From the Departments of Medicine and
§ Surgery, University of Washington School of Medicine,
Seattle, Washington 98104 and the
Department of Molecular
Pharmacology, Isis Pharmaceuticals, Carlsbad, California 92008
Received for publication, January 29, 2001
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ABSTRACT |
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Lipopolysaccharide (LPS) has been implicated as
the bacterial component responsible for much of the endothelial cell
injury/dysfunction associated with Gram-negative bacterial infections.
Protein synthesis inhibition is required to sensitize the endothelium
to lipopolysaccharide-induced apoptosis, suggesting that a constitutive
or inducible cytoprotective protein(s) is required for endothelial
survival. We have identified two known endothelial anti-apoptotic
proteins, c-FLIP and Mcl-1, the expression of which is decreased
markedly in the presence of cycloheximide. Decreased expression of both
proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not
the decreased expression of c-FLIP and Mcl-1, suggesting that they
exert protection upstream of caspase activation. Inhibition of the
degradation of these two proteins with the proteasome inhibitor,
lactacystin, prevented lipopolysaccharide + cycloheximide-induced
apoptosis. Similarly, lactacystin protected against endothelial
apoptosis induced by either tumor necrosis factor- Despite advances in anti-microbial therapy and overall
medical care, Gram-negative bacterial sepsis remains a common,
life-threatening event. The challenge of managing septic patients is
compounded by the development of key vascular complications including,
systemic vascular collapse, disseminated intravascular coagulation, and vascular leak syndromes (1-5). A common denominator to all these complications is endothelial cell
(EC)1 injury and/or
dysfunction. Endotoxin or lipopolysaccharide (LPS), which resides
in the outer membrane of Gram-negative bacteria, has been implicated as
the causative agent responsible for EC dysfunction (1, 5-7). In the
absence of non-endothelial cell-derived host mediator systems, LPS
directly evokes numerous EC responses including: 1) up-regulation of
adhesion molecules; 2) increased production of cytokines, nitric oxide,
and tissue factor; 3) loss of monolayer integrity and barrier function;
and 4) apoptosis (8). In addition to a direct role, LPS stimulates the
production of inflammatory cytokines, including interleukin (IL)-1 LPS-induced EC apoptosis has been observed both in vitro
(10-14) and in vivo (15, 16). LPS directly induces
apoptosis in bovine and ovine EC (10, 12, 13, 17). Sensitization of human EC to LPS-induced apoptosis requires the inhibition of either mRNA or protein synthesis (18). This latter finding suggests that
either a constitutively expressed protein with a relatively short
half-life or an inducible protein is requisite for EC survival following LPS exposure. Similarly, two LPS-inducible cytokines, IL-1 Apoptosis has been implicated as an important mechanism of in
vivo cell death following LPS exposure. Tissues and organs
obtained from either patients who have died of sepsis and multi-organ
failure (19) or animal models of endotoxemia and sepsis reveal enhanced apoptotic cell death (16, 20, 21). The vascular endothelium is one
tissue that is sensitive to LPS-induced apoptosis. In a murine model of
sepsis, apoptotic EC have been detected in pulmonary capillaries (20).
Intravenous administration of endotoxin into rabbits or rats induces EC
death and detachment from the artery wall (22, 23). In mice challenged
with either LPS or TNF- The mechanisms by which LPS activates apoptosis remain unknown. Efforts
to elucidate LPS signaling pathways, apoptotic or otherwise, have
been hampered by the lack of an identifiable membrane-bound receptor
capable of signal transduction. Recently, Toll-like receptor (TLR)-4
has been identified in both cells of monocytic lineage and EC as the
receptor responsible for LPS activation of the NF- The anti-apoptotic pathways utilized by human EC to resist LPS-induced
apoptosis have yet to be elucidated. Human EC sensitivity to
LPS-induced apoptosis is dependent on new protein or mRNA synthesis inhibition, suggesting that either an inducible or constitutively expressed protein is responsible for protection (30). LPS has previously been shown to up-regulate two cytoprotective proteins, the
Bcl-2 homologue, A1, and the zinc-finger protein, A20, in EC (30).
Overexpression of A1, A20, or Bcl-xL confers partial protection against LPS and cycloheximide (CHX)-induced apoptosis. There
is doubt, however, concerning the physiological relevance of these
overexpression studies. Similar to LPS, sensitization of human EC to
TNF- Materials--
The caspase inhibitor peptide,
z-VAD-fluoromethylketone (z-VAD), CHX, and the proteasome
inhibitors, EC Culture--
Human umbilical vein EC were obtained from
collagenase-digested umbilical veins and cultured in RPMI medium
(Biowhittaker, Walkersville, MD) enriched with 20% bovine calf serum
(HyClone Laboratories, Logan, UT), endothelial cell growth factor
prepared from bovine hypothalamus, heparin (90 µg/ml; Sigma),
L-glutamine (2 mM), HEPES (10 mM),
sodium pyruvate (1 mM), and nonessential amino acids, in
the presence of penicillin (100 units/ml), streptomycin (100 µg/ml),
and fungizone (0.25 µg/ml) (all purchased from Biowhittaker). Only
cells from passage 2 or 3 were studied.
