FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.06-7660com.
 (The FASEB Journal. 2007;21:2776-2786.)
© 2007 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.06-7660comv1
21/11/2776    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Birukova, A. A.
Right arrow Articles by Birukov, K. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birukova, A. A.
Right arrow Articles by Birukov, K. G.

HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway

Anna A. Birukova1, Elena Alekseeva, Arsen Mikaelyan and Konstantin G. Birukov

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois, USA

1Correspondence: Section of Pulmonary and Critical Medicine, Department of Medicine, University of Chicago, 929 East 57th St., CIS Bldg., W410, Chicago, IL 60637. E-mail: abirukov{at}medicine.bsd.uchicago.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier-protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac-GTPase and Rac-specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin-induced hyperpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin-induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115-RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac-dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF-induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against thrombin-induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac-dependent mechanism of HGF-induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeability via dynamic interactions between Rho- and Tiam1/Rac-mediated pathways. —Birukova, A., Alekseeva, E., Mikaelyan, A., and Birukov, K. G. HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway.


Key Words: small GTPases • pulmonary endothelium • actin • cytoskeleton


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEPATOCYTE GROWTH FACTOR (HGF) is a multifunctional mesenchyme-derived pleiotropic factor secreted by several cell types. Along with other bioactive substances, it appears in lung circulation under pathological conditions, such as acute lung injury, sepsis, lung inflammation, and ventilator induced lung injury (VILI), and regulates a number of biological events such as cell mitogenesis, morphogenesis, organogenesis, and cell survival (1 2 3) . Novel therapeutic strategies using HGF to fight cardiovascular diseases have been suggested (1 2 3 4 5) . HGF elicits potent angiogenic activities (6 , 7) and exhibits sustained barrier protective effects on human pulmonary endothelial cells (EC) (8) . Barrier protective effects of HGF against vascular leak have also been observed in the cerebral endothelium (9) . We have previously shown that HGF stimulates multiple signaling pathways, including small GTPase Rac, mitogen-activated protein (MAP) kinases Erk1/2 and p38, protein kinase C, and phosphatidylinositol-3-kinase (PI3-kinase) and its downstream effector GSK-3ß (8) . HGF-induced barrier protective effects on the pulmonary endothelium have been associated with remodeling of the actin cytoskeleton as well as the increased interaction between adherens junction proteins {alpha}/ß-catenin and VE-cadherin (8) . However, the precise mechanisms of HGF-mediated EC barrier-protective response are not well understood.

Previous studies have proposed and characterized a working model of EC barrier regulation (10 11 12 13 14) . According to this model, formation of paracellular gaps is regulated by the balance of competing contractile forces imposed by the actomyosin cytoskeleton, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces imposed by focal adhesions and adherens junctions, which together regulate cell shape changes (10 , 15) . Increased EC barrier permeability in response to agonist stimulation is associated with dramatic cytoskeletal rearrangement, activation of EC contraction, paracellular gap formation, and phosphorylation of regulatory myosin light chains (MLC) (16 17 18) . Our previous studies (12 , 16 , 19 , 20) and reports by others (21 22 23) have shown the essential role of small GTPases Rho and Rac in endothelial barrier regulation. Barrier disruptive agonists, such as thrombin, transforming growth factor-ß1, and tumor necrosis factor-{alpha}, activate Rho and Rho-associated kinase, which may directly catalyze MLC phosphorylation or act indirectly by inactivating myosin light chain phosphatase (16 , 17 , 24 25 26) . These events trigger actomyosin contraction resulting in EC barrier dysfunction. In turn, EC barrier enhancement induced by barrier protective factors, such as platelet-derived phospholipid sphingosine-1 phosphate, oxidized phospholipids, HGF, or simvastatin also requires actomyosin remodeling, including formation of a prominent cortical actin rim, disappearance of central stress fibers, and peripheral accumulation of phosphorylated MLC, which is regulated by Rac-dependent mechanisms (8 , 12 , 20 , 27) . Taken together, these studies suggest that the balance between Rho- and Rac-mediated signaling may be a critical component of EC barrier regulation. Rho and Rac GTPases act as molecular switches, cycling between active GTP-bound and inactive GDP-bound states (28 29 30) . This cycling is regulated by guanine nucleotide exchange factors (GEF), which facilitate exchange of GDP for GTP. Rac-specific nucleotide exchange factor Tiam1 belongs to the Dbl family of GEFs, and its nucleotide exchange activity is regulated by diverse mechanisms, including PI3-kinase-dependent, tyrosine kinase-dependent, protein kinase A-dependent, and Epac-Rap1-dependent pathways (29 , 31 32 33 34) . Tiam1 is directly involved in Rac-mediated EC barrier protection induced by sphingosine-1 phosphate (35) .

In this study, we have investigated the role of Rac-dependent mechanisms in HGF-mediated endothelial barrier protective responses and linked these effects to the regulation of Rho-mediated signaling in the model of thrombin-induced EC barrier dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
Human HGF was obtained from R&D Systems (Minneapolis, MN, USA). Texas Red-conjugated phalloidin and Alexa Flour 488 were purchased form Molecular Probes (Eugene, OR, USA). Rac1 and ß-catenin antibodies were purchased from BD Transduction Laboratories (San Diego, CA, USA), Tiam1 and RhoA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), cortactin antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA), and HRP-linked anti-mouse and -rabbit IgG, diphospho-MLC, phospho-p21-activated kinase 1 (PAK1), and PAK1 antibodies were obtained from Cell Signaling (Beverly, MA, USA). The Rac inhibitor NSC-23766 PI3-kinase inhibitor LY294002 and the tyrosine kinase inhibitor genistein were purchased from Calbiochem (La Jolla, CA, USA). Unless otherwise specified, all biochemical reagents, including MLC antibodies, were obtained from Sigma (St. Louis, MO, USA). Human pulmonary artery endothelial cells (HPAEC) were obtained from Cambrex (East Rutherford, NJ, USA). The cells were maintained in complete culture medium consisting of Clonetics EBM basic medium, which contains 10% fetal bovine serum and is supplemented with a set of nonessential amino acids, endothelial cell growth factors, and 100 U/ml penicillin/streptomycin (Cambrex). HPAEC were incubated at 37°C in a humidified 5% CO2 incubator and used for experiments at passages 5–9.

