Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0506319 on October 17, 2006

Published online before print October 17, 2006
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(Journal of Leukocyte Biology. 2007;81:355-363.)
© 2007 by Society for Leukocyte Biology

NF-{kappa}B is required for STAT-4 expression during dendritic cell maturation

Maria Elena Remoli*, Josiane Ragimbeau{dagger}, Elena Giacomini*, Valerie Gafa*, Martina Severa*, Roberto Lande*, Sandra Pellegrini{dagger} and Eliana M. Coccia*,1

* Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy; and
{dagger} Unit of Cytokine Signaling, CNRS URA 1961, Institut Pasteur, Paris, France

1Correspondence: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy. E-mail: e.coccia{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor STAT-4 plays a pivotal role in the IL-12-mediated development of naive CD4+ T cells into the Th1 phenotype. Initially thought to be restricted to the lymphoid lineage, STAT-4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and dendritic cells (DC). Here, we have studied STAT-4 in human monocyte-derived DC, and we demonstrated that its expression can be induced by multiple stimuli, such as the ligands for TLR-4, TLR-2, and TLR-3, different pathogens, CD40 ligand, and the proinflammatory cytokines TNF-{alpha} and IL-1ß. It is interesting that we found that STAT-4 is tyrosine-phosphorylated in response to type I IFN but not IL-12 in human mature DC. Cloning and functional analysis of the STAT-4 promoter showed that a NF-{kappa}B binding site, localized at –969/–959 bp upstream of the transcriptional start site, is involved in the regulation of this gene in primary human DC. EMSAs using a probe containing this NF-{kappa}B binding sequence and chromatin immunoprecipitation indicated that p65/p50 and p50/p50 dimers were the main NF-{kappa}B/Rel proteins involved in STAT-4 gene expression in maturing DC. The mutation of this {kappa}B site or the overexpression of the repressor I{kappa}B{alpha} exerted an inhibitory effect on a STAT-4 promoter-driven reporter as well as on STAT-4 expression. Altogether, these results indicate that STAT-4 can be finely tuned along with DC maturation through NF-{kappa}B activation and that its induction may be involved in preparing the DC to be receptive to the cytokine environment present in lymphoid organs.

Key Words: human primary DC • type I IFN • TLR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are the most important APC and key initiators of immune responses. It is known that DC functional capacities are mainly regulated by their state of maturation. Immature DC capture and process antigens in the periphery and present antigens in a MHC-specific context in the lymph node. Following capture of antigens, DC undergo extensive transformations, down-regulate their antigen-capturing capabilities, and up-regulate the expression of costimulatory molecules and MHC Class I and II molecules as well as that of chemokine receptors, which allow them to home into regional lymphoid organs [1 ]. Inflammatory signals, such as TNF-{alpha} and IL-1ß, cognate T cell signals, such us CD40 ligand (CD40L), and pathogen-associated molecular patterns can all promote a program of maturation in DC [2 3 4 ].

Although the significance of DC as regulators of innate and adaptive immunity is beyond doubt, little is known about intracellular mechanisms regulating DC functions. In the last years, microarray analysis showed that DC maturation is associated with the expression of a complex, stimulus-specific gene profile involving several transcription factors [5 , 6 ]. It is interesting that many inducers of DC maturation are also strong activators of NF-{kappa}B transcription factors [7 8 9 10 ], suggesting that the latter regulate the expression of a specific transcriptome leading to DC maturation. For instance, the classical NF-{kappa}B heterodimer, composed of the p50 and p65 subunits, is a potent activator of genes encoding costimulatory proteins and proinflammatory cytokines, whose expression is finely regulated during the DC maturation process [5 , 11 ]. Moreover, several other transcription factors, such as AP-1 and IFN regulatory factors (IRFs), may play a role in the regulation of genes associated with DC maturation [5 , 12 13 14 15 16 ]. Few data are available about the function of STAT factors in maturing DC [17 ]. Among the STAT factors, we focused on STAT-4, which is activated by two key immunoregulatory cytokines, IL-12 and IFN-ß, released from maturing DC [1 , 18 , 19 ]. Transcriptional and post-transcriptional mechanisms have been described to regulate constitutive and induced STAT-4 expression in human T cells [20 , 21 ]. Conversely, little is known about STAT-4 regulation in activated monocytes, macrophages, and DC [22 ]. Thus, in the present study, we analyzed the regulation of the STAT-4 gene and the activation of the STAT-4 protein during DC maturation. We observed that STAT-4 is tyrosine-phosphorylated in mature DC in response to type I IFN but not IL-12. Moreover, we also found that STAT-4 expression is induced by different maturative stimuli. Cloning of the human STAT-4 promoter showed the presence of binding sites for transcription factors, which are activated during DC maturation, such as NF-{kappa}B and AP-1. A comprehensive analysis of the STAT-4 promoter showed that the {kappa}B site located at –969/–959 bp upstream of the transcriptional start site (TSS) is directly involved in STAT-4 gene regulation in human primary DC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of DC
DC were prepared as described previously [18 ]. Briefly, PBMC were isolated from freshly collected buffy coats obtained from healthy, voluntary blood donors (Blood Bank of University "La Sapienza", Rome, Italy) by density gradient centrifugation using Lympholyte-H (Cedarlane, Hornby, Ontario, Canada). Monocytes were purified by positive sorting using anti-CD14-conjugated magnetic microbeads (Miltenyi, Bergisch Gladbech, Germany). The recovered cells were >99% CD14+, as determined by flow cytometry with anti-CD14 antibody. DC were generated by culturing monocytes in six-well tissue-culture plates (Costar Corp., Cambridge, MA) with 25 ng/ml GM-CSF and 1000 U/ml IL-4 (R&D Systems, Abingdom, UK) for 5 days at 0.5 x 106 cells/ml in RPMI 1640 (BioWhittaker Europe, Verviers, Belgium), supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15% FCS. At day 5, the cells were tested for their differentiation through the expression of CD1a (70–90% CD1a+) and the lack of CD14 (95% CD14–), and then DC were starved from IL-4 and GM-CSF for 20 h before their stimulation.

