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*
Pulmonary Center and Department of Pathology, Boston University School of Medicine, Boston, MA 02118; and
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated sequences (GAS). These binding sites are found within
the promoters of many IFN-responsive genes (reviewed in Ref.
1). The IFN regulatory factor (IRF) family (reviewed in
Ref. 2), which includes IRF1, IRF2, IRF3, IRF4 (previously
termed Pip, LSIRF, and ICSAT), IFN consensus sequence binding protein
(ICSBP), ISGF3/p48, and several viral homologues, mediates cellular
responses to IFNs. The family members IRF1 and ISGF3/p48 have been
reported to activate some IFN-responsive genes (3, 4),
whereas IRF2 and ICSBP have generally been reported to repress
transcription (5, 6). Other family members, such as IRF3
and IRF4, have been reported to function as both activators and
repressors of transcription, depending upon the specific promoter
(7, 8, 9, 10, 11).
Several reports have described the phenotype of mice with a null
mutation of the ICSBP gene. Holtschke et al. (12) reported
that ICSBP-deficient mice display increased susceptibility to
infections with either vaccinia or lymphocytic choriomeningitis
viruses. These mice also spontaneously develop a syndrome similar to
human chronic myelogenous leukemia. Fehr et al. (13)
subsequently reported that ICSBP-deficient mice were highly susceptible
to infection with Listeria monocytogenes, which correlated
with impaired macrophage effector functions and IFN- responsiveness.
Giese et al. (14) and Scharton-Kersten et al.
(15) reported that ICSBP-deficient mice were unable to
control infection with Leishmania major and Toxoplasma
gondii, respectively. This impaired resistance to infection
appears to be a consequence of the inability of these mice to express
the IL-12 p40 subunit, and thereby to mount an effective Th1-type
immune response (15). Impaired IL-12 p40 gene expression
in the ICSBP-deficient mice implicated ICSBP as a transcription factor
that directly or indirectly activates IL-12 p40 gene expression. This
contrasts with earlier studies that concluded that ICSBP is a negative
regulator of several IFN-responsive genes, including MHC class I,
IFN-ß, ISG-54, ISG-15, 2'-5' oligoadenylate synthetase, and the Ig
enhancer (5, 7).
The importance of IRF4 in vivo has been recently demonstrated in mice that lack a functional IRF4 gene. Lymphocyte development in IRF4-deficient mice was found to be blocked at a late stage, leading to a severe accumulation of immature lymphocytes and to defective T cell cytokine production (16). The effects of IRF4 deficiency on myeloid cell development and function have yet to be reported. Recent analyses of ICSBP- and IRF4-deficient mice have revealed both similar and distinct phenotypes. For example, ICSBP-deficient mice spontaneously develop a chronic myelogenous leukemia-like syndrome, whereas IRF4-deficient mice do not (16). In contrast, both IRF4- and ICSBP-deficient mice were unable to mount an effective anti-viral response to lymphocytic choriomeningitis viruses, compared with wild-type mice (12, 16), demonstrating the requirement of these factors in antiviral immunity. A better understanding of the functional roles of IRF4 and ICSBP in vivo will depend upon the identification of genes that are uniquely regulated by each of these transcription factors.
IRF4 and ICSBP, by themselves, possess only weak DNA binding affinity
(17, 18). However, the binding of ICSBP to DNA is
dramatically increased following interaction with IRF1, IRF2, or the
Ets-like transcription factor PU.1 (18, 19). Previous
studies reported that IRF4 could bind to PU.1, and that these IRF4/PU.1
complexes were essential for Ig light chain expression
(19, 20, 21). PU.1 stabilizes the binding of both IRF4 and
ICSBP to a composite PU.1/IRF motif found in both the Ig and 
light chain enhancers in B cells. These interactions between PU.1 and
IRF4 appear to be stabilized, at least in part, by interactions between
DNA binding domains (22). Recent work by Brass et al.
(23) has also demonstrated the importance of direct
physical interaction between PU.1 and IRF4 in transcriptional
regulation. The Ig enhancer composite motif consists of a 5' PU.1
binding site and a 3' sequence that resembles an ISRE half site. Most
recently, a highly similar PU.1/IRF composite element was identified
within the myeloid-specific gp91phox promoter
(24). This gp91phox promoter
element is bound by a complex comprised of PU.1, ICSBP, and
IRF1.
PU.1 regulates the transcription of several myeloid-specific genes in the absence of interaction with either IRF4 or ICSBP. These include the Ig heavy and light chains (19, 20), M-CSF receptor (25), IL-1ß (26), gp91phox (27), and macrophage scavenger receptor (28) genes. A protein kinase CK2 phosphorylation site at serine 148 has been shown to be functionally important (21). Mutation of this serine to an alanine resulted in a complete loss of LPS-induced transcriptional activity. These data demonstrate that serine 148 phosphorylation is critical for inducible transcriptional activation by PU.1. Consistent with these findings, physical and maximal functional interaction between PU.1 and the IRF members IRF4 and ICSBP also requires the presence of serine 148 (20, 21).
