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B, CCAAT/Enhancer-Binding Protein 
, and PU.1 and Identification of a Novel Repressor Element (GA-12) That Responds to IL-4 and Prostaglandin E21
*
Laboratory of Immunology, First Medical Clinic, University of Mainz, Germany; and
Cornell University, New York, NY 14853
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B,
and ETS were only found upon stimulation with LPS and IFN-
. In
contrast, a footprint over a purine-rich sequence at -155, termed
GA-12 (GATA sequence in the IL-12 promoter), was observed in
resting, but not activated, cells. Further characterization of this
site revealed specific complex formation at a protected GATA core motif
in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis
within the GA-12 sequence caused a significant up-regulation of
inducible IL-12 p40 promoter activity in both transient and stable
transfection systems, suggesting a repressor function of this site.
Furthermore, binding activity of the GA-12 binding protein
GAP-12 was increased by treatment with two potent inhibitors of IL-12
expression, IL-4 and PGE2. Finally, we observed that
IL-4-mediated repression of IL-12 p40 promoter activity is critically
dependent on an intact GA-12 sequence. In summary, our data underline
the complex regulation of the human IL-12 p40 promoter and identify
GA-12 as a potent, novel repressor element that mediates IL-4-dependent
suppression of inducible promoter activity in monocytes. Regulation of
GAP-12 binding may thus modulate IL-12 p40 gene
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and 2 chains, which are differentially expressed in Th1 and
Th2 T cells (9, 10, 11); whereas the 1 chain is expressed
in both cell types, the 2 chain is expressed only in Th1 cells.
Thus, it is the expression of the 2 chain that accounts for the
responsiveness of Th1 cells and the nonresponsiveness of Th2 cells to
IL-12. After IL-12 binds to its receptor, it induces activation of
specific members of the STAT family of transcription factors (STAT-3
and STAT-4), which then translocate to the nucleus and bind to genomic
promoter regions, including that governing IFN- (12, 13).
Dysregulated IL-12 gene expression and consecutive Th1 T cell
differentiation may contribute to the initiation and perpetuation of
various autoimmune and chronic inflammatory diseases, such as
rheumatoid arthritis and Crohn’s disease (14, 15, 16). Thus,
it is likely that IL-12 gene expression is tightly controlled at the
transcriptional or posttranscriptional level. Indeed, recent studies
have identified several control elements in the IL-12 p40 promoter that
are necessary for inducible p40 gene expression in myeloid cell lines.
A NF-B element was found to be functionally important for promoter
activation in response to LPS and IFN- (17).
Furthermore, a downstream C/EBP binding site and an ETS-2 element seem
essential for high p40 promoter activity, as mutations of these sites
caused a strong reduction of inducible IL-12 p40 promoter activity in
the RAW264.7 cell line (18, 19, 20). Taken together, these
data suggested that the regulated binding of various
trans-acting factors mediates p40 promoter activation in
monocytic cell lines. However, no repressor elements of the p40
promoter have been identified to date that may be involved in cell
type- or activation-specific suppression of IL-12 p40 promoter
activity.
In the present study, we have analyzed the human IL-12 p40 promoter in
primary human monocytes. An 800-bp fragment of the promoter was found
to drive high inducible and cell type-specific expression of a linked
reporter gene in transient transfections as well as in transgenic mice.
In vivo footprinting and bandshift experiments revealed the binding of
NF-B p50/p65, C/EBP, and PU.1 to the promoter in stimulated
human monocytes. A footprint over a purine-rich sequence at
-155 observed only in resting cells was caused by a specific protein
complex, denoted GATA sequence in the IL-12 promoter
(GA-12)3 binding protein
(GAP-12), thereby repressing
inducible IL-12 p40 gene transcription in monocytes. Interestingly,
GAP-12 binding to this site was enhanced by IL-4 and
PGE2, which have been previously described as
potent inhibitors of IL-12 expression (21, 22, 23, 24, 25). Our data
suggest a model in which the stimulation of monocytes and macrophages
with bacterial cell wall components such as LPS leads to the
displacement of the GAP-12 repressor and subsequently to the binding of
activating transcription factors such as p50/p65, C/EBP, and
PU.1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RAW264.7 cell line was a gift from Dr. X. Ma (Cornell University, New York, NY). The J774, P338, U937, Daudi, J558, K562, THP-1, COS-7, Raji, BW5147, and Jurkat cell lines were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 supplemented with 10% FCS (PAA, Linz, Austria), 20 mM HEPES buffer (Life Technologies), 2 mM L-glutamine (Life Technologies, Paisley, Scotland), and 1000 U/ml penicillin/streptomycin (Biochrom, Berlin, Germany).
Plasmids
For generation of the human IL-12 p40 promoter in the pCRII vector (p40/pCRII), the promoter was amplified by PCR from Jurkat genomic DNA using two gene-specific primer sequences (upstream primer, 5'-CTGTATGCCTCCCTGAGGG-3'; downstream primer, 5'-AGTGCTTACCTTGCTCTGGG-3') derived from previously published sequence data (20). The resulting 813-bp promoter fragment ranging from -747 to +66 relative to the transcriptional start site was cloned into the pCRII vector (Invitrogen, Leek, The Netherlands) by TA cloning according to the manufacturer’s recommendations.
For the luciferase reporter gene vector p40/pXP1, the p40 promoter was amplified by PCR as described above, treated with mung bean nuclease (Amersham, Arlington Heights, IL) to create blunt ends, and cloned into the SmaI site of the promoterless pXP1 luciferase reporter gene vector (26).
The expression vectors for C/EBP, C/EBP
, and C/EBP
were gifts
from Dr. C. Trautwein (27) and Dr. R. Schwartz
(28), respectively.
Isolation and culture of primary human CD14+ blood monocytes
Human PBMC were isolated from healthy volunteers using Ficoll-Hypaque gradients. PBMC were then further purified using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) with immunomagnetic beads specific for CD14 (Miltenyi Biotec). Freshly isolated cells were counted and subjected to FACS analysis using an FITC-labeled CD14 Ab. Only cell populations with a purity >95% were used in the experiments described below. The cells were cultured in RPMI 1640 medium (Biochrom) supplemented as described above in humidified atmosphere with 5% CO2 at 37°C.
Cells were treated with the following reagents as specified in
Results: 100 U/ml human IFN- (Roche, Mannheim, Germany),
100 U/ml mouse IFN- (Genzyme, Cambridge, MA), 1 µg/ml bacterial
LPS (Sigma, St. Louis, MO), 5 ng/ml human IL-4 (BD PharMingen, San
Diego, CA), and 5 ng/ml mouse IL-4 (BD PharMingen).
Isolation of mRNA and RT-PCR
Total RNA was isolated with the High Pure RNA isolation kit
(Roche) according to the manufacturer’s recommendations. Reverse
transcription into cDNA was performed using the Moloney murine leukemia
virus reverse transcriptase (Life Technologies) according
to the manufacturer’s recommendations. PCR was performed using the
following primers derived from previously published sequence data:
human IL-12 p40, 5'-TTTTCTGGCATCTCCCCTCGTG-3' and
5'-TGGGTGGGTCAGGTTTGATGATG-3'; and -actin,
5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and
5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'. PCR products were analyzed on 1%
agarose gels.
Dimethyl sulfate (DMS)-piperidine treatment of primary human monocytes for in vivo footprinting
DMS (0.1%; Sigma) was added directly to the culture medium and incubated for 2 min. Cells were extensively washed, and DNA extraction was performed by an overnight incubation in cell lysis buffer (1 mM Tris-HCl (pH 7.5), 400 mM NaCl, 2 mM EDTA, 0.2% SDS, and 0.2 mg/ml proteinase K) at 37°C. The strand scission reaction was performed by resuspending the DNA in 1 M piperidine (Sigma) with subsequent incubation for 30 min at 90°C. The DNA was finally resuspended in water and diluted at a concentration of 1 µg/µl. For control reactions, naked genomic DNA was treated with DMS in vitro. Therefore, the DNA was incubated for 30 s with 0.1% DMS at room temperature. In vitro methylated control DNA was subsequently treated with piperidine as described above.
Ligation-mediated PCR (LM-PCR)
In vivo footprinting by LM-PCR was conducted essentially as previously described (29, 30, 31). In brief, primer annealing was performed with 0.5 pmol primer 1 for 1 µg genomic DMS-treated and piperidine-cleaved DNA. For primer extension Sequenase 1.0 (U.S. Biochemical, Cleveland, OH) was used. Linker ligation was performed overnight at 15°C. Exponential PCR amplification was performed with primer 2 and the linker primer for 15–22 cycles (94°C for 1 min, 1°C for 2 min, and 76°C for 3 min). Finally, the 32P-labeled third primer (106 cpm) was added together with 2 U Taq DNA polymerase and 2 µl dNTPs (5 mM each), and a final PCR cycle was performed, followed by phenol/chloroform extraction of the samples, ethanol precipitation, and analysis on a 6% denaturing urea/polyacrylamide gel.
Primer sequences specific for LM-PCR of the human IL12 p40 promoter were as follows: primer 1, 5'-GGCTTGGGAAGTGCTTACC-3'; primer 2, 5'-CTCTGGGCAGGACGGAGAGTCC-3'; primer 3, 5'-GGGCAGGACGGAGAGTCCAATGGC-3'; linker (top strand), 5'-GCGGTGACCCGGGAGATCTGAATTC-3'; and linker (bottom strand), 5'-GAATTCAGATC-3'.
The in vivo footprinting ladders were verified by comparison to sequencing ladders of cloned IL-12 p40 promoter DNA (p40/pCRII). Therefore, the p40 promoter was sequenced with the Sequenase sequencing kit (U.S. Biochemical) using LM-PCR primer 3 according to the manufacturer’s instructions.
Isolation of nuclear proteins and in vitro translation
Extraction of nuclear proteins was conducted by the method of Schreiber et al. (32). Protein concentrations were measured with a protein assay kit (Bio-Rad, Munich, Germany). For in vitro translation of C/EBP transcription factors, wheat germ extract, T7 polymerase, and expression vectors as DNA templates were used according to the manufacturer’s recommendations (Promega, Madison, WI).
The EMSA
Oligonucleotides for EMSA were synthesized, annealed,
gel-purified, and end-labeled with [-32P]ATP
(5000 Ci/mmol; Amersham) using T4 polynucleotide kinase (New England
Biolabs, Beverly, MA). Radiolabeled DNA probe (25,000 cpm) were added
to the binding reaction that also contained 1 µg synthetic DNA duplex
of poly(dI-dC) (Pharmacia, Peapack, NJ), 3 µg nuclear proteins, and
binding buffer (25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM DTT, and 10%
glycerol). For supershift assays, 2 µg specific Abs (Santa Cruz
Biotechnology, Santa Cruz, CA) were used. For competition analysis, an
excess of unlabeled oligonucleotides containing consensus binding sites
for transcription factors was added to the binding reaction. Complex
formation was allowed to proceed for 30 min at room temperature.
Finally, the complexes were separated from unbound DNA by native PAGE
on 5% gels. The gels were exposed to Kodak MS films
(Eastman Kodak, Rochester, NY) on intensifying screens at -80°C.
The sequences of oligonucleotides (top strands) for EMSA were as
follows: GA-12,
5'-CCTCGTTATTGATACACACACAGAGA-3'; GA-12 mutant (m)1,
5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m2,
5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m3,
5'-CCTCGTTATTTCTACACACACAGAGA-3'; GA-12 m4,
5'-CCTCGTTATTTCTACACACACAGAGA-3'; GATA (Santa Cruz Biotechnology),
5'-CACTTGATAACAGAAAGTGATAACTCT-3'; OCT (Stratagene, Heidelberg,
Germany), 5'-GATCGAATGCAAATCACTAGCT-3'; p40 NF-B,
5'-GAACTTCTTGAAATTCCCCCAGAAGG-3'; p40 NF-B m1,
5'-GAACTTCTTGAAATTAGCCCAGAAGG-3'; NF-B1 (Stratagene),
5'-GATCGAGGGGACTTTCCCTAGC-3'; NF-B2 (Santa Cruz
Biotechnology), 5'-AGTTGAGGGGACTTTCCCAGGC-3'; AP-1 (Stratagene),
5'-CTAGTGATGAGTCAGCCGGATC-3'; p40 C/EBP,
5'-TGTTTTCAATGTTGCAACAAGTCAGT-3'; p40 C/EBP m1,
5'-TGTTTTCAATGTTCTAACAAGTCAGT-3'; C/EBP (Santa Cruz Biotechnology),
5'-TGCAGATTGCGCAATCTGCA-3'; p40 ETS-L,
5'-GATGTAAACCCAGAGAAATTAGCATCTCCATCTCCTTCCTTATT-CCCCACCCAAAAGTCATTTCCTCTTAGTTCATTA-3';
p40 ETS-S, 5'-CCCAAAAGTCATTTCCTCTTAGTTC-3'; p40 ETS-S m1,
5'-CCCAAAAGTCATTAACTCTTAGTTC-3'; CREB (Stratagene),
5'-GATTGGCTGACGTCAGAGAGCT-3'; ETS (Santa Cruz Biotechnology),
5'-GGGCTGCTTGAGGAAGTATAAGAAT-3'; IFN regulatory factor-1 (Santa
Cruz Biotechnology), 5'-GGAAGCGAAAATGAAATTGACT-3'; STAT-1 (Santa Cruz
Biotechnology), 5'-CATGTTATGCATATTCCTGTAAGTG-3'; STAT-3 (Santa Cruz
Biotechnology), 5'-GATCCTTCTGGGAATCCTAGATC-3'; STAT5/6 (Santa Cruz
Biotechnology) 5'-AGATTTCTAGGAATTCAATCC-3'; and E box
(33), 5'-AGCTTGAACCTGCAGCTGCAGGTGGGGGAGTA -3'.
