 |
INTRODUCTION |
Endothelial cells (EC)1
lining blood vessels form the principal barrier to fluid flux from
blood to tissue. A decrease in barrier properties increases fluid flux
and promotes tissue edema that in vital organs such as lung or brain
could potentially be life threatening. Permeability of the EC barrier
depends largely on the restriction to fluid transport across the
paracellular pathway that contains tight junctions and adherens
junctions (1). Tight junctions are the primary determinants of barrier
function (2). Barrier-deteriorating agents cause EC contraction by
activating Ca2+-dependent myosin light chain
kinase, thereby widening the junctions and causing hyperpermeability of
the EC barrier (3). In a previous study, we reported that exposing lung
capillaries to a 1-min hyperosmolar stimulus caused an immediate
hyperpermeability response followed by a gradual return of barrier
properties to normal (4). These findings suggest that even while
challenged by barrier-deteriorating stimuli, EC institute repair
mechanisms that reestablish adequacy of the barrier. However, these
repair processes remain inadequately understood.
In this regard, the role of focal adhesions requires consideration. EC
exposed to barrier deteriorating stimuli develop focal adhesion
complexes at points of cell-matrix contact (5, 6). Although the barrier
regulatory role of this response remains unclear, evidence from other
cell types indicates that focal adhesion formation causes activation of
focal adhesion kinase (FAK) (1, 6). It is proposed that in EC exposed
to the barrier-deteriorating agent, thrombin, actin-induced
translocation of FAK to focal adhesions reduces barrier deterioration
(7). The actin cytoskeleton may be particularly relevant in this
regard, because receptor-mediated enhancement of actin (8) or actin
stabilization by phallicidin (9) strengthens, whereas actin
depolymerization by cytochalasins deteriorates the barrier (10).
However, the specific mechanisms induced by FAK leading to barrier
strengthening in EC remain unclear.
Here we considered the possibility that FAK may be responsible for
cross-talk between focal adhesions and cadherins. Dynamic regulation of
the EC barrier is attributable to E-cadherin (1, 11, 12) or VE-cadherin
(13). At intercellular junctions, the cadherins form homophilic
interactions between their extracellular domains on adjacent cell
membranes, whereas their cytoplasmic domains bind
-catenin or
plakoglobin (
-catenin) that in turn associates with the
actin-binding protein,
-catenin, thereby establishing a linkage
between the cadherin-catenin complex and the actin cytoskeleton (1,
14). Evidence that this linkage is important for barrier regulation
comes from findings that barrier-deteriorating stimuli deplete both the
cadherin-catenin complex (15) as well as actin (16) from the cell
periphery. The FAK substrate
-actinin also binds
-catenin (17),
thus providing a link between focal adhesions and the cadherin-catenin
complex and thereby raising the possibility that focal adhesion
assembly stabilizes the cadherin complex.
We considered these issues in the context of hyperosmolar exposure that
causes a cell shrinkage-induced activation of focal adhesion proteins
as indicated by increased tyrosine phosphorylation of FAK (18). This
response provided an opportunity to test whether in EC FAK is involved
in the stabilization of the cadherin complex and possibly of barrier
properties. Accordingly, we generated EC expressing a truncated
form of deleted FAK (del-FAK) in which cadherin expression in the
plasma membrane was markedly diminished. Our findings indicate that
although hyperosmolarity increased barrier properties and peripheral
cadherin recruitment in wild type EC, both effects were markedly
blunted in EC-expressing del-FAK, indicating that EC employ
FAK-dependent signaling mechanisms as a means to barrier strengthening.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Chemicals were obtained from Sigma unless
otherwise stated. Cell culture media and growth supplements, M199
medium, Lipofectin, G418, and Opti-MEM were obtained from Invitrogen.
All reagents for immunofluorescence studies were obtained from
Molecular Probes Inc. (Eugene, OR). Anti-phosphotyrosine mAb PY99
(mouse and monoclonal) and protein A/G-agarose beads were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-paxillin mAb was
purchased from Zymed Laboratories Inc. (South San
Francisco, CA). Anti-FAK and E-cadherin mAbs were obtained from
Transduction Laboratories, Inc. (Lexington, KY). Anti-FAK polyclonal
antibody (BC3) was obtained from Upstate Biotechnology (Lake Placid,
NY). Anti-actin rabbit polyclonal antibody and
-tubulin mAb were
obtained from Sigma.
Cell Culture--
Rat lung microvascular endothelial cells
(RLMEC) were cultured as described previously (4, 5) under 5%
CO2 in M199 medium supplemented with 5% fetal bovine serum
and 5% bovine calf serum. Cells were plated at a density of 1 × 105/cm2. EC phenotype was confirmed by cell
uptake of fluorescent labeled-acetylated low density lipoprotein in
imaged monolayers.
