From Institut für Prophylaxe und Epidemiologie
der Kreislaufkrankheiten, Universität München,
Pettenkoferstrasse 9, 80336 München, Germany, ** Max von
Pettenkofer Institut für Medizinische Mikrobiologie,
Pettenkoferstrasse 9a, 80336 München, Germany, and the
¶ Division of Signal Transduction, Nara Institute of Science and
Technology, Ikoma, 630-01, Japan
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ABSTRACT |
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The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.
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INTRODUCTION |
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A variety of pathological conditions including the early stages of
atherosclerosis, acute inflammation, and anaphylactic shock are
associated with increased vascular permeability (1, 2). Thrombin
generated under these pathological conditions induces endothelial cell
contraction and increases vascular permeability through activation of a
specific receptor that is coupled via heterotrimeric G-proteins of the
Gq family to phospholipase C that cleaves
phosphatidylinositol-4,5-bisphosphate to yield inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate3
mobilizes Ca2+ from intracellular stores and thus increases
intracellular Ca2+ concentration (3-5). It is well
established that the thrombin-induced increase in intracellular
Ca2+ concentration leads to activation of
Ca2+/calmodulin-dependent myosin light chain
kinase (MLCK),1 which
phosphorylates Thr-18 and Ser-19 of the light chain of myosin II (MLC)
(3). Phosphorylation induces a conformational change in MLC that
enables actin-myosin interaction and activates the
Mg2+-ATPase activity of myosin (6). Besides MLC kinases,
myosin-associated MLC phosphatase (PP1M) also seems to play a major
role in the control of MLC phosphorylation/dephosphorylation in
endothelial cells. This is demonstrated by the finding that
pharmacological inhibitors of protein phosphatase 1 (PP1) increased MLC
phosphorylation and cell contraction (7), whereas microinjection of
active PP1 decreased MLC phosphorylation and disturbed actin/myosin
interaction (8). PP1 is composed of three components, a 37-38-kDa
catalytic subunit, a 130-kDa regulatory subunit, and a 20-kDa subunit
(9-11). Recently, it was shown that the regulatory subunit can be
phosphorylated and inactivated by Rho kinase, a specific target protein
of the GTPase Rho (12). Previous work had indicated that Rho mediates cell contraction induced by thrombin, but the Rho targets involved have
not been identified thus far (13-15).
The Rho family of Ras-like GTPases that consists of more than 10 members has been implicated in actin cytoskeleton organization and
cellular shape changes in a variety of cell types (16, 17). Rho-GTPases
cycle between a GTP-bound active state and a GDP-bound inactive state,
and this cycle is controlled by guanine nucleotide exchange factors and
GTPase-activating proteins (18-20). ADP-ribosylation and inactivation
of Rho by C3-transferase from Clostridium botulinum specifically inhibits the cellular effects of Rho (21-28).
Microinjection of constitutively active V14Rho induced formation of
stress fibers and focal adhesion sites as well as a contractile
phenotype in fibroblasts (21, 28). It has also been reported that
GTPS- and aluminum flouride-mediated Ca2+ sensitation of
smooth muscle contraction is mediated by Rho (29). A number of target
proteins that interact with GTP-bound but not with GDP-bound Rho have
been identified (16). These include the closely related Ser/Thr kinases
ROK
/Rho kinase and ROK
/p160 (30, 31, 33-37). ROK
/Rho kinase
consists of multiple domains including a catalytic domain at the amino
terminus, a coiled coil domain including the Rho binding domain (RBD),
and a C-terminal pleckstrin homology domain (34). Microinjection of
isolated Rho kinase domains into fibroblasts or HeLa cells indicated
that Rho kinase is the target protein by which Rho forms stress fiber and focal adhesions (36, 37). Consistent with the involvement of Rho
kinase in contractile events, overexpression of constitutively active
V14Rho in fibroblasts caused phosphorylation of the MBS, inactivation
of myosin phosphatase, and an increase in MLC phosphorylation (12).
Here, we provide evidence that thrombin uses the Rho/Rho kinase pathway to inactivate PP1M in human endothelial cells. Inactivation of PP1M seems to be coordinated with activation of Ca2+-calmodulin-dependent MLCK to maximally increase MLC phosphorylation in the early phase of thrombin-induced endothelial cell contraction.
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EXPERIMENTAL PROCEDURES |
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Materials-- The inhibitors okadaic acid, KT5926, and tautomycin were from Calbiochem (Bad Soden, Germany); all other materials not specifically indicated were from Sigma.
