(Received for publication, August 8, 1996, and in revised form, March 10, 1997)
From the Emory Skin Diseases Research Core Center,
Department of Dermatology, Emory University School of Medicine,
Atlanta, Georgia 30322, the § Graduate Division of
Biological and Biomedical Sciences, Emory University, Atlanta, Georgia
30322, the ¶ Department of Dermatology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104, and the
Cancer Institute Chemotherapy Center, 1-37-1, Kami-Ikebukuro,
Toshima-ku 170 Japan
The regulated expression of intercellular
adhesion molecule-1 (ICAM-1) by cytokines such as tumor necrosis factor
(TNF-
) plays an important role in inflammation and immune
responses. Induction of ICAM-1 gene transcription by TNF-
has
previously been shown to be dependent upon a region of the ICAM-1
5
-flanking sequences that contains a modified
B site. We
demonstrate here that this modified
B site alone is insufficient for
induction of transcription by TNF-
. Site-directed mutagenesis of
both the
B site and specific flanking nucleotides demonstrates that
both the specific 5
- and 3
-flanking sequences and the modified
B site are necessary for TNF-
induction. Further, site-directed mutagenesis of this modified
B site to a consensus
B site allows it to mediate transcriptional activation in response to TNF-
, even
in the absence of specific flanking sequences. Transcription through
this minimal ICAM-1 TNF-
-responsive region can be driven by
co-expression of p65, and the minimal response element interacts with
p65 and p50 in supershift mobility shift assays. However, when in
vitro transcription/translation products for the Rel proteins are
used in an electrophoretic mobility shift assay, only p65 is capable of
binding the minimal response element while both p50 and p65 bind a
consensus
B oligonucleotide. Additionally, in the absence of the
specific flanking nucleotides, the ICAM-1
B site is incapable of
DNA-protein complex formation in both electrophoretic mobility shift
assay and UV cross-linking/SDS-polyacrylamide gel electrophoresis
analysis. These results demonstrate the requirement for specific
flanking sequences surrounding a
B binding site for functional
transcription factor binding and transactivation and TNF-
-mediated
induction of ICAM-1.
Intercellular adhesion molecule-1
(ICAM-1)1 is a cell surface glycoprotein
and member of the immunoglobulin superfamily (1, 2) As the
counter-receptor for the leukocyte 2 integrins, ICAM-1 plays a
central role in a number of inflammatory and immune responses. Although
ICAM-1 is constitutively expressed on a variety of cell types,
including hematopoietic cells, fibroblasts, and vascular endothelium
(3), its regulated expression is fundamental to leukocyte trafficking.
Up-regulated ICAM-1 expression on cytokine-activated vascular
endothelial cells controls the targeted transmigration of leukocytes
into specific areas of inflammation. In addition, ICAM-1 can be induced
on some cells types in which it is not constitutively expressed
(e.g. human keratinocytes (4)), further fine tuning the
localized inflammatory reaction once leukocyte transmigration has
occurred.
ICAM-1 expression can be induced by a variety of cytokines, including
interferon- (IFN-
), tumor necrosis factor-
(TNF-
), and
interleukin-1, as well as bacterial lipopolysaccharide (5-8). Both
IFN-
and TNF-
have been shown to mediate this induction at the
level of transcription (5, 9). The molecular events underlying the
transcriptional activation of the ICAM-1 gene in response to TNF-
stimulation are not fully understood. In the up-regulation of other
cellular adhesion molecules by TNF-
, it has been shown that TNF-
induces expression by activation of members of the NF-
B (Rel) family
of transcription factors (10). There are five known members of the Rel
family, NF-
B1 (p50), NF-
B2 (p52), RelA (p65), c-Rel, and RelB
(11). The first NF-
B complex described consisted of p65, p50, and
the inhibitor I
B-
pre-existent in the cytoplasm. Upon stimulation
with TNF-
, I
B-
is degraded, unmasking nuclear translocation
signals on p65 and p50. These proteins translocate to the nucleus, bind
to DNA as a heterodimer (p65/p50) through a decameric consensus
sequence (GGGRNNYYCC), and mediate transactivation (10, 12, 13). Since
this original description, individual subunits of the NF-
B complex
have been shown to regulate transcriptional activity as homodimers or
as heterodimers with other members of the Rel family. Specifically,
both p50 and p65 homodimers, as well as heterodimers with other members
of the Rel family, are capable of modulating transcription. Further,
this activation requires a DNA binding sequence that is specific for
the particular dimer (9), and binding of both subunits is required for
activation (14).
While a B site has been implicated in induction of ICAM-1 gene
expression by TNF-
(11, 15, 16), it is now apparent that this
transcriptional regulatory process is more complex than initially
described. By deletional and mutational analysis, we show that a
variant
B site plus the 5
- and 3
-flanking regions are necessary
and sufficient for induction of ICAM-1 by TNF-
, demonstrating the
requirement of specific flanking nucleotides to a
B site. Thus,
these specific flanking sequences may be necessary for stabilizing Rel
protein binding to this sub-optimal
B site. Further, these data
implicate DNA sequences surrounding nonconsensus
B sites in
directing specific Rel family member binding and may, thus, participate
in directing gene specific transactivation.
