Novel role of microtubules in thrombin-induced ... - The FASEB Journal

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Novel role of microtubules in thrombin-induced endothelial barrier dysfunction ANNA A. BIRUKOVA,*,1 KONSTANTIN G. BIRUKOV,* KSENYA SMUROVA,*,† DJANYBEK ADYSHEV,* KOZO KAIBUCHI,‡ IRINA ALIEVA,† JOE G. N. GARCIA,* AND ALEXANDER D. VERIN* *Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; †Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory, Russia; and ‡Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin-induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombininduced permeability, actin remodeling, and paracellular gap formation and diminished thrombin-induced activation of Rho and Rho-kinase. Expression of activated G␣12/13 subunits involved in thrombin-mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF-specific negative regulator RGS preserved MT from thrombin-induced disassembly. Consistent with these results, expression of activated RhoA and Rho-kinase induced MT disassembly. Conversely, thrombin-induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho-kinase mutants or by pharmacological inhibition of Rho-kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin-induced EC barrier dysfunction and indicate G-protein-dependent mechanisms of thrombin-induced MT alteration.—Birukova, A. A., Birukov, K. G., Smurova, K., Adyshev, D., Kaibuchi, K., Alieva, I., Garcia, J. G. N., Verin, A. D. Novel role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB J. 18, 1879 –1890 (2004)

ABSTRACT

Key Words: thrombin 䡠 G-proteins 䡠 Rho-kinase 䡠 tau 䡠 pulmonary endothelium The vascular barrier is critical to the maintenance of lung function, and thrombin-induced endothelial cell (EC) barrier compromise plays a major role in the pathogenesis of acute lung injury, alveolar flooding, hypoxemia, and respiratory failure (1– 4). Endothelial 0892-6638/04/0018-1879 © FASEB

permeability is regulated by a balance between contractile and tethering forces imposed by cytoskeletal elements (actin filaments, microtubules) and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions) (2, 5). Cross-talk between microtubues (MT) and actin cytoskeleton is essential for regulation of cell dynamics such as migration, locomotion, cytokinesis, and determination of cell polarity (6, 7). Recent studies suggest direct involvement of MT in regulation of endothelial integrity and wound repair (8 –10), as disassembly of MT by MT inhibitors nocodazole and vinblastine results in rearrangement of actin cytoskeleton, increased stress fiber formation, cell contraction, and permeability (8, 9, 11). These effects are associated with activation of small GTPase Rho and its effector Rho-kinase (8, 12, 13) and can be antagonized by cell pretreatment with MT stabilizing agent taxol (9, 12). Thrombin-induced endothelial barrier dysfunction is associated with actin stress fiber formation and actomyosin contraction (2, 5, 14), but the role of MT in thrombin-induced barrier dysfunction has not been yet investigated. Cellular responses to thrombin are mediated by protease-activated receptor PAR1 (15) coupled to heterotrimeric GTP binding proteins G12, G13, Gi, and Gq (16, 17). Thrombin effects on intracellular signaling mediated by PAR1 receptor may be mimicked by thrombin receptor activator oligopeptide with amino acid sequence SFLLRN (18). While the link between MT and thrombin-mediated signaling remains unexplored, recent reports demonstrated the unique ability of tubulin to regulate G-proteinmediated signaling through binding and hydrolysis of GTP. (19, 20). Thrombin-induced signaling mechanisms include G␣12/13-dependent activation of small GTPase Rho mediated by G␣12/13-interacting Rhospecific guanosine nucleotide exchange factor (GEF) 1 Correspondence: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5200 Eastern Ave., MFL Center Tower 660, Baltimore, MD 21224, USA. E-mail: [email protected] doi: 10.1096/fj.04-2328com

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p115RhoGEF (21). The Rho-dependent pathway is directly involved in thrombin-induced alteration of EC barrier properties and endothelial barrier dysfunction (3, 4, 22, 23), and emerging reports suggest that Rho may cause destabilization of MT network in part via Rho kinase-mediated phosphorylation of MT-associated tau proteins, which induces tau dissociation and destabilization of MT (24, 25). Thus, previous findings suggest an essential role for heterotrimeric and small G-proteins in the regulation of MT integrity, which may represent a novel molecular mechanism of thrombininduced EC barrier dysfunction. In this work we studied involvement of MT network in thrombin-induced EC barrier dysfunction and investigated novel functional interactions between heterotrimeric G-proteins G12 and G13, guanosine nucleotide exchange factor p115RhoGEF, small GTPase Rho, and MT network. These interactions may integrate the Rho-GTPase pathway with MT and actin cytoskeletal remodeling in endothelial cells in response to edemagenic agents.

