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 (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 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 PKCdelta, alpha and epsilon isoforms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cytosolic phospholipase A(2), but had little effect on ERK activation induced by basic fibroblast growth factor. GF109203X, calphostin C and the PKCdelta-selective inhibitor, rottlerin, did not inhibit activation of the KDR receptor for VEGF. Inhibition of Ca(2+) fluxes using BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] blocked VEGF-induced PGI(2) 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 PGI(2) synthesis. Wortmannin partially inhibited VEGF stimulation of PGI(2) production, but did not inhibit VEGF-induced ERK activity. VEGF-induced ERK activation and PGI(2) production were blocked by rottlerin, and VEGF increased association of PKCdelta 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 PGI(2) production. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade leading to PGI(2) synthesis and suggest that the PKCdelta isoform may be a key mediator of VEGF-induced activation of the ERK pathway via increased association with Raf-1.

Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field

“…These results are consistent with previous studies that L-NAME does not affect VEGF-induced ERK activity [51,54]. However, the mechanisms for the observed response to eNOS inhibition in the presence of EF are not clear and will be the subject of our future work.…”

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide

“…induced hyperpermeability but failed to block VEGFinduced ERK-1/2 phosphorylation in HUVEC. Although this observation is in contrast to previous findings using porcine coronary venular endothelial cells (30), it is concordant with studies in HUVEC (16). Our results suggest that NO does not modulate VEGFinduced ERK-1/2 phosphorylation.…”

Stimulation‐dependent recruitment of cytosolic phospholipase A2‐α to EA.hy.926 endothelial cell membranes leads to calcium‐independent association

“…In accordance with this, studies on platelets and endothelial cells have shown that this membrane relocation is consistent with an increased PLA 2 activity in membrane fractions [11,12]. Several studies have also shown that cPLA 2 ‐α activity in endothelial cells is regulated by phosphorylation [13–15], however the role of these modifications in the translocation and membrane association of cPLA 2 ‐α is unclear.…”