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INTRODUCTION |
Adhesion molecules such as E-selectin, intercellular adhesion
molecule-1 (ICAM-1),1 and
vascular adhesion molecule-1 (VCAM-1) contribute to the recruitment of
leukocytes not only during the inflammatory or immune response, but
also during the injury following ischemia-reperfusion (1-5) and the
development of atherosclerosis (6, 7). Indeed, there has been an
explosion of interest in these topics, which has been fueled by
remarkable advances in new therapies directed at several adhesion
molecules (reviewed in Ref. 8). Most of these molecules are inducible
and regulated through gene expression. Regulation of adhesion molecule
expression has been largely studied in vitro using human
umbilical vein endothelial cells (HUVEC), in which two monokines, IL-1
and TNF
, can induce these adhesion molecules in the same manner
through NF-
B activation (9-11). However, a study involving dermal
microvascular endothelial cells has shown that ICAM-1 is induced by
either IL-1 or TNF
, whereas VCAM-1 is induced only by TNF
(12),
suggesting that the signals generated by IL-1 and TNF
for adhesion
molecule induction are different in part.
We have examined whether or not there is such tissue-specific
regulatory control of the gene expression of adhesion molecules in vivo, since the expression of chemokine mRNAs
following systemic treatment with proinflammatory cytokines was induced
in a tissue-specific manner in mice (13, 14). Recently, we found that
E-selectin mRNA expression following systemic treatment with
IL-1
was induced in the heart in a tissue-specific manner and that
IL-1
was a much stronger inducer of E-selectin mRNA than TNF
,
while ICAM-1 and VCAM-1 were induced similarly by either IL-1
or
TNF
(15). We assumed the presence of heart-specific
microenvironments as one reason. There are many stimuli that affect
cells in various tissues, including localized cytokines, growth
factors, and specific extracellular matrices. Although the
intracellular signals elicited by them remain to be elucidated, some of
them would induce the activation of protein kinase C (PKC) (16-21).
Since E-selectin mRNA expression is also induced by treatments that
activate PKC (22), we have examined the cooperative effect of PKC
activation on IL-1
- or TNF
-induced gene expression of
E-selectin.
Our results indicate that although the intracellular signals generated
by IL-1
and TNF
themselves are not mediated through classic PKC
activation, the coexistence of activated classic PKC and signals
elicited by IL-1
, but not TNF
, synergistically induces the
expression of E-selectin mRNA via increased transcription. Furthermore, promoter analysis of the E-selectin gene suggests that
NF-ELAM1/activating transcription factor (ATF) element is indispensable
for the synergistic transcription of E-selectin induced by the combined
treatment with IL-1
and PMA.
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EXPERIMENTAL PROCEDURES |
Reagents--
Dulbecco's phosphate-buffered saline, medium 199, and phorbol-12-myristate-13-acetate (PMA) were purchased from Life
Technologies, Inc. Fetal calf serum (FCS) was purchased from
Equitech-Bio (Ingram, TX). The penicillin/streptomycin solution,
agarose, diethyl pyrocarbonate (DEP), dextran sulfate, and MOPS were
from Sigma. Nick translation kits and proteinase K were purchased from
Boehringer Mannheim Yamanouchi (Tokyo, Japan). Formamide and 50×
Denhardt's solution were obtained from Wako Pure Chemical Industries
Inc. (Osaka, Japan). RNase-free DNase was obtained from Promega
(Madison, WI). Restriction endonucleases and T4 polynucleotide kinase
were products of Takara Shuzo Co. (Otsu, Japan). NEN Life Science
Products was the source of [
-32P]dCTP,
[
-32P]ATP, and [
-32P]UTP. Recombinant
human TNF
(9 × 107 units/mg) and recombinant human
IL-1
(2.8 × 108 units/mg) were from Genzyme Corp.
(Cambridge, MA). Human recombinant basic fibroblast growth factor
(bFGF) was obtained from Progen Biotechnik GmbH (Heidelberg, Germany).
Collagen type 4 (Cellmatrix type 4) was from Nitta Gelatin Inc. (Osaka,
Japan). Polyclonal antibodies against ATF-2, ATF-3, c-Jun,
cAMP-responsive element-binding protein (CREB), NF-
B p50, p52, p65,
Rel-B, and c-Rel were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). All other chemicals were obtained from Nacalai Tesque
Inc. (Kyoto, Japan).
Cell Culture--
HUVEC were isolated from human umbilical cords
using a perfusate obtained with trypsin (Life Technologies). HUVEC were
serially passaged (in a 1:5 split ratio) and maintained in medium 199 containing 10% FCS, antibiotics (50 units/ml penicillin and 50 mg/ml
streptomycin) and 10 ng/ml human recombinant bFGF. HUVEC were usually
precultured in medium 199 supplemented with 0.5% FCS and antibiotics
for 24 h before experiments. Tissue culture dishes were precoated
with collagen type 4. HUVEC at the sixth to eighth passage were used for experiments.
