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INTRODUCTION |
The biological effects elicited by cytokines such as
TNF1 and IL-1, which include
inflammation and tissue injury, are initiated by ligand-induced
formation of distinct multiprotein receptor complexes. Activation of
the TNF signal transduction cascade is initiated by the interaction of
TNF with two distinct surface receptors, TNFR1 (55 kDa) and TNFR2 (75 kDa) (reviewed in Ref. 1). Although both TNFR1 and TNFR2 may mediate
the activation of independent downstream signaling pathways (2-4),
there is evidence that TNFR2 functions primarily to bind ligand
rapidly, passing soluble TNF trimers to TNFR1 (5, 6). Ligand-induced clustering of TNFR1 leads to the recruitment of a cytosolic adaptor protein, called TNF receptor 1-associated death domain protein (TRADD),
to the intracellular domain of the receptor (7). Receptor-associated TRADD further recruits a protein first described as Fas-associated death domain protein, thought to lie upstream of the caspase cascade and initiation of apoptosis (8). TRADD also mediates the binding of the
protein kinase RIP (receptor-interacting protein) and the ring/zinc
finger protein, TNF receptor-associated factor-2 (TRAF2) (8, 9). Recent
evidence from studies of rip
/
and
traf2
/
knockout mice suggests that RIP is
essential for the activation of the transcription factor NF
B,
whereas TRAF2 is required for activation of the transcription factor
AP-1 (10-12). More specifically, RIP may activate the
TRAF2-interacting NF
B-inducing kinase (13, 14), which activates two
recently identified kinases, I
B kinase-1 and -2, that exist in a
multiprotein cytosolic complex (15-18). I
B
is normally located
in the cytosol of unstimulated cells in a complex with NF
B
inhibiting the transcriptional activity of NF
B by masking its
nuclear localization sequence (19). Upon phosphorylation by I
B
kinase-1 and -2 (15-18), I
B
is rapidly ubiquitinated and
degraded by the proteosome, thereby releasing NF
B, which is now free
to translocate to the nucleus and induce transcription (20, 21).
IL-1, like TNF, activates NF
B and AP-1 but generally does not
initiate apoptosis. IL-1-stimulated activation of NF
B is also dependent on the formation of a ligand-induced receptor complex. The
occupied type 1 IL-1 receptor interacts with a transmembrane protein
termed IL-1 receptor accessory protein (22) and a cytosolic adaptor
molecule, MyD88 (23). This complex then binds IL-1 receptor-associated kinase (23, 24). Within the assembled type 1 IL-1 receptor complex,
IL-1 receptor-associated kinase becomes autophosphorylated (25) and
subsequently leaves the complex to associate with cytosolic TRAF6 (26).
The IL-1 receptor-associated kinase·TRAF6 complex, like the
TRADD·RIP·TRAF2 complex, lies upstream of the activation of
NF
B-inducing kinase, creating a point of convergence with the TNF
signaling pathway.
Vascular endothelial cells (EC) are a major target for the
pro-inflammatory actions of TNF and IL-1. One of the major
proinflammatory responses in EC initiated by the TNF and IL-1 signal
transduction cascades described above is the expression of the
leukocyte adhesion molecules E-selectin, intercellular adhesion
molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1).
E-selectin is a surface glycoprotein that mediates the initial
tethering and rolling of neutrophils. The
cytokine-dependent transcription of this adhesion molecule
has an absolute requirement for the activation of NF
B (27) and can
be significantly enhanced by the co-binding of ATF2/c-Jun heterodimers
(a form of AP-1) to the E-selectin enhanceosome (28). Inhibition of the
activation of NF
B or AP-1 in response to TNF or IL-1 is therefore a
potential target for novel anti-inflammatory reagents. A wide variety
of of diverse pharmacological agents including sodium salicylate,
arachidonic acid, short chain ceramide (C6-ceramide), and
staurosporine (stauro) have been reported to inhibit TNF-mediated
activation of NF
B, expression of E-selectin, or both in EC (29-32).
