p38 MAPK activation by TGF-beta 1 increases MLC phosphorylation and endothelial monolayer permeability

Peter L. Goldberg, Darren E. MacNaughton, Richard T. Clements, Fred L. Minnear, and Peter A. Vincent

Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor (TGF)-beta 1 increases endothelial monolayer permeability and myosin light chain phosphorylation (MLC-P) beginning 1-2 h posttreatment, suggesting that changes in gene expression may be required for these responses. The role of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38 MAPK) was investigated because both kinases have been implicated in regulating gene expression after TGF-beta 1. ERK1/2 phosphorylation increased threefold above the control level, and the increase was temporally associated with the increase in MLC-P. Inhibition of ERK1/2 phosphorylation with the MAPK kinase inhibitor U-0126 did not prevent the increase in either monolayer permeability or MLC-P. p38 MAPK phosphorylation increased fourfold above the control level, but unlike ERK1/2, this increase peaked 30 min and 1 h post-TGF-beta 1 treatment. Inhibition of p38 MAPK activity with SB-203580 prevented the increases in both monolayer permeability and MLC-P. Treatment of the monolayers with cycloheximide in conjunction with TGF-beta 1-inhibited MLC-P, showing a requirement for protein synthesis. These studies demonstrate that p38 MAPK activation and subsequent protein synthesis are part of the signal transduction pathway leading to the TGF-beta 1-induced increases in monolayer permeability and MLC-P.

transforming growth factor-beta 1; myosin light chain phosphorylation; endothelial cells; cycloheximide; mitogen-activated protein kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSFORMING GROWTH FACTOR (TGF)-beta 1 is a cytokine/growth factor with numerous biological effects that include growth inhibition, bone remodeling, and increased incorporation of extracellular matrix. In the lung, an increase in TGF-beta 1 in bronchoalveolar lavage fluid is temporally correlated with the development of edema in the bleomycin-induced model of acute lung injury (34). In addition, overexpression of active TGF-beta 1 in rat lungs with adenovirus-mediated gene transfer results in an initial period of perivascular and peribronchial edema with no evidence of inflammatory cell accumulation (28), suggesting that TGF-beta 1 has a direct effect on the endothelium. Over time, the continued expression of active TGF-beta 1 resulted in the development of prolonged and severe pulmonary fibrosis (28). The most recent evidence for the importance of TGF-beta 1 in acute lung injury was provided by Pittet et al. (23). These investigators found that expression of a chimeric TGF-beta 1 receptor, which prevents the interaction of TGF-beta 1 with its receptor, protects against bleomycin-induced pulmonary edema. These findings suggest that TGF-beta 1 plays a role in increasing vascular permeability in pathological conditions that result in the development of pulmonary edema and/or vascular injury.

In vitro studies support a role for TGF-beta 1 in the development of pulmonary edema. The addition of TGF-beta 1 to pulmonary endothelial monolayers decreases monolayer integrity as measured by changes in electrical resistance and albumin clearance (12, 15). The increase in the paracellular flux of macromolecules produced by TGF-beta 1 is associated with changes in endothelial cell shape and the formation of intercellular gaps (15). Current models suggest that changes in endothelial cell shape and subsequent gap formation are controlled by signal transduction pathways that alter the balance of competing adhesive and contractile forces (reviewed in Refs. 18, 20). In this model, the loss of cell-cell or cell-matrix attachments and/or the activation of actin-myosin contractile activity will alter the balance of forces within the cell, resulting in cell retraction and a decrease in endothelial monolayer integrity. Hurst et al. (15) found that the decrease in monolayer integrity produced by TGF-beta 1 is temporally associated with an increase in myosin light chain (MLC) phosphorylation and the movement of myosin to an actin-associated pool. In addition, inhibition of MLC kinase by KT-5926 prevented the increase in MLC phosphorylation and the associated increase in monolayer permeability induced by TGF-beta 1, showing that an increase in cell contraction may contribute to the TGF-beta 1-induced decrease in endothelial monolayer integrity.

Although Hurst et al. (15) demonstrated that inhibition of MLC phosphorylation prevented the TGF-beta 1-induced increase in monolayer permeability, other studies have shown that MLC phosphorylation and monolayer permeability are not linked. Indeed, tumor necrosis factor (TNF)-alpha has been shown to increase both MLC phosphorylation and monolayer permeability, but inhibition of MLC phosphorylation did not prevent the TNF-alpha -induced changes in permeability (22). However, the inhibition of MLC phosphorylation did prevent the TNF-alpha -induced increase in apoptosis, suggesting that MLC phosphorylation may regulate processes other than permeability. This is also true for TGF-beta 1 because an increase in cell tension has been implicated as an important component in a number of TGF-beta 1-induced changes in cell function. Increased cell tension has been shown to be an important factor in transforming endothelial cells to a fibroblastic phenotype after Ras activation (35). This transformation also results in the loss of cell-cell adhesion. In addition, cell tension plays an important role in increasing fibronectin matrix deposition, an important chronic effect of TGF-beta 1 activation that may contribute to the development of fibrosis in many tissues (28, 30). Thus tension generation after TGF-beta 1 activation may be an important control point for many of the physiological and pathophysiological effects of TGF-beta 1.

The increases in MLC phosphorylation and endothelial monolayer permeability in response to TGF-beta 1 occur 2 h after the addition of TGF-beta 1 to endothelial monolayers (15). This is in contrast to the changes in MLC phosphorylation and endothelial permeability produced by thrombin that occur within 2-5 min after the addition of thrombin to endothelial monolayers (8). The time course of the TGF-beta 1-induced increases in MLC phosphorylation and endothelial permeability suggests that these increases are the result of changes in gene expression. TGF-beta 1 has been shown to activate the c-Jun NH2-terminal kinase (JNK) and p38 MAPK pathways as well as the ERK1/2 pathway, and all three of these pathways have been implicated in interfacing with the SMAD pathway to regulate TGF-beta 1-induced gene expression (25, 27, 33). In addition to transcriptional regulation, these MAPK pathways have also been implicated in mediating increases in endothelial permeability and MLC phosphorylation (1, 14, 17, 24). Klemke et al. (17) showed that activated ERK1/2 could phosphorylate MLC kinase, resulting in an increase in MLC kinase activity and thus an increase in MLC phosphorylation. Activation of p38 MAPK plays a role in actin rearrangement that is associated with decreased endothelial monolayer integrity after hydrogen peroxide (H2O2) exposure (14). Activation of p38 MAPK has also been shown to be important in the cell signaling pathway for vascular endothelial growth factor (VEGF), a factor known to decrease monolayer integrity (24).

The purpose of these studies was to examine the role of ERK1/2 and p38 MAPK in the TGF-beta 1-induced increases in MLC phosphorylation and endothelial permeability. We show that ERK1/2, although activated by TGF-beta 1, is not involved with the TGF-beta 1-induced endothelial monolayer permeability and MLC phosphorylation. Also, we demonstrate that p38 MAPK is activated by TGF-beta 1 and is potentially linked to these TGF-beta 1 responses. Furthermore, we show here that TGF-beta 1-induced MLC phosphorylation is inhibited by cycloheximide, a protein synthesis inhibitor, demonstrating that de novo protein synthesis is required for increased MLC phosphorylation after TGF-beta 1 treatment. These studies illustrate that p38 MAPK plays a role in the signal transduction pathways initiated by TGF-beta 1 and suggest that activation of transcriptional activity and subsequent de novo protein synthesis regulate the TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cell culture. Calf pulmonary artery endothelial cells (CCL 209) were grown in modified Eagle's medium (MEM) supplemented with 20% fetal bovine serum, 10 mM nonessential amino acids, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The cells were split 1:4 every fifth day. All experiments were performed between passages 18 and 24.

Reagents. Calf pulmonary artery endothelial cells were purchased from the American Type Culture Collection (Manassas, VA). MEM, nonessential amino acids, penicillin, and streptomycin were purchased from GIBCO-BRL (Life Technologies, Grand Island, NY). Fetal bovine serum was purchased from Summit Biotechnology (Chicago, IL). TGF-beta 1 was purchased from R&D Systems. Cycloheximide, puromycin, calyculin A, Triton X-100, formaldehyde, and monoclonal antibodies against MLC were purchased from Sigma-Aldrich (St. Louis, MO). SB-203580 was purchased from Calbiochem (San Diego, CA). U-0126 and PD-98059 were purchased from BIOMOL (Plymouth Meeting, PA). Horseradish peroxidase-conjugated anti-rabbit IgG, horseradish peroxidase-conjugated anti-mouse IgM, and Texas Red-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Phospho-specific MLC antibodies (Strategic Biosolutions, Ramona, CA) were raised in rabbits against peptides corresponding to endogenous nonmuscle MLC diphosphorylated at Ser19/Thr18. The peptide LLRPERATSAVFC was synthesized and provided by Dr. T. Anderson (Albany Medical College, Albany, NY). Oregon Green-conjugated phalloidin and ProLong antifade kit were purchased from Molecular Probes (Eugene, OR). Enhanced chemiluminescence kit was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Antibodies against phosphorylated ERK1/2 MAPK (Thr202/Tyr204), stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185), p38 MAPK (Thr180/Tyr182), and 27-kDa heat shock protein (HSP27; Ser82) and phototope-horseradish peroxidase Western detection kit were purchased from New England Biolabs (Beverly, MA). Antibody against HSP25 was purchased from StressGene.

