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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2759-2765
Lipopolysaccharide Induces the Antiapoptotic Molecules, A1 and
A20, in Microvascular Endothelial Cells
By
Xiaolong Hu,
Esther Yee,
John M. Harlan,
Fred Wong, and
Aly Karsan
From the Department of Pathology and Laboratory Medicine, University
of British Columbia and St Paul's Hospital, Vancouver, British
Columbia, Canada; and the Division of Hematology, University of
Washington, Seattle, WA.
 |
ABSTRACT |
The effect of lipopolysaccharide (LPS) on endothelial cells is a key
component of the inflammatory response seen in Gram-negative sepsis.
LPS does not cause death of cultured human endothelial cells. However,
when the expression of new proteins is inhibited by cycloheximide,
microvascular endothelial cells in culture undergo apoptosis. This
finding suggests that LPS induces apoptotic and antiapoptotic pathways,
with the antiapoptotic response being dependent on the synthesis of new
proteins. Concurrent activation of apoptotic and antiapoptotic pathways
has previously been documented for tumor necrosis factor (TNF). In the
case of TNF, the antiapoptotic signal has been attributed to at least
two cytoprotective proteins: the Bcl-2 homologue, A1, and the
zinc-finger protein, A20. In this study, we demonstrate that both these
molecules are induced in microvascular endothelial cells by LPS.
Enforced overexpression of either A1 or A20 inhibits LPS and
cycloheximide-initiated apoptosis. Induction of A1 and A20 does not
require synthesis of intermediary proteins, but is dependent on the
presence of soluble CD14. In addition, we show that inhibition of
signaling by the transcription factor, NF- B, blocks accumulation of
A1 and A20 mRNA. Taken together, our findings suggest that LPS directly
induces expression of the cytoprotective proteins, A1 and A20, via a
CD14-dependent pathway requiring activation of NF-B.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE INCIDENCE OF Gram-negative sepsis has
increased dramatically through the latter half of the 20th century.
Lipopolysaccharide (LPS) is a critical glycolipid component of the
outer wall of Gram-negative bacteria.1 Many of the
proinflammatory and procoagulant effects of Gram-negative bacteria are
elicited by LPS.2,3 Monocytes, neutrophils, and endothelial
cells are critical for the initiation and potentiation of the
inflammatory response, although several cell types
contribute.4 The effects of LPS on endothelial cells are
diverse and have been demonstrated in animal and human studies in vivo
as well as in vitro.4 Some of the effects on endothelium
include upregulation of adhesion molecules, chemokine release,
increased expression of tissue factor, and decreased expression of
protein C and antithrombin III.4
Apoptosis is a form of cell death requiring the activation of cysteine
proteases termed caspases.5 Endothelial apoptosis has been
associated with several disorders over the last few
years.6-8 In many cases, the significance of endothelial
apoptosis in the etiology of disease is not clear. However, in certain
situations, endothelial apoptosis may be a primary pathogenic event.
For instance, in scleroderma it is felt that the skin lesions are the
result of endothelial apoptosis due to anti-endothelial cell
antibodies.6
Similarly, endothelial damage plays a crucial role in the pathogenesis
of septic shock due to Gram-negative bacteria.9 However, at
the concentrations of LPS measured in patients' sera during sepsis,
there is minimal to no direct toxicity of LPS on cultured human
umbilical vein endothelial cells.10 In contrast, prevention
of new gene expression results in significant amounts of endothelial
death.10 Although umbilical vein endothelial cells are a
well-characterized model, much of the effect of LPS occurs at the level
of the microvasculature.9,11 Thus, we were interested in
determining whether human microvascular endothelial cells
(HMEC-1) showed the same resistance to LPS-induced
toxicity as large vessel endothelium.
We have previously shown that tumor necrosis factor (TNF) induces the
upregulation of the Bcl-2 homologue, A1, which can, in turn, prevent
the apoptotic events mediated by TNF.12,13 Others have
demonstrated similar findings for the zinc finger protein, A20, in
murine fibroblasts.14 However, whether LPS can directly
induce these molecules in HMEC-1 has not been reported. We hypothesized
that, similar to TNF, LPS may induce A1 and A20 in microvascular
endothelial cells.
In this report, we demonstrate that, in the presence of the protein
synthesis inhibitor, cycloheximide (CHX), LPS induces microvascular
endothelial cell apoptosis. However, LPS does not cause endothelial
death in the absence of CHX. We demonstrate further that LPS can
upregulate mRNA of the antiapoptotic molecules A1 and A20, as well as
A20 protein levels. These molecules can inhibit the apoptotic pathway
that is concomitantly induced by LPS. This induction of A1 and A20 is a
direct LPS effect that is dependent on the presence of soluble CD14,
but is independent of TNF or interleukin-1 (IL-1) signaling. Finally,
we show that induction of these cytoprotective molecules by LPS
requires activation of the transcription factor NF-B.
| |
MATERIALS AND METHODS |
Reagents.
LPS and CHX were purchased from Sigma (St Louis, MO) and
TNF from R&D Systems (Minneapolis, MN). FLAG-epitope
antibody (M2) was from IBI (New Haven, CT), rabbit
polyclonal anti-A20 antibody was a gift of V. Dixit (Genentech, Inc,
San Francisco, CA), neutralizing anti-CD14 antibody (60bd)
was a gift of R. Todd (University of Michigan, Ann Arbor,
MI), and the IgG2a isotype control was from Sigma. The
neutralizing anti-TNF antibody (clone 195) was purchased from
Boehringer Mannheim (Indianapolis, IN) and IL-1 receptor antagonist (IL-1RA) was obtained from R&D Systems. The horseradish peroxidase-conjugated secondary antibodies used for immunoblotting were
purchased from Bio-Rad Laboratories (Hercules, CA).
Cell culture.
The HMEC-1 human dermal microvascular endothelial cell
line15 was provided by the Center for Disease Control and
Prevention (Atlanta, GA) and was cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum and 20 µg/mL bovine brain
extract. Cells were maintained at 37°C in 5% CO2.
Viability assay.
For viability assays, transduced or wild-type HMEC-1 cells were seeded
on 96-well plates at a density of 15,000 cells/well. On the following
day, cells were incubated in LPS (concentrations as indicated)
and/or CHX (50 µg/mL). At various time points, viable cell
numbers were estimated by 3-[4 ,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay.16 Briefly, medium
was removed and replaced with medium containing 1 mg/mL MTT (Sigma) and
incubated for 5 hours. The medium was then aspirated and the formazan
product was solubilized with dimethylsulfoxide. Absorbance at 630 nmol/L was subtracted (background absorbance) from absorbance at 570 nmol/L for each well. Viability was expressed as a proportion of cells
incubated in complete medium. In our laboratory, HMEC-1 cells cultured
in complete medium show greater than 98% viability as measured by
trypan blue exclusion or flow cytometry.
Oligonucleosomal banding.
Oligonucleosomal banding was demonstrated by harvesting of total
cellular DNA as previously described.17 Briefly, after the
treatment indicated, cells were harvested, washed with
phosphate-buffered saline (PBS), and lysed in 50 mmol/L
Tris, pH 7.5, 10 mmol/L EDTA, 0.5% Triton-X-100, and 0.5 mg/mL of
proteinase K for 2 hours at 50°C. Samples were then extracted twice
with phenol/chloroform/isoamyl alcohol and precipitated with ethanol.
The pellet was resuspended in Tris-EDTA and 10 µg/mL RNase A, and the
DNA was separated on a 2% agarose gel.
Northern analysis.
HMEC-1 cells were stimulated, as described, with LPS or TNF for various
times as indicated. Total cellular RNA (10 µg) was separated on
agarose-formaldehyde gels, blotted onto nitrocellulose filters, and
hybridized overnight with random-primed 32P-labeled probes
as indicated. The final washing conditions were 0.1× SSC, 0.1%
sodium dodecyl sulfate (SDS) for 15 minutes at room
temperature. Blots were stripped in boiling water before reprobing. A
3 probe was used to identify the approximately 1-kb human A1
transcript, and a 300-bp HindIII fragment of A20 was used to
identify the approximately 4.5-kb A20 mRNA. A  -actin probe
hybridizing to a 2-kb transcript was used to confirm equivalent loading
of RNA samples.
