Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.
- pulmonary endothelium
- guanine nucleotide exchange factor
the vascular endothelial monolayer forms a semiselective permeable barrier between the blood and the interstitial space, which regulates macromolecule transport and leukocyte trafficking through the vessel wall. Small GTPase Rho plays an essential role in the initiation of actomyosin contraction and reorganization of the endothelial cytoskeleton (9, 24, 35), which provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases including asthma, sepsis, and acute lung injury (14, 25, 38). Transition of Rho from GDP-bound to GTP-bound state causes Rho activation and association with effector molecules. In turn, interaction of Rho-GTP with Rho-associated kinase (Rho-kinase) causes activation of Rho-kinase enzymatic activity and phosphorylation of its substrates, regulatory myosin light chains (MLC), and myosin-binding subunit of myosin-associated phosphatase type 1 (MYPT1) (2, 22). MLC phosphorylation triggers stress fiber formation, actomyosin contraction, and paracellular gap formation. Rho-kinase-mediated MYPT1 phosphorylation inhibits MYPT1 phosphatase activity, increases a pool of phosphorylated MLC, and thus further enhances actomyosin contraction and endothelial cell (EC) permeability (7, 9, 34, 36).
The cycling between GTP-bound (active) and GDP-bound (inactive) forms of small GTPases (Rho, Rac, and Cdc42) is tightly controlled by accessory proteins, such as GDI (guanine nucleotide dissociation inhibitor), which captures GDP-bound Rho and maintains Rho in an inactive cytosolic complex, GTPase-activating protein (GAP), which stimulates hydrolysis of GTP to GDP, and guanine nucleotide exchange factor (GEF), which activates GTPases by enhancing the release of bound GDP from the GTPase nucleotide-binding domain (3, 11, 42). Rho family GTPases are activated by the Dbl family of GEF, which play a major role in GTPase regulation by a variety of external stimuli (11, 32, 42). Several members of the Dbl family of GEFs may specifically stimulate Rho, Rac, or Cdc42, whereas other GEFs stimulate all three GTPases (42). GEF-H1 has been recently characterized as a Rho-specific GEF that localizes on microtubules (MT) and exhibits Rho-specific GDP/GTP exchange activity (31). In its MT-bound state, the guanosine-exchange activity of GEF-H1 is suppressed, whereas GEF-H1 release from MT stimulates Rho-specific GEF activity (23). We and others have previously demonstrated the involvement of MT network in the remodeling of actin cytoskeleton, cell contraction, and vascular permeability in lung endothelium in response to stimulation by thrombin, transforming growth factor (TGF)-β, and TNF-α, and MT inhibitors nocodazole and vinblastine (6, 10, 17, 29, 36, 41). Moreover, we have recently reported the protective effect of MT stabilization against thrombin-induced Rho activation and barrier compromise (7). These findings suggest a potential role for MT-associated GEF-H1 in agonist-induced actin cytoskeletal remodeling and permeability changes.
In this study, we utilized models of pulmonary EC barrier compromise induced by inflammatory agonist thrombin and MT inhibitor nocodazole. Both agonists induce EC barrier dysfunction via rearrangement of actin and effects on MT dynamics, which at least in part depend on the Rho cascade (9, 10, 36). To affect GEF-H1 activity, we performed GEF-H1 protein depletion using a small interfering RNA (siRNA) approach or ectopic expression of GEF-H1 functional mutants. Using molecular, immunological, and biochemical approaches, we evaluated the role of GEF-H1 in the regulation of actomyosin cytoskeleton and barrier function in human pulmonary endothelium.
MATERIALS AND METHODS
β-Tubulin antibody was purchased from Covance (Berkeley, CA), di-phospho-MLC antibody was obtained from Cell Signaling (Beverly, MA), phospho-MYPT antibody was purchased from Upstate Biotechnology (Lake Placid, NY), green fluorescent protein (GFP) antibody was from Clontech BD Biosciences (Mountain View, CA); p115Rho GEF, Lbc, and NET1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GEF-H1 antibody was kindly provided by our coauthor Dr. G. M. Bokoch. Texas red-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Unless specified, biochemical reagents were obtained from Sigma (St. Louis, MO).
