Department of Physiology, University of Arizona, and The Benjamin W. Zweifach Microcirculation Laboratories, Department of Veterans Affairs Medical Center, Tucson, Arizona 85723
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The modulation of endothelial barrier function is thought to be
a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA with
Clostridium botulinum exoenzyme C3
transferase (C3), an inhibitor of the small GTP-binding protein RhoA.
Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a
size-selective permeability assay and quantitative digital imaging
measurements of SF/FA. C3 treatment disassembled SF/FA, stimulated
diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY)
content of paxillin and 130- to 140-kDa proteins that included
p125FAK. C3-treated monolayers
displayed a 60-85% decline in F-actin content and a
170-300% increase in EC surface area with enhanced endothelial
barrier function. This activity correlated with reorganization of
F-actin and PY protein(s) to -catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation of
myosin ribbons, SF/FA, and the increased PY content of proteins, these
characteristics were Rho dependent. Our data show that C3 inhibition of
Rho proteins leads to cAMP-like characteristics of reduced SF/FA and
enhanced endothelial barrier function.
Clostridium botulinum exoenzyme C3
transferase; immunofluorescent digital imaging; stress fibers; myosin; -catenin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FAILURE OF THE BLOOD VESSEL wall to retain fluid and plasma proteins in the vascular space is a key characteristic of inflammation. Preservation of a tightly joined endothelial barrier prevents the consequences of acute inflammation, such as interstitial edema and organ dysfunction. Endothelial barrier function is determined by the area available for fluid and solute exchange, which is formed by the paracellular space between adjacent endothelial cell (EC) junctions. The oppositional effects of cytoskeletal tension, produced by actomyosin stress fibers (SF) linked to focal adhesions (FA) (10, 29), and the forces of cell-cell adhesion modulate the size of the paracellular space (6). Thus one mechanism of endothelial barrier failure is thought to involve the formation of SF/FA, which initiates increased cytoskeletal contractile force, enlarging the paracellular space (for reviews see Refs. 9 and 17). An alternative notion is that the actin cytoskeleton is required for barrier maintenance, and filamentous (F)-actin disintegration initiates barrier failure (12).
Small GTP-binding proteins act as triggered amplifiers of signal transduction. Rho proteins are members of the Ras superfamily of small GTP-binding proteins, which modulate the formation of SF/FA in many cell types (3, 5, 13, 15). The activation of Rho proteins appears to stimulate multiple signaling mechanisms linked to the modulation of the F-actin cytoskeleton. GTP-bound Rho activates phosphatidylinositol 4-phosphate 5-kinase and initiates the synthesis of 4,5-bisphosphate, a substrate that regulates a variety of actin-binding proteins, causing the polymerization of F-actin and the formation of FA (4). The rapid transformation of RhoA from its inactive GDP-bound form to its active GTP-bound form allows for the transient activation of Rho kinase (3). Rho kinase causes SF/FA formation by initiating myosin light chain (MLC) phosphorylation (MLC-P) due to two mechanisms: direct phosphorylation of MLC at serine-19 and inhibition of myosin phosphatase activity via phosphorylation of the regulatory myosin binding subunit of myosin phosphatase (3, 15). Phosphorylation of MLC reveals myosin's actin-binding site, initiating myosin II aggregation into spots and ribbons along actin filaments, forming SF/FA (5, 9, 10). FA, which consist of a number of phosphotyrosine (PY)-containing proteins, including paxillin and FA kinase (p125FAK), are seen as streaklike plaques located at the ends of SF (for review see Ref. 5). These integrin-mediated adhesion sites serve as anchoring points between actomyosin filaments and the extracellular matrix, permitting the generation of contractile forces associated with directed cell migration (29). The bacterial toxin Clostridium botulinum exoenzyme C3 transferase (C3) specifically inhibits the small G proteins of the Rho subfamily by ADP-ribosylating the GTP-binding site (19). Inhibition of RhoA leads to a reduction in MLC-P and loss of SF in neurites and human umbilical vein endothelial cells (HUVEC) (8, 13, 29) as well as SF/FA disassembly in Swiss 3T3 cells (20). Signals that cause an elevation in the intracellular free Ca2+ concentration initiate the formation of MLC-P and increased contractile force (for reviews see Refs. 1 and 11). This process appears to be activated by the serine protease thrombin, which stimulates increased MLC-P, leading to myosin ribbon formation and SF/FA (10). These events mediate increases in centripetal tension and endothelial barrier failure (9, 17, 24). It has been proposed that the thrombin-induced development of SF/FA and endothelial barrier dysfunction may be Ca2+/protein kinase C (PKC) dependent (9, 17). However, the thrombin-induced increase in intracellular free Ca2+ concentration returns to baseline levels long before SF/FA and endothelial barrier dysfunction subside. In addition, thrombin-induced SF are unaffected by Ca2+ chelators and PKC inhibitors (27). These data suggest that the thrombin-induced formation of SF/FA may be modulated by other second messenger systems.
