Li Y, Liu J, Zhan X
Department of Experimental Pathology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
J Biol Chem. 2000 Nov 24;275(47):37187-93. doi: 10.1074/jbc.M005301200.
Injury of endothelial cells induced by reactive oxygen species plays an important role in the development of early stages of vascular diseases such as hypertension and atherosclerosis. Exposure of human umbilical vein endothelial cells to hydrogen peroxide (H(2)O(2)), a common form of reaction oxygen species, triggers a series of intracellular events, including actin cytoskeletal reorganization, cytoplasm shrinkage, membrane blebbing and protein-tyrosine phosphorylation. The effect of H(2)O(2) on endothelial cells is dramatically enhanced when a survival pathway involving extracellular signal-regulated kinase is blocked by PD098059. In contrast, the injury of endothelial cells mediated by H(2)O(2) is inhibited by PP2, a selective specific inhibitor for protein-tyrosine kinase Src. Cortactin, a filamentous actin (F-actin)-associated protein, becomes phosphorylated at tyrosine residues upon stimulation by H(2)O(2) in a manner dependent on the activity of Src. The level of tyrosine phosphorylation of cortactin is correlated with the formation of membrane blebs. Overexpression of wild-type cortactin tagged with green fluorescent protein in endothelial cells via a retroviral vector substantiates the H(2)O(2)-induced morphological changes, whereas overexpression of a green fluorescent protein-cortactin mutant deficient in tyrosine phosphorylation renders endothelial cells resistant to H(2)O(2). The functional role of cortactin in H(2)O(2)-mediated shape changes was also evaluated in NIH 3T3 cells. Stable 3T3 transfectants expressing wild-type cortactin in the presence of either H(2)O(2)/PD098059 or H(2)O(2) alone at 200 microm exhibited a dramatic shape change characterized by rounding up or aggregation. However, the similar changes were not detected with cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation. These data demonstrate an important role of the Src/cortactin-dependent actin reorganization in the injury of endothelial cells mediated by reactive oxygen species.
活性氧诱导的内皮细胞损伤在高血压和动脉粥样硬化等血管疾病早期发展中起重要作用。将人脐静脉内皮细胞暴露于过氧化氢（H₂O₂），一种常见的活性氧形式，会引发一系列细胞内事件，包括肌动蛋白细胞骨架重组、细胞质收缩、膜泡形成和蛋白酪氨酸磷酸化。当涉及细胞外信号调节激酶的存活途径被PD098059阻断时，H₂O₂对内皮细胞的作用会显著增强。相反，H₂O₂介导的内皮细胞损伤被PP2抑制，PP2是蛋白酪氨酸激酶Src的选择性特异性抑制剂。皮层肌动蛋白，一种丝状肌动蛋白（F-肌动蛋白）相关蛋白，在H₂O₂刺激下以依赖Src活性的方式在酪氨酸残基处发生磷酸化。皮层肌动蛋白的酪氨酸磷酸化水平与膜泡形成相关。通过逆转录病毒载体在内皮细胞中过表达绿色荧光蛋白标记的野生型皮层肌动蛋白证实了H₂O₂诱导的形态变化，而酪氨酸磷酸化缺陷的绿色荧光蛋白-皮层肌动蛋白突变体的过表达使内皮细胞对H₂O₂具有抗性。还在NIH 3T3细胞中评估了皮层肌动蛋白在H₂O₂介导的形状变化中的功能作用。在200微摩尔的H₂O₂/PD098059或单独的H₂O₂存在下，稳定表达野生型皮层肌动蛋白的3T3转染细胞表现出以变圆或聚集为特征的显著形状变化。然而，在过表达酪氨酸磷酸化缺陷的皮层肌动蛋白突变体的细胞中未检测到类似变化。这些数据表明Src/皮层肌动蛋白依赖性肌动蛋白重组在活性氧介导的内皮细胞损伤中起重要作用。
