From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proliferating human medullary thymocytes can
exhibit characteristic T helper cell type 1 cytokine responses
exemplified by the immediate early expression of interleukin-2,
interferon-, tumor necrosis factor-
, and lymphotoxin-
. Here we
report that cAMP-mediated attenuation of the transcription of T
helper-1-specific cytokine genes in human medullary thymocytes
correlates with the induction of the cAMP-mediated transcriptional
repressor ICER (inducible cAMP early repressor). We show that ICER
binds specifically to several NFAT/AP-1 (nuclear factor of activated T
cells/activating protein-1) composite DNA sites essential for the
activation of the interleukin (IL)-2 promoter as well as to a
homologous DNA motif present in the proximal segment of the
interferon-
promoter. In the presence of the minimal NFAT
DNA-binding domain, which is sufficient for both DNA binding and AP-1
complex formation, ICER and NFAT form NFAT/ICER ternary complexes on
several NFAT/AP-1 DNA composite sites previously identified as
essential for the expression of the immunoregulatory cytokines such as
IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, and tumor
necrosis factor-
. In extracts prepared from human medullary
thymocytes treated with forskolin and ionomycin, these composite sites
bind endogenously expressed ICER either singly or in complexes.
Moreover, in Jurkat cells, ectopically expressed ICER represses
transcription from NFAT-mediated, phorbol ester/ionophore-activated
IL-2, granulocyte-macrophage colony-stimulating factor, and tumor
necrosis factor-
promoters. We present evidence that ICER
interactions with NFAT/AP-1 composite DNA sites correlate with
its ability to repress transcription. These findings provide further
insight into the mechanisms involved in cAMP-mediated
transcriptional attenuation of cytokine expression.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well established that cAMP signaling is inhibitory to T cell proliferation and effector functions. In particular, cAMP inhibits the expression of T helper-1 cytokine genes (1-3). Earlier reported studies of fibroblasts showed that elevated levels of intracellular cAMP inhibit upstream signal transduction pathways involved in cell growth and differentiation (4, 5). In contrast to fibroblasts, in which elevated levels of intracellular cAMP inhibit extracellular signal-regulated kinases 1 and 2 and c-Jun NH2-terminal kinases involved in the signal transduction of mitogen-activated protein kinase pathways, T cell extracellular signal-regulated kinases 1 and 2 are insensitive to elevated levels of intracellular cAMP (6). Moreover, the cAMP-mediated inhibition of c-Jun NH2-terminal kinase in T cells shows delayed kinetics, an observation that correlates with the induction of the cAMP-inducible early repressor ICER1 (7). In addition, overexpression of NFAT achieved by transfection of NFAT-encoding cDNAs to lymphoma cells abrogates the sensitivity of cAMP-mediated inhibition of IL-2 gene expression (8, 9). Importantly, phosphorylation of amino-terminal serines of NFAT by protein kinase A does not prevent calcineurin-mediated translocation of NFAT to the nucleus, despite its ability to prevent IL-2 gene expression (10, 11). The notion that a newly synthesized transcriptional repressor rather than inhibition of upstream signal transduction pathways could be involved in the cAMP-mediated transcriptional attenuation of T helper-1 cytokine expression was further strengthened by the reported alleviation of cAMP-mediated inhibition of IL-2 expression in the presence of inhibitors of both RNA and protein synthesis (6).
ICER is a transcriptional repressor that appears to serve as a generalized negative regulator of the CREB and CREM families of transcription factors as well as other related bZIP family members (12-15). ICER isoforms represent a unique cAMP-inducible CREM subfamily of transcription factors containing cAMP-response elements within an internal P2 promoter. Because of autoregulation of the cAMP-inducible P2 promoter, the expression of ICER can be intrinsically rhythmical. The rhythmical expression of ICER was first described in the pineal gland and in the hypothalamic-pituitary-gonadal axis (16, 17). However, the P2 promoter of ICER is also inducible in organs other than the pineal and hypothalamic-pituitary gonadal axis such as in specific subsets of T lymphocytes including human medullary thymocytes (7). Importantly, in the Jurkat T cell line ectopically expressed ICER can substitute for the inhibitory effects of cAMP on the transcriptional attenuation of IL-2 promoter activity (7).
