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Department of Molecular Preventive Medicine (H.I., T.S., K.M.), University of Tokyo, School of Medicine, 7–3-1, Hongo, Bunkyoku, Tokyo 113-0033, Japan; and the Immunopathology Section, Laboratory of Molecular Immunoregulation (T.Y.), National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201
Address all correspondence and requests for reprints to: Prof. Kouji Matsushima, M.D., Ph.D., Department of Molecular Preventive Medicine, University of Tokyo, School of Medicine, 7–3-1 Hongo, Bunkyoku, Tokyo 113-0033, Japan. E-mail: koujim{at}m.u-tokyo.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IL-1). Addition of E2 to MCF-7 cells inhibited MCP-1
production in a dose-dependent manner. XEs, bisphenol A, and NP also
inhibited MCP-1 production, although the potency was 3–4 orders of
magnitude lower than that of E2. E2, bisphenol
A, and NP inhibited MCP-1 messenger RNA expression in MCF-7 cells. Two
closely located nuclear factor-
B sites, A1 and A2, have been
identified in the promoter of the human MCP-1 gene. A luciferase
construct containing this enhancer region (pGLM-ENH) was activated by
IL-1, and a mutation at either the A1 or A2 site resulted in a loss
of IL-1 responsiveness. Treatment with E2 or XEs
decreased the IL-1-inducible pGLM-ENH luciferase activity
significantly. In an electrophoretic mobility shift assay and
supershift analysis, we found that treatment with E2 or XEs
diminished the IL-1-induced complex formation with both A1 and A2
probes, which was identified immunochemically to consist of nuclear
factor-B, p50, and p65. The IL-1-induced p50/c-Rel complex to the
A2 probe was also, to a lesser extent, decreased by E2 or
XE treatment. The effects of E2 and XEs on the expression
of MCP-1 seem to be much more dramatic than the effects of these agents
on the promoters used in the luciferase assay, suggesting the
involvement of an additional site(s) of the promoter region of the
MCP-1 gene or posttranscriptional regulation of MCP-1 gene expression
by E2 and XEs. This work represents the first report
describing possible regulation of immune system function by XEs through
inhibiting chemokine production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recruitment of macrophages into tissues is an important process in
inflammation and host defense. Although the precise mechanism of
monocyte infiltration has not yet been fully clarified, monocyte
chemoattractant protein-1 (MCP-1), also termed monocyte chemotactic and
activating factor, is thought to play a pivotal role (7, 8, 9). MCP-1 is a
member of the chemokine family and attracts mainly blood monocytes (9, 10). A wide variety of activated cells, including monocytes,
fibroblasts, vascular endothelial cells, and smooth muscle cells,
produce MCP-1 in vitro in response to various stimuli, such
as lipopolysaccharide, interleukin-1 (IL-1), and tumor necrosis
factor- (9, 10). MCP-1 messenger RNA (mRNA) was expressed at high
levels in pathological foci of atherosclerosis (11), glomerulonephritis
(12), pulmonary fibrosis (13), and rheumatoid arthritis (14). Thus, the
mechanism of transcriptional regulation of the MCP-1 gene has drawn
increased clinical interest. Previous studies have indicated that MCP-1
expression is inhibited by glucocorticoid (15) and progesterone (16).
Recent studies have observed that E2 is also able
to block the increase in steady state levels of MCP-1 mRNA in
macrophages and fibroblasts (17, 18). An in vivo study has
also noted that physiological concentrations of
E2 may suppress MCP-1 expression in rabbits (19).
The precise molecular mechanism of this inhibitory effect by
E2 is, however, unknown.
The overall objective of this study was to examine the effects of
E2 and XEs on MCP-1 production in
estrogen-responsive MCF-7 cells, and to investigate the molecular
mechanism. Estrogens are steroid hormones whose effects are mediated by
estrogen receptor (ER). ER belongs to the superfamily of
ligand-activated transcription factors, the nuclear receptors (20).
E2-ER complexes bind to the genomic estrogen
response elements. The estrogen-occupied receptor interacts with
additional transcription factors and components of the transcription
initiation complex to modulate gene transcription (20). In the
5'-flanking regions of the human MCP-1 gene, regions homologous to the
consensus sequence of the estrogen response elements have not been
identified (21, 22). Thus, estrogens may inhibit transcription of the
MCP-1 gene by interacting with other sequences. Previous studies
revealed that nuclear hormone receptors, including glucocorticoid
receptor and progesterone receptor, may repress nuclear factor-B
(NF-B) activity (23, 24). We hypothesized, therefore, that estrogens
may also act on the NF-B sites of the human MCP-1 gene. We observed
that estrogens suppressed IL-1-induced MCP-1 expression in MCF-7
cells, and this inhibition was mediated in part through NF-B sites
of the human MCP-1 gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SA, 2 x
107 U/mg) was a gift from Dainippon
Pharmaceutical Co. Ltd. (Osaka, Japan). The human breast cancer cell
line MCF-7 (25) was obtained from American Type Culture Collection (Manassas, VA). Charcoal, E2,
and BPA were purchased from Sigma (St. Louis, MO). NP was
purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI).
The estrogen antagonist ICI 182,780 was purchased from Tocris (Bristol,
UK). All other chemicals were of the highest purity available from
commercial sources. Rabbit antisera against human p65 (26), p50 (26),
and c-Rel (27) were provided by Dr. Nancy Rice (NCI-Frederick Cancer
Research and Development Center, NIH, Frederick, MD).
