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The Journal of Immunology, 1998, 161: 4736-4744.
Copyright © 1998 by The American Association of Immunologists

p48/STAT-1{alpha}-Containing Complexes Play a Predominant Role in Induction of IFN-{gamma}-Inducible Protein, 10 kDa (IP-10) by IFN-{gamma} Alone or in Synergy with TNF-{alpha}1

Sarmila Majumder2,*, Lucy Z.-H. Zhou*, Priya Chaturvedi3,*, Gerald Babcock*, Sumer Aras4,* and Richard M. Ransohoff5,*,{dagger}

* Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, and {dagger} Neurology Department, Mellen Center for Multiple Sclerosis Treatment and Research, Cleveland Clinic Foundation, Cleveland, OH 44195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human IFN-{gamma}-inducible protein, 10 kDa (hIP-10) and murine IP-10 (mIP-10) genes are induced by IFN-{gamma} alone, and synergistically induced by TNF-{alpha} and IFN-{gamma}. 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-{gamma}. Trans-acting factors mediating the IFN-{gamma} 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-{gamma}, with or without TNF-{alpha}, 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-{gamma}/TNF-{alpha}-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-{alpha}/IFN-{gamma} also required NF{kappa}B binding sites, which were protected in vivo and bound p65 homodimeric NF{kappa}B in vitro. These results documented the essential role of p48 (complexed with STAT-1{alpha}) 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma}-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-{gamma}-treated U937 cells, a histiocytic lymphoma cell line with monocytic characteristics (1). IFN-{gamma} 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, osteoblasts, and endothelial cells (5, 6, 7, 8, 9). IFN-{gamma} (type II IFN) has been studied extensively as a transcriptional regulator. The majority of responsive genes are induced by IFN-{gamma} through the interaction of STAT-1{alpha} homodimers (originally designated the {gamma}-activated factor (GAF) and an inducible palindromic enhancer termed the {gamma}-activated site (GAS) (10).

IFN-{gamma} also induces transcription of a subset of genes in a GAS-independent 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-{gamma} 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-{gamma} induction of the ISRE-dependent ISG54 gene included STAT-1{alpha} and p48 (14). This observation led to the proposal that IFN-{gamma} activated these genes through an alternative form of the IFN-stimulated gene factor 3 (ISGF3) complex (see below), composed of p48 and STAT-1{alpha} homodimers (14). The p48/STAT-1{alpha} 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-{alpha} 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-{alpha}-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).

Early investigations of human IP-10 (hIP-10) regulation disclosed a very prominent synergy between IFN-{gamma} and TNF-{alpha} for its expression in keratinocytes, a major site of synthesis in immune-mediated cutaneous disorders such as tuberculoid leprosy (17, 18). Numerous genes of biological significance are induced synergistically by IFN-{gamma} and TNF-{alpha}, 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-{gamma} plus TNF-{alpha}. Two cis-elements, an ISRE variant termed the IFN consensus sequence (ICS) and a NF{kappa}B 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 NF{kappa}B associated with its recognition site. Another mechanism of synergistic induction by IFN-{gamma} plus TNF-{alpha} was revealed by studies of ICAM-1 transcription (20). Upon TNF-{alpha} treatment, C/EBP (CCAAT/enhancer-binding protein) and NF{kappa}B bound cooperatively to the ICAM-1 NF{kappa}B recognition site. IFN-{gamma} treatment induced activation of STAT-1{alpha} homodimers that bound to its target GAS motif. Therefore, IFN-{gamma} plus TNF-{alpha} can act synergistically through promoters containing recognition sites for NF{kappa}B and elements that respond to IFN-{gamma} (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 NF{kappa}B recognition sites. STAT-1{alpha} was detected in complexes that formed on the ISRE upon IP-10 gene induction by IFN-{gamma}/TNF-{alpha}. 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-{gamma}/TNF-{alpha}. 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-{gamma} alone or in combination with TNF-{alpha} required both p48 and STAT-1{alpha}. We also considered whether other inducible factors such as IRF-1 could be implicated in the maintenance of IFN-{gamma}-inducible transcription and in synergy with TNF-{alpha}. The potential involvement of IRF-1 was addressed because it is an ISRE recognition factor that can be induced by either IFN-{gamma} or TNF-{alpha}; furthermore, IRF-1 can interact physically and functionally with NF{kappa}B (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-{gamma}/TNF-{alpha}, 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-{gamma} alone or in synergy with TNF-{alpha}. These studies demonstrate a novel mechanism of synergy between IFN-{gamma} and TNF-{alpha} that utilizes p48/STAT-1{alpha}-containing complexes and NF{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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/STAT1{alpha}, and U3A/STAT1ß cells were maintained in DMEM supplemented with 10% FCS in the presence of 250 µg/ml hygromycin and 450 µg/ml G418 (25, 26). Purified IFN-{gamma} (1.9 x 107 IU/mg protein) was obtained from Genentech (South San Francisco, CA). TNF-{alpha} (50 mg lyophilized powder) was obtained from Collaborative Biomedical Products (Bedford, MA). IFN-{gamma} was used routinely at 500 IU/ml and TNF-{alpha} 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 pIFN{gamma}-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 [{alpha}-32P]UTP, labeled riboprobes were generated by in vitro transcription as described, using T3 RNA polymerase (Boehringer Mannheim). The {gamma}-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 µg) was hybridized with 250,000 cpm of hIP-10 cRNA probe and 7,000 cpm of {gamma}-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 (5'-GAACCCCATCGTAAATCAACCTG-3') and B7IP-10 (5'-GCAGCAAATCAGAATGGCAGTTTG-3') 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 5'-flanking region of the human IP-10 gene from -875 to +97 (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. 1Go).