The NFAT (nuclear factor of
activated T cells) and AP-1
(activating protein 1) represent two major
transcription factor families implicated in the transcription of the
IL-2 promoter in proliferating T lymphocytes (18-20). To address the
possible mechanism by which ICER down-regulates IL-2 gene expression,
we examined the binding of bacterially expressed ICER to all five NFAT
motifs of the IL-2 promoter reported to be essential for the full
induction of the IL-2 gene (21) either alone or in the presence of the
minimal DNA-binding domain of NFAT (NFAT DBD). The highest affinity of ICER binding was found on a CD28-responsive element (CD28RE; 160 NFAT/AP-1 composite site) and
90 site, which is the motif in the IL-2
promoter that has striking sequence homology with the conserved
proximal region (base pairs
73 to
48) of both the human and mouse
promoters of the IFN
genes (21, 22).
Moreover, certain NFAT/AP-1 composite sites that reside within the
IL-4, GM-CSF, and TNF- promoters resemble those located within the
IL-2 promoter (23-27). It is believed that the mechanism underlying
the actions of NFAT requires the binding of NFAT and/or NFAT/AP-1 to
the NFAT/AP-1 composite binding motifs as ternary complexes (18). These
complexes are believed to be essential for the transcriptional
expression of immunoregulatory cytokines during T cell proliferation,
such as IL-2, IL-4, GM-CSF, and TNF-
(19). Here we demonstrate that
ICER binds to these NFAT/AP-1 composite DNA sites in vitro,
either directly or indirectly via complex formation with the rel
homology region of NFAT (NFAT DBD). Furthermore, we detect the
induction of ICER-immunoreactive complexes in extracts prepared from
human medullary thymocytes treated with forskolin and ionomycin.
Ectopically expressed ICER represses transcription from the IL-2,
GM-CSF, and TNF-
promoters activated by ionomycin and phorbol ester,
suggesting that the induction of ICER in response to cAMP may be
responsible for the observed cAMP-mediated transcriptional attenuation
of T helper-1 cytokine responses.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Human Medullary Thymocytes-- Human thymus glands were obtained from children (ages 3 months to 4 years) undergoing corrective cardiac surgery. Thymocytes were fractionated over discontinuous Percoll gradients (Amersham Pharmacia Biotech) (28). Cells with densities of 1.060 < o < 1.070 and p > 1.070 were collected and classified into large (significantly enriched for medullary thymocytes) and small (cortical) thymocytes according to an established protocol (7). Separated human thymocytes were maintained in short term cultures in RPMI 1640 medium, supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 25 µg/ml streptomycin, and treated as indicated.
RNase Protection Analysis--
RNA extraction was performed as
described (Qiagen). RNA probes hCK1 and hCK3 were purchased from
Pharmingen and labeled with [-32P]CTP using reagents
from an RNA probe kit (Ambion). These probes were used for RNase
protection studies according to the protocol provided by Ambion (RPAII
ribonuclease protection assay kit).
Western Blot Analysis-- Separation of whole cell proteins (50 µg) was carried out by SDS-polyacrylamide gel electrophoresis (10%) for 2 h at 40 mA in Tris/glycine buffer (25 mM Tris, 250 mM glycine, and 0.1% SDS) at room temperature. The proteins were electrotransferred (0.4 A) overnight at 4 °C in 10 mM Tris/glycine buffer with 12% methanol on Immobilon P membrane (Millipore Corp.). The membrane was blocked in a Tris-buffered saline solution containing 0.05% Tween 20 (Sigma) and 5% nonfat dry milk (Bio-Rad) for 1 h with gentle agitation at room temperature. For immunological detection, the same solution without dry milk but containing the ICER or CREM-specific antiserum (CS4) diluted 1:10,000 was agitated for 1 h, followed by three washes, with subsequent incubation with horseradish peroxidase conjugated to a secondary antibody (Amersham Pharmacia Biotech) diluted 1:5000 for 1 h, followed by nine washes, and finally developed using an ECL kit (Amersham Pharmacia Biotech).