However, none of these agents has been shown to interact with any of
the molecules identified to date in the TNF signaling pathway. During
the course of our investigation of the signaling role of arachidonic
acid, we noted that concentrations of this lipid or its structural
analogues, such as the cytosolic phospholipase A2
inhibitors arachidonyl trifluoromethylketone (ATK) and
methylarachidonyl fluorophosphonate, inhibited E-selectin induction but
only at concentrations that induced apoptosis. However, these agents
were able to block the activation of NF
B before morphological
evidence of apoptosis became detectable. These observations led us to
hypothesize that coincident with the induction of apoptosis is a common
mechanism by which diverse pharmacological compounds can selectively
inhibit the EC response to TNF. Here we present evidence in support of this hypothesis and identify the loss of TNFR1 surface expression as
the mechanism by which apoptogenic drugs selectively inhibit TNF responses.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
In accordance with an approved protocol by the
Yale University Human Investigations Committee, human umbilical vein EC
were isolated and cultured as described previously on gelatin (J. T. Baker Inc.)-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in
medium 199 (M199) supplemented with 20% fetal calf serum, 200 µM L-glutamine (all from Life Technologies
Inc.), 50 µg/ml EC growth factor (Collaborative Biomedical Products,
Bedford, MA), 100 µg/ml porcine heparin (Sigma), 100 units/ml
penicillin, and 100 µg/ml streptomycin (Life Technologies Inc.). All
experiments were performed using EC at passage 3 or 4.
Materials--
Recombinant human TNF was a gift from Biogen
(Cambridge, MA). Recombinant human IL-1
was purchased from R & D
Systems Inc. (Minneapolis, MN). zVADfmk was purchased from Enzyme
Systems Products (Dublin, CA). SB203580, C6-ceramide,
stauro, and yVADcmk were purchased from Calbiochem. R32W TNF mutein
protein was a gift from W. Fiers (State University of Ghent, Belgium).
ATK was purchased from Cayman Chemical Co. (Ann Arbor, MI), and a fresh
vial was used each day. A tert-butyl derivative of Tapi was
a gift of the Immunex Corp. (Seattle, WA). All other reagents were
purchased from Bio-Rad or Sigma.
Rabbit anti-human I
B
antibody was purchased from Santa Cruz
Biotechology (Santa Cruz, CA). Mouse mAb to human TRADD was purchased
from Transduction Laboratories (Lexington, KY). Mouse mAb to TNFR1 was
a gift from D. Goeddel (Tularik Inc., South San Francisco, CA). Mouse
mAb to TNFR2 was purchased from Genzyme Diagnostics (Cambridge, MA).
Rabbit anti-human TRAF2 and RIP antibodies were also a gift from D. Goeddel. Mouse mAb to poly(ADP-ribose) polymerase (PARP) was a gift
from G. Poirier, CHUL Research Center (Quebec, Canada). Horseradish
peroxidase-conjugated secondary antibodies for Western blotting were
purchased from Jackson ImmunoResearch (West Grove, PA). For FACS
analysis, fluorescein isothiocyanate-conjugated anti-mouse IgG (H + L)
F(ab')2 was purchased from Roche Molecular Biochemicals.
4',6-Diamidino-2-phenylindole·HCl Staining of EC--
For
experimental manipulation, EC were plated on human plasma
fibronectin-coated multiwell chamber slides (Falcon). After treatment,
cells were fixed by the addition of an equal volume of paraformaldehyde
(1% final) in PBS for 30 min at room temperature. The slide was
subsequently washed twice in PBS, permeabilized for 30 s in PBS
containing 0.1% Triton X-100, washed a further four times in PBS, and
mounted in Gel Mount (Biomedia Corp., Foster City, CA) containing the
nuclear stain 4',6-diamidino-2-phenylindole·HCl (Dapi, ~0.0001%,
Molecular Probes, Eugene, OR). Specimens were examined by
immunofluorescence microscopy using a Nikon diaphot microscope with a
360-nm filter.
