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p48/STAT-1α-Containing Complexes Play a
Predominant Role in Induction of IFN- γ
-Inducible Protein, 10 kDa (IP-10) by IFN-γ
Alone or in Synergy with TNF- α
Sarmila Majumder, Lucy Z.-H. Zhou, Priya Chaturvedi,
Gerald Babcock, Sumer Aras and Richard M. Ransohoff
J Immunol 1998; 161:4736-4744; ;
http://www.jimmunol.org/content/161/9/4736
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
p48/STAT-1a-Containing Complexes Play a Predominant Role
in Induction of IFN-g-Inducible Protein, 10 kDa (IP-10) by
IFN-g Alone or in Synergy with TNF-a1
Sarmila Majumder,2* Lucy Z.-H. Zhou,* Priya Chaturvedi,3* Gerald Babcock,*
Sumer Aras,4* and Richard M. Ransohoff5*†
nterferon-g-inducible protein, 10 kDa (IP-10)6 is a member
of the superfamily of chemokines; the human gene product
mediates a T cell-dependent antitumor response (1, 2). IP-10
is chemotactic for activated T cells and also exhibits angiostatic
effects (3, 4). Expression of IP-10 mRNA and protein has been
documented in a variety of inflammatory and neoplastic disorders
in vivo. The IP-10 gene was initially cloned by differential hybridization using cDNA prepared from IFN-g-treated U937 cells, a
histiocytic lymphoma cell line with monocytic characteristics (1).
IFN-g is a major determinant of IP-10 expression and efficiently
induces its transcription (without intervening protein synthesis) in
varied cell types including macrophages, fibroblasts, astrocytes,
I
*Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, and †Neurology Department, Mellen Center for Multiple Sclerosis Treatment
and Research, Cleveland Clinic Foundation, Cleveland, OH 44195
Received for publication January 29, 1998. Accepted for publication June 22, 1998.
1
This work was supported by grants from the National Institutes of Health (NS 32151
to R.M.R. and CA 62220, G. R. Stark, PI; R.M.R. was leader, project 3) and a
fellowship from the American Heart Association, Northeastern Ohio Affiliate (to
L.Z-H.Z.), and the Williams Family Fund for Multiple Sclerosis Research.
2
Current address: Dept. of Biochemistry, Ohio State University College of Medicine,
Columbus, OH.
3
Current address: Dept. of Molecular Biology, Temple University School of Medicine, Philadelphia, PA.
4
Current address: Dept. of Biology, Ankara University, Ankara, Turkey.
5
Address correspondence and reprint requests to Dr. Richard M. Ransohoff, The
Lerner Research Institute, NC30, Cleveland Clinic Foundation, Cleveland, OH 44195.
E-mail address: [email protected]
Abbreviations used in this paper: IP-10, IFN-g-inducible protein, 10 kDa; mIP-10,
murine IP-10; hIP-10, human IP-10; GL-IP10, IP-10 promoter-reporter construct;
TGL-1P10, truncated GL-IP10; DMS, dimethyl sulfate; ISRE, IFN-stimulated response element; pIP10 mISRE3, IP-10 promoter-reporter containing ISRE mutant-3;
IRF, IFN regulatory factor; GAS, g-activated sequence; ICS, IFN consensus sequence; ISGF3, IFN-stimulated gene factor; IVGF, in vivo genomic footprinting;
EMSA, electrophoretic mobility shift assay.
6
Copyright © 1998 by The American Association of Immunologists
osteoblasts, and endothelial cells (5–9). IFN-g (type II IFN) has
been studied extensively as a transcriptional regulator. The majority of responsive genes are induced by IFN-g through the interaction of STAT-1a homodimers (originally designated the g-activated factor (GAF) and an inducible palindromic enhancer termed
the g-activated site (GAS) (10).
IFN-g also induces transcription of a subset of genes in a GASindependent fashion through a motif termed the IFN-stimulated
response element (ISRE) (11). Mutant cells that lacked a 48-kDa
ISRE-binding protein (p48) were used to show an absolute requirement for this factor for transcriptional responses to IFN-g for
ISRE-dependent promoters (12). This result was confirmed separately by studies using embryonic fibroblast cells derived from
mice rendered p48-deficient by gene targeting (13). Trans-acting
factors required for IFN-g induction of the ISRE-dependent ISG54
gene included STAT-1a and p48 (14). This observation led to the
proposal that IFN-g activated these genes through an alternative
form of the IFN-stimulated gene factor 3 (ISGF3) complex (see
below), composed of p48 and STAT-1a homodimers (14). The
p48/STAT-1a complex has not been characterized in detail with
regard to binding specificity or composition, and the role of other
ISRE recognition factors, such as IFN-regulatory factor (IRF)-1,
was not addressed in these studies.
IFN-a also induces the formation of an ISRE-binding complex,
designated ISGF3 and containing p48 and two members of the
STAT family. The p48 protein is essential for formation of the
IFN-a-induced high-affinity binding complex, but lacks a transactivation domain. STATs are responsible for transcriptional activation and also contribute to sequence-specific contact with DNA
(15). Alternatively, ISRE elements can be bound by p48-related
proteins of the IRF family, including IRF-1, which regulate transcription (positively or negatively) without the participation of
other factors (16).