Expression and Purification of Recombinant Proteins-- Human ICER II cDNA was subcloned into the pGEXKG vector (Amersham Pharmacia Biotech) and expressed in bacteria as a glutathione S-transferase (GST) fusion protein. The pGSTagCREB construct was described previously (29). Purifications of both ICER and CREB were carried out with minor modifications according to the protocol previously established for CREB (29). NFATpXS-(1-187), encompassing the minimal DNA-biding domain of NFATp (gift from Dr. A. Rao), was expressed in bacteria as a hexahistidine-tagged protein and purified as reported previously (30). Recombinant c-Fos (Fos-(139-243)) and c-Jun (Jun-(187-334)) (gift from Dr. T. Kerppola) were purified from Escherichia coli overexpression strains by nickel chelate affinity chromatography (31, 32).
Nuclear Extracts and Gel Mobility Shift Assay--
Whole cell
extracts were prepared by high salt extraction using 50 mM
Hepes, 250 mM NaCl, 5 mM EDTA, 0.5 mM dithiothreitol, and protease inhibitors (20 µM leupeptin, 10 µg/ml aprotinin, 2 mM
phenylmethylsulfonyl fluoride) as described previously (30). Binding
reactions were performed in a 15-µl reaction volume containing 20 mM HEPES, 1 mM MgCl2, 50 mM KCl, 12% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 µg of poly(dI-dC) as an unspecific competitor and recombinant proteins or whole cells extracts as indicated. 32P-Labeled oligonucleotides and, where
indicated, unlabeled competitor oligonucleotides in excess were added
and incubated for 10 min at room temperature. Samples were run on a 4%
polyacrylamide gel in 0.5× TBE at 200 V for 2 h following a 2-h
prerun at 4 °C. The dried gels were exposed for autoradiography
overnight. The oligonucleotides encompassing NFAT composite sites of
the following human promoters were used: IL-2 (45),
5'-ctagaCATTTTGACACCCCCATAATATTTTTCCAGAATTa-3'; IL-2 (
90),
5'-ctagaGTCTTTGAAAATATGTGTAATATGTAAAACATa-3'; IL-2 (
135),
5'-ctagaATCAGAAGAGGAAAAATGAAGGTAATGTTTTa-3'; IL-2 (
160), 5'-ctagaAAAGAATTCCAAAGAGTCATCAGAAa-3'; IL-2 (
280),
5'-ctagaAAGAAAGGAGGAAAAACTGTTTCATACAGa-3'; GM-CSF (
330),
5'-gatccCCCCATCGGAGCCCCTGAGTCAGCATGGa-3'; GM-CSF (
420),
5'-gatccCATCTTTCTCATGGAAAGATGACATCAGGGAa-3'; GM-CSF (
550), 5'-gatccGAAAGGAGGAAAGCAAGAGTCATAATAAGAa-3'; IL-4 (
80),
5'-gatccTAACTGCAATCTGGTGTAATAAAATTTTCCAATGTAAACTCATa-3'; TNF-
(
95), 5'-gatccTTCCTCCAGATGAGCTCATGGGTTTCTCCACGACGGAa-3'. Lowercase letters indicate overhangs for
SpeI/XbaI recognition sites (IL-2) or
BamHI/BglII recognition sites (GM-CSF, IL-4,
TNF-
). As competitors, the following oligonucleotides were used: nf
(mouse IL-4 NFAT, positions
69 to
79), 5'-ATAAAATTTTCCAATGTAAA-3'; ap (human metallothionein IIA AP-1 site, positions
114 to
88), 5'-GAGCCGCAAGTG ACTCAGCGCGGGGCG-3'; and cre (mouse c-fos
gene oligonucleotide, surrounding CRE site in position
60)
5'-gatccCAGTTCCGCCCAGTGACGTAGGAAGTCCATCa-3'. Lowercase letters
indicate overhangs for BamHI/BglII recognition sites.