Quantitation of Cell Death--
EC plated on gelatin-coated 24- or 96-well plates were treated as described. After experimental
manipulation, medium was removed, and cells were washed twice in PBS.
The remaining cells were fixed and stained by the addition of 70%
ethanol containing 100 µg/ml Hoescht 33258 reagent (Molecular Probes)
for 30 min at room temperature. Cells were again washed twice with PBS
before fluorescence was recorded (
ex = 360 nm,
em = 460 nm) using a fluorescence plate reader
(Perspective Biosystems Inc., Framingham, MA).
Immunoprecipitation and Immunoblotting--
For analysis of the
characteristic cleavage of PARP during apoptosis, EC seeded in six-well
plates were treated for 3 h with serum-free M199 containing ATK
(0-50 µM) in the presence and absence of zVADfmk (40 µM). Both floating and attached cells were harvested and
lysed in TNT solution (50 mM Tris-HCl, pH 6.8, 150 mM NaCl, and 1% Triton X-100) containing Pefablock (1 mM), aprotinin (10 µg/ml), pepstatin (1 µg/ml),
leupeptin (10 µg/ml), NaF (10 mM), Na3VO4 (1 mM), and
-glycerophosphate (1 mM) for 20 min on ice. For all
other immunoblots, each well was washed twice in ice-cold PBS and lysed
by the addition of 100 µl of TNT as described above. DNA in cell
lysates was sheared by passing the lysates through a 0.5-cc syringe
fitted with a 28.5-gauge needle two or three times. For each sample, an
equal amount of protein (20 µg) was size-fractionated by
SDS-polyacrylamide gel electrophoresis (33) and then transferred to a
polyvinylidene difluoride membrane (Immobilon P, Millipore, Milford,
MA) and immunoblotted. Detection of the immunogen by enhanced
chemiluminesence was performed according to the manufacturer's
instructions (Pierce). Immunoprecipitation was performed by the lysis
of confluent human umbilical vein EC seeded on three 10-cm dishes
(approximately 107 cells/sample) in a total volume of 3 ml
of TNT. The lysates were centrifuged at 735 × g for 10 min at 4 °C before being precleared by incubation with 25 µl of a
1:1 slurry of Gamma Bind-Sepharose (Amersham Pharmacia Biotech) for
2-3 h at 4 °C on a rocking platform. After centrifugation at 14,000 rpm for 10 s, the cleared lysates were transferred to another tube
and incubated with mAb to TNFR1 (1 µl/sample) overnight at 4 °C on
a rocking platform before the addition of 50 µl of the 1:1 Gamma
Bind-Sepharose slurry to each lysate. Incubation was continued at
4 °C for a further 4-6 h. Immune complexes, collected by
centrifugation at 16,000 × g for 3 s, were washed
once in ice-cold TNT and three to four times in ice-cold PBS and
solubilized by the addition of 25 µl of sample buffer (33). The
entire sample was subjected to SDS-polyacrylamide gel electrophoresis
and Western blot analysis.
Indirect Immunofluorescence and FACS Analysis--
Indirect
immunofluorescence was used to quantify the surface amount of TNFR1 and
TNFR2 after treatment of EC with ATK, C6-ceramide, sodium
salicylate, and stauro. EC were treated as described, harvested by
collagenase (Life Technologies Inc.) digestion, and washed in PBS
containing 1% fetal calf serum before incubation for 30 min at 4 °C
with saturating amounts of each antibody or nonbinding control mAb
(K16/16) as described previously (34). Cells were washed three times
before the addition of fluorescein isothiocyanate-conjugated anti-mouse
secondary Ab and incubation for a further 30 min at 4 °C. Labeled
cells were washed an additional three times, fixed in paraformaldehyde
(2%), and analyzed by FACS (FACSort, Becton Dickinson, San Jose, CA).
Soluble TNFR1 Enzyme-linked Immunosorbent Assay--
EC on
gelatin-coated six-well plates were treated for 2 h with
serum-free M199 ± ATK (50 µM) or sodium salicylate
(20 mM). After incubation the culture medium was harvested,
and the amount of soluble TNFR1 in the medium was determined using a
soluble TNFR1 enzyme-linked immunosorbent assay kit (Immunotech, Miami, FL).
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RESULTS |
Diverse Pharmacological Agents That Induce EC Apoptosis Block TNF
but Not IL-1 Signaling--
Reports from the literature and our own
observations indicate that the cytosolic phospholipase A2
inhibitor ATK, the kinase inhibitor stauro, the anti-inflammatory agent
sodium salicylate, and the cell-permeant ceramide analogue,
C6-ceramide, are all able to inhibit TNF-induced adhesion
molecule expression (29, 31, 32). This inhibition of E-selectin
expression is paralleled by an inhibition of TNF-induced degradation of
inhibitory protein I
B
. Optimal inhibitory concentrations,
determined in preliminary experiments, are 50 µM ATK, 100 nM stauro, 20 mM sodium salicylate, and 50 µM C6-ceramide. We have found that these
concentrations of all of these agents cause EC to undergo apoptosis;
suboptimal inhibitory concentrations were less apoptogenic (Fig.
1 and data not shown). This correlation
between inhibition of TNF responses and induction of apoptosis may have
been missed in prior studies (29, 31, 32) because biochemical and
morphological evidence of apoptosis, such as PARP cleavage or nuclear
condensation, typically did not become apparent for several (3-12)
hours, the precise timing depending on the drug used (data not shown).
More importantly, no evidence of apoptosis was ever detectable in the
first 2 h of treatment, by which time inhibition of TNF-mediated
activation was already evident.

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Fig. 1.
Inhibition of TNF signaling correlates with
induction of programmed cell death. EC were pretreated either for
2 or 5 h with ATK (0-50 µM). After 2 h of
pretreatment, EC were treated for a further 15 min with TNF, and the
degradation of I B was determined by Western blot analysis and
densitometry. After 5 h of treatment, replicate EC cultures were
washed and the remaining cells were quantified by Hoescht
staining.
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Cytokine-induced E-selectin expression is absolutely dependent on the
activation of NF
B (27). Therefore we examined the effect of 2 h
of pretreatment of each apoptosis-inducing reagent on TNF- and
IL-1-dependent degradation of I
B
, a critical step in
the NF
B pathway. As shown in Fig. 2,
A and B, TNF-induced degradation of I
B
is
significantly reduced following 2 h of pretreatment with each
apoptogenic drug. Surprisingly, no inhibition of I
B
degradation
induced by IL-1 is detectable in replicate cultures. The observed
effect of apoptogenic agents on TNF-dependent degradation
of I
B
is not simply a delay in the kinetics of the degradation of
this protein, because a complete time course of TNF treatment from 5 to
60 min revealed that the degradation of I
B
is inhibited at all
times examined (data not shown). These observations led us to
hypothesize that an early common event in the biochemical program of
apoptosis selectively inhibits TNF (but not IL-1) signal
transduction.

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Fig. 2.
Inhibition of TNF but not IL-1-mediated
I B degradation by
diverse initiators of apoptosis. EC were pretreated for 2 h
with M199 in the absence ( ) or presence (+) of apoptosis inducers ATK
(50 µM), C6-ceramide (c-6 cer, 50 µM), sodium salicylate (Na Sal, 20 mM), or stauro (100 nM) before a further
treatment with TNF (50 units/ml) or IL-1 (1 ng/ml) for 15 or 30 min,
respectively. Cell lysates (20 µg) were size-fractionated by
SDS-polyacrylamide gel electrophoresis and analyzed for I B by
Western blotting (A). The mean percentages of inhibition of
the TNF or IL-1 response compared with mock pretreated cells are shown
pooling data from two independent experiments (B).
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Inhibition of TNF-induced I
B
Degradation in EC Is Not
Dependent on the Activation of Caspases or p38 Kinase--
We next
examined whether specific effector molecules implicated in the death
pathway could inhibit TNF signal transduction. Activation of effector
caspases is a common late step in apoptosis (35). To determine
whether the activation of caspases participated in the inhibition of
TNF-mediated NF
B activation by pretreatment with inducers of
apoptosis, we employed the broad spectrum peptide-based caspase
inhibitor zVADfmk. This caspase inhibitor (40 µM) was unable to prevent inhibition of TNF-mediated I
B
degradation when
administered during pretreatment with ATK (50 µM) over a 2-h period (Fig. 3A) even
though it was able to inhibit the characteristic caspase-dependent cleavage of PARP from a 116- to a 85-kDa
fragment over a 3-h incubation with ATK (Fig. 3B). Similar
results using zVADfmk were obtained with the other three initiators of
apoptosis as well as with another broad spectrum caspase inhibitor,
yVADcmk (40 µM) (data not shown). We conclude that
activation of zVADfmk- and yVADcmk-sensitive caspases do not
participate in the inhibition of TNF-mediated NF
B activation.

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Fig. 3.
The caspase inhibitor zVADfmk does not
relieve the inhibition of TNF-induced
I B degradation.
EC were treated with ATK (50 µM) in the presence or
absence of zVADfmk (40 µM) for 2 h prior to the
addition of TNF (50 units/ml). Cell lysates were analyzed for I B
degradation (A). EC were treated with ATK (50 µM) in the presence or absence of zVADfmk (40 µM) for 3 h. Both floating and attached cells were
harvested, lysed, and analyzed for cleavage of PARP
(B).
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Vilcek and colleagues (36, 37) have reported that the p38 kinase
inhibitor SB203580 can prevent both sodium salicylate-induced apoptosis
and inhibition of TNF signaling in fibroblasts. To determine whether
p38 kinase played a role in the inhibition of TNF-dependent activation of NF
B in EC, we employed the same inhibitor. In accord with the fibroblast results, blocking of p38 kinases does relieve the
inhibition of TNF-induced I
B
degradation in EC following pretreatment with sodium salicylate (Fig.
4). However, in contrast to the previous
study, SB203580 was unable to prevent sodium salicylate-induced apoptosis of EC. SB203580 was similarly effective in preventing the
inhibition of TNF-dependent I
B
degradation resulting
from pretreatment with C6-ceramide and stauro. Despite a
corresponding degree of inhibition of TNF responses by ATK compared
with sodium salicylate, SB203580 was ineffective in preventing
inhibition of TNF signaling by ATK. Both ATK and sodium salicylate
treatment resulted in activation of p38 MAPK measured by Western
blotting of the active form of the kinase with a phospho-p38-specific
antibody. However, the activation of p38 in response to sodium
salicylate was detected after only a 15-min treatment with the reagent
and decreased over time, whereas the activation of p38 in respect to
ATK was not increased until after 2 h of treatment with the reagent. At this time point the activation of p38 was slightly stronger
than the activation initially observed with sodium salicylate. The
inability of SB203580 to prevent ATK-dependent inhibition of TNF signaling may therefore result from incomplete blocking of p38
activation. Alternatively, these data may suggest a primary role of p38
kinase for responses to sodium salicylate but a secondary role for
responses to ATK.

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Fig. 4.
Effect of the p38 kinase inhibitor SB203580
on the inhibition of TNF-dependent degradation by
apoptosis-inducing agents. SB203580 ( , 10 µM) was
added to EC for 2 h prior to a further 2-h incubation without or
with ATK (50 µM), C6-ceramide (c-6
cer, 50 µM), sodium salicylate (Na Sal,
20 mM), or stauro (100 nM). After this
preincubation, TNF (50 units/ml) was added for a further 15 min.
Analysis of the degradation of I B was determined by Western
blotting and densitometry. Data are pooled from three independent
experiments and presented as the mean ± S.E. Significance was
assessed by a Student's t test. **, denotes
p < 0.01; ***, denotes p < 0.001. , mock pretreated cells.