MATERIALS AND METHODS Reagents Texas Red-conjugated phalloidin, Alexa Flour 488, and Alexa Flour 594 secondary antibodies were purchased form Molecular Probes (Eugene, OR, USA). ␤-Tubulin antibody was purchased from Covance Inc. (Berkeley, CA, USA). Acetylated tubulin antibody was purchased from Accurate Chemical and Scientific Corporation (Westbury, NY, USA). Site-specific phospho-Ser262tau and pan-tau antibodies were from Biosource International (Camarillo, CA, USA). Phospho-Ser409-tau polyclonal antibody has been described (26). RhoA, HA-tag and c-Myc-tag antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), diphospho-MLC antibody was obtained from Cell Signaling (Beverly, MA, USA), phospho-MYPT antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA), and Rhokinase specific inhibitor Y27632 was obtained from Calbiochem (La Jolla, CA, USA). Thrombin receptor activator for peptide 6 (TRAP-6) with amino acid sequence SFLLRN was obtained from AnaSpec (San Jose, CA, USA). Unless specified, biochemical reagents were obtained from Sigma (St. Louis, MO, USA). Cell culture Human pulmonary artery endothelial cells (HPAEC) were obtained from Cambrex (Walkersville, MD, USA) and used at passages 6 –10. Expression plasmids and transfection protocol Plasmids encoding constitutively active (CA)-RhoA (V14Rho) and CA-Rho-kinase (Rho-kinase/CAT) and dominant negative (DN)-Rho (N19Rho) and (DN)-Rho-kinase (a RB-PH(TT) mutant, the C-terminal fragment of Rho-kinase mutated at Rho binding sites), have been described (27, 28). Constitutively active mutants G␣12-Q229L and G␣13-Q226L, as well as p115RhoGEF and its negative regulator p115RhoGEF-specific RGS vector, were kindly provided by Dr. Voyno-Yasenetskaya (29, 30) and used for transient transfections. Briefly, EC grown in 12-well plates at 70% confluence were incubated 1880

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with 1 mL of OPTI-MEM medium containing 1 ␮g DNA and 3 ␮L of Fugene 6 (Boehringer Mannheim-Roche, Indianapolis, IN, USA) for 4 h in CO2 incubator at 37°C. After washing (EGM-2⫹10% FCS), cells were incubated an additional 24 h and used for experiments with thrombin stimulation. Control transfections were performed with empty vectors. Depletion of endogenous G␣i2, G␣q, G␣12, and G␣13 in EC To reduce the content of endogenous G␣12, G␣i2, G␣13, and G␣q proteins HPAEC were treated with G␣i2 or G␣q-specific small interfering RNA (siRNA) duplex oligonucletides, which guide sequence-specific degradation of the homologous mRNA (31). Predesigned siRNA standard purity was ordered from Ambion, Inc., Austin, Texas in purified, desalted, deprotected, and annealed double strand form. The following 21 base pair duplexes of siRNA were used: for G␣i2: sense 5⬘-GGUGAAGUUGCUGCUGUUGtt-3⬘ and antisense 5⬘-CAACAGCAGCAACUUCACCtc-3⬘, for G␣q: sense 5⬘-GGAGAGAGUGGCAAGAGUAtt-3⬘ and antisense 5⬘-UACUCUUGCCACUCUCUCCtg3⬘, for G␣12: sense 5⬘-GGGCUCAAGGGUUCUUGUUtt-3⬘ and antisense 5⬘-AACAAGAACCCUUGAGCCCtt-3⬘, for G␣13: sense 5⬘-GGAGAUCGACAAAUGCCUGtt-3⬘ and antisense 5⬘-CAGGCAUUUGUCGAUCUCCtt-3⬘. Nonspecific, nonsilencing FIluciferase GL2 duplex fluorescently labeled on the sense strand with 5⬘-fluorescein (Dharmacon Research, Lafayette, CO, USA) was used as a control treatment. HPAEC were grown to 70% confluence and the transfection of siRNA (final concentration 100 nM) was performed using GeneSilencerTM transfection reagent (Gene Therapy Systems, San Diego, CA, USA) according to manufacturer’s protocol. Forty-eight hours later, cells were used to measure transendothelial electrical resistance or for Western blot analysis of siRNA-induced specific G-protein depletion as described below. Introduction of C3 exoenzyme into the cells Introduction of C3 exoenzyme into the cells was performed using lipofectamine-facilitated transfer, as described (32). Immunofluorescent staining Endothelial cells grown on glass coverslips were fixed after agonist treatment in 1.5% glutar aldehyde solution in PBS for 10 min at room temperature, washed three times with PBS, permeabilized with 0.2% triton X-100 in PBS for 60 min at room temperature, and blocked with 1% sodium borohydrite in PBS three times for 10 min. Incubation with antibody of interest was performed in blocking solution (2% BSA in PBS) for 1 h at room temperature, followed by staining with either Alexa 488- or Alexa 594-conjugated secondary antibodies (Molecular Probes). Actin filaments were stained with Texas Red-conjugated phalloidin (Molecular Probes) for 1 h at room temperature. After immunostaining, the glass slides were prepared using mounting medium (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) and analyzed using Nikon video-imaging system (Nikon Instech Co., Japan) consisting of an inverted microscope Nikon Eclipse TE300 with epi-fluorescence module using 60XA/1.40 oil objective connected to SPOT RT monochrome digital camera (temperature of 37°C) and image processor (Diagnostic Instruments, Sterling Heights, MI, USA). The images were acquired using SPOT 3.5 acquisition software (Diagnostic Instruments) and processed with Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA) and Adobe Illustrator CS (Adobe Systems) software.

