Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612
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ABSTRACT |
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Reactive oxygen species have been proposed to signal the
activation of the transcription factor nuclear factor (NF)-B in response to tumor necrosis factor (TNF)-
challenge. In the present study, we investigated the effects of H2O2 and
TNF-
in mediating activation of NF-
B and transcription of the
intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis
showed that TNF-
exposure of human dermal microvascular endothelial
cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface
protein expression. In contrast, H2O2 added at
subcytolytic concentrations failed to activate ICAM-1 expression.
Challenge with H2O2 also failed to induce
NF-
B-driven reporter gene expression in the transduced HMEC-1 cells,
whereas TNF-
increased the NF-
B-driven gene expression
~10-fold. Gel supershift assay revealed the presence of p65 (Rel A),
p50, and c-Rel in both H2O2- and
TNF-
-induced NF-
B complexes bound to the ICAM-1 promoter, with
the binding of the p65 subunit being the most prominent. In vivo
phosphorylation studies, however, showed that TNF-
exposure induced
marked phosphorylation of NF-
B p65 in HMEC-1 cells, whereas
H2O2 had no effect. These results suggest that
reactive oxygen species generation in endothelial cells mediates the
binding of NF-
B to nuclear DNA, whereas TNF-
generates additional
signals that induce phosphorylation of the bound NF-
B p65 and confer
transcriptional competency to NF-
B.
redox state; intercellular adhesion molecule-1 promoter; tumor
necrosis factor-; oxidants; nuclear factor-
B; signaling; hydrogen
peroxide
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INTRODUCTION |
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REACTIVE OXYGEN
SPECIES (ROS) play a critical role in the mechanism of
inflammation associated with septicemia and acute respiratory distress
syndrome (17). ROS can function as second messengers in
the signal transduction pathways activated by the proinflammatory cytokines interleukin-1 and tumor necrosis factor (TNF)-
(28, 31). These cytokines regulate the
redox-sensitive transcription factor nuclear factor (NF)-
B, which
participates in a variety of immune, inflammatory, and acute-phase
responses (4, 6). NF-
B/Rel transcription
factors are composed of five distinct DNA binding subunits: NF-
B1
(p50), NF-
B2 (p100/p52), Rel A (p65), Rel B, and c-Rel
(23). Of these, p65, Rel B, and c-Rel possess both
transactivation and DNA binding domains, whereas p50 and p52 lack the
transactivation domain and serve primarily as DNA binding subunits
(36). The different family members can associate in
homodimer or heterodimer combinations through the highly conserved NH2-terminal NF-
B/Rel/Dorsal or Rel homology domain
(16). NF-
B dimers are most commonly composed of p65 and
p50 or p52 subunits (3, 39). Inactive NF-
B
dimers are sequestered in the cytosol in association with inhibitory
molecules of the I
B family. Stimulation of cells with TNF-
results in phosphorylation of I
B-
on serine-32 and -36 or of
I
B-
on serine-19 and -23 by I
B kinases
and
(13, 46). This targets I
B-
and
I
B-
for polyubiquitination and proteasome-mediated degradation
(11, 43). Release from I
B unmasks the
nuclear localization signal of NF-
B and thus mediates its
translocation to the nucleus (7).
Although phosphorylation of IB is a critical regulatory step in the
activation of NF-
B, studies (8, 36)
suggested that phosphorylation of NF-
B p65 after its release from
I
B may enhance its transactivation potential. Thus phosphorylation
of NF-
B may be an additional mechanism regulating the expression of
NF-
B-dependent genes. In the present study, we addressed the
activation of such a gene, intercellular adhesion molecule-1 (ICAM-1),
an inducible cell surface glycoprotein belonging to the immunoglobulin
supergene family (42). Interaction of ICAM-1 with its
counterreceptors (leukocyte
2-integrins CD11a/CD18 and
CD11b/CD18) is a requirement for the recruitment and extravasation of
leukocytes to sites of tissue injury and infection (40,
41). ICAM-1 mRNA and cell surface expression are
upregulated in response to a variety of inflammatory mediators
including TNF-
(18, 22, 33).
