Synergistic Regulation of the Human Interleukin-12 p40 Promoter by NFkappa B and Ets Transcription Factors in Epstein-Barr Virus-transformed B Cells and Macrophages*

Giorgia Gri, Dawn Savio, Giorgio TrinchieriDagger , and Xiaojing Ma

From the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Monocytes/macrophages produce interleukin-12 (IL-12) in response to pathogenic stimulation, whereas most Epstein-Barr virus-transformed (EBV+) B cells constitutively secrete IL-12. The molecular mechanism regulating the constitutive IL-12 gene expression in EBV+ B cells has not been addressed. In this study, using the EBV+ B cell line RPMI-8866, we localized to the human IL-12 p40 promoter two essential cis elements, the NFkappa B site and the Ets site. The NFkappa B site was shown to interact with members of the NFkappa B family: p50 and c-Rel. The Ets site constitutively bound a multi-component Ets-2-containing complex. While the NFkappa B and Ets sites appear equally critical for inducible p40 promoter activity in macrophage cell lines, NFkappa B plays a more dominant role in the constitutive p40 promoter activity in EBV+ B cells. Transient expression of Ets-2 and c-Rel in B, T, and monocytic cell lines synergistically activated the IL-12 p40 promoter, apparently overcoming the requirement for cell type- or stimulant-specific transcription factors. These data provide new evidence that full activation of the human IL-12 p40 promoter may result primarily from the interplay between NFkappa B and Ets family members.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Interleukin 12 (IL-12),1 a heterodimeric cytokine composed of two disulfide-linked subunits of 35 (p35) and 40 (p40) kDa, was originally identified in the supernatant fluid of Epstein-Barr virus-transformed (EBV+) human B-cell lines (BCL) (1, 2). IL-12 exerts multiple effects, including: (a) induction of cytokine production, particularly interferon-gamma (IFN-gamma ) by T and NK cells, (b) induction of proliferation in T and NK cells and enhancement of their cytotoxic activity, and (c) induction of T helper-type 1 (Th1) responses and inhibition of Th2 responses (3). The finding that IL-12 p40 knockout mice have a severely depressed Th1 response (4) supports the role of IL-12 in Th1 cell differentiation.

IL-12 is produced by phagocytic cells and other antigen-presenting cells in response to bacteria, bacterial products, intracellular pathogens, and viruses (1, 2, 5-11). Normal B cells appear to be relatively poor producers of IL-12 (6). Most EBV+ BCL and EBV+ lymphomas produce IL-12, with the highest levels observed in EBV+ BCL derived from AIDS-associated lymphomas (9, 12). SCID mice injected with human lymphocytes from EBV-seropositive donors developed EBV+ human B cell lymphoma secreting in vivo high levels of human IL-12,2 suggesting that a similar production of IL-12 during initial proliferation of EBV+ B cell in patients may affect the reactivity of their immune cells against the infected cells. Although any extrapolation to the in vivo situation from data observed with established cell lines should be taken with caution, it is tempting to speculate that, in healthy individuals, IL-12-producing EBV+ cells would be easily rejected by immune response and thus only cells that have lost the ability to produce IL-12 would be able to give rise to Burkitt's lymphomas; in immunodeficient AIDS patients, these protective mechanisms would be inefficient and their IL-12-producing EBV-transformed cells could give rise to lymphomas in a relatively high proportion of patients.

The key role of IL-12 in inflammation and in the immune response, and the importance of this cytokine in anti-tumor resistance, have raised considerable interest in the mechanisms of IL-12 gene transcription. We (13) have shown previously that, in lipopolysaccharide (LPS)- and IFN-gamma -treated human monocytes, the expression of IL-12 p40 and p35 is primarily regulated at the transcriptional level. In a luciferase reporter gene construct, a 3300-bp genomic DNA fragment including the upstream sequences of the human p40 gene largely recapitulates its cell type specificity and transcriptional regulation (13). The cis elements in the p40 promoter region responsible for the inducible activation of the genes have been studied extensively in phagocytic cells, but no information is available for the elements controlling the constitutive expression of the gene in EBV+ BCL. We have previously localized a DNA sequence spanning nucleotides -292 to -196 (relative to the transcription start site) responsible for induced promoter activity (13). The core element at position -212 binds a series of IFN-gamma - and LPS-induced nuclear proteins (termed the F1 complex) including Ets-2, IFN-regulatory factor-1 (IRF-1), c-Rel, and Ets-related factors (14). A downstream NFkappa B site between bp -117 and -107 was also characterized as an LPS response element in the murine macrophage J774 cell line (15). Recently, Plevy et al. (16) identified a third cis element located at -96 and -88 (downstream of the NFkappa B site) of the murine IL-12 p40 promoter, also conserved in humans, that binds members of the C/EBP family of transcription factors in activated murine macrophage cell line. The C/EBP element exhibits functional synergy with the upstream NFkappa B site, although no physical interactions between C/EBP and Rel proteins were observed (16). Together, these results suggest that in myeloid cells a complex interplay exists between multiple inducible transcription factors contributing to the regulation of IL-12 p40 gene activation.