C32 melanoma cells (ATCC) were cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 3 mM L-glutamine, 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, 0.25 mg/ml amphotericin B, and 10 µg/ml streptomycin (all from Life
Technologies, Inc.). As described previously (17, 18), human dermal
microvascular endothelial cells (HDMEC) were isolated from neonatal
foreskins and cultured in MCDB131 (Life Technologies, Inc.)
supplemented with 10% normal human serum (Irvine Scientific),
dibutyryl cAMP (5 × 105 M, Sigma), 100 units/ml penicillin, 0.25 mg/ml amphotericin B, and 10 µg/ml
streptomycin (all from Life Technologies, Inc.). Cultures were
maintained at 37 °C in humidified 5% CO2. Experiments with HDMEC were conducted with cells in passage 3-5.
ICAM-1 based heterologous promoter/reporter plasmids were
designed and constructed using techniques and strategies as described previously (19). Briefly, various portions of the ICAM-1 5-flanking region were isolated either by appropriate restriction enzyme digestion
of the ICAM-1 genomic sub-clone pG
6-2.05 (20), by polymerase chain
reaction using primers that incorporated convenient restriction sites
at their 5
ends and were specific for areas of interest within the
ICAM-1 gene or, for smaller fragments, by annealing of complementary
synthesized oligonucleotides (Emory University Microchemical Facility)
spanning areas of interest and incorporating convenient restriction
sites at either end of the double-stranded fragment. All fragments are
designated by their nucleotide location with respect to the identified
transcription initiation site (20). Purified fragments were ligated and
cloned into pBRAMScat2 (21), which contains the CAT reporter gene under the control of the Herpes simplex virus minimal thymidine kinase (TK)
promoter and multiple cloning sites designed for analysis of eukaryotic
enhancers. Single and multiple base mutations within the region of
interest were generated within complementary synthesized oligonucleotides and ligated into pBRAMScat2. Inserts and ligation sites of all vectors generated from either polymerase chain
reaction-generated or oligonucleotide synthesized fragments were
confirmed by sequence analysis via the dideoxynucleotide chain
termination method using the Sequenase 2.0 Quick-Denature Kit (U. S.
Biochemical Corp.).
Site-directed mutagenesis was used to modify the ICAM-1 constructs
199/
170 TK-CAT and
186/
177 TK-CAT such that they contained a
consensus
B site. To generate these point mutants, the constructs were transformed into competent CJ236 Escherichia coli
(prepared according to the method of Chung et al. (22)) and
single-stranded uracil-containing DNA generated by infection with
M13K07 helper phage (Bio-Rad) followed by isolation and precipitation
of DNA. Mutagenic oligonucleotides were 5
phosphorylated by T4
polynucleotide kinase (45 min, 37 °C), and the kinase was
inactivated by incubation at 65 °C for 10 min followed by the
addition of TE buffer (10 mM Tris, 10 mM EDTA)
to a concentration of 6 pmol/µl. Single-stranded DNA (400 ng) and
mutagenic oligonucleotide (6 pmol) were annealed. The mutated DNA
strand was then synthesized and ligated (0.5 units T7 DNA polymerase, 3 units T4 DNA ligase, 0.4 µM each dNTP, 0.75 µM ATP, 17.5 µM Tris-HCl, pH 7.4, 3.75 µM MgCl2, 1.5 µM DTT) on ice
for 5 min, 25 °C for 5 min, 37 °C for 30 min, followed by the
addition of 90 µl of TE buffer (10 mM Tris, 10 mM EDTA). The resultant double-stranded product was then
transformed into uracil-deficient competent XL1blue E. coli
(Stratagene). Colonies were screened by dideoxy sequencing using the
Sequence 2.0 Quick-Denature Kit (U. S. Biochemical Corp.). Mutagenic
oligonucleotides used were the following (mutations are indicated in
bold, underlined letters): for
199/
170 TK-CAT,
5
-GATCCAGCTCGGGAATTTCC-3
, and for
186/
177 TK-CAT,
5
-GGGCCGGATCGGGAATTTCCG-3
.
The p65 expression vector used in cotransfection studies was developed
and generously provided by Dr. Craig Rosen (23, 24). The expression
vector for I-rel (RelB) was provided by Dr. Charles Kunsch (25). The
expression vectors for RC-CMV p65, pcDNAI p50, pcDNAI ATF2, and
pcDNAI c-Jun used for in vitro transcription/translation were provided by Dr. Dmitris Thanos (26). The expression vector pSV3K-IB
was provided by Dr. John Hiscott (27). Expression vectors for MSV-C/EBP
, MSV-C/EBP
, and MSV-C/EBP
were provided by Dr. Steven McKnight (28). Plasmid DNA preparation for transfection and transcription/translation was done using the Qiagen DNA Maxiprep Kit (Qiagen).
For EMSA, a full-length forward oligonucleotide and 10-base pair
reverse primer were annealed, labeled with Klenow (37 °C, 30 min,
100 µCi 32P-dCTP (DuPont), 250 µM dNTPs
(Perkin-Elmer)), and purified by spinning through G25 Sephadex
(Boehringer Mannheim). Oligonucleotides were synthesized by the Emory
University Microchemical Facility. Cold double-stranded DNA was made
identically, except that unlabeled dCTP was substituted for labeled
dCTP and the final oligonucleotide concentration was 100-fold higher.
The oligonucleotides used as probes and cold competition, with
B
sites underlined and primer sites double underlined, were as follows:
ICAM-1
B with ICAM-1 flanking DNA (IC/IC, representing the region
191/
172 upstream of the transcription start site),
5
-TTAGCTTGGAAATTCCGGAGCTCGAGATCCTATG-3
; ICAM-1
B with random flanking sequences (IC/R),
5
-AACCAAAGGAAATTCCGTTAGATCCTATG-3
; consensus
B with ICAM-1 flanking sequences (C/IC),
5
-TTAGCTTGGAAATTCCCGAGCTCGAGATCCTATG-3
; consensus
B with random flanking sequences (C/R),
5
-AACCAAAGGAAATTCCCTTAGATCCTATG-3
(as
shown schematically in Fig. 4A).
Transfections and CAT Assays
Subconfluent C32 cell cultures
(5 × 105 cells/100-mm tissue culture dish) were
transfected with 15 µg of plasmid DNA by the calcium phosphate
precipitation technique (29). After exposure to precipitated plasmids
for 16 h, cells were washed and medium replenished. At 48 h
post-transfection, cells were left untreated or treated with TNF-
(300 units/ml, R&D Systems) for an additional 16 h. Cells were
then harvested and lysates prepared as described previously (30).
Assays for transfection efficiency, normalization of protein, and CAT
expression were performed as described previously (19, 20). HDMEC were
transfected with 20 µg of plasmid DNA (plus 2 µg of p65 expression
vector for cotransfection studies) in the presence of 500 ng/ml
DEAE-dextran (Sigma) at 85% confluence for 30 min at 37 °C, 5%
CO2. After 30 min, medium containing 8 µM
chloroquine was added, and the transfection was continued for an
additional 2 h. After exposure to plasmids for 2.5 h, the
medium was replenished. At 24 h post-transfection, cells were left
untreated or treated with TNF-
(300 units/ml) for an additional
16 h. Cells were then harvested, and lysates were prepared as
above.
Nuclear extracts were prepared
as described previously by Schreiber et al. (31) with
modification by Stahl et al. (32). Briefly, after treatment
with TNF- (300 units/ml), cells were harvested and washed two times
with ice-cold PBS. They were then washed with 400 µl of buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM
MgCl2, 1 mM DTT, 0.1 mM PMSF, 2 µg/ml aprotinin) and incubated on ice for 10 min with 400 µl of
buffer A supplemented with 0.1% Nonidet P-40. Nuclei were pelleted by
centrifugation (4000 rpm, 2 min), the supernatant was discarded, and
the pellet was rinsed in 400 µl of buffer A without Nonidet P-40.
Nuclei were resuspended in 150 µl of buffer C (50 mM
HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF,
10% glycerol, 2.2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml
pepstatin A) and incubated on ice for 30 min. Extracts were harvested
by centrifugation (14,000 rpm, 15 min, 4 °C). The supernatant was
collected and stored at
70 °C for protein assay and EMSA.
Proteins for the
transacting factors p50, p65, RelB, c-Jun, ATF2, C/EBP, C/EBP
,
and C/EBP
, and the Rel inhibitory protein I
B-
were generated
by in vitro transcription/translation of the mammalian
expression vectors described above in the T7 Coupled TNT Rabbit
Reticulocyte Lysate System (Promega) according to the manufacturer
protocol. L-[35S]methionine labeling and
autoradiography or Western blotting were used to verify the integrity
of the proteins generated (data not shown). For EMSA and UV
cross-linking/SDS-PAGE analysis, 2 µl of a 50-µl
transcription/translation reaction was used per condition shown.
The method for EMSA has been described previously (33,
34). In brief, the binding reactions were performed with 5 µg of nuclear extract in binding buffer (12% glycerol, 12 mM
HEPES, pH 7.9, 4 mM Tris-Cl, pH 7.9, 60 mM KCl,
1 mM EDTA, 1 mM DTT, 1 mM PMSF)
containing 2 µg of poly(dI-dC) and 5 µg of bovine serum albumin in
a final volume of 20 µl. After a 15-min incubation at room
temperature, 1 × 105 cpm of radiolabeled probe was
added for an additional incubation period of 30 min. For the
competition assays and supershift experiments, an excess of the
appropriate unlabeled oligonucleotide (50 ng) or antibody (1 µg),
respectively, was added to the binding reaction mixture prior to the
initial 15-min incubation. The samples were electrophoresed at 130 V at
4 °C through 6% native polyacrylamide gels. Gels were dried, and
autoradiography was performed at 70 °C for 1-3 days.
Autoradiographs were scanned on a La Cie scanner (La Cie, Ltd.)
utilizing Adobe Photoshop software (Adobe Systems, Inc.). The digitized
image was subsequently labeled in Microsoft Power Point (Microsoft
Corp.) and printed on a high resolution laser printer. Each figure
represents a computer-generated image of the autoradiograph, and each
is typical of the autoradiograph in the context of relative band and
background densities.
For UV cross-linking/SDS-PAGE
analysis, probes and competitor oligonucleotides were prepared as for
EMSA but substituting 3 mM bromodeoxyuridine (BrdUrd,
Sigma) for dTTP. Binding reactions were performed with 10 µg of
nuclear extract in EMSA binding buffer containing 2 µg of poly(dI-dC)
and 5 µg of bovine serum albumin in a final volume of 20 µl in a
96-well plate. After 15 min of incubation at room temperature, 1 × 105 cpm of radiolabeled BrdUrd-containing probe was
added for an additional incubation period of 30 min. For the
competition assays, an excess of the appropriate unlabeled
oligonucleotide (10-100-fold molar excess) was added to the binding
reaction mixture prior to the initial 15-min incubation. The samples
were exposed to UV irradiation in a Stratalinker (240 nm, 15 min, on
ice). The binding reactions were combined with 5 µl of 6 × SDS-PAGE loading buffer, boiled for 2 min, loaded onto 10% SDS-PAGE,
and electrophoresed until the dye front reached the bottom of the gel.
Gels were dried, and autoradiography was performed at 70 °C for
1-3 days. To demonstrate specificity, after UV cross-linking, excess
DNA was removed by incubation in 0.25 M CaCl2,
0.3 µg of micrococcal nuclease, and 0.05 units of DNase I at 37 °C
for 30 min prior to addition of SDS-PAGE loading buffer.
Our previous work has focused on the
identification and characterization of the ICAM-1 5 regulatory region
(20). Using ICAM-1-based 5
deletion CAT reporter gene constructs, we
observed that inducibility by TNF-
in C32 cells is conferred by DNA
downstream of position
199 (data not shown). These data are in
agreement with the results of others (11, 15, 16). Further, a construct with a 5
deletion to
182 retained no TNF-
inducibility (data not
shown). These results demonstrate that the region below
199 and
perhaps surrounding
182 is necessary for TNF-
induction of ICAM-1
expression.
Sequence analysis of the ICAM-1 199/
170 promoter region identifies
a modified decameric
B site between positions
186 and
177, on
the coding strand in reverse orientation. This sequence differs from
the traditional
B consensus sequence in that it contains a p65
binding site but not the requisite GGG for p50 binding. To test whether
the region bounded by
199/
182 or a region extending further 3
(
199/
170) is sufficient to confer TNF-
inducibility upon a
heterologous promoter, we cloned these fragments into a thymidine
kinase enhancer trap expression vector (19, 21) and assayed their
responsiveness to TNF-
in C32 melanoma cells. Fig.
1A demonstrates that the region bounded by
199/
170 is sufficient to confer responsiveness to TNF-
, but the
region
199/
182 does not confer TNF-
inducibility. Since the
199/
182 construct splits the modified
B site, it is not surprising that it has no ability to respond to TNF-
(Fig.
1A). Interestingly, the construct containing the modified
B site alone (
186/
177) shows no induction by TNF-
(Fig.
1A) while the construct
191/
172 retains TNF-
responsiveness (although to a lower magnitude than
199/
170 because
of its loss of a 5
C/EBP binding site necessary for maximal induction
(35)).
Mutation Analysis of the Minimal ICAM-1 TNF-
Since these results suggest that specific flanking
sequences surrounding the modified B site are necessary for TNF-
induction of transcription through this element, a series of single
base pair mutations within the modified
B site and the surrounding flanking regions were made. As shown in Fig. 1B, mutations
affecting the decameric
B site abolish TNF-
induction of CAT
activity and partially repress constitutive CAT activity. Most
interestingly, however, mutations affecting either the 5
- or
3
-flanking regions also significantly abrogate or abolish
TNF-
-induced CAT activity. These results demonstrate that these
specific flanking sequences are necessary for TNF-
induction of
ICAM-1 gene expression. Further, these specific flanking sequences,
together with the modified
B site, represent the total sequence that
is necessary and sufficient for induction of ICAM-1 expression by
TNF-
. We have demonstrated similar results in HDMEC for both the
minimal region and precise nucleotide sequence (data not shown),
indicating that these results are not cell-type specific and that
induction of ICAM-1 by TNF-
within the context of endothelial cells
also requires these flanking regions.
To test whether the wild-type
ICAM-1 modified B site, which deviates from a consensus
B site by
a single G
C transition, is unable to drive transcriptional
activation in response to TNF-
because of this single nucleotide
difference, we undertook site-directed mutagenesis of this base in the
heterologous promoter constructs
186/
177 TK-CAT and
199/
170
TK-CAT. We then tested the ability of these constructs transfected into
HDMEC to respond to either TNF-
or p65 cotransfection. Fig.
