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Am J Physiol Heart Circ Physiol 293: H366-H375, 2007. First published March 16, 2007; doi:10.1152/ajpheart.01044.2006
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Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

MaryEllen Carlile-Klusacek and Victor Rizzo

Cardiovascular Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 22 September 2006 ; accepted in final form 11 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 µg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

thrombin; endothelial cell; lipid raft; protease-activated receptor-1, myosin light chain

AN EMERGING CONCEPT in the field of G protein signaling is that compartmentalization of G protein receptor and effector molecules into specialized membrane microdomains provides a mechanism by which their function is regulated (20). It has been postulated that membrane rafts, regions of the cell membrane that are enriched in cholesterol and glycosphingolipids, serve as platforms for the organization and concentration of related signaling proteins (17, 39). These regions preferentially attract proteins with posttranslational lipid modifications (myristyl, palmitoyl, farnesyl, and geranylgeranyl groups), as well as glycosylphosphatidylinositol (GPI)-linked molecules (45). A wide variety of signaling molecules, including heterotrimeric G proteins, contain such modifications, and their enrichment in membrane rafts is considered to enhance the efficiency of their activation (17, 39).

Caveolae, a subset of membrane rafts, are characterized by an inverted omega- or flask-shaped morphology and the presence of the structural protein caveolin-1. A portion of the COOH-terminal region of caveolin-1 (aa 81–101, termed the "scaffolding domain") can act as a stable platform for the binding of numerous signaling molecules (6). The scaffolding domain recognizes a unique modular binding motif in the sequence of target proteins, such as G{alpha} subunits (25, 33), the small GTPases Rac and RhoA (14, 29), and the kinase domains of both tyrosine and serine-threonine protein kinases (24, 34), which upon association often impacts their signaling function. In addition to signaling molecules, some G protein-coupled receptors, such as the bradykinin B2 receptor (8) and endothelin-1 ETA receptor (4), show evidence of consensus binding motifs and have been found in complexes with caveolin-1.

The multifunctional serine protease thrombin initiates various signal transduction pathways that regulate endothelial cell morphology and barrier function via reorganization of the actin cytoskeleton (11, 12). Thrombin-induced modifications in actin dynamics are largely achieved via activation of PAR1, a member of the G protein-coupled, protease-activated receptor (PAR) family (7, 43). Cleavage of PAR1 by thrombin stimulates Gq{alpha} to promote phospholipase C (PLC) activity, production of inositol-1,4,5-trisphosphate, and increases in intracellular Ca2+ (27). The Ca2+ release associated with PAR1 activation has been shown to enhance myosin light chain (MLC) kinase activity, resulting in increased MLC phosphorylation and the interaction of myosin with actin filaments to form stress fibers (2, 10). Additionally, stimulation of PAR1 initiates RhoA/Rho kinase signaling through activation of G12/13{alpha} (3) or possibly via Gq{alpha}-mediated induction of Rho guanine nucleotide exchange factors (28). These events also contribute to the redistribution of filamentous actin and augment stress fiber formation and membrane retraction (1, 15, 23, 26).

A number of G protein-coupled receptors and many individual signaling molecules that participate in thrombin signaling are reported to associate with raft and caveolae microdomains in a variety of cell types. Although other reports show that a thrombin receptor is compartmentalized in plasmalemma vesicles on the endothelial cell surface (18, 42), the functional consequence of this localization has not been thoroughly investigated. Here, we provide evidence that PAR1 is present in endothelial cell plasma membrane rafts and caveolae, and that the localization of PAR1 specifically to rafts serves as an important mechanism for the regulation of thrombin-induced cytoskeletal changes in endothelial cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. Unless otherwise specified, chemical reagents were purchased from Sigma (St. Louis, MO) and Fisher Scientific (Fairlawn, NJ). Bovine {alpha}-thrombin was purchased from Enzyme Research Laboratories (South Bend, IN); sphingosine-1-phosphate (S1P) was purchased from Avanti Polar Lipids (Birmingham, AL). Antibodies were obtained from the following commercial sources: RhoA and PAR-1 from Santa Cruz Biotechnology (Santa Cruz, CA); Gq{alpha} from Upstate Biotechnology (Lake Placid, NY); G12{alpha} from Calbiochem/EMD Biosciences (La Jolla, CA); urokinase plasminogen activator receptor (uPAR) from Calbiochem/EMD Biosciences and Imgenex (San Diego, CA); caveolin-1 from BD Transduction Laboratories (San Jose, CA); and beta-actin and beta-cop from Sigma (St. Louis, MO). Diphosphorylated MLC antibody was a kind gift from Dr. Peter Vincent (Albany Medical College, Albany, NY).

