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Originally published In Press as doi:10.1074/jbc.M306441200 on July 22, 2003

J. Biol. Chem., Vol. 278, Issue 41, 39372-39382, October 10, 2003
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Activation of the Murine Interleukin-12 p40 Promoter by Functional Interactions between NFAT and ICSBP*

Chen Zhu {ddagger}, Kavitha Rao §, Huabao Xiong {ddagger}, Khatuna Gagnidze {ddagger}, Fengling Li §, Curt Horvath {ddagger} and Scott Plevy § 

From the {ddagger}Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029 and the §Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, June 18, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-{gamma} and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at –68 to –54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-{gamma}. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-{gamma}-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-{gamma}-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
IL1-12 is a heterodimeric cytokine produced by macrophages and dendritic cells. The key functions of IL-12 are the induction and maintenance of T-helper-1 (Th1) responses (1). As IL-12 is induced in macrophages and dendritic cells by bacteria and bacterial products, it is an important bridge between host innate and adaptive immunity. In addition to its functions in host immune defense against microorganisms, IL-12 is also involved in the pathogenesis of Th1-mediated chronic inflammatory disorders in mice and humans, such as diabetes mellitus, multiple sclerosis, arthritis, and inflammatory bowel disease (2). Therefore, understanding the molecular events that lead to IL-12 gene expression may provide important insight into how a Th1 response is regulated in infectious and inflammatory diseases.

Biologically active IL-12 is a heterodimer composed of two subunits, p40 and p35, which are encoded by two separate genes. The p35 gene is constitutively expressed in many cell types (3). In contrast, the p40 gene is only detected in cells that produce bioactive IL-12 (3) and is strongly induced by intracellular bacteria and bacterial products that in vivo stimulate Th1 responses (4). Furthermore, the IL-12 p40 subunit also may heterodimerize in vivo with a p19 subunit to form the cytokine IL-23. IL-23 influences Th1 development and cytokine secretion and has been demonstrated to have an important role in infectious and inflammatory disease in mice independent of IL-12 (5, 6). Therefore, studies of IL-12 transcriptional regulation have focused on the p40 promoter. An NF-{kappa}B site from –131 to –121 relative to the transcription start site in the murine IL-12 p40 promoter is necessary for induction of promoter activity by LPS (7). In the human p40 promoter, a consensus Ets sequence from –211 and –207 that binds a multiprotein complex was implicated in promoter activation by LPS and IFN-{gamma} (8). We had initially reported a comprehensive functional analysis of the murine and human IL-12 p40 promoters in RAW 264.7 cells. Multiple control elements were involved in heat-killed Listeria monocytogenes- and LPS-activated transcription of the p40 gene. These elements include a C/EBP binding site between –96 and –88 (9), and an AP-1 site from –81 to –75 (10). Furthermore, functional studies suggested that several other cis-acting elements may regulate promoter activation by bacteria and bacterial products (10).

Although much has been learned about the IL-12 p40 promoter, many important biologic questions remain. For example, IFN-{gamma} is required for optimal production of bioactive IL-12 by macrophages and dendritic cells. Mechanisms governing the IFN-{gamma} transcriptional response in the IL-12 p40 promoter remain controversial (9). However, mice deficient in the interferon regulatory factors IRF-1, IRF-2, and ICSBP demonstrated a selective defect in IL-12 production (11, 12). A molecular mechanism for this in vivo phenomenon was suggested when ICSBP was described to activate the IL-12 p40 promoter through the Ets element (13). However, functional analysis from this study demonstrated that other downstream elements may also mediate the effect of ICSBP. Similarly, IL-10 is an important anti-inflammatory cytokine that potently inhibits IL-12 p40 transcription. The effects of IL-10 on the IL-12 p40 promoter remain unknown (14).

In this study, an important control region from –71 to –54 in the murine IL-12 p40 promoter is characterized. Site-directed mutagenesis suggests that this region is important for promoter activation by LPS and IFN-{gamma}. Electrophoretic mobility shift assays (EMSA) and DNA affinity binding experiments demonstrate a multi-protein complex that binds to this region, of which NFAT (–61 to –54) and ICSBP (–71 to –62) are major components. DNA binding of ICSBP is dependent upon NFAT binding. These in vitro interactions are demonstrated on the endogenous promoter by chromatin immunoprecipitation experiments. NFAT and ICSBP protein-protein interactions are also detected by co-immunoprecipitation. Overexpression of NFAT and ICSBP synergistically activates IL-12 p40 gene transcription. Expression of a dominant negative NFAT in RAW264.7 cells attenuates LPS- and IFN-{gamma}-activated endogenous IL-12 p40 mRNA accumulation. IL-10 treatment of RAW264.7 cells prior to LPS and IFN-{gamma} activation abrogates the NFAT-ICSBP interaction. Thus, NFAT-ICSBP may act as a downstream target for IL-10 inhibition of IL-12 p40 gene expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—The murine IL-12 p40 promoter from –101 to +55, or –355 to +55 (relative to the transcription start site), chloramphenicol acetyltransferase (CAT) reporter, and luciferase reporter plasmids have been previously described (10). All two-base pair mutant plasmids described in this paper were generated by two-step PCR using overlapping internal primers containing a mutant site (9). Mammalian expression plasmids for murine wild type NFATc1 (from Tammy Nachman and Melissa Brown) and NFATc2 (from Anjana Rao) were described previously (15, 16). An expression plasmid for the dominant negative NFAT protein, (Dn-NFAT), was obtained from Roger Davis (17). The human ICSBP expression plasmid, the GST mammalian expression vector pEBG, and the human CD4 (hCD4) expression plasmid were provided by Curt Horvath, Patricia Cortes, and Adrian Ting, respectively.

Cell Lines and Reagents—The RAW 264.7 murine macrophage line and the human 293T cell line were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin. Salmonella enteriditis LPS, phorbol 12-myristate 13-acetate (PMA), ionomycin, and cyclosporin A were purchased from Sigma. Murine recombinant IFN-{gamma} and IL-10 were from R&D Inc. Antibodies against NFATc1 (7A6), NFATc1 (K-18), NFATc2 (M-20), NFATc3 (M-75), NFATc4 (C-20), IRF-1 (M-20), IRF-2 (C-19), IRF-4 (M-17), ICSBP (C-19), NF-{kappa}B p50 (NLS), p65 (C-20), c-Rel (C), PU.1 (T-21), and STAT1 (E-23) were purchased from Santa Cruz Biotechnology, Inc.

Transfections—RAW 264.7 cells were transiently transfected using SuperFect Transfection Reagent (Qiagen) as previously described (10). Transient transfections in 293T cells were performed using LipofectAMINE PlusTM reagent (Invitrogen) per the provided protocol. After 36 h, the transfected cells were activated with 5 µg/ml LPS and 10 ng/ml IFN-{gamma} (for RAW264.7 cells), or 50 ng/ml PMA and 1 µM ionomycin (for 293T cells) as indicated under "Results." Ten to 12 h after activation, the cells were harvested using 1x reporter lysis buffer (Promega). CAT assays or luciferase assays were performed as previously described (9). Cells were co-transfected with a plasmid that expresses {beta}-galactosidase under a CMV promoter to correct for differences in transfection efficiency between experiments, as previously described (10).

