Naruse K, Yamada T, Sai X R, Hamaguchi M, Sokabe M
Department of Physiology, Nagoya University School of Medicine, Japan.
Oncogene. 1998 Jul 30;17(4):455-63. doi: 10.1038/sj.onc.1201950.
In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uniaxial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis. We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation. The major phosphorylated proteins had molecular masses of approximately 120-135 kDa, and 70 kDa. Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125FAK) and pp130CAS were included in the 70 kDa and 120-135 kDa bands, respectively. The morphological change was inhibited by herbimycin A and genistein, inhibitors of tyrosine kinases, suggesting that tyrosine phosphorylation was required for the morphological change. In addition, the kinase activation of pp125FAK was observed in response to cyclic stretch. Moreover, suppression of pp125FAK expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125FAK play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.
在本研究中，对人脐静脉内皮细胞（HUVECs）拉伸诱导的形态变化所需的关键信号通路进行了研究。在弹性硅膜上培养的细胞进行单轴循环拉伸（1Hz，长度增加20%），诱导细胞形态逐渐从多边形变为细长的纺锤形，其长轴与拉伸轴垂直排列。我们发现，细胞蛋白的酪氨酸磷酸化在循环拉伸刺激下增加，并在20分钟时达到峰值。用钆（Gd3+，一种拉伸激活通道的有效阻滞剂）处理细胞或去除细胞外Ca2+，均能阻断蛋白的酪氨酸磷酸化，这表明拉伸激活（SA）离子通道调节拉伸特异性酪氨酸磷酸化。主要的磷酸化蛋白分子量约为120 - 135kDa和70kDa。免疫沉淀实验表明，桩蛋白、粘着斑激酶（pp125FAK）和pp130CAS分别包含在70kDa和120 - 135kDa条带中。酪氨酸激酶抑制剂赫比霉素A和染料木黄酮可抑制形态变化，表明形态变化需要酪氨酸磷酸化。此外，还观察到pp125FAK的激酶活性在循环拉伸刺激下被激活。此外，在HUVECs中用反义硫代磷酸酯寡脱氧核苷酸（S-ODN）抑制pp125FAK表达，导致桩蛋白酪氨酸磷酸化受抑制以及拉伸依赖性形态变化受抑制。这些结果表明，细胞内Ca2+增加导致酪氨酸激酶激活以及pp125FAK，在单轴循环拉伸的内皮细胞中特有的独特形态变化中起关键作用。