Experimental protocol. Endothelial cells were seeded (8 × 104 cells/cm2) and grown to confluence (3-5 days) in MEM containing penicillin-streptomycin and 20% fetal bovine serum. The day before the experiment, the medium was changed to MEM containing penicillin-streptomycin and 5% fetal bovine serum. On the day of the experiment, TGF-beta 1 was added to yield a final concentration of 1 ng/ml. In studies with inhibitors, cells were pretreated for 30 min with the respective agent and then TGF-beta 1 was added. The cells were coincubated with the respective agent and TGF-beta 1 for the designated times. The cell lysates were then prepared for assay.

MLC phosphorylation assay. Endothelial monolayers were analyzed for MLC phosphorylation by urea-PAGE with a modification of the method described by Garcia et al. (8) and Verin et al. (32). After treatment, 10% TCA and 0.01 M dithiothreitol were added to the dish. The cells were scraped with a rubber policeman, transferred to a microcentrifuge tube, and placed on ice for 30 min. The tubes were spun at 10,000 g for 7 s at 4°C, the supernatant was removed, and ice-cold ether or acetone was added to remove any remaining TCA from the protein pellet. This process was repeated four times. The samples were then dried, combined with sample buffer (6.7 M urea, 20 mM Tris base, 22 mM glycine, 9 mM dithiothreitol, 0.004% bromphenol blue, and 1% Triton X-100), and sonicated for 30 min. Proteins were separated by charge on a native gel (10% acrylamide, 0.5% bis-acrylamide, 40% glycerol, 20 mM Tris base, 22 mM glycine, and 0.22 µM ammonium persulfate) for 1 h at 400 V and transferred to nitrocellulose for 14 h at 40 V. MLC was detected by immunoblotting with a monoclonal antibody to MLC. After incubation with horseradish peroxidase-conjugated anti-mouse IgM, enhanced chemiluminescence was used for protein detection.

Detection of phosphorylated ERK1/2, SAPK/JNK, and p38 MAPK. Endothelial monolayers were assessed for the phosphorylation of ERK1/2, SAPK/JNK, and p38 MAPK by using a modification of the methods described in the PhosphoPlus antibody kits. After incubation, boiling hot lysis buffer consisting of 30 µM bromphenol blue, 25% Tris base (0.5 M, pH 6.8), 138 mM SDS, 20% glycerol, and 5% 2-mercaptoethanol was added to the dish. The cells were scraped with a rubber policeman and transferred to a microcentrifuge tube, and DNA was sheared by the use of a 1-ml syringe with a 25-gauge (<FR><NU>5</NU><DE>8</DE></FR> in.) needle. The cell lysates were then subjected to sonication for 15 s, boiled for 5 min, and centrifuged at 10,000 g for 5 min. The cell lysates were run on 10% SDS-PAGE gel with a 3% stacker and transferred to nitrocellulose by electroblotting at 100 V for 1 h. Immunoblots were probed for phosphorylation of ERK1/2, SAPK/JNK, and p38 MAPK by using PhosphoPlus ERK1/2 (Thr202/Tyr204), SAPK/JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182), respectively. After incubation with horseradish peroxidase-conjugated secondary antibodies, a phototope-horseradish peroxidase Western detection kit was used for protein detection.

Assessment of endothelial permeability. The continuous measurement of electrical resistance across endothelial monolayers was used to monitor changes in endothelial permeability. Electrical resistance was measured by using an electric cell-substrate impedance sensor (Applied Biophysics) (10, 29). Endothelial cells were grown to confluence on small gold electrodes (5 × 10-4 cm2) in culture medium, which functioned as the electrolyte. The small gold electrode, covered by confluent endothelial cells, and a larger gold counterelectrode (~2 cm2) were connected to a phase-sensitive, lock-in amplifier. A 1-V, 4,000-Hz alternating current was supplied through a 1-MOmega resistor to approximate a constant current source of 1 µA. Treating the cell-electrode system as a simple series resistance-capacitance circuit, the measured changes in the in-phase and out-of-phase voltages can be used to calculate these values for resistance and capacitance. Voltage and phase data were stored and processed with a personal computer. The same computer controlled the output of the amplifier and switched the measurements to different electrodes in each of two 8-well arrays during the course of an experiment. The small size of the cell-seeded electrode is the critical feature of the system. When electrodes of 10-3 cm2 or smaller were used, the impedance at the small electrode dominates the system, allowing this impedance to be measured and also allowing for assessment of cellular morphology. The measurement of electrical impedance was obtained in real time every minute before and for 20 h after treatment of the endothelial cells and is reported as the resistive portion of electrical impedance.

