From the Department of Biology, § College
of Pharmacy, ** Department of Microbiology,
Hormone Research Center, and
§§ Center for Ligand and Transcription, Chonnam
National University, Kwangju 500-757, Korea and the
Wistar
Institute, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Lipopolysaccharide (LPS) increases the production
of interleukin-12 (IL-12) from mouse macrophages via a Interleukin-12
(IL-12),1 a heterodimeric
cytokine composed of two disulfide-linked subunits of 35 (p35) and 40 (p40) kDa encoded by two separate genes, was originally identified in
the supernatant fluid of Epstein-Barr virus-transformed human B cell
lines (1, 2). IL-12 is produced by phagocytic cells and other
antigen-presenting cells in response to stimulation by a variety of
microorganisms as well as their products (3, 4). IL-12 exerts multiple biological activities mainly through T and natural killer cells by
inducing their production of interferon- The transcription factor NF The nuclear receptor superfamily is a group of
ligand-dependent transcriptional regulatory proteins that
function by binding to specific DNA sequences named hormone response
elements in the promoters of target genes (reviewed in Ref. 17). The
superfamily includes receptors for a variety of small hydrophobic
ligands such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands, referred to as
orphan nuclear receptors. In particular, at least six distinct receptors have been extensively characterized for retinoids; retinoic acid receptor (RAR) Interestingly, members of steroid receptors including glucocorticoid
receptor (28, 29), estrogen receptor (30, 31), progesterone receptor
(32), and androgen receptor (33), have been shown to inhibit NF In this report, we found that retinoids inhibit the LPS-stimulated
production of IL-12 from mouse macrophages through direct physical
interactions of retinoid receptors with NF Mice, Cell Lines, Culture Medium, and Transient
Transfection--
Female DBA/2 mice were obtained from Japan SLC, Inc.
(Tokyo, Japan) and used at 6-10 weeks of age. RAW264.7 cells were
cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS)
at 37 °C in a 5% CO2 humidified air atmosphere. CV-1
cells as well as spleen cell populations and macrophages from mice were
maintained in Dulbecco's modified Eagle's medium (DMEM) containing
10% FBS and antibiotics (Life Technologies, Inc.). For transfections, cells were grown in 24-well plates with medium supplemented with 10%
FBS for 24 h and transfected with indicated plasmid in the presence of Superfect according to the manufacturer's protocol (Qiagen). After 12 h, cells were washed and refed with DMEM
containing 10% FBS. Cells were harvested 24 h later, luciferase
activity was assayed as described (34), and the results were normalized to LacZ expression. Similar results were obtained in more
than two separate experiments.
Monoclonal Antibodies (mAbs), Cytokines, and
Reagents--
Anti-IL-12 p40 mAbs C17.8 and C15.6 were purified from
ascitic fluid by ammonium sulfate precipitation followed by
DEAE-Sephagel chromatography (Sigma). Anti-IL-12 p35 mAb
Red-T/G297-289, anti-IL-10 mAbs JES-2A5 and SXC-1, as well as
recombinant mIL-10 were obtained from PharMingen (San Diego, CA).
Recombinant murine IL-12 was generously provided by Dr. Stanley Wolf
(Genetics Institute, Cambridge, MA). Retinoids (9-cis-RA,
TTNPB, and LG69) and LPS (from E. coli 0111:B4) were
purchased from Sigma.
Plasmids--
The Preparation of Splenic Macrophages Stimulated with
LPS--
Spleen cells were cultured at 106 cells/ml for
approximately 3 h at 37 °C. The non-adherent cells were removed
by washing with warm DMEM until visual inspection revealed a lack of
lymphocytes (>98% of the cell population). The adherent cells were
removed from plates by incubating for 15 min with ice-cold
phosphate-buffered saline solution and rinsing repeatedly. The isolated
adherent cell population was stimulated with 5 µg/ml LPS in the
absence or presence of retinoids at 10 Cytokine Assays--
The quantities of IL-12 p40, IL-12 p70, and
IL-10 in culture supernatants were determined by a sandwich
enzyme-linked immunosorbent assay using mAbs specific for each
cytokine, as described previously (36). The mAbs for coating the plates
and the biotinylated second mAbs were as follows: for IL-12 p40, C17.8
and C15.6; for IL-12 p70, Red-T/G297-289 and C17.8; for IL-10, JES-2A5
and SXC-1. Standard curves were generated using recombinant cytokines.
The lower limit of detection was 30 pg/ml for IL-12 p40, 50 pg/ml for
IL-12 p70, and 0.2 ng/ml for IL-10.
Electrophoretic Mobility Shift Assay--
The nuclear extracts
were prepared from the cells, as described previously (37). An
oligonucleotide containing an NF Statistical Analysis--
Student's t test was used
to determine the statistical differences between various experimental
and control groups. A p value of <0.01 was considered as significant.
Retinoids Inhibit IL-12 Production from LPS-activated
Macrophages--
We examined the effect of various retinoids including
9-cis-RA, TTNPB, and LG69 on the production of IL-12 by
primary macrophages stimulated with LPS. 9-cis-RA is a
pan-agonist for both RARs and RXRs, whereas TTNPB and LG69 are specific
agonists for RARs and RXRs, respectively (38). LPS readily induced
production of IL-12 heterodimer as well as the p40 subunit, as
expected. However, retinoids inhibited this LPS-induced IL-12
production in a dose-dependent manner (Fig.
1). Interestingly, 9-cis-RA
and LG69 were significantly more effective than TTNPB
(p < 0.01 at 10 Retinoids Inhibit NF Physical Interaction of Retinoid Receptors with NF NF An Inhibitory Complex of NF The C-terminal AF2 Domain Is Involved with NF The NF Inhibiting the action of IL-12 has been shown to prevent
development and progression of disease in experimental models of autoimmunity (45). These findings have raised great interest in
identifying inhibitors of IL-12 production for the treatment of
Th1-mediated diseases such as type-1 diabetes, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and acute
graft-versus-host disease. Recently, corticosteroids have
been shown to enhance the capacity of macrophages to induce IL-4
synthesis in CD4+ T cells by inhibiting IL-12 production
(46). In addition, captopril and lisinopril, angiotensin-converting
enzyme inhibitors, were also shown to suppress IL-12 production from
human peripheral blood mononuclear cells (47). Phosphodiesterase
inhibitor pentoxifylline (48) and thalidomide (49) inhibited IL-12
production from human monocytes by a mechanism independent of known
endogenous inhibitors of IL-12 production such as IL-10, transforming
growth factor- In this report, we added retinoids to the list of hydrophobic compounds
that inhibit production of IL-12 through specific nuclear receptors
(17), together with corticosteroids (46) and 1,25-dihydroxyvitamin
D3 (51) (Fig. 1). As was the case with corticosteroids and
1,25-dihydroxyvitamin D3, this inhibition was also mapped
to a region in the p40 promoter containing a binding site for NFB site within
the IL-12 p40 promoter. In this study, we found that retinoids inhibit this LPS-stimulated production of IL-12 in a dose-dependent
manner. The NF
B components p50 and p65 bound retinoid X receptor
(RXR) in a ligand-independent manner in vitro, and the
interaction interfaces involved the p50 residues 1-245, the p65
residues 194-441, and the N-terminal A/B/C domains of RXR. Activation
of macrophages by LPS resulted in markedly enhanced binding activities
to the
B site, which significantly decreased upon addition of
retinoids, as demonstrated by the electrophoretic mobility shift
assays. In cotransfections of CV-1 and HeLa cells, RXR also inhibited the NF
B transactivation in a ligand-dependent manner,
whereas a mutant RXR lacking the AF2 transactivation domain, which
serves as ligand-dependent binding sites for transcription
integrators SRC-1 and p300, was without any effect. In addition,
coexpression of increasing amounts of SRC-1 or p300 relieved the
retinoid-mediated inhibition of the NF
B transactivation. From these
results, we propose that retinoid-mediated suppression of the IL-12
