MAP kinases in lung endothelial permeability induced by microtubule disassembly

Anna A. Birukova, Konstantin G. Birukov, Boris Gorshkov, Feng Liu, Joe G. N. Garcia, and Alexander D. Verin

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 1 December 2004 ; accepted in final form 4 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1/2 and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK1/2 and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 µM) and vinblastine (0.1 µM) induced sustained activation of Ras-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 µM, 1 h) attenuated nocodazole-induced ERK1/2 and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 µM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ERK1/2 inhibitor U0126 (5 µM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation.

pulmonary endothelium; actin; extracellular signal-regulated kinases 1/2; mitogen-activated protein


ENDOTHELIAL CELLS (EC) form a semiselective barrier to circulating macromolecules and cellular elements. The integrity of vascular EC monolayer is essential for the maintenance of lung function, whereas compromised EC barrier results in pathological vascular permeability, a key parameter of acute lung injury (13, 17, 32). The actin cytoskeleton is intimately involved in the regulation of EC permeability (13, 17, 32). In addition, we and others have previously described an involvement of the microtubule (MT) network in the regulation of actin cytoskeleton, cell contraction, and vascular permeability, and suggested an important role for the small GTPase Rho-dependent pathway in cellular responses triggered by MT disassembly (8, 14, 15, 44, 47). Cytoskeletal regulation of EC barrier function is mediated by multiple signaling pathways, including mitogen-activated protein kinase (MAPK) cascades (9, 46). Activation of MAPK cascades is involved in a wide variety of cellular responses, including proliferative, inflammatory, apoptotic, and stress response (9, 23, 24). MAPK comprise three major cascades that signal to three major downstream effector kinases: extracellular signal-regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinase (16, 19, 26). Activated MAPK phosphorylate and/or activate various membrane proteins, MAPK-activated protein kinases, nuclear substrates, and cytoskeletal proteins (1, 20, 22, 40). The downstream MAPK targets include focal adhesion protein paxillin, neurofilaments, MT-associated proteins tau and stathmin, and actin cytoskeletal proteins caldesmon and small heat shock actin-capping protein 27-kDa heat shock protein (HSP27) (1, 20, 22, 40). Phosphorylation of HSP27 by MAPK-activated protein kinase II promotes filamentous actin formation and membrane blebbing and mediates actin reorganization and cell migration in human endothelium (22, 39). MAPK signaling plays a critical role in the barrier dysfunction mediated by thrombin, pertussis toxin, TNF-{alpha}, transforming growth factor-{beta}1, hydrogen peroxide (H2O2), and VEGF (2, 9, 10, 18, 21, 27, 28, 37). Moreover, we have recently shown that EC barrier failure induced by a number of inflammatory agonists such as thrombin and TNF-{alpha} is tightly associated with rearrangement and partial dissolution of the MT network (5, 37). These findings suggest a link between MAPK cascades and remodeling of actin and MT cytoskeletons in the agonist-induced EC barrier compromise.

The specific role of ERK1/2 and p38 MAPK in actin remodeling and increased lung endothelial permeability induced by MT disassembly has not yet been investigated. In the present study, we have examined an involvement of MAPK signaling in the lung EC barrier dysfunction induced by MT disassembly. We have studied activation of ERK1/2 and p38 MAPK cascades in bovine and human pulmonary EC induced by MT inhibitors nocodazole and vinblastine, linked these effects with the changes in endothelial barrier properties, and investigated a role of p38 MAPK activation in remodeling of the MT network.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Phospho-ERK1/2 and phospho-p38 MAPK pathway kits, p38 MAPK activity kit, and primary antibodies were purchased from Cell Signaling (Beverly, MA). SB-203580 was obtained from Calbiochem (La Jolla, CA). U0126 was purchased from Promega (Madison, WI). Myosin light chain (MLC) antibody was produced in rabbit against baculovirus-expressed and purified smooth muscle MLC by Biodesign International (Kennebunk, ME). All reagents used for immunofluorescent staining were purchased from Molecular Probes (Eugene, OR). Unless specified, all other reagents were obtained from Sigma Chemical (St. Louis, MO).

Cell cultures. Human pulmonary artery EC were obtained from Cambrex (Walkersville, MD), and bovine pulmonary artery EC (BPAEC) were obtained from American Type Culture Collection (CCL 209; Rockville, MD). They were cultured as described previously (58).

Immunofluorescent staining. EC monolayers plated on gelatin-coated coverslips were treated with agonist and then fixed in 3.7% formaldehyde solution in PBS for 10 min at 4°C, washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS-Tween (PBST) for 30 min at room temperature, and blocked with 2% BSA in PBST for 30 min. Immunofluorescent staining of MT was performed in blocking solution (2% BSA in PBST) for 1 h at room temperature followed by staining with Alexa 488-conjugated secondary antibodies, dilution 1:500 (Molecular Probes). Actin filaments were stained with Texas red-conjugated phalloidin, dilution 1:500 (Molecular Probes), for 1 h at room temperature. After being immunostained, the glass slides were analyzed using a Nikon video imaging system (Nikon Instech, Japan) as described elsewhere (58).