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  		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, {kappa}B2, {kappa}B1; indicated by shaded or hatched boxes), except that the ISRE-{kappa}B2 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 NF{kappa}B motif in {kappa}B2mut are indicated.

 
Site-directed mutagenesis of IP-10 ISRE and IP-10 {kappa}B2 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 (5'-CCAACAGTACCGGAATGCCAAG-3'), complementary to the XhoI end of the DNA fragment, and pIP10 mISRE3-1 (5'-TTCATGTTTTGGAAAtaGAAACCTAATTC-3'); pair 2, IP10-SACI (5'-GCAGGTGCCAGAACATTTCTCTATC-3'), complementary to the SacI end of the DNA fragment, and pIP10 mISRE3-2 (5'-GAATTAGGTTTCtaTTTCCAAAACATG-3').

For mutating the NF{kappa}B2 site, the primers used were: {kappa}B2mut-1 (5'-AAGAGGAGCAGAGtGAAATTaCGTAACTTGGAG-3') and {kappa}B2mut-2 (5'-CCTCCAAGTTACGtAATTTCaCTCTGCTCCTC-3').

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 µg of the plasmid DNA using polybrene (10 µg/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 means ± SD of fold induction, with luciferase expression in control cells set at 1. Bar histograms show the mean fold induction.

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 µl 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 x g for 10 min at 4°C and the supernatant decanted carefully. The nuclear pellet was spun again at 25,000 x 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 µM sodium orthovanadate, and 10 µg/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 µM sodium orthovanadate) and kept at -70°C until use.

Electrophoretic mobility shift assay (EMSA)

For binding reactions, 10 µg of nuclear extracts were incubated in 20 µl 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 µg of poly deoxyinosinic-deoxycytidilic acid (poly(dI:dC); Pharmacia Biotech, Piscataway, NJ) for 15 min at 4°C. Oligonucleotide duplex probes (end labeled with T4 polynucleotide kinase and [{gamma}32P]ATP; 5 x 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 0.5x 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 -70°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 µl 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 NF{kappa}B-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, 5'-TGCAAAGCCATTTTCCCTCC-3'; hIP10-3-2, 5'-CGACTTAGCAAAACCTGCTGGCTG-3'; and hIP10-3-3, 5'-CCTGCTGGCTGTTCCTGGGAAG-3'. 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, 5'-CAAGGAGGACTGTCCAGGTAAATC-3'; hIP10-5-2, 5'-CTGTTCTAATAAT CAGGCACAACTTGC-3'; and hIP10-5-3, 5'-GGCACAACTTGCTGTTACCAA AAAATTAGG-3'. The annealing temperatures were 57°C, 61°C, and 65°C, respectively.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hIP-10 is synergistically induced by IFN-{gamma} and TNF-{alpha}

Accumulation of hIP-10 mRNA was barely detectable in 2fTGH cells treated with either IFN-{gamma} or TNF-{alpha}. However, exposure of 2fTGH cells to IFN-{gamma} in combination with TNF-{alpha} (IFN-{gamma}/TNF-{alpha}) resulted in the robust induction of hIP-10 gene expression as monitored by RNase protection assay (Fig. 2Go, A and B, lanes 1–4). Nuclear run-on experiments indicated that IP-10 mRNA accumulation after IFN-{gamma} or IFN-{gamma}/TNF-{alpha} 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



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  		FIGURE 2. Induction of hIP-10 mRNA in 2fTGH and U2A cells. A, RNase protection assay of total RNA (20 µg) isolated from 2fTGH, U2A, and U2A/p48 cells, treated with IFN-{gamma} (500 U/ml) and/or TNF-{alpha} (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-{gamma} or TNF-{alpha}. C, The densitometric ratio of hIP-10/{gamma}-actin was quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) from data shown in autoradiograms in A (graph ii) and B (graph i).

 
p48 is essential for IFN-{gamma} to induce expression of hIP-10, either alone or in combination with TNF-{alpha}

p48 mediates ISRE-dependent responses to both type I and type II IFNs (10, 17). The role of p48 in synergy with TNF-{alpha} 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 U2A (lacking p48) cells treated with IFN-{gamma} with or without TNF-{alpha} (Fig. 2Go, A and B). In U2A cells, hIP-10 message failed to accumulate upon treatment to IFN-{gamma} alone (Fig. 2Go, A and B, lanes 5–8), and the synergistic response to IFN-{gamma}/TNF-{alpha} was reduced by >80% (Fig. 2GoC). In U2A cells complemented with p48 by transfection (U2A/p48) (20), hIP-10 mRNA induction by IFN-{gamma} alone or in combination with TNF-{alpha} was rescued (Fig. 2Go, A–C). Reduced response of U2A/p48 cells to IFN-{gamma} may be attributed to the inability of IFN-{gamma} to up-regulate expression of the transfected p48 gene, since the endogeous gene is IFN-{gamma} sensitive (34).

A 435-bp hIP-10 genomic fragment governs simple induction by IFN-{gamma} and synergistic response with TNF-{alpha}

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-{gamma}, TNF-{alpha}, and IFN-{gamma}/TNF-{alpha} (Fig. 3Go). 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 NF{kappa}B 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 NF{kappa}B elements of the two promoters. Mutations in the ISRE abrogated induction of the hIP-10 promoter-reporter by IFN-{gamma}, either alone or in combination with TNF-{alpha} (data not shown). Mutations in either of the two putative NF{kappa}B binding sites drastically diminished response to TNF-{alpha} alone or in combination with IFN-{gamma} (data not shown). These results indicated that the response of the hIP-10 and mIP-10 genes to IFN-{gamma} and TNF-{alpha} were dictated by conserved regulatory elements in the respective promoters.



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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-{gamma} (500 U/ml) or IFN-{gamma}/TNF-{alpha} (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-{gamma} is absent in U2A cells, while the simple response to TNF-{alpha} is unaffected. Response to IFN-{gamma}/TNF-{alpha} in U2A is no longer synergistic, but is increased over that induced by TNF-{alpha} alone, possibly because of the action of IRF-1.

 
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-{gamma} alone, 8-fold induction by TNF-{alpha}, and 18-fold induction by IFN-{gamma}/TNF-{alpha} (Fig. 3Go). In U2A cells, there was no induction of luciferase activity by IFN-{gamma} alone, whereas the TNF-{alpha} response remained intact, being mediated by NF{kappa}B rather than p48. The response to IFN-{gamma}/TNF-{alpha} was markedly reduced and not statistically different from induction by TNF-{alpha} alone. In U2A/p48 cells, responses to IFN-{gamma} and IFN-{gamma}/TNF-{alpha} were rescued (data not shown). These data indicated that p48 is essential for the transcription of hIP-10 gene by IFN-{gamma}, alone or in combination with TNF-{alpha}.

STAT-1{alpha} is essential for IP-10 gene induction by IFN-{gamma} in the presence or absence of TNF-{alpha}

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-1{alpha} and STAT-1ß.

Functional involvement of STAT-1{alpha} 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-{gamma} or IFN-{gamma}/TNF-{alpha} (Fig. 4Go). The response to IFN-{gamma} or IFN-{gamma}/TNF-{alpha} was restored in U3A cells complemented with STAT-1{alpha} but not STAT-1ß. It was also shown that STAT2 was not implicated in the response to IFN-{gamma}/TNF-{alpha} 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-1{alpha} in the induction of the hIP-10 gene by IFN-{gamma}/TNF-{alpha}.



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  		FIGURE 4. Induction of hIP-10 in U3A cells complemented with STAT-1{alpha} or STAT-1ß. U3A cells were complemented individually with STAT-1{alpha} (U3A/p91) or STAT-1ß (U3A/p84) and left untreated or treated with (IFN-{gamma} (500 U/ml) or IFN-{gamma}/TNF-{alpha} (50 ng/ml)) for 4 h. hIP-10 and {gamma}-actin was analysed by RNase protection assay. The densitometric ratio of hIP-10 and {gamma}-actin was determined and compared for different cell lines.

 
The pIP10 mISRE3 promoter-reporter, which is relatively deficient for IRF-1 binding, exhibits synergistic response to IFN-{gamma}/TNF-{alpha}

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-{gamma}-dependent transcription, with subsequent replacement by IRF-1, which would directly mediate synergy, along with NF{kappa}B (23). Alternatively, p48 and STAT factors might initiate and sustain the synergistic response, acting in concert with factors bound at the NF{kappa}B 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. 1Go). EMSA was performed with the mISRE3 oligonucleotide as probe to confirm reduced IRF-1 binding. Figure 5Go 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-{gamma}/TNF-{alpha} treatment; these experiments also detected IRF-2 as a minor component of the complex (Fig. 5Go, lanes 9–11). Identical data were obtained in Ab supershift experiments using the wild-type ISRE EMSA probe (data not shown).



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  		FIGURE 5. EMSA analysis of wild-type and mutant-3 ISRE (mISRE3). EMSA analysis of extracts of 2fTGH cells left untreated (0) or exposed to IFN-{gamma} ({gamma}) overnight and then to IFN-{gamma}/TNF-{alpha} ({gamma}/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.

 
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-{gamma}-inducible binding of p48-containing complexes to the 6-16 and 9-27 ISRE elements, with 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-{gamma} and/or TNF-{alpha}. Decreased synergistic response of the pIP10 mISRE3 mutant was anticipated if IRF-1 served as an essential mediator of interaction between IFN-{gamma}/TNF-{alpha}. Synergistic induction of luciferase activity in cells transfected with the pIP10 mISRE3 mutant was clearly demonstrated and was enhanced compared with TGL-IP10 (Fig. 6Go). These results suggested that IRF-1 is not required as a positive regulator of hIP-10 in these cells, either in response to IFN-{gamma} alone or IFN-{gamma}/TNF-{alpha}.



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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-{gamma} (500 U/ml) or IFN-{gamma}/TNF-{alpha} (50 ng/ml)) promoter activity monitored by luciferase assay. The simple response to IFN-{gamma} and to TNF-{alpha} are unaffected by introduction of the mutation in pIP10 mISRE3. Response to IFN-{gamma}/TNF-{alpha} of pIP10 mISRE3 is highly synergistic and is increased over that of the TGL-IP10 construct.

 
hIP-10 ISRE and NF{kappa}B binding sites become occupied in vivo upon treatment with IFN-{gamma}/TNF-{alpha}

Our experiments in mutant cell lines clearly showed that transcriptional initiation of hIP-10 ISRE by IFN-{gamma} or IFN-{gamma}/TNF-{alpha} is dependent on p48 and STAT-1{alpha}. 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-{gamma} and TNF-{alpha}. However, in vitro EMSA experiments indicated that IRF-1 was activated by treatment with IFN-{gamma} or IFN-{gamma}/TNF-{alpha} 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-{gamma}/TNF-{alpha}.

The ISRE core coding strand residue G/-214 (relative to the transcription start site) was strongly protected after IFN-{gamma}/TNF-{alpha} treatment (Fig. 7Go, A–C). This residue is conserved in all ISRE elements that respond to either IFN-{alpha}/ß or IFN-{gamma}. The coding strand G/-216 residue, which would be predicted to be protected by IRF-1 binding based on the in vitro sequence preference of the factor (35, 36, 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. 7GoC, inset). Otherwise, identical protection patterns over the ISRE were observed after 30 min of induction by IFN-{gamma}/TNF-{alpha} (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. 5Go) 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-1{alpha} complexes are responsible for the response of hIP-10 to IFN-{gamma} 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.



