Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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Because activation of the
coagulation cascade and the generation of thrombin coexist with sepsis
and the release of tumor necrosis factor (TNF)-, we determined the
effects of TNF-
on the mechanism of thrombin-induced increase in
endothelial permeability. We assessed Ca2+ signaling in
human umbilical vein endothelial cells. In human umbilical vein
endothelial cells exposed to TNF-
for 2 h, thrombin produced a
rise in the intracellular Ca2+ concentration
([Ca2+]i) lasting up to 10 min. In contrast,
thrombin alone produced a rise in [Ca2+]i
lasting for 3 min, whereas TNF-
alone had no effect on
[Ca2+]i. Thrombin-induced inositol
1,4,5-trisphosphate generation was not different between control and
TNF-
-exposed cells. In the absence of extracellular
Ca2+, thrombin produced similar increases in
[Ca2+]i in both control and TNF-
-exposed
cells. In TNF-
-exposed cells, the thrombin-induced Ca2+
influx after intracellular Ca2+ store depletion was
significantly greater and prolonged compared with control cells.
Increased Ca2+ entry was associated with an approximately
fourfold increase in Src activity and was sensitive to the
Src kinase inhibitor PP1. After TNF-
exposure, thrombin
caused increased tyrosine phosphorylation of junctional proteins and
actin stress fiber formation as well as augmented endothelial
permeability. These results suggest that TNF-
stimulation of
endothelial cells results in amplification of the
thrombin-induced Ca2+ influx by an
Src-dependent mechanism, thereby promoting loss of
endothelial barrier function.
store-operated calcium influx; Src tyrosine kinase
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- released during bacterial infection is a primary
cause of the microvascular complications such as endothelial injury and
tissue edema associated with sepsis (6, 17, 39). TNF-
increases the procoagulant activity by inducing the activation of
coagulation factors such as thrombin and decreases the anticoagulant
activity in the microvascular bed, resulting in fibrin deposition
(5, 6, 13, 22, 26, 27). Because TNF-
generation can
coexist with the activation of thrombin (6, 17, 39), the
effects of these two mediators may be synergistic; however, it remains
unclear whether TNF-
modulates thrombin-mediated responses such as
leakiness of the microvascular endothelial barrier, and if so, by what mechanism.
Thrombin increases endothelial permeability to plasma proteins by
inducing the formation of interendothelial gaps (10, 15, 20, 25,
34, 35, 37) as the direct result of signaling events activated
by increased cytosolic Ca2+ concentration
([Ca2+]i) (4, 18, 19, 34, 35,
41). Thrombin acts on the endothelial cell surface
proteinase-activated receptor-1 to elicit inositol
1,4,5-trisphosphate
[Ins(1,4,5)P3]
generation, which increases [Ca2+]i by
depleting Ca2+ stores and causing Ca2+ influx
(18, 34, 35, 38). The rise in
[Ca2+]i contributes to the mechanism of
permeability increase at multiple levels. Increased
[Ca2+]i is required for phosphorylation of
myosin light chain by activation of
Ca2+/calmodulin-dependent myosin light chain kinase, which,
in turn, results in endothelial cell contracture and increased
paracellular permeability (10, 15, 20, 25, 37). The
increase in [Ca2+]i also induces vascular
endothelial (VE)-cadherin junction disassembly by activation of the
Ca2+-dependent protein kinase C- isoform, which
contributes to the increased endothelial permeability response
(34). In addition, intracellular store
Ca2+ depletion secondary to activation of
Ins(1,4,5)P3 receptors is an
essential requirement for activation of store-operated
Ca2+channels and thus enables the influx of
Ca2+ (14, 21, 30). Prevention of
store-operated Ca2+ entry into endothelial cells reduced
the thrombin-induced increase in transendothelial permeability
(24, 29, 35), indicating an important role for
store-operated Ca2+ channels in the mechanism of the
response. Because Ca2+ signaling is a critical determinant
of endothelial barrier dysfunction, in the present study, we addressed
the effects of TNF-
in regulating the thrombin-activated
Ca2+ influx and how this influences the endothelial
permeability response. We show that TNF-
dramatically augments
thrombin-induced Ca2+ influx in endothelial cells and that
this response is sensitive to Src tyrosine kinase
inhibition. Moreover, the increased Ca2+ influx induces
protein tyrosine phosphorylation of adherens junctional proteins and
increased actin stress fiber formation as well as augmenting the
endothelial permeability response to thrombin. These results show that
TNF-
activates Src tyrosine kinase and thereby induces
Ca2+ influx in response to thrombin, resulting in the
severe loss of endothelial barrier integrity.
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METHODS |
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Materials.