Rahman and colleagues (31, 32) have
shown that TNF- induces ROS generation in endothelial cells through
a protein kinase (PK) C-dependent mechanism and that this event is
critical in signaling the activation of NF-
B and the genes encoding
ICAM-1 and E-selectin. Studies (35, 37) have
also shown that H2O2 mediates TNF-
-induced
NF-
B activation; however, there are reports (10,
21) that ROS may not signal the activation of NF-
B and NF-
B-dependent gene expression. A study (20) has shown
that H2O2 induced the expression of ICAM-1 mRNA
in a human dermal microvessel endothelial cell (HMEC-1) line; in other
studies, H2O2 failed to induce DNA binding
activities of the transcription factors activator protein (AP)-1 and
NF-
B (21) that are known to activate ICAM-1 gene
transcription in endothelial cells (18, 22,
26). In light of these observations, we compared the
effects of H2O2 with those of TNF-
in
mediating the activation of NF-
B in endothelial cells using HMEC-1
cells. We demonstrated that both H2O2 and
TNF-
induced the DNA binding activity of NF-
B; however, the
binding of NF-
B induced by H2O2 was
transcriptionally "silent" as determined by its inability to
activate the NF-
B-driven reporter gene and ICAM-1 expression. In
contrast, the NF-
B binding activity induced by TNF-
was
transcriptionally active. These data suggest that ROS production
signals nuclear NF-
B DNA binding activity; however, the bound
NF-
B may remain transcriptionally inactive. Thus TNF-
activates
additional signaling pathway(s) that induce phosphorylation of NF-
B
p65 and confer transcriptional competency to the DNA-bound NF-
B.
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METHODS |
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Cell culture.
Human dermal microvascular endothelial cells (HMEC-1) were maintained
in culture in MCDB-131 medium (GIBCO BRL, Life Technologies, Gaithersburg, MD) supplemented with 5% fetal bovine serum (FBS), 10 ng/ml of human epidermal growth factor (Sigma, St. Louis, MO), 1 µg/ml of hydrocortisone, 5 mg/ml of L-glutamine, and
antibiotics (penicillin-streptomycin) ("complete" MCDB-131). HMEC-1
cells, an immortalized endothelial cell line transformed by the SV40 large T antigen, retain the endothelial cell phenotype and functional characteristics; they express and secrete von Willebrand factor, take
up acetylated low-density lipoprotein, form tubes when grown in
Matrigel, and express CD31, CD35, ICAM-1 (CD54), and CD44
(1). HMEC-1 cells were passaged in uncoated culture dishes
until they reached confluence. The cells between passages 17 and 21 were used in these studies, and all cell studies were
carried out under the same conditions. Confluent HMEC-1 cells were
serum starved for 2-4 h, washed two times with serum- and phenol
red-free Dulbecco's modified Eagle's medium, allowed to equilibrate
for 30 min, and then incubated with the indicated concentrations of
H2O2 (Sigma) or recombinant human TNF-
(specific activity 2.3 × 107 U/mg protein; Promega,
Madison, WI) for all experiments unless otherwise specified.
Cell viability.
Two methods were used to evaluate cell viability after
H2O2 and TNF- challenge. Trypan blue (Sigma)
exclusion studies were carried out according to manufacturer's
suggested protocol. Confluent cells were treated with
H2O2 or TNF-
for 2 h, washed gently
with PBS two times, trypsinized, resuspended, and washed with
complete MCDB-131. The cell suspension (10 µl) was mixed with
10 µl of 1× trypan blue solution, and 10 µl of the resulting
mixture were loaded onto a hemocytometer. Results showed that
95-98% of the cells were viable after challenge with 100 µM
H2O2 or 100 U/ml of TNF-
, whereas only
70-80% of the cells were viable after 1 mM
H2O2 challenge. Reduction of tetrazolium salt
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was
also used to assay cell viability (19). These results showed that HMEC-1 cells exposed to 100 µM
H2O2 were viable after treatment for 5, 60, or
120 min, whereas cells challenged with 1 mM
H2O2 were not (Lum H, personal communication).
