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

PLoS One. 2010 Jul 6;5(7):e11435. doi: 10.1371/journal.pone.0011435.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Calcimycin / pharmacology
  • Calcineurin / metabolism*
  • Calcium / metabolism*
  • Cell Line
  • Cell Proliferation / drug effects
  • Cells, Cultured
  • DNA-Binding Proteins
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism*
  • Humans
  • Immunoblotting
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinase C-delta / metabolism*
  • RNA, Small Interfering
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / drug effects
  • Tetradecanoylphorbol Acetate / analogs & derivatives
  • Tetradecanoylphorbol Acetate / pharmacology
  • Vascular Endothelial Growth Factor A / pharmacology*


  • Anti-Bacterial Agents
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • Muscle Proteins
  • RCAN1 protein, human
  • RNA, Small Interfering
  • Vascular Endothelial Growth Factor A
  • Calcimycin
  • phorbolol myristate acetate
  • Protein Kinase C-delta
  • Calcineurin
  • Tetradecanoylphorbol Acetate
  • Calcium