Dual-label immunofluorescence microscopy. After incubation, the medium was removed, and the coverslips were washed three times with PBS containing Ca2+ and Mg2+, pH 7.4, on ice. The cells were fixed for 10 min on ice with cold 3% formaldehyde, rinsed with cold PBS, and permeabilized for 10 min on ice with cold 1% Triton X-100. The coverslips were washed three times with cold PBS and subsequently blocked for 1 h at room temperature with 1% BSA (in PBS). Primary antibody (against diphosphorylated MLC) was applied for 1 h at room temperature. The coverslips were washed three times with PBS and incubated at room temperature for 1 h with both Oregon Green-conjugated phalloidin and Texas Red-conjugated goat anti-rabbit antibody. The coverslips were washed three times with PBS and mounted onto glass slides with a ProLong antifade kit. The slides were viewed on an Olympus BX60 microscope with a fluorescence attachment, and pictures were taken with a SPOT digital camera and imaging software.

Statistical analysis. MLC phosphorylation is graphed as a stacked bar such that the entire bar represents the percent of MLC that was phosphorylated. Distribution of the monophosphorylated and diphosphorylated forms of MLC are represented by the open and solid areas, respectively. All data are means ± SE. A one-way ANOVA was used to determine significant increases in MLC phosphorylation and phosphorylation of ERK1/2 and p38 MAPK after treatment with TGF-beta 1. Differences in MLC phosphorylation after cycloheximide treatment were also assessed with ANOVA. Significant differences were further analyzed with a Newman-Keuls post hoc comparison. Significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Time course of TGF-beta 1 activation of ERK1/2 and p38 MAPK. Hurst et al. (15) have previously shown that the increase in endothelial monolayer permeability induced by TGF-beta 1 is temporally associated with an increase in MLC phosphorylation. To determine whether ERK1/2 plays a role in the TGF-beta 1-induced increase in MLC phosphorylation, we first investigated whether TGF-beta 1 activates ERK1/2 (Fig. 1, A and B). The monolayers were incubated with TGF-beta 1 for 0.5, 1, 2, 3, 4, and 6 h, and phosphorylation of ERK1/2 was assessed by using an antibody that only recognizes the phosphorylated form of the kinase. ERK1/2 phosphorylation began to increase 2-3 h after the addition of TGF-beta 1 (Fig. 1, A and B), reaching a maximum of threefold above the control level by 6 h. Independent experiments found that ERK1/2 was not phosphorylated above the control value after treatment with TGF-beta 1 for 10 and 15 min (data not shown). Interestingly, the increase in ERK1/2 phosphorylation was temporally associated with the increase in MLC phosphorylation after TGF-beta 1 treatment (15).


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Fig. 1.   Transforming growth factor (TGF)-beta 1 activates extracellular signal-regulated kinase (ERK) 1/2 (A and B) and p38 mitogen-activated protein kinase (MAPK; C and D) in confluent endothelial monolayers. Calf pulmonary artery endothelial (CPAE) cells were incubated with 1 ng TGF-beta 1/ml for 0.5, 1, 2, 3, 4, and 6 h for analysis of ERK1/2 activation and for 10 and 30 min (m) and 1, 2, 3, 4, and 6 h for analysis of p38 MAPK activation. After incubation, cell lysates were separated with SDS-PAGE, transblotted to nitrocellulose, and detected via Western blotting with either the ERK1/2 or p38 MAPK PhosphoPlus antibody kit. A: representative immunoblot showing TGF-beta 1-induced ERK1/2 phosphorylation with anti-phospho-ERK1/2 (ERK1/2-P; top), which was stripped and reprobed with an anti-ERK1/2 antibody (bottom). Con, control. B: densitometric analysis of the TGF-beta 1-induced increase in phosphorylation of ERK1/2. TGF-beta 1 increased ERK1/2 phosphorylation, which began 2-3 h after treatment and reached a maximum after 6 h. Data are means ± SE; n = 3 experiments. * P < 0.05 from control value. C: representative immunoblot showing TGF-beta 1-induced p38 MAPK phosphorylation with anti-phospho-p38 MAPK antibody (p38 MAPK-P; top), which was stripped and reprobed with anti-p38 MAPK antibody (bottom). D: densitometric analysis of the TGF-beta 1-induced increase in phosphorylation of p38 MAPK. TGF-beta 1 increased p38 MAPK phosphorylation, which began 30 min to 1 h after treatment, decreased by 2 h, and then began an upward trend over the next 4 h. Data are means ± SE; n = 3 experiments. * P < 0.05 from control value.

We next investigated whether SAPK/JNK and p38 MAPK were activated after TGF-beta 1 treatment. Again, the monolayers were incubated with TGF-beta 1 for 10 min and 0.5, 1, 2, 3, 4, and 6 h, and phosphorylation of these MAPKs was assessed with antibodies that only recognize the phosphorylated form of the kinase. TGF-beta 1 did not activate SAPK/JNK above the control level during the first 2 h after treatment (data not shown). As shown in Fig. 1, C and D, p38 MAPK phosphorylation was increased approximately seven- and fivefold above the control level at 30 min and 1 h, respectively, after TGF-beta 1 treatment. This increase in p38 MAPK preceded both the increase in ERK1/2 shown in Fig. 1 and MLC phosphorylation (15). p38 MAPK phosphorylation decreased by 2 h from that at the 30-min and 1-h time points but remained slightly elevated above the control level. The phosphorylation of p38 MAPK then showed an upward trend over the next 4 h that was similar to ERK1/2 phosphorylation and MLC phosphorylation (15).