The purpose of the present study was to test the role of Rho proteins
in the modulation of the EC cytoskeleton and endothelial barrier
function. We used C3 to inhibit Rho protein-dependent formation of
SF/FA, then used measurements of the size-selective permeability of
endothelial monolayers and quantitative immunofluorescence digital
imaging microscopy to characterize the EC cytoskeletal morphology with
EC barrier function. We show that C3 inhibition of RhoA caused the
disassembly of SF/FA and reorganized F-actin and PY-containing proteins
to -catenin-containing cell-cell junctions, a process that enhanced
endothelial barrier function. In addition, C3 prevented
thrombin-induced myosin ribbons and SF/FA formation, indicating that
these processes are Rho dependent. However, thrombin-stimulated endothelial barrier failure was not prevented by C3. These data show
that Rho plays a major role in the modulation of SF/FA and endothelial
barrier function and imply a partial role for noncytoskeletal forces in
thrombin-induced barrier failure.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. EC, isolated from bovine pulmonary arteries as previously described (23), were grown in DMEM containing 10% fetal bovine serum (HyClone, Ogden, UT). Experiments were performed with cells between passages 5 and 11 at 4-6 days postconfluence.
[32P]ADP-ribosylation of
Rho.
EC grown in 60-mm dishes were treated with or without 10 µg/ml of C3
ADP-ribosyltransferase for 16 h (Upstate Biotechnology, Lake Placid, NY
or Calbiochem, San Diego, CA). The cells were lysed in ice-cold buffer
containing 1% NP-40 and 50 mM Tris · HCl, pH 8.0, with 10 µg/ml each of leupeptin, aprotinin, and pepstatin A. The
cells were scraped from the culture dish, passed through a 26-gauge
needle, and centrifuged at 12,400 g
for 5 min at 4°C. Cell lysates (10 µl) were incubated with 12.5 µl of ADP assay buffer containing 50 mM Tris · HCl,
2.6 mM MgCl2, 1 mM EDTA, 10 mM
thymidine, 10 mM dithiothreitol, 1 mM ATP, 100 µM GTP, 5 µl of
[32P]NAD (Amersham
Life Sciences, Arlington Heights, IL), and 1 µg/ml of C3. After 1 h
at room temperature, each reaction mixture was treated with 2×
SDS sample buffer, boiled for 5 min, and run on 12% SDS-PAGE with use
of Tris-Tricine buffer. The dried gel was exposed to X-ray films for
various time intervals to obtain an exposure within the linear range of
the film. Stock solutions of C3 were prepared in sterile, deionized
H2O and stored at 70°C.
Immunoprecipitation and immunoblotting. EC grown in six-well plates were treated as described above and lysed by the addition of 1 ml of 4°C extraction buffer containing 20 mM Tris · HCl, pH 7.6, 1% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, 0.1 mM NaMoO4, 200 µM phenylarsine oxide, 3 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 5 µg/ml each of aprotinin, leupeptin, and pepstatin. Equivalent amounts of supernatant protein (135 µg) were precleared with 20 µl of recombinant protein A-agarose (Oncogenic Sciences, Cambridge, MA) and immunoprecipitated overnight with 2-4 µg of the affinity-purified rabbit antibodies to RhoA, mouse anti-PY (PY99) IgG (Santa Cruz Biotechnology, Santa Cruz, CA), or mouse anti-paxillin antibodies at 4°C, then subjected to capture with 20 µl of protein A-agarose. The washed pellets were diluted using 2× SDS sample buffer with 10% 2-mercaptoethanol, boiled for 5 min, and separated by SDS-PAGE in 12 or 7.5% slab gels with a Laemmli buffer system. The proteins were transferred to 0.2-µm nitrocellulose membranes (Schleicher and Schuell, Keene, NH) in Tobin's solution at 4°C. The membranes were immunoblotted with antibodies to RhoA (Santa Cruz Biotechnology), paxillin, p125FAK (Transduction Laboratories), and anti-PY (4G10, UpState Biotechnology) with enhanced chemiluminescence plus detection (Amersham, Arlington Heights, IL). X-ray films were developed at various time intervals to obtain an exposure within the linear range of the film. Densitometry was performed using MetaMorph software.
Endothelial monolayer barrier function. The techniques listed here have been described in detail elsewhere (23-25). Briefly, the size-dependent passage of fluorescein isothiocyanate-labeled hydroxy ethyl starch macromolecules across bovine pulmonary artery EC monolayers was used to detect the formation or absence of a small "pore" barrier. At 4 days postconfluence, each monolayer was incubated with 10 or 25 µg/ml of C3 for 36 h in conditioned media. Monolayer barrier function in the presence or absence of 10 U/ml of thrombin was performed exactly as previously described (24).
