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(The FASEB Journal. 2005;19:1646-1656.)
© 2005 FASEB

Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and {alpha}-actinin

Patrick A. Singleton, Steven M. Dudek, Eddie T. Chiang and Joe G. N. Garcia1

Division of Pulmonary and Critical Care Medicine, Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

1 Correspondence: Johns Hopkins University, Baltimore, MD 21224, USA. E-mail: drgarcia{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 µM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase {alpha} and ß catalytic subunits, the Rac1 GEF, Tiam1, and {alpha}-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, {alpha}-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and {alpha}-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both {alpha}-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for {alpha}-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.—Singleton, P. A., Dudek, S. M., Chiang, E. T., Garcia, J. G. N. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1 and {alpha}-actinin.


Key Words: cytoskeleton • S1P • S1P1/Edg1 receptor • Tiam1 • PI3 kinase • {alpha}-actinin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ENDOTHELIAL CELLS (EC) provide a semiselective barrier between the blood and underlying tissue interstitium with barrier disruption resulting in increased vascular permeability and organ dysfunction. Agents that enhance EC barrier function are a desirable therapeutic strategy for a variety of inflammatory diseases, tumor angiogenesis, and atherosclerosis (1) . Sphingosine-1-phosphate (S1P) is a platelet-derived phospholipid with multiple EC effects, including promoting barrier integrity in vivo and in vitro, that is dependent on S1P binding to its major cell surface receptor(s), the S1P receptor(s), and activation of the small GTPase, Rac1 (2 3 4) . Concurrently, S1P induces rearrangement of the cortical actin cytoskeleton (2 3 4) . However, the underlying signaling mechanisms by which S1P increases vascular integrity remain poorly understood.

S1P binding to S1P receptor subtypes (also called Edg receptors, endothelial differentiation gene) mediates important biological functions including cell adhesion, barrier regulation, proliferation, differentiation, migration, and survival (5) . S1P binds to the plasma membrane heptahelical S1P receptors 1 (Edg1), 2 (Edg5), 3 (Edg3), 4 (Edg6), and 5 (Edg8) expressed in a variety of cell types including endothelium (4 5 6) . Human EC exhibit high expression of S1P1 and S1P3 with S1P1 signaling coupled to the Gi pathway and Rac1 activation, whereas S1P3 signaling couples to the Gi, Gq/11, and G12/13 pathways and activates RhoA to a much greater extent than Rac1 (4 5 6 7) .

S1P-mediated EC barrier function is dependent on Rac1 activation as overexpression of a Rac1 dominant-negative mutant inhibits S1P-induced EC barrier enhancement and translocation of the cortical actin regulatory molecule cortactin to the EC periphery (3) . Rac1 is involved in cytoskeletal reorganization, and key to the barrier protective effects of agents such as S1P (3) , simvastatin (8) , and HGF (9) . Specific Rho family guanine nucleotide exchange factors (GEFs) catalyze the exchange of Rac1-GDP (inactive) for Rac1-GTP (active) including Tiam1 (T-lymphoma invasion and metastasis gene 1) (10 , 11) . Tiam1 is implicated in diverse functions including regulation of cell-cell adhesion as Tiam1 overexpression increases E-cadherin-dependent cell-cell adhesion (10 11 12 13) . Selective S1P activation of Rac/S1P1 and S1P3/Rho is dependent on the S1P concentration (14) and possibly S1P-induced recruitment of S1P receptors (15) to specialized plasma membrane microdomains (16 , 17) containing the scaffolding protein caveolin-1 (18 19 20) .

The {alpha}-actinin family of cytoskeletal proteins regulates a variety of cellular functions including cell-cell adhesion and migration (21 22 23) . So far there have been four {alpha}-actinin isoforms identified: two nonmuscle (1 and 4) and two muscle (2 and 3) (21) . The structure of {alpha}-actinin reveals an amino-terminal actin binding domain, a central {alpha}-helical spectrin-like repeat region, and a carboxyl-terminal calmodulin-like domain (21) . Two {alpha}-actinin monomers can form a functional dimer that can link certain cell surface adhesion receptors (i.e., ß-integrins, ICAMs, cadherins) to the actin cytoskeleton (21 22 23) . Further, the PI3 kinase pathway plays an important regulatory role in {alpha}-actinin cellular localization and function (21 , 24) .

