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(Received for publication, May 9, 1995; and in revised form, June 7, 1995 ) From the
We investigated the mechanisms by which H
Adhesion of circulating polymorphonuclear leukocytes (PMN) ( Recent studies show
that the reactive oxidant, H In this study, we examined
the basis of H
Confluent cells were washed twice with serum-free
DMEM (without phenol red) containing 20 mM HEPES and incubated
for 2 h before treatment with the agents described below. The
experiments using the inhibitors (PDTC, N-Cys(Ac), or 3-AB)
required a 1-h preincubation period in serum-free medium with each
inhibitor, and treatment was continued during the 1-h
H
The
RNA samples (20 µg/lane) were subjected to gel electrophoresis in
denaturing 1% formaldehyde-agarose gels and transferred overnight in 20
For treatment of HUVEC with antisense and nonsense
oligonucleotides, the cells were rinsed as described above with
serum-free DMEM and incubated for 4 h in serum-free DMEM medium with
addition of 5 µg/ml Lipofectin and an oligonucleotide at
concentrations of 50 and 100 nM. After incubation, the medium
was removed, fresh medium containing Lipofectin and the particular
oligonucleotide was added, and the cells were treated for 1 h with 100
µM H
The
nuclei were incubated for 30 min at 30 °C in 0.3 ml of the assay
mixture (25 mM Tris-HCl, pH 8.0, 1.25 mM concentration each of ATP, CTP, and GTP, 12.5 mM MgCl
Sequence motifs within the oligonucleotide are underlined, the
mutations are in lowercase, and the relative positions of the sequence
motifs are shown in Fig.7and Fig. 8. The NF-
Figure 7:
H
Figure 8:
Localization of the H
Figure 1:
Effects of actinomycin D on
H
Figure 2:
Stability of
H
Treatment
with actinomycin D at the time of H
Figure 3:
Nuclear run-on analysis of
H
Figure 4:
Effect of an antisense oligonucleotide on
the expression of ICAM-1 message induced by H
Figure 5:
Effect of 3-aminobenzamide (3AB),
pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine (NAC) on
H
Figure 6:
H
Unlike H
Figure 9:
AP-1 and Ets binding sites functionally
cooperate to form H
Figure 10:
Characterization of the
H
We introduced point mutations
into the AP-1/Ets repeat to assess the importance of the AP-1 and Ets
binding sites in the induction of these complexes. In the absence of
H In the present study, we examined mechanisms of
H Within the H Similar AP-1/Ets composite elements have been found in the promoters
of other genes including the macrophage scavenger receptor (MSR) gene
(Wu et al., 1994). We demonstrated that the AP-1/Ets elements
from the MSR gene were sufficient to induce transcription in response
to H The AP-1 binding sites of the
AP-1/Ets repeats are also similar to the anti-oxidant response element
(ARE) (Rushmore et al., 1991), a cis-acting sequence
element identified in oxi-protective enzyme genes, glutathione S-transferase Ya subunit (GST Ya) and NAD(P)H:quinone
oxidoreductase (Li and Jaiswal, 1992; Nguyen and Pickett, 1992; Pinkus et al., 1995). Several studies have shown that
H The agent 3-aminobenzamide (3-AB), which
inhibits poly(ADP-ribosyl)ation and prevents oxidant-induced synthesis
of c-Fos (Amstad et al., 1992), prevented the
H Since
the regulation of H In summary, the present results indicate a unique mechanism
of H
Volume 270,
Number 32,
Issue of August 11, pp. 18966-18974, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
O
and Tumor Necrosis Factor-
Activate Intercellular Adhesion
Molecule 1 (ICAM-1) Gene Transcription through Distinct cis-Regulatory Elements within the ICAM-1 Promoter (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O increases intercellular adhesion molecule 1 (ICAM-1; CD54)
expression in endothelial cells. The HO-induced
increase in ICAM-1 mRNA was inhibited by actinomycin D, by the
antioxidant N-acetylcysteine, and by 3-aminobenzamide (which
blocks oxidant-induced AP-1 activity), but not by pyrrolidine
dithiocarbamate (which blocks oxidant-induced NF-
B activity).
Nuclear run-on and transient transfections of ICAM-1 promoter
constructs indicated that HO stimulated ICAM-1
gene transcription by activation of a distinct region of the ICAM-1
promoter. The HO-responsive element was
localized to sequences between -981 and -769 (relative to
the start codon). Located within this region are two 16-base pair
repeats, each containing binding sites for the transcription factors
AP-1 and Ets. A similar composite AP-1/Ets element isolated from the
macrophage scavenger receptor gene conferred HO responsiveness to a minimal promoter. Mutation of the 16-base
pair repeats within the ICAM-1 promoter prevented
HO-induced DNA binding activity, and their
deletion abrogated the HO-induced
transcriptional activity. In contrast, TNF
induced ICAM-1
transcription via activation of promoter sequences between -393
and -176, a region with C/EBP and NF-B binding sites. The
results indicate that HO activates ICAM-1
transcription through AP-1/Ets elements within the ICAM-1 promoter,
which are distinct from NF-B-mediated ICAM-1 expression induced by
TNF.
)to the vascular endothelium is a critical step in the
inflammatory response (Nourshargh and Williams, 1990). PMN adhesion to
the endothelium occurs during reperfusion of tissues when reactive
oxygen intermediates such as HO are generated
(Hernandez et al., 1987). The adhesion event is mediated by
molecules present or expressed on the surface of endothelial cells and
PMN (Lo et al., 1989). Endothelial cells express intercellular
adhesion molecule 1 (ICAM-1; CD54), a counter-receptor for CD11/CD18
integrin (Dustin et al., 1988) that promotes adhesion and
transendothelial migration of PMN (Smith et al., 1989).
Studies using monoclonal antibodies show that increased cell surface
ICAM-1 expression is required for migration of PMN to sites of
inflammation and PMN-mediated endothelial injury associated with
reperfusion (Kukielka et al., 1993). ICAM-1 gene expression is
induced by tumor necrosis factor- (TNF), interferon ,
and interleukin-1
(Myers et al., 1992; Wertheimer et
al., 1992; Look et al., 1994).O, also promotes
ICAM-1 expression in endothelial cells and ICAM-1-dependent adhesion of
PMN (Lo et al., 1993; Bradely et al., 1993; Sellak et al., 1994). HO was recently
reported to also increase ICAM-1 expression on keratinocytes (Ikeda et al., 1994). In human umbilical vein endothelial cells
(HUVEC), we found that oxidant-induced ICAM-1 expression was associated
with increased ICAM-1 mRNA levels occurring 1 h after
HO exposure (Lo et al., 1993).
HO activates transcription factors, AP-1 and
NF-B, in a mouse osteoblastic cell line (Nose et al.,
1991) and in HeLa and Jurkat cells (Meyer et al., 1993). The
ICAM-1 gene contains a number of AP-1-like and NF-B-like binding
sites within its promoter region (Voraberger et al., 1991).
Taken together, these observations suggest that the activation of these
transcription factors by HO may be a mechanism
of endothelial ICAM-1 gene expression.O-induced ICAM-1 expression in
endothelial cells. We showed that HO activated
ICAM-1 gene transcription via a 212-base pair (bp) promoter region
between 981 and 769 bp upstream of the coding sequences. This region
contained two 16-bp repeats which are binding sites for the
transcription factors AP-1 and Ets. AP-1/Ets composite elements were
shown to be sufficient to mediate HO-induced
transcription. Although the AP-1/Ets elements also responded to
TNF, the TNF-induced ICAM-1 expression was mediated by
promoter sequences between 393 and 176 bp upstream of the gene,
containing binding sites for C/EBP and NF-B. Therefore,
HO and TNF activate ICAM-1 gene
transcription in endothelial cells through distinct cis-regulatory elements within the ICAM-1 promoter. The
results identify a novel oxidant response element and indicate that
mediator-specific regulation of ICAM-1 expression involves the
interaction of multiple factors with the ICAM-1 promoter.
Materials
Diethylpyrocarbonate, DMEM, heparin,
HEPES, 3.0% HO, MOPS, PMSF, spermidine,
spermine, pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine (N-Cys(Ac)) were purchased from
Sigma. We purchased 3-aminobenzamide (3-AB) from Pfaltz and Bauer
(Stamford, CT). Guanidine thiocyanate, restriction enzymes, random
primer labeling kit, QuikHyb hybridization mix, and
Duralose-UV
nitrocellulose membranes were purchased from
Stratagene. Human ICAM-1 cDNA was provided by Dr. T. Springer, Harvard
Medical School, Boston, MA. Plasmid containing the cDNA for rRNA was
provided by Dr. M. L. Brown, Boehringer-Ingelheim Pharmaceuticals,
Ridgefield, CT. Plasmid EL1-BS, containing partial human E-selectin
cDNA, was provided by Dr. L. Osborn, Biogen, Cambridge, MA. Agarose,
actinomycin D, LipofectAMINE, and RPMI were purchased from Life
Technologies, Inc. Riboprobe Gemini System II and RNase-free DNase were
purchased from Promega Biotech. Fetal bovine serum was obtained from
Hyclone Laboratories. [
-P]dCTP (3,000
Ci/mmol), [
-
P]ATP (3,000 Ci/mmol), and
[
-P]UTP (3,000 Ci/mmol) were purchased from
DuPont NEN. The antisense oligomer ISIS 1570
(5`-TGGGAGCCATAGCGAGGCTGA-3`) to the 5` end of the ICAM-1 cDNA and a
nonsense oligomer (5`-AGTCGGAGCGATACCGAGGGT-3`) were generous gifts
from Sterling-Winthrop Drug Co., Rensselaer, NY. Oligonucleotides were
purchased from Integrated DNA Technologies Inc. (Coralville, IA).
ICAM-1 luciferase reporter gene plasmids were gifts from Dr. C.
Stratowa, Vienna, Austria.
Cell Cultures
Human umbilical vein endothelial
cells (HUVEC) at the first passage were purchased from Clonetics Corp.
(San Diego, CA). HUVEC were grown on fibronectin-coated flasks or
plates in RPMI medium containing 10-20% fetal calf serum, 6.5
µg/ml endothelial-derived growth factor from bovine neural tissue,
and 75 µg/ml heparin. All experiments used cells under the eighth
passage. EAhy926 cells, a hybrid cell line of HUVEC and A549 cell line
(derived from human lung epithelial type II cells), was provided by Dr.
Edgell (University of North Carolina, Chapel Hill) and cultured as
described (Edgell et al., 1983). EAhy926 cells retain
endothelial cell morphology and express the endothelial cell-specific
marker human factor VIII-related antigen (Edgell et al.,
1983). EAhy926 cells were maintained in DMEM-high glucose, in 5%
CO, 10% fetal calf serum, and passaged by removal in
trypsin-EDTA buffer (0.14 M NaCl, 2.68 mM KCl, 0.42
mM NaHPO
, 0.012 M NaHCO
, 0.01 M dextrose, 0.05% trypsin, 0.53
mM EDTA).O exposure period.Reporter Gene Constructs, Transfections, and Luciferase
Assays
The ICAM-1 LUC reporter plasmid and its 5` deletion
derivatives have been described previously (Voraberger et al.,
1991). The full-length ICAM-1 promoter construct contains approximately
1.4 kb of ICAM-1 5`-flanking DNA linked to the firefly luciferase (LUC)
gene. Transfection into cells showed that this ICAM-1 construct was
responsive to phorbol 12-myristate 13-acetate and TNF (Voraberger et al., 1991). The macrophage scavenger receptor constructs
containing three copies of the AP-1/Ets element or mutations of the
element linked to luciferase have been described previously (Wu et
al., 1994). Cells were plated 24 h prior to transfection at 5
10
cells per 6-cm plate. The cells were refed with
fresh medium containing 10% fetal calf serum 4 h before lipofection
(Malone et al., 1989) with LipofectAMINE as described by Life
Technologies, Inc. Transfection of 100-mm plates at 80% confluency
typically contained 8 µg of reporter plasmid (ICAM-1 LUC) and 2
µg of -gal expression plasmid DNA. The cells were transfected
for 5 to 14 h. After a recovery period, the cells were divided into
five 35-mm plates. At 24 h before treatment with HO (concentration range 100-400 µM), TNF
(100 units/ml) or phorbol 12-myristate 13-acetate (50 ng/ml), the cells
were incubated in medium containing 0.5% fetal calf serum. The cell
extract was prepared and assayed for luciferase activity using Promega
Biotec assay systems and -galactosidase activity using the Tropix
(Bedford, MA) assay system. Protein content was determined using a
Bio-Rad protein determination kit. Mean luciferase activity per µg
of protein extract was normalized to the -galactosidase activity
(which in control experiments was not affected by
HO).RNA Isolation and Northern Analysis
All solutions
used for RNA analysis were treated with diethylpyrocarbonate (0.1%) and
sterilized or prepared in sterile diethylpyrocarbonate-treated water.
Glassware was baked at 240 °C for a minimum of 4 h to remove traces
of RNase. Total RNA was isolated according to the procedure of
Chomczynski and Sacchi(1987). Medium was removed, and the endothelial
cell layer was rinsed with ice-cold, Ca -and
Mg
-free phosphate-buffered saline (PBS), and lysed in
acid guanidine thiocyanate. The lysate was drawn through a 26-gauge
needle and extracted with acid phenol/chloroform (5:1). After a
30-60-min incubation on ice, the mixture was centrifuged for 30
min at 12,000
g. The aqueous phase was collected and
RNA was precipitated with equal volume of ice-cold isopropyl alcohol.
After allowing the RNA to precipitate for 1 h at -70 °C, RNA
was pelleted by centrifugation for 30 min at 12,000
g.
The RNA pellet was washed twice with 75% ethanol, briefly dried, and
dissolved in 0.5% SDS in diethylpyrocarbonate-treated water.
Quantification and purity of RNA were assessed by A
/A
absorption, and RNA
samples with ratios above 1.9 were used for further analysis.
SSC (3 M sodium chloride, 0.3 M sodium
citrate, pH 7.0) to Duralose-UV
nitrocellulose membranes.
The membranes were baked for 2 h in vacuo at 80 °C to fix
the RNA. Blots were prehybridized for 30 min at 68 °C in
QuikHyb
solution and hybridized for 2 h at 68 °C with
random-primed
P-labeled probes. After hybridization, the
blots were washed twice for 15 min each at room temperature in 2
SSC with 0.1% SDS followed by 2 washes for 30 min each at 60
°C in 0.1
SSC with 0.1% SDS. The washed blots were exposed
to Hybond film (Amersham) for 12 to 48 h at -70 °C using an
intensifying screen. The signal intensities were quantified by scanning
the autoradiograms with the Beckman R112 densitometer. All blots were
hybridized with
P-labeled probes of ICAM-1 cDNA (0.96-kb SalI to PstI fragment) and glyceraldehyde-3-phosphate
dehydrogenase (1.1 kb PstI fragment).
Glyceraldehyde-3-phosphate dehydrogenase was used as an internal
control for RNA loading and normalized by densitometry of the ICAM-1
signal.
