Abstract
We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Movement / drug effects
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Cell Movement / physiology
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Cells, Cultured
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Chromones / pharmacology
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Endothelial Cells / cytology
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Endothelial Cells / drug effects*
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Endothelial Cells / metabolism
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Enzyme Inhibitors / pharmacology
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Estrenes / pharmacology
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Focal Adhesion Kinase 2 / drug effects
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Focal Adhesion Kinase 2 / genetics
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Focal Adhesion Kinase 2 / metabolism*
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GTP-Binding Protein alpha Subunits, Gq-G11 / drug effects
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GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
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Humans
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Morpholines / pharmacology
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Neovascularization, Physiologic*
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Oxytocin / pharmacology*
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphodiesterase Inhibitors / pharmacology
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Phosphoinositide-3 Kinase Inhibitors
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Phosphorylation / drug effects
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Phosphorylation / physiology
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Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
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Phosphotransferases (Alcohol Group Acceptor) / metabolism
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Proto-Oncogene Proteins c-akt / drug effects
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Proto-Oncogene Proteins c-akt / metabolism
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Pyrimidines / pharmacology
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Pyrrolidinones / pharmacology
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RNA, Small Interfering / metabolism
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Receptors, Oxytocin / drug effects
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Receptors, Oxytocin / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Sphingosine / analogs & derivatives
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Sphingosine / pharmacology
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Type C Phospholipases / antagonists & inhibitors
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Type C Phospholipases / metabolism*
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Umbilical Veins / cytology
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Umbilical Veins / drug effects
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Vascular Endothelial Growth Factor A / pharmacology
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src-Family Kinases / antagonists & inhibitors
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src-Family Kinases / metabolism*
Substances
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AG 1879
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Chromones
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Enzyme Inhibitors
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Estrenes
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Morpholines
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Phosphodiesterase Inhibitors
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Phosphoinositide-3 Kinase Inhibitors
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Pyrimidines
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Pyrrolidinones
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RNA, Small Interfering
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Receptors, Oxytocin
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Vascular Endothelial Growth Factor A
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1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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Oxytocin
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Phosphotransferases (Alcohol Group Acceptor)
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sphingosine kinase
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Focal Adhesion Kinase 2
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src-Family Kinases
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Proto-Oncogene Proteins c-akt
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Type C Phospholipases
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GTP-Binding Protein alpha Subunits, Gq-G11
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N,N-dimethylsphingosine
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Sphingosine