Gene transfer.
Generation of HMEC-FLAG-A1, HMEC-Bcl-XL, and HMEC-Neo cells has
previously been described.13 HMEC-A20 and HMEC-IBmt
cells were constructed using previously described
methods.18 Briefly, the coding region of A20 (provided by
V. Dixit) or FLAG-IBmt (provided by D. Ballard, Vanderbilt
University, Nashville, TN) was ligated into an engineered
Xho I site (A20) or the HindIII/Hpa I
(FLAG-IBmt) site of the replication-deficient retroviral vector pLNCX (provided by A.D. Miller, Fred Hutchinson Cancer Research Center,
Seattle, WA).19 The viral long terminal repeat drives expression of NeoR, whereas the cytomegalovirus
promoter drives transgene expression. Generation of packaging cell
lines was performed as described.13,18 The pLNCA20
construct or pLNCIBmt was transiently transfected into the ecotropic
packaging line, PE501, by calcium-phosphate precipitation. Viral
supernatants were harvested and used to transduce the amphotropic line
PA317 in the presence of 4 µg/mL of polybrene. Retrovirus-producing
cell lines were obtained by selection in 750 µg/mL G418 (Life
Technologies, Gaithersburg, MD). Retroviral supernatants
from the PA317 cell lines were used to transduce HMEC-1 cells. After
selection in 200 µg/mL of G418 and expansion, transduced HMEC-1 cells
were used in survival assays. Polyclonal HMEC-1 lines were used to
avoid artifacts due to retroviral integration site.
Immunoblotting.
Total cellular extracts were prepared from HMEC-1 cells by lysing
105 cells in 20 mmol/L Tris, 137 mmol/L NaCl, 1%
Triton-X-100, 1 mmol/L AEBSF, 10 µg/mL leupeptin, and 10 µg/mL
aprotinin. Protein was fractionated by 10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) gels and electrotransferred onto
nitrocellulose membranes over 1 hour at 4°C. Filters were blocked
overnight with PBS containing 5% skim milk. Immunostaining steps were
performed in PBS with 3% bovine serum albumin (BSA) and
0.05% Tween 20 at room temperature. Filters were incubated with
primary antibody for 2 hours and secondary antibody for 1 hour. Filters
were washed in PBS and 0.05% Tween 20 four times for 10 minutes
between each step and were developed by chemiluminescence.
| |
RESULTS |
LPS induces HMEC-1 apoptosis in the presence of CHX.
It has been shown previously that LPS can cause toxicity of large
vessel endothelium when protein or mRNA expression is blocked by CHX or
actinomycin D, respectively.10 LPS alone does not kill
human umbilical vein endothelial cells.10 However, in
bovine or ovine large vessel endothelium, LPS can directly cause cell death.20,21 Others have reported LPS-induced toxicity of
human pulmonary arterial endothelial cells.22 Because the
microvasculature plays a key role in the pathogenesis of
sepsis,11 we were interested in determining the effect of
LPS in HMEC-1. As seen in Fig 1A, LPS alone
does not kill HMEC-1. However, when protein synthesis is blocked, there
is a dose-dependent toxicity of LPS on HMEC-1 cells. The mode of cell
death in this model is apoptotic, as demonstrated by the
oligonucleosomal fragmentation of chromosomal DNA (Fig 1B). This
finding is consistent with our studies demonstrating the activation of
caspases in HMEC-1 by LPS in the presence of CHX (Choi et
al22a). Others have also shown the activation
of caspase-1 by LPS in human umbilical vein endothelial
cells.23
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| Fig 1.
LPS initiates endothelial apoptosis only in the presence
of CHX. (A) HMEC-1 cells were treated with LPS for 15 hours in the
presence or absence of CHX (50 µg/mL). Each data point is the
proportion of viable cells with respect to cells treated with medium
only. Results are the means + SD of three experiments.