Intracellular oxidant generation.
Oxidant generation in HMEC-1 cells was measured as previously described
(32). Briefly, confluent HMEC-1 cells were challenged with
TNF- or H2O2 for 15 min in serum-free phenol
red-free DMEM, and all subsequent steps were conducted in the dark.
After challenge, the cells were washed two times with MCDB-131 (2%
FBS) and loaded for 20 min with 1 µM
5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate
bis(acetoxymethyl ester) (C-DCHF-DA; Molecular Probes, Eugene, OR).
The dye solution was freshly prepared in prewarmed MCDB-131 (2% FBS)
for each sample, and the samples were independently stained so that
they were exposed to dye for the same amount of time. After dye loading
at 37°C, the cells were rinsed two times with MCDB-131 (2% FBS)
containing no dye, and the cultures were viewed with a fluorescence
microscope and photographed. Quantitative fluorescence was imaged with
a Nikon Diaphot 200 microscope (Nikon, Garden City, NJ) and
ImagePro Plus software (Media Cybernetics, Silver Spring, MD).
Northern analysis.
Total RNA was isolated from HMEC-1 cells with QIAGEN RNeasy columns
after homogenization through QIAshredder columns (QIAGEN, Chatsworth,
CA). Quantification and purity of RNA were assessed by the ratio of
absorbance at 260 nm to that at 280 nm. An aliquot of RNA (10-20
µg) was analyzed for ICAM-1 mRNA expression as previously described
(30). Autoradiography was performed with an intensifying screen at 70°C for 12-24 h.
Flow cytometric analysis.
HMEC-1 monolayers on six-well tissue culture dishes were left untreated
or were stimulated with H2O2 or TNF- as
indicated, washed two times with cold PBS (Ca2+ and
Mg2+ free), removed by trypsinization, and incubated
in 20% horse serum for 30 min at 4°C to block nonspecific binding.
Samples were pelleted at 4°C and washed two times with cold 3% horse
serum in PBS. The cells were incubated in the same wash buffer with the
anti-human ICAM-1 monoclonal antibody RR1/1 (5-10 µg/ml)
(provided by Dr. Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT) (14), washed two times, and incubated with a
FITC-conjugated goat anti-mouse secondary antibody (10-100
µg/ml; Sigma). After two washes and fixation with 2%
paraformaldehyde while being vortexed, the samples were analyzed by
flow cytometry in a FACScan cytofluorometer (Becton Dickinson, Mountain
View, CA), with mean fluorescence intensity gated above that of the
secondary antibody alone. When purified mouse IgG (1 mg/ml) was used in
place of RR1/1, no significant increase in fluorescence was observed.
Reporter gene construct, endothelial cell transfection, and
luciferase assay.
The NF-B-luciferase plasmid (pNF-
B-Luc) containing five copies of
the consensus NF-
B sequence linked to the minimal E1B promoter-luciferase reporter gene was purchased from Stratagene (La
Jolla, CA). HMEC-1 cells were plated on six-well tissue culture dishes
and transfected the following day at ~70-80% confluence according to the DEAE-dextran method with slight modifications (12). Briefly, 2 µg of DNA were mixed with 0.15 mg/ml of
DEAE-dextran and 0.1 mM chloroquine in serum- and antibiotic-free
MCDB-131 for 1.5 h followed by a 30-s shock with 10% dimethyl
sulfoxide in PBS. The cells were then washed two times with complete
MCDB-131 containing 10% FBS and grown to confluence. To determine
transfection efficiency, HMEC-1 cells were transfected with the plasmid
pGreen Lantern-1, which expresses green fluorescence protein (GIBCO
BRL), and the transfected cells were then subjected to flow cytometry to determine green fluorescence protein expression. Using the above
protocol, we showed that transfection efficiencies were ~50%, with
<5% variability in a given sample. After transfection, the cells were
exposed to H2O2 or TNF-
at the indicated
concentrations and time periods. Cell extracts were prepared, and
luciferase activity was determined with a TD 20/20 luminometer (Turner
Designs, Sunnyvale, CA) with the Promega Biotech luciferase reporter
assay system. Values are expressed as relative light units per
microgram of protein extract, and the protein content was determined
with the Bio-Rad (Hercules, CA) protein determination kit.
