(Received for publication, July 30, 1996, and in revised form, March 3, 1997)
From the Department of Immunology, The Cleveland
Clinic Foundation, Cleveland, Ohio 44195 and the ¶ Center for
Immunology, Department of Pathology, Washington University School of
Medicine, St. Louis, Missouri 63110
Interferon- (IFN
) and tumor necrosis
factor-
(TNF
) cooperate to induce the expression of many gene
products during inflammation. The present report demonstrates that a
portion of this cooperativity is mediated by synergism between two
distinct transcription factors: signal transducer and activator of
transcription 1 (STAT1) and nuclear factor
B (NF-
B). IFN
and
TNF
synergistically induce expression of mRNAs encoding
interferon regulatory factor-1 (IRF-1), intercellular adhesion
molecule-1, Mig (monokine induced by
-interferon), and RANTES
(regulated on activation normal
T cell expressed and secreted) in normal but not STAT1-deficient mouse
fibroblasts, indicating a requirement for STAT1. Transient transfection
assays in fibroblasts using site-directed mutants of a 1.3-kilobase
pair sequence of the IRF-1 gene promoter revealed that the synergy was
dependent upon two sequence elements; a STAT binding element and a
B
motif. Artificial constructs containing a single copy of both a STAT
binding element and a
B motif linked to the herpes virus thymidine
kinase promoter were able to mediate synergistic response to IFN
and
TNF
; such response varied with both the relative spacing and the
specific sequence of the regions between these two sites. Cooperatively
responsive sequence constructs bound both STAT1
and NF-
B in
nuclear extracts prepared from IFN
- and/or TNF
-stimulated
fibroblasts, although binding of individual factors was not
cooperative. Thus, the frequently observed synergy between IFN
and
TNF
in promoting inflammatory response depends in part upon
cooperation between STAT1
and NF-
B, which is most likely mediated
by their independent interaction with one or more components of the
basal transcription complex.
Intercellular communication by cytokines during an inflammatory
reaction is integral to the subsequent orchestration and resolution of
the response. IFN1 and TNF
are
pleiotrophic cytokines that play often critical roles in this process
(1, 2). Although both cytokines independently exert a number of
biological activities in a cell type-specific fashion, they have been
shown in many circumstances to function cooperatively or
antagonistically in controlling expression of a variety of cytokines
and cell surface molecules (3-7).
Much recent work on cytokine-mediated intracellular signaling pathways
has provided a general paradigm for the molecular mechanisms by which
extracellular signals induce transcription of target genes (8-11). A
variety of cytokines, growth factors, and hormones trigger
phosphorylation of latent cytoplasmic transcription factors termed
signal transducers and activators of transcription (STATs) via one or
more members of the Janus (Jak) family of protein tyrosine kinases.
Tyrosine-phosphorylated STATs assemble in dimeric or oligomeric form,
translocate to the nucleus, and bind to specific DNA sequence motifs or
STAT binding elements (SBEs) (12). IFN has been shown to induce
tyrosine phosphorylation of STAT1
, and a homodimeric form of
STAT1
binds to the IFN
-activation sequence (13), an SBE that has
been identified as a critical sequence motif involved in the
transcriptional activation of many IFN-inducible genes including the
IRF-1 and ICAM-1 genes (14-17).
The B sequence motif has been shown to be an essential cis-acting
regulatory element for mediating the TNF-, interleukin-1-, and
lipopolysaccharide-induced transcriptional activation of multiple cytokines and cell surface molecules (18-20). Although this sequence motif is recognized by members of the Rel homology family, including NF-
B1 (p50/p105), NF-
B2 (p52/p100), RelA, c-Rel, and RelB,
various forms of the
B sequence motif have been shown to exhibit
differential affinity for and functional response to different dimeric
combinations of Rel family proteins. Cell type-specific expression of
the Rel family members also mediates specificity for
B-dependent gene expression. Furthermore, members of the
Rel family have been shown to physically and functionally interact with
members of other transcription factor families (21-23). The
combination of these variables generates high potential for diversity
in the control of gene expression during inflammation.
Components of the JAK-STAT and the B signaling pathways appear to be
indispensable for stimulus-dependent, transcriptional activation of many inflammatory genes. Furthermore, SBE and
B motifs
are found in the promoter regions of many inflammatory genes. Many
studies have reported functional synergy between TNF
and IFN
in
promoting inflammatory function and gene expression, some of which
could involve an interplay between STAT1 and
B binding factors
(3-6). The present study was undertaken to determine whether
IFN
-activated STAT1 can cooperate with TNF
-induced NF-
B to
promote enhanced transcription. The results show that IFN
and TNF
synergize to induce expression of several genes that contain both SBE
and
B motifs. The findings indicate that both the SBE and
B
motifs are required for cooperativity and that the synergistic function
of STAT1
and NF-
B appear to result from independent activation
and recognition of cognate nucleotide sequence motifs.