(B) HMEC-1 cells were treated with LPS (100 ng/mL) only, LPS and CHX
(50 µg/mL), or CHX only for 15 hours and harvested DNA was separated
on an ethidium bromide-stained agarose gel. The lane on the left is a
100-bp molecular weight marker.
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|
LPS causes upregulation of the cytoprotectants A1 and A20.
The finding described above suggests that LPS activates an apoptotic
pathway as well as inducing the synthesis of cytoprotective proteins
that prevent LPS-activated apoptosis. This finding is similar to that
described for TNF.8,24-27 We have previously shown that TNF
upregulates the Bcl-2 family member, A1, in endothelial cells.12 It has also been shown that the zinc-finger
protein, A20, is inducible by TNF.14,28 To determine
whether LPS can upregulate the mRNA of these two known cytoprotective
proteins in microvascular endothelial cells, HMEC-1 cells were
stimulated with LPS for various times and Northern blots were
performed. Both A1 and A20 mRNA accumulates in HMEC-1 cells after LPS
stimulation (Fig 2A). We repeated these
experiments in human umbilical vein endothelial cells and found that
LPS also induced A1 and A20 in these primary human endothelial cells
(data not shown). Levels of A20 protein are also induced after LPS
stimulation, and the kinetics of protein induction in HMEC-1 cells
correspond to the mRNA accumulation (Fig 2B). A20 protein levels at 3 to 6 hours are similar to that seen in HMEC-1 cells engineered to
constitutively express A20 (see below).
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| Fig 2.
LPS induces A1 and A20 in HMEC-1 cells. (A) After
treatment with LPS (100 ng/mL) for various times, total RNA from HMEC-1
cells was subjected to Northern analysis. (B) Protein lysates from
HMEC-1 cells were also harvested at times after LPS stimulation as in
(A) and analyzed by immunoblotting. The first lane shows HMEC-1 cells
engineered to constitutively express A20 (HMEC-A20).
|
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Overexpression of A1 or A20 inhibits LPS-mediated cell death.
A1, a Bcl-2 homologue, protects against cell death in some but not all
systems.13,29 Similarly, A20 attenuates TNF-initiated death, but not that induced by Fas ligation or reactive oxygen intermediates.14,30 Studies were performed to determine
whether A1 or A20 could inhibit LPS-mediated apoptosis. HMEC-1 lines
stably overexpressing FLAG-epitope-tagged A1 (HMEC-FLAG-A1) or A20
(HMEC-A20) were generated by retroviral-mediated transfer. Because the
Bcl-2 family member, Bcl-XL, is also able to block various forms of cell death, HMEC-Bcl-XL cells were used as a positive control. Cells
transduced with the empty vector (HMEC-Neo) were used as a negative
control. Immunoblotting confirmed the expression of the specific
protein in each of these cell lines (Fig 3B). Each of
these cell lines was exposed to various concentrations of LPS in the
presence of CHX. Both A1 and A20 were able to inhibit LPS-mediated cell
death (Fig 3A). The partial protection conferred by these proteins has
been noted before for TNF-initiated apoptosis.13,14 In the
case of A1, the short half-life of the protein compared with Bcl-XL may
provide one explanation for the incomplete protection.13 Nevertheless, these experiments demonstrate that A1 and A20 serve to
protect against LPS-initiated apoptosis.
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| Fig 3.
A1 and A20 inhibit LPS-initiated endothelial apoptosis.
(A) HMEC-1 cells stably transduced with various cDNA constructs, as
indicated, were exposed to LPS and CHX (50 µg/mL). MTT assays were
performed at 15 hours after treatment with LPS and CHX. Each data point
is the proportion of viable cells with respect to cells treated with
medium only (Med). Results are the means + SD of three experiments.
(B) Each of the transduced cell lines was tested for expression of the
relevant protein by immunoblotting.
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LPS induction of A1 and A20 is CD14-dependent.
