Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK.
To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase D2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-delta regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-delta pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-beta-cyclodextrin (MbetaCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MbetaCD-treated cells restored caveolar structures. Pretreatment with MbetaCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade.
Cho
Chung-Hyun
CH
National Creative Research Initiatives Center for Endothelial Cells, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea.
Lee
Chang Sup
CS
Chang
Mikyung
M
Jang
Il-Ho
IH
Kim
Soo Jin
SJ
Hwang
Inhwan
I
Ryu
Sung Ho
SH
Lee
Chin O
CO
Koh
Gou Young
GY
D021941:Caveolae / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C105606:phospholipase D2; D010739:Phospholipase D