Dulbecco's modified Eagle's medium, minimum
essential medium nonessential amino acid solution, sodium pyruvate, and
antibiotic were obtained from Life Technologies, Inc. Fetal bovine
serum was purchased from Bio Whittaker (Walkersville, MA). DEAE-dextran and polydeoxyinosinic-deoxycytidylic acid (poly(dI-dC)) were purchased from Pharmacia LKB Ltd. (Uppsala, Sweden). MAGNA Nylon transfer membrane was obtained from Micron Separations Inc. (Westboro, MA).
Restriction enzymes, Klenow fragment of Escherichia coli DNA
polymerase I, T4 kinase, and bovine serum albumin were purchased from
Boehringer Mannheim. UlTma DNA polymerase was obtained from Perkin-Elmer. DuPont NEN was the source of [-32P]dCTP
and [
-32P]ATP.
1-Deoxy-dichloroacetyl-1-[14C]chloramphenicol was
obtained from Amersham Corp. Thin layer chromatography (TLC) plates
(Silica Gel 60) were obtained from Merck (Darmstadt, Germany). Protein
assay reagents were obtained from Bio-Rad. Site-directed mutagenesis
kits, the luciferase reporter plasmid (pGL2-Basic), and luciferase
assay reagents were obtained from Promega Corp. (Madison, WI).
Recombinant mouse IFN
(specific activity, 6.8 × 106 units/mg) was obtained from Life Technologies, Inc.
Recombinant mouse TNF
(specific activity, 2.6 × 107 units/mg) was a generous gift from Genentech Inc.
(South San Francisco, CA). Antisera to mouse p50 (NF-
B1), p65
(RelA), c-Rel, STAT3, and human Sp1 were obtained from Santa Cruz
Biotechnology (Hercules, CA). Mouse monoclonal antibody to human STAT1
(p91/p84) was obtained from Transduction laboratories (Lexington, KY).
Dithiothreitol, HEPES, normal rabbit IgG, chloroquine diphosphate,
dimethyl sulfoxide, leupeptin, antipain, aprotinin, pepstatin, and
phenylmethylsulfonyl fluoride, were obtained from Sigma. Other reagents
were purchased from Mallinckrodt, Inc. (Paris, KY).
Fibroblasts from STAT1-deficient and wild type mice were prepared as described previously (24). These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 mM nonessential amino acid solution, 10 mM sodium pyruvate, 20 mM of L-glutamine. NIH3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin (complete medium) and subcultured twice weekly. Prior to use in experiments, the cells were grown to confluence in 100- or 150-mm diameter culture dishes and then transferred to medium containing 0.2% fetal bovine serum for 24 h in order to deprive growth factor.
Preparation of Plasmid DNAA cDNA encoding mouse IRF-1
(25) was cloned from a mouse macrophage cDNA library (26) using a
reverse transcriptase-PCR fragment as a probe as described
previously.2 The plasmid encoding the
cDNA for mouse ICAM-1 was obtained from the American Type Culture
Collection (Rockville, MD) (27). cDNA fragments for mouse Mig and
RANTES were prepared by reverse transcriptase-PCR using a set of
primers corresponding to the mouse Mig and RANTES cDNA sequences
obtained from the GenBankTM data base (28-30) and
subcloned into pBluescript (Stratagene, La Jolla, CA). The nucleotide
sequences were independently confirmed. The plasmid encoding GAPDH was
obtained from Dr. David Stern (Columbia University, New York, NY).
Methods for plasmid DNA preparations were as described in Sambrook
et al. (31). One µg of plasmid DNA or 100 ng of PCR
products were radiolabeled by random priming with
[-32P]dCTP. The resultant specific activity was
approximately 108 cpm/µg, which was used at
107 cpm/blot.
Each
assay utilized confluent monolayer of fibroblasts cultured in 100-mm
diameter plastic Petri dishes for preparation of total RNA. After
treatment of the cells with the indicated stimuli, total cellular RNA
was extracted by the guanidine isothiocyanate-cesium chloride method
(32). Samples of total RNA (5 µg) were separated on a 1% agarose,
2.2 M formaldehyde gel and subsequently blotted onto MAGNA
nylon membrane with 20 × SSC by capillary transfer according to
previously published methods (31). The RNA was cross-linked to the
membrane with a UV cross-linker (Stratagene). The blots were
prehybridized for 8-12 h at 42 °C in 50% formamide, 1% SDS,
5 × SSC, 1 × Denhardt's solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.25 mg/ml denatured salmon sperm DNA, and 50 mM sodium phosphate (pH
6.5) and then hybridized with 1 × 106 cpm/ml of
radiolabeled cDNA plasmid probe at 42 °C for 16-24 h. After
hybridization, blots were washed with 0.1% SDS, 2 × SSC for 30 min at room temperature followed by two washes at 55 °C. The blots
were then exposed using XAR-5 x-ray film with intensifying screens at
70 °C.
The luciferase
reporter constructs containing the 1.3-kb IRF-1 promoter was kindly
provided by Dr. Bryan Williams (Department of Cancer Biology, Cleveland
Clinic Foundation). The SBE site at positions 123 to
113 and the
B site at positions
49 to
40 of the IRF-1 promoter (14) were
respectively mutated in the 1.3-kb 5
-flanking sequence of the IRF-1
gene by oligonucleotide-directed, site-specific mutagenesis as
described previously (33). The mutant sequence utilized for the SBE and
the
B were TTCCCtcc and GtGGAATCaC, respectively. Lowercase letters
represent the mutant nucleotides.
One or two copies of the IRF-1 SBE or the IP-10 B2 were placed in
front of the
105 or the
81 base pair herpes simplex virus-thymidine kinase (TK) promoter (34) linked to the chloramphenicol acetyl transferase (CAT) gene (pTK-105 CAT) (35) or the luciferase gene
(pTK-105 Luc or pTK-81 Luc) (36). These constructs were prepared by
ligating the synthetic oligonucleotides (see below) into restriction
enzyme sites of the reporter plasmids. To generate constructs
containing different nucleotide spacing between the IRF-1 SBE and the
B2 sites, one or more SalI linkers (GGTCGACC) were placed
between the two sites. The sequences of these reporter gene constructs
were confirmed.
Transfection of the luciferase and
the CAT reporter genes into fibroblasts or NIH3T3 cells were as
described previously (5). Briefly, the cells were seeded at a density
of 3 × 106 cells/150-mm diameter dish 24 h prior
to transfection. 30 µg of reporter luciferase construct plasmid DNA
were transfected by the DEAE-dextran method (300 µg/ml DEAE-dextran)
for 20 min at room temperature. After the incubation, the cells were
subjected to dimethyl sulfoxide shock for 1 min (10% dimethyl
sulfoxide in phosphate-buffered saline), washed with phosphate-buffered saline, replenished with fresh culture medium, and cultured for 2 h. To standardize transfection efficiencies, the transfected cells were
then harvested in trypsin-EDTA solution, pooled, and seeded in four
100-mm diameter Petri dishes. The cells were cultured in medium
containing 0.2% fetal bovine serum for 24 h to deprive growth
factors and then stimulated with IFN and/or TNF
for 16 h for
the CAT reporter gene and for 8 h for the luciferase reporter gene, respectively. After stimulation, the cells were washed and extracted in lysis buffer (Promega), and luciferase activity was assayed using reagents provided by Promega according to the
manufacturer's instructions. Twenty µg of extract protein were
utilized in each assay. CAT activity was assessed by determination of
the conversion of [14C]chloramphenicol into acetylated
forms detected by thin layer chromatography as described previously
(35). The acetylated products were quantified using a phosphorescence
detection system (Molecular Dynamics, Sunnyvale, CA).
The following oligonucleotides were used in this study.
IRF-1 SBE | 5![]() ![]() |
3![]() ![]() |
|
mut1 SBE | 5![]() ![]() |
(m1 SBE) | 3![]() ![]() |
mut2 SBE | 5![]() ![]() |
(m2 SBE) | 3![]() ![]() |
IRF-1
![]() |
5![]() ![]() |
3![]() ![]() |
|
mutIRF-1![]() |
3![]() ![]() |
IP-10
![]() |
5![]() ![]() |
3![]() ![]() |
|
mutIP-10
![]() |
5![]() ![]() |
3![]() ![]() |
|
OLIGONUCLEOTIDES 1-7 |
The nucleotide sequences of IRF-1 SBE and B were taken from
Sims et al. (14). The IP-10
B2 sequence was taken from
Ohmori and Hamilton (33, 37). Lowercase letters represent the bases included for creating restriction sites. Underlined sequences represent
the consensus sequences for the SBE and
B elements, respectively.
Boldface type indicates the substituted bases for mutation.
Oligonucleotides were synthesized using an Applied Biosystem DNA
synthesizer (model 381A) or obtained from Ransom Hill Bioscience Inc.
(Ramona, CA). Double-stranded oligonucleotides were prepared by
annealing the complementary single strands. A DNA fragment corresponding to the region between
129 and
37 of the IRF-1 promoter (14) was generated by PCR using a sense oligonucleotide of the
IRF-1 SBE and an antisense oligonucleotide of the IRF-1
B as
primers, and the luciferase reporter plasmid containing the 1.3-kb
IRF-1 promoter was used as a template. A mutant fragment was also
generated by using a sense oligonucleotide of mut1 SBE and antisense
oligonucleotide mutIRF-1
B as described above. Double-stranded oligonucleotides were radiolabeled with the Klenow fragment of DNA
polymerase I and [
-32P]dCTP in a fill-in reaction for
5
protruding ends. PCR-amplified DNA fragments were radiolabeled
with T4 kinase and [
-32P]ATP.
Nuclear extracts were prepared using a modification of the method of Dignam et al. (38) as described previously (5, 37). After stimulation, the cells were washed with ice-cold phosphate-buffered saline three times, harvested, and resuspended in 300 µl of hypotonic buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of leupeptin, antipain, aprotinin, and pepstatin) for 10 min on ice. The cells were then lysed in 0.6% Nonidet P-40 by vortexing for 10 s. Nuclei were separated from cytosol by centrifugation at 12,000 × g for 30 s, washed with 300 µl of buffer A, and resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of leupeptin, antipain, aprotinin, and pepstatin) and briefly sonicated on ice. Nuclear extracts were obtained by centrifugation at 12,000 × g for 10 min. Protein concentration was measured by the method of Bradford (39) by using the protein dye reagent (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)For binding
reactions, nuclear extracts (5 µg of protein) were incubated in 12.5 µl of total reaction volume containing 20 mM HEPES (pH
7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 5% glycerol, 200 µg/ml bovine serum albumin, and
1.25 µg of poly(dI-dC) for 15 min at 4 °C. The
32P-labeled oligonucleotide (~5 × 105
cpm) was then added to the reaction mixture and incubated for 20 min at
room temperature. In some experiments, antibodies against NF-B1,
RelA, c-Rel, STAT1, STAT3, and Sp1 were included in the reaction
mixture. The reaction products were analyzed by electrophoresis in a
4% polyacrylamide gel with 0.25 × TBE buffer (22.3 mM Tris, 22.2 mM borate, 0.5 mM
EDTA). The gels were dried and analyzed by autoradiography.
IFN and TNF
have been shown
to cooperatively regulate transcription of many inflammatory genes
(3-6, 40). Previous studies have demonstrated that the IFN
-induced
transcriptional activation of the IRF-1, ICAM-1, and Mig genes depends
upon IFN
response elements or SBEs in the promoter region of the
genes (Fig. 1), which are recognized by STAT1 or
STAT1-containing factor(s) (14-17, 41-43). The promoter regions of
these IFN
-inducible genes also contain one or more
B sequence
motifs, and their transcriptional activation in response to TNF
is
dependent upon activation of
B binding activities (44, 45). On the
basis of these observations, we postulated that IFN
-induced STAT1
and TNF
-induced NF-
B cooperatively regulate transcription of
genes containing both SBE and
B motifs. Analysis of RANTES gene
expression was also included, since IFN
and TNF
can cooperatively
induce expression of this chemokine gene, although no SBE has been
identified in the promoter (6, 30, 46-48). Levels of endogenous
mRNA expression were determined in fibroblasts from wild-type mice
or from mice in which the STAT1 gene has been deleted through
homologous recombination (24). Serum-starved cultures were stimulated
with IFN
and TNF
either alone or in combination for 3 h
prior to isolation of RNA and Northern analysis. While the sensitivity
of normal cells to IFN
or TNF
alone varied with each gene, all
four genes were strongly expressed in cells stimulated with both agents
(Fig. 2A). IFN
and TNF
cooperativity
was markedly reduced (>90%) in STAT1-deficient fibroblasts without
affecting the sensitivity to TNF
alone (Fig. 2B). These
four responses were not mechanistically identical; the synergistic
enhancement of RANTES mRNA expression was blocked in cells
co-treated with cycloheximide (CHX), while expression of
IRF-1, ICAM-1, and Mig was unaltered (Fig. 2C). Thus, the
synergy between IFN
and TNF
may involve protein
synthesis-dependent and -independent mechanisms, both of
which require IFN
-induced STAT1.
IFN
IFN-induced
transcription of the IRF-1 gene has been shown to depend upon the SBE
located in positions
123 to
113 (14, 15). A highly conserved
B
motif has also been identified, although its functional significance
remains equivocal (Fig. 1) (14, 49, 50). To determine if cooperation
between IFN
and TNF
for induction of the IRF-1 gene depends upon
one or both of these sites, luciferase reporter gene constructs of the
IRF-1 promoter (1.3 kb) were prepared in which the SBE and/or the
proximal
B site were individually mutated. These constructs were
transiently transfected into wild type or STAT1-deficient fibroblasts,
and their activity was assessed following treatment with IFN
and/or TNF
(Fig. 3). As reported previously, IFN
markedly
induced luciferase activity in normal fibroblasts transfected with the
wild type 1.3-kb IRF-1 promoter construct (14, 15). TNF
also
modestly induced luciferase activity (5-7-fold induction). When cells
were simultaneously stimulated with IFN
and TNF
, the promoter
activity was synergistically enhanced. Mutation of the SBE site nearly abolished the IFN
sensitivity and markedly reduced the cooperative response to the combination of IFN
and TNF
(85% reduction in magnitude) without affecting the TNF
-induced luciferase activity. Similarly, the mutation of the
B site abolished the response to
TNF
alone and reduced the cooperativity for IFN
and TNF
(77%
reduction in magnitude). Mutation of both the SBE and the
B site in
the same construct nearly eliminated sensitivity to either stimulus
alone, and the cooperative response, although evident, was only 3% of
that seen in cells transfected with the intact promoter. Furthermore,
in STAT1-deficient fibroblasts, response to IFN
either alone or in
combination with TNF
was less than 5% of the response seen in wild
type cells. Taken together, these results indicate that both the SBE
and the
B sequence motifs are required for optimal cooperativity
between IFN
and TNF
and that activation of STAT1, at least, is
also necessary.
While the magnitude of cooperative response is markedly reduced in wild
type cells transfected with mutations in individual motifs (either SBE
or B) and in STAT1-deficient cells, there is residual cooperativity
evident in both circumstances. This apparent leakiness may derive from
multiple sources. For example, the mutated motifs may retain some low
affinity interaction with individual factors. Alternatively, there may
be other sites that are able to participate, providing the lower
magnitude cooperativity. Indeed, low but detectable cooperativity is
evident using promoter constructs in which both the SBE and the
proximal
B sites are mutated. The very low but reproducible response
to IFN
seen in STAT1-deficient cells (both in Fig. 1 and Fig. 3) may
reflect minor compensatory action of IFN
functioning through
STAT1-independent systems. Indeed, in EMSA experiments using nuclear
extracts from IFN
-treated STAT1-deficient fibroblasts, we detected
low but significant levels of STAT3 that were not seen in wild type
cells (data not shown). Since STAT3 can act to modulate transcription through IFN
activation sequence motifs, this could account for the
leaky response to IFN
. It should be emphasized that these low level
responses may detectable in our experimental system due to the very
high sensitivity of the luciferase reporter gene.
IFN is well documented to stimulate the
phosphorylation and nuclear localization of STAT1
homodimers
(8-11). Similarly, TNF
is a potent stimulus of the nuclear
translocation of various members of the Rel homology family (18-20).
The functional cooperativity between IFN
and TNF
might result
from cooperative effects on DNA binding activities of the respective
transcription factors. Thus, we next compared the binding activities of
STAT1 and NF-
B to their respective sequence motifs using nuclear
extracts prepared from cells stimulated with IFN
and/or TNF
for
30 min by EMSA (Fig. 4). Although nuclear extracts from
fibroblasts stimulated with IFN
showed little or no inducible
B
binding activity, cells stimulated with TNF
exhibited two inducible
complexes designated as C1 and C2 in Fig. 4A. When cultures
were co-stimulated with IFN
and TNF
, the magnitude of the binding
activity and pattern of complex formation were essentially the same as
seen in cells stimulated with TNF
alone. As shown in Fig.
4B, the C1 complex was fully reactive with antiserum
specific for NF-
B1, while the C2 complex showed partial reactivity
with anti-NF-
B1 and full reactivity with antiserum specific for
RelA. Antibodies specific for c-Rel and STAT1 did not recognize any of
the IFN
- and/or TNF
-induced
B binding activities. These
results suggest that the binding activity induced by TNF
that
recognizes the IRF-1
B site is composed of NF-
B1/RelA
heterodimers and RelA homodimers. Specificity for the Rel protein
binding was further assessed by oligonucleotide competition assays
(Fig. 4C). Oligonucleotides containing the wild type IRF-1
B motif competed effectively for the binding of these Rel proteins,
while a mutant oligonucleotide was inactive (lanes 2 and
3). Interestingly, oligonucleotides containing a wild type
IRF-1 SBE or a mutant SBE in which two adenine residues in the 3
half
of the inverted repeat were changed (m1 SBE) also partially competed
for the binding of NF-
B1 and RelA (lanes 4 and
5). Another mutant SBE (m2 SBE), in which the intervening
sequence between the inverted repeats was also altered, could not
compete for binding to the
B motif. Since the adenine residues in
the inverted repeat have been previously shown to be critical for
recognition by STAT1 (14), sequence preferences for STAT1 and NF-
B
appear to be distinct. This ability of SBE to compete for NF-
B
recognition may result from the
B-like site in the 5
portion of the
IRF-1 SBE motif. In addition it may reflect the low affinity
recognition of SBEs by NF-
B as previously reported (51).
Consistent with previous reports, nuclear extracts from IFN-treated
fibroblasts contained a prominent stimulus-dependent DNA
binding activity specific for the IRF-1 SBE (Fig.
5A), and this complex is fully reactive with
antibody to STAT1 (data not shown). Interestingly, TNF
also induced
a DNA binding activity that recognized the IRF-1 SBE forming a complex
that migrated at a slightly different mobility. This complex was
reactive with antibodies specific for NF-
B1 and RelA (Fig.
5B, lanes 3 and 4). These findings are also
consistent with the results in Fig. 4C showing competition
between the SBE and NF-
B on the IRF-1
B site. When nuclear
extracts from cells stimulated with both IFN
and TNF
were
analyzed, a single broad band was observed, consistent with the
presence of both the STAT1 and NF-
B complexes seen with IFN
or
TNF
stimulation alone. The most prominent component in this complex
was STAT1, as indicated by immunoreactivity with anti-STAT1 (Fig.
5B, lane 6). The more slowly migrating complex, which was not reactive with anti-STAT1, was reactive with anti-NF-
B1 and anti-RelA (lanes 8 and 9). Competition assays
showed that an oligonucleotide containing a wild type SBE effectively
competed for the binding of all complexes (Fig. 5C,
lane 4), while the wild type
B motif either did not
compete or did so poorly (lane 2). The m1 SBE did not
compete, indicating that most of the binding activity present was
STAT1, since this mutation appears to affect primarily the formation of
STAT1 complexes and not NF-
B. A large DNA fragment containing both
the SBE and the
B sites was also able to compete complex formation
in response to treatment with IFN
and TNF
. When this larger
fragment (spanning positions
129 and
37 of the IRF-1 promoter) was
used as a probe in EMSA, each complex was formed independently, and no
evidence was obtained for cooperativity in binding between factors
activated independently by IFN
or TNF
(data not shown).
SBE and
To
explore the generality of the B motif and SBE functional
cooperativity, we asked whether transcriptional synergy could be
reconstituted using isolated sequence elements placed in a heterologous
promoter. Initially, one copy of the IRF-1 SBE and/or the
B2 motif
from the mouse IP-10 gene (33, 37) were placed in front of the TK
promoter (TK-105) linked to the CAT reporter gene and tested for
sensitivity to IFN
and/or TNF
following transient transfection in
NIH3T3 cells (Fig. 6). Although one copy of the
B2
motif exhibited little sensitivity to IFN
or TNF
either alone or
in combination with the SBE, one copy of the IRF-1 SBE motif was
sensitive to IFN
or IFN
and TNF
. When a construct containing
one copy each of the
B2 and the SBE was analyzed, a strong
synergistic response was seen in cells stimulated with the combination
of agents. Mutation of the SBE site abolished all stimulus sensitivity
of the combination construct and was essentially identical to that of a
construct containing only a single
B site. Thus cooperativity was
not due to creation of fortuitous binding sites in the region where
inserted sequences are coupled. Interestingly, cooperativity between
IFN
and TNF
was also seen using the construct containing only the
IRF-1 SBE and using the construct containing wild type SBE and mutant
B. The synergistic response of such constructs to IFN
and TNF
appears to depend upon the distal portion of the TK promoter, which
contains a GC box and a CCAAT box; no cooperativity was seen in a
truncated form of the TK promoter in which the distal GC box and CCAAT
box have been deleted (pTK-81, see Fig. 7).
The spacial relationship between the two cooperating sites may be an
important determinant of their synergistic interaction. To examine this
possibility, reporter constructs in which the sequence motif
orientation and the nucleotide spacing between motifs were varied were
prepared and examined in transient assays (Fig. 7). For these
experiments, a truncated form of the TK-luciferase vector (pTK-81) was
utilized in which both a GC box (Sp1 binding site) and a CCAAT box have
been deleted. When a single copy of either a B site or the IRF-1 SBE
were linked to this reporter plasmid, no cooperative response was
obtained. As mentioned above, this result suggests that the cooperative
response seen with constructs containing a single SBE site (see Fig. 6)
requires one or both of the sites deleted from the TK promoter. When a
construct containing a single copy each of the SBE and the
B motif
was examined, a strong synergistic response was obtained. The synergy
was not dependent upon the relative order of sites. Although constructs containing the SBE in either a distal or proximal relationship to the
TK promoter exhibited variable response to IFN
alone, cooperative
responses were comparable (6-7-fold). The variability in sensitivity
to IFN
is also observed in Fig. 7 when comparing the response of pTK
SBE and pTK m
B2 + SBE, where the spacing of the SBE relative to the
TK promoter is comparably altered. When the spacing between the two
sites was incrementally increased, sensitivity to individual and
combination stimulation was reduced. An increase of 5 nucleotides only
modestly reduced the cooperativity, indicating that the orientation of
bound factors relative to each other and the turn of the helix was not
a limiting feature of the response. As the spacing interval was
increased, the response was much more dramatically reduced.
Interestingly, when the sites were separated by 64 nucleotides, a
distance equivalent to that separating the SBE and
B sites in the
endogenous IRF-1 promoter, sensitivity to stimulation was lost
entirely. These results indicate that while spacing may influence the
magnitude of cooperativity, other features of the sequence between
sites are probably of more critical importance.
IFN and TNF
utilize distinct signaling pathways leading to
altered gene transcription (8-11, 52). When these cytokines have been
used in combination, both cooperative and antagonistic effects on gene
transcription have been observed (3-7, 40). The present study was
undertaken to define the mechanisms involved in such a synergistic
response. The results demonstrate that STAT1 activation by IFN
and
NF-
B activation by TNF
are the principle events necessary for
cooperative induction of genes containing appropriate SBE and
B
sequence motifs. Independent interaction of STAT1 and NF-
B with
their cognate binding sites is sufficient for mediating the
cooperativity. These conclusions are based on the following
observations. 1) IFN
and TNF
synergized strongly to promote
expression of multiple genes that contain at least one copy of an SBE
and a
B site, including the IRF-1, ICAM-1, and Mig genes. 2) This
activity was abolished in fibroblasts prepared from mice in which the
STAT1 gene has been deleted by homologous recombination. 3) Synergistic
transcription induced by IFN
and TNF
was observed in normal
fibroblasts transfected with a reporter gene under control of a 1.3-kb
fragment of the IRF-1 gene promoter. 4) The synergistic induction of
the IRF-1 promoter activity was nearly abolished in a STAT1-deficient
cell line. 5) Site-directed mutagenesis of the SBE and the proximal
B site in the IRF-1 gene promoter significantly reduced the
magnitude of the synergistic response. 6) IFN
and TNF
independently activated STAT1 and NF-
B (NF-
B1/RelA),
respectively, as measured by binding to their cognate sequence motifs.
7) No cooperative effects on DNA binding activities were observed. 8)
The SBE and
B motifs could confer transcriptional synergy in
response to IFN
and TNF
when examined in a heterologous promoter.
IFN-induced transcriptional synergy appears to be mediated by
multiple pathways involving both protein
synthesis-dependent and -independent mechanisms (5, 53).
The results presented in this study indicate that cooperative effects
involving IFN
and TNF
exhibit similar behavior (Fig. 2). An
important observation is that both protein
synthesis-dependent and -independent cooperativity still
depends largely on STAT1, consistent with the recent reports showing
STAT1 to be obligatory for IFN-mediated biological activities (24, 54).
The requirement for protein synthesis during IFN
/TNF
-mediated RANTES gene expression suggests that some IFN
-induced protein(s) (e.g. IRF-1) might be necessary for cooperativity in this
circumstance, consistent with such roles for other genes (24, 53, 55). Inspection of the RANTES promoter sequence suggests the presence of
IRF-binding motifs (56). Furthermore, functional
B motifs have been
identified in the promoter (Fig. 1) (46) and cooperative regulation of
transcription by IRF-1 and NF-
B has been previously reported (57,
58). In contrast, direct activation of STAT1, which may include the
formation of STAT1
homodimers, heterodimers, or other oligomeric
interactions, appears to be involved in the cooperative induction of
IRF-1, ICAM-1, and Mig gene expression. IFN
-dependent
transcription of the IRF-1, ICAM-1, and Mig genes has been shown to
depend upon SBE motifs that bind STAT1 in homo- or heterodimeric forms
(14-17, 41-43). Furthermore, synergistic induction of IP-10 gene
transcription by IFN
and TNF
also depends on an IFN
-inducible
factor that contains STAT1 and binds the IFN-stimulated response
element found in the IP-10 promoter (5).
TNF is well documented as a potent inducer of NF-
B and has been
reported elsewhere to cooperate functionally with other transcription
factors (5, 21, 23, 57, 58). The results in the present study indicate
that NF-
B (NF-
B1/RelA) can cooperate with STAT1 to promote
synergistic transcriptional activity. The proximal
B site in the
IRF-1 promoter is a functional
B motif, which is recognized by a
combination of NF-
B1 and RelA in fibroblasts. TNF
-mediated ICAM-1
gene transcription has been shown to depend upon a
B motif
recognized by Rel family members (44, 45). Interestingly, despite the
fact that the Mig gene is not independently induced by stimuli that
activate NF-
B (e.g. TNF
and lipopolysaccharide) (28),
the cooperative induction of this gene by IFN
and TNF
suggests
that the
B motifs found in the Mig promoter are functional when
STAT1 is also available.
Interestingly, we noted that the IRF-1 SBE appeared able to mediate a
synergistic response to stimulation with IFN and TNF
independently of the proximal
B site (see Figs. 3 and 6). Since the
SBE site was also recognized by STAT1 and NF-
B, this dual recognition might contribute to the functional synergy. While this
possibility cannot be ruled out, several considerations suggest that
the cooperativity observed derives from other sources. For example, in
Fig. 3, the constructs containing mutations in the SBE, in the
B
site, and in the double mutant all showed some synergistic
response to the stimulus combination. Because this fragment is large
(1.3 kb), there are apparently other independent sites that can
cooperate with the SBE, the
B site, or each other. Second, although
the cooperative behavior of the artificial construct utilized in Fig. 6
(pTK SBE) appears to depend solely upon the SBE, data shown in Fig. 7
illustrate that such cooperativity is dependent upon a 25-base pair
fragment of the TK promoter between positions
105 and
81. When the
pTK-81 promoter was used with the isolated SBE, no cooperativity was
evident. While we do not understand the mechanism(s) through which
cooperativity occurs in this setting, the results suggest that the SBE
is not independently capable of mediating cooperative
response to IFN
and TNF
.
The molecular mechanisms involved in functional synergy between
distinct transcription factors appear to be multifactorial (23,
59-63). In some cases, direct protein-protein interaction between
activator proteins has been observed (23, 59). The physical interaction
may result in cooperative DNA binding, more stable protein-DNA
interactions, and/or increased affinity of one or both activator
proteins, ultimately creating a highly stable multiprotein complex that
has markedly enhanced functional properties (23, 63). In this regard,
members of the NF-B and the STAT families have been observed to
interact with members of other distinct factor families (21-23, 64,
65) although not with each other. Both NF-
B and STAT1 formed
complexes on the IRF-1 SBE, but these appeared to be independent
interactions between individual factors and DNA, since each complex
exhibited a distinct mobility in EMSA. Furthermore, the presence of one
factor did not alter the interaction of the other with its cognate
site, nor did the presence of both factors promote the formation of any
unique complexes not detected in cells treated with either stimulus
alone. Nevertheless, we cannot completely rule out the possibility that
a weak interaction between STAT1 and NF-
B in vivo might
produce the observed functional cooperativity, since in
vitro study of protein-protein interaction will only detect relatively high affinity interactions. Furthermore, analysis of nucleotide spacing between these sequence motifs indicated that, although spacial distances may quantitatively modify the response, the
specific intervening nucleotide sequences were more important. This
latter observation may suggest a role for other factors or an influence
of flanking sequence on the functional behavior of transacting factors
bound to DNA. This possibility is also supported by the finding that a
single SBE motif could mediate moderate cooperative response to IFN
and TNF
when other stimulus-insensitive sites are present.
An alternative mechanism for transcriptional synergy might involve independent interaction of the activation domains of individual factors with components of the general transcription machinery such as the TATA-binding protein, TATA-binding protein-associated factors, TFIIA, and TFIIB (61, 62). The same activator domain may interact with more than one component of the RNA polymerase complex. These multiprotein interactions could facilitate assembly of a preinitiation complex, stabilize the complex on promoter DNA, and thus promote the frequency of transcriptional initiation and elongation. Members of the Rel family have been reported to interact directly with TATA-binding protein and TFIIB (66, 67), and thus it is conceivable that the activation domains of these factors and of STAT1 may differentially interact with basal transcription components.