Optimal activation of endothelial cells by LPS requires the presence of
serum.31 Although a cell-surface receptor for LPS has not
been identified on endothelial or epithelial cells, many signaling
functions are dependent on the presence of soluble CD14 contained in
plasma.32,33 It has been suggested that many of the effects
of LPS are secondary to TNF and IL-1, and various sequelae of
Gram-negative sepsis have been attributed to TNF or IL-1.34-36 LPS has been shown to induce the expression of
TNF and IL-1 in endothelial cells.37,38 Both TNF and IL-1
are capable of inducing A1 and A20 expression.12,28 Given
the slightly delayed induction of A1 and A20 by LPS compared with TNF,
it is conceivable that LPS-mediated induction is secondary to
TNF or IL-1. To delineate the route of LPS-mediated upregulation of A1 and A20 in endothelial cells, HMEC-1 cells were pretreated with neutralizing antibodies (anti-CD14, 20 µg/mL; anti-TNF, 20 µg/mL) or IL-1 receptor antagonist (IL-1RA, 500 ng/mL) at concentrations sufficient to block signaling by these various pathways (data not
shown). Only the anti-CD14 neutralizing antibody was capable of
attenuating A1 and A20 expression (Fig 4A), suggesting
that this induction is mediated by a CD14-dependent, but
IL-1-independent and TNF-independent mechanism.
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| Fig 4.
LPS-initiated accumulation of A1 and A20 mRNA is an early
gene response dependent on CD14. (A) Northern analysis of HMEC-1 cells
treated with neutralizing antibodies to CD14 and TNF, an IL-1
antagonist (IL-1RA), or an anti-CD14 isotype control (IgG2a) for 1 hour
before stimulation with LPS (100 ng/mL) for 3 hours. (B) Northern
analysis of HMEC-1 cells after 3 hours of treatment with LPS (100 ng/mL) only, CHX (50 µg/mL) only, or LPS and CHX.
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To clarify whether any intermediary proteins were required for A1 and
A20 upregulation, HMEC-1 cells were treated with CHX to block protein
synthesis before LPS stimulation for 3 hours. As seen in Fig 4B, CHX
pretreatment did not block A1 or A20 induction. This finding in
endothelial cells is consistent with A1 and A20 being early response
genes to LPS stimulation.39 As described for other early
response genes,39 treatment with CHX alone also resulted in
accumulation of A1 and A20 mRNA.
LPS induction of A1 and A20 is NF-B-dependent.
Recently, several groups have demonstrated that TNF initiates an
antiapoptotic pathway by activation of the transcription factor
NF-B.25-27 NF-B is normally sequestered in the
cytoplasm by an inhibitor, IB.40,41 After activation by
TNF or other stimuli, IB is phosphorylated on two serine residues,
Ser32 and Ser36.42,43 This phosphorylation results in the
ubiquitination and degradation of IB and subsequent translocation of
NF-B to the nucleus.42,43 A mutant IB with Ser32 and
Ser36 mutated to alanine residues resists degradation, thereby
preventing translocation of NF-B to the nucleus.42,43 It
was shown that inhibition of NF-B by overexpression of this IB
mutant (IBmt) sensitized normally resistant cells to TNF-initiated
apoptosis.25-27 To determine whether the LPS induction of
the cytoprotectants A1 and A20 was directed via NF-B activation,
HMEC-1 cells were transduced with the mutant IB construct to
generate HMEC-IBmt cells. To confirm that HMEC-IBmt cells were
able to abrogate activation of NF-B, these cells were tested for
TNF-inducible expression of the cell adhesion molecule, vascular cell
adhesion molecule-1 (VCAM-1), which is an
NF-B-dependent event, and were found to prevent this expression
compared with the HMEC-Neo cells (data not shown). Figure 5A demonstrates that
LPS-mediated induction of both A1 and A20 is blocked by overexpression
of IBmt. It has previously been shown that A20 induction by TNF is
NF-B-dependent in fibroblasts.44 Hence, the
demonstration that TNF induction of A20 is also blocked by IBmt
confirms that NF-B activation is blocked in the HMEC-IBmt cells.
This finding suggests that LPS signals upregulation of A1 and A20 in
microvascular endothelial cells by an NF-B-dependent mechanism.
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| Fig 5.