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Initiation of Cell Death Reduces TNFR1 Expression and Recruitment
of TRADD--
The differential effects of apoptosis inducers on TNF-
and IL-1-dependent degradation of I
B
suggest that the
point of inhibition caused by the signaling cascade leading to
apoptosis must be upstream of the convergence of TNF and IL-1 signal
transduction cascades. In EC, TNF signaling begins with ligand binding
to TNFR2, which passes the TNF trimer to TNFR1. Previous studies in EC
have established that the R32W TNF mutein protein directly and
exclusively signals through TNFR1, bypassing the role of TNFR2 and
ligand passing (34). Sodium salicylate inhibits both R32W TNF- and wild
type TNF-dependent degradation of I
B
(Fig.
5). Similar results were obtained with
all other inducers of apoptosis (data not shown). These data indicate
that the point of inhibition of TNF-induced I
B
degradation caused
by apoptosis-inducing drugs is downstream of TNFR2.

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Fig. 5.
Sodium salicylate inhibits R32W TNF-mediated
I B degradation.
EC were pretreated for 2 h with M199 in the absence ( ) or
presence (+) of sodium salicylate (Na Sal, 20 mM) before treatment for a further 15 min with R32W TNF
(0-10 ng/ml). Cell lysates were analyzed for I B degradation by
Western blotting.
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We next examined the level of expression of cytosolic TNF signaling
components. Two h of pretreatment with apoptogenic drugs does not cause
a change in the size or quantity of the TRADD, TRAF2, or RIP adaptor
proteins (data not shown). A similar approach (i.e. Western
blotting) is not useful for evaluation of TNFR1 because the
preponderance of this receptor is found in a nonsignaling, Golgi-associated pool (38). However, FACS analysis (Fig.
6 and Table
I) show that 2 h of treatment with
apoptogenic agents reduces the surface expression of TNFR1 by at least
50%. Under these same conditions, the effects on surface expression of
TNFR2 were not statistically significant. Consistent with a reduction
in surface TNFR1, we observed that pretreatment with sodium salicylate
reduced TNF-induced recruitment of TRADD to the TNFR1 complex (Fig.
7). These data suggest that the common
mechanism shared by apoptogenic drugs is the reduction in TNFR1 surface
expression with consequent inhibition of receptor complex assembly.

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Fig. 6.
Reduction in surface expression of TNFR1 by
diverse apoptogenic agents. EC were treated for 2 h with M199
alone (A) or M199 in the presence of ATK (50 µM, B), C6-ceramide (c-6
cer, 50 µM, C), sodium salicylate
(Na Sal, 20 mM, D), or stauro (100 nM, E). Cells were stained by indirect
immunofluorescence and FACS-analyzed for TNFR1 (dashed line)
or irrelevant control mAb (K16/16, solid line)
binding.
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Table I
Effect of diverse apoptogenic agents on surface expression of TNFR1 and
TNFR2
EC were mock treated or treated for 2 h with apoptogenic agents
ATK (50 µM), C6-ceramide (50 µM),
sodium salicylate (20 mM), and stauro (100 nM).
Cells were harvested by brief collagenase digestion and FACS-stained
for TNFR1 and TNFR2. Data shown are pooled means ± S.E. for four
independent experiments corrected for background staining (K16/16) for
each treatment. Statistical significance observed using Student's
t test: *, p < 0.05; **, p < 0.01.
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Fig. 7.
Effect of sodium salicylate on
TNF-dependent recruitment of TRADD to TNFR1. Following
treatment with M199 in the absence ( ) or presence (+) of sodium
salicylate (Na Sal, 20 mM) for 2 h, EC were
stimulated with TNF (50 units/ml) for 5 min. The recruitment of TRADD
to TNFR1 is demonstrated by immunoprecipitation (IP) with
anti-TNFR1 and Western blotting for TRADD.
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Inhibition of TNFR1 Shedding Maintains TNF Signaling--
To
determine whether the decrease in surface TNFR1 accounted for the
inhibition of TNF signaling, we manipulated surface TNFR1 in the
presence of apoptosis-inducing agents using a tert-butyl derivative of Tapi. This agent is an inhibitor of TNF processing and is
comparable to that described as an effective inhibitor of the shedding
of TNFR1 (39). We observed that the decrease in surface staining of
TNFR1 resulting from treatment with apoptosis-inducing agents was
paralleled by an increase in soluble TNFR1 detected in culture
supernatant by an enzyme-linked immunosorbent assay (data not shown).
