(Received for publication, September 26, 1994; and in revised form, November 1, 1994)
From the
Intercellular adhesion molecule-1 (ICAM-1) is greatly
up-regulated on endothelial cells at sites of inflammation and is
involved in leukocyte attachment and extravasation. Previously, we had
shown that the ICAM-1 gene expression in human umbilical vein
endothelial cells (HUVECs) was transcriptionally regulated by tumor
necrosis factor- (TNF-
) (Wertheimer, S. J., Myers, C. L.,
Wallace, R. W., and Parks, T. P.(1992) J. Biol. Chem. 267,
12030-12035). In the present investigation, TNF-
induced
transcription was found to be initiated exclusively at two sites, 40
and 41 base pairs upstream of the translation start site. Deletion
analysis of the 5` regulatory region of the ICAM-1 gene revealed a
92-base pair sequence which was both necessary and sufficient to confer
TNF-
responsiveness to a linked luciferase reporter gene in
transient transfection assays. This TNF-
-responsive region
contained a variant NF-
B site at -187 to -178, which
when mutated, completely abolished ICAM-1 promoter activation by
TNF-
, interleukin-1
, and lipopolysaccharide. Two inducible
nuclear protein complexes bound to the ICAM-1
B site and were
identified as the NF-
B p65 homodimer and p65/p50 heterodimer.
Overexpression of p65, but not p50, transactivated the ICAM-1 promoter
in a
B site-dependent manner in HUVECs. In addition, p65-mediated
transactivation was suppressed by co-expression of p50. Our results
suggest that cytokine activation of the ICAM-1 promoter in HUVECs may
critically depend on p65 homodimers binding to a variant
B site.
Intercellular adhesion molecule-1 (ICAM-1) ()is an
inducible cell surface glycoprotein belonging to the immunoglobulin
su-pergene family(1, 2) . By serving as a
counter-receptor for the leukocyte
integrins, LFA-1
and Mac-1, ICAM-1 participates in a wide range of inflammatory and
immune responses(3, 4) . The expression of ICAM-1 on
activated endothelium, for example, plays a key role in the recruitment
and extravasation of circulating leukocytes at sites of tissue injury
or infection(3) . ICAM-1 is expressed basally at low levels on
vascular endothelium and lymphocytes, and at moderate levels on
monocytes (5, 6, 7) , but can be induced to
high levels on a number of cell types by stimulation with bacterial
lipopolysaccaride (LPS), phorbol esters, or inflammatory cytokines,
such as TNF-
, IL-1
, and IFN-
. Up-regulation of ICAM-1
expression by these mediators has been reported on endothelial
cells(5, 6, 7, 8, 9) ,
keratinocytes(5) , synovial cells(10) , epithelial
cells(11) , fibroblasts(5) , hepatocytes(12) ,
myocytes (13) , as well as activated lymphocytes(5) .
The induction of ICAM-1 cell surface expression requires de novo mRNA and protein synthesis(5, 14) . Following
treatment with TNF-, ICAM-1 expression on human umbilical vein
endothelial cells (HUVECs) can be detected within several hours,
reaches maximal levels at 8-10 h, and remains elevated for at
least 72 h in the presence of
cytokine(6, 7, 9) . ICAM-1 message levels are
elevated within 30 min following TNF-
stimulation and peak at 2
h(14) . Unlike cell surface expression, ICAM-1 mRNA returns to
basal levels within 24 h. Nuclear run-on assays demonstrated that
TNF-
transiently activated ICAM-1 transcription which preceded and
paralleled the changes in steady state mRNA levels. Therefore,
regulation of ICAM-1 gene expression by TNF-
in HUVECs is
controlled primarily at the transcriptional level.
The molecular
events underlying the transcriptional activation of the ICAM-1 gene in
response to cytokine stimulation are poorly understood. Several
transcription start sites for the ICAM-1 gene have been reported, and
two TATA boxes identified, but their use was dependent on the cell type
and stimulus studied(15, 16, 17) . The
5`-flanking region of the ICAM-1 gene has been cloned and shown to
possess potent constitutive and inducible promoter activity when linked
to a luciferase or CAT reporter gene and transfected into a variety of
primary cell cultures or cell
lines(15, 16, 17, 18) . Sequence
analysis of the 5`-flanking sequence has revealed numerous potential
regulatory elements which could be involved in the cytokine activation
of the ICAM-1 promoter. Recent reports have begun to identify
tissue-specific and cytokine responsive regions within the ICAM-1
promoter. Look et al.(19) identified a responsive
element present in the ICAM-1 5`-flanking sequence, -116 to
-106 relative to the start of translation, that allowed the
promoter to respond to IFN- in primary epithelial cells. Another
group of investigators identified four regions of the ICAM-1 promoter
which conferred PMA responsiveness in human embryonal kidney
cells(20) . One of these regions, -267 to -215
relative to the start of translation, also appeared to be involved in
TNF-
responsiveness, and mediated dexamethasone repression of both
the PMA and TNF-
responses.