O. RNA was isolated and
processed for Northern analysis.Nuclear Run-on Assay
Nuclei were isolated from
HUVEC (3-5 10
) according to the procedure
described by Clayton and Darnell(1983). Cells were washed twice with
cold PBS, harvested by scraping, and centrifuged for 10 min at 1,000
rpm. The cell pellet was washed once with ice-cold PBS and once with
RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and
3.0 mM MgCl). The cells were suspended in 10 ml of
RSB and incubated on ice for 10 min. The cell pellets were collected by
centrifugation, when the cells were sufficiently swollen as monitored
by microscopy. Cell pellets were resuspended in 5 ml of RSB and
homogenized with a dounce type B homogenizer. The homogenate was
treated briefly with 0.1% Triton X-100 to remove cytoplasmic tags. The
nuclear pellet was collected by centrifugation for 10 min at 1500 rpm
at 4 °C. The nuclear pellet was resuspended in 210 µl of
freezing buffer (50 mM Tris-HCl, pH 8.0, 5 mM
MgCl, 0.5 mM dithiothreitol, and 40% glycerol),
flash frozen in liquid nitrogen, and stored at -70 °C., 325 mM KCl, and 250 µCi of
[-P]UTP). RNase-free DNase (20 µl of 2
µg/ml) was added and incubated for an additional 15 min at 30
°C. The run-on reaction was terminated by the addition of 36 µl
of 10
SET buffer (10% SDS, 100 mM Tris-HCl, pH 7.5,
and 50 mM EDTA). Proteinase K (100 µg) was added and
incubated for 45 min at 37 °C, and the reaction mixture was
extracted once with buffer-saturated phenol/chloroform (1:1) and once
with chloroform/isoamyl alcohol (24:1). The aqueous phase was
collected, ammonium sulfate (final concentration of 2.3 M) was
added, and the RNA was precipitated with an equal volume of isopropyl
alcohol. After 1 h at -70 °C, the RNA was pelleted and washed
twice with 75% ethanol. The pellet was dissolved in 100 µl of TE
buffer (10 mM Tris-HCl, pH 7.4, and 1 mM EDTA) and
passed through a Sephadex G-50 column to remove any unincorporated
nucleotides. Filters were prepared for hybridization by application of
denatured plasmids (5 µg/slot) using a slot blot apparatus.
Plasmids containing cDNAs for ICAM-1, E-selectin, ribosomal RNA (rRNA),
and glyceraldehyde-3-phosphate dehydrogenase were used for the
experiments. Baked filters were hybridized with the RNA in the run-on
assay as described for Northern analysis, and autoradiograms were
developed.
Nuclear Protein Extracts
HUVEC were prepared for
nuclear extracts as described by Shapiro et al.(1988).
Briefly, the cells were treated with HO,
TNF (100 units/ml) or medium for 1 h prior to harvesting. The
cells were washed twice with ice-cold PBS and collected by
centrifugation (Sorvall RT6000) for 5 min at 2,000 rpm. The cell pellet
was resuspended in 5 volumes of hypotonic buffer (10 mM HEPES,
pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.1
mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 10 mM KCl, 0.5 mM PMSF) and incubated on ice for 10 min to
allow the cells to swell. Cells were collected by centrifugation
(Sorvall RT6000) for 7.5 min at 3,000 rpm in the cold. The cell pellet
was resuspended in twice the original volume of ice-cold hypotonic
buffer. Cells were homogenized with 30 strokes of a Wheaton dounce
glass homogenizer (pestle B), followed by the addition of one-tenth
volume of restore buffer (2.2 M sucrose, 10 mM HEPES
pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.1
mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, 10 mM KCl, and 0.5 mM PMSF). Nuclei were collected by
centrifugation (Sorvall RC2-B) at 10,000 rpm in a Sorvall HB4 rotor in
the cold for 3.5 min. The nuclei pellet was resuspended in 3 ml of
nuclei lysis buffer (20 mM HEPES, pH 7.9, 0.42 M NaCl, 0.75 mM spermidine, 0.15 spermine, 0.2 mM EDTA, 0.2 mM EGTA, 2.0 mM DTT, 25% glycerol, and
0.5 mM PMSF). Nuclear debris was removed by
ultracentrifugation (Beckman L8-M ultracentrifuge) at 40,000 rpm in a
Beckman Ti80 fixed angle rotor for 90 min at 1 °C, and 0.33 g of
finely powdered ammonium sulfate was added to each milliliter of the
collected supernatant and mixed gently by rocking in the cold for 60 to
90 min until the ammonium sulfate was completely dissolved. The
precipitated nuclear protein was collected by ultracentrifugation
(Beckman L8-M) at 30,000 rpm in a Beckman Ti80 fixed angle rotor for 20
min at 1 °C. Nuclear protein pellets were resuspended in 200 µl
of nuclear dialysis buffer (20 mM HEPES, pH 7.9, 20% glycerol,
100 mM KCl, 0.2 mM EDTA, 2.0 mM DTT, and 0.1
mM PMSF) and dialyzed twice for 90 min against 200 ml of NDB
in the cold. Nuclear extracts were cleared of insoluble material by
microcentrifugation for 10 min, and 30 µl of nuclear protein
extracts were aliquoted and stored in a liquid nitrogen freezer until
use. Protein concentrations were determined by the Bio-Rad assay kit.Gel Mobility Shift Assay
The electrophoretic
mobility shift assay was performed as described by Roebuck et
al.(1993). Nuclear extracts prepared from HUVEC by the method of
Shapiro et al.(1988) were incubated with 50,000 cpm (0.1
to 0.5 ng) of various
P-end-labeled double-stranded
synthetic deoxyoligonucleotide probes for 30 min at 25 °C in a
20-µl reaction volume containing 12% glycerol, 12 mM
HEPES-NaOH (pH 7.9), 60 mM KCl, 5 mM MgCl
, 4 mM Tris-HCl (pH 7.9), 0.6 mM EDTA (pH 7.9), 0.6 mM DTT, and 1 µg of
poly(dI)
(dC). DNA probes were end-labeled with
[-
P]ATP (3,000 µCi/mmol) and T4
polynucleotide kinase. The labeled DNA probe was purified on push
columns (Stratagene). Protein-DNA complexes were resolved in 5% native
polyacrylamide gels pre-electrophoresed for 30-60 min at room
temperature in 0.25
TBE buffer (22.5 mM Tris borate
and 0.5 mM EDTA, pH 8.3). Dried gels were exposed overnight to
x-ray film with an intensifying screen at -70 °C.
Oligonucleotides used for the gel shift analysis were as follows:
ICAM-1 AP-1/Ets, 5`-GCTGCTGCCTCAGTTTCCC-3`; ICAM-1 NF-
B,
5`-GCCCGGGGAGGATTCCTGGGCCCC-3`; ICAM-1 TRE, 5`-GACCGTGATTCAAGCTTA-3`;
ICAM-1 AP-1 motif, 5`-TGGCCAGTGACTCGCAGCCCCAGC-3`; AP-1 m/Ets,
5`-GCTGCgtaagacGTTTCCCAGC-3`; AP-1/Ets-m, 5`-GCTGCTGCCTCAGTcagtCAGC-3`. B
oligonucleotide corresponds to the element upstream of the AP-3 site
and downstream of the AP-1/Ets repeats.
O activates the
ICAM-1 gene promoter. A, structure of the ICAM-1 promoter
luciferase reporter gene construct. Rectangles indicate the
location (relative to the start site of translation) of binding sites
for the transcription factors AP-1, AP-3, NF-B, C/EBP, and Ets. A
12-O-tetradecanoylphorbol-13-acetate responsive element (TRE)
is located at -321. The arrow downstream of the TATA box
indicates the start site of translation (ATG). B, ICAM-1
promoter activity in HUVEC. The ICAM-1 LUC construct was transfected
into HUVEC as described under ``Experimental Procedures.'' At
24 h post-transfection, the cells were exposed to 100, 200, or 400
µM HO or to 100 units/ml TNF.
