Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Transforming growth
factor (TGF)-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-
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-
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-
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-
1-induced increases in monolayer permeability and
MLC-P.
transforming growth factor-1; myosin light chain
phosphorylation; endothelial cells; cycloheximide; mitogen-activated
protein kinase
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INTRODUCTION |
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TRANSFORMING GROWTH
FACTOR (TGF)-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-
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-
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-
1 has a direct effect on the endothelium. Over
time, the continued expression of active TGF-
1 resulted in the
development of prolonged and severe pulmonary fibrosis
(28). The most recent evidence for the importance of
TGF-
1 in acute lung injury was provided by Pittet et al.
(23). These investigators found that expression of a
chimeric TGF-
1 receptor, which prevents the interaction of TGF-
1
with its receptor, protects against bleomycin-induced pulmonary edema.
These findings suggest that TGF-
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-1 in the development of
pulmonary edema. The addition of TGF-
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-
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-
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-
1, showing that an increase in cell contraction may contribute
to the TGF-
1-induced decrease in endothelial monolayer integrity.
Although Hurst et al. (15) demonstrated that inhibition of
MLC phosphorylation prevented the TGF-1-induced increase in monolayer permeability, other studies have shown that MLC
phosphorylation and monolayer permeability are not linked. Indeed,
tumor necrosis factor (TNF)-
has been shown to increase both MLC
phosphorylation and monolayer permeability, but inhibition of MLC
phosphorylation did not prevent the TNF-
-induced changes in
permeability (22). However, the inhibition of MLC
phosphorylation did prevent the TNF-
-induced increase in
apoptosis, suggesting that MLC phosphorylation may regulate
processes other than permeability. This is also true for TGF-
1
because an increase in cell tension has been implicated as an important
component in a number of TGF-
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-
1 activation that may contribute to
the development of fibrosis in many tissues (28, 30). Thus tension generation after TGF-
1 activation may be an important control point for many of the physiological and pathophysiological effects of TGF-
1.
The increases in MLC phosphorylation and endothelial monolayer
permeability in response to TGF-1 occur 2 h after the addition of TGF-
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-
1-induced increases in MLC
phosphorylation and endothelial permeability suggests that these
increases are the result of changes in gene expression. TGF-
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-
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-1-induced increases in MLC phosphorylation and
endothelial permeability. We show that ERK1/2, although activated by
TGF-
1, is not involved with the TGF-
1-induced endothelial monolayer permeability and MLC phosphorylation. Also, we demonstrate that p38 MAPK is activated by TGF-
1 and is potentially linked to
these TGF-
1 responses. Furthermore, we show here that
TGF-
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-
1
treatment. These studies illustrate that p38 MAPK plays a role in the
signal transduction pathways initiated by TGF-
1 and suggest that
activation of transcriptional activity and subsequent de novo protein
synthesis regulate the TGF-
1-induced increases in MLC
phosphorylation and endothelial monolayer permeability.
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METHODS |
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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-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-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-
1 was added. The cells were
coincubated with the respective agent and TGF-
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 (
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 × 104 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-M
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-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.
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RESULTS |
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Time course of TGF-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-
1 is
temporally associated with an increase in MLC phosphorylation. To
determine whether ERK1/2 plays a role in the TGF-
1-induced increase
in MLC phosphorylation, we first investigated whether TGF-
1
activates ERK1/2 (Fig. 1, A
and B). The monolayers were incubated with TGF-
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-
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-
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-
1 treatment (15).
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Role of ERK1/2 in TGF-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-
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-
1 and U-0126 for 4 h. U-0126 inhibited the increase in ERK1/2 phosphorylation after TGF-
1 treatment (normalized phospho-ERK: 2.36 ± 0.28 after TGF-
1 treatment and 0.084 ± 0.00 after TGF-
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-
1-induced
increase in MLC phosphorylation (Fig.
2A). In addition, U-0126 did
not inhibit the TGF-
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-
1-induced increase in ERK1/2 phosphorylation but did not prevent
the TGF-
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-
1 treatment, this pathway does
not play a role in regulating endothelial monolayer permeability and MLC phosphorylation produced by TGF-
1.
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Role of p38 MAPK in TGF-1-induced increases in MLC
phosphorylation and endothelial monolayer permeability.
Because p38 MAPK phosphorylation was increased after TGF-
1 treatment
(Fig. 1, C and D), we determined whether p38 MAPK
was involved in the TGF-
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-
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-
1 treatment, and SB-203580 inhibited the
TGF-
1-induced HSP27 phosphorylation (data not shown). In addition
(Fig. 3B), SB-203580 (20 µM) inhibited the
TGF-
1-induced decrease in endothelial monolayer integrity as
assessed by a decrease in electrical resistance.
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TGF-1-induced MLC phosphorylation is dependent on protein
synthesis.
Because MLC phosphorylation does not increase until 2 h after
treatment with TGF-
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-
1 for 4 h. As shown in Fig.
5, treatment with cycloheximide completely blocked the TGF-
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-
1-induced increase in MLC phosphorylation
requires protein synthesis.
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DISCUSSION |
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The MAPK pathway has been shown to mediate transcriptional
activation and the subsequent protein expression of cytokines and growth factors including TGF-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-
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-
1 to endothelial monolayers. Surprisingly, the increase in
ERK1/2 phosphorylation did not begin until 2-3 h after the
addition of TGF-
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-
1, whereas p38 MAPK activity was increased
30 min and 2 h post-TGF-
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-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-
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-
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-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-
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-
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
MAPKAP-2
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-
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-
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-
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-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-
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-
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-1-induced increase in
MLC phosphorylation, indicating that protein synthesis is required for
this response. This inhibition of TGF-
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-
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-1 treatment. TGF-
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-
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-
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-
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-
1.
In summary, we have shown that TGF-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-
1-induced changes in gene expression are required for this
response. Furthermore, inhibition of p38 MAPK prevented the TGF-
1-induced increase in MLC phosphorylation and the decrease in
endothelial monolayer integrity, demonstrating an important role for
p38 MAPK in these responses.
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ACKNOWLEDGEMENTS |
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We thank Kari L. Shephard for excellent technical assistance and Wendy Hobb for excellent secretarial assistance in the preparation of this manuscript.
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FOOTNOTES |
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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.
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