Preparation of Plasmid DNA and Oligonucleotide Probes--
The
plasmids encoding the genes for human ICAM-1 and E-selectin were
purchased from British Biotechnology Ltd. (Oxford, United Kingdom). For
Northern hybridization, plasmid DNA (1 mg) was radiolabeled by nick
translation with [32P]dCTP to a specific activity of
approximately 108 cpm/mg and was used at 7 × 106 cpm/blot. Double-stranded oligonucleotides containing
the NF-ELAM1/ATF cis-element (
166 to
129)
(5'-GAGACAGAGTTTCTGACATCATTGTAATTTTAAGCATC-3'), the oligonucleotides
that introduced the mutations in the NF-ELAM1/ATF cis-element
(5'-GAGACAGAGTTTCgtcgAgCcTTGTAATTTTAAGCATC-3'), the distal two
B consensus sequences (
128 to
101)
(5'-GTGGATATTCCCGGGAAAGTTTTTGGAT-3'), or the proximal
B site (
100
to
75) (5'-GCCATTGGGGATTTCCTCTTTACTGG-3') in the E-selectin promoter
region were synthesized. The oligonucleotides (10 pmol) were
radiolabeled with T4 polynucleotide kinase and [
-32P]
ATP to a specific activity of over 106 cpm/pmol.
Preparation of RNA and Northern Hybridization--
Total RNA was
isolated from confluent HUVEC using guanidine isothiocyanate cesium
chloride according to the previously published method (23). Equal
amounts of RNA (20 µg) were denatured and subjected to
electrophoresis in a 1% agarose-formaldehyde gel as described
previously (24, 25). The RNA was then blotted by capillary transfer
onto nylon membranes (Boehringer Mannheim Yamanouchi). The blots were
prehybridized for 6 h at 42 °C in 50% formamide, 1% SDS, 5×
SSC, 1× Denhardt's solution (0.02% Ficoll, 0.02% bovine serum
albumin, and 0.02% polyvinylpyrrolidone), 0.25 mg/ml denatured herring
testes DNA and 50 mM sodium phosphate buffer, pH 6.5. Hybridization was carried out at 42 °C for 12-18 h with 7 × 106 cpm of the denatured probe. The filters were washed for
30 min at room temperature in 0.1% SDS, 2× SSC and then for 15-30
min at 55 °C in the same solution. The blots were then exposed to XAR-5 x-ray film (Eastman Kodak Co.) with DuPont Cronex Lightening Plus
intensifying screens at
70 °C. In some experiments, the blots were
reutilized by stripping and rehybridization with different probes.
Expression of
-actin was used as an internal control and was applied
in all experiments. In addition, the RNA load per lane was assessed by
ethidium bromide staining of the original agarose gel after capillary
transfer. In some experiments, the autoradiograms were quantified by
videodensitometry, and the levels of mRNA were normalized relative
to those of
-actin.
Nuclear Run-on Transcription Assay--
HUVEC were treated as
indicated in the text, and nuclei were isolated as described previously
(26). Transcription that started in intact cells was allowed to reach
completion in the presence of [
-32P]UTP, and then the
RNA was isolated and hybridized to slot-blotted plasmids containing
specific cDNA inserts (5 µg of DNA/slot) essentially as described
elsewhere (27, 28). The blots were autoradiographed for 24-72 h at
70 °C as described above. The autoradiograms were quantified by
videodensitometry, and the levels of transcripts were normalized
relative to those of
-actin.
Promoter-Luciferase Construction--
The wild-type fragment
(
800 to +50) of the human E-selectin promoter was generated by
polymerase chain reaction using two gene-specific primers and HUVEC
genomic DNA as the template. The two primers carried base pair
substitutions that introduced a BglII site at the 5'-end of
the fragment (BglII (
800) primer, 5'-GCGAAGATCTGAGATGGCGTTTCTCCATGT-3') and a
HindIII site at the 3'-end (HindIII primer,
5'-AGAGAAGCTTTGTCTCAGGTCAGTATAGGA-3'). The resulting
fragment was cloned into the BglII and HindIII
sites of the luciferase expression vector, PicaGene Basic Vector (Toyo Ink, Tokyo, Japan). The 5'-flanking restriction sites, SspI,
AccI, NsiI, and SmaI, were used to
generate
554,
382,
232, and
116 chimeras, respectively.
Deletion mutants with end points at
166 and
129 were generated
using the HindIII primer, and the BglII (
166)
primer (5'-GCGAAGATCTGAGACAGAGTTTCTGACATCATTG-3')
and BglII (
129) primer
(5'-GCGAAGATCTCGTGGATATTCCCGGGAAAGT-3'), respectively. A
mutant that introduced the mutations in the NF-ELAM1/ATF cis-element, mNF-ELAM1/ATF, was generated using the HindIII primer and
the BglII (
166m) primer
(5'-GCGAAGATCTGAGACAGAGTTTCgtcgAgCcTTG-3'). The resulting
fragments were also cloned into the PicaGene Basic Vector. The 4×
NF-ELAM1/ATF construct was made as follows. The double-stranded
oligonucleotide carrying a XhoI site at the 5'-end and a
BglII site at the 3'-end
(5'-TCGAGTTTCTGACATCATTTTCTGACATCATTTTCTGACATCATTTTCTGACATCATA-3') was inserted into the XhoI/BglII sites of the
PicaGene Promoter Vector (Toyo Ink), which only contains the SV40 virus
minimal promoter.
