条件下的激活。
This study was designed to characterize the direct effects of hyperglycemia on plasminogen activator inhibitor-1 (PAI-1) expression in cultured vascular smooth muscle cells, Glucose induced dose- and time-dependent increases of PAI-1 mRNA expression in rat aortic smooth muscle (RASM) cells in vitro, Using a series of luciferase reporter gene constructs containing PAI-1 5'-flanking sequence (from -6.4 kilobase to -42 base pairs (bp)) transfected into RASM, we found that glucose (25 mM) consistently induced a 4-fold increase in luciferase activity, with the response localized to sequence between -85 and -42 bp. Mutagenesis of two putative Sp1-binding sites located in the region of interest essentially obliterated the glucose-response. Electrophoretic mobility shift assays with radiolabeled oligonucleotides containing the two putative Sp1-binding sites from PAI-1 promoter and nuclear extracts from RASM cells revealed that glucose treatment markedly changed the mobility pattern of the major protein-DNA complexes. Supershift assay showed that transcription factor Sp1 was present in the complexes under control and hyperglycemic conditions. These results suggest that glucose regulates PAI-1 gene expression in RASM cells through an effect on two adjacent Sp1 sites located between -85 and -42 bp of the PAI-1 5'-flanking region and that the release of a transcriptional repressor from the Sp1 complexes may explain the activation of the PAI-1 gene under high glucose conditions in RASM cells.