The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta.
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 [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]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 [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha 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-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.
Wellner
M
M
Franz Volhard Clinic and Max Delbrück Center, Virchow Klinikum, Humboldt University, Berlin, Germany.
Maasch
C
C
Kupprion
C
C
Lindschau
C
C
Luft
F C
FC
Haller
H
H
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division; D004533:Egtazic Acid / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008222:Lymphokines / Q000494:pharmacology*; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000099221:Protein Kinase C zeta
D002118:Calcium; D004533:Egtazic Acid; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008222:Lymphokines; D009281:Naphthalenes; D020319:Oligodeoxyribonucleotides, Antisense; D037342:Pertussis Toxin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D019311:Staurosporine; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D010566:Virulence Factors, Bordetella; D000099221:Protein Kinase C zeta; C496628:PRKCA protein, human; C058819:calphostin C