Cells were harvested 24 h after HO or TNF
treatment, and cell extracts were assessed for luciferase activity. C, ICAM-1 promoter activity in EAhy926 cells. Cells were
transfected as described under ``Experimental Procedures.''
Phorbol 12-myristate 13-acetate (50 ng/ml)- and TNF (100
units/ml)-treated cells were included for comparison. Luciferase
activity is expressed as relative light units (RLU)/10
s/µg of protein normalized to -galactosidase activity
expressed by a cotransfected -galactosidase expression
vector.
O responsive region of the ICAM-1 gene promoter. The structure of
the different ICAM-1 promoter luciferase constructs is shown to the left. Rectangles indicate the location of various DNA binding
motifs. The AP-1/Ets repeats are indicated by solid
rectangles. The nucleotide position of the 5` end of each
construct is given relative to the translation initiation codon of the
gene. EAhy926 cells were transfected with the ICAM-1 luciferase
constructs and treated with HO (400
µM) or TNF (100 units/ml) as described under
``Experimental Procedures.'' Luciferase activity normalized
to -galactosidase activity is expressed as mean fold increase
relative to the untreated medium control of each ICAM-1 promoter
construct. Results are shown as mean ± S.D. of 3 to 5 separate
experiments.
H
We have previously reported that exposure of
human umbilical vein endothelial cells (HUVEC) to 100 µM HOInduces de Novo mRNA
Synthesis
O for 1 h resulted in maximal
accumulation of steady-state ICAM-1 message, which could be detected as
early as 30 min after oxidant exposure (Lo et al., 1993). To
determine whether the HO-induced increase in
ICAM-1 mRNA was the result of increased de novo synthesis of
the message or decreased rate of message degradation, we examined the
effects of actinomycin D, a RNA synthesis inhibitor. We carried out two
experiments: (i) actinomycin D was added to the cells at the same time
as HO exposure (Fig.1) and (ii) cells
were first pretreated with HO for 1 h to
maximize the expression of ICAM-1 message followed by treatment with
actinomycin D to block new mRNA synthesis (Fig.2).
O-induced expression of ICAM-1 mRNA. Confluent
HUVEC were treated with 50 µM (lanes 2 and 4) or 100 µM HO (lanes 3 and 5) for 1 h either in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of actinomycin D. Total RNA was isolated and analyzed by
Northern blot. Control cells (lane 1) did not receive any
treatment. A, autoradiogram; B, bar graph
representing the relative intensities of the ICAM-1 mRNA signals
(representative of 4 separate experiments).
O-induced ICAM-1 mRNA. HUVEC were treated with
100 µM HO for 1 h to achieve peak
RNA synthesis (lane 2) followed by addition of actinomycin D
(50 µM) for 0.5 to 2 h (lanes 3-6). Total
RNA was isolated from cells at the times indicated and analyzed by
Northern blot. A, autoradiogram; B, bar graph
presenting the relative intensities of the ICAM-1 mRNA signals
(representative of 4 separate experiments).
O exposure
(50 µM or 100 µM) abrogated ICAM-1 message
induction (Fig.1; compare lanes 2 and 3 with 4 and 5). To examine the effect of
HO on mRNA stability, endothelial cells were
first exposed to 100 µM HO for 1 h
to maximize ICAM-1 expression, and this was followed by treatment with
actinomycin D. Total RNA was isolated at 0.5, 1, 1.5, and 2 h after
actinomycin D, and steady-state levels of ICAM-1 mRNA were analyzed by
Northern blotting (Fig.2). The
HO-induced mRNA level returned to baseline
level at 0.5 h (lane 3) and remained at this level up to 2 h (lanes 4-6). Both actinomycin D experiments indicated
that HO increased the synthesis of ICAM-1 mRNA. H
Fig.3compares the
transcription rate of ICAM-1 with that of E-selectin, ribosomal RNA
(rRNA), and glyceraldehyde-3-phosphate dehydrogenase as determined by
nuclear run-on analysis. The transcription rates of E-selectin, rRNA,
and glyceraldehyde-3-phosphate dehydrogenase were unaffected by
HOIncreases Rate of
ICAM-1 Gene Transcription
O over the 2-h time course. In contrast,
HO increased the rate of ICAM-1 transcription
at 1 h, and the rate remained high at 2 h, a finding that correlates
with the HO induction of ICAM-1 message (Lo et al., 1993). These results indicate that
HO activates ICAM-1 gene transcription.
O-induced ICAM-1 mRNA. Slot-blot analysis of
the ICAM-1 RNA transcription rates of nuclei isolated from control
HUVEC and HUVEC treated with HO (100
µM) for 1 and 2 h (slot 3). Labeled RNA isolated
from the nuclei was hybridized to immobilized DNA as indicated. For
comparison, E-selectin (slot 2), glyceraldehyde-3-phosphate
dehydrogenase (slot 4), and ribosomal RNA (slot 1)
were also analyzed (representative of 4 separate
experiments).
Antisense Oligonucleotide Prevents
H
We transfected a complementary ICAM-1
oligonucleotide that targets the 5` end of the ICAM-1 mRNA (Fig.4), to determine whether
HO-induced ICAM-1 mRNA
Expression
O-induced ICAM-1 mRNA expression was sensitive
to antisense deoxyoligonucleotides. The transfected HUVEC were exposed
for 1 h with 100 µM HO and
analyzed by Northern blot for ICAM-1 mRNA expression. Lipofection with
the antisense oligonucleotide produced a concentration-dependent
reduction in the ICAM-1 message (Fig.4, lanes 3 and 5). Neither control (nonsense oligonucleotide used as a
negative control in lanes 4 and 6 or lipofection
alone in lane 1) affected ICAM-1 mRNA expression. These data
indicated that antisense oligonucleotides targeted to the ICAM-1 mRNA
prevented the HO-induced ICAM-1 transcription.
O (100 µM) for 1 h. Total RNA was isolated from cells
incubated with 50 (lane 3) or 100 µM (lane
5) antisense oligonucleotide (AS) (see
``Experimental Procedures'' for sequence of the
oligonucleotide). For specificity, the effect of a nonsense
oligonucleotide (NS) was assessed (lanes 4 and 6). Control RNA was isolated from cells incubated with
Lipofectin alone (lane 1). A, autoradiogram of the
Northern blot; B, bar graph presenting relative intensities of
the ICAM-1 mRNA signal (representative of 4 separate
experiments).
Antagonists of H
We used three agents to investigate
possible mechanisms underlying the induction of ICAM-1. These agents
were selected to study the DNA binding proteins AP-1 (Jun/Fos) and
NF-O-induced
ICAM-1 mRNA Expression
B, transcription factors known to be modulated by redox
mechanisms (Abate et al., 1990; Meyer et al., 1993).
We used 3-aminobenzamide (3-AB), an inhibitor of
poly(ADP-ribosyl)ation, to target AP-1 since 3-AB inhibited
oxidant-induced c-fos expression and AP-1 binding activity
(Amstad et al., 1992). The anti-oxidant pyrrolidine
dithiocarbamate (PDTC) was used to target NF-B since PDTC
inhibited oxidant-induced NF-B activity without affecting AP-1
binding activity (Schreck et al., 1992). N-Acetylcysteine (N-Cys(Ac)), a general antioxidant
and precursor of glutathione (Toledano and Leonard, 1991; Abate et
al., 1990), was used to alter the redox state of cells. As shown
in Fig.5, 3-AB abrogated the induction of ICAM-1 message (lane 3), whereas PDTC had no effect (lane 4)
suggesting a role for AP-1 in the induction of endothelial ICAM-1
transcription by HO. Pretreatment of
endothelial cells for 1 h with N-Cys(Ac) also prevented the
HO-induced mRNA expression (lane 5).