ELISA for IL-12 p40 and p70
To measure IL-12 protein production, 106 primary monocytes/well were seeded out in 1 ml culture medium in triplicate in 48-well tissue culture plates and incubated at 37°C in humidified 5% CO2 atmosphere in the presence or absence of different stimuli as indicated above. After 48 h cell-free culture supernatants were removed and assayed for p70 and p40 concentrations by ELISA (34).
Site-directed mutagenesis
Site-directed mutagenesis was performed with the QuikChange Site-Directed Mutagenesis Kit (catalog no. 200518, Stratagene) according to the manufacturer’s instructions. Mutant primer sequences (top strands) were as described above.
Transfections and reporter gene analysis
Thee p40/pXP1 reporter gene vector (10 µg) along with 2 µg
of a -galactosidase expression vector were transfected into
107 RAW264.7 cells using the DEAE transfection
method. For cotransfection studies, 0.5–4 µg expression vector was
used. After 18 h, the cells were stimulated as described above.
The stimulation was allowed to proceed for 8 h before the cells
were harvested, washed in PBS, and lysed in cell lysis buffer
(Promega). Luciferase activity was measured as light emission over a
period of 10 s after addition of luciferase assay buffer (Promega)
with a standard luminometer (Sirius, Berthold Detection Systems,
Pforzheim, Germany). Luciferase activity was normalized to the
-galactosidase expression level of the lysate or where applicable to
the protein content of the solution.
To generate cell lines stably transfected with p40/pXP1 constructs, a cotransfection strategy with the pEGFP-C1 vector (CLONTECH Laboratories, Heidelberg, Germany) followed by geneticin-based selection of clones was used. In an initial series of experiments we performed dose-response studies with geneticin (G418, obtained from Roche) in RAW264.7 cells and observed that the lethal concentration of geneticin for these cells was 500 µg/ml. Stable transfectants were obtained by electroporating (Bio-Rad apparatus; 500 V, 2.5 ms) RAW264.7 cells with 20 µg linearized p40/pXP1 reporter gene vectors along with 2 µg of the linearized pEGFP-C1 vector carrying the expression cassettes for neomycin resistance and enhanced green fluorescent protein. Individual clones were identified by fluorescence microscopy (IX 70, Olympus, New Hyde Park, NY), transferred to new petri dishes, and subcultivated. Finally, cells of each clone were lysed and analyzed for luciferase expression.
Statistical analysis
Data from transfection experiments were analyzed by Student’s t test using the program Excel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze functional aspects of the IL-12 p40 promoter, we cloned
an 800-bp fragment of the human IL-12 p40 promoter upstream of the
luciferase gene of the promoterless pXP1 vector yielding the p40/pXP1
construct. Consistent with previous observations in tumor cell lines
(17, 18, 19, 20), transient transfection assays showed that this
fragment of the promoter is synergistically induced in RAW264.7 cells
upon stimulation with LPS plus IFN- (9.5 ± 1.5-fold compared
with unstimulated cells). These studies suggested that the proximal
promoter directs temporal and spatial expression of IL-12 p40. However,
as the reporter gene vector remains episomal, transient transfections
do not necessarily reflect the complex biochemical processes involved
in endogenous IL-12 p40 gene regulation in the nucleus
(35). Furthermore, recent studies indicate that promoter
regulation in primary cells and tissue culture cell lines may differ
significantly (13, 31). To explore whether potential
binding sites for regulatory nuclear proteins in the 800-bp p40
promoter fragment were occupied in intact primary human
CD14+ monocytes in vivo, we next performed in
vivo genomic footprinting studies via LM-PCR. In these studies
CD14+ monocytes were highly purified from
peripheral blood by immunomagnetic beads. These primary monocytes were
analyzed by ELISA and RT-PCR analysis and displayed a highly inducible
and transient expression of IL-12 p40 protein and mRNA in response to
LPS plus IFN- (data not shown). In vivo methylated DNA from
unstimulated and LPS- plus IFN--stimulated
CD14+ monocytes was then isolated and subjected
to the LM-PCR procedure (see Materials and Methods). An
altered DMS reactivity in stimulated monocytes, characterized by
hyperreactive A and G and protected G nucleotides (as shown by
densitometric analysis), was found at the previously described NF-B,
C/EBP, and ETS sites, suggesting that these sites contribute to
promoter regulation in primary monocytes in vivo (Fig. 1
). In contrast, we observed protected G
residues at a potentially novel DNA binding site at -155 bp relative
to the transcriptional start site in unstimulated, but not in
stimulated, CD14+ monocytes (Fig. 1A),
as discussed below. No altered DMS reactivities were detected between
-260 and -555 bp upstream of the transcriptional start site (Fig. 1B) in primary CD14+ monocytes,
indicating that the proximal (up to -260 bp) rather than the distal
part of the IL-12 p40 promoter is the target for multiple protein/DNA
interactions in vivo.
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in primary human
monocytes coincides with inducible protein/DNA interactions at the
proximal p40 promoter (Fig. 2A).
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are key activators of
IL-12 p40 gene expression
To assess the potential functional relevance of the in
vivo-protected DNA sequences, reporter gene experiments were performed.
Therefore, we generated various constructs of the IL-12 p40 promoter
carrying 2-bp mutations at in vivo-protected sites. These mutant
promoter constructs were then transfected into RAW264.7 macrophages,
and the resulting luciferase activity was compared with that of the p40
wild-type construct (Fig. 2
B). Site-directed mutagenesis of
the C/EBP, NF-B, and ETS sites of the human IL-12 p40 promoter
caused a significant reduction of inducible promoter activity in
RAW264.7 cells. Furthermore, double mutations of the C/EBP and NF-B
sites or the C/EBP and ETS sites (but not the NF-B plus ETS sites)
resulted in lower reporter gene activity compared with the single
mutants, suggesting synergistic effects of these sites on p40 promoter
activity. Finally, we observed that in a construct carrying mutations
at all three sites, LPS- plus IFN--dependent p40 promoter activity
was almost completely abrogated, suggesting that proteins binding to
the C/EBP, ETS, and NF-B sites are synergistic key regulators of
IL-12 p40 promoter activity.
Mutation of the C/EBP site led to a strongly impaired promoter
activation in response to LPS and IFN-, suggesting a crucial role
for C/EBP in IL-12 p40 expression. Cotransfection studies were
performed to verify whether ectopic overexpression of C/EBP proteins
could trans-activate the human IL-12 p40 promoter (Fig. 3). Strikingly, overexpression of
C/EBP, but not C/EBP, trans-activated the p40 promoter
25-fold in unstimulated and 6-fold in LPS- plus IFN--stimulated
cells (Fig. 3A). In contrast, overexpression of C/EBP had
adverse effects. The trans-activation of unstimulated
macrophages by C/EBP was strongly dose dependent (Fig. 3B), indicating that C/EBP alone may efficiently drive
the human IL-12 p40 promoter without the need of further stimulation.
To rule out the possibility that C/EBP binding sites other than the
site at -80 bp may contribute to the trans-activation
potential of C/EBP, C/EBP was cotransfected together with the
wild-type and mutant promoter constructs. Strikingly, the promoter
carrying a mutation in the -80 C/EBP site was not
trans-activated by overexpressing C/EBP, thus providing
evidence for a single functional C/EBP binding site in the human
IL-12 p40 promoter (Fig. 3C).
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In vivo footprinting at the IL-12 p40 promoter indicated the
simultaneous occupancy of the NF-B, C/EBP, and ETS sites in
stimulated human monocytes (Fig. 1A). In contrast, we
observed protected G residues around -155 bp relative to the
transcriptional start site in unstimulated CD14+
monocytes (Fig. 1A). However, this protection was not
observed after stimulation of monocytes with LPS plus IFN- in
several independent experiments, suggesting the potential
binding of a regulatory protein to this site in resting, but not
activated, human monocytes. Interestingly, sequence alignment revealed
that the observed footprint corresponds to a consensus motif that is
characteristic for binding of proteins of the GATA family of
transcription factors. This novel promoter element was denoted
GA-12.
Mutagenesis of the -155 GA-12 motif results in increased IL-12 p40 promoter activity in both transient and stable transfection systems
Our in vivo footprinting results implicated the possible binding
of a regulatory protein to the GATA core of the GA-12 motif at -155 of
the IL-12 p40 promoter in unstimulated cells, but not in cells
stimulated with LPS/IFN-. To explore whether the protected promoter
sequence could be functionally important for inducible promoter
activity, reporter gene experiments were performed with the wild-type
p40/pXP1 reporter gene vector and the p40/pXP1 vector carrying
mutations within the GA-12 motif. Interestingly, in transient
transfection assays mutations at the GATA core sequence of the GA-12
motif (GA-12 m1, GA12 m2) resulted in 2-fold higher inducible p40
promoter activity than that of the wild-type construct or mutant
constructs with base pair exchanges elsewhere in the p40 promoter (Fig. 4, mut 5–7). Interestingly, base pair
exchanges directly up- and downstream of the core GATA sequence (GA-12
m3, GA-12 m4) also resulted in an increased promoter activity,
suggesting that the complex binding at this site may need some spacing
around the core motif. Taken together, these data suggest the potential
binding of a repressor protein to the GA-12 site in the IL-12 p40
promoter.