0022-1767/98/$02.00
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Human IFN-g-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-g alone, and synergistically induced by TNF-a and IFN-g. Upstream regions of the human and murine genes contain conserved regulatory motifs,
including an IFN-stimulated response element (ISRE), which governs response of the mIP-10 gene to IFN-g. Trans-acting factors
mediating the IFN-g response via ISRE remain incompletely defined. We examined ISRE-binding factors in the regulation of the
hIP-10 gene. The requirement of p48 for hIP-10 induction by IFN-g, with or without TNF-a, was demonstrated using p48-deficient
U2A cells. An hIP-10 promoter-reporter mutant (mISRE3) that was relatively deficient for binding a related factor, IFN regulatory factor-1 (IRF-1) but competent for binding p48, was induced as well as the wild-type hIP-10 promoter, supporting the
interpretation that p48 played a necessary and sufficient role in hIP-10 transcription. Genomic in vivo footprinting revealed
IFN-g/TNF-a-inducible binding at the ISRE consistent with the presence of p48 and associated factors, but not with IRF-1.
Induction of hIP-10 by TNF-a/IFN-g also required NFkB binding sites, which were protected in vivo and bound p65 homodimeric
NFkB in vitro. These results documented the essential role of p48 (complexed with STAT-1a) for induction and sustained
transcription of the IP-10 gene, strongly suggesting that IRF-1 is not required for IP-10 induction by these inflammatory
cytokines. The Journal of Immunology, 1998, 161: 4736 – 4744.
The Journal of Immunology
Materials and Methods
Cell cultures and cytokines
Human fibrosarcoma 2fTGH cells and the mutants U2A and U3A, described earlier (12, 24), were grown in complete medium: DMEM with
10% FCS (Life Technologies, Gaithersburg, MD) and 2 mM L-glutamine;
U2A/p48, U3A/STAT1a, and U3A/STAT1b cells were maintained in
DMEM supplemented with 10% FCS in the presence of 250 mg/ml hygromycin and 450 mg/ml G418 (25, 26). Purified IFN-g (1.9 3 107 IU/mg
protein) was obtained from Genentech (South San Francisco, CA). TNF-a
(50 mg lyophilized powder) was obtained from Collaborative Biomedical
Products (Bedford, MA). IFN-g was used routinely at 500 IU/ml and
TNF-a at 50 ng/ml, unless otherwise indicated.
RNA isolation and RNase protection assay
Total cellular RNA was isolated from 70% confluent cells using TRIzol
(Life Technologies) according to manufacturer’s instructions (27) and
RNase protection assay was performed as described previously (28).
hIP-10 probe protects a 500-base fragment of hIP-10. The 1-kb hIP-10
cDNA insert from pIFNg-31.7 plasmid (a generous gift from Dr. J. V.
Ravetch, Dewitt Wallace Research Laboratory, Memorial Sloan-Kettering
Cancer Center, New York, NY) was excised and subcloned at the PstI site of
pBluescript and designated pBS-hIP10. The hIP-10 probe was generated after
cleaving plasmid pBS-hIP10 with restriction enzyme BglII. From the linearized plasmids [a-32P]UTP, labeled riboprobes were generated by in vitro transcription as described, using T3 RNA polymerase (Boehringer Mannheim).
The g-actin probe which protects a 160-base fragment was generated in a
similar manner using SP6 RNA polymerase (Boehringer Mannheim, Indianapolis, IN) (21). Total cellular RNA (10–20 mg) was hybridized with 250,000
cpm of hIP-10 cRNA probe and 7,000 cpm of g-actin probe.
Promoter-reporter plasmid construction and mutagenesis
A 972-bp DNA fragment was generated from CRT astrocytoma cell DNA
by PCR, using F4IP-10 (59-GAACCCCATCGTAAATCAACCTG-39) and
B7IP-10 (59-GCAGCAAATCAGAATGGCAGTTTG-39) for forward and
backward primers, respectively. The 972-bp fragment was cloned into the
pCRII vector (Invitrogen, San Diego, CA), excised with SacI and XhoI, and
inserted into the promoterless pGL3-basic vector (Promega, Madison, WI).
Orientation was verified by restriction digestion, and the vector insert
boundaries were verified by sequence analysis. The promoter-reporter construct, designated GL-IP10, has the 59-flanking region of the human IP-10
gene from 2875 to 197 (relative to the transcriptional start site) including
the native context initiation site.
A 440-bp fragment was excised from GL-IP10 by digestion with SpeI
and AspI, blunt ended with Klenow enzyme (Boehringer Mannheim), and
then religated to generate the truncated GL-IP10, designated TGL-IP10
(Fig. 1).
Site-directed mutagenesis of IP-10 ISRE and IP-10 kB2 sites was done
by multiple rounds of PCR using the TGL-IP10 plasmid and appropriate
primers with altered bases, following essentially the method of Aiyar and
Leis (29). Mutagenic bases are indicated below by lower case letters. The
primers used to make pIP10 mISRE3 mutant were: pair 1; IP10-XHOI
(59-CCAACAGTACCGGAATGCCAAG-39), complementary to the XhoI
end of the DNA fragment, and pIP10 mISRE3-1 (59-TTCATGTTTTG
GAAAtaGAAACCTAATTC-39); pair 2, IP10-SACI (59-GCAGGTGCC
AGAACATTTCTCTATC-39), complementary to the SacI end of the
DNA fragment, and pIP10 mISRE3-2 (59-GAATTAGGTTTCtaTTTCCAA
AACATG-39).
For mutating the NFkB2 site, the primers used were: kB2mut-1 (59AAGAGGAGCAGAGtGAAATTaCGTAACTTGGAG-39) and kB2mut-2
(59-CCTCCAAGTTACGtAATTTCaCTCTGCTCCTC-39).
The first two rounds of PCR generated two fragments of DNA, with 30
bp of overlap, extending both upstream and downstream of the ISRE site.
These two fragments were gel purified and used as the templates for a third
PCR with IP10-XHOI and IP10-SACI primers, to generate a full-length
mutagenized DNA fragment. This mutated hIP-10 DNA was then subcloned in pGL3-basic and verified by sequence analysis.
Transient transfection assay
2fTGH and its mutants were grown to 70 to 80% confluency in 100-mm
plates and transfected with 20 mg of the plasmid DNA using polybrene (10
mg/ml) for 6 h at 37°C as described (30). After incubation, the cells were
subjected to DMSO shock for 100 s (30% DMSO in DMEM), washed,
allowed to recover overnight from DMSO shock, pooled from several
plates to adjust for differential transfection efficiency, counted, and equally
redistributed in several plates. The cells were reserved as controls or treated
with cytokines for 6 h, washed and incubated overnight to allow luciferase
protein to accumulate, harvested, and lysed, and luciferase activity was
assayed (Promega), in a Luminometer (Dynatech Laboratories,
Chantilly, VA).
Transfection experiments were performed two to four times each, as
indicated in the figure legends. Data are presented in tabular form as
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Early investigations of human IP-10 (hIP-10) regulation disclosed a very prominent synergy between IFN-g and TNF-a for its
expression in keratinocytes, a major site of synthesis in immunemediated cutaneous disorders such as tuberculoid leprosy (17, 18).
Numerous genes of biological significance are induced synergistically by IFN-g and TNF-a, but the mechanism underlying this
effect has been evaluated in detail for only a few cases. Johnson
and Pober (19) analyzed synergistic induction of MHC class I
heavy chain mRNA in HeLa cells treated with IFN-g plus TNF-a.
Two cis-elements, an ISRE variant termed the IFN consensus sequence (ICS) and a NFkB binding site, were shown to be essential
for this response. It was proposed that transcriptional synergy for
this MHC class I promoter resulted from the sum of individual
interactions between IRF-1 bound to the ICS and NFkB associated
with its recognition site. Another mechanism of synergistic induction by IFN-g plus TNF-a was revealed by studies of ICAM-1
transcription (20). Upon TNF-a treatment, C/EBP (CCAAT/enhancer-binding protein) and NFkB bound cooperatively to the
ICAM-1 NFkB recognition site. IFN-g treatment induced activation of STAT-1a homodimers that bound to its target GAS motif.
Therefore, IFN-g plus TNF-a can act synergistically through promoters containing recognition sites for NFkB and elements that
respond to IFN-g (ISRE, ICS, or GAS).
Synergistic induction of the murine IP-10 (mIP-10) gene in NIH
3T3 cells has been examined (21). This induction is dependent on
the ISRE and on one of two NFkB recognition sites. STAT-1a was
detected in complexes that formed on the ISRE upon IP-10 gene
induction by IFN-g/TNF-a. However, the roles of ISRE recognition factors such as p48 and other IRF family members (IRF-1,
IRF-2) were not characterized in these studies.
In the current report, we address primarily the ISRE-binding
factors that govern synergistic induction of hIP-10 by IFN-g/
TNF-a. Our experiments made use of of specific reagents such as
human fibrosarcoma cell line 2fTGH and its derivative mutants
U2A and U3A, which selectively lack IFN signaling components
p48 and STAT1, respectively (22). Results from these experiments
demonstrated that induction of the hIP-10 gene by IFN-g alone or
in combination with TNF-a required both p48 and STAT-1a. We
also considered whether other inducible factors such as IRF-1
could be implicated in the maintenance of IFN-g-inducible transcription and in synergy with TNF-a. The potential involvement of
IRF-1 was addressed because it is an ISRE recognition factor that
can be induced by either IFN-g or TNF-a; furthermore, IRF-1 can
interact physically and functionally with NFkB (23). For these
experiments, an hIP-10 promoter-reporter mutant with decreased
affinity for IRF-1 was constructed; this construct was robustly expressed after treatment with IFN-g/TNF-a, suggesting that IRF-1
binding was not essential for synergistic induction of hIP-10. Consistent with this interpretation, in vivo genomic footprinting
(IVGF) analysis demonstrated a pattern of inducible protection of
the hIP-10 ISRE that would be predicted to occur after binding of
p48. Furthermore, this in vivo protection pattern was sustained
from 30 min through 2 h postinduction, i.e., during the time course
of inducible transcription of hIP-10. In summary, genetic, functional, and biochemical experiments indicated that p48 was required for hIP-10 induction by IFN-g alone or in synergy with
TNF-a. These studies demonstrate a novel mechanism of synergy
between IFN-g and TNF-a that utilizes p48/STAT-1a-containing
complexes and NFkB.
4737
4738
CYTOKINE INDUCTION OF HUMAN IP-10 GENE
FIGURE 1. Putative regulatory elements in
the hIP-10 promoter. Putative regulatory elements are shown in schematic form, with sequence content indicated above. Comparison
of hIP-10 and mIP-10 indicates identity of
three of these elements (ISRE, kB2, kB1; indicated by shaded or hatched boxes), except
that the ISRE-kB2 spacing in mIP-10 is 40 bp.
In the TGL-IP10 construct, a 440-bp fragment
is eliminated by truncation mutagenesis. Sequences of mutated ISRE motif in pmISRE3
and NFkB motif in kB2mut are indicated.
Preparation of nuclear extract
Nuclear extract was prepared according to a modification of the method of
Dignam (31). 2fTGH cells were grown to 70 to 80% confluency and treated
with cytokines for varying lengths of time or reserved as controls. The cells
were washed three times with ice-cold PBS, harvested, and resuspended in
500 ml of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.0 mM
MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, and 0.5 mM PMSF)
and homogenized by 10 strokes in a microglass homogenizer. The lysed
cell suspension was spun at 1,000 3 g for 10 min at 4°C and the supernatant decanted carefully. The nuclear pellet was spun again at 25,000 3
g for 20 min at 4°C. The supernatant was removed, and nuclear proteins
from the pellet were extracted with high salt buffer (20 mM HEPES, pH
7.9, 25% glycerol, 0.42 M NaCl, 1.0 mM MgCl2, 0.2 mM EDTA, 0.2 mM
EGTA, 1.0 mM DTT, 0.5 mM PMSF, 200 mM sodium orthovanadate, and
10 mg/ml each of leupeptin and antipain), first by 10 strokes in the microglass homogenizer, followed by incubation at 4°C for 30 min with intermittent vortexing. The nuclear extract was clarified by centrifugation and
dialyzed against low salt buffer (20 mM HEPES, pH 7.9, 20% glycerol, 75
mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM DTT, 0.5 mM PMSF,
200 mM sodium orthovanadate) and kept at 270°C until use.
Electrophoretic mobility shift assay (EMSA)
For binding reactions, 10 mg of nuclear extracts were incubated in 20 ml of
reaction mixture containing 20 mM HEPES (pH 7.9), 37.5 mM KCl, 0.2
mM EDTA, 1.0 mM DTT, 10% glycerol with 5 mg of poly deoxyinosinicdeoxycytidilic acid (poly(dI:dC); Pharmacia Biotech, Piscataway, NJ) for
15 min at 4°C. Oligonucleotide duplex probes (end labeled with T4
polynucleotide kinase and [g32P]ATP; 5 3 104 cpm) were then added to
reaction mixtures, which were incubated for 20 min at room temperature.
Reaction products were analyzed by nondenaturing electrophoresis in a 6%
polyacrylamide gel with 0.53 TBE buffer (44.6 mM Tris, 44.4 mM borate,
0.5 mM EDTA) at room temperature. Gels were dried and exposed to
X-ray film at 270°C for autoradiography. For competition experiments,
unlabeled oligonucleotides were added in molar equivalence or excess at
room temperature for 15 min before the addition of radiolabeled probe. For
supershift experiments, transcription factor Abs (Santa Cruz Biotechnology, Santa Cruz, CA) in 1 ml were incubated with the nuclear extract in
presence of poly(dI:dC) for 30 min on ice before the oligonucleotide probe
was added into the reaction mixture.
In vivo genomic foot printing
In vivo methylation of cellular DNA and DNA preparation was done as
described by Mueller et al. (32). Ligation-mediated PCR was carried out
according to the procedure of Mueller et al., with modifications as described by Ping et al. (33). The ISRE- and NFkB-binding sites were analyzed using one set of upper strand and one set of lower strand primers.
Primers used to read the upper strand were: hIP10-3-1, 59-TGCAAAGC
CATTTTCCCTCC-39; hIP10-3-2, 59-CGACTTAGCAAAACCTGCTG
GCTG-39; and hIP10-3-3, 59-CCTGCTGGCTGTTCCTGGGAAG-39. The
annealing temperatures for this set of primers were 59°C, 66°C, and 69°C,
respectively.
The sequences of primers to read the lower strand were: hIP10-5-1,
59-CAAGGAGGACTGTCCAGGTAAATC-39; hIP10-5-2, 59-CTGT
TCTAATAAT CAGGCACAACTTGC-39; and hIP10-5-3, 59-GGCA
CAACTTGCTGTTACCAA AAAATTAGG-39. The annealing temperatures were 57°C, 61°C, and 65°C, respectively.
Results
hIP-10 is synergistically induced by IFN-g and TNF-a
Accumulation of hIP-10 mRNA was barely detectable in 2fTGH
cells treated with either IFN-g or TNF-a. However, exposure of
2fTGH cells to IFN-g in combination with TNF-a (IFN-g/TNF-a)
resulted in the robust induction of hIP-10 gene expression as monitored by RNase protection assay (Fig. 2, A and B, lanes 1– 4).
Nuclear run-on experiments indicated that IP-10 mRNA accumulation after IFN-g or IFN-g/TNF-a treatment was determined by
increased transcriptional initiation, beginning after 30 to 60 min of
cytokine exposure (50). IP-10 transcription returned to undetectable levels after three hours of cytokine treatment.7
p48 is essential for IFN-g to induce expression of hIP-10, either
alone or in combination with TNF-a
p48 mediates ISRE-dependent responses to both type I and type II
IFNs (10, 17). The role of p48 in synergy with TNF-a has not been
addressed, and its requirement for the expression of IP-10 has not
been established. To determine whether p48 is needed for hIP-10
gene expression. We performed RNase protection assays in mutant
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means 6 SD of fold induction, with luciferase expression in control cells
set at 1. Bar histograms show the mean fold induction.