production from LPS-activated macrophages may involve both inhibition
of the NF
B-DNA interactions and competitive recruitment of
transcription integrators between NF
B and RXR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
(IFN-
), which in turn
augments their cytotoxicity, and by enhancing their proliferation potential. IL-12 production is critical for the development of T helper
type 1 (Th1) cells and the initiation of cell-mediated immune responses
(reviewed in Ref. 5). The key role of IL-12 in inflammation as well as
the cell-mediated immune responses (6, 7) have raised considerable
interest in the mechanisms of IL-12 gene transcription. Inducible
expression of IL-12 has been documented in macrophages and dendritic
cells after stimulation by microbial antigens or via CD40-CD40L
interaction (8, 9). In lipopolysaccharide (LPS)- and IFN-
-treated
monocytes, the expression of IL-12 p40 has been shown to be primarily
regulated at the transcriptional level, which involved at least two
transcription factors that belong to the NF
B and Ets families
(10-12). Expression of IL-12 p35 is also known to be subject to
similar transcriptional regulation, although characterized to a much
lesser extent than p40 (13, 14).
B is important for the inducible
expression of a wide variety of cellular and viral genes (reviewed in
Ref. 15). NF
B is composed of homo- and heterodimeric complexes of
members of the Rel (NF
B) family of polypeptides. In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB. These proteins share a 300-amino acid region, known as the Rel homology domain, which binds to DNA and mediates homo- and heterodimerization. This domain is also a target of the I
B inhibitors, which include I
B
, I
B
, I
B
, Bcl-3, p105, and p100 (16). In the
majority of cells, NF
B exists in an inactive form in the cytoplasm,
bound to the inhibitory I
B proteins. Treatment of cells with various inducers results in the degradation of I
B proteins. The bound NF
B
is released and translocates to the nucleus, where it activates appropriate target genes.
,
, and
and retinoid X receptor (RXR)
,
, and
(reviewed in Ref. 18). Functional analysis of nuclear receptors has shown that there are two major activation domains. The
N-terminal domain (AF1) contains a ligand-independent activation function, whereas the extreme C-terminal region of the ligand binding
domain (AF2) exhibits ligand-dependent transactivation and
undergoes an allosteric change upon ligand binding. The AF2 region
plays a critical role in mediating transactivation by a ligand-dependent interaction with transcription
coactivators such as functionally conserved proteins CREB-binding
protein (CBP) and p300 (reviewed in Ref. 19) and SRC-1 (20).
Accordingly, deletion or point mutations in this region impair
transcriptional activation without changing ligand and DNA binding
affinities (21-23). CBP/p300 and SRC-1 have also been shown to be
essential for the activation of transcription by a large number of
regulated transcription factors, including CREB, NF
B, STATs, SRF,
p53, and AP-1 (19, 24-27). Based on this broad spectrum of action, these coactivator proteins were termed transcription integrators.
B
activity and can physically interact with NF
B in vitro.
Since RelA represses ligand-dependent activation of steroid
receptor-regulated promoters, a mutually inactive complex formed by a
direct protein-protein interaction of steroid receptors and RelA has
been proposed.
B, like steroid receptors.
The experimental results indicated that retinoid-mediated suppression
of the IL-12 production from LPS-activated macrophages may involve both
inhibition of the NF
B-DNA interactions and competitive recruitment
of CBP/p300 and SRC-1 between NF
B and RXR.
EXPERIMENTAL PROCEDURES
689/+98 fragment of mIL-12 p40 promoter from
pXP2 (11) was subcloned into KpnI/XhoI sites of
pGL3-basic luciferase vector (Promega Co., Madison, WI). All the
deletion mutants were generated by polymerase chain reaction (PCR)
using an upstream primer containing BamHI site. A
linker-scanning mutant was generated by a two-step PCR procedure with
overlapping internal primers that contain mutated sequences for the
NF
B site. A vector expressing Gal4/p65 fusion protein was
constructed by subcloning an appropriate p65 PCR fragment into
EcoRI/XhoI sites of pCMXGal4/N (35). Mammalian expression vectors for SRC-1, p300, p50, p65, RXR
and RXR
AF2, the
reporter constructs TREpal-LUC and
B-LUC, and the transfection indicator construct pRSV-
-gal were as described previously
(25-27).
8,
10
7, and 10
6 M at 1 × 105 cells/well in 96-well culture plates for 48 h.
B-binding site within the Ig
-chain (5'-CCG GTT AAC AGA GGG GGC TTT CCG AG-3') was used as a
probe. Labeled oligonucleotides (10,000 cpm) were incubated for 30 min
at room temperature, along with 10 µg of nuclear extracts, in 20 µl
of binding buffer (10 mM Tris·HCl, pH 7.6, 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng of
poly(dI-dC), and 1 mM dithiothreitol). The reaction mixture
was analyzed by electrophoresis on a 4% polyacrylamide gel in 0.5×
Tris borate buffer. Specific binding was confirmed by competition
experiments with a 50-fold excess of unlabeled, identical
oligonucleotides or cAMP response element-containing oligonucleotides.
RESULTS
6 M RA). In
contrast, treatment with retinoids did not influence IL-10 production
from LPS-stimulated macrophages, suggesting that the retinoid effects
were not the result of a general dampening of cellular activation.
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Fig. 1.
Inhibition of IL-12 production in primary
macrophages by retinoids. Macrophages were stimulated with LPS (5 µg/ml) in the absence or presence of different concentrations of
retinoids. Cytokine levels were evaluated by enzyme-linked
immunosorbent assay, and results are presented as mean ± standard
deviations of the percentage response of cytokine production of
retinoid-treated macrophages compared with untreated control
macrophages stimulated with LPS. Mean cytokine levels in the absence of
retinoids were as follows: IL-12 p70, 650 pg/ml; IL-12 p40, 1.9 ng/ml;
IL-10, 1.2 ng/ml. Closed square, open
square, and closed triangle indicate
the IL-12 p70 heterodimer, IL-12 p40, and IL-10, respectively.
B-mediated Activation of IL-12 p40 Promoter
by LPS--
An IL-12 p40 subunit was known as the highly inducible and
tightly regulated component of IL-12 (5). To identify the region involved in these retinoid actions, we generated a series of luciferase reporter constructs containing the p40 promoter sequences from positions
689,
231, and
185 to +98 relative to the transcription initiation site (Fig. 2A).
Mouse RAW264.7 monocytic cells were transfected with each of these
constructs and stimulated with LPS either in the absence or presence of
retinoids, and the luciferase activity was determined. All of these
constructs showed strong stimulation with LPS in the absence of
retinoids but impaired stimulation with retinoids (Fig. 2B).
In particular, deleting sequences to -185 (p40/185) did not diminish
the LPS-dependent promoter activities and the inhibitory
effect of retinoids was still observed, suggesting that the target site
for retinoids should reside within this region. To directly test the
role of a
B site found between -121 and -131 of the p40 promoter
in the retinoid-mediated inhibitory actions, we introduced a linker
scanning mutation into the
B site within the context of the
689/+98 construct (p40/LS). The LPS-dependent promoter
activation was still observed with p40/LS, although significantly
reduced (Fig. 2B), consistent with the previous findings in
which the
B site was shown to be important for the LPS induction of
p40 promoter (10). However, addition of retinoids to LPS-stimulated
cells did not have any repressive effects with p40/LS, clearly
indicating that the inhibitory effect of retinoids on IL-12 production
was mediated through the
B site.