Human pulmonary artery endothelial cells (HPAEC) were obtained from Cambrex (Walkersville, MD) and used at passages 5–9 as described elsewhere (5).
Measurement of transendothelial electrical resistance.
Measurements of transendothelial electrical resistance (TER) across confluent HPAEC monolayers were performed using electrical cell-substrate impedance sensing system (ECIS; Applied Biophysics, Troy, NY) as previously described (9, 36).
Depletion of GEF-H1 in EC.
To reduce the content of endogenous GEF-H1, HPAEC were treated with GEFF-H1-specific siRNA duplexes, which guide sequence-specific degradation of the homologous mRNA (15). siRNA was ordered from Dharmacon Research (Lafayette, CO) in ready-to-use, desalted, 2′-deprotected, and duplexed form. Duplex of sense 5′-UGUGACUAUCCACAACCGCdTdT-3′ and antisense 5′-PGCGGUUGUGGAUAGUCACAdTdT-3′ siRNA was used for targeting sequences that are part of the coding region for GEF-H1: 5′-AAUGUGACUAUCCACAACCGC-3′ as described previously (4). Nonspecific, nontargeting siRNA duplex #1 (Dharmacon Research, Lafayette, CO) was used as a control treatment. HPAEC were grown to 70% confluence, and the transfection of siRNA (final concentration 50 nM) was performed using DharmaFECT1 transfection reagent (Dharmacon Research, Lafayette, CO) according to manufacturer's protocol. Forty-eight hours posttransfection cells were harvested and used for experiments. Additional control experiments using EC transfections with fluorescently labeled nonspecific RNA showed that this protocol allowed us to achieve 90–100% transfection efficiency.
Semiquantitative RT-PCR analysis.
To estimate the levels of GEF-H1 RNAs in the lung EC after siRNA treatment, total RNA (0.5 μg) isolated from control and treated cells was subjected to PCR in 25-μl reaction mixture using reagents from Superscript One Step RT-PCR kit (Invitrogen, Carlsbad, CA). 18S ribosomal RNA 187-bp fragment, used as an internal control for normalization, was amplified with 40 nM primers from TaqMan Gold RT-PCR Core Reagents Kit (Applied Biosystems, Foster City, CA). To amplify the 224-bp fragment of GEF-H1 cDNA, 200 nM each of the following primers were used: forward, 5′-ACACGCTTCCTCAGCCAGCTATTA-3′; reverse, 5′-AATTGCTGGAAGCGTTTGTCTCGG-3′. The amplified cDNA corresponds to nucleotides 899–1122 in Homo sapiens GEF-H1 mRNA (accession no. NM-004723). The PCR products were analyzed by agarose gel electrophoresis.
Expression plasmids and transient transfection protocol.
Plasmids encoding human GEF-H1 (1–894), GEF-H1 (1–572), and GEF-H1 (DH-mutant) bearing EGFP-tag have been previously described (23) and were used for transient transfections according to protocol described previously (7, 9). Control transfections were performed with empty vectors.
For more effective introduction of cDNA into the cell, we used Nucleofector kit from Amaxa Biosystems (Gaithersburg, MD). An optimized protocol of nucleofection was provided by manufacturer and used with minor modifications. In brief, EC grown in T75 tissue culture flasks at 100% confluence were trypsinized, counted, and pelleted at 200 g for 10 min. Then, samples, 5 × 105 cells, were resuspended in 100 μl of Nucleofector solution available from manufacturer, mixed with 2 μg of DNA, and transfected using manufacturer's recommended program (S-05) with an Amaxa Nucleofector device. Next, cells were plated on D35 dishes and incubated for 18 h in a CO2 incubator at 37°C. Expression of GEF-H1 (DH-mutant) was confirmed by immunoblotting GFP antibody.
EC were plated on glass coverslips, grown to 70% confluence, and transfected with siRNA or specific plasmid DNA followed by stimulation with thrombin or nocodazole. Then cells were subjected to immunofluorescent staining for F-actin or di-phospho-MLC as previously described in detail (7–9).
After stimulation cells were lysed, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with specific antibodies as previously described (8). Intensities of immunoreactive protein bands were quantified using Image Quant software.
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 analysis of variance followed by the post hoc Fisher's test were used. P < 0.05 was considered statistically significant.