Unlike most other IRF family members, ICSBP expression is mainly
restricted to cells of the immune system, including lymphocytes and
macrophages (5). However, recent work by Li et al.
(29, 30) demonstrated that ICSBP is expressed in the
retinal epithelia and ocular lens. ICSBP is constitutively expressed in
macrophages, although its expression can be further up-regulated by
IFN-, but not significantly by IFN
ß (31, 32, 33).
Moreover, LPS and IFN- synergistically enhance ICSBP steady-state
mRNA and protein levels (34). IRF4 has been reported to
only be expressed in lymphocytes (7, 10, 16, 35, 36), and
its expression was not altered by IFN- treatment (10, 36). In contrast to these earlier reports, we demonstrate here
that macrophages express both IRF4 mRNA and protein. The capacity of
macrophages to express both IRF4 and ICSBP raises the possibility that
they perform distinct functions in these cells. To assess the function
of these IRF proteins in macrophages, we examined their ability to
regulate the transcription of selected promoters. Here, we report that
IRF4 and ICSBP can function as both transcriptional activators and
repressors of different promoters in a cell type- and promoter-specific
manner. Our data suggest a complex molecular mechanism that regulates
the expression of genes by IRF4 and ICSBP in macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypsin, proteinase K, antipain, aprotinin, chymostatin, and
leupeptin were purchased from Sigma (St. Louis, MO). Recombinant murine
IFN- was purchased from R&D Systems (Minneapolis, MN). LPS,
Escherichia coli serotype 055:B5, was purchased from Sigma.
Polyclonal rabbit antisera that specifically recognize PU.1, IRF1, or
IRF2, as well as polyclonal goat antisera that specifically recognize
IRF4 or ICSBP, were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Normal, preimmune goat and rabbit sera were purchased from
Pierce (Rockford, IL).
Cell lines and tissue culture conditions
The RAW264.7 murine macrophage, NIH-3T3 murine fibroblast, and
Sup-T1 murine T cell lines were purchased from the American Type
Culture Collection (ATCC; Manassas, VA). RAW264.7 and NIH-3T3 cells
were maintained in DMEM culture medium (BioWhittaker, Walkersville, MD)
supplemented with 10% heat-inactivated FBS (HyClone Laboratories,
Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (BioWhittaker). Sup-T1 cells
were cultured in RPMI 1640 culture medium (BioWhittaker) supplemented
as described above. Murine peritoneal exudate macrophages (PEC) were
elicited by thioglycollate injection of BALB/c mice (The Jackson
Laboratory, Bar Harbor, ME). Following elicitation (72 h), peritoneal
cells were harvested, and macrophages were obtained by adherence
purification. Macrophages were used within 5 days of harvest. Murine
splenic B cells were obtained from BALB/c mice using
complement-mediated lysis to remove T cells and adherence to deplete
macrophages, as previously described (37). Human monocytes
were obtained by adherence purification from total PBMC, as we
described previously (38). All cells were cultured at
37°C, 5% CO2, in a humidified incubator.
Endotoxin levels in all medium components were <10 pg/ml final
concentration, as indicated by BioWhittaker or measured by
Limulus amebocyte lysate kit (BioWhittaker). Cells were
stimulated with LPS or recombinant IFN- at a final concentration of
100 ng/ml for the times indicated in the text.
Nuclear extraction and cytosolic lysate preparation
Nuclear extracts were prepared essentially as described by Schreiber et al. (39). Approximately 1.0 x 107 cells were harvested, washed with Ca2+ and Mg2+-free PBS (BioWhittaker), and pelleted by centrifugation at 800 x g for 10 min at 4°C. The resulting cell pellets were resuspended in 400 µl of buffer I (10 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 0.3 M sucrose, 10 mM ß-glycerol phosphate, 0.1 mM EGTA, 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, chymostatin, and antipain), then incubated on ice for 10 min. Subsequently, 25 µl of 10% Nonidet P-40 (Sigma) was added to each sample and vortexed. The nuclei were pelleted by centrifugation for 1 min at 5000 x g. Supernatants were collected and stored for later use as cytosolic lysates. Nuclear pellets were resuspended in a nuclear extraction buffer (buffer II) containing 320 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 10 mM ß-glycerol phosphate, 0.1 mM EGTA, 25% glycerol, 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, antipain, and chymostatin. Samples were extracted on ice for 15 min followed by centrifugation at 16,000 x g for 10 min at 4°C. Protein concentration was determined using the Bio-Rad (Hercules, CA) assay kit. All nuclear extracts were stored at -70°C, and multiple freeze-thawing cycles were avoided.
EMSA and DNA probes
A double-stranded oligonucleotide containing a single copy of
the Ig 3' enhancer (Ig ) composite PU.1/IRF sequence
(5'-CTTTGAGGAACTGAAAACAGAACCT-3';
(40)) was utilized as an EMSA probe. Italicized sequences
represent PU.1 (5') and IRF (3') binding sites. Mutant oligonucleotides
were also generated where either the PU.1 (GGAA to TTCA) or IRF (AAAC
to ATCA) binding sites were previously described (20). An
unlabeled double-stranded oligonucleotide containing a single copy of
the human IL-1ß cap site-proximal PU.1 binding site (GCAGAAGT;
(26)) or IL-2R -chain NF-B site (GGGGAATTCC) were
utilized as competitor DNA. DNA probes were labeled with
[-32P] deoxynucleotide triphosphates
(DuPont-NEN, Boston, MA) using E. coli DNA polymerase Klenow
fragment (United States Biochemicals, Cleveland, OH), as recommended by
the manufacturer. Unincorporated nucleotides were removed using
Sephadex G-25 columns (5 Prime 
3 Prime, Boulder, CO). Nuclear
extracts (typically 3 µg) were incubated with radiolabeled probe DNA
(0.1 ng, typically 10,000 cpm) in the presence of 2 µg poly dI-dC
(Pharmacia, Piscataway, NJ), 1.0 mM EDTA, 10 mM Tris-HCl (pH 7.9), 25
mM glycerol, and 0.5 mM DTT in a final volume of 20 µl, as previously
described (41, 42). Binding reactions were incubated at
room temperature for 30 min. In competition experiments, unlabeled DNA
was added at a 100-fold molar excess in addition to the binding
reaction. For supershift experiments, 2 µg of antiserum was added as
indicated in the text in addition to the binding reaction. Following
incubation, a portion of each binding reaction (7 µl) was
electrophoresed through a 7% nondenaturing low ionic strength
polyacrylamide gel, dried, and visualized by autoradiography.
Western blot analysis
Nuclear extracts and cytosolic lysates were prepared from cells as described above. Samples (50–150 µg total protein) were electrophoresed through either a 15 or 10% SDS-polyacrylamide gel (noted in figure legend), transferred to a nitrocellulose membrane (Bio-Rad), and blocked with 5% nonfat dry milk (Carnation) in TBST containing 0.1% Tween 20 (Sigma). PU.1 protein was detected using a 1:2000 dilution of anti-PU.1 antisera and developed using a 1:4500 dilution of a protein A conjugated to HRP (Amersham, Arlington Heights, IL). IRF4 and ICSBP proteins were visualized using a 1:3000 dilution of specific antisera and developed with a 1:1500 dilution of protein G-HRP (Pierce). Membranes were visualized using an enhanced chemiluminescence (ECL) reagent (CL-HRP substrate system; Pierce), according to the manufacturer’s instructions.
RNA isolation and RT-PCR
Total RNA from macrophages, fibroblasts, and murine B cells was purified using RNA STAT-60 (Leedo Medical Laboratories, Houston, TX), as recommended by the manufacturer. RNA was converted to cDNA using avian myeloblastosis virus RT (Promega, Madison, WI). PCR were performed using between 100 ng and 2 µg of cDNA, 0.5 µM oligonucleotide primers (each), 1.5 mM MgCl2, 150 µM dNTPs and 2.5U Taq polymerase in a final reaction volume of 75 µl. Thirty amplification cycles were performed (95°C denaturation, 30 s; 55°C annealing, 1 min; 72°C extension, 1.5 min). Intron-spanning IRF4, CD19, and ß-actin PCR primers used in this study are listed below. The IRF4 primers correspond to sequences that do not share sequence similarity with ICSBP. Following amplification, a portion of the PCR reactions were electrophoresed through a 1.2% agarose gel. The 554-bp IRF4, 747-bp CD19, and 285-bp ß-actin products were visualized using ethidium bromide. Sense-strand IRF4 primer: 5'-GCT GCA TAT CTG CCT GTA TTA CCG-3'; anti-sense strand IRF4 primer: 5'-GTG GTA ACG TGT TCA GGT AAC TCG TAG-3'; sense-strand CD19 primer: 5'-CCC CAG AAG TCC TTA CTG-3'; anti-sense CD19 primer: 3'-GCC TCT CGA TGG TCA GGT TT-3'; sense-strand ß-actin primer: 5'-TCA TGA AGT GTG ACG TTG ACA TCC GT-3'; anti-sense strand ß-actin primer: 5'-CCT AGA AGC ATT TGC GGT GCA CGA TG-3'.
