From the Cardiff School of Biosciences, Cardiff University, Museum Avenue, P. O. Box 911, Cardiff CF10 3US, United Kingdom
Received for publication, February 14, 2003
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ABSTRACT |
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Interferon- Transcriptional regulation coupled to the
cAMP-dependent signal transduction pathways is mediated
through a related family of DNA-binding proteins comprising the
cAMP-response element-binding protein
(CREB),1 cAMP-response
element modulator protein (CREM), and activating transcription factors
(ATF) (1, 2). These proteins belong to the basic leucine zipper (bZIP)
class of transcription factors and bind to the cAMP-response elements
(CREs) in the promoter and enhancer regions of target genes (1, 2). The
increased level of cAMP induces the trans-activation
potential of these proteins by a protein kinase A-mediated
phosphorylation of a critical serine residue (Ser-133 in CREB) (1, 2).
However, activation can also be triggered via phosphorylation by other
kinases, including protein kinase C,
calcium-calmodulin-dependent protein kinases, casein kinase
2, and mitogen-activated protein kinases (1, 2). In addition to
phosphorylation, heterodimerization between the different family
members has a profound effect on the regulation of cAMP-mediated gene
transcription (1, 2).
The CREM gene encodes multiple members by alternative splicing and
internal transcriptional initiation through the use of two promoters
(1-3). These proteins act as both activators and antagonists of
cAMP-mediated gene transcription, with inducible cAMP early repressor
(ICER) being the most potent repressor (1-3). ICER is produced from an
internal CREM gene promoter (P2), which, in contrast to the
P1 promoter that produces all CREM isoforms, contains CREs
(1-3). The ICER protein consists mainly of the bZIP domain and,
therefore, represses transcription either by heterodimerization with
activating forms of CREB/CREM/ATF or other bZIP-containing
transcription factors, or by competition with these proteins for DNA
binding (1, 2). The kinetics of cAMP-induced ICER expression is typical
of early response genes, with maximal expression levels being attained
within 3-6 h of stimulation (1-3). ICER then inhibits both its own
transcription and the expression of other CRE-containing promoters
(1-3). Indeed, the induced expression of ICER through the cAMP
signaling pathway has been implicated in several responses, including
the suppression of T lymphocyte function (4, 5), attenuation of Fas
ligand expression in T and NK lymphocytes (6), glucagon-mediated
suppression of insulin gene expression in pancreatic B cells (7), and
the noradrenaline-triggered suppression of Our laboratory is interested in understanding the mechanisms involved
in the regulation of macrophage gene expression by cytokines, particularly interferon- During the course of our studies on the regulation of macrophage gene
expression by IFN- Reagents--
The mouse macrophage J774.2 cell line was from the
European Collection of Animal Cell Cultures. Recombinant human and
mouse IFN- Cell Culture--
The J774.2 cell line was maintained in
Dulbecco's modified Eagle's medium, which was supplemented with 10%
(v/v) heat-inactivated fetal calf serum (56 °C, 45 min), 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin (designated as complete medium). The cultures
were maintained at 37 °C in a humidified atmosphere containing 5%
(v/v) CO2 in air. For human monocyte-derived macrophages,
blood was collected from healthy volunteers into a syringe containing
an equal volume of sterile 2% (w/v) dextran in 1× PBS containing
0.8% (w/v) trisodium citrate and was allowed to stand for 30 min to
permit erythrocyte sedimentation. The upper layer was collected,
underlayered with LymphoprepTM (Nycomed Pharmaceuticals)
(2:1 (v/v) supernatant:LymphoprepTM) and centrifuged at
800 × g for 20 min. The resultant interface was
collected and washed six to eight times with PBS, 0.4% (w/v) trisodium
citrate to remove contaminating platelets. Monocytes were plated out in
six-well plates (1 × 106 cells/well) in complete
medium, as above except containing 10% (v/v) human serum, and were
allowed to adhere to the surface for 6 h in a humid incubator at
37 °C containing 5% (v/v) CO2. The cells were then
washed twice with PBS to remove any other mononuclear cells, and fresh
medium was added. The homogeneity of macrophages was verified by
morphological analysis.
Before stimulation with the mediators, the cells were washed twice with
complete medium and then incubated for the requisite time in fresh
complete medium. For experiments involving the use of inhibitors
against key components of known signal transduction pathways, these
were added to the cells 1 h before the mediator (i.e.
pretreatment). The mediators or inhibitors were used at the following
concentration: 8-Br-cAMP (300 µM), A23187 (5 µM), AG490 (100 µM), apigenin (0-40
µM), bisindoylmaleimide (1 µM), forskolin
(20 µM), H89 (20 µM), IFN- RT-PCR--
Total cellular RNA was prepared using the
RNeasyTM total RNA isolation kit (Qiagen) according to the
instructions from the manufacturer. Each isolated RNA sample (1 µg)
was then subjected to RT-PCR essentially as described before (16,
30-32), except that the annealing temperature and the total numbers of
cycles varied: 20 cycles and 55 °C for murine ICERI/I
The PCR products were size-fractionated on a 2% (w/v) agarose gel,
photographed using a Syngene gel documentation system (GRI), converted
to an uncompressed TIFF file format, and quantified using the
Quantiscan computer package (Biosoft).
Cloning of ICER I and I In Vitro Transcription/Translation Reactions and Preparation of
Whole Cell Extracts--
In vitro transcription and
translation was carried out using the TNTTM
coupled reticulocyte lysate system using the protocol recommended by
the manufacturer (Promega). The recombinant protein was labeled with
[35S]methionine (40 µCi) to aid detection. Nuclear and
whole cell extracts were prepared essentially as described previously
(12, 15, 16, 40, 42). Protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 10 µg/ml
aprotinin, 10 µg/ml I-S soybean trypsin inhibitor) and DTT (0.5 mM) were added to all buffers before use.
Where the maintenance of proteins in the phosphorylated state was
desired (e.g. immunodetection of phosphorylated protein), whole cell extracts were made in a buffer containing both phosphatase and protease inhibitors (43). Briefly, the cells were washed twice with
ice-cold PBS, containing 10 mM sodium fluoride and 100 µM sodium orthovanadate, and then scraped off in 500 µl
of phosphatase-free whole cell extraction buffer (10 mM
Tris-HCl, pH 7.05, 50 mM NaCl, 50 mM sodium
fluoride, 1% (v/v) Triton X-100, 30 mM sodium
pyrophosphate, 5 µM zinc chloride, 100 µM
sodium orthovanadate, 1 mM DTT, 2.8 µg/ml aprotinin, 2.5 µg/ml each of leupeptin and pepstatin, 0.5 mM
benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride).
After vortexing for 45 s at 4 °C, the lysates were cleared by
centrifugation at 10,000 × g for 10 min at 4 °C.
Aliquots were immediately frozen at
The concentration of proteins in extracts was determined using the
MicroBCA protein assay kit as described by the manufacturer (Pierce).