Role of GEF-H1 in thrombin- and nocodazole-induced lung EC permeability.
To evaluate involvement of GEF-H1 in thrombin- and nocodazole-induced EC barrier dysfunction, we depleted endogenous GEF-H1 in lung EC by siRNA techniques described in materials and methods and measured changes in TER upon thrombin or nocodazole stimulation. Treatment with GEF-H1-specific siRNA described in previous studies (4) caused 2.5-fold decrease in GEF-H1 mRNA expression compared with cells treated with nonspecific RNA duplex oligonucleotides (Fig. 1A). Next, to evaluate the levels of protein expression in transfected cells we performed Western blot analysis of cells treated with siGEF-H1, nonspecific RNA, or nontreated EC. The results presented in Fig. 1B demonstrate 52.3 ± 4.5% decrease in GEF-H1 expression in siGEF-H1-treated cells, whereas expression of other Rho-regulating GEFs, such as p115-Rho GEF, Lbc, and NET1, remained unaffected.
In the presented studies, the basal transendothelial resistance of cell monolayers treated with siGEF-H1 RNA was slightly lower (1,317 ± 94 Ohm) compared with cells transfected with nonspecific RNA (1,620 ± 98 Ohm). TER decline was associated with significantly decreased GEF-H1 protein expression as result of siGEF-H1 RNA treatment. These changes likely reflect an essential role for GEF-H1 in the maintenance of basal resistance as indication of EC basal barrier function. A number of reports by several groups show an essential role for MT in many cellular processes including vesicle and organelle transport, focal adhesion remodeling, cell migration, cytokinesis (19, 28, 37), and regulation of Rho-mediated endothelial permeability (7, 24, 36). Because recent reports convincingly demonstrate that MT-associated GEF-H1 can be involved in stabilization of MT (23), decreased expression of GEF-H1 may lead to partial microtubule destabilization resulting in decreased cell-substrate interactions and lower levels of basal electrical resistance.
Data expressed as normalized resistance (Fig. 1, C and D) show attenuation of thrombin-induced TER decline in EC transfected with GEF-H1-specific siRNA compared with cells transfected with nonspecific RNA duplexes. Depletion of GEF-H1 significantly attenuated permeability increase in response to thrombin (0.02 U/ml, Fig. 1C) or nocodazole (0.2 μM, Fig. 1D) compared with agonist-stimulated cells transfected with nonspecific RNA. Remarkably, downregulation of GEF-H1 expression not only abolished the initial drop in EC resistance but also significantly enhanced the recovery phase observed 30 min after agonist challenge. These data clearly indicate an essential role of GEF-H1 in mediating agonist-induced lung EC permeability.
Involvement of GEF-H1 in agonist-induced EC cytoskeletal remodeling.
Compromise of EC barrier function is tightly associated with remodeling of cell cytoskeleton and accompanied by actin rearrangement manifested by formation of stress fibers and increased MLC phosphorylation leading to cell contraction (9, 14, 35). In the next series of experiments we analyzed the effect of GEF-H1 depletion on human endothelial actin cytoskeleton. HPAEC were subjected to transfection with nonspecific RNA duplex oligonucleotides (Fig. 2, A and B, top) or GEF-H1-specific siRNA (Fig. 2, A and B, bottom) followed by 15-min treatments with either thrombin (0.02 U/ml) or nocodazole (0.2 μM). Double immunofluorescent staining using Texas red phalloidin to visualize F-actin and di-phospho-MLC antibody to detect phosphorylated MLC was performed, as described in materials and methods. Nonstimulated EC transfected with GEF-H1-specific siRNA showed no significant difference in the organization of actin cytoskeleton and levels of MLC phosphorylation compared with control EC treated with nonspecific RNA. However, thrombin or nocodazole stimulation of EC treated with nonspecific RNA induced robust stress fiber formation and accumulation of di-phospho-MLC, which was almost completely prevented by GEF-H1 depletion. These results indicate an essential role for GEF-H1 in the regulation of the endothelial actomyosin cytoskeleton.
Effect of functional GEF-H1 mutants on agonist-induced EC cytoskeletal rearrangement.