Immunoblotting--
EC monolayers were washed once with
phosphate-buffered saline (PBS), lysed with ice-cold modified
radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl
(pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, protease inhibitor mixture
tablet (Roche Molecular Biochemicals), 1 mM vanadate, 50 mM NaF), scraped, transferred to microcentrifuge tubes, and centrifuged (16,000 × g, 10 min, 4 °C). Total
protein was determined using the BCA protein assay (Pierce). The
supernatants were combined with 5× sample buffer (Genomic Solutions
Inc., Chelmsford, MA), boiled for 3 min, and 20 µg of protein/lane
were resolved by SDS-polyacrylamide gel electrophoresis on a 4-20%
Tris-glycine gradient gel (Novex Inc., San Diego, CA). Protein was
subsequently transferred for 1 h at 100 v to polyvinylidene
fluoride (PVDF) membrane (Millipore Corp, Bedford, MA). Blots were
blocked with 5% dry milk and then incubated with anti-Bcl-2 (0.5 µg/ml), anti-Bcl-xL (0.25 µg/ml), anti-Bax (1.0 µg/ml) (all purchased from Transduction Laboratories Inc., Lexington,
KY), anti-Akt (1:1000 dilution; New England Biolabs, Inc., Beverly,
MA), anti-Mcl-1 (1:1000 dilution; PharMingen International, San Diego,
CA), or anti-c-FLIP (NF6; 1:20 dilution; generous gift of Dr. Peter H. Krammer of the German Cancer Research Center, Heidelberg, Germany) (34)
antibodies for 1 h at room temperature. The blots were incubated
with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit
immunoglobulin G (IgG) (0.13 µg/ml; Transduction Laboratories),
developed with enhanced chemiluminescence (Amersham Pharmacia
Biotech), and exposed to Kodak X-Omat Blue film (PerkinElmer Life
Sciences). To ensure equal protein loading, select blots were stripped
with 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate,
62.5 mM Tris-HCl (pH 6.7), at 70 °C for 30 min, washed,
blocked, and re-probed with anti- Detection of Apoptosis--
EC viability was measured with
Alamar Blue vital dye (35, 36), and apoptosis was assayed by the
detection of poly(ADP-ribose) polymerase (PARP) cleavage (37, 38) and
histone release into the cytoplasm (39). To assess viability, EC were
seeded into 96-well plates at a density of 20,000 cells/well. After
48 h in culture, cells were subjected to various experimental
conditions and 3 h prior to the end of treatment, Alamar Blue
(10% final concentration; BIOSOURCE
International, Inc., Camarillo, CA) was added. Monolayers were assayed
in a Cytofluor Series 4000 fluorescence plate reader (Perseptive
Biosystems Inc., Framingham, MA) at 530 nm excitation and 590 nm
emission and viability expressed in arbitrary fluorescent units.
For the detection of PARP cleavage, EC were scraped into ice-cold PBS,
briefly centrifuged, and an equal number of EC resuspended in lysis
buffer (62.5 mM Tris-HCl (pH 6.8), 6 M urea,
10% glycerol, 2% sodium dodecyl sulfate, 5% Vascular Cell Adhesion Molecule-1 (VCAM-1) Enzyme-linked
Immunosorbent Assay--
EC were seeded into 96-well plates at a
density of 20,000 cells/well and cultured for 48 h. Following
treatment, cells were washed twice with RPMI media supplemented with
2.5% bovine calf serum and incubated with 5 µg/ml anti-VCAM-1
monoclonal antibody (clone 4B9) for 1 h at 37 °C. Monolayers
were washed as above, fixed for 10 min with 0.05% glutaraldehyde in
PBS, washed again, and incubated with HRP-conjugated anti-mouse IgG (1 µg/ml) (Transduction Laboratories) for 1 h at 37 °C. EC were
then washed five times with PBS, developing solution (1 mg/ml
o-phenylaminediamine in 0.1 M sodium citrate (pH
4.5) with 0.003% hydrogen peroxide) added for 10 min at room
temperature, and plates analyzed at 490 nm on a microplate reader
(Bio-Tec Instruments, Inc.).
Adenoviral Transduction--
The I Oligonucleotide Design and
Synthesis--
2'-O-Methoxyethyl/2'-deoxynucleotide
chimeric antisense oligonucleotides (32, 41) were used in all
experiments. Chimeric oligonucleotides were used to support an RNase
H-dependent mechanism of action, which results in a
selective loss of target mRNA (42). All oligonucleotides were
synthesized and purified as described previously (32, 41). The c-FLIP
and Mcl-1 antisense oligonucleotides, respectively, were identified by
screening a series of different antisense oligonucleotides designed to
hybridize to their respective targets using quantitative reverse
transcription-polymerase chain reaction and Northern blot assays (43).
ISIS 23296 and ISIS 20408 were found to be the most effective
inhibitors of c-FLIP and Mcl-1 expression, respectively. The control
antisense oligonucleotide contained eight or four mismatches, as
compared with FLIP and Mcl-1 antisense oligonucleotides, respectively.
Sequence of the oligonucleotides used are shown in Table
I.
Oligonucleotide Transfection of EC--
EC were seeded into
60-mm dishes and grown to ~80% confluence. EC were rinsed twice with
Opti-MEM I (Life Technologies, Inc.) followed by incubation with 200 nM oligonucleotides premixed with 10 µg/ml Lipofectin
reagent (Life Technologies, Inc.) in Opti-MEM I. After a 4-h incubation
at 37 °C, the oligonucleotide solution was replaced with normal
growth medium and EC allowed to recover for 8 h. EC were then
treated with LPS in the presence or absence of CHX for 12 h and
assayed for protein expression and apoptosis.
Statistical Methods--
Analyses of variance was used to
compare the mean responses among experimental and control groups. The
Tukey post hoc comparison test was used to determine between
which groups significant differences existed. All statistical analyses
were performed using GraphPad Prism version 3.00 for Windows 95 (GraphPad Software, Inc., San Diego, CA). A p value of
<0.05 was considered significant.