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Image analysis of gap formation, stress fiber formation, and MT structure Quantitative analysis of assembled MT, paracellular gap and stress fiber formation was performed as described (9, 22). Texas Red-stained EC monolayers stimulated with either thrombin or vehicle were viewed under microscope using 60XA/1.40 objective and images were captured as described above. The 16-bit images were analyzed using MetaVue 4.6 (Universal Imaging, Downington, PA, USA). Images were differentially segmented between gaps (black) and cells (highest gray value) based on image gray scale levels. The gap formation was expressed as a ratio of the gap area to the area of the whole image. For assessment of stress fiber formation, actin fibers were marked out and the ratio to the cell area covered by stress fibers to the whole cell area was determined. At least 20 microscopic fields for each experimental condition were analyzed. Similar technique was used to monitor MT assembly. The values were statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago, IL, USA) software. Immunoblotting Protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with specific antibodies as described (8). Immunoreactive proteins were detected using enhanced chemiluminescent detection system according to the manufacturer’s protocol (Amersham, Little Chalfont, UK), and intensities of immunoreactive protein bands were quantified using Image Quant software. Isolation of microtubules and MAPs Cells grown on 100 mm Petri dishes were stimulated with either vehicle (media) or thrombin. Separation of MT-enriched, cytoskeletal (CSK), and cytosolic (CSL) fractions was performed as described elsewhere (33). Supernatants containing cytosolic proteins and pellets containing particulate fraction were solubilized in 3⫻ SDS sample buffer; specific protein content in cytosolic and particulate fractions was analyzed by Western immunoblotting. Measurement of transendothelial electrical resistance (TER) Cellular barrier properties were measured using electrical cell substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY, USA). HPAEC were seeded onto plates with small gold electrodes (10⫺4 cm2) and measurements of transendothelial electrical resistance across confluent HPAEC monolayers were performed as described elsewhere (8, 22, 34). Rho activation assay Rho activation in EC culture was analyzed using a Rho assay kit available from Upstate Biotechnology as described (22, 34). Statistical analysis Results are expressed as means ⫾ sd of three to five independent experiments. Stimulated samples were compared with controls by unpaired Student’s t test. For multiple group comparisons, one-way ANOVA followed by the post hoc Fisher’s test was used. P ⬍ 0.05 was considered statistically significant.

RESULTS Involvement of MT in thrombin-induced pulmonary endothelial cell barrier dysfunction Measurements of transendothelial electrical resistance (TER) reflecting endothelial monolayer permeability changes showed that thrombin-induced TER decline was partially attenuated by MT stabilization achieved by pretreatment of human pulmonary artery endothelial cells (HPAEC) with taxol (5 ␮M, 1 h) before thrombin (50 nM) challenge (Fig. 1A). EC treatment with thrombin receptor activator oligopeptide (TRAP-6) with amino acid sequence SFLLRN that binds to and activates PAR1 receptor (18) caused TER decline, also attenuated by taxol pretreatment (Fig. 1B). These results strongly suggest barrier-protective effects of MT stabilization on thrombin-induced HPAEC permeability. Immunofluorescent detection of F-actin and quantitative analysis of paracellular gap formation in agoniststimulated HPAEC monolayers indicate that MT stabilization by taxol caused a significant attenuation of thrombin-induced stress fiber formation (Fig. 1C, D) and gap formation (Fig. 1E), and consistent with preservation of HPAEC barrier properties (Fig. 1A, B). Immunoflurorescent analysis of MT structure indicated significant disassembly of peripheral MT network in HPAEC upon thrombin (50 nM, 15 min) stimulation (Fig. 2A), which was confirmed by quantitation of assembled MT pools in control and thrombin-stimulated HPAEC, as described in Materials and Methods (Fig. 2B). Because stable MT pools undergo posttranslational modifications such as acetylation and detyrosination (35) and these modifications may reflect stability of MT network under particular conditions (36 –38), we next analyzed a pool of acetylated MT in thrombin-stimulated HPAEC. Thrombin treatment significantly decreased the amount of acetylated microtubules, as detected by Western blot analysis and immunofluorescent staining (Fig. 2C and inset), and further substantiated by quantitative analysis of immunofluorescent images (Fig. 2D). Together, these results reflect increased MT disassembly upon thrombin stimulation. Identification of heterotrimeric G-proteins involved in thrombin-induced EC barrier dysfunction Because thrombin receptor PAR1 is coupled to G12, G13, Gi, and Gq proteins (16), we tested the effects of specific G-protein depletion on thrombin-induced signal transduction and barrier dysfunction using siRNAmediated knockdown of G␣ subunits. Depletion of G␣12 and G␣13 subunits partially attenuated TER decline induced by thrombin stimulation (Fig. 3A), whereas depletion of G␣i and G␣q (Fig. 3B) or treatment with nonspecific RNA duplex oligonucleotide (Fig. 3A, B) did not affect thrombin-induced TER decline. Depletion of G␣12, G␣13, G␣i2, and G␣q by treatment with corresponding siRNA was confirmed by Western blots with the appropriate antibody (Fig. 3C).