We examined S1P-induced signaling in human lung EC caveolin-rich microdomains (CEMs) resulting in cytoskeletal rearrangement and barrier enhancement. Our findings indicate that S1P actively recruits S1P1 and S1P3, PI3 kinase catalytic subunits p110 {alpha} and ß, Tiam1 and {alpha}-actinin-1/4 to CEM fractions in a PI3 kinase-dependent manner resulting in cortical actin rearrangement and EC barrier enhancement. Reductions in expression of either S1P1 receptor or Tiam1 (siRNA), but not S1P3 inhibited S1P-induced, {alpha}-actinin-1/4 CEM translocation, Rac1 activation, cortical actin reorganization, and increased EC barrier function. These data indicate the critical importance of the PI3 kinase pathway in S1P signaling to the EC cytoskeleton and barrier enhancement through the S1P1 receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
Human pulmonary artery EC were obtained from Clonetics (Walkersville, MD, USA) and cultured as described previously (4) in EBM-2 complete medium (Clonetics) at 37°C in a humidified atmosphere of 5% CO2, 95% air, with passages 6–10 used for experimentation. Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO, USA). Reagents for SDS-PAGE electrophoresis were purchased from Bio-Rad (Richmond, CA, USA), Immobilon-P transfer membrane from Millipore (Millipore Corp., Bedford, MA, USA), and gold microelectrodes from Applied Biophysics (Troy, NY, USA). Rabbit anti-caveolin-1 antibody, rabbit anti-p110 {alpha}, ß, {gamma}, {delta} PI3 kinase antibodies, rabbit anti-Tiam1, and mouse anti-vimentin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-KDR (VEGF receptor 2) antibody and mouse anti-{alpha}-actinin-1 antibody were purchased from Chemicon International (Temecula, CA, USA). Rabbit anti-S1P1 receptor antibody and S1P1 receptor blocking peptide were purchased from Affinity Bioreagents (Golden, CO, USA). Mouse anti-S1P3 receptor antibody, S1P3 receptor blocking peptide, and S1P1 receptor transfected cell-positive control lysate were purchased from Exalpha Biologicals (Watertown, MA, USA). Mouse anti-Rac1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Rabbit anti-{alpha}-actinin-4 antibody was purchased from Alexis Biochemicals (San Diego, CA, USA). Biotintylated mouse anti-PIP3 antibody was purchased from Echelon Biosciences Inc. (Salt Lake City, UT, USA). Secondary horseradish peroxidase (HRP) -labeled antibodies were purchased from Amersham Biosciences (Piscataway, NJ, USA). Texas Red-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR, USA). LY294002 was purchased from Calbiochem (San Diego, CA, USA).

Caveolin-enriched microdomain (CEM) isolation and cholesterol quantitation
CEMs were isolated from human lung EC as we have described previously (17 , 25) . Briefly, EC were scraped in PBS, centrifuged at 2000 rpm at 4°C and lysed with 0.2 mL of TN solution [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, protease inhibitors, 10% sucrose,1% Triton X-100] for 30 min on ice. Triton X-100-insoluble materials were then mixed with 0.6 mL of cold 60% OptiprepTM and overlaid with 0.6 mL of 40%, 30%, and 20% OptiprepTM in TN solution. The gradients were centrifuged at 35,000 rpm in SW60 rotor for 12 h at 4°C and different fractions were collected. Cellular proteins or lipids associated with each fraction were precipitated according to the procedures described previously and analyzed by SDS-PAGE plus immunoblotting and/or immunoprecipitation. In some cases, cholesterol content associated with different fractions was also measured using Amplex RedTM cholesterol assay kit (Molecular Probes).

Immunoprecipitation and immunoblotting
Cellular materials associated within the 20% OptiprepTM fractions (CEM fraction) were incubated with IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1% Nonidet P-40 (NP-40), 0.4 mM Na3VO4, 40 mM NaF, 50 µM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem protease inhibitor mixture 3). Samples were then immunoprecipitated with anti-S1P1 receptor, anti-S1P3 receptor, anti-Tiam1, or anti-PI3 kinase p110 IgG followed by SDS-PAGE in 4-15% polyacrylamide gels, transfer onto ImmobilonTM membranes, and developed with specific primary and secondary antibodies. Visualization of immunoreactive bands was achieved using enhanced chemiluminescence (Amersham Biosciences). In some cases, Arbitrary Units for immunoreactive bands in CEMs were generated using the calculation (standardized average gray value (S.A.G.V., obtained from ImageQuantTM software (Amersham Biosciences) immunoreactive band of interest divided by S.A.G.V. caveolin-1 immunoreactive band) x 100.