Image analysis of gap formation and stress fiber formation. The fluorescent images were analyzed using MetaVue 4.6 (Universal Imaging, Downington, PA), as previously described (58). Texas red-stained EC monolayers stimulated with agonist of interest were viewed under a fluorescent microscope using an 60XA/1.40 objective. The 16-bit images were analyzed using MetaVue 4.6. Paracellular gaps were manually marked out, and images were differentially segmented between gaps (black) and cells (highest gray value) based on image grayscale levels. The gap formation was expressed as a ratio of the gap area to the area of the whole image. Similarly, actin fibers or MT were marked out, and the ratio to the cell area covered by stress fibers or assembled MT to the whole cell area was determined. The values were statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago, IL) software.

Ras GTPase activation assay. Ras GTPase activation was determined using in vitro pulldown assays available from Upstate Biotechnology (Lake Placid, NY) according to the manufacturer's protocols, as we have previously described (45).

p38 MAPK activation assay. p38 MAPK activation in EC culture was analyzed using a p38 MAPK assay kit available from Cell Signaling (Beverly, MA). All procedures were done according to the manufacturer's protocol. Briefly, after stimulation, EC were harvested under nondenaturing conditions and subjected to immunprecipitation with immobilized phospho-p38 MAPK antibody. Next, using provided buffers and activating transcription factor-2 (ATF-2) as p38 MAPK substrate, the kinase assay was performed. ATF-2 phosphorylation was then detected by Western blotting using phospho-ATF-2 antibody.

In-gel ERK1/2 MAPK activation assay. EC grown to confluent monolayers were preincubated with U0126 followed by stimulation with nocodazole or vinblastine (0.2 and 0.1 µM, respectively). In-gel ERK1/2 MAPK assays were performed using polyacrylamide gels with incorporated myelin basic protein as an ERK1/2 substrate according to our previously described protocol (4).

Determination of HSP27 phosphorylation. Phosphorylation of HSP27 induced by MT disruption was measured in HSP27 immunoprecipitates obtained from 32P-labeled cells. EC monolayers were serum starved in PO43–-free DMEM for 16 h, followed by radioactive labeling with [{gamma}-32P]orthophosphate for 4 h. After being rinsed with PO43–-free DMEM, cells were preincubated with taxol (5 µM) or vehicle controls for 30 min and then treated with nocodazole (0.2 µM) for 30 min. Next, cells were lysed in lysis buffer containing 10 mM Tris, pH 7.4, 1 mM sodium orthovanadate, and 1% SDS. HSP27 was immunoprecipitated from supernatants using 20 µg of monoclonal anti-HSP27 antibody followed by 1-h incubation with preequilibrated Protein G 4 Fast Flow Sepharose (Amersham) at 4°C with gentle agitation. After four washings with immunoprecipitation buffer (20 mM Tris, pH 7.4, 300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.4 mM sodium orthovanadate, 2% Triton X-100, 1% Nonidet P-40), the supernatant was subjected to SDS-PAGE, and protein was transferred to nitrocellulose and exposed to a phosphoimager plate overnight. The intensity of phosphorylated HSP27 bands was quantified using Molecular Dynamics Phosphoimager 445 SI, and the total HSP27 from the same blot was detected by Western blotting using anti-HSP27 antibody.

MLC phosphorylation assay. Monophosphorylated, diphosphorylated, and nonphosphorylated MLC in bovine pulmonary EC were separated by urea gel electrophoresis followed by Western blot with pan-MLC antibody, as described elsewhere (8, 17, 44).

Western immunoblotting. After being stimulated, cells were washed with PBS and lysed with cell lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.2 mM vanadate, 0.2 mM PMSF, and 0.5% phosphatase inhibitor cocktail. Total cell lysates were cleared by centrifugation and boiled with the same amount of 3x SDS sample buffer for 5 min. Protein extracts were separated by SDS-PAGE on 10% gels for detection of MEK1/2, ERK-1/2, MKK3/6, p38, and MAPKAPK2; 7.5% gels for detection of Raf, p90RSK, Elk, and ATF-2; and 12.5% gels for detection of HSP27. The separated proteins were transferred to nitrocellulose membranes by electrotransfer (30 V for 18 h or 90 V for 2 h). The blots were subsequently blocked with 5% nonfat dry milk in PBST at room temperature for 1 h and then incubated at 4°C overnight with primary specific antibodies of interest. After being washed three times for 10 min with PBST, the membrane was incubated with horseradish peroxidase-linked IgG secondary antibody at room temperature for 1 h, followed by three washes for 10 min with PBST. Immunoreactive proteins were detected using an enhanced chemiluminescent detection system according to the manufacturer's protocol (Amersham, Little Chalfont, UK). The amount of detected proteins was analyzed using Image Quant software.

Measurement of transendothelial electrical resistance. The cellular barrier properties were measured using the highly sensitive biophysical assay with an electrical cell-substrate impedance sensing system (Applied Biophysics, Troy, NY) as described previously (7, 8, 44).

Statistical analysis. Results are expressed as means ± SD of three to eight 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 were used. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of MT disruption on ERK1/2 MAPK cascade activation. EC were treated with nocodazole. Phosphorylation of c-Raf, MEK1/2, ERK1/2, p90RSK, and Elk was detected by immunoblotting with corresponding phosphospecific antibodies (Fig. 1A). Equal loadings were confirmed by reprobing of the membranes with pan-ERK antibody. Nocodazole-induced MT disassembly caused rapid and sustained activation of c-Raf, observed after 5 min, and reached maximal levels by 15 min and remained elevated up to 60 min. Activation of c-Raf was followed by activation of its downstream signaling cascade, which included MEK1/2, ERK1/2 MAPK, and ERK1/2 substrates p90RSK and Elk with maximal activation at 30 and 45 min. Importantly, activation of ERK1/2 MAPK cascade correlated with the onset of MT disassembly in lung EC, which was observed as early as 5 min after nocodazole treatment (Figs. 1A and 3C). Because small GTPase Ras may serve as upstream activator of c-Raf, we next measured effects of MT depolymerization on Ras activity directly using in vitro pulldown assay described previously (45). On the basis of previous findings from our laboratory showing PMA-induced Ras and ERK1/2 activation (45), treatment of EC with PMA was used as a positive control in Fig. 1, B and C. EC treatment with nocodazole caused a 150% increase in Ras activity, which was almost completely attenuated by the MT stabilizer taxol (Fig. 1B). Consistent with these findings, MT stabilization by taxol significantly attenuated nocodazole-induced ERK1/2 phosphorylation (Fig. 1C). MT inhibitor-induced ERK1/2 activation was further evaluated by in-gel ERK1/2 activation assay as described in MATERIALS AND METHODS. Figure 1D shows that EC treatment with nocodazole or vinblastine dramatically increased phosphorylation of the ERK1/2 substrate myelin basic protein. These effects were significantly attenuated by the pharmacological MEK inhibitor U0126 (5 µM, 1 h). Thus our results demonstrate that MT depolymerization by nocodazole and vinblastine induced activation of ERK1/2-mediated signaling in the lung EC.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 1. Effect of microtubule (MT) inhibitors on extracellular signal-regulated kinases (ERK) 1/2 MAPK cascade activation. A: human pulmonary artery EC (HPAEC) were treated with either vehicle or nocodazole (0.2 µM) for the indicated periods of time. Activation of ERK1/2 MAPK cascade was analyzed by Western blot with a panel of phosphospecific antibodies (p-Raf, p-MEK1/2, p-ERK1/2, p-p90RSK, p-Elk) as described in MATERIALS AND METHODS. Equal protein loading was confirmed by reprobing of membranes with a pan-ERK antibody. B: bovine pulmonary artery EC (BPAEC) were preincubated with either vehicle or taxol (5 µM, 1 h) and then stimulated with nocodazole (0.2 µM, 30 min). As a positive control, cells were stimulated with 100 nM PMA for 10 min. Ras activation assay was performed as described in MATERIALS AND METHODS. Shown are representative results from 3 independent experiments. *P < 0.05. C: BPAEC monolayers were preincubated with either vehicle or taxol (5 µM, 1 h) and then treated with nocodazole (0.2 µM) for the indicated time periods. As a positive control, cells were stimulated with 100 nM PMA for 10 min. Cells were lysed and subjected to Western blot analysis with phospho-ERK1/2 antibody. D: BPAEC were preincubated with either vehicle or U0126 (5 µM, 1 h) followed by treatment with nocodazole (0.2 µM) or vinblastine (0.1 µM) for 30 min. MAPK activation assay was performed as described in MATERIALS AND METHODS. Cells were lysed and subjected to Western blot analysis with phospho-ERK1/2 antibody. Quantitative analysis of protein phosphorylation was performed by scanning densitometry of the membranes and was expressed in relative density units (RDU). Shown are representative results from 3 independent experiments. *P < 0.05. ND, nocodazole; VB, vinblastine.

 


View larger version (134K):
[in this window]
[in a new window]
 
Fig. 3. Effect of ERK and p38 MAPK inhibition on EC permeability induced by MT disruption. EC were plated on gold microelectrodes and cultured to confluence. A: EC were treated with vehicle or U0126 (5 µM; first arrow). At the time indicated by second arrow, cells were treated with vehicle or nocodazole (0.2 µM), and transendothelial resistance (TER) was monitored for 4 h. Shown are pooled data of 8 independent experiments. B: at the time indicated by first arrow, BPAEC were pretreated with either vehicle or SB-203580 (20 µM). At the time indicated by second arrow, cells were treated with vehicle or nocodazole (0.2 µM), and TER was monitored for 4 h. Shown are pooled data of 8 independent experiments. C: HPAEC grown on glass coverslips were treated with nocodazole (0.2 µM) for indicated time periods. Cells were then fixed and stained for {beta}-tubulin with specific antibody or for filamentous (F) actin with Texas red phalloidin. Shown are representative results of 3 independent experiments.