EMSA—RAW 264.7 cell nuclear extracts were prepared as previously described (9). For EMSA, 200 ng of double-stranded oligonucleotide DNA probes were labeled with [{alpha}-32P]dGTP and [{alpha}-32P]dCTP by Klenow, and purified with Quick Spin columns (Sephadex G-50) (Roche). Our numerical nomenclature of EMSA probes denotes the sequences spanned within the murine IL-12 p40 promoter with respect to the transcription start site. The sequence of the EMSA probe 77/46 is 5'-gatcTCAGTTTCTACTTTGGGTTTCCATCAGAAAGT, consensus NFAT probe is 5'-gatcGCCCAAAGAGGAAAATTTATTTCATA, and consensus IFN-stimulated response element (ISRE) probe is 5'-gatcACTTTCAGTTTCAT. The sequence of the IL-18 p1 promoter EMSA probe is 5'-gatcTCAGTTGAAGTCTGGAAAGAGAAACTTAGTGAGA, and the murine inducible nitric-oxide synthetase probe is 5'-gatcACTGACACTGTCAATATTTCACTTTCATAATGGAAAAT. Other probes spanning the IL-12 p40 C/EBP site (112/78), NF-{kappa}B site (146/107), AP-1 element (88/63), and a consensus NF-{kappa}B DNA binding element have been described previously (9, 10). Sequences for other mutant probes are available from the authors. EMSAs were performed as in previous publications (9, 10).

DNA Affinity Binding Assay—A biotinylated IL-12 p40 promoter from –101 to –36 was amplified by PCR using a 5'-biotin-labeled upstream primer, and conjugated to Dynabeads (M-280 streptavidin, Dynal®). The NFAT site mutant (from –62 to –57), ICSBP site mutant (–68 to –63), and AP-1 site mutant (–80 to –75) promoter sequences were also PCR amplified by the same method (mutant sequences are available from the authors). The experimental procedure has been described in previous studies (13) and the Technical Handbook for Dynal® with some modifications. 5'-Biotin-labeled promoters were conjugated with 200 µg of streptavidin beads in TE buffer (pH 7.9). The DNA-conjugated beads were blocked by 0.5% bovine serum albumin in TGED buffer (10 mM Tris, pH 8.0, 1 mM EDTA, and 0.1 M NaCl) overnight at 4 °C. 500 µg of nuclear extracts from IFN-{gamma} and LPS-activated RAW264.7 cells were then added, and TGED was used to adjust the final volume to 500 µl with the final concentration of NaCl at 100 mM. The mixtures were subsequently incubated at 4 °C for 2–4 h. Beads were extensively washed with TGED buffer, and DNA-binding proteins were eluted from the DNA affinity column using TGED with 1 M NaCl. Proteins were separated by SDS-PAGE for immunoblot detection with specific antibodies.

Murine IL-12 p40 mRNA Expression—1.5 x 107 RAW 264.7 cells in 150-mm plates were transiently transfected with 15 µg of Dn-NFAT and 15 µg of human CD4 expression plasmids. As a control, 15 µgofthe empty expression plasmid pCDNA3 was used instead of Dn-NFAT. The plasmids were suspended in 2 ml of DMEM and mixed with 120 µl of Superfect® (Qiagen) at room temperature for 5–10 min, then added to PBS-washed RAW264.7 cells with an additional 13 ml of complete DMEM (containing 10% fetal bovine serum, and 1% penicillin and streptomycin) for 4 h. The cells were then washed with PBS, and 50 ml of complete DMEM was added. After 36 h, CD4-positive cells were selected using the CD4 Positive Isolation Kit (Dynal®). CD4-positive cells were cultured for an additional 12 h. Cells were then pretreated with 10 ng/ml murine IFN-{gamma} for 1 h and activated with 5 µg/ml LPS for an additional 4 h. Total RNA was harvested using RNeasy® Mini Kits (Qiagen). Finally, reverse transcriptase-PCR was performed to detect IL-12 p40 mRNA expression as previously described (9).

Co-immunoprecipitation Experiments—RAW 264.7 cells were cultured to 90% confluence in 150-mm plates. IFN-{gamma} (10 ng/ml) was then added 1 h before LPS (5 µg/ml) activation. In some experiments, IL-10 (10 ng/ml) was added 1 h before IFN-{gamma} activation. Four hours later, cells were harvested for nuclear extract preparation, as previously described (10). 500 µg of nuclear extract was incubated with 2 µg of antibody to ICSBP or NFATc1 in presence of 50 µg/ml ethidium bromide at 4 °C overnight. Protein G-Sepharose® (Amersham Biosciences) was then added for another 3 h at 4 °C. Beads were extensively washed with WCE buffer (50 mM Tris, pH 8.0, 280 mM NaCl, 0.5% Nonidet P-40, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM dithiothreitol), and boiled with 1x SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE for immunoblot detection.

GST Pull-down Experiments—For precipitation with GST-NFAT fusion proteins in mammalian cells, deleted NFATc1 cDNA constructs were subcloned in the mammalian expression vector pEBG in-frame. 293T cells were transfected with GST-NFATc1 fusion and ICSBP expression plasmids for 36 h and either left unactivated or activated with PMA (50 ng/ml) and ionomycin (1 µM) for another 4 h. Cells were harvested and suspended in WCE buffer for 10 min on ice. Lysates were then centrifuged at 14,000 rpm for 10 min. Supernatant containing 2 µg of total protein was incubated with 30 µl of glutathione-Sepharose® beads (Amersham Biosciences) in the presence of 50 µg/ml ethidium bromide at 4 °C overnight. Beads were extensively washed with WCE buffer. Proteins were eluted with Elution Buffer (25 mM Tris, pH 8.3, 300 mM NaCl, 10% glycerol, 2 mM reduced glutathione, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride), then analyzed by SDS-PAGE and immunoblot.

Chromatin Immunoprecipitation Assays—These experiments were performed using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Inc.), following the provided protocol with modifications. RAW264.7 cells were cultured in complete DMEM medium to 80% confluence. 2–5 x 106 cells (unactivated, activated with LPS, IFN-{gamma}/LPS, or cyclosporin A (CsA)/IFN-{gamma}/LPS) were fixed with 1% formaldehyde (final concentration) for 30 min at room temperature. CsA was used at a concentration of 10 µg/ml. Cells were washed with cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/ml pepstatin A), then collected by centrifugation and resuspended into 200 µl of SDS lysis buffer for 10 min on ice. The cell lysate was sonicated to dissociate chromatin into 200–1000-base pair fragments. Debris was removed by centrifugation. The supernatant was diluted in Chromatin Immunoprecipitation Dilution Buffer containing protease inhibitors. One µg of DNA was used for immunoprecipitation with 4 µg of anti-ICSBP or anti-NFATc1 antibodies, and protein G-agarose beads (Amersham Biosciences). The beads were washed with Radioimmune Precipitation Assay Buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1 SDS), Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, and TE in sequence. DNA-protein complexes were eluted with 250 µl of Elution Buffer. DNA-protein cross-links were reversed at 65 °C in the presence of 20 µl of 5 M NaCl. Proteins were digested with proteinase K, and DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation. Immunoprecipitated DNA was utilized for PCR with primers spanning –146 to +40 of the murine IL-12 p40 promoter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of an NFAT Binding Site in the IL-12 p40 Promoter—Our previous studies demonstrated a functional control element around the –62 to –57 region of the murine IL-12 p40 promoter (9, 10). To better characterize this element, a series of 2-base pair substitution mutants from –73 to –54 was generated in the context of a minimal inducible IL-12 p40 promoter from –101 to +55, linked to a CAT reporter. Promoter activities were analyzed following transient transfection in unactivated and LPS-activated RAW 264.7 cells. Mutants from –71 to –60 reduced LPS-induced promoter activity up to 80% (Fig. 1A). Mutants downstream of –59 and upstream of –72 had minimal affects on promoter activity (Fig. 1A). A bioinformatic search for potential cis-regulatory domains suggested that this small region is relatively complex (18). The sequence from –63 to –54 has strong homology to a consensus NFAT (for nuclear factor of activated T cells) DNA binding site ((T/A)GGAAAN) (19), and the promoter region from –74 to –62 has weaker homology to an ISRE ((A/G)NGAAANNGAAACT) (20), which is a binding site for interferon regulatory factor (IRF) family members.