Human -thrombin was obtained from Enzyme Research
Laboratories (South Bend, IN). Endothelial growth medium (EBM-2) was
obtained from Clonetics (San Diego, CA). Dulbecco's modified Eagle's
medium (DMEM), Hanks' balanced salt solution (HBSS),
L-glutamine, phosphate-buffered saline (PBS), and trypsin
were obtained from Life Technologies (Grand Island, NY). Fetal bovine
serum (FBS) was obtained from HyClone Laboratories (Logan, UT). Fura
2-AM was purchased from Molecular Probes (Eugene, OR). Anti-c-Src
polyclonal antibody (Ab), anti-v-Src monoclonal antibody (MAb), and
protein A/G plus agarose were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). The Src tyrosine kinase-specific inhibitor
4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was purchased from Calbiochem (La Jolla, CA). An
anti-phosphotyrosine MAb was from PharMingen (San Diego, CA). An
Src kinase assay kit was obtained from Upstate Biotechnology
(Lake Placid, NY). [
-32P]ATP was purchased from
Amersham Pharmacia Biotech (Piscataway, NJ).
Endothelial cell culture. Human umbilical vein endothelial cells (HUVECs) obtained from Vec Technologies (Rensselaer, NY) were grown in EBM-2 supplemented with 10% FBS. Cells were cultured on tissue culture dishes coated with 0.1% gelatin and used between passages 4 and 8.
[Ca2+]i. The thrombin-induced increase in [Ca2+]i was measured with the Ca2+-sensitive fluorescent dye fura 2 (38). Cells were grown to confluence on 0.1% gelatin-coated 22-mm glass coverslips and then washed two times with serum-free medium and incubated for 2 h at 37°C in culture medium containing 1% FBS. They were washed two times with HBSS and then loaded with 3 µM fura 2-AM for 30 min at 37°C. After the loading, the cells were washed two times with HBSS. The cells were imaged with an Attoflor Ratio Vision digital fluorescence microscopy system (Atto Instruments, Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluar ×40, 1.3 numerical aperture oil-immersion objective. Regions of interest in individual cells were marked and excited at 334 and 380 nm, with emission at 520 nm, at 5-s intervals. At the end of each experiment, 10 µM ionomycin was added to obtain the fluorescence of Ca2+-saturated fura 2 (high [Ca 2+]i) and 10 mM EGTA was added to obtain the fluorescence of free fura 2 (low [Ca2+]i). [Ca2+]i was calculated based on a dissociation constant of 225 nM with a two-point fit curve.
Ins(1,4,5)P3 generation.
Ins(1,4,5)P3 generation in response
to thrombin was measured by using a Biotrak
D-myo-Ins(1,4,5)P3
3H assay kit from Amersham Pharmacia Biotech. Endothelial
cells grown to confluence on 100-mm culture dishes were washed two
times with medium containing 0.1% FBS and incubated for 2 h at
37°C in similar medium. After incubation, the cells were incubated with and without TNF- (250 U/ml) for 2 h and then stimulated with thrombin (5 U/ml) for 15 and 30 s. The reaction was stopped by the addition of ice-cold 15% (vol/vol) trichloroacetic acid. The
endothelial cells were scraped off the dishes, after which the cells
were centrifuged for 15 min at 2,000 g. The supernatant was
washed three times with 10 volumes of water-saturated diethyl ether and
neutralized by titration to pH 7.5 with NaHCO3. Total Ins(1,4,5)P3 generation was
determined following the protocol described by Amersham Pharmacia Biotech.
Immunoprecipitation and immunoblotting.
HUVECs grown in 100-mm culture dishes were incubated with medium
containing 1% FBS for 2 h at 37°C and exposed to TNF- for 2 h before stimulation with thrombin. The cells were lysed with lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1%
Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1 µg/ml of
pepstatin) at 4°C for 30 min. The lysates were collected and
centrifuged at 14,000 rpm at 4°C for 30 min. The clear supernatants
were precleared with protein A/G agarose beads for 60 min at 4°C. The
precleared supernatants were incubated with rabbit polyclonal
anti-c-Src Ab at 4°C overnight with constant shaking. After
incubation, protein A/G agarose beads were added, incubated for an
additional hour at 4°C with constant shaking, and centrifuged at
14,000 rpm for 5 min. Beads were washed three times with ice-cold PBS
containing 1 mM sodium orthovanadate and finally dissociated by boiling
in SDS sample buffer for 5 min. The proteins were resolved by SDS-PAGE
and transferred to Duralose membranes (Stratagene). The membranes were
blocked with 5% nonfat dry milk in 10 mM Tris · HCl, pH 7.7, 150 mM NaCl, and 0.05% Tween 20 for 1 h at 22°C. The membranes
were incubated with the indicated primary Abs (diluted in blocking
buffer) at 22°C for 1 h. After three washes with wash buffer,
the membrane was incubated with horseradish peroxidase-conjugated
goat anti-rabbit or anti-mouse Ab for 1 h. The protein bands were
detected by enhanced chemiluminescence (Pierce Chemical,
Rockford, IL).
Src kinase activity.
Src activity was measured with the in vitro kinase assay kit
from Upstate Biotechnology. Src was immunoprecipitated
as described in Immunoprecipitation and
immunoblotting, and the immunoprecipitate was washed three
times with ice-cold PBS and suspended in kinase assay buffer (50 mM
HEPES, pH 7.5, containing 0.1 mM EDTA and 0.015% polyoxyethylene
glycol dodecyl ether). The final reaction volume (60 µl) contained 50 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.015% polyoxyethylene glycol dodecyl
ether, 15 mM MgCl2, 1 mM Na3VO4, 150 µM ATP, and 33 µCi of [-32P]ATP with and
without 150 µM peptide substrate (KVEKIGEGTYGVVYK). The reaction was
continued for 10 min at 30°C and terminated by adding 1% phosphoric
acid. The samples were spotted on P81 filter paper and washed five
times with 1% phosphoric acid and one time with methanol, and then the
radioactivity associated with the filter was determined in a
scintillation counter.
Phosphotyrosine immunostaining.
HUVECs were grown to confluence on glass coverslips coated with 0.1%
gelatin. The cells were washed three times with serum-free medium and
kept for 2 h at 37°C in medium containing 1% FBS. Then the
cells were primed with TNF- (250 U/ml) in medium containing 1% FBS
for 2 h at 37°C and then stimulated with thrombin. The cells
were washed three times with ice-cold HBSS and fixed with 2%
paraformaldehyde for 15 min at 22°C. The cells were then blocked with
1% bovine serum albumin in HBSS at 22°C for 60 min and then incubated with the anti-phosphotyrosine MAb (5 µg/ml) for 60 min at
22°C. The cells were washed two times with HBSS and incubated with
rhodamine-labeled goat anti-mouse IgG (5 µg/ml) for 45 min at 22°C.
After three washes with HBSS, the coverslips were mounted on glass
slides with the ProLong antifade mounting medium kit (Molecular
Probes). Total cell fluorescence was visualized with digital-imaging
fluorescence microscopy as previously described (4).
Distribution of actin stress fibers. HUVECs were grown to confluence on gelatin-coated glass coverslips. The cells were washed three times with HBSS and fixed with 4% paraformaldehyde for 20 min at 22°C and then permeabilized with 0.1% Triton X-100 for 30 min at 22°C. The cells were stained with Alexa 568-phalloidin for 30 min at 22°C, washed two times with HBSS, mounted with ProLong antifade mounting medium, and viewed under a digital fluorescence microscope (Diapshot 200, Nikon Instruments, Fair Lawn, NJ).
Transendothelial cell electrical resistance.
The thrombin-induced endothelial cell contractile response was measured
as previously described (37). HUVECs were seeded on a
gelatin-coated gold electrode (5.0 × 104
cm2) and grown to confluence. The small electrode and the
larger counterelectrode were connected to a phase-sensitive lock-in
amplifier. An approximate constant current of 1 µA was supplied by a
1-V, 4,000-Hz AC signal connected serially to a 1-M
resistor between the small electrode and the larger counterelectrode. The voltage between the small electrode and the large electrode was monitored by
lock-in amplifier, stored, and processed with a personal computer. The
same computer controlled the output of the amplifier and switched the
measurement to different electrodes in the course of an experiment. Before the experiment, the confluent endothelial monolayer was kept in
medium containing 1% FBS for 2 h. The cells were incubated with
and without TNF-
for 2 h, and then the thrombin-induced change
in resistance of the endothelial monolayer was measured. In some
experiments, the TNF-
-induced change in resistance was measured
directly. The data are presented as resistance normalized to its value
at time 0 as previously described (3, 4, 37).
Transendothelial 125I-albumin
permeability.
Permeability of 125I-albumin across the HUVEC monolayer was
determined with Costar Transwell units as previously described
(34, 35). This system measures transendothelial flux
(luminal to abluminal) of tracer macromolecules in the absence of
hydrostatic and oncotic pressure gradients. The system consists of
luminal and abluminal compartments separated by a polycarbonate filter (0.4-µm pore size, 6.5-mm diameter). The luminal (upper) side of
filters was coated with gelatin. HUVECs were seeded at 105
cells/filter and grown for 4-5 days to attain confluence. The cells were washed and incubated with medium containing 1% FBS for
2 h before TNF- exposure for 2 h at 37°C. HUVEC
monolayers were then transferred to 10 mM HEPES-DMEM, pH 7.4, containing 5 mg/ml of bovine serum albumin. Both the luminal and
abluminal compartments contained the same medium at volumes of 0.2 and
1.0 ml, respectively. Thrombin (5 U/ml) was added to the upper chamber containing tracer 125I-albumin (5 × 106
counts · min
1 · ml
1). After
the addition of thrombin, 0.05-ml samples from the lower compartment
were collected at 10-min intervals for 90 min for determination of the
transendothelial clearance rate of 125I-albumin as
previously described (34, 35).
Statistical analysis. Statistical comparisons were made with two-tailed Student's t-test. Values are reported as means ± SE. Differences in mean values between and among groups were measured by one-way analysis of variance with Bonferroni correction. Values were considered significant at P < 0.05.
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RESULTS |
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TNF- augments the thrombin-induced increase in
[Ca2+]i.
We exposed HUVECs to varying concentrations of TNF-
(0.5-3
ng/ml, i.e., 50-300 U/ml) for 2 h and measured the
transendothelial monolayer electrical resistance to assess endothelial
monolayer integrity. We observed no significant changes in
transendothelial monolayer electrical resistance compared with that in
the control HUVEC monolayer. To study whether TNF-
exposure produces
a synergistic effect on thrombin-activated responses in HUVECs, we
first measured the changes in [Ca2+]i. We
exposed TNF-
(250 U/ml) for 30 and 60 min and 2 h and then
measured the thrombin-induced increase in
[Ca2+]i. In control cells, thrombin increased
the [Ca2+]i (peak value of 1,070 ± 75 nM) followed by a gradual decline to the basal level (67 ± 5 nM)
within 3 min after thrombin challenge (Fig.