GST Pull-down Assays-- GST-ICER (GST-CREB) Sepharose beads prepared as described above were diluted 1:10 in 50 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10 mM Na3 (PO4)2, 10 mM NaF, 0.1% Triton X-100, 2.5 mM leupeptin, 20 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotinin, and recombinant protein(s) were added to a final volume of 250 µl and incubated at 4 °C on a nutator for 1.5 h. The beads were then washed three times with the same buffer, resuspended directly in Laemmli buffer, and loaded on 10% SDS-polyacrylamide gel. Retained NFAT DBD protein was visualized by Western blotting described below using R59 anti-NFAT DBD antiserum.
Transient Overexpression in Jurkat T Cells--
Transfection
assays were performed by the DEAE-dextran technique. Typically,
107 Jurkat cells were transfected with 2 µg of the
reporter and the same amount of ICER expression vector and treated
18 h posttransfection with phorbol ester (105 mg/ml)
and ionomycin (1 µg/ml) for 48 h. Luciferase and chloramphenicol acetyltransferase assays and quantification methods are described elsewhere (Promega; Ref. 33). The percentage of conversion of [14C]chloramphenicol to its acetylated forms was
quantified using ImageQuant (Molecular Dynamics).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cAMP-mediated Transcriptional Attenuation of T Helper-1-responsive
Cytokine Genes in Human Medullary Thymocytes--
Under the
experimental conditions used, human medullary thymocytes exhibited the
characteristics of naive T helper-0 and T helper-1 cells with
predominant IL-2 and IFN expression documented by RNase protection
used for the evaluation of mRNA levels of multiple cytokines (IL-2,
IL-4, IL-5, IL-9, IL-10, IL-13, IL-14, IL-15, interferon-
, IFN
,
human migration inhibitory factor, TNF-
, TNF-
, Lt
, and
transforming growth factors
1,
2, and
3) (Fig.
1). Stimulation of medullary thymocytes
with a combination of phorbol ester and ionomycin significantly induced
the synthesis of mRNAs encoding IL-2, IFN
and to a lesser extent
also TNF-
and Lt
(Fig. 1A, lane 4; Fig.
1B, lane 4). At the same time, cotreatment with
forskolin or 8-bromo-cAMP reduced the cellular mRNA levels of IL-2,
IFN
, TNF-
, and Lt
(Fig. 1A, lanes 5 and 6; Fig. 1B, lanes 5 and 6).
We propose that at least part of this transcriptional attenuation is
based on cAMP-mediated expression of the transcriptional repressor ICER
and a subsequent blockade of NFAT/AP-1 composite DNA sites essential
for T helper-1 cytokine expression. The inhibition by ICER may occur
either directly through binding to the DNA element or indirectly via
protein-protein interactions such as to the rel homology domain of NFAT
(NFAT DBD).
|
Cyclic AMP-mediated Attenuation of T Helper-1 Cytokine
Transcription in Human Medullary Thymocytes Correlates with
cAMP-mediated Induction of the Transcriptional Repressor ICER--
To
test the proposed mechanism involved, we first sought evidence for the
presence of ICER protein in human medullary thymocytes after forskolin
treatment to explore whether ICER may interact with important NFAT/AP-1
enhancer motifs of the IL-2 and IFN promoters. Western
immunoblotting analysis using an ICER-specific antiserum (7) confirmed
that ICER protein is indeed detectable in human medullary thymocytes
but is absent in cortical thymocytes after 3 h of forskolin
treatment (Fig. 1C). This finding is in agreement with the
previously observed delayed appearance of ICER mRNA in medullary
thymocytes after their exposure to forskolin and its subsequent
deinduction (7). These observations suggest that the cAMP-mediated
inhibition of cytokine expression may occur in a stage-specific manner
in cells of T cell lineage.