Role of ERK1/2 in TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability. Klemke et al. (17) demonstrated that ERK1/2 can directly induce the phosphorylation of MLC kinase, resulting in a subsequent enhanced phosphorylation of MLC. To determine whether ERK1/2 plays a similar role after TGF-beta 1 treatment, endothelial monolayers were pretreated with 10 µM U-0126, a specific inhibitor of the upstream kinase MAPK kinase (MEK)1/2 (6), for 30 min and then coincubated with TGF-beta 1 and U-0126 for 4 h. U-0126 inhibited the increase in ERK1/2 phosphorylation after TGF-beta 1 treatment (normalized phospho-ERK: 2.36 ± 0.28 after TGF-beta 1 treatment and 0.084 ± 0.00 after TGF-beta 1 plus U-0126 treatment) and decreased the control level of phospho-ERK (normalized phospho-ERK: 0.75 ± 0.18 in control cells and 0.04 ± 0.01 after U-0126 treatment). The addition of U-0126, however, did not inhibit the TGF-beta 1-induced increase in MLC phosphorylation (Fig. 2A). In addition, U-0126 did not inhibit the TGF-beta 1-induced decrease in endothelial electrical resistance (Fig. 2B). Pretreatment with PD-98059, another inhibitor of the upstream kinase MEK1/2, also inhibited the TGF-beta 1-induced increase in ERK1/2 phosphorylation but did not prevent the TGF-beta 1-induced increase in MLC phosphorylation or the decrease in electrical resistance (data not shown). These studies demonstrate that although ERK1/2 is activated by TGF-beta 1 treatment, this pathway does not play a role in regulating endothelial monolayer permeability and MLC phosphorylation produced by TGF-beta 1.


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Fig. 2.   ERK1/2 inhibitor U-0126 does not inhibit TGF-beta 1-induced increases in myosin light chain (MLC) phosphorylation and endothelial monolayer permeability. A: CPAE cells were pretreated with U-0126 for 1 h and then coincubated with 1 ng TGF-beta 1/ml and U-0126 for 4 h. MLC phosphorylation assays and TGF-beta 1 treatment were performed as described in METHODS. The percentage of monophosphophorylated (open bars) and diphosphorylated (solid bars) MLC was normalized to total MLC. Data are means ± SE; n = 3 experiments. ERK1/2 inhibitor U-0126 did not inhibit TGF-beta 1-induced MLC phosphorylation. * P < 0.05 from control value. B: CPAE cells were pretreated with U-0126 (left arrow) for 1 h followed by coincubation with U-0126 and TGF-beta 1 (right arrow), and electrical resistance was measured for 20 h. Data are means at 30-min intervals; n = 4 experiments. TGF-beta 1-induced increase in monolayer permeability was not blocked by ERK1/2 inhibitor U-0126. The decrease in electrical resistance induced by TGF-beta 1 between 1.5 and 2 h was not blocked by U-0126.

Role of p38 MAPK in TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability. Because p38 MAPK phosphorylation was increased after TGF-beta 1 treatment (Fig. 1, C and D), we determined whether p38 MAPK was involved in the TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability by using the specific inhibitor SB-203580. Pretreatment of the monolayers with SB-203580 followed by coincubation with TGF-beta 1 inhibited the increase in MLC phosphorylation in a dose-dependent manner, with maximal inhibition of MLC phosphorylation resulting from 20 µM SB-203580 (Fig. 3A). To document that SB-203580 was inhibiting p38 MAPK, we assessed changes in HSP27 phosphorylation, a stress protein that is phosphorylated after increases in p38 MAPK activity. HSP27 phosphorylation was increased at 1 h post-TGF-beta 1 treatment, and SB-203580 inhibited the TGF-beta 1-induced HSP27 phosphorylation (data not shown). In addition (Fig. 3B), SB-203580 (20 µM) inhibited the TGF-beta 1-induced decrease in endothelial monolayer integrity as assessed by a decrease in electrical resistance.