O-induced ICAM-1 message. Confluent HUVEC were
pretreated with 3-AB (lane 3), PDTC (lane 4), or N-Cys(Ac) (lane 5) for 1 h as described under
``Experimental Procedures'' followed by exposure to 100
µM HO for 1 h in the presence of
inhibitor. Control RNA (lane 1) and RNA from TNF (100
units/ml)-treated cells (lane 6) were also analyzed. A, autoradiogram of the Northern blot; B, bar graph
presenting relative intensities of the ICAM-1 mRNA signals
(representative of 3 separate experiments).
H
We prepared nuclear
protein extracts from endothelial cells treated with
HOStimulates AP-1 DNA
Binding Activity in Endothelial Cells
O for 1 h and examined DNA binding by
electrophoretic mobility shift assay to study the effects of
HO on AP-1 and NF-B binding activities (Fig.6). HO stimulated DNA binding
activity on AP-1-like binding sites of the ICAM-1 promoter (lanes
1-9), but this was not the case with NF-B binding
activity (lanes 10-12) consistent with the inhibitor
studies above. In contrast, TNF increased the binding activity on
both the AP-1-like and NF-B-like sequences.
O induces AP-1
but not NF-B binding activity in endothelial cells. Nuclear
protein extracts of HUVEC exposed for 1 h to 100 µM HO or TNF (100 units/ml) were
incubated with TRE (lanes 1-3), AP-1/Ets (lanes
4-6), AP-1 (lanes 7-9), or NF-B (lanes 10-12) binding site oligonucleotides of the
ICAM-1 promoter (see ``Experimental Procedures'' for
oligonucleotide sequences). Gel shift complexes indicated by the arrow were resolved by electrophoresis and DNA binding
activity assessed by autoradiography.
H
We determined the ability of an ICAM-1
promoter construct to respond to HOIncreases ICAM-1
Promoter Activity
O activation
signals in transient transfection assays. Fig.7A shows
the structure of the ICAM-1 promoter construct and the positions of DNA
binding sites of several inducible transcription factors that were
identified by visually scanning the promoter. The full-length wild type
construct containing nearly 1.4 kb of the ICAM-1 promoter linked to a
luciferase reporter gene (ICAM-1 LUC) (Voraberger et al.,
1991) was transfected into HUVEC. Fig.7B shows that
the ICAM-1 promoter was maximally activated by 100 µM HO in HUVEC, which was in agreement with
ICAM-1 mRNA expression. HO also induced ICAM-1
promoter activity in EAhy926 cells in a concentration-dependent manner (Fig.7C). These results indicated that
HO increases gene transcription through
activation of the ICAM-1 promoter.H
We examined a nested set of 5`
deletion mutation constructs containing different lengths of the ICAM-1
promoter to identify the DNA sequences within the ICAM-1 promoter
required for the HOActivates Distinct
Regions of ICAM-1 Promoter
O response. Fig.8shows that deletion of ICAM-1 promoter sequences 769 bp
upstream of the ATG start codon (construct B) abrogated
HO-induced ICAM-1 transcription. In contrast,
the construct containing promoter sequences up to 981 bp upstream of
the gene (Fig.8, construct A) was as responsive to
HO as the full-length ICAM-1 promoter
(-1393 ICAM-1 construct) even though this deletion construct (A) lacked an AP-1-like binding site. Therefore, DNA sequences
between nucleotides 981 and 769 upstream of the gene are required for
HO-induced ICAM-1 transcription. Within this
212-bp DNA segment are two identical 16-bp repeats, each containing
binding sites for the transcription factors AP-1 and Ets (solid
rectangles).O, the TNF
response decreased only slightly (about 2-fold) with increasing
deletion of the promoter to nucleotide position -769, indicating
that these promoter sequences containing AP-1 binding sites, although
contributing to a maximal TNF response, are not essential for
TNF-mediated ICAM-1 transcription. The distal NF-B binding
site was also not essential since deletion of sequences containing this
element (Fig.8, construct C) had little effect on the
TNF response. Indeed, a significant TNF response of at least
3-fold persisted until sequences between -393 (construct
D) and -176 (construct E) containing adjacent
NF-B and C/EBP binding sites were removed. This result is
consistent with the findings of Hou et al.(1994) demonstrating
cooperativity between the proximal NF-B and C/EBP binding sites
for the TNF response in endothelial and epithelial cells.AP-1/Ets Composite Sites Are H
The DNA binding studies coupled
with the functional analysis of the ICAM-1 promoter suggest that the
AP-1/Ets binding site repeats might be oxidant response elements.
AP-1/Ets composite sites are present in a number of viral and cellular
promoters including the macrophage scavenger receptor (Wu et
al., 1994). AP-1/Ets elements are also known as Ras responsive
elements (Westwick et al., 1994), since they can mediate
activation signals transduced through the GTP-binding protein Ras, an
early signaling intermediate of the AP-1 signal transduction pathway.
We transfected a heterologous promoter (3 OResponse Elements
AP-1/Ets-LUC)
containing three copies of the AP-1/Ets element from the macrophage
scavenger receptor (MSR) gene linked to a minimal prolactin
promoter-luciferase reporter gene (Wu et al., 1994) to
determine whether AP-1/Ets composite elements similar to the ICAM-1
repeats could function as H
O response elements.
As shown in Table1, the MSR AP-1/Ets element is nearly identical
with the ICAM-1 AP-1/Ets repeats and is also similar to anti-oxidant
response elements (ARE) present in oxiprotective enzyme genes. As shown
in Fig.9, AP-1/Ets-directed promoter activity increased when
the transfected cells were exposed to either HO or TNF. However, mutation of either the AP-1 or Ets binding
sites prevented these responses, indicating that the AP-1 and Ets
binding sites are essential for HO-induced
promoter activity. These data define AP-1/Ets composite sites as
oxidant response elements and indicate that the AP-1 and Ets binding
sites functionally cooperate to activate
HO-mediated transcription.
O responsive elements. Three
copies of wild type or mutant AP-1/Ets element from the macrophage
scavenger receptor gene (Wu et al., 1994) linked to a
prolactin TATA box luciferase construct were transfected into EAhy926
cells together with a -actin--galactosidase expression
plasmid (internal control) as described under ``Experimental
Procedures.'' EAhy926 cells were exposed to HO (400 µM), TNF (100 units/ml), or medium
(control) for 24 h, and cell extracts were assessed for luciferase
activity. The wild type AP-1/Ets composite element is double
underlined. Mutations in either the AP-1 or Ets binding sites are
delineated by a single underline. Results are expressed as
mean fold increase (n = 3) of luciferase activity
normalized to the -galactosidase activity ±
S.D.
Effect of Mutations of AP-1/Ets Repeat on
H
We characterized the DNA binding activity on the
AP-1/Ets repeats to determine whether redox-sensitive DNA binding
proteins complex with these elements. Gel shift analysis revealed the
formation of two specific gel shift complexes that competed with the
AP-1/Ets repeat (Fig.10, lanes 1 and 5). Like
TNFO-induced DNA Binding
Activity (lane 4), HO increased the
binding activity of these complexes (lane 2), whereas in the
presence of N-Cys(Ac) HO did not
stimulate their binding activity (lane 3). These data indicate
that HO stimulated redox-sensitive AP-1/Ets
binding activity of nuclear extracts.