Time-dependent Effect of LPS-induced EC Apoptosis in
the Absence of New Protein Synthesis--
Exposure to LPS+CHX induced
EC death in a time-dependent manner (Fig.
1A). An 8-h
stimulus-to-response lag time was observed following LPS+CHX treatment.
EC exposed to CHX alone demonstrated no change in viability over this
time period, consistent with the ability of CHX to inhibit de
novo protein synthesis and cell proliferation without inducing EC
death. Increased metabolic activity in EC exposed to medium or LPS
alone reflected continued EC proliferation over time. To confirm that
the decreased EC viability following administration of LPS+CHX was due
to apoptosis, cleavage of PARP and histone release into the cytoplasm
were assayed (Fig. 1, B and C). PARP is a known
substrate of caspases, a family of highly specific proteases activated
during apoptosis (38). Western blot analysis revealed PARP cleavage
only in lysates derived from EC exposed to LPS in combination with CHX.
Consistent with these data, Hu et al. (30) reported that EC
treated with LPS alone or CHX alone demonstrate no evidence of
apoptosis, but upon co-administration of both LPS and CHX, cleavage of
DNA into repeating oligonucleosomal fragments of 180 base pairs, a
hallmark of apoptosis, was observed. Cleavage of PARP was
time-dependent and observed at exposures of
The requisite inhibition of de novo protein synthesis
necessary for EC sensitization to LPS-induced apoptosis has been
reported for other inflammatory stimuli as well, including TNF- Western Blot Analysis of Anti-apoptotic Proteins following EC
Exposure to LPS and CHX--
That inhibition of de novo
protein synthesis sensitizes EC to LPS-induced apoptosis suggests that
either a rapidly degraded constitutive protein and/or an inducible
protein confer resistance under physiological conditions. We,
therefore, screened EC exposed to medium, LPS, CHX, or LPS+CHX for
changes in the expression of known anti-apoptotic proteins
constitutively expressed in human EC, including Akt (44), c-FLIP (45),
Mcl-1(32), Bcl-xL (32), and Bcl-2 (46). The expression of
two proteins, FLIP and Mcl-1, was shown to decrease in the presence of
CHX (Fig. 2A). The expression of all other cytoprotective proteins assayed, as well as the
pro-apoptotic protein, Bax, remained stable even after a 16-h
exposure. Decreases in the protein levels of both FLIP and Mcl-1 were
demonstrated within 2 h of CHX exposure, a time frame that
precedes the onset of LPS+CHX-induced apoptosis (Fig.
2B).
Interestingly, exposure of EC to both LPS and CHX resulted in a greater
decrease in Mcl-1 expression at longer time points (
FLIP is an anti-apoptotic protein with significant homology to
caspase-8 (47). A substitution of two amino acids in the region of FLIP
that corresponds to the catalytic active site of caspase-8 renders it
incapable of proteolysis. The role of FLIP in the inhibition of FAS
death signaling has been well elucidated. Upon Fas ligand binding, the
adapter protein FADD is recruited to the Fas receptor via an
interaction between the DD of each protein (48). FADD, in turn, can
recruit FLIP and pro-caspase-8 via protein-protein interactions of
death effector domains contained within all three proteins.
Pro-caspase-8 has intrinsically low levels of proteolytic activity,
which enables it to cleave other pro-caspase-8 proteins brought into
close proximity following their recruitment to FADD (49). Activation of
caspase-8 initiates a proteolytic cascade resulting in the activation
of downstream effector caspases (50). FLIP has been proposed to inhibit
activation of upstream initiator caspases, including caspase-8, by
competitively binding to FADD and blocking assembly of a functional
death signaling complex (51). Alternatively, FADD recruitment of both
pro-caspase-8 and FLIP may occur; however, the lack of intrinsic FLIP
proteolytic activity prevents the transactivation of pro-caspase-8
(34).
The TLR family of receptors has been implicated in LPS signaling (25,
52). An adapter protein that plays an integral role in TLR signaling,
MyD88, contains a conserved DD (28). We have previously reported that
another DD-containing protein, FADD, is required for LPS+CHX-induced
apoptosis (11). A recent report suggests that MyD88 can interact with
FADD through reciprocal binding of their DD (53). Analogous to the Fas
pathway, the involvement of FADD in the LPS death pathway is compatible
with a protective role for FLIP.
Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins
primarily localized to mitochondrial membranes (54). The Bcl-2 family
contains both pro- and anti-apoptotic members capable of
heterodimerization. In response to certain apoptotic stimuli, the
relative ratios of these pro- and anti-apoptotic proteins can dictate
whether a cell will survive or undergo apoptosis (55). Bcl-2 exerts its
protection by preventing the release of cytochrome c from
the mitochondrion, the latter being a co-factor with Apaf-1 in the
activation of pro-caspase-9 (56, 57). Activation of caspase-9 results
in a cascade effect on the activation of effector caspases culminating
in apoptosis. Reportedly, Bcl-2 can also block killing downstream of
and independent of cytochrome c release (58).
Mcl-1, unlike other Bcl-2 members, contains two PEST sequences, which
target it for rapid turnover via proteasome-mediated degradation (54).
Correspondingly, inhibition of new protein synthesis resulted in
decreased expression of Mcl-1, but no change in the expression levels
of other Bcl-2 family members, including Bcl-2, Bcl-xL, and
Bax (Fig. 2A). The implication of a cytoprotective role for
Mcl-1 is consistent with previous reports that overexpression of other
Bcl-2 homologues, including Bcl-xL and A1, confers partial protection against LPS- or TNF- Caspase Inhibition Blocks LPS-induced Apoptosis but Fails to
Protect against Decreased FLIP and Mcl-1 Expression--
The
cell-permeable peptide, z-VAD, a highly specific caspase inhibitor with
broad selectivity for several members of the caspase family (59, 60),
completely protected against LPS+CHX-induced EC apoptosis (Fig.
3, A and B).
Caspase inhibition blocked decreased EC viability (Fig. 3A),
cleavage of the caspase substrate, PARP (Fig. 3B), and
histone release (data not shown) in response to LPS+CHX exposure. These
findings are in agreement with previous reports that LPS+CHX-induced EC
death is apoptotic in nature and mediated through caspase activation
(11).
During LPS-induced EC apoptosis, cellular proteins are cleaved after
caspase activation (10). To determine whether the decreased levels of
FLIP and Mcl-1 could be attributed to caspase-mediated proteolytic
degradation, EC were exposed to LPS+CHX in the presence or absence of
z-VAD (Fig. 3C). Caspase inhibition did not block decreased
FLIP expression and offered only modest protection against changes in
Mcl-1 levels. This latter observation is consistent with the findings
in Fig. 2 that the decrement in Mcl-1 expression following CHX exposure
is enhanced with the co-administration of LPS, suggesting that Mcl-1 is
also a substrate for activated caspases. The caspase-independent
decrease of FLIP and Mcl-1 is consistent with the decrease in their
expression preceding both cleavage of PARP, a known caspase substrate,
and the onset of apoptosis. Furthermore, CHX exposure alone decreased
the expression levels of both FLIP and Mcl-1 in the absence of
detectable apoptosis. These data suggest that decreased EC levels of
FLIP and Mcl-1 following CHX or LPS+CHX exposure are due to
intrinsically short half-lives of these proteins and not apoptotic processes.
Proteasome Inhibition Protects against FLIP and Mcl-1 Degradation
and LPS+CHX-induced EC Apoptosis--
There are two predominant
mechanisms for intracellular degradation of proteins, one involving the
lysosomal apparatus and the other involving the proteasome (61). The
lysosomal pathway is primarily involved in the proteolytic degradation
of membrane-bound proteins and extracellular proteins, whereas the
proteasome pathway is primarily responsible for degradation of
cytosolic proteins. To determine whether CHX-induced decreases in the
levels of FLIP and Mcl-1 could be attributed to rapid turnover via the
proteasome, EC were pre-treated with a highly specific proteasome
inhibitor, lactacystin, and its derivative
Based upon the findings that 1) decreased levels of FLIP and Mcl-1
occur independently of caspase activation, 2) changes in FLIP and
Mcl-1 expression precede the onset of apoptosis, and 3) inhibition of
new protein synthesis is required for EC sensitization to LPS, a
consequence of which is decreased expression of these two
cytoprotective proteins, we hypothesized that preventing FLIP and/or
Mcl-1 degradation would block LPS+CHX-induced apoptosis. Therefore, EC
were pre-treated with lactacystin and
The requisite inhibition of new gene expression necessary for EC
sensitization to LPS-induced apoptosis suggests that either a
constitutive or inducible protein is required for cytoprotection. That
inhibition of proteasome-dependent protein degradation in the absence of protein synthesis blocks LPS+CHX-evoked apoptosis implicates a cytoprotective role for a constitutively expressed protein(s). In contrast to this finding, a previous study reports that
overexpression of an inducible protein, A1, partially protects against
LPS+CHX- and TNF- Proteasome Inhibition Confers Protection against Apoptosis Elicited
by TNF-
A common signaling pathway shared by LPS, TNF-
Lactacystin, by virtue of its ability to inhibit proteasome activity,
should block NF-
The role of NF- FLIP Antisense Sensitizes EC to LPS-induced Apoptosis at Low CHX
Concentration--
To elucidate a definitive anti-apoptotic role
for either FLIP and/or Mcl-1 against LPS-induced apoptosis, antisense
oligonucleotides were designed to specifically reduce the expression of
each protein. Western blot analysis of EC transfected with either FLIP
or Mcl-1 antisense revealed a dramatic decrement in the expression of
FLIP and Mcl-1, respectively, compared with EC transfected with
mismatch oligonucleotide controls (Fig.
7, A and C). Under
identical conditions, we exposed the transfected cells to medium or LPS
(100 ng/ml) for 12 h and assayed for histone release (Fig. 7,
B and D). The decrease in FLIP or Mcl-1 with
antisense oligonucleotides failed to directly sensitize EC to
LPS-induced apoptosis. Since the antisense oligonucleotides reduced the
level of each protein but failed to completely ablate expression, we
postulated that minimal threshold level expression of either protein
could confer protection. To test this hypothesis, we exposed EC
transfected with FLIP or Mcl-1 antisense to LPS and a low concentration
of CHX (2 µg/ml) and assayed for protein expression levels (Fig. 7,
A and C) and apoptosis (Fig. 7, B and
D). FLIP antisense in combination with low dose CHX (2 µg/ml) resulted in almost complete knockout of FLIP expression (Fig.
7A) and sensitized EC to LPS killing (Fig. 7B).
In contrast, EC transfected with mismatch control oligonucleotides had
detectable levels of FLIP expression and were resistant to LPS killing.
Doubling the concentration of CHX sensitized these cells to LPS
killing, albeit to a lesser extent than those transfected with FLIP
antisense (Fig. 7B). These data implicate a role for the
constitutively expressed protein FLIP in conferring protection against
LPS-induced EC apoptosis. Using a low dose of CHX in combination with
FLIP antisense, cellular levels of FLIP were reduced to barely
detectable limits, and sensitivity to LPS killing was conferred.
Importantly, this concentration of CHX alone failed to sensitize
control EC to LPS killing, indicating that the specific reduction of
FLIP sensitized EC to LPS-induced apoptosis. FLIP levels in
control-transfected EC exposed to low dose CHX (2 µg/ml) were
comparable to those in EC transfected with FLIP antisense without CHX
exposure (Fig. 7A). In both cases, sensitivity to LPS
killing was not observed, suggesting that a threshold level of FLIP
conferred cytoprotection. At higher concentrations of CHX, a
dose-dependent increase in apoptosis among
control-transfected EC was demonstrated (Fig. 7B),
consistent with Fig. 2, in which high concentrations of CHX completely
diminished FLIP levels. Although this finding clearly implicates a
cytoprotective role for FLIP, it does not preclude the requisite
involvement of another unidentified cytoprotective protein. It is
possible that sensitivity to LPS killing was conferred to EC
transfected with FLIP antisense, not only by reducing FLIP expression
below minimal threshold levels necessary for protection, but also by
reducing the level of another cytoprotective protein.
Similar to FLIP antisense, Mcl-1 antisense in the presence of low
dose CHX (2 µg/ml) decreased Mcl-1 expression below detectable levels
(Fig. 7C). However, in contrast to FLIP antisense, Mcl-1 antisense neither sensitized EC to LPS-induced apoptosis nor enhanced killing compared with mismatch oligonucleotides in the presence of any
concentration of CHX tested (Fig. 7D).
In summary, we have characterized a cytoprotective pathway in EC that
confers resistance against LPS-induced apoptosis (Fig. 8). First, we established that inhibition
of gene expression sensitizes EC to LPS-evoked killing by blocking
de novo synthesis of a constitutively expressed
cytoprotective protein(s), not an inducible protein. Second, this
cytoprotective protein(s) has a relatively short half-life and is
degraded through the proteasome. Third, EC apoptosis induced by two
other inflammatory mediators, TNF- or
interleukin-1
in the presence of cycloheximide. That apoptosis could
be blocked in the absence of new protein synthesis by inhibition of the
proteasome degradative pathway implicates the requisite involvement of
a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and tumor necrosis factor (TNF)-
, which also elicit an altered
pathophysiological endothelial state (9).
and TNF-
, induce human EC apoptosis only when new gene expression is
blocked (18).
, disseminated EC apoptosis has been
reported in the lung, thymus, and intestine (15, 16, 24). Finally,
injection of a broad spectrum caspase inhibitor following LPS
administration decreased EC apoptosis in the lung and improved survival
in a murine model of acute lung injury (24). Together, these in
vitro and in vivo studies indicate that the vascular
endothelium is a key target of LPS-induced apoptosis.
B signaling
pathway (25-27). Interestingly, MyD88, a TLR-4-binding protein that
has a requisite role in the downstream activation of NF-
B in EC,
contains a death domain (DD) (28, 29). DD are conserved regions of
amino acids, which facilitate protein-protein interaction. In the well
characterized TNF pathway, the TNF receptor-binding protein, TRADD,
recruits another adapter protein, FADD, through the interaction of
their respective death domains. An additional conserved sequence in
FADD, the death effector domain (DED), enables the recruitment of
caspase 8, an upstream cysteine protease whose activation initiates a
cascade of proteolytic events characteristic of apoptosis. We have
previously reported that LPS-induced EC apoptosis is
FADD-dependent (11). Whether FADD is recruited to MyD88
following LPS stimulation via binding of their respective DD remains unknown.
-induced apoptosis requires inhibition of gene expression.
Further, overexpression of A1 or Bcl-xL protects EC against
TNF-
and CHX induced-apoptosis (31). The selective inhibition of A1
or Bcl-xL gene expression, however, fails to sensitize
human EC to direct TNF-
-induced apoptosis, indicating that these
proteins are not critical for protection (32). Another proposed
mechanism of protection is through NF-
B-dependent gene expression of certain cytoprotective proteins, including cellular inhibitor of apoptosis proteins (cIAP) (33). Selective blockade of the
NF-
B signaling pathway by expression of a mutant I
B sensitizes EC
to apoptosis induced by prolonged exposure to TNF-
, but not LPS
(14). This finding suggests that cell survival following LPS exposure
is NF-
B-independent. In the present report, we have attempted to
elucidate the mechanism of human EC resistance to LPS-induced apoptosis.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-lactone and lactacystin, were purchased from
Calbiochem-Novabiochem Corp. (La Jolla, CA). Staurosporine was obtained
from Kamiya Biomedical Co. (Seattle, WA). LPS from Escherichia
coli serotype 055:B5 and dimethyl sulfoxide (Me2SO)
were purchased from Sigma. Recombinant human TNF
and IL-1
were purchased from R&D Systems, Inc.
-tubulin murine monoclonal antibody
(0.5 µg/ml; Roche Molecular Biochemicals) followed by HRP-conjugated
anti-mouse IgG (0.13 µg/ml) (Transduction Laboratories).
-mercaptoethanol, and
.002% bromphenol blue). EC lysates were then sonicated on ice for
15 s, incubated for 15 min at 65 °C, loaded on a 4-20%
Tris-glycine gradient gel, and transferred to PVDF membrane. After
blocking, the blots were incubated with anti-PARP antibody (1 µg/ml;
Biomol Research Laboratories, Inc., Plymouth Meeting, MA), subsequently
incubated with HRP-conjugated anti-mouse IgG, and developed as above.
For the quantitation of apoptosis, EC were seeded into 96-well
plates at a density of 20,000 cells/well, cultured for 48 h,
treated, and histone release measured with the Cell Death Detection
ELISAPlus assay according to the manufacturer's
instructions (Roche Molecular Biochemicals). The plates were analyzed
at 405 and 450 nm (reference wavelength) on a microplate reader
(Bio-Tec Instruments, Inc., Winooski, VT). Background readings defined
as EC exposed to medium alone were subtracted from each experimental treatment.
B
mutant (I
B
M) and
-galactosidase recombinant adenoviruses were gifts of Dr. C. B. Wilson (University of Washington, Seattle, WA). The I
B
M
(S32A/S36A) cDNA was generously provided by Drs. J. DiDonato and M. Karin (University of California, San Diego, CA) (40). Construction and
purification of I
B
M and control (
-galactosidase) recombinant
adenovirus were performed as previously described (14). For adenoviral
transduction, EC were seeded into 96-well plates at a density of 8,000 cells/well for 24 h, washed once with medium, and incubated for
72 h at a multiplicity of infection of 1000 with control or
I
B
M adenovirus in complete EC medium. EC were washed once with
complete medium and subjected to experimental treatment.
Sequence of the c-FLIP, Mcl-1, and control oligonucleotides
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
12 h. Another
hallmark of apoptosis is the release of histones into the cytoplasm
(39). Using a highly sensitive ELISA-based assay, the presence of
cytosolic mono- and oligonucleosomes were only detected in EC exposed
to both LPS and cycloheximide for
12 h (Fig. 1C),
consistent with the detection of PARP cleavage.
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Fig. 1.
Time-dependent effect of LPS+CHX
on EC apoptosis. EC were incubated with medium or LPS (100 ng/ml)
in the presence or absence of CHX (40 µg/ml) for increasing exposure
times (A-C). EC viability was measured using the Alamar
Blue assay of metabolic activity (A). Mean (± S.E.)
metabolic activity is reported in arbitrary fluorescent units. *,
significantly decreased compared with all other conditions at the same
time point as well as the same treatment at the 4-h time point. In
other experiments, EC lysates were immunoblotted with an antibody
raised against PARP, a caspase substrate cleaved during apoptosis
(B). Molecular mass (in kDa) is indicated. A quantitative
assay for apoptosis, which measures histone release into the cytosol,
was performed (C). Mean (± S.E.) histone release is
reported in OD units. *, significantly increased compared with all
other conditions at the same time point.
and
IL-1
(14). Further, LPS or TNF-
in the presence of actinomycin D, an inhibitor of mRNA synthesis, induces EC apoptosis suggesting that inhibition of gene expression regardless of whether it occurs at
the protein or mRNA level is sufficient for sensitization
(18). The ability of LPS to induce EC apoptosis only in the absence of
new gene expression implies that either an inducible or constitutively expressed cytoprotective protein is required for EC resistance to
LPS-evoked cell death.
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Fig. 2.
Time-dependent effect of protein
synthesis inhibition on changes in the expression levels of known EC
anti-apoptotic proteins. EC were incubated with medium or LPS (100 ng/ml) in the presence or absence of CHX (40 µg/ml) for increasing
exposure times (A and B). EC lysates were
immunoblotted with antibodies raised against the anti-apoptotic
proteins, Akt, FLIP, Mcl-1, Bcl-2, Bcl-xL, or the
pro-apoptotic protein, Bax. To demonstrate equal protein loading, blots
were reprobed with an antibody against -tubulin. Molecular mass (in
kDa) is indicated.
4 h) than EC
exposed to CHX alone. This latter finding may suggest that, after
activation of EC apoptosis by LPS, Mcl-1 becomes a downstream target of
caspases. In contrast, diminished levels of FLIP were comparable in EC
exposed to CHX alone or in combination with LPS.
-induced EC apoptosis (30, 31). An essential protective role, however, for Bcl-xL and A1 at
physiological levels is uncertain as selective inhibition of their
expression by antisense oligonucleotides fails to sensitize EC to
TNF-
-mediated killing (32).
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Fig. 3.
Effect of caspase inhibition on
LPS+CHX-induced apoptosis. EC were pre-treated for
1 h with Me2SO or the caspase inhibitor peptide, z-VAD
(100 µM), and subsequently exposed to medium or LPS (100 ng/ml) in the presence or absence of CHX (40 µg/ml) for 12 h
(A-C). Viability was assayed with the Alamar Blue vital dye
(A). Vertical bars represent mean (± S.E.) metabolic activity in arbitrary fluorescent units. *,
significantly decreased compared with all other conditions. **,
significantly increased compared with LPS+CHX. In other experiments, EC
lysates were immunoblotted with either an antibody raised against the
caspase substrate, PARP (B) or antibodies raised against
FLIP or Mcl-1 (C). Molecular mass (in kDa) is indicated.
-lactone (62, 63), and
subsequently exposed to medium, LPS, CHX, or LPS+CHX (Fig.
4A). Lactacystin protected
against decreased levels of both FLIP and Mcl-1 in the presence of CHX,
indicating that, once protein synthesis is inhibited with CHX, FLIP and
Mcl-1 levels are rapidly decreased through a degradation process
involving the proteasome.
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Fig. 4.