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Fig. 2.
Analysis of retinoid-mediated transcriptional
repression of p40 promoter constructs activated by LPS.
A, schematic representation of the mouse p40 promoter
constructs as well as a linker-scanning mutant for NF B site are as
shown, along with Ets and NF
B binding sites. The nucleotide sequence
numbers for each construct are shown. B, transient
transfection of RAW264.7 cells with the p40 promoter constructs,
followed by stimulation with LPS either in the absence or presence of
10
7 M retinoids. The results are expressed as
induction (n-fold) over the value obtained with the
unstimulated RAW264.7 cells transfected with the
689/+98 construct,
which was given an arbitrary value of 1. Closed,
open, striped, checked, and
stippled boxes indicate no LPS added, 5 µg/ml
LPS, 5 µg/ml LPS plus 10
7 M
9-cis-RA, 5 µg/ml LPS plus 10
7 TTNPB, and 5 µg/ml LPS plus 10
7 LG69, respectively. The data are
representative of three similar experiments.
B--
With
the precedent of direct physical interactions of NF
B with steroid
receptors (28-33), we hypothesized that associations of NF
B with
retinoid receptors may have led to the NF
B-inhibitory action of
retinoids. Indeed, in vitro translated, labeled RXR interacted with GST fusions to the NF
B components p50 and p65 but
not with GST alone, in a ligand-independent manner (Fig.
3A). Similarly, RXR
AF2,
deleted for the C-terminal AF2 domain (39), and RXR-ABC containing only
the N-terminal A/B/C domains also interacted with GST fusions to p50
and p65. In contrast, RXR-LBD, which contains only the hinge and ligand
binding domains, did not bind any of these GST proteins. The RXR
interaction domains were also mapped to the p50 residues 1-245 and the
p65 residues 194-441 by using a series of C-terminal deletions
introduced into p50 and p65 (Fig. 3B). Similarly, RAR also
interacted with the p50 residues 1-245 and the p65 residues 194-441
(data not shown).
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Fig. 3.
Interactions of RXR with p50 and p65
in vitro. A, RXR, RXR AF2, RXR-LBD,
and RXR-ABC were labeled with [35S]methionine by in
vitro translation and incubated with glutathione beads containing
GST alone or GST fusions to p50 and p65, either in the absence or
presence of 10
7 M 9-cis-RA, as
indicated. Beads were washed, and specifically bound material was
eluted with reduced glutathione and resolved by SDS-polyacrylamide gel
electrophoresis. Approximately 10-20% of total input was typically
retained. B, schematic representations of p65 and p50 and
their deletion constructs. The Rel homology domains are indicated in
stripes, and the amino acid residues are as indicated.
C, the full-length p50 and p65 and their deletions were
labeled with [35S]methionine by in vitro
translation and incubated with glutathione beads containing GST alone
or GST fusions to RXR, either in the absence or presence of
10
7 M 9-cis-RA, as
indicated.
B Binding to the
B Site Inhibited by Retinoid--
Steroid
receptors have been shown to inhibit NF
B binding to
B sites in a
ligand-dependent manner (28-33). To examine whether retinoid-mediated inhibition of the NF
B transactivation also exploits similar mechanisms, we analyzed the
B binding activity present in nuclear extract of unstimulated or LPS-stimulated primary macrophages, either in the absence or presence of retinoids. As expected, nuclear extracts from LPS-stimulated macrophages exhibited strong
B binding activity in the electrophoretic mobility shift assays using a labeled oligonucleotide containing a consensus Ig-
B
site (40) (Fig. 4A). The
binding was specific as it was competed with an unlabeled, identical
oligonucleotide, but not with unrelated, nonspecific oligonucleotide,
and was absent with nuclear extracts from nonstimulated cells. Similar
to steroid receptors, nuclear extracts from macrophages stimulated by
LPS in the presence of various retinoids showed much diminished
B binding activities in a retinoid-dose dependent manner (Fig.