Pretreatment with Tapi (25 µM) for 30 min prior to the
addition of the apoptosis-inducing agents ATK and sodium salicylate
maintained surface expression of TNFR1 (Fig.
8A and data not shown).
Consistent with this observation, Tapi pretreatment also maintained
TNF-dependent degradation of I
B
(Fig. 8B).
However, Tapi did not protect EC from apoptosis in response to either
ATK or sodium salicylate (Fig.
8C). This evidence is
consistent with our hypothesis that TNFR1 shedding causes loss of TNF
signaling in response to apoptosis-inducing agents and suggests that
the signaling cascade leading to apoptosis in response to these agents is not dependent on the loss of TNFR1 for its progression.

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Fig. 8.
Reduction in surface TNFR1 and inhibition of
TNF signaling is blocked by Tapi without change in the induction of
apoptosis. A, EC were pretreated for 30 min with M199
in the absence (a-c) or presence of Tapi (25 µM, d-f) before the addition of vehicle
(a and d) or ATK (50 µM,
b and e) or sodium salicylate (20 mM,
c and f). Cells were stained by indirect
immunofluorescence and FACS-analyzed for TNFR1 (dashed line)
or irrelevant control mAb (K16/16, solid line) binding. The
numbers in parentheses indicate the mean
fluorescence of TNFR1 staining corrected for by subtraction of K16/16.
B, to determine the effect of Tapi on TNF signaling, EC were
pretreated with M199 in the presence or absence of Tapi before the
addition of vehicle without or with ATK (50 µM) or sodium
salicylate (Na Sal, 20 mM) for a further 2 h. Subsequent to this manipulation, EC were further treated by the
addition of vehicle without ( ) or with (+) TNF (50 units/ml) for 15 min. Analysis of the degradation of I B was determined by Western
blotting. C, EC were pretreated for 30 min with M199 in the
presence or absence of Tapi (25 µM) before the addition
of vehicle without or with ATK (50 µM) or sodium
salicylate (Na Sal, 20 mM) for a further 3 or
6 h, respectively. Cell survival was quantified by Hoescht
staining. CTR, control.
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|
 |
DISCUSSION |
In EC, the activation of NF
B in response to cytokines such as
TNF and IL-1 leads to the increased transcription of many genes including those of adhesion molecules such as E-selectin, ICAM-1, and
VCAM-1 as well as cytokines IL-6 and IL-8 (reviewed in Ref. 40). The
activation of these genes is critical in the recruitment of leukocytes
into inflammatory reactions. Inhibition of the signal transduction
cascade leading to the activation of NF
B is therefore a potential
target for novel anti-inflammatory reagents, and many drugs have been
reported as displaying such activities. In this study, we confirm that
a diverse range of chemical compounds can indeed inhibit TNF signaling.
Specifically we observed that ATK, C6-ceramide, sodium
salicylate, and stauro all inhibited TNF (and R32W TNF)- but not
IL-1-dependent degradation of I
B
. Such apparent specificity has often been interpreted as evidence for pharmacological inhibition of signaling. Significantly, none of these agents has been
shown to interact with any of the molecules known to mediate TNF
activation of NF
B-inducing kinase, namely TNFR2, TNFR1, TRADD, TRAF2, and RIP. However, we also noted that continued incubation with
each of the reagents causes apoptosis, leading us to propose an
alternative explanation, namely that the inhibition of
TNF-dependent degradation of I
B
results from an early
and common step in the signaling cascade leading to apoptosis rather
than from direct pharmacological targeting.