Transendothelial Electrical Resistance (TER)--
For EC barrier
quantification, we determined TER in RLMEC monolayers grown on sterile
polycarbonate inserts held at 37 °C (Endohm, World Precision
Instruments, Sarasota, FL). After a 30-min base-line period,
experimental solutions were added and TER was determined every 15 s for the first 10 min and then at 1-min intervals for the subsequent
20 min. The data were corrected for the resistance of the insert alone.
Plasmid Construction Del-FAK--
The plasmid pBS-FAK (a gift of
Dr. James Parsons, Department of Microbiology, School of Medicine,
University of Virginia, VA) was prepared by cloning the full-length FAK
cDNA into the pBluescript, K5 vector (Stratagene, La Jolla, CA)
(19). This plasmid was used for the generation of del-FAK DNA by
deleting sequences among the EaeI sites at 1176 and 2793 bp
(amino acids 392-931) in the FAK gene (Fig.
1A). This deletion includes a
segment containing tyrosine residues that are critical for FAK
activation. These residues include Tyr-397 at which FAK
autophosphorylates (20), Tyr-576 and Tyr-577 at which phosphorylation
determines the kinase activity of FAK (21), and Tyr-925 at which
phosphorylation leads to activation of Src (22). Del-FAK DNA (1.6 kb)
was subcloned into the plasmid vector pBK-CMV (7.76 kb) at
BamH1-XhoI sites and then introduced into the
bacterial strain MV10. We selected the clone (kanamycin, 50 µg/ml)
containing the 1.6-kb segment (pSLRCU33) corresponding to del-FAK
variant (Qiagen, Valencia, CA) using restriction enzymes
(BamH1-XhoI). Each 2 µg of plasmid pSLRCU33 or
the empty vector was stably transfected in RLMEC using nominal
procedures (Lipofectin, Invitrogen).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 1.
Transfections of del-FAK construct.
A, a map of FAK gene shows a segment
deleted in the del-FAK construct. The tyrosine (Y) residues,
amino acids, and the sites of the sense (S) and antisense
(As) primers are indicated. B, G418-resistant EC
clones of stably transfected RLMEC were subjected to RT-PCR. Gel shows
RT-PCR products for PCR buffer (PCR mix), wild type EC
(wt), and cells containing empty vector (vec) or
del-FAK.
|
|
To confirm transfection of the del-FAK construct in RLMEC, we prepared
primers based on the full-length FAK cDNA (GenBankTM
accession number M86656) (19): sense primer (873-895 nucleotides), 5'-CCC AGA GGA AGG AAT CAG CTA C-3', and antisense primer (3085-3065 nucleotides), 5'-GCT GGT CAT GAC GTA CTG CTG-3' (Fig. 1A).
For PCR, the parameters were: denaturing at 95 °C for 15 min
followed by 35 cycles of denaturing, annealing, and extension at 95, 59, and 72 °C for 1, 1, and 2 min, respectively, in 3 mM
MgCl2. In the presence of these primers, amplification by
RT-PCR is expected to yield a 600-bp product only in cells containing
the del-FAK construct (Fig. 1B), confirming successful
transfection of the del-FAK construct. In wild type and empty
vector-transfected cells, the same primers yielded only the expected
RT-PCR product of 2.2 kb (Fig. 1B).
V12Rac1GFP and N17Rac1GFP--
V12Rac1GFP and N17Rac1GFP
constructs were generously provided by Dr. P. Jurdic (Laboratoire de
Biologie Moléculaire et Cellulaire, Ecole Normale
Supérieure de Lyon, Lyon, France) (23). Chimeras between
enhanced GFP and GTPases were derived by insertion of the mutated
GTPase open reading frames between
EcoR1/SalI into the pEGFP-Cl expression vector
downstream to the enhanced green fluorescent protein (GFP) coding
sequence (Clontech, Palo Alto, CA). The resulting
cDNA encoded chimeric GTPase-GFP expression vector. Plasmid DNA,
V12Rac1GFP, N17Rac1GFP, and vector pEGFP-Cl were amplified by the
standard protocol and adjusted at a final concentration of 1 mg/ml in
water. All of the plasmids were expressed by stable transfection using
Lipofectin reagent and following the manufacturer's instructions.
Lysate Preparation--
These procedures are routine in our
laboratory (4, 5). Cells were exposed to isosmolar medium, or medium
was made hyperosmolar by the addition of sucrose (except where stated)
at 37 °C under 5% CO2 in M199 for indicated periods.
Subsequently, cells were washed twice with ice-cold PBS and lysed in
radioimmune precipitation assay buffer (50 mM Tris-HCl, pH
7.6, 150 mM NaCl, 2 mM EDTA, 50 mM
NaF, 0.5% SDS, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) containing 1 mM sodium orthovanadate, 10 mM sodium
pyrophosphate, 25 mM
-glycerophosphate, 0.1% SDS, and
1% Triton X-100 at 4 °C. Total cell lysate was clarified by centrifugation at 10,000 × g for 10 min. Protein
concentrations were determined using a protein analysis kit (BCA, Pierce).
