Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-δ and by mobilization of intracellular Ca2+

We reported previously that vascular endothelial growth factor (VEGF) stimulates prostacyclin (PGI₂) production via activation of the extracellular signal-regulated kinase (ERK) cascade. In this paper, we examined the role of protein kinase C (PKC) in this pathway. VEGF-induced PGI₂ generation and arachidonic acid release in human umbilical vein endothelial cells were inhibited by the PKC inhibitors GF109203X and calphostin C. VEGF increased PKC activity and immunoreactivity of the PKCδ, α and ε isoforms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cytosolic phospholipase A₂, but had little effect on ERK activation induced by basic fibroblast growth factor. GF109203X, calphostin C and the PKCδ-selective inhibitor, rottlerin, did not inhibit activation of the KDR receptor for VEGF. Inhibition of Ca²⁺ fluxes using BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] blocked VEGF-induced PGI₂ production but did not inhibit ERK activation. Neither activation nor inhibition of the NO/cGMP pathway had any effect on VEGF induction of ERK activity and PGI₂ synthesis. Wortmannin partially inhibited VEGF stimulation of PGI₂ production, but did not inhibit VEGF-induced ERK activity. VEGF-induced ERK activation and PGI₂ production were blocked by rottlerin, and VEGF increased association of PKCδ with Raf-1, the upstream activator of MEK. The PKC-selective inhibitor Go6976 did not inhibit ERK activation and had only a partial effect on PGI₂ production. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade leading to PGI₂ synthesis and suggest that the PKCδ isoform may be a key mediator of VEGF-induced activation of the ERK pathway via increased association with Raf-1.