Construction and transfection of siRNA against S1P1, S1P3, Tiam1, {alpha}-actinin-1, and {alpha}-actinin-4
The siRNA sequence(s) targeting human S1P1, S1P3, Tiam1, {alpha}-actinin-1, or {alpha}-actinin-4 were generated using mRNA sequences from Gen-BankTM (gi:13027635, gi:38788192, gi:897556, gi:12025669 or gi:34452697 respectively). For each mRNA (or scrambled), two targets were identified. Specifically, S1P1 target sequence 1 (5'-AAGCTACACAAAAAGCCTGGA-3'), S1P1 target sequence 2 (5'-AAAAAGCCTGGATCACTCATC-3'), S1P3 target sequence 1 (5'-AACAGGGACTCAGGGACCAGA-3'), S1P3 target sequence 2 (5'-AAATGAATGTTCCTGGGGCGC-3'), Tiam1 target sequence 1 (5'-AAACAGCTTCAGAAGCCTGAC-3'), Tiam1 target sequence 2 (5'-AATGCTCTGAATCCTAGTCTC-3'), {alpha}-actinin-1 target sequence 1 (5'-AAGAAATCCAGACCCTAGCAC-3'), {alpha}-actinin-1 target sequence 2 (5'-AACGATTACATGCAGCCAGAA-3'), {alpha}-actinin-4 target sequence 1 (5'-AACATTGATGAGGACTTCCGA-3'), {alpha}-actinin-4 target sequence 2 (5'-AATCAACAATGTGAACAAAGC-3'), scrambled sequence 1 (5'-AAGAGAAATCGAAACCGAAAA-3'), and scrambled sequence 2 (5'-AAGAACCCAATTAAGCGCAAG-3') were used. Sense and antisense oligonucleotides were provided by the Johns Hopkins University DNA Analysis Facility. For construction of the siRNA, a transcription-based kit from Ambion was used (SilencerTM siRNA construction kit). Human lung EC were then transfected with siRNA using siPORTamineTM as the transfection reagent (Ambion, Austin, TX, USA) according to the protocol provided by Ambion. Cells (~40% confluent) were serum-starved for 1 h, followed by incubated with 3 µM (1.5 µM of each siRNA) of target siRNA (or scrambled siRNA or no siRNA) for 6 h in serum-free media. The serum-containing media was then added (1% serum final concentration) for 42 h before biochemical experiments and/or functional assays were conducted.

PI3 kinase activation assay and dot immunoblot analysis
Cellular materials associated with the CEM fraction were incubated with IP buffer (as described above). The samples were then divided into four equal volumes and incubated with a 1:100 dilution of anti-p110 PI3 kinase {alpha}, ß, {gamma}, {delta} antibodies and secondary anti-rabbit IgG conjugated agarose beads on a rotating mixer at 32°C for 1 h. The immunobeads were washed in IP buffer three times. The resulting p110 PI3 kinase-conjugated immunobeads were incubated in 50 µL of IP buffer (0.01% NP-40) containing 20 µg of sonicated PIP2 (Sigma) and 1 mM ATP for 6 h at 37°C. The reactions were subsequently terminated by the addition of acidified chloroform:methanol (1:1, v/v) (26 , 27) . Extracted lipids were then vacuum-spotted onto ImmobilonTM membranes presoaked in methanol. The resulting dot membranes were blocked in 5% nonfat bovine serum albumin and probed with anti-PIP3 antibody conjugated to biotin, followed by secondary avidin HRP. In some cases, CEM fractions were directly vacuum-blotted onto ImmobilonTM membranes and probed for caveolin-1 or PIP3. Visualization of the resulting dot immunoreactive areas was achieved using enhanced chemiluminescence (Amersham Biosciences).

Tiam1 activation assay
Cellular materials associated with the CEM fraction were incubated with IP buffer (as described above). Samples were then incubated with anti-Tiam1 antibody and secondary anti-rabbit IgG-conjugated agarose beads on a rotating mixer 32°C for 1 h, then washed in IP buffer three times. A portion of the Tiam1 immunobeads was kept for quantitation of total Tiam1. Purified Escherichia coli-derived GST-tagged Rac1 was preloaded with GDP as described (28) . The GDP-Rac1 was added to Tiam1-associated immunobeads in IP buffer in the presence of 1 mM GTP and incubated at 32°C for 30 min. The immunobeads were centrifuged on a tabletop centrifuge. Supernatants were collected, added to p21 binding domain (PBD) -conjugated beads (Calbiochem) and incubated at room temperature for 30 min. The mixtures were centrifuged in a tabletop centrifuge, supernatants removed, and GTP-Rac1 bound PBD beads were washed in IP buffer three times. Samples were then run on SDS-PAGE, transferred to ImmobilonTM membranes, and probed with mouse anti-Rac1 antibody.

Rac1 activation assay
Rac activity in human lung EC was performed as described previously (4) .

Measurement of EC electrical resistance
EC were grown to confluence in polycarbonate wells containing evaporated gold microelectrodes and TER measurements were performed using an electrical cell substrate impedance sensing system obtained from Applied Biophysics (Troy, NY, USA) as described in detail (4) . TER values from each microelectrode were pooled at discrete time points and plotted vs. time as the mean ± SE.

