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INTRODUCTION |
The vascular endothelium consisting of the monolayer of
endothelial cells and the extracellular matrix represents the
major barrier for the exchange of liquid and solutes across the vessel wall (1-4). Thrombin by binding to the endothelial cell surface protease-activated receptor-1 induces a repertoire of signaling events
that result in the development of minute gaps among cells, thereby
mediating increased vascular permeability, a hallmark of tissue
inflammation (5-7). Loss of endothelial barrier function primarily
occurs as a result of cell shape change via actinomyosin driven
contraction activated by myosin light chain phosphorylation and actin
polymerization (3, 8-11).
Studies have shown an important role of the small GTPase, Rho, in the
regulation of cytoskeletal dynamics, actin stress fiber formation, and
myosin light chain-phosphorylation, and thus by inference, in
the control of endothelial barrier function (11-13). The multiple
functions of Rho are mediated through the tightly regulated
GTP-binding/GTPase cycle (14-16). Three different classes of proteins
are required for this regulation: (i) guanine nucleotide exchange
factors (GEFs),1 which
stimulate the GTP-GDP exchange reaction; (ii) GTPase-activating proteins (GAPs), which stimulate the GTP-hydrolytic reaction; and (iii)
guanine nucleotide dissociation inhibitors (GDIs), which by binding to
Rho block the dissociation of GDP from Rho GTPases (17). Furthermore,
GDI is also capable of inhibiting GTP hydrolysis by Rho family GTPases
as well as stimulating the release of Rho-GTPases from cellular
membranes, thereby shutting off the Rho cycle (18). Thus, GDI plays a
critical role in the signaling events regulated by Rho-GTPases
(19).
The GDP-bound form of Rho complexed with GDI is not activated by
Rho-GEFs, suggesting that Rho activation critically depends upon
upstream factors mediating the dissociation of GDI from Rho (19-21).
The mechanisms activating the dissociation of Rho-GDI from the Rho-GDP
complex remain to be determined. It has been suggested that the
dissociation of Rho-GDI might be facilitated by members of
ezrin/radixin/moesin family of proteins (22, 23). However, Rho-GDI was
found to interact only with the N-terminal fragment of radixin but not
the full-length radixin, indicating the need of upstream effectors that
are required to induce the unfolding of radixin (23). Furthermore,
several studies indicate that the translocation and activation of
ezrin/radixin/moesin proteins to the membrane are critically dependent
on Rho, thereby indicating the intervention of other molecules that
activate dissociation of GDI from Rho (24, 25).
Rho-GDI is a family consisting of Rho-GDI-1, Ly/D4-GDI, and Rho
GDI-III. Of these, Rho-GDI is ubiquitously expressed (19, 22). The
structure of Rho-GDI-1 indicates that it contains sequences for
phosphorylation by serine-threonine kinases, raising the possibility that Rho-GDI is regulated by signaling mechanisms that induce its phosphorylation.
Protein kinase C (PKC) isozymes are serine-threonine kinases that
induce phosphorylation of multiple proteins, which in turn regulate
intracellular signaling (26). A PKC-dependent
pathway is important in the mechanism of thrombin-induced increase in endothelial permeability (3, 27-30). Because of the possibility that
PKC may activate Rho by mediating Rho-GDI phosphorylation, we
investigated the role of PKC in the mechanism of thrombin-induced Rho
activation and in signaling the loss of endothelial barrier function in
human umbilical venular endothelial (HUVE) cells. The present findings
suggest the existence of a novel pathway by which thrombin can
stimulate Rho activation. This pathway involves PKC-
-mediated
phosphorylation of GDI, which may stimulate GDI dissociation, thereby
resulting in Rho activation and increased endothelial permeability.
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EXPERIMENTAL PROCEDURES |
Materials--
Human
-thrombin was obtained from Enzyme
Research Laboratories (South Bend, IN). HUVE cells and endothelial
growth medium (EBM-2) were obtained from Clonetics (San Diego, CA).
Phosphate-buffered saline (PBS) and trypsin were obtained from Life
Technologies, Inc. Anti-Rho-A, anti-Rho-GDI, and anti-PKC-
,
-
, -
, and -
polyclonal antibodies were obtained from Santa
Cruz Biotechnology (San Diego, CA). Purified GST-Rho-GDI was purchased
from Cytoskeleton, Inc. (Denver, CO).
Endothelial Cell Culture--
HUVE cells were cultured in a T-75
flask coated with 0.1% gelatin in EBM-2 medium supplemented with 10%
fetal bovine serum. Cells were maintained at 37 °C in a
humidified atmosphere of 5% CO2 and 95% air until they
formed a confluent monolayer. Cells from each of the primary flasks
were detached with 0.05% trypsin, 0.02% EDTA and resuspended in fresh
culture medium and passaged as described below. In all experiments,
unless otherwise indicated, a confluent monolayer of HUVE cells was
washed twice with serum-free medium and incubated in serum-free medium
for 3-4 h before treatment with the drug. In all experiments, cells
between passages 4 and 8 were used.
Transendothelial Cell Resistance--
The time course of
endothelial cell retraction, a measure of increased endothelial
permeability, was measured according to the procedures described
previously (31). HUVE cells grown to confluence on a gelatin-coated
small gold electrode (4.9 × 10
4cm2)
were pretreated with 500 nM phorbol 12-myristate 13-acetate (PMA) overnight or with 10 µg/ml of C3 transferase for 16 h in EBM-10% fetal bovine serum medium. After serum deprivation, cells were
stimulated with thrombin to measure change in electrical resistance of
the endothelial monolayer. The small electrode and larger counter
electrode were connected to a phase-sensitive lock-in amplifier. A
constant current of 1 µA was supplied by a 1-V 4000-Hz alternating
current connected serially to a 1-megohm resistor between the
small electrode and the larger counter electrode. The voltage between
the small and large electrode was monitored by a lock-in amplifier,
stored, and processed on a computer. Data are presented as change in
resistive (in phase) portion of impedance normalized to its initial
value at time zero.
Measurement of Rho Activity--
pGEX-2T containing rhotekin-Rho
binding domain was provided by Dr. M. A. Schwartz (Scripps
Research Institute, La Jolla, CA). Bacterial-expressed GST-rhotekin-Rho
binding domain (RBD) protein was purified from
isopropyl-1-thio-
-D-galactopyranozide (1 mM)-induced DH5
cells previously transformed with the
appropriate plasmid as described previously (32). Confluent HUVE cells
grown in 100-mm dishes were stimulated for the indicated times with 50 nM thrombin or 100 nM PMA. Cells were then
quickly washed with ice-cold Tris-buffered saline and lysed in lysis
buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 µg/ml each of aprotinin and leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Cell lysates were
clarified by centrifugation at 14,000 × g at 4 °C for 2 min, and equal volumes of cell lysates were incubated with GST-RBD beads (15 µg) at 4 °C for 1 h. The beads were washed
3 times with wash buffer (50 mM Tris, pH 7.4, 1% Triton
X-100, 150 mM NaCl, 10 mM MgCl2, 10 µg/ml each of aprotinin and leupeptin, and 0.1 mM PMSF),
and bound Rho was eluted by boiling each sample in Laemmli sample
buffer. Eluted samples from beads and total cell lysate were then
electrophoresed on 12.5% SDS-polyacrylamide gel electrophoresis gels,
transferred to nitrocellulose, blocked with 5% nonfat milk, and
analyzed by Western blotting using a polyclonal anti-Rho-A antibody.
The amount of RBD-bound Rho was normalized to the total amount
of Rho in cell lysates for quantitation of Rho activity in different
samples using scanning densitometry.
Phosphorylation of Rho-GDI--
A serum-starved confluent
monolayer of HUVE cells was labeled with 300 µCi/ml 32P
for 4 h in phosphate-free medium, after which they were stimulated with 50 nM thrombin or 100 nM PMA at indicated
times. Cells were quickly rinsed twice with ice-cold PBS and then lysed
for 20 min on ice with 300 µl of radioimmune precipitation buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 1% Triton-X, 1 mM
sodium orthovanadate, 1 mM PMSF, and 1 µg/ml each of
leupeptin, pepstatin A, and aprotinin). After clearing the lysate by
centrifuging at 4 °C at 14,000 × g for 10 min, the
lysate was incubated with anti-rabbit polyclonal Rho-GDI antibody for 1 h followed by the addition of protein A-agarose beads overnight at 4 °C. The beads were collected by centrifugation, washed 4 times
with radioimmune precipitation buffer, electrophoresed on 4-15%
gradient SDS-polyacrylamide gels, and transferred to nitrocellulose for
visualization of GDI phosphorylation by autoradiography and for Western
blotting with Rho-GDI antibody to verify equal protein loading.
