VEGF stimulates RCAN1.4 expression in endothelial cells via a pathway requiring Ca2+/calcineurin and protein kinase C-delta.
BACKGROUND
Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.


RESULTS
We show that VEGF is able to induce RCAN1.4 expression during cellular proliferation and differentiation, and that VEGF-mediated expression of RCAN1.4 was inhibited by the use of inhibitors to protein kinase C (PKC) and calcineurin. Further analysis revealed that siRNA silencing of PKC-delta expression partially inhibited VEGF-stimulated RCAN1.4 expression. Knockdown of RCAN1.4 with siRNA resulted in a decrease in cellular migration and disrupted tubular morphogenesis when HDMECs were either stimulated with VEGF in a collagen gel or in an endothelial/fibroblast co-culture model of angiogenesis. Analysis of intracellular signalling revealed that siRNA mediated silencing of RCAN1.4 resulted in increased expression of specific nuclear factor of activated T-cells (NFAT) regulated genes.


CONCLUSIONS
Our data suggests that RCAN1.4 expression is induced by VEGFR-2 activation in a Ca(2+) and PKC-delta dependent manner and that RCAN1.4 acts to regulate calcineurin activity and gene expression facilitating endothelial cell migration and tubular morphogenesis.
Holmes
Katherine
K
Department of Pharmacology and Therapeutics, School of Biomedical Sciences, University of Liverpool, Liverpool, United Kingdom.
Chapman
Elinor
E
See
Violaine
V
Cross
Michael J
MJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C108612:RCAN1 protein, human; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D000001:Calcimycin; C016658:phorbolol myristate acetate; D051745:Protein Kinase C-delta; D019703:Calcineurin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium