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Am J Physiol Lung Cell Mol Physiol 292: L487-L499, 2007. First published September 29, 2006; doi:10.1152/ajplung.00217.2006
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Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction

Natalia V. Bogatcheva, Djanybek Adyshev, Bolot Mambetsariev, Nurgul Moldobaeva, and Alexander D. Verin

University of Chicago, Department of Medicine, Chicago, Illinois

Submitted 14 June 2006 ; accepted in final form 28 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.

permeability; tubulin reorganization

2-METHOXYESTRADIOL (2ME), a derivative of the main natural estrogen, 17beta-estradiol, has a potential to be added soon to the pharmacopeia of cancer treatment. Currently, it is tested in the therapy of several cancers to assess the maximum-tolerated doses and clinical efficiency (9, 22). The first reported biological activity of 2ME was its effect on the mitotic spindles (19). This effect was elicited independently of estrogen receptors and was later attributed to the direct interaction with tubulin. 2ME competes with colchicine for the binding to tubulin and exerts its action in vivo via the inhibition of microtubule (MT) formation (10). Thus, unlike MT stabilizer taxol, another effective drug used for cancer treatment, 2ME destabilizes MT. Aside from cancer cells, 2ME was shown to affect the proliferation of smooth muscle cells and endothelial cells (EC). The latter fact holds further promise for the treatment of cancer, as regulators of the angiogenesis were shown to be effective anti-cancer therapeutic tools (15). 2ME effect on EC is not confined to the functions related to angiogenesis. 2ME was shown to stimulate the production of prostacyclin and decrease the production of endothelin-1 by EC (11), connecting 2ME to the regulation of blood pressure. However, the effect of 2ME on one of the major endothelial functions, the control of transendothelial permeability, remained unstudied to date.

We (1, 3, 24) have previously described the critical role of MT cytoskeleton for the maintenance of endothelial barrier function. Inhibition of tubulin polymerization by nocodazole and vinblastine dramatically increased transendothelial permeability (24). Aside from destabilization of MT, nocodazole and vinblastine were also shown to elicit their effects indirectly, via the cross-talk with actin cytoskeleton, inducing the rearrangement of actin cytoskeleton and stress fiber formation (24). The latter event was a consequence of the increase in myosin light chain (MLC) phosphorylation, which was linked to the Rho kinase (ROCK) activation (24). The special role in the signaling cascade, initiated by the MT disruptors, is played by mitogen-activated protein kinases (MAPKs) (2). MAPKs comprise three major kinases: extracellular signal-regulated kinases (ERK1/2), p38, and c-Jun NH2-terminal kinase. The most studied cytoskeletal targets of MAPKs cascades are caldesmon, regulator of actomyosin ATPase, and 27-kDa heat shock protein (HSP27), actin-stabilizing chaperone.

In this study we have analyzed the effect of 2ME on endothelial permeability in human pulmonary artery EC (HPAEC). We have studied the role of MT dynamics and microfilament contractility in 2ME-induced increase in endothelial permeability. We examined the involvement of several protein kinases, including ROCK and MAP kinases, in 2ME-induced barrier dysfunction. This study may help to assess the potential side effects of 2ME therapy and to analyze the molecular mechanisms of the 2ME-induced barrier disruption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. 2ME was obtained from Tocris (Ellisville, MO). MLC kinase (MLCK) inhibitor ML-7, inhibitors Y27632, U0126, and SB203580 were purchased from Calbiochem (La Jolla, CA). JNK inhibitor 1 was from Alexis Biochemicals (San Diego, CA). MLC inhibitor PIK (17, 27) was a generous gift from Dr. J. R. Turner (University of Chicago, Chicago, IL). Phospho-ERK1/2, phospho-p38, phospho-JNK, diphospho-MLC, phospho-HSP27, and HSP27 antibodies were purchased from Cell Signaling (Beverly, MA). Phospho-caldesmon and anti-JNK antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Monoclonal anti-caldesmon (clone C21) and anti-MLC antibodies were from Sigma. Monoclonal anti-tubulin antibodies were purchased from Covance (Berkeley, CA). Anti-GAPDH antibodies were from Ambion (Austin, TX). ROCK1/ROCKbeta and ROCK2/ROCK{alpha} antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA). All reagents used for immunofluorescent staining were obtained from Invitrogen (Carlsbad, CA).

Cell culture. HPAEC were purchased from Cambrex (Walkersville, MD) and used at passages 6–10. They were cultured in complete media and maintained at 37°C in a humidified atmosphere of 5% CO2-95% air.

Depletion of endogenous ROCK1 and ROCK2 in EC. To reduce the content of endogenous ROCK1 and ROCK2, HPAEC were treated with ROCK1- and ROCK2-specific small interfering RNA (siRNA) duplexes, which guide sequence-specific degradation of the homologous mRNA (12). Functionally validated siRNA was ordered from Qiagen (Valencia, CA) and used for targeting sequences that are part of coding region for ROCK1 and ROCK2. Nonspecific, nonsilencing siRNA duplex (Qiagen) 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. At 48 h posttransfection, cells were harvested and used for experiments.

Measurement of transendothelial permeability. Transendothelial electrical resistance (TER) was measured using the highly sensitive biophysical assay with an electrical cell-substrate impedance sensor (Applied Biophysics, Troy, NY) as described previously (24). Transendothelial permeability for high molecular weight molecules was measured with Chemicon in vitro vascular permeability assay utilizing 2,000-kDa FITC-dextran. For both assays, cells grown to confluence on gold electrodes or collagen-coated 24-well inserts were serum-starved for 1 h before addition of any inhibitor/stimulator. Statistical analysis was performed using one-way ANOVA test; results with P < 0.05 were considered significantly different.

EC imaging and image analysis. EC monolayers plated on gelatin-coated coverslips were serum-starved for 1 h before the addition of any inhibitor/stimulator. Cells were fixed, permeabilized, and stained with anti-tubulin antibody, Alexa 594-phalloidin, or 4,6-diamidino-2-phenylindole (DAPI) as described previously (2). After mounting to the glass slides, the coverslips were viewed and photographed using Nikon video imaging system (Nikon Instech, Japan). Images were converted to 8-bit grayscale and processed with Image J software (National Institutes of Health, Bethesda, MD). To assess tubulin distribution within one cell, the image grayscale level profile was analyzed along the line, connecting centrosome area to the cell periphery. The line was parallel to the longitudinal axis of the cell and did not cross the nucleus area; cells of similar size from different microscopic fields were chosen for analysis. Statistical analysis was performed using a one-way ANOVA test; results with P < 0.05 were considered significantly different.

Western Immunoblotting. Cells grown in six-well plates were starved for 1 h before the addition of any inhibitor/stimulator. After stimulation, cells were washed with phosphate-buffered saline (PBS) and lysed with PBS containing 1% SDS and 1% beta-mercaptoethanol. After aspiration through 25-gauge needle and boiling, protein extracts were separated by SDS-PAGE on 5% gel for the detection of ROCK1 and ROCK2, 10% gel for the detection of myosin phosphatase targeting subunit (MYPT), ERK, p38, and caldesmon, or 12% gel for MLC or HSP27. Following transfer, nitrocellulose membranes were blocked with 5% BSA solution in Tris-buffered saline with 0.1% Tween (TBST) and incubated with phospho-specific antibodies overnight. After incubation with secondary antibodies and development, membranes were stripped in 10% acetic acid solution, reblocked, and probed with pan-specific antibodies.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of 2ME on pulmonary EC barrier properties. To evaluate the potential effect of 2ME on endothelial barrier properties, we employed two methods assessing transendothelial permeability. Measurement of the electrical resistance of EC monolayer, grown on gold electrode, revealed that 2ME induces significant hyperpermeability of HPAEC (Fig. 1, A and B). 2ME-induced TER decrease reached its maximum within the first 15 min after HPAEC challenge and gradually resolved over a period of time, returning to the value approaching baseline after 3–4 h (Fig. 1A). 2ME-induced disruption of endothelial integrity was confirmed in the experiments with cells grown on semipermeable membranes and tested for 2,000-kDa FITC-dextran permeability. In both sets of experiments, the changes in HPAEC permeability were significant in micromolar range, although increase in 2,000-kDa FITC-dextran permeability required higher threshold concentration of 2ME than increase in ion permeability assessed by means of electrical resistance measurement (Fig. 1, B and C).


Figure 1
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  		Fig. 1. The effect of 2-methoxyestradiol (2ME) on endothelial permeability. A and B: human pulmonary artery endothelial cells (EC) (HPAEC) grown on gold microelectrodes were challenged with 0.1% DMSO [control (Cntr)], 60 µM 2ME (A), or with indicated concentrations of 2ME (B). A: transendothelial electrical resistance (TER) values are normalized to the time point before 2ME addition. Shown is a representative tracing from 3 independent experiments. B: maximal HPAEC TER declines evoked by different concentrations of 2ME are presented as means ± SE (n = 3). C: HPAEC grown on semipermeable collagenated membrane to 100% confluence were challenged with indicated concentrations of 2ME; permeability of FITC-dextran across cell monolayer was measured 2 h after 2ME challenge. FITC-dextran fluorescence in lower chamber media is presented as means ± SE (n = 3). *P < 0.05. Norm., normalized; ru, relative units.