Nuclear protein isolation.
Cells were washed three times with ice-cold Tris-buffered saline (TBS)
and resuspended in 400 µl of buffer A [10 mM HEPES (pH
7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml of leupeptin, and 5 µg/ml of aprotonin]. After 15 min, Nonidet P-40 (NP-40) was added to
a final concentration of 0.6%. Nuclei were pelleted and suspended in
50 µl of buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 5 µg/ml of leupeptin, and 5 µg/ml of aprotonin]. After 30 min of agitation at 4°C, the lysates
were centrifuged, and the supernatants containing the nuclear proteins
were transferred to fresh vials in 15-µl aliquots and stored at
70°C until used.
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA) was performed as previously
described (31). Briefly, nuclear extract (10 µg) was
incubated with 1 µg of poly(dI-dC) in 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. End-labeled, double-stranded oligonucleotides (30,000 counts/min each) were then
added in the absence and presence of a 25- to 100-fold molar excess of
cold wild-type (ICAM-1NF-B) or mutant (mut-ICAM-1NF-
B) competitor
and incubated for 15 min at room temperature. In antibody supershift
experiments, nuclear extracts were incubated for 15 min at room
temperature with polyclonal rabbit antibody to various NF-
B proteins
(2 µg/20 µl; Santa Cruz Biotechnology, Santa Cruz, CA) before
incubation with the labeled probe for another 15 min at room
temperature. The DNA-protein complexes were resolved by 5-6%
native polyacrylamide gel electrophoresis (PAGE) in low ionic strength
buffer (0.25× Tris-borate-EDTA). Oligonucleotides used for the gel
shift analysis were 5'-AGCTTGGAAATTCCGGAGCTG-3' for ICAM-1NF-
B and 5'-AGCTTccAAATTCCGGAGCTG-3' for
mut-ICAM-1NF-
B. The oligonucleotide designated as ICAM-1NF-
B
represents the 21-bp sequence of ICAM-1 promoter encompassing the
downstream NF-
B binding site located 223 bp upstream from the
transcription initiation site (18, 22). The
oligonucleotide mut-ICAM-1NF-
B is similar to ICAM-1NF-
B except
that it has 2-bp mutations in the NF-
B binding site. NF-
B
sequence motifs within the oligonucleotides are underlined and
mutations are shown in lowercase. In separate experiments, nuclear
proteins were incubated with the oligonucleotide Ig-
B
(5'-AGTTGAGGGGACTTTCCCAGGC-3'), which contains the
consensus NF-
B binding site present in the Ig gene and pNF-
B-Luc construct.
In vivo labeling of cells and immunoprecipitation.
In vivo labeling of cells and immunoprecipitation were carried out as
described by Ollivier et al. (27) with slight
modifications. Briefly, confluent monolayers of HMEC-1 cells on
six-well tissue culture dishes were washed two times with
phosphate-free medium and incubated for 2 h before being loaded
with 200-500 µCi [32P]orthophosphate/ml for
3-4 h, followed by stimulation with TNF- or
H2O2. Cells were needle lysed in 500 µl of
cold radioimmunoprecipitation assay buffer [1% Triton X, 1%
deoxycholate, 150 mM NaCl, 10 mM Tris · HCl (pH 7.5), 1 mM
EGTA, 1 mM PMSF, 1 mM Na3VO4, 0.1% SDS, 5 µg/ml of aprotonin, and 1 nM calyculin A]. NF-
B p65 was recovered by immunoprecipitation (after preclearance with 50 µl of protein A
agarose beads) with an anti-p65 antibody (1 µg, 4-18 h at 4°C; Santa Cruz Biotechnology) and protein A agarose beads (2 h at 4°C;
Boehringer Mannheim, Indianapolis, IN). The precipitated proteins were
washed three times, including one high salt wash (radioimmunoprecipitation assay with 500 mM NaCl), and were then pelleted. The antibody-p65-bead complexes were boiled in sample buffer
containing 25 mM DTT for 5 min and spun, and the supernatants were
separated on 10-12.5% SDS-polyacrylamide gels for 4.5 h (20 mA). Coomassie blue staining of the gels revealed the presence of a
single band corresponding to a molecular mass of 65 kDa. The
gels were then either dried and visualized by autoradiography or
transferred to polyvinylidene difluoride membranes for Western blotting
and confirmation of NF-
B p65.
