Synergy between Interferon-gamma and Tumor Necrosis Factor-alpha in Transcriptional Activation Is Mediated by Cooperation between Signal Transducer and Activator of Transcription 1 and Nuclear Factor kappa B*

(Received for publication, July 30, 1996, and in revised form, March 3, 1997)

Yoshihiro Ohmori Dagger §, Robert D. Schreiber and Thomas A. Hamilton Dagger

From the Dagger  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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Interferon-gamma (IFNgamma ) and tumor necrosis factor-alpha (TNFalpha ) 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 kappa B (NF-kappa B). IFNgamma and TNFalpha synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by gamma -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 kappa B motif. Artificial constructs containing a single copy of both a STAT binding element and a kappa B motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFNgamma and TNFalpha ; 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 STAT1alpha and NF-kappa B in nuclear extracts prepared from IFNgamma - and/or TNFalpha -stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFNgamma and TNFalpha in promoting inflammatory response depends in part upon cooperation between STAT1alpha and NF-kappa B, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.


INTRODUCTION

Intercellular communication by cytokines during an inflammatory reaction is integral to the subsequent orchestration and resolution of the response. IFNgamma 1 and TNFalpha 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). IFNgamma has been shown to induce tyrosine phosphorylation of STAT1alpha , and a homodimeric form of STAT1alpha binds to the IFNgamma -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 kappa 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-kappa B1 (p50/p105), NF-kappa B2 (p52/p100), RelA, c-Rel, and RelB, various forms of the kappa 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 kappa 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 kappa B signaling pathways appear to be indispensable for stimulus-dependent, transcriptional activation of many inflammatory genes. Furthermore, SBE and kappa B motifs are found in the promoter regions of many inflammatory genes. Many studies have reported functional synergy between TNFalpha and IFNgamma in promoting inflammatory function and gene expression, some of which could involve an interplay between STAT1 and kappa B binding factors (3-6). The present study was undertaken to determine whether IFNgamma -activated STAT1 can cooperate with TNFalpha -induced NF-kappa B to promote enhanced transcription. The results show that IFNgamma and TNFalpha synergize to induce expression of several genes that contain both SBE and kappa B motifs. The findings indicate that both the SBE and kappa B motifs are required for cooperativity and that the synergistic function of STAT1alpha and NF-kappa B appear to result from independent activation and recognition of cognate nucleotide sequence motifs.


EXPERIMENTAL PROCEDURES

Reagents

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 [alpha -32P]dCTP and [gamma -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 IFNgamma (specific activity, 6.8 × 106 units/mg) was obtained from Life Technologies, Inc. Recombinant mouse TNFalpha (specific activity, 2.6 × 107 units/mg) was a generous gift from Genentech Inc. (South San Francisco, CA). Antisera to mouse p50 (NF-kappa 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).

Cell Culture

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 DNA

A 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 [alpha -32P]dCTP. The resultant specific activity was approximately 108 cpm/µg, which was used at 107 cpm/blot.

Preparation of RNA and Northern Hybridization Analysis

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.

Preparation of Reporter Gene Plasmid DNA

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 kappa 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 kappa 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 kappa 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 kappa B2 sites, one or more SalI linkers (GGTCGACC) were placed between the two sites. The sequences of these reporter gene constructs were confirmed.

Transient Transfection

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 IFNgamma and/or TNFalpha 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).

Preparation of Oligonucleotides and PCR-amplified DNA

The following oligonucleotides were used in this study.
IRF-1 SBE 5'-tcgaGCCTGATTTCCCCGAAATGACGGC-3'
    3'-CGGACTAAAGGGGCTTTACTGCCGagct-5'
mut1 SBE 5'-tcgaGCCTGATTTCCCCGCCATGACGGC-3'
  (m1 SBE)     3'-CGGACTAAAGGGGCGGTACTGCCGagct-5'
mut2 SBE 5'-tcgaGCCTGATTGAATTCCAATGACGGC-3'
  (m2 SBE)     3'-CGGACTAACTTAAGGTTACTGCCGagct-5'
IRF-1 kappa B 5'-tcgaGCTGGGGAATCCCGCT-3'
    3'-CGACCCCTTAGGGCGAagct-5'
mutIRF-1kappa B (antisense) 3'-GCCGGTCCCGACACCTTAGTGCGATTCACAAA-5'
IP-10 kappa B2 5'-gatcGAGGGGAGAGGGAAATTCCAAGTTCATG-3'
    3'-CTCCCCTCTCCCTTTAAGGTTCAAGTACctag-5'
mutIP-10 kappa B2 5'-gatcGAGGGGAGAGTGAAATTACAAGTTCATG-3'
    3'-CTCCCCTCTCACTTTAATGTTCAAGTACctag-5'
OLIGONUCLEOTIDES 1-7