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Fig. 3.   p38 MAPK inhibitor SB-203580 (SB) blocks TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability. A: CPAE cells were pretreated with indicated concentrations of SB-203580 for 30 min and then coincubated with 1 ng TGF-beta 1/ml and SB-203580 for 4 h. MLC phosphorylation assays and TGF-beta 1 treatment were performed as described in METHODS. The percentage of monophosphorylated (open bars) and diphosphorylated (solid bars) MLC was normalized to total MLC. Data are means ± SE; n = 3 experiments. p38 MAPK inhibitor SB-203580 attenuated TGF-beta 1-induced MLC phosphorylation. * P < 0.05 from control value. B: CPAE cells were pretreated with SB-203580 (left arrow) for 30 min followed by coincubation with SB-203580 and TGF-beta 1 (right arrow), and electrical resistance was measured for 20 h. Data are means ± SE at 30-min intervals; n = 4 experiments. TGF-beta 1-induced increase in monolayer permeability was blocked by p38 MAPK inhibitor SB-203580. SB-203580 compound blocked the decrease in electrical resistance induced by TGF-beta 1.

Hurst et al. (15) previously demonstrated that TGF-beta 1 caused a rearrangement of actin characterized by a loss of the peripheral band, an increase in actin stress fibers in a time course similar to that for MLC phosphorylation, and a decrease in endothelial monolayer integrity. In the present study, we used immunofluorescence microscopy to determine whether SB-203580 could prevent the TGF-beta 1-induced changes in actin distribution and the colocalized distribution of MLC phosphorylation (Fig. 4). Phosphorylated MLC was visualized with an antibody specific to diphosphorylated MLC, and actin was labeled with Oregon Green-conjugated phalloidin. Four hours of TGF-beta 1 treatment resulted in a loss of the peripheral actin band, an increase in actin stress fibers, and formation of intercellular gaps (arrows, Fig. 4, C and D). Diphosphorylated MLC was colocalized with the actin filaments and showed an increase in intensity consistent with increases in diphosphorylated myosin found in immunoblots. Pretreatment with 20 µM SB-203580 preserved the peripheral band and attenuated the formation of stress fibers after 4 h of TGF-beta 1 treatment. The experiments shown in Figs. 3 and 4 demonstrate that p38 MAPK is an important mediator in the signal transduction pathways that lead to TGF-beta 1-induced increases in MLC phosphorylation and endothelial monolayer permeability.


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Fig. 4.   Inhibition of TGF-beta 1-induced stress fiber formation by SB-203580. CPAE cells were treated with either untreated (control; A and B), treated with 1 ng/ml of TGF-beta 1 for 4 h (C and D), or pretreated with 20 µM SB-203580 for 30 min followed by TGF-beta 1 for 4 h (E and F) and then dual-labeled for diphosphorylated MLC (A, C, and E) or actin (B, D, and F). A 4-h treatment with TGF-beta 1 resulted in the loss of the peripheral band of actin, increased formation of stress fibers, and the formation of intercellular gaps (arrows, C and D). SB-203580 (20 µM) pretreatment preserved the peripheral band of actin and prevented the increase in stress fibers and the formation of intercellular gaps (E and F).

TGF-beta 1-induced MLC phosphorylation is dependent on protein synthesis. Because MLC phosphorylation does not increase until 2 h after treatment with TGF-beta 1 (15), we hypothesized that an increase in protein synthesis is required for this delayed response. We tested this hypothesis by pretreating endothelial monolayers with cycloheximide for 30 min and then coincubating them with cycloheximide and TGF-beta 1 for 4 h. As shown in Fig. 5, treatment with cycloheximide completely blocked the TGF-beta 1-induced increase in MLC phosphorylation. Similar results were obtained with puromycin, another inhibitor of protein synthesis (data not shown). As shown in Fig. 6A, cycloheximide treatment did not prevent MLC phosphorylation induced by inhibiting the primary phosphatase responsible for the dephosphorylation of MLC, protein phosphatase 1 (4, 9, 32), with calyculin A. Treatment for 4 h with cycloheximide also did not prevent the increase in MLC phosphorylation produced by thrombin, which increases MLC phosphorylation through an MLC kinase/Rho kinase-dependent pathway. The results in Fig. 6 demonstrate that the kinases and phosphatases responsible for the phosphorylation and dephosphorylation of MLC are not directly inhibited after a 4-h treatment with cycloheximide and can still be activated by other mediators. Thus the data in Figs. 5 and 6 demonstrate that the TGF-beta 1-induced increase in MLC phosphorylation requires protein synthesis.


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Fig. 5.   Inhibition of TGF-beta 1-induced MLC phosphorylation by cycloheximide (CHX). CPAE cells were pretreated with 10 µg/ml of CHX for 30 min and then coincubated with 1 ng TGF-beta 1/ml and CHX for 4 h. MLC phosphorylation assays and TGF-beta 1 treatment were performed as described in METHODS. The percentage of monophosphorylated (open bars) and diphosphorylated (solid bars) MLC was normalized to total MLC. Data are means ± SE; n = 3 experiment. * P < 0.05 from control value.



