TY - JOUR

T1 - VEGF stimulates RCAN1.4 expression in endothelial cells via a pathway requiring Ca2+/calcineurin and protein kinase C-delta

AU - Holmes, Katherine

AU - Chapman, Elinor

AU - See, Violaine

AU - Cross, Michael J

PY - 2010/7/6

Y1 - 2010/7/6

N2 - 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.METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

AB - 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.METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

KW - Anti-Bacterial Agents/pharmacology

KW - Calcimycin/pharmacology

KW - Calcineurin/metabolism

KW - Calcium/metabolism

KW - Cell Line

KW - Cell Proliferation/drug effects

KW - Cells, Cultured

KW - DNA-Binding Proteins

KW - Endothelial Cells/drug effects

KW - Humans

KW - Immunoblotting

KW - Intracellular Signaling Peptides and Proteins/genetics

KW - Muscle Proteins/genetics

KW - Phosphorylation/drug effects

KW - Protein Kinase C-delta/metabolism

KW - RNA, Small Interfering

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Signal Transduction/drug effects

KW - Tetradecanoylphorbol Acetate/analogs & derivatives

KW - Vascular Endothelial Growth Factor A/pharmacology

U2 - 10.1371/journal.pone.0011435

DO - 10.1371/journal.pone.0011435

M3 - Article

C2 - 20625401

VL - 5

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 7

M1 - e11435

ER -