DNA Transfection and Luciferase Assay--
HUVEC were plated
onto six-well collagen-coated culture dishes (2 × 105
cells/well) 24 h prior to transfection. Transfection was performed with Lipofectin (Life Technologies) according to the manufacturer's recommendations. Briefly, reporter DNA (20 µg) was mixed with 2 µl
of Lipofectin and up to 200 µl of serum-free medium (Opti-MEM; Life
Technologies). After a 30-min incubation, further Opti-MEM (800 µl)
was added, and the mixture was applied to cells that had been washed
twice with Opti-MEM. Four hours later, the medium was changed to medium
199 containing 10% FCS and 10 ng/ml bFGF. After a 24-h culture, the
medium was changed to medium 199 containing only 0.5% FCS, followed by
culture for a further 24 h. After the indicated treatment for
6 h, the cells were harvested. The cells were lysed in 150 µl of
lysis buffer (Toyo Ink), and 20 µl of the resultant extract was used
for the luciferase assay (Toyo Ink). The luciferase activities were
normalized relative to total protein concentrations of the cell extracts.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared by a modification of the procedure of Dignam et al.
(29). Cells were washed three times with phosphate-buffered saline,
scraped off, and then harvested by centrifugation. The cells were
resuspended and then incubated on ice for 15 min in hypotonic buffer A
(10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride)
and then vortexed for 10 s with 0.6% Nonidet P-40. Nuclei were
separated from the cytosol by centrifugation at 12,000 × g for 60 s and then resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride)
and shaken for 30 min at 4 °C. Nuclear extracts were obtained by
centrifugation at 12,000 × g. Protein concentrations were measured by means of the Bradford assay (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)--
For the binding
reaction, nuclear extracts (10 µg of protein) were incubated in a
total reaction mixture of 25 µl comprising 20 mM HEPES,
pH 7.9, 80 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 8% glycerol, and 2.5 µg of
poly(dI-dC) (Amershan Pharmacia Biotech, Uppsala, Sweden). The
radiolabeled oligonucleotide (105 cpm) was added to the
mixture after preincubation for 15 min at 4 °C, and then the total
reaction mixture was incubated for 20 min at room temperature. For
antibody "supershift" analysis, 2 µg each of the indicated
antibodies was added, followed preincubation for 1 h at 4 °C.
Samples were loaded on 6% polyacrylamide gels in low ionic strength
buffer (25 mM Tris, 22.5 mM borate, and 0.25 mM EDTA) and run at 15 V/cm with cooling. The gels were
then dried and analyzed by autoradiography.
UV Cross-linking--
UV cross-linking was performed as
described previously (30). Briefly, 80 mg of nuclear extract was
incubated in a 100-µl total reaction mixture comprising 4 mM HEPES, pH 7.9, 5 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.2 mM dithiothreitol, 5% glycerol,
80 mM NaCl, and 20 µg of poly(dI-dC). The radiolabeled
bromodeoxyuridine-substituted oligonucleotide (8 × 105 cpm) was then added to the reaction mixture. The
preincubation and incubation times were as described above. The
reaction mixture was then subjected to electrophoresis in a 6%
polyacrylamide gel. After separation, the gel was placed in contact
with the filter surface of a 302-nm UV transilluminator and irradiated
for 10 min. The optimal exposure conditions for cross-linking were
determined experimentally. The gel was then autoradiographed for 30 min. The region corresponding to the specific protein-DNA complex was located by inspection and excised. To each slice, 500 µl of a mixture
comprising 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol was added, followed by equilibration for 40 min. The gel slices were then loaded onto 1.5-mm-thick 10%
polyacrylamide gels. The gels were stained with Coomassie Brilliant
Blue, destained, dried under vacuum, and then subjected to autoradiography.
 |
RESULTS |
Induction of E-selectin mRNA Expression Is Synergized by the
Combined Treatment with IL-1
/PMA, but Not with TNF
/PMA, through
Elevated Transcriptional Activity--
Initially, we wished to
determine if classic PKC activation was involved in the intracellular
signaling pathway through which IL-1
or TNF
induces E-selectin
mRNA in HUVEC, since the PKC dependence of IL-1
or
TNF
-induced E-selectin mRNA expression has not been well defined
(31). One reason for the discrepancy is that available PKC inhibitors
such as H7 are not strictly specific for that kinase (32), and another
reason is whether or not the culture medium during the period of HUVEC
stimulation includes FCS and bFGF, which can activate classic PKC.
Therefore, we examined the effect of PKC down-regulation, due to
pretreatment with PMA, on the gene expression of E-selectin and ICAM-1
induced by IL-1
or TNF
in the presence or absence of FCS and
bFGF. HUVEC were precultured for 24 h with PMA (1 µg/ml) in
medium 199 containing 10% FCS and 10 ng/ml of bFGF or containing only
0.5% FCS. Then the HUVEC were washed three times with
phosphate-buffered saline and stimulated with IL-1
(400 pg/ml) or
TNF
(500 pg/ml) for 3 h. Total RNA was isolated and analyzed by
Northern hybridization as to the specific mRNA content using
radiolabeled probes for the E-selectin, ICAM-1, and
-actin genes
(Fig. 1). In the presence of FCS/bFGF,
IL-1
-induced E-selectin mRNA expression was strongly decreased
after a 24-h pretreatment with PMA, whereas ICAM-1 mRNA induction
was decreased only a little by such PKC down-regulation. Interestingly,
TNF
-induced gene expression of E-selectin and ICAM-1 was unaffected
by the classic PKC down-regulation. In contrast, in the absence of
FCS/bFGF, pretreatment with PMA did not affect the inducibility of
these genes upon treatment with either IL-1
or TNF
. These data
demonstrate that PKC-mediated pathways are not involved in the
intracellular signaling pathways through which IL-1
or TNF
induces gene expression of E-selectin and ICAM-1 and that classic PKC
activation by FCS/bFGF and IL-1
(but not TNF
) cooperatively
affects the selective induction of E-selectin mRNA expression in
HUVEC.

