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INTRODUCTION |
A critical mediator of several inflammatory and ischemic
conditions (1), tumor necrosis factor
(TNF
)1 promotes leukocyte
adhesion to vascular endothelium both in vitro and in
vivo; such adhesion is mediated by the complex interplay of
adhesion receptors on both leukocytes and endothelial cells. TNF
induces the transcription of the leukocyte adhesion molecules ICAM,
VCAM, and E-selectin by activating the transcription factor NF
B.
Multiple vascular cell types, including macrophages, cardiac myocytes,
mast cells, and neutrophils release TNF
during ischemia and
inflammation (1-3). In addition to its proinflammatory effect, TNF
can cause cell death by apoptosis and promote cell survival (4). These
diverse effects can result in apparently contradictory experimental
findings, as experimental data support both deleterious and beneficial
roles for TNF
in, for example, preservation of myocardial function
following ischemia and reperfusion (5-8).
The complexity of the physiologic effects of TNF
is mimicked by the
mechanisms by which it generates signals to produce those effects.
TNF
interacts with two cell surface receptors, TNFR1 and TNFR2,
ubiquitously expressed on cells of the cardiovascular system. TNFR1 is
dominant in most cell types; an independent role for TNFR2 is yet to be
established (4). After ligand binding, a conformational change in the
pretrimerized TNF receptor complex causes dissociation of the SODD
protein and, in a series of protein-protein interactions, intracellular
mediators are recruited to the cytosolic portion of the receptor (9).
TNFR1 activates two broad signaling pathways, both initiated by the
recruitment of TRADD to TNFR1. One pathway, mediated by the subsequent
recruitment of FADD, results in caspase 8 activation and initiation of
apoptosis. Conversely, the survival pathway involves association of RIP
and TRAF-2 with TRADD. The TRADD·RIP·TRAF-2 complex recruits
the IKK signalosome to TNFR1 thus activating NF
B. In addition, the
TRADD·RIP·TRAF-2 complex also activates MAPK pathways that lead to
AP-1 activation (4, 10).
During inflammation, multiple cytokines and stimulants of vascular
endothelium are released. Thus, any pathophysiological effect produced
by TNF
must take place in the context of "cross-talk" with those
mediators. The most common class of receptor for these mediators are G
protein-linked receptors, multiple forms of which are expressed on
vascular cells. One mediator of particular importance in ischemia and
inflammation is the eicosanoid thromboxane A2 (TXA2), released from activated platelets and damaged
vessel walls. Local and systemic elevations in TXA2 are
reported in several thrombotic and vascular diseases (11).
TXA2 mediates vascular damage by inducing platelet
activation, vasoconstriction, vascular smooth muscle
hypertrophy/hyperplasia, and by converting the endothelial surface to a
"prothrombotic" state. These biological actions of TXA2
are mediated by a G protein-linked receptor (TP) of which two
alternatively spliced subtypes, TP
and TP
, exist in humans (12,
13). Although the intracellular tail of TP
differs from that of
TP
, these isoforms couple to 80% of the same G proteins (14).
Differences in signaling reported so far mediate receptor trafficking
and desensitization (15-17).
In this study, we sought to investigate whether stimulation of TP, a
prototypical G protein-coupled receptor that is activated during
ischemia and inflammation, could alter TNF
-induced endothelial cell
apoptosis and leukocyte adhesion in vivo. Furthermore, we tested the possibility that such an effect resulted from a selective effect on specific signal transduction pathways employed by TNF
. The
data present a currently unrecognized interaction between TNF
and
ligands for G protein-coupled receptors that may provide a mechanism by
which endothelial cell death occurs during inflammation.
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EXPERIMENTAL PROCEDURES |
Chemicals--
The G protein inhibitors NF023
(G
i), NF449 (G
s), GP antagonist 2A
(G
q), pertussis toxin, and GDP were all purchased from Calbiochem-Novabiochem. All other chemicals were of suitable grade and
purchased from Sigma unless otherwise stated.
Cell-based Receptor Enzyme-linked Immunosorbent Assay and
Fluorescence-activated Cell Sorter--
Human endothelial cells (HEC)
isolated from umbilical veins were cultured as reported previously
(18). HEC were stimulated for up to 24 h with media alone
(Control), recombinant human TNF
or IL-1
(100 units/ml in all
experiments unless otherwise stated), the TXA2 mimetic IBOP
(0-200 nM), or the TP blocker SQ29548 (5 µM)
(Cayman Chemical, Ann Arbor, MI). Expression of ICAM-1, VCAM-1, and
E-selectin was detected by enzyme-linked immunosorbent assay (19) or
flow cytometry (18) using commercially available monoclonal antibodies
(Dako Corp., Carpinteria, CA).
Leukocyte Adhesion Assays--
Confluent monolayers of first
passage HEC were stimulated with TNF
, IBOP, or the two in
combination for 8 h and were washed extensively before the
addition of leukocytes. U937 cells, a leukocyte cell line, were labeled
with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (2 µM) for 30 min and 1 × 106 cells were
added to stimulated HEC monolayers for 2 h. At the conclusion,
cells were washed with phosphate-buffered saline and incubated with
lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.1 mM phenylmethylsulfonyl
fluoride) for 30 min. U937 attachment was quantitated in a fluorometric
plate reader using fluorescein isothiocyanate filters.
Analysis of Leukocyte Adhesion and Extravasation in
Vivo--
The modulation of TNF
by TXA2 in
vivo was analyzed by monitoring the passage of leukocytes along
postcapillary venules in the externalized cremaster muscle as
previously described (20). C57-black mice were injected
intraperitoneally with murine TNF
(150 pM), the
TXA2 mimetic IBOP (50 nM), or the two together
and prepared for intravital microscopy after 4 h. Adherent
leukocytes were quantified per 100 µm of vessel length. Extravasated
leukocytes were determined as the number of interstitial leukocytes
adjacent (within 30 µm) to venules.
Immunoprecipitation and Immunoblotting--
Confluent HEC were
treated with IBOP, IL-1
, or TNF
alone or in combination for 5 min. Monolayers were washed twice in phosphate-buffered saline
containing 1 mM phenylmethylsulfonyl fluoride and scraped into immunoprecipitation lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium
deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml
aprotinin, 1 mg/ml leupeptin). Cell suspensions were sonicated,
incubated on ice for 30 min, and clarified by centrifugation. For
immunoprecipitation, protein content was determined and 300 µg of
total protein was incubated for 16 h at 4 °C with protein
G-agarose beads coated with saturating amounts of antibodies to TNFR1,
TNFR2, NF
B (p65RelA) (Santa Cruz Biotechnology, Santa
Cruz, CA), RIP, or TRADD (Transduction Laboratories). The resulting
immune complexes were recovered after centrifugation by boiling for 10 min in SDS-PAGE loading buffer. Nuclear proteins were isolated as
previously reported (21).
For immunoblotting, aliquots of whole cell lysates (30 µg) or
isolated immune complexes were separated by SDS-PAGE under reducing conditions using 10% acrylamide gels. Proteins were transferred onto
polyvinylidene difluoride membrane and analyzed by immunoblotting as
previously described (18) using antibodies against MADD, STAT1, NIK
(Pharmingen), Ask1, MEKK1, FADD, TRAF-2, I
B, IKK
, and the
phosphorylated forms of p38, ERK1/2, JNK (Santa Cruz), STAT1
(Tyr701) (Upstate Biotechnology), and I
B
(Calbiochem-Novabiochem Corp.) in addition to those described above. An
-tubulin monoclonal antibody was used to control for loading.
Annexin V Staining--
For annexin V staining, HEC stimulated
with IBOP, TNF
, or the two in combination (16 h) were harvested
using trypsin/EDTA and stained for annexin V using the Pharmingen
annexin-fluorescein isothiocyanate staining kit (Pharmingen). Annexin V
staining was quantitated using flow cytometry.
Caspase Activity Assays--
Following treatment with TNF
,
IBOP, or the two in combination for 16 h, HEC were scraped into
lysis buffer and incubated on ice for 30 min. Lysates were clarified by
centrifugation and protein concentration was estimated. Protease
reactions utilized cleavage of the chromogenic peptide substrates
Z-DEVD-p-nitroanilide and Ac-IETD-p-nitroanilide
for caspase 3- and 8-like activity, respectively (Biomol Research
Laboratories, Plymouth Meeting, PA). Caspase activity assays were
carried out in reaction buffer (100 mM HEPES, pH 7.5, 5 mM dithiothreitol, 20% (v/v) glycerol, 0.5 mM
EDTA, 0.1% (w/v) bovine serum albumin, 10 mM caspase
substrate) using 300 µg of total protein. Reactions were incubated at
30 °C for 30 min and cleavage of colorimetric substrates quantitated at 415 nm.
Cell Transfection and Luciferase Assays--
HEC were
transfected with the NF
B-dependent luciferase reporter
construct pBII-Luc(2× Igk
B-fos-luciferase) (22) or STAT1 oligonucleotides using the GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA). Transfected HEC were stimulated with
TNF
or IL-1
alone or in combination with IBOP for 16 h and
relative luciferase activity was determined using the luciferase gene
reporter kit (Roche Molecular Biochemicals). STAT1 sense (5'-ggTggCAggATgTCTCAgTgG 3') and antisense
(5'-CCACTgAgACATCCTgCCACC-3') oligonucleotides were transfected into
cells 24 h prior to assay; preliminary experiments established
that this time was sufficient for the ablation of STAT1 expression.
RNA Isolation and Northern Blot Analysis--
Confluent HEC were
exposed to TNF
or IBOP or both agents for up to 8 h. HEC were
washed twice with phosphate-buffered saline and total mRNA was
isolated using TRIzol reagent (Invitrogen). RNA (5 µg of each
sample) was separated on a denaturing 1% agarose gel, transferred to
nylon membrane (Amersham Biosciences), and blotted for leukocyte
adhesion molecule (LAM) expression as previously described (23).
Full-length human cDNA probes to ICAM-1, VCAM-1, E-selectin, and
GAPDH mRNAs were made from all cDNAs by random priming using
the Megaprime Labeling kit (Amersham Biosciences). After blotting,
membranes were dried and autoradiographed for the appropriate period of
time with changes in mRNA levels quantitated by scanning densitometry.
Statistical Analysis--
Data were pooled and statistical
analysis was performed using the Mann-Whitney U test.
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RESULTS |
TXA2 Mimetics Stimulate LAM Expression on
HEC--
Stimulation with the TXA2 mimetic IBOP (50 nM) increased expression of LAM molecules on HEC, with
maximal induction after 16 h for ICAM-1 and 6-8 h for VCAM-1 and
E-selectin (p
0.001, Fig.
1A). Induction of LAM
expression by IBOP was concentration-dependent. ICAM-1 and
VCAM-1 expression on HEC were maximally induced at IBOP concentrations
25 nM (IC50 12 nM,
p
0.005) at 16 and 6 h, respectively.
E-selectin was maximally induced at IBOP concentrations
50
nM (IC50 25 nM) after 6 h
(p
0.01, Fig. 1B). U46619, another TP
agonist, also stimulated LAM expression on HEC with maximal induction
at 400 nM and time courses similar to those shown for IBOP
(data not shown). Inclusion of the TP antagonist SQ29548 (5 µM) abrogated IBOP-induced LAM expression (data not shown), indicating the effect was mediated by the TXA2
receptor (TP).

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Fig. 1.
Effect of the TXA2 mimetic IBOP
on LAM expression by HEC. A, representative flow
cytometer tracing indicating the levels of ICAM staining on HEC treated
with media alone ( ), IBOP ( ), or IBOP in the presence of the TP
blocker SQ29548 ( ). Staining with preimmune IgG is also shown ( ).
B, time course for the induction of LAM by IBOP. HEC were
stimulated with IBOP (50 nM) for up to 16 h and probed
for E-selectin ( ), VCAM ( ), and ICAM ( ) expression. Control
cultures were incubated with media alone. C, cells were
stimulated for the appropriate time with IBOP (0-400 nM)
and the expression of E-selectin ( ), VCAM ( ), and ICAM
( ) was determined. All data generated using the flow cytometric
assay are expressed as geometric mean of the curve (mean ± S.D.)
from three different experiments. * indicates significance
(p 0.05) from control. D, NF B in
nuclear extracts of IBOP-stimulated cell lines expressing TP or
TP . To determine the G protein(s) involved in activation of NF B,
cells were incubated with GDP (50 µM) or antagonists of
specific G proteins (10 µM, G q,
G i, G s, and pertussis toxin
(G iPT)) for 30 min prior to the addition of
stimulants. Histone H1 is shown as a loading control. These data are
representative of three individual experiments.
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LAM expression is regulated by increased transcription, which in turn
is mediated by NF
B (24). HEC express both TP isoforms, TP
and
TP
. In cell lines overexpressing each of the two TP isoforms on a
null background, NF
B nuclear translocation (Fig. 1C) and LAM expression (data not shown) were found to follow ligation of TP
,
but not of TP
. NF
B nuclear translocation in response to IBOP in
TP
expressing cells was inhibited by loading the cells with GDP,
indicating the process was mediated by G proteins, and by two
pharmacological inhibitors of the heterotrimeric G protein G
i (Fig. 1C). Antagonists of
G
q and G
s were ineffective, consistent with the concept that this process was mediated by G
i signaling.
IBOP Prevents the Induction of LAM on HEC by TNF
, but Not
IL-1
--
We hypothesized that TXA2 may regulate
expression of LAM induced by other proinflammatory mediators, such as
IL-1
or TNF
. TNF
and IL-1
increased expression of ICAM-1,
VCAM-1, and E-selectin by an average of 5.5-, 12-, and 15-fold,
respectively (p
0.001) (Fig.
2A). Inclusion of IBOP with
IL-1
did not significantly alter the induction of LAMs, suggesting
that IL-1
and TP stimulation were neither synergistic nor additive.
In contrast, stimulation with both TNF
and IBOP abrogated the
induction of LAM (p
0.001 versus TNF
)
(Fig. 2A). IBOP-mediated inhibition of TNF
-induced expression of ICAM-1 (p
0.001, Fig. 2B),
VCAM-1, and E-selectin (data not shown) was blocked by SQ29548 (5 µM), indicating that TP binding was a requirement for
this effect. SQ29548 did not alter basal or TNF
-induced LAM
expression (Fig. 2B).

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Fig. 2.
Inhibition of the induction of LAM on
TNF , but not by IL-1 ,
stimulated HEC by TXA2 mimetics. A, HEC
were stimulated with IBOP (100 nM), TNF or IL-1 (100 units/ml) alone or in combination and LAM expression was quantitated by
flow cytometry after 2, 6, or 16 h for expression of E-selectin
( ), VCAM ( ), and ICAM ( ), respectively. B, HEC were
stimulated with IBOP, TNF , or the TP blocker SQ29548 (5 µM) either alone or in combination and ICAM-1 expression
was measured. C, HEC were stimulated with TNF and
increasing concentrations of IBOP (0-200 nM) and ICAM-1
expression was quantitated after 16 h. D, HEC were
stimulated with media alone ( ), TNF ( ), IBOP ( ), or both
agents together ( ) and ICAM-1 expression was quantitated over
24 h. Control cultures were incubated with media alone. Data
(B-D) are expressed as OD measured at 415 nm
(mean ± S.D.) from four different experiments. * indicates
significance (p 0.05) from control, # indicates
significance (p 0.05) from TNF or IBOP
treatment.
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The inhibition of LAM expression by IBOP in TNF
-stimulated HEC was
also concentration-dependent (Fig. 2C). TNF
treatment induced a 4.5-fold increase in ICAM-1 expression (Fig.
2C), above that seen with untreated HEC. Increasing
concentrations of the TXA2 mimetic IBOP attenuated the
induction of ICAM-1 by TNF
with inhibition greatest at IBOP
concentrations
100 nM (p
0.01, IC50 = 50 nM) (Fig. 2C). The
induction of E-selectin and VCAM-1 expression were similarly inhibited
by IBOP in TNF
-stimulated HEC (data not shown). Inhibition of LAM
expression on TNF
-stimulated HEC was not because of alterations in
the time course for maximal expression, as cells stimulated with both
agents displayed only basal expression of ICAM-1 (Fig. 2D),
VCAM-1, and E-selectin (data not shown) at all time points. Thus, the
prevention of TNF
-induced LAM expression by TXA2
mimetics did not result from cross-talk between the two agents
in which the time to peak expression was altered.
Consistent with these observations, IBOP-mediated inhibition of LAM
expression prevented leukocyte adhesion to TNF
-stimulated HEC.
Stimulation with either the TXA2 mimetic IBOP or TNF
induced a 3-4-fold increase in adhesion of the monocytic cell line
U937 to HEC (p
0.005, Fig.
3A). Simultaneous treatment
with both agents resulted in U937 adhesion similar to that in untreated controls (p
0.4, Fig. 3A). Similar
results were observed when the adhesion of leukocytes to the vessel
wall and their extravasation from the vasculature into surrounding
tissues was examined in vivo. Injection of recombinant
murine TNF
(150 pM) into mice resulted in a 7- and
11-fold increase in leukocyte adhesion to and extravasation from,
respectively (p
0.0001), the venous network of
cremaster muscle preparations after 4 h (Fig. 3B). Co-stimulation with IBOP (50 nM) resulted in an attenuation
of TNF
-mediated leukocyte adhesion and extravasation
(p
0.005 versus TNF
alone), with
leukocyte rolling increased 3-fold (data not shown). Consistent with
the observation above that ligation of TP
induces LAM expression in
EC, IBOP stimulation did not cause an increase in leukocyte trafficking
in vivo (Fig. 3B), as a mouse homologue for TP
has not been identified, and the murine TP resembles the human TP
isoform.

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Fig. 3.
Prevention of the adhesion of leukocytes to
TNF -stimulated endothelium both in
vitro and in vivo through TP-stimulated
inhibition of transcription. A, adhesion of labeled
U937 cells to HEC stimulated with IBOP (100 nM), TNF
(100 units/ml), or the two in combination was quantitated after 2 h by measuring the fluorescence of the resulting lysates. Data
(mean ± S.D.) are from four different experiments. B,
effect of TNF and IBOP alone and in combination on leukocyte
adhesion and extravasation in the mouse cremaster muscle in
vivo. Vessel segments were examined by video microscopy and the
number of leukocytes adherent to the vessel wall ( ) or in the
extravascular tissue ( ) were counted. Data are expressed as cells
per 100-µm vessel length and are the average (mean ± S.E.) of
five mice for each group. * indicates significance (p 0.01) from control, # indicates significance (p 0.01) from TNF . C, HEC were stimulated for 3 h
(E-selectin and VCAM-1) or 8 h (ICAM-1) with IBOP (100 nM), TNF (100 units/ml), or the two in combination.
Total RNA was extracted and E-selectin, VCAM-1, and ICAM-1 expression
were analyzed by Northern blotting. GAPDH mRNA was probed as a
loading control. Autoradiographs are from a single experiment and are
representative of three experiments.
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Regulation of ICAM-1, VCAM-1, and E-selectin by TNF
depends upon
increased transcription (24). To determine how TP inhibits LAM
expression in response to TNF
, we examined the pattern of LAM
mRNA expression. Fig. 3C shows that only ICAM-1 mRNA
was detectable in untreated endothelial cells by Northern blot
analysis. TNF
and IBOP both induced ICAM-1 expression 9-fold over
controls after stimulation for 8 h (Fig. 3C). Both
agents also induced robust expression of VCAM-1 and E-selectin after
3 h. The equivalent induction of ICAM-1, VCAM-1, and E-selectin
mRNA by TNF
and TP stimulation is consistent with the pattern of
protein expression determined by flow cytometry (Fig. 2A).
Consistent with the results in panels A and B,
incubation with both TNF
and IBOP prevented the increased mRNA
expression for ICAM-1, VCAM-1, and E-selectin observed with either
agent alone (Fig. 3C).
TP Stimulation Prevents Activation of NF
B by
TNF
--
Comparison of the ICAM-1, VCAM-1 promoters, and E-selectin
promoters indicated that NF
B and AP1 were factors that possibly indicated transcriptional regulation by TNF
. To examine whether TNF
-stimulated NF
B activity is regulated by TXA2
mimetics, we transfected HEC with a NF
B-sensitive luciferase
reporter construct. TNF
, IL-1
, and IBOP treatment induced
luciferase activity in transfected HEC to similar levels when used
individually (Fig. 4A,
p
0.005). In contrast, IBOP abrogated the
transcriptional activity of NF
B when used in combination with
TNF
, but not IL-1
(Fig. 4A).

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Fig. 4.
Prevention of gene expression in response to
TNF by TP-mediated inhibition of the nuclear
translocation of NF B. A, HEC
were treated with IBOP, IL-1 , or TNF alone or in combination for
16 h and luciferase activity in transfected HEC lysates was
determined. Data are from four different experiments (mean ± S.D.). * and # indicate significance (p 0.01) from
control and TNF , respectively. B, I B phosphorylation
and NF B nuclear translocation in response to TNF and IL-1 ,
with or without IBOP co-stimulation, after 30 min, was determined using
specific antibodies for p65RelA and phosphorylated I B.
In panels B and C, histone H1 is shown as a
loading control. C, NF B in nuclear extracts of HEC
stimulated with TNF and IBOP was measured. To determine the G
protein(s) involved in the inhibition of TNF induced activation of
NF B by IBOP, cells were incubated with GDP (50 µM), or
antagonists of specific G proteins (10 µM,
G q, G i, and G s) for 30 min
prior to the addition of stimulants. D, IBOP reverses the
activation of NF B by TNF . HEC were incubated with IBOP or TNF
alone or in combination for 15 or 30 min. In addition, HEC were
incubated with IBOP 15 min after TNF treatment had begun (lane
6); in the reverse experiment, TNF was added 15 min after
treatment with IBOP (lane 8). The activation of NF B was
examined by nuclear translocation of NF B, phosphorylation of I B,
and complex formation between NF B and I B. In the bottom two
subpanels, immunoprecipitation (IP) of NF B is
followed by immunoblotting (WB) with I B or NF B, the
latter as a control. Histone H1 blotting was used to control for
loading on nuclear protein preparations. The blots shown are
representative of four individual experiments.
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Nuclear translocation and full transcriptional activity of NF
B
require phosphorylation of the NF
B inhibitor protein, I
B (4).
Treatment with TNF
, IL-1
, or IBOP for 20 min increased I
B
phosphorylation in HEC lysates 10-12-fold and induced nuclear translocation of p65RelA, the most common NF
B subunit.
Untreated endothelial cells had little or no phosphorylated I
B or
nuclear p65RelA as assessed by immunoblotting (Fig.
4B). In accordance with the luciferase data (showing a lack
of NF
B-mediated transcriptional activity), co-stimulation with IBOP
prevented the nuclear translocation of p65RelA and the
enhanced I
B
phosphorylation observed with TNF
, but not
IL-1
, alone (Fig. 4B). The inhibition of TNF
-induced
NF
B activation by TP was prevented both in GDP-loaded cells, showing that the effect was G protein-mediated, and was also blocked by an
antagonist of G
q signaling, but not that of
G
s or G
i (Fig. 4C). IBOP
stimulation also abrogated TNF
-induced NF
B activation in cell
lines expressing TP
or TP
alone (data not shown), consistent with
the previously reported data that both TP isoforms are coupled to
G
q (14).
Interestingly, IBOP not only inhibited TNF
-induced NF
B nuclear
translocation but also reversed the process (Fig. 4D).
Simultaneous addition of TNF and IBOP (30 min) inhibited the nuclear
accumulation of NF
B observed with either agent alone. TNF
stimulation for 15 min induced nuclear NF
B staining and I
B
phosphorylation similar to that obtained after 30 min (Fig.
4D, lane 3 versus lane 5). Addition of
IBOP (to TNF
-stimulated HEC) for a further 15 min resulted in an
absence of I
B phosphorylation and nuclear NF
B staining
(lane 6). In addition, NF
B was found to be reassociated with I
B in HEC treated with TNF
for 15 min prior to the addition of the TP agonist for a further 15 min, indicating that the levels of
free NF
B were returned to baseline (Fig. 4D, lane
6 versus lane 5). The reciprocal was also true, as
addition of TNF
after IBOP stimulation reversed the activation of
NF
B, decreased I
B phosphorylation, and increased I
B·NF
B
complexes (Fig. 4D, lane 8 versus lane
7). These data indicate that TP agonists both inhibit the
activation of NF
B by TNF
and also reverse previously activated NF
B.
TP Specifically Inhibits the Activation of NF
B, but Not Other
TNF
Pathways, by Disrupting Early Events in Receptor
Signaling--
NF
B activation by IL-1
and TNF
converge at the
IKK signalosome and share all distal elements. The inability of
TXA2 mimetics to prevent IL-1
-mediated NF
B activation
indicates that the pathways involved may be specific to TNF
.
Stimulation with either IBOP or TNF
alone, or together, did not
alter the cellular expression of any of the proteins investigated (data
not shown). Thus, we immunoprecipitated TNFR1 and TNFR2 and blotted for
the expected proteins to investigate whether TP stimulation altered the
protein-protein interactions evoked by TNF
signaling. None of the
proteins tested were recruited to the cytoplasmic tail of TNFR1 or
TNFR2 in the absence of TNF
(Fig. 5,
lanes 1 and 2). TNF
stimulation induced robust
association of TRADD, RIP, TRAF-2, and IKK
with TNFR1. TRADD
associated with TNFR1 to a similar degree in the presence or absence of
IBOP. In contrast, IBOP co-stimulation prevented association of RIP,
TRAF-2, and IKK
with TNFR1 (Fig. 5A, lane 4).
Furthermore, neither TNFR1 nor TRADD co-immunoprecipitated with either
anti-RIP or TRAF-2 antibodies in the presence of TNF
and IBOP (data
not shown). This indicates that the interaction of RIP and TRAF-2 with
TRADD, which occur independently, were both inhibited by IBOP. STAT-1
phosphorylation at Tyr701 was recently reported to be part
of the TNFR1·TRADD complex, and inhibited TRADD-TRAF-2/RIP
interactions without influencing TRADD-FADD interactions (25).
Interestingly, the TNF
-induced STAT1-TNFR1 interaction was increased
3-fold by co-stimulation with the TXA2 mimetic IBOP (Fig.
5A). TNF
was found to increase STAT1 phosphorylation
(Fig. 5B), which was augmented 3-fold by co-stimulation with
IBOP. IBOP alone did not stimulate STAT1 phosphorylation (Fig.
5B), however, suggesting that the enhancement of
TNF2-induced STAT1 phosphorylation by IBOP and TNF
is likely a
result of another mechanism, such as inhibition of phosphatase
activity. As is the case with inhibition of NF
B, the IBOP-enhanced
phosphorylation of STAT1 was reduced by inhibition of G
q
signaling (Fig. 5B). Thus, the abrogation of TNFR1 NF
B
signaling by IBOP may result from a TP-mediated enhancement of STAT1
activation by TNF
.

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Fig. 5.
IBOP-mediated prevention of the association
of proteins responsible for NF B signaling with
TNFR1 and TNFR2 upon TNF stimulation. The
binding of downstream effectors to TNFR1 (A) and TNFR2
(C) was analyzed after stimulation for 5 min with IBOP,
TNF , or the two in combination. TNFR1 or TNFR2 were
immunoprecipitated and immune complexes were probed with antibodies to
the proteins as indicated in the figure. In addition, lysates from
stimulated HEC were probed using antibodies against STAT1
phosphorylated at Tyr701 (B). Blots were
reprobed with a monoclonal STAT1 antibody to assess loading
(n = four experiments). The role of STAT1 in regulating
the association of proteins with TNFR1 and TNFR2 was assessed using an
antisense oligonucleotide to deplete HEC of STAT1 with the sense
oligonucleotides used as a control (panels A and
C). The blots shown are representative of three individual
experiments.
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When potential alterations in TNFR2 signaling were examined, we
observed that TRAF-2 bound to TNFR2 and associated with NIK in
TNF
-stimulated HEC (Fig. 5C). Co-stimulation with IBOP
and TNF
prevented association of NIK and TRAF-2, but not TRAF-2 and TNFR2. Furthermore, TP stimulation induced the association of RIP with
TNFR2, which does not occur following TNF
alone, in contrast to the
case with TNFR1 (Fig. 5C). Thus, TXA2 mimetics may enhance those aspects of TNFR2 signaling that result in apoptosis, as well as inhibit NF
B activation, by redirecting RIP to associate with TNFR2.
In this model, the effects of TP stimulation on the recruitment of
signaling intermediates to TNFR1 and TNFR2 depend upon the increased
association of STAT1 with TNFR1. To explore further the causative role
of STAT1, we used antisense oligonucleotides to antagonize STAT1
expression. Transfection of HEC with the STAT1 antisense
oligonucleotides abrogated STAT1 expression over a period of 16 h,
and did not affect HEC viability. STAT1 sense oligonucleotides did not
affect STAT1 expression during the same time course (data not shown).
When STAT1 oligonucleotide-treated HEC were stimulated, those HEC
treated with sense oligonucleotides were not found to differ from
untransfected cells (Fig. 5, A and C). In
contrast, STAT1 antisense oligonucleotides ablated the interfering
effects of TP stimulation, thus allowing RIP, TRAF-2, and IKK
to be
recruited to TNFR1. Similarly, the recruitment of NIK to TNFR2 was also restored and the recruitment of RIP did not occur. Thus, the enhanced phosphorylation and recruitment of STAT1 to TNFR1 in the presence of
IBOP is a crucial step in the antagonism of TNF
-induced NF
B activation by TP stimulation.
The importance of MAPK activation to LAM transcription has recently
become apparent (26-29). To determine whether the effects of
TXA2 were specific for NF
B, we probed for MAPK
activation in extracts of HEC stimulated with IBOP, TNF
, or both
agents with phosphospecific antibodies. TNF
treatment induced a 4-, 8.5-, and 13-fold increase in p38, ERK1/2, and JNK phosphorylation, respectively (Fig. 6A). The
increased MAPK phosphorylation was not diminished by IBOP
co-stimulation. Because IBOP disrupts TNF
-mediated TRAF-2-TNFR1
interactions, we posited that MAPK activation following TNF
was a
response to either MADD/TNFR1 or TRAF-2/TNFR2 signaling. TRAF-2
associated with Ask1 and MAKK1 in TNF
-stimulated HEC (Fig. 6B) and co-stimulation with IBOP did not influence their
recruitment. Similarly, the association of MADD with TNFR1 in response
to TNF
was unaffected by co-stimulation with IBOP (Fig.
6C). Thus, TP stimulation induces a functional bifurcation
in TNFR2 signaling that maintains MAPK activation but inhibits NF
B
activation.

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Fig. 6.
Lack of inhibition of
TNF -mediated MAPK activation by TP stimulation
in HEC. In panel A, lysates from stimulated HEC were
probed using monoclonal antibodies to the phosphorylated forms of p38,
ERK1/2, and JNK following stimulation for 5 min with IBOP, TNF , or
the two in combination. Blots were reprobed with a monoclonal
-tubulin antibody to assess loading (n = four
experiments). The interaction of TNFR2 (B) and TNFR1
(C) with downstream effectors that influence MAPK activation
were determined after TNFR1 or TNFR2 were immunoprecipitated and immune
complexes were probed with antibodies against MADD, Ask1, or
MEKK1.
|
|
TP Does Not Inhibit Caspase Activation by TNF
and Enhances
Endothelial Cell Apoptosis--
Concurrent TP stimulation inhibits
TNF
-induced activation of a major cell survival pathway. TNF
also
promotes cell death by activating caspases. Thus, we examined the
effect of TXA2 mimetics on TNF
-mediated caspase
activation and cell survival. TNF
-mediated caspase activation causes
the association of TRADD and FADD (4). Immunoprecipitation of TNFR1
showed that FADD associated with the receptor when TNF
was present;
this association was not altered by co-stimulation with IBOP (Fig.