4A). To rule out the possibility that these inhibitory
actions of retinoids are the results of retinoid-directed gene
expressions of a third component, retinoids were directly added to the
binding reactions, along with nuclear extracts from LPS-stimulated
macrophages. In these experiments, the
B binding activities
decreased in a retinoid-dose dependent manner, suggesting that the
retinoid-bound receptor may directly modulate the NF
B-DNA
interactions by forming a complex with NF
B that is unable to bind
B sites (Fig. 4B).
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Fig. 4.
Retinoid-mediated inhibition of
B binding by NF
B.
A, nuclear extracts prepared from macrophages stimulated by
LPS either in the absence or presence of retinoids (10
7
and 10
8 M each) were examined for
B
binding activity in the electrophoretic mobility shift assays using a
labeled oligonucleotide containing a consensus Ig-
B site, as
indicated. S and NS indicate the presence of an
unlabeled, identical oligonucleotide and nonspecific oligonucleotide,
respectively. The specific NF
B complexes are as indicated.
B, retinoids were directly added to nuclear extracts
prepared from macrophages stimulated by LPS in the absence of
retinoids, and
B binding activity was examined in the
electrophoretic mobility shift assays. Increasing amounts of
9-cis-RA (10
8, 10
7,
10
6, and 10
5 M) or carrier
(ethanol) were used as indicated.
B-RXR--
To test if this
retinoid-mediated inhibition of NF
B activities in macrophages are
generally observed in other cell types, we employed a reporter
construct
B-LUC, previously characterized to efficiently mediate the
NF
B-dependent transactivations in various cell types,
that consists of a minimal promoter from the IL-2 gene and four
upstream
B sites from the IL-6 gene (41). Cotransfection of CV-1
cells with RXR had a minimal effect on the p65-induced reporter gene
expression in the absence of retinoids. In the presence of retinoids,
however, increasing amounts of cotransfected RXR inhibited the reporter
gene expression in an RXR dose-dependent manner (Fig.
5A). Similar results were also
obtained with the LPS- or TNF
-induced level of transactivations in
various cell types, including HeLa cells (data not shown). Similarly,
cotransfection of increasing amounts of p50 or p65 also inhibited the
9-cis-RA-dependent transactivation by RXR (Fig.
5B). These results suggest that the interactions of
NF
B-RXR may lead to a formation of transcriptionally inactive
complex in vivo, regardless of the nature of DNA binding sites.
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Fig. 5.
Transcriptionally inhibitory complex of
NF B and RXR. A, CV-1 cells
were transfected with p65 (50 ng) and increasing amounts of RXR (10, 50, 100, and 200 ng) expression vectors along with a reporter gene
B-LUC in the absence or presence of RA. Closed,
open, striped, and checked
boxes indicate no hormone added and the presence of
10
7 M 9-cis-RA, 10
7
M TTNPB, and 10
7 M LG69,
respectively. B, CV-1 cells were transfected with increasing
amounts of p50 or p65 expression vectors (10, 50, 100, 200, and 300 ng)
along with TREpal-LUC, as indicated. Normalized luciferase expressions
from triplicate samples are presented relative to the LacZ
expressions, and the standard deviations are less than 5%.
B
Inhibition--
Interestingly, cotransfection of CV-1 cells with
RXR
AF2, a mutant RXR lacking the C-terminal AF2 domain, moderately
enhanced the reporter gene expression in the absence of retinoids,
whereas it appeared unable to inhibit the NF
B transactivation in the presence of 9-cis-RA (Fig.
6A). The AF2 domain serves as
ligand-dependent binding sites for a number of
transcription coactivator molecules such as SRC-1 (20) and CBP (19),
which were also shown to function as transcription coactivators for
NF
B (25, 42, 43). Thus, competition for limiting amounts of SRC-1
and p300 could account for the mutual inhibitions between NF
B and
liganded RXR. Indeed, the inhibitory effects of NF
B by liganded RXR
were largely relieved upon addition of increasing amounts of SRC-1 and
p300 expression vectors (Fig. 6B). Similar results were also
obtained with NF
B-mediated inhibition of the RXR transactivation
(data not shown).