Western blot analysis.
The immunoprecipitated NF-B p65 samples were subjected to
SDS-PAGE (10%) as described in In vivo labeling of cells and
immunoprecipitation and then transferred to polyvinylidene
difluoride membranes (Bio-Rad). The membranes were blocked for 1 h
with 5% (wt/vol) nonfat dry milk solution in 10 mM Tris base,
150 mM NaCl, and 0.05% Tween 20 before the membranes were incubated
for 1 h with rabbit polyclonal anti-human NF-
B p65 antibodies
(Santa Cruz Biotechnology) diluted 1:1,000. The membranes were washed
three times with TBS-Tween 20 and incubated for another hour with goat
anti-rabbit horseradish peroxidase-linked IgG (Amersham, Arlington
Heights, IL) diluted 1:5,000. After the washes, antibody-labeled
proteins were detected by the enhanced chemiluminescence method
(Amersham) according to manufacturer's recommendations.
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RESULTS |
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H2O2 failed to mimic TNF--induced
expression of ICAM-1 in HMEC-1 cells.
We compared the effects of H2O2 with those of
TNF-
on NF-
B-dependent ICAM-1 gene expression. Northern blot
analysis showed that H2O2 failed to activate
ICAM-1 transcription in HMEC-1 cells at all concentrations studied,
whereas TNF-
induced robust ICAM-1 mRNA expression (Fig.
1). HMEC-1 cells were subjected
to fluorescence-activated cell-sorting analysis with the anti-ICAM-1
monoclonal antibody RR1/1 (14) to determine whether
H2O2 altered the cell surface expression of
ICAM-1 independent of increased mRNA expression. As shown in Fig.
2, H2O2 failed to
induce ICAM-1 cell surface expression. In contrast, TNF-
induced a
marked increase in ICAM-1 cell surface expression within 2 h,
which increased further after 18 h of exposure (Fig. 2).
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TNF- and H2O2 challenges induced similar
intracellular oxidant generation.
To determine whether differences in ICAM-1 induction by
H2O2 and TNF-
could be ascribed to
differences in the oxidant-generating capacity of the mediators,
confluent HMEC-1 cells were challenged with TNF-
(100 U/ml) or
H2O2 (subcytolytic concentrations ranging from
10 to 500 µM) for 15 min and loaded with the oxidant-sensitive dye
C-DCHF-DA as described in METHODS. After incorporation and subsequent cleavage by cellular esterases, C-DCHF-DA was trapped in
cells and converted to the fluorescent species
carboxydichlorofluorescein after oxidation. Results showed that 100 U/ml of TNF-
and 100 and 500 µM H2O2 both
induced comparable C-DCHF-DA fluorescence compared with that in control
cells, whereas the fluorescence activated by 10 µM
H2O2 was similar to the control values (Fig. 3A). Figure 3B
shows the quantification of the relative fluorescence intensity of each
field shown in Fig. 3A. Total relative fluorescence for each
image was divided into three classes of brightness where class
1 represents the area of each cell with lowest brightness intensity and class 3 represents the area of each cell with
highest brightness intensity. Control cells exhibited fluorescence in brightness class 1, and treatment with 100 and 500 µM
H2O2 or 100 U/ml of TNF-
caused a shift to a
higher brightness, with maximum fluorescence occurring in brightness
class 2 (Fig. 3B). These results indicate that
differences in ICAM-1 expression are not secondary to a differential
oxidant-generating capacity of the two mediators.
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H2O2 failed to mimic TNF--induced
transcriptional activity of NF-
B.