 
Effect of MT disruption on p38 MAPK cascade activation. To examine the effects of MT depolymerization on p38 MAPK signaling, cells were treated with nocodazole for the periods of time indicated above, and activation of p38 MAPK cascade was evaluated by monitoring the phosphorylation status of p38-related MAPK cascade MKK3/6-p38 MAPK-MAPKAPK2 and downstream substrate ATF-2. As shown in Fig. 2A, MT disassembly induced activation of MKK3/6, p38 MAPK, MAPKAPK2, and ATF-2 phosphorylation, which increased over time with maximal levels at 30 min. Equal loading was confirmed by reprobing membranes with pan-p38 antibody. To confirm that p38 MAPK cascade activation was due to MT depolymerization, EC were pretreated with taxol before nocodazole or vinblastine challenge. MT stabilization by taxol significantly attenuated nocodazole- and vinblastine-induced p38 MAPK phosphorylation (Fig. 2B). Moreover, additional in vitro p38 MAPK activity assays showed that SB-203580 and taxol prevented nocodazole- or vinblastine-induced phosphorylation of p38 MAPK downstream target ATF-2 (Fig. 2C). These findings clearly indicate activation of the p38 MAPK cascade by MT disassembly.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2. Effect of MT inhibitors on p38 MAPK cascade activation. A: HPAEC were treated with either vehicle or nocodazole (0.2 µM) for the indicated periods of time. Activation of p38 MAPK cascade was analyzed by Western blot analysis with a panel of phosphospecific antibodies [p-MKK3/6, p-p38, p-MAPKAPK2, p-activating transcription factor (ATF)] as described in MATERIALS AND METHODS. Equal protein loading was confirmed by reprobing of membranes with a pan-p38 antibody. B: BPAEC monolayers were preincubated with either vehicle or taxol (5 µM, 1 h) and then stimulated with nocodazole (0.2 µM, 30 min) or vinblastine (0.1 µM, 30 min). Cells were lysed and subjected to Western blot analysis with phospho-p38 or pan-p38 antibodies. C: BPAEC were preincubated with either vehicle, taxol (5 µM, 1 h), or SB-203580 (20 µM, 1 h) and then treated with nocodazole (0.2 µM, 30 min) or vinblastine (0.1 µM, 30 min). In vitro p38 MAPK activation assay was performed using ATF-2 as a substrate, as described in MATERIALS AND METHODS. Cells were lysed and subjected to Western blot analysis with phospho-ATF-2 antibody. Quantitative analysis of protein phosphorylation was performed by scanning densitometry of the membranes and expressed in RDU. Shown are representative results from 3 independent experiments. *P < 0.05.

 
Different roles for ERK1/2 and p38 MAPK in EC permeability induced by MT disruption. Previous reports have described agonist- and cell-specific effects of ERK1/2 and p38 MAPK activation on barrier regulation (11, 3335, 39, 42). To examine a role of ERK1/2- and p38-dependent MAPK pathways in EC barrier dysfunction induced by MT disassembly, we measured transendothelial resistance (TER) across EC monolayers. Treatment of lung EC monolayers with nocodazole dramatically decreased electrical resistance, reflecting endothelial barrier compromise. Importantly, EC pretreatment with U0126 had no effect on nocodazole-induced permeability increase (Fig. 3A), whereas inhibition of p38 MAPK with SB-203580 significantly attenuated drop in TER in response to nocodazole (Fig. 3B). These findings suggest involvement of p38 MAPK, but not ERK1/2, in nocodazole-induced lung EC barrier dysfunction.

As a complementary approach, we have shown the correlation between permeability changes, EC cytoskeletal remodeling, and MAPK activation in response to nocodazole. Our results demonstrate that initial permeability increase (5 min) (Fig. 3B) was accompanied by shortening and partial disassembly of MT (Fig. 3C), formation of actin fibers and paracellular gaps (Fig. 3C), and activation of ERK1/2 and p38 MAPK cascades (Figs. 1A and 2A). Further increase in permeability (15, 30 min, Fig. 3B) was associated with significant changes in actin and MT structure and pronounced MAPK activation (Figs. 1A, 2A, 3C, 4A, and 5A, top). These results suggest a correlation between activation of ERK1/2 and p38 MAPK cascades, cytoskeletal remodeling, and permeability changes. However, inhibitory analyses have demonstrated that only p38 MAPK cascade activation is tightly linked to cellular responses mediated by MT disassembly (Fig. 3, A and B).



View larger version (137K):
[in this window]
[in a new window]
 
Fig. 4. Effect of ERK and p38 MAPK inhibition on nocodazole-induced actin cytoskeleton remodeling. EC grown on glass coverslips were pretreated with vehicle (Veh), SB-203580 (20 µM), or U0126 (5 µM) for 1 h before being stimulated with nocodazole (0.2 µM, 30 min). After being stimulated, cells were fixed, and F-actin was visualized by staining with Texas red phalloidin (A). Paracellular gaps are marked by open arrows and stress fibers are marked by gray arrows. Results are representative of 3 independent experiments. Stress fiber formation (B) and gap formation (C) were quantitated by morphometric analysis of Texas red phalloidin-stained BPAEC, as described in MATERIALS AND METHODS. Shown are representative results of 3 independent experiments. *P < 0.05. U0, U0126.