ICER Binds to NFAT/AP-1 Composite Sites of IL-2 Promoter--
To
further address the possible mechanism by which ICER down-regulates
IL-2 gene expression, we examined the binding of bacterially expressed
ICER to all five NFAT motifs of the IL-2 promoter reported to be
essential for the full induction of the IL-2 gene (21) (Fig.
2A). Four out of five NFAT
sites in positions 90,
135,
160, and
280 were previously
characterized as NFAT/AP-1 composite sites due to their inherent
ability to bind to the NFAT/AP-1 complex in a cooperative fashion (34).
The fifth NFAT site in the most proximal position, NFAT
45, does not
bind to the NFAT/AP-1 complex and was determined to be exclusively an
NFAT binding site (34). Bacterially expressed ICER, as well as ICER
expressed in COS cells (data not shown), binds to all five NFAT sites,
although to different extents (Fig. 2B). The strongest
binding was observed to the NFAT/AP-1 composite sites in positions
90
and
160, an intermediate binding to the
135 NFAT/AP-1 composite
site, and weak binding to the most proximal and distal NFAT and
NFAT/AP-1 sites in positions
45 and
280, respectively. The binding
specificity of recombinant ICER was evaluated using a CREM-specific
antiserum (CS4) that "supershifts" ICER bound to specific
oligonucleotides containing individually the five DNA motifs, leaving
nonspecific binding unaffected (Fig. 2B, lanes
1-10) as well as by using control oligonucleotide encompassing
the first 21-base pair repeat (H21) of the human T-cell
lymphotrophic virus type I long terminal repeat promoter containing the
CRE-like motif (35) (Fig. 2B, lanes 11 and
12).
|
ICER Binding to NFAT/AP-1 Composite Sites in the Presence of NFAT
DBD--
To better understand the role of the interactions of ICER
with NFAT/AP-1 composite DNA motifs, we examined the effectiveness of
ICER binding to the DNA motifs in the presence of NFAT DBD. This domain
of NFAT, which has the highest degree of conservation among NFAT family
members (36), is both necessary and sufficient for DNA binding as well
as to associate AP-1 with NFAT (30). All NFAT motifs on the IL-2
promoter examined, with the exception of the NFAT/AP-1 composite site
in position 90, bound NFAT efficiently (Fig. 2C). The most
pronounced ability of NFAT to associate with ICER was observed on the
sites with the highest DNA binding efficiencies to both proteins,
particularly at site position
160 (CD28RE), and to a lesser extent to
the NFAT site in position
45. Interestingly, this NFAT
45 site
tends to show a stronger binding of ICER to NFAT in the NFAT/ICER
complex compared with ICER by itself (Fig. 2C, lanes
1-3). Because the NFAT
45 of the IL-2 promoter is the only one
of the five sites examined without an adjacent AP-1 site, the finding
that this site can form an NFAT/ICER complex suggests that ICER itself
may tether to NFAT by a protein-protein interaction in addition to a
protein-DNA action.
ICER Can Interact Directly with NFAT DBD--
To examine whether
or not ICER and NFAT may directly interact in the absence of DNA, GST
pull-down assays were performed using a GST-ICER fusion protein linked
to a Sepharose matrix in the presence of a truncated NFAT consisting of
the NFAT DBD (Fig. 3, lane 1).