The progression of cellular apoptosis is known to be dependent on the
activation of a family of aspartic acid proteases termed caspases
(reviewed in Refs. 3 and 5), which cleave substrates such as PARP,
caspase-activated DNase, nuclear lamin, and focal adhesion kinase
(41-44) to cause DNA fragmentation, nuclear condensation, and
cytoplasmic shrinkage. Caspases have also recently been implicated in
the activation of signal transduction cascades by cleavage and the
activation of enzymes such as mitogen-activated protein kinase kinase
kinase-1 and cytosolic phospholipase A2 (45, 46). We
therefore questioned whether the caspase-dependent cleavage of a TNFR complex component could account for the inhibition of TNF
signaling. However, we could not show either that any TNF complex
component changed in concentration or molecular weight or that broad
spectrum caspase inhibitors zVADfmk or yVADcmk could relieve inhibition
due to apoptogenic agents. We next considered the role of the p38
protein kinase, which has been implicated in apoptosis in response to a
number of stimuli and in a number of cell types (36, 47-50). Recent
evidence has implicated p38 not only in apoptosis (36, 47, 49) but also
in the negative regulation of NF
B by sodium salicylate (37). As
described in previous studies in COS cells (37), the inhibitory effect
of sodium salicylate on the TNF signaling pathway could be completely reversed by pretreatment with SB203580. The effect on ATK was less
striking, possibly indicative of alternative mechanisms linking apoptosis to loss of signaling.
From our experiments with R32W TNF, we determined that the point of
inhibition targeted by apoptosis inducers did not involve TNFR2 or
ligand passing and so must lie at or between TNFR1- and NF
B-inducing
kinase. All four apoptogenic drugs caused a significant reduction in
surface TNFR1 expression, suggesting that an early event in the
initiation of programmed cell death results in the decrease of
expression of this receptor. To support the interpretation that the
decrease in surface TNFR1 accounted for the inhibition of TNF
signaling, we considered the effect of sodium salicylate on the
TNF-dependent recruitment of TRADD to the receptor complex. Our results demonstrate an early inhibition of the recruitment of this
signaling molecule. Thus the common mechanism of TNF signal disruption
by apoptogenic drugs appears to be loss of TNFR1. These new data also
provide an explanation of a prior observation that H2O2, an endogenous mediator of apoptosis,
inhibits TNF binding and TNF signaling in EC (51).
We established that the reduction of TNFR1 expression results from
receptor shedding, which can be effectively blocked by a derivative of
the metalloproteinase inhibitor Tapi. Soluble forms of both TNF
receptors have been identified in the body fluids of patients with
various diseases that are proteolytically derived from the cell surface
molecules (52, 53). These soluble receptors can also be detected in the
supernatant of cultured cells following both physiological and
nonphysiological activation (52, 54-56). A shed receptor can
neutralize the bioactivity of circulating TNF by binding to this
cytokine, thereby restricting its binding to surface-bound receptors
(57, 58). The observation that sodium salicylate is a potent inducer of
receptor shedding suggests that the neutralization of circulating TNF
may account in part for the action of sodium salicylate as an
anti-inflammatory. Shedding of TNFR1 is known to be independent of the
cytoplasmic region of the receptor (54) but largely dependent on the
spacer region between the transmembrane and a conserved extracellular
cysteine-rich domains (58). Most critically, residues Val-173 and
Lys-174 within the spacer region have been identified as essential for shedding, possibly allowing recognition of the receptor by a
metalloproteinase that ultimately cleaves the receptor from the surface
(58, 59). To date, the protease that specifically cleaves TNFR1 from
the surface of the cell has not been identified, and therefore it is
not known how the activity of the protease is regulated. Some evidence
suggests that the shedding of both TNF receptors may be dependent on a
phosphorylation event (60, 61). Such a mechanism could be implicated in
the shedding of TNFR1 induced by apoptogenic agents when p38 kinase is activated.
In conclusion, our observations provide a toxicological rather than
direct pharmacological explanation for the effects of many structurally
diverse agents on TNF signaling. Evidence points to a loss of TNFR1
from the cell surface as the key step involved in apoptosis-induced
inhibition of TNF signal transduction. We speculate that
down-regulation of TNF signaling through this mechanism may serve
to limit inflammation during apoptosis.