 
Involvement of MT in 2ME-induced pulmonary artery EC barrier dysfunction. 2ME is known to directly interact with tubulin and inhibit MT polymerization (10). To assess the state of MT network in 2ME-challenged HPAEC, we performed immunofluorescent studies utilizing anti-tubulin antibody. Immunofluorescent analysis of MT structure indicated significant morphological changes in MT network appearance upon 50 µM 2ME stimulation (Fig. 2). Moderate MT rearrangement was evident as early as 5 min after 2ME addition; 15–30 min-exposure led to a significant reduction of MT amount while remaining MT appear disoriented and collapsed toward the centrosome area (Fig. 2A). Similar results were obtained with cells treated with lower 2ME concentration (7 µM, data not shown). The quantitative analysis of tubulin distribution reveals that most of the tubulin staining in the quiescent cell localizes within the centrosome area, with tubulin amount gradually decreasing through the internal part of the cell toward the cell periphery. Thirty minutes of exposure to 2ME results in dramatic decline of tubulin staining both in internal and peripheral parts of cell (Fig. 2C).


Figure 2
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  		Fig. 2. 2ME induces tubulin rearrangement in HPAEC. HPAEC grown on glass coverslips were treated with 50 µM 2ME for varying time, fixed, probed with antitubulin antibody, stained by Alexa 488 secondary antibody (A), and examined by immunofluorescence microscopy using a x60 oil objective. The rectangles mark the portions of the images that are enlarged on the right. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (B). C: the tubulin distribution in control cells (squares) and cells challenged with 50 µM 2ME for 30 min (circles) was assessed using Image J software. Five images of cells from different microscopic fields were analyzed to create each profile. Points between "#" symbols are significantly different from control with P < 0.05.

 
The MT dynamics are known to play a role in the regulation of endothelial barrier function (1, 3, 24). We examined the involvement of MT dynamics in 2ME-induced barrier compromise. Measurement of TER in the presence of MT stabilizer taxol revealed that taxol pretreatment significantly attenuated 2ME-induced TER decline (Fig. 3A). Moreover, TER recovery after 2ME treatment was facilitated by the later addition of taxol (Fig. 3B). These results strongly indicate that MT stabilization counteracts 2ME-induced increase in HPAEC permeability. To evaluate the effect of taxol on 2ME-induced MT rearrangement, we assessed the MT state by immunofluorescence. Pretreatment of EC with taxol led to a certain MT reorganization resulting in the partial loss of centrosomal staining and thickening of the tubulin peripheral network (Fig. 4, A and B). The morphology of taxol-stabilized MT noticeably differed from MT morphology in untreated cells; this acquired morphology was not changed by 2ME stimulation (Fig. 4A). It is significant that taxol prevented 2ME-induced MT destabilization in the peripheral and internal parts of cell, although the loss of centrosomal staining was exacerbated by the 2ME (Fig. 4, A and C).


Figure 3
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  		Fig. 3. The role of microtubule (MT) dynamics in 2ME-induced HPAEC barrier dysfunction. HPAEC grown on gold microelectrodes were pretreated with 5 µM taxol (txl) or 0.1% DMSO and then challenged with 20 µM 2ME (A), or challenged with 20 µM 2ME or 0.05% DMSO and then treated with 5 µM taxol (B). Shown are representative tracings from 3 (A) and 2 (B) independent experiments.

 

Figure 4
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  		Fig. 4. The effect of MT stabilizer taxol on 2ME-induced tubulin rearrangement in HPAEC. A: HPAEC grown on glass coverslips were pretreated with 0.1% DMSO (a, c) or 5 µM taxol (b, d) for 30 min and then challenged with 0.05% DMSO (a, b) or 50 µM 2ME (c, d) for 30 min, fixed, probed with anti-tubulin antibody, stained by Alexa 488 secondary antibody, and examined by immunofluorescence microscopy using a x60 oil objective. B: tubulin distribution in cells treated with 0.1% DMSO (squares) or 5 µM taxol (triangles) was assessed using Image J software. C: tubulin distribution in cells pretreated with 0.1% DMSO (squares) or 5 µM taxol (triangles) and challenged with 50 µM 2ME for 30 min was assessed using Image J software. Five images of cells from different microscopic fields were analyzed to create each profile. Points marked with "*" are significantly different from control with P < 0.05. Points between "#" symbols are significantly different with P < 0.05.