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  		FIG. 1.
Characterization of a functional element at –73 to –54 region of the p40 promoter. A, 2-base pair substitution mutants were created between –73 and –54 in the context of a minimal inducible IL-12 p40 promoter (–101 to +55 with respect to the transcription start site). Two µg of each promoter-CAT reporter plasmid and 0.25 µg of CMV-{beta}-galactosidase reporter plasmid were co-transfected into RAW264.7 cells. After 24 h, cells were activated with LPS (5 µg/ml) for 10–12 h. CAT activity was normalized to {beta}-galactosidase activity. Each result represents the mean ± standard deviation from three to five experiments. Mutations from –59 to –54 and –73 to –72 had minor effects on promoter activity, whereas mutations from –71 to –60 significantly reduced LPS-induced p40 promoter activity. B, to determine effects of mutants on synergistic promoter activation by LPS and IFN-{gamma}, RAW264.7 cells were transiently transfected with murine IL-12 p40 promoter-luciferase plasmids (–101 to +55, –68/–62D, –62/–57D) and a CMV-{beta}-galactosidase reporter. After 24 h, cells were stimulated with LPS (5 µg/ml) with or without IFN-{gamma} (10 ng/ml) for 10–12 h. Luciferase activity was normalized to {beta}-galactosidase activity, and results expressed as percentage of promoter activity compared with LPS plus IFN-{gamma} activation for the wild type –101 to +55 IL-12 p40 promoter-luciferase plasmid (100%). Each result represents the mean ± standard deviation from four experiments.

 

As members of the IRF family are induced in macrophages by IFN-{gamma} (13, 21–23), this region of the IL-12 p40 promoter was next tested for functional significance upon activation with IFN-{gamma} and LPS. We have previously demonstrated that IL-12 p40 promoter-CAT reporter plasmids are not induced by IFN-{gamma} in RAW 264.7 cells (9). Therefore, for this analysis, IL-12 p40 luciferase reporter plasmids that respond to IFN-{gamma} (9, 24) were utilized. When transfected into RAW 264.7 cells, the IL-12 p40 luciferase plasmid, –101 to +55, was activated by LPS, and strong synergistic activation was demonstrated when cells are stimulated with IFN-{gamma} plus LPS (Fig. 1B). Mutants in the predicted NFAT binding element (–62/–57D) and ISRE (–68/–62D) diminished LPS-induced promoter activity. Significantly, mutants in either region markedly attenuated the synergistic promoter activation seen in cells treated with IFN-{gamma} plus LPS (Fig. 1B).

To characterize specific DNA-protein interactions in this region, EMSAs were performed. Two DNA-protein complexes, I and II, were observed when the probe 77/46 (–77 to –46 relative to p40 promoter transcription start site) was incubated with nuclear proteins prepared from RAW264.7 cells (Fig. 2A). Complex II was slightly enhanced by LPS- or IFN-{gamma}-activated nuclear extracts, and clearly enhanced by LPS and IFN-{gamma} co-activation (Fig. 2A, lanes 1–4). Protein binding was eliminated when probe 77/46m, containing a mutated region from –62 to –57 that correlates with decreased functional activity in reporter assays, was used (Fig. 2A, lanes 5–8). Competition with unlabeled double stranded oligonucleotides containing a consensus NFAT binding site and the unlabeled probe 77/46 in 100-fold molar excess completely inhibited the formation of complex II in EMSAs (Fig. 2B, lanes 3, 4, 8, and 9). Double-stranded oligonucleotides containing a consensus NF-{kappa}B or an ISRE binding site did not inhibit the formation of complex II (Fig. 2B, lanes 2, 5, 7, and 10). These results suggest that an NFAT binding site is located in the IL-12 p40 promoter roughly from –62 to –54, and NFAT DNA binding contributes to the formation of complex II in EMSAs. This experiment also suggested that the IL-12 p40 promoter sequence from –74 to –62, although homologous, is not a classical ISRE element (Fig. 2B, lanes 5 and 10). The significance of complex I is less clear. Complex I was not competed by unlabeled probe (Fig. 2B, lanes 4 and 9). However, this complex was not present when the mutant probe 77/46m (Fig. 2A, lanes 5–8) was used for EMSA. These finding suggest that complex I represents an abundant protein in nuclear extracts whose identity is thus far unknown.



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  		FIG. 2.
Demonstration of NFAT site at –62 to –57 in the murine IL-12 p40 promoter. A, LPS and IFN-{gamma} enhanced protein binding to –77 to –46 of the IL-12 p40 promoter, and mutations that decreased LPS-induced IL-12 p40 promoter activity in reporter assays eliminated DNA binding. EMSA probe 77/46 spans the IL-12 p40 promoter sequence –77 to –46. The EMSA probe 77/46m contains a mutated sequence from –62 to –57. 32P-Labeled probes 77/46 (lanes 1–4) and 77/46m (lanes 5–8) were incubated with 5 µg of nuclear extracts from untreated (lanes 1 and 5), LPS-activated (lanes 2 and 6), IFN-{gamma}-activated (lanes 3 and 7), and IFN-{gamma}- and LPS-activated (lanes 4 and 8) RAW 264.7 cells for 30 min prior to electrophoresis. Two protein-DNA complexes (I and II) were detected with probe 77/46. LPS or IFN-{gamma} activation enhanced protein binding to probe 77/46, and IFN-{gamma} and LPS co-activation further enhanced protein binding. In contrast, the mutant probe 77/46m abrogated complex I and II binding. B, EMSAs were performed using nuclear extracts from untreated (lanes 1–5) and IFN-{gamma}/LPS-activated (lanes 6–10) RAW 264.7 cells. 100-fold molar excess of unlabeled consensus NF-{kappa}B (lanes 2 and 7), NFAT (lanes 3 and 8), and ISRE (lanes 5 and 10) double-stranded oligonucleotides and unlabeled probe 77/46 (lanes 4 and 9) were added to compete protein binding to the 32P-labeled probe 77/46. Complex II was competed by unlabeled probe 77/46 and an NFAT consensus oligonucleotide. However, either ISRE or NF-{kappa}B consensus sequences did not affect complex II. Complex I was not competed by any of the competitor oligonucleotides. C, identification of specific NFAT and IRF family members and ICSBP comprising complex II by supershift experiments. 32P-Labeled probe 77/46 was incubated with 5 µg of nuclear extracts from IFN-{gamma}/LPS-treated RAW 264.7 cells at room temperature for 30 min. Then, 1.5 µl of polyclonal antibodies against Rel family (p50, c-Rel) (lanes 2 and 3), IRF family (IRF-1, IRF-2, IRF-4, ICSBP) (lanes 4–7), NFAT family members (NFATc1, NFATc2, NFATc3, NFATc4) (lanes 8–12), and PU.1 (lane 13) were added. Antibodies to NFAT and IRF family members caused supershift or inhibition of complex II, among which NFATc1, NFATc2, NFATc4, IRF-4, and ICSBP (lanes 6, 7, 9, 10, and 12) are identified as components. Combination of antibodies to ICSBP and NFATc1 (K18 or 7A6) (or NFATc2, NFATc4) further inhibited complex II formation (lanes 14–17). However, an anti-PU.1 antibody did not further attenuate protein-DNA binding by anti-NFATc1 (7A6) antibody (lane 18). As negative controls, antibodies against Rel (lanes 2 and 3) family members and PU.1 (lane 13) did not inhibit or shift complex II. D, CsA eliminated NFAT and ICSBP binding to the –77 to –46 region of the IL-12 p40 promoter. Nuclear extracts were obtained from RAW264.7 cells that were either untreated, or treated with LPS/IFN-{gamma}, CsA, CsA/LPS, CsA/IFN-{gamma}, and CsA/IFN-{gamma}/LPS. EMSAs were performed using 77/46 and consensus ISRE probes. CsA treatments virtually eliminated complex II formed with probe 77/46 (lanes 3–6); ICSBP binding was also eliminated (lane 7). However, CsA did not affect binding of IRF family members to consensus ISRE probe (compare lane 9 and lanes 11 and 12) including ICSBP (lane 14). Each of these experiments is representative of three to five experiments with similar results.

 

To identify specific proteins that bind to this promoter region, supershift experiments were performed. Antibodies against NFATc1, -c2, and -c4, and an antibody recognizing NFAT family members c1, c2, c3, and c4 ({alpha}NFATc1; K18) strongly inhibited or supershifted complex II (Fig. 2C, lanes 8–10 and 12). Antibodies recognizing different IRF family members were also tested. Interestingly, an anti-ICSBP antibody strongly inhibited formation of complex II, and an antibody to IRF-4 attenuated this DNA-protein complex (Fig. 2C, lanes 6 and 7). Antibodies against IRF-1 and IRF-2 did not affect complex II formation (Fig. 2C, lanes 4 and 5). As the Rel family of transcription factors and NFAT share a similar DNA binding domain (19), anti-c-Rel and p50 antibodies were tested and also did not affect complex II formation (Fig. 2C, lanes 2 and 3). The Ets family member PU.1 may recruit ICSBP/IRF-4 to PU.1/IRF composite elements (25–27). However, PU.1 is not a component of the DNA binding complex present at –77 to –46 of the p40 promoter (Fig. 2B, lane 13). A combination of antibodies for NFATc1 (or NFATc2 or NFATc4) and ICSBP further attenuated the binding of complex II. In contrast, antibodies to PU.1 and NFATc1 together did not have same effect (Fig. 2B, lanes 14–18). To test whether antibodies against IRF-1 and IRF-2 used in these experiments functioned properly in supershift, another EMSA was performed using a consensus ISRE probe. Both antibodies strongly supershifted a DNA binding complex (data not shown). Therefore, ICSBP and IRF-4 are the predominant IRF family members bound to this region. In summary, ICSBP/IRF-4 and NFAT are the two major transcription factor groups bound to the –77 to –46 region of the IL-12 p40 promoter.

ICSBP Binding to p40 Promoter Is Dependent upon NFAT DNA Binding—ICSBP does not independently bind to DNA, but requires the assistance of other proteins, such as PU.1, IRF-1, or IRF-2 (28, 29). Therefore, EMSA results may suggest an interaction between ICSBP and NFAT. To understand how ICSBP gains access to this p40 promoter element, the following experiment was performed. CsA is a potent immunosuppressant that binds to the intracellular protein cyclophilin, inhibits the phosphatase activity of calcineurin, and blocks the dephosphorylation and nuclear translocation of NFAT (19). RAW264.7 cells were pretreated with CsA and then activated with LPS, IFN-{gamma}, and IFN-{gamma}/LPS, respectively. Nuclear extracts were prepared from these cells for EMSA. CsA treatment completely inhibited complex II (Fig. 2D, lanes 3–6). In contrast, CsA did not inhibit, and in fact enhanced, C/EBP, NF-{kappa}B, and AP-1 DNA binding to IL-12 p40 promoter (data not shown). Importantly, CsA did not abrogate ICSBP binding to a consensus ISRE probe (Fig. 2D, lanes 10–14). Therefore, ICSBP binding to this promoter region is NFAT-dependent, and NFAT may function similar to PU.1, IRF-1, and IRF-2 in recruiting ICSBP to a promoter.

To further confirm EMSA observations, a complementary approach was developed to study DNA-protein interactions. DNA affinity binding assays were performed using a PCR-amplified biotinylated IL-12 p40 promoter sequence from –101 to –36 conjugated to streptavidin beads. After incubating with nuclear extracts from IFN-{gamma}- and LPS-activated RAW264.7 cells, DNA-conjugated beads were washed and proteins eluted for Western blot analysis. As expected, both ICSBP and NFAT were detected (Fig. 3, lane 2). When a promoter sequence containing an NFAT site (–62 to –57) mutation was tested in the same experiment, binding of both NFAT and ICSBP was eliminated (Fig. 3, lane 3). Interestingly, a p40 promoter mutant from –68 to –63 lost ICSBP binding activity, but retained NFAT binding (Fig. 3, lane 4), suggesting that this region is required for ICSBP but not NFAT DNA binding. A promoter sequence with an AP-1 site mutation (–80 to –75) did not affect the interaction of either NFAT or ICSBP with the p40 promoter (Fig. 3, lane 5). In conclusion, these results again suggest that ICSBP binding to the IL-12 p40 promoter is dependent upon NFAT. The sequence from –68 to –63 is necessary for ICSBP binding to IL-12 p40 promoter, and this region, when mutated, abrogates functional activity in reporter assays (Fig. 1).



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  		FIG. 3.
Demonstration of ICSBP-NFAT interactions on the IL-12 p40 promoter by DNA affinity binding assay. Dynal® beads were conjugated with a PCR-generated 5'-biotin-labeled wild type p40 promoter sequence (–101 to –36; lane 2) and three mutant p40 promoter sequences-NFAT site mutation (–62 to –57; lane 3), AP-1 site mutation (–80 to –75; lane 5), and a presumed ICSBP binding site mutation (–68 to –63; lane 4), respectively, and then incubated with 500 µg of nuclear extract from IFN-{gamma}- and LPS-activated RAW264.7 cells at 4 °C for 4 h. Beads were washed extensively, and proteins were eluted for Western blot analysis for ICSBP and NFAT. The wild type p40 promoter bound both NFATc1 and ICSBP (lane 2). The promoter sequence containing an NFAT site mutant lost binding to both NFAT and ICSBP (lane 3), whereas a promoter mutated within a presumed ICSBP binding sequence abrogated ICSBP binding but retained the ability to interact with NFAT (lane 4). A mutant promoter at the AP-1 site did not affect either NFAT or ICSBP binding (lane 5). A template containing the multiple cloning site from the BlueScript® plasmid did not bind to either ICSBP or NFAT (lane 6). An antibody to STAT1 was also used in the Western blot, and no signal was detected on the p40 probes (lanes 2–5), but was abundant in the input nuclear extract (lane 1), demonstrating specificity of NFAT and ICSBP DNA binding for the IL-12 p40 promoter. This experiment is representative of five experiments with similar results.

 

ICSBP and NFAT Interact on the Endogenous IL-12 p40 Promoter—To determine whether the ICSBP-NFAT interaction characterized through in vitro experiments occurs in vivo, chromatin immunoprecipitation experiments were performed. In these studies, proteins cross-linked to DNA in intact nuclei were immunoprecipitated as sheared chromatin fragments with specific antibodies. The IL-12 p40 promoter region from –146 to +50 was then detected by PCR amplification. When RAW264.7 cells were activated by LPS, or IFN-{gamma} and LPS, antibodies against ICSBP and NFATc1 precipitated this p40 promoter region (Fig. 4, lanes 2, 3, 6, and 7). Anti-NFATc1, but not anti-ICSBP, precipitated the p40 promoter from unactivated cells (Fig. 4, lanes 1 and 5), indicating that NFAT localizes to the nucleus and to the p40 promoter in unactivated cells. However, CsA pretreatment abrogated p40 promoter immunoprecipitation by either anti-ICSBP or anti-NFATc1 antibodies (Fig. 4, lanes 4 and 8). This result demonstrates that NFAT and ICSBP interact with the endogenous IL-12 p40 promoter in vivo, and once again, that ICSBP DNA binding to the p40 promoter is NFAT-dependent.



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  		FIG. 4.
Demonstration of NFAT-ICSBP interactions on the endogenous IL-12 p40 promoter by chromatin immunoprecipitation assays. RAW264.7 cells were either untreated or treated with LPS, IFN-{gamma}/LPS, and CsA/IFN-{gamma}/LPS for 4 h. Formaldehyde was added to 1% final concentration for 30 min at room temperature. In cell extracts, chromatin was sheared by sonication and purified for immunoprecipitation with antibodies against NFATc1 and ICSBP. After reversal of cross-linking, DNA was amplified by PCR with primers spanning –146 to +40 of IL-12 p40 promoter. ICSBP and NFAT occupied the p40 promoter in cells activated by LPS or IFN-{gamma}/LPS (top panels). However, CsA pretreatment eliminated their interaction with the promoter. NFATc1 bound to the promoter even in untreated cells (top right panel). As controls, when goat IgG and mouse IgG were used as species-matched control antibodies for immunoprecipitation, the p40 promoter was not detected by PCR. This experiment is representative of three experiments with similar results.

 

Demonstration of a Direct Protein-Protein Interaction between ICSBP and NFAT in Mammalian Cells—To study whether ICSBP may interact with NFAT independent of DNA binding, RAW264.7 cells were activated with IFN-{gamma} and LPS. Nuclear extracts were prepared for co-immunoprecipitation of endogenous ICSBP and NFAT in the presence of ethidium bromide. A direct protein-protein interaction between endogenous ICSBP and NFAT was demonstrable in RAW264.7 cells (Fig. 5A).



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  		FIG. 5.
Demonstration of an in vivo protein-protein interaction between NFAT and ICSBP. A, RAW264.7 cells were stimulated with IFN-{gamma}/LPS for 5 h. Nuclear extracts were prepared and immunoprecipitated overnight with 2 µg of antibodies to NFATc1 (7A6, left), or ICSBP (right) and 20 µl of protein G-agarose beads. The beads were washed extensively and boiled with 1x SDS-PAGE running buffer, separated by SDS-PAGE, and immunoblotted with the antibodies against ICSBP (left) and NFATc1 (right). Mouse IgG and goat IgG were used as the negative controls. This result is representative of three experiments with identical results (B). GST pull-down experiments to map domains of NFAT that interact with ICSBP in mammalian cells. Diagram of deletion constructs of NFATc1 (a GST tag was fused to the N-terminal of each construct). In this and subsequent panels (C–E), arrows are used to denote the deletion that contains the relevant interaction domain. C–E, ICSBP was co-transfected with each of the N-terminal GST-tagged deletion constructs of NFATc1 into 293T cells. After 24 h, cells were activated with PMA/ionomycin for additional 4 h. Cell lysates were incubated with 30 µl of glutathione-Sepharose® beads for 5 h at 4 °C. Beads were extensively washed, and proteins were eluted, separated by SDS-PAGE, and assayed by immunoblot with antibodies against ICSBP and GST (lower panels in C–E). Three regions of NFATc1, 190–219 (C, domain I), 465–495 (D, domain II), and 620–718 (E, domain III), contributed to the interaction with ICSBP. ICSBP and GST-NFATc1 fusion expression in each transfection was determined in whole cell extracts by Western blot with antibodies to ICSBP and GST (upper panels in C–E), and demonstrates that protein expression from each sample were similar.

 

To map the domain(s) of NFAT necessary for ICSBP binding, GST pull-down experiments were performed. Full-length NFATc1a and NFATc1 deletions were subcloned into the GST mammalian expression vector pEBG to generate GST-NFATc1 fusions (Fig. 5B). Each of these NFAT constructs and ICSBP were co-expressed in 293T cells to roughly equivalent levels (Fig. 5, C,Fig. 5 D–E, whole cell extracts). Cells were either unactivated or activated with PMA/ionomycin, and whole cell lysates incubated with glutathione-Sepharose® beads (Amersham Biosciences). Proteins eluted from beads were analyzed by SDS-PAGE and Western blot for ICSBP. It appears that three domains of NFATc1 are required for ICSBP binding. ICSBP-interacting domains include the peptide sequences from 190 to 219 (Fig. 5C; note constructs from lanes 6–9; domain I), 465 to 495 (Fig. 5D; note constructs from lanes 4–6; domain II), and 650 to 718 (Fig. 5E; note constructs from lanes 4–6; domain III). Peptide sequences from 106 to 190, and from 425 to 465, may also have a minor contribution to this interaction. GST pull-down results were identical in PMA/ionomycin-activated cells and untreated cells (data not shown), suggesting that post-translational modification of NFAT is not necessary for this interaction.