Immunofluorescence microscopy
Polymerized actin rearrangement was assessed with Texas Red-conjugated phalloidin (Molecular Probes) and analyzed using a Nikon Eclipse TE 300 microscope as we have described (4) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of S1P-induced human EC signaling within caveolin-enriched microdomains (CEMs)
S1P receptor-specific ligation promotes a variety of important EC biological functions including vascular barrier enhancement (5 , 6) . We explored the role of S1P-induced signaling within CEM fractions resulting in EC barrier enhancement. Using specific antibodies (Fig. 1 A), modest levels of S1P1 (but not S1P3) were observed in CEM fractions in control EC monolayers (Fig. 1B ) whereas S1P induced strong and specific recruitment of both S1P1 and S1P3 into CEMs (Fig. 1B-b, c ). S1P failed to recruit VEGF receptor 2 (KDR) or the intermediate filament protein vimentin (Fig. 1B-d, e ) to CEMs. Methyl-ß-cyclodextrin (MßCD), a cholesterol depletion agent that abolishes CEM formation (29) (Fig. 1B-D ), abolished S1P-induced recruitment of S1P receptors to CEM fractions (Fig. 1B-b, c ). These effects appear to be specific for S1P since sphingosine challenge did not affect CEM recruitment of S1P1 or S1P3 (Fig. 1E, F ).



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  		Figure 1. Analysis of caveolin-enriched microdomains (CEM) in S1P-challenged human EC. A) Antibody specificity using immunoblotting with anti-S1P1 (A-a, b, c) antibody, anti-S1P3 antibody (A-d, e, f), or Tiam1 antibody (A-g, h) on EC lysates or S1P1 transfected cell lysates (provided as a positive control for S1P1 receptor antibody). In some cases, S1P1 or S1P3 blocking peptides were incubated with primary antibody for 30 min before immunoblotting (A-b, c, e, f). B, C) Confluent EC were either untreated (control) or challenged with 1 µM S1P (5 min) in the presence or absence 5 mM methyl-ß-cyclodextrin (MßCD, 1 h), a cholesterol depletion agent that abolishes CEM formation. CEM fractions were prepared as described in Materials and Methods as the 20% OptiprepTM layer (*) (17) . B, C) Detection of immunoreactivity within the 20–60% OptiprepTM layers or Triton X-100 soluble material was performed to verify caveolin-1 (B-a, C-a), S1P1 (B-b), S1P3 (B-c), VEGF receptor 2 (B-d), vimentin (B-e), p110{alpha} PI3 kinase (C-b), p110ß PI3 kinase (C-c), p110 PI3{gamma} kinase (C-d), p110{delta} PI3 kinase (C-e), Tiam1 (C-f), {alpha}-actinin-1 (C-g), or {alpha}-actinin-4 (C-h). Experiments were performed in triplicate with highly reproducible findings (representative data shown). D) Cholesterol content in CEMs (the 20% OptiprepTM layer) isolated from untreated cells or 5 mM methyl-ß-cyclodextrin-treated cells. Graph represents the results of 3 independent experiments. E) Confluent EC were either untreated (control) or challenged with 1 µM sphingosine (5 min, a negative control for S1P) and CEM fractions were prepared as described in Materials and Methods as the 20% OptiprepTM layer (*) (17) . Immunoblots were performed on the 20% OptiprepTM layer (CEM) using anti-caveolin-1 (E-a, C-a), anti-S1P1 (E-b), anti-S1P3 (E-c), anti-Tiam-1 (E-d), anti-{alpha}-actinin-1 (E-e), or anti-{alpha}-actinin-4 (E-f) antibody. Experiments were performed in triplicate with highly reproducible findings (representative data shown).F) Depicts graphical quantitation of immunoreactive bands from experiments depicted in panels B, C, and E, which were analyzed using ImageQuantTM software (see Materials and Methods). Arbitrary Units on the y-axis refer to % (standardized average gray value (S.A.G.V.) immunoreactive band of interest divided by S.A.G.V. caveolin-1 immunoreactive band).

The role of PI3 kinase in S1P1-mediated CEM signaling
As the PI3 kinase pathway participates in S1P receptor signaling (30) , S1P-induced CEM fractions were next analyzed for the presence of the catalytic PI3 kinase subunits. Recruitment of PI3 kinase p110{alpha} and ß (but not p110{gamma} or {delta}) to these specialized microdomains were observed (Fig. 1C ) and isolation of p110 PI3 kinase catalytic subunits revealed kinase activation and PIP3 production via p110 PI3 kinase {alpha} subunit activation (see Fig. 4A ) in S1P-induced CEM fractions (Fig. 2 B). These effects were abolished by disrupting CEM formation (MßCD) or by PI3 kinase inhibition (LY294002). Finally, reduction in S1P1 protein expression (but not S1P3) with siRNA targeting (Fig. 3 A, B) significantly attenuates S1P-induced p110{alpha} PI3 kinase activation (Fig. 4B ).