Depletion of Rac1 and Tiam1 in EC
To reduce the content of endogenous Tiam1, HPAEC were treated with specific silencing RNA (siRNA) duplexes, which guide sequence-specific degradation of homologous mRNA (36) . Predesigned Rac1-specific siRNA has been previously described and was ordered from Ambion (Austin, TX, USA) in purified, desalted, deprotected, annealed double-strand form (19 , 20) . Specific siRNAs to Tiam1 (sense 5'-GAGCAAGCGAAGGAGCAGGTTTTCTTT-3' and antisense 5'-AGAAAACCTGCTCCTTCGCTTGCTCTT-3') (37) have been previously described and were purchased from Invitrogen (Carlsbad, CA, USA) in ready to use, desalted, deprotected, annealed double-strand form. Non-specific, nontargeting siRNA duplex #1 (Dharmacon Research, Lafayette, CO, USA) was used as a control treatment. HPAEC were grown to 70% confluence, and siRNA transfection (final concentration=50 nM) was then performed using DharmaFECT1 transfection reagent (Dharmacon) according to manufacturer's protocol. Seventy-two hours after transfection, cells were harvested and used for experiments.

Rho and Rac activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology (Lake Placid, NY, USA), as we have previously described (20) . In brief, after stimulation, cell lysates were collected, and GTP-bound Rac or Rho was captured using pull-down assays with immobilized PAK1-PBD and Rhotekin-RBD, respectively, according to the manufacturer's protocols. The levels of activated small GTPases as well as total Rac and Rho content were evaluated by Western blot analysis and quantified by scanning densitometry of autoradiography films. The levels of activated proteins Rac or Rho were normalized to total Rac or Rho level for densitometry evaluations.

Measurement of transendothelial electrical resistance
Measurements of transendothelial electrical resistance (TER) across confluent HPAEC monolayers were performed using the electrical cell-substrate impedance sensing system (ECIS; Applied Biophysics, Troy, NY, USA) as described previously (20 , 38) .

Immunofluorescent staining
Endothelial monolayers plated on glass coverslips were treated with the agonist of interest, fixed in 3.7% formaldehyde solution in PBS for 10 min at 4°C, washed three times with PBS, permeabilized with 0.1% Rriton X-100 in PBS-Tween (PBST) for 30 min at room temperature, and blocked with 2% BSA in PBST for 30 min. Incubation with ß-catenin or cortactin antibodies was performed in blocking solution (2% BSA in PBST) for 1 h at room temperature followed by staining with Alexa 488-conjugated secondary antibodies. Actin filaments were stained with Texas Red- conjugated phalloidin. After immunostaining, slides were analyzed using a Nikon video imaging system (Nikon Instech, Tokyo, Japan) as described elsewhere (16 , 20) .

Immunoblotting
After stimulation, cells were lysed, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with specific antibodies as described previously (39) . Intensities of immunoreactive protein bands were quantified using Image Quant software.

Statistical analysis
Results are expressed as means ± SD of three to five independent experiments. Stimulated samples were compared to controls by unpaired Student's t tests. For multiple-group comparisons, a one-way ANOVA, followed by the post hoc Fisher's test, were used. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HGF attenuates thrombin-induced pulmonary EC barrier dysfunction
In this study, we tested the effects of HGF on EC barrier dysfunction caused by proinflammatory agonist thrombin. Human pulmonary EC monolayers we treated with HGF (30 ng/ml) or thrombin (0.5 U/ml), or stimulated with HGF for 15 min followed by thrombin challenge, after which changes in electrical resistance across the EC monolayers (TER) were monitored over time. EC pretreatment with HGF significantly attenuated thrombin-induced permeability increase, as was reflected by decline in TER and dramatically accelerated barrier recovery in thrombin-stimulated EC (Fig. 1 A). Furthermore, HGF added after the thrombin challenge (0.5 U/ml, 15 min) also accelerated EC barrier restoration, as was characterized by more rapid recovery of TER to basal levels (Fig. 1B ). The protective effects of HGF against thrombin-induced EC permeability changes were then linked to effects on cytoskeletal remodeling. EC pretreated with HGF or vehicle were challenged with thrombin (15 or 50 min), and the effects of HGF on thrombin-induced actin stress fiber formation, paracellular gap formation, and adherens junction disruption were examined by double immunofluorescent staining with Texas Red-conjugated phalloidin and ß-catenin antibodies. Consistent with the previous observations (8) , HGF stimulation increased peripheral F-actin staining and also resulted in partial disappearance of central stress fibers (Fig. 2 A, top). Remarkably, HGF dramatically attenuated paracellular gap formation and noticeably decreased levels of actin stress fiber formation observed during the acute-phase of thrombin stimulation (15 min; Fig. 2A , middle). It also promoted monolayer restoration, as was characterized by the nearly complete disappearance of stress fibers and paracellular gaps during the recovery phase of thrombin stimulation (50 min) as opposed to EC monolayers stimulated with thrombin alone (Fig. 2A , bottom). Analysis of adherens junction remodeling judged by immunofluorescent staining with antibodies to ß-catenin further demonstrated the protective effects of HGF against thrombin-induced disruption of EC monolayer integrity. Moreover, HGF promoted quick re-establishment of adherens junctions during EC recovery after thrombin challenge (Fig. 2B ).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. Effect of HGF on thrombin-induced EC hyperpermeability. A) Human pulmonary EC were treated with HGF (30 ng/ml, marked by first arrow). At time point indicated by second arrow, cells were stimulated with thrombin (0.5 U/ml), and TER was monitored over 2 h. B) EC were stimulated with thrombin (0.5 U/ml, marked by first arrow) followed by addition of HGF (30 ng/ml, marked by second arrow), and TER was monitored over 2 h. Shown are cumulative data of 5 independent experiments. Results are mean ± SD.