The nucleotide sequences of IRF-1 SBE and kappa B were taken from Sims et al. (14). The IP-10 kappa 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 kappa 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 kappa 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-1kappa B as described above. Double-stranded oligonucleotides were radiolabeled with the Klenow fragment of DNA polymerase I and [alpha -32P]dCTP in a fill-in reaction for 5' protruding ends. PCR-amplified DNA fragments were radiolabeled with T4 kinase and [gamma -32P]ATP.

Preparation of Nuclear Extracts

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-kappa 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.


RESULTS

STAT1 Is Essential for the Synergistic Induction of IFNgamma and TNFalpha -mediated Gene Expression

IFNgamma and TNFalpha have been shown to cooperatively regulate transcription of many inflammatory genes (3-6, 40). Previous studies have demonstrated that the IFNgamma -induced transcriptional activation of the IRF-1, ICAM-1, and Mig genes depends upon IFNgamma 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 IFNgamma -inducible genes also contain one or more kappa B sequence motifs, and their transcriptional activation in response to TNFalpha is dependent upon activation of kappa B binding activities (44, 45). On the basis of these observations, we postulated that IFNgamma -induced STAT1 and TNFalpha -induced NF-kappa B cooperatively regulate transcription of genes containing both SBE and kappa B motifs. Analysis of RANTES gene expression was also included, since IFNgamma and TNFalpha 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 IFNgamma and TNFalpha either alone or in combination for 3 h prior to isolation of RNA and Northern analysis. While the sensitivity of normal cells to IFNgamma or TNFalpha alone varied with each gene, all four genes were strongly expressed in cells stimulated with both agents (Fig. 2A). IFNgamma and TNFalpha cooperativity was markedly reduced (>90%) in STAT1-deficient fibroblasts without affecting the sensitivity to TNFalpha 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 IFNgamma and TNFalpha may involve protein synthesis-dependent and -independent mechanisms, both of which require IFNgamma -induced STAT1.


Fig. 1. Schematic representation of the promoter region of the IRF-1, ICAM-1, Mig, and RANTES genes. Potential cis-regulatory elements and critical defined sequences are schematically shown based on gene sequences obtained from the GenBankTM data base and the following references; human IRF-1 (14, 15, 49); human ICAM-1 (16, 17, 44, 45, 68); mouse Mig (41, 42); mouse RANTES (30, 46). The numbers above the promotor regions refer to the nucleotide position relative to the transcription start site.
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Fig. 2. STAT1 is essential for optimal synergy between IFNgamma and TNFalpha . Serum-starved confluent monolayers of fibroblasts from wild type mice (A) or STAT1-deficient mice (B) were either untreated (UT) or stimulated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) for 3 h in the presence or absence of cycloheximide (C, CHX; 5 µg/ml) prior to preparation of total RNA and analysis of specific mRNA levels by Northern hybridization as described under "Experimental Procedures." Five µg of total RNA were analyzed in each lane. Blots were hybridized with the indicated radiolabeled cDNA probes. Similar results were obtained in three independent experiments.
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IFNgamma /TNFalpha -induced Cooperative Transcription of the IRF-1 Gene Depends upon both the SBE and kappa B Sites

IFNgamma -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 kappa B motif has also been identified, although its functional significance remains equivocal (Fig. 1) (14, 49, 50). To determine if cooperation between IFNgamma and TNFalpha 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 kappa 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 IFNgamma and/or TNFalpha (Fig. 3). As reported previously, IFNgamma markedly induced luciferase activity in normal fibroblasts transfected with the wild type 1.3-kb IRF-1 promoter construct (14, 15). TNFalpha also modestly induced luciferase activity (5-7-fold induction). When cells were simultaneously stimulated with IFNgamma and TNFalpha , the promoter activity was synergistically enhanced. Mutation of the SBE site nearly abolished the IFNgamma sensitivity and markedly reduced the cooperative response to the combination of IFNgamma and TNFalpha (85% reduction in magnitude) without affecting the TNFalpha -induced luciferase activity. Similarly, the mutation of the kappa B site abolished the response to TNFalpha alone and reduced the cooperativity for IFNgamma and TNFalpha (77% reduction in magnitude). Mutation of both the SBE and the kappa 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 IFNgamma either alone or in combination with TNFalpha was less than 5% of the response seen in wild type cells. Taken together, these results indicate that both the SBE and the kappa B sequence motifs are required for optimal cooperativity between IFNgamma and TNFalpha and that activation of STAT1, at least, is also necessary.


Fig. 3. The kappa B and SBE sites in the 1.3-kb promoter of the IRF-1 gene are required for optimal IFNgamma and TNFalpha -induced transcriptional activity. The diagram at the left shows wild type and mutant 1.3-kb IRF-1 luciferase constructs. Potential enhancer elements are shown schematically. The numbers above the enhancer elements refer to the nucleotide position relative to the transcription start site of the IRF-1 gene (14). Fibroblasts from wild type mice (top part) or from STAT1-deficient mice (bottom part) were transfected with the indicated luciferase constructs as described under "Experimental Procedures." Twenty-four hours after transfection, the cells were either left untreated or stimulated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) for 8 h prior to analysis of luciferase activity. The relative luciferase activity is expressed as -fold induction in stimulated as compared with unstimulated samples. Mean arbitrary luciferase units (units/µg) for different constructs in unstimulated cultures are as follows: pGL Basic, 0.2; pGL IRF-1, 1.3; mut SBE, 2.8; mut kappa B, 3.9, mut SBE + kappa B, 1.2. Each column and bar represents the mean ± S.E. from four independent experiments.
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While the magnitude of cooperative response is markedly reduced in wild type cells transfected with mutations in individual motifs (either SBE or kappa 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 kappa B sites are mutated. The very low but reproducible response to IFNgamma seen in STAT1-deficient cells (both in Fig. 1 and Fig. 3) may reflect minor compensatory action of IFNgamma functioning through STAT1-independent systems. Indeed, in EMSA experiments using nuclear extracts from IFNgamma -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 IFNgamma activation sequence motifs, this could account for the leaky response to IFNgamma . 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.

Binding of IFNgamma -induced STAT1 and TNFalpha -induced NF-kappa B Is Not Cooperative

IFNgamma is well documented to stimulate the phosphorylation and nuclear localization of STAT1alpha homodimers (8-11). Similarly, TNFalpha is a potent stimulus of the nuclear translocation of various members of the Rel homology family (18-20). The functional cooperativity between IFNgamma and TNFalpha 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-kappa B to their respective sequence motifs using nuclear extracts prepared from cells stimulated with IFNgamma and/or TNFalpha for 30 min by EMSA (Fig. 4). Although nuclear extracts from fibroblasts stimulated with IFNgamma showed little or no inducible kappa B binding activity, cells stimulated with TNFalpha exhibited two inducible complexes designated as C1 and C2 in Fig. 4A. When cultures were co-stimulated with IFNgamma and TNFalpha , the magnitude of the binding activity and pattern of complex formation were essentially the same as seen in cells stimulated with TNFalpha alone. As shown in Fig. 4B, the C1 complex was fully reactive with antiserum specific for NF-kappa B1, while the C2 complex showed partial reactivity with anti-NF-kappa B1 and full reactivity with antiserum specific for RelA. Antibodies specific for c-Rel and STAT1 did not recognize any of the IFNgamma - and/or TNFalpha -induced kappa B binding activities. These results suggest that the binding activity induced by TNFalpha that recognizes the IRF-1 kappa B site is composed of NF-kappa 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 kappa 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-kappa 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 kappa 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-kappa B appear to be distinct. This ability of SBE to compete for NF-kappa B recognition may result from the kappa 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-kappa B as previously reported (51).


Fig. 4. NF-kappa B1 (p50) and RelA (p65) bind to the IRF-1 proximal kappa B site in TNFalpha -induced fibroblasts. A, fibroblasts from wild type mice were either untreated (UT) or treated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) as indicated for 30 min prior to the preparation of nuclear extracts. Five µg of each nuclear extract were analyzed for kappa B binding activity by EMSA using a radiolabeled oligonucleotide containing the IRF-1 kappa B sequence motif. Two major complexes are indicated as C1 and C2. B, nuclear extracts from IFNgamma and TNFalpha -stimulated cells were incubated with the indicated antibodies (1 µg) before analysis of the kappa B binding activity as described above. C, competition analysis of IRF-1 kappa B binding activity. Specificity of binding was assessed by competition with a 50-fold molar excess of unlabeled wild type or mutant oligonucleotide corresponding to the kappa B or SBE motifs as shown at the bottom. Mutated nucleotides are indicated in italic type. Underlined sequences represent the kappa B and SBE motifs, respectively. The overlined sequence indicates a potential kappa B motif contained within the SBE site. An oligonucleotide fragment corresponding to the region between -129 and -37 (SBE-kappa B) or an oligonucleotide fragment in which both the SBE and the kappa B sites have been mutated (mSBE-mkappa B) was also used as a competitor. Nuclear extracts (5 µg) from wild type fibroblasts treated with IFNgamma (100 units/ml) and TNFalpha (10 ng/ml) for 30 min were analyzed. Similar results were obtained from three independent experiments.
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Consistent with previous reports, nuclear extracts from IFNgamma -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, TNFalpha 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-kappa 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-kappa B on the IRF-1 kappa B site. When nuclear extracts from cells stimulated with both IFNgamma and TNFalpha were analyzed, a single broad band was observed, consistent with the presence of both the STAT1 and NF-kappa B complexes seen with IFNgamma or TNFalpha 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-kappa 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 kappa 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-kappa B. A large DNA fragment containing both the SBE and the kappa B sites was also able to compete complex formation in response to treatment with IFNgamma and TNFalpha . 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 IFNgamma or TNFalpha (data not shown).


Fig. 5. SBE binding activity in fibroblasts treated with IFNgamma and TNFalpha . A, fibroblasts from a wild type mouse were either untreated (UT) or treated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) as indicated for 30 min prior to the preparation of nuclear extracts. Five µg of each nuclear extract were analyzed for DNA binding activity by EMSA using radiolabeled oligonucleotides containing the IRF-1 SBE sequence motif. B, nuclear extracts (5 µg) from wild type fibroblasts treated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) for 30 min were incubated with the indicated antibodies before analysis of the SBE binding activity. C, competition analysis of IRF-1 SBE binding activity. Specificity of binding was assessed by competition with a 50-fold molar excess of unlabeled wild type or mutant oligonucleotide corresponding to the kappa B or SBE motifs as described under "Experimental Procedures" and as shown in Fig. 4. A double-stranded oligonucleotide corresponding to the region between -129 and -37 (SBE-kappa B) or an oligonucleotide in which the SBE and the kappa B sites have been mutated (mSBE-mkappa B) was also used as a competitor. Nuclear extracts (5 µg) from wild-type fibroblasts treated with IFNgamma (100 units/ml) and TNFalpha (10 ng/ml) for 30 min were analyzed. Similar results were obtained from three independent experiments.
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SBE and kappa B Sequences Cooperate in an Artificial Promoter

To explore the generality of the kappa 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 kappa 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 IFNgamma and/or TNFalpha following transient transfection in NIH3T3 cells (Fig. 6). Although one copy of the kappa B2 motif exhibited little sensitivity to IFNgamma or TNFalpha either alone or in combination with the SBE, one copy of the IRF-1 SBE motif was sensitive to IFNgamma or IFNgamma and TNFalpha . When a construct containing one copy each of the kappa 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 kappa B site. Thus cooperativity was not due to creation of fortuitous binding sites in the region where inserted sequences are coupled. Interestingly, cooperativity between IFNgamma and TNFalpha was also seen using the construct containing only the IRF-1 SBE and using the construct containing wild type SBE and mutant kappa B. The synergistic response of such constructs to IFNgamma and TNFalpha 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).


Fig. 6. kappa B and SBE motifs confer functional cooperativity on a heterologous promoter in response to IFNgamma and TNFalpha . One copy of a oligonucleotide corresponding to the wild type or mutant form of the IRF-1 SBE or the IP-10 kappa B2 site (see "Experimental Procedures") was linked upstream of the TK-105 promoter (pTK-105) containing the CAT gene. The nucleotide length between the SBE and the kappa B sites was 25 bases. The combination of the SBE and the kappa B sequences are schematically indicated. These CAT constructs were transiently transfected into NIH3T3 cells as described under "Experimental Procedures." Twenty-four hours after transfection, the cells were either untreated or stimulated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) for 16 h and assayed for CAT activity. The relative CAT activity is shown as -fold induction of stimulated versus unstimulated samples. Mean percentage of acetylation for different constructs in unstimulated cultures ranged from 0.2 to 0.4%. The maximum CAT activity was 27% acetylation, which was obtained in cultures transfected with pTK kappa B2 + SBE and stimulated with IFNgamma and TNFalpha . Each column and bar represents the mean ± S.E. from three independent experiments.
[View Larger Version of this Image (22K GIF file)]


Fig. 7. Effect of motif order and spacing on IFNgamma and TNFalpha -induced cooperativity. One copy of an oligonucleotide corresponding to the IRF-1 SBE or the kappa B2 motif from the IP-10 gene (see "Experimental Procedures") was placed in front of the TK-81 promoter linked to the luciferase gene (pTK-81 Luc) as indicated schematically. Constructs with increasing nucleotide spacing between the SBE and the kappa B site were prepared as described under "Experimental Procedures." These constructs were transiently transfected into NIH3T3 cells, and following a 24-h rest, the cells were either untreated or stimulated with IFNgamma (100 units/ml) and/or TNFalpha (10 ng/ml) for 8 h prior to harvest and determination of luciferase activity. The relative luciferase activity is presented as a percentage of activity obtained in cells transfected with the pTK-81 SP25 kappa B + SBE plasmid stimulated with IFNgamma and TNFalpha . The -fold induction of stimulated versus unstimulated samples is also indicated. Each column and bar represents the mean ± S.E. from three independent experiments.
[View Larger Version of this Image (26K GIF file)]

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 kappa 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 kappa 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 IFNgamma alone, cooperative responses were comparable (6-7-fold). The variability in sensitivity to IFNgamma is also observed in Fig. 7 when comparing the response of pTK SBE and pTK mkappa 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 kappa 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.


DISCUSSION

IFNgamma and TNFalpha 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 IFNgamma and NF-kappa B activation by TNFalpha are the principle events necessary for cooperative induction of genes containing appropriate SBE and kappa B sequence motifs. Independent interaction of STAT1 and NF-kappa B with their cognate binding sites is sufficient for mediating the cooperativity. These conclusions are based on the following observations. 1) IFNgamma and TNFalpha synergized strongly to promote expression of multiple genes that contain at least one copy of an SBE and a kappa 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 IFNgamma and TNFalpha 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 kappa B site in the IRF-1 gene promoter significantly reduced the magnitude of the synergistic response. 6) IFNgamma and TNFalpha independently activated STAT1 and NF-kappa B (NF-kappa 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 kappa B motifs could confer transcriptional synergy in response to IFNgamma and TNFalpha when examined in a heterologous promoter.

IFNgamma -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 IFNgamma and TNFalpha 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 IFNgamma /TNFalpha -mediated RANTES gene expression suggests that some IFNgamma -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 kappa B motifs have been identified in the promoter (Fig. 1) (46) and cooperative regulation of transcription by IRF-1 and NF-kappa B has been previously reported (57, 58). In contrast, direct activation of STAT1, which may include the formation of STAT1alpha homodimers, heterodimers, or other oligomeric interactions, appears to be involved in the cooperative induction of IRF-1, ICAM-1, and Mig gene expression. IFNgamma -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 IFNgamma and TNFalpha also depends on an IFNgamma -inducible factor that contains STAT1 and binds the IFN-stimulated response element found in the IP-10 promoter (5).

TNFalpha is well documented as a potent inducer of NF-kappa 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-kappa B (NF-kappa B1/RelA) can cooperate with STAT1 to promote synergistic transcriptional activity. The proximal kappa B site in the IRF-1 promoter is a functional kappa B motif, which is recognized by a combination of NF-kappa B1 and RelA in fibroblasts. TNFalpha -mediated ICAM-1 gene transcription has been shown to depend upon a kappa 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-kappa B (e.g. TNFalpha and lipopolysaccharide) (28), the cooperative induction of this gene by IFNgamma and TNFalpha suggests that the kappa 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 IFNgamma and TNFalpha independently of the proximal kappa B site (see Figs. 3 and 6). Since the SBE site was also recognized by STAT1 and NF-kappa 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 kappa 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 kappa 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 IFNgamma and TNFalpha .

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-kappa 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-kappa 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-kappa 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 IFNgamma and TNFalpha 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.


FOOTNOTES

*   This work was supported by U. S. Public Health Service Grant CA62220.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Immunology, NN10, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-4669; Fax: 216-444-9329.
1   The abbreviations used are: IFN, interferon; TNF, tumor necrosis factor; STAT, signal transducer and activator of transcription; SBE, STAT binding element; IRF-1, interferon regulatory factor-1; ICAM-1, intercellular adhesion molecule-1; PCR, polymerase chain reaction; IP-10, IFN-inducible protein, 10 kDa; kb, kilobase pair; NF-kappa B; nuclear factor kappa B; TK, thymidine kinase; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay.
2   Y. Ohmori and T. A. Hamilton, submitted for publication.

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