Department of Pharmacology, College of Medicine, The University of Illinois, Chicago, Illinois 60612
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ABSTRACT |
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Because reactive oxygen species (ROS) can
function as second messengers and regulate the activation of the
transcription factor nuclear factor (NF)-B, we investigated the
possible role of tumor necrosis factor-
(TNF-
)-induced ROS
generation in endothelial cells in signaling E-selectin gene
transcription. We demonstrated that stimulation of human pulmonary
artery endothelial cells with TNF-
(100 U/ml) resulted in ROS
production using the oxidant-sensitive dye 5 (and
6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester. Pretreatment with
N-acetyl-L-cysteine (NAC)
or pyrrolidine dithiocarbamate (PDTC) for 0.5 h inhibited TNF-
-induced generation of ROS as well as activation of NF-
B and
E-selectin mRNA and the cell surface protein expression. These findings
indicate that TNF-
induces NF-
B activation and the resultant
E-selectin gene expression by a pathway that involves formation of ROS
and that E-selectin expression can be inhibited by the antioxidant
action of NAC or PDTC. The results support the hypothesis that
generation of ROS in endothelial cells induced by proinflammatory
cytokines such as TNF-
is a critical signal mediating E-selectin
expression.
endothelium; tumor necrosis factor-; reactive oxygen species; nuclear factor-
B
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INTRODUCTION |
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E-SELECTIN (CD62E), a member of the selectin family of
cell surface glycoproteins, is an endothelial cell-specific adhesion protein involved in the initial vessel wall binding and rolling of
polymorphonuclear leukocytes (PMN) and other leukocytes at sites of
endothelial activation (34). E-selectin ligands on PMN are fucose-rich
glycoproteins containing sialylated Lewisx and related
oligosaccharides (14, 20). E-selectin is expressed on the endothelial
cell surface in response to cytokines such as tumor necrosis factor-
(TNF-
) and interleukin-1
(IL-1
; see Refs. 6 and 7).
Transcription of the E-selectin gene is tightly regulated in a
cell-specific manner because it is expressed exclusively on the
endothelial cell surface and its expression is dependent in major part
on activation of the transcription factor nuclear factor (NF)-
B
after exposure to TNF-
(11, 21, 27, 35, 38, 45). NF-
B, a
heterodimer of 50-kDa (p50) and 65-kDa (p65) subunits, exists in the
cytoplasm of most cells, including endothelial cells in an inactive
form bound to the inhibitory protein I-
B (I
B) through the p65
molecule (3, 5). Treatment of cells with TNF-
leads to activation of
I
B kinases
and
(IKK
and IKK
; see Refs. 12 and
46), which phosphorylate I
B
and I
B
, respectively, at
two conserved NH2-terminal
residues and which in turn targets them for rapid polyubiquitination
followed by degradation through the 26S proteasome (8-10, 43).
This results in release of NF-
B dimer and its translocation to the
nucleus to activate transcription of genes involved in immune and
inflammatory responses (2-4). Genetic analysis has identified
three NF-
B sites in the human E-selectin gene that are essential for
maximal promoter activity after cytokine exposure (38): NF-
B
sites 1 and
2 (5'-CGTGGATATTCCCGGGAAAGTTTTT-3',
element underlined, within
129 to
105 bases from the
transcriptional start site) and NF-
B site
3 (5'-CA
TCTTT-3',
element underlined, within
99 to
80 bases from the
transcriptional start site). Studies have shown that NF-
B binds
preferentially to sites 2 and
3 and that mutations within these
sites are more specific in inhibiting E-selectin expression than
site 1 mutations (38).
TNF- stimulates the production of reactive oxygen species (ROS):
superoxide anion, hydrogen peroxide, and hydroxyl radicals in a variety
of cell types (19, 23, 32). ROS generated in excess amounts damages
cells by peroxidizing lipids and disrupting proteins and nucleic acids.