Digital imaging immunofluorescence microscopy. An Olympus IMT-2 microscope was used with the following attachments: 1) an Olympus ×60, 1.4 NA oil-immersion objective, 2) a 150-W xenon lamp and an achromatic focusing lens (Optiquip, Highland Mills, NY), 3) an eight-position MetalTek filter wheel and shutter containing 490 ± 10, 555 ± 15, and 635 ± 15 nm excitation filters (Chroma Technology, Brattleboro, VT), 4) a quad filter set polychromatic beam splitter/emitter (series 84, Chroma Technology), and 5) a z-axis controller (Ludl Electronic Products, Hawthorne, NY). Epifluorescent digital images were collected with a charge-coupled device camera (model PXL, Photometrics, Tucson, AZ). MetaMorph software (version 2.5, Universal Imaging) was used to color encode the separately obtained fluorochrome (fluorescein, tetramethylrhodamine, and CY-5) images of each cell.
Labeling of cytoskeletal structures.
EC grown to 4 days postconfluence on gelatin/fibronectin-coated
eight-well plastic (Nunc, Naperville, IL) slides or Transwell membranes
were stained with the following monoclonal antibodies: 1) paxillin [monoclonal
antibody (MAb) 3060, Chemicon International], 2) -catenin (no. 13-8400, Zymed
Laboratories), and 3)
affinity-purified rabbit antibodies to nonmuscle myosin II (BT-561,
myosin II, Biomedical Technologies, Stoughton, MA), anti-RhoA, or
anti-PY PY99 (Santa Cruz Biotechnology) and anti-PY 4G10 (Upstate
Biotechnology, Lake Placid, NY). Oregon Green 488-phalloidin (Molecular
Probes, Eugene, OR) was used to label F-actin. The cells were fixed and
permeabilized with 4% formaldehyde in PBS for 1 min, then for 30 min
with 2% formaldehyde, 0.2% Triton X-100, and 0.5% deoxycholate, as
described by Goeckeler and Wysolmerski (10). Nonspecific binding was
minimized by incubation with PBS containing 1% IgG/protease-free BSA
in PBS for 15 min at room temperature. EC were stained with
anti-paxillin (1:200 dilution), anti-
-catenin (1:50 dilution), or
4G10 (1:50 dilution) in 0.1% BSA-PBS for 60 min, washed, and incubated
with anti-myosin II (1:25 dilution) or RhoA (1:50 dilution) for 60 min.
After they were washed and incubated with 5% goat serum, the cells
were incubated with TRITC-labeled anti-rabbit IgG (1:25 dilution) and
goat-Cy5 anti-mouse IgG (1:25 dilution; Jackson Immunoresearch Labs,
Westgrove, PA) at room temperature for 45 min. After the cells were
washed again, F-actin was stained with Oregon Green
488-phalloidin. The slide was covered with 30 µl of Vectashield
mounting medium (Vector Laboratories, Burlingame, CA) and a no. 1 coverslip and sealed with nail polish. We did not observe changes in
these staining patterns in the presence or absence of mouse or rabbit IgG.
Quantification of endothelial surface area and F-actin content.
After endothelial barrier function measurements, each Transwell
membrane was fixed and double stained for F-actin (tetramethylrhodamine isothiocyanate-phalloidin) and -catenin, as described above. Blinded
digital images for both fluorochromes were collected simultaneously (10 images/treatment) and at random for each Transwell membrane of each
treatment group. MetaMorph software was used to trace the
-catenin
outline of each cell and compute EC surface area (µm2). The F-actin content was
computed for the identical cells as the total tetramethylrhodamine
isothiocyanate fluorescence intensity divided by the surface area of
each EC. We divided each random image into two distinct populations of
C3-treated EC on the basis of the following morphological criteria:
1) the most responsive (MR)
C3-treated EC were large EC that lacked F-actin SF, but a thin rim of
F-actin was present at cell-cell junctions; and
2) the least responsive (LR)
C3-treated EC exhibited a linear shape and displayed thinner, less
prominent actin filaments than control cells, as well as reorganization
of F-actin to a thin junctional rim. The percentages of the MR and LR
EC were computed from the total number of cells counted.
Statistical analysis.
Values are means ± SE. The 2 = 0.05 level was selected for
statistical significance. A paired Student's
t-test and an ANOVA (Hewlett-Packard
41CV Stat Pack) were used to compare each test interval with baseline
values for selected measurements.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoprecipitation and
[32P]ADP-ribosylation.
RhoA was detected as an ~22-kDa protein by Western blotting of EC
lysates with or without immunoprecipitation (Fig.
1, lanes 4 and 3,
respectively). C3-dependent
[32P]ADP-ribosylation
of EC lysates revealed a protein band with an apparent molecular mass
of ~25 kDa (Fig 1, lane 1). The
appearance of this
[32P]ADP-ribosylated
protein was prevented by pretreatment with C3 (Fig. 1,
lane 2).
|
Effects of C3, C3 + thrombin, and thrombin on
endothelial barrier function.
We used a high-performance size exclusion chromatography technique (25)
to quantitatively measure the size and number of holes formed across
endothelial monolayers by analyzing their size-selective solute
permeability characteristics. Restricted diffusion, displayed by
control monolayers (Fig.