 


View larger version (102K):
[in this window]
[in a new window]
 
Fig. 5. Effect of ERK and p38 MAPK inhibition on MT rearrangement induced by nocodazole. Lung EC grown on glass coverslips were pretreated with vehicle, SB-203580 (20 µM), or U0126 (5 µM) for 1 h before being stimulated with nocodazole (0.2 µM, 30 min). A: after being stimulated, cells were fixed, and {beta}-tubulin was visualized by immunofluorescent staining with {beta}-tubulin antibody. B: microtubule dissolution induced by nocodazole was assessed by morphometric analysis of {beta}-tubulin-stained EC, as described in MATERIALS AND METHODS. Shown are representative results of 3 independent experiments. *P < 0.05.

 
Effects of ERK1/2 and p38 MAPK inhibition on actin cytoskeletal rearrangement induced by MT disruption. We and others (8, 17, 32) have previously shown a key role for actin remodeling in the regulation of EC barrier properties. In the next series of experiments, we examined effects of ERK1/2 and p38 MAPK inhibition on the nocodazole-induced actin and MT remodeling using immunofluorescent staining of EC monolayers. MT depolymerization by nocodazole induced formation of massive stress fibers (Fig. 4, filled arrows) and paracellular gaps (Fig. 4, open arrows), whereas p38 MAPK inhibition significantly reduced these effects (Fig. 4A, middle). In contrast, inhibition of ERK1/2 MAPK did not affect nocodazole-induced stress fibers and gap formation (Fig. 4A, bottom). Next, we quantitatively assessed gap and stress fiber formation and performed morphometric analysis of Texas red phalloidin-stained BPAEC using MetaVue software, as described in MATERIALS AND METHODS and in our previous studies (58). Inhibition of p38 MAPK by SB-203580 decreased amount of stress fibers by 53% (Fig. 4B) and formation of paracellular gaps by 78% (Fig. 4C) compared with cells treated with nocodazole alone. In contrast, EC pretreatment with U0126 had no effect on nocodazole-induced stress fiber and gap formation (Fig. 4, B and C). These data are highly consistent with the effects of ERK1/2 and p38 MAPK on nocodazole-induced EC permeability changes (Fig. 3) and suggest an essential role for p38 MAPK in the EC barrier dysfunction induced by MT disassembly.

Effects of ERK1/2 and p38 MAPK inhibition on MT disassembly induced by nocodazole. To study a potential role of MAPK signaling in MT remodeling, EC were preincubated with ERK1/2 or p38 MAPK inhibitors before nocodazole stimulation. Inhibition of p38 MAPK activity by SB-203580 significantly preserved MT structure against nocodazole-induced disassembly (Fig. 5A, middle), whereas inhibition of ERK1/2 MAPK by U0126 was without effect (Fig. 5A, bottom). Quantitative analysis of assembled MT showed that SB-203580 pretreatment before nocodazole stimulation preserved 26 ± 9% of the total MT pool, compared with nearly complete MT depolymerization in EC stimulated with nocodazole alone or cells pretreated with U0126 and stimulated with nocodazole (Fig. 5B).

MT disassembly causes p38 MAPK-mediated phosphorylation of actin cytoskeletal proteins. HSP27 is a terminal p38 MAPK substrate involved in actin remodeling and barrier regulation (22, 25). Treatment of EC with nocodazole induced phosphorylation of HSP27 in a time-dependent manner, which was detected at 5 min and reached maximal levels at 45–60 min (Fig. 6A). Inhibition of p38 MAPK by SB-203580 abolished nocodazole-induced HSP27 phosphorylation (Fig. 6A). Importantly, pretreatment of EC monolayers with taxol abolished HSP27 phosphorylation, as detected in immunoprecipitates from 32P-labeled cells (Fig. 6B). Because MLC phosphorylation is crucial for activation of actomyosin contraction and increased EC permeability associated with MT disassembly (6, 8), we next examined effects of MT stabilization and p38 MAPK inhibition on nocodazole- and vinblastine-induced MLC phosphorylation. Pretreatment of BPAEC with SB-203580 or taxol significantly decreased MLC phosphorylation levels in response to nocodazole or vinblastine treatment (Fig. 6C). These results support an essential role for p38 MAPK-dependent signaling in actin remodeling triggered by MT disassembly.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6. Effects of MT disassembly on phosphorylation of p38 MAPK, 27-kDa heat shock protein (HSP27), and myosin light chain (MLC). A: HPAEC were preincubated with either vehicle or SB-203580 (20 µM, 1 h) followed by treatment with nocodazole (0.2 µM) for the indicated periods of time. Phosphorylation of HSP27 was analyzed by Western blot with phospho-HSP27 antibody. Equal protein loading was confirmed by reprobing of the membrane with a pan-HSP27 antibody. B: BPAEC were pretreated with either vehicle or taxol (5 µM, 30 min) followed by treatment with nocodazole (0.2 µM, 30 min). Measurement of HSP27 phosphorylation in EC preloaded with 32P-labeled phosphate was performed as described in MATERIALS AND METHODS. C: BPAEC monolayers were preincubated with vehicle, SB-203580 (20 µM), or taxol (5 µM) for 1 h followed by stimulation with nocodazole (0.2 µM) or vinblastine (0.1 µM) for 30 min. Shown are immunoblots of MLC species separated by urea gel electrophoresis and probed with anti-MLC antibody. Quantitative analysis of protein phosphorylation was performed by scanning densitometry of the membranes. Measurements of HSP27 phosphorylation are expressed in RDU, and content of mono- (mono-P) and diphosphorylated (di-P) MLC is calculated as a percent of total MLC content. Shown are representative results from 3 independent experiments. *P < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Studies from different groups suggest that p38 and ERK1/2 MAPK may exhibit different effects on the cell cytoskeleton and barrier properties in a context of specific cell type and specific agonist. For example, inhibition of p38 MAPK, but not ERK1/2 MAPK, reduced TNF-{alpha}-induced permeability in human umbilical vein EC (34), whereas in human lung microvascular EC, both p38 and ERK1/2 MAPK were essential for TNF-{alpha}-induced permeability (35). In BPAEC and lung microvascular EC, only p38 MAPK inhibition attenuated H2O2-induced permeability, but both p38 and ERK1/2 MAPK were involved in VEGF-mediated permeability increase (2, 33, 43). These results emphasize physiological and biochemical diversity among different cell types or EC of different origins.

