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-delta and by mobilization of intracellular Ca2+

We reported previously that vascular endothelial growth factor (VEGF) stimu lates prostacyclin (PGI(2)) 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 PGI(2) generation a nd 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 PKC delta, alpha and epsilon isofo rms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cy tosolic phospholipase A,, but had little effect on ERK activation induced b y basic fibroblast growth factor. GF109203X, calphostin C and the PKC delta -selective inhibitor, rottlerin, did not inhibit activation of the KDR rec eptor for VEGF, Inhibition of Ca2+ fluxes using BAPTA/AM [1,12-bis-(o-amino phenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] b locked VEGF induced PGI, production but did not inhibit ERK activation. Nei ther activation nor inhibition of the NO/cGMP pathway had any effect on VEG F induction of ERK activity and PGI(2) synthesis. Wortmannin partially inhi bited VEGF stimulation of PGI(2) production. but did not inhibit VEGF-induc ed ERK activity. VEGF-induced ERK activation and PGI, production were block ed by rottlerin, and VEGF increased association of PKC delta with Raf-l, th e upstream activator of MEK. The PKC-selective inhibitor Go6976 did not inh ibit ERK activation and had only a partial effect on PGI, production. These findings indicate that activation of PKC plays a crucial role in VEGF sign alling via the ERK cascade leading to PGI, synthesis and suggest that the P KC delta isoform may be a key mediator of VEGF-induced activation of the ER K pathway via increased association with Raf-l.