Cell Fractionation--
Cells were rinsed 2× PBS and
solubilized in Triton X-100 buffer (50 mM NaCl, 10 mM Pipes, pH 6.8, 3 mM MgCl2, 0.5%
Triton X-100, 300 mM sucrose, 1.2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) for 20 min at
4 °C on a rocking platform. The cells were scraped from the plate
and centrifuged (10 min). The supernatant was collected. The cell
pellet was suspended in 100 µl of SDS immunoprecipitation buffer (15 mM Tris, pH 7.5, 5 mM EDTA, 2.5 mM
EGTA, 1% SDS) and then boiled for 10 min and diluted to 300 µl with
Triton X-100 buffer. Equal amounts of extracted proteins were
immunoprecipitated and loaded for SDS-PAGE gel electrophoresis.
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were performed as described previously (4). Cell
lysates containing equal amounts of protein were precleared for 30 min
with 20 µl of protein A/G-agarose beads followed by incubation with
primary antibodies (4 µg for 2 h). Antibody-antigen complexes
were precipitated with 30 µl of protein A/G-agarose beads overnight
at 4 °C. Nonspecific bound proteins were removed by washing the
agarose beads three times with radioimmune precipitation assay buffer
and one time with PBS. Bound proteins were eluted in 40 µl of 4×
Laemmli's loading buffer. The proteins were resolved by
SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose
membrane, and analyzed by immunoblotting.
FAK Activity--
For the analysis of FAK autophosphorylation,
we used the reported immune complex kinase assay (21). We
immunoprecipitated p125FAK from wild type or del-FAK-transfected RLMEC.
The immunoprecipitated complexes were washed three times with
radioimmune precipitation assay buffer and once with kinase buffer (pH
7.4, 20 mM Hepes, 50 mM NaCl, 5 mM
MgCl2, 5 mM MnCl2). The pellet was resuspended in 30 µl of the kinase buffer containing 10 µCi of
[
-32P]ATP (6000 Ci/mmol), and the sample was incubated
for 30 min at 37 °C with frequent agitation. The reaction was
terminated by the addition of 10 µl of 4× SDS-PAGE gel sample buffer
followed by boiling for 5 min, and all of the products were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane (Bio-Rad). Direct exposures were used to
visualize FAK autophosphorylation. The amount of FAK present in the
reactions was visualized by immunoblotting of the same membrane with
anti-FAK monoclonal antibody.
Rac Activation Assay--
Using glutathione
S-transferase-tagged p21- activated kinase-p21
binding domain (PAK-PBD) protein beads that specifically bind active Rac1, we obtained immunoprecipitates from EC lysates as per the
methods reported previously (24) using the assay kit from Cytoskeleton
Inc. (Denver, CO). For positive and negative controls for the active
and inactive small GTPases, the non-hydrolyzable GTP analog, GTP
S,
and GDP were used, respectively. Cell lysates obtained from untreated
cells were incubated with 100 µM GTP
S or 1 mM GDP in the presence of 10 mM EDTA for 15 min
at 30 °C to ensure efficient loading with the added nucleotide. To
terminate the reaction, the lysates were placed on ice and supplemented with 60 mM MgCl2. These control samples were
then incubated with the glutathione S-transferase-tagged
PAK-PBD protein beads and washed in the same manner as the other
samples. Captured proteins were removed from the beads by boiling the
samples in Laemmli buffer, and the samples were subjected to SDS-PAGE
and Western blotting.
Immunofluorescence and Confocal Microscopy--
RLMEC monolayers
grown on glass coverslips were fixed (4% formaldehyde in PBS, pH 7.4, 20 min, 22 °C), rinsed (3× PBS), permeabilized (0.1% Triton
X-100), and stained using rhodamine-phalloidin. For immunofluorescence,
cells were incubated with diluted primary antibodies (1:50) in blocking
solution, 4% goat serum in PBS (1 h at 22 °C), and washed with 3×
PBS. Fluorescence-conjugated antibodies then were added (1:500, 1 h at 22 °C) and washed with 3× PBS. The glass coverslips were
mounted upside down on object slides using fluorescent-mounting medium
(Dako Corporation Carpinteria, CA). Confocal images were obtained by
means of a laser-scanning microscope (Pascal LSM, Carl Zeiss) and
subjected to image analysis as described below (MCID 5, Imaging
Research, St. Catherine, Canada).
 |
RESULTS |
Barrier Response--
TER of RLMEC monolayers was stable at
37 ± 3 ohm/cm2 for up to 40 min (mean ± S.E.,
n = 6). Exposure of monolayers to isosmolar medium (300 mosM) did not change TER from base line (Fig.
