Silencing of directional migration in roundabout4 knockdown endothelial cells.
BACKGROUND
Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro.


RESULTS
Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.


CONCLUSIONS
This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.
Kaur
Sukhbir
S
Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA. kaurs@mail.nih.gov
Samant
Ganesh V
GV
Pramanik
Kallal
K
Loscombe
Philip W
PW
Pendrak
Michael L
ML
Roberts
David D
DD
Ramchandran
Ramani
R
D002465:Cell Movement; D002478:Cells, Cultured; D002633:Chemotaxis; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D012313:RNA; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein; C462951:ROBO4 protein, human