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Figure 1. Role of MT in thrombin-induced EC barrier dysfunction. A) Effect of taxol pretreatment on thrombin-induced changes in TER. HPAEC were pretreated with vehicle (0.1% DMSO) or taxol (5 ␮M, 1 h) followed by thrombin stimulation (50 nM) in the time marked by arrow. B) Effect of taxol pretreatment on TER changes induced by thrombin receptor activator peptide TRAP-6. HPAEC were pretreated with vehicle (0.1% DMSO) or taxol (5 ␮M, 1 h) followed by TRAP-6 stimulation (10 ␮M) in the time marked by arrow. C) Effect of taxol pretreatment on thrombin-induced actin remodeling and paracellular gap formation. Confluent HPAEC monolayers grown on coverslips were preincubated with vehicle (0.1% DMSO) or taxol (5 ␮M, 1 h) followed by thrombin stimulation (50 nM, 15 min). F-actin was visualized by staining with Texas Red phalloidin. Representative results of 3 independent experiments. Bar ⫽ 10 ␮m. D) Stress fiber formation induced by thrombin was assessed using morphometric image analysis, as described in Materials and Methods. Data are expressed as a ratio of the intracellular area occupied by stress fibers to the whole cell area and represent results of 3 independent experiments. *P ⬍ 0.05. E) Gap formation induced by thrombin. Data are expressed as a ratio of the gap area to the area of the whole image and represent results of 3 independent experiments. *P ⬍ 0.05.

Besides the effects on thrombin-induced HPAEC permeability, protein depletion of G␣12 and G␣13 attenuated thrombin-induced MLC phosphorylation whereas depletion of G␣i and G␣q was without effect (Fig. 3D). Effect of activated G12/13 on MT and actin cytoskeletal arrangement To test the involvement of G12/13 in thrombin-induced MT remodeling and actin stress fiber formation,

we used ectopic expression of HA-tagged activated G␣12 and G␣13 subunits. Transfected cells (Fig. 4A) were subjected to double immunofluorescent staining to visualize ␤-tubulin and HA-tagged activated G␣12 (Fig. 4A, left panels) or F-actin and HA-tagged G␣12 (Fig. 4A, right panels). Similarly, cells were transfected with HA-tagged activated G␣13 (Fig. 4B) and subjected to staining for ␤-tubulin and HA-tagged G␣13 (Fig. 4B, left panels) or F-actin and HA-tagged G␣13 (Fig. 4B, right panels). Figure 4C, D depicts results of quantitative analysis of assembled MT and actin stress fibers in control

Figure 2. Effect of thrombin on MT structure in human pulmonary EC. A) Cells grown on coverslips were stimulated with thrombin (50 nM, 15 min) followed by immunofluorescent staining with antibody against ␤-tubulin. B) Quantitation of thrombin-induced microtubule disassembly was performed using morphometric analysis of HPAEC monolayers stained with anti-␤-tubulin antibody, as described in Materials and Methods. Data are expressed as a ratio of the cell area covered by assembled microtubules to the whole cell area. Results are mean ⫾ sd of 3 independent experiments. *P ⬍ 0.05. C) After stimulation with thrombin (50 nM, 15 min), cells were stained with antibody against acetylated tubulin. Inset shows results of Western blot detection of acetylated tubulin in control (left band) and thrombin-stimulated (right band) HPAEC. Bar ⫽ 10 ␮m. B) Quantitation of thrombin effects on the pool of acetylated tubulin. Morphometric analysis of HPAEC monolayers stained with antibody against acetylated tubulin was performed, as described in Materials and Methods. Data are expressed as a ratio of the cell area covered by microtubules containing acetylated tubulin to the whole cell area. Results are mean ⫾ sd of 3 independent experiments. *P ⬍ 0.05. 1882

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Figure 3. Effect of inhibition of G␣12, G␣13, G␣i, and G␣q expression by siRNA on thrombin-induced permeability changes and MLC phosphorylation. A) HPAEC grown on gold microelectrodes were incubated with siRNA to G␣12 and G␣13 or treated with nonspecific RNA duplex oligonucelotide as described in Materials and Methods and used for TER measurements. Cells were stimulated with thrombin (50 nM) in the time marked by arrow. B) Cells grown on gold microelectrodes were incubated with siRNA to G␣i and G␣q or treated with nonspecific RNA duplex oligonucelotide and used for TER measurements. Cells were stimulated with thrombin (50 nM) in the time marked by arrow. Shown are results of 3 independent experiments. C) Cells grown in D35 culture plates were incubated with siRNA to G␣12, G␣13, G␣i2, G␣q or treated with nonspecific RNA duplex as described in Materials and Methods, and protein depletion of G␣ subunits was examined by immunoblotting with corresponding antibody. Control staining was performed with anti-␤-tubulin antibody. D) HPAEC incubated with indicated siRNA were stimulated with thrombin (50 nM, 15 min), and phospho-MLC levels were determined by Western blot of cell lysates, as described in Materials and Methods. Graph represents quantitation of MLC phosphorylation by scanning densitometry of the membranes and is expressed in relative density units (RDU). Results are mean ⫾ sd of 3 independent experiments. *P ⬍ 0.05.