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  		Figure 4. S1P1-dependent p110{alpha} PI3 kinase activity in human EC.A) EC either untreated (control) or challenged with 1 µM S1P (5 min) in the absence or presence of 10 µM LY294002 or 5 mM methyl-ß-cyclodextrin. B) EC transfected with scrambled siRNA, S1P1 siRNA, S1P3 siRNA, or Tiam1 siRNA. The 20% OptiprepTM CEM-containing fractions were collected, solublized in NP-40 immunoprecipitation buffer, and immunoprecipitated with either anti-p110{alpha} PI3 kinase, anti-p110ß PI3 kinase, anti-p110{gamma} PI3 kinase or anti-p110{delta} PI3 kinase antibody followed by secondary antibody-conjugated agarose beads. The resulting immunobeads were subjected to PI3 kinase activity assays using PIP2 as a substrate as described in Materials and Methods. The resulting phospholipids were extracted and vacuum-immobilized onto ImmobilonTM membranes. The resulting dot blots were immunoblotted with anti-PIP3 antibody (A, B). Experiments were performed in triplicate with reproducible findings with representative data shown.



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  		Figure 2. Effect of PI3 kinase inhibition (LY294002) on S1P-treated human EC CEMs. Confluent EC were either untreated (control) or challenged with 1 µM S1P (5 min) in the presence or absence of either LY294002 (a specific PI3 kinase inhibitor) (10 µM, 1 h) or 10 mM methyl-ß-cyclodextrin (MßCD) to disrupt CEM formation (1 h). A) Detection of immunoreactive protein in 20–60% OptiprepTM layers using anti-caveolin-1 (A-a), anti-S1P1 (A-b), anti-S1P3 (A-c), anti-Tiam1 (A-d), anti-{alpha}-actinin-1 (A-e), or anti-{alpha}-actinin-4 (A-f) antibodies. B) Dot immunoblot analysis of the 20% OptiprepTM CEM-containing fractions using anti-PIP3 (B-a) or anti-caveolin-1 (B-b) as described in Materials and Methods. C) Complex formation between Tiam1 and S1P1using anti-S1P1 or anti-S1P3 immunoprecipitates generated from control (untreated), 1 µM S1P-treated (5 min), 5 mM MßCD-treated (1 h) followed by 1 µM S1P (5 min) or 10 µM LY294002 (1 h) followed by 1 µM S1P (5 min). Immunoprecipitated cellular material-associated with CEMs was immunoblotted with anti-Tiam1 antibody (a), anti-S1P1 antibody (b), or anti-S1P3 antibody (c). Experiments were performed in triplicate, each with similar results. Representative data are shown.



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  		Figure 3. Characterization of siRNA treatment of human EC. Immunoblot analysis of siRNA-treated or untreated human EC. Cellular lysates from untransfected (control, no siRNA), scrambled siRNA (siRNA that does not target any known human mRNA), S1P1 siRNA, S1P3 siRNA, or Tiam1 siRNA-transfection were analyzed using immunoblotting with anti-S1P1 antibody (A), anti-S1P3 antibody (B), anti-Tiam1 antibody (C), anti-{alpha}-actinin-1 antibody (D), anti-{alpha}-actinin-4 antibody (E), anti-actin antibody (F), anti-VEGF receptor antibody (G), or anti-caveolin-1 antibody (H) as described in Materials and Methods. Experiments were performed in triplicate each with similar results. Representative data are shown.

Regulation of S1P-induced, PI3 kinase-dependent Tiam1/Rac1 activity
Rac1 activation is crucial for certain S1P-induced barrier enhancement (3 , 4) , likely via specific Rac1 guanine nucleotide exchange factors (GEFs) such as Tiam1 (10 , 11) . We observed Tiam1 to be present in untreated CEMs (Fig. 1C-f ) with significantly greater Tiam1 reactivity after S1P challenge (Fig. 1C-f ). Consistent with the role of PI3 kinase in regulating Rac1 activation, PI3 kinase inhibition reduced S1P-stimulated Tiam1 accumulation in CEM fractions (Fig. 2A-d ) without affecting S1P1 or S1P3 recruitment (Fig. 2A-a, b ) (31) . We next explored potential complex formation between Tiam1, S1P1, and S1P3 and observed selective PI3 kinase-dependent association of Tiam1 with S1P1, but not S1P3 within S1P-induced CEM fractions (Fig. 2C ). Tiam1 siRNA-mediated reductions in Tiam1 expression (Fig. 3C ) did not affect S1P-induced p110{alpha} PI3 kinase activity (Fig. 4B ).

We next determined that S1P induces isolated Tiam1-mediated Rac1 GDP-GTP exchange in vitro (Fig. 5 A), which was abolished by LY294002 (Fig. 5A ) as well as by siRNA-mediated reductions in either S1P1 or Tiam1 expression (but not S1P3 expression) (Fig. 3A-C , Fig. 5A ). Tiam1 activity in vitro was directly linked to total EC Rac1 activation (Fig. 5B ), providing evidence for the critical importance of Tiam1 during S1P-induced Rac1 activation in EC.