Cell culture. Bovine aortic endothelial cells (BAEC) were purchased from Cell Applications (San Diego, CA). Cells were cultured in MCDB-131 medium (Sigma) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 0.05 mg/ml gentamycin sulfate (Cambrex Biosciences, Walkersville, MD) and were maintained at 37°C, 97% humidity, and 5% carbon dioxide. All experiments were performed using cells between passages 5 and 10.

Sucrose density centrifugation. Membrane rafts were fractionated from BAEC as described in our previous work (44). Briefly, BAEC were scraped into ice-cold, detergent-free Tricene buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tricene, pH 7.4) and centrifuged to precipitate nuclear material. The resulting supernatant was mixed with 30% Percoll in Tricene buffer and subjected to ultracentifugation for 25 min (Beckman MLS50 rotor, 77,000 g, 4°C). The separated plasma membranes were collected, sonicated (3 x 30 s bursts), and mixed with 60% sucrose (to a final concentration of 40%) before being overlaid with a 35–5% step sucrose gradient and subjected to overnight ultracentrifugation (Beckman MLS50 rotor, 87,400 g, 4°C). Fractions were collected every 400 µl from the top sucrose layer, and proteins were precipitated using a solution of 0.1% wt/vol deoxycholic acid in 100% wt/vol trichloroacetic acid. Samples were then subjected to SDS-PAGE and immunoblotted using indicated antibodies.

Caveolae immunoaffinity isolation. Isolation of caveolar organelles was performed according to the method published by Oh and Schnitzer (32). Briefly, goat anti-mouse IgG-coated magnetic beads (Dynal Biotech) were preincubated with a specific monoclonal antibody that recognizes the oligomeric form of caveolin-1 (clone 2234, Transduction Laboratories) for 2 to 4 h at room temperature. Sonicated plasma membranes prepared as described above were added to prepared beads and incubated for 1 h at 4°C. Bound material, representative of caveolae vesicles, was separated magnetically from unbound, noncaveolar membranes, subjected to SDS-PAGE and immunoblotted using indicated antibodies.

Cholesterol depletion. Membrane rafts were disrupted via treatment of cell monolayers with cholesterol-sequestering agent filipin III (Sigma). Filipin (1–5 µg/ml) was prepared in serum-free media and applied to the cells for 5 min at 37°C, followed by a brief wash with serum-free media before additional treatments or cell lysis.

Caveolin-1 siRNA. BAEC at 85–90% confluence were transfected with 100 nM caveolin-1 SMARTpool small interferring RNA (siRNA) or siCONTROL (a nontargeting pool of siRNAs that contains at least four mismatches for all known gene sequences) using DharmaFECT-1 (Dharmacon, Lafayette, CO) according to the manufacturer's protocol. Cells were used for experiments 48 h posttransfection. Knockdown of caveolin-1 was confirmed by Western analysis and/or immunofluorescent labeling of cells for caveolin-1.

Microscopy. For colocalization studies, BAEC were grown to confluency on glass coverslips coated with 30 µg/ml Vitrogen collagen in solution (Cohesion Technologies, Palo Alto, CA). Cells were washed with PBS, fixed in cold 80% ethanol, and incubated with uPAR and PAR1 primary antibodies for 1 h on ice. All subsequent steps were performed at room temperature in a humidified chamber. Cells were permeabilized with 100 µg/ml saponin in HEPES buffer (10 mM HEPES, 100 mM KCl, and 5 mM MgCl2) for 5 min, washed with PBS, and blocked in 10% goat serum in PBS for 20 min. Caveolin-1 primary antibody was applied for 1 h, followed by the desired Alexa-conjugated secondary antibodies (Molecular Probes/Invitrogen, Carlsbad, CA). Cells were imaged using a Leica TCS confocal microscope operated by LCS software (Leica). Individual red and green images of the same field were overlaid and processed using Adobe Photoshop software; areas of colocalization were confirmed by presence of yellow in the merged images.