 
Effect of ME on the state of actomyosin cytoskeleton. Several MT-destabilizing agents are known to induce actin cytoskeleton reorganization and cause actomyosin contraction in EC. Immunofluorescence data demonstrated that 2ME causes induction of stress fiber formation, which starts primarily on the cell periphery but eventually progresses across the cell (Fig. 5A). Stress fiber formation coincides with the increase in MLC phosphorylation (Fig. 5B) and initial changes in tubulin organization (Fig. 2). We examined if HPAEC response to 2ME is driven by the elevated MLCK or ROCK activity. Measurement of transendothelial resistance revealed that, unlike MLCK inhibitors PIK and ML-7, ROCK inhibitor Y27632 significantly attenuated 2ME-induced TER decline (Fig. 6A). Y27632 pretreatment effectively suppressed the phosphorylation of both MYPT and MLC (Fig. 6B) and 2ME-induced actin reorganization (Fig. 7). MLCK inhibitor PIK, although attenuating basal level of MLC phosphorylation, failed to change diphospho-MLC level in 2ME-challenged cells (Fig. 6B) and prevent 2ME-induced stress fiber induction (Fig. 7). To confirm the critical role of ROCK in 2ME-induced endothelial hyperpermeability and evaluate the involvement of ROCK isoforms, we employed small RNA interference approach. As shown on Fig. 8, A and B, ROCK1 and ROCK2 siRNA successfully suppressed the expression of corresponding proteins in HPAEC. Surprisingly, permeability measurements revealed that only ROCK2 siRNA attenuated 2ME-induced TER decline (Fig. 8B). In accordance with those results, we observed that the effect of ROCK1 and ROCK2 siRNA on MLC and MYPT phosphorylation was significantly different. ROCK1 siRNA pretreatment had very moderate effect on basal MLC (Fig. 8C) and MYPT (data not shown) phosphorylation level, with no effect on 2ME-induced MLC and MYPT phosphorylation. On the contrary, ROCK2 siRNA pretreatment markedly reduced basal MLC phosphorylation level. Importantly, 2ME-induced increase in diphospho-MLC (Fig. 8C) and phospho-MYPT (data not shown) content was almost completely prevented by ROCK2 siRNA.


Figure 5
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  		Fig. 5. The effect of 2ME on actin cytoskeleton. A: HPAEC grown on glass coverslips were treated with 50 µM 2ME for the time indicated, fixed, stained by Alexa 594-phalloidin, and examined by immunofluorescence microscopy using a x60 oil objective. B: HPAEC grown in 6-well plates were treated with 50 µM 2ME for the time indicated, lysed, and analyzed by Western blot with anti-diphospho-myosin light chain (pp-MLC) and anti-MLC antibodies. Challenge of HPAEC with 2ME results in significant induction of stress fiber formation and increase in MLC phosphorylation.

 

Figure 6
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  		Fig. 6. The effect of myosin light chain kinase (MLCK) and Rho kinase (ROCK) inhibition on 2ME-induced barrier dysfunction. A: HPAEC grown on gold microelectrodes to 100% confluence were pretreated with MLCK inhibitors ML-7 (3 µM) and PIK (100 µM) or ROCK inhibitor Y27632 (Y; 3 µM) for 30 min and then challenged with 20 µM 2ME. Maximal TER decrease was assessed in 3 experiments and expressed as a ratio to maximal TER decrease in the absence of inhibitor. Data are presented as means ± SE; *P < 0.05. B: HPAEC grown in 6-well plates were pretreated with PIK (100 µM) or Y27632 (3 µM) for 30 min, challenged with 50 µM 2ME for 30 min, lysed, and analyzed by Western blot with anti-phospho-myosin phosphatase targeting subunit (pMYPT) and anti-diphospho-MLC antibodies. Probing with anti-MLC antibodies was used as a loading control. Shown are representative Western blots from 3 independent experiments.

 

Figure 7
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  		Fig. 7. Y27632 pretreatment prevents 2ME-induced actin rearrangement in HPAEC. HPAEC grown on glass coverslips were pretreated with 0.1% DMSO (A and D), 100 µM PIK (B and E), or 3 µM Y27632 (C and F) for 30 min and then challenged with 0.05% DMSO (A–C) or 50 µM 2ME (D–F) for 30 min, fixed, probed with Alexa 594-phalloidin, and examined by immunofluorescence microscopy using a x60 oil objective.