SDS-PAGE and Western Blot Analysis--
Samples (10 µg of
whole cell extracts or 5 µl of a 50-µl in vitro
transcription/translation reaction) were size-fractionated under
reducing conditions using 10-15% (w/v) polyacrylamide gels containing
SDS (12, 13, 42). The in vitro translated protein was
detected by fluorography (31, 44). For Western blot analysis, the
proteins on the gel were transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore) by blotting (12, 42). The blotted
membranes were incubated first for 1 h at room temperature in
blocking solution (1× TBS containing 5% (w/v) nonfat milk powder and
0.05% (w/v) Tween 20) to reduce any nonspecific interaction of the
antiserum with the membrane. Following three washes of 5 min each with
1× TBS containing 0.05% (v/v) Tween 20, the membrane was incubated
with the primary antibody for 1 h in 1× TBS containing 5% (w/v)
nonfat milk powder and 0.1% (v/v) Tween 20. After washes as above, the
membrane was incubated with secondary horseradish peroxidase-conjugated
antibodies (goat anti-rabbit IgG or mouse anti-goat IgG) for 30 min at
room temperature in 1× TBS containing 5% (w/v) nonfat milk powder and
0.05% (v/v) Tween 20. After washing once more as above, the membranes
were developed using an enhanced chemiluminescence detection kit
(Amersham Biosciences) and XAR sensitive film (Eastman Kodak Co.). The
sizes of the proteins were determined by comparison with Rainbow
molecular weight markers (Amersham Biosciences) that had been subjected
to electrophoresis and blotting on the same gel as the test samples.
Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs were
carried out essentially as reported previously (12, 15, 16, 30-32, 40,
42). The sequences of the oligonucleotides used for EMSAs were: CRE,
5'-CTTGGCTGACGTCAGAGA-3' and 5'-GTCTCTCTGACGTCAG-3'; C/EBP,
5'-TGCAGATTGCGCAAT-3' and 5'-TGCATTGCGCAATCT-3'; and Sp1, 5'-TAGGTCCCTCCCCCCAACTT-3' and 5'-AAGTTGGGGGGAGGGACCTA-3'.
The oligonucleotides were radiolabeled by "fill-in" reactions using
[ Measurement of Nitrite Formation Using the Greiss
Reagent--
Determination of nitrite levels, an indicator of nitric
oxide (NO) production by inducible nitric-oxide synthase, was carried out as described by Ruetten and Thiemermann (45) with minor modifications. J774.2 cells were grown in 24-well plates with 500 µl
of culture medium and exposed for 24 h with the respective mediators (see "Results"). The nitrite levels were then determined by adding 100 µl of Greiss reagent (Sigma) to 100 µl of conditioned medium in a 96-well plate. Optical density at 560 nm was measured in a
MRX microplate reader (Dynex Technologies), and the nitrite concentration was calculated by comparison with the
A560 of sodium nitrite standards prepared in the
cell culture media.
Casein Kinase 2 Assay--
The method used was a combination of
those employed by Sung et al. (46) and Lodie et
al. (47) with minor modifications. The cells were incubated with
the mediators for the requisite time, and whole cell extracts were
prepared using phosphatase-free buffer as detailed above. CK2 was then
immunoprecipitated as follows. The extracts were first precleared by
the addition of the protein A/G-agarose beads (20 µl; Santa Cruz)
with incubation and gentle rolling at 4 °C for 1 h, followed by
centrifugation at 13,000 × g for 3 min to remove the
beads. The precleared supernatant was mixed with the anti-CK2 ICER Gene Expression in the Macrophage J774.2 Cell Line--
The
expression of ICER has been shown to be induced rapidly in several cell
types in response to increase in intracellular cAMP levels
(e.g. Refs. 3, 7, 9, and 10). However to our knowledge, the
expression of ICER isoforms in macrophages has, as yet, not been
determined. To examine ICER expression, murine J774.2 macrophages were
incubated with the membrane-permeable, non-hydrolyzable cAMP anologue,
8-Br-cAMP, and forskolin, a cell-permeable activator of adenylyl
cyclase, for various periods of time. Total RNA was isolated and
subjected to RT-PCR using primers specific for ICER and the control
ICER levels in the rat pancreatic cell line AR42H have been shown
recently to be modulated by the action of PMA (10), a protein kinase C
agonist. We decided to perform a similar experiment on the J774.2 cell
line because the effect of this signaling cascade on ICER expression in
macrophages had not previously been carried out. As shown in Fig.
1E, the addition of PMA to the cells also resulted in an
immediate increase in ICERI/I
To confirm that the amplification products shown in Fig. 1 were indeed
derived from the ICER transcripts, the DNA was subcloned into the
pcDNA3 expression vector as described under "Experimental Procedures." Sequence analysis confirmed that the products
corresponded to ICER I and ICER I
We next examined whether the induction of ICER I/I
Finally, the effect of 8-Br-cAMP on ICER DNA binding was investigated
using extracts that were employed for Western blot analysis (Fig.
2A). The ICER protein binds, either as homodimers or
heterodimers with other CREB/CREM/ATF family members, to CRE
recognition sites in the regulatory regions of target genes to exert
the transcriptional repressive effects. A CRE binding site
oligonucleotide, such as that from the somatostatin gene promoter has
been, therefore, used extensively to study the DNA binding properties
of ICER, and also other members of the CREB/CREM/ATF family, from
different cell types (3, 10, 51). The binding of the ICER homodimers is
identified by their fastest migration during gel electrophoresis because ICER is the smallest member of the CREB/CREM/ATF family (consisting essentially of the CREM bZIP region), similar migration to
that obtained using in vitro or bacterially produced ICER, and the inhibition of binding by an anti-CREM antiserum, which recognizes the protein. As shown in Fig. 2B, at least three
DNA-protein complexes were obtained when extracts from J774.2
macrophages were used. None of these complexes were present when only
the radiolabeled probe was used (data not shown). All these three complexes could be competed out using a 250- or 500-fold molar excess
of the unlabeled somatostatin CRE element but not with the same
excesses of unrelated, unlabeled C/EBP or Sp1 binding site
oligonucleotides (Fig. 2C). This indicates that all three complexes represent specific interaction of proteins with the CRE
binding site oligonucleotide (i.e. belong to the
CREB/CREM/ATF family). From these, the signal from the fastest
migrating complex was judged to be composed of ICER because its
migration was consistent with that expected from the smallest
CRE-binding protein. Indeed, this complex migrated only marginally more
slowly than the single complex produced by in vitro
translated ICER protein (Fig. 2B) (the slightly slower
migration could be caused by some form of post-translational
modification such as phosphorylation; see, e.g., Ref. 52).
In addition, the use of the CREM-1 antiserum, which also recognizes
ICER, in EMSA could completely inhibit the formation of this complex
and that formed by the in vitro translated protein (Fig.
2B). The kinetics of the formation of this complex in
response to 8-Br-cAMP were similar to that seen at the level of the
ICER protein (Fig. 2A), being absent with extracts from untreated cells but increasing dramatically in a
time-dependent manner following addition of 8-Br-cAMP to
the cells, with maximal binding seen at 24 h (Fig.
2B).
IFN-
The IFN-
Having established that IFN- Signal Transduction Pathway(s) Involved in the IFN-
Fig. 1 showed that forskolin, an activator of adenylyl cyclase, and
thus protein kinase A-mediated phosphorylation of CREB (1-2), could
induce ICER expression. We, therefore, investigated whether IFN- The Role of CK2 in the IFN-
Because the level of phosphorylated CREB, an inducer of ICER
transcription, was found to increase when macrophages were incubated with IFN- The Action of Intracellular Ca2+ Levels on the
IFN-
We next investigated whether the synergism between IFN- The studies presented in this paper show for the first time
several findings that are relevant to the regulation of macrophage gene
transcription: (i) ICER is expressed in macrophages; (ii) the levels of
ICER mRNA and proteins in macrophages along with their binding to
CRE are induced dramatically when the cells are stimulated with
IFN- IFN- It is interesting to note that there is a differential expression of
the different ICER transcripts in macrophages (e.g. Fig. 1B). Such differential expression has been noted previously
(39, 51, 91), but its significance remains unclear as all four isoforms
are thought to function identically as potent transcriptional repressors (1-3). The ICER protein levels do not peak until 12-24 h,
at least 6 h after the peak for ICER I mRNA expression (Figs. 1-3). Only a few previous studies on ICER gene expression have
analyzed both mRNA and protein expression (3, 7, 9). In most of these studies, a close correlation was observed between mRNA and protein expression except for forskolin-treated AtT20 cells (3) and, as
shown in this study, in macrophages. However, such an expression
profile correlates well with the kinetics of IFN- Several extracellular mediators have been shown to increase the
intracellular ICER levels, including NGF, thyroid-stimulating factor,
follicle-stimulating hormone, glucagons, noradrenaline, gastrin, and
cholecystokinin (7-10, 48, 51, 92). Although most of these factors act
by increasing intracellular cAMP levels, the use of pharmacological
agents has shown that the action of NGF and gastrin was not mediated
through this route (9, 10). Similarly, the use of a range of
pharmacological agents showed that the IFN- The importance of CREB phosphorylation at Ser-133 for the initiation of
transcription from the CREM P2 promoter is well documented (1-3). It should, however, be noted that CREB phosphorylation does not
necessarily constrain a cell to ICER production (9). For example,
treatment of PC12 cells with either epidermal growth factor or NGF
leads to increased Ser-133 phosphorylation of CREB, but only NGF
treatment leads to an increase in ICER expression (9). As IFN- Another interesting finding to emerge from studies presented in this
manuscript is the synergism between intracellular Ca2+
elevating agents and IFN- In conclusion, we have identified a novel induction of ICER expression
in macrophages by IFN- (IFN-
) is a
pleiotropic cytokine that modulates the immune function, cell
proliferation, apoptosis, macrophage activation, and numerous other
cellular responses. These biological actions of IFN-
are
characterized by both the activation and the inhibition of gene
transcription. Unfortunately, in contrast to gene activation, the
mechanisms through which the cytokine suppresses gene transcription
remain largely unclear. We show here for the first time that exposure
of macrophages to IFN-
leads to a dramatic induction in the
expression of the inducible cAMP early repressor (ICER), a potent
inhibitor of gene transcription. In addition, a synergistic action of
IFN-
and calcium in the activation of ICER expression was
identified. The IFN-
-mediated activation of ICER expression was not
blocked by H89, bisindoylmaleimide, SB202190, PD98059, W7, and AG490,
which inhibit protein kinase A, protein kinase C, p38 mitogen-activated
protein kinase, extracellular signal-regulated kinase,
calcium-calmodulin-dependent protein kinase, and Janus
kinase-2, respectively. In contrast, apigenin, a selective casein
kinase 2 (CK2) inhibitor, was found to inhibit response. Consistent
with this finding, IFN-
stimulated CK2 activity and the level of
phosphorylated cAMP response element-binding protein, which is known to
induce ICER gene transcription, and this response was inhibited in the
presence of apigenin. These studies, therefore, identify a previously
uncharacterized pathway, involving the IFN-
-mediated stimulation of
CK2 activity, activation of cAMP response element-binding
protein, and increased production of ICER, which may then play
an important role in the inhibition of macrophage gene transcription by
this cytokine.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenoreceptor in brown adipocytes (8). The expression of ICER has also recently been shown to
be induced by cAMP-independent pathways, and includes the action of
nerve growth factor, epidermal growth factor, and the gastrointestinal
peptides gastrin and cholecystokinin (9-11).
(IFN-
) (e.g. Refs. 12-16).
This cytokine is a key modulator of the immune and inflammatory
responses along with numerous other cellular actions during
physiological and pathophysiological conditions (17, 18). Such
biological responses are characterized by the transcriptional
activation and the inhibition of numerous genes (17, 18). The
mechanisms through which IFN-
induces gene transcription have been
investigated over the last decade and identified a key role for the
Janus kinase (JAK)-signal transducer and activator of transcription
(STAT) pathway (18-20). However, several recent studies, including
gene expression profiling on STAT-1-deficient cells, have revealed the
existence of STAT-1-independent pathways (20-22), which remain to be
deciphered in detail. In addition, although IFN-
suppresses the
expression of numerous genes, little is understood of the corresponding
mechanisms, which may be the result, at least in part, of limited
research on this aspect that has been carried out to date. Further
studies are required, especially in the light of the potential
biological importance of such regulation. For example, IFN-
has been
shown to inhibit macrophage foam cell formation, a critical early event
in atherogenesis, at least in part by suppressing the expression of
several genes that are involved in the uptake of lipoproteins,
including lipoprotein lipase (LPL), very low density lipoprotein
receptor, low density lipoprotein receptor-related protein, and the
scavenger receptors A and CD136 (12, 23-27).
, we were intrigued by the presence of ICER
binding sites in the promoter regions of many IFN-
inhibited genes,2 the co-regulation of
some of these genes by cAMP (e.g. LPL) (28), and a study
demonstrating the ability of IFN-
to suppress CRE- or the
bZIP-containing AP1-regulated promoters (29), and wondered whether this
cytokine induces ICER expression. We present here studies that
investigate the IFN-
-ICER link in detail. We show for the first time
that IFN-
produces a dramatic increase in ICER expression in
macrophages, and acts in a synergistic manner with agents that induce
intracellular calcium levels. In addition, we identify the mechanisms
through which IFN-
induces ICER expression. The studies, therefore,
indicate the existence of a potentially novel pathway for
IFN-
-mediated suppression of macrophage gene transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was from Peprotech. All the cell culture reagents were
purchased from Greiner, Helena Biosciences, or Invitrogen. Antisera
against CREM-1 (X-12), phospho-CREB, and CK2-
subunit along with the protein A/G-agarose were from Santa Cruz. The purified CK2 enzyme and
the TNTTM coupled in vitro
transcription/translation kit were from Promega. The pharmacological
agents SB202190, PD98059, AG490, thapsigargin, A23187, forskolin,
bisindoylmaleimide, and H89 were from Calbiochem whereas the secondary
horseradish peroxidase-conjugated antibodies, 8-bromo-cAMP (8-Br-cAMP),
phorbol 12-myristate 13-acetate (PMA), apigenin,
-casein, and the
Greiss reagent were purchased from Sigma.
(1000 units/ml), LPS (100 ng/ml), PD98059 (50 µM), PMA (0.16 or
1 µM), SB202190 (10 µM), thapsigargin (0.5 µM), and W7 (25 µM).
