Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.
BACKGROUND
Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis.


OBJECTIVE
We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism.


RESULTS
Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis.


CONCLUSIONS
These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.
Tawa
Hideto
H
Division of Signal Transduction, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
Rikitake
Yoshiyuki
Y
Takahashi
Motonori
M
Amano
Hisayuki
H
Miyata
Muneaki
M
Satomi-Kobayashi
Seimi
S
Kinugasa
Mitsuo
M
Nagamatsu
Yuichi
Y
Majima
Takashi
T
Ogita
Hisakazu
H
Miyoshi
Jun
J
Hirata
Ken-ichi
K
Takai
Yoshimi
Y
D000818:Animals; D017209:Apoptosis; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D006614:Hindlimb; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism; D007511:Ischemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000098:blood supply*; D018919:Neovascularization, Physiologic; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015861:Retinal Neovascularization / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D008246:Lysophospholipids; D008840:Microfilament Proteins; C107953:RAC1 protein, human; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C108835:afadin; C060506:sphingosine 1-phosphate; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D013110:Sphingosine