 

Figure 8
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  		Fig. 8. ROCK2 depletion inhibits 2ME-induced barrier dysfunction and MLC phosphorylation. HPAEC grown on gold microelectrodes were pretreated with nonsilencing RNA (nsRNA) (squares and circles, A and B), ROCK1 small interfering RNA (siRNA) (triangles and inverted triangles, A), or ROCK2 siRNA (triangles and inverted triangles, B) for 48 h, and then challenged with 0.05% DMSO (squares and triangles) or 20 µM 2ME (circles and inverted triangles). TER values are normalized to the time point before 2ME addition. Data are presented as means ± SE from 3–5 parallel experiments. Points between "#" symbols are significantly different from control (nsRNA) with P < 0.05. Blots at top of A and B show ROCK1 and ROCK2 expression in cells pretreated with transfection reagent (No Tr), nsRNA, and ROCK1 and ROCK2 siRNAs. Probing with anti-tubulin antibodies was used as a loading control. C: HPAEC grown in 6-well plates were pretreated with transfection reagent (No Tr), nsRNA, ROCK1, or ROCK2 siRNA for 48 h, and then challenged with 20 µM 2ME for 20 min, lysed, and analyzed by Western blot with anti-diphospho-MLC antibodies. Probing with anti-GAPDH antibodies was used as a loading control.

 
Role of MAPK cascade in 2ME-induced EC cytoskeletal remodeling and permeability. Previous studies (14, 21), including our own (2), have demonstrated a key role of MAP kinases in signaling events, initiated by MT inhibitors. Indeed, phosphorylation of ERK1/2, p38, and JNK was detected in 2ME-treated EC by immunoblotting with corresponding phospho-specific antibodies (Fig. 9A). To evaluate the involvement of MAP kinases in EC barrier regulation, we employed the inhibitors of ERK1/2 upstream kinase (MEK), p38 and JNK, and studied their effect on 2ME-induced TER decline. Inhibition of p38 activity with SB203580 attenuated 2ME-induced endothelial permeability, whereas MEK inhibitor U0126 and JNK inhibitor did not have a significant effect on 2ME-induced TER decline (Fig. 9B). SB203580 was shown to be a specific p38 inhibitor as it failed to inhibit 12 other protein kinases in vitro and lacked the effect on other MAP kinase cascades in vivo (8, 13).


Figure 9
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  		Fig. 9. The involvement of p38 cascade in 2ME-induced HPAEC response. A: HPAEC grown in 6-well plates were challenged with 50 µM 2ME for the time indicated, lysed, and analyzed by Western blot with anti-phospho-ERK1/2, anti-phospho-p38, and anti-phospho-JNK. Probing with corresponding total protein antibodies (tot) was used as a loading control. Shown are representative Western blots from 2–3 independent experiments. ph, phosphorylated samples. B: HPAEC grown on gold microelectrodes to 100% confluence were pretreated with upstream kinase MAP ERK kinase (MEK) inhibitor U0126 (U0; 5 µM), p38 inhibitor SB203580 (SB; 20 µM), or JNK inhibitor-1 (JNKinh; 5 µM) for 30 min, and then challenged with 20 µM 2ME. Maximal TER decrease was assessed in 3 experiments and expressed as a ratio to maximal TER decrease in the absence of inhibitor. Data are presented as means ± SE; *P < 0.05. C: HPAEC grown on gold microelectrodes to 100% confluence were pretreated with p38 inhibitor SB203580 (10 µM), ROCK inhibitor Y27632 (3 µM), or both SB203580 and Y27632 (SB+Y) for 30 min, and then challenged with 20 µM 2ME. Maximal TER decrease was assessed in 3 experiments and expressed as a ratio to maximal TER decrease in the absence of inhibitor. Data are presented as means ± SE; *P < 0.05.

 
To establish whether p38 pathway contributes to 2ME-induced barrier disruption independently of ROCK pathway, we compared the effect of p38 inhibitor SB203580 on 2ME-induced TER decline in the presence and absence of ROCK inhibitor Y27632. We observed that in the presence of Y27632, the effect of SB203580 was significantly intensified, although not exactly additive (Fig. 9C).

To examine the involvement of the members of p38 cascade in HPAEC response to 2ME, we monitored the phosphorylation status of MAPKAP2, MAPKAP2 substrate HSP27, and p38 substrate caldesmon. As positive control, we used a known stimulator of p38 cascade, nocodazole. Our data clearly reveal the time-dependent increase in phosphorylation of MAPKAP2 and its substrate HSP27; however, p38 substrate caldesmon did not manifest elevated phosphorylation level in 2ME-treated cells (Fig. 10, A and B).