O-induced binding activity on the AP-1/Ets
repeat. AP-1/Ets repeat oligonucleotide or oligonucleotides with
mutation of either the AP-1 (AP-1 m/Ets) or Ets (AP-1/Ets-m) binding site were incubated with nuclear extracts
of EAhy926 cells treated with TNF (100 units/ml) or
HO (400 µM) in the presence or
absence of N-Cys(Ac) (30 mM) as indicated above each
lane. The proteinDNA complexes were resolved by gel
electrophoresis and detected by autoradiography. Binding specificity
was assessed by 50 ng of unlabeled homologous competitor
oligonucleotide.
O, the mutation of either the AP-1 or Ets
binding sites had no effect on the constitutive binding activity (Fig.10, lanes 6 and 10). However, in the
HO-treated cell extracts these mutations in
either the AP-1 or Ets binding sites prevented the
HO-induced binding activity (lanes 7 and 11), indicating that an intact AP-1/Ets repeat was
essential for the HO-mediated binding activity.
These muations also abrogated the TNF-induced binding activity (Fig.10, lanes 8 and 12). These data suggest
that the AP-1 and Ets binding sites cooperated to form redox sensitive
gel shift complexes on the AP-1/Ets repeats.
O-mediated induction of ICAM-1 mRNA expression
in endothelial cells. The level of expression induced by
HO was consistently 2- to 3-fold greater than
basal ICAM-1 expression. This effect was detectable at 0.5 h, peaked at
1 h, and was sustained for at least 2 h. HO did
not activate the transcription of E-selectin, and it has not been shown
to increase expression of vascular cell adhesion molecule 1 (Bradely et al., 1993). Deletional analysis of ICAM-1 promoter
sequences identified a 212-bp region required for the
HO-mediated activation of ICAM-1 transcription.
Although this region from -981 to -769 (relative to the
start of translation) contributed to the TNF response, it was not
essential for activation of ICAM-1 transcription by TNF. The major
TNF responsive region was localized to promoter sequences more
than 300 bp downstream between -393 and -176, binding sites
for C/EBP and NF-B.O responsive region of the ICAM-1 promoter, we identified two 16-bp
repeats located 865 and 940 bp upstream of the coding region. These
repeats are binding sites for the inducible transcription factors AP-1
(composed of Jun and Fos protein dimers) and Ets. No other known
binding sites for nuclear regulatory factors were apparent within this
HO responsive region. An oligonucleotide of the
AP-1/Ets repeats formed HO-induced gel shift
complexes that were sensitive to mutation of either the AP-1 or Ets
binding sites, suggesting these elements mediated the
HO-induced transcription of the ICAM-1 gene. O. Both the AP-1 and Ets binding sites were
essential since mutation of these sequences reduced the induced
response to HO suggesting these two binding
sites functionally cooperated to form the HO response element. Wu et al.(1994) have shown that the
AP-1/Ets composite elements form ternary complexes containing c-Jun,
JunB, and Ets-2, which cooperate to mediate the response to phorbol
ester. We also found that the AP-1/Ets elements functionally cooperated
to mediate responses to TNF, suggesting that HO and TNF activate a similar set of transcription factors.
However, in the context of the ICAM-1 promoter, the AP-1/Ets repeats
mediated primarily the HO response, indicating
that the AP-1/Ets elements are not necessary for the TNF response
even though TNF has been shown to activate transcription through
oxidant-mediated signals (Meyer et al., 1993) and through AP-1
binding sites (Brenner et al., 1989). Indeed, we found that N-Cys(Ac) could prevent the TNF-induced binding to the
AP-1/Ets elements. ()O activates ARE sequences (Friling et
al., 1992; Choi and Moore, 1993; Li and Jaiswal, 1994). Sequence
comparisons of mammalian ARE and the ICAM-1 repeats revealed
similarities between the AP-1/Ets repeats and the human NAD(P)H:quinone
oxidoreductase and mouse GST Ya ARE (Table1). The mouse GST Ya
contains two functional ARE sequences, one of which cooperates with an
adjacent inverse Ets binding site to activate redox responses via the
promoter (Bergelson and Daniel, 1994). The ICAM-1 promoter may utilize
a similar mechanism to respond to HO since
mutations in either the AP-1 or Ets binding sites abrogated the
HO-induced binding activity. The family of AP-1
proteins (i.e. JunD, c-Fos, and JunB) have been shown to be
involved in the activation of ARE (Bergelson et al., 1994;
Nguyen et al., 1994), even though the ARE is functionally
distinguishable from consensus AP-1 binding sites suggesting non-AP-1
proteins may also play a role in their activation (Nguyen et
al., 1994; Wang and Williamson, 1994). We have shown that
overexpression of JunB in epithelial cells stimulated the ICAM-1
promoter 5-fold suggesting the importance of AP-1 proteins in the
response.O-induced ICAM-1 expression. In contrast,
pyrrolidine dithiocarbamate (PDTC) (which prevents NF-B activation
(Schreck et al., 1992)) did not alter the
HO-induced ICAM-1 message. HO also did not activate NF-B binding activity as reported by
Bradely et al.(1993), consistent with the lack of effect of
PDTC on HO-induced ICAM-1 expression. In
contrast, TNF does activate NF-B (Schreck et al.,
1992), and TNF-induced ICAM-1 has recently been shown to be under
NF-B control (Ledebur and Parks, 1995; Hou et al., 1994).
Taken together, these data indicate that
HO-mediated ICAM-1 transcription does not
involve the activation of NF-B. In the ICAM-1 promoter studies, we
showed that TNF activated a region between 393 and 176 bp upstream
from the start codon which contained the C/EBP and NF-B binding
sites. Hou et al.(1994) showed that these two transcription
factors cooperated to activate ICAM-1 transcription in response to
TNF. However, since we did not selectively block the TNF
response by specifically mutating the NF-B and C/EBP sites, we
cannot rule out the possibility that the AP-1/Ets repeats are also
mediators of the TNF response, nor can we rule out the possibility
that the NF-B and C/EBP sites contribute to the
HO response. Although HO and TNF apparently function through distinct cis-regulatory elements to activate transcription, additional
studies will be required to further elucidate the specific roles these
elements play in the complex regulation of the ICAM-1 gene.O-induced ICAM-1 expression
appears to be the result of the redox activity of
HO, we determined the effects of N-acetylcysteine (N-Cys(Ac)), an anti-oxidant that
increases intracellular glutathione levels (Meyer et al.,
1993). The results showed that N-Cys(Ac) inhibited AP-1/Ets
binding activity and the induction of ICAM-1 expression, consistent
with the findings that glutathione regulates AP-1 activity (Bergelson et al., 1994). However, the mechanism by which
HO activation is transmitted to the ICAM-1
promoter is yet unknown. HO can activate the
AP-1 signal transduction pathway in T-cells through tyrosine
phosphorylation of kinase intermediates (Nakamura et al.,
1993) raising the possibility that HO stimulates the ICAM-1 gene by a similar signal transduction
mechanism. HO has also been shown to increase
endothelial permeability via a protein kinase C-dependent mechanism
(Siflinger-Birnboim et al., 1992). HO has also been shown to induce c-fos and c-jun gene expression and increase AP-1 activity (Li et al.,
1994).O-induced activation of a cis-regulatory domain of the ICAM-1 promoter. This region
situated between -981 and -769 (relative to the start
codon) contains two 16-bp repeats similar to a functional AP-1/Ets
composite binding site capable of transmitting HO activation signals to a minimal promoter. In contrast, TNF
activated ICAM-1 transcription through a domain between -393 and
-176 containing the C/EBP and NF-B binding sites of the
promoter. These results indicate that HO and
TNF may mediate ICAM-1 expression by distinct intracellular
mechanisms involving unique sequence elements within the promoter
region of the gene.