Reagents and microorganisms
A final concentration of 1000 U/ml IFN-ß (Avonex, Biogen Inc., Cambridge, MA) or IFN-{alpha} (IFN-{alpha}2, Roferon-A, F. Hoffmann-La Roche Ltd., Basel, Switzerland), 10 ng/ml IL-6, 100 ng/ml TNF-{alpha}, 10 ng/ml IL-1ß, and 100 ng/ml IL-12 (PeproTech EC Ltd., London, UK) human cytokines was generally used. DC maturation was obtained with 1 µg/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO), 50 µg/ml polyinosinic:polycytidylic acid (poly I:C; Sigma-Aldrich), and 10 µg/ml macrophage-activating lipopeptide-2 (MALP-2; a kind gift of Dr. Alberto Visintin, University of Massachusetts Medical School, Worcester). Human soluble CD40L (Alexis Corp., San Diego, CA) was used at a concentration of 200 ng/ml, together with 1 µg/ml enhancer. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich. The drug was dissolved in RPMI 1640, and the pH was adjusted to 7.4 by the addition of NaOH. NAC was used at the optimal concentration of 25 mM, which did not affect cell viability. Antihuman TNF-{alpha} antibody (R&D Systems) was used at a concentration of 0.15 µg/ml. Candida albicans, Strain IP 3153 (Institut Pasteur, Paris, France), was used at a yeast:DC ratio of 1:1. Mycobacterium tuberculosis (Mtb) H37Rv (ATCC27294) was used at a ratio of 1:5. Sendai virus (a kind gift of Dr. Ilkka Julkunen, National Public Health Institute, Helsinki, Finland) was used at a concentration of 60 hemagglutination (HA) U/ml. All pathogen preparations were analyzed for LPS contamination by the Limulus lysate assay (BioWhittaker Europe) and contained less than 10 pg/ml LPS.

Cell extracts
Whole cell extracts were prepared as described previously [23 ]. Briefly, cells (1x107) were lysed in 30–50 µl ice-cold whole cell extraction buffer [20 mM Hepes, pH 7.9, 50 mM NaCl, 0.5% Nonidet P-40 (NP-40), 1 mM DTT, 10 mM EDTA, 2 mM EGTA, 10 µg/ml leupeptin, 100 mM NaF, 0.5 mM PMSF, 10 mM sodium orthovanadate, and sodium molybdate]. The lysate was incubated for 30 min on a shaker at 4°C, insoluble debris was removed by centrifugation (13,000 rpm, 4°C, 10 min), and the lysate was stored at –80°C.

Nuclear cell extracts were prepared as described previously [23 ]. Briefly, cell pellets (5x106) were resuspended in 1 ml buffer A (0.5% NP-40, 10 mM EDTA, 10 mM EGTA, 10 mM KCl, 10 mM Hepes, pH 7.9), to which 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml trypsin inhibitor, and 1 µg/ml antipain were freshly added and incubated on ice for 10 min. Nuclei were sedimented by centrifuging the lysates at 1200 g for 10 min. The nuclear pellets were resuspended in 30–40 µl with buffer C [1 mM EDTA, 1 mM EGTA, 0.4 M NaCl, 20 mM HEPES (pH 7.9), 5 mM MgCl2, 25% glycerol, with fresh addition as above] and incubated for 10 min on ice with occasional mixing. The suspensions were clarified by centrifugation at 15,000 g for 10 min. The supernatants were recovered as nuclear extracts and were frozen rapidly on crushed dry ice and stored at –80°C.

Western blot analysis
Whole cell extracts (30 µg) were separated by 7% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with rabbit polyclonal antibodies against STAT-4 (Santa Cruz Biotechnology, CA) and tyrosine-phosphorylated STAT-4 (Zymed Laboratories Inc., South San Francisco, CA) and reacted with antirabbit HRP-coupled secondary antibody (Amersham Pharmacia Biotech, Little Chalfont, UK) using an ECL system. Blots, after stripping, were incubated with ß-actin antibodies (Sigma-Aldrich) to evaluate the content. Bands were revealed with an ECL detection system (Amersham Pharmacia Biotech) and quantified with the Kodak Image Station 440CF.

EMSA
To measure the association of DNA-binding proteins with different DNA sequences, synthetic, double-stranded oligonucleotides were end-labeled with [{gamma}32P] ATP by T4 polynucleotide kinase. For the analysis of NF-{kappa}B complexes, nuclear cell lysates (10 µg) were used in EMSA experiments. Binding reaction mixture (20 µl final vol) contained labeled oligonucleotide probes (50,000 cpm) in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 µg poly(dI)-poly(dC), Amersham Pharmacia Biotech]. Nuclear lysates were added, and the reaction mixture was incubated for 30 min at room temperature. The samples were analyzed on 5% PAGE gels with 0.25x Tris-boric acid-EDTA buffer [TBE; 1x TBE is 50 mM Tris-borate (pH 8.2) and 1 mM EDTA] for 3 h at 150 V at 18°C.

For the analysis of AP-1 complexes, whole cell lysates (10 µg) were used in EMSA experiments. The labeled oligonucleotide probe (50,000 cpm) was mixed with 1 µg poly(dI)-poly(dC) and 1 µg BSA (Sigma-Aldrich) and incubated for 30 min at 20°C in a final volume of 20 µl binding buffer (75 mM KCl, 20 mM Tris-HCl, pH 7.5, 1 mM DTT) containing cell extract. Glycerol was added to 13% (v/v), and samples were analyzed on 5% PAGE gels with 0.5x TBE for 1.5 h at 300 V at 18°C. For supershift analysis, 1 µg anti-p50, anti-p65, or anti-c-Jun/AP-1 (Santa Cruz Biotechnology) was added to the reaction for 30 min at room temperature. For competition analysis, a 100-fold molar excess of cold wild-type or noncanonical sequence was added to the binding reaction.

The sequences of oligonucleotides were as follows: {kappa}B STAT-4 #2 for 5'-CGGTGGGATTTTACTGTG-3'; {kappa}B STAT4 #2 rev 5'-CACAGTAAAATCCCACCG-3'; m{kappa}B STAT-4 #2 for 5'-CGGTGGTATGTTACTGTG-3'; m{kappa}B STAT-4 #2 rev 5'-CACAGTAACATACCACCG-3'; AP-1 for 5'-CGCTTGATGACTCAGCCGCCGGAA-3'; AP-1 rev 5'-TTCCGGCGGCTGAGTCATCAAGCG-3'; STAT-4_AP-1 #4 for 5'-AAATTGAGTGACACTTTCTACC-3'; STAT-4_AP-1 #4 rev 5'- GGTAGAAAGTGTCACTCAATTT-3'; STAT-4_AP-1 #5 for 5'-CCTACTGTGAATCAAGGGGT-3'; STAT-4_AP-1 #5 rev 5'-ACCCCTTGATTCACAGTAGGG-3'.

RNA ligase-mediated rapid amplification of cDNA ends (RACE)-PCR
Total RNA (10 µg) from purified human DC treated for 16 h with LPS was used for cDNA synthesis with the FirstChoice RML-RACE kit (Ambion, Austin, TX). The following STAT-4-specific primers were used to amplify full-length 5' cDNA fragments of human (h) STAT-4: h STAT-4 outer 5'-GAAACACGACCTAACTGTTCATCC-3'; h STAT-4 inner 5'-CGCAAGCTTGAAGCTGCCTCCCAGTCTTGA-3'. PCR products were cloned into pBluescript II Phagemid Vectors (Stratagene, La Jolla, CA), and multiple clones were sequenced.

Plasmids
To analyze the 5'-flanking region of the human STAT-4 gene, a 2447 bp PCR product corresponding to the region from –2437 nt to +10 nt was amplified from genomic DNA (h ST4 2.4 Kb). Progressive deletion clones (h ST4 1.4 Kb, from –1476 nt to +10 nt, and h ST4 0.6 Kb, from –654 nt to +10 nt) were generate by PCR using h ST4 2.4 Kb as template.

All STAT-4 constructs were generated with MluI and XhoI linkers at their 5' and 3' ends, respectively; once cleaved, these were subcloned directionally into the promoterless luciferase reporter vector pGL3basic (Promega, Madison, WI), digested with the same restriction enzymes, and sequenced.

Two point mutations in the potential NF-{kappa}B binding site, localized at –969 bp and –959 bp upstream of the TSS, were introduced by two-step, PCR-mediated mutagenesis of h ST4 2.4 Kb plasmid. In the primary PCR reaction, specific STAT-4 promoter primers were used to generate the overlapping fragments, which were subsequently used as templates for a further PCR reaction to generate the mutation. For the PCR amplification, the reactions were performed in a total volume of 50 µl PCR buffer (10 mM Tris HCl, pH 8.3, 50 mM KCl) containing 1 U Vent enzyme (New England BioLabs, Ipswich, MA), 0.2 mM each deoxy-unspecified nucleoside 5'-triphosphate (Invitrogen, Paisley, UK), and 1 µM each forward and reverse primers. The reaction mixture was subjected to 25 cycles of PCR with the following conditions: 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The I{kappa}B{alpha} expression plasmid was a kind gift of Dr. Catherine Dargemont (Institut Curie-CNRS UMR144, Paris, France). All plasmid DNA for transient tranfections was prepared using Endofree plasmid maxi kits (Qiagen Inc., Valencia, CA).

Transfection and luciferase assays
DC were transfected using Nucleofector technology, according to the recommendations of the manufacturer (Amaxa Nucleofector, Koeln, Germany). Two hours after transfection, cells were treated for 16 h with LPS, and luciferase activity was analyzed using the Dual-Luciferase reporter assay system (Promega). The efficiency of transfection, determined by Renilla luciferase activity in the lysates, was used to normalize the activity of firefly luciferase activity.

Chromatin immunoprecipitation (ChIP) assay
The protocol used for ChIP was performed as described previously [24 ]. In brief, 12 x 106 cells were cross-linked by incubation with 1% formaldehyde for 15 min, and after fixation, cells were resuspended in lysis buffer (5 mM Pipes, pH 8, 85 mM KCl, and 0.5% NP-40 with protease inhibitors). After a 10-min incubation on ice, the nuclei were resuspended in sonication buffer [0.1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8), and 0.5% deoxycholic acid with protease inhibitors] and were sonicated to an average length of 1000 bp. Chromatin from 6 x 106 cells was incubated in immunoprecipitation buffer (sonication buffer plus 150 mM LiCl) overnight at 4°C with 4 µg anti-p50 polyclonal antibodies (Santa Cruz Biotecnology). DNA–protein complexes were collected with Ultralink immobilized protein A/G-Sepharose (Pierce, Rockford, IL), followed by sequential washes with immunoprecipitation buffer, low salt buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 150 mM NaCl], high salt buffer [0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl (pH 8.1)], and 10 mM Tris-HCl and 1 mM EDTA (pH 8). Samples were then eluted, and after reverse cross-linking, proteins were digested with protease K, and RNA was removed by RNase A. DNA was purified with the QIAquick PCR purification kit (Qiagen) and resuspended in 30 µl water. Five microliters was used for PCR amplification, and forward and reverse primers (5'-GAATCCAGGGTGTACCTG-3'; 5'-GCTGAAGTGGAAGGACTCCTTG-3') flanked the NF-kB #2 site of the STAT-4 promoter. The PCR product was separated by agarose gel electrophoresis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
STAT-4 expression and activation in mature DC
First, we characterized the expression of STAT-4 in human DC treated with different maturative stimuli. DC were treated for 24 h with LPS, MALP-2, or poly I:C, agonists of TLR-4, TLR-2, and TLR-3, respectively. DC were also stimulated with three different pathogens: C. albicans, Mtb, and Sendai virus, which are all able to stimulate DC maturation [25 26 27 ]. Finally, to mimick the maturation induced by the interaction with activated T lymphocytes, DC were stimulated with soluble CD40L. In unstimulated cells, STAT-4 protein was not detectable, and all treatments induced the STAT-4 content, although to a different extent (Fig. 1A ). Next, we addressed the question of whether STAT-4 was induced directly by these stimuli or whether secondary factors secreted by DC in the course of maturation were involved. For this, we analyzed STAT-4 expression following a 24-h treatment of DC with the principal regulatory and proinflammatory cytokines: TNF-{alpha} plus IL-1ß, IL-6, type I IFN, and IL-12. Only the combined TNF-{alpha} and IL-1ß treatment stimulated STAT-4 expression (Fig. 1B , left panel). To evaluate the autocrine effect of the released TNF-{alpha} on STAT-4 expression in LPS-stimulated DC, neutralization experiments were conducted. DC were stimulated with LPS in the presence or absence of neutralizing anti-TNF-{alpha} antibody (Fig. 1B , right panel). Blockade of TNF-{alpha} resulted in a clear reduction of STAT-4, suggesting a cooperative effect of LPS treatment with the released TNF-{alpha}.


Figure 1
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  		Figure 1. Induction and activation of STAT-4 in maturing DC, which were left untreated or treated for 24 h with LPS (1 µg/ml), MALP-2 (10 ng/ml), poly I:C (50 µg/ml), C. albicans (yeast:DC ratio, 1:1), Mtb (bacterium:DC ratio, 1:5), Sendai virus (60 HA U/ml), and CD40L (200 ng/ml plus 1 µg/ml enhancer; A). (B, Left panel) TNF-{alpha} + IL-1ß (100 ng/ml and 10 ng/ml, respectively), IL-6 (10 ng/ml), IL-12 (100 ng/ml), IFN-{alpha} (1000 U/ml), and IFN-ß (1000 U/ml) and (B, right panel) with LPS in the presence or absence of neutralizing anti-TNF-{alpha} antibody (0.15 µg/ml). (C, Left panel) DC from one donor were incubated for 24 h with TNF-{alpha} and IL-1ß and treated with exogenous IFN-ß. (C, Middle panel) Cells from a different donors were treated with LPS for 24 h and then were washed three times with complete medium to remove the secreted cytokines. Treatment with IL-12 or IFN-ß for 30 min was then performed. (C, Right panel) As control, PBMC were treated for 30 min with exogenous IL-12 and IFN-ß. Whole cell lysates (30 µg) were separated on a 7% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted sequentially with the indicated antibodies to evaluate STAT phosphorylation status (STAT-4P) and protein content (STAT-4C). After stripping, the blots were reprobed with ß-actin as an internal loading control. The results shown are from one of three experiments that yielded similar results.

 
Together, these results suggested that maturative stimuli known to activate transcription factors, such as NF-{kappa}B or AP-1, could be involved in STAT-4 regulation during DC maturation. Indeed, both transcription factors may modulate each other and cooperate in inducing DC maturation [12 , 14 ].

Next, we characterized the profile of STAT-4 tyrosine phosphorylation by IFN-ß and IL-12, conventional STAT-4-activating cytokines, in human DC matured with TNF-{alpha} plus IL-1ß or LPS treatment. In DC that underwent maturation following the combined treatment with TNF-{alpha} and IL-1ß, a clear STAT-4 phosphorylation was observed upon a 30-min stimulation with IFN-ß (Fig. 1C , left panel). A similar activation profile was observed in LPS-matured DC. However, in this case, to avoid signaling saturation as a result of autocrine-acting IFN-ß [28 ], LPS-matured DC were first washed and then challenged with cytokines. Although a clear STAT-4 activation was observed in response to IFN-ß, no induction occurred in response to IL-12 in mature-washed DC (Fig. 1C , middle panel). In contrast, in PBMC used as control, STAT-4 phosphorylation was induced by IFN-ß and IL-12 (Fig. 1C , right panel).

Kinetics of STAT-4 induction
To define the kinetics of STAT-4 induction, DC were stimulated with LPS, MALP-2, poly I:C, TNF-{alpha} + IL-1ß, or CD40L for different times, and STAT-4 expression was analyzed by immunoblotting. All stimuli induced STAT-4 expression as early as 3 h with a further increase at 8 h (Fig. 2A and 2B ). The level remained sustained thereafter.


Figure 2
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  		Figure 2. Kinetics of STAT-4 expression in mature DC and effects of NAC. Whole cell extracts were prepared from cells treated with LPS, MALP-2, and poly I:C (A) and TNF-{alpha} + IL-1ß and CD40L (B) at different time-points. (C) DC were treated for 24 h with LPS alone or in the presence of NAC (25 mM). Extracts (30 µg) were analyzed by immunoblot with anti-STAT-4 antibody. The filters were reblotted with ß-actin antibody as an internal loading control. These are representative experiments, which were repeated three additional times with cell extracts from different DC cultures.

 
Given the nature of the stimuli and the rapid kinetics of induction of STAT-4, we investigated the possible involvement of transcription factors such as AP-1 and NF-{kappa}B. First, we tested whether the inhibition of the NF-{kappa}B and AP-1 pathways might affect STAT-4 expression. To this purpose, DC were treated for 24 h with LPS in the presence of NAC, an antioxidant molecule known to inhibit the activation of different signaling pathways involving c-Jun N-terminal kinase, p38 MAPK, and NF-{kappa}B [29 ]. In addition, it is known that NAC can inhibit the LPS-induced activity of the transcription factor NF-{kappa}B in human DC [30 ]. As shown in Figure 2C , we observed a clear inhibition of STAT-4 expression, when DC were stimulated with LPS in the presence of NAC. These data suggested a possible requirement of AP-1 and NF-{kappa}B activities in the regulation of STAT-4 gene transcription in response to stimuli that promote DC maturation.

