Title: 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 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 ∼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, Gαq 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. 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 ∼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, Gαq 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. Caveolae are specialized plasmalemmal microdomains that were originally described on the surface of endothelial and epithelial cells (1Palade G.E. J. Appl. Physiol. 1953; 24: 1424-1436Google Scholar, 2Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-458Crossref PubMed Scopus (527) Google Scholar). First described as endocytic structures, caveolae have been identified as sites for the sequestration of diverse membrane-targeted signaling proteins (for review, see Refs. 3van Deurs B. Roepstorff K. Hommelgaard A.M. Sandvig K. Trends Cell Biol. 2003; 13: 92-100Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 4Parton R.G. Nat. Rev. Mol. Cell. Biol. 2003; 4: 162-167Crossref PubMed Scopus (140) Google Scholar, 5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (926) Google Scholar). Caveolae are characterized by the presence of the scaffolding/regulatory protein caveolin (6Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar, 7Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar) 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 (6Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar). The most extensively characterized member of this protein family, caveolin-1, has been shown to interact with and modulate the function of many signaling proteins in caveolae, including the epidermal growth factor receptor, the endothelial isoform of nitric oxide synthase (eNOS), 1The abbreviations used are: eNOS, endothelial nitric-oxide synthase; MAP, mitogen-activated protein; PI3-kinase, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; Cav-1 siRNA, small interfering RNA targeted to the bovine caveolin-1 mRNA; VEGF, vascular endothelial growth factor; ERK, extracellular signal-regulated kinase; BAEC, bovine aortic endothelial cells; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; S1P, sphingosine-1 phosphate; GSK3β, glycogen synthase kinase 3β; ANOVA, analysis of variance. Src family tyrosine kinases, Gα proteins, diverse serine/threonine protein kinases, and the insulin receptor (8Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar, 9Yamamoto M. Toya T. Schwencke C. Lisanti M.P. Myers Jr M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). On the other hand, caveolin-1 also interacts with acting binding cytoskeletal proteins; these interactions have been proposed to be required for the spatial organization of caveolin-associated membrane domains and caveolae internalization (10Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (557) Google Scholar, 11Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (266) Google Scholar). Much of our understanding about the role of caveolin-1 in cellular signaling derives from overexpression experiments in heterologous systems. However, in cell types in which caveolin-1 is already robustly expressed, the complementary experimental approach of attenuating caveolin-1 expression has proven more challenging. Standard antisense methods have not been broadly applied (12Griffoni C. Spisni E. Santi S. Riccio M. Guarnieri T. Tomasi V. Biochem. Biophys. Res. Commun. 2000; 276: 756-761Crossref PubMed Scopus (81) Google Scholar) because of their intrinsic limitations, particularly in endothelial cells, where caveolin-1 is so abundant. Caveolin-1 knock-out mice have recently been characterized and provide a promising experimental system for exploring the role of caveolae in physiological processes (13Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar, 14Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 15Zhao Y.Y. Liu Y. Stan R.V. Fan L. Gu Y. Dalton N. Chu P.H. Peterson K. Ross Jr., J. Chien K.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11375-11380Crossref PubMed Scopus (392) Google Scholar, 16Razani B. Lisanti M.P. J. Clin. Investig. 2001; 108: 1553-1561Crossref PubMed Scopus (204) Google Scholar). Cells derived from these mice lack caveolae membranes, and a prominent phenotype is observed in the vascular system of these mice with abnormalities in permeability and contractile functions (13Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar, 14Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). However, biochemical analyses in vascular tissues prepared from these mice are hampered by the limited quantities of tissue available for analysis. In particular, the role of caveolin-1 in the regulation of receptor-modulated kinase pathways has been difficult to ascertain, particularly with respect to the differential control of MAP kinase and phosphatidylinositol-3 (PI3) kinase pathways, both of which have regulatory components localized in caveolae (8Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar, 17Liu P. Ying Y. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13666-13670Crossref PubMed Scopus (193) Google Scholar). Antisense RNA studies have implied that caveolin-1 has an inhibitory effect on the MAP-kinase cascade in NIH-3T3 cells (18Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R.G. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (432) Google Scholar). The role of caveolin-1 in the PI3-kinase/Akt pathway is more controversial; some reports suggest that overexpression of caveolin-1 down-regulates the PI3-kinase pathway and sensitizes fibroblast and epithelial cells to apoptotic stimuli (19Liu J. Lee P. Galbiati F. Kitsis R.N. Lisanti M.P. Am. J. Physiol. 2001; 280: C823-C835Crossref PubMed Google Scholar, 20Zundel W. Swiersz L.M. Giaccia A. Mol. Cell. Biol. 2000; 20: 1507-1514Crossref PubMed Scopus (162) Google Scholar), whereas other studies have reported that overexpression of caveolin-1 activates Akt signaling (21Shack S. Wang X.T. Kokkonen G.C. Gorospe M. Longo D.L. Holbrook N.J. Mol. Cell. Biol. 2003; 23: 2407-2414Crossref PubMed Scopus (80) Google Scholar, 22Li L. Ren C.H. Tahir S.A. Ren C. Thompson T.C. Mol. Cell. Biol. 2003; 23: 9389-9404Crossref PubMed Scopus (258) Google Scholar). In this article, we describe the results of experiments using transfection of small interfering RNA (siRNA) duplexes to specifically and efficiently knockdown caveolin-1 expression in cultured bovine aortic endothelial cells (BAEC). We explore the effects of siRNA-mediated caveolin-1 knockdown on signaling pathways initiated by the agonists S1P and vascular endothelial growth factor (VEGF), and the consequence of caveolin-1 knockdown on the subcellular targeting of key signaling proteins in endothelial cells. Materials—Fetal bovine serum was purchased from Hyclone (Logan, UT). Cell culture reagents, media, and LipofectAMINE 2000 transfection reagent were from Invitrogen. S1P was purchased from BIOMOL (Plymouth Meeting, PA). VEGF, bradykinin, and wortmannin were from Calbiochem (La Jolla, CA). Polyclonal antibodies directed against phospho-eNOS (Ser-1179), phospho-Akt (Ser-473), Akt, phospho-glycogen synthase kinase 3β (GSK3β) (Ser-9), phospho-ERK1/2 (Thr-202/Tyr-204) and ERK1/2 were from Cell Signaling Technologies (Beverly, MA). Caveolin-1 polyclonal antibody, eNOS monoclonal antibody, GSK3β monoclonal antibody, and flotillin-1 monoclonal antibody were from BD Transduction Laboratories. Polyclonal antibodies for Src, the insulin receptor (β subunit), and Gαq were from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488 anti-mouse IgG antibody, Alexa Fluor 568 anti-rabbit IgG antibody, anti-Alexa Fluor 488 antibody, bovine serum albumin labeled with Alexa Fluor 488, phalloidin labeled with Alexa Fluor 488 and 4′,6-diamino-2-phenylindole, dihydrochloride were from Molecular Probes Inc. (Eugene, OR). Rac activation assay kit was from Upstate Biotechnology (Lake Placid, NY). Super Signal substrate for chemiluminescence detection and secondary antibodies conjugated with horseradish peroxidase were from Pierce. Tris-buffered saline and phosphate-buffered saline were from Boston Bioproducts (Ashland, MA). Other reagents were from Sigma. Cell Culture—Bovine aortic endothelial cells (BAEC) were obtained from Cell Systems (Kirkland, WA) and maintained in culture in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10% v/v) as described previously (23Michel T. Li G.T. Busconi L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6252-6256Crossref PubMed Scopus (305) Google Scholar). Cells were plated onto 0.2% gelatin-coated culture dishes and studied before cell confluence between passages 5 and 9. siRNA Preparation and Transfection—Based upon established characteristics of siRNA targeting constructs (24Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar, 25Dykxhoorn D.M. Novina C.D. Sharp P.A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 457-467Crossref PubMed Scopus (1024) Google Scholar), we designed a caveolin-1 siRNA duplex corresponding to bases 223–241 from the open reading frame of the bovine caveolin-1 mRNA: 5′-CCA GAA GGA ACA CAC AGU U-dTdT-3′, and a Rac siRNA duplex corresponding to bases 78–96 from the open reading frame of the bovine Rac mRNA: 5′-UGC GUU UCC UGG AGA AUA U-dTdT-3′. The RNA sequence used as a negative control for siRNA