Inhibition of NF-B activation blocks LPS-mediated A1
and A20 induction. (A) HMEC-1 cells transduced with a FLAG-IBmt
construct (HMEC-IBmt) or empty vector (HMEC-Neo) were treated with
LPS or TNF for 3 hours and subjected to Northern analysis. (B)
Transduced cell lines were immunoblotted with the FLAG (M2) antibody to
confirm expression of the FLAG-IBmt protein.
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| |
DISCUSSION |
Integrity of the endothelium is critical for modulating the
inflammatory response.4 Endothelial injury plays a crucial role in the pathogenesis of septic shock due to Gram-negative bacteria.9 Endothelial cell death initiated by inflammatory mediators is species-dependent. In general, ovine and bovine endothelia are highly sensitive to LPS, whereas human cells are
not.10,20,21 However, when RNA transcription or protein
synthesis is inhibited, LPS does cause death of human endothelial
cells, suggesting that LPS can induce parallel cell death and survival
pathways.10
Our findings demonstrated that, similar to large vessel endothelium,
the human dermal microvascular line, HMEC-1, only undergoes apoptosis
in response to LPS in the presence of CHX. Thus, LPS must stimulate the
expression of genes that provide protection against apoptosis. This is
not a surprising finding given the critical importance of maintaining
an intact vascular barrier that provides selective permeability to
macromolecules. We have previously shown that human endothelial cells
express several antiapoptotic members of the Bcl-2
family.17 It is likely that, under different physiologic or
pathologic circumstances, different members of the Bcl-2 family play a
key role. For instance, in ML-1 myeloid leukemia cells induced to
undergo monocytic/macrophage differentiation, Mcl-1 may play a role in
permitting survival.45 Similarly, A1 may also play an
antiapoptotic role in differentiating hematopoietic
cells.46 In endothelial cells, A1 may be the critical Bcl-2
homologue promoting viability during inflammation.8,13
Because LPS-initiated endothelial death only occurs in the presence of
CHX, this model cannot directly be applied to in vivo findings during
sepsis. However, there is evidence to suggest that the survival pathway
may fail due to inhibition by other inflammatory mediators such as
interferon- or that the death pathway overwhelms the survival
pathway by synergism with other cytokines during
sepsis.47,48 It has also been suggested that LPS-induced
nitric oxide may synergize with IL-1 to render endothelial cells more
susceptible to apoptosis.49 Others have reported that CHX
upregulates Fas mRNA.50 However, Fas ligation does not kill
endothelial cells,51,52 and we have not found the combination of LPS and Fas-ligation to cause endothelial death (A. Karsan, unpublished observations).
The situation in vivo is further complicated by the fact that LPS has
functional effects on leukocytes that can then directly or indirectly
affect the viability of endothelium.53 For instance, it has
been shown that cytokine-activated neutrophils and LPS-activated mononuclear cells can cause endothelial damage.53,54 The
apoptotic effect of LPS-treated monocytes has been attributed to
membrane-bound TNF as well as an unidentified secreted
factor.47,54 It has also been demonstrated in vivo that TNF
and subsequent ceramide generation mediate the LPS-induced endothelial
apoptosis.55
We were interested in determining the direct effects of LPS on HMEC-1.
Because the initial findings suggested the stimulation of a
cytoprotective pathway, we looked for induction of the cytoprotective molecules A1 and A20. mRNA for both proteins accumulated after LPS
stimulation, although at slightly later times compared with TNF
induction. In contrast to TNF, where peak induction of A1 is seen after
3 hours of exposure, the LPS-induced A1 expression is delayed, reaching
a maximum at 6 hours or later.12 Similarly, A20 expression
in fibroblasts after TNF exposure peaks at 1 hour,28 whereas LPS-induced A20 expression is maximal at 3 to 6 hours in
microvascular endothelial cells. Enforced expression of either A1 or
A20 protected HMEC-1 cells from apoptosis induced by LPS and CHX. Thus,
these molecules may account in part for the resistance of human
endothelial cells to the direct toxic effects of LPS.