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Fig. 6.
Involvement of the AF2 domain in
retinoid-directed repression. A, CV-1 cells were
transfected with p65 (50 ng) and increasing amounts of RXR AF2 (0, 10, 50, and 100 ng) expression vectors along with a reporter gene
B-LUC. B, CV-1 cells were transfected with p65, RXR, and
increasing amounts of SRC-1 or p300 expression vectors (0, 100, and 200 ng) along with
B-LUC, as indicated. Normalized luciferase
expressions from triplicate samples are presented relative to
LacZ expression, and the standard deviations are less than
5%. Closed and open boxes indicate no
hormone added and the presence of 10
7 M
9-cis-RA, respectively.
B-inhibitory Actions of Retinoids Independent of
B
Sites--
Since the RXR binding sites involved the DNA binding Rel
domain of both p50 and p65 (Fig. 3), we tested whether the
NF
B-inhibitory actions of retinoids require
B site bindings.
Thus, we expressed a Gal4 fusion protein to p65 (Gal4/p65) in CV-1
cells, along with a reporter construct controlled by upstream Gal4
sites (35). Consistent with previous findings (44), Gal4/p65 directed a strong activation of the reporter gene expression (Fig.
7). Cotransfection of increasing amount
of RXR expression vector was without any significant effects in the
absence of retinoids. In contrast, however, liganded RXR directed
inhibition of the Gal4/p65 transactivation in an RXR
dose-dependent manner (Fig. 7). These results, along with
the results shown in Fig. 6, suggest that the inhibitory actions of
retinoids can also operate without
B site bindings and involve the
AF2-dependent factors.
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Fig. 7.
Retinoid-mediated transrepression of p65 in
the absence of B binding. CV-1 cells were
transfected with Gal4/p65 and RXR expression vectors along with a
reporter gene Gal4-LUC, as indicated. Normalized luciferase expressions
from triplicate samples are presented relative to LacZ
expression, and the standard deviations are less than 5%.
Closed and open boxes indicate no
hormone added and the presence of 10
7 M
9-cis-RA, respectively.
DISCUSSION
, or prostaglandin E2.
2-Adrenergic compounds including salbutamol inhibited
IL-12 production from human monocytes or dendritic cells by increasing
intracellular cAMP levels, leading to inhibition of the development of
Th1 cells while promoting Th2 cell differentiation (50). Interestingly,
1,25-dihydroxyvitamin D3 was also shown to inhibit IL-12
production, presumably by down-regulating the NF
B activities from
human IL-12 p40 gene (51).
B
(Fig. 2) and may involve direct physical interactions of retinoid
receptors with NF
B (Fig. 3). However, it is interesting to note that
NF
B constitutively interacted with RXR (Fig. 3), whereas the
inhibitory actions were absolutely ligand-dependent (Figs.
1, 2, and 5). Thus, NF
B may exist constitutively associated with RXR
in vivo, and this complex becomes transcriptionally inactive upon addition of retinoids, in which ligand-dependent
interactions with transcription coactivators may play important roles
(as summarized in Fig. 8). This notion is
consistent with the inability of the AF2-mutant RXR to inhibit the
NF
B transactivation as well as derepression of the inhibitory
actions by coexpressed SRC-1 and p300 (Fig. 6). However, it is not
certain whether these coactivators remain complexed with NF
B in the
presence of liganded RXR. In addition, retinoids also inhibited the
B binding activities of NF
B in vitro (Fig. 4),
suggesting that the liganded RXR/NF
B complex is unable to recognize
B sites. However, it is not currently known why this liganded
RXR/NF
B complex loses its ability to bind
B sites. It is possible
that conformational change brought into this complex, along with the
transcription coactivators SRC-1 and CBP/p300, upon addition of
retinoids may become propagated to the Rel homology domain of NF
B,
resulting in inability to bind
B sites. However, the inhibitory
actions of retinoids can also operate in the absence of
B site
bindings by NF
B, as demonstrated by the results shown in Fig. 7, in
which transactivation mediated by Gal4/p65 was shown to be inhibited by
retinoids. Overall, these results are similar to previously described
results with steroid receptors (28-33), in which the mutual
inhibitions between GR and RelA involved the DNA and the ligand binding
domains of the GR (Fig. 3). Since the NF
B interactions were
subsequently mapped to the DNA binding C domain of the GR, the
requirement for the GR LBD may have also reflected the importance of
the AF2-dependent transcription coactivators
(i.e. SRC-1 and CBP/p300).
