Korenaga R, Yamamoto K, Ohura N, Sokabe T, Kamiya A, Ando J
Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
Am J Physiol Heart Circ Physiol. 2001 May;280(5):H2214-21. doi: 10.1152/ajpheart.2001.280.5.H2214.
Endothelial purinoceptors play an important role in vascular responses to extracellular adenine nucleotides and hemodynamic forces. Here we report that P2X4 purinoceptor expression in human umbilical vein endothelial cells is transcriptionally downregulated by fluid shear stress. When human umbilical vein endothelial cells were subjected to a laminar shear stress of 15 dyn/cm(2), P2X4 mRNA levels began to decrease within 1 h and further decreased with time, reaching 60% at 24 h. Functional analysis of the 1.9-kb P2X4 5'-promoter indicated that a 131-bp segment (-112 to +19 bp relative to the transcription start site) containing a consensus binding site for the Sp1 transcription factor was critical for the shear stress responsiveness. Mutations of the Sp1 site decreased the basal level of transcription and abolished the response of the P2X4 promoter to shear stress. Electrophoretic mobility shift assays showed a marked decrease in binding of Sp1 to the Sp1 consensus element in shear-stressed cells, suggesting that Sp1 mediates the shear stress-induced downregulation of P2X4 gene transcription.
内皮嘌呤受体在血管对细胞外腺嘌呤核苷酸和血流动力学力的反应中起重要作用。在此我们报告，人脐静脉内皮细胞中P2X4嘌呤受体的表达在转录水平上受到流体剪切力的下调。当人脐静脉内皮细胞受到15达因/平方厘米的层流剪切力作用时，P2X4 mRNA水平在1小时内开始下降，并随时间进一步降低，在24小时时降至60%。对1.9 kb P2X4 5'-启动子的功能分析表明，一个包含Sp1转录因子共有结合位点的131 bp片段（相对于转录起始位点为-112至+19 bp）对剪切力反应性至关重要。Sp1位点的突变降低了基础转录水平，并消除了P2X4启动子对剪切力的反应。电泳迁移率变动分析显示，在受到剪切力作用的细胞中，Sp1与Sp1共有元件的结合显著减少，这表明Sp1介导了剪切力诱导的P2X4基因转录下调。