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  		FIG. 5.
Demonstration of an in vivo protein-protein interaction between NFAT and ICSBP. A, RAW264.7 cells were stimulated with IFN-{gamma}/LPS for 5 h. Nuclear extracts were prepared and immunoprecipitated overnight with 2 µg of antibodies to NFATc1 (7A6, left), or ICSBP (right) and 20 µl of protein G-agarose beads. The beads were washed extensively and boiled with 1x SDS-PAGE running buffer, separated by SDS-PAGE, and immunoblotted with the antibodies against ICSBP (left) and NFATc1 (right). Mouse IgG and goat IgG were used as the negative controls. This result is representative of three experiments with identical results (B). GST pull-down experiments to map domains of NFAT that interact with ICSBP in mammalian cells. Diagram of deletion constructs of NFATc1 (a GST tag was fused to the N-terminal of each construct). In this and subsequent panels (C–E), arrows are used to denote the deletion that contains the relevant interaction domain. C–E, ICSBP was co-transfected with each of the N-terminal GST-tagged deletion constructs of NFATc1 into 293T cells. After 24 h, cells were activated with PMA/ionomycin for additional 4 h. Cell lysates were incubated with 30 µl of glutathione-Sepharose® beads for 5 h at 4 °C. Beads were extensively washed, and proteins were eluted, separated by SDS-PAGE, and assayed by immunoblot with antibodies against ICSBP and GST (lower panels in C–E). Three regions of NFATc1, 190–219 (C, domain I), 465–495 (D, domain II), and 620–718 (E, domain III), contributed to the interaction with ICSBP. ICSBP and GST-NFATc1 fusion expression in each transfection was determined in whole cell extracts by Western blot with antibodies to ICSBP and GST (upper panels in C–E), and demonstrates that protein expression from each sample were similar.

 

NFAT and ICSBP Synergistically Activate the IL-12 p40 Promoter—To study functional interactions between ICSBP and NFAT in IL-12 p40 promoter activation, ICSBP and/or NFATc1 were co-transfected into RAW264.7 cells with murine IL-12 p40 promoter-luciferase plasmids (–350 to +55, and –101 to +55). In unactivated cells, expression of NFATc1 alone did not increase promoter activity, whereas ICSBP augmented –350 to +55 (containing NF-{kappa}B and Ets element) (Fig. 6A) and –101 to +55 (lacking NF-{kappa}B and Ets element) (Fig. 6B) p40 promoter activity. Co-expression of NFATc1 and ICSBP synergistically activated the –350 to +55 and –101 to +55 p40 promoters (Fig. 6, A and B). Activation by NFATc1 and ICSBP was not observed with control promoters, including a multimerized SP1 DNA binding element, a minimal promoter containing a TATAA box plus initiator, and the pGL2 basic luciferase reporter (data not shown). When an IL-12 p40 promoter with a mutated NFAT binding site (–62/–57) was tested, NFATc1 and ICSBP did not synergistically activate the mutant promoter (Fig. 6C). Similar results were observed with co-expression of NFATc2 and ICSBP (data not shown). Therefore, the synergistic effect between NFAT and ICSBP is specific for the murine IL-12 p40 promoter and requires an intact NFAT binding site.



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  		FIG. 6.
ICSBP and NFAT functionally cooperate to activate the IL-12 p40 promoter. ICSBP and/or NFATc1 were co-transfected into RAW264.7 cells with murine IL-12 p40 promoter-luciferase constructs (–350 to +55, and –101 to +55) and a CMV-{beta}-galactosidase reporter. After 24 h, cells were stimulated with LPS (5 µg/ml) for 10–12 h. Luciferase activity was normalized to {beta}-galactosidase activity, and results expressed as percentage of promoter activity compared with LPS activation (100%). Each result represents the mean ± standard deviation from three to five experiments. A, –350 to +55 p40 promoter activity was enhanced to 10% of the LPS-activated promoter activity by transient transfection of 0.5 µg of ICSBP expression plasmid. NFATc1 (0.5 µg) did not affect promoter activity. However, co-expression of ICSBP and NFAT synergistically activated promoter activity. B, similarly, with the –101 to +55 minimal inducible p40 promoter, NFATc1 and ICSBP expression alone did not or slightly enhance promoter activity, respectively, whereas co-expression of NFAT and ICSBP synergistically activated the promoter. C, an NFAT DNA binding site mutant from –62 to –57 in the –350 to +55 IL-12 p40 promoter abrogated the functional synergy observed when ICSBP and NFATc1 were co-expressed.

 

As RAW264.7 cells contain nuclear NFAT in the unactivated state, this functional analysis was repeated HEK 293T cells, as endogenous NFAT and ICSBP are undetectable by Western blot (data not shown). The purpose of these experiments is to determine whether NFAT may have a direct ability to activate the IL-12 p40 promoter or whether ICSBP is required for transcriptional activation. After transfection with ICSBP and/or NFAT expression plasmids for 24 h, 293T cells were either activated with PMA/ionomycin for additional 10 h or left untreated. In both unactivated and PMA/ionomycin-activated cells, the expression of ICSBP alone activated the IL-12 p40 promoter (–101 to +55) 8-fold. Co-expression of ICSBP and NFATc1 synergistically activated the p40 promoter 30-fold compared with control cells transfected with the empty pCDNA3 plasmid. NFATc1 and NFATc2 had similar effects on the synergistic up-regulation of the p40 promoter when expressed with ICSBP (data not shown). However, neither NFATc1 nor NFATc2 alone affected promoter activity, as demonstrated in RAW264.7 cells. Thus, these experiments suggest a model for IL-12 p40 transcription where NFAT functions as a DNA-binding protein that recruits the ICSBP transactivation domain to the promoter.

A Role for NFAT in Endogenous IL-12 p40 mRNA Expression—To determine the functional role of NFAT in endogenous IL-12 p40 gene expression, Dn-NFAT and hCD4 expression plasmids were co-transfected into RAW264.7 cells. Dn-NFAT (17) is an NFAT deletion construct generated from NFATc3 which retains the N-terminal transactivation domain. Expression of Dn-NFAT prevents nuclear localization of NFAT and therefore inhibits NFAT-mediated gene expression (17). Transfected cells were positively selected for expression of hCD4. After culturing for additional 12 h, cells were activated by IFN-{gamma} and LPS. By reverse transcriptase-PCR, IL-12 p40 mRNA accumulation in cells expressing Dn-NFAT was decreased compared with control cells transfected with empty pCDNA3 and hCD4 plasmids (Fig. 7, left panel). Expression of Dn-NFAT did not affect {beta}-actin mRNA accumulation in the same cells (Fig. 7, right panel). Furthermore, expression of Dn-NFAT in RAW 264.7 cells inhibited LPS and LPS plus IFN-{gamma} induced promoter activation in a dose-dependent manner (data not shown). Therefore, these experiments suggest that NFAT plays a role in endogenous IL-12 p40 gene expression activated by IFN-{gamma} and LPS.



