title: Regulation of endothelial signaling and migration by v-ATPase.
abstract: The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach.
authors.affiliation: Department of Pharmacy, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Rath
authors.forename: Johanna
authors.initials: J
authors.lastname: Liebl
authors.forename: Robert
authors.initials: R
authors.lastname: Fürst
authors.forename: Angelika M
authors.initials: AM
authors.lastname: Vollmar
authors.forename: Stefan
authors.initials: S
authors.lastname: Zahler
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: C541389:ATP6V0C protein, human; D004791:Enzyme Inhibitors; D018942:Macrolides; D021122:Protein Subunits; D051880:Receptors, Notch; C036978:concanamycin A; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D025262:Vacuolar Proton-Translocating ATPases; D020830:rac1 GTP-Binding Protein