Proteasome inhibition protects against
decreased expression of FLIP and Mcl-1 and LPS-induced apoptosis in the
absence of new protein synthesis. EC were pre-treated for 30 min
with either Me2SO or a combination of lactacystin (10 µM) and its aqueous derivative -lactone (10 µM) (LAC), and subsequently exposed to either
medium or LPS (100 ng/ml) in the presence or absence of CHX (40 µg/ml) for 12 h (A and B). EC lysates were
immunoblotted with anti-FLIP or anti-Mcl-1 antibodies (A).
Molecular mass (in kDa) is indicated. In other experiments, EC
viability was measured with Alamar Blue (B).
Vertical bars represent mean (± S.E.) metabolic
activity in arbitrary fluorescent units. *, significantly decreased
compared with all other conditions. **, significantly increased
compared with LPS+CHX.
-lactone, exposed to medium,
LPS, CHX, or LPS+CHX, and viability assessed (Fig. 4B).
Under identical experimental conditions, proteasome inhibition, which
prevented the degradation of FLIP and Mcl-1, also conferred complete
protection against LPS+CHX-induced decrements in viability.
Measurement of histone release confirmed a protective effect of
proteasome inhibition against LPS+CHX-induced apoptosis (data not shown).
+CHX-induced EC apoptosis (30, 31). Since the
authors report that overexpression of two other cytoprotective proteins, Bcl-xL and A20, also confer partial protection,
there is some question as to the physiological implications of these overexpression data. In fact, selective inhibition of A1 gene expression fails to sensitize EC to direct TNF-
killing, suggesting EC sensitization following CHX exposure is not attributed to the inhibition of A1 induction (32).
+CHX or Il-1
+CHX--
TNF-
and Il-1
are established
mediators of the acute phase response to Gram-negative bacterial
infections (64-66). Similar to LPS, their ability to induce EC
apoptosis requires the inhibition of new gene expression (Fig.
5A). To determine whether a
common cytoprotective pathway is operative in the resistance of EC to these inflammatory mediators, EC were pre-treated with lactacystin and
subsequently exposed to either TNF-
or Il-1
in the presence of
CHX (Fig. 5B). Lactacystin completely protected against
apoptosis elicited by these agents. Interestingly, lactacystin
conferred only marginal protection against apoptosis elicited by
staurosporine (Fig. 5B), which evokes direct EC killing
without requisite inhibition of new gene expression (Fig.
5A).
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Fig. 5.
Effect of caspase and proteasome inhibition
on TNF- +CHX-,
IL-1
+CHX-, and staurosporine-induced
apoptosis. EC were pre-treated for 1 h with Me2SO
or the caspase inhibitor peptide, z-VAD (100 µM)
(A), or pre-treated for 30 min with either Me2SO
or a combination of lactacystin (10 µM) and its aqueous
derivative,
-lactone (10 µM) (LAC)
(B). EC were subsequently exposed for 12 h to medium,
TNF-
(10 ng/ml), IL-1
(10 ng/ml), or staurosporine
(STA; 1 µM) in the presence or absence of CHX
(40 µg/ml) (A and B). EC viability was measured
with Alamar Blue (A and B). Vertical
bars represent mean (± S.E.) metabolic activity in
arbitrary fluorescent units. *, significantly decreased compared with
medium and CHX. **, significantly increased compared with TNF-
+CHX,
IL-1
+CHX, and staurosporine.
, and IL-1
involves
the activation of the transcription factor, NF-
B (67). NF-
B
regulates the expression of several gene products involved in
inflammatory responses, including up-regulation of TNF-
, Il-1
, IL-6, IL-8, VCAM-1, and E-selectin (67, 68). In unstimulated cells,
NF-
B is sequestered in the cytoplasm by members of the I
B family.
Upon cell activation, I
B is phosphorylated on two serine residues,
targeting it for proteasome-mediated degradation. The resultant free
NF-
B is then able to translocate to the nucleus, where it promotes
new gene expression.
B activation by blocking the degradation of its
inhibitor, I
B. As predicted, lactacystin completely blocked LPS-,
TNF-
-, and IL-1
-induced up-regulation of VCAM-1, the expression of which is dependent on NF-
B activation (Fig.
6A). These data are in
agreement with a previous report that lactacystin inhibits EC NF-
B
activation assayed by an electrophoretic mobility shift (69).
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Fig. 6.
Effect of inhibition of the proteasome and
NF- B activation on VCAM-1 expression and
apoptosis. EC were pre-treated with Me2SO or the
caspase inhibitor peptide, z-VAD (100 µM) for 1 h,
or pre-treated for 30 min with the proteasome inhibitor lactacystin (10 µM), and its aqueous derivative
-lactone (10 µM) (LAC) (A). EC were subsequently
exposed to either medium, LPS (100 ng/ml), TNF-
(10 ng/ml), or
IL-1
(10 ng/ml), for 4.5 h. VCAM-1 cell surface expression was
assayed by ELISA. In other experiments, EC were infected with control
adenovirus (
-gal) or I
B
M adenovirus for
72 h (multiplicity of infection of 1000; B and
C). EC were either treated for 4.5 h with medium, LPS,
TNF-
, or IL-1
, and assayed for VCAM-1 as above (B) or
treated for 12 h with the same reagents in the presence or absence
of CHX (40 µg/ml) and assayed for viability with Alamar Blue
(C). Vertical bars represent mean (± S.E.) VCAM-1 expression (A and B) or metabolic
activity in arbitrary fluorescent units (C). *,
significantly increased compared with medium alone.