Plasmids
Expression plasmids encoding the full-length, wild-type and
S148A mutant murine PU.1 proteins were previously described
(21). An expression plasmid encoding the full-length
murine ICSBP protein was provided by Dr. Keiko Ozato (National
Institutes of Health, Bethesda, MD) and was previously described
(43, 44, 45). Expression plasmids encoding the murine IRF4
proteins were previously described (35). The control
vector pcDNA3.1 was purchased from Invitrogen (Carlsbad, CA), and was
used to maintain a constant quantity of plasmid DNA in each
transfection. The (PU.1/IRF)4 chloramphenicol acetyl transferase (CAT)
(wild type), (mPU.1/IRF)4 CAT (PU.1 binding site mutant), and
(PU.1/mIRF)4 CAT (IRF binding site mutant) reporter constructs
containing four copies of the composite PU.1/IRF binding site derived
from the Ig 3' enhancer were previously described
(20). The IL-1ß luciferase reporter construct was
generated from the previously described 7 mXT-CAT reporter plasmid
(26). Briefly, the XbaI to TaqI
fragment (positions -3759 to +11) from the human IL-1ß promoter was
subcloned into the promoterless pGL3 luciferase reporter plasmid
(Promega). The H-2Ld MHC class I reporter plasmid
was provided by Dr. Keiko Ozato, and was previously reported
(5).
Transient transfections
Transient transfections were performed using SuperFect reagent (Promega) as per the manufacturer’s instructions. Briefly, cells were plated on 6-well dishes and transfected when cells reached 80% confluence. Plasmid DNA was added to 100 µl of Opti-Mem reduced serum media (Life Technologies, Rockville, MD). All transfections utilized a total of 4 µg of plasmid DNA consisting of 2 µg of reporter plasmid, 1 µg of each expression vector, and the balance was made up with empty vector described above unless otherwise noted in the text. A total of 10 µl of SuperFect was added to the DNA-media mixture, incubated for 10 min, diluted with 600 µl of serum-containing media, and added to individual wells. Each reaction was prepared separately and in triplicate. Fresh media containing serum was added 2–3 h after transfection. All conditions were incubated for an additional 16–24 h. CAT and luciferase assays were performed as described below. All transfection experiments were repeated at least three times using different plasmid preparations, and a single representative experiment is shown. Each single experiment represents triplicate independent transfections, and data are expressed as average values ± SD.
Reporter gene assays
CAT reporter activity was assessed using a two-phase fluor diffusion CAT assay as previously described (46). Equal concentrations of cell lysates (15–35 µg total protein per sample) were used in the assay. Protein concentration was determined utilizing the Bio-Rad protein assay kit, according to the manufacturer’s instructions. Data were calculated by plotting total cpm of acetylated chloramphenicol vs time, and the slopes for each reaction were calculated within a linear kinetic range. Luciferase activity was measured using the luciferase assay system (Promega), according to manufacturer’s instructions and performed as previously described (47). Lysates were assayed for total protein using the Bio-Rad protein assay. Luciferase activity was measured using 5–20 µg total protein as measured by light emissions in a scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We performed a series of EMSA analyses to determine whether
ICSBP/PU.1 complexes could be formed in RAW264.7 macrophages and
primary B cells. The EMSA probe used in these experiments is a PU.1/IRF
composite binding site (see Materials and Methods for
sequence) that contains an ISRE half site located downstream and
adjacent to a consensus PU.1 binding site. Such sites have been
identified within the Ig light chain enhancers and the
gp91phox promoter (19, 20, 24). IRF
proteins alone do not bind to this composite site, although concomitant
binding of PU.1 to the site stabilizes IRF binding to DNA as a
consequence of its interaction with PU.1 (7). As shown in
Fig. 1
, PU.1 and IRF proteins that were
present in unstimulated macrophage and B cell nuclear extracts
specifically bound to the composite site. Abs against PU.1
(lane 7) and ICSBP (lane
3) could supershift the DNA-protein complexes, whereas Abs
against IRF1 (lane 5) and IRF2 (lane
6) did not supershift the complexes. Abs that recognize PU.1
blocked the binding of both PU.1 and ICSBP to the DNA, suggesting that
ICSBP could not bind to DNA in the absence of PU.1. Unexpectedly,
specific Abs for IRF4 (Fig. 1A, lane 4) could
also supershift the DNA-protein complexes generated using macrophage
extracts. Like ICSBP, Abs that recognize PU.1 blocked the binding of
both PU.1 and IRF4 to the DNA, indicating that IRF4 could not bind to
DNA in the absence of PU.1. The IRF4 Ab was also able to supershift a
complex when incubated with B cell nuclear extracts (Fig. 1B, lane 4). Furthermore, normal preimmune goat
and rabbit sera used at similar concentrations as all other antisera
did not produce any supershifted complexes when incubated with either
macrophage or B cell nuclear extracts (lanes 8 and
9). In addition, when EMSA analysis was performed upon
primary murine and human macrophage nuclear extracts, similar results
were obtained (data not shown). Mutation of the IRF binding site
abolished binding of the PU.1/IRF complexes, but did not affect the
binding of PU.1 alone (data not shown), while mutation of the PU.1 site
blocked the binding of both PU.1 and IRF proteins to the
oligonucleotide (data not shown). Together, our data suggest that
macrophages express IRF4, or a related protein that is also recognized
by the Ab, and that this protein can bind to the PU.1/IRF composite
sites in the presence of PU.1.