Several intracellular molecules have been implicated in transducing LPS
signals. Activation of NF-B, the Jak-STAT pathway, mitogen-activated
protein kinases, and phosphatidylinositol 3-kinase have all been
demonstrated to play a role in the intracellular signaling of
LPS-mediated events.31,56,57 LPS complexed with a serum
protein, LPS binding protein, signals through membrane-bound CD14 on
monocytes and myeloid cells. In contrast, endothelial and epithelial
cells, which are CD14 but still respond to LPS,
require soluble CD14 present in serum to transduce LPS
signals.31,33,58 It is still unclear how the LPS-CD14
complex actually transmits a signal across the cell membrane. Evidence
has been presented to suggest the presence of a signaling transmembrane
receptor recognizing the LPS-CD14 complex.59 Transmembrane
signaling by LPS has also been shown to be mediated by CD11/CD18
integrins independently of CD14.60,61 However, others have
postulated that LPS is internalized by a vesicular transport mechanism
and mediates signals, at least partly, by structurally mimicking
ceramide.62,63 In addition, many of the in vivo effects of
LPS have been attributed to secretion of TNF and/or IL-1 by
other cells.34-36 However, our results demonstrate that
induction of A1 and A20 is independent of the expression of any
secondary molecules, demonstrating that the cytoprotective activity is
a direct effect of LPS stimulation. We also show that soluble CD14 is
required for this activity. Previously, we have demonstrated that
exogenous ceramide does not induce A1 mRNA, suggesting that the
LPS-mediated A1 induction is likely not due to molecular mimicry of
this lipid second messenger.13
Recently, it was demonstrated that inhibition of NF-B by expressing
an IB mutant sensitizes cells to the apoptotic effects of
TNF.25-27 This finding led to the proposal that TNF induces cytoprotective proteins by activation of NF-B. To determine whether the LPS induction of A1 and A20 was also secondary to NF-B
activation, we overexpressed the mutant IB in HMEC-1 cells.
Inhibition of NF-B by overexpression of the IB mutant, in
microvascular endothelial cells, inhibits expression of A1 and A20 by
both TNF and LPS. Interestingly, concomitant expression of A20 with
IBmt was not able to reverse the sensitization of cells to TNF in
one study.26 Furthermore, inhibition of NF-B did not
sensitize cells to Fas-mediated death.25 We have shown that
LPS signals HMEC-1 death via an intracellular death-domain-containing
protein, FADD (Choi et al22a). FADD signals
death initiated by TNF receptor 1 as well as by Fas.8 It is
interesting to note that, whereas A20 protects against both TNF and
LPS-mediated death, it does not protect against Fas-mediated apoptosis.14,30 In other studies, we have shown that
IBmt overexpression does not sensitize endothelial cells to IL-1-
or LPS-mediated apoptosis, but it does sensitize to TNF-initiated death
(Zen et al, manuscript submitted). Thus, although there are several downstream molecules in common that are involved in pathways signaling apoptosis and protection against apoptosis, it is
clear that many differences in these pathways remain to be elucidated.
| |
FOOTNOTES |
Submitted February 17, 1998;
accepted June 5, 1998.
Supported by grants from the Medical Research Council of Canada
(CLN-1002-42547 and MT-14373) with funds from the British Columbia Lung
Association. A.K. is a Clinician-Scientist of the Medical Research
Council of Canada.
Address reprint requests to Aly Karsan, MD, UBC McDonald Research
Laboratories, Room 292, St Paul's Hospital, 1081 Burrard St,
Vancouver, BC, Canada V6Z 1Y6; e-mail: akarsan{at}prl.pulmonary.ubc.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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167(12):
7044 - 7051.
[Abstract]
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K. HEERMEIER, W. LEICHT, A. PALMETSHOFER, M. ULLRICH, C. WANNER, and J. GALLE
Oxidized LDL Suppresses NF-{{kappa}}B and Overcomes Protection from Apoptosis in Activated Endothelial Cells
J. Am. Soc. Nephrol.,
March 1, 2001;
12(3):
456 - 463.
[Abstract]
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B. Levkau, K. J. Garton, N. Ferri, K. Kloke, J.-R. Nofer, H. A. Baba, E. W. Raines, and G. Breithardt
xIAP Induces Cell-Cycle Arrest and Activates Nuclear Factor-{{kappa}}B : New Survival Pathways Disabled by Caspase-Mediated Cleavage During Apoptosis of Human Endothelial Cells
Circ. Res.,
February 16, 2001;
88(3):
282 - 290.