In this study, we focused upon the role of cis-acting regulatory elements in the transcriptional activation of the human IL-12 p40 gene in EBV+ BCL. In these cells, we showed constitutive binding of multiple transcription factors to the NFkappa B and Ets sites of the p40 promoter. We found that cotransfection of NFkappa B and Ets-2 transcription factors strongly and specifically synergized in the induction of p40 promoter activity in EBV+ BCL as well as in both inducible IL-12-producing macrophage cell lines and IL-12-nonproducing EBV- and T cell lines.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines and Reagents-- Human EBV-transformed B lymphoblastoid cell line (EBV+), RPMI-8866, PA682BM-2, and AS283A (9), EBV- Burkitt's lymphoma cell line BJAB (a gift from E. Kieff, Harvard Medical School, Boston, MA), T cell line Jurkat, and murine macrophage cell line RAW264.7 were all grown in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum (Irvine Scientific, Santa Ana, CA). All tissue culture media and supplements were endotoxin-free. All polyclonal antibodies used in supershift experiments were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant murine (rmIFN-gamma ) was a gift from Dr. G. Garotta (Human Genome Sciences, Inc., Rockville, MD). Anti-CD3 monoclonal antibody (OKT3, ascites) was produced from cells obtained from America Type Culture Collection (ATCC, Rockville, MD). LPS from Escherichia coli and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma.

Nuclear Run-on Assay-- Untreated RPMI-8866 cells, TPA-treated BJAB cells (50 ng/ml, 4 h), and TPA- and anti-CD3-treated Jurkat cells (OKT3, ascites 1:1000, 4 h) were harvested and washed with phosphate-buffered saline (BioWhittaker, Walkersville, MD). Isolation of nuclei and in vitro transcription in the presence of [32P]UTP (3000 Ci/nmol, DuPont) were performed as described (17, 18).

Plasmids-- A 3300-bp fragment of the human IL-12 p40 promoter was cloned into the luciferase reporter construct pXP2 (13) at the PstI site. All deletion mutant constructs were generated by PCR and fully sequenced for verification as described previously (13). NFkappa B expression vectors were a gift from Dr. K. M. Murphy (Washington University School of Medicine, St. Louis, MO). The CMV-based murine Ets-2 expression vector was a gift from Dr. R. Maki. The TNF-alpha promoter was obtained from a TNF cosmid library (ATCC, catalog no. 57590) by PCR amplification of a 1294-bp fragment. The sequence of the 5' primer used is 5'-TCTCTGAAATGCTGTCTGCTTGT-3' and the 3' primer used is 5'-CCTCTTAGCTGGTCCTCTGC-3'. The PCR product was cloned into pXP2 expression vector. All plasmids were banded twice in cesium chloride and verified by restriction mapping.

Transient Transfection-- DNA transfection experiments were performed by electroporation as described (13). Briefly, 0.4 ml of the cell suspension was mixed with 60 µg of DNA (10 µg of p40-luc/3.3 kb plus 3 µg of pCMV-beta -galactosidase (transfection efficiency control) and 47 µg of BS/KSII+) and electroporated in 0.45-cm electroporation cuvettes (Gene Pulser, Bio-Rad) at 960 microfarads and 250 V for BJAB and Jurkat cells, 300 V for RPMI-8866 cells, and 350 V for RAW264.7 cells. Cells were harvested 24 h post-transfection, pelleted by centrifugation, and resuspended in 100 µl of lysis buffer (Promega, Madison, WI). Lysates were used for both luciferase and beta -galactosidase assay (13).

Where indicated, RAW264.7 cells were treated with 1.2% Me2SO for 24 h and with rmIFN-gamma (1000 units/ml) for 8 h, stimulated with LPS (1 µg/ml) for 8 h, and harvested.

For cotransfection studies, pRSV-beta -galactosidase was used instead of pCMV-beta -galactosidase, and the molar ratio between the reporter plasmid (luciferase p40 promoter constructs) and expression vector was 1:2.5 for Rel family members and 1:1 for the Ets-2.

