Shyy J Y, Lin M C, Han J, Lu Y, Petrime M, Chien S
Department of Bioengineering, University of California, San Diego, La Jolla 92093-0412, USA.
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):8069-73. doi: 10.1073/pnas.92.17.8069.
Vascular endothelial cells, serving as a barrier between vessel and blood, are exposed to shear stress in the body. Although endothelial responses to shear stress are important in physiological adaption to the hemodynamic environments, they can also contribute to pathological conditions--e.g., in atherosclerosis and reperfusion injury. We have previously shown that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells and that the regulation is at the transcriptional level. These observations led us to functionally analyze the 550-bp promoter region of the MCP-1-encoding gene to define the cis element responding to shear stress. The shear stress/luciferase assay on the deletion constructs revealed that a 38-bp segment (-53 to -90 bp relative to the transcription initiation site) containing two divergent phorbol ester "12-O-tetradecanoylphorbol 13-acetate" (TPA)-responsive elements (TRE) is critical for shear inducibility. Site-specific mutations on these two sites further demonstrated that the proximal one (TGACTCC) but not the distal one (TCACTCA) was shear-responsive. Shear inducibility was lost after the mutation or deletion of the proximal site. This molecular mechanism of shear inducibility of the MCP-1 gene was functional in both the epithelial-like HeLa cells and bovine aortic endothelial cells (BAEC). In a construct with four copies of the TRE consensus sequences TGACTACA followed by the rat prolactin minimal promoter and luciferase gene, shear stress induced the reporter activities by 35-fold and 7-fold in HeLa cells and BAEC, respectively. The application of shear stress on BAEC also induced a rapid and transient phosphorylation of mitogen-activated protein kinases. Pretreatment of BAEC with TPA attenuated the shear-induced mitogen-activated protein kinase phosphorylation, suggesting that shear stress and TPA share a similar signal transduction pathway in activating cells. The present study provides a molecular basis for the transient induction of MCP-1 gene by shear stress.
血管内皮细胞作为血管与血液之间的屏障，在体内承受剪切应力。尽管内皮细胞对剪切应力的反应在对血流动力学环境的生理适应中很重要，但它们也可能导致病理状况，例如动脉粥样硬化和再灌注损伤。我们之前已经表明，剪切应力介导血管内皮细胞中单核细胞趋化蛋白1（MCP-1）基因表达的双相反应，并且这种调节发生在转录水平。这些观察结果促使我们对编码MCP-1的基因的550 bp启动子区域进行功能分析，以确定响应剪切应力的顺式元件。对缺失构建体进行的剪切应力/荧光素酶测定表明，一个包含两个不同佛波酯“12-O-十四烷酰佛波醇13-乙酸酯”（TPA）反应元件（TRE）的38 bp片段（相对于转录起始位点为-53至-90 bp）对剪切诱导至关重要。对这两个位点进行的位点特异性突变进一步表明，近端位点（TGACTCC）而非远端位点（TCACTCA）对剪切有反应。近端位点发生突变或缺失后，剪切诱导性丧失。MCP-1基因剪切诱导的这种分子机制在上皮样HeLa细胞和牛主动脉内皮细胞（BAEC）中均起作用。在一个含有四个TRE共有序列TGACTACA拷贝，随后是大鼠催乳素最小启动子和荧光素酶基因的构建体中，剪切应力在HeLa细胞和BAEC中分别使报告基因活性诱导35倍和7倍。对BAEC施加剪切应力还诱导丝裂原活化蛋白激酶的快速和短暂磷酸化。用TPA预处理BAEC可减弱剪切诱导的丝裂原活化蛋白激酶磷酸化，表明剪切应力和TPA在激活细胞方面共享相似的信号转导途径。本研究为剪切应力对MCP-1基因的瞬时诱导提供了分子基础。