1A, Table
1). TNF-
exposure up to 60 min had
no significant effect on the thrombin-induced increase in
[Ca2+]i (Table 1). Also, TNF-
alone had no significant effect on [Ca2+]i
(Fig. 1B, inset). However, in cells pretreated
with TNF-
for 2 h, thrombin produced an initial peak increase
in the [Ca2+]i (peak value of 1,085 ± 78 nM; Fig. 1B, Table 1), but the value remained elevated
(380 ± 25 nM 3 min after thrombin addition) and reached the basal
level ~10 min after thrombin challenge (Fig. 1B). Thus
TNF-
exposure for 2 h markedly amplified the thrombin-induced increase in [Ca2+]i in HUVECs. Further
experiments were carried out exposing HUVECs with TNF-
for 2 h to
study its synergistic effects with thrombin.
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TNF- has no effect on thrombin-induced
Ins(1,4,5)P3 generation.
To address whether the effects of TNF-
on thrombin-induced increase
in [Ca2+]i are the result of differences in
the production of Ins(1,4,5)P3, we
measured thrombin-induced
Ins(1,4,5)P3 generation in control and TNF-
-exposed cells. Basal
Ins(1,4,5)P3 levels were not
significantly changed in these two groups (Table
2). Moreover, thrombin exposure produced
similar increases in Ins(1,4,5)P3
concentration in both control and TNF-
-pretreated cells (Table 2),
indicating that the increased
Ins(1,4,5)P3 production does not
account for the greater rise in [Ca2+]i.
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TNF- amplifies thrombin-induced
Ca2+ influx.
Because thrombin-induced
Ins(1,4,5)P3 generation was not
affected by TNF-
exposure, we measured the thrombin response in the
absence of extracellular Ca2+ to assess Ca2+
influx. In the absence of extracellular Ca2+, thrombin
produced an initial peak increase in [Ca2+]i
that rapidly returned to the baseline value in control cells (Fig.
2A, Table 2). The addition of
Ca2+ to the extracellular medium after thrombin-induced
Ca2+ store depletion caused Ca2+ entry; the
value peaked at 615 ± 55 nM and returned to the basal level as in
control cells (Fig. 2A, Table 2). In TNF-
-primed cells in
the absence of extracellular Ca2+, thrombin produced an
initial peak increase in [Ca2+]i similar to
that in control cells; however, the addition of Ca2+ to the
extracellular medium after store depletion produced a significantly
greater increase in the [Ca2+]i (peak value
of 948 ± 70 nM; Fig. 2B, Table
3), and the elevated [Ca2+]i persisted for a longer period
compared with the control value (Fig. 2, A vs.
B). The addition of Ca2+ in the extracellular
medium in control or TNF-
-exposed cells alone did not induce
Ca2+ influx (data not shown). These results suggest that
thrombin-induced intracellular store depletion induces Ca2+
entry in HUVECs and, moreover, that TNF-
exposure augmented the
Ca2+ influx in response to thrombin.
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TNF- increases Src activity in
endothelial cells.
Because the Src tyrosine kinase-specific inhibitor prevented
the store depletion-mediated Ca2+ entry in both control and
TNF-
-exposed cells, we measured the autophosphorylation of
Src to assess Src activation. The cell lysates
were immunoprecipitated with anti-Src Ab and blotted with anti-phosphotyrosine MAb (see METHODS). TNF-
or thrombin
treatment alone increased tyrosine phosphorylation of Src by
approximately twofold (Fig.
3A). The addition of thrombin
to TNF-
-exposed cells further increased tyrosine phosphorylation of
Src (Fig. 3A). Because tyrosine phosphorylation
of Src can either activate (Tyr416
phosphorylation) or inhibit (Tyr527 phosphorylation)
Src activity (12), we measured Src
activity with a peptide substrate (see METHODS). HUVECs
were incubated with TNF-
(250 U/ml) for 2 h and lysed, and the
cell lysates were immunoprecipitated with anti-Src Ab. The
immunoprecipitated proteins were used for the in vitro Src
kinase assay. Thrombin increased the phosphorylation of the
peptide substrate by approximately twofold over the basal value (Fig.
3B). TNF-
treatment alone increased the phosphorylation
of the peptide substrate by approximately twofold over the control
value (Fig. 3B). The addition of thrombin to TNF-
-exposed
cells further increased phosphorylation of the peptide substrate (Fig.
3B).
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TNF- increases thrombin-induced tyrosine
phosphorylation of adherens junction proteins.
We next determined the thrombin-induced tyrosine phosphorylation
of adherens junction proteins in control and TNF-
-exposed cells.
Thrombin markedly increased tyrosine phosphorylation of junction
proteins along with the formation of intercellular gaps in control
cells (Fig. 4, A vs.