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  		Figure 5. S1P promotes S1P1 /PI3 kinase-dependent Tiam1/Rac1 activation in human EC. Human EC were either untreated (control) or challenged with 1 µM S1P (5 min) in the absence or presence of 10 µM LY294002, 5 mM methyl-ß-cyclodextrin, or transfected with scrambled siRNA, S1P1 siRNA, S1P3 siRNA, or Tiam1 siRNA and solublized in NP-40 immunoprecipitation buffer. A) Immunoreactivity of anti-Tiam1 immunoprecipitates immunoblotted with anti-Tiam1 antibody (A-b) or subjected to Tiam1-mediated Rac1 GDP-GTP exchange followed by precipitation of Rac1-GTP with p21 binding domain (PBD) -conjugated beads, run on SDS-PAGE and immunoblotted with anti-Rac1 antibody (A-b) as described in Materials and Methods. B) Solublized cellular material either run on SDS-PAGE and immunoblotted with anti-Rac1 antibody to measure total Rac1 (B-a) or incubated with PBD-conjugated beads, run on SDS-PAGE, and immunoblotted with anti-Rac1 antibody to determine activated (GTP-bound) Rac1 (B-b). Experiments were performed in triplicate with reproducible findings with representative data shown.

Regulation of S1P-induced {alpha}-actinin 1 and 4 translocation to EC CEMs
Cytoskeletal proteins, including nonmuscle {alpha}-actinin 1 and 4, play an important role in cell-cell and cell-ECM adhesions (21 , 23 , 32 , 33) . We observed that in control (unchallenged) conditions, {alpha}-actinin 1 and 4 isoform expression in EC CEMs was undetectable (Fig. 1C ). S1P challenge induced a rapid translocation of both {alpha}-actinin isoforms 1 and 4 into CEMs, which was inhibited by cholesterol depletion (Fig. 1C ), PI3 kinase inhibition (Fig. 2A ) or silencing of either Tiam1 or S1P1 (but not S1P3) expression (Fig. 6 ).



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  		Figure 6. S1P-induced {alpha}-actinin-1 and 4 translocation to CEM is dependent on S1P1 receptor and Tiam1. Immunoblot analyses of 20% OptiprepTM CEM fractions from either untreated (control) or S1P challenged (1 µM, 5 min) EC transfected with scrambled siRNA, S1P1 siRNA, S1P3 siRNA, or Tiam1 siRNA. CEM material was run on SDS-PAGE and immunoblotted with anti-caveolin-1 (A), anti-{alpha}-actinin-1 (B), or anti-{alpha}-actinin-4 (C) antibody.

S1P1- and CEM-dependent regulation of EC barrier function and cortical actin rearrangement
Measurements of EC barrier function in vitro (transendothelial cell electrical resistance, TER) revealed that inhibition of CEM formation via cholesterol depletion (MßCD) or PI3 kinase inhibition (LY294002) reduced S1P-induced EC barrier enhancement as did reduction in expression of S1P1 (but not S1P3), Tiam1 or both {alpha}-actinin-1 and 4 (siRNA) (Fig. 7 ). Finally, as cytoskeletal reorganization is a common feature of virtually all EC barrier regulatory responses, we examined phalloidin staining of S1P-challenged EC to visualize cellular F-actin localization and observed that the prominent cortical actin ring formation previously reported after S1P (3 , 4) (Fig. 8 A) was abolished by disruption of CEM formation (MßCD) or PI3 kinase inhibition (LY294002) in favor of increased F-actin stress fiber formation. Reduction of S1P1 (but not S1P3), Tiam1 or both {alpha}-actinin-1 and 4 expression also significantly attenuated S1P-induced cortical actin ring formation (Fig. 8B ), suggesting that S1P-induced recruitment of S1P1 to CEMs promotes p110 {alpha}-regulated PI3 kinase activity resulting in Tiam1/Rac1 activation events and {alpha}-actinin-1/4 translocation, which are required for S1P-induced cortical actin rearrangement and EC barrier enhancement (Fig. 9 ).



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  		Figure 7. Regulation of S1P-regulated transendothelial electrical resistance (TER). A) EC were plated on gold microelectrodes, serum starved for 1 h, and either untreated (control) or treated with vehicle (PBS, pH=7.4), 0.1 mM methyl-ß-cyclodextrin (MßCD), 1 mM MßCD, or 5 mM MßCD for 1 h followed by 1 µM S1P addition. The TER tracing represents pooled data ± SE from 3 independent experiments as described in Materials and Methods. B) Bar graph inset demonstrates the effects of 5 mM MßCD treatment on S1P- and VEGF-mediated EC barrier regulation. The bar graph represents the % MßCD-treated TER vs. untreated (no MßCD) TER (y-axis) with S1P and VEGF challenge. Disruption of CEM formation with MßCD inhibits S1P- but not VEGF-induced changes in TER (at least n=3 for each condition). C) EC on gold microelectrodes were either untreated (control), treated with vehicle (DMSO), 0.1 µM LY294002, 1 µM LY294002, or 10 µM LY294002 for 1 h followed by 1 µM S1P addition. The TER tracing represents pooled data ± SE from 3 independent experiments as described in Materials and Methods. D) EC were plated on gold microelectrodes and treated with scrambled siRNA, S1P1 receptor siRNA, S1P3 receptor siRNA, or Tiam1 siRNA for 48 h. EC were then serum starved for 1 h, followed by addition of 1 µM S1P. The TER tracing represents pooled data ± SE from 3 independent experiments as described in Materials and Methods. E) Bar graph inset demonstrates the effects of scrambled (control) siRNA or {alpha}-actinin-1 and/or {alpha}-actinin-4 siRNA on S1P-induced maximal EC TER (at least n=3 for each condition). Silencing both {alpha}-actinin-1 and 4 is required for maximal inhibition of S1P-induced TER in EC.