EC were transiently transfected with plasmids encoding GFP-tagged full-length GEF-H1 (amino acid residues 1–894), truncated GEF-H1 mutant with deleted MT binding site, and high intrinsic nucleotide exchange activity [GEF-H1 (1–572)], or GEF-H1 DH-mutant lacking Rho-specific GEF activity. Transfected cells were stimulated with thrombin (0.02 U/ml) or nocodazole (0.2 μM) for 15 min, and F-actin structure was examined by immunofluorescent staining with Texas red phalloidin. Cells overexpressing GEF-H1 were detected by GFP fluorescence. In agreement with previous reports in HeLa cells (23), human lung EC overexpressing GEF-H1 (1–894) revealed MT-like GEF-H1 localization. Overexpression of wild-type GEF-H1 slightly increased stress fibers in nonstimulated cells. These effects were likely due to GEF-H1 saturation in overexpressing EC and appearance of active cytosolic GEF-H1, which could result in modest Rho activation. Thrombin or nocodazole treatment of nontransfected cells caused stress fiber formation and moderate cell retraction, whereas EC expressing GEF-H1 (1–894) exhibited dramatically increased stress fiber formation and were overcontracted (Fig. 3A). EC expressing the GEF-H1 (1–572) mutant revealed significant stress fiber formation even without agonist stimulation (Fig. 3B). Further stimulation with thrombin or nocodazole caused collapse of transfected cells. In contrast to the activated GEF-H1 mutant, ectopic expression of the dominant negative GEF-H1(DH) mutant deficient in nucleotide exchange activity inhibited formation of actin fibers in response to thrombin or nocodazole (Fig. 3C). Similar to full-length GEF-H1 (1–894), the GEF-H1(DH) mutant revealed MT-like intracellular localization (Fig. 3C, bottom).
To characterize effects of the dominant negative GEF-H1 mutant on MLC phosphorylation, which reflects the levels of actomyosin contraction and barrier disruption, we transfected EC with the GEF-H1(DH) mutant and stained them with di-phospho-MLC antibody after thrombin and nocodazole stimulation. Figure 4 represents images of transfected (depicted by GFP fluorescence) and nontransfected HPAEC subjected to immunofluorescent staining for di-phospho-MLC. Consistent with results shown above, thrombin or nocodazole stimulation dramatically increased accumulation of phosphorylated MLC, which was significantly attenuated in cells expressing GEF-H1(DH) mutant.
Effect of downregulation of GEF-H1 nucleotide exchange activity on agonist-induced EC cellular signaling.
To further examine involvement of GEF-H1 in EC barrier regulation, we performed biochemical analysis of MLC and MYPT1 phosphorylation, the important markers of EC contraction and barrier function (9, 14, 35). HPAEC were transfected with GEF-H1-specific siRNA or with nonspecific RNA duplex oligonucleotides followed by 15-min stimulation with thrombin (0.02 U/ml) or nocodazole (0.2 μM) (Fig. 5A). The protein phosphorylation profile was monitored using immunoblotting with di-phospho-MLC or phospho-MYPT1 (Thr850) antibodies. In the next experiments, cells were transfected with plasmids encoding GFP-tagged GEF-H1(DH) mutant or with an empty vector (Fig. 5B) using the nucleofection protocol described in materials and methods. MLC and MYPT1 phosphorylation after agonist treatment was analyzed as described above. High levels of transfection efficiency were confirmed by reprobing membranes with GFP antibody (Fig. 5B, bottom). These results suggest that downregulation of GEF-H1 nucleotide exchange activity by either GEF-H1 protein depletion or expression of dominant negative GEF-H1 mutant significantly attenuates agonist-induced MLC and MYPT1 phosphorylation and actomyosin contraction. These results are consistent with immunofluorescence and permeability data and strongly suggest a critical role of GEF-H1 in the Rho-mediated regulation of lung endothelial barrier function.
The essential role of the Rho-dependent pathway in the regulation of EC barrier function is well recognized. We and others have described Rho activation in response to EC stimulation with barrier-disruptive agonists, such as thrombin, cytokines (TGF-β1, TNF-α), and growth factors (VEGF), mechanical strain, and MT inhibitors (5, 9, 10, 33, 39). However, upstream mechanisms of regulation of Rho activity in pulmonary EC are not well understood. We and others have previously shown involvement of another Rho-specific GEF, p115Rho-GEF, in thrombin-induced stress fiber formation and MT disassembly and demonstrated that p115Rho-GEF-regulator of G protein signaling (RGS), a negative regulator of p115Rho-GEF, inhibited thrombin effects on EC cytoskeletal remodeling (7, 21). Furthermore, our and others’ data suggest an important role for MT dynamics in the regulation of Rho, actin cytoskeleton, and endothelial permeability (7, 16, 30, 41).