For visualization of the cytoskeleton, BAEC were grown to confluency on Vitrogen-coated glass coverslips. Cells were serum starved overnight (0.1% FBS) before stimulation. Immediately after treatment, cells were fixed, permeabilized and blocked as detailed above, followed by incubation with Texas red-conjugated phalloidin (Sigma) to label filamentous actin. Cells were imaged using an inverted fluorescent microscope (Nikon Eclipse TE300) with a x100 PlanApo objective. Images were captured using a Nikon DMX1200 digital camera operated by ACT-1 software (vs. 2.20).

MLC phosphorylation assay. Confluent BAEC monolayers were serum starved for 4 h, treated as indicated, and processed for detection of MLC diphosphorylation as described previously (21). Briefly, cells were scraped into a reducing lysis buffer (8 mM Tris, pH 6.8, 4% SDS, 20 mM EDTA, 0.1% glycerol, 0.01% beta-mercaptoethanol) supplemented with 100 mM NaF. Equal volumes of the samples were subjected to SDS-PAGE and immunoblotted with an affinity-purified antibody to the Ser-19/Thr-18 diphosphorylated form of MLC (see Reagents and antibodies for source).

Western analysis. Nitrocellulose membranes were incubated with primary antibody, followed by anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL). Membranes were exposed to SuperSignal enhanced chemiluminescence substrate (Pierce), and immunoblots were scanned, digitized, and quantified using Image J software.

Statistical analysis. Data points were pooled according to group, with differences between groups determined by ANOVA, followed by post hoc analysis using Bonferroni's multiple comparison test (GraphPad Prism software). Data are presented as means ± SE. Differences between control and experimental groups were deemed significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PAR1 signaling components are localized in caveolin-rich membrane rafts. It has been suggested that the clustering of separate rafts and their resident proteins can, in a localized area of the plasma membrane, enrich specific signaling molecules such that signal propagation is significantly amplified (39). Several properties of membrane rafts and caveolae, namely their resistance to nonionic detergents and lower density compared with surrounding membranes, have been exploited to study them in isolation. To examine the localization of key components of the PAR1 signaling cascade in membrane rafts, we have used a detergent-free method of cellular subfractionation.

Consistent with our past findings (44), both caveolin-1 and endothelial nitric oxide synthase (eNOS) were enriched at the plasma membrane compared with the whole cell homogenate (data not shown) and were localized to those fractions of the plasma membrane corresponding to light buoyant density membrane rafts (Fig. 1A). Urokinase plasminogen-activated receptor (uPAR), a glycosylphosphatidyl-inositol (GPI)-linked protein that has been previously localized to membrane rafts (37, 40), was also detected in the raft fractions (Fig. 1A). More importantly, here we confirm that the thrombin receptor PAR1 is localized to the same plasma membrane compartment as caveolin-1 (Fig. 1A). Consistent with previous reports (14, 29, 33), we detected the {alpha} subunits of Gq and G12, as well as the small GTPase RhoA in our raft isolates (Fig. 1B). These findings demonstrate that key molecules of the thrombin signaling cascade are enriched or concentrated in rafts relative to other portions of the plasma membrane.


Figure 1
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  		Fig. 1. Isolation of caveolin-rich membrane rafts. Bovine aortic endothelial cells (BAEC) were subfractionated as described in MATERIALS AND METHODS to generate purified plasma membranes that were sonicated, overlaid with a 35–5% step sucrose gradient and subject to differential ultracentrifugation overnight. Equal fractions were collected beginning at the top sucrose layer and precipitated proteins were analyzed via Western blotting. A: results indicate that caveolin-1 is enriched in the light bouyant density membrane raft fractions at the interface of the sucrose step gradient. Endothelial nitric oxide synthase (eNOS) and urokinase protein-activated receptor (uPAR), a glycerylphosphatidyl inositol (GPI)-linked protein commonly found in membrane rafts, colocalize with caveolin-1 in these raft fractions. PAR1 is also detected in these same raft fractions. B: key downstream components of the PAR1 signaling cascade, Gq{alpha}, G12{alpha}, and RhoA are also compartmentalized in membrane rafts. Results are representative of at least three independent experiments.

