(Received for publication, May 9, 1995; and in revised form, June 7, 1995 )
From the
We investigated the mechanisms by which HO
increases intercellular adhesion molecule 1 (ICAM-1; CD54)
expression in endothelial cells. The H
O
-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 H
O
stimulated ICAM-1
gene transcription by activation of a distinct region of the ICAM-1
promoter. The H
O
-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 H
O
responsiveness to a minimal promoter. Mutation of the 16-base
pair repeats within the ICAM-1 promoter prevented
H
O
-induced DNA binding activity, and their
deletion abrogated the H
O
-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 H
O
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
.
Adhesion of circulating polymorphonuclear leukocytes (PMN) ()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 H
O
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).
Recent studies show
that the reactive oxidant, HO
, 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). H
O
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
H
O
exposure (Lo et al., 1993).
H
O
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 H
O
may be a mechanism
of endothelial ICAM-1 gene expression.
In this study, we examined
the basis of HO
-induced ICAM-1 expression in
endothelial cells. We showed that H
O
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 H
O
-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,
H
O
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.
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
HO
exposure period.
The
RNA samples (20 µg/lane) were subjected to gel electrophoresis in
denaturing 1% formaldehyde-agarose gels and transferred overnight in 20
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.
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 HO
. RNA was isolated and
processed for Northern analysis.
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, 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.
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-B
oligonucleotide corresponds to the element upstream of the AP-3 site
and downstream of the AP-1/Ets repeats.
Figure 7:
HO
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 H
O
or to 100 units/ml TNF
.
Cells were harvested 24 h after H
O
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.
Figure 8:
Localization of the HO
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 H
O
(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.
Figure 1:
Effects of actinomycin D on
HO
-induced expression of ICAM-1 mRNA. Confluent
HUVEC were treated with 50 µM (lanes 2 and 4) or 100 µM H
O
(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).
Figure 2:
Stability of
HO
-induced ICAM-1 mRNA. HUVEC were treated with
100 µM H
O
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).
Treatment
with actinomycin D at the time of HO
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
H
O
on mRNA stability, endothelial cells were
first exposed to 100 µM H
O
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
H
O
-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 H
O
increased the synthesis of ICAM-1 mRNA.
Figure 3:
Nuclear run-on analysis of
HO
-induced ICAM-1 mRNA. Slot-blot analysis of
the ICAM-1 RNA transcription rates of nuclei isolated from control
HUVEC and HUVEC treated with H
O
(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).
Figure 4:
Effect of an antisense oligonucleotide on
the expression of ICAM-1 message induced by HO
(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).
Figure 5:
Effect of 3-aminobenzamide (3AB),
pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine (NAC) on
HO
-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 H
O
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).
Figure 6:
HO
induces AP-1
but not NF-
B binding activity in endothelial cells. Nuclear
protein extracts of HUVEC exposed for 1 h to 100 µM H
O
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.
Unlike HO
, 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.
Figure 9:
AP-1 and Ets binding sites functionally
cooperate to form HO
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 H
O
(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.
Figure 10:
Characterization of the
HO
-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
H
O
(400 µM) in the presence or
absence of N-Cys(Ac) (30 mM) as indicated above each
lane. The protein
DNA complexes were resolved by gel
electrophoresis and detected by autoradiography. Binding specificity
was assessed by 50 ng of unlabeled homologous competitor
oligonucleotide.
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
HO
, 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
H
O
-treated cell extracts these mutations in
either the AP-1 or Ets binding sites prevented the
H
O
-induced binding activity (lanes 7 and 11), indicating that an intact AP-1/Ets repeat was
essential for the H
O
-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.
In the present study, we examined mechanisms of
HO
-mediated induction of ICAM-1 mRNA expression
in endothelial cells. The level of expression induced by
H
O
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. H
O
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
H
O
-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.
Within the HO
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
H
O
responsive region. An oligonucleotide of the
AP-1/Ets repeats formed H
O
-induced gel shift
complexes that were sensitive to mutation of either the AP-1 or Ets
binding sites, suggesting these elements mediated the
H
O
-induced transcription of the ICAM-1 gene.
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 HO
. Both the AP-1 and Ets binding sites were
essential since mutation of these sequences reduced the induced
response to H
O
suggesting these two binding
sites functionally cooperated to form the H
O
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 H
O
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 H
O
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. (
)
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
HO
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 H
O
since
mutations in either the AP-1 or Ets binding sites abrogated the
H
O
-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.
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
HO
-induced ICAM-1 expression. In contrast,
pyrrolidine dithiocarbamate (PDTC) (which prevents NF-
B activation
(Schreck et al., 1992)) did not alter the
H
O
-induced ICAM-1 message. H
O
also did not activate NF-
B binding activity as reported by
Bradely et al.(1993), consistent with the lack of effect of
PDTC on H
O
-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
H
O
-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
H
O
response. Although H
O
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.
Since
the regulation of HO
-induced ICAM-1 expression
appears to be the result of the redox activity of
H
O
, 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
H
O
activation is transmitted to the ICAM-1
promoter is yet unknown. H
O
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 H
O
stimulates the ICAM-1 gene by a similar signal transduction
mechanism. H
O
has also been shown to increase
endothelial permeability via a protein kinase C-dependent mechanism
(Siflinger-Birnboim et al., 1992). H
O
has also been shown to induce c-fos and c-jun gene expression and increase AP-1 activity (Li et al.,
1994).
In summary, the present results indicate a unique mechanism
of HO
-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 H
O
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 H
O
and
TNF
may mediate ICAM-1 expression by distinct intracellular
mechanisms involving unique sequence elements within the promoter
region of the gene.