[Abstract]
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C. W. Xiao, K. Ash, and B. K. Tsang
Nuclear Factor-{{kappa}}B-Mediated X-Linked Inhibitor of Apoptosis Protein Expression Prevents Rat Granulosa Cells from Tumor Necrosis Factor {{alpha}}-Induced Apoptosis
Endocrinology,
February 1, 2001;
142(2):
557 - 563.
[Abstract]
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B. D'Souza, M. Rowe, and D. Walls
The bfl-1 Gene Is Transcriptionally Upregulated by the Epstein-Barr Virus LMP1, and Its Expression Promotes the Survival of a Burkitt's Lymphoma Cell Line
J. Virol.,
July 15, 2000;
74(14):
6652 - 6658.
[Abstract]
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T. Stefanec
Endothelial Apoptosis: Could It Have a Role in the Pathogenesis and Treatment of Disease?
Chest,
March 1, 2000;
117(3):
841 - 854.
[Abstract]
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S. T. Grey, M. B. Arvelo, W. Hasenkamp, F. H. Bach, and C. Ferran
A20 Inhibits Cytokine-Induced Apoptosis and Nuclear Factor {kappa}B-Dependent Gene Activation in Islets
J. Exp. Med.,
October 18, 1999;
190(8):
1135 - 1146.
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K. Zen, A. Karsan, A. Stempien-Otero, E. Yee, J. Tupper, X. Li, T. Eunson, M. A. Kay, C. B. Wilson, R. K. Winn, et al.
NF-kappa B Activation Is Required for Human Endothelial Survival during Exposure to Tumor Necrosis Factor-alpha but Not to Interleukin-1beta or Lipopolysaccharide
J. Biol. Chem.,
October 1, 1999;
274(40):
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H. H. Lee, H. Dadgostar, Q. Cheng, J. Shu, and G. Cheng
NF-kappa B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes
PNAS,
August 3, 1999;
96(16):
9136 - 9141.
[Abstract]
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H. Sugiyama, J. S. Savill, M. Kitamura, L. Zhao, and E. Stylianou
Selective Sensitization to Tumor Necrosis Factor-alpha -induced Apoptosis by Blockade of NF-kappa B in Primary Glomerular Mesangial Cells
J. Biol. Chem.,
July 9, 1999;
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D. M. Stroka, A. Z. Badrichani, F. H. Bach, and C. Ferran
Overexpression of A1, an NF-kappa B-Inducible Anti-Apoptotic Bcl Gene, Inhibits Endothelial Cell Activation
Blood,
June 1, 1999;
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3803 - 3810.
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A. Stempien-Otero, A. Karsan, C. J. Cornejo, H. Xiang, T. Eunson, R. S. Morrison, M. Kay, R. Winn, and J. Harlan
Mechanisms of Hypoxia-induced Endothelial Cell Death. ROLE OF p53 IN APOPTOSIS
J. Biol. Chem.,
March 19, 1999;
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D. D. Bannerman, J. C. Tupper, W. A. Ricketts, C. F. Bennett, R. K. Winn, and J. M. Harlan
A Constitutive Cytoprotective Pathway Protects Endothelial Cells from Lipopolysaccharide-induced Apoptosis
J. Biol. Chem.,
April 27, 2001;
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P. J. Duriez, F. Wong, K. Dorovini-Zis, R. Shahidi, and A. Karsan
A1 Functions at the Mitochondria to Delay Endothelial Apoptosis in Response to Tumor Necrosis Factor
J. Biol. Chem.,
June 9, 2000;
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R. Hofer-Warbinek, J. A. Schmid, C. Stehlik, B. R. Binder, J. Lipp, and R. de Martin
Activation of NF-kappa B by XIAP, the X Chromosome-linked Inhibitor of Apoptosis, in Endothelial Cells Involves TAK1
J. Biol. Chem.,
July 14, 2000;
275(29):
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Abstract
Introduction
Abstract