GST-ICER formed a complex with NFAT DBD as demonstrated by retention of
NFAT DBD. Interestingly, GST-CREB failed to associate with NFAT DBD
under the same conditions (Fig. 3, lane 10). To examine the
specificity of both components in the NFAT/ICER ternary complex found
on the CD28RE (160 motif) of the IL-2 promoter, we used both
CREM-specific antiserum (CS4) and antiserum raised against the minimal
DNA-binding domain of NFAT (R59). Both antisera either prevented or
reduced the amount of the NFAT/ICER complex (Fig. 2D,
lanes 4 and 5, respectively). Competition with
unlabeled oligonucleotides containing NFAT (nf oligonucleotide spanning mouse IL-4 NFAT, positions
69 to
79) or the AP-1 motif (ap
oligonucleotide spanning the human metallothionein IIA AP-1 site,
positions
114 to
88) eliminated the NFAT/ICER complex (Fig.
2D, lanes 6 and 7, respectively),
suggesting that both ICER and NFAT are essential components of the
observed complex.
|
NFAT/ICER Ternary Complex Shows Distinct Features in Comparison
with NFAT/AP-1 Complex on Various NFAT Motifs of IL-2
Promoter--
Analogous experiments performed with the NFAT DBD and
truncated forms of Fos and Jun confirmed in vitro that both
NFAT/AP-1 and NFAT/ICER complexes could exist in the context of the
CD28RE (160 NFAT/AP-1 composite site; Fig. 2D, lanes
8-14). Interestingly, in the presence of NFAT DBD, the majority
of ICER protein was retained in the complex with NFAT DBD, although
NFAT/ICER and NFAT/AP-1 complexes appear to show unequal binding
affinities to different NFAT/AP-1 motifs. For example, the
280 motif
of the IL-2 promoter, known from NFAT studies as the principal site for
binding of the NFAT/AP-1 complex, binds NFAT/AP-1 complexes effectively
(32, 37), showing little or no detectable formation of an NFAT/ICER
complex (Fig. 2C, lanes 13-15). In contrast, the CD28RE (
160 NFAT/AP-1 composite site) of human IL-2 promoter creates
a ternary NFAT/ICER complex with an equal or slightly higher efficiency
than the NFAT/AP-1 complex (Fig. 2D).
ICER Binds Directly to the Conserved Proximal Motif of IFN
Promoter--
Unlike the CD28RE (
160 motif), which shows equally
high affinity for both ICER and NFAT, the NFAT/AP-1 motif of the IL-2 promoter in position
90, which has striking homology to the conserved proximal element of the INF
promoter, does not interact with NFAT
DBD (Fig. 4). Studies performed on the
conserved proximal motifs of both human and mouse IFN
promoters
demonstrated high affinity ICER binding and a lack of NFAT binding or
NFAT/ICER complex formation in the presence of NFAT/DBD (Fig. 4), a
situation similar to that observed on the homologous
90 motif of the
IL-2 promoter (Fig. 2).
|
NFAT/AP-1 Composite Sites in the Context of the GM-CSF, IL-4, and
TNF- Promoters Bind ICER either Alone or in
Complexes--
NFAT/AP-1 binding sites have been shown previously to
be essential for the efficient activation of the GM-CSF, IL-4, and
TNF-
promoters. Therefore, we examined the binding to these sites of ICER and NFAT, both as purified recombinant proteins and in extracts prepared from human medullary thymocytes treated with forskolin and
ionomycin (Fig. 5). These studies
demonstrated that ICER can bind either by itself or in complexes with
NFAT DBD to these composite sites in the promoters of the GM-CSF, IL-4,
and TNF-
promoters, similar to the experiments using the binding
site motifs of the IL-2 and IFN
promoters.
|
Ectopically Expressed ICER Represses NFAT-mediated Activation of
IL-2, GM-CSF, and TNF- Promoters--
To determine whether ICER
expression could supplant the effect of forskolin in transcriptional
attenuation of various cytokine promoters observed in medullary
thymocytes, ICER (isoform II) was expressed in Jurkat T cells in
transient transfection assays. Expression of ICER down-regulated the
human IL-2, GM-CSF, and TNF-
promoters activated by the combined
treatment of the cells with PMA and ionomycin, whereas ectopic
expression of neither isoform of ICER did not prove to have any
significant effect on VP16-mediated transactivation of (3×
GAL4)-CR-CAT under the same conditions (Fig.