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  		FIG. 7.
A dominant negative NFAT molecule inhibits IFN-{gamma}- and LPS-activated IL-12 p40 mRNA expression in RAW264.7 cells. Human CD4 and Dn-NFAT plasmids were co-transfected into RAW264.7 cells. As a control, pCDNA3 was used instead of Dn-NFAT. After 24 h, cells were positively selected for CD4 expression by Dynal® beads. The eluted CD4+ cells were resuspended into fresh complete DMEM and cultured for additional 12 h, then activated with IFN-{gamma} and LPS for 4 h. Total RNA were prepared, and reverse transcriptase-PCR was performed to detect IL-12 p40 (left panel) and {beta}-actin (right panel) mRNA levels. Expression of Dn-NFAT inhibited IFN-{gamma}- and LPS-activated endogenous IL-12 p40 mRNA accumulation, but did not affect {beta}-actin mRNA levels. This experiment is representative of three experiments with similar results.

 

NFAT-ICSBP Interactions May Be Important for the Inhibition of IL-12 Expression by IL-10 —This study suggests that ICSBP and NFAT functionally cooperate to activate IL-12 p40 transcription. Disruption of the functional interaction between NFAT and ICSBP could therefore inhibit activation on p40 gene expression by LPS and IFN-{gamma}. IL-10 is potent inhibitor of IL-12 production and inhibits IL-12 p40 transcription (14). However, molecular mechanisms of this important biologic effect are unknown. Therefore, whether IL-10 may inhibit the interaction between NFAT and ICSBP was next addressed. RAW264.7 cells were pretreated with IL-10 and subsequently activated with IFN-{gamma} and LPS. After 4 h, cells were harvested for nuclear extract preparation. Co-immunoprecipitation was performed to detect ICSBP and NFAT immunoprecipitated by antibodies to NFATc1 and ICSBP. A strong protein-protein interaction between ICSBP and NFAT was demonstrated in IFN-{gamma}- and LPS-activated cells. Interestingly, IL-10 priming abrogated the ICSBP-NFAT interaction (Fig. 8). Furthermore, IL-10 pretreatment attenuated the ICSBP-NFAT EMSA complex on the p40 promoter (data not shown). Therefore, IL-10 may inhibit IL-12 p40 promoter activity through the molecular disruption of the interaction between ICSBP and NFAT.



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  		FIG. 8.
The interaction between NFAT and ICSBP is disrupted by IL-10 priming. RAW264.7 cells were activated with IFN-{gamma}/LPS for 4 h with or without IL-10 priming (10 ng/ml), and then nuclear extracts were prepared. Immunoprecipitation was performed using 2 µg of antibodies against ICSBP or NFATc1, and 20 µl of protein G-agarose beads. Beads were washed extensively and boiled with 1x SDS-PAGE running buffer, separated by SDS-PAGE, and immunoblotted with antibodies to NFATc1 (top left panel) or ICSBP (top right panel). Goat IgG and mouse IgG were used as species matched negative control antibodies. Nuclear extracts were also used to detect total amounts of ICSBP and NFATc1 (bottom panels), and ICSBP or NFATc1 protein levels in nuclear extracts were not affected by IL-10 priming. This result is representative of three experiments with similar results.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In summary, a composite ICSBP-NFAT DNA binding element is demonstrated in the murine IL-12 p40 promoter from –68 to –54. Binding of ICSBP to the promoter is dependent upon interaction with NFAT. ICSBP and NFAT also associate independent of DNA. Three domains on NFAT contribute to the interaction between these proteins. Co-expression of NFAT and ICSBP synergistically activates the p40 promoter. Promoter mutants in the NFAT site abrogate this functional synergy. Expression of a dominant negative NFAT inhibits IFN-{gamma} and LPS-activated endogenous IL-12 p40 mRNA expression. Furthermore, disruption of the ICSBP-NFAT interaction may be a molecular mechanism behind the inhibitory effect of IL-10 on IL-12 p40 expression.

The IL-12 p40 promoter has been extensively studied, and what has emerged is an increasingly complex model for inducible gene expression characterized by numerous protein-DNA and protein-protein interactions. An NF-{kappa}B binding site, a C/EBP element (–96 to –88), and an AP-1 (–81 to –75) element were characterized to be important for activation of the murine IL-12 p40 promoter (9, 10). An Ets site at –211 to –207 of the human IL-12 p40 promoter, which binds a multiprotein complex including Pu.1, IRF-1, c-Rel, and GLp109 (8, 30), is involved in activation by LPS and IFN-{gamma}. The existence of multiple DNA-protein interactions in the proximal region of murine IL-12 p40 promoter was demonstrated in primary macrophages by in vivo genomic footprinting (31). Although not discussed in the paper, this study reveals a LPS-plus IFN-{gamma}-induced hypersensitive site at –60 relative to the transcription start site that corresponds in location to the element characterized in this report.

IFN-{gamma} is critical for the synergistic induction of IL-12 p40 by bacteria and bacterial products in macrophages. The IRF family is a group of transcription factors that respond to signals from type I and type II IFNs. They share significant homology in their N-terminal domains that interact with a consensus DNA sequence, the ISRE (32). ICSBP is one of the family members specifically expressed in hematopoietic cells, and it is induced by IFN-{gamma} at the protein and mRNA level in macrophages (22, 33). LPS has a modest up-regulatory effect on ICSBP protein in either primary macrophages or RAW267.4 cells (data not shown). Both IRF-1- and ICSBP-deficient mice are vulnerable to bacterial infections (12, 34). These mice do not generate Th1 cell-mediated immunity against intracellular pathogens, and IL-12 production in response to LPS and IFN-{gamma} activation in the macrophages from these mice is seriously compromised (35, 36). ICSBP-deficient macrophages display selective impairment of IL-12 p40 expression, but not other cytokines such as IL-1, IL-6, IL-10, and tumor necrosis factor, when activated by bacteria and bacterial products. Therefore, ICSBP is directly involved in IL-12 p40 gene regulation. Recently, ICSBP was identified as a component of the protein complex bound to the Ets site in the human IL-12 p40 promoter (13). In this study, a deleted promoter construct lacking the Ets site significantly abrogated LPS- or LPS and IFN-{gamma}-induced activation of the human p40 promoter. Overexpression of ICSBP could not rescue the defect. These results suggest that Ets element in the human p40 promoter is necessary for the response to LPS or LPS/IFN-{gamma} stimulation. However, even in the absence of the Ets site, residual promoter activity was noted, raising the possibility that another IFN-{gamma}/ICSBP-responsive element exists downstream. Furthermore, there may be species-specific differences in the hierarchical importance of these various cis-acting control elements. Two recent papers functionally characterized an ISRE at this location in the IL-12 p40 promoter (37, 38). These groups suggest a role for IRF-1 binding to this element. One study also suggests functional involvement of ICSBP in IL-12 p40 promoter activation through this element by overexpression studies, although interactions with an NFAT element were not described (37). In the present study, IRF-1 was not a component of the IL-12 p40 promoter EMSA complex II (see Fig. 2C). However, slight inhibition of EMSA complex I was seen in supershift experiments with IRF-1 antibodies (see Fig. 2C). Therefore, we cannot exclude a functional role for IRF-1 in IL-12 p40 promoter activation.