, 30 cycles
and 65 °C for murine ICER I/II, 27 cycles and 60 °C for IL-1
,
30 cycles and 60 °C for Suppressor of cytokine signaling-1
(SOCS-1), 8 cycles and 62 °C for 28 S rRNA, 16 cycles at
60 °C for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18 cycles at 58 °C for
-actin. These conditions were in the
exponential phase of amplification and, therefore, provided a direct
correlation between the amount of products and RNA template abundance
in the samples. The sequences of the primers were
5'-CAAAAGCCCAACATGGCTG-3' and 5'CCAATTCACACTCTACAGCAG-3' for
murine ICER I/I
(7), 5'-ATGGCTGTAACTGGAGATGAAACT-3' and 5'-CTAATCTGTTTTGGGAGAGCAAATGTC-3' for murine ICER I/II (33), 5'-CTGATGAGGAAACTGAACTTG-3' and 5'-TCGGCTCTCCAGACATTTTAC-3' for human
ICERs (34, 35), 5'-ATGGCAACTGTTCCTGAACTCAACT-3' and 5'-CAGGACAGGTATAGATTCTTTCCTTT-3' for IL-1
(36),
5'-CTCGAGTAGGATGGTAGCACGCAA-3' and 5'-CATCTTCACGCTGAGCGCGAAGAA-3' for
SOCS1 (37), 5'-TGAACTATGCTTGGGCAGGG-3' and 5'-AGCGCCATCCATTTTCAGGG-3'
for 28 S rRNA (38), 5'-CCCTTCATTGACCTCAACTACATGG-3' and
5'-AGTCTTCTGGGTGGCAGTGATGG-3' for GAPDH, and
5'-TGGAGAAGAGCTATGAGCTGCCTG-3' and 5'-GTGCCACCAGACAGCACTGTGTTG-3' for
-actin (16, 30-32).
into the pcDNA3
Vector--
J774.2 macrophages were stimulated for 12 h with
8-Br-cAMP and, following isolation of RNA and cDNA synthesis as
above, the ICER I/I
isoforms were amplified using primers containing
BamHI and EcoRI restriction sites to facilitate
the cloning into the pcDNA3 vector (Invitrogen) in the correct
orientation (5'-ACTTTAGGATCCACTGTGGTACGGCCAAC-3' and
5'-GAGCTCGAATTCCCAATTCACACTCTACAGCAG-3', with the
restriction endonuclease sites shown in bold) (39). The amplification
was carried out using the high fidelity Pwo DNA polymerase
to minimize PCR-generated mutations (40, 41), and this was confirmed by sequencing. The amplification product (~530 bp) was purified, digested with BamHI and EcoRI, and subcloned into
the pcDNA3 vector that had been previously digested with the same enzymes.
70 °C until use.
-32P]dCTP and Klenow DNA polymerase. Whole cell
extracts (2.5-30 µg) or in vitro translated protein (3 µl of a 50-µl reaction) were incubated in a total reaction volume
of 20 µl containing 34 mM potassium chloride, 5 mM magnesium chloride, 0.1 mM DTT, and 3 µg
of poly(dI-dC). After 10 min on ice, 32P-labeled probes
(50,000 cpm) were added and the incubation continued for 30 min at room
temperature. Following the addition of 5 µl of a 20% (w/v) Ficoll
solution to each sample, the free probe and the DNA-protein complexes
were resolved on 4-6% (w/v) polyacrylamide gels in 0.5× TBE (45 mM Tris base, 45 mM boric acid, 1 mM EDTA). The gels were then dried under vacuum and exposed
to x-ray film. For antibody supershift assays, samples of nuclear or
whole cell extracts were incubated with the appropriate antiserum for
30 min on ice prior to the addition of the radiolabeled probe (12, 15,
16, 30, 32, 40, 42). For competition assays, a 250-500-fold molar
excess of the double-stranded oligonucleotides was added to the samples
of proteins prior to the addition of the radiolabeled probe (12, 15,
16, 30-32, 40, 42).
-chain
antiserum (2 µg/ml) and incubated overnight at 4 °C with gentle
rolling. The resulting protein-antibody complex was isolated by the
addition of protein A/G-agarose (20 µl) with gentle rotary mixing for
2 h at 4 °C. The beads, containing the immunocomplex, were
collected by centrifugation (13,000 × g for 3 min at
4 °C) and washed once with phosphatase-free cell extraction buffer
(see above). The pellet was then resuspended in 25 µl of kinase
buffer (100 µM sodium orthovanadate, 100 mM Tris-HCl (pH 8), 100 mM sodium chloride, 20 mM
magnesium chloride, 50 mM potassium chloride) containing 5 µCi of [
-32P]ATP and 5 mg/ml
-casein. Kinase
reactions were incubated for 15 min at 37 °C and stopped by the
addition of 10 µl of reducing solubilizing buffer (50 mM
Tris-HCl (pH 6.8), 100 mM DTT, 2% (w/v) SDS, 0.1% (w/v)
bromphenol blue, 10% (v/v) glycerol). Samples were boiled for 10 min
and subjected to SDS-PAGE using 15% (w/v) acrylamide gels. After
electrophoresis, the gels were fixed for 20 min in a solution
containing 40% (v/v) methanol and 10% (v/v) acetic acid. These were
washed once with distilled water before being dried under vacuum
and visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin gene. For mouse ICER, four functionally indistinct isoforms,
ICER I, ICER I
, ICER II, and ICER II
, have been identified that
arise as a result of alternative splicing of a transcript produced from
the CREM P2 promoter (see Fig.
1A for a schematic
representation of the different ICER isoforms). Our initial RT-PCR was
carried out using representative time points (0, 6, 12, and 24 h)
from 8-Br-cAMP-treated cells using primer sets that allow amplification
of all four ICER transcripts (primers 1 and
2 in Fig. 1A). As shown in Fig. 1B,
the mRNA levels of all ICER isoforms was increased by 8-Br-cAMP
treatment, with the induction of ICER I/I
being more dramatic than
that for ICER II/II
. As a result, all further studies on J774.2
macrophages were restricted to the examination of ICER I/I
expression (primers 1 and 3 in Fig.
1A). A detailed time course experiment was carried out with cells exposed to 8-Br-cAMP or forskolin. The expression of ICER I/I
RNA was induced immediately following incubation of the cells with
8-Br-cAMP, peaking at ~3 h and being maintained at similar or
slightly reduced levels until 24 h (Fig. 1C). On the
other hand, the levels of ICERI/I
were induced immediately following addition of forskolin to the cells, peaking at 2 h, and declining thereafter to reach almost undetectable levels at 6 h (Fig.
1D). The induction in these experiments was specific to the
addition of 8-Br-cAMP or forskolin and not the result of any
alterations in the status of the cells over time because no ICER
mRNA expression was observed in untreated cells at 4, 12, and
24 h in the experiment.
View larger version (50K):
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Fig. 1.
Regulation of ICER mRNA expression by
8-bromo-cAMP, forskolin, and PMA. A, schematic
representation of the ICER-encoding region of the murine CREM gene and
the four ICER isoforms. The boxes represent the different
exons (ICER, , H, DBI, and DBII from left to
right). ICERI mRNA isoforms (ICERI and ICERI
) contain
the exons encoding DNA-binding domains I and II (DBI and DBII,
respectively), whereas ICERII (ICERII and ICERII
) isoforms contain
only the DNA binding domain II exon. The presence of a TAA stop codon
in the DBI sequence prevents the inclusion of the DBII domain in the
final ICERI protein. ICERI
and ICERII
both lack the
exon. The
ATG initiation codon, the TAA and the TAG stop codons, and the
P2 promoter are also shown. Use of primers (pr)
1 and 2 in RT-PCR leads to amplification of mRNA encoding all four
ICER isoforms, whereas primers 1 and 3 are specific for ICERI and
ICERI
. B-E, time-course RT-PCR using RNA from cells
exposed for the indicated times to 300 µM 8-bromo-cAMP
(panels B and C), 20 µM
forskolin (panel D), and 1 µM PMA
(panel E). For ICER, PCR was carried out using
primers 1 and 2 (B) or 1 and 3 (C-E) and
-actin was used as a control. 4h
, 12h
, and
24h
represent samples from untreated cells taken at 4, 12, and 24 h of the experimental period.
RT denotes a
reaction where no reverse transcriptase was added during the cDNA
synthesis step (cDNA from 24 h (B-D) or 6 h
(E) were used). The products were analyzed by
electrophoresis on a 2% (w/v) agarose gel along with molecular size
markers (M). The results shown are representative of two to
three independent experiments.
mRNA levels that peak at 1 h
and then decline gradually to reach levels at 6 h that were
similar to those seen in untreated cells.
; the sequences have been deposited
in the EMBL nucleotide sequence data base under the accession numbers AJ311667 and AJ292222, respectively. In addition, in vitro transcription and translation of the recombinant plasmid, in the presence of [35S]methionine, followed by SDS-PAGE of the
protein and fluorography showed a protein with a relative molecular
mass (~13.5 kDa) that was consistent with the size expected from the
cDNA insert (data not shown). Furthermore, this in vitro
translated protein was demonstrated to interact specifically with a CRE
(see below). Moreover, transient transfection assays showed that the
LPL promoter, which contains a number of CRE-like sequences and
expression of which in J774.2 macrophages is suppressed by both
8-Br-cAMP3 and IFN-
(12-14), was inhibited in a dose-dependent manner by an
ICER expression plasmid (data not shown).
mRNA seen in
the experiments shown in Fig. 1 was also accompanied by an increase in
the expression of the ICER protein. Whole cell protein extracts from
J774.2 macrophages were, therefore, subjected to Western blot analysis
using an anti-CREM-1 primary antibody, which recognizes ICER. Anti-CREM
antiserum has been used in a number of studies to investigate ICER
expression (3, 4, 6, 11, 33, 39, 48-50). As shown in Fig.
2A, the levels of the 13.5-kDa ICER protein were induced following exposure of the cells to 8-Br-cAMP. The level of the protein at 3 h following treatment of the cells with 8-Br-cAMP was substantially greater than untreated cells, and this
is likely to be of major functional significance given that ICER
represses the action of the activating members of the CREB/CREM/ATF
family at substoichiometric levels (1-3). The maximal levels were
attained at 12-24 h and were, therefore, delayed compared with that
for the mRNA (see Fig. 1C). A delay in the induction of
ICER protein compared with the corresponding RNA has also been reported
previously for forskolin-treated AtT20 cells (3).
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Fig. 2.
The action of 8-bromo-cAMP on the expression
of the ICER protein and DNA binding activity in macrophages.
A, Western blot analysis was carried out using whole cell
extracts from cells exposed to 8-Br-cAMP for the indicated time
periods. The position of the 13.5-kDa ICER protein is shown.
B, EMSA was carried out using radiolabeled somatostatin CRE
oligonucleotide, which recognizes ICER along with other members of the
CREB/CREM/ATF family, and extracts from J774.2 macrophages that were
either left untreated (lane 1) or incubated with
8-Br-cAMP for 3 h (lane 2), 6 h
(lane 3), 12 h (lane
4), or 24 h (lane 5). Complexes
formed by the ICER-I protein produced by in vitro
transcription and translation are shown in lane
7. Supershift experiments using the anti-CREM-1 antibody,
which also recognizes ICER, are shown with extracts from cells treated
with 8-Br-cAMP for 24 h (lane 6) or the
in vitro translated protein (lane 8).
C, competition EMSA to demonstrate the specificity of DNA
binding. The lanes marked 0h and 24h
show the profile obtained using extracts from cells that were untreated
or stimulated with 8-Br-cAMP for 24 h, respectively. The remaining
lanes show the outcome of binding reactions using extracts
of cells treated for 24 h with 8-Br-cAMP and the indicated molar
excesses of unlabeled somatostatin CRE, C/EBP, or Sp1 oligonucleotides.
In both B and C, the free probe has migrated off
the gel. Vertical lines indicate the position of
the fastest migrating ICER-CRE (ICER) complex. All the
results are representative of at least two independent
experiments.
Induces ICER Expression in Macrophages--
Having
established that ICERs are expressed in macrophages, we next determined
whether their expression could be regulated by IFN-
. Time-course
RT-PCR showed that exposure of J774.2 macrophages to IFN-
results in
a dramatic induction of ICER I/I
mRNA expression, peaking
between 1 and 4 h, and declining gradually thereafter (Fig.
3A). Western blot analysis
using an anti-CREM-1 antiserum showed that the activation of ICER
I/I
mRNA expression was followed by the corresponding protein
(Fig. 3B). However, similar to the case with cells exposed
to 8-Br-cAMP (Fig. 2A), there was a lag between mRNA and
protein expression, and this may be peculiar to macrophages. EMSA
showed that IFN-
induced the binding of the fastest migrating
ICER-CRE complexes in a time-dependent manner, and that the
formation of these complexes was inhibited by the inclusion of the
anti-CREM antiserum but not the nonimmune serum (Fig. 3C).
The presence of two closely migrating ICER-CRE complexes is consistent
with the possibility of a number of similar sized ICER proteins being
produced from the various transcripts generated by alternative splicing
(see Fig. 1A). The kinetics of the induction of the ICER-CRE
complexes in response to IFN-
treatment of the cells was similar to
that seen at the level of the ICER protein (Fig. 3B).
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Fig. 3.
The action of IFN-
on ICER mRNA expression, protein levels, and DNA binding in
J774.2 macrophages. A, J774.2 macrophages were treated
with IFN-
for the indicated period of time and total RNA was
subjected to RT-PCR using primers for ICERI/I
and
-actin. RNA
from untreated cells at 4-, 12-, and 24-h periods of the experiment
were also analyzed (designated as 4h
, 12h
,
and 24h
, respectively).
RT denotes a reaction
where no reverse transcriptase was added during the cDNA synthesis
step (cDNA from the 0-h sample was used). B, Western
blot analysis of proteins from whole cell extracts of J774.2
macrophages treated with IFN-
for the indicated time. The position
of the 13.5-kDa ICER protein is shown. C, EMSA was carried
out using radiolabeled somatostatin CRE oligonucleotide, which
recognizes ICER along with other members of the CREB/CREM/ATF family,
and extracts from J774.2 macrophages that were either left untreated
(lane 1) or incubated with IFN-
for 1 h
(lane 2), 3 h (lane
3), 6 h (lane 4), 12 h
(lane 5), or 24 h (lane
6). Supershift experiments using the anti-CREM-1 antibody,
which also recognizes ICER, and the nonimmune serum are shown in
lanes 7 and 8, respectively. The free
probe has migrated off the gel. The vertical line
indicates the position of the fastest migrating ICER-CRE
(ICER) complexes. All the results are representative of at
least two independent experiments.