 
Immunoaffinity isolation of caveolae from the plasma membrane. To determine whether PAR1-related signaling components are localized to membrane rafts in general or more specifically to caveolae, magnetic beads conjugated to an antibody that recognizes the oligomeric form of caveolin-1 were used to separate caveolar vesicles from other raft and non-raft membranes (32). Figure 2 illustrates that nearly all of the caveolin-1 was detected in the bead-bound material, indicating efficient organelle isolation. Consistent with our past findings, eNOS was also sequestered in the bound fraction (36). As expected, the raft-marker uPAR was found only in noncaveolar membranes (Fig. 2A).


Figure 2
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  		Fig. 2. Immunoaffinity isolation of caveolae from the plasma membrane. Caveolae organelles were isolated from BAEC plasma membrane preparations using magnetic beads (Dynal Biotech) precoated with caveolin-1 antibody, as described in MATERIALS AND METHODS. The bound material, representative of isolated caveolae, was separated magnetically from unbound, noncaveolar membranes, subjected to SDS-PAGE and immunoblotted using indicated antibodies. A: results indicate a near-complete isolation of caveolae, as shown by the abundance of caveolin-1 in the bound fraction. eNOS, a known resident protein in caveolae, is also sequestered completely in the bound fraction. The raft-marker uPAR is found in noncaveolar membranes only, whereas the PAR1 receptor resides in both caveolae and noncaveolar membranes. B: PAR1 signaling molecules Gq{alpha}, G12{alpha}, and RhoA localize to caveolar and noncaveolar membranes, with Gq{alpha} notably enriched in the caveolae. Blots are representative of three independent experiments.

 
Examination of isolated caveolae organelles for the presence of signaling molecules related to the PAR1 signaling cascade revealed the presence of PAR1 in both the bound and unbound fractions (Fig. 2A), indicating that the receptor localizes to both caveolae and noncaveolar plasma membranes. Similarly, G12{alpha} and RhoA were detected in both the unbound and bound fractions (Fig. 2B). However, Gq{alpha} was comparatively enriched in bound caveolae fraction, consistent with a past report (33).

PAR1 partitions into membrane rafts and caveolae. To confirm our biochemical findings, we evaluated the localization of PAR1 in intact BAEC. As illustrated in Fig. 3A, cells that were dual-labeled for PAR1 and the raft-marker uPAR revealed colocalization between these two proteins (overlapping red and green signals were confirmed by the presence of yellow in the merged image and detailed in the inset by arrowheads). In cells labeled for PAR1 and caveolin-1, both proteins exhibited a punctate staining pattern that colocalized predominantly at the edges of the cell (Fig. 3B). These data are consistent with our biochemical observations (Figs. 1 and 2) and indicate that PAR1 is generally localized to membrane rafts, with a portion of the PAR1 pool distributed more discretely in caveolae.


Figure 3
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  		Fig. 3. PAR1 colocalizes with uPAR and/or caveolin-1 at the cell membrane. Confluent BAEC were stained with caveolin-1 and uPAR or PAR1 antibodies as detailed in MATERIALS AND METHODS and imaged accordingly. Colocalization was confirmed by signal overlap (yellow) in the merged images and detailed by arrowheads in inset. A: PAR1 (green) colocalizes with uPAR (red), a GPI-linked protein commonly found in membrane rafts. B: dual staining for PAR1 (green) and caveolin-1 (red) indicates colocalization of the two proteins, particularly at the cell edges. Image is representative of two independent experiments.

 
Raft disruption attenuates PAR1-induced MLC phosphorylation. To address the functional significance of the localization of PAR1 in membrane rafts verses caveolae, we first sought to determine the role of membrane rafts in PAR1-mediated alteration of the endothelial cell cytoskeleton. Since MLC phosphorylation on residues Ser-19/Thr-18 is necessary for acto-myosin interaction and stress fiber formation (2, 12), we used an affinity-purified antibody to the dual-phosphorylated form of MLC (5) to provide a biochemical assessment of cytoskeletal reorganization in response to PAR1 activation.