2A). However, in monolayers
exposed to medium-made hyperosmolar by the addition of sucrose (350 mosM), TER decreased in the first minute and then increased
in the subsequent ~10 min (Fig. 2A). The increase was
sustained at a steady level for 5-7 min after which TER gradually
returned to base line in 20-30 min. The increase of TER was abrogated
by the addition of isosmolar medium (Fig. 2B), and it could
be repeated subsequently in the same monolayer (data not shown). These
findings indicated that subsequent to an initial decrease of barrier
properties, the dominant effect of hyperosmolar exposure was to enhance
the EC barrier.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
TER responses to hyperosmolarity in
RLMEC. A and B, single tracings exemplify
responses to sucrose medium (added at arrows) at indicated
levels of hyperosmolarity and replicated six times each. C,
group responses to 350 mosM sucrose medium in monolayers
treated for 30 min with each of the indicated treatments. Each bar is
mean ± S.E. for four experiments. D, maximum TER
increase is in relation to base-line value. Each point is mean ± S.E. for seven experiments.
|
|
Because hyperosmolar exposure increases protein tyrosine
phosphorylation in EC (18) and because actin polymerization is proposed
to cause barrier strengthening (8, 9), we exposed monolayers to
inhibitors of tyrosine kinase and actin polymerization. The TER
increase was inhibited by the tyrosine kinase inhibitor, genistein, and
by the inhibitors of actin polymerization, latrinculin B (Fig.
2C) and cytochalasin D (data not shown). Although
hyperosmolar exposure is reported to increase the cytosolic
Ca2+ (25), the intracellular calcium chelator,
BAPTA-AM had no effect on the present TER response to
hyperosmolarity (Fig. 2C). Not shown are results from
experiments in which we incubated monolayers separately with the p38
mitogen-activated protein kinase blocker SB203580 (25 µM), the protein kinase C blocker calphostin C (500 nM), or the phosphatidylinositol 3-kinase blocker
wortmannin (50 nM) (n = 3 for each
inhibitor). A single monolayer was pretreated with the nitric-oxide
synthase inhibitor L-NAME (30 µM).
None of these treatments affected the TER response to hyperosmolarity. The maximum increase in TER correlated non-linearly with an osmolar concentration (Fig. 2D) with 80% of the response being
established at 350 mosM. Hence, for the studies described
below, we exposed EC to this hyperosmolar concentration for 15 min.
Focal Adhesion Proteins--
Because genistein blocked the TER
response to hyperosmolarity, we considered that hyperosmolar cell
shrinkage might increase cell-matrix interactions, leading to
activation of focal adhesion proteins. By confocal microscopy of wild
type cells under base-line conditions, immunofluorescence of the focal
adhesion marker protein paxillin was largely localized to the nuclear
and perinuclear regions (Fig.
3A, left image).
Following hyperosmolar exposure, the fluorescence became pronounced as
aggregates localized to the cell periphery in a pattern characteristic
of focal adhesion formation (Fig. 3A, right
image) (6). This focal adhesion response was absent in
del-FAK-expressing monolayers (Fig. 3B). Exposure of plated
EC to hyperosmolar sucrose also increased tyrosine phosphorylation of
FAK and paxillin (Fig. 3C). FAK activity, which was less in del-FAK-transfected monolayers than in wild type monolayers under base-line conditions (Fig. 3, D and E), increased
3-fold in wild type monolayers exposed to hyperosmolar medium (Fig. 3,
D and E). By contrast, hyperosmolar exposure
caused no enhancement of FAK activity in del-FAK-transfected cells
(Fig. 3, D and E). Under non-stimulated
conditions, TER was 30 ± 5% less in del-FAK-expressing monolayers than in wild type monolayers or in monolayers expressing vector alone (p < 0.05; n = 4).
Following hyperosmolar exposure, the increase of TER was blunted in
del-FAK-expressing monolayers to 60 ± 5% of that of wild type
monolayers (Fig. 3F). Taking these findings together, we
interpret that hyperosmolar exposure increased focal adhesion formation
and that inhibition of this response inhibited the
hyperosmolarity-induced barrier enhancement.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
Responses to hyperosmolarity in
del-FAK-transfected and wild type RLMEC. EC were exposed to
isosmolar (300 mosM) or hyperosmolar (350 mosM)
conditions for 15 min each as indicated. A and B,
immunofluorescence of paxillin (arrows) shown in images
obtained by confocal microscopy in wild type (A) and
del-FAK-expressing (B) cells. Monolayers were fixed and
permeabilized and then stained with mouse anti-paxillin mAb followed by
Alexa Red-linked anti-mouse IgG with each set replicated four
times. C, plated EC were subjected to immunoprecipitation
(IP) using indicated antibodies. Immunoblots were obtained
using anti-phosphotyrosine antibody. Immunoblots using the indicated
protein-specific antibodies confirm loading of equal protein amounts in
each lane with each set replicated four times. D, gel shows
enzymatic activity of FAK autophosphorylation as determined by immune
complex kinase assay of FAK immunoprecipitates from EC lysates.