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Fig. 1.
Different effects of PKC down-regulation on
E-selectin gene expression induced by IL-1 or TNF treatment in
the presence or absence of FCS and bFGF. A, HUVEC were
pretreated with PMA (1 µg/ml) for 24 h in the presence or
absence of FCS and bFGF and then washed and stimulated with IL-1
(400 pg/ml) or TNF (500 pg/ml) for 3 h. At the end of the
treatment, the cells were collected and subjected to Northern
hybridization. B, the resulting autoradiographs were
quantified by videodensitometry. The data are presented as the ratios
of the optical density values to those of -actin.
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|
To determine whether or not activated PKC synergizes with IL-1
, but
not with TNF
, to induce E-selectin mRNA expression, HUVEC were
treated with IL-1
or TNF
, either alone or in combination with PMA
(100 ng/ml), in medium 199 containing only 0.5% FCS, without bFGF.
Following autoradiography, the expression levels of E-selectin and
ICAM-1 mRNA were normalized relative to that of
-actin (Fig.
2). Treatment with IL-1
, TNF
, or
PMA stimulated equivalent gene expression of E-selectin and ICAM-1. The
combined treatment with IL-1
and PMA synergistically induced
E-selectin mRNA expression, although the cotreatment had only an
additive effect on the induction of ICAM-1 mRNA. In contrast, when
TNF
was substituted for IL-1
, the cotreatment with PMA had no
synergistic effect on the gene expression of E-selectin or ICAM-1.
These data demonstrate that classic PKC activation and the
IL-1
-induced intracellular signaling pathway, which differs from the
TNF
-induced one, are necessary for full induction of E-selectin
mRNA.

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Fig. 2.
Simultaneous treatment with IL-1 and PMA
synergistically induces E-selectin gene expression. Northern
hybridization was performed with RNA from cells untreated or treated
for 3 h with IL-1 (400 pg/ml) or TNF (500 pg/ml) either
alone or in combination with PMA (100 ng/ml). The resulting
autoradiographs were quantified by videodensitometry as described in
the legend to Fig. 1B.
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|
To confirm the markedly different effects of IL-1
and TNF
on
E-selectin mRNA expression in the presence of PMA, dose-response profiles for IL-1
and TNF
were examined. HUVEC were treated with
increasing doses of IL-1
, ranging from 16 to 400 pg/ml, or TNF
,
from 20 to 500 pg/ml, in the presence or absence of PMA (100 ng/ml)
(Fig. 3). The dose curve for
IL-1
-mediated induction of E-selectin mRNA revealed that in the
presence of PMA, E-selectin was cooperatively induced by IL-1
in a
dose-dependent manner. In contrast, E-selectin mRNA
expression was only slightly increased by treatment with increasing
doses of TNF
in the presence or absence of PMA.

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Fig. 3.
IL-1 cooperatively increases E-selectin
mRNA expression in a dose-dependent manner in the
presence of PMA. HUVEC were treated with increasing amounts of
IL-1 (0, 16, 80, and 400 pg/ml) or TNF (0, 20, 100, and 500 pg/ml) with or without PMA (100 ng/ml) for 3 h. At the end of the
treatment, the cells were collected and subjected to Northern
hybridization. The resulting autoradiographs were quantified as
described in the legend to Fig. 1B.
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|
The synergistic effect of combined treatment with IL-1
and PMA on
E-selectin mRNA induction is independent of de novo
protein synthesis, because it was potentiated and not inhibited by the presence of cycloheximide at the concentration (10 µg/ml) sufficient to inhibit protein synthesis by 95% (33) (data not shown).
To determine if the effects of IL-1
, TNF
, and PMA on the gene
expression of adhesion molecules are mediated by increased transcriptional activity of the genes, nuclear run-on studies were
performed. HUVEC cultures were treated with IL-1
and TNF
, either
alone or in combination with PMA for 2 h; nuclei were isolated; and the transcription that had started in intact cells was allowed to
reach completion in the presence of [32P]UTP. The
radiolabeled RNA transcripts were subsequently hybridized to
slot-blotted plasmid DNA fragments encoding E-selectin, ICAM-1, or
-actin (Fig. 4). While either IL-1
,
TNF
, or PMA weakly stimulated transcription of the E-selectin gene,
the combination of IL-1
and PMA synergistically increased E-selectin
transcription. In contrast, cotreatment with TNF
and PMA did not
have a marked cooperative effect. On the other hand, ICAM-1
transcription was induced by IL-1
, TNF
, or PMA, and treatment
with either IL-1
or TNF
combined with PMA had only an additive
effect on the transcription of the gene. These results are largely
consistent with the levels of specific mature mRNA found in total
cellular RNA, indicating that the coexistence of classic PKC activation
and the intracellular signals generated by IL-1
, but not by TNF
,
synergize E-selectin mRNA induction principally via the activation
of specific transcription.

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Fig. 4.
Synergistic induction of E-selectin
transcription by IL-1 and PMA. A, HUVEC were
untreated or treated for 2 h with IL-1 (400 pg/ml) or TNF
(500 pg/ml), either alone or in combination with PMA (100 ng/ml).