HPAEC and in cells expressing activated G␣12 or G␣13 performed as described in Materials and Methods. We noticed that cells with the highest G␣12 or G␣13 expression levels detected by strong anti-HA-tag immunoreactivity became rounded and detached from the substrate. These cells were excluded from morphometric analysis, and only cells with modest HA-tag signal were analyzed. Results of these experiments demonstrate that overexpression of activated G␣12 and G␣13 subunits induced significant disassembly of MT network, with a loss of

fibrillar MT structure that was accompanied by dramatic stress fiber formation. Transfection of HPAEC with empty vectors did not affect MT or F-actin cytoskeletal structures (data not shown). Involvement of p115RhoGEF in thrombin induces MT rearrangement To evaluate a potential role of G␣12/13-coupled Rho-specific guanosine nucleotide exchange factor

Figure 4. Effect of expression of activated G␣12 and G␣13 on actin cytoskeleton and MT structure. A) HPAEC were transiently transfected with activated HA-tagged G␣12 mutant, as described in Materials and Methods and stained with ␤-tubulin and HA-tag antibody (left panels) or with Texas Red phalloidin and HA-tag antibody (right panels). B) HPAEC were transiently transfected with activated HA-tagged G␣13 mutant and stained with ␤-tubulin and HA-tag antibodies (left panels) or Texas Red phalloidin and HA-tag antibody (right panels). C) Morphometric analysis of MT disassembly induced by expression of activated G␣12 and G␣13. D) Morphometric analysis of stress fiber formation induced by expression of activated G␣12 and G␣13. Data are expressed as a ratio of the cell area covered by assembled MT or stress fibers to the whole cell area. Results are mean ⫾ sd of 3 independent experiments. *P ⬍ 0.05. Bar ⫽ 10 ␮m.


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p115RhoGEF in thrombin-induced MT alteration, we transiently transfected HPAEC with plasmid encoding wild-type p115RhoGEF, which results in constitutive activation of Rho-specific GDP/GTP exchange activity (39). Cells overexpressing p115RhoGEF were detected by immunofluorescent staining with myc-tag antibody. MT and F-actin structure was examined by staining with anti-␤-tubulin antibody and Texas Red phalloidin, respectively. Cells overexpressing p115RhoGEF revealed a significant disassembly of MT network (Fig. 5A and insets) but dramatically increased stress fiber formation (Fig. 5B). Guanosine nucleotide exchange activity of p115RhoGEF is negatively regulated by specific regulator of G-protein signaling (RGS) (39). We next examined effects of p115RhoGEF-RGS expression on MT disassembly and F-actin changes in human pulmonary EC induced by thrombin challenge (50 nM, 15 min).

Cells expressing p115RhoGEF-RGS (detected by antimyc-tag staining, shown by arrows) revealed preservation of peripheral MT network after thrombin challenge compared with nontransfected cells (Fig. 5C and insets). Expression of p115RhoGEF-RGS attenuated stress fiber formation induced by thrombin (Fig. 5D). These results show direct involvement of p115RhoGEF in thrombin-induced MT disassembly and actin remodeling. Role of small GTPase RhoA in thrombin-mediated alteration of MT structure HPAEC were transiently transfected with dominant negative RhoA mutant (N19Rho) and stimulated with thrombin (50 nM, 15 min), followed by double immu-

Figure 5. Involvement of p115RhoGEF in thrombin-induced regulation of MT dynamics. HPAEC transfected with plasmid encoding p115RhoGEF were stained with ␤-tubulin antibody (A) or Texas Red phalloidin (B). Cells expressing p115RhoGEF (shown by arrows) were detected by staining with myc-tag antibody (lower panels). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. C) HPAEC transfected with plasmid encoding p115RhoGEF-RGS were stimulated with thrombin (50 nM, 15 min, right panels) or left untreated (left panels), and MT structure was visualized by immunofluorescent staining with ␤-tubulin antibody. D) F-actin was visualized by staining with Texas Red phalloidin. Cells expressing p115RhoGEF (shown by arrows) were detected by staining with myc-tag antibody (lower panels). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. Shown are representative data of 3 independent experiments. Bar ⫽ 10 ␮m. 1884

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nofluorescent staining with antibody against ␤-tubulin (upper), to visualize MT network and with antibody against HA-tag (lower) to detect N19Rho-expressing cells. Expression of dominant negative Rho mutant did not affect MT structure in nonstimulated HPAEC cultures (Fig. 6A, left panels) but significantly attenuated thrombin-induced dissolution of peripheral MT network in transfected cells (shown by arrows) compared with nontransfected cells (Fig. 6A, right panels and insets). In contrast, expression of constitutively active RhoA mutant (V14Rho) promoted disassembly of peripheral MT network even without thrombin stimulation (Fig. 6B and insets), suggesting direct involvement of Rho in the regulation of MT network stability. To further investigate interactions between thrombin-induced Rho activation and MT disassembly, MT were stabilized by taxol pretreatment (5 ␮M, 1 h), and Rho activation was measured upon thrombin stimulation (50 nM, 15 min). Figure 6C shows that taxol pretreatment significantly attenuated thrombin-induced Rho activation. Consistent with these results, stabilization of MT network by taxol pretreatment (5 ␮M, 1 h) attenuated thrombin-induced phosphorylation of Rho/Rho-

kinase target MYPT and decreased phospho-MLC levels (Fig. 6D, E). Role of Rho-kinase in thrombin-induced alteration of MT structure To examine a role of Rho-kinase activity in control of MT integrity in quiescent and thrombin-challenged EC monolayers, cells were transiently transfected with plasmids encoding dominant negative and constitutively active Rho-kinase mutants. Cells expressing dominant negative Rho-kinase mutant [RB-PH(TT)] were further stimulated with thrombin (50 nM, 15 min). Double immunofluorescent staining for ␤-tubulin and myc-tag revealed a prominent protective effect of dominant negative Rho-kinase mutant on MT structure against thrombin-induced disassembly (Fig. 7A, right panels and insets). Cells overexpressing dominant negative Rho-kinase mutant are marked by arrows. In contrast to dominant negative Rho-kinase mutant, expression of constitutively active Rho-kinase (Rho-kinase-CA) induced MT disassembly in nonstimulated HPAEC (Fig.