C). TNF-
alone slightly increased tyrosine
phosphorylation of junctional proteins but failed to induce gap
formation (Fig. 4E). Stimulation of TNF-
-exposed cells with thrombin produced a greater increase in tyrosine phosphorylation of junctional proteins than thrombin alone and greater interendothelial gap formation over thrombin alone (Fig. 4, G vs.
C). The Src tyrosine kinase inhibitor PP1
prevented thrombin-induced tyrosine phosphorylation in both control and
TNF-
-primed cells (Fig. 4, B, D, F,
and H). Furthermore, PP1 treatment significantly reduced the
size of the interendothelial gaps in response to thrombin (Fig. 4,
B, D, F, and H).
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TNF- augments thrombin-induced stress fiber
formation.
Because TNF-
priming enhanced Ca2+ entry in response to
thrombin, we measured actin stress fiber formation in control and
TNF-
-treated cells. In control as well as in TNF-
(250 U/ml for
2 h)-exposed cells, only peripheral actin bands were observed
(Fig. 5). In control cells, thrombin
increased the actin stress fiber formation within 5 min, and actin
filamentous changes returned to normal within 2 h after thrombin
challenge (Fig. 5, top). In TNF-
-exposed cells, thrombin
induced a marked increase in stress fiber formation (Fig. 5,
bottom) and prolonged the recovery time compared with control cells (Fig. 5, bottom vs. top).
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TNF- augments thrombin-induced transendothelial
resistance and transendothelial albumin permeability.
We measured the thrombin-induced changes in transendothelial monolayer
electrical resistance and transendothelial albumin clearance. Exposure
of HUVECs to TNF-
(250 U/ml) alone for 2 h had no significant
effect on transendothelial monolayer resistance (Fig.
6A). Thrombin produced an
~40% maximum decrease in transendothelial monolayer electrical
resistance and resistance returned to normal within 2 h after
thrombin challenge (Fig. 6). In TNF-
-primed cells, thrombin produced
an ~40% maximum decrease in transendothelial monolayer electrical
resistance, but the monolayer resistance recovery to the basal level
was markedly delayed (Fig. 6). The Src kinase inhibitor PP1
prevented the delayed recovery of endothelial monolayer resistance in
TNF-
-primed cells (Fig. 6). PP1 had no significant effect on the
initial drop in resistance (i.e., 30 min after thrombin challenge)
induced by thrombin (Fig. 6) in either control or TNF-
-primed
HUVECs. These results indicate that Src activation-mediated
Ca2+ influx prolonged the thrombin-induced endothelial
contractile response, consistent with the greater actin stress fiber
formation.
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DISCUSSION |
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Thrombin is a procoagulant as well as a potent proinflammatory
mediator that increases vascular endothelial permeability (10, 15, 20, 25, 31, 34, 35). Thrombin by binding to its receptor,
proteinase-activated receptor-1, in endothelial cells increases
[Ca2+]i. This is the result of the generation
of Ins(1,4,5)P3 and the release of
intracellular Ca2+ as well as of the influx of
Ca2+ (18, 34, 35, 38). Recent studies
(14, 21, 30) indicate that agonist-dependent intracellular
store Ca2+ depletion is directly coupled to
Ca2+ influx to rapidly refill the intracellular
Ca2+ stores. Ca2+ influx secondary to
intracellular store depletion (i.e., "capacitative Ca2+
entry") is mediated by activation of store-operated Ca2+
channels localized in the plasma membrane (14). The rise
in intracellular Ca2+ signaling induced by thrombin is
critically involved in the mechanism of the increase in endothelial
permeability (18, 34, 35, 38). Sandoval et al.
(35) and others (24, 29) have shown that thrombin-induced Ca2+ influx is, in fact, an essential
requirement for the increase in endothelial permeability. In the
present study, we addressed the possibility that another
proinflammatory mediator, TNF-, generated during sepsis (6,
17, 39), may synergistically affect the thrombin-induced
increase in endothelial permeability by pathways that activate
Ca2+ entry. To address the actions of TNF-
and thrombin,
we exposed endothelial cells to TNF-
for defined periods and studied
the thrombin-induced alterations in Ca2+ signaling and
endothelial barrier function.
We observed that thrombin challenge of endothelial cells in the
presence of nominal extracellular medium Ca2+ (1.26 mM)
produced a characteristic initial peak increase in the
[Ca2+]i followed by a decline to basal
levels. However, in endothelial cells exposed to TNF-, thrombin
produced a similar initial peak increase in
[Ca2+]i, but surprisingly, the concentration
remained elevated for up to 10 min. TNF-
alone had no effect on the
endothelial [Ca2+]i. Because the
thrombin-induced Ca2+ influx is functionally coupled to
intracellular store Ca2+depletion, we also measured
thrombin-induced Ins(1,4,5)P3
generation in control and TNF-
-primed cells.
Ins(1,4,5)P3 generation activated by thrombin was not different in these cells, suggesting that the
sustained increase in [Ca2+]i due to a
TNF-
"priming effect" was not the result of
Ins(1,4,5)P3-mediated intracellular
store depletion. Because
Ins(1,4,5)P3 generation occurs
secondary to the activation of thrombin receptors (18, 35,
38), the finding of similar thrombin-induced
Ins(1,4,5)P3 generation after
TNF-
also suggests that the differential Ca2+ signaling
response cannot be explained by increased thrombin receptor activation
in the TNF-
-primed endothelial cells.
