Slit2-Robo4 signalling promotes vascular stability by blocking Arf6 activity.
Slit-Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4-paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2-Robo4-paxillin-GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.
Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA.
Christopher A
CA
Jones
Naoyuki
N
Nishiya
Nyall R
NR
London
Weiquan
W
Zhu
Lise K
LK
Sorensen
Aubrey C
AC
Chan
Chinten J
CJ
Lim
Haoyu
H
Chen
Qisheng
Q
Zhang
Peter G
PG
Schultz
Alaa M
AM
Hayallah
Kirk R
KR
Thomas
Michael
M
Famulok
Kang
K
Zhang
Mark H
MH
Ginsberg
Dean Y
DY
Li
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors*; D000818:Animals; D001808:Blood Vessels / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement; D006224:Cricetinae; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D000098552:Slit Homolog 2 Protein; D000097905:Roundabout Proteins
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D000098552:Slit Homolog 2 Protein; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse