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Deletional Analysis of the Murine IL-12 p35 Promoter Comparing IFN- and Lipopolysaccharide Stimulation¹

Jutta Kollet^*, Christian Witek^*, John D. Gentry^*, Xiaojuan Liu^*, Steven D. Schwartzbach and Thomas M. Petro^2,*

^* Department of Oral Biology, University of Nebraska Medical Center, and School of Biological Sciences, University of Nebraska, Lincoln, NE 68583

	Abstract

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IL-12, pivotal to the development of Th1 cells and formed by association of p35 and p40 subunits, is made by macrophages and the macrophage cell line RAW264.7. In this study, the promoter for p35 was cloned and analyzed. The murine IL-12 p35 gene has promoters upstream from each of the first two exons. The exon 1 and exon 2 promoters, cloned into a reporter vector, were responsive to LPS or IFN-/CD40 ligation in transfected RAW264.7 cells. The exon 2 promoter containing bp -809 to +1 has significant homology to the human p35 promoter. Thus, deletion analysis was performed to determine the regions required for responsiveness to LPS, CD40, and/or IFN-. Base pairs -809 to -740 influenced responsiveness to LPS. In contrast, bp -740to -444 and bp -122 to -100 were required for responses to IFN-, IFN-/LPS, or IFN-/CD40 ligation. Removal of bp -444 to -392 increased the response of the exon 2 promoter to each stimulant. IFN regulatory factor (IRF)-1 is involved in the activity of this promoter at bp -108 to -103 because levels of nuclear IRF-1 correlated with exon 2 promoter activity in response to IFN- and IRF-1 overexpression stimulated and enhanced exon 2 promoter activity. Also, site or deletion mutation of the IRF-1 element at bp -108 to -103 reduced the responsiveness of the promoter and IRF-1 bound to an oligonucleotide containing bp -108 to -103. The data suggest that the response of the p35 promoter to IFN- requires a distinct IRF-1 positive regulatory element at bp -108 to -103.

	Introduction

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Cytokines from APCs control T cell development during immune responses (1, 2, 3). The cytokine IL-12 brings about the development of Th1 CD4⁺ T cells from naive CD4⁺ T cells (1). This cytokine is different from others because it is a heterodimer of p35 and p40 subunits (4); thus, different genes encode each IL-12 subunit. The rate of p35 gene expression limits the amount of IL-12 formed because the level of p40 expression exceeds that of p35 (5, 6). Therefore, to elucidate the important transcriptional factors in IL-12 expression, the active sites of the p35 promoter must be determined.

Significant production of bioactive IL-12 occurs during both innate and acquired immune responses. First, microbial molecules such as LPS induce IL-12 in a prototypical innate immune response of APCs to common microbial pattern molecules (7). IFN- and CD40 ligand (CD40L)³ produced by NK cells or T cells in response to IL-12 continue induction of IL-12 during adaptive immune responses (8, 9). While studies of p40 expression have observed a single species of p40 mRNA, studies of p35 expression have observed p35 mRNA of different sizes. We know now that the murine p35 gene has eight exons. However, the first murine p35 cDNA clone was derived from the last seven exons and encoded a 215-aa pre-p35 containing a 22-aa signal peptide (5). In contrast, Tone et al. (10) isolated two groups of p35 cDNAs from murine monocytic cells differing in their 5' untranslated region (UTR). One of the monocytic mRNAs was derived from eight exons, with the 5' end formed by splicing an untranslated exon 1 to exon 2. Conceptual translation of the mRNA containing exon 1 revealed an additional in-frame initiation codon upstream from the pre-p35 initiation codon. Initiation at the first ATG of this murine transcript would produce a longer version of pre-p35 containing a presequence with 21 additional aa. It is unclear whether the upstream exon 1 ATG is used, producing the longer isoform of murine pre-p35. The 5' UTR of the second group of cDNAs isolated by Tone et al. (10) were similar to the 5' UTR of the cDNAs isolated by Yoshimoto et al. (11) and Schoenhaut et al. (5) in that they lacked exon 1. This suggested a second p35 promoter within intron 1 driving the expression of these transcripts. However, there was also heterogeneity in this set of transcripts. The 5' end of a group of these p35 mRNAs corresponded to sequence within intron 1 and contained two ATGs upstream from the initiation codon for pre-p35 (10). The first upstream ATG is out-of-frame to pre-p35 ATG and encodes a theoretical 40-aa peptide. There is evidence that the presence of this out-of-frame ATG represses translation from the downstream pre-p35 initiation site (12). The second upstream ATG encodes a single amino acid and does not influence translation of pre-p35. The second subset of p35 mRNAs derived from p35 promoter 2 did not contain upstream ATGs and is translated into pre-p35. Thus, efficient synthesis of p35 depends upon where transcription initiates at the p35 exon 2 promoter. IL-12 p35 transcripts initiate within the first exon, an alternate first exon (exon 1a), or second exon. We have seen that the proportion of each set of transcripts differs depending upon the stimulant. Exon 1 transcripts are constitutively expressed, LPS stimulates more of the transcripts with exon 1 than without exon 1, while IFN-/CD40 ligation stimulates more exon 1a/exon 2 transcripts than exon 1 transcripts (13).

The human p35 gene also produces two sets of transcripts from the proximal p35 promoter region in a region equivalent to the proximal murine exon 2 promoter. However, instead of an out-of-frame upstream ATG, some of the human p35 transcripts contain an upstream, but in-frame, ATG protein initiation codon that translates into a pre-p35 with an extended presequence containing 35 additional amino acids. Unlike the longer murine p35 transcripts, human transcripts with upstream ATGs are indeed translated (14). Nevertheless, the evidence indicates that the p35 promoters in mice and humans function in a similar fashion.

Eukaryotic promoters have DNA sequences proximal to the transcription start site that recognize the basal transcriptional complex and have upstream DNA sequences that recognize regulatory transcription factors that are either enhancers or repressors of the basal complex (15). The basal transcriptional machinery for many genes is located near a site termed the TATA box that binds the TFIID transcription factor. Therefore, the p35 exon 2 promoter should have regions for binding the basal transcriptional complex as well as enhancers and repressors. Examination of the sequences of the human and murine proximal p35 promoters (10, 11) reveals that transcripts containing the upstream ATG initiation codon result from TATA box-independent transcription, while the transcripts without upstream ATG initiation codons probably result from TATA box-dependent transcription. Therefore, stimulation of the p35 promoter may involve mechanisms to switch from TATA box-independent to TATA box-dependent transcription.