Upper panel shows FAK activity. Lower panel shows
the amount of FAK present in the reactions as indicated by
immunoblotting the same membrane with anti-FAK mAb. E, bar
diagram shows FAK activity as optical densities of bands relative to
protein content (n = 3 for each bar). F, TER
responses to hyperosmolar (350 mosM) exposure in wild type
(wt) monolayers and in monolayers transfected with vector
alone (vec) or FAK mutant (del-FAK).
n = 4 for each bar; mean ± S.E., *,
p < 0.01 against bar at left.
|
|
To further consider the role of the cytosolic Ca2+, we
carried out immunoprecipitations from monolayers treated with the
intracellular Ca2+ chelator BAPTA-AM. Predictably,
BAPTA-AM blocked the tyrosine phosphorylation of the
Ca2+-dependent focal adhesion protein Pyk2 (Fig.
4A) (26), but it did not
modify phosphorylation responses for FAK or paxillin (data not shown).
These results together with the above lack of an inhibitory effect on
TER by BAPTA-AM indicate that the hyperosmolarity-induced TER
increase was Ca2+-independent. In EC lysates, bands were
evident at 125 and 68 kDa (Fig. 4, B and C),
corresponding to FAK and paxillin, respectively (Fig. 3A).
However, as opposed to the responses in plated EC, no increase of
tyrosine phosphorylation was evident in lysates prepared either from
suspended EC exposed to hyperosmolar sucrose (Fig. 4B,
lanes 3 and 4) or from plated EC exposed to
hyperosmolar urea (Fig. 4C, lane 3). The
implications of these findings are discussed below.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of cell suspension, urea, and
intracellular Ca2+ chelation in RLMEC.
A, plated EC exposed for 15 min to conditions shown
were subjected to immunoprecipitation (IP) using anti-Pyk2
mAb antibodies. Immunoblots are for phosphotyrosine (upper
panels) and protein (lower panels) contents. BAPTA-AM
(100 µM) with each set replicated three times.
B and C, data shown for plated EC
(monolayer) or EC held in suspension in medium
(suspended). Medium was made hyperosmolar by adding sucrose
except where stated (urea). Cell lysates were subjected to
SDS-PAGE, transfer, and immunoblotting (IB) using
anti-phosphotyrosine antibody (Tyrp), and each set
replicated three times each.
|
|
E-cadherin--
In considering the barrier regulatory function of
FAK, we addressed the role of cadherins. Immunoprecipitation studies
using mAbs against either VE- or E-cadherin indicated that although E-cadherin was well expressed in RLMEC (Fig.
5A), the expression of
VE-cadherin was weak (Fig. 5B), indicating that E-cadherin was the dominant cadherin type expressed in these EC. We confirmed that
the mAb against VE-cadherin was capable of recognizing rat antigens
(Fig. 5B) and that as reported by others (27) it recognized a band in human umbilical vein endothelial cells (Fig.
5B).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
E-cadherin expression in RLMEC.
A and B, gels show immunoprecipitation
(IP) and immunoblot (IB) with anti E-cadherin
(A) or anti-VE-cadherin (B) mAb. Wild type
(wt), del-FAK-transfected (del-FAK), and
vector-transfected (vec) cells were exposed to isosmolar
(300 mosM) or hyperosmolar (350 mosM) sucrose
medium for 15 min. Monolayer lysates were detergent-fractionated into
cytosolic (C) and membrane (M) fractions, and
each set replicated three times. C, bar diagram shows
E-cadherin as optical densities of bands (n = 3 for
each bar). p < 0.05 compared with corresponding value
for 300 mosM (*) or the corresponding cytosolic value
(#). D and E, E-cadherin distribution
(arrows) shown in images obtained by confocal microscopy in
wild type (D) and del-FAK-expressing (E) cells.
Monolayers were fixed and permeabilized and then stained with mouse
anti-E-cadherin mAb followed by Alexa Red-linked anti-mouse IgG. Each
set replicated four times. HUVEC, human umbilical vein
endothelial cells.
|
|
Detergent-based fractionation of EC lysates into cytosolic and membrane
fractions followed by quantitative immunoprecipitation and
immunoblotting revealed that in wild type EC, E-cadherin content was
greater in the membrane than the cytosolic fraction (Fig. 5A, lanes 1 and 2). Hyperosmolar
exposure increased the membrane content (Fig. 5A,
lanes 2 and 4) while decreasing the cytosolic content (Fig. 5A, lanes 1 and 3).