The results of our study show that nocodazole treatment causes activation of Ras-c-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 signaling cascades (Figs. 1 and 2), whereas only the p38 MAPK cascade is involved in nocodazole-induced EC barrier dysfunction and actin remodeling (Figs. 3 and 4). Furthermore, our results show a direct link between nocodazole-induced MT depolymerization and p38 MAPK-mediated phosphorylation of cytoskeletal target proteins HSP27 and MLC, which are intimately involved in actin remodeling and EC contraction (10, 18, 21). Phosphorylation of HSP27 may be achieved via p38 MAPK effector MAPKAPK2 (41). In addition to phosphorylation of HSP27, MAPKAPK2 mediates MLC phosphorylation at Ser19 and activates Mg2+-ATPase activity of myosin II (29).

Our data show that pharmacological inhibition of p38 MAPK partially protected MT structure from nocodazole-induced MT disassembly, which was associated with partial attenuation of nocodazole-induced permeability changes. Conversely, MT stabilization by taxol attenuated phosphorylation of p38 MAPK and its downstream targets HSP27 and MLC. These results are also consistent with previous studies, which demonstrated the barrier-protective effect of MT stabilization in TNF-{alpha} and thrombin models of lung endothelial permeability (5, 37) and indicate the tight link between p38 MAPK activity and MT remodeling.

We have previously described the involvement of the Rho-dependent pathway in barrier dysfunction mediated by MT inhibitors (8, 44). Based on the results of this study and previous reports, we speculate that p38 MAPK-mediated signaling represents an additional pathway linking MT disassembly and EC barrier dysfunction. The primary EC cytoskeletal response to MT disassembly may involve activation of p38 MAPK cascade by release and additional activation of MT-associated MKK3/6 pool (12). These signaling events lead to phosphorylation of HSP27 and MLC, which triggers actin remodeling, cell contraction, and increased vascular endothelial permeability. In addition, a positive feedback mechanism of MT disassembly may involve p38 MAPK-mediated phosphorylation of MT-associated regulatory proteins, which promote further destabilization of the MT network. For example, MT-associated protein tau, in its unphosphorylated form, stabilizes MT and promotes MT assembly, whereas phosphorylation of tau at multiple serine/threonine sites by a number of kinases, including p38 MAPK, decreases tau capacity to bind MT and leads to MT disassembly (20, 31, 38). Another potential mechanism of p38 MAPK-mediated alteration of MT structure involves a regulator of MT dynamics, stathmin (3, 30). Similar to tau family proteins, phosphorylation of stathmin by p38{delta} MAPK isoform alters stathmin binding to MT and results in MT destabilization (36). Studies addressing involvement of tau and stathmin in p38 MAPK-mediated MT dynamics and EC barrier regulation are currently underway in our laboratory.

In conclusion, this study examined specific involvement of ERK1/2 and p38 MAPK signaling in regulation of the actin cytoskeleton, MT stability, and permeability in the lung EC. Our results show involvement of p38 MAPK, but not ERK1/2 MAPK, in MT-mediated EC barrier regulation. In addition, our data suggest a similar role for p38 MAPK-mediated signaling in cytoskeletal and barrier regulation observed in human and bovine EC. Thus phosphorylation of cytoskeletal regulatory proteins such as HSP27, MLC, tau, or stathmin by p38 MAPK may represent an important mechanism for regulation of MT network stability and signaling from MT to actin cytoskeleton, which is critical for fine tuning of the lung endothelial barrier regulation.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67307, HL-68062, and HL-58064 (A. D. Verin) and American Heart Association Scientist Development Grant (A. A. Birukova).