We next evaluated the ability of H2O2 to induce
transcriptional activation of NF-
B because oxidants generated in
response to TNF-
are critical in signaling NF-
B activation and
ICAM-1 (31). We transiently transfected HMEC-1 cells with
the plasmid pNF-
B-Luc containing five copies of the consensus
NF-
B site linked to a minimal adenovirus E1B promoter-luciferase
reporter gene. NF-
B-directed luciferase activity increased
~10-fold when the transfected cells were exposed to TNF-
(Fig.
4). In contrast, H2O2 failed to induce NF-
B-directed
luciferase expression (Fig. 4). Because H2O2
failed to induce transcriptional activation of pNF-
B-Luc, we focused
our attention on NF-
B to explain the basis for the lack of ICAM-1
induction.
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H2O2 and TNF- induced NF-
B DNA
binding activity.
Because the antioxidant actions of N-acetylcysteine and
pyrrolidine dithiocarbamate prevented TNF-
-induced NF-
B DNA
binding activity (31, 32), we determined
whether the inability of H2O2 to activate
ICAM-1 transcription could be explained by the failure of NF-
B to
bind to the ICAM-1 promoter. We performed the EMSA using the
oligonucleotide ICAM-1NF-
B (21 bp), which contains the downstream
NF-
B sequence in the ICAM-1 promoter required for ICAM-1 expression
(18, 22). Both H2O2
and TNF-
induced marked binding of NF-
B to the ICAM-1 promoter
(Fig. 5A). These results also
showed that H2O2 induced NF-
B binding in a time-dependent manner, with the maximum response occurring after 30-60 min (Fig. 5B).
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H2O2- and TNF--induced NF-
B complexes
were predominantly composed of NF-
B p65.
The gel supershift assay was carried out to determine whether
differences in the H2O2 and TNF-
responses
could be explained by differences in the NF-
B complex bound to the
ICAM-1 promoter. Antibody directed against p50 (which lacks the
transactivation domains), p65, or c-Rel (which contain the
transactivation domains) were incubated with nuclear extracts isolated
from H2O2- or TNF-
-treated cells before
addition of the labeled ICAM-1NF-
B oligonucleotide. Antibody to p65
significantly reduced both H2O2 (Fig.
7A, lane 4)- and
TNF-
(Fig. 7, A, lane 6, and B,
lanes 6 and 7)-induced NF-
B binding to the
ICAM-1 promoter. In contrast, antibodies against p50 and c-Rel had less
effect on DNA binding activities (Fig. 7, A, lanes
2, 3, and 7, and B, lanes
4 and 5). Therefore, differential binding of NF-
B
subunits to DNA induced by H2O2 and TNF-
fails to explain the observed differences in the transcriptional activation responses induced by the two mediators.
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TNF- and H2O2 differentially
phosphorylated NF-
B p65 subunit.
Because phosphorylation of NF-
B p65 may regulate
transcriptional activation of NF-
B (2,
45), we determined whether alterations in the
phosphorylation status of NF-
B p65 after
H2O2 and TNF-
stimulation could explain the
differential response. Cells were loaded with
[
-32P]orthophosphate in phosphate-free medium and
stimulated with H2O2 or TNF-
for 30 min.
Immunoprecipitation of NF-
B p65 followed by SDS-PAGE showed marked
phosphorylation of p65 in response to TNF-
(Fig.
8A, lane 3) but not
to H2O2 (Fig. 8A, lane
2). Similar results were obtained after 15 min of
H2O2 or TNF-
challenge (data not shown). To
rule out the possibility that the phosphorylated proteins observed did
not represent coimmunoprecipitation of other NF-
B subunits
associated with p65, Western blot analysis was performed with the
anti-p65 antibody. These results indicated the selective recovery of
p65 after immunoprecipitation (Fig. 8B).
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DISCUSSION |
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Oxidants have been proposed to function as second messengers in
the activation of transcription factors (5,
24, 28). Studies have shown that
H2O2 induces the activation of NF-B and the
transcription of NF-
B-dependent genes in several cell types (37). Although oxidants can induce binding of NF-
B in
endothelial cells (5), it remains unclear whether
additional signals are involved in the transcriptional activation of
NF-
B-dependent genes such as ICAM-1.