Specificity of the Rho-GDI antibody was confirmed by using peptide
immunogen as a negative control.
Reporter Gene Constructs, Endothelial Cell Transfections, and
Luciferase Assay--
Rho has been shown to be required primarily for
agonist-induced SRE reporter gene activity (33). Therefore, we
determined using the SRE reporter gene activity, the role of PKC
isozymes in the mechanism of Rho activation. The pSRE-luciferase
plasmid was kindly provided by Dr. T. Kozasa (University of Illinois, Chicago, IL). C3 transferase, produced by Clostridium
botulinum that specifically ADP-ribosylates and inhibits Rho
protein (34), was purified from an Escherichia coli
pGEX-2r-bcr recombinant vector expression system as described
previously (35).
The expression vector pcDNA3-containing tagged dominant-negative
form of PKC-
and PKC-
isozymes were provided by Dr.
I. B. Weinstein (Columbia University, New York, NY). The
dominant-negative PKC-
, PKC-
, and PKC-
mutants lacking a
functional catalytic domain were generated by a substitution of lysine
368, 437, or 376 for arginine, respectively (36). Transfections were
performed with DEAE-dextran method (37). 5 µg of DNA were mixed
with 50 µg/ml DEAE-dextran in serum-free EBM medium, and the mixture
was added onto 70-80% confluent cells. pTKRLUC plasmid (0.125 µg) (Promega Corp., Madison, WI) containing Renilla luciferase
gene driven by the constitutively active thymidine kinase promoter was
added to normalize the transfection efficiencies. After 1 h, cells
were incubated for 4 min with 10% dimethyl sulfoxide (Me2SO) in serum-free medium, washed twice with
EBM-containing 10% fetal bovine serum, and grown to confluence. Cell
extracts were prepared and assayed for luciferase activity using the
Dual Luciferase Reporter Assay System (Promega, Madison, WI).
SRE-luciferase activity was expressed as the ratio of firefly and
Renilla luciferase activity. Cell viability (>95%) after
transfection was confirmed using trypan blue (Sigma) exclusion assay.
Immunocomplex Protein Kinase Assay--
Phosphorylation of
Rho-GDI in vitro was performed using immunocomplexes of
PKC-
or PKC-
obtained after immunoprecipitation of cell lysate
with respective PKC antibodies as described previously (38). Confluent
cells grown in 100-mm dishes were stimulated for 1 min with 50 nM thrombin, washed quickly with ice-cold PBS, and lysed in
radioimmune precipitation buffer containing 50 mM Tris, pH
7.4, 150 mM NaCl, 0.25 mM EDTA, pH 8.0, 1%
deoxycholic acid, 1% Triton-X, 5 mM NaF, 1 mM
sodium orthovanadate, 1 mM PMSF, and 5 µg/ml each of
leupeptin and aprotinin, and 1 µg/ml pepstatin A. The lysate was
scraped and cleared by centrifugation at 4 °C at 14,000 × g for 10 min. Cell lysate containing an equal amount of
protein was then incubated with anti-rabbit polyclonal PKC-
or
PKC-
antibody for 1 h followed by an addition of protein
A-agarose beads overnight at 4 °C.
Beads from each sample were collected by centrifugation, washed twice
with ice-cold lysate buffer, then washed 3 times with PBS, and once
with PKC kinase assay buffer (25 mM Tris, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, and 20 µg of
phosphatidylserine/reaction). The purified GST-Rho-GDI fusion proteins
(3 µg) were incubated with immunocomplexes of PKC dissolved in kinase
assay buffer for 10 min at 30 °C followed by an addition of cold ATP
(20 µM) and 10 µCi of [
-32P]ATP, after
which the mixture was incubated for an additional 30 min. The reaction
was stopped by the addition of Laemmli sample buffer, and each sample
was electrophoresed on 10% SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membrane, and exposed to x-ray films. The
blots were then subjected to Western blotting with anti-PKC-
or
PKC-
antibodies to verify the equal amount of the protein in each reaction.
Statistical Analysis--
Comparisons among experimental groups
were made by ANOVA or Kruskal-Wallis one-way analysis of variance using
SigmaStat software. Differences in mean values were considered
significant at p < 0.05.
 |
RESULTS |
Thrombin and PMA Activate Rho in HUVE Cells--
We used
GST-rhotekin fusion protein, which specifically binds to activated Rho,
to quantitate Rho activation (32, 39). Thrombin induced a 3-4-fold
increase in Rho activity in a time-dependent manner with a
maximum response occurring at 1 min followed by a decline at 10 min
(Fig. 1, A and B).
We also used PMA, a direct activator of PKC, to determine the role of
PKC activation in mediating Rho activation. As shown in Fig. 1,
C and D, PMA induced a 2-3-fold increase in Rho
activity in a time-dependent manner with maximum activation
at 10 min.

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Fig. 1.
Rho activation induced by thrombin and
PMA. HUVE cells grown to confluence were serum-starved, after
which they were stimulated with thrombin or PMA. Rho activity was
assayed at the indicated times. Rho activity is indicated by the amount
of RBD-bound Rho (top) normalized to the amount of Rho in
whole cell lysates (bottom). A and C,
Western blots from a representative experiment showing activation of
Rho in response to thrombin or PMA. B and D, data
from multiple experiments shown as means ± S.E. -fold increase in
Rho activation (n = 3). Rho activation in response to
thrombin was quantitated as -fold increase over activity at time 0. *,
values different from unstimulated cells (p < 0.05);
-T, -thrombin; , absence;
+, presence.
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Thrombin-induced Rho Activation Is Secondary to PKC
Activation--
To investigate whether PKC acts upstream of Rho
activation, we used chelerythrine chloride, a specific (but not
isozyme-selective) PKC inhibitor belonging to a new class of PKC
inhibitors that interfere with the phosphate acceptor site and
non-competitively inhibit the ATP binding site (40, 41). Chelerythrine
pretreatment of HUVE cells prevented thrombin-induced Rho activation,
indicating that PKC is an upstream regulator of Rho activation (Fig.
2).

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Fig. 2.
Protein kinase C regulates thrombin-induced
Rho activation. HUVE cells were serum-starved, after which they
were pretreated without or with chelerythrine for 30 min. Rho activity
was assayed after a 1-min thrombin challenge of cells. Rho activity is
indicated by the amount of RBD-bound Rho (top) normalized to
the amount of Rho in whole cell lysates (bottom). Data are
representative of three independent experiments.
-T, -thrombin; , absence;
+, presence.
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|
To determine whether PKC isozymes involved in thrombin-induced Rho
activation were phorbol-sensitive, we studied the effects of PKC
depletion by PMA on thrombin-induced Rho activation. HUVE cells were
pretreated without or with 500 nM PMA overnight, after which they were stimulated with thrombin to measure Rho activation. Fig. 3A shows that overnight
pretreatment of HUVE cells with PMA prevented Rho activation in
response to thrombin. Western blot analysis of the cell lysate from
these samples showed that exposure of HUVE cells to phorbol esters
resulted in the depletion of PKC-
, PKC-
, and PKC-
with PKC-
and PKC-
being most sensitive to phorbol esters, whereas residual
levels of PKC-
remained detectable. In contrast phorbol ester
treatment had no effect on PKC-
(Fig. 3B). Thus, these
results indicate that thrombin-induced Rho activation in HUVE cells is
regulated by phorbol-sensitive PKC isozymes but not by the atypical PKC
isozymes.

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Fig. 3.
Phorbol ester-sensitive PKC isozymes regulate
thrombin-induced Rho activation. HUVE cells were treated overnight
with PMA to deplete conventional and novel PKC isozymes as described
under "Experimental Procedures." Cells were then lysed after a
1-min thrombin challenge to assay Rho activity. A, Rho
activity is indicated by the amount of RBD-bound Rho (top)
normalized to the amount of Rho in whole cell lysates
(bottom). B, Western blot analysis of PKC
isozymes in total cell lysate after overnight treatment without or with
PMA. Data are representative of three independent experiments.
-T, -thrombin; , absence;
+, presence.
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|
Using LY379196 and rottlerin, which inhibit PKC-
and
PKC-
isozymes, respectively (42-44), we ruled out the
involvement of PKC-
or PKC-
isoforms in regulating
thrombin-induced Rho activation (data not shown). As these data and the
results in Fig. 2 pointed to the phorbol ester-sensitive PKC isozymes,
such as PKC-
, as being responsible for thrombin-induced Rho
activation, we studied the specific role of PKC-
in the mechanism of
thrombin-induced Rho activation.
PKC-
Mediates Rho-dependent Thrombin-induced SRE
Reporter Activity in HUVE Cells--
Using the SRE reporter gene
activity, we determined the role of PKC isozymes in the mechanism of
Rho activation. We used the dominant-negative mutant of PKC-
to address its role in thrombin-induced Rho activation. The
dominant-negative mutants of PKC-
and PKC-
were also included in
these experiments. In addition, we studied the effect of C3
transferase, which inhibits Rho activation (34), on thrombin-induced
SRE activation.
HUVE cells were cotransfected with SRE-luciferase reporter gene
construct together without or with C3 transferase and assayed for
thrombin-induced SRE luciferase activity. Thrombin increased SRE
reporter gene activity by 4-fold, whereas it failed to increase SRE
activation in HUVE cells that were cotransfected with C3 transferase. These results indicate that thrombin-induced SRE reporter gene activation is mediated by Rho (Fig.
4A).

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Fig. 4.
C3 transferase and dominant-negative
PKC- prevent thrombin-induced
Rho-dependent SRE reporter gene activity. A,
HUVE cells transfected with SRE-Luc plasmid in the presence or absence
of C3 transferase (2 µg) were assessed for SRE-luciferase activity.
Cells were stimulated with thrombin for 5 h prior to SRE activity
measurement. SRE-Luc activity is expressed as the ratio of firefly and
Renilla luciferase activity. Data are mean ± S.E.
(n = 3 for each condition). B, HUVE cells
were cotransfected with SRE-Luc plasmid without or with
dominant-negative mutants of PKC- , PKC- , and PKC- . Cells were
stimulated with thrombin for 5 h prior to SRE activity
measurement. SRE-Luc activity is expressed as the ratio of firefly and
Renilla luciferase activity. Data are mean ± S.E.
(n = 3 for each condition). *, values
different from unstimulated cells (p < 0.05);
-T, -thrombin; Luc, luciferase;
, absence; +, presence.
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|
The results of thrombin-induced SRE reporter gene activity in cells
cotransfected with SRE-luciferase reporter without or with
dominant-negative mutants of PKC-
, PKC-
, or PKC-
are shown in
Fig. 4B. Thrombin-induced SRE reporter gene activity was
completely prevented in cells transfected with the dominant-negative
mutant of PKC-
. In contrast, dominant-negative PKC-
or PKC-
had no significant effect on thrombin-induced SRE activation. Thus,
these results, which show that PKC-
is critical in stimulating Rho activation, are in accord with the above findings, which were obtained
using pharmacological inhibitors.
Thrombin Induces Rapid Association of Rho with PKC-
--
To
determine whether thrombin-induced regulation of Rho by PKC-
occurs
as a result of their physical interaction, lysates from cells
stimulated without or with thrombin (1-min challenge period) were
incubated with GST-rhotekin fusion protein to pull down activated Rho,
after which they were subjected to Western blotting with anti-Rho,
PKC-
, or PKC-
antibody. Fig. 5
shows that thrombin induced the rapid association of PKC-
and Rho, whereas under similar conditions, Rho and PKC-
did not
associate.

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Fig. 5.
Thrombin induces rapid association of Rho
with PKC- . Cells stimulated without or
with thrombin (for 1 min) were lysed, and the cell lysate was incubated
with GST-rhotekin fusion protein to pull down activated Rho. Lysates
were then electrophoresed, transferred to nitrocellulose, and Western
blotted with anti-Rho-A or anti-PKC- antibody. Western blots from a
representative experiment shows the association of Rho with PKC- .
-T, -thrombin; , absence;
+, presence.
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Thrombin and PMA Induces Rho-GDI Phosphorylation in HUVE
Cells--
The GDP-bound form of Rho complexed with GDI is not
activated by Rho-GEFs (19), suggesting that Rho activation critically depends on upstream factors that activate the dissociation of GDI.
Since Rho-GDI contains sequence for phosphorylation, we addressed the
possibility that the activation of HUVE cells results in the phosphorylation of GDI. Fig. 6 shows the
autoradiograph of GDI phosphorylation in 32P-labeled HUVE
cells in response to thrombin or PMA stimulation. Thrombin as well as
PMA induced the rapid phosphorylation of GDI that returned to near
basal level at 5 min after challenge (Fig. 6).

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Fig. 6.
Thrombin and PMA induce Rho-GDI
phosphorylation in HUVE cells. HUVE cells grown to confluence were
serum-starved, after which they were labeled with 32P for
4 h in phosphate-free medium. Cells were then stimulated with
thrombin or PMA for the indicated times and lysed, and Rho-GDI was
immunoprecipitated as described under "Experimental Procedures."
Proteins bound to protein A-agarose beads were eluted from the beads by
boiling them, after which they were electrophoresed and transferred to
nitrocellulose membrane for analysis of phosphorylation
(top) and Rho-GDI content by Western blotting
(bottom). Data are representative of three independent
experiments. -T, -thrombin;
P-GDI, phosphorylated GDI; , absence;
+, presence.
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Thrombin-induced GDI Phosphorylation Is Secondary to PKC
Activation--
Since thrombin-induced Rho activation was blocked by
either PKC depletion of cells or by the inhibition of PKC using
chelerythrine, we determined the role of PKC in mediating the
phosphorylation of GDI in response to thrombin. Fig.
7 shows the role of PKC in mediating
thrombin-induced GDI phosphorylation. We found that depletion of PKC by
overnight pretreatment with PMA abrogated thrombin-induced
phosphorylation of Rho-GDI. Similarly, the inhibition of PKC by
chelerythrine pretreatment prevented the phosphorylation of GDI in
response to thrombin stimulation of endothelial cells. Thus, these
results demonstrate that phorbol-sensitive PKC isozymes are involved in
regulating the phosphorylation of GDI in response to thrombin.

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Fig. 7.
PKC regulates thrombin-induced Rho-GDI
phosphorylation. HUVE cells were depleted of phorbol-sensitive PKC
isozymes by overnight treatment with 500 nM PMA.
Serum-starved cells were then labeled with 32P for 4 h
in phosphate-free medium. In parallel, labeled cells were treated with
chelerythrine for 30 min. Cells were then stimulated with thrombin for
1 min and lysed, and Rho-GDI was immunoprecipitated as described under
"Experimental Procedures." Proteins bound to protein A-agarose
beads were eluted from the beads by boiling them, after which they were
electrophoresed and transferred to nitrocellulose membrane for analysis
of phosphorylation (top) and Rho-GDI content by Western
blotting (bottom). Data are representative of three
independent experiments. -T, -thrombin;
P-GDI, phosphorylated GDI; , absence;
+, presence.
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|
PKC-
Phosphorylate Rho-GDI--
As Rho activation was regulated
by PKC-
, we next performed an immunocomplex protein kinase assay to
determine whether PKC-
can directly phosphorylate GDI in
vitro. HUVE cells were stimulated without or with thrombin,
and the lysates were immunoprecipitated with anti-PKC-
antibody.
These immunocomplexes were used for kinase assay using GST-GDI as a
substrate. In parallel, control kinase reactions were performed using
immunocomplexes obtained from cells immunoprecipitated using
anti-PKC-
antibody. As shown in Fig.