Following the isolation of nuclei, transcription was allowed to proceed
in the presence of radiolabeled UTP, and then the generated RNA was
detected by hybridization to slot-blotted plasmids containing specific
cDNA inserts for E-selectin, ICAM-1, and -actin. B,
the transcript levels for E-selectin and ICAM-1 were quantified by
videodensitometry and normalized relative to those for -actin. The
value for each gene in untreated cells was taken as 1.
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The NF-ELAM1/ATF Site in the 5' Promoter Region of the E-selectin
Gene Is Required for the Synergistic Expression Induced by Cotreatment
with IL-1
and PMA--
To identify the cis-regulatory sequences
required for the synergistic effect of IL-1
and PMA on induction of
E-selectin mRNA expression, HUVEC were transiently transfected with
a series of 5'-flanking deletion mutants inserted into a firefly
luciferase expression vector. Twenty-four hours after transfection, FCS
in the medium was reduced to 0.5%, and the HUVEC were cultured further for 24 h. Then the cells were treated for 6 h with IL-1
or
TNF
, either alone or in combination with PMA (100 ng/ml), and
harvested for the luciferase assay. A transient transfection study on
HUVEC with a deletion mutant with 800 bp upstream of the transcription start site demonstrated that simultaneous treatment with IL-1
and
PMA induced the luciferase activity synergistically, while cotreatment
with TNF
and PMA caused only additive induction (Fig. 5B), as found in nuclear
run-on studies (Fig. 4). This result demonstrates that the promoter
region from
800 to +50 bp of the E-selectin gene is sufficient for
the synergistic effect of IL-1
and PMA. As shown in Fig.
5C, all of the deletion mutants from 800 to 166 bp upstream
of the transcription start site induced luciferase activity to
equivalent levels in response to cotreatment with IL-1
and PMA.
However, the deletion mutant with 116 bp of the upstream sequence was
not able to induce the luciferase gene synergistically in response to
the cotreatment, indicating that a critical promoter region involved in
the synergistic E-selectin gene expression on cotreatment with IL-1
and PMA lies within the
166 to
116 bp region. As shown in Fig.
5A, this region between
166 and
116 bp of the E-selectin
promoter gene contains one NF-ELAM1/ATF site (34, 35) and one NF-
B
site, which was recently identified (11, 36, 37). To further delineate
the precise element(s) within the
166 to
116 bp region required for
the synergism between IL-1
and PMA, we constructed a deletion mutant with 129 bp of the upstream sequence in which the NF-ELAM1/ATF site was
deleted and examined its luciferase inducibility (Fig. 6A). The 166-bp of the
upstream sequence was able to induce the synergistic expression of the
luciferase gene in response to simultaneous treatment with IL-1
and
PMA, whereas only additive induction was seen when TNF
was
substituted for IL-1
. However, a reporter construct with 129 bp of
the upstream sequence did not exhibit synergistic induction of the
luciferase gene at all. To further confirm that the synergistic effect
of IL-1
and PMA is mediated through the NF-ELAM1/ATF site, we
created a reporter construct with 166 bp of the upstream sequence into
whose NF-ELAM1/ATF site a mutation was introduced. It has been reported
that the dATP residues at
151 and
146 bp are important for the
binding of ATF proteins to the NF-ELAM1/ATF site of the E-selectin
promoter (34). So we introduced the same mutations at the NF-ELAM1/ATF site (mNF-ELAM1/ATF) and assayed the luciferase activity (Fig. 6B). As expected, the reporter construct with mNF-ELAM1/ATF
did not cause synergistic induction of the luciferase gene in response to combined treatment with IL-1
and PMA. These results clearly demonstrate that the NF-ELAM1/ATF site is indispensable for the synergistic transcription of E-selectin induced by simultaneous treatment with IL-1
and PMA. Next, to clarify that only the
NF-ELAM1/ATF site is necessary and sufficient for the synergism between
IL-1
and PMA, we made a reporter construct driven by a 4 times
repeated NF-ELAM1/ATF site (4× NF-ELAM1/ATF) and assayed the
luciferase activity (Fig. 6C). Although the activity was
induced a little by each treatment, synergistic induction in response
to combined treatment with IL-1
and PMA was never observed. This
indicates that the NF-ELAM1/ATF site is necessary but not sufficient
for the synergistic transcription of E-selectin induced by simultaneous treatment with IL-1
and PMA.

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Fig. 5.
Identification of the promoter elements
critical for the synergistic induction by IL-1 and PMA.
A, schematic representation of the E-selectin promoter. The
previously described 5'-flanking elements containing binding sites for
NF-ELAM1 and NF- B are shown. The positions of the 5' end point used
in the deletion analysis are indicated with arrows.
B, HUVEC were transfected transiently with the wild type
( 800) reporter construct. After a 24-h incubation with 0.5% FCS,
HUVEC were left untreated or treated with IL-1 (400 pg/ml) or TNF
(500 pg/ml), either alone or in combination with PMA (100 ng/ml) for
6 h. HUVEC extracts were prepared and assayed for luciferase
activity. The value obtained from untreated cells was taken as 1. C, luciferase activity was assayed in HUVEC transiently
transfected with each reporter construct and untreated or treated with
IL-1 /PMA for 6 h. The value obtained from the untreated cells
transfected with the 800 construct was taken as 1. The results in
B and C are presented as the means ± S.E.
of the means for triplicate samples, and at least three independent
experiments were performed with similar results.