Confluent BAEC were acutely challenged with 100 nM bovine {alpha}-thrombin, which induced MLC phosphorylation in a time-dependent manner, consistent with previous reports (10). The response was robust and occurred rapidly, achieving a 2.73 ± 0.1-fold increase in phosphorylation over control after only 1 min stimulation, before returning to baseline levels by 10 min (Fig. 4, A and B). Stimulation with a peptide agonist for PAR1 (SFLLRN) also significantly increased MLC diphosphorylation (2.17 ± 0.23-fold change, data not shown).


Figure 4
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  		Fig. 4. Disruption of lipid rafts attenuates thrombin-induced myosine light chain (MLC) phosphorylation. Confluent BAEC were pretreated with vehicle or the cholesterol-sequestering agent filipin III before stimulation with 100 nM bovine {alpha}-thrombin for the desired length of time. Cells were lysed immediately and samples were generated as described in MATERIALS AND METHODS. A: results indicate that thrombin significantly increases MLC diphosphorylation at 1 and 5 min, with responses returning to basal levels by 10 min. Raft disruption by filipin attenuated both basal and thrombin-induced MLC phosphorylation. beta-Actin was used as a control to verify equal loading of samples and normalize changes in phosphorylation. B: results are representative of at least four independent experiments and are expressed as fold change in MLC diphosphorylation over nontreated control. *P < 0.05 vs. nontreated control and ^P < 0.05 vs. time-matched nonfilipin-pretreated sample. There were no significant differences within the filipin-treated group. C: to confirm that filipin did not generally decrease the ability of myosin to be phosphorylated, cells were treated with calyculin A, a serine/threonine phosphatase inhibitor. When applied following filipin treatment, calyculin A was able to restore MLC diphosphorylation levels. *P < 0.05 vs. nontreated control. NS, no significant difference.

 
BAEC were then pretreated with 5 µg/ml filipin, a statin antibiotic that binds cholesterol with high affinity and interdigitates into the membrane to effectively disrupt raft structure (31). Treatment with filipin before thrombin challenge significantly attenuated (0.73 ± 0.28-fold change) thrombin-induced MLC phosphorylation at all time points examined (Fig. 4, A and B). As with thrombin, filipin prevented any significant increase in PAR1 agonist-induced MLC phosphorylation (data not shown). Our results indicate that intact lipid rafts are necessary for proper thrombin-induced rearrangement of the cytoskeleton.

Since basal levels of MLC phosphorylation were also reduced following filipin pretreatment, we treated BAEC with calyculin A, a serine-threonine phosphatase inhibitor, to prevent dephosphorylation of MLC via myosin phosphatase. Calyculin A alone stimulated a 1.6-fold ± 0.05 increase in phosphorylated MLC (Fig. 4C). When applied after filipin pretreatment, calyculin A induced MLC diphosphorylation levels without addition of stimuli. These data confirm that MLC is still capable of being phosphorylated in the presence of filipin and serve as a control for generalized filipin effects.

Disruption of lipid rafts inhibits the formation of actin stress fibers and paracellular gaps induced by PAR1 activation. Although the thrombin-stimulated redistribution of actin microfilaments and the subsequent formation of gaps between vascular endothelial cells have been well characterized (11, 12), the regulation of the initiating events in this cascade has not been established. Our data thus far illustrates the importance of membrane rafts in maintaining proper signaling leading to MLC phosphorylation events. To extend these findings, we examined the morphological changes in endothelial cells exposed to thrombin and PAR1 agonists. BAEC were grown to confluency on Vitrogen-coated coverslips and treated with filipin or vehicle alone before stimulation with 100 nM thrombin for 5 min. Thrombin stimulated a rearrangement of the web-like actin network into thick bundles of stress fibers transversing the cell (Fig. 5). In addition, large paracellular gaps were evident in the monolayer (arrows). Stimulation with the PAR1 agonist SFLLRN produced similar changes in the cytoskeleton (data not shown). Filipin pretreatment prevented both of these effects, supporting the hypothesis that rafts play an important role in PAR1-mediated cytoskeletal modification.