The Journal of Immunology
4739
response of U2A/p48 cells to IFN-g may be attributed to the inability of IFN-g to up-regulate expression of the transfected p48
gene, since the endogeous gene is IFN-g sensitive (34).
A 435-bp hIP-10 genomic fragment governs simple induction by
IFN-g and synergistic response with TNF-a
FIGURE 2. Induction of hIP-10 mRNA in 2fTGH and U2A cells. A,
RNase protection assay of total RNA (20 mg) isolated from 2fTGH, U2A,
and U2A/p48 cells, treated with IFN-g (500 U/ml) and/or TNF-a (50 ng/
ml) as indicated for 4 h. The dried gel was exposed to film for 2 h to obtain
the hIP-10 signal for synergistic induction. B, the same gel as shown in A
was exposed to film for 24 h to demonstrate hIP-10 expression in response
to IFN-g or TNF-a. C, The densitometric ratio of hIP-10/g-actin was quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) from
data shown in autoradiograms in A (graph ii) and B (graph i).
U2A (lacking p48) cells treated with IFN-g with or without TNF-a
(Fig. 2, A and B). In U2A cells, hIP-10 message failed to accumulate upon treatment to IFN-g alone (Fig. 2, A and B, lanes 5– 8),
and the synergistic response to IFN-g/TNF-a was reduced by
.80% (Fig. 2C). In U2A cells complemented with p48 by transfection (U2A/p48) (20), hIP-10 mRNA induction by IFN-g alone
or in combination with TNF-a was rescued (Fig. 2, A–C). Reduced
We addressed the role of p48 in hIP-10 gene transcription by transient transfection experiments. TGL-IP10, a promoter-reporter
containing 435 bp of hIP-10 sequence upstream of the transcriptional start site, recapitulated the regulation of the endogenous
gene in response to IFN-g, TNF-a, and IFN-g/TNF-a (Fig. 3).
Computer-assisted inspection of this region of the hIP-10 promoter
and sequence alignment with the well-characterized mIP-10 promoter revealed conservation of potential regulatory elements: an
ISRE homology and two NFkB binding site motifs. The organization and sequence content of these potential regulatory motifs in
hIP-10 and mIP-10 were precisely conserved; strikingly, the remainder of ;0.5 kb near the transcription start site was divergent.
Several preliminary experiments were performed to establish the
functional equivalence of the ISRE and NFkB elements of the two
promoters. Mutations in the ISRE abrogated induction of the
hIP-10 promoter-reporter by IFN-g, either alone or in combination
with TNF-a (data not shown). Mutations in either of the two putative NFkB binding sites drastically diminished response to
TNF-a alone or in combination with IFN-g (data not shown).
These results indicated that the response of the hIP-10 and mIP-10
genes to IFN-g and TNF-a were dictated by conserved regulatory
elements in the respective promoters.
To address the role of p48 in hIP-10 gene transcription, we
analyzed the regulation of TGL-IP10 in parental 2fTGH and U2A
cells. In 2fTGH cells, we observed an ;3-fold induction of luciferase activity by IFN-g alone, 8-fold induction by TNF-a, and
18-fold induction by IFN-g/TNF-a (Fig. 3). In U2A cells, there
was no induction of luciferase activity by IFN-g alone, whereas the
TNF-a response remained intact, being mediated by NFkB rather
than p48. The response to IFN-g/TNF-a was markedly reduced
and not statistically different from induction by TNF-a alone. In
U2A/p48 cells, responses to IFN-g and IFN-g/TNF-a were rescued (data not shown). These data indicated that p48 is essential
for the transcription of hIP-10 gene by IFN-g, alone or in combination with TNF-a.
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FIGURE 3. Transient transfection analysis of TGL-IP10 in U2A cells.
TGL-IP10 was transiently transfected into 2fTGH cells or U2A cells, and
cytokine-inducible (IFN-g (500 U/ml) or IFN-g/TNF-a (50 ng/ml)) promoter activity monitored by luciferase assay. Table on right shows means
and SD of four independent experiments, with means indicated by bar
histograms at left. The simple response to IFN-g is absent in U2A cells,
while the simple response to TNF-a is unaffected. Response to IFN-g/
TNF-a in U2A is no longer synergistic, but is increased over that induced
by TNF-a alone, possibly because of the action of IRF-1.
4740
CYTOKINE INDUCTION OF HUMAN IP-10 GENE
complemented with STAT-1a but not STAT-1b. It was also
shown that STAT2 was not implicated in the response to IFN-g/
TNF-a by demonstrating that regulation of the TGL-IP10 promoter-reporter in U6A cells (lacking STAT2) was indistinguishable
from 2fTGH cells (data not shown). These results indicated a specific requirement for STAT-1a in the induction of the hIP-10 gene
by IFN-g/TNF-a.
The pIP10 mISRE3 promoter-reporter, which is relatively
deficient for IRF-1 binding, exhibits synergistic response to
IFN-g/TNF-a
STAT-1a is essential for IP-10 gene induction by IFN-g in the
presence or absence of TNF-a
It has been documented that p48 exhibits low-affinity binding activity toward the ISRE unless it is complexed with the activated
STAT components. Further, p48 does not possess transactivation
competence unless associated with STATs (34). We addressed the
role of STAT1 proteins in hIP-10 induction through the use of
U3A cells and in cells complemented with either of the two alternatively spliced isoforms STAT-1a and STAT-1b.
Functional involvement of STAT-1a in hIP-10 gene induction
was explored by nuclease protection experiments in U3A cells
lacking the STAT-1 gene. IP-10 mRNA accumulation failed to
occur in U3A cells stimulated by IFN-g or IFN-g/TNF-a (Fig. 4).
The response to IFN-g or IFN-g/TNF-a was restored in U3A cells
FIGURE 5. EMSA analysis of wildtype and mutant-3 ISRE (mISRE3).
EMSA analysis of extracts of 2fTGH
cells left untreated (0) or exposed to
IFN-g (g) overnight and then to IFN-g/
TNF-a (g/T) for 4 h as indicated. Oligodeoxynucleotide probes correspond
to wild-type hIP-10 ISRE (ISREw) or
mutant-3 (mISRE3). Each probe supports the formation of two inducible
comigrating complexes, which contain
IRF-1 or p48, by Ab supershift analysis.
The abundance of the IRF-1-containing
complex was substantially decreased in
analysis of the mISRE probe (left panel), while the p48-complex was
unaffected.
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FIGURE 4. Induction of hIP-10 in U3A cells complemented with
STAT-1a or STAT-1b. U3A cells were complemented individually with
STAT-1a (U3A/p91) or STAT-1b (U3A/p84) and left untreated or treated
with (IFN-g (500 U/ml) or IFN-g/TNF-a (50 ng/ml)) for 4 h. hIP-10 and
g-actin was analysed by RNase protection assay. The densitometric ratio of
hIP-10 and g-actin was determined and compared for different cell lines.
Synergistic induction of hIP-10 gene was significantly reduced in
U2A cells that lacked p48. Two possible pathways of p48-dependent synergy could therefore be envisioned. In one scenario, p48
and associated STAT factors initiate IFN-g-dependent transcription, with subsequent replacement by IRF-1, which would directly
mediate synergy, along with NFkB (23). Alternatively, p48 and
STAT factors might initiate and sustain the synergistic response,
acting in concert with factors bound at the NFkB recognition sites.
To address this issue, we constructed a mutant of TGL-IP10 in
which two core nucleotides of the ISRE element were altered from
CG to TA (pIP10 mISRE3) to decrease IRF binding selectively
while retaining affinity for p48 (35) (Fig. 1). EMSA was performed
with the mISRE3 oligonucleotide as probe to confirm reduced
IRF-1 binding. Figure 5 shows that IRF-1 binding to the mISRE3
mutant oligonucleotide is substantially reduced, whereas p48 binding is not affected (compare lane 2 with lane 3 and lane 4 with lane
5). Ab supershift analysis shows availability of both p48 and IRF-1
for mISRE3 binding during the course of IFN-g/TNF-a treatment;
these experiments also detected IRF-2 as a minor component of the
complex (Fig. 5, lanes 9 –11). Identical data were obtained in Ab
supershift experiments using the wild-type ISRE EMSA probe
(data not shown).
Participation of two positive transactivators (p48, IRF-1) in
IP-10 induction could be rationalized, based on analogy with other
ISRE-containing promoters. For example, prior work demonstrated rapid (within 15–30 min) IFN-g-inducible binding of p48containing complexes to the 6-16 and 9-27 ISRE elements, with
The Journal of Immunology
subsequent (2– 4 h) binding of IRF-1 (36). These investigators proposed that p48 (with associated factors) was involved in initiating
transcription, which was subsequently sustained by IRF-1.
2fTGH cells were transiently transfected with the pIP10
mISRE3 mutant or TGL-IP10 and treated with IFN-g and/or
TNF-a. Decreased synergistic response of the pIP10 mISRE3 mutant was anticipated if IRF-1 served as an essential mediator of
interaction between IFN-g/TNF-a. Synergistic induction of luciferase activity in cells transfected with the pIP10 mISRE3 mutant
was clearly demonstrated and was enhanced compared with TGLIP10 (Fig. 6). These results suggested that IRF-1 is not required as
a positive regulator of hIP-10 in these cells, either in response to
IFN-g alone or IFN-g/TNF-a.
hIP-10 ISRE and NFkB binding sites become occupied in vivo
upon treatment with IFN-g/TNF-a
Our experiments in mutant cell lines clearly showed that transcriptional initiation of hIP-10 ISRE by IFN-g or IFN-g/TNF-a is dependent on p48 and STAT-1a. Further, results of transient transfections with the pIP10 mISRE3 mutant provided support for the
concept that IRF-1 is not required for synergistic induction of
hIP-10 by IFN-g and TNF-a. However, in vitro EMSA experiments indicated that IRF-1 was activated by treatment with IFN-g
or IFN-g/TNF-a and could bind to the hIP-10 ISRE homology
motif. Therefore, IVGF was performed to define the occupancy of
the ISRE site during the induction of the hIP-10 gene by
IFN-g/TNF-a.