Cloning and characterization of the human STAT-4 promoter
To verify the presence of potential AP-1 and NF-{kappa}B-binding sites, a region of 2437 bp upstream of the putative TSS was analyzed using the MatInspector program (Fig. 3 ). Among the multiple predicted transcription factor binding sites, two NF-{kappa}B and five AP-1 binding sites were found.


Figure 3
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  		Figure 3. Nucleotide sequence of the human STAT-4 promoter. A region up to –2437 bp upstream of TSS was analyzed by the MatInspector program. Potential transcription factor binding sites are underlined. The major TSS was identified by 5' RACE, and +1 (indicated with an arrow) refers to the first nucleotide of the coding region.

 
To identify the TSS, we performed 5'-RACE using mRNA from LPS-matured DC. Sequencing analysis of the RACE products identified a major TSS (nt 66,678 of GenBank locus AC079777.5), 62 bp upstream of the translational start codon (nt 63,875 of GenBank locus AC079777.5). It is interesting that this predominant initiation sequence did not resemble an initiator welement nor did it contain an upstream TATA box. However, this sequence corresponds to the TSS identified by Yap and colleagues [20 ] in T cells. By RACE analysis, minor transcripts were identified ~200 bp upstream of the major TSS, indicating that the STAT-4 gene contains multiple start sites, similar to other TATA-less genes (data not shown) [31 ].

To ascertain the functional significance of the putative NF-{kappa}B and AP-1 binding elements in the STAT-4 promoter, a series of STAT-4 promoter-luciferase constructs were made: h ST4 2.4 Kb (from –2437 to +10); h ST4 1.4 Kb (from –1476 to +10); and h ST4 0.6 Kb (from –654 to +10; Fig. 4 ). Transient transfection experiments in primary human DC showed that after LPS treatment, h ST4 2.4 Kb and h ST4 1.4 Kb constructs drove a twofold increase in luciferase activity, and the expression of h ST4 0.6 Kb construct, lacking NF-{kappa}B and AP-1 binding sites, was not affected by LPS treatment (Fig. 4) . Moreover, the lower basal level of luciferase activity from the 2.4 Kb construct observed in untreated cells suggested that the region from –2437 bp to –1476 bp might contain negative regulatory elements. The comparable fold induction of the h ST4 2.4 Kb and h ST4 1.4 Kb constructs in LPS-treated cells suggested that the potential NF-{kappa}B and AP-1 binding sites (here referred as seq. #2, #3, #4, and #5), present in the intermediary h ST4 1.4 Kb construct, were necessary for the regulation of LPS-inducible STAT-4 gene transcription.


Figure 4
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  		Figure 4. Functional analysis of the human STAT-4 promoter. The schematic organization of the STAT-4 gene promoter is shown with the putative NF-{kappa}B and AP-1 binding sites. The structure of the constructs containing deletions of the 5'-flanking region of the human STAT-4 gene used to transfect human primary DC is shown. Two hours after transfection, the cells were treated with LPS for 16 h. The luciferase activity (LUC) was normalized by cotransfection with the Renilla-thymidine kinase expression plasmid. The promoterless pGL3 basic plasmid was included to obtain background values. Data are reported as the fold induction of luciferase activity over background values. The results represent the means ± SE of three independent experiments.

 
Characterization of NF-{kappa}B and AP-1 binding sites within the h ST4 1.4 Kb promoter region
To determine whether the potential {kappa}B and AP-1 sites located within the intermediary hST4 1.4 Kb region could bind these transcription factors, EMSA was performed. Among the predicted AP-1 binding sites, only two (seq. #5 and seq. #4, localized at nt –832/–825 and nt –1077/–1067, respectively) showed high similarity to the AP-1 consensus sequence (5'TGACTCA3') [13 ]. These two sequences were analyzed for their capacity to bind AP-1 complexes by EMSA using nuclear extracts from DC treated for 3 h with LPS. As control, a canonical AP-1 binding sequence was used. As shown in Figure 5A , a distinct, complex binding to the canonical AP-1 consensus sequence was detected following LPS treatment. A noninduced complex, with a faster mobility, was visible using the oligonucleotide seq. #4, whereas no complex was detected with seq. #5. To investigate the composition of the AP-1 complex binding to seq. #4, competition experiments and supershift assays were carried out (Fig. 5B) . Complex formation was not inhibited by the addition of an excess of unlabeled consensus oligonucleotides, and no supershift was observed when antibodies, recognizing different Jun family members, were added.