2A) and
created by predominantly small "pores" (<250-Å pore
radius), is characterized by a decline in the size-selective
permeability (P)-to-free diffusion coefficient
(Do) ratio with
increasing solute molecular radius (ae) (23). In
contrast, the formation of large holes (>2,000-Å pore
radius) is characterized by an increase in
P/Do for solutes of increased ae
(24). C3 treatment (25 µg/ml for 36 h) significantly ( = P < 0.05; Fig.
2A) enhanced this endothelial
barrier function characteristic, an effect that was not seen when 10 µg/ml of C3 was used (data not shown). In contrast, endothelial
monolayers exposed to 10 U/ml thrombin exhibited barrier failure, as
characterized by a shift in the
P/Do vs.
ae curve upward
and to the right in comparison to control (Fig.
2B). Pretreatment of endothelial
monolayers with C3 (25 µg/ml for 36 h) did not inhibit the barrier
failure characteristic of thrombin alone (Fig.
2B).
|
Heterogeneous F-actin morphology of C3-treated EC with or without
thrombin.
Adherens junctions, characterized by homophilic binding of
Ca2+-dependent adhesion
(cadherins) molecules, are linked to the F-actin cytoskeleton by the
accessory proteins -,
-, and
-catenins (6). We previously
showed enhanced EC barrier function in association with reorganization
of F-actin to a thin junctional rim after treatment with cAMP (22). To
test whether C3-induced reorganization of F-actin to a thin rim
colocalized at adherens junctions, Transwell membranes from the barrier
function assays listed above were fixed and double stained for F-actin
and
-catenin. Control cells displayed actin-containing SF and
prominent dense peripheral bands of F-actin (Fig.
3a).
Two morphologically distinct populations of C3-treated EC were
observed. The MR C3-treated EC were large cells that exhibited no
distinct F-actin SF, but a thin rim of F-actin was observed at
-catenin-containing cell-cell junctions (Fig. 3,
b and
f). In contrast, the LR cells in our
C3-treated population exhibited reorganization of F-actin to the cell
border in conjunction with thinly attenuated actin filaments. In the MR
and LR C3-treated EC, the rim of F-actin (arrows, Fig. 3,
b and
c) colocalized with
-catenin
(arrows, Fig. 3, f and
g). In addition, the
-catenin fingerlike staining pattern in control (arrow, Fig.
3e) and thrombin-treated EC (arrows,
Fig. 3h) appeared linked to F-actin
filaments (Fig. 3a; arrow, Fig.
3d). Thrombin stimulated
the formation of actin filament bundles that was prevented by C3
pretreatment, although these cells displayed large paracellular holes
that did not show
-catenin staining. Thrombin-induced hole formation
was unaffected by C3 pretreatment (Fig. 3,
c and
g, and Fig. 3,
d and
h).
|
Heterogeneous effects of C3 on F-actin content and EC surface area.
The MR C3-treated EC (18.5 ± 3%) showed a 300% increase in EC
surface area compared with the controls (1,018 ± 84 and 329 ± 14 µm2, respectively; Fig.
4A). In
contrast, the LR C3-treated EC displayed a 170% increase in EC surface
area (549 ± 29 µm2,
P < 0.001) compared with the control
or thrombin-treated groups. The same characteristics were seen in the
group treated with C3 + thrombin. EC surface area after thrombin
treatment was not significantly different from that of the control
group.
|
C3 stimulates diffuse myosin II immunostaining and prevents
thrombin-induced myosin ribbon formation.
Activated Rho kinase and thrombin are known to stimulate the
phosphorylation of MLC on serine-19 (MLC-P), a biochemical process reduced by C3 (2, 8, 29). Because MLC-P initiates the reorganization of
myosin spots into ribbons along actin filament bundles (10, 29), we
tested whether thrombin-induced myosin spots and ribbons were
inhibitable by C3. In addition, we determined whether C3 treatment
alone caused a diffuse pattern of myosin II immunostaining, an indirect
characteristic of reduced MLC-P (10). Thrombin stimulated the creation
of myosin ribbons that traversed in a focal plane below the nucleus
across the base of most cells (Fig.
5b, arrows
and inset). In contrast, control EC showed random myosin spots (Fig. 5a,
arrows and inset). The MR EC
stimulated with C3 and C3 + thrombin displayed a diffuse, perinuclear, myosin staining pattern (Fig. 5, c and
d, respectively). Because each image
was collected at the focal plane of F-actin at the base of the cell,
the appearance of the nucleus with perinuclear myosin staining in these
C3-treated EC indicates that these cells had flattened. Similar
reorganization of the myosin focal plane was seen in the LR and MR EC;
however, the loss of myosin spots was less marked in the LR
C3-stimulated EC (data not shown).
|
C3 disassembles SF and FA, reorganizes F-actin to a thin junctional
rim, and prevents thrombin-induced SF/FA.