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Fig. 8.
A model for the
NF B-RXR interactions. NF
B may
constitutively bind RXR through the N-terminal ABC domains of RXR and
the Rel homology domain of p50 and p65 in vivo. Retinoid
binding may lead to two changes within this complex, which might be
responsible for formation of ligand-dependent inhibitory
complex between NF
B and RXR. First, retinoids bind and induce
significant conformational change with RXR, leading to inhibition of
the NF
B-
B site interactions. Second, SRC-1 and CBP/p300 may
constitutively bind NF
B but recognize RXR only in the presence of
ligand.
The retinoid receptor-NFB interactions are likely to have wide
implications in various aspects of retinoid and NF
B biology, not
limited to the regulation of IL-12 production in macrophages described
in this study. Both retinoids and NF
B have been shown to be involved
with a wide variety of biological processes, including immunomodulation, embryonic development, spermatogenesis, and inhibition of cancer cell proliferation (reviewed in Ref. 52). In
particular, this negative cross-talk could be relevant for tumor
inhibitory actions of retinoids. Several lines of evidence suggested
that constitutive activation of NF
B contributes to the malignant
phenotype of tumor cells. A naturally occurring splice variant of RelA
was shown to transform Rat-1 cells (53), whereas antisense
oligonucleotides to RelA were shown to inhibit proliferation and
tumorigenicity of several tumor cell lines, including the human breast
cancer cell lines MCF7 and T47D (54). In addition, activation of NF
B
through the disruption of I
B
regulation was shown to result in
malignant transformation (55).
In conclusion, we have shown that retinoid receptors form a
transcriptionally inhibitory complex with NFB. With the NF
B transactivation, in particular, this retinoid-mediated inhibitory action appeared to involve inhibition of the NF
B-DNA interactions as
well as competitive recruitment of transcription integrators between
NF
B and RXR. This transrepression between NF
B and retinoid receptors could play an important role in a large variety of biological processes.
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ACKNOWLEDGEMENTS |
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We thank Drs. Stanley Wolf and Yong Kyung Choe for reagents.
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FOOTNOTES |
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* This work was supported in part by grants from the Korea Science and Engineering Foundation (HRC) and from the Korean Research Foundation (to T. S. K. and S.-Y. I.).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.
¶ Recipient of an intern fellowship from the Korean Ministry of Science and Technology.
¶¶ Supported by the National Creative Research Initiative from the Korean Ministry of Science and Technology.
|| To whom correspondence should be addressed. Tel.: 82-62-530-2935; Fax: 82-62-530-2949; E-mail: taekim{at}chonnam.chonnam.ac.kr.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; Th1, T helper type 1; LPS, lipopolysaccharide; RAR, retinoic acid receptor; RXR, retinoid X receptor; mAb, monoclonal antibody; 9-cis-RA, 9-cis-retinoic acid; TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl]benzoic acid; PCR, polymerase chain reaction; GST, glutathione S-transferase; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; CBP, CREB-binding protein; GR, glucocorticoid receptor; LBD, ligand-binding domain.
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REFERENCES |
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