In mammalian cells, ICSBP interacts with other transcription factors, such as IRF-1, IRF-2, and PU.1, to bind to promoter elements (39). In the current study, an interaction between ICSBP and NFAT is characterized in the murine IL-12 p40 promoter. In this model, NFAT does not appear to have an inherent ability to activate p40 transcription but, similar to the described role of PU.1, serves to recruit ICSBP to the promoter. As IFN-{gamma} is important for the synergistic activation of IL-12 production in macrophages, this promoter region may contribute to the IFN-{gamma} response in IL-12 p40 transcription. We present functional evidence implicating this region of the promoter in the IFN-{gamma} response. The existence of two putative ICSBP sites in the IL-12 p40 proximal promoter region, the induction of promoter activity by overexpressed ICSBP, the abrogation of LPS plus IFN-{gamma}-induced promoter activity by specific mutants in the NFAT site and ISRE, and the inhibition of IFN-{gamma}- and LPS-induced endogenous p40 mRNA accumulation by a dominant negative NFAT in RAW264.7 cells provide compelling evidence that this region mediates the IFN-{gamma} response at least in part. However, the possibility that an IFN-{gamma}-responsive element lies outside of this IL-12 p40 region is certainly not excluded by this and other studies. Resolution of these issues will require further experiments to assess IFN-{gamma} responses on the endogenous IL-12 p40 promoter in primary macrophages and dendritic cells from wild type, and NFAT- and/or IRF-deficient mice.

IL-10 is a potent anti-inflammatory cytokine that inhibits IL-12 p40 gene expression at the level of transcription (14). However, its molecular effects upon p40 transcription remain unknown. In this study, we demonstrate that IL-10 pretreatment of RAW264.7 cells blocks the molecular interaction between NFAT and ICSBP without affecting levels of these proteins. EMSAs also reveal attenuation of the NFAT-ICSBP DNA binding complex in nuclear extracts from cells pretreated with IL-10 (data not shown). We (data not shown) and others (14) cannot demonstrate an effect of IL-10 on the proximal p40 promoter region using a transfection-reporter assay model system. This may reflect limitations of this type of transfection-based promoter analysis in cell lines, but it is also possible that IL-10-responsive elements exist elsewhere within the IL-12 p40 gene and promoter.

NFAT family members are transcription factors that mediate inducible gene expression during immune responses. Five NFAT family members had been identified. NFATc1 (NFAT2, NFATc) and NFATc2 (NFAT1, NFATp) are predominantly expressed in hematopoietic cells. Although the functional role of NFAT has been well described in T cells, its role in macrophage- and dendritic cell-specific gene expression is virtually unknown. NFAT is expressed in primary macrophages (40, 41) (data not shown). NFAT activates many genes through a close functional and physical interaction with AP-1 family members (19). However, NFAT also can function independently of AP-1 (42). NFAT, except for NFAT5, is activated by extracellular signals such as antigen receptors on B cells, T cells, and Fc receptors that lead to calcium mobilization. Unactivated NFAT is phosphorylated and located in the cytoplasm. When an extracellular signal activates Ca2+ channels, calcineurin, a phosphatase of NFAT, is activated. Dephosphorylated NFAT then translocates into nucleus and activates its target genes (19). The regulation of NFAT5 is calcium flux-independent (43). It is generally believed that Ca2+ flux induced by Fc{gamma} receptors cause inhibition of IL-12 production in response to LPS (44), or IFN-{gamma} and LPS (45). Whether LPS can trigger Ca2+ mobilization is still controversial (46, 47). In macrophages, calcineurin may be constitutively active; therefore, NFAT is predominantly located in nuclei (41). In our experiments, NFAT binds to IL-12 p40 promoter in unactivated cells, and its nuclear localization is not affected by LPS or by LPS plus IFN-{gamma} activation. Therefore, it is unlikely that NFAT is involved in the IL-12 inhibitory effect of Fc{gamma}.

Several lines of evidence support a role for NFAT in macrophage-specific gene transcription. The African swine fever virus inhibits inflammatory gene expression in infected macrophages. A viral protein, A238L, binds to calcineurin, and abrogates NFAT-dependent gene transcription (40, 48). Calcineurin also may function as a negative regulator of gene expression downstream of Ca2+ flux and IFN-{gamma} in macrophages. It constitutively acts on macrophages to suppress expression of proinflammatory cytokines in the absence of specific activation (41). Calcineurin inhibitors, FK506 and CsA, synergize with Ca2+ flux and IFN-{gamma} to enhance IL-12 protein production in peritoneal macrophages. However, in this study, at least 8-fold increase in NF-{kappa}B activity was detected, which likely explains the activation of IL-12 expression (41). Because of the effect CsA on NF-{kappa}B, to study the regulation of endogenous IL-12 p40 mRNA expression by NFAT, we utilized a dominant negative NFAT molecule that inhibits the nuclear localization of all NFAT family members. Expression of Dn-NFAT in RAW264.7 cells suppresses endogenous p40 mRNA activated by IFN-{gamma} and LPS. Therefore, this result suggests that NFAT plays an activating role in IL-12 p40 gene expression and that the previously described effects of CsA on IL-12 are likely through NFAT-independent mechanisms.

In mapping NFATc1-ICSBP interactions, complex interactions were demonstrated and three domains in NFAT may be important. ICSBP-interacting domains include the peptide sequences from 190 to 219 (Fig. 4C), 465 to 495 (Fig. 4D), and 650 to 718 (Fig. 4E). Interestingly, the region between 190 and 210 contains SP motifs required for NFAT-calcineurin association (16). Furthermore, the peptide sequences from 465 to 495 within the DNA binding domain are highly conserved in NFATc1 through NFATc4. Therefore, it is likely that ICSBP can interact with NFAT family members other than NFATc1. Accordingly, we had demonstrated similar functional interactions between NFATc2 and ICSBP in IL-12 p40 promoter activation.

Recently, an interaction between IRF4 and NFATc2 was demonstrated in T cells (49). IRF4 and NFATc2 synergized to activate the IL-4 promoter and endogenous IL-4 production in T cells. This functional association was dependent upon physical interactions. Naive T helper cells from IRF4-deficient mice were severely compromised in their ability to make IL-4 and other T-helper-2 cytokines. In this study, NFATc2 associated with IRF4 but not ICSBP (IRF8) (49). In our experiments, a functional and physical interaction between NFATc1 and ICSBP was demonstrated. Functional synergy was also observed between NFATc2 and ICSBP in IL-12 p40 promoter activation, although their physical interaction was not studied. Furthermore, we demonstrated the presence of IRF4 in the IL-12 p40 EMSA complex. This study did not address the functional relevance of IRF-4. However, the interaction between NFATc2 and IRF4, as well as NFATc1 and ICSBP, may be cell type-specific. Irregardless, IRF4 and NFAT interactions in IL-4 gene activation substantiate the hypothesis that NFAT and IRF cooperativity may represent an important mechanism of cytokine gene regulation in immune responses.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom all correspondence should be addressed: Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Scaife Hall, Rm. S857, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9573; Fax: 412-648-9731; E-mail: sep1{at}pitt.edu.

1 The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; IFN, interferon; Th1, T-helper-1; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; Dn-NFAT, dominant negative NFAT; CsA, cyclosporin A; hCD4, human CD4; PBS, phosphate-buffered saline; GST, glutathione S-transferase; CMV, cytomegalovirus; ISRE, interferon-stimulated response element; IRF, interferon regulatory factor; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; NF-{kappa}B, nuclear factor-{kappa}B. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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