Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4
Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4
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DOI：
10.1016/j.bbrc.2015.12.075
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发表时间：
2016-01-22
影响因子：
3.1
通讯作者：
Hamakubo, Takao
中科院分区：
文献类型：
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作者：
Enomoto, Satoshi;Mitsui, Kenichi;Hamakubo, Takao
已知狭缝蛋白及其受体Roundabout （Robo）家族在血管系统中起着关键作用。Slit2/Robo1调控人脐静脉内皮细胞（HUVECs）和肿瘤相关内皮细胞的迁移。Robo4，内皮特异性的Robo，也被认为参与了血管细胞的迁移。然而，Slit/Robo信号通路仍不清楚。通过Boyden室实验，我们发现在Robo4敲低的条件下，Slit2诱导HUVECs迁移。这种效应在Robo1敲除细胞中消失。Robo1的n端胞外部分的共存阻断了slit2诱导的HUVECs迁移，而Robo4的共存则没有影响。这些结果表明，Slit2信号是通过Robo1转导的，而Robo4的负调控是胞内事件。使用抗Robo1单克隆抗体的靶向蛋白质组学鉴定出CdGAP，一种粘附定位的rac1和cdc42特异性GTPase激活蛋白，作为Slit2/Robo1信号传导的候选蛋白。从共转染Robo1和CdGAP基因的CHO细胞中共免疫沉淀出Robo1和CdGAP。这些结果表明，Slit2/Robo1结合对细胞迁移有影响，而Robo4对细胞迁移有负调控作用，并且Robo1可能通过与HUVECs中的CdGAP相互作用发挥作用。(C) 2015爱思唯尔公司版权所有。
Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs. (C) 2015 Elsevier Inc. All rights reserved.