, tumor necrosis factor-; N-Cys(Ac), N-acetylcysteine; PDTC, pyrrolidine dithiocarbamate; 3-AB,
3-aminobenzamide; AP-1, activator protein-1; NF-B, nuclear factor
B; ARE, anti-oxidant response element; MSR, macrophage scavenger
receptor; bp, base pair(s); kb, kilobase(s); DMEM, Dulbecco's
modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid;
PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline;
DTT, dithiothreitol; GST, glutathione S-transferase.
We thank Chris Glass for plasmids and Alison Finnegan
for critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 M. Satoh, I. Date, M. Nakajima, K. Takahashi, K. Iseda, T. Tamiya, T. Ohmoto, Y. Ninomiya, S. Asari, and R. L. Macdonald Inhibition of Poly(ADP-Ribose) Polymerase Attenuates Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbits Editorial Comment Stroke, January 1, 2001; 32(1): 225 - 231. [Abstract] [Full Text] [PDF]
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V. Zaninovic, A. S. Gukovskaya, I. Gukovsky, M. Mouria, and S. J. Pandol Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells Am J Physiol Gastrointest Liver Physiol, October 1, 2000; 279(4): G666 - G676. [Abstract] [Full Text] [PDF]
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A. Niemann-Jonsson, P. Dimayuga, S. Jovinge, F. Calara, M. P. S. Ares, G. N. Fredrikson, and J. Nilsson Accumulation of LDL in Rat Arteries Is Associated With Activation of Tumor Necrosis Factor-{alpha} Expression Arterioscler. Thromb. Vasc. Biol., October 1, 2000; 20(10): 2205 - 2211. [Abstract] [Full Text] [PDF]
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 C. D. Kim, Y. K. Kim, S. H. Lee, and K. W. Hong Rebamipide Inhibits Neutrophil Adhesion to Hypoxia/Reoxygenation-Stimulated Endothelial Cells via Nuclear Factor-kappa B-Dependent Pathway J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 864 - 869. [Abstract] [Full Text]
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A. L. True, A. Rahman, and A. B. Malik Activation of NF-kappa B induced by H2O2 and TNF-alpha and its effects on ICAM-1 expression in endothelial cells Am J Physiol Lung Cell Mol Physiol, August 1, 2000; 279(2): L302 - L311. [Abstract] [Full Text] [PDF]
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Y. Amrani, A. L. Lazaar, R. Hoffman, K. Amin, S. Ousmer, and R. A. Panettieri Jr. Activation of p55 Tumor Necrosis Factor-alpha Receptor-1 Coupled to Tumor Necrosis Factor Receptor-Associated Factor 2 Stimulates Intercellular Adhesion Molecule-1 Expression by Modulating a Thapsigargin-Sensitive Pathway in Human Tracheal Smooth Muscle Cells Mol. Pharmacol., July 1, 2000; 58(1): 237 - 245. [Abstract] [Full Text]
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Y. Suzuki, K. Nishio, K. Takeshita, O. Takeuchi, K. Watanabe, N. Sato, K. Naoki, H. Kudo, T. Aoki, and K. Yamaguchi Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells Am J Physiol Lung Cell Mol Physiol, February 1, 2000; 278(2): L245 - L252. [Abstract] [Full Text] [PDF]
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 C. Kunsch and R. M. Medford Oxidative Stress as a Regulator of Gene Expression in the Vasculature Circ. Res., October 15, 1999; 85(8): 753 - 766. [Abstract] [Full Text] [PDF]
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 A. Schawalder, B. Oertli, B. Beck-Schimmer, and R. P. Wuthrich Regulation of hyaluronan-stimulated VCAM-1 expression in murine renal tubular epithelial cells Nephrol. Dial. Transplant., September 1, 1999; 14(9): 2130 - 2136. [Abstract] [Full Text] [PDF]
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H. Kobuchi, S. Roy, C. K. Sen, H. G. Nguyen, and L. Packer Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway Am J Physiol Cell Physiol, September 1, 1999; 277(3): C403 - C411. [Abstract] [Full Text] [PDF]
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Y. Amrani, A. L. Lazaar, and R. A. Panettieri Jr. Up-Regulation of ICAM-1 by Cytokines in Human Tracheal Smooth Muscle Cells Involves an NF-{kappa}B-Dependent Signaling Pathway That Is Only Partially Sensitive to Dexamethasone J. Immunol., August 15, 1999; 163(4): 2128 - 2134. [Abstract] [Full Text] [PDF]
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D. Elewaut, J. A. DiDonato, J. Mogg Kim, F. Truong, L. Eckmann, and M. F. Kagnoff NF-{kappa}B Is a Central Regulator of the Intestinal Epithelial Cell Innate Immune Response Induced by Infection with Enteroinvasive Bacteria J. Immunol., August 1, 1999; 163(3): 1457 - 1466. [Abstract] [Full Text] [PDF]
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A. Rahman, K. N. Anwar, A. L. True, and A. B. Malik Thrombin-Induced p65 Homodimer Binding to Downstream NF-{kappa}B Site of the Promoter Mediates Endothelial ICAM-1 Expression and Neutrophil Adhesion J. Immunol., May 1, 1999; 162(9): 5466 - 5476. [Abstract] [Full Text] [PDF]
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T. J. Kalogeris, F. S. Laroux, A. Cockrell, H. Ichikawa, N. Okayama, T. J. Phifer, J. S. Alexander, and M. B. Grisham Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells Am J Physiol Cell Physiol, April 1, 1999; 276(4): C856 - C864. [Abstract] [Full Text] [PDF]
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A. Rahman, M. Bando, J. Kefer, K. N. Anwar, and A. B. Malik Protein Kinase C-Activated Oxidant Generation in Endothelial Cells Signals Intercellular Adhesion Molecule-1 Gene Transcription Mol. Pharmacol., March 1, 1999; 55(3): 575 - 583. [Abstract] [Full Text]
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T. J. Kalogeris, C. G. Kevil, F. S. Laroux, L. L. Coe, T. J. Phifer, and J. S. Alexander Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells Am J Physiol Lung Cell Mol Physiol, January 1, 1999; 276(1): L9 - L19. [Abstract] [Full Text] [PDF]
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V. Lakshminarayanan, E. A. Drab-Weiss, and K. A. Roebuck H2O2 and Tumor Necrosis Factor-alpha Induce Differential Binding of the Redox-responsive Transcription Factors AP-1 and NF-kappa B to the Interleukin-8 Promoter in Endothelial and Epithelial Cells J. Biol. Chem., December 4, 1998; 273(49): 32670 - 32678. [Abstract] [Full Text] [PDF]
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 L. Yang, L. Cohn, D.-H. Zhang, R. Homer, A. Ray, and P. Ray Essential Role of Nuclear Factor kappa B in the Induction of Eosinophilia in Allergic Airway Inflammation J. Exp. Med., November 2, 1998; 188(9): 1739 - 1750. [Abstract] [Full Text] [PDF]
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L. H. McKinlay, M. J. Tymms, R. S. Thomas, A. Seth, S. Hasthorpe, P. J. Hertzog, and I. Kola The Role of Ets-1 in Mast Cell Granulocyte-Macrophage Colony-Stimulating Factor Expression and Activation J. Immunol., October 15, 1998; 161(8): 4098 - 4105. [Abstract] [Full Text] [PDF]