activity was: 5′-GCG CGC UUU GUA GGA UUC G-dTdT-3′. Small interfering RNA duplex oligonucleotides were purchased from Dharmacon, Inc. (Lafayette, CO). In preliminary experiments, we optimized conditions for the efficient transfection of BAEC using siRNA. We found that optimal conditions for siRNA knock-down involved transfecting BAEC at 50–70% confluence maintained in Dulbecco's modified Eagle's medium/10% fetal bovine serum; transfections with siRNA (30 nm, or as noted) used LipofectAMINE 2000 (0.15%, v/v), following protocols provided by the manufacturer. Fresh medium was added 5 h after transfection, and experiments were conducted 48 h after transfection. Drug Treatment, Cell Lysates, and Immunodetection—Culture medium was changed to serum-free medium, and incubations proceeded overnight before all experiments (26Lee H. Goetzl E.J. An S. Am. J. Physiol. 2000; 278: C612-C618Crossref PubMed Google Scholar, 27Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). S1P was solubilized in methanol and stored at –20 °C; the same volume of methanol was used as a vehicle control, and the final concentration of methanol did not exceed 0.4% (v/v). VEGF was solubilized in Tris-buffered saline containing 0.1% bovine serum albumin and stored at –70 °C. Bradykinin was solubilized in water and stored at –20 °C. After drug treatments, BAEC were washed with phosphate-buffered saline (PBS) and incubated for 10 min in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA, 2 mm Na3VO4, 1 mm NaF, 2 μg/ml leupeptin, 2 μg/ml antipain, 2 μg/ml soybean trypsin inhibitor, and 2 μg/ml lima trypsin inhibitor). Cells were harvested by scraping and then centrifuged for 15 min at 4 °C. For immunoblot analyses, 20 μg of cellular protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed with antibodies using protocols provided by the suppliers. Densitometric analyses of the Western blots were performed using a ChemiImager 4000 (Alpha Innotech). Rac Activity Assay—BAEC in 100-mm dishes were transfected with control or caveolin-1-specific siRNA. Cells were starved overnight before experiments, and Rac activity was measured 48 h after transfection. After stimulation with S1P, cells were washed with ice-cold PBS and lysed in lysis buffer (25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Igepal CA-630, 10 mm MgCl2, 1 m m EDTA, 10% glycerol, 2 mm Na3VO4, 1mm NaF, 2 μg/ml leupeptin, 2 μg/ml antipain, 2 μg/ml soybean trypsin inhibitor, and 2 μg/ml lima trypsin inhibitor). Pull-down of GTP-bound Rac was performed by incubating the cell lysates with glutathione S-transferase fusion-protein corresponding to the p21-binding domain of PAK-1 bound to glutathione agarose (Upstate Biotechnology) for 1 h at 4 °C following instructions provided by the suppliers. The beads were washed three times with lysis buffer, and the protein bound to the beads was eluted with Laemmli buffer and analyzed for the amount of GTP-bound Rac by immunoblotting using a Rac monoclonal antibody (Upstate Biotechnology). Migration Assay—Cell migration was assayed (30Wang F. Van Brocklyn J.R. Hobson J.H. Movafagh S. Zukowska-Grojec Z. Milstien S. Spiegel S. J. Biol. Chem. 1999; 274: 35343-35350Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) using a Transwell cell culture chamber containing polycarbonate membrane inserts with 8-μm pore (Corning Costar Corp.) coated with 0.2% gelatin. BAEC were transfected with control or caveolin-1-specific siRNA, and experiments were carried out 48 h after transfection; the transfected cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.4% fetal bovine serum for 18 h before migration experiments. The cells were briefly incubated with trypsin to obtain a single-cell suspension, and 5 × 104 cells in Dulbecco's modified Eagle's medium/0.4% fetal bovine serum were added to the upper Transwell chamber. The bottom chamber was filled with 600 μl of media, and the assembly was incubated at 37 °C for 1 h. S1P (100 nm) was added to the lower chamber, and the assembly was incubated at 37 °C for 3 h to allow cell migration. In some experiments, wortmannin (500 nm) was added to the lower chamber 30 min before the addition of S1P. After incubation, the membranes were washed with PBS, and the cells were fixed in 3.7% formaldehyde (10 min), permeabilized in 0.5% Triton X-100 in PBS (3 min), and stained with 4′,6-diamino-2-phenylindole, dihydrochloride according to the manufacturer's protocols. Cells that did not migrate through the membrane were gently removed from the upper surface and the membranes were mounted between slide and coverslips using the SlowFade antifade kit (Molecular Probes Inc.) Cell migration was scored under light microscopy in four