The cellular response to thrombin represents a complex mechanism, which involves activation of thrombin receptor protease-activated receptor (PAR) 1 and associated heterotrimeric proteins G12/13 and Gq, which in turn trigger downstream events such as activation of phosphoinositide turnover, protein kinase C, and ERK/2 MAP kinase cascades, Ca2+-calmodulin-dependent kinase, MLC kinase, and Rho-mediated signaling (13, 18, 25, 27). Several mechanisms leading to Rho activation in thrombin-stimulated endothelium have been described so far. First, activated protein kinase C-α may phosphorylate and thus inactivate Rho-GDP dissociation inhibitor (Rho-GDI), leading to increased Rho-GDP/GTP turnover and Rho activation (27). Second, thrombin stimulation induces G12/13-mediated activation of another Rho-specific GEF, p115RhoGEF (20, 21). Inactivation of p115RhoGEF by siRNA-based protein depletion or expression of p115 RhoGEF negative regulator p115RhoGEF RGS attenuated thrombin-induced Rho activation and Rho-mediated stress fiber formation, thus leading to attenuation of EC contraction and decreased permeability (7, 21). This study demonstrates for the first time that another Rho-specific GEF, microtubule-associated GEF-H1, is also involved in thrombin-induced mechanisms of endothelial permeability. SiRNA-based GEF-H1 protein depletion or expression of dominant negative mutants lacking nucleotide exchange activity significantly attenuated thrombin-induced permeability and Rho-mediated intracellular events, such as stress fiber formation, MLC phosphorylation, and Rho-kinase-mediated site-specific phosphorylation of MYPT. This study, taken together with previously described mechanisms of thrombin-induced Rho activation, suggests that regulation of Rho activity is precisely balanced by inhibitory (RhoGAPs, Rho-GDI) and stimulatory (p115RhoGEF, GEF-H1) proteins and that interference with one member of this group may dramatically shift the total balance toward Rho activation or inhibition. It is also important to note that Rho-mediated mechanisms do not rule out a role for other mechanisms of thrombin-induced permeability described above.
GEF-H1 specificity toward Rho and involvement of MT in the regulation of GEF-H1 activity has been previously described (4, 23, 26, 31). Expression of the activated GEF-H1 mutant lacking MT-binding domain in Madin-Darby canine kidney (MDCK) cells induced more than a twofold increase in Rho but not Rac activity, whereas overexpression of full-length GEF-H1 caused a 1.5-fold increase in Rho activity (4). In addition, measurements of transepithelial resistance and macromolecule passage in epithelial cells revealed increased permeability for small molecules (4-kDa dextran) in GEF-H1-overexpressing cells compared with wild-type MDCK, which reflect the important role of GEF-H1 in the regulation of paracellular permeability (4). In turn, expression of the GEF-H1(DH) mutant inhibited stress fiber formation induced by pathogenic substrates from Escherichia coli, EspG, and Orf3 (26), as well as by MT disassembly induced by nocodazole (23). GEF-H1(DH) expression in Cos-1 and HeLa cells also attenuated Rho-dependent SRE activation and stress fiber formation in response to MT dissolution induced by nocodazole or colchicines (23, 26). Consistent with these findings, we observed robust stress fiber formation in human pulmonary EC overexpressing activated, MT-unbound GEF-H1 (1–572) truncated mutant and modest increase in stress fiber formation in nonstimulated EC expressing full-length GEF-H1 (1–894), whereas expression of inactive GEF-H1(DH) mutant preserved EC monolayers against thrombin- and nocodazole-induced stress fiber formation (Fig. 3). Thrombin-induced activation of GEF-H1 activity may occur as the result of thrombin-induced partial MT disassembly (7) and release of MT bound GEF-H1, or alternatively it may be mediated by thrombin receptor-coupled G proteins. An elegant study by Krendel and coauthors (23) showed that expression of the GEF-H1(DH) mutant did not inhibit SRE activation induced by activation of Gα12/13 subunits, which are also associated with thrombin PAR1. These results indicate that thrombin-induced GEF-H1 activation results from thrombin-induced MT disassembly rather then it being directly regulated by Gα12/13, as it has been described for p115Rho-GEF activation mechanism. Thus these results further support our novel hypothesis about a critical involvement of MT dynamics in thrombin-induced barrier failure.