    ACKNOWLEDGMENTS
 
The authors thank Nurgul Moldobaeva for superb laboratory assistance and Maria Birukova for technical assistance in preparation of the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Verin, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Univ. School of Medicine, 5200 Eastern Ave., MFL Center Tower, Rm. 676, Baltimore, MD 21224 (E-mail: averin1{at}jhmi.edu)

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


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adam LP, Haeberle JR, and Hathaway DR. Phosphorylation of caldesmon in arterial smooth muscle. J Biol Chem 264: 7698–7703, 1989.[Abstract/Free Full Text]
  2. Becker PM, Verin AD, Booth MA, Liu F, Birukova A, and Garcia JG. Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 281: L1500–L1511, 2001.[Abstract/Free Full Text]
  3. Belmont L, Mitchison T, and Deacon HW. Catastrophic revelations about Op18/stathmin. Trends Biochem Sci 21: 197–198, 1996.[CrossRef][ISI][Medline]
  4. Birukov KG, Lehoux S, Birukova AA, Merval R, Tkachuk VA, and Tedgui A. Increased pressure induces sustained protein kinase C-independent herbimycin A-sensitive activation of extracellular signal-related kinase 1/2 in the rabbit aorta in organ culture. Circ Res 81: 895–903, 1997.[Abstract/Free Full Text]
  5. Birukova A, Birukov K, Smurova K, Adyshev D, Kaibuchi K, Alieva I, Garcia JG, and Verin A. Novel role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB J 18: 1879–1890, 2004.[Abstract/Free Full Text]
  6. Birukova A, Liu F, Garcia J, and Verin A. Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am J Physiol Lung Cell Mol Physiol 287: L86–L93, 2004.[Abstract/Free Full Text]
  7. Birukova AA, Smurova K, Birukov KG, Kaibuchi K, Garcia JG, and Verin AD. Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvasc Res 67: 64–77, 2004.[CrossRef][ISI][Medline]
  8. Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms. J Cell Physiol 201: 55–70, 2004.[CrossRef][ISI][Medline]
  9. Bogatcheva NV, Dudek SM, Garcia JG, and Verin AD. Mitogen-activated protein kinases in endothelial pathophysiology. J Investig Med 51: 341–352, 2003.[ISI][Medline]
  10. Borbiev T, Birukova A, Liu F, Nurmukhambetova S, Gerthoffer WT, Garcia JG, and Verin A. p38 MAP kinase-dependent regulation of endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol 287: L911–L918, 2004.[Abstract/Free Full Text]
  11. Breslin JW, Pappas PJ, Cerveira JJ, Hobson RW II, and Duran WN. VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide. Am J Physiol Heart Circ Physiol 284: H92–H100, 2003.[Abstract/Free Full Text]
  12. Cheung PY, Zhang Y, Long J, Lin S, Zhang M, Jiang Y, and Wu Z. p150(Glued), Dynein, and microtubules are specifically required for activation of MKK3/6 and p38 MAPKs. J Biol Chem 279: 45308–45311, 2004.[Abstract/Free Full Text]
  13. Dudek SM and Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91: 1487–1500, 2001.[Abstract/Free Full Text]
  14. Elbaum M, Chausovsky A, Levy ET, Shtutman M, and Bershadsky AD. Microtubule involvement in regulating cell contractility and adhesion-dependent signalling: a possible mechanism for polarization of cell motility. Biochem Soc Symp 65: 147–172, 1999.[Medline]
  15. Enomoto T. Microtubule disruption induces the formation of actin stress fibers and focal adhesions in cultured cells: possible involvement of the rho signal cascade. Cell Struct Funct 21: 317–326, 1996.[ISI][Medline]
  16. Errede B, Cade RM, Yashar BM, Kamada Y, Levin DE, Irie K, and Matsumoto K. Dynamics and organization of MAP kinase signal pathways. Mol Reprod Dev 42: 477–485, 1995.[CrossRef][ISI][Medline]
  17. Garcia JG, Davis HW, and Patterson CE. Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J Cell Physiol 163: 510–522, 1995.[CrossRef][ISI][Medline]
  18. Garcia JG, Wang P, Schaphorst KL, Becker PM, Borbiev T, Liu F, Birukova A, Jacobs K, Bogatcheva N, and Verin AD. Critical involvement of p38 MAP kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability. FASEB J 16: 1064–1076, 2002.[Abstract/Free Full Text]
  19. Garrington TP and Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11: 211–218, 1999.[CrossRef][ISI][Medline]
  20. Goedert M, Hasegawa M, Jakes R, Lawler S, Cuenda A, and Cohen P. Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases. FEBS Lett 409: 57–62, 1997.[CrossRef][ISI][Medline]
  21. Goldberg PL, MacNaughton DE, Clements RT, Minnear FL, and Vincent PA. p38 MAPK activation by TGF-{beta}1 increases MLC phosphorylation and endothelial monolayer permeability. Am J Physiol Lung Cell Mol Physiol 282: L146–L154, 2002.[Abstract/Free Full Text]