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To confirm that IRF4 is expressed by macrophages, we used
sequence-specific, intron-spanning oligonucleotide primers to amplify
cDNA synthesized from both primary and cell line murine mRNA. These
primers were selected from regions of the IRF4 mRNA that lack sequence
similarity with ICSBP transcripts. Using RT-PCR, these primers
generated a 554-bp product using cDNA synthesized from RAW264.7 cells
and primary murine PEC (Fig. 2,
lanes 2 and 4). A PCR product of identical size
was generated using cDNA synthesized from primary murine B cells
(lane 3). Meanwhile, no PCR product was generated
from NIH-3T3 fibroblast cDNA (lane 1), cells that do
not express IRF4, ICSBP, or PU.1. To confirm that the PCR product
generated from primary murine macrophages was not due to B cell
contamination, we performed PCR using primers to detect expression of
the B cell-specific gene CD19. A PCR product of the expected size (747
bp) was generated only from the B cell cDNA. These data confirmed that
the IRF4 PCR product generated using primary macrophage RNA was not due
to the presence of contaminating B cells. In all cases, a 285-bp PCR
product was generated using ß-actin primers. The PCR products
generated from macrophage cDNA were subsequently sequenced and found to
be identical to the sequence of IRF4 (data not shown).
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To determine whether this IRF4 mRNA was translated into protein,
Western blot analysis was performed. Nuclear extracts were prepared
from primary murine macrophages, RAW264.7 macrophages, and primary
human monocytes. These samples were fractionated by SDS-PAGE and probed
using non-cross-reactive Abs against either IRF4 or ICSBP. Nuclear
extracts from primary murine B cells were also included in the figure,
because they express both IRF4 and ICSBP. As shown in Fig. 3A, immunoreactive IRF4 and
ICSBP protein were present in both the macrophage and B cell lysates.
To confirm that the Abs were non-cross-reactive and recognized proteins
of the predicted molecular size, three duplicate lanes were transferred
to nitrocellulose membranes and probed with the Abs singly, or in
combination. As shown in Fig. 3B, the IRF4 and ICSBP Abs
each identified a single protein with the expected molecular size (42
and 45 kDa, respectively). Together, these data demonstrate that
macrophages express both IRF proteins.
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Yamagata et al. (10) reported that IRF4 expression
and function in lymphocytes were not activated by IFN treatment. In
contrast, ICSBP is expressed constitutively at low levels in
macrophages, and this expression can be further up-regulated by IFN-
treatment (32, 33, 34). We sought to determine whether IRF4
expression could be regulated by IFN- in macrophages. The capacity
of IFN- to up-regulate ICSBP gene expression in the RAW264.7
macrophages was confirmed by Western blot analysis of nuclear extracts
(Fig. 4), and densitometric
quantification of the Western blots revealed that 6 h of IFN-
stimulation increased the nuclear levels of ICSBP by 
26-fold. In
contrast to ICSBP, levels of nuclear IRF4 protein in the macrophages
was not altered by IFN- treatment, consistent with previous
findings. Cytosolic levels of IRF4 and ICSBP mirrored that observed in
the nucleus (data not shown), consistent with previous findings in
lymphocytes (13, 36). In addition, the levels of nuclear
PU.1 protein were not altered by IFN- treatment (Fig. 4).
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IRF4 activity has been reported to be induced in activated
lymphocytes (10, 36). Therefore, we sought to determine
whether LPS stimulation of the RAW264.7 cells could similarly activate
IRF4. As shown in Fig. 5A,
levels of nuclear IRF4 protein increased within 2 h following LPS
stimulation, with a concomitant decrease in the cytosolic levels of
this protein. In contrast, ICSBP protein levels in both the nucleus and
cytosol were not measurably altered following LPS stimulation within
the time period examined (data not shown). As a control for
cross-contamination of the samples, we found that these nuclear
extracts did not contain a protein found solely in the cytosol (p105
NF-B; data not shown). Densitometric quantification of the Western
blots revealed that 6 h of LPS stimulation increased the nuclear
levels of IRF4 by 3-fold (Fig. 5B). While our data are
only suggestive of nuclear translocation, they are consistent with a
previous study by Lin et al. (8). These investigators
reported that stimulation of various fibroblast cell lines with Sendai
virus resulted in the rapid nuclear translocation of IRF3, a
ubiquitously expressed IRF family member.
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One objective of these studies was to determine whether IRF
proteins could form functional associations with PU.1 in macrophages.