These compartmental changes were approximately equal as quantified by
band densitometry (Fig. 5C). Accordingly, hyperosmolarity
increased the membrane-cytosol ratio for E-cadherin (Fig.
5C). In del-FAK-expressing cells, E-cadherin content was
less under control conditions than in wild type cells (p < 0.01) (Fig. 5, A and C).
Moreover, the membrane content was less than the cytosolic (Fig.
5A, lanes 5 and 6, and C).
Furthermore, in contrast to the wild type response, in
del-FAK-expressing cells, E-cadherin failed to increase following
hyperosmolar exposure (Fig. 5A, lanes 6 and
8), resulting in similar membrane-cytosol ratios under
control and hyperosmolar conditions (Fig. 5C). Responses in
monolayers expressing the empty vector were similar to those of wild
type monolayers (Fig. 5A, lanes 9-12, and
C).
In wild type cells under base-line conditions, confocal microscopy
revealed the distribution of E-cadherin as a discontinuous line of
fluorescence that marked the cell periphery (Fig. 5D, left image). Following hyperosmolar exposure, this
peripheral fluorescence became pronounced and was now evident as a
continuous line (Fig. 5D, right image),
indicating increased E-cadherin expression. In del-FAK-expressing
monolayers, the peripheral fluorescence was poorly developed under both
base-line and hyperosmolar conditions (Fig. 5E), indicating
abrogation of E-cadherin expression and pointing to FAK as
critical factor in the hyperosmolarity-induced enhancement of
E-cadherin.
Actin--
Because latrinculin B inhibited the
hyperosmolarity-induced TER increase (Fig. 2C), we
considered the involvement of the actin cytoskeleton in the present
barrier response. Confocal microscopy of untreated wild type cells
revealed actin distribution as reflected in rhodamine-phalloidin
fluorescence as a thin band at the cell periphery and a perinuclear
condensation (Fig. 6A,
left image). A 15-min hyperosmolar exposure markedly
enhanced the density of filamentous actin in wild type cells but not in
del-FAK-expressing cells (Fig. 6, A-C). Immunoblotting with
an actin-recognizing mAb indicated that, as expected, actin content was
higher in the membrane than in the cytosolic fraction under control
conditions (Fig. 7, A and
B). In wild type cells, hyperosmolar exposure increased membrane actin content almost 2-fold (Fig. 7B, compare
second and fourth bars) while decreasing the
cytosolic content (Fig. 7B, compare first and
third bars). However, in del-FAK-expressing cells, these
responses were completely blocked (Fig. 7, A and B).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
Immunofluorescence of actin in RLMEC.
A and B, images obtained by confocal microscopy
images show fluorescence of rhodamine-phalloidin depicting
distribution of actin in wild type (A) and
del-FAK-transfected (B) cells. As indicated, monolayers were
exposed to isosmolar (300 mosM) or hyperosmolar (350 mosM) sucrose medium for 15 min. Increase of filamentous
actin is indicated by double-headed arrow. Each set
replicated four times. C, determinations of fluorescence
intensity by image analysis. Linear fluorescence was determined along a
5-µm-wide band that followed the cell contour midway between the
periphery and the center (white line in inset).
Data are for wild type (wt) and del-FAK-expressing
(del-FAK) cells exposed to isosmolar (300 mosM)
or hyperosmolar (350 mosM) conditions. Mean ± S.E.;
n = 4 for each bar. *, p < 0.05 compared with control (300 mosM).
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Compartmental actin distribution in
RLMEC. A, monolayer lysates were detergent-treated to
obtain cytosolic (C) and membrane (M) fractions.
Gels show immunoblots (IB) conducted sequentially on same
membrane with anti-actin (actin) and anti- -tubulin
(tubulin) antibodies. Immunoblot with anti- -tubulin mAb
confirms equal protein loading in each lane. B, bars show
densitometric analyses of bands in A expressed as optical
density normalized to -tubulin density. For each paired
determination, density was higher for membrane than cytosolic fractions
(p < 0.05). *, p < 0.05 compared with
300 mosM. Mean ± S.E.; n = 4 for each
bar.
|
|
To determine mechanisms underlying actin assembly, we considered the
role of the small GTPase, Rac1 that regulates actin polymerization (8,
28). We immunoprecipitated increased amounts of active Rac1 from cells
exposed to hyperosmolar sucrose (Fig.
8A), which indicated that
hyperosmolar exposure increased Rac1 activity in these cells. To modify
Rac1 activity, we expressed GFP-tagged constitutively active (V12Rac1)
or dominant negative (N17Rac1) Rac1 mutants in EC monolayers. These
isoforms enhance and reduce actin filament formation, respectively (8,
28). Responses in RLMEC are exemplified in the images shown in Fig.
