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.

Gonzalez E ，Nagiel A ，Lin AJ ，Golan DE ，Michel T ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells.

Gonzalez E ，Kou R ，Michel T 《JOURNAL OF BIOLOGICAL CHEMISTRY》

Transforming growth factor-beta1 downregulates caveolin-1 expression and enhances sphingosine 1-phosphate signaling in cultured vascular endothelial cells.

Igarashi J ，Shoji K ，Hashimoto T ，Moriue T ，Yoneda K ，Takamura T ，Yamashita T ，Kubota Y ，Kosaka H ... -  《-》

Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells. Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway.

Levine YC ，Li GK ，Michel T 《JOURNAL OF BIOLOGICAL CHEMISTRY》

Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction.

Igarashi J ，Michel T 《JOURNAL OF BIOLOGICAL CHEMISTRY》