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+

Abstract

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.