The ISRE core coding strand residue G/2214 (relative to the
transcription start site) was strongly protected after IFN-g/TNF-a
treatment (Fig. 7, A–C). This residue is conserved in all ISRE
elements that respond to either IFN-a/b or IFN-g. The coding
strand G/2216 residue, which would be predicted to be protected
by IRF-1 binding based on the in vitro sequence preference of the
factor (35–37), was only weakly protected after 30 min of cytokine
treatment and was unprotected after 2 h, at peak transcriptional
activation of the gene (Fig. 7C, inset). Otherwise, identical protection patterns over the ISRE were observed after 30 min of induction by IFN-g/TNF-a (at an early time point after hIP-10 transcription is detected) and after 2 h. These observations argue that
IRF-1 may not bind the hIP-10 ISRE under these conditions of
synergistic induction. In vitro EMSA experiments (Fig. 5) detected
high-level IRF-1 binding activity in nuclear extracts of 2fTGH
cells under these conditions of cytokine treatment, indicating that
the factor was available in the nucleus. The in vivo protection
pattern on the hIP-10 ISRE homology under conditions of optimal
transcriptional activation of hIP-10 was therefore consistent with
the presence of p48 rather than IRF-1. This observation, taken in
the context of our results using U2A and U3A mutant cell lines and
the pIP10 mISRE3 promoter, indicates that p48/STAT-1a complexes are responsible for the response of hIP-10 to IFN-g in these
cells. Participation of IRF-1 or other family members via weak
binding not detected in these assays cannot, however, be addressed
by these experiments.
The GG/2220/2221 residues in the ISRE coding strand became
inducibly DMS resistant; G/2238 upstream of the ISRE is also
partially protected under conditions in which hIP-10 is synergistically induced (Fig. 7). In the noncoding strand, ISRE residues
GG/2209/2210 become DMS resistant upon treatment with IFNg/TNF-a (Fig. 7B). Additionally, G/2229 and G/2241 upstream
of the ISRE are protected (Fig. 7, B and C). Thus, IFN-g/TNF-a
treatment leads to in vivo protection of an extended, asymmetric
ISRE motif, consistent with the binding of p48 and associated
STAT factors.
We also observed IFN-g/TNF-a-inducible footprinting of the
kB2 NFkB binding site, on both the coding strand (G/2174/2175/
2176) and the noncoding strand (G/2168/2167) (Fig. 7). By transient transfection studies with mutant promoter-reporters, the kB2
site was found to be required for full synergistic induction of
hIP-10 (data not shown) as it was in mIP-10 (21). EMSA and
supershift analysis indicated that the protein complex associating
with kB2 site is composed of p65 homodimers (Fig. 8). The failure
of other rel family Abs to supershift this complex was not technical in nature, as these Abs are of demonstrated competence in
other supershift experiments. Further, the pattern of complex formation on the kB2 element was identical regardless of whether
cells were treated with TNF-a alone or with TNF-a/IFN-g (data
not shown). The kB1 NFkB recognition site also became inducibly
DMS resistant in IFN-g/TNF-a-treated cells, at G/2118/2119/
2120 on the coding strand (data not shown) and at G/2111/2112
on the noncoding strand (Fig. 7). These observations complemented transfection studies with mutant promoters that showed a
contribution of both kB2 and kB1 to full synergistic induction of
hIP-10 and mIP-10 by IFN-g/TNF-a (data not shown) (21).
Discussion
The current report documents a novel mechanism of synergy between two proinflammatory cytokines, IFN-g and TNF-a, for transcriptional induction of the hIP-10 gene. This transcriptional synergy operates through two well-characterized cis elements, the
hIP-10 ISRE homology and NFkB binding sites. The protein factors that mediate transcriptional synergy for hIP-10 induction are
the p48 ISRE recognition factor, STAT-1a, and p65-homodimeric
NFkB. Data that support these conclusions are as follows: 1) there
is little or no hIP-10 response to IFN-g in U2A cells lacking p48
and in U3A cells lacking STAT-1a; 2) integrity of the ISRE is
absolutely required for response to IFN-g; 3) the kB2 site is important for TNF-a to contribute to transcriptional synergy with
IFN-g; 4) IRF-1 is not essential, as indicated by intact IFN-g/TNFa-induced expression of pIP10 mISRE3, a promoter mutant that is
deficient for IRF binding; and 5) IVGF demonstrated binding consistent with p48 and associated STATs.
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FIGURE 6. Transient transfection analysis of the pIP10 mISRE3 mutant. TGL-IP10 or pIP10 mISRE3 were transiently transfected into 2fTGH
cells and cytokine-inducible (IFN-g (500 U/ml) or IFN-g/TNF-a (50 ng/
ml)) promoter activity monitored by luciferase assay. The simple response
to IFN-g and to TNF-a are unaffected by introduction of the mutation in
pIP10 mISRE3. Response to IFN-g/TNF-a of pIP10 mISRE3 is highly
synergistic and is increased over that of the TGL-IP10 construct.
4741
4742
CYTOKINE INDUCTION OF HUMAN IP-10 GENE
Previous analysis of transcriptional synergy for the mIP-10
gene in fibroblasts indicated a requirement for the ISRE and for
one NFkB binding site (21). This region corresponds to an IFNg-inducible DNase I hypersensitive site that was identified be-
tween residues 2260 and 260 in the hIP-10 gene, using U937
cells (38). The activation and promoter binding of STAT-1a
was also demonstrated in studies of mIP-10 induction by IFNg/TNF-a (21). Our investigation of hIP-10 regulation confirms
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FIGURE 7. IVGF analysis of hIP-10 promoter in cells treated with IFN-g or IFN-g/TNF-a. A, Proximal regulatory elements of the hIP-10 promoter.