  22. Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, and Landry J. Regulation of actin filament dynamics by p38 MAP kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 110: 357–368, 1997.[Abstract/Free Full Text]
  23. Herlaar E and Brown Z. p38 MAPK signalling cascades in inflammatory disease. Mol Med Today 5: 439–447, 1999.[CrossRef][ISI][Medline]
  24. Hoefen RJ and Berk BC. The role of MAP kinases in endothelial activation. Vascul Pharmacol 38: 271–273, 2002.[CrossRef][ISI][Medline]
  25. Huot J, Houle F, Rousseau S, Deschesnes RG, Shah GM, and Landry J. SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. J Cell Biol 143: 1361–1373, 1998.[Abstract/Free Full Text]
  26. Johnson GL and Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298: 1911–1912, 2002.[Abstract/Free Full Text]
  27. Kevil CG, Oshima T, and Alexander JS. The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability. Endothelium 8: 107–116, 2001.[Medline]
  28. Kiemer AK, Weber NC, Furst R, Bildner N, Kulhanek-Heinze S, and Vollmar AM. Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-{alpha}-induced actin polymerization and endothelial permeability. Circ Res 90: 874–881, 2002.[Abstract/Free Full Text]
  29. Komatsu S and Hosoya H. Phosphorylation by MAPKAP kinase 2 activates Mg2+-ATPase activity of myosin II. Biochem Biophys Res Commun 223: 741–745, 1996.[CrossRef][ISI][Medline]
  30. Lawler S. Microtubule dynamics: if you need a shrink try stathmin/Op18. Curr Biol 8: R212–R214, 1998.[CrossRef][ISI][Medline]
  31. Li Y, Liu L, Barger SW, and Griffin WS. Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway. J Neurosci 23: 1605–1611, 2003.[Abstract/Free Full Text]
  32. Lum H and Malik AB. Mechanisms of increased endothelial permeability. Can J Physiol Pharmacol 74: 787–800, 1996.[CrossRef][ISI][Medline]
  33. Niwa K, Inanami O, Ohta T, Ito S, Karino T, and Kuwabara M. p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not. Free Radic Res 35: 519–527, 2001.[ISI][Medline]
  34. Nwariaku FE, Chang J, Zhu X, Liu Z, Duffy SL, Halaihel NH, Terada L, and Turnage RH. The role of p38 MAP kinase in tumor necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability. Shock 18: 82–85, 2002.[ISI][Medline]
  35. Nwariaku FE, Rothenbach P, Liu Z, Zhu X, Turnage RH, and Terada LS. Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability. J Appl Physiol 95: 1889–1895, 2003.[Abstract/Free Full Text]
  36. Parker CG, Hunt J, Diener K, McGinley M, Soriano B, Keesler GA, Bray J, Yao Z, Wang XS, Kohno T, and Lichenstein HS. Identification of stathmin as a novel substrate for p38 delta. Biochem Biophys Res Commun 249: 791–796, 1998.[CrossRef][ISI][Medline]
  37. Petrache I, Birukova A, Ramirez SI, Garcia JG, and Verin AD. The role of the microtubules in tumor necrosis factor-{alpha}-induced endothelial cell permeability. Am J Respir Cell Mol Biol 28: 574–581, 2003.[Abstract/Free Full Text]
  38. Reynolds CH, Nebreda AR, Gibb GM, Utton MA, and Anderton BH. Reactivating kinase/p38 phosphorylates tau protein in vitro. J Neurochem 69: 191–198, 1997.[ISI][Medline]
  39. Rousseau S, Houle F, Landry J, and Huot J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15: 2169–2177, 1997.[CrossRef][ISI][Medline]
  40. Roux PP and Blenis J. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68: 320–344, 2004.[Abstract/Free Full Text]
  41. Stokoe D, Engel K, Campbell DG, Cohen P, and Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313: 307–313, 1992.[CrossRef][ISI][Medline]
  42. Usatyuk PV and Natarajan V. Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells. J Biol Chem 279: 11789–11797, 2004.[Abstract/Free Full Text]
  43. Usatyuk PV, Vepa S, Watkins T, He D, Parinandi NL, and Natarajan V. Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells. Antioxid Redox Signal 5: 723–730, 2003.[CrossRef][ISI][Medline]
  44. Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation. Am J Physiol Lung Cell Mol Physiol 281: L565–L574, 2001.[Abstract/Free Full Text]
  45. Verin AD, Liu F, Bogatcheva N, Borbiev T, Hershenson MB, Wang P, and Garcia JG. Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 279: L360–L370, 2000.[Abstract/Free Full Text]
  46. Yuan SY. Protein kinase signaling in the modulation of microvascular permeability. Vascul Pharmacol 39: 213–223, 2002.[CrossRef][ISI][Medline]
  47. Zhang Q, Magnusson MK, and Mosher DF. Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rho-dependent actin stress fiber formation and cell contraction. Mol Biol Cell 8: 1415–1425, 1997.[Abstract]