Endothelial cell cortactin phosphorylation by src contributes to polymorphonuclear leukocyte transmigration in vitro
Endothelial cell cortactin phosphorylation by src contributes to polymorphonuclear leukocyte transmigration in vitro
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DOI：
10.1161/01.res.0000201958.59020.1a
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发表时间：
2006-02-17
影响因子：
20.1
通讯作者：
Luscinskas, FW
中科院分区：
文献类型：
--
作者：
Yang, L;Kowalski, JR;Luscinskas, FW
调节白细胞通过血管内皮跨内皮迁移的潜在机制仍不清楚。 Cortactin 是 Src 酪氨酸激酶的底物和细胞骨架动力学的调节剂。先前的研究证明 Cortactin 的 Src 磷酸化在 E-选择素和细胞间细胞粘附分子 1 围绕贴壁白细胞聚集的过程中发挥作用。在当前的研究中，我们使用体外流动模型来研究Src诱导的内皮皮质蛋白磷酸化在多形核白细胞（PMN）通过肿瘤坏死因子-α预激活的人脐静脉内皮（HUVEC）单层细胞迁移过程中的作用。使用 Src 激酶抑制剂 PP2 和 SU6656 抑制 HUVEC 中的 Src，分别使 PMN 迁移减少 45 +/- 8% 和 36 +/- 6%。 HUVEC 中绿色荧光蛋白标记的皮质蛋白的活细胞成像揭示了皮质蛋白在迁移中性粒细胞周围区域的重新分布。小干扰RNA对HUVEC中cortactin的敲低也同样程度地损害了轮回，并且通过野生型cortactin的重新表达来挽救这种表型。对粘附于 HUVEC 的 PMN 的初始停滞和运动位置的分析表明，抑制 Src 酪氨酸激酶或用 cortactin 小干扰 RNA 预处理可减少 PMN 在内皮细胞与细胞连接处的迁移，但不会减少粘附。 Cortactin 的酪氨酸磷酸化对于轮回很重要，因为酪氨酸磷酸化位点突变为苯丙氨酸（cortactin3F）的突变体的表达未能挽救 PMN 轮回。此外，cortactin3F 的单独表达部分阻断了 PMN 的迁移。这些数据表明 Src 家族激酶对 Cortactin 的酪氨酸磷酸化调节 PMN 迁移的模型。
The underlying mechanisms that regulate leukocyte transendothelial migration through the vascular endothelium remain unclear. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of cortactin in clustering of E-selectin and intercellular cell adhesion molecule-1 around adherent leukocytes. In the current study, we used an in vitro flow model to investigate the role of Src-induced cortactin phosphorylation in endothelium during polymorphonuclear leukocyte (PMN) transmigration through human umbilical vein endothelium (HUVEC) monolayers preactivated with tumor necrosis factor-alpha. Inhibition of Src in HUVEC using Src kinase inhibitors PP2 and SU6656 reduced PMN transmigration by 45 +/- 8% and 36 +/- 6%, respectively. Live cell imaging of green fluorescent protein-tagged cortactin in HUVEC revealed redistribution of cortactin in the region surrounding transmigrating PMN. Knockdown of cortactin in HUVEC by small interfering RNA also impaired transmigration to a similar degree, and this phenotype was rescued by reexpression of wild-type cortactin. Analysis of the location of initial arrest and locomotion of PMN adherent to HUVEC demonstrated that inhibition of Src tyrosine kinases or pretreatment with cortactin small interfering RNA reduced PMN transmigration at endothelial cell-to-cell junctions and not adhesion. Tyrosine phosphorylation of cortactin was important for transmigration, because expression of a mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortactin3F), failed to rescue PMN transmigration. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby tyrosine phosphorylation of cortactin by Src family kinases regulates PMN transmigration.