Figure 10
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  		Fig. 10. Identification of p38 targets in 2ME-treated EC. A: HPAEC grown in 6-well plates were challenged with 50 µM 2ME or 1 µM nocodazole (NCD; positive control) for the time indicated, lysed, and analyzed by Western blot with anti-phospho-p38 (p-p38), anti-phospho-MAPKAP2 (pMAPKAP2), anti-phospho-27-kDa heat shock protein (pHSP27), and anti-phospho-caldesmon (pCD). Probing with corresponding total protein antibodies was used as a loading control. Shown are representative Western blots from 2–3 independent experiments. B: Western blots from A were scanned and analyzed using Image J software. The intensity of phospho-proteins bands was normalized to the intensity of total proteins bands. Phospho-content after 60 min of treatment with 50 µM 2ME (gray bars) is expressed as fold of control in the absence of treatment (white bars). Data are presented as means ± SE; *P < 0.05. C: taxol pretreatment prevents activation of p38 cascade in 2ME-challenged cells. HPAEC grown in 6-well plates were pretreated with taxol (5 µM) for 30 min, challenged with 50 µM 2ME for 30 min, lysed, and analyzed by Western blot with anti-phospho-p38 and anti-phospho-HSP27. Probing with anti-HSP27 antibodies was used as a loading control. Shown are representative Western blots from 2 independent experiments.

 
To confirm that activation of p38 cascade was due to the changes in MT dynamics, we pretreated EC with taxol before 2ME challenge. MT stabilization significantly attenuated p38 and HSP27 phosphorylation level, indicative of p38 cascade activation (Fig. 10C).

To analyze the role of p38 in MT dynamics, we assessed the effect of SB203580 on 2ME-induced tubulin rearrangement. Inhibition of p38 in untreated cells caused a thickening of MT network while centrosome area was not affected (Fig. 11, A and B). Pretreatment of HPAEC with SB203580 attenuated severe MT destabilization in 2ME-treated HPAEC (Fig. 11, A and C).


Figure 11
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  		Fig. 11. A: SB203580 pretreatment prevents 2ME-induced MT disassembly in HPAEC. HPAEC grown on glass coverslips were pretreated with 0.1% DMSO (a and c) or 20 µM SB203580 (b and d) for 30 min, and then challenged with 0.05% DMSO (a and b) or 50 µM 2ME (c and d) for 30 min, fixed, probed with anti-tubulin antibody, stained by Alexa 488 secondary antibody, and examined by immunofluorescence microscopy using a x60 oil objective. B: tubulin distribution in cells treated with 0.1% DMSO (squares) or 20 µM SB203580 (circles) was assessed using Image J software. C: tubulin distribution in cells pretreated with 0.1% DMSO (squares) or 20 µM SB203580 (circles) and challenged with 50 µM 2ME for 30 min was assessed using Image J software. Five images of cells from different microscopic fields were analyzed to create each profile. Points between "#" symbols are significantly different from control with P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
2ME is a major endogenous metabolite of estradiol with both antiproliferative and antiangiogenic activity. The most studied 2ME effects on vasculature are the inhibition of EC proliferation and tube formation in vitro and angiogenesis in vivo. The molecular mechanism of antiangiogenic effect of 2ME seems to include upregulation of proapoptotic factors Fas and Bcl-2, and downregulation of proangiogenic transcription factor HIF-1 (16). Those effects require long-term (20 h) treatment with pharmacological concentrations of 2ME (25, 26).

In this study, we demonstrate that 2ME is able to elicit rapid (minutes to several hours) response in EC, and this response is associated with acute but reversible loss of endothelial barrier function (Fig. 1). The changes in transendothelial permeability are evoked by the pharmacological concentrations of 2ME (more than 100 nM), as opposed to the physiological concentrations (less than 100 nM). The recent study on the pharmacokinetics of 2ME revealed poor oral bioavailability of this compound. Plasma level after oral intake of the daily 2ME dose (up to 3,000 mg) did not exceed 10–13 ng/ml (~40 nM) (9, 22). Nonetheless, reported drug toxicities included cases of angioedema and respiratory failure (9). As new formulations to increase 2ME bioavailability are being discussed, it's important to understand that loss of endothelial barrier function may represent one of the potential side effects of 2ME therapy; therefore, the issue of 2ME tolerance should be addressed with caution. Of course, our conclusion is derived from findings in cultured cells and will require further in vivo experiments to assess 2ME effect on endothelial hyperpermeability.