Because the formation of SF/FA appears to be Rho dependent (13), we
tested whether C3 disassembled SF/FA and prevented thrombin-induced SF/FA formation by triple immunostaining EC monolayers for F-actin (green), paxillin (red), and nonmuscle myosin II (blue; Fig.
6). Control EC showed prominent dense
peripheral bands of F-actin (Fig. 6a,
double arrows) that appear to be above the focal plane. Colocalized
myosin II and F-actin appear as a few blue-green filaments that cross
the base of the cell (Fig. 6a, arrow).
Paxillin-containing FA (arrowheads) are seen at the ends of these
filaments as red and orange streaklike plaques. Thrombin stimulated the
formation of thick blue-green-appearing actomyosin SF (arrow, Fig.
6c) that spanned the entire base of
the cell and terminated in paxillin-containing FA. These FA colocalized
paxillin with F-actin, as indicated by the color transition from green
to yellow to red (arrowhead, Fig. 6c) at the ends of these filaments.
The dense peripheral bands of F-actin (double arrows, Fig.
6c) that appear out of focus above the base of this EC indicate the rounded, "contractile" nature of
the thrombin-treated EC. These morphological characteristics were not
displayed in the C3-treated EC. The MR C3 and C3 + thrombin-treated EC
displayed disintegrated F-actin SF and redistribution of F-actin (green) to a thin rim at the perimeter of these cells (arrow, Fig. 6,
b and
d). The image plane of these EC
displayed a prominent nucleus with a diffuse myosin II staining pattern
and small disorganized streaks of paxillin in the nuclear region,
characteristic of cell flattening. A few paxillin-containing streaks
(arrowhead, Fig. 6d) were associated
with thin, blue-green actomyosin filaments.
|
C3 stimulates reorganization of PY-containing proteins to cell-cell
junctions.
Because FA appear as streaklike plaques containing a variety of
PY-containing proteins, we tested whether treatment with C3, C3 + thrombin, and thrombin altered the immunofluorescence-staining pattern
of these PY-containing proteins compared with controls (Fig.
7). Control EC displayed PY-containing
proteins at random locations around the perimeter of the cell (arrows)
and a few streaklike plaques localized at the ends of SF (arrowheads,
Fig. 7a). In contrast, thrombin
stimulated a marked increase in the number of streaklike PY-containing
proteins at the base of the cell (arrowheads, Fig. 7,
b and
inset) and at the cell edge
(arrows). This pattern corresponds to the paxillin-containing plaques
seen at the ends of SF (arrowhead, Fig.
6c). In the MR and LR C3-treated EC,
there was complete loss of these streaklike plaques, and PY-containing proteins were reorganized to the cell-cell junctions (arrows, Fig.
7c). A similar effect was observed
in EC treated with C3 + thrombin (arrows, Fig.
7d).
|
C3 inhibits tyrosine phosphorylation of the FA protein paxillin.
We next identified the PY-containing proteins in control EC and EC
stimulated with C3, C3 + thrombin, and thrombin by immunoprecipitation with anti-paxillin and anti-PY (PY99) antibodies followed by Western blot analyses with anti-PY (4G10) and anti-paxillin IgG (Fig. 8). Although C3 did not affect the quantity
of immunoprecipitated paxillin (Fig.
8B), this inhibitor stimulated a
decline in the PY content of two protein bands, paxillin and a 130- to
140-kDa band, with or without thrombin treatment (Fig. 8,
A, C, and
D). Densitometry of the
thrombin-stimulated PY-containing proteins revealed an increase in the
PY content of paxillin and the 130- to 140-kDa protein band (Fig. 8,
C and
D), which included
p125FAK (data not shown). Because
C3 prevented these changes in the presence or absence of thrombin,
these tyrosine phosphorylation events were Rho dependent.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major finding of this study is that C3-dependent ADP-ribosylation
of Rho proteins caused SF/FA disassembly and enhanced endothelial
barrier function. This process reflects a reduction in basal
cytoskeletal tension, as indicated by the loss of actin SF and punctate
myosin staining, a reduction in F-actin content, and an increase in EC
surface area, which mediated a decline in the size-selective
permeability of endothelial monolayers. Hallmarks of these cAMP-like
events were reorganization of F-actin and PY-containing proteins to a
thin band that colocalized with -catenin-containing cell-cell
junctions and reduction in the size of the paracellular space. Because
the thrombin-stimulated formation of myosin ribbons and assembly of
SF/FA were inhibitable by C3, these events appear to be Rho dependent.
However, C3 did not inhibit thrombin-induced barrier dysfunction,
implying that this process is partially mediated by noncytoskeletal forces.
We used recombinant C3 to test the role of Rho proteins in the SF/FA-dependent modulation of endothelial barrier function. The identity of the 22- to 25-kDa protein that we detected in our EC is probably RhoA for the following reasons: 1) this protein was detected by a polyclonal antibody to RhoA at an apparent molecular mass similar to that described in a recent report (2), 2) this band was [32P]ADP-ribosylated by C3, and 3) detection of this activity was inhibited by exposure to C3 for 24 h.