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E. N. Atochina, I. V. Balyasnikova, S. M. Danilov, D. N. Granger, A. B. Fisher, and V. R. Muzykantov Immunotargeting of catalase to ACE or ICAM-1 protects perfused rat lungs against oxidative stress Am J Physiol Lung Cell Mol Physiol, October 1, 1998; 275(4): L806 - L817. [Abstract] [Full Text] [PDF]
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M. Mietus-Snyder, C. K. Glass, and R. E. Pitas Transcriptional Activation of Scavenger Receptor Expression in Human Smooth Muscle Cells Requires AP-1/c-Jun and C/EBPß : Both AP-1 Binding and JNK Activation Are Induced by Phorbol Esters and Oxidative Stress Arterioscler. Thromb. Vasc. Biol., September 1, 1998; 18(9): 1440 - 1449. [Abstract] [Full Text] [PDF]
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A. Rahman, J. Kefer, M. Bando, W. D. Niles, and A. B. Malik E-selectin expression in human endothelial cells by TNF-alpha -induced oxidant generation and NF-kappa B activation Am J Physiol Lung Cell Mol Physiol, September 1, 1998; 275(3): L533 - L544. [Abstract] [Full Text] [PDF]
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C. Hellerbrand, C. Jobin, L. L. Licato, R. B. Sartor, and D. A. Brenner Cytokines induce NF-kappa B in activated but not in quiescent rat hepatic stellate cells Am J Physiol Gastrointest Liver Physiol, August 1, 1998; 275(2): G269 - G278. [Abstract] [Full Text] [PDF]
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B. Zingarelli, A. L. Salzman, and C. Szabo Genetic Disruption of Poly (ADP-Ribose) Synthetase Inhibits the Expression of P-Selectin and Intercellular Adhesion Molecule-1 in Myocardial Ischemia/Reperfusion Injury Circ. Res., July 13, 1998; 83(1): 85 - 94. [Abstract] [Full Text] [PDF]
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S. Sugiyama, K. Kugiyama, N. Ogata, H. Doi, Y. Ota, M. Ohgushi, T. Matsumura, H. Oka, and H. Yasue Biphasic Regulation of Transcription Factor Nuclear Factor-{kappa}B Activity in Human Endothelial Cells by Lysophosphatidylcholine Through Protein Kinase C–Mediated Pathway Arterioscler. Thromb. Vasc. Biol., April 1, 1998; 18(4): 568 - 576. [Abstract] [Full Text] [PDF]
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R. Kacimi, J. S. Karliner, F. Koudssi, and C. S. Long Expression and Regulation of Adhesion Molecules in Cardiac Cells by Cytokines : Response to Acute Hypoxia Circ. Res., March 23, 1998; 82(5): 576 - 586. [Abstract] [Full Text] [PDF]
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Y. Zhu, J. H.-C. Lin, H.-L. Liao, O. Friedli Jr, L. Verna, N. W. Marten, D. S. Straus, and M. B. Stemerman LDL Induces Transcription Factor Activator Protein-1 in Human Endothelial Cells Arterioscler. Thromb. Vasc. Biol., March 1, 1998; 18(3): 473 - 480. [Abstract] [Full Text] [PDF]
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 A Clayton, R. Evans, E Pettit, M Hallett, J. Williams, and R Steadman Cellular activation through the ligation of intercellular adhesion molecule-1 J. Cell Sci., January 2, 1998; 111(4): 443 - 453. [Abstract] [PDF]
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 J.-J. Cheng, B.-S. Wung, Y.-J. Chao, and D. L. Wang Cyclic Strain-Induced Reactive Oxygen Species Involved in ICAM-1 Gene Induction in Endothelial Cells Hypertension, January 1, 1998; 31(1): 125 - 130. [Abstract] [Full Text] [PDF]
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V. Lakshminarayanan, D. W. A. Beno, R. H. Costa, and K. A. Roebuck Differential Regulation of Interleukin-8 and Intercellular Adhesion Molecule-1 by H2O2 and Tumor Necrosis Factor-alpha in Endothelial and Epithelial Cells J. Biol. Chem., December 26, 1997; 272(52): 32910 - 32918. [Abstract] [Full Text] [PDF]
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J.J. Chiu, B.S. Wung, J. Y.J. Shyy, H.J. Hsieh, and D.L. Wang Reactive Oxygen Species Are Involved in Shear Stress-Induced Intercellular Adhesion Molecule-1 Expression in Endothelial Cells Arterioscler. Thromb. Vasc. Biol., December 1, 1997; 17(12): 3570 - 3577. [Abstract] [Full Text]
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C. Szabo, L. H.K. Lim, S. Cuzzocrea, S. J. Getting, B. Zingarelli, R. J. Flower, A. L. Salzman, and M. Perretti Inhibition of poly (ADP-ribose) Synthetase Attenuates Neutrophil Recruitment and Exerts Antiinflammatory Effects J. Exp. Med., October 6, 1997; 186(7): 1041 - 1049. [Abstract] [Full Text] [PDF]
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J. R. Michael, B. A. Markewitz, and D. E. Kohan Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells Am J Physiol Lung Cell Mol Physiol, October 1, 1997; 273(4): L768 - L774. [Abstract] [Full Text] [PDF]
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B. S. Wung, J. J. Cheng, H. J. Hsieh, Y. J. Shyy, and D. L. Wang Cyclic Strain–Induced Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells Involves Reactive Oxygen Species Activation of Activator Protein 1 Circ. Res., July 19, 1997; 81(1): 1 - 7. [Abstract] [Full Text]
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 S. Song, H. Ling-Hu, K. A. Roebuck, M. F. Rabbi, R. P. Donnelly, and A. Finnegan Interleukin-10 Inhibits Interferon-gamma -Induced Intercellular Adhesion Molecule-1 Gene Transcription in Human Monocytes Blood, June 15, 1997; 89(12): 4461 - 4469. [Abstract] [Full Text] [PDF]
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M. Introna and A. Mantovani Early Activation Signals in Endothelial Cells: Stimulation by Cytokines Arterioscler. Thromb. Vasc. Biol., March 1, 1997; 17(3): 423 - 428. [Abstract] [Full Text]
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S. Jovinge, A. Hultgardh-Nilsson, J. Regnstrom, and J. Nilsson Tumor Necrosis Factor-{alpha} Activates Smooth Muscle Cell Migration in Culture and Is Expressed in the Balloon-Injured Rat Aorta Arterioscler. Thromb. Vasc. Biol., March 1, 1997; 17(3): 490 - 497. [Abstract] [Full Text]
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J.-J. Cheng, B.-S. Wung, Y.-J. Chao, and D. L. Wang Cyclic Strain Enhances Adhesion of Monocytes to Endothelial Cells by Increasing Intercellular Adhesion Molecule-1 Expression Hypertension, September 1, 1996; 28(3): 386 - 391. [Abstract] [Full Text]
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J. Roy, M. Audette, and M. J. Tremblay Intercellular Adhesion Molecule-1 (ICAM-1) Gene Expression in Human T Cells Is Regulated by Phosphotyrosyl Phosphatase Activity. INVOLVEMENT OF NF-kappa B, Ets, AND PALINDROMIC INTERFERON-gamma -RESPONSIVE ELEMENT-BINDING SITES J. Biol. Chem., April 27, 2001; 276(18): 14553 - 14561. [Abstract] [Full Text] [PDF]
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G. Szabo, S. Bahrle, N. Stumpf, K. Sonnenberg, E. Szabo, P. Pacher, T. Csont, R. Schulz, T. J. Dengler, L. Liaudet, et al. Poly(ADP-Ribose) Polymerase Inhibition Reduces Reperfusion Injury After Heart Transplantation Circ. Res., January 11, 2002; 90(1): 100 - 106. [Abstract] [Full Text] [PDF]
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