Cytokine activation of the
promoters of two other inducible adhesion proteins, E-selectin and
VCAM-1, in endothelial cells has been shown to critically rely on the
transcription factor
NF-B(21, 22, 23, 24, 25, 26) .
Six potential NF-
B sites have been identified in the ICAM-1
5`-flanking sequence and first intron, but which, if any, are involved
in cytokine activation of the ICAM-1 promoter in endothelial cells has
yet to be firmly
established(15, 16, 17, 20, 27) .
The NF-
B/rel family of transcription factors consists of five
proteins, p65 (relA), p50 (NFKB-1), c-Rel (c-rel), Rel B (relB), and
p52 (NFKB-2), which are related to each other through a N-terminal
stretch of
300 amino acids called the rel homology
domain(28) . DNA binding is achieved through dimerization of
the family members, resulting in numerous homo- and heterodimeric
combinations of NF-
B/rel proteins. In most cells, NF-
B/rel
dimers are retained in the cytoplasm in an inactive state by binding to
one of several inhibitor molecules which belong to the I
B
family(29) . Activation of NF-
B involves the release of
I
B and subsequent translocation of NF-
B/rel dimers to the
nucleus. Once NF-
B has entered the nucleus it is capable of
binding to target DNA sequences and activating transcription. The
C-terminal regions of p65, c-Rel, and RelB harbor transcriptional
activation domains and the in vivo transcriptional activity is
attributed to p65-, c-Rel-, and RelB-containing
dimers(30, 31, 32, 33, 34, 35) .
p50 homodimers have been shown to transactivate in vitro, but
there is no in vivo evidence for this phenomenon(36) .
Not all NF-
B/rel dimers can bind to the same NF-
B sequence.
It appears that variations of the NF-
B consensus sequence can
exclude or recruit different homo- or heterodimeric combinations of
NF-
B/rel to the site(28, 37, 38) .
The present investigation was conducted to define the molecular mechanisms involved in cytokine-induced transcription of the ICAM-1 gene in vascular endothelium. Using primary cultures of human endothelial cells as our model system, we sought to determine 1) the transcription start site(s) of the ICAM-1 gene, 2) the cis-elements within the ICAM-1 5`-flanking sequence involved in transcriptional activation of the gene, 3) the transcription factors that bind to these cis-elements, and 4) the functional relevance of these transcription factors for ICAM-1 promoter activation.
DNA fragments
were end-labeled with [-
P]dATP and dGTP,
dCTP, and dTTP by incubation with Klenow fragment at 37 °C for 30
min. The labeled probes were separated from unincorporated nucleotides
using STE SELECT-D G-25 spin columns from 5`
3`, Inc. Nuclear
extract (10-20 µg protein) was incubated with 0.5-1 ng
(
30,000 cpm) of labeled probe for 30 min at room temperature in a
binding buffer containing 10 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM
-mercaptoethanol, 75 mM NaCl,
0.1 mg/ml poly(dI-dC)
poly(dI-dC), and 5% glycerol.
Electrophoresis was carried out using 4% native acrylamide gels (50
mM Tris base (pH 8.5), 380 mM glycine, 2.5 mM EDTA, 2.5% glycerol). Gels were vacuum-dried and exposed to x-ray
film overnight. In some experiments, nuclear extracts were preincubated
with antibodies or unlabeled oligonucleotides to antagonize the binding
reactions. Rabbit polyclonal antibodies directed against various
transcription factors were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Double-stranded oligonucleotides containing
transcription factor consensus binding sites were obtained from Life
Technologies, Inc. (Sp1), Santa Cruz Biotechnology (consensus and
mutant NF-
B), or synthesized and annealed by Midland Certified
Reagent (Midland, TX). Recombinant p65 for EMSA was produced in
vitro using the plasmid pRc/CMV p65 and the TNT T7 Coupled Wheat
Germ Extract System from Promega.
Figure 1:
Identification of the ICAM-1 gene
transcription start sites. Total RNA was isolated from HUVECs following
a 2-h stimulation with TNF- (T; 100 units/ml), a 4-h
stimulation with PMA (P; 50 ng/ml), or from unstimulated
cells(-). Primer extension analysis on the RNA was performed
using the ICAM-1-specific oligonucleotide primers PE2 or PE4, and
subjected to electrophoresis through a denaturing polyacrylamide gel
(see ``Materials and Methods''). Lengths of the extended
products were determined by comparison with a sequence ladder of
M13mp19; sizes in bases are indicated. Arrows mark the
position of extension products on the ICAM-1 sequence from -30 to
-53 (relative to the start of translation). The numbers on the top and bottom of the ICAM-1 sequence
indicate the product length if extension was terminated at that
residue. Similar results were obtained with three different RNA
preparations.
Figure 2:
Identification of a critical
TNF--responsive region of the ICAM-1 promoter. A series of 5`
deletions (A and B) and an internal deletion (C) of the 1.3-kilobase fragment of the ICAM-1 promoter were
fused to a promoterless luciferase reporter gene, or an internal
fragment (-227 to -136) was placed upstream of the SV40
promoter-linked luciferase reporter gene as described under
``Materials and Methods.'' The deletion constructs were
transiently transfected into HUVECs with the control plasmid,
pRSV-lacZ, using DEAE-dextran. Twenty-four hours after
transfection, one-half of the transfectants were stimulated with
TNF-
(100 units/ml) for 8 h. The cells were then lysed and assayed
for luciferase and
-galactosidase activity. The luciferase
activity of individual transfectants was subsequently normalized to
-galactosidase activity. Transfections were performed in
triplicate (stimulated and unstimulated) and the normalized luciferase
activities averaged. Average stimulated values were divided by average
unstimulated values to give the induction ratios reported. The results
presented were representative of three separate
experiments.
The 92-bp ICAM-1 TNF--responsive region was
cloned into the luciferase reporter vector, pGL2 Prom, upstream of the
SV40 early promoter (pGL2 Prom SASB). The empty promoter vector, pGL2
Prom, exhibited very low basal activity and was not responsive to
TNF-
treatment when transfected into HUVECs. However, the 92-bp
ICAM-1 TNF-
-responsive region conferred TNF-
inducibility to
the SV40 promoter similar to that obtained with the shortest inducible
deletion construct, pGL SmaIA (Fig. 2B). Conversely,
removal of the 92-bp responsive region from pGL 1.3 (pGL 1.3Sma
)
completely eliminated the ability of the full-length construct to
respond to TNF-
(Fig. 2C). It appeared, then, that
this 92-bp region of the ICAM-1 5`-flanking sequence was both
sufficient and necessary for the ICAM-1 promoter to drive expression of
a luciferase gene in response to TNF-
.
Figure 3:
Sequence of the TNF--responsive
region of the ICAM-1 promoter. ICAM-1 sequence from -227 to
-136 relative to the start of transcription, comprising the
TNF-
regulatory region, is shown. Potential transcription factor
binding sites were identified based on sequence homologies to their
published consensus sequences: Sp1 (-96 to -199), C/EBP
family (opposite strand, -201 to -206), NF-
B
(-176 to -187), and the ets family (-150 to
-153).
Figure 4:
Nuclear protein complexes which bind to
the TNF--responsive region of the ICAM-1 promoter. Nuclear
extracts were prepared from HUVECs incubated in the presence or absence
of TNF-
(100 units/ml) for 20 min. A radiolabeled probe
(-227 to -136, relative to the start of transcription) was
incubated with nuclear extract (10 µg of protein) for 30 min at
room temperature and protein-DNA complexes resolved by electrophoresis. A, nuclear extracts from unstimulated (lanes 1 and 2) and TNF-
-stimulated (lanes 3 and 4)
HUVECs were analyzed for binding activity. Lanes 2 and 4 contained a 50-fold molar excess of unlabeled probe as a
competitor. Specific protein-DNA complexes (A, B, and C) are indicated by arrows. B, competition
EMSA using nuclear extract prepared from TNF-
-treated HUVECs and
various unlabeled double-stranded oligonucleotides. The
oligonucleotides contained portions of the ICAM-1 regulatory element
identified in Fig. 3and other transcription factor binding
sites. They were present in the binding reactions at a 50-fold molar
excess over probe as follows: lane 1, no competitor; lane
2, ICAM-1-responsive region -227 to -136; lane
3, consensus Sp1; lane 4, consensus C/EBP
; lane
5, ICAM-1 NF-
B site -189 to -177; lane
6, consensus NF-
B site; lane 7, consensus NF-
B
site with a single point mutation; lane 8, ICAM-1 promoter
region -177 to -166; lane 9, ICAM-1 ets site
-160 to -148.
To confirm that the inducible complexes
bound to the NF-B element, methylation interference footprint
assays were performed. The binding site for the upper inducible
complex, Complex A, was determined to be between -187 and
-178, corresponding to the NF-
B site identified above (Fig. 5). Evident from the protection pattern was the importance
of 4 guanine residues for binding of complex A to the site. In
addition, the lower inducible complex, Complex B, produced a footprint
identical to that obtained for Complex A (data not shown), indicating
that these two distinct protein complexes recognized and bound to the
same NF-
B site. No binding site for the constitutive complex was
evident by methylation interference footprinting (data not shown).
Figure 5:
Methylation interference footprinting of
complex A to the ICAM-1 B site. Coding and noncoding strands were
individually radiolabeled and used in binding reactions with nuclear
extract from TNF-
-treated HUVECs. Reactions were scaled up 8-fold
to permit the isolation of probe bound to complex A (B) and
unbound free probe (U). Isolated complexes were then cleaved
with piperidine and subjected electrophoresis on a denaturing
polyacrylamide gel as described in detail in (40) . ICAM-1
sequences (-196 to -169) of both the coding and noncoding
strands are shown. Specific residues are marked with an asterisk to
represent those residues that when methylated interfere with the
binding of Complex A. The ICAM-1
B site identified in Fig. 3is indicated with brackets.
Figure 6:
Functional consequences of mutating the
ICAM-1 B site. A, the ICAM-1
B and ets sites were
mutated in the context of the full-length construct, pGL 1.3, as
detailed under ``Materials and Methods.'' pGL 1.3, pGL
1.3
B
, pGL 1.3 Ets
, and pGL2
Basic were transiently transfected into HUVECs and the cells
subsequently stimulated with TNF-
(100 units/ml) for 8 h. B, pGL 1.3 and pGL 1.3
B
were
transiently transfected into HUVECs and stimulated with either
TNF-
(100 units/ml), PMA (20 ng/ml), IL-1
(200 ng/ml), or LPS
(1 µg/ml) for 8 h. The induction values given in A and B were calculated as described in the legend to Fig. 2and are representative of three separate experiments, each
performed in triplicate.
The ICAM B mutant construct was also used to
assess the importance of this element for induction of the ICAM-1
promoter in response to other proinflammatory stimuli. In addition to
TNF-
, pGL 1.3 responded to other proinflammatory agents known to
up-regulate ICAM-1 expression in HUVECs, including IL-1
, PMA, and
LPS (Fig. 6B). It was evident that the NF-
B
mutation eliminated the ability of the reporter construct to respond to
TNF-
and IL-1
. Although the mutation significantly lowered
PMA induction, the reporter construct still retained a low level of PMA
induction. The induction observed in response to LPS treatment was
small, but when the NF-
B site was mutated, there was a consistent
decrease to unstimulated levels. Therefore, in HUVECs, mutation of the
NF-
B site between -187 and -178 completely eliminated
the induction of the ICAM-1 promoter by TNF-
, IL-1
, and LPS,
and largely, but not completely, suppressed induction by PMA.
Figure 7:
Selective disruption of inducible complex
formation using anti-p65. A, nuclear extracts from
unstimulated (lanes 1 and 2) and TNF--stimulated (lanes 3 and 4) HUVECs were incubated with a 38-bp
radiolabeled probe (ICAM-1 -200 to -163) containing the
ICAM-1
B site. Lanes 2 and 4 contained a 50-fold
molar excess of unlabeled probe as a competitor. The inducible
complexes are indicated by arrows. B, competition of
the upper and lower inducible complexes with unlabeled 38-bp fragment (lanes 2-4), a mutant ICAM-1
B
38-bp fragment (containing the same
B site mutation as Fig. 6; lanes 5-7), and a 10-bp fragment
containing only the
B site (-187 to -178) (lanes
8-10). The molar excess of competitor over probe is
indicated above each lane. Lane 1 did not contain competitor. C, specific antisera to various members of the NF-
B/rel
family of transcription factors were tested: p65 (lane 2),
c-Rel (lane 3), RelB (lane 4), p50 (lane 5),
and p52 (lane 6). As controls, lane 1 did not contain
antibody and lane 7 contained c-fos antibody. D,
antisera to various members of the bZIP family of transcription factors
were tested: c-jun (lane 3), C/EBP
(lane
4), C/EBP
(lane 5), ATF-1 (which cross-reacts with
CREB-1 and CREM-1, lane 6), ATF-2 (lane 7), ATF-3 (lane 8), and CREB-2 (which cross-reacts with ATF-4, lane
9). Lane 1 did not contain antibody and anti-p65 was
included in lane 2 as a positive
control.
To determine which proteins were
responsible for the formation of the two inducible nuclear complexes,
we initially attempted to disrupt binding using specific antisera
directed against members of the NF-B/rel family (p65, p50, p52,
c-Rel, and RelB) (Fig. 7C). An antiserum against
c-fos was included as a control. Although the addition of
serum protein tended to diminish binding of the complexes in a
nonspecific manner, only the antiserum directed against p65 had a
dramatic effect on the complexes. It eliminated the formation of both
the upper and lower complexes. None of the other NF-
B/rel
antibodies affected the formation of inducible complexes in a
reproducible manner, although p50 and c-rel proteins and p100 (p52)
message have been detected in HUVECs(48, 49) .
NF-
B/rel proteins have recently been shown to interact with other
proteins, particularly members of the bZIP family of transcription
factors (50, 51, 52) . In order to
investigate the possibility that one of the complexes resulted from an
interaction between p65 and a non-rel protein, antisera to various bZIP
proteins were tested in EMSAs (Fig. 7D). None of the
antibodies tested, including those directed against c-jun,
C/EBP
, C/EBP
, ATF-1, ATF-2, ATF-3, and CREB-2, had any effect
on inducible complex formation. From these experiments, we concluded
that p65 was a component of both the upper and lower complexes, but we
were unable to identify other factors that might contribute to the
formation of the two complexes.
To further define the polypeptide composition of the inducible complexes, the proteins of the upper and lower complexes were UV-cross-linked to a BrdUrd-substituted 38-bp probe. For comparison, recombinant p65 (rp65) was included in the cross-linking experiments. Recombinant p65 produced a single retarded complex with a mobility similar to that of the upper complex when analyzed by EMSA (Fig. 8A), and like the upper complex, could be eliminated by addition of p65 antibody or unlabeled probe (data not shown). The polypeptides of the upper and lower complexes and rp65 were cross-linked to the probe by exposure to UV light, excised from the gel, and analyzed on a SDS-polyacrylamide gel (Fig. 8B). The upper complex and rp65 contained a single cross-linked polypeptide of approximately 75 kDa. The lower complex also contained a 75-kDa cross-linked polypeptide, as well as a smaller 59-60 kDa adduct. Based on the comparison of antibody cross-reactivity and cross-linking experiments with rp65, we concluded that the larger polypeptide present in both inducible complexes was p65. These results indicated that the upper complex was due to the binding of a p65 homodimer, and the lower complex was a heterodimer of p65 and another polypeptide approximately 15 kDa smaller than p65. Thus, the smaller polypeptide exhibited a molecular weight similar to that expected for p50.
Figure 8:
UV
cross-linking of the inducible complexes. A photoreactive 38-bp probe
was prepared as described under ``Materials and Methods.'' A, EMSA using the photoaffinity probe and nuclear extract
prepared from TNF- treated HUVECs (lane 1) and
recombinant p65 (lane 2). B, the upper and lower
complexes were UV-cross-linked to the probe, excised from the gel, and
subjected to SDS-PAGE. Molecular weights of the cross-linked products
were estimated by comparison with molecular weight
standards.
Since it was possible that the p50 antiserum
did not react strongly with the p50/p65 heterodimer in the EMSA binding
reaction, p50 or p65 was immunodepleted from nuclear extracts using
peptide-specific p50 or p65 antisera conjugated to agarose beads and
the depleted supernatant used in the EMSA binding reaction. Antibody
specificity was demonstrated by pretreating the antiserum with the
peptide to which the antiserum was raised or an irrelevant peptide (Fig. 9A). Immunodepletion of p50 specifically
eliminated the lower complex, an effect not observed when the antiserum
was preincubated with the p50 peptide. In contrast, the p65 peptide did
not block immunodepletion of the lower complex by anti-p50. As
expected, both the upper and lower complexes were eliminated when
nuclear extracts were depleted of p65. This effect was reversed by
pretreatment of the antiserum with p65 peptide, but not p50 peptide.
Immunodepletion of other NF-B/rel family members, i.e. c-rel, relB, and p52, had no effect on either of the inducible
complexes (data not shown).
Figure 9:
Immunochemical identification of p50 as a
component of the lower complex. A, immunodepletion of the
lower complex. Peptide-specific antibodies conjugated to agarose beads
were used to immunodeplete p50 or p65 from nuclear extracts prepared
from TNF--treated HUVECs. To demonstrate specificity, conjugated
antibody (25 µl) was treated with an excess of specific peptide to
which the antibody was raised, an irrelevant peptide, or PBS for 2 h at
room temperature. The conjugated antibodies were pelleted by
centrifugation and incubated with nuclear extract (88 µg) in the
presence of the appropriate peptide or PBS (50 µl total volume) for
4 h at 4 °C. The conjugated antibodies were then collected by
centrifugation and 10 µl of the immunodepleted supernatant analyzed
by EMSA. The antisera and peptides used are indicated at the top of the figure. Untreated nuclear extract was included in the EMSA
as an additional control (lane 4). B,
immunopurification of the lower complex. Nuclear extract prepared from
TNF-
-treated HUVECs (500 µg) was incubated with 60 µl of
anti-p50 conjugated to agarose beads for 4 h at 4 °C. The
conjugated antibody was collected by centrifugation and washed five
times with PBS. The protein retained on the conjugated antibody was
eluted using two sequential 2-h incubations with 100 µl of the p50
peptide. The elutates were combined, concentrated to a final volume of
10 µl using an Amicron Microcon Microconcentrator with a 30-kDa
molecular weight cutoff, and analyzed by EMSA using the 38-bp
photoaffinity probe (lane 2). Unfractionated nuclear extract
was included for comparison (lane 1). C, polypeptide
composition of the immunopurified complex. The immunopurified complex (lane 2) and lower complex from unfractionated nuclear extract (lane 1) were UV-cross-linked to the photoaffinity probe and
analyzed by SDS-PAGE as described under ``Materials and
Methods.''
Based on the results of the
immunodepletion experiment above, the lower complex from nuclear
extracts of TNF--treated HUVECs was immunopurified using
agarose-conjugated p50 antibody as an affinity matrix, and the bound
material eluted with p50 peptide. When analyzed by EMSA, the eluted
material yielded a single major retarded complex whose mobility was
similar to that of the lower complex (Fig. 9B). When
cross-linked to the photoaffinity probe and subjected to SDS-PAGE, the
immunopurified complex was found to contain similar cross-linked
polypeptides as the ``native'' lower complex from
unfractionated nuclear extracts (Fig. 9C). These
results clearly established that p50 was the smaller cross-linked
polypeptide of the lower complex. Therefore, we concluded that the
lower complex was a p65/p50 heterodimer bound to the NF-
B site.
Figure 10:
Functional effects of p65 and p50
overexpression. HUVECs were transfected with the indicated amounts of
p65 (pRc/CMV p65) or p50 (pRc/CMV p50) expression vectors, 0.5 µg
of luciferase reporter construct, 1 µg of pRSV-lacZ, and
pBR322 to a total of 2 µg of DNA/well. Cells were lysed 16 h after
transfection and assayed for luciferase and -galactosidase
activities. Luciferase activity was normalized to
-galactosidase
activity. Induction values were expressed as the ratio of normalized
luciferase activity in cells transfected with and without the indicated
expression vector. Experiments shown were representative of three
independent transfections carried out in triplicate. A,
reporter constructs: pGL 1.3 (filled bars) and pGL
1.3
B
(open bars). B, pGL2 Prom
38bpwt (filled bars) and pGL2 Prom 38bp
B
(open bars), described under ``Materials and
Methods.''
To determine the effect of overexpressing
both p65 and p50 in the same cell, we co-transfected p65 and p50
expression vectors into HUVECs along with the reporter pGL2 Prom
38bpwt. The amount of one expression vector was varied while the other
was held constant. In the first set of transfections, the concentration
of the p50 expression vector was held constant and increasing amounts
of pRc/CMV p65 or the empty expression vector, pRc/CMV, were
transfected into HUVECs (Fig. 11A). Increasing amounts
of the p65 expression vector transactivated the reporter construct in a
concentration-dependent manner, whereas the control expression vector
had no effect. In the converse experiment, the amount of the p65
expression vector was held constant and increasing amounts of control
or p50 expression vector were transfected into the cells. In addition,
an expression vector for IB
(pRc/CMV Mad3) was included as a
control (Fig. 11B). The control vector had no effect on
p65-mediated transactivation, whereas increasing amounts of p50 or
I
B
expression vector strongly suppressed transactivation.
Using the full-length construct, pGL 1.3, similar results were obtained
except that p50 could only partially suppress p65 activation at the
concentrations tested (data not shown). Thus, under no condition was
p50 found to augment transcription induced by p65.
Figure 11:
Functional effects of p65 and p50
coexpression. HUVECs were transfected with 0.5 µg of pGL2 Prom
38bpwt, 1 µg of pRSV-lacZ, 50 ng of one expression vector
(pRc/CMV p65 or pRc/CMV p50), and increasing amounts of the other (0,
50, 100, 200, and 400 ng), and pBR322 to bring the total amount of DNA
to 2 µg. Luciferase activity was determined and normalized to
-galactosidase activity. Values are given in normalized relative
light units (RLU). A, cells were transfected with 50
ng of pRc/CMV p50 alone, or with increasing amounts of pRc/CMV p65, or
the empty expression vector, pRc/CMV, as indicated. B, cells
were transfected with 50 ng of pRc/CMV 65 alone or with increasing
amounts of pRc/CMV, pRc/CMV p50, or the I
B
expression vector,
pRc/CMV MAD3, as indicated.
We began our studies on ICAM-1 transcriptional regulation by
determining the transcription start site of the ICAM-1 gene in HUVECs.
Primer extension analysis of total RNA isolated from TNF- and PMA
treated cells clearly demonstrated that transcription began 40 and 41
bp upstream from the start of translation. These two start sites were
25 and 24 base pairs downstream, respectively, of the proximal TATA box
identified earlier by others(15, 16) . We were unable
to locate the start site of basal transcription in unstimulated cells,
probably because basal ICAM-1 message levels were below the limit of
detection (14) . However, no alternative start sites were
detected, even under conditions known to stabilize ICAM-1 mRNA in
HUVECs(14) , such as treatment with PMA or cycloheximide, (
)suggesting that basal and induced transcripts may be
initiated from the same start site. Other studies on the
transcriptional start site(s) of the ICAM-1 gene have been published,
although they were conducted in other cell
types(15, 16, 17) . In human keratinocytes
and A431 cells, a human epidermoid squamous cell carcinoma line,
stimulated with IFN-
, ICAM-1 transcription was found to begin at
-39 and -40 relative to the start of
translation(15) . Voraberger et al.(16) found
two start sites in Hs913T fibrosarcoma cells at 319 and 41 bp upstream
from the translational start site. They also reported differential use
of these sites in A549 cells in response to TNF-
and PMA. ICAM-1
transcripts in unstimulated and TNF-
stimulated A549 cells were
initiated at -41, whereas PMA-induced transcription began at
-319. Another start site was detected at -84 in the Burkitt
lymphoma cell line Raji(17) . Therefore, basal and induced
ICAM-1 transcription have been reported to initiate at multiple sites
in different cell types, but we found no evidence for this in
endothelial cells.
In this study, we identified a single NF-B
site within a 92-bp responsive region that was essential for the ICAM-1
promoter to respond to TNF-
, IL-1
, PMA, and LPS in
endothelial cells. van de Stolpe et al.(20) recently
identified this site based on deletion analysis of the ICAM-1
5`-flanking sequence in human 293 embryonal kidney cells. Their
evidence suggested that this NF-
B site was largely responsible for
mediating PMA and TNF-
responsiveness of the ICAM-1 promoter in
this cell type. The ICAM-1 NF-
B site, TGGAAATTCC, deviates from
the published NF-
B consensus sequence of GGGRNNYYCC at the extreme
5` end of the sequence where the conserved guanine residue is replaced
by a thymine residue(46) . Although this type of substitution
within the NF-
B consensus sequence is uncommon, it has been
observed previously in the promoters of the urokinase, G-CSF, GM-CSF,
tissue factor, and IL-8
genes(53, 54, 55, 56, 57) .