Previous reports suggest that the effects of C3 on cellular functions may be limited by the slow uptake of C3 into cells, which results in the heterogeneous F-actin SF morphology (2, 12). In this regard, Aepfelbacher et al. (2) reported that although many C3-treated HUVEC showed no SF, a significant percentage of these cells displayed substantial F-actin filaments. They attributed this effect to inefficient endocytosis of C3 by the SF-containing subpopulation. In the present study, our C3-treated monolayers displayed similar heterogeneous F-actin morphologies. However, all C3-treated EC exhibited diffuse myosin II staining, decreased F-actin content, reduced PY of FA plaques, and increased surface area. These data suggest that the activity of C3 is greater in EC than that revealed by F-actin staining alone.
Activated Rho proteins have been reported to modulate the direct and indirect phosphorylation of serine-19 of MLC (3, 15), a process that reveals myosin's actin-binding site and initiates myosin II aggregation from a diffuse cloud into spots and ribbons along actin filaments, forming SF/FA (5, 10, 29). These findings are confirmed by recent studies in EC that show that C3 reduces MLC-P and prevents thrombin from generating this product (8, 29). This activity is similar to that initiated by cAMP or the MLC kinase inhibitor KT-5926 (13, 21, 22). Our morphological data provide indirect evidence of this C3 activity. We show that this exoenzyme stimulated a diffuse myosin-staining pattern with reorganization of F-actin and PY-containing proteins to cell-cell adherens junctions, flattening of EC, and increased surface area with enhanced monolayer barrier function. These findings indicate that C3 reduces MLC-P, leading to diffuse myosin, EC flattening, and an increase in EC surface area.
How inhibition of active Rho proteins affects monolayer barrier function remains unclear. Using Clostridium difficile toxin B to UDP-glucosylate and inhibit Rho proteins, Hippenstiel et al. (12) reported an 80% decline in F-actin and complete barrier failure in porcine aortic EC monolayers. cAMP-increasing agents did not prevent this barrier defect, indicating a noncontractile mechanism. C. difficile toxin B appears to affect multiple GTPase-linked processes because of its inhibition of a variety of small G proteins of the Rho superfamily, including Rho, Rac, and CDC42 (26). In the present study, C3 stimulated an increase in the size selectivity of EC monolayers, which indicates an enhancement of endothelial barrier function. These data are similar to those of two recent reports in which C3 initiated a decline in HUVEC monolayer permeability (8, 28). Thus the selective inhibition of Rho proteins by C3 initiates a decline in F-actin and reduced endothelial monolayer permeability to small solutes, whereas inactivation of Rho, Rac, and CDC42 appears to initiate monolayer barrier failure.
RhoA appears to regulate the formation of integrin-dependent FA sites in lymphocytes and EC (2, 16). In the present study we show that C3 prevents the thrombin-induced increase in the PY content of paxillin and PY-containing FA. In addition, C3 alone caused EC to lose paxillin and PY-containing proteins at FA and reorganized tyrosine phosphorylated proteins to intercellular junctions. This process was associated with a decline in the tyrosine phosphorylation of paxillin and a 130- to 140-kDa band that included p125FAK in C3-treated EC with and without exposure to thrombin. Similar tyrosine phosphorylation signals were observed when EC were treated with C3 in the presence or absence of cyclic strain (30). In these experiments, cyclic strain caused increased tyrosine phosphorylation of FA, possibly by activation of p125FAK. C3 pretreatment prevented these tyrosine phosphorylation events and reduced the PY content of paxillin and p125FAK. Together, these data suggest that Rho plays an important but perhaps indirect role in the modulation of the PY content of FA (3).
C3-induced changes in cell shape resulted in the formation of tyrosine-phosphorylated proteins at endothelial cell-cell attachment sites. This effect has been seen in vivo in the endothelium of guinea pig aorta, where significant shear stress and cell stretch occur (14). In addition, we previously showed that an expansion of EC surface area by cAMP-increasing agents or KT-5926 initiates similar characteristics (22). These data are consistent with the notion that the C3-induced reorganization of tyrosine-phosphorylated proteins to cell-cell junctions appears linked to an increase in endothelial surface area, producing increased cell-cell apposition.
Thrombin is known to stimulate actomyosin-based SF contractile forces, causing cell retraction and rounding in neurites and EC (9, 12, 21). These effects were prevented by C3 pretreatment of thrombin-stimulated HUVEC or neurites (8, 13). Because C3 treatment of neurites did not prevent thrombin-stimulated Ca2+ release, these data suggest that C3 does not inhibit thrombin-induced signal transduction (13). In the present study we extend this view, showing that inhibition of RhoA prevents thrombin-induced formation of myosin ribbons and PY-containing SF/FA, since these effects were not seen in the EC treated with C3 + thrombin. Thus thrombin-induced formation of SF/FA is RhoA dependent.