Luciferase activity was corrected for transfection efficiency by normalizing to the measured beta -galactosidase value.

Gel Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were isolated from RPMI-8866 as described (19) and from RAW264.7 also described (20). EMSA and supershift were performed as described previously (14).

Oligonucleotides Used for EMSAs-- Oligonucleotides -292/-196, -243/-196, and -222/-196 have been described (14). Oligonucleotide -135/-99 used as a probe has the following sequence (plus strand): 5'-AAACAAAAAAGGAACTTCTTGAAATTCCCCCAGAAGG-3' and encompasses the potential PU.1 and NFkappa B sites (underlined) of the human IL-12 p40 promoter found, respectively, in positions -143 and -132 of the murine IL-12 p40 promoter (15). Oligonucleotide -135/-99 m differs from oligonucleotide -135/-99 by a CC right-arrow GG transversion at position -108 and -107. Oligonucleotide -123/-99 has the following sequence: 5'-AACTTCTTGAAATTCCCCCAGAAGG-3'. An IRF-1 repeated consensus binding sequence was used as nonspecific oligonucleotide in competition assays (21). NFkappa B oligonucleotide is a high affinity binding site for NFkappa B/c-Rel homodimeric and heterodimeric complexes (22).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Constitutive Transcription of the Human IL-12 p40 Gene in EBV-transformed B Cells-- Based on our previous observation that EBV+ BCL constitutively produce IL-12 protein (1, 6, 9), we tested whether the p40 and p35 genes were also constitutively transcribed in nuclear run-on assays performed with unstimulated EBV+ BCL (Fig. 1A). Nascent p40 transcripts were constitutively present in RPMI-8866 (Fig. 1A) and in all other EBV+ BCL tested (PA682BM-2 and AS283A), and expression was not further enhanced by TPA treatment (data not shown). Jurkat T cells and BJAB EBV- BCL, which do not express p40 mRNA or protein, were used as negative controls (Fig. 1A). Transcription of the IL-12 p40 gene, unlike that of the TNF-alpha gene, was not detectable in unstimulated cells or in the EBV- BCL or T cell lines stimulated with TPA alone or TPA in combination with alpha -CD3, respectively. However, TNF-alpha transcription was shown to be further up-regulated by TPA treatment of all cells examined. Nascent p35 transcripts were present in all cell lines and under all conditions tested. Thus, the IL-12 p40 gene appears to be constitutively transcribed in EBV+ but not in EBV- BCL or T cells. We then analyzed the IL-12 p40 promoter activity in the same EBV+, EBV- and T cell lines using a full-length promoter construct linked to a luciferase reporter gene (-3300/+108) in a transient transfection assay. The promoter construct was active in EBV+ BCL, but poorly or not active at all in EBV- and T cell lines (Fig. 1B), confirming that the construct contains adequate sequence information for appropriate cell type-specific expression (13).


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Fig. 1.   IL-12 p40 and p35 gene transcription in EBV+ BCL, EBV- BCL, and T cell line. A, transcription rate was analyzed by nuclear run-on assay performed on isolated nuclei prepared from unstimulated EBV+ cells (RPMI-8866) or EBV- cells (BJAB) treated with TPA and T cells (Jurkat) treated with TPA plus anti-CD3. [alpha -32P]UTP-labeled nuclear RNA was hybridized to PCR-amplified p40, p35, TNF-alpha , and beta -actin cDNAs (500 ng/slot) immobilized on nylon membranes. Results are from one of two experiments with superimposable results. B, transfection of EBV+, EBV-, and T cell line. RPMI-8866, BJAB, and Jurkat were transfected by electroporation with the IL-12 p40 promoter-luciferase construct (-3300/+108) or the promoterless control plasmid (pXP2). After 24 h, luciferase activity was determined by normalizing the relative light units from each transfectant for transfection efficiency, using beta -galactosidase activity determined in the same cell extract. Results are mean (± S.E.) from four independent experiments.

Important cis-Regulatory Elements Located in the -222/+108 bp Region of the IL-12 p40 Promoter-- To delineate the cis-acting elements regulating the constitutive IL-12 p40 expression in EBV+ B cells, we first examined the activity of the full-length promoter (-3300/+108) and a series of nested 5' deletion constructs (Fig. 2) in EBV+ BCL. Luciferase activity of the -3300/+108 construct was 65-fold that of the parental, promoterless pXP2 control vector (Fig. 3). Promoter activity was progressively decreased when the full-length promoter was deleted from the 5' end at position -471 (32-fold over pXP2 control vector), -292 (38-fold), -265 (28-fold), and -243 (29-fold). Surprisingly, the activity of -222 bp plasmid was only slightly reduced compared with the full-length promoter. An additional 100-bp deletion to -position 122 abolished half of the full-length promoter activity. Truncation of the promoter to -28 bp (containing the TATA box) markedly diminished the constitutive promoter expression to a level only 10-fold greater than the promoterless control plasmid pXP2. These data suggest that the region between nucleotide positions -222 and -28 is most critical for constitutive promoter activity in the EBV+ BCL.