ISRE, kB2, and kB1 elements are indicated by hatched regions, with CCAAT and TATA elements indicated by open boxes. Bases at the 59-terminus of
each cytokine-governed regulatory element are indicated, with reference to the transcriptional start site defined by Luster and Ravetch (38). B, Schematic
of cytokine-inducible DMS protection in the hIP-10 promoter. The nucleotide sequence of the hIP-10 promoter, from 2242 to 2110 and encompassing
the ISRE, kB2, and kB1 elements, is shown. Putative regulatory elements are delimited by boxes. Residues that become DMS-resistant after treatment with
IFN-g/TNF-a are indicated with vertical arrows. ISRE residue 2214, which is strongly protected, is indicated with an asterisk. C, IVGF analysis of hIP-10
promoter. 2fTGH cells were reserved as controls or treated for 30 min with IFN-g (500 U/ml), with or without pretreatment for 4 h with TNF-a (50 ng/ml),
as indicated, before preparation of genomic DNA and IVGF analysis. The autoradiograms shows the coding strand (left) and noncoding strand (right). In
each set of four lanes, the first lane shows genomic DNA analyzed in vitro. The remaining three lanes represent analysis of DNA methylated in vivo; the
second lane from left shows DNA from untreated cells; the remaining lanes show analysis of DNA from cells treated with IFN-g or IFN-g/TNF-a. The
ISRE and kB2 sequences on the coding strand are shown at left. In the sequence presentation, asterisks indicate protected residues and double asterisks show
the strongly protected residue 2214. The plus sign (1) indicates unprotected residue 2216. Arrows to the right of the autoradiograms indicate protected
residues and a bolded arrow shows strongly protected residue 2214. The open arrow indicates a guanidine residue (G/2216), which would be predicted
to be protected by IRF-1 binding, but which remains methylation sensitive in this analysis. TNF-a-inducible protection of the kB1 and kB2 sites is also
demonstrated. Numbers to the right of individual arrowheads correspond to numbering of residues upstream of the transcription start site, as shown in A.
The inset shows IVGF analysis of the coding strand ISRE region. For this experiment, cells were reserved as controls, or treated with IFN-g (500 U/ml)
for 2 h in the presence or absence of TNF-a (50 ng/ml). Lanes show analysis of DNA methylated in vivo: the first lane shows DNA from untreated cells;
the remaining lanes show analysis of DNA from cells treated with IFN-g, TNF-a, and IFN-g/TNF-a. Designation of arrows and numbering of G residues
is the same as in C. The results indicate that residue 2216, the putative IRF-1 contact site, remains unprotected from the initiation through the peak of
hIP-10 transcription.
The Journal of Immunology
4743
these prior observations and provides complementary new information about the ISRE-binding factors that mediate synergistic induction of hIP-10. In particular, the current report documents a requirement for p48 in the expression of hIP-10. In
studies of the mIP-10 gene, p50/p65 NFkB was formed in response to IFN-g/TNF-a; in the current experiments, only p65/
p65 complexes were detected. Cell type-specific expression of
the varied species of NFkB has been described frequently, and
p65 homodimers have been previously reported as mediators of
synergistic response to IFN-g/TNF-a for induction of the human IL-6 gene (39).
IVGF experiments demonstrated IFN-g/TNF-a-inducible asymmetric DMS resistance over the ISRE consensus, a binding activity
consistent with p48/STAT-1a complexes (2221/220 and 2214 on
the coding strand and 2210/209 on the noncoding strand). Results
from these IVGF experiments suggested a specific contact between
p48 and the 39 ISRE half-site and were consistent with the p48
recognition site defined by Qureshi et al. using UV cross-linking
(40).
The functions of p48 and IRF-1 are distinct, although both factors recognize ISRE motifs through similar N termini. In particular, IRF-1 appears to have transactivating competence, while p48
operates only by recruiting STATs to the target DNA element; the
p48-associated STAT factors promote high-affinity binding and
provide transactivation function (22). In this regard, IFN-g and
TNF-a can also act synergistically through NFkB recognition sites
and STAT-1a/GAS interactions, as shown convincingly for
ICAM-1. The detailed mechanisms by which these varied factors
mediate transcriptional synergy remain uncertain. In vitro mixing
experiments were used to demonstrate physical interaction between IRF-1/2 and NFkB components, but evidence that such contacts occur in vivo has not been reported (41, 42). For synergistic
induction of MHC class I heavy chain, IRF-1 and NFkB were
implicated (19). The MHC class I heavy chain and hIP-10 regulatory regions are structurally comparable because both promoters
contain ISRE/ICS elements that cooperate functionally with NFkB
binding-sites. Therefore, the current report indicates at least two
mechanisms by which IFN-g and TNF-a can activate similar promoter elements.
Structurally similar inducible promoters, containing ISRE-like
elements that cooperate with NFkB recognition sites, have been
studied in some detail. For example, the IFN-b gene contains tandem ICS-like recognition sites for IRF-1, flanked by binding motifs for NFkB. IRF-1 and NFkB factors act to induce IFN-b transcription in concert with a nonhistone chromosomal component,
high mobility group (HMG) protein I(Y), that appears to stabilize
interactions between NFkB and DNA target sequences (43– 47). A
striking mechanistic similarity between virus induction of IFN-b
and TNF-a-mediated induction of E-selectin has been described
(48). It is clear, therefore, that several stimuli can activate inducible promoters through the combined action of IRF-1 and NFkB.
The coordinated action of p48, STAT-1a, and NFkB is likely to be
restricted to circumstances of immune-mediated inflammation in
which IFN-g plays a cardinal role (49).
Acknowledgments
We thank Dr. Tom Hamilton for helpful comments on the manuscript.
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