Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4

• Published in: Biochemical and biophysical research communications Vol. 469; no. 4; pp. 797 - 802
• Main Authors: Enomoto, Satoshi, Mitsui, Kenichi, Kawamura, Takeshi, Iwanari, Hiroko, Daigo, Kenji, Horiuchi, Keiko, Minami, Takashi, Kodama, Tatsuhiko, Hamakubo, Takao
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.01.2016
• Subjects: CdGAP; Cell Line; Cell Movement - physiology; Endothelial Cells - cytology; Endothelial Cells - physiology; GTPase-Activating Proteins - metabolism; Humans; HUVEC; Intercellular Signaling Peptides and Proteins; Migration; Nerve Tissue Proteins - metabolism; Phosphoproteins - metabolism; Receptors, Cell Surface - metabolism; Receptors, Immunologic - metabolism; Robo1; Robo4; Roundabout Proteins; Signal Transduction - physiology; Slit2; LC/MS/MS; Migration; Slit2; CdGAP; CHO; Robo; EDTA; Robo1; SDS-PAGE; DMEM; HEPES; FBS; HUVEC; Robo4; EGM-2; HUVECs; srGAP; EBM-2
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.12.075

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. •Slit2/Robo1 signaling intrinsically exerts a migratory effect on HUVEC and Robo4 regulates the effect.•The migratory response to HUVEC by Slit2 is through its binding to Robo1, not to Robo4.•CdGAP is suggested as a potential intracellular partner of Robo1 by proteomics and immunochemical analyses.