-mediated induction of ICER mRNA expression detailed
above was shown in the macrophage J774.2 cell line. To rule out that
this was not a peculiar property of this cell line, the experiment was
repeated using primary cultures of human monocyte-derived macrophages.
Thus, RNA from untreated macrophages and those exposed to IFN-
were
subjected to RT-PCR using primers that have been used previously to
study ICER expression in pituitary adenomas and hyperfunctioning
thyroid adenomas (34, 35). These primers have been shown to amplify
products of 657 and 257 bp, which correspond to ICER I and II,
respectively, along with additional product(s) that contain partial
sequences of the 657-bp product, which have been postulated to be the
result of alternative splicing (35). As shown in Fig.
4, the expression of ICER-I and ICER-II
was increased when the cells were treated with IFN-
.
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Fig. 4.
The action of IFN-
on ICER mRNA expression in primary cultures of human
monocyte-derived macrophages. The cells were either left untreated
or stimulated with IFN-
for the indicated period of time, and total
RNA was subjected to RT-PCR using primers that recognize human ICERs
and GAPDH.
RT denotes a reaction where no reverse
transcriptase was added during the cDNA synthesis step (cDNA
from the 2-h sample was used). The products were analyzed by agarose
gel electrophoresis along with molecular size markers (M).
The positions of the ICER I and ICER II products of approximately 657 and 257 bp, respectively, are shown. * indicates a smaller
product that has been observed previously with these primers and
contains partial sequences of ICER I (35).
induces the expression of ICER in both
the murine J774.2 cell line and primary cultures of human monocyte-derived macrophages, our further studies focused on
understanding the potential mechanisms that are involved in this action
of the cytokine.
-mediated
Induction of ICER Expression--
As described in the Introduction,
the activation of CREB by phosphorylation at Ser-133 is a critical step
in the production of ICER by virtue of its ability to transactivate the
CREM gene P2 promoter (1-3). It was, therefore, possible
that IFN-
also induces the cellular levels of CREB phosphorylated at
this residue. This possibility was investigated by time-course Western
blot analysis using antisera specific for Ser-133-phosphorylated CREB. As shown in Fig. 5, IFN-
treatment of
J774.2 macrophages does indeed lead to an immediate and marked increase
in the level of phosphorylated CREB.
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Fig. 5.
The effect of IFN-
on Ser-133-phosphorylated CREB in macrophages. J774.2
macrophages were incubated with IFN-
for the indicated time and
subjected to Western blot analysis using a phospho-CREB antiserum as
described under "Experimental Procedures." The position of the
43-kDa phospho-CREB protein is shown. Similar results were obtained in
three independent experiments.
also acted through this pathway in the activation of ICER expression
using H89, an inhibitor of cyclic-nucleotide dependent protein kinase.
The action of all the pharmacological agents employed in this study was
demonstrated not to be the result of any cytotoxic effect because they
had no effect on both the viability of the cells, as judged by the
trypan blue exclusion assay (data not shown), and the expression of the
mRNA for the constitutively expressed marker (see Figs.
6-9). As shown in Fig. 6A,
incubation of the cells with H89 did not inhibit the IFN-
-mediated induction of ICER mRNA expression, indicating that the protein kinase A signaling cascade was not involved. As expected, H89 did
prevent the forskolin-mediated induction of ICER mRNA levels, thereby proving that the inhibition of ICER gene expression by H89 can
be measured using this experimental technique (Fig. 6A). In
the light of this finding, the potential role of the other signaling
pathways that are involved in the phosphorylation, and thereby
activation of CREB, was investigated using a panel of inhibitors. These
included: (i) SB202190, an inhibitor of p38 MAP kinase (55); (ii)
PD98059, an inhibitor of the extracellular signal-regulated kinase MAP
kinase pathway (56); (iii) W7, an inhibitor of
calcium-calmodulin-dependent protein kinase (36); (iv)
bisindoylmaleimide, an inhibitor of protein kinase C (58); and (v)
apigenin, an inhibitor of CK2 (59-63). In addition, a JAK2 inhibitor,
AG490 (64, 65), was included because its activation is required for the
expression of numerous IFN-
-regulated genes (17-20). The
concentration of all the inhibitors used was based on an extensive
literature survey on their action in macrophages. Overall, the
IFN-
-induced ICER mRNA expression was not affected by incubation
of the cells with SB202190, PD98059, W7, bisindoylmaleimide, and AG490
(Fig. 6, B-E). The functionality of these inhibitors was
confirmed by investigation of their ability to affect a previously reported response (Fig. 7,
A-D). Thus, W7 could inhibit the previously reported
LPS-induced expression of IL-1
in macrophages (36) (Fig.
7A). In addition, bisindoylmaleimide could inhibit the
PMA-mediated induction of LPL in the macrophage THP-1 cell line, which
is mediated through protein kinase C (66) (Fig. 7B).
Similarly, AG490 could successfully inhibit the LPS-induced nitrite
production in J774.2 macrophages (45) or the IFN-
-mediated
stimulation of SOCS1 mRNA, which is known to require the JAK-STAT
pathway (20) (Fig. 7, C and D). Furthermore,
PD98059 and SB202190 were found to inhibit the PMA-induced
differentiation of THP-1 monocytes and the reduced serum
concentration-mediated activation of LPL mRNA
expression.4 In contrast to
all these pharmacological agents, incubation of macrophages with the
CK2 inhibitor apigenin prevented the IFN-
-induced expression of ICER
(Fig. 6F). This suggests that CK2 plays an important role in
this action of the cytokine and was, therefore, investigated in
detail.
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Fig. 6.
The action of pharmacological inhibitors on
the IFN- -mediated induction of ICER
expression. RT-PCR was carried out using RNA from J774.2
macrophages that were either left untreated or exposed to IFN-
for
2 h in the absence or the presence of the inhibitor H89
(panel A), SB202190 and PD98059 (panel
B), W7 (panel C), bisindoylmaleimide
(bis) (panel D), AG490
(panel E), and apigenin (panel
F). Panel A also shows the positive
control for H89, the previously noted inhibition of forskolin-mediated
ICERI/I
expression.
RT denotes a reaction where no
reverse transcriptase was added during the cDNA synthesis step
(cDNA from untreated control cells was used). The products were
analyzed by electrophoresis on a 2% (w/v) agarose gel along with
molecular size markers (M). The results shown are
representative of two to three independent experiments.
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Fig. 7.
Demonstration of the positive action of W7,
bisindoylmaleimide, and AG490. A, B, and
D, RT-PCR was carried out using RNA from J774.2
(panels A and D) and THP-1
(panel B) macrophages that were either left
untreated or exposed to LPS for 24 h (panel
A), PMA for 2 days (panel B), or
IFN- for 2 h (panel D) in the absence or
the presence of the inhibitor W7 (panel A),
bisindoylmaleimide (bis; panel B), and
AG490 (panel C). The nature of the amplification
product is indicated on the right side of the
figure with
RT showing the outcome of a
reaction where no reverse transcriptase was added during the cDNA
synthesis step (cDNA from untreated control cells was used). The
products were analyzed by agarose gel electrophoresis along with
molecular size markers (M). Panel C,
J774.2 macrophages were either left untreated or exposed to LPS for
18 h in the absence or the presence of the AG490. The production
of nitrite was determined as described under "Experimental
Procedures." All the results shown are representative of two
independent experiments.