B in apoptosis has been predominantly anti-apoptotic
through its ability to up-regulate cytoprotective gene products,
including cIAPs (70, 71). Recently, however, a requisite role for
NF-
B in promoting apoptosis has been reported (72). Therefore, it
was important to rule out that lactacystin was protecting against
LPS+CHX-induced apoptosis by blocking NF-
B activation through
inhibition of proteasome-mediated degradation of the NF-
B inhibitor,
I
B. Using an adenovirus transduction system, EC were transduced with
either a gene for
-galactosidase or I
B
M with serine to alanine
mutations at residues 32 and 36 (14). These serine to alanine mutations
prevent signal-induced phosphorylation and proteasome-mediated
degradation of the I
B
M protein (14, 40). LPS-, TNF-
, or
IL-1
each up-regulated VCAM-1 expression in non-transduced EC and EC
transduced with a control gene, but not in those transduced with the
I
B
M (Fig. 6B). In contrast, I
B
M transduction did
not affect the sensitivity of EC to LPS-, TNF-
-, or IL-1
-induced
killing at 12 h in the absence of de novo protein
synthesis (Fig. 6C). These data suggest that the protective
effect elicited by lactacystin is through inhibition of the degradation
of a cytoprotective protein, and not through the blockade of a
pro-apoptotic NF-
B signaling pathway. Further, the presumed
mechanism of the pro-apoptotic properties of NF-
B reported by Ryan
et al. (72) is through the promotion of new gene expression,
similar to its anti-apoptotic role and the induction of cIAPs. The
finding that LPS, TNF-
, and IL-1
all stimulate EC apoptosis in
the absence of new protein synthesis argues against a pro-apoptotic
role for NF-
B involving new gene expression. Finally, transduction
of EC with the I
B
M gene sensitizes these cells to apoptosis
induced by long term exposure (
24 h) to TNF-
, implicating an
anti-apoptotic role for NF-
B and not a pro-apoptotic role
(14).
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Fig. 7.
Effect of FLIP and Mcl-1 antisense on
LPS-induced EC apoptosis. EC transfected with either FLIP
antisense, Mcl-1 antisense, or appropriate mismatch control
oligonucleotides were exposed to LPS (100 ng/ml) in the presence or
absence of CHX (2 µg/ml) for 12 h. EC lysates were immunoblotted
with anti-FLIP or anti-Mcl-1 antibodies (A and
C). Molecular mass (in kDa) is indicated. In other
experiments, EC transfected as above were exposed to a fixed
concentration of LPS (100 ng/ml) in the presence of increasing
concentrations of CHX for 12 h, and assayed for cytosolic histone
release (B and D). Mean (± S.E.) histone release
is reported in OD units. *, significantly increased compared with
simultaneous mismatch oligonucleotide control.
and IL-1
, in combination with
a protein synthesis inhibitor is blocked by proteasome inhibition,
suggesting a common cytoprotective pathway dependent upon constitutive
expression of an anti-apoptotic protein(s). Fourth, by screening EC for
known constitutively expressed anti-apoptotic proteins, the expression
of which is acutely sensitive to inhibition of new gene expression and
proteasome degradation, we identified a potential cytoprotective role
against LPS-induced apoptosis for two proteins, FLIP and Mcl-1.
Finally, using antisense oligonucleotides, we have identified a
definitive role for FLIP in conferring protection against
LPS-induced apoptosis in human EC.
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Fig. 8.
Schematic diagram of a constitutive
cytoprotective pathway that protects EC from inflammatory
mediator-induced apoptosis. Under physiological conditions,
constitutive expression of a cytoprotective protein(s) confers
resistance to LPS-, TNF- -, and IL-1
-induced apoptosis in human
EC. Inhibition of de novo protein synthesis with CHX
sensitizes EC to apoptosis elicited by each of these inflammatory
mediators. In the absence of new protein synthesis, pre-existing levels
of a cytoprotective protein(s) are rapidly diminished via
proteasome-mediated degradation. Decreased levels of this
cytoprotective protein(s) enable LPS-, TNF-
-, or IL-1
-induced
caspase activation resulting in EC apoptosis. Proteasome inhibition
with lactacystin blocks the degradation of the cytoprotective
protein(s), thus, preventing CHX sensitization of EC to inflammatory
mediator-induced apoptosis. In addition to a constitutive
cytoprotective pathway, an inducible pathway dependent upon NF-
B
activation has been reported which confers human EC resistance to
TNF-
-, but not LPS or IL-1
-induced apoptosis.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM42686, GM07037, HL59969, and HL03174.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.
¶ To whom correspondence should be addressed: Div. of Hematology, Box 359756, Harborview R&T Bldg., Rm. 521, 325 9th Ave., University of Washington, Seattle, WA 98104-2499. Tel.: 206-341-5319; Fax: 206-341-5322; E-mail: dbannerm@u.washington.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M100819200
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ABBREVIATIONS |
---|
The abbreviations used are:
EC, endothelial
cell;
LPS, lipopolysaccharide;
TNF-, tumor necrosis factor-
;
IL, interleukin;
TLR, Toll-like receptor, CHX, cycloheximide;
z-VAD, z-VAD-fluoromethylketone;
PBS, phosphate-buffered saline;
PVDF, polyvinylidene fluoride membrane;
HRP, horseradish peroxidase;
IgG, immunoglobulin G;
PARP, poly(ADP-ribose) polymerase;
DD, death domain;
cIAP, cellular inhibitor of apoptosis;
ELISA, enzyme-linked
immunosorbent assay;
VCAM-1, vascular cell adhesion molecule-1;
TRADD, TNF receptor-associated death domain;
FADD, Fas-associated death
domain.
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