Activation of diacylglycerol kinase alpha is required for VEGF-induced angiogenic signaling in vitro.
Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.
Baldanzi
Gianluca
G
Department of Medical Sciences, University Amedeo Avogadro of Piemonte Orientale, v. Solaroli 17, 28100, Novara, Italy.
Mitola
Stefania
S
Cutrupi
Santina
S
Filigheddu
Nicoletta
N
van Blitterswijk
Wim J
WJ
Sinigaglia
Fabiola
F
Bussolino
Federico
F
Graziani
Andrea
A
D000818:Animals; D001011:Aorta; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae; D013552:Swine; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D019852:Diacylglycerol Kinase