Oxidant generation induced by TNF- in endothelial cells contributes
to the mechanism of NF-
B activation and transcription of the genes
encoding ICAM-1 and E-selectin (31, 32). In
the present study, we determined whether H2O2
mimics the effects of TNF-
in mediating the activation of NF-
B
and ICAM-1 expression. We demonstrated that
H2O2 promoted the binding of the NF-
B p65 subunit to its cognate site; interestingly, the bound NF-
B remained transcriptionally "silent." In contrast, the TNF-
-induced
binding of NF-
B p65 promoted a robust NF-
B-driven reporter gene
activation and ICAM-1 expression. The results suggest that the
transcriptional activity of NF-
B induced by TNF-
is conferred by
phosphorylation of the NF-
B p65 subunit, whereas
H2O2 has no effect on the phosphorylation of
this subunit. Thus phosphorylation of NF-
B p65 appears to be
critical in mediating the transcriptional activation of
NF-
B-dependent genes in endothelial cells.
H2O2 is a permeable oxidant that rapidly enters
cells but is also rapidly degraded by intracellular antioxidants. To
ensure that intracellular antioxidants did not scavenge
H2O2 and interfere with the response, we made
studies in which HMEC-1 cells transfected with pNF-B-Luc were
exposed to freshly prepared H2O2 every 2 h
for up to a 6-h period. Replenishment of H2O2
in these studies failed to induce transcriptional activity of NF-
B
(data not shown); thus the present results cannot be explained on the
basis of degradation of H2O2.
There may be various reasons for the failure of
H2O2 to activate NF-B-dependent
transcription in HMEC-1 cells. One possibility is that
H2O2 inhibits NF-
B-dependent transcription
by oxidizing the critical cysteine residue required for NF-
B DNA
binding activity (25). We observed that
H2O2 was capable of promoting NF-
B DNA binding activity but that the bound NF-
B was transcriptionally inactive; thus under the conditions of our experiment, oxidation of
NF-
B does not appear to be responsible for failure of
H2O2 to induce NF-
B activity. Another
possibility is that there may be differences in the redox state
resulting from differences in oxidant generation. To address this
question, endothelial cells were loaded with the oxidant-sensitive dye
C-DCHF-DA with which we assayed oxidant generation in response to
H2O2 and TNF-
. Because challenge of cells
with H2O2 and TNF-
resulted in comparable dye fluorescence, any difference in the redox state cannot explain the
failure of H2O2 to activate NF-
B in HMEC-1
cells. Another possibility is that H2O2 and
TNF-
differentially activate the mitogen-activated protein kinase
(MAPK) pathway regulating NF-
B-dependent gene expression
(44). However, in other studies, we have shown that both
H2O2 and TNF-
induced comparable and rapid
phosphorylation of MAPK in HMEC-1 cells (True A, Lum H, Beno DW, and
Malik AB, unpublished results); thus differences in activation
of this pathway also cannot explain the present observations.
The question arises as to what confers transcriptional competency to
the DNA-bound NF-B after TNF-
challenge. Previous studies (22, 38) showed that overexpression of
NF-
B p65 in endothelial cells transactivated the ICAM-1 and vascular
cell adhesion molecule-1 promoters in a
B site-dependent manner.