8B. Under isosmolar conditions, actin density was higher in
V12Rac1-expressing monolayers than in monolayers expressing N17Rac1 or
vector alone. Following hyperosmolar exposure, actin density increased
in cells expressing empty vector, which is consistent with the above
immunoprecipitation data in which hyperosmolar exposure increased
membrane actin. Hyperosmolarity caused greater increases in actin
filaments in V12Rac1-expressing cells than in vector controls. The
overlay of rhodamine on GFP resulted in yellow discoloration along the cell margin in V12Rac1- and N17Rac1-transfected cells, indicating that
transfected Rac1 was targeted to the cell periphery. In contrast, hyperosmolarity failed to increase actin density in cells expressing N17Rac1, indicating that Rac1 activation was critical for the actin
response to hyperosmolar exposure (28). However, despite these
differences, TER was unaffected by the expression of either V12Rac1 or
N17Rac1 in both base-line and hyperosmolar conditions (Fig.
8C). We interpret from these findings that actin assembly was irrelevant in the barrier-enhancing response to
hyperosmolarity.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 8.
Rac activity in RLMEC. Wild type
(wt), constitutively active Rac1 (V12Rac1GFP) or
dominant negative Rac1 (N17Rac1GFP) or empty vector pEGFP
(vec) transfected cells were exposed to isosmolar (300 mosM) or hyperosmolar (350 mosM) sucrose medium
for 15 min. A, upper panel shows bands for active
Rac1 immunoprecipitated from EC lysates using glutathione
S-transferase-tagged PAK-PBD protein beads. Bands for
GDP and GTP S denote negative and positive controls obtained,
respectively, in untreated cells. Immunoblots using anti-Rac1
polyclonal antibody in the lower panel indicate that lysate
samples contained equal amounts of Rac1 protein prior to
immunoprecipitation. Each set replicated three times. B,
images show fluorescence in single cells of monolayers for
rhodamine-phalloidin (rhod) and GFP (GFP).
Arrows point to actin filaments. Double-headed
arrows indicate site of increased actin density. Yellow
pseudocolor in the overlay panel indicates co-localization of
filamentous actin with the transfected vector (double
arrows). Each set replicated three times. C,
TER responses to hyperosmolar (350 mosM) exposure in
wild type-, vec-, V12Rac1-, and N17Rac1-transfected cells.
n = 4 for each bar; mean ± S.E.
|
|
 |
DISCUSSION |
Our findings may be summarized as follows. A 15-min exposure
of EC to hyperosmolarity resulted in (i) an immediate decrease and then
a relatively sustained increase of TER, (ii) increases in focal
adhesion formation and FAK activity, (iii) increased membrane
accumulation of E-cadherin, (iv) increased Rac1 activity, and (v)
increased density of filamentous actin.
We conclude from these findings that the dominant effect of
hyperosmolarity was to enhance EC barrier properties. This
counterintuitive interpretation opposes the expectation that in the
presence of external hyperosmolarity, cell water extraction leading to
cell shrinkage should widen intercellular junctions and consequently decrease the barrier (4). Although such an effect did in fact occur as
indicated by the initial decrease in TER, the effect was short-lived
and lasted for only the first minute of the response. However, this
initial cell shrinkage was critical for the subsequent barrier
enhancement as was evident when we replaced sucrose with urea, the
membrane-permeable osmolyte that does not extract cell water.
Hyperosmolar urea failed to alter EC barrier properties, indicating
that cell shrinkage initiated the complex barrier response to
hyperosmolar sucrose. An important consequence of cell shrinkage was
the induction of tyrosine phosphorylation in plated EC. Although this
result is similar to previous findings that we (4) as well as others
(18) have reported in lung microvascular and aortic EC, no
hyperosmolarity-induced increase of tyrosine phosphorylation occurred
in EC suspended in buffer. These results indicate that cell shrinkage
alone was not sufficient to initiate signaling but that cell-matrix
interactions were a key element in this response.
The Role of FAK--
In blood vessels, mechanical stresses such as
flow-induced shear and mechanical stretch cause EC-matrix interactions,
leading to activation of focal adhesion proteins and enhanced protein tyrosine phosphorylation (29). Here, hyperosmolar exposure enhanced focal adhesion formation, tyrosine phosphorylation of FAK and paxillin,
and FAK activity. These responses suggest that focal adhesions were
induced following hyperosmolarity-induced cell shrinkage, possibly as a
result of displacement of the cell membrane on the underlying matrix.
In an analogous mechanism, cell contraction induces focal adhesion
formation in tracheal smooth muscle cells (30). In del-FAK-expressing
EC, the hyperosmolarity-induced enhancement of the barrier was markedly
blunted, indicating that FAK was critical in this barrier-strengthening response.