8, GST-GDI was slightly phosphorylated by
PKC-
in unstimulated cells, but GDI phosphorylation increased
markedly in the kinase reaction in cells treated with thrombin. In
contrast, PKC-
failed to induce the phosphorylation of GST-GDI in
kinase reaction regardless of whether the immunocomplex was obtained
from stimulated or unstimulated cells. The amounts of PKC-
or
PKC-
in each reaction were equivalent (Fig. 8). Thus, these results
indicate that activated PKC-
is directly capable of phosphorylating
Rho-GDI in vitro.

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Fig. 8.
PKC- phosphorylates
purified GST-GDI in vitro. HUVE cells were
serum-starved, after which they were stimulated with thrombin and lysed
to immunoprecipitate PKC- or PKC- as described under
"Experimental Procedures." PKC immunocomplex was incubated with
GST-GDI in kinase buffer containing [ -32P]ATP at
30 °C for 30 min. Reactions were then stopped by boiling the samples
in SDS sample buffer, after which they were electrophoresed and
transferred on nitrocellulose for analysis of GDI phosphorylation by
PKC (top) and PKC content by Western blotting
(bottom). -T, -thrombin;
P-GDI, phosphorylated GDI; , absence;
+, presence.
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PKC Depletion or Rho Inhibition Inhibits Thrombin-induced Decrease
in Transendothelial Electrical Resistance--
The biochemical events
by which PKC activation regulates thrombin-induced barrier dysfunction
are incompletely understood (3, 27-30). Because the results of this
study directly implicate PKC as the upstream regulator of Rho, we
measured changes in transendothelial electrical resistance (the basis
of increased paracellular endothelial permeability) in PKC-depleted
cells or in cells treated with C3 transferase to block Rho activation.
As shown in Fig. 9, thrombin caused a
significant decrease in resistance in untreated cells, whereas the
decreases in resistance in response to thrombin were significantly
reduced in cells, which were depleted of phorbol ester-sensitive PKC
isozymes, as well as in cells treated with C3 transferase.

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Fig. 9.
Effects of inhibition of Rho and depletion of
PKC on thrombin-induced endothelial cell retraction. HUVE cells
grown to confluence on gold electrodes were either pretreated overnight
with 500 nM PMA to deplete phorbol ester-sensitive PKC
isozymes (top) or pretreated for 16 h with 10 µg/ml
C3 transferase (bottom). Cells were then serum-deprived
after, which they were stimulated with 50 nM thrombin to
measure the changes in transendothelial electrical resistance in real
time. Similar results were obtained in two other experiments.
T, -thrombin.
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|
 |
DISCUSSION |
Rho activation plays an important role in the mechanism of
increased transendothelial permeability induced by mediators such as
thrombin (11-13, 39); however, the mechanisms of activation of Rho,
thereby the loss of endothelial barrier integrity, are not elucidated
(39). The dissociation of GDI from Rho-GDP complex is a prerequisite
for the activation of Rho by Rho-GEF (19, 22, 45, 46). As GDI may play
a critical role in mediating thrombin-induced Rho activation and thus
in signaling increased endothelial permeability, we addressed in this
study the basis of Rho activation and its contribution in mediating the
loss of endothelial barrier function induced by thrombin.
The present results provide several lines of evidence that Rho-GDI
phosphorylation and Rho activation are regulated by a
PKC-dependent pathway in endothelial cells. We showed that
thrombin as well as the direct activation of PKC by PMA induced the
phosphorylation of Rho-GDI and that the Rho-GDI phosphorylation
occurred concurrently with the thrombin-induced activation of Rho.
Furthermore, the inhibition of PKC by chelerythrine (a specific but not
isozyme-selective inhibitor of PKC) abrogated not only thrombin-induced
Rho activation but also Rho-GDI phosphorylation. We also showed that
the phosphorylation of GDI and Rho activation is regulated by phorbol
ester-sensitive isozymes as the depletion of these isozymes by exposing
HUVE cells to phorbol esters in the standard manner prevented
thrombin-induced GDI phosphorylation and Rho activation. Because the
PKC isozymes, -
, -
, -
, and -
, expressed in endothelial
cells are all phorbol ester-sensitive, we used both pharmacological and
genetic approaches to further identify the specific PKC isozyme
regulating GDI phosphorylation and Rho activation.
