Lin M C, Almus-Jacobs F, Chen H H, Parry G C, Mackman N, Shyy J Y, Chien S
Department of Bioengineering and Institute for Biomedical Engineering, University of California, San Diego, La Jolla 92093-0412, USA.
J Clin Invest. 1997 Feb 15;99(4):737-44. doi: 10.1172/JCI119219.
Using flow channel, we report that the application of a laminar shear stress induced a transient increase of tissue factor (TF) procoagulant activity in human umbilical vein endothelial cells (HUVEC), which was accompanied by a rapid and transient induction of the TF mRNA in the HUVEC. Functional analysis of the 2.2 kb TF 5' promoter indicated that a GC-rich region containing three copies each of the EGR-1 and Sp1 sites was required for induction. Mutation of the Sp1 sites, but not the EGR-1 sites, attenuated the response of TF promoter to shear stress. Thus, Sp1 is a newly defined shear stress responsive element. Electrophoretic mobility shift assays showed there was no increase in binding of nuclear extracts from sheared cells to an Sp1 consensus site. In contrast, immunoblotting of these nuclear extracts with antibody against transcription factor Sp1 demonstrated that shear stress increased the phosphorylation of Sp1. We also showed that shear stress, like the phosphatase inhibitor okadaic acid, increased the transcriptional activity of Sp1. These findings suggest that the shear stress induction of TF gene expression is mediated through an increased Sp1 transcriptional activity with a concomitant hyperphosphorylation of Sp1.
利用流动通道，我们报告称，层流切应力的施加会导致人脐静脉内皮细胞（HUVEC）中组织因子（TF）促凝活性出现短暂增加，这伴随着HUVEC中TF mRNA的快速且短暂诱导。对2.2 kb TF 5'启动子的功能分析表明，富含GC的区域包含三个EGR - 1和Sp1位点拷贝，是诱导所必需的。Sp1位点而非EGR - 1位点的突变减弱了TF启动子对切应力的反应。因此，Sp1是一个新定义的切应力反应元件。电泳迁移率变动分析表明，剪切细胞的核提取物与Sp1共有位点的结合没有增加。相反，用抗转录因子Sp1抗体对这些核提取物进行免疫印迹显示，切应力增加了Sp1的磷酸化。我们还表明，切应力与磷酸酶抑制剂冈田酸一样，增加了Sp1的转录活性。这些发现表明，TF基因表达的切应力诱导是通过Sp1转录活性增加以及Sp1的伴随超磷酸化介导的。