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Fig. 6.
The NF-ELAM1/ATF site is required for the
synergistic transcription of E-selectin induced by cotreatment with
IL-1 and PMA. A, HUVEC transfected with 166,
129, or 116 deleted construct as described in the legend to Fig.
5A were untreated or treated with IL-1 (400 pg/ml) or
TNF (500 pg/ml), either alone or in combination with PMA (100 ng/ml)
for 6 h. The value obtained with the 166 construct in untreated
cells was taken as 1. B, the 166 deleted construct with
(mNF-ELAM1/ATF) or without the point mutations at the NF-ELAM1/ATF site
was transfected into HUVEC and analyzed as described for A.
The value obtained with the wild type 166 construct in untreated
cells was taken as 1. C, the construct containing 4×
NF-ELAM1/ATF sites was transfected and analyzed as described in
A. The value obtained from untreated cells was taken as 1. The
results shown in A, B, and C are
representative of three identical experiments performed. The results
are the means ± S.E.
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|
Synergistic Transcription Induced by Combined Treatment with
IL-1
and PMA Does Not Occur through the Changed DNA Binding Activity
of NF-ELAM1 and NF-
B--
The NF-ELAM1/ATF site is recognized by
members of the ATF family of transcription factors, including ATFa,
ATF2, and ATF3 (35). It has been reported that homo- or heterodimers
consisting of not only these members of the ATF family but also c-Jun
and CREB bind to the ATF site of the E-selectin promoter (35, 38). Furthermore, Kaszubska et al. have demonstrated that each of
the three ATF members has a different effect on E-selectin promoter activity (35). Therefore, we examined if the nuclear proteins that bind
to the NF-ELAM1/ATF site of the E-selectin promoter differed
quantitatively or qualitatively, depending on the stimulant, such as
IL-1
, TNF
, and PMA, either alone or in combination. Nuclear
extracts were prepared from HUVEC treated for 2 h with IL-1
or
TNF
, either alone or in combination with PMA (100 ng/ml), and then
the EMSA was performed using a oligonucleotide probe including the
NF-ELAM1/ATF site at
166 to
129 bp of the E-selectin promoter (Fig.
7, A and B). As
reported previously (38), three distinct complexes (C1-C3) were
detected constitutively. Although the presence of PMA decreased the
amount of the C1 complex, there was neither quantitative nor
qualitative difference in the binding of NF-ELAM1 to the specific DNA
sequence between the combined treatments with IL-1
/PMA and
TNF
/PMA. Because the reporter construct with mNF-ELAM1/ATF sequence
did not cause synergistic induction of the luciferase gene in response
to the cotreatment with IL-1
and PMA (Fig. 6B), we next
examined the effect of mNF-ELAM1/ATF sequence on the EMSA using the
nuclear extracts from HUVEC treated with IL-1
/PMA and TNF
/PMA
(Fig. 7C). Only the faint binding activity to the
mNF-ELAM1/ATF oligonucleotides was seen using both nuclear proteins,
and the binding activities of both nuclear proteins to the NF-ELAM1/ATF
site was slightly inhibited by the mNF-ELAM1/ATF sequence to the same
extent, while they were completely inhibited by the NF-ELAM1/ATF
oligonucleotides. These results demonstrate that the binding of
NF-ELAM1 to the corresponding cis-element is indispensable for the
synergistic transcriptional activity of E-selectin induced by the
cotreatment with IL-1
/PMA and that the binding affinities of both
the NF-ELAM1 activated by the treatment with IL-1
/PMA and TNF
/PMA
were the same.

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Fig. 7.
DNA binding activity and protein composition
of NF-ELAM1 are not different between both treatments with IL-1 /PMA
and TNF /PMA. HUVEC were either left untreated or treated with
IL-1 (400 pg/ml) or TNF (500 pg/ml) either alone or in
combination with PMA (100 ng/ml) for 2 h, and then nuclear
extracts were prepared. A, electrophoretic mobility shift
assay with a radiolabeled oligonucleotide containing the NF-ELAM1/ATF
site. B, quantification of the complexes (C1-C3) by
videodensitometry using the resulting autoradiographs. C,
binding affinity to the wild type and mutated NF-ELAM1/ATF site on the
EMSA using the nuclear extracts treated with IL-1 or TNF in
combination with PMA. w and m, wild type and
mutated NF-ELAM1/ATF site, respectively. D, supershift
analysis with polyclonal antibodies against ATF2, ATF3, c-Jun, and
CREB, using nuclear extracts of HUVEC treated with IL-1 /PMA for
2 h. E, protein compositions of C2 and C3 complexes
from nuclear extracts in HUVEC treated with IL-1 /PMA and TNF /PMA.
C2 and C3 complexes obtained from EMSA (A) were analyzed
using the UV cross-linking method described under "Experimental
Procedures." Molecular mass markers are indicated at the
left in kilodaltons. The arrows show location of
the proteins involved.