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  		FIGURE 7. IVGF analysis of hIP-10 promoter in cells treated with IFN-{gamma} or IFN-{gamma}/TNF-{alpha}. A, Proximal regulatory elements of the hIP-10 promoter. ISRE, {kappa}B2, and {kappa}B1 elements are indicated by hatched regions, with CCAAT and TATA elements indicated by open boxes. Bases at the 5'-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 -242 to -110 and encompassing the ISRE, {kappa}B2, and {kappa}B1 elements, is shown. Putative regulatory elements are delimited by boxes. Residues that become DMS-resistant after treatment with IFN-{gamma}/TNF-{alpha} are indicated with vertical arrows. ISRE residue -214, 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-{gamma} (500 U/ml), with or without pretreatment for 4 h with TNF-{alpha} (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-{gamma} or IFN-{gamma}/TNF-{alpha}. The ISRE and {kappa}B2 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 -214. The plus sign (+) indicates unprotected residue -216. Arrows to the right of the autoradiograms indicate protected residues and a bolded arrow shows strongly protected residue -214. The open arrow indicates a guanidine residue (G/-216), which would be predicted to be protected by IRF-1 binding, but which remains methylation sensitive in this analysis. TNF-{alpha}-inducible protection of the {kappa}B1 and {kappa}B2 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-{gamma} (500 U/ml) for 2 h in the presence or absence of TNF-{alpha} (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-{gamma}, TNF-{alpha}, and IFN-{gamma}/TNF-{alpha}. Designation of arrows and numbering of G residues is the same as in C. The results indicate that residue -216, the putative IRF-1 contact site, remains unprotected from the initiation through the peak of hIP-10 transcription.

 
The GG/-220/-221 residues in the ISRE coding strand became inducibly DMS resistant; G/-238 upstream of the ISRE is also partially protected under conditions in which hIP-10 is synergistically induced (Fig. 7Go). In the noncoding strand, ISRE residues GG/-209/-210 become DMS resistant upon treatment with IFN-{gamma}/TNF-{alpha} (Fig. 7GoB). Additionally, G/-229 and G/-241 upstream of the ISRE are protected (Fig. 7Go, B and C). Thus, IFN-{gamma}/TNF-{alpha} 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-{gamma}/TNF-{alpha}-inducible footprinting of the {kappa}B2 NF{kappa}B binding site, on both the coding strand (G/-174/-175/-176) and the noncoding strand (G/-168/-167) (Fig. 7Go). By transient transfection studies with mutant promoter-reporters, the {kappa}B2 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 {kappa}B2 site is composed of p65 homodimers (Fig. 8Go). 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 {kappa}B2 element was identical regardless of whether cells were treated with TNF-{alpha} alone or with TNF-{alpha}/IFN-{gamma} (data not shown). The {kappa}B1 NF{kappa}B recognition site also became inducibly DMS resistant in IFN-{gamma}/TNF-{alpha}-treated cells, at G/-118/-119/-120 on the coding strand (data not shown) and at G/-111/-112 on the noncoding strand (Fig. 7Go). These observations complemented transfection studies with mutant promoters that showed a contribution of both {kappa}B2 and {kappa}B1 to full synergistic induction of hIP-10 and mIP-10 by IFN-{gamma}/TNF-{alpha} (data not shown) (21).



