Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells.
Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells.
Cardiovascular, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Eva
E
Gonzalez
Aaron
A
Nagiel
Alison J
AJ
Lin
David E
DE
Golan
Thomas
T
Michel
D000199:Actins / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015151:Immunoblotting; D008246:Lysophospholipids / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000199:Actins; D000418:Albumins; D051242:Caveolin 1; D022461:Caveolins; D008246:Lysophospholipids; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C060506:sphingosine 1-phosphate; D019001:Nitric Oxide Synthase; D011972:Receptor, Insulin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; D013110:Sphingosine