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Fig. 2.   Map of the human IL-12 p40 promoter. Proximal sequences (-407/+108) of the human IL-12 p40 promoter are shown (Ma et al.; Ref. 13). The promoter sequences are numbered relative to the transcription start sites. The 5' boundaries of plasmids containing various truncations of the promoter are marked with arrows, and putative motifs for binding of transcription factors are underlined.


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Fig. 3.   Deletion analysis of the human IL-12 p40 promoter in EBV+ BCL. Luciferase activity, in untreated EBV+ BCL RPMI-8866, induced by transient transfection of the indicated constructs, was measured in the cell lysate, and values were normalized against the internal control (CMV-beta -galactoside construct). Activity is expressed as -fold above the background of cells transfected with the promoterless control plasmid pXP2. Data here are mean (± S.E.) of four independent assays.

Functional Role of the NFkappa B and Ets Sites-- Since the -222/+108 promoter region was shown to exhibit significant promoter activity, we analyzed the activity of this construct in further detail. To evaluate the functional role of the putative NFkappa B half-site at -117 and the Ets site at -212, we prepared a series of additional promoters with defined mutations. The -222NFkappa Bm construct has a substitution of the core -109CC-108 with -109GG-108. The -222ets-2Delta construct has a 5-bp deletion that eliminates the -211TTTCC-207 sequence (13). The same mutations were made in the context of the -3300/+108 full-length promoter and were designated respectively, -3300NFkappa Bm and -3300ets-2Delta . Analysis of the activity of these constructs in transiently transfected EBV+ BCL (RPMI-8866) (Fig. 4A) revealed a dramatic decrease in the luciferase activity of promoter constructs mutated at the NFkappa B site (-3300NFkappa Bm and -222NFkappa Bm) (80-88% less than their corresponding wild-type constructs), but only a 50% decrease for the Ets-2 deletion mutants. In contrast, analysis of these constructs in the macrophage cell line RAW264.7 (Fig. 4B) demonstrated that the mutations of both the NFkappa B and Ets sites almost completely abolished the inducibility of the p40 promoter by IFN-gamma and LPS (reduced to 12-20% of the -3300/+108 wild-type promoter activity), the latter result being consistent with our previous finding that the Ets site plays an important role in this system (13). Together, these results suggest that the B cells may use the same cis elements and transcription factors as do macrophages, but the relative contribution of each factor may vary.


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Fig. 4.   Effect of NFkappa B and Ets-2 core mutations on constitutive and inducible promoter activity of the IL-12 p40 gene. A, the EBV+ BCL (RPMI-8866) was transiently transfected with the indicated luciferase plasmid. Luciferase activity was measured in the cell lysate, and values were normalized against the internal control (CMV-beta -galactosidase construct) and expressed as percentage of the activity of the full-length promoter (control), -3300/+108. Results are mean (± S.E.) from four independent experiments. B, macrophage RAW264.7 cells were transfected with the indicated luciferase plasmid. At 16 h after transfection, cells were treated with 1.2% Me2SO for 24 h and with rmIFN-gamma (1000 U/ml) for 12 h before the addition of LPS (1 µg/ml, 8 h). Luciferase reporter gene activity, corrected for transfection efficiency, is expressed as percentage of the activity of the IFN-gamma plus LPS-treated full-length promoter (control), -3300/+108. Results are mean (± S.E.) from six independent experiments.

Characterization of the DNA-Protein Complexes Associated with the NFkappa B Site-- To establish the relationship between promoter activity and specific DNA-protein interactions at the NFkappa B site (-117/-107) in EBV+ BCL, EMSA were performed using a 37-mer oligonucleotide probe (-135/-99) encompassing the putative NFkappa B and PU.1 sites. Three DNA-protein complexes were observed via EMSA (Fig. 5A); two of them indicate specific binding to nuclear proteins since they were competed by an excess of the unlabeled oligonucleotide corresponding to the probe -135/-99, but not by an unrelated (NS) oligonucleotide competitor. Competition of the slower mobility complex was observed using a 25-mer oligo (-123/-99) containing the NFkappa B site, but not the putative upstream PU.1 site located at -127 of the promoter. Use of an oligonucleotide with recognized binding activity for members of the NFkappa B/Rel family (22) as competitor resulted in complete disappearance of the top band, indicating that this DNA-protein complex likely involves members of the NFkappa B/Rel family. Further characterization of the NFkappa B protein members bound to the -135 to -99 promoter region (Fig. 5B) revealed a slowly migrating supershifted complex in the presence of NFkappa B anti-p50 and anti-c-Rel antibodies. The appearance of this supershifted band was accompanied by a partial disappearance of the upper complex. Thus, p50 and c-Rel-containing complexes constitutively bind to a transcriptionally active 6B site in the IL-12 p40 gene in EBV+ BCL. Anti-PU.1 antibody decreased the intensity of the faster-migrating band, suggesting the presence of PU.1 in this complex. No supershifted bands were detected with anti-c-Fos used as a control.


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Fig. 5.   Binding of nuclear protein extracts from EBV+ cell RPMI-8866 to the -135/-99 region of the IL-12 p40 promoter. Nuclear extracts (5 µg) from EBV+ BCL, and RPMI-8866 were incubated with the -135/-99 probe and analyzed by EMSA. A, the indicated competitor DNAs (oligonucleotides) were mixed at 40-fold molar excess with the probe. B, before addition of the probe, nuclear extract was mixed with affinity-purified polyclonal antibodies directed against the indicated transcription factors. Arrows identify complexes detected in each panel.

We then examined the ability of nuclear extract from IFN-gamma - and LPS- treated RAW264.7 macrophages to bind the probe -135/-99. In unstimulated cells, a series of three rapidly migrating complexes were observed (group III, Fig. 6A). However, only the slowest migrating complex of the three was consistently seen, suggesting that the faster migrating complexes may be proteolytic products of the upper complex. IFN-gamma treatment did not yield any additional binding, in contrast to an earlier report that IFN-gamma treatment of the murine macrophage cell line J774 resulted in augmented NFkappa B binding to this region of the murine p40 promoter (15). LPS, however, induced two major complexes (I and II). Pretreatment with IFN-gamma did not affect the binding activity of these two complexes. Competitive EMSA to assess the specificity of the three complexes showed that each of the three groups of complexes was abolished specifically by an excess of nonradiolabeled oligomer -135/-99, but not by an unrelated oligonucleotide (NS). The smaller oligonucleotide -123/-99 and an NFkappa B consensus oligonucleotide completely blocked the formation of complexes I and II (Fig. 6B), but had little effect on complex III. An anti-p50 antibody inhibited the formation of complexes I and II (Fig. 6C). Neither two distinct antibodies against the amino terminus (N) or the carboxyl terminus (C) of RelA (p65), nor an anti-c-Rel antibody affected the formation of the doublet band, although the combination of an anti-RelA and anti-c-Rel partially reduced the formation of complex I. Therefore, complex I appears to include mixed heterodimers p50/c-Rel and p50/RelA (p65) as well as c-Rel/RelA, while complex II is composed predominantly of p50. The faster-migrating complex (complex III) was decreased following IFN-gamma and LPS stimulation (Fig. 6A) and was competed by an anti-PU.1 antibody (Fig. 6C), thus confirming the presence of PU.1 in complex III.


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Fig. 6.   Binding of nuclear protein extracts from an IFN-gamma - and LPS-stimulated macrophage cell line to the -135/-99 region of the IL-12 p40 promoter. A, EMSA was performed with the -135/-99 probe and nuclear extracts isolated from the macrophage cell line, RAW264.7, either unstimulated or stimulated with IFN-gamma for 8 h followed by LPS for 8 h. B, binding of IFN-gamma - and LPS-treated extract and the probe was carried out in the absence (none) or presence of a 40-fold molar excess of the indicated oligomers. C, before addition of the probe (-135/-99), nuclear extracts were preincubated in the absence (no antibody) or presence of affinity-purified polyclonal antibodies recognizing p50, the amino terminus of RelA (p65(N)), the carboxyl terminus of RelA (p65(C)), c-Rel, PU.1, Ets-2, or combinations thereof.

