Small Interfering RNA-mediated Down-regulation of Caveolin-1 Differentially Modulates Signaling Pathways in Endothelial Cells

Abstract

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptormodulated 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 "knockeddown" the expression of caveolin-1 by ϳ90%, 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, G␣q 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 knockdown also significantly enhanced the basal and agonistinduced 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.Caveolae are specialized plasmalemmal microdomains that were originally described on the surface of endothelial and epithelial cells (1, 2). First described as endocytic structures, caveolae have been identified as sites for the sequestration of diverse membrane-targeted signaling proteins (for review, see Refs. 3-5). Caveolae are characterized by the presence of the scaffolding/regulatory protein caveolin (6, 7) and by a distinctive lipid composition notable for high concentrations of cholesterol and sphingolipids; the presence of caveolin distinguishes caveolae from other "lipid raft" domains that have a similar lipid composition but do not necessarily contain caveolin.The three caveolin isoforms in mammalian cells are 22-24-kDa integral membrane proteins; caveolin-1 and caveolin-2 are co-expressed in most cell types and are particularly abundant in endothelial cells, whereas caveolin-3 is an isoform that is specific to muscle cells (6). The most extensively characterized member of this protein family, caveolin-1, has been shown to interact with and modulate ...

Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

“…Cells were maintained in 0.2% gelatin-coated culture dishes and starved in serum-free media overnight before treatment. BAECs were transfected with specific siRNA-targeting constructs as described previously (43), and analyzed 48 h after transfection. For PEG-catalase treatment (27), BAECs were incubated overnight with PEG-catalase (100 U/mL) or vehicle.…”

Agonist-modulated Regulation of AMP-activated Protein Kinase (AMPK) in Endothelial Cells

“…A nonspecific control siRNA from Dharmacon (Lafayette, CO) was used as a negative control (5Ј-AUU GUA UGC GAU CGC AGA CdTdT-3Ј). BAEC were transfected with siRNA as described previously (24) and analyzed 48 h after transfection.…”

“…VEGF and S1P were prepared as previously reported (24). Genistein, PP2, cyclosporin A, SB203580, wortmannin, STO-609, and Compound C were solubilized in Me 2 SO and FIGURE 2.…”

“…The scaffolding/ regulatory protein of caveolae, caveolin-1 (22,23), is known to interact with and modulate the function of eNOS in endothelial cells (24). We have demonstrated that caveolin-1 negatively regulates the small GTPase Rac1 (25,26), which in turn modulates the PI3K/Akt/ eNOS pathway and regulates migration in endothelial cells (24,27). It is therefore possible that AMPK regulates agonist-mediated eNOS activation and endothelial migration by interacting with caveolin-1 or Rac1 in endothelial cells.…”

Receptor-regulated Dynamic S-Nitrosylation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

“…Cell fixing, permeabilization, incubation with polyclonal anti-HA antibody (1:250 dilution), Alexa Fluor 568 anti-rabbit IgG secondary antibody (1:850 dilution), mounting, and imaging at the Nikon Imaging Center at Harvard Medical School were performed as described previously (27). Images were processed using two-dimensional no-neighbors deconvolution by MetaMorph (Universal Imaging, Downington, PA) before threedimensional reconstruction.…”