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C).
Interestingly, clones carrying the promoter with a mutant GA-12 motif
produced, on the average, an almost 100-fold higher luciferase activity
compared with wild-type clones. This finding indicates an important
function for the GA-12 site for promoter regulation in the nucleosomal
context. A specific nuclear factor, GAP-12, binds to the -155 GATA sequence of the IL-12 p40 promoter
Subsequently, EMSA experiments were performed to determine whether
the footprint observed over the GA-12 motif in unstimulated primary
monocytes might be caused by binding of a sequence-specific nuclear
protein. As shown in Fig. 5A,
retarded complexes were detected using EMSA. The observed complex was
present in nuclear extracts from unstimulated primary monocytes (Fig. 5A, lanes 1–3) and RAW264.7 macrophages (Fig. 5B), but was only weakly detectable or not detectable in
cells stimulated with LPS alone or LPS plus IFN-. In contrast,
specific complexes at the NF-B and C/EBP binding sites (using
extracts from primary monocytes or RAW264.7 cells) were unchanged or
even increased upon such stimulation (data not shown). The protein
complex responsible for the EMSA bands in the unstimulated extracts was
termed GAP-12. Because the -155 GA-12 motif contains a consensus
binding sequence characteristic for the GATA family of transcription
factors we considered the possibility that GAP-12 might be a GATA-like
transcription factor. Interestingly, the GAP-12 complex comigrated
and was even coregulated with the GATA complex obtained with a GATA
reference binding site and extracts of primary human monocytes (Fig. 5A). The specificity of the complex was demonstrated by
competition studies; GAP-12 binding was specifically competed with
unlabeled probe and a reference GATA site, but not with unrelated
competitor DNA or a mutated -155 GA-12 probe (Fig. 5C).
Mutant GA-12 oligonucleotides as probes (for sequences see Fig. 4B) resulted in the abrogation of GAP-12 binding (Fig. 5D). Therefore, consistent with the results of reporter gene
analysis (Fig. 4) the binding of GAP-12 to the -155 GATA sequence may
need some spacing. Subsequently, complex formation at the IL-12 p40
promoter GA-12 site was analyzed in a variety of cell lines. Binding to
the -155 GA-12 site was found in all cell lines analyzed
(Fig. 5E), implicating a ubiquitous, rather than a
tissue-restricted, expression of GAP-12. In an attempt to directly
identify the protein binding to this site in primary human monocytes,
Abs against GATA-1 to GATA-6 were added to the EMSA reactions. None of
them produced a supershift under our experimental conditions (Fig. 5F and data not shown). Thus, we were not able to identify
previously described GATA transcription factors in the GAP-12
complex.
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IL-4 is a potent inhibitor of IL-12 expression
(21, 22, 23). To investigate whether IL-4 may act by
modulating binding of the GAP-12 repressor to the -155 GATA site,
primary blood CD14+ monocytes and RAW264.7 cells
were stimulated with LPS plus IFN- in the presence or absence of
IL-4. IL-4 stimulation led to suppression of inducible IL-12 p40
promoter activity that was accompanied by reduced IL-12 p40 mRNA and
protein levels (Fig. 6). Moreover, after
1 h of LPS/IFN- stimulation no GAP-12 binding was detectable,
but after 3 h (and even more pronounced after 5 h), binding
of GAP-12 to the -155 GATA site was increased in IL-4-treated cells
compared with controls (Fig. 6D, left panel). In contrast,
binding at the IL-12 p40 promoter C/EBP and NF-B sites was unchanged
after IL-4 treatment, as assessed by EMSA (data not shown).
Furthermore, the addition of PGE2, another
inhibitor of IL-12 expression (24, 25) produced in
Th2-type immune responses, to the culture medium of human monocytes
strongly increased GAP-12 binding to its site (Fig. 6D,
right panel). Our data thus provide evidence that the
inhibitory pathway initiated by IL-4 and PGE2 may
involve the modulation of GAP-12 binding to the -155 GA-12
site.