Figure 5
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  		Fig. 5. Disruption of membrane rafts inhibits the formation of actin stress fibers and paracellular gaps induced by PAR1 activation. BAEC were grown to confluency on Vitrogen-coated coverslips and pretreated with filipin before stimulation with bovine {alpha}-thrombin (100 nM, 5 min). Cells were fixed and labeled with Texas red-conjugated phalloidin to stain the actin cytoskeleton. PAR1 activation stimulated a rearrangement of the actin network into stress fibers transversing the cell, as well as the formation of paracellular gaps in the monolayer (arrows). Disruption of membrane rafts with filipin prevented both of these effects. Shown is a representative image from one of three independent experiments.

 
Caveolin-1 is not required for PAR1-induced MLC phosphorylation or downstream changes in cytoskeletal dynamics. To determine whether caveolae, in addition to membrane rafts in general, are essential for thrombin signaling, cells were depleted of caveolin-1. BAEC were transfected (48 h) with either caveolin-1 siRNA (cav-1 SMARTpool) or a pool of nontargeting siRNAs (siCONTROL) before stimulation with 100 nM thrombin. Figure 6A, right, demonstrates a 90% reduction in caveolin-1 protein expression by this method. In contrast to cells pretreated with filipin, MLC diphosphorylation in response to thrombin was not significantly affected by caveolin-1 knockdown (Fig. 6A). Similarly, there was no change in the pattern of the MLC response to PAR1 agonist challenge after loss of caveoln-1 expression (data not shown).


Figure 6
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  		Fig. 6. Knockdown of caveolin-1 expression does not prevent thrombin-induced MLC phosphorylation. BAEC at 85–90% confluence were treated with transfection reagent alone, a pool of nontargeting small interferring RNA (siRNAs) (siCONTROL) or caveolin-1 SMARTpool siRNA as described in MATERIALS AND METHODS. Cells were stimulated with thrombin 48 h after initial transfection and processed for Western analysis. A: MLC diphosphorylation in response to thrombin was not affected by caveolin-1 knockdown. Shown is a representative blot of four independent experiments. B: to confirm that caveolin-1 knockdown was sufficient to interrupt signaling, cells were stimulated with sphinogsine-1-phosphate (S1P). The MLC diphosphorylation response to S1P was inhibited following cav-1 siRNA treatment. Blots are representative of at least two independent experiments.

 
These results are inconsistent with our expectation that caveolin-1 depletion and filipin treatment would affect thrombin signaling in the same manner. To rule out the possibility that this lack of response was due to insufficient knockdown of caveolin-1 expression, we performed an additional experiment involving an independent signaling pathway leading to MLC phosphorylation. Sphingosine-1-phosphate (S1P) has been shown to induce the rapid phosphorylation of MLC in HUVEC (9), and recent studies demonstrate that its receptor Edg-1 has been localized to caveolae (19). Caveolin-1-depleted BAEC were stimulated with 1 µM S1P for 10 min. Figure 6B shows that S1P induced a threefold increase in MLC diphosphorylation in siCONTROL-treated cells, whereas cells transfected with cav-1 siRNA showed no response to S1P. These data indicate that S1P-induced MLC diphosphorylation is sensitive to caveolin-1 depletion and illustrates the effectiveness of caveolin-1 knockdown in mediating signaling leading to alterations in the endothelial cytoskeleton.

Finally, we evaluated the impact of caveolin-1 knockdown on PAR1-stimulated changes in cell morphology. BAEC were transfected with siCONTROL (Fig. 7A) or cav-1 SMARTpool siRNA (Fig. 7B), followed by stimulation with vehicle or thrombin. Cells were then fixed and dual-labeled with Texas red-conjugated phalloidin and an antibody to caveolin-1 (to confirm knockdown). Whereas the knockdown of cav-1 expression was ~90%, the transfection efficiency for our system was 85–90%, leaving a portion of the cells on each coverslip with residual expression of cav-1. Fields of view containing at least one untransfected cell were purposely selected to emphasize the fact that both caveolin-positive and caveolin-negative cells from the same sample responded to thrombin in the same manner. In caveolin-1-depleted cells (marked by an asterisk), PAR1 activation by thrombin (Fig. 7B) or SFLLRN (data not shown) produced stress fibers and gaps in the monolayer (arrows) comparable to those seen in control-transfected (Fig. 7A) or untransfected cells.