random fields by a blinded observer; each experimental treatment was analyzed in triplicate. Albumin Uptake—BAEC were transfected with control or caveolin-1-specific siRNA; 48 h after transfection, cells were incubated with Alexa Fluor 488-conjugated albumin (10 μg/ml) at 37 °C. Cells were washed with PBS, and cell lysates were prepared as described above. Albumin uptake was analyzed in immunoblots probed with an antibody against Alexa Fluor 488. Isolation of Caveolae-enriched Fractions—Caveolae-enriched fractions were separated by ultracentrifugation in a discontinuous sucrose gradient system as reported previously (28Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). In brief, transfected BAEC from two 100-mm dishes were scraped together into 2 ml of carbonate buffer containing 500 mm sodium carbonate, pH 11, and the cells were homogenized (40 strokes in a Dounce homogenizer) and sonicated (three 20-s bursts in a Branson Sonifier 450). The resulting cell suspension was brought to 45% sucrose by adding 2 ml of 90% sucrose prepared in MES-buffered saline (25 mm MES, pH 6.5, 150 mm NaCl) and placed at the bottom of a 12-ml ultracentrifuge tube. A discontinuous gradient was formed above the 45% sucrose bed by adding 4 ml each of 35% and 5% sucrose solutions (prepared in MES-buffered saline containing 250 mm of sodium carbonate) and centrifuging at 39,000 rpm for 16–20 h in a TH-641 rotor (Sorvall, Asheville, NC). Twelve 1-ml fractions were collected starting at the top of each gradient, and an equal volume of each fraction was analyzed by SDS-PAGE and immunoblotting as described above. Confocal Fluorescence Microscopy—BAEC grown on coverslips were transfected with control or caveolin-1-specific siRNA using LipofectAMINE 2000. 48 h after transfection, cells were fixed with 3.7% paraformaldehyde in PBS for 10 min, rinsed with PBS, permeabilized in 0.1% Triton X-100/0.1% bovine serum albumin in PBS for 5 min, and blocked with 10% horse serum in PBS for 1 h. Incubations with primary antibodies were performed in blocking solution for 1 h at room temperature. After washing with PBS, cells were incubated with appropriate secondary antibodies conjugated to immunofluorescent dyes (Alexa Fluor 488 anti-mouse IgG or Alexa Fluor 568 anti-rabbit IgG) in blocking solution for 1 h at room temperature. After washing three times with PBS, coverslips were mounted on slides using the SlowFade antifade kit (Molecular Probes Inc.). For staining of F-actin, fixed cells were permeabilized in 0.1% Triton X-100 in PBS for 3 min and blocked with 1% bovine serum albumin in PBS for 30 min. Cells were incubated with phalloidin-Alexa Fluor 488 in PBS containing 1% bovine serum albumin for 20 min at room temperature. After washing with PBS, the coverslips were mounted on slides as described previously. Microscopy analysis of samples was performed in the Nikon Imaging Center at Harvard Medical School, using a Nikon TE2000U inverted microscope in conjunction with a PerkinElmer Ultraview spinning disk confocal system equipped with a Hamamatsu Orca ER cooled-CCD camera. Images were acquired using a 60× differential interference contrast oil immersion objective lens and analyzed using Metamorph software from Universal Imaging, Inc. (Downingtown, PA). siRNA-mediated Down-regulation of Caveolin-1 Expression in BAEC—To selectively "knockdown" the expression of caveolin-1 protein in BAEC, we designed a siRNA duplex targeted to the bovine caveolin-1 mRNA (Cav-1 siRNA, sequence 5′-CCAGAAGGAACACACAGUU-dTdT-3′, corresponding to bases 223–241 from the open reading frame of bovine caveolin-1 mRNA). We decided to use siRNA duplexes rather than plasmid-based RNA interference techniques because preliminary experiments (and our past experience) showed that plasmid-based methods had an unacceptably low transfection efficiency in endothelial cells. After optimizing conditions for duplex siRNA transfection, we assayed the ability of this caveolin-1-specific siRNA to knock down caveolin expression by transfecting BAEC with increasing concentrations of this siRNA duplex. Fig. 1A shows an immunoblot probed for caveolin-1 protein in BAEC 48 h after transfection with caveolin-1-specific siRNA. Caveolin-1 expression was efficiently knocked-down in a dose-dependent manner by transfection with Cav-1 siRNA; transfection with a control random sequence siRNA did not affect the expression of caveolin-1 (Fig. 1). Under both experimental conditions, levels of β-actin protein in cell lysates remained constant. To further characterize the effect of caveolin-1 siRNA on caveolin-1 expression levels and protein distribution, we performed immunofluorescence analysis of caveolin-1 in