The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ

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Abstract
Abstract —The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca 2+ ] i regulation and [Ca 2+ ] i -dependent messenger systems; and (2) these mechanisms are important for VEGF’s proliferative effects. [Ca 2+ ] i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca 2+ ] i followed by a sustained elevation. The sustained [Ca 2+ ] i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms α, δ, ε, and ζ were identified in endothelial cells. VEGF induced a translocation of PKC-α and PKC-ζ toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-δ and PKC-ε was not influenced. Cell exposure to TPA led to a down-regulation of PKC-α and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-α and PKC-ζ with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca 2+ influx. VEGF leads to the translocation of the [Ca 2+ ] i -sensitive PKC isoform α and the atypical PKC isoform ζ. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms α and ζ are important for VEGF’s angiogenic effects.

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