Figure 6. Involvement of Rho in thrombin-induced regulation of MT dynamics. A) HPAEC transfected with plasmid encoding dominant negative Rho mutant (Rho-DN) were stimulated with thrombin (50 nM, 10 min, right panels) or left untreated (left panels), and MT structure was visualized by immunofluorescent staining with ␤-tubulin antibody. Cells expressing dominant negative Rho mutant (shown by arrows) were detected by staining with HA-tag antibody (lower panels). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. B) HPAEC transfected with plasmid encoding constitutively active Rho mutant (Rho-CA), and MT structure was visualized by immunofluorescent staining with ␤-tubulin antibody. Cells expressing Rho-CA (shown by arrows) were detected by staining with HA-tag antibody (lower panel). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. Shown are representative data of 3 independent experiments. Bar ⫽ 10 ␮m. C) HPAEC were preincubated with vehicle (0.1% DMSO) or taxol (5 ␮M, 1 h) followed by thrombin stimulation (50 nM, 15 min). Rho activity in cell lysates was measured using Rho activation pulldown assay, as described in Material and Methods. Lower panel depicts results of quantitative analysis of Rho activation expressed in relative density units (RDU) and performed by scanning densitometry of the membranes. D) Effect of taxol pretreatment on thrombin-induced MYPT phosphorylation was detected by immunoblotting with phospho-Thr850 MYPT1 antibody. E) Effect of taxol pretreatment on thrombin-induced MLC phosphorylation was detected by immunoblotting with diphospho-MLC antibody. Rearranged lanes from the same blot are outlined by vertical lines. The lower panels depict results of quantitative analysis of MLC phosphorylation and MYPT1 phosphorylation expressed in relative density units (RDU) and performed by scanning densitometry of the membranes. Results are representative of 3 independent experiments. MICROTUBULES AND THROMBIN-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION

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Figure 7. Involvement of Rho-kinase in thrombininduced regulation of MT dynamics. A) HPAEC transfected with plasmid encoding dominant negative Rho-kinase (Rho-kinase-DN) were stimulated with thrombin (50 nM, 10 min, right panels) or left untreated (left panels), and MT structure was visualized by immunofluorescent staining with ␤-tubulin antibody. Cells expressing dominant negative Rho mutant (shown by arrows) were detected by staining with myc-tag antibody (lower panels). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. B) HPAEC were transfected with plasmid encoding constitutively active Rho-kinase (Rhokinase-CA), and MT structure was visualized by immunofluorescent staining with ␤-tubulin antibody. Cells expressing Rho-CA (shown by arrows) were detected by staining with myc-tag antibody (lower panel). Magnified images (insets) show details of MT structure in transfected and nontransfected cells. Shown are representative data of 3 independent experiments. Bar ⫽ 10 ␮m.

7B). Consistent with these results, pharmacological inhibition of Rho-kinase by Y27632 (5 ␮M, 1 h) significantly attenuated MT disassembly in response to thrombin stimulation (50 nM, 15 min) (Fig. 8A). Quantitation of assembled MT (Fig. 8B) confirmed

protective effect of pharmacological inhibition of Rhokinase activity on the pool of assembled MT in thrombin-stimulated HPAEC. Pretreatment with Y27632 increased a pool of acetylated (stable) MT in thrombinstimulated HPAEC (Fig. 9C).

Figure 8. Effects of Rho-kinase inhibition on MT remodeling in thrombin-stimulated human pulmonary EC. A) HPAEC monolayers were preincubated with vehicle (0.1% DMSO) or Rho-kinase specific inhibitor Y27632 (5 ␮M, 1 h) followed by stimulation with thrombin (50 nM, 15 min). MT were visualized by immunofluorescent staining with ␤-tubulin antibody. Bar ⫽ 10 ␮m. B) Morphometric analysis of MT structure in thrombin-stimulated HPAEC with and without Y27632 pretreatment was assessed as described in Materials and Methods. Data are expressed as a ratio of the cell area covered by assembled microtubules to the whole cell area and represent results of 3 independent experiments. *P ⬍ 0.05. C) Effect of Y27632 and thrombin on the levels of acetylated tubulin was detected by immunoblotting with antibody against acetylated tubulin followed by membrane reprobing with ␤-tubulin antibody. Quantitative analysis of acetylated tubulin was performed by scanning densitometry of the membranes and expressed in relative density units (RDU) and. Results are representative of 3 independent experiments. *P ⬍ 0.05. 1886

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Figure 9. Subcellular distribution of tau and effect of thrombin on site-specific tau phosphorylation. A) Microtubule (MT), F-actin-enriched (CSK), and cytosolic (CSL) fractions were isolated from HPAEC, as described in Materials and Methods, and proteins of interest were detected by immunoblotting. Site-specific tau phosphorylation in fractions was detected using anti-phospho-Ser262 tau antibody. B) HPAEC were pretreated with C3 exotoxin (2.5 ␮g/mL), Y27632 (5 ␮M), or taxol (5 ␮M) followed by treatment with vehicle or thrombin (50 nM, 15 min), and site-specific tau phosphorylation was examined using phospho-Ser409 and phospho-Ser262 tau antibodies. Control membrane reprobing was performed with ␤-tubulin antibody. Quantitative analysis of phospho-Ser409 and phospho-Ser262 tau was performed by scanning densitometry of the membranes and expressed in relative density units (RDU). Results are representative of 3 independent experiments. *P ⬍ 0.05.