The signaling mechanisms that lead to EC barrier compromise have been a subject of intense investigation for years. Increasing evidence had suggested that balance between forces opposing the cell collapse (cell periphery tethering forces and intracellular cytoskeleton struts) and forces providing centripetal tension (contraction provided by actomyosin filaments) defines the integrity of endothelial monolayer and transendothelial permeability. Recently, the critical role of MT cytoskeleton and its cross-talk with actomyosin network was emphasized in several studies. It has been demonstrated that barrier-disruptive agents such as thrombin and TGF-beta, thought to act primarily via stress fiber induction and contraction, also evoke MT destabilization (1, 3). Vice versa, when endothelial permeability was induced by MT inhibitors, an additional barrier-disrupting mechanism was provided by stress fiber formation and contraction (2, 24).

To investigate the molecular mechanisms involved in 2ME-induced permeability increase, we first examined 2ME effect on MT organization. Our data indicate that 2ME-treated HPAEC exhibit severe MT destabilization (Fig. 2). These results are in accordance with earlier observation that 2ME elicits its effect via the inhibition of tubulin polymerization. The rapid rearrangement of MT after 2ME addition is consistent with a role for the MT in 2ME-induced barrier dysfunction. To confirm the involvement of MT in this process, we studied the effect of MT stabilizer taxol on HPAEC response to 2ME. In the presence of taxol, HPAEC exhibit certain decline in TER (Fig. 3), which may be attributed to the rearrangement of MT network seen by immunofluorescent microscopy (Fig. 4). Importantly, taxol pretreatment significantly decreases 2ME-induced TER decline (Fig. 3) and prevents 2ME-induced peripheral MT destabilization, whereas centrosomal organization was lost in the presence of 2ME (Fig. 4). These facts suggest that intact peripheral MT network rather than unimpaired centrosome-MT organization provides the basis for the barrier-protective effect of taxol.

MT network remodeling is often linked to the reorganization of another important cytoskeletal element, namely actomyosin network. As edemagenic agents are known to induce both contractile (1, 3) and noncontractile (4, 5) actin rearrangements in EC, we first assessed the nature of actin reorganization in 2ME-treated cells. Immunofluorescent studies and Western blotting with anti-diphospho-MLC antibodies revealed that 2ME induces stress fiber formation and increases MLC phosphorylation, evidence of elevated contractility (Fig. 5). We further examined the role and mechanism of contractile response in 2ME-induced hyperpermeability. Elevated contractility can be a result of MLC phosphorylation due to the increased MLCK activity or the inhibition of MLC phosphatase by ROCK. We evaluated the effects of MLCK inhibitors PIK and ML-7 and ROCK inhibitor Y27632 on 2ME-induced barrier dysfunction (Fig. 6). We observed that, unlike MLCK inhibitor, ROCK inhibitor was able to attenuate 2ME-induced TER decline. Y27632 pretreatment effectively suppressed 2ME-induced increase in MLC phosphorylation and stress fiber formation (Figs. 6 and 7). These results are in good accordance with our previous data (24), indicating that MT disruptor nocodazole induces increase in MLC phosphorylation via ROCK-dependent rather than MLCK-dependent mechanism. Our current and previous data suggest that Rho/ROCK pathway preferential involvement differentiates MT disruptor-induced contractility from that induced by calcium influx-initiating edemagenic agents, as the latter act primarily through the activation of MLCK. To analyze the involvement of ROCK isoforms in 2ME-induced hyperpermeability, we employed siRNA approach. 2ME-induced TER decline was attenuated in the presence of ROCK2 siRNA but not ROCK1 siRNA. Consistent with those data, the increase in MLC phosphorylation in 2ME-treated HPAEC was significantly suppressed by ROCK2 siRNA (Fig. 8). Based on these results, we conclude that 2ME-induced hyperpermeabilty is provided in part by ROCK2-specific contractile response. The differential effect of ROCK2 vs. ROCK1, observed in our study, was very interesting, although not entirely surprising. Recent generation of mice deficient in each ROCK isoform suggested different roles for ROCK1 and ROCK2 in embryonic development (20, 23). Loss of ROCK1 results in the eyelid open at birth and ventral body wall defect, whereas loss of ROCK2 led to placental dysfunction, growth retardation, and fetal death. Moreover, it was shown that at least some mechanisms of regulation of ROCK1 and ROCK2 activity differ significantly. ROCK1, but not ROCK2, is cleaved during apoptosis by activated caspases, generating a truncated kinase with increased intrinsic activity (7, 18). It is not unlikely therefore that, in EC, ROCK1 and ROCK2 are regulated dissimilarly and respond to the agonist challenge in different ways.