Figure 2
View larger version (99K):
[in this window]
[in a new window]

  		Figure 2. Effect of HGF on thrombin-induced EC remodeling of actin cytoskeleton, adherens junctions, and monolayer disruption. EC grown on glass coverslips were preincubated with HGF (30 ng/ml 15 min), followed by thrombin treatment (0.5 U/ml) for 15 min or 50 min and double immunofluorescent staining with Texas Red phalloidin to detect actin filaments (A) and with ß-catenin antibody to visualize adherens junctions (B). Paracellular gaps are marked by arrows. Results represent 3 independent experiments.

HGF modulates thrombin-induced activation of Rho signaling
To explore the molecular mechanisms of HGF protective effects against thrombin-induced barrier dysfunction, we studied the effects of HGF on thrombin-induced activation of the Rho pathway. We have previously shown that thrombin-induced activation of Rho at early time points is driven by Rho-specific nucleotide exchange factor p115-RhoGEF and is accompanied by decreases in basal Rac activity, whereas at later time points relative to EC barrier restoration, the return of Rho activity to basal level is counteracted by an increase in Rac activity, which is essential for resealing of paracellular gaps (16 , 19) . In comparison with thrombin alone, HGF pretreatment (30 ng/ml, 15 min) significantly attenuated thrombin-induced Rho activation at the 15 min time point and Rho association with its activator p115-RhoGEF (Fig. 3 A). In addition, HGF pretreatment elevated Rac activity in thrombin-challenged EC (Fig. 3B ).


Figure 3
View larger version (31K):
[in this window]
[in a new window]

  		Figure 3. Effect of HGF and thrombin-induced modulation of Rho- and Rac-dependent pathways. Pulmonary EC were preincubated with HGF (30 ng/ml, 15 min), followed by treatment with thrombin (0.5 U/ml) for 15 and 30 min. A) Rho activation assays were performed after 15 min of thrombin stimulation. Top: p115Rho-GEF associated with activated Rho shown in middle. Bottom: Rho content in the total lysates detected by Western blot. B) Rac activation assays of EC pretreated with HGF (30 ng/ml, 15 min) or vehicle followed by 15 min of thrombin stimulation. Top depicts activated Rac, and bottom represents total lysates probed with Rac antibodies. C) Phosphorylation of MLC in EC pretreated with HGF (30 ng/ml, 15 min) or vehicle on thrombin stimulation (0.2 or 0.5 U/ml) for 15 or 30 min was detected by Western blot with diphospho-MLC specific antibodies. Bottom: Membrane reprobed with pan-MLC antibody. D)Site-specific phosphorylation of PAK1 in EC subjected to thrombin (0.5 U/ml) stimulation for 15 or 30 min with or without HGF pretreatment was detected by Western blot with phospho-Thr423-specific antibodies. E) EC were stimulated with a combination of HGF (30 ng/ml) and thrombin (0.5 U/ml) for 15 or 30 min, followed by detection of phosphorylated MLC by immunoblotting with diphospho-MLC specific antibodies. Equal protein loading was confirmed by reprobing of membranes with antibodies to nonphosphorylated proteins. Graphs represent quantitative analysis of GTPase activation and MLC and PAK phosphorylation by scanning densitometry of membranes and are expressed in relative density units (RDU). Results are mean ± SD of 3 independent experiments. *P < 0.05.

Next, we analyzed the effects of HGF on thrombin-induced phosphorylation of regulatory MLC and PAK1, the downstream targets of the Rho and Rac pathways, respectively. Reduction in thrombin-induced Rho activation in EC pretreated by HGF before thrombin challenge was associated with pronounced decreases in MLC phosphorylation levels after 15 and 30 min of thrombin stimulation (Fig. 3C ). In turn, Rac activity in HGF-pretreated EC after 15 or 30 min of thrombin challenge was increased, as compared to EC stimulated with thrombin alone (Fig. 3B ) and was associated with increased autophosphorylation of PAK1 at Thr423 in HGF-pretreated cells (Fig. 3D ), which indicates Rac-induced PAK1 activation (40) . Simultaneous treatment of the pulmonary EC with HGF and thrombin (30 ng/ml and 0.5 U/ml, respectively) did not significantly affect MLC phosphorylation at 15 min, in comparison with EC treated with thrombin alone (Fig. 3E ), but did cause a significant reduction of MLC phosphorylation levels after 30 min of costimulation. These data are consistent with results presented in Fig. 1B and show that addition of HGF after thrombin treatment accelerated recovery of EC barrier. Thus, using different models of HGF and thrombin administration in the pulmonary EC cultures we have described the barrier protective effects of HGF against thrombin-induced EC barrier dysfunction. Taken together, our results suggest that HGF effects are associated with the stimulation of the Rac pathway involved in EC barrier recovery and attenuation of Rho-dependent mechanisms of thrombin-induced EC barrier disruption.

Rac and Tiam1 mediate protective effects of HGF on thrombin-induced EC permeability
To further examine involvement of the Rac-dependent pathway in HGF-mediated barrier protective effects, human pulmonary EC were transfected with Rac-specific siRNA to inhibit endogenous Rac expression. Agonist-induced permeability responses in Rac-depleted EC monolayers and control cells treated with non-specific RNA were monitored by changes in TER. Rac depletion dramatically attenuated HGF-induced increases in TER, when compared to cells transfected with non-specific RNA (Fig. 4 A). Attenuation of HGF-induced barrier-protective response was also observed in EC pretreated with pharmacological Rac inhibitor NSC-23766 (200 µM, 30 min) before HGF stimulation (Fig. 4B ). The inhibitory effect of NSC-23766 was dose-dependent with maximal response reached at 200 µM (Fig. 4B , inset). This concentration of NSC-23766 was used in the next experiments.