Because Src tyrosine kinase activation has been implicated
in the mechanism of Ca2+ influx after activation of G
protein-coupled receptors (2), we assessed this
possibility using the Src inhibitor PP1. In the absence of
Ca2+ in the extracellular medium, thrombin produced an
initial peak increase in [Ca2+]i that was not
different between control and TNF--primed cells. The addition of
extracellular Ca2+ after store depletion induced by
thrombin, however, produced a significantly greater rise in the
increase in [Ca2+]i in the TNF-
-exposed
endothelial cells. Importantly, this increase was prevented by PP1,
suggesting that the thrombin-induced Ca2+ influx is
regulated by Src activation. We also showed that TNF-
exposure of endothelial cells amplified Src kinase activity
after thrombin challenge; thus the increased Ca2+ influx in
response to thrombin in the TNF-
-primed cells may reflect the
Src activation.
The mechanism by which Src can regulate Ca2+
influx occurring secondary to store depletion is unclear. One
possibility is that Src phosphorylates plasma membrane
transient receptor potential (Trp) channels expressed in endothelial
cells (9, 23), which mediate the Ca2+ influx
during Ins(1,4,5)P3-sensitive
intracellular store depletion (21, 30). Although
Src was activated in TNF--exposed cells, there was no
change in [Ca2+]i without thrombin
stimulation, suggesting that the greater Ca2+ influx
mediated by thrombin in TNF-
-primed cells is coupled to
intracellular store depletion. These results are consistent with
Src-induced activation of Trp-like channels and thus could account for the increased Ca2+ influx observed in the
TNF-
-exposed endothelial cells. The mechanism may be analogous to
the Src-dependent tyrosine phosphorylation of
Ca2+-sensitive K+ channels, resulting in
increased channel activity (16).
To address the functional significance of the greatly amplified
Ca2+ influx after TNF- exposure of endothelial cells, we
assessed tyrosine phosphorylation of adherens junctional proteins
implicated in the mechanism of increased endothelial permeability
(8, 36, 40). Thrombin increased the tyrosine
phosphorylation of junctional proteins and the formation of
interendothelial gaps that are characteristically associated with the
loss of barrier function (10, 15, 20, 25, 31, 37). In the
present study, we showed that the addition of thrombin to
TNF-
-primed cells markedly augmented both tyrosine phosphorylation
and gap formation. Moreover, PP1 prevented these alterations. Because PP1 treatment had no effect on the initial peak increase in
[Ca2+]i resulting from intracellular store
depletion, whereas it inhibited the Ca2+ influx component,
the results suggest that the tyrosine phosphorylation of adherens
junction proteins is dependent on the augmented Ca2+ influx.
We measured the transendothelial monolayer electrical resistance [an
assessment of endothelial cell shape change induced by Ca2+
signaling (37)] and transendothelial albumin
permeability, a direct measure of endothelial barrier function
(20, 34, 35, 41). Ca2+ signaling is critical
in the mechanism of thrombin-induced myosin light chain phosphorylation
and subsequent actomysin cross bridging (which induces actin stress
fiber formation) (11, 34, 35, 41). In control cells,
thrombin markedly increased stress fibers within 5 min, and the cells
recovered within 2 h after thrombin exposure. TNF- alone failed
to increase stress fibers; however, thrombin-induced actin stress fiber
formation was markedly enhanced after TNF-
exposure, and the cells
failed to recover even 2 h after thrombin challenge. Thus the
prolonged intracellular Ca2+ elevation may account for this
atypical alteration of stress fiber formation in the TNF-
-primed
cells. The changes in transendothelial electrical resistance paralleled
the actin stress fiber formation. The addition of thrombin to control
monolayers produced a decrease in endothelial monolayer resistance, and
the value returned to the normal range within 2 h after the
addition of thrombin. However, in TNF-
-primed cells, thrombin
produced a similar maximum decrease in resistance, but the endothelial
cell monolayer resistance recovery time was significantly prolonged.
Moreover, the thrombin-induced increase in transendothelial
125I-albumin clearance in TNF-
-primed cells was also
greater. PP1 prevented these responses, demonstrating the importance of
Src in mediating the loss of endothelial barrier function
after TNF-
priming. These results suggest that the Src
activation-dependent Ca2+ influx is an important factor
signaling endothelial barrier dysfunction.
The mechanism of Src activation in TNF--primed cells in
the absence of thrombin is unclear. Src may be activated by
reactive oxygen species (ROS) (1, 28). Moreover, ROS
production in response to TNF-
exposure occurs in endothelial cells
(32, 33). In the present study, we observed that
Src activation occurred in HUVECs 2 h after TNF-
exposure. This time frame is consistent with the
H2O2-induced Src activation in
cardiac myocytes (28). Thus it is possible that
TNF-
-mediated ROS generation (1, 28, 32, 33) in
endothelial cells induces Src activation, and this, in turn,
is responsible for augmentation of Ca2+ influx on challenge
with thrombin.