Figure 5
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  		Figure 5. NF-{kappa}B but not AP-1 binding activity to the STAT-4 promoter is present in LPS mature DC. (A) Whole extract (10 µg) from untreated DC or DC stimulated for 3 h with LPS were analyzed by EMSA. An oligonucleotide containing an AP-1 consensus sequence (cs) and two oligonucleotides specific for the AP-1 sequences within the STAT-4 promoter (#4 and #5) were used as probes. (B) For competition studies, 100x excess of cold oligonucleotides containing an AP-1 consensus sequence, the AP-1 putative binding site within the STAT-4 promoter (#4), and a sequence unrelated to AP-1 (unr) was added to the binding reaction. For supershift analysis, 1 µg anti-c-Jun/AP-1 antibody was preincubated with 10 µg whole extracts. Results were representative of at least three independent experiments. (C) Nuclear extracts (10 µg) from untreated DC or from DC treated with LPS for 1 h were analyzed by EMSA using radiolabeled oligonucleotides corresponding to the putative NF-{kappa}B binding site present within the STAT-4 promoter ({kappa}B STAT-4 #2) and to a mutated version of the {kappa}B site (m{kappa}B STAT-4 #2). Supershift assay was performed with anti-p50 and anti-p65 antibodies. (D) Proteins were cross-linked to DNA in control DC and in DC treated for 1 h with LPS. DNA-bound NF-{kappa}B was immunoprecipitated with anti-p50 antibody. The chromatin fragments were amplified by PCR using primers flanking the {kappa}B STAT-4 #2. Input refers to amplification of 1/10 of the total amount of DNA prior to immunoprecipitation.

 
To analyze NF-{kappa}B binding to the sequence located at –969/–959 ({kappa}B site #2), EMSA was carried out with double-stranded oligonucleotides corresponding to this {kappa}B site and nuclear extracts from LPS-treated DC. As shown in Figure 5C , this {kappa}B site was able to bind a NF-{kappa}B complex following LPS treatment. The identity of the induced complex was assessed by using antibodies raised against p50 and p65 subunits (Fig. 5C) . The same oligonucleotide, containing two mutations (–967 nt G{Rightarrow}T, –964 nt T{Rightarrow}G), destroyed the NF-{kappa}B binding activity (Fig. 5C) . To test in vivo the interaction of NF-{kappa}B with the STAT-4 promoter, ChIP assay was performed in control and LPS-stimulated DC (Fig. 5D) . Proteins were cross-linked to DNA, and the chromatin was immunoprecipitated with specific anti-p50 antibody. In LPS-stimulated DC, the region flanking the {kappa}B site #2 was amplified from the DNA coimmunoprecipitated with the anti-p50 antibody, thus confirming the critical role of this NF-{kappa}B binding site in the regulation of the STAT-4 gene during DC maturation.

The NF-{kappa}B site #2 is required for LPS-inducible STAT-4 gene expression
To investigate the functional significance of the NF-{kappa}B binding site, DC were cotransfected with the h ST4 1.4 Kb construct and an expression vector for I{kappa}B{alpha}, which is an inhibitory protein that retains NF-{kappa}B dimers in the cytoplasm. As shown in Figure 6 , in DC overexpressing the I{kappa}B{alpha} construct, LPS-induced STAT-4 promoter activity was prevented. A marked decrease in the LPS-induced luciferase activity was also observed when DC were transfected with a mutated h ST4 1.4 Kb construct containing two substitutions in the NF-{kappa}B site #2 (h ST4m 1.4 Kb in Fig. 6 ). The mutations of this site also abolished LPS-induced promoter activity of the h ST4 2.4 Kb construct, indicating that this element is required for full reporter activity (data not shown). As predicted, we also observed a clear inhibition of STAT-4 protein increase following LPS treatment in DC overexpressing I{kappa}B{alpha} compared with those transfected with an empty control vector (Fig. 6B) . Altogether, these results indicate that NF-{kappa}B is directly involved in the induction of STAT-4 gene transcription.


Figure 6
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  		Figure 6. NF-{kappa}B is required for transactivation of the STAT-4 gene promoter. (A) h ST4 1.4 Kb luciferase reporter construct was cotransfected with a plasmid encoding I{kappa}B{alpha} or with an empty vector (EV), as indicated. DC were also transfected with a mutated construct, h ST4 m1.4 Kb, containing two point mutations within the NF-{kappa}B site #2 (–967 nt G{Rightarrow}T, –964 nt T{Rightarrow}G). Transfection experiments were conducted as described in the legend of Figure 4 . Cells were lysed after 16 h of LPS treatment, and luciferase activity was measured. The results represent the means ± SE of three independent experiments. (B) DC were transfected with a plasmid encoding I{kappa}B{alpha} or with EV. Whole cell extracts were prepared 16 h after LPS treatment, and 30 µg extracts were analyzed by immunoblot with anti STAT-4 antibody. After stripping, the blots were reprobed with ß-actin as an internal loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
STAT-4 is a key transcription factor regulating Th1 development [32 , 33 ]. Initially thought to be restricted to the lymphoid lineage, STAT-4 was subsequently shown to be expressed in the myeloid compartment, mainly in activated monocytes, macrophages, and DC [22 ]. Despite these scattered observations, STAT-4 transcriptional regulation and activation signals in DC remain poorly understood. In the present study, we showed that different pathogens, TLR agonists, and proinflammatory cytokines can induce STAT-4 expression in primary DC. To elucidate the mechanism underlying this induction, the promoter of the human STAT-4 gene was cloned and sequenced. Among the cis-elements identified in the STAT-4 promoter, five AP-1 binding sequences and two NF-{kappa}B regulatory elements were found. NF-{kappa}B and AP-1 are ubiquitous factors that can regulate the expression of many genes. Indeed, AP-1 elements are often found in the enhancer or promoter of many NF-{kappa}B-regulated genes. Moreover, in DC, these transcription factors are activated simultaneously by a multitude of maturative stimuli [12 13 14 ]. Among the predicted AP-1 binding sites, only one (seq. #4) was bound constitutively by a factor, which remains to be identified. Its binding was not inhibited by an excess of unlabeled AP-1 consensus oligonucleotide or by the addition of antibodies recognizing different Jun family members. Our EMSA and ChIP data showed that the NF-{kappa}B binding site #2, located at –969/–959 bp upstream of the TSS, binds the p50/65 NF-{kappa}B heterodimer. Transient transfection of different promoter constructs confirmed that this {kappa}B site is required for STAT-4 gene induction in primary DC. In line with this, mutation of {kappa}B site #2 and overexpression of the repressor I{kappa}B{alpha} exerted an inhibitory effect on the STAT-4 promoter-driven reporter and accordingly, on the STAT-4 protein.

An important role has been attributed to NF-{kappa}B in DC development and survival as well as in cytokine production [8 , 34 ]. It has been reported that a large-scale change in gene expression occurs along DC maturation, which parallels the marked transformation in cellular phenotype and function [5 ]. These changes reflect a reprogramming of the transcriptional activity, which is dependent on modification in the expression of transcription factors and in the responsiveness to cytokines and chemokines. In particular, STAT-4 is activated by IL-12, IL-23, IL-27, and type I IFN [35 36 37 38 39 ]. These cytokines are induced in DC in response to several extracellular stimuli [18 , 23 , 25 ], and in turn, they might act in an autocrine manner. Nagayama and colleagues [40 ] showed that IL-12 acts directly on human DC through the engagement of the IL-12 receptor (IL-12R). In our experimental system, we could neither detect expression of IL-12Rß1 and -ß2 receptor chains (data not shown) nor STAT-4 phosphorylation following IL-12 treatment (Fig. 1C) . No data are presently available about the ability of IL-23 to activate STAT-4 in human DC. Conversely, we could show STAT-4 activation in response to type I IFN (Fig. 1C) , an event that may occur when mature DC reach lymphoid organs.

The STAT-4-dependent transcriptome of mature DC has not yet been identified. Conversely, the STAT-4-dependent transcriptome induced by IL-12 in differentiated Th1 cells has been reported [41 , 42 ], and IFN-{gamma} is the main target gene. Although the ability of APCs to produce IFN-{gamma} has been described [43 44 45 46 ], we did not detect IFN-{gamma} production in our experimental model (data not shown). Another possible STAT-4 target gene could be IRF-1, encoding a key transcription factor involved in the regulation of several genes of the immune response [47 ]. Indeed, we have previously shown that STAT-4 induces IRF-1 gene expression in human Th1 cells and that this accounts for the regulation of some IL-12-induced effector functions [48 ]. Altogether, these data suggest the following scenario: under inflammatory conditions, mature DC activate NF-{kappa}B, which in turn, induces STAT-4 gene expression; activation of STAT-4 by type I IFN may ultimately regulate the transcription of specific target genes, such as IRF-1. The understanding of this signaling cascade should help to better manipulate DC, considering their pivotal role in the initiation of immune responses and the promising strategies for their therapeutical use [49 , 50 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Istituto Superiore di Sanità (ISS)-National Institutes of Health program (#5303) and from AIDS Research (#50F/G) to E. M. C. and a grant from the Association pour la Recherche sur le Cancer (#3387) to S. P. We are grateful to I. Julkunen (Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland), A. Visintin (University of Massachusetts Medical School, Worcester), and C. Dargemont (Institut Curie-CNRS UMR144, Paris, France) for providing reagents. We are grateful to E. Perrotti and R. Ilari (Department of Infectious, Parasitic and Immune-Mediated Diseases, ISS, Rome, Italy) for help with the ChIP assay, to L. Cianetti (Department of Hematology, Oncology and Molecular Medicine, ISS) for RACE, and to E. Morassi for preparing drawings.

Received May 10, 2006; revised August 16, 2006; accepted September 22, 2006.


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