What could cause this switch in TATA box dependence at a promoter is unclear. It is possible that the interaction of the TATA box-dependent basal transcriptional machinery with other transcription factors could play a role in the differential use of the TATA box. Inspection of DNA sequences in the p35 exon 2 promoter reveals three potential IFN-stimulated response elements (ISRE) that could recognize IFN regulatory factor (IRF) transcription factors (10, 11). Because IFN- induces the expression of both p35 and IRF-1 (16), and mice with a disrupted IRF-1 gene exhibit reduced p35 expression (17), IRF-1 is probably involved in p35 expression. Since there is a single report that IRF-1 supports TATA box-independent transcription (18), it is conceivable that IRF-1 is involved in the differential use of the TATA box at the p35 promoter.

Due to the prominence of IL-12 in the development of the Th1 CD4⁺ T cell subset in resistance (1) and autoimmune (19) disorders and the similarities between the proximal human p35 promoter and the p35 exon 2 promoter, we analyzed the murine p35 exon 2 promoter. To gain insight into the DNA sequences used by the basal transcriptional complex, enhancer, and repressor transcription factors in the expression of p35 in response to these stimuli, we cloned the entire p35 promoter, the exon 1 promoter, and the exon 2 promoter into the pGL3 reporter vector. The results from transfected RAW264.7 cells show that the IFN--stimulated response of the p35 promoter requires two regions of the p35 exon 2 promoter. One of the regions contains an ISRE, is required for IRF-1-dependent exon 2 promoter activity, and is upstream of the TATA box. The response of the p35 exon 2 promoters to LPS required different positive regulatory regions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abs, recombinant proteins, and cells

Anti-CD40 (clone 3/23; rat IgG2a), recombinant mouse IFN-, IL-12 p70 (clone 9A5), rIL-12, and biotinylated Ab to mouse IL-12 p40/p70 (clone C17.8) were purchased from BD PharMingen (San Diego, CA). RAW264.7 cells from a mouse monocyte-macrophage line obtained from American Type Culture Collection (Manassas, VA) were maintained in cell culture medium (CCM) that contained DMEM (Life Technologies, Gaithersburg, MD) with 10% FBS and 50 µg/ml gentamicin.

Induction of IL-12

Spleens were extracted under aseptic conditions, and cells were gently dispersed using 90-mesh stainless steel screens into cold CCM. The cell suspensions were centrifuged at 1200 rpm at 4°C for 11 min. Erythrocytes were lysed with Gey’s reagent for 5 min, then the remaining cells were washed twice with CCM. The viability and concentration of mononuclear cells were determined using trypan blue and a hemocytometer. Adherent splenic macrophages were obtained by adjusting the cells to 2 x 10⁷/2 ml CCM in six-well culture dishes and incubating for 4 h at 37°C in 5% CO₂/air. After 4 h nonadherent cells were removed from the splenic or peritoneal cell populations by repeatedly (five times) adding CCM warmed to 37°C, swirling, and then aspirating away suspended cells. In a separate set of six-well culture dishes, 1 x 10⁶ RAW264.7 cells were added to 2 ml of CCM. RAW264.7 cells and adherent splenic macrophages were left unstimulated or were stimulated with 1 µg/ml LPS (Sigma-Aldrich, St. Louis, MO), 5 µg/ml anti-CD40 (BD PharMingen) to cross-link CD40, 10 ng/ml IFN-, or combinations thereof. After 24 h of stimulation supernatants were collected.

IL-12 ELISA

Mouse IL-12 p70 was quantified by coating 96-well assay plates with 8 µg/ml purified unconjugated anti-IL-12 p70 (clone 9A5). After two washes with PBS/0.05% Tween 20 (Sigma-Aldrich), the plate was blocked with PBS/10% FBS and washed again, and then supernatants were added. For standards, serial dilutions of rIL-12 p70 (BD PharMingen) were added to additional wells coated with the respective anti-IL-12. The plates were incubated at room temperature for 2 h. After washing twice with PBS/Tween 20, the plates were incubated with 4 µg/ml biotinylated anti-IL-12 p40/p70 (clone C17.8) for 2 h. After four washes with PBS/Tween 20, the plates were incubated with diluted (1/400) avidin/peroxidase (Sigma-Aldrich) at room temperature for 30 min. The plates were washed five times with PBS/Tween 20 and incubated with 3,3',5,5'-tetramethylbenzindine substrate/hydrogen peroxide solution. Readings were taken at OD 450 nm wavelength with a reference of OD 570 nm using an ELISA spectrophotometric plate reader.