This physical and functional interaction has been previously
demonstrated for Ig light chain enhancer expression in B cells
(19, 20). We sought to determine whether such composite
sites were also functional in macrophages, cells that express PU.1
constitutively. We transfected RAW264.7 macrophage cells transiently
with a reporter plasmid containing the CAT reporter gene under the
control of a HSV thymidine kinase promoter ligated downstream of four
tandem copies of a PU.1/IRF composite element (plasmid designated
(PU.1/IRF)4), or reporter constructs containing composite elements in
which either the IRF (designated (PU.1/mIRF)4) or the Ets (designated
(mPU.1/IRF)4) binding sites were mutated. EMSA analysis was used to
demonstrate that the (PU.1/mIRF)4 mutant was still capable of binding
PU.1 in vitro (data not shown). In the absence of PU.1 binding, as in
the case of the (mPU.1/IRF)4 mutant, the affinity of IRF binding is
markedly reduced (20). As shown in Fig. 6, the (PU.1/IRF)4 reporter plasmid was
constitutively expressed in RAW264.7 macrophages, whereas mutation of
the IRF binding site completely abolished this activity. Similarly,
mutation of the PU.1 binding site substantially reduced transcription.
These data demonstrate that a PU.1/IRF composite element can activate
transcription in macrophages, that neither the PU.1 nor the IRF binding
site alone is sufficient for full activation of transcription, and that
both proteins appear to be required for this activation.
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, over-expression of
ICSBP resulted in a 4-fold enhancement in basal promoter activity.
Meanwhile, over-expression of ICSBP had no effect upon the basal
expression of either the Rous sarcoma virus long terminal repeat, or
the NF-B-dependent E-selectin (ELAM-1) reporters. Reporter plasmids,
in which either the IRF or PU.1 binding sites were mutated, lacked
basal activity and could not be activated by over-expression of ICSBP.
In contrast, over-expression of IRF4 did not result in enhanced
transcription of the promoter, as was seen with ICSBP. We also found
that transfecting the cells with increasing amounts of the ICSBP
expression plasmid (100–2000 ng) resulted in dose-dependent activation
of the reporter, whereas increasing amounts of IRF4 had no effect on
reporter gene expression (data not shown). Moreover, these data also
demonstrate that IRF4 over-expression in macrophages does not activate
the (PU.1/IRF)4 reporter plasmid, which contrasts with its ability to
activate this reporter in fibroblast cell lines (19, 20). Functional synergy between PU.1 and IRF proteins
Interactions between PU.1 and IRF4, or between PU.1 and ICSBP,
cannot be easily examined in macrophages because these cells
constitutively express all three transcription factors. To study these
interactions in isolation, we expressed these factors in fibroblast
lines that do not intrinsically express PU.1, IRF4, or ICSBP. We
cotransfected NIH-3T3 cells with the (PU.1/IRF)4 reporter plasmid and
various combinations of PU.1 and IRF expression plasmids. As shown in
Fig. 7, neither IRF4 nor ICSBP alone
could activate the promoter, although PU.1 alone could activate a low
level of transcription. Coexpression of PU.1 and either IRF4 or ICSBP
synergized to induce a high level of promoter activity in the NIH-3T3
cells. It is likely that this synergy only became apparent in the
fibroblasts because the basal level of reporter gene activity in these
cells was extremely low due to the absence of endogenous PU.1, IRF4,
and ICSBP.
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Our data demonstrate that IRF4 and ICSBP can activate
transcription of a promoter controlled by PU.1/IRF composite elements.
Previous studies have reported that IRF4 and ICSBP function as a
transcriptional repressor in various cell types (5, 10, 18). Therefore, it was important to confirm that IRF4 and ICSBP
could also suppress transcription in our model system. The
PU.1-independent H-2Ld MHC class I promoter had
been previously reported to be negatively regulated by both IRF4 and
ICSBP (5). We cotransfected RAW264.7 cells with a CAT
reporter plasmid under the control of the H-2Ld
promoter and an expression plasmid encoding either IRF4 or ICSBP. As
shown in Fig. 8, over-expression of
either IRF4 or ICSBP resulted in reduced basal promoter activity,
compared with cells transfected with the reporter alone. Similar
results were obtained using transfected RAW264.7 cells (Fig. 8A) and NIH-3T3 cells (Fig. 8B). Together, these
findings demonstrate that IRF4 and ICSBP can repress transcription of
the PU.1-independent H-2Ld promoter in both cell
types, whereas only ICSBP can activate transcription of the
PU.1-dependent (PU.1/IRF)4 reporter in both cell lines. Furthermore,
repression of the H-2Ld promoter in RAW264.7
cells (that constitutively express PU.1) and NIH-3T3 cells (that lack
PU.1) by both IRF4 and ICSBP suggest that repression by these two IRF
family members is independent of their ability to interact with
PU.1.
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The studies described above assessed the ability of IRF4 and ICSBP
to interact with PU.1 and to activate a synthetic promoter containing
tandem copies of a PU.1/IRF composite site. We subsequently sought to
determine whether IRF4 and ICSBP could activate transcription of a
natural PU.1-dependent promoter. We selected IL-1ß as a potential
gene that could be regulated by both PU.1 and IRF proteins in
macrophages. The IL-1ß promoter has been previously shown to be
regulated by both LPS and IFN-, and to require a functional PU.1
binding site within the cap site-proximal promoter (46).
We cotransfected RAW264.7 cells with an IL-1ß reporter plasmid and
increasing amounts of expression plasmids encoding either IRF4 or ICSBP
(100–2000 ng). As shown in Fig. 9, we
found that over-expression of ICSBP resulted in a consistent 40–50%
increase in basal transcriptional activity, which was not further
enhanced by increasing the quantities of ICSBP expression plasmid used.
In contrast, over-expression of IRF4 activated the IL-1ß reporter in
a dose-dependent fashion, with more than 7-fold activation at the
highest amount of IRF4 expression plasmid used. These results are
qualitatively distinct from those obtained using the (PU.1/IRF)4
reporter plasmid (Fig. 6), suggesting that the two promoters are
regulated differently by these IRF proteins.
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A, IRF4, PU.1, and
S148A alone were incapable of activating expression of the reporter
plasmid. Synergistic activation of the IL-1ß promoter was observed
when cells were cotransfected with IRF4 and PU.1 expression plasmids.
Unlike wild-type PU.1, the S148A mutant failed to synergize with IRF4
to activate the reporter plasmid. Similar results were obtained using
this promoter in cells that over-expressed ICSBP (Fig. 10B).
However, cotransfection of ICSBP with the S148A mutant resulted in a
complete loss of synergy, as compared with the incomplete loss of
activity seen with coexpression of IRF4 with S148A. These data
demonstrate that a natural PU.1-dependent promoter can be
synergistically activated by these IRF proteins and PU.1. This
synergistic activation with ICSBP requires a critical serine residue on
PU.1, whereas IRF4 may be less dependent upon this serine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Together, these data suggest a complex molecular mechanism that
regulates the expression of IFN-responsive genes by IRF4 and ICSBP.
|
). When normalized to ß-actin expression, it appears that
macrophages express 25–35% of the IRF4 message expressed in B cells.
The lack of CD19 transcripts in RNA prepared from primary
thioglycollate-elicited macrophages demonstrates the lack of B cell
contamination (Fig. 2). These data support the conclusion that
macrophages produce IRF4 message. In summary, confirmation that
macrophages express IRF4 came from: 1) the sequence of the RT-PCR
product (data not shown), 2) specific binding of a 45-kDa protein by
non-cross-reactive anti-IRF4 Abs (Fig. 3B), and 3)
supershifting of a specific DNA-protein complex by both anti-PU.1
and anti-IRF4 Abs (Fig. 1). Therefore, we cannot fully explain why
previous studies failed to identify macrophages as cells that can
produce IRF4, although the paucity of IRF4 message in macrophages may
have been below the level of detection of detection techniques used in
previous studies.
Several features indicate that IRF4 and ICSBP are differentially
regulated in macrophages. First, only ICSBP expression was increased
following IFN- treatment. This is consistent with data obtained
using lymphocytes where ICSBP, but not IRF4, expression was
up-regulated by IFN- (10, 33). Second, LPS stimulation
appeared to induce nuclear translocation of IRF4, but not of ISCBP
(Fig. 5). Third, over-expression of ICSBP activated transcription of
the (PU.1/IRF)4 reporter plasmid (Fig. 6) and weakly activated
transcription of the IL-1ß reporter plasmid (Fig. 9). In contrast,
over-expression of IRF4 only enhanced transcription of the IL-1ß
reporter plasmid. However, both IRF4 and ICSBP were capable of
repressing the PU.1-independent H-2Ld promoter
(Fig. 8). Together, these data suggest that the expression and function
of IRF4 and ICSBP are regulated by different mechanisms in macrophages.
Furthermore, such differences suggest that these IRF proteins serve
nonredundant functions in macrophages which may, in part, be related to
their ability to interact with PU.1.
Our findings also extend several previous reports on the functions of both ICSBP and PU.1/IRF complexes. Nelson et al. (5) demonstrated that ICSBP repressed several ISRE-containing promoters in macrophages. In contrast, Eklund et al. (24) recently reported that the PU.1/IRF composite site within the gp91phox promoter was positively regulated by ICSBP in U937 cells. These investigators found that PU.1, ICSBP, and IRF1 functioned together to activate transcription of a reporter plasmid containing tandem copies of this composite element. Our data agree with these results, and support a model in which ICSBP functions to repress transcription when bound to ISRE sequences, but instead activates transcription when bound to PU.1. Thus, the capacity of ICSBP to activate or repress transcription appears to be determined in a promoter-specific context, defined by the proteins with which it interacts.