Moy et al. (18) proposed that endothelial barrier failure is determined by centripetal cytoskeletal forces generated by MLC-P-dependent actomyosin filaments, and these stresses are opposed by centrifugal forces generated by cell-cell adhesion. Whether Rho-dependent phosphorylation of MLC-P and SF formation cause thrombin-induced endothelial monolayer hyperpermeability remains controversial. In HUVEC, thrombin-induced increases in permeability were prevented or attenuated by pretreatment with C3 for 24 h (8, 28). Our data contrast these findings. Although C3 pretreatment of EC monolayers blocked thrombin-induced SF/FA, C3 did not prevent thrombin-induced barrier dysfunction in bovine pulmonary artery EC. These data suggest that thrombin-induced monolayer hyperpermeability is independent of MLC-P and SF/FA formation. Similar results were reported with use of serine-threonine phosphatase inhibitors to modulate monolayer barrier function (7). Blockade of these phosphatases initiated maximal MLC-P, which, in turn, was inhibited by the specific MLC kinase inhibitor KT-5926. Although this inhibitor prevented MLC-P, it did not block monolayer hyperpermeability, suggesting that changes in cell-cell adhesion may be involved in this process. These data suggest that thrombin-induced monolayer barrier dysfunction involves MLC-P and cell-cell adhesive mechanisms.
In conclusion, our data indicate that Rho GTPases modulate the thrombin-induced formation of myosin ribbons, SF, and tyrosine-phosphorylated FA in EC. Enhanced barrier function stimulated by C3 appears to be due to the disassembly of SF/FA, reorganization of F-actin and tyrosine-phosphorylated proteins to cell-cell junctions, and increased EC surface area.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Dr. Murray A. Katz for support of this work and David Mucha, Alex Cohen, Glenn Soja, and Max Gratrix for excellent technical contributions to this study.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants RO1 HL-48816-5 and DK-55151-01 and a Merit Review Grant from the Veterans Affairs Medical Research Service.
This study was presented by José M. Carbajal as a senior thesis in partial fulfillment of the requirements for the degree Bachelor of Science in the Department of Biochemistry at the University of Arizona, Tucson, AZ.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. C. Schaeffer, Jr., Research Service (151), VA Medical Center, Tucson, AZ 85723 (E-mail: rcs{at}u.arizona.edu).
Received 12 February 1998; accepted in final form 2 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adelstein, R. S.,
M. D. Pato,
and
M. A. Conti.
The role of phosphorylation in regulating contractile proteins.
Adv. Cyclic Nucleotide Res.
14:
361-373,
1981[Medline].
2.
Aepfelbacher, M.,
M. Essler,
E. Huber,
M. Sugai,
and
P. C. Weber.
Bacterial toxins block endothelial wound repair: evidence that Rho GTPases control cytoskeletal rearrangements in migrating endothelial cells.
Arterioscler. Thromb. Vasc. Biol.
17:
1623-1629,
1997
3.
Amano, M.,
K. Chihara,
K. Kimura,
Y. Fukata,
N. Nakamura,
Y. Matsuura,
and
K. Kaibuchi.
Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase.
Science
275:
1308-1311,
1997
4.
Chong, L. D.,
A. Traynor-Kaplan,
G. M. Mokoch,
and
M. A. Schwartz.
The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells.
Cell
79:
507-513,
1994[Medline].
5.
Craig, S. W.,
and
R. P. Johnson.
Assembly of focal adhesions: progress, paradigms and portents.
Curr. Opin. Cell Biol.
8:
74-85,
1996[Medline].
6.
Dejana, E.
Endothelial adherens junctions: implications in the control of vascular permeability and angiogenesis.
J. Clin. Invest.
98:
1949-1953,
1996
7.
Diwan, A. H.,
R. E. Honkanen,
R. C. Schaeffer, Jr.,
S. J. Strada,
and
W. J. Thompson.
Inhibition of serine-threonine protein phosphatases decreased barrier function of rat pulmonary artery endothelial cells.
J. Cell. Physiol.
171:
259-270,
1997[Medline].
8.
Essler, M.,
M. Amano,
H.-J. Kruse,
K. Kaibuchi,
P. C. Weber,
and
M. Aepfelbacher.
Thrombin inactivates myosin light chain phosphatase via rho and its target rho kinase in human endothelial cells.
J. Biol. Chem.
273:
21867-21874,
1998
9.
Garcia, J. G. N.,
A. D. Verin,
and
K. L. Schaphorst.
Regulation of thrombin-mediated endothelial cell contraction and permeability.
Semin. Thromb. Hemost.
22:
309-315,
1996[Medline].
10.
Goeckeler, Z. M.,
and
R. B. Wysolmerski.
Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization and myosin phosphorylation.
J. Cell Biol.
130:
613-627,
1995[Abstract].
11.
Hartshorne, D. J.,
and
T. Kawamura.
Regulation of contraction-relaxation in smooth muscle.
News Physiol. Sci.
7:
59-64,
1992.
12.