Isolation of the promoter for the IL-12 p35 gene

The mouse IL-12 p35 promoter was cloned using the Promoter Finder DNA Walking kit (Clontech Laboratories, Palo Alto, CA) as described by the manufacturer. The primary PCR reaction used the gene-specific antisense oligonucleotides 5'-GGTAGCGTGATTGACACATGCT-3' (University of Nebraska Medical Center, Molecular Biology Core Laboratory, Lincoln, NE) corresponding to IL-12 p35 cDNA starting at base 145 using 5 U of a Taq/Pfu polymerase mix. The secondary (nested) PCR used 1 µl of a 1/50 dilution of the primary PCR product and primers 5'-AGG ATC CAG ATC TCT GGA CCG GCA CTG AGA GGA-3' (University of Nebraska Medical Center, Molecular Biology Core Laboratory) that corresponded to antisense IL-12 p35 genomic DNA starting at bp 1429 of the sequence reported (DNA accession no. S82412). The secondary gene-specific primers contained BamHI and BglII 5' restriction enzyme sites. The PCR reaction products were size fractionated on 1.0% agarose/0.5x Tris-acetate-EDTA buffer and visualized by ethidium bromide fluorescence. A band of approximately 1700 bp was isolated and cloned as a SalI and BamHI fragment into pBluescript II KS⁺ vector, which was used to transform competent Escherichia coli JM109. Sequencing of the inserted DNA was performed at the core facilities of the Beadle Center for Biotechnology (University of Nebraska, Lincoln, NE). Once the inserted sequence was confirmed as the murine p35 promoter, it was excised as a KpnI and SacI fragment and ligated into the pGL3 enhancer vector. Plasmids were isolated from transformed E. coli JM109 using a Qiagen Plasmid Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s specifications. The exon 1 and exon 2 p35 promoters were made from the full p35 promoter by using directional PCR cloning to remove the region upstream from and including exon 1 of the p35 gene or to remove the region downstream from but not including exon 1 of the p35 gene. Each PCR product contained sites for SacI and XhoI restriction enzymes and cloned into the pGL3 enhancer vector (Promega, Madison, WI). The sequence of each insert was verified at the core facility of the Beadle Center for Biotechnology, University of Nebraska. The mouse IL-12 p35 promoter was found to be a region spanning 1672 bp upstream from the p35 start codon. 5' deletions were made in the cloned p35 exon 2 promoter to remove potential exon 2 promoter transcriptional regulatory elements. In addition, site-directed mutagenesis of the IRF-1 site within the p35 exon 2 promoter was performed using PCR such that GAAAGT at bp -108 to -103 was mutated to GACTCA. In one of the initial PCR reactions, sense primer (P1) 5'-CGAGCTCTAAAAGTCCAGAAAGGCTAAG-3' with a SacI overhang and antisense primer 5'-AGCCGGCAGGTGAGTCCCAGGACTGTGTCT-3' with the site mutation (underline) were used, while in the other initial PCR reaction the sense primer 5'-AGACACAGTCCTGGGACTCACCTGCCGGCT-3' with the site mutation (underline) and the antisense primer (P2) 5'-CCGCTCGAGTGGACCGGCACTGAGAGGAGCT-3' with an XhoI overhang were used. The products of the initial PCR reactions were isolated from agarose gels and mixed to serve as templates for the secondary PCR reactions in which primers P1 and P2 were used. Following isolation from an agarose gel the secondary PCR product was cut with SacI and XhoI and then ligated into precut pGL3 enhancer vector. The sequence of each insert was verified at the core facility of the Center for Biotechnology, University of Nebraska.

Murine IRF-1 expression vector

Murine IRF-1 cDNA was produced by RT-PCR from RNA isolated from murine RAW264.7 cells that were stimulated with 10 ng/ml IFN- for 24 h. PCR product was cloned into the pTARGET (Promega) mammalian expression vector using 3'-T overhangs according to the recommendations provided by the manufacturer (Promega). PCR amplification to detect IRF-1 was performed by adding 2 µl of cDNA with sense primer 5'-GCCATGCCAATCACTCGAATGCGGA-3' (Life Technologies) and antisense primer 5'-CTATGGTGCACAAGGAATGGCC-3' (Life Technologies). PCR was conducted in 50-µl reaction volumes with 20 mM Tris-HCl (pH 8.0); 2 mM MgCl₂; 10 mM KCl; 6 mM (NH₄)2SO₄; 0.1% Triton X-100; 10 µg/ml nuclease-free BSA; 0.2 mM each dATP, dGTP, dCTP, and dTTP; and 1.25 U Taq polymerase (Life Technologies). PCR amplifications were performed for 30 cycles of 94°C for 1 min, 60°C for 30 s, and 75°C for 2 min. The PCR reaction products were size fractionated on 1.5% agarose/0.5x Tris-acetate-EDTA buffer and visualized by ethidium bromide fluorescence. A band of approximately 1000 bp was isolated and cloned into precut pTARGET, derived from the parent vector pCIneo (Promega), which was used to transform competent E. coli JM109. Several transformed clones were isolated, and plasmids were prepared from each. The sequence of the inserted DNA in several plasmid constructs made at the core facilities of Beadle Center for Biotechnology, University of Nebraska, confirmed IRF-1 cDNA in the pTARGET vector (IRF1-pTARGET). One clone contained IRF-1 cDNA inserted in reverse orientation (1FRI-pTARGET).

Transfections

To determine the responsiveness of the pGL3 reporter constructs, RAW264.7 cells were seeded at 3 x 10⁵/2 ml culture medium. After 24 h cells were transfected using a total of 3 µg of pGL3 reporter construct DNA, 10 µg of Lipofectamine (Life Technologies), and 0.1 µg pRL-SV40 that constitutively expresses Renilla luciferase. For experiments with IRF-1pTARGET, cells were transfected with 1.5 µg of pGL3 reporter construct DNA, 1.5 µg of expression vector DNA, 10 µg of Lipofectamine, and 0.1 µg of pRL-SV40. After 24 h, transfected cells were incubated with or without E. coli LPS O127:B8 (1 µg/ml), CD40 ligation (5 µg/ml anti-CD40), and IFN- (10 ng/ml), alone or in combination. Twenty-four hours after stimulation, transfected RAW264.7 cells were washed twice with PBS and treated with passive lysis buffer according to the manufacturer’s specifications (Promega). Cell lysates were frozen, vortexed, and centrifuged at 12,000 x g for 15 s before collection of supernatant. Twenty microliters of supernatant was used to detect firefly luciferase, Renilla luciferase, using the dual luciferase detection kit (Promega), and luminescence was measured with a Turner Design luminometer (Turner Designs, Palo Alto, CA). Firefly luminescence for each culture was normalized to the Renilla luciferase luminescence in each culture.

PAGE and Western blot analysis

Cell extracts of unstimulated and stimulated RAW264.7 cells or peritoneal macrophages were made using reporter lysis buffer (Promega). The cell extracts were added to a sample buffer containing 63 mM Tris-HCl, 10% glycerol, 2% SDS, 2.5% 2-ME, and 0.0025% bromophenol blue. Twenty microliters of each sample was applied to a 4–12% SDS, Tris-glycine-polyacrylamide gel in a running buffer of 25 mM Tris base, 192 mM glycine, and 0.1% SDS. Current was applied at 125 V for 90 min. Transfer at 25 V for 1 h was made to a polyvinylidene difluoride membrane in a 12 mM Tris base, 96 mM glycine, and 20% methanol transfer buffer. The membrane was blocked with TBS-10 mM Tris-HCl (pH 8.0) and 150 mM NaCl containing 0.05% Tween 20/5% milk for 1 h at room temperature, followed by incubation in a 1/1000 dilution of rabbit IgG anti-mouse IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer without Tween 20 overnight, followed by an incubation in 0.2 µg/ml peroxidase-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology) in blocking buffer for 1 h. The membrane was washed three times with TBS containing 0.05% Tween 20 and once with TBS, then exposed to Luminol reagent (Santa Cruz Biotechnology) for 1 min before detecting chemiluminescence with Kodak BioMax Light Film (Eastman Kodak, Rochester, NY).

Nuclear extract preparation

RAW264.7 cells or peritoneal macrophages were seeded at 2 x 10⁷ cells in 10 ml DMEM with 10% FBS and 50 µg/ml gentamicin and allowed to adhere for at least 6 h before stimulation with 10 ng/ml IFN- (BD PharMingen) and 2 µg/ml LPS. Cells were harvested after 2, 16, or 20 h of stimulation, and nuclear extract preparation was conducted according to previously published methods (20, 21). After two washes with PBS cells were resuspended in PBS and pulse centrifuged at 13,000 rpm for 5 s at 4°C. The cell pellet was resuspended in buffer A (10 mM HEPES (pH 7.9), 10 mM KC, 1.5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1% aprotinin), pulse-centrifuged again for 5 s at 4°C, and resuspended in 800 µl of buffer A with 0.1% Nonidet P-40. The suspension was incubated at 4°C with gentle rotation for 10 min before nuclei were centrifuged at 3500 rpm for 10 min at 4°C. The nuclear pellet was washed with 100 µl of buffer A without Nonidet P-40 and pulse-centrifuged. Nuclei were resuspended in 15 µl of buffer C (20 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1 mM DTT, 1 mM PMSF, and 1% aprotinin) per 5 x 10⁶ cells and incubated at 4°C with gentle rotation for 40 min. The nuclear extract was removed after centrifugation at 12,000 x g for 10 min at 4°C and diluted with a 3.75-fold excess of buffer D (20 mM HEPES (pH 7.9), 50 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20% glycerol, 1 mM PMSF, and 1 mM DTT). Protein concentration was determined by measuring the absorption at 280 nm. Nuclear extracts were stored at -80°C.

Magnetic bead/DNA affinity assay

The magnetic bead affinity assay described previously (22, 23) was used to determine whether IRF-1 bound to the p35 promoter. A double-stranded DNA probe homologous to the -137/-93 bp fragment of the p35 exon 2 promoter (5'-GAGAGAGAAAGCAAGAGACACAGTCCTGGGAAAGTCCTGCCGGCT-3') and a double-stranded control DNA probe containing an Oct-2 element (5'-GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3') were labeled with digoxigenin (DIG) as described by the manufacturer in the DIG Gel Shift kit (Roche, Indianapolis, IN). Briefly, 3 pM DIG-labeled DNA corresponding to nt -137/-93 of the p35 exon 2 promoter region or DIG-labeled control oligonucleotide corresponding to the consensus Oct-2 promoter element (Roche) were conjugated to 10 µl of washed anti-DIG magnetic beads (Roche) under rotation for 30 min at room temperature in 50 µl of TEN100 buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 100 mM NaCl). The conjugated beads were then washed twice in TEN1000 buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1 M NaCl) and equilibrated to binding buffer TGED (20 mM HEPES (pH 7.9), 1 mM EDTA, 10% glycerol, 0.01% Triton X-100, and 100 mM NaCl) with the help of the magnetic particle separator (Roche). The DNA beads were blocked for 30 min at room temperature with TGED plus 0.5% BSA. After the incubation of nuclear extracts with DIG-oligo-conjugated beads, the nuclear extracts were combined. Nuclear extract (500 µg) derived from unstimulated or stimulated cells was used for protein pull-down. For competition experiments a 50-fold excess (150 pM) of unlabeled specific oligonucleotide -137/-93 was added before adding the nuclear extract. All samples were completed with 400 µl of binding-TGED buffer and incubated under rotation at 4°C for 2–4 h. Unspecific bound proteins were washed out with TGED (20 mM HEPES (pH 7.9), 1 mM EDTA, 10% glycerol, and 0.01% Triton X-100) three times. Elution was performed with 30 µl of elution-TGED buffer (TGED plus 1 M NaCl) for 30 min on ice with rocking. Eluates were kept at -80°C. IRF-1 detection was performed by Western blot with 15 µl of eluate or 15 µl of control input of raw nuclear extract (5 µg of protein) for PAGE.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LPS and IFN-/CD40 ligation stimulate production of IL-12 from splenic and RAW264.7 macrophages

IL-12 expression from macrophages arises from a dual promoter system in which the exon 1 promoter is constitutively expressed, but LPS enhances exon 1 promoter activity and the exon 2 promoter is stimulated by both LPS and IFN- (13). To establish a suitable cell line to analyze the p35 promoter activity, IL-12 production from splenic macrophages in response to LPS, IFN-/CD40 ligation, and IFN-/LPS was compared with that from RAW264.7 cells. While splenic macrophages produced a small background level of IL-12, LPS and IFN-/CD40 ligation stimulated the production of IL-12 by 7- and 15-fold, respectively (Table I). The combination of IFN-/CD40 ligation results in substantially more (100-fold) IL-12 compared with unstimulated splenic macrophages. In contrast to splenic macrophages, the amount of IL-12 from unstimulated RAW264.7 cells was undetectable. While the overall IL-12 production was lower from RAW264.7 cells compared with splenic macrophages, as with splenic macrophages RAW264.7 cells produced more IL-12 in response to IFN-/CD40 ligation than in response to LPS. In contrast to splenic macrophages, the combined stimulus of IFN- and LPS did not substantially increase the production of IL-12 by RAW264.7 cells compared with cells stimulated by IFN-/CD40 ligation. Therefore, RAW264.7 cells appear to be a suitable cell line to analyze p35 promoter activity.

LPS and IFN-/CD40 ligation stimulate the mouse p35 exon 2 promoter

To establish the active murine p35 promoter, the 1672-bp region of the p35 gene upstream from the ATG start site of pre-p35 was cloned into the pGL3 enhancer vector that encodes the firefly luciferase reporter gene (10, 11). Except for an additional 242 bp at the 5' end, the sequence of the cloned p35 region was identical with the published sequence (10). The two sets of transcripts from the murine p35 gene suggest two p35 promoter regions. To verify the activity of two murine p35 promoters, the regions from bp -1672 to -813 (exon 1 promoter) and from bp -809 to +1 (exon 2 promoter; Fig. 1) were inserted separately into the pGL3 enhancer vector. IL-12 p35 has been reported to be constitutively expressed with increased expression during innate and adaptive immune responses. To determine the constitutive, LPS-induced (innate), and IFN-/CD40L-induced (adaptive) activities of each promoter, RAW264.7 cells were transfected with the promoter reporter constructs along with a positive control promoter Renilla luciferase construct (pRL-SV40). To determine the relative activity of the promoters, unstimulated and stimulated activities of each promoter normalized to Renilla luciferase activity (RLA) were compared with the unstimulated activity of the full promoter normalized to RLA (Fig. 2). In unstimulated cells the exon 2 promoter exhibited a similar level of activity as the full promoter (Fig. 2). However, the exon 1 promoter in the absence of the intron 1 region (Fig. 2) exhibited 5-fold greater activity (Fig. 2) than the full promoter in unstimulated cells. All three promoters were expressed at higher levels in cells stimulated by LPS. The activity of each stimulated promoter could also be compared with its own activity in unstimulated cells. Using this comparison, the full p35 promoter exhibited 4-fold responsiveness to LPS compared with its activity in unstimulated RAW264.7 cells (Fig. 2). The activity of the exon 1 and exon 2 promoters was stimulated by LPS 3- and 4-fold, respectively, compared with their activity in unstimulated RAW264.7 cells (Fig. 2).

View larger version (77K): [in this window] [in a new window]   FIGURE 1. Map of the mouse IL-12 p35 exon 2 promoter region aligned with the human proximal p35 promoter region showing potential transcription factor binding sites in bold type. The nested cut sites for the deletional mutants used in the present work are denoted by a down arrow. The out-of-frame ATG start site at bp -71 and the conventional pre-p35 ATG start sites are designated.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. The exon 1 promoter of the p35 gene exhibits constitutive expression in the absence of intron 1 compared with the full promoter. LPS or IFN- plus CD40 ligation stimulates the full promoter, exon 1 promoter, and exon 2 promoter. Fold enhancement of luciferase activity generated by RAW264.7 cells transfected with the full, exon 1, and exon 2 p35 promoter constructs at 24 h in response to stimulation with LPS (1 µg/ml) or the combination of CD40 ligation (5 µg/ml anti-CD40) plus IFN- (10 ng/ml) compared with unstimulated cells transfected with the full p35 promoter construct is shown. The absolute luminescence values for the unstimulated full, exon 1, and exon 2 p35 promoters were 1.41 ± 0.14, 17.2 ± 0.71, and 1.43 ± 0.05, respectively.

Analysis of the p35 exon 2 promoter response to IFN-, LPS, and CD40 ligation

Because there is significant homology between the murine exon 2 p35 promoter and the proximal human p35 promoter region, with bp -215 and -55 having 83% homology (Fig. 1), the murine p35 exon 2 promoter was analyzed further by deletional analysis. The first step in this analysis was to determine the locations of potential regulatory elements based on published consensus sequences. Our analysis of the sequence of the exon 2 p35 promoter as performed by the Transfac 3.2 program (Fig. 1) reveals a potential glucocorticoid response element (GRE) and AP-2 elements between bp -809 and -740; C/EBP and Sp1 elements between bp -740 and -652; GRE, Oct-1, and AP-2 elements between bp -652 and -444, AP-1; C/EBP, AP-1, and cAMP response element CRE between bp -444 and -392; -associated sequence (GAS), IRF-1, and NF-B elements between bp -392 and -172; an IRF-1 element between bp -172 and -122; NF-B and IRF-1 elements between bp -122 and -100; and a TFIID recognition sequence (TATA box) between bp -100 and -72.

We created 5' deletional mutants of the p35 exon 2 promoter to eliminate these segments before reinsertion into the pGL3 enhancer reporter vector (Fig. 1). The responsiveness of the exon 2 promoter to LPS declined significantly upon elimination of bp between -809 and -740, a region that contained GRE and AP-2 recognition sequences (Fig. 3A). Elimination of regions between bp -740 and -444 did not restore responsiveness. However, elimination of bp between -444 and -392 completely restored responsiveness to LPS. Responsiveness of the exon 2 promoter was not reduced by elimination of sequence to bp -100. These results suggest that GR and AP-2 are positive regulatory transcription factors, while C/EBP, CREB, and AP-1 are negative regulatory transcription factors in LPS-induced expression from the p35 exon 2 promoter.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. The responsiveness of the p35 exon 2 promoter to LPS (A), IFN- (B), IFN-/LPS (C), and IFN-/CD40 (D) is dependent upon distinct regions of the exon 2 promoter. The p35 exon 2 promoter (bp -809 to +1) or the indicated 5' deletion mutants of the p35 exon 2 promoter were cloned into the pGL3 enhancer reporter vector at the multiple cloning site. The activity of the promoters in transfected RAW264.7 cells cotransfected with the pRL-SV40 Renilla luciferase control reporter vector were determined after no stimulation (UNS) or stimulation (STIM) with 1 µg/ml LPS (A), 10 ng/ml IFN- (B), LPS plus IFN- (C), or IFN- plus CD40 ligation (5 µg/ml anti-CD40) for 24 h. Promoter activity was measured as firefly luciferase-dependent luminescence of stimulated cells normalized to Renilla luciferase-dependent luminescence in 24-h cell extracts divided by values obtained from unstimulated cells normalized to Renilla luciferase. Data are the mean ± SE. *, The mean is significantly different from the mean generated by the response of the full-length promoter; p 0.05.

IFN- stimulates IRF-1 production in RAW264.7 cells

The overall response pattern of the p35 exon 2 promoter to stimuli containing IFN- was very similar, suggesting that addition of LPS or CD40 ligation merely augmented the basic IFN--induced response. These results also imply that the IFN--induced response of the p35 exon 2 promoter is influenced by a distal enhancer region containing potential C/EBP and/or SP-1 recognition sites, followed by a potent central negative regulatory region that is then followed by a powerful proximal region exhibiting core promoter activity, possibly due to NF-B and/or IRF-1 recognition elements.

Because of this and the fact that mice with disrupted IRF-1 gene expression exhibit a significant reduction in production of bioactive IL-12, p40, and p35 mRNA (17), the ability of IRF-1 to stimulate the p35 promoter was analyzed. IRF-1 is a member of a family of transcription factors induced by IFNs that have the ability to bind to ISRE with the GAAAGC/T oligonucleotide motif within promoters (24, 25). The p35 exon 2 has three potential IRF-1 binding motifs, two as GAAAGC and one as GAAAGT. However, only bp -122 to -100 containing the GAAAGT influenced the basal response to IFN-. To determine which stimuli induced the expression of IRF-1 in RAW264.7 cells and peritoneal macrophages, we examined the cellular and nuclear levels of IRF-1 by Western blot of extracts from cells stimulated with LPS, IFN-, CD40 ligation, IFN-/CD40 ligation, or IFN-/LPS. LPS (Fig. 4) or CD40 ligation (data not shown) failed to induce IRF-1 protein expression. However, IFN- stimulated IRF-1 expression by 2 h in RAW264.7 cells and by 20 h in peritoneal macrophages (Fig. 4). LPS increased IRF-1 levels by 2 h in both RAW264.7 cells and peritoneal macrophages. LPS reduced, but did not eliminate, IFN--induced IRF-1 expression in RAW264.7 cells by 20 h. In addition, detectable IRF-1 levels were present at 2 h in the nuclear extracts of both RAW264.7 cells and peritoneal macrophages in response to IFN- or IFN-/LPS. Induced nuclear IRF-1 levels declined by 20 h, except in RAW264.7 cells stimulated by IFN-. These data show that IRF-1 is present in detectable levels by 2 h in the nucleus of RAW264.7 cells and peritoneal macrophages following IFN- stimulation and could be available to stimulate p35 exon 2 promoter activity.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. IFN- does, but LPS does not, induce cellular (C) or nuclear (N) expression of IRF-1 in RAW264.7 cells or peritoneal macrophages after 2 and 20 h. Cell extracts and nuclear extracts of unstimulated or IFN--, LPS-, or IFN-/LPS-stimulated RAW264.7 cells were run on a 4–12% SDS, Tris-glycine-polyacrylamide gel. Following transfer to a polyvinylidene difluoride membrane and incubation in a 1/1000 dilution of rabbit IgG anti-mouse IRF-1, chemiluminescence detection of IRF-1 protein onto film was made using peroxidase-labeled goat anti-rabbit IgG and Luminol reagent (Santa Cruz Biotechnology).

To determine the degree to which IRF-1 alone influences p35 exon 2 promoter activity, we cloned the cDNA of IRF-1 into the pTARGET mammalian expression vector in the sense (pIRF-1) and antisense (p1-FRI) directions. The responses of the p35 full, exon 1, and exon 2 promoters were analyzed in unstimulated or stimulated RAW264.7 cells cotransfected with pIRF-1, p1-FRI, or pCIneo, the parent vector for pTARGET, plus pRL-SV40 for normalization. The full and exon 2 p35 promoters, but not the exon 1 p35 promoter, were responsive to transfection with pIRF-1 alone (Fig. 5). Transfection with p1-FRI or pCIneo has no effect on the activity of any of the promoters. The responsiveness of the p35 full and exon 2 promoters to LPS, IFN-, or IFN-/CD40 ligation was greatly enhanced by overexpression of IRF-1. In contrast, surprisingly, the responsiveness of the p35 exon 1 promoter to LPS, IFN-, or IFN-/CD40 ligation, but not constitutive activity, was significantly reduced by overexpression of IRF-1 (Fig. 5). This suggests that IRF-1 alone or in conjunction with constitutively activated transcription factors can stimulate the p35 exon 2 promoter. Overexpression of IRF-1 did not augment the response of the exon 2 promoter in the presence of anti-CD40 (data not shown), suggesting that synergism between IFN- and CD40 ligation is not through cooperation between IRF-1- and CD40-induced transcription factors. Together these data indicate that IFN--induced IRF-1, in association with basal or induced regulatory transcriptional complexes, can stimulate the p35 exon 2 promoter and is probably an important transcription factor in the IFN--induced response of the p35 exon 2 promoter.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. The p35 exon 2 promoter responds to overexpression of IRF-1. The activities of the full, exon 1, and exon 2 promoters in pGL3 were determined in unstimulated (UNS) and LPS-, IFN--, or IFN-/CD40 ligation-stimulated (STIM) RAW264.7 cells cotransfected with pRL-SV40 plus with pCIneo, pTARGET-reverse IRF-1 (1FRI), or pTARGET-sense IRF-1 (IRF-1). Promoter activity was measured as firefly luciferase-dependent luminescence of stimulated cells normalized to Renilla luciferase-dependent luminescence in 24-h cell extracts divided by values obtained from unstimulated cells transfected with the full p35 promoter construct plus pCIneo normalized to Renilla luciferase. Data are the mean ± SE. *, The mean is significantly different from the mean generated by the response of the promoter in cells transfected with pCIneo under the same conditions; p 0.05.

The original consensus IRF-1 binding motifs is GAAAGT (25), and the p35 exon 2 promoter is responsive to overexpression of IRF-1. This region of the p35 gene has two potential variants of the IRF-1 recognition sequences, GAAAGC at bp -194 to -189 and bp -131 to -126 and one potential exact IRF-1 sequence GAAAGT at bp -108 to -103. However, the response of the p35 exon 2 promoter to IFN- depended upon the region from bp -122 to -100. To determine whether this region is also required for responsiveness to IRF-1, the activities of the 5' deletion constructs of the p35 exon 2 promoter were analyzed in RAW264.7 cells cotransfected with pIRF-1. The activity of the exon 2 promoter in response to overexpressed IRF-1 was not significantly affected by 5' deletions from bp -809 to -122, but was significantly affected by deletions from bp -122 to -100, suggesting that IRF-1 influences the p35 exon 2 promoter through the IRF-1 site at bp -108 and -103 (Fig. 6).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. The responsiveness of the p35 exon 2 promoter to overexpressed IRF-1 is dependent upon bp -122 to -100 of the exon 2 promoter. The activities of the p35 exon 2 promoter (bp -809 to +1) with no deletions or mutated promoters with the indicated 5' deletions in the pGL3 enhancer reporter vector were determined in transfected RAW264.7 cells cotransfected pIRF-1 plus pRL-SV40. Promoter activity was measured as firefly luciferase-dependent luminescence of stimulated cells normalized to Renilla luciferase-dependent luminescence in 24-h cell extracts divided by values obtained from unstimulated cells normalized to Renilla luciferase. Data are the mean ± SE. *, The mean is significantly different from the mean generated by the response of the full-length exon 2 promoter; p 0.05.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. The ability to stimulate the p35 exon 2 promoter is decreased by mutating the IRF-1 site within the region bp -122 to -100 from GAAAGT to GACTCA. The activities of the wild-type and mutated p35 exon 2 promoters were determined in transfected RAW264.7 cells cotransfected with pRL-SV40 and left unstimulated or stimulated with LPS, IFN-, IFN-/CD40 ligation, or IFN-/LPS (A), or cotransfected with pCIneo or expression vector IRF-1pTARGET (B). Promoter activity was measured as firefly luciferase-dependent luminescence of stimulated cells normalized to Renilla luciferase-dependent luminescence in 24-h cell extracts divided by values obtained from unstimulated cells transfected with the wild-type p35 exon 2 promoter that was also normalized to Renilla luciferase. Data are the mean ± SE. *, The mean is significantly different from the mean generated by the response of the wild-type promoter; p 0.05.

IRF-1 is recruited to bp -137 to -93 of the exon 2 promoter

To further evaluate the potential involvement of IRF-1 at the predicted IRF-1 site (-108 to -103 bp) of the exon 2 promoter in closest proximity to the TATA box (-81 to -75 bp) we have employed an established DNA affinity binding assay with magnetic beads (22, 23). DIG-labeled DNA probes corresponding to bp -137 to -93 containing the IRF-1 site of the p35 exon 2 promoter were conjugated to magnetic beads and incubated with nuclear extracts derived from RAW264.7 cells stimulated with or without IFN-/LPS for 2 or 16 h. Input IRF-1 and DNA-bound IRF-1 were detected by Western immunoblot. The amount of nuclear IRF-1 was very low in untreated RAW cells (Fig. 8). In contrast, stimulation with IFN-/LPS increased the level of nuclear IRF-1 by 2 h similar to that seen in previous reports (17, 26). As shown previously, the level of IRF-1 in the nucleus of IFN-/LPS-stimulated RAW264.7 cells declined after 16 h. Furthermore, at 2 and 16 h nuclear IRF-1 were recruited to the -137/-93 bp DNA probe upon stimulation with IFN-/LPS. The interaction was specific, because reduced binding was observed after competition with a 50-fold excess of specific unlabeled -137/-93 DNA probe. IRF-1 also did not bind to a DIG-labeled control oligonucleotide with the recognition motif for Oct-2. This strongly suggests an involvement of IRF-1 at the predicted IRF-1 site (-108 to -103 bp) after induction with IFN-/LPS in initiating p35 transcription.

View larger version (36K): [in this window] [in a new window]   FIGURE 8. Nuclear extracts of RAW264.7 cells stimulated with IFN-/LPS have IRF-1 that binds to an oligonucleotide corresponding to bp -137 to -93 of the p35 exon 2 promoter. RAW264.7 cells (2 x 107 cells in 10 ml of culture medium) were stimulated for 2 or 16 h with 10 ng/ml IFN- and 2 µg/ml LPS. Cells were harvested after 16 h of stimulation, and nuclear extract was prepared. Three picomoles of DIG-labeled DNA corresponding to nt -137/-93 of the p35 exon 2 promoter region or DIG-labeled control oligonucleotide corresponding to the consensus Oct-2 promoter element were conjugated to anti-DIG magnetic beads. Nuclear extract (500 µg) derived from unstimulated or stimulated cells was mixed with conjugated DIG-DNA with or without a 50-fold excess of unlabeled oligonucleotide -137/-93. Following elution, IRF-1 detection was performed by Western blot with 15 µl of eluate or 15 µl of control input of raw nuclear extract (15 µg of protein).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The ability of diverse molecules such as LPS and IFN- to stimulate the production of the same cytokine is the result of different transcriptional factor complexes that interact with different regions of the same promoter (31, 32). Therefore, it was not surprising that the data here show that stimulation of p35 expression by LPS requires different regions of the p35 promoter compared with IFN-. By way of 5' deletional mutations of the p35 promoters in reporter constructs we show herein that the response of the exon 2 promoter to LPS requires two regions; one region is distal to and one is proximal to the +1 ATG. These two positive regulatory regions flank a powerful negative regulatory region. LPS stimulation of the exon 2 promoter does not appear to be affected by removal of potential NF-B sites that are located at bp -302 to -293 and bp -112 to -103. A distal, LPS-sensitive, positive regulatory region in the p35 exon 2 promoter located between bp -809 and -740 contains potential AP-2 and GR elements. Thus, LPS stimulation of the p35 exon 2 promoter could be influenced by transcription factors such as AP-2 and GR, by TFIID that can bind to the TATA box, and by the ability of its transcripts to be efficiently translated.

Despite the fact that the regions required for stimulation of the exon 2 promoter by LPS are different from that required for stimulation by IFN-, the data herein suggest that a major negative regulatory region is situated in the middle of the promoter. This region between bp -444 and -392 contains AP-1, C/EBP, and CREB recognition sequences. While AP-1 and C/EBP have been associated with activation of numerous cytokine genes, CREB has been associated with down-regulation of cytokine gene expression (33). It is unclear how CREB could be activated when only positive stimuli were added to the culture medium. However, it could be that PGE, which accumulates in the culture medium of stimulated cells and increases CREB activity, or TGF-, which also increases CREB activity and is found in FBS, is responsible for regulating p35 exon 2 promoter activity (34, 35). Thus, CREB may be involved in decreasing IL-12 production through its action upon the promoter for the IL-12 p35 subunit.

Understanding the complexity of the murine p35 promoter will provide meaningful information for understanding the human p35 promoter. First, the murine exon 2 p35 promoter region has significant homology to the human proximal p35 promoter. In fact, the promoter area surrounding -71 ATG is identical in mice and humans. Second, a protein initiation ATG codon at bp -71 is embedded in the TATA box. In mice this alternate ATG codon is out-of-frame with the pre-35 ATG at bp +1, and translation of a theoretical alternate 40-aa peptide is possible. Use of the TATA box diverts transcription so that transcripts do not contain the alternate ATG initiation codon. However, in humans this upstream ATG is in-frame to the pre-p35 ATG at +1 bp, and translation of a different isoform of pre-p35 with an additional 35 aa in the presequence is possible. It was shown recently that translation initiation from both human ATGs is possible (14). Finally, even with differences in the more distal regions of the murine p35 exon 2 promoter compared with the human p35 promoter, it is likely that similar elements evolved elsewhere to recognize the same transcription factors. For instance, while the human p35 promoter has not conserved the CREB recognition sequence in the region equivalent to the murine bp -444 to -392, it does have the exact same sequence in the human promoter at bp -667. Therefore, the murine p35 promoter system described here should prove to be a useful model of the human p35 promoter system.