ROS in lower concentrations may function as second messengers in
mediating TNF-
- and IL-1
-activated signal transduction pathways
that regulate the transcription factors NF-
B and activator protein
(AP)-1 (22, 33). However, there are conflicting reports regarding
oxidant-induced expression of E-selectin through NF-
B activation
(16). Studies have shown that the antioxidant pyrrolidine
dithiocarbamate (PDTC) inhibited cytokine-induced vascular cell
adhesion molecule-1 (VCAM-1) expression by preventing NF-
B
activation but surprisingly failed to prevent E-selectin expression
(24, 44). Because p65 alone or in concert with the p50 subunit of
NF-
B is essential for regulation of these adhesion molecules in
cytokine-activated endothelial cells (11, 38, 41, 45), it is expected
that there would be a common redox-sensitive mechanism controlling the
expression of both VCAM-1 and E-selectin. The differential sensitivity
of E-selectin and VCAM-1 expression to antioxidants may be ascribed to
factors such as 1) specific
arrangement and number of NF-
B sites present in promoters of
E-selectin versus VCAM-1 (17, 38),
2) codependency of NF-
B on other
transcription factors in E-selectin gene (21, 31, 36),
3) nature of the antioxidant, and
4) differences in DNA binding sites
in the promoter of these genes for antioxidant-responsive transcription
factors. In view of the conflicting evidence on the signaling function
of oxidants in inducing E-selectin expression, in the present study, we
have reevaluated the role of TNF-
in stimulating ROS production in
endothelial cells and in regulating NF-
B activation and the
E-selectin mRNA and protein expression.
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MATERIALS AND METHODS |
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Cell culture. Human pulmonary artery
endothelial (HPAE) cells at the first passage were obtained from
Clonetics and were grown on gelatin-coated flasks or plates in
endothelial cell growth medium (EGM) containing 10% fetal calf serum
(FCS) and 3.0 mg/ml of endothelial-derived growth factor from bovine
brain extract protein. Human recombinant TNF- with a specific
activity of 2.3 × 107 was
purchased from Promega (Madison, WI). All experiments were performed on
cells under the 10th passage except where otherwise indicated.
Confluent HPAE cells were washed two times with serum-free DMEM
(without phenol red) containing 20 mM HEPES or EGM containing 2% FCS
and then were incubated in the same medium with the indicated concentrations of
N-acetyl-L-cysteine (NAC)
or PDTC for 0.5 h before TNF-
was added. TNF-
was added directly
to the medium containing NAC or PDTC for the times and at the
concentrations indicated in each experiment. NAC was neutralized to pH
7.0 before use in all experiments.
Northern blot analysis. Total RNA was
isolated from HPAE cells with TRIzol (GIBCO BRL) according to
manufacturer's recommendations. Quantification and purity of RNA were
assessed by the ratio of absorbance at 260 nm to that at 280 nm, and an aliquot of RNA (20 µg) from samples with a
ratio above 1.6 was fractionated using a 1% agarose-formaldehyde gel.
The RNA was transferred to a Duralose-ultraviolet (UV)
nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently linked by UV irradiation using a Stratalinker UV cross-linker (Stratagene). Human E-selectin (1.35-kb
EcoR I fragment; see Ref. 7) and rat
glyceraldehyde-3-phosphate dehydrogenase (1.1-kb Pst I fragment) were labeled with
[-32P]dCTP using
the random-primer kit (Stratagene), and hybridization was carried out
as described (37). Briefly, the blots were prehybridized for 30 min at
68°C in QuikHyb solution (Stratagene) and hybridized for 2 h at
68°C with random-primed
32P-labeled probes. After
hybridization, the blots were washed two times for 30 min at room
temperature in 2× saline-sodium citrate (SSC) with
0.1% SDS followed by two washes for 30 min each at 60°C in
0.1× SSC with 0.1% SDS. Autoradiography was performed with an
intensifying screen at
70°C for 12-24 h. The signal
intensities were quantified by scanning the autoradiograms with a laser
densitometer (Howtek, Hudson, NH) linked to a computer analysis system
(PDI, Huntington Station, NY). The nitrocellulose membrane was soaked for stripping the probe with boiled water or 0.1× SSC with 0.1% SDS.