6). Thus, ICER can be induced by, and
substituted for, forskolin in the transcriptional down-regulation of
the calcineurin-dependent, NFAT/AP-1-mediated
transactivation of IL-2, GM-CSF, and TNF-
promoters when induced by
PMA and ionomycin.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism of cAMP-mediated inhibition of cytokine expression in proliferating T lymphocytes has been attributed to cAMP-mediated inactivation of upstream signal transduction pathways directing the proliferation of T lymphocytes. Although this hypothesis was supported by studies of fibroblasts (5), it was not confirmed by studies of T cells (6). Surprisingly, several protein kinases required for T cell proliferation were found to be insensitive or to exhibit a delayed response to high levels of intracellular cAMP (6). Here we provide evidence that in human medullary thymocytes expression of the transcriptional repressor ICER correlates with a delayed cAMP-mediated transcriptional attenuation of T helper-1 cytokine responses.
Footprinting and electrophoretic mobility shift analysis of the IL-2
promoter revealed (34) that the originally defined AP-1 site at
position 150 of the IL-2 promoter (37, 38), a major CD28RE that
contains an upstream NFAT binding site (39, 40), represents a novel
NFAT/AP-1 composite site at position
160 (34). A reexamination of the
original observations in which NF-
B was identified as a major
component of the complex (41, 42) determined that NFAT is a prevalent
component of the complex that binds the CD28RE in vivo (34,
43). Our findings indicate that the CD28RE (
160 composite site of
IL-2 promoter) effectively binds ICER either alone or in an NFAT/ICER
complex. These findings may be important for obtaining a better
understanding of a direct cAMP-mediated transcriptional attenuation of
IL-2 expression. In addition, a potential indirect role of ICER has
been demonstrated in transgenic mice overexpressing the dominant
negative CREB mutant (a functional homologue of ICER), which impairs
the expression of IL-2 in thymocytes (44). Since the induction of IL-2
and IFN
expression is dependent on the activity conferred by each of
the individual DNA motifs (22, 34), a demonstration of a direct binding
of ICER and/or the formation of an inhibitory NFAT/ICER complex on any
of these NFAT/AP-1 composite sites could provide an explanation for the
mechanism involved in the transcriptional attenuation of IL-2 and
IFN
expression mediated by cAMP. These findings correlate with
observations in which the conserved proximal motif of the IFN
promoter was reported to be inhibited by forskolin in proliferating
thymocytes of mice made transgenic with an IFN
promoter-luciferase
reporter gene (45). These findings further suggest that both NFAT/AP-1
motifs, either those that directly bind ICER or those that form
NFAT/ICER complexes, could convey ICER-mediated transcriptional
attenuation.
It appears that numerous NFAT/AP-1 composite sites previously
identified in the context of GM-CSF, IL-4, and TNF- promoters as
essential determinants of their expression (23, 25, 27) can associate
with ICER. This property does not seem to be a universal feature shared
by all NFAT/AP-1 composite sites tested because we find that relatively
minor differences in DNA sequences have a profound effect on both the
binding of ICER and the formation of NFAT/ICER complexes. An example of
this circumstance is represented by the GM-CSF promoter in which the GM
420 element shows binding for ICER and NFAT/ICER complexes, whereas
both neighboring motifs GM
330 and GM
550 show only a modest
binding of ICER and/or formation of NFAT/ICER complexes. The strong
binding of ICER to the GM
420 element, previously defined by deletion
analysis as the essential core of the GM-CSF enhancer (25), suggests
that ICER may play an important role in transcriptional attenuation of
GM-CSF expression. Similar binding studies performed with several NFAT/AP-1 composite sites important in the context of IL-4 and TNF-
promoters show that these sites, previously shown to be essential for
efficient expression (23, 26, 27), bind ICER either alone or in
complexes similarly to the motifs of the IL-2 and IFN
promoters. It
remains to be determined whether the induction of ICER can selectively
modulate T helper-1 versus T helper-2 cytokine expression in
peripheral blood T lymphocytes.