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  		Figure 8. Regulation of S1P-mediated EC cortical actin rearrangement. A) EC were serum starved for 1 h and either untreated, treated with 5 mM MßCD or treated with LY294002 for 1 h followed by either no (control) or 1 µM S1P treatment (5 min). B) EC treated with scrambled siRNA, S1P1 siRNA, S1P3 siRNA, Tiam1 siRNA, {alpha}-actinin-1 siRNA or {alpha}-actinin-4 siRNA for 48 h. EC were then serum starved for 1 h followed by addition of 1 µM S1P (5 min). Cells were then fixed and stained with TRITC-phalloidin (to visualize F-actin) and analyzed using fluorescent microscopy. These observations are representative of the entire cell monolayer and were reproduced in multiple independent experiments (at least n=3 for each condition).



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  		Figure 9. Proposed model for S1P-mediated cortical actin rearrangement and human EC barrier enhancement via CEM recruitment of PI3 kinase, Tiam1, and {alpha}-actinin-1/4. We propose that S1P1 ligation results in recruitment of S1P1 into CEM microdomains (1) to join limited preexisting S1P1 receptors in this locale. In this spatially defined compartment, S1P1 promotes recruitment and activation of p110{alpha} PI3 kinase and production of PIP3 (2) . S1P-induced PIP3 generation promotes recruitment of Tiam1 to CEM (3) , which regulates {alpha}-actinin-1 and 4 CEM translocation (4) . CEM-localized Tiam1 catalyzes the formation of Rac1-GTP from Rac1-GDP (5) . S1P1-mediated signaling in CEMs and activation of subsequent downstream targets (i.e., PI3 kinase, Tiam1, {alpha}-actinin-1/4) are required for S1P-induced cortical actin rearrangement and increased EC barrier function (5) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Agents that exhibit the capacity to reverse increases in vascular permeability, a prominent feature in diverse inflammatory syndromes, tumor angiogenesis, and atherosclerosis, have obvious therapeutic applications (1 , 34) . S1P decreases EC permeability in vitro and in vivo and is the primary barrier-enhancing agent released from platelets (3 , 4 , 35) . As the exact mechanisms of S1P-mediated barrier protection remain poorly understood, we examined the role of caveolin-enriched microdomains, Tiam1, and PI3 kinase activities in S1P-mediated signaling and human EC barrier regulation.

CEMs, also known as lipid rafts or caveolae, though controversial, have been implicated in EC migration, proliferation, adhesion, endocytosis, cholesterol and calcium regulation, and signal transduction (20 , 25 , 36 , 37) . Deletion of caveolin-1 expression in mice inhibits CEM (caveolae) formation in EC and promotes lung fibrosis (38) . Monocrotaline-induced rodent pulmonary hypertension results in a loss of lung EC caveolin-1 expression (39) . A criticism of the functional significance of CEMS has been related to the use of detergents (i.e., Triton X-100) to extract lipid rafts, potentially resulting in cellular artifacts (40) . Used extensively in the present study to deplete cholesterol and abolish CEM formation, MßCD has been implicated in nonspecific cellular effects including PIP2 dispersion and actin cytoskeletal changes (41) . While we did not use FRET (fluorescence resonance energy transfer) to measure lipid raft dynamics in living cells (42) , we observed that S1P requires the existence of CEM fractions for PI3 kinase activation, Rac1 signaling, and EC barrier enhancement, in agreement with earlier studies (25 , 43) and the role of CEMs as sites for receptor clustering and consequent EC signal transduction (44) . We previously reported that human lung EC primarily express S1P1 (Edg1) and S1P3 (Edg3) (4) with S1P1, clearly the dominant barrier-enhancing S1P receptor (14 , 35) . We observed that S1P1 (but not S1P3) is endogenously present within CEM fractions with S1P recruiting additional S1P1 receptors and a fraction of cellular S1P3 receptors to these EC plasma membrane microdomains. Targeted use of siRNA to differentially reduce expression of either S1P1 or S1P3 revealed that S1P ligation of S1P1 is responsible for subsequent signaling to the EC cytoskeleton and barrier enhancement (via PI3 kinase activation and Rac1 signaling). We and others have reported differential signaling evoked by S1P1 and S1P3 (4 , 5 , 7 , 45) with S1P1 signaling coupled to Gi and Rac1, and S1P3 signaling coupled to Gi, Gq/11, and G12/13 pathways, preferentially activating RhoA over Rac1 (4 , 5 , 7 , 45 , 46) . The importance of S1P1 is demonstrated in Edg1 (S1P1) receptor knockout mice that succumb in utero to incomplete vascular maturation with progressive vascular hemorrhage (47) . Consistent with the well-appreciated barrier disruptive role of Rho (48) , silencing S1P3 expression enhanced the initial TER and cortical actin rearrangement induced by S1P. Though not directly tested, it is plausible that S1P3 recruitment to CEMs after S1P challenge and may serve as a negative regulator of specific S1P1-mediated functions.