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Fig. 6.   Neither protein phosphatase inhibitor calyculin (Cal) A nor thrombin-induced MLC phosphorylation could be inhibited by the protein synthesis inhibitor CHX. A: CPAE cells were pretreated with 10 µg/ml of CHX for 4 h and then coincubated with Cal A for 10 min. MLC phosphorylation induced by the protein phosphatase inhibitor Cal A was not blocked by the protein synthesis inhibitor CHX. B: CPAE cells were pretreated with 10 µg/ml of CHX for 4 h and then coincubated with 1 µM thrombin for 10 min. Assessment of MLC phosphorylation was performed as described in METHODS. The percentage of monophosphorylated (open bars) and diphosphorylated (solid bars) MLC was normalized to total MLC. Data are means ± SE; n = 3 experiments. Thrombin-induced MLC phosphorylation was not blocked by protein synthesis inhibitor cycloheximide. * P < 0.05 from control value.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The MAPK pathway has been shown to mediate transcriptional activation and the subsequent protein expression of cytokines and growth factors including TGF-beta 1 (21); however, these studies have been primarily performed in transformed or immortalized cells. We assessed the role of three MAPK pathways (ERK1/2, SAPK/JNK, and p38 MAPK) in regulating the TGF-beta 1-induced increases in MLC phosphorylation and monolayer permeability in primary cultures of pulmonary endothelial cells. We found no increase in SAPK/JNK phosphorylation (data not shown); however, both ERK1/2 phosphorylation (Fig. 1, A and B) and p38 MAPK phosphorylation (Fig. 1, C and D) were increased by the addition of TGF-beta 1 to endothelial monolayers. Surprisingly, the increase in ERK1/2 phosphorylation did not begin until 2-3 h after the addition of TGF-beta 1 and was temporally associated with the increase in MLC phosphorylation and the decrease in electrical resistance. This time course of MAPK activation (Fig. 1) is similar to that reported by Finlay et al. (7) for primary cultures of fibroblasts. These investigators found that the increase in ERK1/2 activation and the subsequent increase in AP-1 activation began between 2 and 6 h after the addition of TGF-beta 1, whereas p38 MAPK activity was increased 30 min and 2 h post-TGF-beta 1 treatment (7).

ERK1/2 has been shown to directly phosphorylate MLC kinase, resulting in enhanced phosphorylation of MLC (17), implicating a role for ERK phosphorylation in regulating cell contraction. The temporal association of MLC and ERK1/2 phosphorylation shown in Fig. 1, A and B, and Ref. 15, respectively, suggested that ERK1/2 could play a similar role after TGF-beta 1 treatment. However, the inhibition of ERK1/2 phosphorylation by U-0126, a specific inhibitor of the upstream kinase MEK1/2, did not prevent the TGF-beta 1-induced increases in MLC phosphorylation (Fig. 3A) or endothelial monolayer permeability (Fig. 3C). Alternatively, the temporal association of ERK1/2 activation and MLC phosphorylation may reflect the activation of common upstream regulators of ERK1/2 and MLC phosphorylation. Ras has been shown to activate ERK1/2 through Raf and MEK1 (reviewed in Ref. 19). Ras can activate the Rho family of small GTPases, with a hierarchy of Ras activating Rac, which activates Rho (reviewed in Ref. 31). Rho activation results in MLC phosphorylation through an increase in Rho kinase activity and a decrease in myosin phosphatase activity (2, 16). Thus activation of Ras could play a central role in both the activation of ERK1/2 and MLC phosphorylation after TGF-beta 1 treatment, explaining the temporal association between these two events and the inability of U-0126 to prevent increases in MLC phosphorylation and monolayer permeability.

Inhibition of p38 MAPK by the specific inhibitor SB-203580 prevented both the TGF-beta 1-induced increase in MLC phosphorylation and the decrease in electrical resistance (Fig. 3). The increase in p38 MAPK phosphorylation (Fig. 1, C and D) showed a biphasic response. The largest increase occurred at 30 min and 1 h, time points that preceded both the increase in MLC phosphorylation and monolayer permeability. At 2 h post-TGF-beta 1 treatment, p38 MAPK phosphorylation began to drop but then showed an upward trend over the next 4 h. Two mechanisms may allow p38 MAPK to be involved in mediating the changes in monolayer integrity after the addition of TGF-beta 1. p38 MAPK has been shown to activate a downstream kinase, MAPK activator protein (MAPKAP)-2, resulting in the phosphorylation of HSP27 (14). Activation of this signaling cascade (p38 MAPK right-arrow MAPKAP-2 right-arrow HSP27) has been implicated in the formation of actin stress fibers after treatment of endothelial monolayers with H2O2 or VEGF (14, 24). In addition, the inhibition of p38 MAPK has been shown to prevent both H2O2-induced and VEGF-induced decreases in monolayer integrity (14, 24, 24), similar to the results shown in Fig. 3B. In the present study, TGF-beta 1 also caused a rearrangement of actin from a peripheral band to stress fibers (Fig. 4), and diphosphorylated MLC was associated with these stress fibers. TGF-beta 1 also caused the predicted increase in HSP27 phosphorylation that was inhibited by SB-203580 (data not shown). Because SB-203580 prevented the increase in MLC phosphorylation, actin rearrangement, and monolayer permeability induced by TGF-beta 1 as well as by VEGF and H2O2 (14, 24), p38 MAPK may have a common role in regulating the changes in barrier function after treatment with these three agents.

