The Proliferative Effect of Vascular Endothelial Growth Factor Requires Protein Kinase C-α and Protein Kinase C-ζ

Abstract

Abstract-The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca 2ϩ ] i regulation and [Ca 2ϩ ] i -dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca 2ϩ ] i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca 2ϩ ] i followed by a sustained elevation. The sustained [Ca 2ϩ ] i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms ␣, ␦, ⑀, and were identified in endothelial cells. VEGF induced a translocation of PKC-␣ and PKC-toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-␦ and PKC-⑀ was not influenced. Cell exposure to TPA led to a down-regulation of PKC-␣ and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-␣ and PKC-with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca 2ϩ influx. VEGF leads to the translocation of the [Ca 2ϩ ] i -sensitive PKC isoform ␣ and the atypical PKC isoform . Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms ␣ and are important for VEGF's angiogenic effects.

Utilization of Distinct Signaling Pathways by Receptors for Vascular Endothelial Cell Growth Factor and Other Mitogens in the Induction of Endothelial Cell Proliferation

“…In contrast with observations made in BAEC, Wellner et al (52) associated activation of PKC-␣ and PKC-with the proliferative response induced by VEGF in HUVEC. We were unable to detect expression of PKC␤ in HUVEC and found that by acting through KDR, VEGF induced membrane translocation of PKC-⑀ but not PKC-␣ or PKC-.…”

Activation of diacylglycerol kinase α is required for VEGF-induced angiogenic signaling in vitro

“…Indeed VEGF-A stimulates PLC-g-mediated hydrolysis of PI(4,5)P 2 to generate DG, which then leads to activation of PKC (Matsumoto and Claesson-Welsh, 2001). PKC-a and -b isoforms are required for VEGF-stimulated cell proliferation and migration in vitro, as well as for neovascularization in vivo (Wellner et al, 1999;Wang et al, 2002).…”

“…However, in endothelial cells, the increased activity of PKCa, which indeed is activated by VEGF-A in a DG-mediated manner, is expected to enhance rather than inhibit VEGF-A-induced angiogenic response (Wellner et al, 1999;Matsumoto and Claesson-Welsh, 2001;Wang et al, 2002). Conversely, the role of activation of Dgk-a in VEGF-A angiogenic signaling may consist in the generation of PA-mediated signals.…”

“…Indeed, VEGF-A-induced angiogenic signaling requires both phosphorylation and hydrolysis of PI(4,5)P 2 , mediated, respectively, by phosphatidylinositol 3-kinase (PI 3-kinase) and phospholipase C-g (PLC-g to generate phosphatidylinositol(3,4,5)tris-phosphate (PI(3,4,5)P 3 ) and diacylglycerol (DG). While PI(3,4,5)P 3 is required for activation of Akt, which mediates VEGF-A-induced survival signaling (Gerber et al, 1998), DG is required for activation of PKC-a, which mediates VEGF-Ainduced proliferative and chemotactic signaling (Wellner et al, 1999;Matsumoto and Claesson-Welsh, 2001). Diacylglycerol kinase (Dgk) enzymes phosphorylate diacylglycerol to generate phosphatidic acid (PA), regulating in a reciprocal manner the level of two lipid second messengers (Topham and Prescott, 1999).…”

Vascular Endothelial Growth Factor KDR Receptor Signaling Potentiates Tumor Necrosis Factor-induced Tissue Factor Expression in Endothelial Cells

“…Stimulation with PMA alone resulted in a striking increase in TF expression, indicating that PKC plays a central role in VEGF/TNF-induced up-regulation of TF. Previously, several isoforms of PKC such as PKC-␣, -␤, -␥, -, and -⑀ have been shown to be activated by VEGF stimulation (34,(37)(38). Because TNF has also been shown to activate PKC (39), the synergistic up-regulation of TF induced by VEGF and TNF may occur at the level of PKC.…”