Abstract
Distribution of vascular endothelial cell growth factor A (VEGF-A) as a gradient determines microvascular endothelial cell (EC) fate during organogenesis. While much is understood about mechanisms of differential distribution, less is known about how EC perceive and interpret a graded VEGF-A signal to generate positional target gene activation. Using microvascular EC, we analyzed the effect of time and graded VEGF-A input on VEGFR2 autophosphorylation, signal kinase activation and induction of immediate-early genes. The threshold and time to peak activation of VEGFR2 were dependent on signal strength over a 50-fold range in concentration with 3-fold concentration differences readily distinguished. Longer duration of exposure did not compensate for low concentration of VEGF-A, suggesting intensity and duration of signal were not interpreted equivalently. With the same conditions, graded and time-sensitive information was transduced through the PLCgamma/p44/p42MAPK signal pathway but not the parallel AKT pathway. Analysis of MAPK-induced angiogenic immediate-early genes determined that EGR-1, EGR-3, and NR4A1 were dependent on graded input while NR4A2 and DSCR1 were independent with 'switch-like' induction. These data demonstrate rapid, linear integration of VEGF-A levels but independent interpretation of duration of signal and identify potential nodes for segregation of gradient-dependent and -independent responses. These results describe how microvascular EC fate decisions can be determined by comparatively moderate changes in VEGF signal strength, resulting in combinatorial changes in the repertoire of immediate-early genes for transcription effectors.
Copyright 2010 Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Animals
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Cell Movement / drug effects
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Cell Proliferation / drug effects
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Cells, Cultured
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DNA-Binding Proteins
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Dose-Response Relationship, Drug
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Early Growth Response Protein 1 / genetics
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Early Growth Response Protein 3 / genetics
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Embryo, Mammalian / cytology
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Embryo, Mammalian / metabolism
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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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Epithelial Cells / cytology
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Epithelial Cells / metabolism
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Female
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Gene Expression / drug effects
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Gene Expression / genetics
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Gene Expression Regulation, Developmental / physiology*
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Humans
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Intracellular Signaling Peptides and Proteins / genetics
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Kinetics
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Lung / cytology
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Lung / metabolism
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Mice
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Mice, Inbred Strains
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Mice, Transgenic
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / metabolism
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Muscle Proteins / genetics
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Nuclear Receptor Subfamily 4, Group A, Member 1 / genetics
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Nuclear Receptor Subfamily 4, Group A, Member 2 / genetics
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Phospholipase C gamma / metabolism
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Phosphorylation / drug effects
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Proto-Oncogene Proteins c-akt / metabolism
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Tyrosine / metabolism
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Vascular Endothelial Growth Factor A / genetics
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Vascular Endothelial Growth Factor A / pharmacology*
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Vascular Endothelial Growth Factor Receptor-2 / metabolism
Substances
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DNA-Binding Proteins
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EGR1 protein, human
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Early Growth Response Protein 1
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Intracellular Signaling Peptides and Proteins
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Muscle Proteins
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NR4A1 protein, human
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NR4A2 protein, human
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Nuclear Receptor Subfamily 4, Group A, Member 1
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Nuclear Receptor Subfamily 4, Group A, Member 2
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RCAN1 protein, human
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VEGFA protein, human
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Vascular Endothelial Growth Factor A
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vascular endothelial growth factor A, mouse
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Early Growth Response Protein 3
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Tyrosine
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Vascular Endothelial Growth Factor Receptor-2
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Proto-Oncogene Proteins c-akt
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Phospholipase C gamma