2 shows that the wild-type
186/
177 TK construct does
not respond to either TNF-
or p65 cotransfection. However, when a
single base C
G mutation is created at position
177, the resulting
186/
177 Cmut TK-CAT construct, which converts the wild-type
decameric ICAM-1 modified
B site to a consensus
B site, is fully
capable of responding to TNF-
and, at a somewhat lesser extent than
the wild-type construct, to p65 cotransfection. Further, when the same
change is made within the context of the
199/
170 construct that is
maximally inducible by TNF-
, TNF-
responsiveness is maintained,
but induction by p65 cotransfection is also decreased in comparison
with the wild-type construct. These data support a transactivation
pathway with the wild-type ICAM-1 TNF-
-response region that is
optimally driven by p65 homodimers, and conversion of the wild-type
ICAM-1 decameric
B site to a consensus
B site results in an
element that likely is best driven by p50/p65 heterodimers. These
results are also supported by those of Ledebur and Parks (11) who
demonstrate that the cotransfection of increasing amounts of p50
expression vector at a constant level of p65 expression vector and a
wild-type ICAM-1 TNF-
-responsive region CAT construct results in
reduced levels of transactivation in comparison to p65 expression
vector alone. We, too, have observed a suppressive effect with p50
expression vector cotransfection when assaying expression by a fully
responsive wild-type ICAM-1 TNF-
-responsive region construct
(
199/
170 TK CAT) after treatment with TNF-
or joint
cotransfection with a p65 expression vector. Like other consensus
B
elements (such as that in the HIV
B site), our consensus
B
construct (
186/
177 Cmut TK-CAT) is less responsive to p65
cotransfection alone and is probably driven optimally by a combination
of p65 and p50. What appears to be unique concerning transactivation
via the full ICAM-1 TNF-
-responsive region is that specific flanking
regions surrounding the wild-type decameric modified
B site are
required in order for TNF-
or p65 to drive expression, whereas
conversion to a consensus
B site by mutation of a single base
removes the necessity of these specific flanking sequences.
The ICAM-1 TNF-
To
demonstrate the formation of a specific DNA-protein complex, the newly
identified TNF--response element (
191/
172) was used in an EMSA.
Two DNA-protein complexes were formed when labeled probe was incubated
with nuclear extracts from TNF-
-treated HDMEC (Fig.
3, bottom complexes). The specificity of
these complexes is demonstrated by their competition with excess cold
identical oligonucleotide but not with excess cold irrelevant
oligonucleotide (data not shown). Both anti-p65 antibody (Fig. 3,
lane 5) and anti-p50 antibody (lane 3) supershift
these complexes, and irrelevant antibody (lane 8) and
antibodies to c-Rel, RelB, and p52 (lanes 4, 6, 7) have no
effect. A similar set of results was obtained using untreated or
TNF-
-treated nuclear extracts from HUVEC and C32 melanoma cells
(data not shown). These results show that a specific DNA-protein
complex is formed between TNF-
-treated HDMEC, HUVEC, or C32 nuclear
extract proteins NF-
B p65 and NF-
B p50 and the identified
TNF-
-response element (
191/
172) of the ICAM-1 gene. Thus,
although cotransfection data by us and others (11) strongly suggest
that p65 alone best drives expression through the wild-type ICAM-1
TNF-
-responsive region and that p50 serves to inhibit expression
through this element, these in vitro binding data indicate
that both p65 and p50 can bind to the complete TNF-
-responsive region.
To further explore the ability of Rel proteins to bind both the ICAM-1
TNF--responsive region and the comparable consensus probe, we
generated in vitro transcription/translation products for
p50, p65, RelB, and p52, the non-Rel TNF-
-inducible transcription factors ATF2 and c-Jun, the Rel inhibitor molecule I
B-
, and the
high mobility group protein HMG-I(Y). We then used these proteins in
EMSA and UV cross-linking/SDS-PAGE studies. When the wild-type ICAM-1
TNF-
-responsive region (
191/
172) was used as a labeled probe in
an EMSA, HDMEC nuclear extracts showed two inducible bands when cells
had been treated with TNF-
(Fig. 4A,
lane 2). These bands are present in low amounts in untreated
cells (lane 1) and are specifically competed by the addition
of excess unlabeled DNA (lane 3). In addition, these
base-line complexes, as well as TNF-
-induced complexes, are competed
by the addition of in vitro-generated I
B-
(lane
4). Of all of the transcription factor proteins generated, only
p65 is capable of forming a distinct DNA-protein complex (lane
6) with the wild-type ICAM-1 TNF-
-responsive region, and
binding of in vitro-generated p65 is also inhibited by
addition of I
B-
(lanes 13 and 14). All
other in vitro generated proteins show essentially base-line
complex levels formed by the apparent presence of background amounts of
a p65-like protein in the rabbit reticulocyte lysate, as indicated by
the ability of I
B-
to inhibit formation of this basal complex
either alone (lane 17) or when added with other proteins
(lanes 12-14). Furthermore, addition of rabbit reticulocyte
lysate alone forms the same basal complex that is also competed away
with the addition of in vitro generated I
B-
. When a
consensus
B site with ICAM-1 flanking DNA sequence (C/IC as
described under "Materials and Methods"), in which the wild-type
ICAM-1 modified
B site is mutated to a consensus
B site by a
single C
G transition, is used as the radiolabeled probe,
TNF-
-induced HDMEC nuclear extracts demonstrate inducible complex
formation (Fig. 4B, lane 2; see further
discussion below) in comparison with untreated cells (lane
1). These complexes are specific and contain Rel proteins as
indicated by their competition by excess unlabeled DNA (lane
3) and the addition of in vitro-generated I
B-
(lane 4). However, unlike the wild-type ICAM-1 probe
(panel A), the consensus
B probe is capable of binding
both p50 (panel B, lanes 5 and 7) and
p65 (panel B, lanes 6 and 7). Like the
wild-type ICAM-1 site, the consensus
B probe does not bind other Rel
family members or other TNF-
-inducible transcription factors. Again, I
B-
alone (lane 17) or in combination with other
proteins (lanes 12-14) inhibits p65 present in the
reticulocyte lysate from forming DNA-protein complexes. In combination
with the p65 cotransfection data and EMSA supershift analysis, these
data strongly suggest that complex formation is dependent upon the
ability of NF-
B, particularly p65, to bind to DNA. They further
suggest that the wild-type ICAM-1 TNF-
-responsive region
preferentially binds a p65 homodimer while a consensus
B site,
created by a single base mutation, binds both p50 and p65
(i.e. a classical NF-
B complex).