These previous studies used reporter plasmids containing tandem copies
of the Ig light chain enhancer or gp91phox
PU.1/IRF composite elements to drive transcription (19, 20, 24). Here, we showed that a natural promoter (i.e., IL-1ß)
could be activated by IRF4, and weakly by ICSBP, in RAW264.7
macrophages (Fig. 9). Furthermore, this promoter is activated
synergistically by both IRF4 and ICSBP with PU.1 in NIH-3T3 fibroblasts
(Fig. 10). The IL-1ß promoter is not known to contain functional
PU.1/IRF composite sites, and future studies will be needed to identify
the elements within this 3.7-kb IL-1ß promoter fragment that mediates
this synergy. The function of ICSBP/PU.1 complexes has been previously
examined in NIH-3T3 fibroblasts (35). One study reported
that ICSBP/PU.1 complexes failed to activate the same (PU.1/IRF)4
reporter plasmid used also in our study. This finding contrasts with
our data in these same cells, but the differences may be due to the IRF
expression plasmids used in each study. Brass et al. (35)
used an epitope-tagged ICSBP expression plasmid, whereas the
experiments reported here were performed using a wild-type ICSBP cDNA
that lacked an epitope tag.
Phosphorylation of ICSBP has been reported to affect its ability to
associate with other transcription factors (18). While the
regulation of IRF4 function by phosphorylation has not been reported,
it is likely to share this regulatory mechanism with ICSBP. PU.1 that
is phosphorylated at serine 148 by protein kinase CK2 has a 9- to
10-fold higher binding affinity for IRF4, compared with
unphosphorylated PU.1 (21, 35). Similarly, Eklund et al.
(24) showed that the ability of ICSBP to active
transcription from a synthetic promoter containing four copies of the
gp91phox composite element also required PU.1
phosphorylation at serine 148. Thus, both IRF4 and ICSBP appear to
require PU.1 phosphorylation at the same site for maximal
protein-protein interaction. Like Eklund et al. (24), we
also found that ICSBP and PU.1 could synergistically activate
transcription from a PU.1/IRF composite element. These data are
consistent with earlier studies showing that IRF4 shares this capacity
in NIH-3T3 cells, and that serine 148 on PU.1 is required for both IRF
proteins to activate transcription (48). We have extended
these findings by showing that the natural IL-1ß promoter could be
synergistically activated by these IRF proteins in conjunction with
PU.1. In macrophages, over-expression of ICSBP modestly enhanced basal
transcription of the IL-1ß reporter plasmid, and IRF4 was able to
activate basal transcription in a dose-dependent manner (Fig. 9). In
NIH-3T3 fibroblasts, serine 148 of PU.1 was required for maximal
synergy with these IRF protein in the activation of transcription of
the IL-1ß promoter. However, we propose that the interaction between
PU.1 and ICSBP is more dependent upon serine 148 phosphorylation than
the interaction between PU.1 and IRF4. This is based on our findings:
1) that only IRF4 could increase IL-1ß reporter activity in a
dose-dependent manner in unstimulated RAW264.7 macrophages (i.e., cells
that contain little phosphorylated PU.1), and 2) that only IRF4 was
capable of synergizing with the S148A PU.1 mutant (albeit at lower
levels than wild-type PU.1).
A role for ICSBP in the regulation of the gp91phox and IL-12 p40 promoters has been reported, whereas the specific role of IRF4 in macrophages remains to be determined. However, mice lacking either a functional IRF4 or ICSBP gene do exhibit impaired resistance to intracellular infection (13, 14, 15, 16). Mice deficient in IRF4 do not generate mature, functional lymphocytes (16), although the effects of IRF4 deficiency on macrophage function have not yet been reported. Future studies will be needed to determine precisely how these transcription factors contribute to the phenotype observed in these knockout mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, R-220, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118. E-mail address:
3 Abbreviations used in this paper: ISRE, IFN-stimulated response elements; GAS, IFN--activated sequence; IRF, IFN regulatory factors; ICSBP, IFN consensus sequence binding protein; PEC, peritoneal exudate macrophage; CAT, chloramphenicol acetyl transferase; ECL, enhanced chemiluminescence.
Received for publication March 4, 1999. Accepted for publication June 25, 1999.
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), as described in the text. Extracts (150 µg
per lane) were fractionated on 15% denaturing polyacrylamide gels,
transferred to nitrocellulose, and probed with either the anti-IRF4
or anti-ICSBP Abs. The membranes were then developed using protein
G-HRP, and visualized using ECL. The molecular sizes of the protein
bands are indicated in the figure. B, To confirm that
the IRF4 and ICSBP Abs were non-cross-reactive, three identical
membranes were prepared from RAW264.7 cytosolic lysates (30 µg),
fractionated on 10% SDS-PAGE gels, and probed with each Ab singly or
together. The membranes were developed using protein G-HRP, and
visualized using ECL. The molecular sizes of the protein bands are
indicated in the figure.