-mediated Induction of ICER
Expression--
A single concentration of apigenin was used in the
experiments shown in Fig. 6F. A dose-response experiment
was, therefore, carried out and showed that apigenin inhibited the
IFN-
induced activation of ICER mRNA expression in a
concentration-dependent manner (Fig.
8A). These results, therefore,
confirmed that the activation of CK2 was a key event in the
IFN-
-induced ICER expression. However, to our knowledge, no studies
have been published with respect to IFN-
and increased CK2 activity
and was thus investigated. For this, macrophages were stimulated with
IFN-
for 2 h, a time point when the major increase in ICER
expression were seen (see Fig. 3A), in the absence or the
presence of apigenin. After preparation of whole cell extracts using a
buffer containing phosphatase and protease inhibitors, CK2 was
immunoprecipitated using an antibody raised against the
-subunit and
subjected to the in vitro kinase assay as described under
"Experimental Procedures." As shown in Fig. 8B, a
dramatic increase in the phosphorylation of the
-casein substrate
was seen with extracts from cells treated with IFN-
and this was
inhibited in the presence of apigenin. The increased phosphorylation of
-casein in this experiment could be caused by a cytokine-mediated
increase in either the activity or the steady state levels of the
protein. Western blot analysis was carried out to distinguish between
these two possibilities and showed that IFN-
induced CK2 activity in
macrophages (Fig. 8C).
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Fig. 8.
The role of CK2 in the
IFN- -mediated induction of ICER
expression. A, RT-PCR showing the effect of IFN-
on
ICERI/I
and
-actin mRNA expression in J774.2 macrophages in
the absence (Cont) or the presence of the indicated
concentration of apigenin.
RT and M denote a
reaction where no reverse transcriptase was added during the cDNA
synthesis step (cDNA from the control sample was used) and markers,
respectively. B, the action of IFN-
and apigenin on CK2
activity of J774.2 macrophages. The cells were either untreated or
exposed to IFN-
for 2 h in the absence or the presence of
apigenin (40 µM). CK2
was immunoprecipitated and
subjected to in vitro kinase assay as described under
"Experimental Procedures." Also shown are a positive control
(+ve) with purified CK2 enzyme and a negative control
(-ve) without any enzyme. The signal from phosphorylated
-casein substrate (24 kDa) is shown. C, Western blot
analysis of CK2-
from cells that were either untreated or stimulated
with IFN-
for 2 h in the absence or the presence of apigenin.
The position of the 42-kDa CK2-
polypeptide is shown. D,
effect of apigenin on IFN-
-mediated phosphorylation of CREB. Western
blot analysis was carried out on extracts from cells that were either
untreated or stimulated with IFN-
for 2 h, in the absence or
the presence of apigenin (40 µM), using the phospho-CREB
antiserum as described under "Experimental Procedures." All the
results are representative of two independent experiments.
(Fig. 5), it was possible that the activation of CK2 played an important role. We, therefore, investigated whether the
IFN-
-triggered increase in the level of phospho-CREB was inhibited
in the presence of apigenin. Western blot analysis showed that this was
indeed the case (Fig. 8D).
-induced Expression of ICER in Macrophages--
Krueger and
co-workers (67) have shown recently that elevation of intracellular
calcium levels can prevent the forskolin-mediated induction of ICER
mRNA expression in the murine WEHI7.2 thymocyte cell line.
Furthermore, they demonstrated that this inhibition was mediated by a
83-kDa Ca2+-activated repressor protein that bound to the
ICER promoter region (67). We therefore investigated whether a similar
mechanism for the prevention of IFN-
-induced ICER mRNA
expression also existed in macrophages. For this, the cells were
treated with IFN-
for 2 h in the absence or the presence of two
different agents that raise intracellular Ca2+ levels:
thapsigargin, which releases endoplasmic reticulum-stored Ca2+; or the ionophore A23187, which passively transfers
extracellular calcium as well as triggering release from intracellular
stores. Total RNA was then isolated and subjected to RT-PCR with
ICERI/I
- and
-actin-specific primers. Fig.
9 (panels A and
B) shows that, instead of inhibiting the
IFN-
-mediated ICER mRNA expression, the presence of A23187
or thapsigargin enhanced it in a synergistic manner. This was
particularly apparent in cells treated with IFN-
and A23187, where
the increase in ICER mRNA expression in the presence of both
factors was approximately 2-fold greater than that expected from a
simple additive action. These studies, therefore, reveal a novel
synergistic action of IFN-
and Ca2+ in the activation of
ICER mRNA expression in macrophages.
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Fig. 9.
The effect of A23187 and thapsigargin on the
IFN- -induced expression of ICER. J774.2
macrophages were exposed to IFN-
for 2 h in the absence or the
presence of A23187 (5 µM) (panel A)
or thapsigargin (0.5 µM) (panel B).
Total RNA was isolated and subjected to RT-PCR using primers against
ICER-I/I
and
-actin as described under "Experimental
Procedures."
RT denotes a reaction where no reverse
transcriptase was added during the cDNA synthesis step (cDNA
from untreated control cells was used). C, EMSA was carried
out using extracts from J774.2 macrophages that were either left
untreated or exposed for 12 h to IFN-
, thapsigargin, or
apigenin in the absence or presence of anti-CREM-1 antiserum
(anti-CREM-1) or nonimmune serum (NIS), as
indicated. The vertical line indicates the
position of the fastest migrating ICER-CRE (ICER) complex.
All the results are representative of two independent
experiments.
and
intracellular Ca2+ elevating agents seen with ICER mRNA
expression was also manifested at the level of ICER-CRE DNA binding.
Because of a longer incubation period required in such experiments as a
result of the noted delay between ICER mRNA and protein expression
(e.g. Figs. 2A and 3B), such studies
could only be carried out for IFN-
and thapsigargin (A23187 was
found to be toxic to the cells at 12 h). As shown in Fig.
9C, the signal from the fastest migrating ICER-CRE-binding complex, which was inhibited by the inclusion of the anti-CREM-1 antiserum, was substantially greater when extracts from cells treated
with both IFN-
and thapsigargin were used compared with those
obtained with either agent alone. In these experiments, we also
examined whether the IFN-
-mediated induction of ICER-CRE complex was
inhibited by apigenin, as found at the level of ICER mRNA
expression (Figs. 6F and 8A). As shown in Fig.