Figure 4
View larger version (39K):
[in this window]
[in a new window]

  		Figure 4. Involvement of the Rac-dependent pathway in HGF-mediated TER enhancement. A) Endothelial cells were transfected with Rac-specific siRNA or with non-specific RNA 72 h before TER measurements. At the time indicated by arrow, cells were stimulated with HGF (30 ng/ml). The inset represents Western blot analysis of Rac depletion induced by Rac1-specific siRNA in comparison with to EC treated with non-specific RNA duplexes. B) At the time indicated by first arrow, confluent EC monolayers were treated with Rac inhibitor NSC-23766 (200 µM), then stimulated by HGF (30 ng/ml) marked by second arrow. Inset: EC were preincubated with NSC-23766 (50, 100, or 200 µM) followed by HGF-stimulation. C) Cells grown in plastic dishes were transfected with Tiam1-specific or non-specific RNA. After 72 h, cells were stimulated with HGF (30 ng/ml, 15 min), and Rac activation assays were performed as described in Materials and Methods. D) EC transfected with siRNA to Tiam1 or non-specific RNA were used for TER measurements. At the time point indicated by arrow, cells were treated with HGF (30 ng/ml). Inset: Western blot analysis showing Tiam1 protein depletion induced by Tiam1-specific siRNA. E) Cells were transfected with siTiam1 or non-specific RNA. After 72 h of trasnfection, EC were preincubated with HGF (30 ng/ml, indicated by first arrow), followed by thrombin treatment (0.5 U/ml, indicated by second arrow). Results are representative data of 3 independent experiments. F) Cells were preincubated with LY294002 (25 µM), genistein (100 µM), or NSC-23766 (200 µM) for 30 min followed by HGF (30 ng/ml, 15 min) challenge. Rac and Rho activities were determined using pull-down assays. Graphs represent quantitative analysis of GTPase activation by scanning densitometry of the membranes and are expressed in relative density units (RDU). Results are mean ± SD of 3 independent experiments. *P < 0.05.

Because NSC-23766 inhibits Rac activity by preventing of Rac interaction with Rac-specific GEFs (41) , we next investigated the involvement of Rac-specific GEF Tiam1 in the HGF-mediated Rac activation and EC barrier enhancement. Depletion of endogenous Tiam1 using EC transfection with Tiam1-specific siRNA abolished HGF-induced activation of Rac (Fig. 4C ). Furthermore, depletion of Tiam1 dramatically decreased HGF-induced barrier-protective EC responses (Fig. 4D ) and significantly attenuated the HGF-mediated protective effect against thrombin-induced hyperpermeability (Fig. 4E )

PI3-kinase involvement in HGF-mediated barrier protective responses in the pulmonary EC has been previously reported (8) . The results of this study show that pharmacological inhibition of PI3-kinase by LY294002 (25 µM, 30 min) as well as inhibition of tyrosine kinases by the general tyrosine kinase inhibitor genistein (100 µM, 30 min) dramatically attenuated HGF-induced Rac activation (Fig. 4F ). Treatment of EC with NSC-23766 (200 µM, 30 min) before HGF stimulation was used as a control for Rac inhibition. These data suggest that PI3-kinase and tyrosine kinases may act as upstream activators of the Rac-mediated pathway for the HGF-induced EC barrier protective response.

Tiam1 inhibition abolishes HGF protective effects against thrombin-induced disruption of EC monolayer integrity
The following experiments examined the role of Tiam1 in HGF-mediated protection of EC monolayer integrity in thrombin-challenged human pulmonary EC monolayers. After transfection with Tiam1-specific or non-specific siRNAs, EC were preincubated with HGF (30 ng/ml, 15 min) and then stimulated with thrombin for 15 min (the acute-phase of thrombin-induced barrier disruption) or the 50 min (the recovery of EC monolayer integrity). Thrombin-induced stress fiber formation, paracellular gap formation, and Rac-mediated cortical actin remodeling were visualized using double immunofluorescent staining for F-actin and Rac-regulated activator of peripheral actin polymerization cortactin (42 , 43) . Similar to nontransfected controls, EC transfected with non-specific RNA and pretreated with HGF exhibited a significantly reduced number of paracellular gaps during the acute-phase (15 min) of thrombin challenge, in contrast with cells stimulated with thrombin alone (Fig. 5 A, top). Furthermore, HGF pretreatment accelerated disappearance of paracellular gaps and actin stress fibers during EC monolayers recovery after thrombin challenge (50 min), as opposed to EC treated with thrombin alone (Fig. 5A , top). Consistent with HGF-induced enhancement of the peripheral actin cytoskeleton and EC monolayer barrier properties, EC stimulated with HGF exhibited increased cortactin peripheral accumulation (Fig. 5A , bottom, insets). The acute phase of thrombin-induced EC barrier dysfunction (15 min) was characterized by the disappearance of cortactin from the cell periphery as well as increased cortactin peripheral localization after 50 min of thrombin treatment. In contrast, EC preincubation with HGF significantly enhanced accumulation of cortactin at the cell periphery at both time points of thrombin treatment (Fig. 5A , bottom, insets). Depletion of Tiam-1 using Tiam1-specific siRNA attenuated HGF protective effects against thrombin-induced paracellular gap formation and delayed resealing of paracellular gaps during the recovery phase (Fig. 5B , top). In addition, Tiam1 depletion abolished peripheral accumulation of cortactin on HGF stimulation and during EC recovery after thrombin challenge (Fig. 5B , bottom, insets). High transfection efficiency was confirmed by cotransfection of pulmonary EC with Tiam1-specific siRNA and fluorescein-labeled RNA duplexes (Fig. 5C ).


Figure 5
View larger version (100K):
[in this window]
[in a new window]

  		Figure 5. Effect of Tiam1 knockdown on agonist-induced EC cytoskeletal remodeling and monolayer integrity. EC grown on glass coverslips were incubated with non-specific RNA (A) or treated with Tiam1-specific siRNA for 72 h (B). After incubation with HGF (30 ng/ml, 15 min), cells were stimulated with thrombin (0.5 U/ml) for 15 or 50 min, followed by double immunofluorescent staining using Texas Red phalloidin to visualize actin filaments (A, B, top). Paracellular gaps are shown by arrows. Cortactin translocation was analyzed using immunofluorescent staining with cortactin antibody (A, B, bottom). Shown are results representative of 3 independent experiments. C) To confirm high transfection efficiency, pulmonary EC were cotransfected with Tiam1-specific siRNA and control fluorescein-labeled RNA duplexes using DharmaFECT1 transfection reagent. Number of transfected cells were determined by live fluorescent microscopy (right image) and compared to a total cell number determined by contrast microscopy of the same microscopic field (left image).