Phosphorylation of serine-529 in the transactivation domain 1 of
NF-
B p65 in HeLa cells exposed to TNF-
may promote NF-
B
transcriptional activation (45). Phosphorylation at this
residue was specific in that it did not interfere with nuclear
translocation or DNA binding activity. Phosphorylation of
transactivation domain 2 of NF-
B in phorbol ester-stimulated COS-7
cells also resulted in enhanced NF-
B transcriptional activity
(36). In the present study, we observed that the
TNF-
-challenged cells contained the phosphorylated p65 species,
consistent with a critical role for NF-
B p65 phosphorylation in
mediating NF-
B activation. The kinase responsible for p65
phosphorylation in endothelial cells is unknown. Casein kinase-II and
MAPKs have been proposed to phosphorylate p65 in vitro (8,
44). PKC-
may also regulate phosphorylation and
transcriptional activity of NF-
B p65 in endothelial cells
(2) and thus may mediate ICAM-1 gene transcription after
TNF-
stimulation (31). In addition, phosphorylation of
NF-
B p65 by the catalytic subunit of PKA may activate NF-
B
(47). NF-
B p65 phosphorylation may also be necessary for association with the coactivator p300/cAMP-responsive element binding protein (CBP), a requirement for NF-
B transactivation (15, 29). PKA-induced phosphorylation of
NF-
B p65 exposed the binding site for interaction with p300/CBP
(48), suggesting a mechanism by which p65 phosphorylation
can induce transcriptional activation. Thus failure of phosphorylation
of NF-
B p65 by one or more of these kinases could explain the
inability of H2O2 to induce NF-
B gene
expression in HMEC-1 cells.
Another explanation for our results is that
H2O2 fails to activate transcription factors
that may coordinately regulate transcription of the ICAM-1 gene. For
example, CAAT enhancer binding protein (C/EBP) was reported to
cooperate with NF-B in the mechanism of TNF-
-induced ICAM-1
expression (18). We showed that
H2O2 did not induce transcriptionally active
NF-
B in cells transfected with the pNF-
B-Luc construct. Because
this construct is driven by NF-
B sequences and C/EBP does not bind
to these sequences, it is unlikely that the failure of
H2O2 to activate C/EBP can explain our
observations. The role of AP-1 was also proposed in the mechanism of
ICAM-1 transcription (26), although the ability of
H2O2 to activate AP-1 in endothelial cells
remains controversial (21). The results obtained with
pNF-
B-Luc reporter construct argue that transcriptionally silent
NF-
B is the more likely explanation for the absence of ICAM-1
induction by H2O2. A recent study
(9) has shown that p50 can act as a negative regulator of
NF-
B-dependent gene expression. Because we did not find that
H2O2 preferentially induced p50 binding, it is
unlikely that H2O2 prevented the
transcriptional activation of NF-
B and expression of ICAM-1 by this mechanism.
The present observations differ from those in previous reports
(20, 34). The cell culture conditions may
have been different such that there may be differences in the redox
state of cells. This could result in oxidant-induced activation of
NF-B and, thereby, ICAM-1 expression. The present results are
important because they unmask a phosphorylation-dependent mechanism
that may signal transcriptional activation of NF-
B after NF-
B
binding to DNA.
Although H2O2 did not induce transcriptionally
active NF-B, the present results are fully consistent with our
observations that TNF-
-induced ICAM-1 expression can be blocked by
antioxidants (31). Because the results point to an
important role for oxidants generated by TNF-
in signaling the
binding of NF-
B to the promoter, it would be expected that
antioxidants would prevent TNF-
-induced NF-
B binding and the
resultant ICAM-1 expression as demonstrated (31).
In summary, the present results show that H2O2
induces the nuclear DNA binding activity of NF-B and that the
binding of NF-
B induced by H2O2 alone is
insufficient to activate NF-
B-dependent reporter gene activation and
ICAM-1 transcription. These results suggest that the
H2O2-induced DNA binding activity of NF-
B in the absence of phosphorylation of NF-
B p65 fails to activate ICAM-1
gene transcription. Thus TNF-
-induced phosphorylation of NF-
B p65
may be an important factor regulating expression of the
NF-
B-dependent ICAM-1 gene in endothelial cells.
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ACKNOWLEDGEMENTS |
---|
We thank Tina Leisner for assistance with fluorescence-activated cell-sorting analysis and Khandaker N. Anwar for technical support.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-27016, HL-46350, and HL-45638.
Address for reprint requests and other correspondence: A. Rahman, Dept. of Pharmacology, College of Medicine, Univ. of Illinois, 835 South Wolcott Ave., Chicago, IL 60612-7343 (E-mail: ARahman{at}uic.edu).
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.
Received 23 February 2000; accepted in final form 28 March 2000.
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