9C, this was indeed found to be the case.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and this is inhibited by the CK2 inhibitor apigenin; (iii)
this cytokine stimulates CK2 activity and increases the level of
activated CREB phosphorylated at Ser-133; and (iv) IFN-
synergizes
with Ca2+ in the induction of ICER expression. These
results, therefore, reveal the existence of a potentially novel pathway
for the IFN-
-mediated suppression of macrophage gene transcription
that involves increased activation of CK2 and CREB leading to
transcriptional stimulation from the CREM P2 promoter,
thereby culminating in increased ICER production.
has been shown to suppress the expression of numerous genes;
for example, analysis of the published literature shows at least 30 genes for which expression is suppressed by the cytokine (LPL; very low
density lipoprotein receptor; scavenger receptor A; CD136; low density
lipoprotein receptor-related protein; C/EBP
; matrix
metalloproteinase 2, 9, and 13; cathepsin K; histidine decarboxylase;
neu/Her-2; collagenase-1; angiotensin AT1a receptor; cyclin A; CXCR4
(fusin);
1(I)- and
1(III)-procollagen; IL-4 receptor;
stromelysin; chemokine receptor CCR2; long pentraxin PTX3; fibronectin;
-amyloid precursor; renin; IL-8; epithelial neutrophil activating
protein 78; thyrotrophin receptor; IL-1
; and c-Myc) (12, 16, 23-27,
68-89). The expression of some of these genes has been investigated in
monocytes/macrophages (12, 16, 23-27, 77, 81, 82, 86, 88). In
addition, another 12 IFN-
-inhibited genes have been identified
recently from a limited expression profiling on the human fibrosarcoma
HT1080 cell line treated for 6 h with the cytokine (90). However,
studies aimed at delineating the mechanisms through which IFN-
suppresses gene expression have been limited and restricted to a few
genes: (i) matrix metalloproteinase-9 and -13 (does not occur in the absence of STAT-1) (70, 71); (ii) stromelysin (requires an AP1 binding
site) (80); (iii) scavenger receptor A (mediated by competition between
STAT1 and AP1/ets transcription factors for a limiting amount of shared
co-activator) (25); (iv)
1(I) procollagen (requires a region
containing overlapping binding sites for Sp1, Sp3, and nuclear
factor-1) (78); and LPL (our recent studies showing a novel role for
Sp1 and Sp3) (12). Thus, disparate mechanisms have so far been
identified for the IFN-
-mediated regulation of such genes. However,
it is possible that regulation through ICER may turn out to be a more
common mechanism, given that it is a potent repressor that acts at
substoichiometric levels (1-3). Detailed characterization of the
promoter regions of a large number of genes for which transcription is
inhibited by IFN-
in the future will clarify this issue. It is also
possible that, besides acting as a direct repressor of genes that are
expressed constitutively at high levels and inhibited by IFN-
, ICER
may also play an important role in the transcriptional suppression of
immediate early genes that have been induced transiently by this
cytokine through other pathways. For example, the NGF-mediated expression of ICER has been proposed to be involved in the inhibition of c-fos gene transcription once it has been induced
immediately by the mediator (9).
-inhibited genes,
such as LPL (12), with maximum decreases seen between 12 and 24 h.
-induced ICER expression
was not affected by inhibitors of cyclic
nucleotide-dependent protein kinase, MAP kinases,
Ca2+/calmodulin-dependent protein kinase,
protein kinase C, and JAK2 but required activation of CK2 (Fig. 6). The
lack of the effect of the JAK2 inhibitor AG490 may be of interest in
light of several recent studies indicating the potential existence of
JAK-STAT-independent pathways in IFN-
action (20-22). For example,
although IFN-
induces major histocompatibility complex class II and
intracellular adhesion molecule-I (ICAM-I) gene expression in human
corneal epithelial cells, incubation of the cells with a global protein
tyrosine kinase inhibitor prior to IFN-
prevents the expression of
major histocompatibility complex class II but not ICAM-1, thus
inferring that ICAM-1 transcription is activated in this instance
through a JAK-independent pathway (93).
was
shown to modulate ICER mRNA expression, we also decided to
investigate whether there was a concominant CREB phosphorylation.
Indeed, an increased level of Ser-133-phosphorylated CREB was found
when the cells were incubated with IFN-
and this was inhibited in
the presence of apigenin (Figs. 5 and 8). However, CK2 is not currently
thought to phosphorylate CREB at Ser-133 but instead at five closely
spaced sites (Ser-108, Ser-111, Ser-114, Ser-117, and Thr-119) (1, 2,
94). Nevertheless, given the general paucity of research in this
particular area, it is entirely possible that this situation does not
occur in macrophages, and that CK2 can indeed directly phosphorylate
CREB at Ser-133. Alternatively, because of co-operation between the
numerous consensus protein kinase sites that exist in the
"phosphorylation P-box" domain of CREB (1, 2), it is possible that
a CK2-mediated phosphorylation may initiate a processive
phosphorylation cascade, catalyzed by kinases other than CK2, that
potentially results in Ser-133 phosphorylation. Although the precise
"CREB kinase" responsible for Ser-133 phosphorylation remains to be
identified, classical kinases, such as cAMP-dependent
protein kinase, protein kinase C , and
Ca2+-calmodulin-dependent protein kinases, can
be excluded (Fig. 6). In this respect, it is interesting to note that
basic fibroblast growth factor has recently been shown to activate a
novel 120-kDa CREB kinase during differentiation of immortalized
hippocampal cells, which then triggers the phosphorylation of Ser-133
in CREB (46).
(Fig. 9). A synergism between
Ca2+ and the ICER inducer cAMP has been seen previously in
the regulation of lysozyme gene expression in chicken myelomonocytic
HD11 cell line, c-fos in the corticotroph cell line AtT20,
and apoptosis of WEH17.2 cells (53, 54, 57). Interestingly, the
synergism in relation to the apoptosis of WEH17.2 cells and the
induction of c-fos expression has been demonstrated to be
the result of the convergence of the protein kinase A and
Ca2+ signaling pathways, via activation of
calcium-calmodulin kinase IV, at CREB phosphorylation on Ser-133 (54,
57). Extrapolating from these findings, it is thus likely that the
synergism between IFN-
and Ca2+ is the result of
increased CREB phosphorylation modulated by the convergence of
IFN-
-induced CK2 activity and thapsigargin/A23187-induced calcium-calmodulin kinase activity.
that is mediated through the activation of
CK2 and CREB. Future studies should seek to identify all the components
from the interaction of IFN-
to its cell-surface receptors to the
activation of ICER gene transcription, and the potential role of this
potent inhibitor in the suppression of downstream genes by this cytokine.
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ACKNOWLEDGEMENT |
---|
We thank Maurice Hallett for help in the determination of intracellular Ca2+ levels.
![]() |
FOOTNOTES |
---|
* This work was supported by the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel./Fax:
44-29-20876753; E-mail: ramji@cardiff.ac.uk.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M301602200
2 J. R. Mead and D. P. Ramji, unpublished observations.
3 J. R. Mead and D. P. Ramji, unpublished data.
4 J. R. Mead and D. P. Ramji, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
CREB, cAMP-response
element-binding protein;
8-Br-cAMP, 8-bromo-cAMP;
ATF, activating
transcription factor;
AP, activator protein;
bZIP, basic leucine
zipper;
C/EBP, CCAAT/enhancer-binding protein;
CRE, cAMP-response
element;
CREM, cAMP-response element modulator protein;
CK2, casein
kinase 2;
DB, DNA-binding domain;
DTT, dithiothreitol;
EMSA, electrophoretic mobility shift assay;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
ICAM, intracellular adhesion molecule;
ICER, inducible
cAMP early repressor;
IFN-, interferon-
;
IL, interleukin;
JAK, Janus kinase;
LPL, lipoprotein lipase;
LPS, lipopolysaccharide;
MAP, mitogen-activated protein;
NGF, nerve growth factor;
PBS, phosphate-buffered saline;
PMA, phorbol 12-myristate 13-acetate;
RT, reverse transcription;
SOCS, suppressor of cytokine signaling;
STAT, signal transducers and activators of transcription;
TBS, Tris-buffered
saline.
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