In summary, we studied the synergistic effects of TNF- on the
thrombin-induced increase in [Ca2+]i and
endothelial barrier function. Exposure of endothelial cells to TNF-
increased the activation of Src tyrosine kinase and markedly increased thrombin-induced Ca2+ influx. These results
suggest that the generation of thrombin and TNF-
during sepsis can
synergistically activate Ca2+ influx to cause endothelial
barrier dysfunction. Thus drugs designed to interfere with
Src-activated Ca2+ influx in endothelial cells
may be useful in treating inflammation and tissue injury associated
with sepsis.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grant GM-58531 and National Heart, Lung, and Blood Institute Grants HL-45638, P01-HL-60678, and T32-HL-07829.
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FOOTNOTES |
---|
* Chinnaswamy Tiruppathi and Tabassum Naqvi contributed equally to this work.
Address for reprint requests and other correspondence: C. Tiruppathi, Dept. of Pharmacology (M/C 868), College of Medicine, The Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612 (E-mail: tiruc{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 March 2001; accepted in final form 4 June 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aikawa, R,
Komuro I,
Yamazaki T,
Zou Y,
Kudoh S,
Tanaka M,
Shiojima I,
Hiroi Y,
and
Yazaki Y.
Oxidative stress activated extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats.
J Clin Invest
100:
1813-1821,
1997
2.
Babningg, G,
Bowersox SR,
and
Villereal ML.
The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels.
J Biol Chem
272:
29434-29437,
1997
3.
Ellis, CA,
Malik AB,
Gilchrist A,
Hamm H,
Sandoval R,
Voyno-Yasenetskaya T,
and
Tiruppathi C.
Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked ras/mitogen-activated protein kinase pathway.
J Biol Chem
274:
13718-13727,
1999
4.
Ellis, CA,
Tiruppathi C,
Sandoval R,
Niles WD,
and
Malik AB.
Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression.
Am J Physiol Cell Physiol
276:
C38-C45,
1999
5.
Esmon, CT.
The roles of protein C and thrombomodulin in the regulation of blood coagulation.
J Biol Chem
264:
4743-4746,
1989
6.
Esmon, CT,
Fukudome K,
Mather T,
Bode W,
Regan LM,
Stearns-Kurosawa DJ,
and
Kurosawa S.
Inflammation, sepsis, and coagulation.
Haematologica
84:
254-259,
1999[ISI][Medline].
7.
Fleming, I,
Fisslthaler B,
and
Busse R.
Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases.
Circ Res
76:
522-529,
1995
8.
Fleming, I,
Fisslthaler B,
and
Busse R.
Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells.
J Biol Chem
271:
11009-11015,
1996
9.
Freichel, M,
Suh SH,
Pfeifer A,
Schweig U,
Trost C,
Weibgerber P,
Biel M,
Philip S,
Freise D,
Droogmans G,
Hofmann F,
Flockerzi V,
and
Nilius B.
Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.
Nat Cell Biol
3:
121-127,
2001[ISI][Medline].
10.
Garcia, JG,
Davis HW,
and
Patterson CE.
Regulation of endothelial gap formation and barrier dysfunction: role of myosin light chain phosphorylation.
J Cell Physiol
163:
510-522,
1995[ISI][Medline].
11.
Goeckeler, ZM,
and
Wysolmerski RB.
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].
12.
Gonfloni, S,
Weijland A,
Kretzschmar J,
and
Superti-Furga G.
Crosstalk between the catalytic and regulatory domains allows bi-directional regulation of Src.
Nat Struct Biol
7:
281-286,
2000[ISI][Medline].
13.
Griffin, JH,
Mosher DF,
Zimmerman TS,
and
Kleiss AJ.
Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation.
Blood
60:
261-264,
1982[Abstract].
14.
Holda, JR,
Klishin A,
Sedova M,
Huser J,
and
Blatter LA.
Capacitative calcium entry.
News Physiol Sci
13:
157-163,
1998
15.
Laposata, M,
Dovnarsky DK,
and
Shin HS.
Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro.
Blood
62:
549-556,
1983[Abstract].
16.
Ling, S,
Woronuk G,
Sy L,
Lev S,
and
Braun AP.
Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase.
J Biol Chem
275:
30683-30689,
2000
17.
Lo, SK,
Everitt J,
Gu J,
and
Malik AB.
Tumor necrosis factor mediates experimental pulmonary edema by ICAM-1 and CD18-dependent mechanisms.
J Clin Invest
89:
981-988,
1992[ISI][Medline].
18.
Lum, H,
Aschner JL,
Phillips PG,
Fletcher PW,
and
Malik AB.
Time course of thrombin-induced increase in endothelial permeability: relationship to [Ca2+]i and inositol polyphosphates.
Am J Physiol Lung Cell Mol Physiol
263:
L219-L225,
1992
19.
Lum, H,
Del Vecchio PJ,
Schneider AS,
Goligorsky MS,
and
Malik AB.
Calcium dependence of the thrombin-induced increase in endothelial albumin permeability.
J Appl Physiol
66:
1471-1476,
1989
20.