A second explanation for how p38 MAPK inhibition may prevent changes in permeability and MLC phosphorylation relates to p38 MAPK acting as a transcription factor mediating TGF-beta 1-induced changes in gene expression (13). The early increase in p38 MAPK phosphorylation would support this second mechanism. As seen in Fig. 1, p38 MAPK phosphorylation increased 30 min after TGF-beta 1 treatment and preceded the increases in permeability, MLC phosphorylation, and ERK1/2 phosphorylation. A previous study (13) has found that p38 MAPK activates transcription factors that lead to changes in gene expression after TGF-beta 1 treatment. Inhibition of p38 MAPK could prevent the transcriptional activation and thereby prevent the expression of de novo protein synthesis required for increased permeability and MLC phosphorylation.

The concept that changes in gene expression are required for the increase in MLC phosphorylation is supported by the studies in Fig. 5 showing that cycloheximide inhibited the TGF-beta 1-induced increase in MLC phosphorylation, indicating that protein synthesis is required for this response. This inhibition of TGF-beta 1-induced MLC phosphorylation is not due to cycloheximide directly inhibiting the kinase pathways known to phosphorylate MLC. Increases in MLC kinase activity and Rho kinase activity have been implicated in mediating the thrombin-induced increase in MLC phosphorylation (5, 8, 11). As shown in Fig. 6, thrombin treatment in the presence of cycloheximide still resulted in an increase in MLC phosphorylation, demonstrating that the acute regulation of MLC phosphorylation by MLC kinase and/or Rho kinase is operative after cycloheximide treatment. Calyculin A, an inhibitor of myosin phosphatase, also increased MLC phosphorylation in the presence of cycloheximide, further demonstrating that the kinases involved in MLC phosphorylation are operative after 4 h of cycloheximide treatment. We conclude that TGF-beta 1-induced gene expression and subsequent protein synthesis are required for the increased MLC phosphorylation.

A number of proteins may be responsible for the increase in MLC phosphorylation and monolayer permeability found in endothelial cells after TGF-beta 1 treatment. TGF-beta 1 has been shown to mediate the transdifferentiation of epithelial cells to a mesenchymal cell phenotype, and this transformation has been shown to be dependent on activation of RhoA signaling pathways (3). Shen et al. (26) have demonstrated that the increase in RhoA activity after TGF-beta 1 treatment results from an increase in the expression of NET1, a guanine nucleotide exchange factor for RhoA. A second potential mediator that may be upregulated after TGF-beta 1 treatment is basic fibroblast growth factor. Finlay et al. (7) recently demonstrated that increases in ERK1/2 activation and subsequent AP-1 activation are due to an increase in basic fibroblast growth factor release into the medium after treatment of primary fibroblast cultures with TGF-beta 1. Studies are currently underway to determine whether similar mediators are responsible for the increase in MLC phosphorylation and monolayer integrity after treatment of endothelial cells with TGF-beta 1.

In summary, we have shown that TGF-beta 1 treatment of endothelial monolayers results in an increase in MLC phosphorylation. This increase is temporally associated with an increase in endothelial monolayer permeability and with activation of ERK1/2. Although increased MLC phosphorylation and endothelial permeability are temporally associated with ERK1/2 activation, this is not a cause-and-effect relationship because inhibition of ERK1/2 activation did not prevent the changes in monolayer integrity or MLC phosphorylation. Increased phosphorylation of MLC is dependent on protein synthesis, suggesting that TGF-beta 1-induced changes in gene expression are required for this response. Furthermore, inhibition of p38 MAPK prevented the TGF-beta 1-induced increase in MLC phosphorylation and the decrease in endothelial monolayer integrity, demonstrating an important role for p38 MAPK in these responses.


    ACKNOWLEDGEMENTS

We thank Kari L. Shephard for excellent technical assistance and Wendy Hobb for excellent secretarial assistance in the preparation of this manuscript.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-54206 (to P. A. Vincent) and American Heart Association Grant AHA-97-127A (to F. L. Minnear).

Address for reprint requests and other correspondence: P. A. Vincent, Center for Cardiovascular Sciences (MC-8), Albany Medical College, Albany, NY 12208 (E-mail: vincenp{at}mail.amc.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.

Received 14 June 2001; accepted in final form 6 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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