7A). Association of FADD and TNFR1 activates caspase 8 and subsequently caspase 3. Thus, we next
examined caspase activity in stimulated HEC lysates. Control cells
contained little caspase 3- or 8-like enzyme activity; however, robust
caspase 3 and 8 activation were observed in TNF
-treated cells, which
was not altered by IBOP (Fig. 7B). Thus, proapoptotic signaling from TNFR1 remained intact in the presence of
TXA2. We next examined the effect of simultaneous TNF
and TP stimulation on endothelial cell survival. Stimulation with IBOP
or TNF
alone increased annexin V staining by 2-fold in untreated HEC
after 16 h (p
0.01, Fig. 7C). In
contrast, the use of both agents induced a 5-fold increase in
endothelial cell apoptosis (p
0.001). This
synergistic increase in endothelial cell death is likely the result of
the preservation of the proapoptotic cascade of TNFR1, with the
inhibition of prosurvival pathways of both TNFR1 and TNFR2, such as
NF
B activation. Indeed, when HEC that had been transfected with
oligonucleotides with the NF
B consensus binding site were treated
with TNF
, endothelial cell apoptosis was increased 2-fold and
ICAM-1, VCAM-1, and E-selectin expression was ablated (data not shown).
Thus, the inhibition of TNF
-induced NF
B activity by
TXA2 stimulation appears to be sufficient to promote
endothelial cell apoptosis.

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Fig. 7.
Lack of inhibition of
TNF -mediated caspase activation by TP
stimulation and promotion of endothelial cell apoptosis.
A, binding of FADD to TNFR1 was determined by Western
blotting of immune complexes from HEC stimulated with IBOP, TNF , or
the two in combination for 5 min (n = three
experiments). B and C, TP stimulation and TNF
both activate caspases in HEC, which results in increased apoptosis.
After stimulation with IBOP, TNF , or the two in combination for
16 h, cell lysates were assayed for caspase 3 ( ) and 8 ( )
activity (B) or stained using fluorescein
isothiocyanate-conjugated annexin V as a marker of apoptosis
(C). The percentage of cells binding annexin V was
determined by flow cytometry. Data are expressed as
A415 nm (B) or percentage of cells
binding annexin V (C) (mean ± S.D.) and represent the
average of three experiments. * and # indicate significance
(p 0.01) from control and TNF ,
respectively.
|
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 |
DISCUSSION |
In this study we found that ligands for TP, the G protein-coupled
receptor for TXA2, abrogated the enhanced expression of proinflammatory markers on TNF
-stimulated endothelial cells and inhibited leukocyte adhesion to TNF
-treated endothelium in
vitro and in vivo. Furthermore, in endothelial cells
incubated with both agents, apoptosis was markedly increased, as
TXA2 mimetics prevented TNF
from initiating pathways
linked to cell survival, but not those linked to apoptosis.
TP stimulation prevented NF
B activation by both TNF
receptors.
The cross-talk between the TXA2 and TNF
receptors is
unlikely to occur at a point downstream of the convergence of the TNFR1 and TNFR2 signaling pathways, as activation of NF
B by IL-1
, which
shares these pathways, was unaffected by TP stimulation. Thus,
mechanisms that universally inhibit NF
B activation, such as
induction of A20 and inhibition of I
B kinase (4), do not appear to
be involved. In addition, TRAF-2 bound to TNFR2, but not TNFR1, in HEC
stimulated with TNF
and IBOP. Together these data indicate that
TP-mediated inhibition of both TNFR1 and TNFR2 is facilitated via novel mechanisms.
Association of RIP and TRAF-2 with TNFR1 are necessary for NF
B
activation and cells deficient in either protein do not activate NF
B
fully in response to TNF
(30, 31). Overexpression of STAT1 inhibits
the association of RIP and TRAF-2 with TNFR1·TRADD complexes, but
does not affect FADD·TRADD complex formation, after TNF
stimulation (25). Concurrent stimulation with TNF
and IBOP also
interfered with TRADD-RIP/TRAF-2 interactions but not FADD binding or
caspase activation. Furthermore, stimulation with TNF
and IBOP
increased STAT1 phosphorylation on Tyr701 and the
association of TNFR1 and STAT1. Thus, TP enhanced TNF
-mediated STAT1
activation; this may inhibit NF
B activation by TNFR1, most likely by
competitively inhibiting RIP/TRAF-2 binding to a TNFR1·TRADD complex.
The TXA2- and G
q-mediated enhancement of
TNF
-stimulated STAT1 activation is another novel finding of the
present study. This model of antagonism by TP ligands is an extension
of the findings of Wang et al. (25), who showed that STAT1
overexpression prevented the recruitment of RIP to TNFR1 in a
concentration dependent fashion. Other G protein-linked receptor
ligands, such as angiotensin II and
1 adrenergic
receptors, have also been reported to stimulate STAT1 (32, 33). Whereas
the pathways of STAT1 activation may differ, all these receptors are
linked to G
q, which suggests the possibility of a common
mechanism of G protein-mediated inhibition of TNF-stimulated NF
B
activation during inflammation and reperfusion.
TP stimulation also inhibited TNFR2 activation of NF
B. TP
stimulation did not inhibit TRAF-2-TNFR2 interactions, however; TP
induced a functional bifurcation in TNFR2 signaling, leading to
activation of MAPK, but not NF
B. This division of TNFR2 signaling was mediated by selective inhibition of NIK-TRAF-2 interactions after
TRAF-2 binds to TNFR2. We propose that recruitment of RIP to TNFR2 in
the presence of IBOP mediates the selective inhibition of NIK binding.