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To identify the protein complexes binding to the NF-ELAM1/ATF site in
the nuclear extracts of HUVEC treated with IL-1
and TNF
in
combination with PMA, we performed supershift analysis with polyclonal
antibodies against ATF2, ATF3, c-Jun, and CREB. In HUVEC treated with
IL-1
/PMA, the antibodies against ATF2 and ATF3 caused shifts in the
mobilities of the C2 and C3 complex in part, respectively, and
anti-c-Jun antibody caused shifts in both the C2 and C3 complexes,
while the anti-CREB antibody caused no shift (Fig. 7D). With
the nuclear extracts of HUVEC stimulated with TNF
/PMA, the
antibodies had the same effects on the mobilities of the C2 and C3
complexes as mentioned above (data not shown), suggesting that the
composition of the proteins binding to the NF-ELAM1/ATF site was not
different between HUVEC treated with IL-1
and TNF
in combination
with PMA. Furthermore, UV cross-linking experiments on the C2 and C3
complexes from HUVEC treated with IL-1
/PMA and TNF
/PMA showed
that the C2 and C3 complexes contained four proteins and two proteins,
respectively, whose protein compositions on both treatments appeared to
be identical on the basis of SDS-PAGE mobility (Fig.
7E).
Direct protein-protein associations are important mechanisms by which
transcription factors synergistically cooperate. Since ATF family
members and c-Jun have been shown to physically interact with NF-
B,
a quantitative or qualitative difference in NF-
B may be responsible
for the NF-ELAM1/ATF-dependent synergistic transcription of
the E-selectin gene induced by IL-1
/PMA but not by TNF
/PMA
treatment. EMSA involving an oligonucleotide probe containing two
distal
B sites (
128 to
101 bp) or the other proximal one (
100
to
75 bp) of the E-selectin promoter sequence showed that while two
complexes (C
1 and C
2) were induced on treatment with IL-1
,
TNF
, or PMA in the same manner, combined treatment with IL-1
/PMA
or TNF
/PMA did not cause synergistically quantitative or qualitative
change in NF-
B activation (Fig. 8, A and B). Supershift analyses involving the
antibodies against p50, p52, p65, Rel-B, and c-Rel with nuclear
extracts of HUVEC stimulated with IL-1
/PMA (Fig. 8C) and
TNF
/PMA (data not shown) at the proximal
B site revealed that
C
1 was a p50/p65 heterodimer and C
2 was a p50 homodimer. In
addition, UV cross-linking experiments on the C
1 and C
2 complexes
showed that there was no difference between nuclear extracts from HUVEC
treated with IL-1
/PMA and TNF
/PMA (data not shown).

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Fig. 8.
Analysis of DNA binding activity to the B
sites in the E-selectin promoter. Nuclear extracts were prepared
as described in Fig. 7, and the electrophoretic mobility shift assay
was performed with a radiolabeled oligonucleotide containing distal
B sites ( 128 to 101 bp) (A) or the other proximal one
( 100 to 75 bp) (B) of the E-selectin promoter.
C, supershift analysis of the proximal B site with
polyclonal antibodies against p50, p52, p65, Rel-B, and c-Rel, using
nuclear extracts of HUVEC treated with IL-1 /PMA.
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DISCUSSION |
While phorbol ester PMA principally activates classic protein
kinase C (31, 39, 40) and is capable of inducing the expression of
E-selectin as well as other adhesion molecules (22, 31), whether or not
IL-1
and TNF
induce the expression through PKC activation has not
been well defined (31). In this study, we have clarified that the
IL-1
- or TNF
-provoked intracellular signaling pathway that
induces gene expression of E-selectin and ICAM-1 is not through classic
PKC activation (Fig. 1). To further confirm this, we also assayed PKC
activation, which was assessed as PKC translocation in HUVEC following
treatment with IL-1
, TNF
, or PMA. Although PMA caused a rapid
decrease in PKC activity in the cytoplasmic fraction and a proportional
increase in the membrane fraction, such PKC activation was not detected
upon treatment with IL-1
or TNF
(data not shown).
However, our results demonstrated that PKC activation, which occurred
in the presence of FCS and bFGF as well as PMA, induced E-selectin gene
expression synergistically with IL-1
but not with TNF
, while such
a synergistic effect was not observed on the expression of ICAM-1
(Figs. 1-3). Since HUVEC are usually cultured in a medium containing
FCS and FGF, the synergistic effect of activated PKC and signals
elicited by IL-1
is one reason why the PKC dependence of E-selectin
mRNA expression induced by proinflammatory cytokines has been
misunderstood. Although it has been reported that IL-1 and TNF
induce NF-
B activation, which is indispensable for the expression of
adhesion molecules (reviewed in Ref. 41) through ceramide formation
(42), some studies have suggested that signals generated by both
proinflammatory cytokines differ, at least in part (12, 43). Swerlick
et al. have shown that ICAM-1 was induced on cultured dermal
microvessel endothelium by either IL-1 or TNF
, whereas VCAM-1 was
induced only by TNF
(12). Treatment of HUVEC with the combination of
IFN
and TNF
induced the surface expression of ICAM-1 and
E-selectin, whereas the combination of IL-1
and IFN
had a minimal
effect (43). Furthermore, our recent study demonstrated that IL-1
is
a much stronger inducer of E-selectin mRNA than TNF
in the
heart, although both proinflammatory cytokines induce ICAM-1 and VCAM-1
mRNAs to similar extents (15). From these observations, there
appear to be both common (NF-
B activation) and specific pathways of endothelial adhesion molecule expression induced by IL-1
and TNF
,
although what kinds of signals generated by both cytokines result in
stimulus-specific induction of adhesion molecules has never been
clarified. Since the promoter regions of endothelial adhesion molecule
genes contain
B elements in common and specific regulatory elements
such as the NF-ELAM1/ATF site in E-selectin, the Sp1 and interferon
regulatory factor binding sites in VCAM-1 and the CCAAT
enhancer-binding protein element and
-activated sequences in ICAM-1
(reviewed in Ref. 41), the different effects of IL-1
and TNF
on
the full expression of adhesion molecules may result from the different
modulation of nuclear factors that can bind to these specific
regulatory elements.