BAEC transfected with Cav-1 and control siRNA. As illustrated in Fig. 2, cells transfected with caveolin-1 siRNA showed a marked reduction of caveolin-1 expression levels compared with control cells (Fig. 2). The subcellular distribution pattern of the small amount of residual caveolin-1 remaining after siRNA-mediated knockdown paralleled the caveolin-1 distribution pattern of control cells: despite the quantitative reduction in caveolin-1 expression, the distribution of the expressed protein seemed to be unchanged (Fig. 2). We next explored whether down-regulation of caveolin-1 expression would affect the subcellular distribution of caveolae-targeted proteins. Fig. 2 shows the results of immunofluorescence microscopy analyses of BAEC transfected with control or caveolin-1 siRNA and stained for the key caveolae-targeted signaling protein eNOS. The subcellular distribution of eNOS was not affected by siRNA-mediated knockdown of caveolin-1; the enzyme was detected as a membrane-associated protein localized at the plasma membrane and around the perinuclear region, as described previously in native BAEC (29Prabhakar P. Thatte H.S. Goetz R.M. Cho M.R. Golan D.E. Michel T. J. Biol. Chem. 1998; 273: 27383-27388Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Because caveolin-1 has been reported to interact with a variety of actin-binding cytoskeletal proteins (10Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (557) Google Scholar, 11Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (266) Google Scholar), we also explored whether actin organization would be affected in caveolin-1 knockdown cells. Fig. 2 shows siRNA-transfected BAEC stained with Alexa Fluor 488-labeled phalloidin to visualize the actin cytoskeleton. As seen in this figure, caveolin-1 siRNA-treated cells showed both a notable increase in the abundance of lamellipodia and augmented cortical actin rings compared with control cells, suggesting that cytoskeletal structure is altered in endothelial cells in which caveolin-1 is knocked down. Down-regulation of Caveolin-1 Expression Enhances S1P-induced Migration of BAEC—Because siRNA-mediated down-regulation of caveolin-1 significantly altered the cytoskeletal structure of BAEC, we next explored whether caveolin-1 knock-down affected agonist-induced endothelial cell migration. Fig. 3 shows the results of a cell motility assay using a Transwell cell culture chamber. As reported previously in wild-type BAEC (30Wang F. Van Brocklyn J.R. Hobson J.H. Movafagh S. Zukowska-Grojec Z. Milstien S. Spiegel S. J. Biol. Chem. 1999; 274: 35343-35350Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar), we observed a chemotactic response to S1P in BAEC treated with the control siRNA. BAEC transfected with caveolin-1-specific siRNA displayed significantly greater S1P-mediated migration compared with control cells. The PI3-kinase inhibitor wortmannin significantly attenuated S1P-induced cell migration in control and caveolin-1 siRNA-treated cells (Fig. 3), suggesting that the augmentation in BAEC migration induced by caveolin-1 siRNA is mediated by PI3-kinase pathways. Down-regulation of Caveolin-1 Expression Impairs Albumin Uptake in BAEC—Previous reports have suggested that the transport of albumin across the endothelial barrier is mediated by caveolae (31Schubert W. Frank P.G. Razani B. Park D.S. Chow C.W. Lisanti M.P. J. Biol. Chem. 2001; 276: 48619-48622Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 14Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). We analyzed the effects of siRNA-mediated caveolin-1 knockdown on albumin uptake in cultured BAEC. Control or caveolin-1 siRNA transfected BAEC were incubated with Alexa Fluor 488-labeled albumin, and albumin uptake was analyzed in the cell lysates at different time points by immunoblot analyses using an antibody against the exogenous labeled albumin. As shown in Fig. 4, the uptake of exogenous albumin was significantly attenuated in caveolin-1 siRNA-treated cells, demonstrating that down-regulation of caveolin-1 expression is sufficient to impair caveolae-mediated albumin endocytosis in BAEC. Down-regulation of Caveolin-1 Expression Does Not Impair Targeting of Signaling Molecules—Diverse signaling molecules are targeted to caveolae (5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (926) Google Scholar), where caveolin-1 represents the dominant protein component and is essential for the structure of these plasma membrane microdomains (6Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar, 7Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar, 14Razani B. Engelman J.A. Wang X.B. Schubert W. Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H.