To explore further the requirement of the
ICAM-1 modified B site for specific flanking sequences, we used a
series of oligonucleotide probes, as detailed under "Materials and
Methods" and shown schematically in Fig.
5A. These oligonucleotides contained the
ICAM-1
B with ICAM-1 flanking sequences (IC/IC), a consensus
B
with ICAM-1 flanking sequences (C/IC), the ICAM-1
B with random
flanking sequences (IC/R), or a consensus
B with random flanking
sequences (C/R). The sequences for the random flanking nucleotides used in these studies were designed to include a maximum number of mutations
relative to the wild-type ICAM-1 flanking sequences. Similar results
have been obtained with two additional sets of arbitrarily random
flanking sequences (data not shown). When each of these four
oligonucleotides was labeled and used as a probe in an EMSA, only IC/R
failed to form any TNF-
-induced complexes (Fig. 5B,
lanes 4-6). In addition, although IC/IC and the two consensus oligonucleotides (C/IC and C/R) formed TNF-
-induced complexes, the composition of complexes formed with the wild-type ICAM-1 modified
B sequence versus the consensus
B
sequence was different, as indicated by their different mobilities
(Fig. 5B, lanes 1-3 versus lanes 7-9 and
10-12, arrows). These differences in complex
mobility cannot be attributed to differences in oligonucleotide size.
Indeed, the IC/IC and C/IC probes are identical in length and differ in
sequence only by a single nucleotide. Furthermore, complex mobility is
identical with both consensus
B oligonucleotides (C/IC and C/R)
though the C/R probe is five nucleotides shorter. These results show
that the ICAM-1 specific flanking sequences are required for
DNA-protein complex formation to occur. They also suggest that the
nature of the DNA-protein complexes that form with the ICAM-1
B site
differs from that which binds to the consensus
B site.
To study the nature of the proteins that interact with these
oligonucleotides, each of these four oligonucleotides was labeled and
used as probe in UV cross-linking/SDS-PAGE. Similar to our EMSA
results, while the ICAM-1 wild-type (IC/IC) and B consensus (C/IC
and C/R) oligonucleotides all demonstrate cross-linking of specific
proteins, the IC/R oligonucleotide is incapable of cross-linking any
proteins (Fig. 5C, lanes 4-6). Although the exposure shown here is light, all DNA-protein bands present using the
consensus probes (lanes 7-9 and 10-12) are also
seen with the ICAM-1 wild-type sequence, IC/IC (lanes 1-3).
All detected bands are also specific, as shown by incubation of the
samples with a mixture of micrococcal nuclease/DNase I after UV
cross-linking and before addition of SDS-PAGE loading buffer (data not
shown).
Coupled with our reporter assays, these analyses of TNF--induced
DNA-protein complex formation indicate that specific flanking sequences
are required for the ICAM-1
B site to be biologically functional for
both transcription factor optimal binding and activation of
transcription. However, these flanking sequences that are required for
the ICAM-1 modified
B site to be functionally active become irrelevant when the
B site is changed by one nucleotide to form a
classic consensus
B site. Our data also support a model in which
specific flanking sequences, coupled to the wild-type ICAM-1 modified
B site, preferentially form a binding site for p65 homodimers, with
minimal involvement of p50, while a consensus
B site binds, regardless of flanking sequences, traditional p65/p50 heterodimer (i.e. NF-
B) complexes.
The present study identifies the TNF--responsive region of the
ICAM-1 gene as
191/
172 upstream of the transcription start site by
deletional mutation of the ICAM-1 5
-flanking region. Previous studies
(11, 15, 16) have also identified this region and have further
suggested that a modified
B site contained within the region is
sufficient for up-regulation of ICAM-1 expression, as has been shown
for the TNF-
responsiveness of other adhesion molecules (36-39).
Through mutational analysis, cotransfection studies, supershift EMSA,
and in vitro transcription/translation, our results confirm
the importance of this modified
B site and demonstrate the
involvement of p65 in the DNA binding complex. However, in addition to
previous studies, we find that specific 5
- and 3
-flanking sequences
are necessary for TNF-
induction and that the wild-type ICAM-1
decameric modified
B site alone is insufficient. Further, conversion
of the wild-type ICAM-1 decameric modified
B site to a consensus
B site results in restoration of the ability of the 10-base pair
B site alone to respond to TNF-
but decreases its ability to
respond to p65 cotransfection. The requirement of these specific
B
flanking regions for TNF-
responsiveness represents a novel finding
for
B-mediated gene transactivation.
The NF-B proteins p65 and p50, first described in the
transcriptional activation of the B cell
immunoglobulin gene, form the classical NF-
B complex that binds to the consensus sequence GGGRNNYYCC (10). In the promoter of the ICAM-1 gene, however, the
identified TNF-
-response element contains a modified
B sequence CGGAATTTCC, differing at the first position by a G
C transition. This
transition, although relatively uncommon, has also been shown to occur
in the promoters of GM-CSF, G-CSF, tissue factor, urokinase, and
interleukin-8 genes (40-44). It has been suggested that this modified
sequence is capable of selectively binding members of the Rel family
other than the classical p65/p50 heterodimer (45). Previous
investigators have shown that one complex forming with this ICAM-1
region on EMSA consists of either p65 homodimers (11) or p65/c-Rel
heterodimers (15), whereas the other complex consists of the classical
p65/p50 heterodimers (11, 15). The present study confirms the
preferential involvement of p65, and to a lesser extent p50, in these
in vitro complexes by EMSA but fails to demonstrate involvement of c-Rel in either complex using lysates from C32 melanoma
cells, HDMEC, and HUVEC. In addition, only p65 in vitro transcription/translation product, and not other Rel protein products including p50, is capable of binding the ICAM-1 minimal
TNF-
-responsive region. Further, the ability of p65 co-expression to
drive transcriptional activation through the TNF-
-response element
supports the involvement of p65 homodimers in TNF-
-induced
transcriptional activation.
Our results demonstrate that the molecular requirements for induction
of ICAM-1 gene expression by TNF- are more complex than initially
described (11, 15, 16). Ledebur and Parks (11) described a
TNF-
-responsive region of
227 to
136, while both van de Stolpe
et al. (16) and Jahnke and Johnson (15) further limit the
TNF-
-responsive region to
227 to
173 in transcriptional activation studies (CAT assays). All three groups proceed to show that
a shorter fragment (
200 to
163 (11) or
190/
173 (15, 16)) is
capable of forming protein-DNA complexes on EMSA, but they do not
address the ability of these shorter fragments to activate
transcription. Most interestingly, when van de Stolpe et al.
(16) used a shorter fragment (
189/
174) containing the wild-type
modified
B site but deleting portions of the flanking sequences in
transcriptional activation studies, these investigators could not
demonstrate any TNF-
-inducible increase in reporter gene expression
(16). However, an engineered construct containing a tri-tandem repeat
of the wild-type ICAM-1
B site was shown to display TNF-
inducibility (16). Relevant to our present studies, however, this
engineered tri-tandem construct coincidentally restored the wild-type
specific flanking sequences around the central repeated decameric
element, though the contribution of these specific sequences was not
further explored (16).
These data, as well as our own initial reporter gene analyses,
certainly suggested that the wild-type modified B site alone may be
capable of forming complexes with NF-
B proteins in TNF-
-activated nuclear extracts. However, our subsequent data definitively show that,
in the absence of the specific ICAM-1 flanking sequences, the ICAM-1
modified
B site is incapable of forming DNA-protein complexes (EMSA)
or binding any proteins (UV cross-linking/SDS-PAGE). Significantly,
conversion of this ICAM-1 modified
B site to a consensus decameric
B site restores its ability to mediate transactivation in response
to TNF-
, to form DNA-protein complexes even in the absence of any
specific flanking DNA sequence, and to bind p50 protein in addition to
p65.
The data demonstrating the involvement of specific flanking sequences
for the ICAM-1 modified B site in TNF-
-induced ICAM-1 transcriptional activation suggested a potential involvement of additional nuclear proteins or transcription factors. However, our
results show that, potentially, five proteins of similar molecular weight and with similar intensity are bound and UV cross-linked to both
the full wild-type ICAM-1 probe and both consensus
B probes, either
with ICAM-1-specific or random flanking sequences. While these results
suggest that the proteins bound to these probes are the same, they
cannot rule out the involvement of additional proteins in the ICAM-1
complex for several reasons. First, the mobility of the complexes
formed with the ICAM-1 probe differs from that of the consensus probes
on EMSA, which suggests that different proteins may be involved in
binding to these probes. Also, UV cross-linking requires protein
proximity to DNA and cross-linking to BrdUrd. Thus, additional proteins
present in the ICAM-1 complex may not lie in close enough proximity to
the DNA to be cross-linked, may not lie near BrdUrd-containing
stretches of DNA, or may be sterically prevented from cross-linking by
other members of the complex.
The interaction of NF-B proteins with other nuclear proteins and
transcriptional activators (and repressors) has recently become
evident. Precedent for direct protein-protein interaction between
transcription factors comes from the discovery of the interaction of
AP-1 binding proteins with the glucocorticoid receptor, which
down-regulates expression of the individual target genes of both
transcription factors (46-48). A similar interaction was proposed for
TNF-
/NF-
B-mediated gene expression on the basis of a similar
down-regulation (42, 49, 50). Recently, LeClair et al. (51)
have shown a direct protein-protein interaction of NF-
B,
specifically p50, with the interleukin-1-activated transcription factor
NF-IL6, and Matsusaka et al. (52) have shown that such an
interaction is important in TNF-
and IL-1 regulation of IL-8 gene
expression. In addition, Neish et al. (53) have shown that cooperation of NF-
B with interferon regulatory factor 1 is necessary for maximal TNF-
induction of VCAM-1 expression. Furthermore, binding of the high mobility group I(Y) (HMG-I(Y)) protein in the minor
groove of the NF-
B sequence of the human interferon-
gene (54)
and the E-selectin gene (55) is necessary for optimal activation of
these genes.
We have explored the involvement of candidate transcription factors in
TNF--induced complex formation and transcriptional activation of
ICAM-1. HMG I(Y) binds to AT rich regions of DNA and has no specific
consensus sequence for binding. We have ruled out a possible role for
HMG I(Y) in ICAM-1 transcription regulation in both supershift EMSA and
in vitro transcription/translation and EMSA. Supershift EMSA
does not detect any immunoreactive HMG I(Y) in the TNF-
-induced
complexes in HDMEC (data not shown). An in vitro
transcription/translation product for HMG I(Y) does not bind to the
ICAM-1 TNF-
-responsive region either alone or in combination with
other Rel family members (Fig. 4), nor does it enhance Rel protein
binding to this region as has been the case for IFN-
and E-selectin
(54-55). In addition, a C/EBP binding site located upstream of the
modified
B site in the ICAM-1 5
regulatory region and binding the
factors C/EBP
or C/EBP
in various homo- or heterodimers has been
shown to be necessary for maximal inducibility of ICAM-1 by TNF-
(56). However, the requirement for flanking sequences described in this
manuscript does not rely on interaction of Rel proteins with C/EBP
family members since antibodies to these C/EBP proteins (
,
, and
) do not supershift EMSA complexes formed with the ICAM-1 TNF-
minimal response region nor do a consensus C/EBP oligonucleotide or the
ICAM-1 C/EBP oligonucleotide compete for TNF-
-induced complex
formation with this probe (data not shown). Finally, the sequences 5
and 3
of the ICAM-1 NF-
B binding site do not resemble any known
transcription factor consensus sequence.
Hansen et al. (57) have shown that flanking sequences for
the HIV-1 and urokinase promoter NF-B binding sites are important in
determining the specificity of NF-
B interactions at those sites.
Specifically, these flanking sequences dictate c-Rel/RelA binding and
thus control transcriptional activation through these specific Rel
family members. Our results support the results of that study in that
specific flanking sequences for the ICAM-1 modified
B site allow p65
(RelA) homodimer binding. They further demonstrate that these specific
flanking sequences are absolutely required for DNA-protein complex
formation with the modified
B site and transcription activation
through the ICAM-1 TNF-
-responsive region. Unlike the study by
Hansen et al. (57), however, in the context of a consensus
B site (like that present in HIV-1), flanking sequences are
dispensable both for DNA-protein complex formation and transcriptional
activation in response to TNF-
. Further, in either the presence or
absence of the ICAM-1 flanking sequences, the consensus
B site bound
p65/p50 heterodimers (classic NF-
B) with high affinity. Thus,
specific flanking sequences for the ICAM-1 TNF-
-responsive region
are unable to confer p65 homodimer binding on a consensus
B
site.
The present study has established a TNF--responsive region for the
ICAM-1 gene, which contains a modified
B site and interacts with the
Rel family member p65. However, this modified
B site, while
necessary, is not sufficient for TNF-
-mediated up-regulation of gene
expression. We demonstrate a novel requirement for specific 5
- and
3
-flanking sequences surrounding this modified
B site. Further, we
demonstrate that the G
C transition present in the ICAM-1
B site
dictates binding of p65 homodimers. The requirement for specific 5
-
and 3
-flanking sequences and the single-base modification make ICAM-1
a member of a unique subset of
B-regulated genes. Because ICAM-1 is
an attractive target for therapeutic intervention as a result of its
involvement in the pathogenesis of various inflammatory processes,
understanding the precise mechanisms by which its expression is
regulated is important. The present study further refines the current
understanding of the subtleties of gene-specific transcriptional
activation by the pleiotropic and rather promiscuous group of
transcriptional activators, the Rel family, possibly allowing for the
development of highly specific therapeutics for intervention in
inflammatory processes.
The assistance of Dr. Jens Gille and Kimberly Quinlan with technical aspects and the preparation of this manuscript is gratefully acknowledged.