Lum, H,
and
Malik AB.
Regulation of vascular endothelial barrier function.
Am J Physiol Lung Cell Mol Physiol
267:
L223-L241,
1994
21.
Ma, HT,
Patterson RL,
van Rossum DB,
Birnbaumer L,
Mikoshiba K,
and
Gill DL.
Requirement of inositol trisphosphate receptor for activation of store-operated Ca2+channels.
Science
287:
1647-1651,
2000
22.
Moore, KL,
Andreoli SP,
Esmon NL,
Esmon CT,
and
Bang NU.
Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro.
J Clin Invest
79:
124-130,
1987[ISI][Medline].
23.
Moore, TM,
Brough GH,
Babal P,
Kelly JJ,
Li M,
and
Stevens T.
Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.
Am J Physiol Lung Cell Mol Physiol
275:
L574-L582,
1998
24.
Moore, TM,
Norwood NR,
Creighton JR,
Babal P,
Brough GH,
Shasby DM,
and
Stevens T.
Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability.
Am J Physiol Lung Cell Mol Physiol
279:
L691-L698,
2000
25.
Moy, AB,
Van Engelenhoven J,
Bodmer J,
Kamath J,
Keese C,
Giaever I,
Shasby S,
and
Shasby DM.
Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.
J Clin Invest
97:
1020-1027,
1996
26.
Nawroth, PP,
Handley DA,
Esmon CT,
and
Stern DM.
Interleukin-1 induces endothelial cell procoagulant while suppressing cell surface anticoagulant activity.
Proc Natl Acad Sci USA
83:
3460-3464,
1986[Abstract].
27.
Nawroth, PP,
and
Stern DM.
Modulation of endothelial cell hemostatic properties by tumor necrosis factor.
J Exp Med
163:
740-745,
1986[Abstract].
28.
Nishida, M,
Maruyama Y,
Tanaka R,
Kontani K,
Nagao T,
and
Kurose H.
Gi and G
o are target proteins of reactive oxygen species.
Nature
408:
492-495,
2000[ISI][Medline].
29.
Norwood, N,
Moore TM,
Dean DA,
Bhattacharjee R,
Li M,
and
Stevens T.
Store-operated calcium entry and increased endothelial cell permeability.
Am J Physiol Lung Cell Mol Physiol
279:
L818-L824,
2000.
30.
Putney, JW.
TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry.
Proc Natl Acad Sci USA
96:
14669-14671,
1999
31.
Rabiet, MJ,
Plantier JL,
Rival Y,
Genoux Y,
Lampugnani MG,
and
Dejana E.
Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization.
Arterioscler Thromb Vasc Biol
16:
488-496,
1996
32.
Rahman, A,
Bando M,
Kefer J,
Anwar KN,
and
Malik AB.
Protein kinase C-activated oxidant generation in endothelial cells signals intracellular adhesion molecule-1 gene transcription.
Mol Pharmacol
55:
575-583,
1999
33.
Rahman, A,
Kefer J,
Bando M,
Niles WD,
and
Malik AB.
E-selectin expression in human endothelial cells by TNF--induced oxidant generation and NF-
B activation.
Am J Physiol Lung Cell Mol Physiol
275:
L533-L544,
1998
34.
Sandoval, R,
Malik AB,
Minshall RD,
Kouklis P,
Ellis CA,
and
Tiruppathi C.
Ca2+ signaling and PKC activate increased endothelial permeability by disassembly of VE-cadherin junctions.
J Physiol (Lond)
533:
433-445,
2001
35.
Sandoval, R,
Malik AB.,
Naqvi T,
Mehta D,
and
Tiruppathi C.
Requirement of Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability.
Am J Physiol Lung Cell Mol Physiol
280:
L239-L247,
2001
36.
Schaphorst, KL,
Pavalko FM,
Patterson CE,
and
Garcia JGN
Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation.
Am J Respir Cell Mol Biol
17:
443-455,
1997
37.
Tiruppathi, C,
Malik AB,
Del Vecchio PJ,
Keese CR,
and
Giaever I.
Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.
Proc Natl Acad Sci USA
89:
7919-7923,
1992[Abstract].
38.
Tiruppathi, C,
Yan W,
Sandoval R,
Naqvi T,
Pronin AN,
Benovic JL,
and
Malik AB.
G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells.
Proc Natl Acad Sci USA
97:
7440-7445,
2000
39.
Tracey, KJ,
Beutler B,
Lowery SF,
Merryweather J,
Wolpe S,
Milsark IW,
Hariri RJ,
Fahey TJ, III,
Zentella A,
Albert JD,
Shires GT,
and
Cerami A.
Shock and tissue injury induced by recombinant human cachectin.
Science
234:
470-474,
1986[ISI][Medline].
40.
Ukropec, JA,
Hollinger MK,
Salva SM,
and
Woolkalis MJ.
SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin.
J Biol Chem
275:
5983-5986,
2000
41.
Wysolmerski, RB,
and
Lagunoff D.
Involvement of myosin light chain kinase in endothelial cell retraction.
Proc Natl Acad Sci USA
87:
16-20,
1990[Abstract].