Figure 7
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  		Fig. 7. Knockdown of caveolin-1 does not alter PAR1-mediated cytoskeletal rearrangement or gap formation. BAEC were grown as before on Vitrogen-coated coverslips and treated with siCONTROL (A) or cav-1 SMARTpool (B) siRNA as previously described. Cells were stimulated with bovine {alpha}-thrombin (100 nM, 5 min) and fixed and dual-labeled with Texas red-conjugated phalloidin and an antibody to caveolin-1 (to confirm knockdown). Results indicate that in transfected cells lacking caveolin-1 expression (marked by *), PAR1 activation by thrombin produced stress fibers and gaps in the monolayer (arrows) comparable to those seen in control-transfected cells (A). Shown is a representative image from one of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Selective sequestration of receptors and lipid-modified signaling molecules into membrane rafts and caveolae provides the cell with a means of regulating signal transduction events in an efficient manner (39). Evidence suggests that some members of the G protein-coupled class of receptors (GPCRs), in particular the B2 bradykinin (8) and ETA endothelin-1 receptors (4), are regulated in this manner. Early studies detected the GPCR PAR1, at that time known only as the functional thrombin receptor, on the plasmalemma of human umbilical vein cells (HUVEC) (18). Presently, there has not been a recent investigation into the functional consequences of PAR1 localization in the endothelial plasma membrane.

In this study we found that PAR1 is localized to endothelial cell membrane rafts (Fig. 1) and is present in immunoaffinity isolated caveolae (Fig. 2). Although some G protein-coupled receptors possess a consensus binding motif for caveolin-1 (4, 8), examination of the amino acid sequence for PAR1 did not reveal such a motif. It is likely that PAR1 is targeted to membrane rafts via palmitoylation or some other lipid modification; however, the exact means of receptor localization is unresolved. In confirmation of our biochemical findings, immunofluorescent studies illustrate that PAR1 colocalizes with caveolin-1 (Fig. 3B), as well as a GPI-linked protein marker for membrane rafts (uPAR, Fig. 3A), suggesting the receptor is distributed between membrane raft and caveolar compartments within the plama membrane. Our findings that the {alpha}-subunits of heterotrimeric G proteins also target to caveolin-rich, low-density membrane fractions and, more specifically, to caveolae organelles support previous observations of G{alpha}-subunit subcellular localization (33). In addition, our data confirm earlier reports showing that the small GTPase RhoA is likewise localized to membrane rafts and caveolae (14, 22, 29). Collectively, these findings demonstrate that key players involved in detecting and propagating thrombin responses are present in the same compartment of the endothelial cell membrane.

To evaluate the functional consequences of the raft- versus caveolae-oriented compartmentalization of this signaling cascade, we manipulated the structure of both membrane rafts and caveolae via the pharmacological sequestration of cholesterol or the depletion of the structural protein caveolin-1, respectively. Thrombin induced MLC phosphorylation (Fig. 4) and enhanced the formation of stress fibers and interendothelial gaps in BAEC monolayers (Fig. 5). Whereas not directly evaluated, we expect that the existence of paracellular gaps following thrombin treatment would compromise endothelial barrier function (2, 13, 26). Pretreatment of cells with the cholesterol-sequestering agents filipin or methyl-beta-cyclodextrin (data not shown) produced inhibitory effects on cytoskeletal dynamics. Interestingly, these compounds reduce basal levels of MLC phosphorylation, suggesting that raft integrity may be necessary for maintaining the normal molecular machinery governing actin dynamics. Cholesterol sequestration alone did not appear to visibly alter endothelial cell shape or cytoskeletal structure (Fig. 5), thus the functional consequence of this reduction of baseline phosphorylation is unknown.

We then evaluated whether caveolae, more specifically, play a role in thrombin-mediated signaling. It has been well documented, in knockout animals and in cell-based models, that caveolae fail to form in the absence of caveolin-1 (35, 38). Using a pool of rationally designed siRNAs custom targeted to bovine caveolin-1 (cav-1 SMARTpool), we were able to achieve a 90% reduction in the expression of the protein. Although our work here implicates a role for membrane rafts in maintaining proper thrombin signaling, and a link between caveolae and the cytoskeleton has been suggested in previous studies (30, 41), this considerable knockdown of caveolin-1 expression did not have a significant effect on PAR1-mediated signaling leading to cytoskeletal reorganization (Figs. 6 and 7).