HGF attenuates thrombin-induced Rho activation via Tiam1 and Rac
Previous experiments revealed some antagonism between HGF and thrombin in the regulation of the Rho pathway (Fig. 3) . The following experiments examined the role of Tiam1 and Rac in the inhibitory effects of HGF on thrombin-induced Rho activation. EC monolayers pretreated with NSC-23766 (200 µM, 30 min) were stimulated with HGF (30 ng/ml) for 15 min, followed by thrombin challenge (0.5 U/ml, 15 min), and activation of Rho and MLC phosphorylation was determined as described in Materials and Methods. In agreement with the results described above (Fig. 3) , HGF significantly decreased thrombin-induced Rho activation (Fig. 6 A). Down-regulation of Tiam1-dependent Rac activity using NSC-23766 (200 µM, 30 min) abolished the inhibitory effects of HGF on thrombin-induced Rho activation.


Figure 6
View larger version (43K):
[in this window]
[in a new window]

  		Figure 6. Effect of Rac and Tiam1 inhibition on modulation of thrombin-induced Rho signaling by HGF. A) EC preincubated with vehicle or NSC-23766 (200 µM, 30 min), were treated with HGF (30 ng/ml, 15 min) and stimulated with thrombin (0.5 U/ml, 15 min). Control cells were treated with HGF or thrombin alone. Rho activation pull-down assays were performed as described in Materials and Methods. B) Pulmonary EC transfected with Tiam1-specific siRNA or non-specific RNA duplexes were stimulated with vehicle or HGF (30 ng/ml, 15 min), followed by thrombin addition (0.5 U/ml, 15 min). Phosphorylated MLC was determined by immunoblotting using MLC phospho-specific antibodies. Bottom: Membrane reprobed with pan-MLC antibody. Graphs represent quantitative analysis of GTPase activation and MLC phosphorylation by scanning densitometry of the membranes and are expressed in relative density units (RDU). Results are mean ± SD of 3 independent experiments. *P < 0.05.

To directly examine the role of Tiam1 in HGF-induced down-regulation of thrombin-induced Rho signaling, pulmonary EC were transfected with Tiam1-specific siRNA or control non-specific RNA, preincubated with HGF (30 ng/ml, 15 min), and stimulated with thrombin (0.5 U/ml, 15 min). HGF dramatically attenuated thrombin-induced MLC phosphorylation in cells transfected with non-specific RNA. However, depletion of Tiam1 completely abolished the inhibitory effects of HGF on thrombin-induced MLC phosphorylation (Fig. 6B ). Collectively, our data demonstrate that HGF modulates the thrombin-activated Rho pathway via Tiam1/Rac-dependent mechanisms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Alterations in vascular permeability are a defining feature of several diverse processes, including inflammation, ischemia/reperfusion, and ventilator-induced lung injury. Increased endothelial permeability causes alveolar flooding, hypoxemia, and tissue leukocyte infiltration, which triggers inflammatory cytokine production and may lead to increased morbidity and mortality. Increased levels of circulating HGF have been observed in experimental models and in patients with acute lung injury and ARDS syndrome (44 , 45) . Importantly, HGF up-regulation by pathogenic stimuli has been linked to pulmonary recovery after pathological insult (44 , 45) , suggesting that HGF may serve as a protective agonist in lung reparative processes and in restoration of vascular permeability to normal levels. We (8) and others (9) have previously reported involvement of HGF in endothelial barrier enhancement. However, molecular mechanisms underlying effects of HGF on pulmonary EC barrier regulation are not yet well understood.

The main finding of this study is the involvement of the Tiam1/Rac-dependent pathway in HGF-mediated barrier protective effects on EC monolayers. Our experiments showed that knockdown of either Rac or Rac-specific nucleotide exchange factor Tiam1 abolished HGF-induced cytoskeletal remodeling and EC barrier protective responses. Rac- and Rho-dependent mechanisms differentially regulate agonist-mediated cytoskeletal remodeling, which is essential for cellular contraction, motility, and EC barrier control (16 , 19 , 20 , 38 , 46 47 48) . Nucleotide exchange activity of Tiam1, which mediates HGF-induced Rac activation and EC barrier protective response, can be regulated by diverse mechanisms, including phosphorylation by tyrosine kinases, Ca2+/calmodulin-dependent kinase II (CaMKII) or related kinases, interaction with PI3-kinase product PtdIns(3,4,5)P3, binding to cell surface molecule CD44 or cytoskeletal protein ankyrin, as well as direct binding to activated Ras (29 , 31 , 34) . Our data show that the HGF-induced EC barrier-protective response is abolished when Rac is inhibited by PI3-kinase and tyrosine kinase inhibitors (8) . These results suggest a signaling pathway of HGF-induced EC barrier protection via PI3-kinase and tyrosine kinase-dependent activation of Tiam1 and stimulation of Rac.

The other key observation of this study is the ability of HGF to attenuate the thrombin-induced Rho pathway of EC barrier dysfunction. Our results show that HGF significantly attenuated Rho-mediated MLC phosphorylation and hyperpermeability in pulmonary endothelium challenged with thrombin. Furthermore, protective effect of HGF in the thrombin-induced EC barrier dysfunction model was associated with reduction of thrombin-induced Rho activation and with stimulation of Rac signaling critical for EC barrier recovery (19 , 20 , 49) .

Mechanisms of Rho-Rac crosstalk are the focus of current studies by several groups (19 , 50 , 51) . One potential mechanism may involve HGF-activated signal protein kinases (PI3K, PKC, Src) modulating Rho-specific guanine nucleotide exchange factors and resulting in reduction of the RhoGTP pool (8 , 52 53 54) . Another possible mechanism includes Rac-mediated regulation of RhoA activity via direct interaction of Rac with Rho inhibitor RhoGDI (55) . Alternatively, activated Tiam1/Rac may down-regulate Rho activity via stimulation of Rho-specific GTPase activating protein p190-RhoGAP (56) . In addition, activation of Rac downstream target PAK1 may lead to a further increase in Rac activity, therefore enhancing its inhibitory effects on Rho pathway (56) . Our results also indicate a critical involvement of Tiam1 in HGF-mediated inhibition of Rho signaling in response to thrombin (Figs. 5 and 6) . However, precise mechanisms of crosstalk between Rac and Rho in HGF-stimulated endothelial cells remain to be determined.