Numerous studies indicated the critical role of MAP kinases in cell responses, induced by MT disruptors (2, 14, 21). The interest in MAP kinases pathways and their role in endothelial permeability is greatly induced by the knowledge that certain MAP kinases substrates are cytoskeletal proteins, involved in the regulation of microfilament assembly or contractility. We analyzed the phosphorylation status of major MAPK family members, ERK1/2, p38, and JNK, in 2ME-treated cells and revealed that all three members are phosphorylated and activated upon 2ME challenge. To examine the role of MAP kinases in increased permeability, we analyzed the effect of specific inhibitors on 2ME-induced TER decline. Out of three kinases studied, only p38 seems to be critical for 2ME-induced barrier dysfunction (Fig. 9B). To ascertain independent involvement of p38 pathway in this process, we compared the effects of p38 and ROCK inhibition on 2ME-induced TER decline. Our data indicate that p38 and ROCK pathways contribute separately to barrier dysfunction, although certain cross-regulation cannot be excluded.

We further analyzed p38 activation in 2ME-treated HPAEC and found that this process is followed by the phosphorylation of p38 substrate MAPKAP2 and MAPKAP2 substrate HSP27 (Fig. 10, A and B). Caldesmon, the direct substrate of p38, was not significantly phosphorylated in 2ME-treated cells. Earlier, we (6) had shown that caldesmon can be a target of p38 phosphorylation in thrombin-treated EC. Our current results indicate that p38 activation is not always accompanied by caldesmon phosphorylation. Significant increase in HSP27 phosphorylation implies that HSP27 rather than caldesmon conveys p38 signal toward actin filaments. Altogether, these data suggest that 2ME can modulate both stress fiber assembly and actomyosin interaction via p38- and ROCK-dependent mechanisms, respectively.

The cross-talk between p38 pathway and MT dynamics became the area of intensive research recently (2, 14, 21). Here we show that inhibition of p38 pathway attenuates 2ME-induced MT destabilization (Fig. 11), and, vice versa, stabilization of MT with taxol significantly suppresses activation of p38 cascade (Fig. 10). As similar results were obtained earlier in cells treated with another MT inhibitor, nocodazole (2), we hypothesize that inhibition of MT polymerization in EC initiates activation of p38 cascade, which, in turn, facilitates further MT destabilization. The precise role of p38 pathway and delineation of p38 cascade substrates leading to MT rearrangement will require further experiments.

In summary, this study examined for the first time the effect of 2ME on barrier function in pulmonary EC. Our results reveal that 2ME increase transendothelial permeability in HPAEC, with the primary mechanism involving direct inhibition of MT polymerization. MT destabilization leads to increased MLC phosphorylation and stress fiber formation via a ROCK2-dependent mechanism (Fig. 12). We show that p38, but not ERK1/2 and JNK, pathways are critical for the 2ME-induced barrier dysfunction. p38 activation is accompanied by the phosphorylation of downstream targets MAPKAP2 and HSP27. Our results suggest that both MT depolymerization and elevated HPAEC contractility contribute to the barrier disruptive response to 2ME (Fig. 12). We conclude therefore that molecular mechanism of 2ME-induced hyperpermeability is close to that induced by other MT inhibitors, vinblastine and nocodazole.


Figure 12
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  		Fig. 12. Schematic representing major pathways activated by 2ME in endothelial cells. Directly interacting with tubulin, 2ME causes MT destabilization. MT network remodeling affects p38 activation, induced by 2ME. Vice versa, activation of p38 pathway positively regulates further tubulin reorganization. Phosphorylation of HSP27 by p38 substrate MAPKAP2 is thought to facilitate stress fiber formation in EC. Contraction is induced by 2ME via ROCK2/MYPT/MLC-dependent mechanism. Thus 2ME destabilizes intracellular struts (MT), maintaining cell in spread position, and activates centripetal forces (actomyosin), providing cell contractility. Altogether, those effects lead to the loss of endothelial monolayer integrity.

 

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This work was supported by National Heart, Lung, and Blood Institute Grants HL-80675, HL-083327, HL-067307, and HL-58064.


   ACKNOWLEDGMENTS
 
We thank Dr. J. R. Turner (University of Chicago) for the generous gift of MLCK inhibitor PIK. We are grateful to Yevgeniy Kovalenkov for the assistance in manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. D. Verin, Univ. of Chicago, Dept of Medicine, 929 E. 57th St., CIS W412, Chicago, IL 60637 (e-mail: averin{at}medicine.bsd.uchicago.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.


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