We found that the treatment of HUVE cells with LY379196 or rottlerin,
which inhibits PKC-
or PKC-
isozymes, respectively, failed to
prevent Rho activation in response to thrombin in endothelial cells.
Using dominant-negative mutant constructs, we showed that dominant-negative PKC-
failed to prevent thrombin-induced SRE reporter gene activity that is regulated by Rho. Furthermore, we found
that Rho-mediated SRE reporter gene activity in response to thrombin
was completely prevented in endothelial cells transfected with the
dominant-negative mutant of PKC-
, whereas PKC-
had no significant
effect on thrombin-induced SRE activation. Thus, these data demonstrate
that PKC-
is the major kinase regulating Rho activation in
endothelial cells. As the above findings indicate the critical role of
PKC-
activation in the regulation of Rho activation, we used
in vitro kinase assay to test the possibility that PKC-
can directly phosphorylate GDI. Results of the in vitro kinase assay using PKC-
and PKC-
immunoprecipitates from
unstimulated and stimulated cells indicated that only PKC-
from
activated cells was capable of inducing phosphorylation of GST-GDI.
Thus, these findings indicate that the activation of PKC-
is
required for phosphorylation of Rho-GDI, although the possibility
cannot be ruled out that PKC may also activate another protein kinase controlling the phosphorylation state of GDI in HUVE cells.
We also found in the pull-down assay that stimulation of endothelial
cells with thrombin leads to a rapid association of PKC-
with the
activated Rho, although it failed to associate with PKC-
. The
association of PKC-
with Rho after activation, although not in
resting cells, indicates that the protein complex formation is probably
important in targeting and regulating Rho function (14, 47). However,
our results do not distinguish between the possibility that association
of PKC-
with Rho can occur directly or whether it is mediated by
intermediate factors.
There is little information regarding the role of different PKC
isoforms in regulating the activation of Rho. Studies in endothelial and epithelial cells have implicated Rho in PMA-induced recruitment of
PKC-
to the cell membrane (48); however, these observations were not
confirmed in bovine arterial endothelial cells (49). A permissive role
of PKC-
but not PKC-
in sphingosine 1-phosphate-induced Rho-A
translocation from cytosol to membrane (an indirect measure of Rho
activation) was also recently reported in C2C12 myoblasts (50). Thus,
on the basis of using multiple approaches our results provide
unequivocal evidence that PKC-
is a key upstream regulator of Rho activation.
Several studies have implicated a critical role of
PKC-dependent pathway in regulating thrombin-induced
increase in endothelial permeability (3, 27-30). We, therefore,
measured changes in transendothelial electrical resistance (the basis
of increased paracellular endothelial permeability) (31) using cells
depleted of PKC by PMA treatment or cells treated with C3 transferase
to inhibit Rho. We used these cells to address the possibility that PKC-induced barrier dysfunction can be explained by Rho activation. The
results showed that thrombin caused a decrease in transendothelial electrical resistance, whereas depletion of PKC or inhibition of Rho
reduced the response. Thus, these observations indicate that PKC
induces the permeability increase activated by thrombin via the
Rho-mediated pathway.
What are the implications of PKC-
-induced GDI phosphorylation in the
mechanism of Rho activation? It has been shown that the cytoplasmic
pool of Rho-GTPase is complexed with GDI proteins (45); thus, GDI can
influence both the cellular localization and cycling of Rho proteins
between GDP- and GTP-bound states. The dissociation of GDI from the Rho
protein is a prerequisite for membrane association and its activation
by Rho-GEFs (19, 20). In bovine neutrophils,
phosphorylation/dephosphorylation events have been implicated in the
regulation of the dissociation of the Rho/Rho-GDI complex (51). Based
on our results of thrombin-induced phosphorylation of GDI and Rho
activation through a PKC-dependent pathway, we hypothesize
that phosphorylation/dephosphorylation of GDI may play a role in the
mechanism of PKC-
-induced activation of Rho. Thus, the results of
this study describe a novel pathway of GDI phosphorylation and Rho
activation regulated by PKC-
and in signaling PKC-induced loss of
endothelial barrier function.