One of the dissimilarities between the two p35 promoter systems is the strategy employed by each to obtain transcripts with an upstream ATG in-frame to the pre-p35 protein initiation ATG (12). A distal murine exon 1 promoter adds an untranslated exon 1 to produce transcripts with the upstream ATG, while the human p35 promoter initiates transcription at two proximal locations, one upstream of the alternate ATG and one downstream of the alternate ATG initiation codon. The existence and significance of the alternate pre-p35 isoform are unclear.

IFN- can induce gene expression in either direct or indirect ways. IFN- directly activates Stat1 dimers that localize to GAS elements of genes (36). The exon 2 promoter contains one potential GAS element between bp -366 and -357. However, removal of this region did not have any influence on p35 exon 2 promoter activity in response to IFN-. Therefore, IFN- does not induce the p35 exon 2 promoter through GAS elements. IFN- also stimulates gene expression indirectly by inducing the expression of other transcription factors such as IRF-1 (24, 36). IRF-1 then interacts with regulatory elements that include the GAAAGT/C motifs (24, 25). Both human and mouse p35 promoters have three potential IRF-1 recognition sequences in a region that is highly conserved between mouse and man. Elimination of the two distal IRF-1 recognition sequences did not influence the response of the exon 2 promoter to IFN-. However, elimination of the proximal IRF-1 recognition sequence abolished the response to IFN-. Involvement of the IRF-1 recognition site at -108/-103 bp is likely, since we also show here that overexpression of IRF-1 substantially increases the constitutive response of p35 exon 2 promoter and its response to IFN- or LPS. In addition, we show here that detectable levels of IRF-1 enter the nucleus within 2 h after stimulation, and this IRF-1 associates with an oligonucleotide containing the -108/-103 IRF-1 site. Finally, we show that if this IRF-1 site is disrupted, then responsiveness of the p35 exon 2 promoter is substantially decreased. Whether IRF-1 binding to DNA occurs directly or indirectly by recruitment through other factors is currently under investigation. Due to the synergistic effect of LPS plus IFN- or LPS plus ectopically expressed IRF-1 on the promoter activity in this specific region, it is expected that IRF-1 cooperatively acts with other transcription factors such as NF-B or other IRFs to activate transcription of the p35 gene. Therefore, IFN- stimulates the p35 promoter through IRF-1 recognition elements, but not GAS elements.

The data here also suggest that at least a portion of the response of the exon 2 promoter is not dependent upon the TATA box starting at bp -82, while another portion is dependent upon the TATA box. The TATA box-independent promoter activity induced by IFN- may be related to its induction of IRF-1 and the ability of IRF-1 to interact directly with the transcriptional preinitiation complex (18). In the present report all responses to IFN- localized to the same region of the p35 exon 2 promoter, a proximal 22-bp region containing IRF-1 and NF-B elements. Eukaryotic general transcription requires, in addition to RNA polymerase II, one or more general transcription factors, TFIIA through TFIIJ. While TFIID interacts with TATA box regions, other factors assemble subsequently (31). Regulatory transcription factors either would enhance or reduce the assembly of the general transcription factors. However, IRF-1 has been shown to directly interact with TFIIB and is capable of supporting TATA box-independent transcription (18). Because overexpression of IRF-1 alone induces activation of the p35 exon 2 promoter, the data suggest that IRF-1 is interacting with general transcription factors in TATA box-independent transcription.

The results here demonstrate that IFN- can synergize with either CD40 ligation or LPS in the induction of the p35 exon 2 promoter. Since both CD40 ligation (37, 38) and LPS (39) stimulate NF-B activation, it is possible that there is cooperation between IFN--induced IRF-1 and CD40- or LPS-induced NF-B. This is consistent with other reports showing IRF-1 collaboration with NF-B in the activation of IL-6 (40), MHC genes, and VCAM-1 genes (41, 42). However, IRF-1 overexpression did not synergize with CD40 (data not shown), but did with LPS stimulation to enhance p35 exon 2 promoter activity. Therefore, IRF-1/NF-B cooperation is not the basis for IFN-/CD40 ligation synergism in activation of the p35 exon 2 promoter.

It is interesting that the regions required for IFN-/LPS stimulation of the exon 2 promoter were different from the regions required for LPS-alone stimulation. The regions required for IFN-/LPS stimulation were similar to the regions required for IFN- and IFN-/CD40 stimulation. This suggests that LPS or CD40 ligation merely augments the basic IFN- response.

Because IL-12 induces development of the T cell lineage to the Th1 phenotype during the acquired immune response (1, 43) and is necessary for the development of cellular, antiviral, and anti-cancer immunity (43) and several autoimmune disorders (17), it is important to examine the requirements for the expression of both subunits of IL-12. Since the synthesis of the IL-12 p35 subunit limits the amount of bioactive IL-12 formed, the transcription factors responsible for the expression of p35 could be better targets for therapy than transcription factors responsible for p40 expression. We have shown here that three regions of each p35 promoter and several transcription factors, including IRF-1, could also be used as targets for the therapeutic manipulation of p35 expression. A more precise analysis of the regulatory domains, DNA binding domains, and protein interacting domains of these transcriptional factors could lead to the development of therapeutic agents, such as antagonistic peptides, that block p35 expression.

	Footnotes

 
¹ This work was supported by the Smokeless Tobacco Research Council (Grant 0692) and the National Multiple Sclerosis Society (Grant PP0777).

² Address correspondence and reprint requests to Dr. Thomas M. Petro, Department of Oral Biology, University of Nebraska Medical Center, 40th and Holdrege Avenue, Lincoln, NE 68583-0740. E-mail address: tpetro{at}unmc.edu

³ Abbreviations used in this paper: CD40L, CD40 ligand; CCM, cell culture medium; DIG, digoxigenin; GAS, -associated sequence; GRE, glucocorticoid response element; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; UTR, untranslated region; RLA, Renilla luciferase activity.

Received for publication September 29, 2000. Accepted for publication September 18, 2001.

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