Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.
Biochem Biophys Res Commun. 2012 Feb 24;418(4):708-13. doi: 10.1016/j.bbrc.2012.01.083. Epub 2012 Jan 24.
Paxillin, a structural and signaling scaffold molecule in focal adhesions (FAs), is considered to be important in intracellular signaling transduction and the cell shape changes in response to cyclic stretching. However, the detailed role of paxillin in stretch-induced morphological changes of endothelial cells (ECs) has not fully determined until date. In this study, in order to understand the role of paxillin in the orientation of ECs exposed to cyclic stretching, we examined the time course of changes in the shape and distribution of FA proteins of paxillin knockdown ECs. Non-treated ECs subjected to 20% cyclic stretching at 0.5Hz oriented perpendicularly to the direction of stretching after 10min of exposure. On the other hand, the orientation of paxillin knockdown ECs was abolished at 10min, but it was observed after 60min of cyclic stretching exposure. Immunofluorescent microscopy revealed that accumulation and redistribution of FA proteins, including focal adhesion kinase (FAK) and integrin β1, were observed at 10min of exposure to cyclic stretching in non-treated ECs. The accumulation of FAK and integrin β1 was not prominent in paxillin knockdown ECs under static conditions and after 10min of exposure to cyclic stretching. However, we found that accumulation of FA proteins in paxillin knockdown ECs at 30 and 60min was similar to that in non-transfected ECs. Because paxillin is an adaptor protein offering binding sites for FAK and integrin β1, which are critical molecules for the early signaling events of focal adhesion formation in ECs, these results suggest that paxillin is required for the early phase of EC orientation in response to cyclic stretching by scaffolding for accumulation of FA proteins.
桩蛋白（paxillin）是黏着斑（focal adhesions，FAs）中的一种结构和信号支架分子，被认为在细胞内信号转导和细胞形状变化以响应循环拉伸中很重要。然而，到目前为止，桩蛋白在拉伸诱导的内皮细胞（endothelial cells，ECs）形态变化中的详细作用尚未完全确定。在这项研究中，为了了解桩蛋白在暴露于循环拉伸的 ECs 定向中的作用，我们检查了 FA 蛋白形状和分布变化的时间过程在被敲低桩蛋白的 ECs 中。未经处理的 ECs 在 0.5Hz 的 20%循环拉伸下，在暴露 10 分钟后垂直于拉伸方向定向。另一方面，在 10 分钟时敲低桩蛋白的 ECs 的定向被消除，但在 60 分钟的循环拉伸暴露后观察到。免疫荧光显微镜显示，在未经处理的 ECs 中，在暴露于循环拉伸 10 分钟时，FA 蛋白，包括黏着斑激酶（focal adhesion kinase，FAK）和整合素β1 的积累和再分布。在静态条件下和在暴露于循环拉伸 10 分钟后，在被敲低桩蛋白的 ECs 中，FAK 和整合素β1 的积累不明显。然而，我们发现，在被敲低桩蛋白的 ECs 中，在 30 和 60 分钟时 FA 蛋白的积累与非转染的 ECs 相似。由于桩蛋白是 FAK 和整合素β1 的衔接蛋白，提供了黏着斑形成的早期信号事件中关键分子的结合位点，这些结果表明，在响应循环拉伸时，桩蛋白通过为 FA 蛋白的积累提供支架，是 EC 定向的早期阶段所必需的。