We have reported previously that human medullary but not cortical
thymocytes synthesize ICER mRNA after 3 h of forskolin
treatment (7). Western immunoblot analysis using an ICER-specific
antiserum confirmed that in these conditions the ICER mRNA is
translated efficiently into ICER protein. Moreover, endogenously
expressed ICER protein was detected in extracts prepared from human
medullary thymocytes treated with forskolin and ionomycin using
oligonucleotide probes containing NFAT/AP-1 DNA motifs that are able to
form NFAT/ICER complexes in vitro. In contrast to
bacterially expressed ICER, endogenously expressed ICER in medullary
thymocytes shows an altered mobility in gel shift assays, suggesting
that posttranslational modification(s) may be involved in the
regulation of the binding properties of ICER and/or degradative
pathways involved in its proteolysis in vivo. The
ICER-containing complexes that are immunoreactive to ICER-supershifting
antisera are efficiently competed by oligonucleotides containing CRE or
NFAT motifs. NFAT antisera that are unable to recognize directly bound
ICER still affect the mobility of ICER-containing complexes, suggesting
the possibility of the formation of NFAT/ICER complexes in
vivo. The ambiguity of the DNA-protein complexes in extracts of
thymocytes observed on gel shift assays may be due to posttranslational
modifications of the proteins involved (data not shown) and/or their
potential consequences for DNA binding. At this point, the possibility
cannot be excluded that proteins other than ICER and NFAT that contain
homologous bZIP or rel homology regions may also participate in the
formation of ICER-containing complexes. Finally, ectopic
expression of ICER in Jurkat cells demonstrates that ICER, in agreement
with its binding capabilities, can also effectively inhibit
NFAT-mediated, phorbol ester/ionophore-induced expression of IL-2,
GM-CSF, and TNF- promoters.
In conclusion, inducible ICER expression in developing human medullary thymocytes as well as in certain subset(s) of human peripheral blood lymphocytes (7) and monocytes (work in progress) could significantly influence their respective effector functions(s). The proposed inhibitory effects on effector function of the immune system mediated by ICER may be related to its ability to bind (mask) a wide range of CRE and AP-1 motifs and/or its ability to inactivate certain transcription complexes via protein-protein interactions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Rao for the generous gift of truncated NFAT DBD proteins and NFAT antibodies; Dr. T. K. Kerppola for the gift of truncated Fos and Jun proteins; Drs. J. L. Strominger, David Weng, Peter A. Cohen, K. Shaw, P. McCaffrey, Cathy Bare, and Ronald E. Gress for stimulating discussions; E. Burgeon for expert advice on GST pull-down assays; and T. Budde for manuscript preparation.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK 25532 (to J. F. H.).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. Present address:
Experimental Immunology Branch, Division of Basic Sciences, National Cancer Institute, Bldg. 10, R4B14, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892-1360. Tel.: 301-496-6898; Fax: 301-496-0887; E-mail: Bodorj{at}exchange.nih.gov.
§ An Investigator with the Howard Hughes Medical Institute.
1
The abbreviations used are: ICER, inducible cAMP
early repressor; AP-1, activating protein 1; bZIP, basic region/leucine
zipper; CRE, cAMP-response element; CREB, CRE-binding protein; CREM,
CRE modulator protein; GM-CSF, granulocyte-monocyte colony-stimulating factor; IFN, interferon-
; IL, interleukin, NFAT, nuclear factor of activated T cells; NFAT DBD; DNA binding domain of NFAT; PMA, 12-O-tetradecanoylphorbol 13-acetate; GST, glutathione
S-transferase; TNF, tumor necrosis factor; CD28RE, CD28-responsive
element; CAT, chloramphenicol acetyltransferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|