It has been suggested that NF-
B sites differing at this base can
selectively recruit other members of the NF-
B/rel family besides
the classical p65/p50 heterodimer(58) .
Apart from an A to T
transition, the IL-8 NF-B site, TGGAATTTCC, is identical to the
ICAM-1 NF-
B site, and our studies on the ICAM-1
B site
parallel those obtained with the IL-8 promoter
B site. Both
elements bound p65 homodimers and p65/p50 heterodimers when nuclear
extracts from TNF-
-treated cells were analyzed by
EMSA(59, 60) . In co-transfection experiments, p65 and
p50 did not synergistically transactivate reporter constructs
containing the IL-8 or ICAM-1
B sites(59) . Rather, in
both cases, increasing amounts of p50 expression vector resulted in a
substantial decrease in p65-mediated transactivation. In contrast,
co-expression of p50 and p65 synergistically transactivated reporter
constructs containing the human immunodeficiency virus or IL-2 receptor
B sites, which do not deviate from the NF
B consensus
sequence(61, 62) . These results suggest that
activation of the IL-8 and ICAM-1 promoters through their respective
B sites depends primarily on p65 homodimers. This is further
supported by the finding that antisense oligonucleotides to p65, but
not p50, completely inhibited PMA-stimulated IL-8 production (59) .
The IL-8 B site was found to cooperatively
interact with an adjacent C/EBP site, resulting in the synergistic
activation of the IL-8 promoter(51, 60) . Considering
the high degree of similarity between the IL-8 and ICAM-1
B sites,
we anticipated that the potential C/EBP site adjacent to the ICAM-1
B site might also be important. EMSAs using C/EBP specific
antibodies and unlabeled competitors containing the ICAM-1 C/EBP site
did not suggest the involvement of C/EBP in inducible complex
formation. However, since C/EBP involvement in transcriptional
regulation may not be readily apparent in gel shift experiments, we
cannot rule out the possibility of a C/EBP/NF-
B interaction until
functional studies using a mutated C/EBP site are performed.
The
promoters for two other cytokine-inducible adhesion molecules, VCAM-1
and E-selectin, have been examined in
detail(21, 22, 23, 24, 25, 26, 63) .
Unlike the single essential NF-B site present in the ICAM-1
promoter, the VCAM-1 and E-selectin promoters contained two and three
critical
B sites, respectively. Mutation or deletion of these
essential
B elements rendered their respective promoters
unresponsive to cytokine stimulation in endothelial cells. The five
B sites from these two promoters have different decameric
sequences, but all five possess the characteristic triplet of guanine
residues present at the 5` end of the NF-
B consensus sequence. In
contrast to ICAM-1 reporter constructs, co-expression of p65 and p50
augmented rather than inhibited transactivation of the E-selectin and
VCAM-1 reporter constructs(48, 49, 64) ,
providing functional evidence that p65/p50 heterodimers may be involved
in the regulation of these two promoters. Therefore, all three adhesion
molecule promoters critically rely on the presence of NF-
B sites,
but the ICAM-1 gene differs from the E-selectin and VCAM-1 genes, in
that it appears to be selectively activated by p65 homodimers.
Considering the pathophysiological importance of ICAM-1, E-selectin,
and VCAM-1 in the progression of inflammatory responses, these adhesion
proteins have become attractive therapeutic targets(3) .
Despite an enormous effort to identify small molecule inhibitors of
cell adhesion, little progress has been made in this area. One
relatively untapped aspect of cellular adhesion is the transcriptional
regulation of these inducible genes. Dissection of the ICAM-1,
E-selectin, and VCAM-1 promoters has identified specific elements that
confer responsiveness to inflammatory cytokines in endothelial cells.
Although cytokine up-regulation of each promoter is distinct, all three
promoters depend on the presence of upstream NF-B sites,
suggesting that selective inhibitors of NF-
B might be particularly
useful in the treatment of acute and chronic inflammatory diseases.
In summary, our findings indicate that TNF--induced
transcription of the ICAM-1 gene in endothelial cells is directed
exclusively from the proximal TATA box and critically depends on a
single atypical NF-
B located 187 bp upstream of the transcription
start site. Transcriptional activation by IL-1
and LPS, and to a
lesser extent PMA, is also mediated through this element. Even though
endogenous p65 homodimers and p65/p50 heterodimers bind to the ICAM-1
B site, functional studies implicate the p65 homodimer as the key
transactivator working through this site on the ICAM-1 promoter.