Detection of ROS generation with C-DCHF-DA
staining. Confluent HPAE monolayers were pretreated for
0.5 h with NAC (30 mM) or PDTC (100 µM) and stimulated for 1 h with
TNF- (100 U/ml) in EGM containing 2% serum. After treatment, cells
were washed two times with EGM (with 2% serum) and stained for 20 min
with 2.5 µM 5 (and
6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester (C-DCHF-DA; Molecular Probes, Eugene, OR)
in EGM (with 2% serum) as described (15). Cells were viewed with
fluorescence microscopy and photographed. Quantitative fluorescence images were obtained on a Nikon Diaphot 200 Microscope (Nikon, Garden
City, NJ) using Image Pro Plus software (Media Cybernetics, Silver
Spring, MD). Each sample was independently stained so that the samples
were exposed to the dye for the same time. The dye solution was freshly
prepared in prewarmed EGM (with 2% serum) for each sample. After being
stained for 20 min at 37°C, samples were rinsed two times with EGM
(with 2% serum) containing no dye and were scanned on a fluorescence
microscope. Samples were epi-illuminated by a 150-watt Hg lamp and
viewed with fluorescein filters (B2E cube). Fields were observed at
×20 numerical aperture 0.4 and were acquired with a
charge-coupled device imaging array (Photometrics, Tucson, AZ) under
computer control with 1-s integration time. Illumination
caused increased fluorescence because of oxidation of the dye;
therefore, each field was exposed to light for exactly the same time.
The image size for scanning was 768 horizontal × 468 vertical.
The average relative fluorescence intensity for every cell in each
field was determined using Imag Proplus software (Media Cybernetics).
Nuclear extract preparation. HPAE
cells were pretreated for 0.5 h without or with NAC (30 mM) and then
were left untreated or stimulated for 1 h with TNF- (100 U/ml).
Cells were washed two times with ice-cold Tris-buffered saline and
resuspended in 400 µl of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF). After 15 min, Nonidet P-40 was
added to a final concentration of 0.6%. Nuclei were pelleted and
resuspended in 50 µl of 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA,
1 mM EGTA, 1 mM DTT, and 1 mM PMSF. After 30 min at 4°C, the
lysates were centrifuged, and supernatants containing the nuclear
proteins were transferred to new vials. The protein concentration of
the extract was measured using a Bio-Rad protein determination kit (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assays were performed by incubating 10 µg of nuclear extract with 1 µg of poly(dI-dC) in a
binding buffer [10 mM Tris · HCl (pH
7.5), 50 mM NaCl, 0.5 mM DTT, and 10% glycerol (20 µl final volume)] for 15 min at room temperature. Next, end-labeled double-stranded oligonucleotides containing the NF-B binding site of
E-selectin promoter (30,000 counts/min each) were added in
the absence or presence of 25- or 100-fold molar excess of cold
competitor, and the reaction mixtures were incubated for 15 min at room
temperature. The DNA-protein complexes were resolved in 5% native
polyacrylamide gel electrophoresis in low-ionic-strength buffer
(0.25× Tris-borate-EDTA). Oligonucleotides used for the gel shift analysis were as follows: E-selectin
NF-
BA,
5'-TTTTAAGCATC
TTTTT-3' (underlined sequences represent sites
1 and 2,
respectively); mut-E-selectin
NF-
BA,
5'-TTTTAAGCATCGTGGATATTggCGGccgccTTTTT-3'; E-selectin
NF-
BB, 5'-TTTTTGGATGCCATT
TCTTT-3'
(underlined sequence represents site
3); mut-E-selectin
NF-
BB,
5'-TTTTTGGATGggcggccGccTTTCCTCTTT-3'; Ig
B,
5'-AGTTGAGGGGACTTTCCCAGGC-3';
Oct-1 DNA,
5'-AATTGCATGCCTGCAGGTCGACTCTAGAGGAT
GCCGGGTACCGAGCTC-3'.
E-selectin promoter is characterized by the presence of three NF-B
sites located 126 (site 1), 116 (site 2), and 94 (site 3) bp upstream relative to the
transcription start site (38). The oligonucleotides E-selectin
NF-
BA and E-selectin
NF-
BB represent the 35 (
139 to
105)- and 30 (
110 to
81)-bp sequences, respectively, of E-selectin promoter. The
oligonucleotide E-selectin
NF-
BA encompasses NF-
B
site 1 (
126) and NF-
B
site 2 (
116), whereas the
oligonucleotide E-selectin
NF-
BB contains the NF-
B site (
94) of the promoter. The oligonucleotides mut-E-selectin
NF-
BA and mut-E-selectin
NF-
BB represent the
NF-
B-mutated versions of E-selectin
NF-
BA and E-selectin
NF-
BB oligonucleotides,
respectively. Similarly, Ig
B oligonucleotide contains the consensus
NF-
B binding site present in the immunoglobulin gene. The
oligonucleotide Oct-1, which contains a binding site for Oct-1, was
used to serve as a negative control in competition experiments.