That our method of caveolin-1 depletion was sufficient to prevent other, similar signaling events leading to changes in cytoskeletal dynamics was confirmed by additional experiments in which cells were challenged with S1P. Previous studies have shown that the localization of the S1P receptor Edg-1 to caveolae has consequences for the regulation of signal transduction events (19). Likewise, in our system, S1P-induced MLC phosphorylation was inhibited following depletion of caveolin-1 (Fig. 6). These findings demonstrate that some cytoskeletal dynamics, although not necessarily those mediated by PAR1 activation, are dependent on intact caveolae.

There is also some controversy over whether targeting to caveolae is essential for certain G protein receptor-linked cellular signaling events. Whereas the loss of caveolin/caveolae has been shown to impede GPCR signaling in response to angiotensin II in vascular smooth muscle (46), a recent study in endothelial cells showed that cav-1 siRNA treatment enhances S1P-induced Akt phosphorylation but has no effect on S1P-induced ERK phosphorylation (16). This suggests that cav-1 knockdown has differential effects on signaling, even when the same stimulus is used. Our data are certainly in line with the idea that loss of caveolin-1 may affect some signaling pathways (S1P-induced MLC phosphorylaion) but not others (thrombin-induced MLC phosphorylation). Since caveolin knockdown does not alter PAR1 signaling, yet cholesterol depletion does have an effect, it appears that noncaveolar rafts, but not specifically caveolae, are critical for the mediation of MLC phosphorylation and stress fiber formation following thrombin stimulation. Whether a compensatory mechanism involving raft-based PAR1 is at work in caveolin-depleted endothelial cells remains to be tested. Evaluation of membrane raft structure and signaling properties in endothelial cells derived from caveolin-1 null mice may serve as a good experimental model to assess this concept.

Investigation of the molecular mechanisms underlying the involvement of membrane rafts in MLC phosphorylation and cytoskeleton regulation is a logical extension of the work presented here and is the subject of current studies in the lab. Preliminary findings suggest that cholesterol depletion does indeed attenuate thrombin-induced Rho activity. Whether this is also true for small G protein activation and whether this effect is dependent on intact caveolae will require further investigation.

Given that caveolin-1 depletion has an effect on thrombin-induced MLC signaling, but not S1P-induced signaling, a particular area of interest would be whether differences in the membrane localization of these receptors are responsible for their differential regulation. Differential compartmentalization of receptors (i.e., raft vs. caveolae) could lead to the activation of distinct co-resident G proteins with different functional outcomes. In support of this concept, our caveolae isolations illustrate that whereas RhoA and G12{alpha} are found in both caveolar and noncaveolar membranes, Gq{alpha} partitions more selectively and is enriched in caveolae (see Fig. 2). It would be interesting to see whether a difference exists in the state of Rho or G protein activation following S1P or thrombin stimulation in cholesterol-depleted versus caveolin-depleted endothelial cells.

In summary, we have shown that PAR1 and several of its downstream effectors are localized to the same compartment in the endothelial plasma membrane and have demonstrated a novel mechanism for the regulation of thrombin- and PAR1 agonist-induced cytoskeletal rearrangement and changes in endothelial monolayer integrity. Membrane rafts, though not necessarily caveolae, are critical for the organization of PAR1-associated signaling molecules and proper signal cascade propagation leading to the changes in cytoskeletal structure and cell morphology in the endothelium. Whether the discrete localization of PAR1 is responsible for the selective activation of differential signaling pathways, and whether this accounts for thrombin-induced endothelial cell dysfunction in a pathological situation, is an avenue of future study that may provide insight into a number of vascular disease processes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a National Heart, Lung, and Blood Institute Grant HL-66301.


   ACKNOWLEDGMENTS
 
We thank Dr. Tracee Panetti (Temple University) for technical assistance in confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Rizzo, Cardiovascular Research Center and Dept. of Anatomy & Cell Biology, Temple Univ. School of Medicine, 3420 N. Broad St, MRB 826, Philadelphia, PA 19140 (e-mail: rizzov{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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