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.

Anti-chemorepulsive Effects of Vascular Endothelial Growth Factor and Placental Growth Factor-2 in Dorsal Root Ganglion Neurons Are Mediated via Neuropilin-1 and Cyclooxygenase-derived Prostanoid Production

“…Given that NP-1 does not have a known signaling function, we next examined other signaling pathways that could be responsible for mediating VEGF-dependent inhibition of DRG growth cone collapse. Prostaglandins exert direct effects on sensory neurons via specific EP and IP receptors (37)(38)(39)(40) and also mediate biological functions of VEGF (12,16). The role of prostanoids in regulating neurotrophic or growth cone activity has not been examined previously, however (Fig.…”

“…The cells were grown in Ham's F-12 medium containing 10% fetal bovine serum and 25 g/ml hygromycin B. PAE cells expressing either KDR (PAE/KDR) or Flt-1 (PAE/Flt-1) were provided by Professor Lena Claesson-Welsh and grown in Ham's F-12 medium containing 10% fetal bovine serum and 250 g/ml gentamicin G418. Dorsal root ganglion (DRG) explants from newborn rats were cultured as described (33, 34) on 24-well tissue culture plates precoated with poly-D-lysine (10 g/ml) and laminin (10 g/ml) in serum-free DMEM/F-12 medium supplemented with 100 g/ml transferrin, 16 g/ml putrescine, 5 g/ml insulin, 50 ng/ml thyroxine, 50 ng/ml triiodothyronine, 39 ng/ml sodium selenite, 100 g/ml crystallized bovine serum albumin, and with or without nerve growth factor (50 ng ml Ϫ1 , mouse 7 S form, Alomone Labs). Cytosine arabinofuranoside (10 M) was added after 1 day in culture to kill non-neuronal cells.…”

The zinc-finger transcription factor, early growth response 3, mediates VEGF-induced angiogenesis

“…VEGF exerts its diverse biological effects in endothelial cells through high-affinity binding with two tyrosine-kinase receptors, Flt-1 (VEGFR1) and KDR (VEGFR2) (Neufeld et al, 1999;Zachary, 2003). KDR is activated through ligandstimulated receptor dimerization and trans(auto)phosphorylation of multiple tyrosine residues in the cytoplasmic domain (Dougher and Terman, 1999;Takahashi et al, 2001), triggering an array of early signalling events mediating cellular biological responses, including production of prostacyclin and nitric oxide, increased cell survival, migration, proliferation and angiogenesis (Waltenberger et al, 1994;Xia et al, 1996;Abedi and Zachary, 1997;Laitinen et al, 1997;Wheeler-Jones et al, 1997;Gerber et al, 1998;AbuGhazaleh et al, 2001;Gliki et al, 2001;Takahashi et al, 2001;Zachary, 2003). The role of gene expression in endothelial functions regulated by VEGF-A 165 is still poorly understood, but recent findings from cDNA and oligonucleotide array screening have identified the endothelial gene expression profile induced by VEGF-A 165 (Abe and Sato, 2001;Liu et al, 2003).…”

VEGF Receptor Signaling and Endothelial Function

“…Expression of a dominantnegative ERK2 mutant blocks either JNK or ERK activity, indicating a cross-talk between ERK and JNK (72). It has been reported that VEGF-A induced activation of ERK kinase is dependent on the Ras-Raf-MEK-ERK pathway (70) or PLC-yinduced PKC-activation of Raf-MEK-ERK pathway (75)(76)(77). Two reports also suggest another novel pathway for VEGF-A-induced activation involving NO-mediated Raf-1 activation (71,95).…”