Sequence motifs within the oligonucleotides are underlined, and the
mutations are shown in lowercase letters.
Reporter gene constructs, endothelial cell
transfection, and luciferase assay. The E-selectin
luciferase (Luc) plasmid containing ~782 bp of E-selectin
5'-flanking DNA, linked to the firefly Luc reporter gene, has
been described previously (38). Cells under the fifth passage were
plated into six-well Primaria culture dishes 18-24 h before
transfection. Transfections were performed with lipofectine (GIBCO BRL)
as described (37). Briefly, reporter DNA (1 µg) was mixed with 2 µl
of lipofectine in 200 µl of Opti-MEM I (GIBCO BRL). We used the
plasmid pSVgal (0.2 µg) containing the -galactosidase gene driven
by the constitutively active SV40 promoter to normalize the
transfection efficiencies. Because we did not observe any significant
difference in transfection efficiencies in the initial experiments, we
did not cotransfect the pSVgal construct in the later experiments.
After a 30-min incubation, Opti-MEM I (800 µl) was added, and the
mixture was applied on the cells that had been washed two times with
Opti-MEM I. At 3 h later, the medium was changed to EGM containing 10%
serum, and the cells were harvested 24-48 h after transfection.
Using this protocol, we achieved a transient transfection efficiency of
15 ± 3% (mean ± SD; n = 3 experiments) for HPAE cells. Recombinant TNF-
was used
at a concentration of 100 U/ml for 12-18 h before the cells were
harvested. Cell extract was prepared and assayed for Luc activity using
the Promega Biotech assay system (Promega) and
-galactosidase
activity using the Tropix assay system (Bedford, MA). Luc activity per
microgram of protein extract was normalized to
-galactosidase
activity. The protein content was determined using a Bio-Rad protein
determination kit.
Flow cytometry analysis. HPAE cells
cultured in six-well Primaria tissue culture dishes were pretreated for
0.5 h with 30 mM NAC or 100 µM PDTC and then 100 U/ml TNF- were
added without removing the antioxidant. After completion of the
incubation period, cells were washed two times with cold PBS, removed
by careful trypsinization, incubated in 20% FCS for 30 min at 4°C,
and washed two times with cold PBS. Cells were incubated at 4°C for
30 min with monoclonal antibody against E-selectin (BBA1
obtained from R&D Systems, Minneapolis, MN). Samples were analyzed by
flow cytometry in a FACScan cytofluorometer (Becton Dickinson, Mountain
View, CA).
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RESULTS |
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TNF- induces ROS generation in HPAE
cells. We used the oxidant-sensitive dye C-DCDHF-DA to
detect ROS production in TNF-
-stimulated HPAE cells. After uptake,
intracellular esterase hydrolyzes the ester bonds, releasing the intact
nonfluorescent substrate. This reduced substrate is oxidized by ROS to
the fluorescent species carboxydichlorofluorescein, which is retained
by living cells. Confluent HPAE monolayers were stimulated with TNF-
(100 U/ml) for 1 h and then loaded for 20 min with 2.5 µM C-DCDHF-DA
in EGM (with 2% serum) containing no TNF-
. Cells were examined for
fluorescence intensity by viewing under a fluorescence microscope.
Figures 1 and 2
show the fluorescence images of HPAE cells after C-DCDHF-DA staining.
Control cells showed a low intensity of fluorescence. In contrast,
cells treated with TNF-
showed an increased level of fluorescence
(the average fluorescence intensity ranged from 5- to 6-fold of
control). Preincubation of cells for 0.5 h with NAC (30 mM) or PDTC
(100 µM) reduced the TNF-
-induced average fluorescence intensity
to 1.9- and 2.7-fold, respectively, above the control value. Figures 1
and 2 use histograms showing the results of the above experiment in
which fluorescence imaging was used to quantitate the relative
fluorescence intensity. The results indicate that TNF-
treatment of
HPAE cells leads to production of ROS (as indicated by a greater
percentage of cells having higher fluorescence intensities) and that
this was prevented by preincubation of cells with NAC or PDTC.
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TNF--induced ROS generation mediates E-selectin
mRNA expression. We used NAC, which readily enters
cells and serves as both a scavenger for oxidants (1) and a precursor
for glutathione (26), to determine the role of ROS in TNF-
-induced
E-selectin mRNA expression. Preincubation of HPAE monolayers with NAC
(5-30 mM) for 0.5 h caused a dose-dependent decrease in
TNF-
-induced E-selectin mRNA expression (Fig.
