Korenaga R, Ando J, Kosaki K, Isshiki M, Takada Y, Kamiya A
Department of Cardiovascular Biomechanics, Faculty of Medicine, University of Tokyo, Japan.
Am J Physiol. 1997 Nov;273(5 Pt 1):C1506-15. doi: 10.1152/ajpcell.1997.273.5.c1506.
To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between -694 and -329 bp upstream from the transcription initiation site. Gel shift assay using overlapping oligonucleotide probes of this region showed that oligonucleotides containing a double AP-1 consensus sequence (TGACTCA) formed distinct complexes with nuclear proteins extracted from shear-stressed cells. Mutation of either one or both of two AP-1 consensus sequences completely abolished the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the VCAM-1 gene via an upstream cis-element, a double AP-1 consensus sequence, in murine lymph node venule ECs.
为了探究剪切应力诱导小鼠内皮细胞（ECs）中血管细胞黏附分子1（VCAM-1）表达下调的机制，我们检测了剪切应力对VCAM-1基因转录的影响，并评估了参与此现象的顺式作用元件。如通过核转录分析和VCAM-1启动子-荧光素酶基因构建体的瞬时转染所定义，VCAM-1 mRNA表达在转录水平上被下调。对VCAM-1缺失突变体的荧光素酶分析显示，顺式作用元件包含在转录起始位点上游-694至-329 bp之间。使用该区域的重叠寡核苷酸探针进行的凝胶迁移分析表明，含有双重AP-1共有序列（TGACTCA）的寡核苷酸与从受剪切应力作用的细胞中提取的核蛋白形成了不同的复合物。两个AP-1共有序列中的一个或两个发生突变完全消除了启动子对剪切应力作出反应的能力。这些结果表明，流体剪切应力通过上游顺式元件——双重AP-1共有序列，下调小鼠淋巴结小静脉ECs中VCAM-1基因的转录。