EC CEMs are enriched in the phospholipid, PIP2, which serves as a substrate for the class I PI3 kinases (27 , 49) whose activity is often modulated by regulatory subunits (26 , 50 , 51) . The catalytic PI3 kinase subunit p110{alpha} is the primary PI3 kinase recruited to and activated within CEMs by S1P and is consistent with increased PIP3 production in CEM fractions. The exact regulatory subunit(s) that interact with p110{alpha} PI3 kinase during S1P stimulation of EC is currently being explored. An important downstream target of PI3 kinase signaling is the serine/threonine kinase, AKT (52 , 53) , which regulates cellular functions including survival, proliferation, and migration (30 , 53) . AKT directly phosphorylates threonine residues within S1P1 (T236), an event critical to S1P-mediated EC Rac1 activation, cortical actin reorganization, and migration (30) .

We demonstrated that S1P promotes PI3 kinase-dependent Tiam1/Rac1 activation in CEM fractions, findings consistent with earlier studies (13 , 31 , 54 , 55) . Previous reports that Tiam1 is involved in cell-cell adhesion and acts downstream of the Edg2 receptor (12 , 56) are consistent with our findings that Tiam1 expression is required for S1P/S1P1-induced Rac1 activation and EC barrier enhancement. S1P induced significant recruitment of Tiam1 into CEM fractions and increased total cellular Tiam1 activation, indicating either post-translational modifications of Tiam1 or that localization within CEMs enhances Tiam1 activity. Tiam1 is phosphorylated by serine/threonine kinases, including Ca2+/calmodulin-dependent protein kinase II and protein kinase C-dependent mechanisms (57 , 58) . Studies examining S1P-induced Tiam1 post-translational modification are currently in progress.

We observed clear evidence for a complex formation between Tiam1 and S1P1 (but not S1P3) in CEM fractions after S1P. It remains unknown whether this interaction is direct or mediated through an adaptor protein. Tiam1 contains two pleckstrin homology (PH) domains (11) with the N-terminal PH domain of Tiam1 involved in Tiam1 translocation to the plasma membrane, binding to PIP2 and/or PIP3, and interaction with various signaling molecules (59 , 60) . Whether S1P-induced PIP3 production in EC CEMs promotes Tiam1 recruitment to CEMs where S1P1 complex formation may occur is unknown.

Although there is a high degree of homology between {alpha}-actinin 1 and 4 (>80% similarity), researchers have suggested a nonredundant role for these {alpha}-actinin nonmuscle isoforms in certain cellular functions (21 , 61) . Our data indicate it is necessary to silence both isoforms of {alpha}-actinin for complete abolition of S1P-induced EC TER and cytoskeletal rearrangement. Further, we demonstrate a novel role of Tiam1 in {alpha}-actinin translocation to CEM. The {alpha}-actinins can bind to a host of molecules including actin, vinculin, catenin, PI3 kinase, and PIP3 (21 , 24) . Our data support a regulatory role for the PI3 kinase pathway in {alpha}-actinin CEM translocation. {alpha}-Actinins can link certain cell surface adhesion receptors (i.e., ß-integrins, ICAMs, cadherins) to the underlying actin cytoskeleton (21 , 23) . This suggests a potential involvement of adhesion receptors in S1P-induced S1P1 receptor-mediated EC signaling and cytoskeletal reorganization via {alpha}-actinin.

In summary, while mechanisms of vascular barrier enhancement by agonists such as S1P are poorly understood, we now show that S1P-mediated cortical actin rearrangement and barrier regulation are critically dependent on S1P1 localization within CEMs, CEM-dependent PI3 kinase activation, and PI3 kinase-dependent Tiam1 recruitment to CEMs. The recruitment of Rac GTPase and PI3 kinase within CEM fractions may be a common feature of barrier-enhancing stimuli.

Received for publication March 7, 2005. Accepted for publication June 1, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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