HGF-induced assembly of adherens junctions observed in this study is precisely regulated via Rac-dependent association of transmembrane protein VE-cadherin with the intracellular {alpha}-ß-{gamma}-catenin complex, which links VE-cadherin to the cytoskeleton and establishes the physical integrity of the AJ complex (57) . The VE-cadherin–ß-catenin interaction is negatively regulated by actin- and ß-catenin-binding protein IQGAP1. Activated Rac binds its effector IQGAP1 and promotes the VE-cadherin–ß-catenin interaction (58) . Thus, enhanced Rac activation observed in HGF-pretreated cells after 15 min of thrombin stimulation (Fig. 3) may promote AJ assembly and enhance AJ integrity (Fig. 2B ). On the other hand, increased Rac activity leads to activation of PAK1, which is a principal mediator of Rac-dependent signaling to the cytoskeleton, and results in activation of cytoskeletal effector proteins Arp2/3, N-Wasp and cortactin. These proteins trigger peripheral actin polymerization (43 , 59 60 61) and formation of the peripheral actin rim, which is essential for EC response to HGF (8) or other barrier protective stimuli (8 , 20 , 27 , 42 , 62 , 63) . Therefore, Rac-dependent enhancement of the cortical actin cytoskeleton and AJ assembly may represent two complementary Rac-mediated mechanisms involved in the HGF-induced EC barrier recovery after challenge with barrier-disruptive agonists.

The results of the current study show that HGF exhibits protective effects on the pulmonary vascular endothelial barrier under acute pathological conditions by stimulating intrinsic EC barrier protective mechanisms dependent on crosstalk between Rac and Rho pathways. Based on previous reports and the results of this study, we propose a scheme of HGF-induced small GTPase regulation and EC barrier protection (Fig. 7 ). HGF, via yet to be identified mechanisms, activates PI3-kinase and tyrosine kinases (34 , 35 , 54 , 64) , leading to stimulation of Tiam1 and Rac GTPase, which in turn stimulate PAK1 and Rac cytoskeletal effector proteins and induce cortical actin polymerization and thus EC barrier enhancement. In addition, activation of Rac signaling results in inhibition of Rho activity and Rho-mediated barrier disruption and contributes to the maintenance of EC monolayer integrity in the injured lungs. These findings may represent a fundamental mechanism of endothelial cellular response to a spectrum of barrier-protective agonists.


Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		Figure 7. Proposed mechanisms of HGF-induced endothelial barrier protection and modulation of Rho pathway of EC barrier dysfunction. HGF stimulates tyrosine kinases and PI3-kinase, which activate Rac-specific GEF Tiam1. Tiam1-mediated activation of Rac results in activation of cytoskeletal and cell adhesion-associated Rac-effectors, which promote enhancement of adherens junctions and peripheral actin cytoskeleton and thus increase EC monolayer barrier properties. In addition, HGF attenuates the Rho pathway of endothelial barrier dysfunction via Tiam1- and Rac-dependent reduction of Rho activity, which leads to decreased myosin light chain phosphorylation, EC contraction, and less severe endothelial barrier dysfunction. HGF may further contribute to the restoration of EC barrier properties after challenge with edemagenic agonists by promoting Rac-mediated signaling essential for EC monolayer recovery.

In conclusion, in the present study we examined signaling pathways involved in HGF-induced protection of pulmonary endothelial barrier and provided novel mechanistic insights into regulation of EC permeability via dynamic interactions between Rho- and Rac-mediated signaling. Because the molecular basis of acute lung injury is poorly understood and no specific pharmacologic therapies are currently available, these studies may not only support the potential therapeutic significance of HGF in the management of VILI but also delineate a role of Tiam1/Rac-dependent signaling in EC barrier maintenance. We believe that these studies will enhance our understanding of lung vascular barrier function and may lead to new therapeutic approaches in ARDS.


   ACKNOWLEDGMENTS
 
This work was supported by the AHA Scientist Development Grant for A. A. Birukova and Grants from National Heart, Lung, and Blood Institutes (HL-076259 and HL-075349) for K. G. Birukov. The authors also thank N. Moldobaeva and M. Birukova for superb laboratory assistance.