Cell Culture--
Human umbilical vein endothelial cells (HUVEC)
were obtained and cultured as described previously (24). Briefly, cells
harvested from umbilical cords were plated onto collagen-coated (24 h,
100 µg/ml collagen G; Biochrom, Berlin, Germany) plastic culture
flasks and cultured in endothelial growth medium (Promo Cell,
Heidelberg, Germany), containing endothelial cell growth
supplement/heparin and 10% fetal calf serum. For all experiments,
cells were plated at a density of 2 × 104
cells/cm2 and grown to confluency for 10 days. Confluent
monolayers were stimulated for the indicated time periods with
-thrombin from human plasma (Boehringer, Mannheim, Germany).
Measurement of Endothelial Permeability-- Horseradish peroxidase diffusion through HUVEC monolayers was determined as described previously with some modifications (38). Briefly, cells were plated (2 × 104 cells/cm2) on collagen-coated polyethylene terephtalate cell culture inserts (3-µm pore size, Becton Dickinson), which were set into 24-well Falcon companion TC plates (Becton Dickinson). HUVEC were cultured for 10 days with medium changes every 2 days. For thrombin stimulation, medium was replaced with 500 µl of culture medium containing thrombin. For controls, thrombin was omitted, but otherwise cells were treated identically. After 15 min of stimulation, 500 µl of medium was filled into the lower compartment, and the medium in the upper compartment was replaced with fresh medium containing horseradish peroxidase (0.34 mg/ml, IV-A type, 44,000 Mr; Sigma, Deisenhofen, Germany). After 1 min, 60 µl of medium was collected from the lower compartment and mixed with 860 µl of reaction buffer (50 mM NaH2PO4, 5 mM Guaiacol) and 100 µl of freshly made H2O2 solution (0.6 mM in H2O). The reaction was allowed to proceed for 15 min at room temperature, and absorbance was measured at 470 nm.
Immunofluorescence--
For fluorescence staining, HUVEC were
plated (2 × 104 cells/cm2) on Eppendorf
Cellocate glass coverslips (Eppendorf, Hamburg, Germany) coated with
100 µg/ml collagen G (24 h) and grown to confluency for 10 days. To
label F-actin, cells were fixed for 10 min with 3.7% formaldehyde
solution in PBS containing 1 mM Ca2+ and 1 mM Mg2+, permeabilized for 5 min in cold
acetone (20 °C), and air dried. Coverslips were then incubated for
20 min with rhodamine phalloidin (Molecular Probes; 1/20 in PBS) and
mounted in Mowiol (Calbiochem, Bad Soden, Germany) containing 0.2%
p-phenylenediamine (Sigma, Deisenhofen, Germany) as
anti-fading agent. All steps were performed at room temperature with
three washes in PBS/2% BSA between antibody incubations. Fluorescence
microscopy was performed with a Leica RDM 3 microscope, and
microphotographs were recorded on Kodak T-Max 400 film.
Recombinant Proteins--
Recombinant C3-transferase and V14
RhoA and RBD were expressed as glutathione S-transferase
fusion proteins in Escherichia coli and purified on
glutathione-Sepharose beads as described (21, 39). The fusion proteins
were cleaved by thrombin. Thrombin was removed by incubation for 2 h with benzamidine beads, and the released proteins were concentrated
and dialyzed against microinjection buffer (see below). Purity and
complete removal of thrombin was checked by SDS-PAGE and Coomassie
staining. Extracellular application of V14Rho even at amounts exceeding
the microinjected volume by 9 orders of magnitude (1015
versus 10
6 liters) did not produce any
thrombin-like effect, excluding thrombin contamination of recombinant
protein. Protein concentrations were determined with the BCA protein
assay kit (Pierce) using BSA as a standard. As tested by SDS-PAGE and
Coomassie staining, protein preparations showed essentially only one
band. Rho kinase was expressed in SF9 cells as described previously
(37).
Microinjection--
Microinjection was performed using an
Eppendorf Transjector 5246 and an Eppendorf Micromanipulator 5171. Cells were plated and cultured on Cellocate coverslips (Eppendorf,
Hamburg, Germany) as described above. V14Rho, recombinant catalytic
domain of Rho kinase, RBD from Rho kinase, and C3-transferase were
diluted with microinjection buffer (150 mM NaCl, 50 mM Tris, 5 mM MgCl2, pH 7.5) and
injected at a concentration of 0.4 µg/µl (V14Rho), 0.64 µg/µl
(RBD), and 0.025 µg/µl (C3-transferase) into the cytoplasm of
HUVEC. The microinjected volume was about 1-3 × 1015 liters per cell. PP1 catalytic subunit (
-isoform;
Calbiochem, Bad Soden, Germany) was diluted with phosphatase buffer
(100 mM K+ glutamate, 39 mM
K+ citrate, pH 7.3) and was injected at a concentration of
200 units/ml. Control injections carried out with microinjection or
phosphatase buffer, respectively, did not produce any significant
effect on cell morphology or actin organization. After injection, cells were returned to the incubator for 30 min. Injected cells were identified by labeling a coinjected marker protein (5 mg/ml rat IgG)
with fluorescein isothiocyanate-conjugated goat anti-rat IgG (Dianova,
Hamburg, Germany) and by relocation on the Cellocate coverslips using
the microgrid. For each experiment, about 100 cells were injected and
examined by fluorescence microscopy.
Myosin Light Chain Phosphorylation-- MLC phosphorylation was analyzed by urea PAGE separation of the mono- and diphosphorylated forms as described in detail elsewhere (40). HUVEC were stimulated with thrombin as indicated, and reaction was terminated by immediate addition of 1.5 ml of ice-cold 10% trichloroacetic acid. Cells were scraped and then centrifuged for 20 min at 14,000 × g. Supernatants were discarded, and pellets were washed with ddH20 to remove trichloroacetic acid and resolved in 1.5 ml of sample buffer (6.7 M urea, 20 mM Tris, 22 mM glycine, 10 mM dithiothreitol, pH 9.0). Samples (75 µg per lane) were applied to urea gel electrophoresis (top gel 3.5% acrylamide, bottom gel 10% acrylamide) and run at 9 mA for approximately 45 min until the marker dye had come out of the bottom gel. The gels were stained for 1 h with 0.05% Coomassie Brilliant Blue 250, 10% acetic acid, 30% methanol, 60% H2O. The stoichiometry of MLC phosphorylation (mol phosphate/mol MLC) was determined by densitometric analysis of the wet gels, using a Sharp XL-325 densitometer and Pharmacia Image Master software, and calculated using the formula P1 + 2xP2/P0 + P1 + P2.
Measurement of Cytosolic Ca2+
Concentration--
Cytosolic
[Ca2+]i was measured as described
previously (41). Cells grown on collagen-coated glass coverslips were loaded for 30 min at 37 °C with fura-2 AM (2.5 µM;
Calbiochem, Bad Soden, Germany) and resuspended in HEPES buffer (20 mM HEPES, 120 mM NaCl, 2.7 mM KCl,
1.4 mM MgSO4, 0.5 mM
CaCl2, 1.4 mM KH2PO4, 25 mM NaHCO3, 10 mM glucose, pH
7.4). Ca2+-dependent fluorescence was measured
using a double excitation spectrofluorimeter (AMKO Light Technology,
Tornesch, Germany) with the emission wavelength set at 510 nm and the
emission wavelength rapidly alternating between 340 and 380 nm.
[Ca2+]i was quantified applying
the equation [Ca2+]i = Kd(R Rmin)/(Rmax
R) × (Sf2/Sb2).
Preparation of Myosin-enriched Cell
Fractions--
Myosin-enriched fractions of HUVEC were prepared as
described previously (7). Briefly, HUVEC were plated on collagen-coated 100-mm plates (Falcon) and cultivated for 10 days. Monolayers were
washed two times with ice-cold PBS (Sigma), and 200 µl of homogenization buffer containing 50 mM Tris-aminomethane,
pH 7.5, 0.1 mM EDTA, 28 mM -mercaptoethanol,
and 1 µg/ml each of leupeptin, pepstatin, pefabloc, and aprotinin as
protease inhibitors (1 µg/ml each) were added to the cells. Plates
were immediately cooled down to
80 °C, scraped with a rubber
policeman, and homogenized by passing the suspension several times
through a syringe. Homogenates were then treated with high salt buffer
(0.6 M NaCl, 0.1% Tween 20), containing protease
inhibitors (1 µg/ml of each) leupeptin, pepstatin, and pefabloc for
1 h at 4 °C and subsequently centrifuged at 4500 × g for 30 min at 4 °C. The supernatant was diluted 10-fold with assay buffer (50 mM Tris, 0.1 mM EDTA, 28 mM
-mercaptoethanol, pH 7.0) and centrifuged at
10,000 × g at 4 °C. The resulting pellet was
resolved in 10 µl of high salt buffer. This myosin-enriched cell
fraction contains only PP1 and essentially no PP2 activity (7). To
inhibit residual PP2 activity, the myosin-enriched cell fractions were
supplemented with okadaic acid (1 nM) to completely inhibit
PP2 (IC50 0.1 nM), whereas PP1 is only affected
at concentrations 100-fold higher (IC50 10 nM).
Measurement of Myosin-associated Phosphatase Activity--
For
measuring myosin-associated phosphatase activity in myosin-enriched
cell fractions, we used the protein phosphatase assay system (Life
Technologies, Inc.) according to the instructions of the manufacturer.
This assay system is based on the method described by Cohen et
al. (42). Briefly, phosphorylase b (0.1 mM) was in vitro phosphorylated by
phosphorylase kinase (0.1 mg/ml) in the presence of
[-32P]ATP (5 mCi/ml) in phosphorylation buffer (250 mM Tris-HCl, pH 8.2, 16.7 mM MgCl2,
1.67 mM ATP, 0.83 mM CaCl2, 133 mM 2-mercaptoethanol) for 1 h at 30 °C. Reaction
was stopped with 90% ammonium persulfate solution (4 °C). Reaction
tube was then kept on ice for 1 h and subsequently centrifuged at
12,000 × g for 10 min. The resulting protein pellet
was resuspended with ammonium persulfate solution (45% saturated). The
protein pellet was washed four times in this way. Protein solution was
then concentrated to a final concentration of 3 mg/ml using Amicon
Centricon-30 concentrators.
Western Blot Analysis of PP1/MBS Load of Myosin-enriched Cell Fractions-- The PP1 and MBS load in myosin-enriched cell fractions was tested by Western blot analysis. Briefly, myosin-enriched cell fractions were dissolved in Laemmli buffer containing protease inhibitors (1 µg/ml leupeptin, pepstatin, aprotinin) and applied to SDS, 10% PAGE. Proteins were then blotted onto polyvinylene difluoride membranes for 1 h at 80 V. Membranes were blocked with 10% low-fat milk dissolved in Tris-buffered saline. PP1 was detected using rabbit polyclonal anti-PP1 antibody (Upstate Biotechnology, Lake Placid, NY) or rabbit polyclonal anti-MBS antibody (12), both diluted 1/1000 in Tris-buffered saline, and horseradish peroxidase-labeled secondary antibody (Amersham, Braunschweig, Germany), diluted 1/4000 in Tris-buffered saline, developed with enhanced chemiluminescence reagent (Amersham), and then exposed to hyperfilm ECL (Amersham).
Detergent Solubility Assay of Catenins--
Triton X-100
solubility of catenins was assayed as described elsewhere (43) Briefly,
Triton X-100 soluble and insoluble cell fractions were analyzed be
Western blots using anti--catenin (American Research Products,
Belmont, MA) or anti-plakoglobin (clone PG5.1; American Research
Products, Belmont, MA) monoclonal antibodies.
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RESULTS |
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Inactivation of Rho by C3-transferase Blocks Thrombin-induced Endothelial Cell Contraction-- To test whether Rho is involved in endothelial cell contraction, we measured the thrombin-stimulated increase of transendothelial horseradish peroxidase diffusion in the absence and presence of the specific Rho inactivator C3-transferase from C. botulinum (45, 46). The results presented in Fig. 1 demonstrate that confluent monolayers of HUVEC show a low transendothelial diffusion of horseradish peroxidase. Stimulation with thrombin (0.1-1 unit) dose dependently increased horseradish peroxidase permeability up to 10-fold. The thrombin-induced increase in permeability could almost completely be abolished by pretreatment of the cells with C3-transferase (24 h, 5 µg/ml). To confirm that the underlying mechanism for this C3-transferase effect was inhibition of cell contraction, we performed actin staining. As can be seen in Fig. 2a, actin is mainly concentrated in a dense peripheral band along cell-cell contacts in intact endothelial monolayers. When stimulated with thrombin, cells contracted, expressed actin fibers, and formed numerous intercellular gaps (Fig. 2b). In cells pretreated with C3-transferase, these thrombin effects were abolished, and cells remained flat and spread out (Fig. 2c). The actin band at cell-cell contacts was thinned out by the C3 treatment but remained essentially intact. These results suggest that activation of Rho is a key mechanism in thrombin-induced endothelial cell contraction/increase of endothelial permeability. The Rho subtype involved likely is RhoA because we recently found that RhoA is by far the predominant substrate of C3-transferase in HUVEC and is ADP-ribosylated to about 70-80% under the conditions employed (27). Consistent with this, microinjection of constitutively active V14RhoA produced cell contraction and stress fiber formation similar to thrombin (Fig. 2f).
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Evidence for the Involvement of Rho Kinase and Myosin PP1M in Thrombin-induced Cell Contraction-- To obtain evidence that the Rho target protein Rho kinase mediates the thrombin/Rho effect on contraction, we microinjected isolated RBD of Rho kinase, which has been shown to inhibit interaction of Rho with Rho kinase (37). Microinjection of RBD blocked thrombin-induced cell contraction similar to C3-transferase (Fig. 2d). Conversely, when we microinjected the recombinant catalytic domain of Rho kinase, endothelial cells contracted and showed a shape change (Fig. 2g) similar to stimulation with thrombin (Fig. 2b), microinjection of V14RhoA (Fig. 2f), or treatment with the PP1M inhibitor tautomycin (not shown). These data suggest that Rho kinase is the Rho target protein by which thrombin exerts its effects on cell contraction. To investigate whether inhibition of the Rho kinase target protein PP1M contributes to thrombin-induced endothelial cell contraction, we microinjected the constitutively active catalytic domain of PP1. As can be seen in Fig. 2e, microinjection of PP1 inhibited the thrombin-induced cell contraction similar to C3-transferase (Fig. 2c) or RBD (Fig. 2d). These data are consistent with the idea that thrombin uses a pathway that involves activation of Rho and Rho kinase as well as inactivation of PP1M to regulate cell contraction.
Thrombin Inactivates PP1M Activity via Rho-- We reasoned that if Rho kinase is in fact activated by thrombin, an inhibition of PP1M activity should be detected. We therefore determined PP1M activity in cells stimulated with thrombin for different time periods by assaying dephosphorylation of phosphorylase b (9). The results presented in Fig. 3 clearly show that thrombin produced a transient decrease of PP1M activity between 30 s and 3 min, which was followed by a return to base-line values after 5 min (Fig. 3a). In the C3-transferase-treated cells, the thrombin-induced decrease in PP1M activity was abolished, further supporting the notion that Rho regulates PP1M. To unambiguously demonstrate that [32P]phosphorylase b dephosphorylation reflects MLC-phosphatase (PP1C) activity, we also used 32P-MLC as a substrate. The inset in Fig. 3a shows that PP1C activity determined with 32P-MLC as substrate was essentially identical to the PP1C activity determined with [32P]phosphorylase b as substrate. Furthermore, we tested by Western blot whether thrombin caused dissociation of the catalytic subunit (PP1C) or the regulatory subunit (MBS) of PP1M from the myosin-enriched fractions. The data in Fig. 3c show that PP1C and MBS did not dissociate from myosin, suggesting that the MLC-phosphatase activity measured was because of the holoenzyme.
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Inhibition of Rho Blocks Thrombin-stimulated MLC Phosphorylation-- Phosphorylation of the light chain of myosin II (MLC) is a crucial mechanism by which thrombin signals are converted into the mechano-chemical force for cell contraction (47, 48). To investigate whether the observed thrombin-induced inhibition of PP1M is relevant for MLC phosphorylation, we stimulated control and C3-pretreated (24 h, 5 µg/ml) endothelial cells for different times with thrombin (1 unit/ml) and separated un-, mono-, and diphosphorylated MLC on 10% urea polyacrylamide gels. Densitometric quantitation of these MLC forms revealed that thrombin caused phosphate incorporation into MLC with a peak after 1 min (Fig. 3c). The level of phosphorylated MLC then dropped to a plateau above base line between 5 and 15 min, indicating that MLC is partly dephosphorylated after the initial peak. The peak in phosphorylation exactly correlated with maximal PP1M inhibition, whereas the drop to plateau phosphorylation correlated with the increase of PP1M activity back to base line. In cells pretreated with C3, the thrombin-stimulated peak in MLC phosphorylation was essentially abolished (Fig. 3c). These data suggest that inhibition of PP1M activity via Rho/Rho kinase is an essential mechanism by which thrombin yields a peak level in MLC phosphorylation.
Rho Is Not Involved in Thrombin-stimulated Ca2+ Mobilization in Endothelial Cells-- It is well established that thrombin elevates intracellular Ca2+ concentration and thereby activates Ca2+/calmodulin-dependent MLCK (48). Interestingly, C3-transferase was shown to inhibit thrombin-stimulated Ca2+ mobilization in fibroblasts (32). To test whether this is the mechanism by which C3-transferase prevents thrombin-induced MLC phosphorylation in HUVEC, we loaded control or C3-treated endothelial cells with the Ca2+ indicator fura-2 AM and determined cytosolic-free Ca2+ concentration using fluorescence spectrometry. We found that the C3 treatment affected neither basal Ca2+ concentration nor the thrombin-stimulated increase in peak (after 30 s) or plateau (after 3 min) Ca2+ concentration (Fig. 6). We conclude that Rho is not involved in the thrombin-induced cytosolic Ca2+ increase in endothelial cells.
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C3-transferase Does Not Prevent Thrombin-induced Release of
Catenins from the Cytoskeleton--
In tightly confluent HUVEC, the
VE-cadherin/catenin-based adherens junctions are associated with the
actin cytoskeleton to stabilize the endothelial barrier. It has been
suggested that thrombin-induced increase in endothelial permeability
might be partly because of a release of catenins from the Triton X-100 insoluble cytoskeletal cell fraction (43, 44). To exclude that the
effect of C3-transferase on thrombin-induced increase in endothelial
permeability was the result of prevention of this shift, we performed
detergent solubility assays of catenins. As shown in Fig.
7, we found that plakoglobin and
-catenin are associated with the Triton X-100 insoluble fraction in
confluent HUVEC. After thrombin treatment, both
-catenin and
plakoglobin (
-catenin) lost their association with the
cytoskeleton and shifted to the cytoplasm. This shift was not prevented
by C3-transferase treatment. This result indicates that prevention of
the release of catenins from the cytoskeleton is not the mechanism by
which C3-transferase inhibits thrombin-induced increase in
endothelial permeability.
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DISCUSSION |
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Our data suggest an additional signal pathway by which thrombin
regulates myosin light chain phosphorylation and the subsequent increase in endothelial cell contraction/vascular permeability. We
propose that thrombin activates Rho, which then interacts with its
target Rho kinase that in turn inactivates PP1M, most likely by
phosphorylation of the 130-kDa regulatory subunit (12). The transient
inactivation (within 0.5-5 min) of PP1M demonstrated here most likely
produces the peak in MLC phosphorylation seen within the first 5 min of
thrombin stimulation. Others have found a similar peak of MLC
phosphorylation in thrombin-stimulated cells (49). Interestingly, the
peak in MLC phosphorylation also correlates with the transient peak in
intracellular Ca2+ elevation obtained after thrombin
stimulation (41). After 5 min of thrombin stimulation, MLC phosphatase
activity reversed to near base-line values and, in parallel MLC
phosphorylation, dropped to a plateau. This plateau MLC phosphorylation
correlates with a plateau in intracellular Ca2+
concentration (41). Taken together, these data suggest that the
Rho-induced inhibition of MLC phosphatase activity is coordinated with
a peak in Ca2+ mobilization to produce maximal MLC
phosphorylation. Along this line, it has been reported that GTPS-
and aluminum flouride-mediated Ca2+ sensitation of smooth
muscle cell contraction is dependent on Rho (29). In these smooth
muscle cells, myosin light chain phosphatase was inhibited by a
Triton-soluble membrane-bound effector, which was not Rho. This
effector could be Rho kinase.
We demonstrated that C3-transferase could completely block thrombin-stimulated MLC phosphorylation and cell contraction as well as the increase in endothelial permeability. We want to emphasize that this does not contradict the idea that Rho-induced inhibition of MLC phosphatase is mainly responsible for the transient peak in MLC phosphorylation. As shown in Fig. 5, C3-transferase by itself increased MLC phosphatase activity in unstimulated cells, most likely by inhibiting a basal Rho activity. This somewhat artificially elevated MLC phosphatase activity could blunt MLC kinase activity brought about by the thrombin-induced Ca2+ signal. The fact that the MLC phosphatase inhibitor tautomycin completely reversed the C3-transferase effect on cell contraction also supports this idea.
Besides phosphorylating and inactivating PP1M, it was demonstrated that Rho kinase can directly phosphorylate MLC in vitro, i.e. can act as a MLC kinase (50). At present, we have no indication that this mechanism is relevant in endothelial cells.
Recently, it was reported that C3-transferase inhibited lysophosphatidic acid-induced MLC phosphorylation and contraction in fibroblasts. It was speculated that this inhibition is because of enhanced myosin phosphatase activity (13). Our results obtained in thrombin-stimulated endothelial cells seem to support this notion. Furthermore, we noticed that contraction induced by thrombin, V14Rho, or active Rho kinase precedes formation of stress fibers,2 which is consistent with the idea that contraction drives stress fiber formation (13). We noticed, however, that stress fibers were not as efficiently produced by Rho kinase as by V14Rho, indicating that additional Rho targets contribute to efficient stress fiber formation.
Endothelial cells flatten and spread out when Rho is inactivated and contract when Rho is activated. A similar behavior has been found in neuronal cells (15), human and mouse macrophages (51, 52), and HeLa cells (36). In contrast, other cells including NIH 3T3 fibroblasts and Vero cells round up when Rho is inactivated and spread out when it is activated (53, 54). Presumably, this behavior depends on the relative importance of Rho-dependent focal adhesion/integrin cluster formation versus Rho-dependent contractility in the respective cell type.
In fibroblasts, Rho seems to directly trigger Ca2+ mobilization, most likely by providing phosphatidylinositol 4,5-bisphosphate through stimulation of a PI(5) kinase activity (32). The reason why we did not find an effect of Rho inhibition on Ca2+ mobilization in endothelial cells might lie in the recruitment of different target proteins by Rho, depending on the cell type (16).
Fig. 8 depicts the presumptive signal pathway by which thrombin induces cell contraction. The Ca2+-triggered activation of MLCK induces in concert with inhibition of MLC phosphatase by Rho/Rho kinase an increase in MLC phosphorylation and finally cell contraction.
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The mechanism by which Rho is activated by thrombin in HUVEC remains to be determined. One possibility is that thrombin activates Rho via G13 and the epidermal growth factor receptor tyrosine kinase, as was recently shown for lysophosphatidic acid in fibroblasts (55).
Here, we describe a pathway involving Rho/Rho kinase by which thrombin inactivates PP1M and thus controls MLC phosphorylation and contraction in human endothelial cells. This pathway is coordinated with the well established Ca2+/calmodulin-dependent pathway of MLC kinase activation and presumably with yet another pathway regulating adherens junction disassembly. A complex signaling network of thrombin-controlled increase in endothelial permeability is evolving.
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ACKNOWLEDGEMENTS |
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We thank Barbara Böhlig for expert technical assistance, Manfred Schliwa (Institut für Zellbiologie, LMU München) for help with microinjection, Alan Hall for providing V14Rho, Wolfgang Siess for helpful discussions, Jürgen Heesemann (Institut für Medizinische Mikrobiologie, LMU München) for support, and Markus Bauer for help with densitometry.
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FOOTNOTES |
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* This study was supported by Deutsche Forschungsgemeinschaft Grants Ae11/5-1 and SFB413, by August Lenz Stiftung, and by Wilhelm Sander Stiftung.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.
§ To whom correspondence and reprint requests may be addressed. Markus Essler: Tel.: 49-89-5160-4371; E-mail: messler{at}klp.med.uni-muenchen.de. Martin Aepfelbacher: Tel.: 49-89-5160-5264; E-mail: aepfelbacher{at}m3401.mpk.med.uni-muenchen.de.
Present address: Universitätsklinikum Carl Gustav Carus
der TU Dresden, Med. Klinik III/Angiologie, Fetscherstr. 74, 01307 Dresden, Germany.
The abbreviations used are:
MLCK, myosin
light chain kinase; HUVEC, human umbilical vein endothelial cells; MLC, myosin light chain; MBS, myosin binding subunit of myosin light chain
phosphatase; PP1, protein phosphatase 1; PP2, protein phosphatase 2; PP1C, catalytic subunit of PP1; PP1M, myosin-bound PP1; RBD, Rho-binding domain of Rho kinase; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; GTPS, guanosine
5'-3-O-(thio)triphosphate.
2 M. Essler, M. Amano, H.-J. Kruse, K. Kaibuchi, P. C. Weber, and M. Aepfelbacher, unpublished observation.
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REFERENCES |
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