Hippenstiel, S.,
S. Tannert-Otto,
N. Vollrath,
M. Krull,
I. Just,
K. Aktories,
C. Von Eichel-Streiber,
and
N. Suttorp.
Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L38-L43,
1997
13.
Jalink, K.,
E. J. van Corven,
T. Hengeveld,
N. Morii,
S. Narumiya,
and
W. H. Moolenaar.
Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.
J. Cell Biol.
126:
801-810,
1994[Abstract].
14.
Kano, K.,
K. Katoh,
M. Masuda,
and
K. Fujiwara.
Macromolecular composition of stress fiber-plasma membrane attachment sites in endothelial cells in situ.
Circ. Res.
79:
1000-1006,
1996
15.
Kimura, K.,
M. Ito,
M. Amano,
K. Chihara,
Y. Fukata,
M. Nadafuku,
B. Yamamori,
J. Feng,
T. Nakano,
K. Okawa,
A. Iwamatsu,
and
K. Kaibuchi.
Regulation of myosin phosphatase by Rho and Rho-associated kinase (rho-kinase).
Science
273:
245-248,
1996[Abstract].
16.
Laudanna, C.,
J. J. Campbell,
and
E. C. Butcher.
Role of Rho in chemoattractant-activated leukocyte adhesion through integrins.
Science
271:
981-983,
1996[Abstract].
17.
Lum, H.,
and
A. B. Malik.
Mechanisms of increased endothelial permeability.
Can. J. Physiol. Pharmacol.
74:
787-800,
1996[Medline].
18.
Moy, A. B.,
J. Van Engelenhoven,
J. Bodmer,
J. Kamath,
C. Keese,
I. Girener,
S. Shasby,
and
D. M. Shasby.
Histamine and thrombin increase endothelial focal adhesion through centripedal and centrifugal forces.
J. Clin. Invest.
97:
1020-1027,
1996
19.
Nemoto, Y.,
T. Namba,
S. Kozaki,
and
S. Narumiya.
Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli.
J. Biol. Chem.
266:
19312-19319,
1991
20.
Ridley, A. J.,
and
A. Hall.
The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70:
389-399,
1992[Medline].
21.
Schaeffer, R. C., Jr.,
and
M. S. Bitrick, Jr.
Effects of human -thrombin and 8-bromo-cAMP on large and microvessel endothelial monolayer equivalent "pore" radii.
Microvasc. Res.
49:
364-371,
1995[Medline]
22.
Schaeffer, R. C., Jr.,
A. W. Cohen,
C. J. Pusch,
and
K. A. Seitz.
Cyclic AMP and KT5926 initiate p125FAK phosphorylation and paxillin appearance at bovine pulmonary artery endothelial cell-cell junctions (Abstract).
Mol. Biol. Cell
7:
386A,
1996.
23.
Schaeffer, R. C., Jr.,
F. C. Gong,
and
M. S. Bitrick, Jr.
Restricted diffusion of macromolecules by endothelial monolayers and small pore filters.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L27-L36,
1992
24.
Schaeffer, R. C., Jr.,
F. C. Gong,
M. S. Bitrick, Jr.,
and
T. L. Smith.
Thrombin and bradykinin initiate discrete endothelial solute permeability mechanisms.
Am. J. Physiol.
264 (Heart Circ. Physiol. 33):
H1798-H1809,
1993
25.
Schaeffer, R. C., Jr.,
R. R. Renkiewicz,
S.-M. Chilton,
D. Marsh,
and
R. W. Carlson.
Preparation and high performance size-exclusion chromatographic analysis of fluorescein isothiocyanate-hydroxy ethyl starch: macromolecular probes of the blood-lymph barrier.
Microvasc. Res.
32:
230-243,
1986[Medline].
26.
Somlyo, A. P.,
and
A. V. Somlyo.
From pharmacomechanical coupling to G-proteins and myosin phosphatase.
Acta Physiol. Scand.
164:
437-448,
1998[Medline].
27.
Thurston, G.,
and
D. Turner.
Thrombin-induced increase of F-actin in human umbilical vein endothelial cells.
Microvasc. Res.
47:
1-20,
1994[Medline].
28.
Van Niew Amerongen, G. P.,
R. Draijer,
M. A. Vermeer,
and
V. W. M. van Hinsbergh.
Transient and prolonged increase in endothelial permeability induced by histamine and thrombin.
Circ. Res.
83:
1115-1123,
1998
29.
Verkhovsky, A. B.,
T. M. Svitkina,
and
G. G. Borisy.
Myosin II filament assembly in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles.
J. Cell Biol.
131:
989-1002,
1995[Abstract].
30.
Yano, Y.,
Y. Saito,
S. Narumiya,
and
B. E. Sumpio.
Involvement of Rho p21 in cyclic strain-induced tyrosine phosphorylation of focal adhesion kinase (pp125FAK), morphological changes and migration of endothelial cells.
Biochem. Biophys. Res. Commun.
224:
508-515,
1996[Medline].