Received for publication November 8, 2006. Accepted for publication March 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Matsumoto, K., Nakamura, T. (1996) Emerging multipotent aspects of hepatocyte growth factor. J. Biochem. (Tokyo) 119,591-600[Abstract/Free Full Text]
  2. Rosen, E. M., Goldberg, I. D. (1995) Scatter factor and angiogenesis. Adv. Cancer Res. 67,257-279[Medline]
  3. Zhang, L., Himi, T., Morita, I., Murota, S. (2000) Hepatocyte growth factor protects cultured rat cerebellar granule neurons from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway. J. Neurosci. Res. 59,489-496[CrossRef][Medline]
  4. Tomita, N., Morishita, R., Higaki, J., Ogihara, T. (2000) Novel molecular therapeutic approach to cardiovascular disease based on hepatocyte growth factor. J. Atheroscler. Thromb. 7,1-7[Medline]
  5. Aoki, M., Morishita, R., Taniyama, Y., Kaneda, Y., Ogihara, T. (2000) Therapeutic angiogenesis induced by hepatocyte growth factor: potential gene therapy for ischemic diseases. J. Atheroscler. Thromb. 7,71-76[Medline]
  6. Bussolino, F., Di Renzo, M. F., Ziche, M., Bocchietto, E., Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L., Coffer, A., Comoglio, P. M. (1992) Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol. 119,629-641[Abstract/Free Full Text]
  7. Grant, D. S., Kleinman, H. K., Goldberg, I. D., Bhargava, M. M., Nickoloff, B. J., Kinsella, J. L., Polverini, P., Rosen, E. M. (1993) Scatter factor induces blood vessel formation in vivo. Proc. Natl. Acad. Sci. U. S. A. 90,1937-1941[Abstract/Free Full Text]
  8. Liu, F., Schaphorst, K. L., Verin, A. D., Jacobs, K., Birukova, A., Day, R. M., Bogatcheva, N., Bottaro, D. P., Garcia, J. G. (2002) Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J. 16,950-962[Abstract/Free Full Text]
  9. Date, I., Takagi, N., Takagi, K., Kago, T., Matsumoto, K., Nakamura, T., Takeo, S. (2004) Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction. Biochem. Biophys. Res. Commun. 319,1152-1158[CrossRef][Medline]
  10. Dudek, S. M., Garcia, J. G. (2001) Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91,1487-1500[Abstract/Free Full Text]
  11. Garcia, J. G., Davis, H. W., Patterson, C. E. (1995) Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J. Cell. Physiol. 163,510-522[CrossRef][Medline]
  12. Garcia, J. G., Liu, F., Verin, A. D., Birukova, A., Dechert, M. A., Gerthoffer, W. T., Bamberg, J. R., English, D. (2001) Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J. Clin. Invest. 108,689-701[CrossRef][Medline]
  13. Garcia, J. G., Verin, A. D., Schaphorst, K. L. (1996) Regulation of thrombin-mediated endothelial cell contraction and permeability. Semin. Thromb. Hemost. 22,309-315[Medline]
  14. Garcia, J. G., Schaphorst, K. L., Verin, A. D., Vepa, S., Patterson, C. E., Natarajan, V. (2000) Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation. J. Appl. Physiol. 89,2333-2343[Abstract/Free Full Text]
  15. Lum, H., Malik, A. B. (1996) Mechanisms of increased endothelial permeability. Can. J. Physiol. Pharmacol. 74,787-800[CrossRef][Medline]
  16. Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G. N., Verin, A. D. (2004) Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc. Res. 67,64-77[CrossRef][Medline]
  17. van Nieuw Amerongen, G. P., van Delft, S., Vermeer, M. A., Collard, J. G., van Hinsbergh, V. W. (2000) Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases. Circ. Res. 87,335-340[Abstract/Free Full Text]
  18. Birukov, K. G., Csortos, C., Marzilli, L., Dudek, S., Ma, S. F., Bresnick, A. R., Verin, A. D., Cotter, R. J., Garcia, J. G. (2001) Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src). J. Biol. Chem. 276,8567-8573[Abstract/Free Full Text]
  19. Birukova, A. A., Chatchavalvanich, S., Rios, A., Kawkitinarong, K., Garcia, J. G., Birukov, K. G. (2006) Differential regulation of pulmonary endothelial monolayer integrity by varying degrees of cyclic stretch. Am. J. Pathol. 168,1749-1761[Abstract/Free Full Text]
  20. Birukov, K. G., Bochkov, V. N., Birukova, A. A., Kawkitinarong, K., Rios, A., Leitner, A., Verin, A. D., Bokoch, G. M., Leitinger, N., Garcia, J. G. (2004) Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac. Circ. Res. 95,892-901[Abstract/Free Full Text]
  21. Essler, M., Amano, M., Kruse, H. J., Kaibuchi, K., Weber, P. C., Aepfelbacher, M. (1998) Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells. J. Biol. Chem. 273,21867-21874[Abstract/Free Full Text]
  22. Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., Brown, J. H. (1998) Requirement for Rho-mediated myosin light chain phosphorylation in thrombin-stimulated cell rounding and its dissociation from mitogenesis. J. Biol. Chem. 273,10099-10106[Abstract/Free Full Text]
  23. Sanders, L. C., Matsumura, F., Bokoch, G. M., de Lanerolle, P. (1999) Inhibition of myosin light chain kinase by p21-activated kinase (see comments). Science 283,2083-2085[Abstract/Free Full Text]
  24. Birukova, A. A., Birukov, K. G., Adyshev, D., Usatyuk, P., Natarajan, V., Garcia, J. G., Verin, A. D. (2005) Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction. J. Cell. Physiol. 204,934-947[CrossRef][Medline]
  25. Petrache, I., Crow, M. T., Neuss, M., Garcia, J. G. (2003) Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis. Biochem. Biophys. Res. Commun. 306,244-249[CrossRef][Medline]
  26. Vouret-Craviari, V., Boquet, P., Pouyssegur, J., Van Obberghen-Schilling, E. (1998) Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function. Mol. Biol. Cell 9,2639-2653[Abstract/Free Full Text]
  27. Jacobson, J. R., Dudek, S. M., Birukov, K. G., Ye, S. Q., Grigoryev, D. N., Girgis, R. E., Garcia, J. G. (2004) Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. Am. J. Respir. Cell Mol. Biol. 30,662-670[Abstract/Free Full Text]
  28. Boguski, M. S., McCormick, F. (1993) Proteins regulating Ras and its relatives. Nature 366,643-654[CrossRef][Medline]
  29. Zheng, Y. (2001) Dbl family guanine nucleotide exchange factors. Trends. Biochem. Sci. 26,724-732[CrossRef][Medline]
  30. Bishop, A. L., Hall, A. (2000) Rho GTPases and their effector proteins. Biochem. J. 348,241-255[CrossRef][Medline]