3, A and
B), with maximum inhibition of
>90% occurring with 30 mM NAC. At a lower concentration of 5 mM, NAC
showed no inhibitory effect on TNF-
-induced E-selectin mRNA
expression, whereas 15 mM NAC prevented ~30% of the TNF-
response
(Fig. 3, A and
B). We determined the effect of NAC
on the time course of TNF-
-induced E-selectin transcripts. As shown in Fig. 3C, NAC prevented the
expression of E-selectin mRNA at all time points tested.
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NF-B inhibitor PDTC prevents TNF-
-induced
E-selectin mRNA expression. We determined the effects
of PDTC, which functions as an inhibitor of oxidant-induced NF-
B
activity through its ability to chelate metal ions and deliver thiol
groups to cells (39, 40), on TNF-
-induced E-selectin mRNA
expression. As shown in Fig. 4, PDTC
prevented in a dose-dependent manner E-selectin mRNA expression,
suggesting the involvement of ROS in mediating TNF-
-induced
activation of NF-
B and E-selectin gene transcription.
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TNF--induced ROS generation mediates E-selectin
cell surface expression. We evaluated the effects of
both NAC and PDTC to determine whether TNF-
-induced ROS generation
is critical in mediating E-selectin expression on endothelial cell
surface. As shown in Fig. 5, exposure of
HPAE cells to TNF-
for 4 h resulted in a significant increase in
E-selectin cell surface expression. Pretreatment for 0.5 h with NAC
(Fig. 5A) or PDTC (Fig.
5B) prevented the TNF-
-induced
cell surface expression of E-selectin.
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TNF--induced ROS generation mediates E-selectin
promoter activation. To determine whether
TNF-
-induced ROS generation mediated E-selectin expression by
activating gene transcription, we assessed the effects of NAC on
transcriptional activity of the E-selectin promoter after transfection
of HPAE cells with an expression plasmid containing firefly Luc gene as
a reporter driven by the E-selectin promoter. Treatment with TNF-
resulted in an approximately fourfold increase in the activity of
E-selectin promoter (Fig.
6A).
Pretreatment with NAC prevented TNF-
-induced activation of the
E-selectin promoter (Fig. 6A). As a
control, we transfected HPAE cells with constitutively active
cytomegalovirus (CMV) promoter-Luc reporter gene construct (CMV-Luc)
and determined the effects of NAC on its transcriptional activity. As
shown in Fig. 6B, NAC had no effect on
CMV-Luc activity. These results indicate that NAC exerted its
inhibitory effects on TNF-
-induced E-selectin expression by
preventing activation of its promoter.
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TNF--induced ROS production mediates NF-
B
activation. To address whether NAC regulates E-selectin
promoter activity by preventing TNF-
-induced ROS-dependent
activation of NF-
B, we performed electrophoretic mobility shift
assays using oligonucleotides E-selectin NF-
BA (35 bp) or E-selectin
NF-
BB (30 bp) containing the
E-selectin promoter NF-
B sites
1-3. Incubation
of these oligonucleotides with the nuclear extracts prepared from
TNF-
-stimulated HPAE cells revealed the presence of two closely
migrating bands corresponding to p65 homodimers (slow-migrating band)
and a heterodimeric mixture of p65 and p50 (fast- migrating band; Fig.
7,
A and
B, lane
2, and Ref. 38). The DNA binding
activity of NF-
B was more pronounced with E-selectin
NF-
BB compared with E-selectin
NF-
BA, consistent with previous
findings (38). It should be noted that the NF-
B complex interaction
may be influenced by subtle differences in sequence (18). Because
NF-
B site 3 (GGGGATTTCC) in E-selectin NF-
BB probe differs from the
consensus NF-
B sequence (GGGACTTTCC) in two bases shown in italics
compared with NF-
B site 1 (GGATATTCCC) and site 2 (GGGAAAGTTT)
in E-selectin NF-
BA probes,
which differ in four and five bases, respectively, from the consensus
NF-
B sequence, the difference in the binding activities and the
binding patterns between NF-
BA
and NF-
BB is not surprising. In
competition experiments, these binding activities were effectively
competed by the presence of excess unlabeled wild-type E-selectin
NF-
BA or E-selectin
NF-
BB oligonucleotide in a
dose-dependent manner (Fig. 7, A and
B, lanes
3 and 4) or by the
presence of wild-type Ig
B oligonucleotides containing NF-
B
consensus sequences (Fig. 7, A and
B, lane
7). However, the bands were not eliminated by the
presence of either an irrelevant probe or an oligonucleotide bearing
mutations in NF-
B binding sites (Fig. 7,
A and
B, lanes 5 and 6).
|
Pretreatment of cells for 0.5 h with NAC inhibited
TNF- induction of NF-
B activity (Fig.