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  		FIGURE 8. EMSA analysis of the hIP-10 {kappa}B2 element. A, Sequences of the hIP-10 {kappa}B2 site and {kappa}B2 mutant. Sequence comparison of hIP-10 promoter and {kappa}B2 mutant are shown. At center is the {kappa}B2 motif (GGGAAATTCC). Substitutions in the {kappa}B2 mutant, designed to disrupt NF{kappa}B binding, are indicated in lowercase. B, EMSA of hIP-10 {kappa}B2 site and mutant. The autoradiogram shows EMSA analysis of nuclear extracts of untreated 2fTGH cells (0) or cells exposed to IFN-{gamma}/TNF-{alpha} ({gamma}/T) for 30 to 120 min, as indicated, using wild-type hIP-10 {kappa}B2 probe as shown in A. The probe supports the formation of two inducible complexes (T1, T2: arrows at left). Both complexes were efficiently competed by unlabeled {kappa}B2 (or {kappa}B1) oligo, as indicated at top. Neither nonspecific octamer factor binding site competitor oligo nor the {kappa}B2 mutant competed this binding. Supershift analysis indicated that both complexes contained p65/RelA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current report documents a novel mechanism of synergy between two proinflammatory cytokines, IFN-{gamma} and TNF-{alpha}, for transcriptional induction of the hIP-10 gene. This transcriptional synergy operates through two well-characterized cis elements, the hIP-10 ISRE homology and NF{kappa}B binding sites. The protein factors that mediate transcriptional synergy for hIP-10 induction are the p48 ISRE recognition factor, STAT-1{alpha}, and p65-homodimeric NF{kappa}B. Data that support these conclusions are as follows: 1) there is little or no hIP-10 response to IFN-{gamma} in U2A cells lacking p48 and in U3A cells lacking STAT-1{alpha}; 2) integrity of the ISRE is absolutely required for response to IFN-{gamma}; 3) the {kappa}B2 site is important for TNF-{alpha} to contribute to transcriptional synergy with IFN-{gamma}; 4) IRF-1 is not essential, as indicated by intact IFN-{gamma}/TNF-{alpha}-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.

Previous analysis of transcriptional synergy for the mIP-10 gene in fibroblasts indicated a requirement for the ISRE and for one NF{kappa}B binding site (21). This region corresponds to an IFN-{gamma}-inducible DNase I hypersensitive site that was identified between residues -260 and -60 in the hIP-10 gene, using U937 cells (38). The activation and promoter binding of STAT-1{alpha} was also demonstrated in studies of mIP-10 induction by IFN-{gamma}/TNF-{alpha} (21). Our investigation of hIP-10 regulation confirms 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 NF{kappa}B was formed in response to IFN-{gamma}/TNF-{alpha}; in the current experiments, only p65/p65 complexes were detected. Cell type-specific expression of the varied species of NF{kappa}B has been described frequently, and p65 homodimers have been previously reported as mediators of synergistic response to IFN-{gamma}/TNF-{alpha} for induction of the human IL-6 gene (39).

IVGF experiments demonstrated IFN-{gamma}/TNF-{alpha}-inducible asymmetric DMS resistance over the ISRE consensus, a binding activity consistent with p48/STAT-1{alpha} complexes (-221/220 and -214 on the coding strand and -210/209 on the noncoding strand). Results from these IVGF experiments suggested a specific contact between p48 and the 3' 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-{gamma} and TNF-{alpha} can also act synergistically through NF{kappa}B recognition sites and STAT-1{alpha}/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 NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B binding-sites. Therefore, the current report indicates at least two mechanisms by which IFN-{gamma} and TNF-{alpha} can activate similar promoter elements.

Structurally similar inducible promoters, containing ISRE-like elements that cooperate with NF{kappa}B recognition sites, have been studied in some detail. For example, the IFN-ß gene contains tandem ICS-like recognition sites for IRF-1, flanked by binding motifs for NF{kappa}B. IRF-1 and NF{kappa}B factors act to induce IFN-ß transcription in concert with a nonhistone chromosomal component, high mobility group (HMG) protein I(Y), that appears to stabilize interactions between NF{kappa}B and DNA target sequences (43, 44, 45, 46, 47). A striking mechanistic similarity between virus induction of IFN-ß and TNF-{alpha}-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 NF{kappa}B. The coordinated action of p48, STAT-1{alpha}, and NF{kappa}B is likely to be restricted to circumstances of immune-mediated inflammation in which IFN-{gamma} plays a cardinal role (49).


   Acknowledgments
 
We thank Dr. Tom Hamilton for helpful comments on the manuscript.


   Footnotes
 
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. Back

2 Current address: Dept. of Biochemistry, Ohio State University College of Medicine, Columbus, OH. Back

3 Current address: Dept. of Molecular Biology, Temple University School of Medicine, Philadelphia, PA. Back

4 Current address: Dept. of Biology, Ankara University, Ankara, Turkey. Back

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: Back

6 Abbreviations used in this paper: IP-10, IFN-{gamma}-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, {gamma}-activated sequence; ICS, IFN consensus sequence; ISGF3, IFN-stimulated gene factor; IVGF, in vivo genomic footprinting; EMSA, electrophoretic mobility shift assay. Back

Received for publication January 29, 1998. Accepted for publication June 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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