Previous reports using highly conservative human GEF-H1 revealed MT-associated localization of both endogenous and overexpressed GEF-H1 in nonpolarized cells (31). On the basis of deletion analysis, the carboxy terminus of GEF-H1 was identified as an MT-binding site (23). In further studies GEF-H1 properties were characterized using models of cells transfected with DNA constructs and/or siRNA (1, 4, 12, 23, 26, 31, 40), and MT-associated localization of overexpressed GEF-H1 was demonstrated for nonpolarized cells (12, 23, 26, 31, 40). However, localization of GEF-H1 may also depend on the cell type (1, 4, 23, 31). For example, in Cos, NIH/3T3, and HeLa cells, GEF-H1 colocalizes with MT (23, 31), whereas in human fibroblasts MRC-5 GEF-H1 was associated with actin filaments (4). In the epithelial MDCK cell line, GEF-H1 was colocalized with tight junctions, and in Caco-2 cells GEF-H1 was colocalized with F-actin adjacent to cell junctions but not with F-actin along stress fibers (4). Our experiments show MT-like localization of wild-type GEF-H1 (1–894) and GEF-H1(DH) mutant, whereas truncated GEF-H1 (1–597) mutant lacking MT-binding domain exhibited cytosolic localization (Fig. 3). Thus, consistent with other nonpolarized cells (12, 23, 31, 40), ectopic expression of GEF-H1 in the human lung EC targets it to the MT.
Agonist stimulation induces short-term and reversible activation of Rho. However, potential mechanisms of negative feedback regulation of GEF-H1 and Rho activities after thrombin stimulation are not clear. GEF-H1 has been identified as a substrate for p21 activated kinases (PAK) 1 and 4 (12, 40). It was suggested that in HeLa cells PAK1-mediated phosphorylation of GEF-H1 at Ser885 leads to inhibition of GEF-H1 activity (40). Furthermore, MT-associated GEF-H1 formed a complex with PAK4 via a GEF-H1 interaction domain (12). PAK4-induced GEF-H1 phosphorylation at Ser810 downregulated GEF-H, initiated dissociation of GEF-H1 from MT to cytoplasm, and led to stress fiber disassembly. A very plausible hypothesis suggests that unphosphorylated and PAK-unbound GEF-H1 activates Rho, whereas GEF-H1 interaction with PAK and PAK-mediated phosphorylation suppresses GEF-H1 activity, which leads to downregulation of Rho and activation of Rac. Thus PAK-mediated GEF-H1 phosphorylation may be an important switch between Rho- and Rac-mediated signaling pathways in cytoskeletal regulation of endothelial permeability. Further studies in our laboratory are underway to examine potential role of PAK-mediated GEF-H1 phosphorylation in pulmonary EC barrier regulation.
In summary, this study demonstrates for the first time a critical role for MT-associated Rho GTPase regulator GEF-H1 in the Rho activation in response to thrombin and nocodazole. Our results also demonstrate inhibitory effect of GEF-H1(DH) mutant on thrombin-induced stress fiber formation, MLC, and MYPT1 phosphorylation and thus further support our novel hypothesis about a critical involvement of MT dynamics and MT-bound Rho activator GEF-H1 in thrombin-induced Rho stimulation, actin cytoskeleton remodeling, and EC barrier compromise.
This work was supported by an American Heart Association Scientist Development grant (to A. A. Birukova) and National Heart, Lung, and Blood Institute Grants HL-67307, HL-68062, and HL-58064 (to A. Verin), and HL-076259 and HL-075349 (to K. G. Birukov).
The authors thank Nurgul Moldobaeva for superb laboratory assistance and Maria Birukova for technical assistance in preparation of the manuscript.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 the American Physiological Society