  31. Welch, H. C., Coadwell, W. J., Stephens, L. R., Hawkins, P. T. (2003) Phosphoinositide 3-kinase-dependent activation of Rac. FEBS Lett. 546,93-97[CrossRef][Medline]
  32. Arthur, W. T., Quilliam, L. A., Cooper, J. A. (2004) Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors. J. Cell Biol. 167,111-122[Abstract/Free Full Text]
  33. O'Connor, K. L., Mercurio, A. M. (2001) Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma cells. J. Biol. Chem. 276,47895-47900[Abstract/Free Full Text]
  34. Servitja, J. M., Marinissen, M. J., Sodhi, A., Bustelo, X. R., Gutkind, J. S. (2003) Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src. J. Biol. Chem. 278,34339-34346[Abstract/Free Full Text]
  35. Singleton, P. A., Dudek, S. M., Chiang, E. T., Garcia, J. G. (2005) Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin. FASEB J. 19,1646-1656[Abstract/Free Full Text]
  36. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411,494-498[CrossRef][Medline]
  37. Malliri, A., van Es, S., Huveneers, S., Collard, J. G. (2004) The Rac exchange factor Tiam1 is required for the establishment and maintenance of cadherin-based adhesions. J. Biol. Chem. 279,30092-30098[Abstract/Free Full Text]
  38. Birukova, A. A., Birukov, K. G., Smurova, K., Adyshev, D. M., Kaibuchi, K., Alieva, I., Garcia, J. G., Verin, A. D. (2004) Novel role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB J. 18,1879-1890[Abstract/Free Full Text]
  39. Birukova, A. A., Birukov, K. G., Gorshkov, B., Liu, F., Garcia, J. G., Verin, A. D. (2005) MAP kinases in lung endothelial permeability induced by microtubule disassembly. Am. J. Physiol. Lung. Cell. Mol. Physiol. 289,L75-L84[Abstract/Free Full Text]
  40. Bokoch, G. M. (2003) Biology of the p21-Activated Kinases. Annu. Rev. Biochem. 72,743-781[CrossRef][Medline]
  41. Gao, Y., Dickerson, J. B., Guo, F., Zheng, J., Zheng, Y. (2004) Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc. Natl. Acad. Sci. U. S. A. 101,7618-7623[Abstract/Free Full Text]
  42. Dudek, S. M., Jacobson, J. R., Chiang, E. T., Birukov, K. G., Wang, P., Zhan, X., Garcia, J. G. (2004) Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase. J. Biol. Chem. 279,24692-24700[Abstract/Free Full Text]
  43. Uruno, T., Liu, J., Zhang, P., Fan, Y., Egile, C., Li, R., Mueller, S. C., Zhan, X. (2001) Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat. Cell Biol. 3,259-266[CrossRef][Medline]
  44. Ware, L. B., Matthay, M. A. (2002) Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. Am. J. Physiol. Lung. Cell Mol. Physiol. 282,L924-L940[Abstract/Free Full Text]
  45. Geiser, T. (2003) Mechanisms of alveolar epithelial repair in acute lung injury–a translational approach. Swiss Med. Wkly. 133,586-590[Medline]
  46. Manser, E. (2002) Small GTPases take the stage. Dev. Cell. 3,323-328[CrossRef][Medline]
  47. Parsons, J. T. (2003) Focal adhesion kinase: the first ten years. J. Cell Sci. 116,1409-1416[Abstract/Free Full Text]
  48. Turner, C. E., West, K. A., Brown, M. C. (2001) Paxillin-ARF GAP signaling and the cytoskeleton. Curr. Opin. Cell Biol. 13,593-599[CrossRef][Medline]
  49. Mehta, D., Malik, A. B. (2006) Signaling mechanisms regulating endothelial permeability. Physiol. Rev. 86,279-367[Abstract/Free Full Text]
  50. DerMardirossian, C., Schnelzer, A., Bokoch, G. M. (2004) Phosphorylation of RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol. Cell. 15,117-127[CrossRef][Medline]
  51. Tamma, G., Klussmann, E., Procino, G., Svelto, M., Rosenthal, W., Valenti, G. (2003) cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI. J. Cell Sci. 116,1519-1525[Abstract/Free Full Text]
  52. Wojciak-Stothard, B., Ridley, A. J. (2002) Rho GTPases and the regulation of endothelial permeability. Vascul. Pharmacol. 39,187-199[CrossRef][Medline]
  53. Royal, I., Lamarche-Vane, N., Lamorte, L., Kaibuchi, K., Park, M. (2000) Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol. Biol. Cell 11,1709-1725[Abstract/Free Full Text]
  54. Mertens, A. E., Roovers, R. C., Collard, J. G. (2003) Regulation of Tiam1-Rac signalling. FEBS Lett. 546,11-16[CrossRef][Medline]
  55. Wong, K. W., Mohammadi, S., Isberg, R. R. (2006) Disruption of RhoGDI and RhoA regulation by a Rac1 specificity switch mutant. J. Biol. Chem. 281,40379-40388[Abstract/Free Full Text]
  56. Herbrand, U., Ahmadian, M. R. (2006) p190-RhoGAP as an integral component of the Tiam1/Rac1-induced downregulation of Rho. Biol. Chem. 387,311-317[CrossRef][Medline]
  57. Dejana, E. (2004) Endothelial cell-cell junctions: happy together. Nat. Rev. Mol. Cell Biol. 5,261-270[CrossRef][Medline]
  58. Noritake, J., Watanabe, T., Sato, K., Wang, S., Kaibuchi, K. (2005) IQGAP1: a key regulator of adhesion and migration. J. Cell Sci. 118,2085-2092[Abstract/Free Full Text]
  59. Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., Parsons, J. T. (2000) Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex. J. Cell Biol. 151,29-40[Abstract/Free Full Text]
  60. Weed, S. A., Parsons, J. T. (2001) Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 20,6418-6434[CrossRef][Medline]
  61. Borisy, G. G., Svitkina, T. M. (2000) Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12,104-112[CrossRef][Medline]
  62. Birukov, K. G., Birukova, A. A., Dudek, S. M., Verin, A. D., Crow, M. T., Zhan, X., DePaola, N., Garcia, J. G. (2002) Shear stress-mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells. Am. J. Respir. Cell Mol. Biol. 26,453-464[Abstract/Free Full Text]
  63. Vouret-Craviari, V., Bourcier, C., Boulter, E., van Obberghen-Schilling, E. (2002) Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells. J. Cell Sci. 115,2475-2484[Abstract/Free Full Text]
  64. Gonzalez, E., Kou, R., Michel, T. (2006) Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells. J. Biol. Chem. 281,3210-3216[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.06-7660comv1
21/11/2776    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Birukova, A. A.
Right arrow Articles by Birukov, K. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Birukova, A. A.