Differences in Ets Site Binding Activities between B and Macrophage Cell Lines-- We previously demonstrated the functional predominance of the Ets site at -212/-207 in macrophage cells and the characteristics of the nuclear complexes formed around this site in response to stimulation (13, 14). To extend this analysis to EBV+ BCL with respect to differences in the relative importance of the Ets site between EBV+ and macrophage cells, we performed EMSAs using the same probe (spanning -292 to -196) as in our previous studies (14). Nuclear extract from IFN-gamma - and LPS-stimulated macrophage (RAW264.7) cells formed the previously characterized complex F1, whereas extract from EBV+ BCL (RPMI-8866) formed a slightly faster migrating complex, designated F1a. F1a was efficiently competed by a 50-fold molar excess of the -292/-196 DNA (Fig. 7A). Furthermore, F1a was supershifted by an anti-Ets-2 and an anti-c-Rel affinity-purified polyclonal antibody (Fig. 7B), as observed previously for the F1 complex (14). However, formation of the F1a complex was blocked by an anti-IRF-2 antibody and not, as in the case of the F1 complex, by an anti-IRF-1. These data suggest that Ets-2, c-Rel, and IRF-2, but not IRF-1, may be constituents of the F1a complex in EBV+ BCL.


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Fig. 7.   Differential binding of EBV+ BCL and macrophage cell extracts to the -292/-99 probe. Nuclear extracts from EBV+ BCL (RPMI-8866) and from IFN-gamma - and LPS-stimulated macrophages (RAW264.7gamma +L) were mixed with the -292/-196 probe and analyzed by EMSA. A, competitive EMSA was performed either in the absence (none) or presence of a 50 or 100 molar excess of the unlabeled oligonucleotide -292/-196. B, supershift EMSA was performed with the indicated affinity-purified polyclonal antibodies.

Transactivation of the IL-12 p40 Promoter by NFkappa B and Ets-2-- To assess the ability of several NFkappa B proteins and the Ets-2 protein to activate the p40 promoter, we transfected EBV+ BCL with the luciferase-based p40 reporter construct (-3300/+108) together with an expression vector for either NFkappa B RelA (p65), NFkappa B c-Rel, NFkappa B p50, or Ets-2. No transactivation of the -3300/+108 construct was detected by c-Rel, RelA, and p50, alone or any combination. Ets-2 alone was able to activate the promoter 4-fold, similarly to its actions in macrophages (13). Interestingly, co-expression of Ets-2 and c-Rel, but not RelA or p50, synergistically enhanced the p40 promoter activity (Fig. 8A). The decreased activity seen in Ets-2 and p50 cotransfection may have been due to the formation of the p50/p50 homodimer, which acts as a repressor, and may be a default method of regulation, as we have observed high levels of endogenous p50 produced by EBV+ BCL (data not shown). Transactivation by Ets-2 and c-Rel was dependent upon the integrity of their respective sites, since a 5-base deletion of the Ets site (-3300ets2Delta ) or a CC right-arrow GG substitution in the NFkappa B core sequence (-3300NFkappa Bm) in the context of the 3.3-kb promoter resulted in the failure of the promoter to respond to Ets-2 and c-Rel (Fig. 8B). In addition, mutation at the NFkappa B site abolished the transactivating effect of exogenous Ets-2 (Fig. 8B). This effect, seen with the wild type construct, may have resulted from a moderate level of synergy between exogenous Ets-2 and constitutively present endogenous NFkappa B in EBV+ BCL.


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Fig. 8.   Rel proteins synergize with Ets-2 in mediating activation of the p40 promoter in EBV+ BCL. A, EBV+ BCL, and RPMI-8866 were transiently cotransfected with different combinations of RelA (p65), c-Rel, and Ets-2 and the luciferase-based promoter (-3300/+108). B, cotransfection was performed with the Ets-2 and c-Rel expression vectors and either the wild-type or the mutated -3300/+108 constructs (-3300NFkappa Bm and -3300ets2Delta ). After 24 h, cells were assayed for luciferase expression. Luciferase counts were normalized using beta -galactosidase activity determined in the same cell extract (normalized luciferase counts). The empty expression vectors for all transcription factors were tested and produced background levels of luciferase activity (data not shown). Results are mean (± S.E.) from three independent experiments.

Cell Type and Promoter Specificity of the Synergistic Activation by Ets-2 and c-Rel-- To determine whether the same combination of expression vectors for the NFkappa B and Ets family could transactivate the p40 promoter in uninduced macrophage cells and in Jurkat T cells (which do not produce IL-12), c-Rel and Ets-2 expression vectors were transiently cotransfected in these cells. In both systems, the expression vectors strongly induced the activity of the full-length p40 promoter reporter construct (-3300/+108) (Fig. 9). Mutations of the NFkappa B or the Ets site dramatically decreased the level of transactivation by the effector constructs (Fig. 9, A and C). The remaining activity observed in macrophage cells using the -3300NFkappa Bm reporter construct, and in T cells using the -3300ets2Delta , might reflect the presence of additional noncanonical NFkappa B or Ets-2 sites, or a cooperativity with other endogenous transcription factors. Thus, the synergy between co-transfected NFkappa B and Ets-2 transcription factors in IL-12 p40 gene regulation is observed regardless of whether the endogenous gene is expressed in different cell types. This synergy is not observed in macrophage cells transfected with a luciferase construct under the control of the TNF-alpha promoter (Fig. 9B), also known to contain functional NFkappa B and Ets sites (23-25). In addition, overexpression of the two transcription factors failed to transactivate the thymidine kinase minimal promoter, lacking NFkappa B and Ets sites (data not shown). Taken together, these results demonstrate that the functional synergy between NFkappa B and Ets-2 is promoter-specific.


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Fig. 9.   C-Rel and Ets-2 synergize in macrophage and T cell line. A, macrophage cells, RAW264.7, were transiently transfected with the p40 promoter construct (-3300/+108) or its mutant forms (-3300NFkappa Bm and -3300ets2Delta ), together with c-Rel and/or Ets-2 expression vectors, without further stimulation. B, transient cotransfections were performed as in A, except that the reporter used was under the control of the TNF-alpha promoter. C, transient cotransfections were performed in T cell line, Jurkat, with the IL-12 p40 promoter constructs (-3300/+108). After 24 h, cells were assayed for luciferase expression. Luciferase counts were normalized using beta -galactosidase activity determined in the same cell extract (normalized luciferase counts). The empty expression vectors for all transcription factors were tested and produced background levels of luciferase activity (data not shown). Results are mean (± S.E.) from three independent experiments.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The present study was performed to delineate the molecular mechanisms by which EBV transformation of B lymphocytes results in the activation of IL-12 gene expression. We demonstrate that two regulatory elements, Ets at -212/-207 and NFkappa B at -117/-107, play a role in the high level expression of the human IL-12 p40 promoter in EBV+ BCL and in its inducible expression in macrophages. In EBV+ BCL, activity of a 3300-bp p40 promoter parallels transcription of the endogenous p40 gene, whereas no promoter activity was detectable in EBV- BCL or in Jurkat T cells in which the endogenous p40 gene is inactive. In EBV+ BCL, a segment of the p40 promoter containing 222 bp upstream and 108 bp downstream of the transcription start site (-222/+108) retains most of the activity of the full-length promoter, indicating that constitutive transcription from the p40 promoter is regulated by transcription factors acting on this region. This basal promoter activity is dependent mostly on the integrity of the NFkappa B site (-117/-107), as demonstrated by mutation analysis. A DNA probe encompassing this site binds the p50 and c-Rel NFkappa B family members, consistent with previous reports that NFkappa B proteins in mature B cell lines are constitutively present in the nucleus (26-28). Similar experiments on activated macrophages revealed that the NFkappa B site, bound by p50/RelA, p50/c-Rel, and RelA/c-Rel heterodimers, is also critical for p40 promoter activity. When this site is mutated, macrophage cells completely lose their ability to activate the p40 promoter after IFN-gamma and LPS stimulation. The data provided here are corroborated by the studies of Murphy et al. (15) on the murine p40 promoter in activated macrophages. Our studies indicate that NFkappa B transcription factors are essential for constitutive IL-12 p40 gene expression in EBV+ BCL and for p40 gene inducibility in macrophage cells. However, in both B and macrophage cells, p50, c-Rel, and RelA individually are unable to transactivate the IL-12 p40 promoter, suggesting the requirement for additional signals. Ets-2 appeared to be a likely candidate as the IL-12 p40 promoter contains a functional Ets motif, TTTCCT or AGGAAA (-212 to -207) essential for promoter activity in stimulated macrophage cells (13). Complete activation of the IL-12 p40 promoter in Ets-2 transiently transfected macrophages has been shown to require IFN-gamma or LPS stimulation (13), suggesting that, although vital, Ets-2 alone is not sufficient to achieve optimal p40 promoter activation. Here, we clearly demonstrate, in both B and unstimulated macrophage cells, that Ets-2 exerts a strong enhancing effect on p40 promoter activity only in the presence of the NFkappa B c-Rel. Ets-2 and c-Rel are sufficient for potent transactivation of the p40 promoter even in T and EBV- BCL (data not shown), where the p40 promoter is normally inactive. The specificity of this response is demonstrated by mutation or deletion of the NFkappa B or Ets binding sites resulting in greatly diminished promoter activation. Cooperativity between these transcription factors seems to be specific for the IL-12 p40 promoter, since no synergistic activation was observed for the TNF-alpha promoter, also known to contain NFkappa B and Ets sites (23-25).