Indeed, our results (Fig. 6) demonstrated that the NF-ELAM1/ATF site of
the E-selectin promoter is required for the synergistic expression
induced by the cotreatment with IL-1
and PMA and that the individual
transcriptional activities of NF-
B and NF-ELAM1 are not involved in
the synergistic effect of treatment with IL-1
/PMA.
Various publications on E-selectin reporter constructs containing
multiple point mutations or deletions that abrogate NF-ELAM1 binding
differ in the effects described (11, 34-38). The results range from
unchanged to reduced basal reporter activity or to severely decreased
induction by IL-1
or TNF
. Variations in transfection protocols as
well as reporter genes, leading to differences in the detection limits
of the respective reporter assays, and whether or not the NF-ELAM1/ATF
site was upstream of the wild-type E-selectin promoter or of a minimal
heterologous promoter (SV40) might explain the divergent results and
interpretations. Although the effects of the two proinflammatory
stimulants were never compared in these publications, a survey
comparing the effects of IL-1
and TNF
under similar experimental
conditions, mentioned in the publications, led us to the suggestion
that the NF-ELAM1/ATF site is much more necessary for full
transcription of the E-selectin gene when IL-1
is used as the
stimulant than when TNF
is used.
Since it has been reported that cAMP inhibits TNF-induced E-selectin
promoter activity through alteration of the composition of nuclear
factors that bind to the NF-ELAM1/ATF element (38), we have considered
that the synergistic effect of combined treatment with IL-1
and PMA
might occur through the same mechanism. However, EMSAs together with
supershift experiments and UV cross-linking studies involving HUVEC
nuclear extracts showed no differences in the binding activity and the
protein composition of NF-ELAM1 between combined treatment with
IL-1
/PMA and TNF
/PMA (Fig. 7, A-C), demonstrating
that the synergistic effect of IL-1
and PMA on the induction of
E-selectin mRNA expression is not dependent on the binding activity
or protein composition of NF-ELAM1. Since direct physical interaction
between the proteins (ATFa, ATF2, ATF3, and c-Jun) bound to the
NF-ELAM1/ATF element and NF-
B subunits (p50 and p65) has been
demonstrated and reported to be the mechanism underlying the
cooperativity yielding maximal levels of E-selectin gene transcription
(34, 35), the binding activity and protein composition of NF-
B
activated by combined treatment with IL-1
and PMA have been examined
in comparison with NF-
B induced by cotreatment with TNF
/PMA (Fig.
8). The results showed no differences in the binding activity or
protein composition of NF-
B activated by both combined treatments
and the direct transcriptional activity mentioned above. Therefore,
increased transactivation of NF-ELAM1 and/or NF-
B in conjunction
with each other, which cannot be detected on EMSA, is most likely the
mechanism through which E-selectin expression is induced
synergistically upon combined treatment with IL-1
and PMA but not
with TNF
/PMA.
Recently, it was reported that ATF2 is phosphorylated as well as c-Jun
by proinflammatory cytokines or ultraviolet radiation through the c-Jun
NH2-terminal protein kinase cascade and that the
transcriptional activity of the phosphorylated ATF2 increases without
changes in its DNA binding activity (44). In the same report, it was
suggested that ATFa is also a substrate of c-Jun NH2-terminal protein kinase. Although IL-1
and TNF
,
as well as phorbol ester, were all reported to be potent activators of c-Jun NH2-terminal protein kinase in several cells
(45-48), the effects of the two proinflammatory cytokines alone or in
combination with PMA have not been compared. Another subgroup of
mitogen-activated protein (MAP) kinases, the p38 family, also strongly
phosphorylate ATF2 but phosphorylate c-Jun very weakly (49-51).
Interestingly, at least two members of the p38 family (p38 and p38-2)
have been shown to be activated more strongly by IL-1
than TNF
or
PMA in COS cells (50), corresponding to the finding in this study that
IL-1
is a more potent stimulant in combination with PMA for
E-selectin transcription than TNF
in vascular endothelial cells.
This is also consistent with our finding in a recent in vivo
study that IL-1
is a much stronger inducer of E-selectin in the
heart (15). Furthermore, p38 MAP kinases such as p38, p38-2, and p38
have been shown to be expressed at very high levels in the heart in a
tissue-specific manner (49, 50), in accord with our observation of
E-selectin induction in vivo (15). Therefore, it is
suggested that p38 MAP kinases play key roles in tissue- and
stimulus-specific expression of E-selectin through phosphorylation and
increased transactivational ability of ATF2 and/or its family. However,
since other groups have reported that p38 MAP kinases are activated to
similar extents by IL-1
and TNF
in HeLa cells (49, 51) or that
both p38 and c-Jun NH2-terminal protein kinase are
activated with different time courses by the two proinflammatory cytokines (52), it is necessary to study, using vascular endothelial cells, the effects of IL-1
and TNF
in combination with PMA not only on the activation of MAP kinases (p38 and c-Jun
NH2-terminal protein kinase) but also on the
phosphorylation and transactivational abilities of ATF2 and its family
as well as c-Jun.