Effects of thrombin on tau phosphorylation Results of subcellular fractionation indicate noticeable tau expression levels in human endothelium and show that major pool of tau is associated with MT (Fig. 9A). Tau phosphorylated at Ser262 was mostly detected in cytosolic and membrane/actin cytoskeletal fractions, but not in MT fraction (Fig. 9A, lower panel). Thrombin stimulation of HPAEC induced tau phosphorylation at Ser262 and Ser409, another Rho-kinase phosphorylation site (26) (Fig. 9B). Consistent with biochemical analysis, immunofluorescent staining of thrombin-stimulated HPAEC monolayers revealed accumulation of phospho-Ser262 tau in submembrane compartment (data no shown), which correlated with disassembly of peripheral MT network in thrombin-stimulated cells. To further examine the involvement of the Rho/Rhokinase pathway and MT dynamics in thrombin-induced tau phosphorylation, HPAEC were pretreated with C3exotoxin, Y27632, or taxol before thrombin challenge. Figure 9B shows that thrombin induced tau phosphorylation at Ser262 and Ser409 and that inhibition of Rho/Rho-kinase pathway or MT stabilization with taxol significantly attenuated thrombin-induced tau phosphorylatioorn at Ser262 and Ser409.

DISCUSSION Extensive data have accumulated about the effects of physiological inflammatory and edemagenic agonists on EC actin cytoskeletal remodeling and barrier regulation. However, agonist-induced regulation of MT dynamics and cross-talk between MT and actin cytoskeleton are not well understood, nor has the involvement of MT dynamics in thrombin-induced permeability changes been described. We recently showed that EC barrier regulation is critically dependent on coordinated functioning of two components of cytoskeleton: microtubules and microfilaments (8, 9, 40). This study demonstrates for the first time that thrombin stimulation induces rapid disassembly of peripheral MT network, which has been linked to thrombin-induced EC

barrier compromise. These observations are consistent with previous reports demonstrating thrombin-induced decrease of dynamic MT pool in embryonic chick dorsal root ganglion neurons and attenuated response to thrombin in platelets isolated from ␤1-tubulin deficient mice (41, 42). Our results show that stabilization of MT network by taxol significantly attenuates thrombin-induced permeability increases, and thus further emphasize a critical role for MT dynamics in thrombinmediated barrier failure. Taxol is a well-characterized MT stabilizing agent widely used as chemotherapeutic drug. Taxol binds avidly to ␤-tubulin with Kd ⬃10 nM and shifts the dynamic equilibrium between disassembly and assembly of MT in a favor of assembly (35). Separate from its effects on microtubules, taxol can induce genes encoding proinflammatory cytokines (TNF-␣, interleukins, COX2) via interaction with LPS receptor complex containing CD18/CD11/TLR4 (43). No reports suggesting other effects of taxol on cell cultures independent of MT stabilization have been described. Consistent with this notion, our earlier studies and this work indicate that taxol alone neither activated Rho nor induced phosphorylation of Rho-kinase, MLC, or MYPT (9, 34) or affected HPAEC barrier properties (8, 9). When added alone, Taxol exhibit no effect on other signaling pathways such as c-Raf, p38, JNK, and Erk-1,2 MAP kinases (40, 44), PKA signaling (34), or phosphorylation of ␤-catenin, HSP 27, and CaD (A. Birukova, unpublished data). Thus, available information does not indicate any direct effects of taxol on signal transduction in pulmonary endothelial cells. Therefore, attenuation of thrombin-induced EC barrier dysfunction and Rho/Rho-kinase activation by taxol are most likely attributed to MT stabilizing properties of taxol. Thrombin stimulation triggers several signaling cascades in EC, which are initiated by PAR1-mediated activation of heterotrimeric G-proteins (15, 16, 45). Effects of activated G␣ subunits on MT dynamics have been shown in cells stimulated with nerve growth factor (46). Our experiments using siRNA-based depletion of endogenous G␣12, G␣13, G␣i2, and G␣q proteins, and ectopic expression of G␣12 and G␣13 strongly suggest