RIP overexpression alters the signaling pathway of TNFR2,
"switching" the downstream effects from NF
B activation to a
cascade inducing programmed cell death (34). TP signaling prevents the
RIP-TRADD interaction in response to TNF
by inducing translocation
of STAT1 to TNFR1. In this model, RIP displaced from TNFR1 by STAT1
binds to TNFR2. Thus, the enhanced association of RIP with TNFR2 may
aid the induction of apoptosis and prevent NF
B activation by
TNF
in the presence of TP stimulation. Indeed, the importance of
STAT1 to this pathway of inhibition was highlighted by the experiments
with antisense STAT1 oligonucleotides, indicating that the recruitment
of RIP to TNFR2 was abrogated in the absence of STAT1.
These data present a model for the inhibition of TNF
-mediated NF
B
activation by IBOP. The effects of IBOP are more complicated than just
mere inhibition of the NF
B pathway, however. Initiating IBOP
treatment after TNF
stimulation had achieved maximal
expression reduced NF
B activation to baseline. This response
is characterized by the movement of NF
B of the nucleus, decreased
I
B phosphorylation, and reassociation of the two proteins. Whereas
the inhibition of proximal events in TNF
signaling by IBOP explain
why no further NF
B activation occurs, it does not fully explain the
rapid reversion to baseline for NF
B activated during the previous
period. One possibility, proposed by Ghosh and Karin (35), is that
I
B may be recruited to the nucleus to recomplex to NF
B and cause
its redistribution to the cytoplasm. In the case of the present
experiments, TP might be suspected of triggering a phosphatase that
leads to dephosphorylation of I
B. The ability to inhibit previously
activated NF
B is a powerful proapoptotic effect of TP stimulation;
regardless of the order that HEC receive the stimuli, the result is the
inhibition of NF
B and the induction of apoptosis.
We found that TP stimulation does not affect TNF
-induced activation
of JNK, p38, and ERK1/2. Activation of MAPK by TNF
is dependent upon
TRAF-2 (36, 37). As the TNFR2-TRAF-2-Ask1, but not the
TNFR1-TRADD-TRAF-2, pathway is preserved in the presence of TP
stimulation, it is most likely the pathway that leads to MAPK
activation. Induction of LAM requires MAPK activation and p38 is
required for full transcriptional activity of NF
B (26, 38). The
inhibition of TNF
-stimulated LAM expression, despite activation of
all three MAPKs, indicates either that AP-1 and NF
B activation are
both required for LAM expression, or instead that NF
B alone is
necessary and sufficient. Interestingly, ERK, JNK, and p38 all promote
EC apoptosis (39) and have direct links to caspase activation as well
as other cell death pathways, such as the functional suppression of
Bcl-2 (40). Thus, p38, JNK, and ERK activation in the presence of
TNF
and TXA2 may enhance the caspase activation mediated
by the TRADD-FADD pathway, thus leading to the enhanced apoptosis
observed in endothelial cells treated with both agents.
Although not emphasized in this report, the inhibition of NF
B
activation in HEC treated with TNF
and TXA2 mimetics is
reciprocal. The ability of TXA2 mimetics to induce NF
B
activation is specific for a single TP isoform; TP
, through a
G
i-coupled signaling pathway not used by TP
,
activates NF
B in response to IBOP and U46619. In contrast, the
inhibition of TNF
signaling is potentially mediated through either
isoform, as both couple to G
q. TNF
stimulation prevented the activation of NF
B in TP-stimulated HEC. Furthermore, TNF
could also reverse the activation of NF
B if cells were
stimulated with IBOP prior to TNF
. The mechanism by which TNF
controls G
i-mediated NF
B activation is not yet identified.
Circulating levels of TXA2 and TNF
are increased during
ischemia. In addition, ischemic episodes increase the number of
circulating endothelial cells by stimulating apoptosis (41). These
endothelial cells were macrovascular in origin and did not express
markers of "activation" such as LAM. We have found that
co-stimulation with TXA2 and TNF
produces a similar
phenotype in macrovascular endothelial cells in vitro.
Although TXA2 does not inhibit NF
B induction by all
cytokines, e.g. IL-1
, similar mechanisms might prevent
activation of endothelial cell NF
B by such cytokines. The reciprocal
inhibition of NF
B activation, such as that which occurs with
TXA2 and TNF
, is sufficient for the induction of endothelial cell apoptosis and thus may increase the number of circulating endothelial cells during ischemia. Consistent with this
hypothesis is the recent observation that blocking TNF
decreases the
number of circulating endothelial cells (42).
Apoptosis mediates inflammation and leukocyte extravasation associated
with renal injury by cleavage of the monocyte-activating polypeptide II
protein (43). We speculate that apoptotic endothelial cells, exposed to
TNF
and TXA2, could mediate leukocyte infiltration into
ischemic tissues by a similar mechanism. Co-stimulation by TXA2 and TNF
may propagate an inflammatory response by
perturbing the vessel wall, inducing injury to the vasculature, and
exposing the subendothelial matrix following loss of endothelial cells because of apoptosis. In addition, the induction of endothelial apoptosis may attenuate an angiogenic response, thus reducing revascularization in inflamed or ischemic tissues.
Taken together, these data describe a previously unrecognized
interaction between two mediators of inflammation and reperfusion injury that may provide a mechanism by which endothelial cell injury
occurs during these conditions. In addition to elucidating this
mechanism, a rationale is provided for the use of a TP antagonist to
preserve the cell survival properties of TNF
during inflammation and reperfusion.