Physical interactions between NFkappa B and Ets-like protein have been reported to regulate expression of the IL-2 receptor alpha  gene (29) and the activation of the human immunodeficiency virus enhancer (30). The Ets domain was shown to be necessary and sufficient to mediate this interaction, suggesting that the highly conserved Ets domain may act broadly to facilitate multiple protein-protein interactions. Indeed, it is now well established that Ets family proteins can activate transcription in conjunction with other proteins (31-35). Here, we have shown an association between Ets-2 and c-Rel in the F1a complex in EBV+ BCL similar to the F1 complex of activated macrophages (14). We are currently investigating whether the F1a and NFkappa B complexes physically interact at the p40 promoter.

The composition of the F1a complex differs from the previously characterized F1 of activated macrophages (14), in that the former contains the IRF-2 protein but not IRF-1. IRF-1-deficient mice fail to produce IL-12, resulting in a severely compromised Th1 immune response in vivo and in vitro (36, 37) strongly implicating a role for IRF-1 in IL-12 activation. IRF-1 and IRF-2 are highly homologous and bind to common DNA sequence elements (38, 39) with similar affinities. As a consequence of mutating the Ets site, we demonstrated here, in EBV+ BCL, only a partial reduction in the constitutive p40 promoter activity. In contrast, mutation of the same site in macrophages resulted in total abrogation of inducible p40 promoter activity. In other promoters, such as that of IFN-beta , it has been shown that IRF-2 antagonizes the activating function of IRF-1 (40). Therefore, the possibility that IRF-2 binding to the Ets site in EBV+ BCL may function as a repressor of IRF-1 activity by preventing its binding; thus, decreasing the relative participation of this site in the constitutive activation of the p40 promoter in EBV+ BCL should be considered. However, the ability of IRF-2 to act as a suppressor is by no means absolute, as both IRF-1 and IRF-2 have been shown to up-regulate expression of two genes, the histone H4 gene FO108 and the EBNA 1 gene (41, 42).

We provided evidence of a pivotal role of the NFkappa B site in constitutive IL-12 p40 gene expression in EBV+ BCL and a functional cooperativity between NFkappa B and Ets-2 transcription factors, critical for the constitutive and inducible activation of the IL-12 p40 gene. Parallel to our findings, it was recently demonstrated that a synergy exists between C/EBP and NFkappa B in human and mouse p40 promoter regulation (16), but whether C/EBP plays a role in constitutive p40 gene expression in EBV+ BCL remains to be determined. Together, this information suggests that the expression of IL-12 p40 gene requires the coordinated interactions between multiple regulatory proteins. The B cell lines used in our studies expressed the EBV latent-infection membrane protein 1 (LMP1) capable of activating the NFkappa B transcription factors (43-46). However, transient or stable transfection of LMP1 in EBV- B cells was insufficient to transactivate the p40 promoter.3 This suggests that it is likely that EBV employs more elaborate mechanisms stimulating multiple transcription factors critical for the activation of the IL-12 p40 gene.

    ACKNOWLEDGEMENTS

We thank Dr. Paolo Salomoni, Dr. Susan Robertson, and Dr. Frank Rauscher III for helpful suggestions during this work. We thank Sana Chehimi for excellent technical assistance and Marina Hoffman for editing the manuscript.

    FOOTNOTES

* This work was supported by Public Health Service Grants CA10815, CA20833, CA32898, and AI34412.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.

Dagger To whom correspondence should be addressed: Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3992; Fax: 215-898-2357; E-mail: trinchieri{at}wista.wistar.upenn.edu.

1 The abbreviations used are: IL, interleukin; EBV, Epstein-Barr virus; BCL, B-cell line; Th1, T helper-type 1; EMSA, electrophoretic mobility shift assay; LPS, lipopolysaccharide; IFN-gamma , interferon-gamma ; TPA, 12-O-tetradecanoylphorbol-13-acetate; bp, base pair(s); TNF, tumor necrosis factor; CMV, cytomegalovirus; PCR, polymerase chain reaction; IRF, IFN-regulatory factor; C/EBP, CCAAT-enhancer-binding protein.

2 R. Baiocchi, G. Gri, and M. Caligiuri, unpublished results.

3 G. Gri, D. Savio, G. Trinchieri, and X. Ma, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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