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To directly test the above hypothesis that IL-4 mediates
suppression of IL-12 p40 promoter activity by modulating GAP-12 binding
to the GA-12 sequence element, we performed additional experiments
using stably transfected RAW264.7 cell lines. RAW264.7 macrophages that
were transfected with the wild-type or GA-12 m1 mutant IL-12 p40
reporter gene constructs were stimulated with LPS plus IFN- in the
presence or absence of IL-4, and luciferase activity was analyzed in
the cell lysates (Fig. 6E). It was found that IL-4 inhibited
luciferase expression from the wild-type p40 promoter construct by
60% (Fig. 6E). In contrast, luciferase expression driven
by the p40 promoter carrying a mutation in the GA-12 motif was
repressed by only 5–20%, suggesting an important function for the
GA-12 site in IL-4-mediated IL-12 p40 promoter repression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, NF-B p50/p65, and PU.1. Furthermore, we
have identified a novel repressor element in the IL-12 p40 promoter,
bound by a specific nuclear complex termed GAP-12 in monocytes and
macrophages, that is critical for IL-4-mediated suppression of
inducible IL-12 p40 promoter activity. Suppression of monocytic GAP-12
binding may thus be relevant for activating IL-12 p40 cytokine gene
transcription in response to bacterial Ags and autoimmune diseases.
The IL-12 p40 promoter was previously characterized by transfection
studies in various cell lines (17, 18, 19, 20, 39, 40, 41). Data
derived from these studies suggested that several important regulatory
proteins, such as C/EBP, NF-B, and ETS-2/GLp109, have functionally
active binding sites in the promoter. However, insights into the
contribution of these sites to promoter regulation in primary monocytes
and macrophages in vivo have been limited. Interestingly, recent
studies have shown that promoter regulation in primary cells in vivo
may differ significantly compared with data obtained from studies in
tissue culture cell lines in vitro (13, 31). Moreover,
results often differ depending on the cell line used for these promoter
studies. In an approach toward the goal to understand p40 promoter
regulation in vivo, we have used in vivo genomic footprinting in
primary CD14+ blood monocytes to investigate
occupancy of cis-activating elements of the IL-12 p40
promoter in living cells. We observed in vivo-protected and
hyperreactive residues in LPS- plus IFN- activated primary monocytes
at an NF-B half site (-122 to -132) and a downstream C/EBP site,
suggesting that these sites may contribute to promoter regulation in
vivo in monocytes. Depending of the cell line used, recent
investigation of these sites have demonstrated the binding of
p50/c-Rel, p50/p65, and C/EBP and -, respectively (17, 18, 42). Because in vivo footprinting suggested protein/DNA
interactions at the C/EBP site after cell stimulation only, we assume
that in vivo, upon stimulation, C/EBP has to be modified
post-translationally to bind to the IL-12 p40 promoter site at -80.
Recent studies have indeed revealed that C/EBP is targeted by
several signaling pathways, including LPS-mediated signaling and that
phosphorylation of serine and threonine residues is essential for its
DNA binding and trans-activation (27, 28, 43, 44, 45, 46). The functional relevance of the NF-B and C/EBP sites
in monocytes was further underlined by the striking reduction of
inducible p40 promoter activity in RAW264.7 macrophages upon specific
site-directed mutation of these sites described by different groups
(17, 18) and in the present manuscript. Furthermore, we
observed that simultaneous mutations of these sites resulted in lower
inducible promoter activity compared with the single mutant constructs,
suggesting synergistic effects of both sites on LPS- plus
IFN--dependent p40 promoter activity. Such functional synergy of
NF-B and C/EBP has recently been described for several promoters,
including that of the IL-6 and IL-8 genes (47, 48). In our
experiments overexpression of C/EBP strongly
trans-activated the human IL-12 p40 promoter even without
the need of further stimulation. The protein appears to act via the
previously described C/EBP site on IL-12 p40 promoter activity, because
mutation of this motif completely abolished the
trans-activation potential of overexpressed C/EBP. In
contrast, promoter activity was suppressed upon overexpression of
C/EBP, another member of the C/EBP family of transcription factors.
Because C/EBP was recently shown to be highly expressed in
proliferating myelomonocytic cells and down-regulated during cell
maturation (49, 50), one may speculate that C/EBP might
serve to repress the IL-12 p40 promoter in undifferentiated cells,
whereas upon maturation, C/EBP is replaced by C/EBP. Further
support for an important function of C/EBP for IL-12 p40 promoter
regulation comes from studies in C/EBP-knockout mice. In these mice,
defective activation of macrophages and diminished IL-12 production was
observed (51).
Dysregulated IL-12 levels have a strong impact on cell-mediated immune responses, suggesting the potential existence of negative regulatory elements that control IL-12 gene expression. In the present study, using in vivo footprinting in live monocytes, we have identified a novel purine-rich sequence element denoted GA-12 in the human IL-12 p40 promoter. GA-12 was protected from in vivo methylation in unstimulated, but not activated primary human monocytes suggesting protein binding to this site in the former, but not the latter cells. Sequence alignments showed that the protected G residues correspond to a GATA consensus sequence at -155 bp that is well preserved between the human and murine p40 promoter. Gel retardation assays demonstrated sequence specific binding of a complex, termed GAP-12, to this site in monocytes and macrophages. Interestingly, activation of both RAW264.7 cells and primary human CD14+ monocytes resulted in a strong reduction of nuclear GAP-12 binding activity associated with an abrogation of protein/DNA interactions at the -155 bp GA-12 site in vivo. The GAP-12 complex in human monocytes was competed by a reference GATA site. Furthermore, using a reference GATA site as probe in bandshift experiments with extracts of unstimulated human monocytes resulted in the appearance of a complex that comigrated and even was coregulated with the GAP-12 complex. Because GAP-12 can bind to a reference GATA site, one may speculate that GAP-12 might be a GATA-related protein or at least a protein that can bind to a GATA site. Unfortunately, only little is known about GATA proteins in monocytes and macrophages, and down-regulation of GATA-1 and GATA-2 seems to be critical for myeloid development (52, 53, 54, 55). Interestingly, several myeloid promoters appear to have GATA sites that bind GATA proteins (56). The PU.1 promoter also contains a site that can bind GATA proteins, and cotransfection studies overexpressing GATA-1 and GATA-2 have shown to repress the PU.1 promoter 2-fold (57). In primary CD14+ human monocytes neither GATA-1 nor GATA-2 mRNA was detectable by RT-PCR (our unpublished observations). However, using RT-PCR with degenerated primers specific for the highly conserved GATA zinc finger region we are currently investigating potentially novel GATA proteins expressed in monocytes.
The repressor function of GAP-12 on p40 promoter activity in monocytes was shown by several findings. In initial experiments we observed that site-directed mutagenesis within the GA-12 motif resulted in a considerable up-regulation of inducible promoter activity compared with the wild-type construct in both transient transfection assays and stable transfection experiments. In particular, promoter activity was induced nearly 100-fold in stably transfected cell lines carrying mutations at the GA-12 site, strongly suggesting that GA-12 plays an important role in controlling inducible IL-12 p40 promoter activity in the nucleosomal context. Mutations a few base pairs up- and downstream of the GATA core sequence were also effective, suggesting that GAP-12 binding to the GATA sequence may need some spacing around the core motif. This hypothesis was supported by bandshift experiments using mutated GA-12 oligonucleotides as probes. Furthermore, we found that binding of the GAP-12 complex was strongly increased in extracts of monocytes stimulated with IL-4 or PGE2, two mediators of Th2-like immune responses that have been shown to suppress IL-12 production (21, 22, 23, 24, 25). Our data demonstrated that IL-4-mediated repression of promoter activity was strongly impaired in stable transfectants carrying a mutant GA-12 site, implicating an important function of this site in the regulation of IL-12 gene expression. The correlation between GAP-12 binding observed in the bandshift experiments and repressor function as determined in reporter gene assays underlines the role of GAP-12 for the inhibition of IL-12 p40 promoter activity.
Taken together, our data support a model in which in unstimulated human
monocytes GAP-12 binds to the IL-12 p40 promoter, thereby suppressing
gene expression. Upon stimulation, GAP-12 is displaced from the
promoter, and NF-B p50/p65, C/EBP, and PU.1 promote the induction
of IL-12 p40 gene expression. Because LPS stimulation led to
suppression of GAP-12 binding activity in RAW264.7 without inducing p40
promoter activity, one can assume that other elements (outside the
proximal promoter) must exist that function to prevent LPS alone from
driving p40 gene transcription. However, in any case, the GA-12 element
is the first regulatory site identified in the IL-12 p40 promoter that
functions as a repressor element. Furthermore, this site is critical
for IL-4-mediated suppression of promoter activity and thus appears to
play an important role in regulating IL-12 p40 gene transcription.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Markus F. Neurath, Laboratory of Immunology, First Medical Clinic, University of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. E-mail address: neurath{at}1-med.klinik.uni-mainz.de
3 Abbreviations used in this paper: GA-12, GATA sequence in the IL-12 promoter; GAP-12, GA-12 binding protein; LM-PCR, ligation-mediated PCR; DMS, dimethyl sulfate; m, mutant.
Accepted for publication July 9, 2001.
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) and
stimulated (•) primary human monocytes. Hyperreactivities are marked
with an arrowhead. One representative experiment of three using the
same LM-PCR amplification products in gels A and
B is shown. C, Densitometric analysis of
protected G residues around the GA-12 core motif
(left panel) and the NF-