8, A and
B). Because NAC can also serve as a
reducing agent, we evaluated the effect of a reducing agent, DTT, on
TNF-
activation of NF-
B. Because NF-
B binding was more
pronounced on NF-
B site 3 (Figs.
7B and
8B), we used only E-selectin
NF-
BB probe in this experiment.
At a lower concentration of 1 mM, DTT potentiated the TNF-
-induced
NF-
B binding to the E-selectin promoter (Fig.
8C). At a higher concentration of 2 mM, DTT reduced the slow-migrating binding activity, which was associated with an increase in the fast-migrating binding activity to
E-selectin promoter (Fig. 8C). These
results indicate that NAC and DTT have differential effects on TNF-
induction of NF-
B activity, and thus the effect of NAC in preventing
E-selectin expression could not be ascribed to its reducing activity.
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DISCUSSION |
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In the present study, we have demonstrated that TNF--induces ROS
production in HPAE cells. We present data showing that TNF-
-induced ROS generation serves as a critical signal in the activation of NF-
B
and E-selectin gene expression in HPAE cells. These data include the
inhibitory effects of NAC on TNF-
induction of
1) ROS production,
2) NF-
B activity,
3) E-selectin promoter activity, and
4) E-selectin mRNA and
cell surface protein expression. We also demonstrated that PDTC, an
antioxidant structurally unrelated to NAC, had similar effects in
preventing TNF-
-induced ROS production and the increase in
E-selectin mRNA and cell surface expression; thus the effects of NAC
are likely secondary to its antioxidant action and cannot be ascribed
to its ability to increase the intracellular glutathione concentration
(26). It is also unlikely that NAC exerted its inhibitory effects by
interfering with TNF-
receptor binding, since Suzuki et al. (42)
have shown that inhibition of NF-
B activation by NAC in Jurkat cells
did not occur at the level of binding of TNF-
to its receptor. The
possibility that NAC could directly interact with NF-
B to interfere
with its DNA binding activity can also be ruled out. If this is the
case, NAC should prevent the activation of NF-
B irrespective of
whether it occurs by an oxidant-dependent or -independent mechanism.
Moynagh et al. (28) showed that IL-1
activation of NF-
B in human
glial cells did not involve ROS, and the response was insensitive to NAC. Moreover, NAC did not prevent the ROS generation-independent NF-
B activation induced by the inhibitors of serine/threonine protein phosphatases 1 and 2A, calyculin A, and okadaic acid (42). Therefore, the inhibitory effect of NAC in preventing NF-
B
activation cannot be attributed to a direct interaction of NAC with
NF-
B. Another possibility is that NAC selectively stimulates the
rate of E-selectin mRNA degradation; however, this does not seem to be
the case, since pretreatment with NAC, which prevents the TNF-
induction of E-selectin transcript, slightly induced E-selectin mRNA
expression, which was undetectable in control untreated cells (Fig. 3,
A and
C). Finally, it is unlikely that
reducing activity of NAC was responsible for the inhibitory action of
NAC, since DTT, a reducing agent, failed to prevent the TNF-
-induced
activation of NF-
B.
The present data indicate that downregulation of transcriptional
activity of E-selectin promoter is the likely basis of the NAC-mediated
reduction in E-selectin mRNA expression. This assertion is
supported by a number of observations. In gel shift assays, NAC
prevented TNF--induced binding of NF-
B to the E-selectin promoter. Furthermore, in transient transfection studies, we found that
inhibition of E-selectin promoter-directed Luc expression correlated
with suppression of E-selectin mRNA expression (maximal inhibition of
~90%; compare Figs. 3B and
6A). In control experiments, however, NAC failed to inhibit CMV-promoter-directed Luc expression, indicating that the inhibitory effect of NAC on E-selectin promoter activity was not secondary to Luc mRNA/protein degradation.