The presence of cross-talk between FAK and the cadherin complex was
indicated in that hyperosmolarity also induced the increased expression
of E-cadherin at the cell periphery. This increase occurred within the
duration of the TER increase, suggesting that the peripheral E-cadherin
enrichment was responsible for the strengthening of adherens junctions
(11, 12). The link to focal adhesions was indicated in that in
del-FAK-expressing cells, the hyperosmolarity-induced peripheral
E-cadherin enrichment was markedly diminished. Taking this result
together with the diminished barrier enhancement response in
del-FAK-expressing cells, we interpret that FAK-dependent
junctional E-cadherin enrichment induced barrier enhancement.
According to the cell contraction hypothesis of vascular permeability,
deterioration of the EC barrier is determined importantly by myosin
light chain kinase activation that is itself induced by an
increase in the cytosolic Ca2+ (3). The intracellular
Ca2+ chelator, BAPTA-AM, blocks increases of cytosolic
Ca2+ and inhibits Ca2+-induced barrier
deterioration (3). However, in the present experiments, BAPTA-AM
modified neither the hyperosmolarity-induced TER response nor the
associated enhancements of tyrosine phosphorylation on FAK and
paxillin. Nevertheless, the Ca2+-blocking effect of this
agent was evident in that BAPTA-AM blocked the hyperosmolarity-induced
enhancement of tyrosine phosphorylation of the
Ca2+-sensitive kinase Pyk2. These findings indicate
that the present barrier enhancement occurred through
Ca2+-independent mechanisms.
Actin--
A striking result in these EC was that hyperosmolar
exposure increased the density of filamentous actin as indicated in the immunoprecipitation data for membrane actin as well as by confocal microscopy of actin immunofluorescence. Increases in filamentous actin
have been reported following exposure of Swiss T3 cells to
platelet-derived growth factor (31), neutrophils to hyperosmolarity (24), and pulmonary artery EC to sphingosine 1-phosphate (8). The
polymerization of actin is attributable to Rac1, a member of the Rho
family of small GTPases that is activated by phosphatidylinositol 3-kinase (32). Cell expression of a constitutively active form of Rac
(V12Rac1) increases filamentous actin by activating LIM kinase that
phosphorylates cofilin, thereby inhibiting actin depolymerization (33).
The findings in mouse fibroblasts indicate that Rac1 also stabilizes
the cadherin-catenin complex by inhibiting the interaction of
IQGAP1 with
-catenin (34, 35) and by activating the
p21-activated protein kinase (36). However, the understanding of these
interactions in the context of EC barrier regulation is complicated by
the proposal that the expression of either constitutively active or inactive Rac deteriorates barrier properties in aortic EC (37) and that
overexpression of Rac destabilizes actin filaments in transformed
epithelial cells (38).
Here, in agreement with reported findings in neutrophils (24),
hyperosmolar exposure increased Rac1 activity in EC monolayers. Furthermore, the hyperosmolarity-induced increase of actin density was
inhibited in EC-expressing N17Rac1 but augmented in EC-expressing V12Rac1, consistent with the interpretation that Rac activation was
responsible for the actin enhancement (8, 28). However, the
phosphatidylinositol 3-kinase inhibitor, wortmannin, failed to
modify the hyperosmolarity-induced TER enhancement, thereby suggesting
that this kinase played no role in the present barrier response.
Moreover, in V12Rac1- and N17Rac1-expressing cells, TER was not
affected either under unstimulated conditions or during hyperosmolar
exposure. We conclude from these findings that despite the present
evidence for Rac1 activation and actin enhancement, these factors
played no role in the hyperosmolarity-induced barrier strengthening.
The bulk of the understanding of actin involvement in barrier
regulation comes from the application of the actin inhibitors, the
latrinculins and the cytochalasins (7, 10, 27). Several groups have
reported that the reduction of polymerized actin in EC caused by
these agents associates with the deterioration of EC barrier properties
(8, 10). Our findings are consistent with these reports, because both
agents not only reduced TER at base-line (data not shown) but they also
abrogated the TER increase during hyperosmolar exposure. However, these
pharmacological data were not supported by TER responses in V12Rac1-
and N17Rac1-expressing cells, suggesting that pharmacological
inhibitors of actin polymerization may induce barrier effects through
nonspecific mechanisms.
In conclusion, our results provide the first evidence that focal
adhesion formation and FAK involvement are critical events leading to
barrier-strengthening processes in EC. Although the role of the
associated increase in actin filaments remains unclear, we believe that
this response did not determine the barrier strengthening. It is
possible that these EC developed the actin response to increase cell
rigidity and thereby oppose imposed shape changes forced by
hyperosmolar cell shrinkage (39). The ability of FAK to regulate E-cadherin accumulation at the cell periphery suggests that FAK serves
as a determining factor in the induction of barrier integrity. Although
a further understanding of this signaling pathway is required, we
propose that the present evidence for EC barrier enhancement may
provide a basis for the consideration of hyperosmolar therapy in the
treatment of vascular leak syndromes.