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an involvement of G12/13 in regulating MT dynamics, and show disassembly of peripheral MT network and increased stress fiber formation induced by activated G␣12 and G␣13 subunits. We speculate that alteration of MT structure by activated G␣ subunits may occur via direct interaction between MT and G-proteins as well as by G12/G13-driven activation of Rho-dependent signaling cascade. G12/13-mediated Rho activation may be achieved via G12/13-dependent guanosine nucleotide exchange factor p115RhoGEF (21). Our results show that increased p115RhoGEF expression, which induces GDP/ GTP exchange and activation of Rho GTPase, caused disassembly of peripheral MT whereas a negative regulator of p115RhoGEF activity (p115RhoGEF-RGS) preserved MT network against thrombin-induced MT disassembly. The role of Rho-dependent pathway in thrombin-induced EC barrier dysfunction has been described and is generally associated with remodeling of actin cytoskeleton and cell adhesive structures (3, 4, 22, 23), whereas potential cross-talk between Rhomediated signaling and MT network in agonist-induced EC barrier regulation remains virtually unexplored. Consistent with the proposed role for Rho-mediated pathway in thrombin-induced regulation of MT dynamics, overexpression of activated Rho or its activated downstream target Rho-kinase induced MT disassembly in pulmonary EC. Recent reports further support the role of Rho in regulation of MT dynamics and demonstrated that injection of active Rho in fibroblasts or overexpression of wild-type Rho in neuroblastoma cells induces MT disassembly (24, 26). This study demonstrates that Rho activation in human pulmonary EC causes MT disassembly and stress fiber formation similar to neuronal cells. In contrast to endothelial and neuroblastoma cells, Rho activation in NIH3T3 fibroblasts induces stabilization of a subset of microtubules in the lamella via another Rho effector, mDia (38). Thus, our results and published data emphasize the importance of cell-specific interactions between Rho and microtubules. A potential mechanism of Rho-mediated MT disassembly involves Rho-kinase-mediated phosphorylation of the MT-associated protein tau, which results in tau dissociation from MT and MT destabilization (25). Tau was originally described in the neuronal cells, but several reports have demonstrated its expression in non-neuronal cells such as fibroblasts and lymphocytes (47, 48). It was described that phosphorylation of tau by several kinases decreases its capacity to bind MT, which leads to MT disassembly (49, 50). Thrombin causes hyperphosphorylation of tau and reduces the capacity to promote MT assembly in neurites (51, 52). MTassociated proteins tau and MAP2 have been described recently as novel substrates of Rho-kinase and MYPT1, and tau amino acid residues (Thr245, Thr377, Ser262, and Ser409) have been identified as Rho kinase-mediated phosphorylation sites (26). Moreover, it was shown that Rho-kinase-mediated tau phosphorylation decreased MT assembly (26). Consistent with these find1888

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ings, we show thrombin-induced phosphorylation of tau at Ser262 and Ser409 that was attenuated by inhibition of Rho, Rho-kinase, or MT stabilization with taxol. On the other hand, expression of dominant negative Rho-kinase and pharmacological inhibition of Rhokinase by Y27632 protected MT from thrombin-induced depolymerization (Figs. 7, 8). Thus, taken together these results strongly suggest the involvement of tau-dependent mechanisms in thrombin-mediated regulation of MT assembly. Our results indicate that thrombin-induced disassembly of peripheral MT network was mediated by Rho activation and attenuated by inhibition of the Rho/ Rho-kinase pathway using C3-exotoxin, Rho-kinase inhibitor Y27632, dominant negative Rho and Rho-kinase mutants, or a negative regulator of p115-RhoGEF, RGS. In turn, MT stabilization by taxol partially attenuated thrombin-induced Rho/Rho-kinase activation. Thus, our data suggest reciprocal relations between Rho/ Rho-kinase signaling pathway and MT dynamics. Recent reports demonstrate that Rho GTPase activity may be regulated by MT-associated guanosine nucleotide exchange factors GEF-H1/Lfc and p190RhoGEF (53– 55). In MT-bound form, guanosine-exchange activity of GEF-H1 is suppressed whereas release of GEF-H1 from MT dramatically increases its Rho-specific GEF activity (55). Thus, published data and results of this study suggest a novel potential mechanism of secondary Rho activation by release of MT-bound Rho-specific GEFs upon thrombin-induced disassembly of peripheral MT network. Studies are under way in our laboratory to clarify a role of MT-bound GEFs in positive feedback regulation of Rho activity by thrombin-induced MT disassembly. Based on our findings and published reports, we speculate that thrombin-induced microtubule disassembly and barrier dysfunction may occur in two phases. The first includes thrombin-induced engagement of heterotrimeric G-proteins, which activate Rho via p115RhoGEF-dependent mechanism. Activation of Rho effector Rho-kinase results in activation of MLC phosphorylation, which triggers actomyosin contractile mechanisms of EC barrier dysfunction. Rho kinase activation induces phosphorylation of microtubule-associated cytoskeletal targets (tau proteins) and causes dissociation of phospho-tau from microtubules and microtubule disassembly. In the second phase, MT disassembly may result in the release of MT-associated GEFs such as p190RhoGEF and GEF-H1. These events induce a second wave of Rho activation resulting in more sustained cytoskeletal changes and EC barrier dysfunction. These studies characterize for the first time a specific role for heterotrimeric G-proteins in the thrombin-induced MT rearrangement and permeability and define important structure/function relationships between heterotrimeric G-proteins, Rho GTPase, microtubule reorganization, and thrombin-induced barrier compromise. The examination of cross-talk between actin and microtubule cytoskeletons provides novel information about

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molecular mechanisms of EC signaling and morphological changes relevant to vascular barrier regulation, EC migration and angiogenesis.

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This work was supported by grants from National Heart, Lung, and Blood Institutes (HL67307, HL68062, and HL58064). The authors thank Dr. Voyno-Yasenetskaya, Chicago, IL, USA, for providing of G␣12, G␣13, p115RhoGEF, and p115RhoGEF-RGS constructs. The authors also thank Nurgul Moldobaeva for superb technical assistance.

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Received for publication May 13, 2004. Accepted for publication August 25, 2004.

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