Faruqi et al. (13) have reported that the NAC-mediated inhibition of
E-selectin gene transcription in IL-1-activated human umbilical vein
endothelial cells (HUVEC) did not involve inhibition of NF-
B binding
to E-selectin promoter, whereas NAC notably inhibited NF-
B binding
to VCAM-1 promoter. These results are surprising since the E-selectin
promoter NF-
B binding site (GGGATTTCCC) used in this study is
identical to at least one of the two NF-
B binding sites in the
VCAM-1 promoter (GGGTTTCCC and GGGATTTCC). Thus the basis for the
different result from the present study is not clear; it may reflect
differences in activation of NF-
B and other transcription factors
that may cooperate to mediate E-selectin expression in HUVEC compared
with HPAE cells.
Marui et al. (24) and others (44) have reported that the
oxidant-regulated pathway controlling cytokine-induced VCAM-1 expression is distinct from E-selectin expression. Given that p65
homodimer or p50-p65 heterodimer activates transcription of these genes
(11, 38, 39, 45), it is plausible that a common redox-sensitive
mechanism regulates expression of both VCAM-1 and E-selectin in
cytokine-activated endothelial cells. However, the sensitivity to
oxidants in regulation of E-selectin and VCAM-1 expression may be
different due to the following factors. First, the 5' regulatory
region of the gene encoding VCAM-1 has two versus three functional
NF-B binding sites in the E-selectin gene (17, 38). Second,
induction of these genes in response to cytokines requires
participation of a specific repertoire of transcription factors in
concert with NF-
B; that is, interferon regulatory factor-1 (31) is
required for maximal VCAM-1 induction, whereas full cytokine
responsiveness of E-selectin gene requires activation of high-mobility
group protein-1 (Y) (21), activating transcription factor-2, and c-Jun
(36). Third, antioxidants can differentially activate transcription
factors; for example, PDTC induces activation of a number of
transcription factors, including AP-1 (25, 29, 30). In support of this
concept, we demonstrated that, as in the case of VCAM-1 (24), PDTC
prevented the expression of E-selectin mRNA in a
concentration-dependent manner (Fig. 4). However, unlike VCAM-1 (23),
the expression of E-selectin mRNA became insensitive to PDTC after a
2-h exposure period (data not shown). Upon extended exposure, PDTC
itself induced E-selectin transcript in a time-dependent fashion,
although weakly and with much slower kinetics compared with the TNF-
response (data not shown). Munoz et al. (29) have also recently
reported that PDTC mediated a delayed induction of intercellular
adhesion molecule-1 mRNA expression through the activation of AP-1.
These results suggest that PDTC initially affects a component that is
common in the expression of these adhesion molecules, most likely
activation of NF-
B, whereas extended exposure influences another
step required for expression of E-selectin but not of VCAM-1. Because
AP-1 activation is required for maximal expression of E-selectin in
response to TNF-
(36) and because PDTC also induces AP-1 activation
in endothelial cells, these findings are consistent with the concept
that PDTC initially inhibits NF-
B activation and hence E-selectin
expression, but this effect is masked at later time points because of
the ability of PDTC to cooperate with TNF-
in inducing E-selectin
expression through the activation of AP-1.
In summary, the present data indicate that TNF- induces NF-
B
activation and the resultant E-selectin gene expression by a pathway
that involves formation of ROS and that can be inhibited by the
antioxidant actions of NAC or PDTC. The results support the hypothesis
that generation of ROS by inflammatory cytokines in endothelial cells
is a critical signaling mechanism mediating E-selectin expression.
Pharmacological manipulation of the redox equilibrium in endothelial
cells may provide novel means of preventing activation of the
E-selectin gene and hence of inappropriate endothelial hyperadhesiveness after TNF-
stimulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Vijai Baichwal of Tularik for E-selectin-luciferase plasmid and Asma Naqvi for technical assistance.
![]() |
FOOTNOTES |
---|
A. Rahman and J. Kefer contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-27016, HL-46350, and HL-45638.
Present addresses: M. Bando, Dept. of Pulmonary Medicine, Jichi Medical School, Tochigi, Japan 329-04; W. D. Niles, Aurora Biosciences Corporation, 11010 Torreyana Rd., San Diego, CA 92121.
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. §1734 solely to indicate this fact.
Address for reprint requests: A. Rahman, Dept. of Pharmacology (M/C 868), The Univ. of Illinois, College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612-7343.
Received 30 March 1998; accepted in final form 13 May 1998.
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