Cell culture and treatment
Cells were grown in MEM (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% FCS. Cells were subcultured at
approximately 80% confluence. For induction experiments, cells were
cultured in phenol red-free medium (28) containing 10% FCS treated
with dextran-coated charcoal (29). Stock solutions of
E2, BPA, and NP were prepared in ethanol; the
final ethanol concentration was 0.1% or less. Cells (1 x
105) were plated in 24-well plates and cultured
overnight before being treated with 10 ng/ml IL-1. The indicated
concentrations of E2 or XEs were added
contemporaneously with IL-1.
Enzyme-linked immunosorbent assay (ELISA)
The MCP-1 concentration was measured by a sandwich ELISA as
previously described (30). Briefly, 96-well plates were coated with
monoclonal antibody at 4 C overnight and blocked with 1% BSA in PBS at
37 C for 1 h. After washing, samples (100 µl) in duplicate or
triplicate were added, followed by incubation for 2 h at 37 C. The
plates were washed, then rabbit anti-MCP-1 polyclonal IgG was added as
the second antibody, and incubation was performed for 2 h at 37 C.
Subsequently, alkaline phosphatase-conjugated antirabbit IgG (1:10,000;
Biosource International, Camarillo, CA) in 1% BSA-PBS was
added and incubated for an additional 2 h at 37 C. After plates
were washed, 100 µg/well p-nitrophenyl phosphate
(Sigma) at a concentration of 1 mg/ml in 1
M diethanolamine (pH 9.8) was added and allowed
to react for 45 min at room temperature. The reaction was terminated by
the addition of 100 µl of 1 N NaOH. The OD at
405 nm was measured using an ELISA plate reader (Tosoh Co., Tokyo,
Japan). For standards, human recombinant MCP-1 at concentrations
ranging from 10 pg/ml to 10 ng/ml was used. The detection limit of this
assay was 50 pg/ml.
Northern blot analysis
After treatment of the cells, total RNA was extracted with
RNAzol B (Biotex Laboratories, Inc., Houston, TX)
according to the protocol from the manufacturer. Northern blot analysis
was performed in 1% agarose gel containing formaldehyde. Filters were
hybridized at 42 C overnight in 50% formamide, 5 x Denhardt’s
solution, 50 µg/ml sheared denatured salmon sperm DNA, 1% SDS, and
1 x 106 dpm/ml
32P-labeled complementary DNA (cDNA) probe.
Filters were washed twice with 2 x SSC (standard saline
citrate)-0.5% SDS at room temperature for 15 min and with 1 x
SSC-0.5% SDS at 60 C for 30 min before autoradiographic exposure. The
probes used were human MCP-1 cDNA (31) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Densitometer analysis was performed for data
quantification.
Plasmid construction, cell transfection, and luciferase assay
The plasmid vector pGLM-ENH and its mutated constructs pGLM-MA1,
pGLM-MA2, and pGLM-MA1A2 were prepared as previously described (32).
Wild-type pGLM-ENH reporter gene was constructed by conjugation of the
230-bp human MCP-1 enhancer region between -2742 and -2513 to the
167-bp human MCP-1 promoter region between -107 and +60 (32). Two
NF-B-binding sites, A1 and A2, were identified between -2742 and
-2513 (32, 33). The mutations in the A2 or A1 sequence or in both
sites were prepared by a two-step PCR mutagenesis method and designated
pGLM-MA1, pGLM-MA2, and pGLM-MA1A2, respectively (32). When cell
density reached 80% confluence, MCF-7 cells were transiently
transfected with 2 µg luciferase plasmid DNA/35-mm tissue culture
plate by Lipofectamine reagent (Life Technologies, Inc.)
according to the protocol from the manufacturer. After incubation with
the DNA-Lipofectamine complex for 18 h, the cells were washed with
PBS and stimulated with IL-1 in fresh medium for 24 h in the
presence or absence of E2 or XEs. The luciferase
assay was performed with PicaGene (Toyo Ink Co., Tokyo, Japan). The
light intensity was measured with a Lumat model LB950 luminometer
(Berthold, Germany). The luciferase activities were normalized
for the total protein concentration of the cell extracts. Results were
confirmed by three independent transfections, and representative data
are shown.
Preparation of nuclear extracts and electrophoretic mobility shift
assay (EMSA)
Nuclear extracts were prepared by a method previously reported
(34), and aliquots were frozen at -80 C. Protein concentrations were
measured by means of the Bradford assay (Bio-Rad Laboratories, Inc., Richmond, CA). EMSA was carried out using 5%
polyacrylamide gels in 0.5 x TBE buffer (90 mM Tris
borate and 2 mM EDTA). The oligonucleotide sequences used
in EMSAs were as follows: A1, 5'-GATCTGGGAACTTCCAAAGC-3'; A1 mutant
(MA1), 5'-ACGGGATCTAGAAACTTCCA-3'; A2, 5'-AGAGTGGGAATTTCCACTCA-3'; and
A2 mutant (MA2), 5'-GAGTGGGAATTCGGACTCACTTCTCT-3', all of which were
annealed with each antisense oligonucleotide. For the binding reaction,
nuclear extracts (10 µg protein) were incubated in a total reaction
mixture of 20 µl comprising 12 mM HEPES (pH 7.9), 60
mM KCl, 4 mM MgCl2, 1
mM EDTA, 12% glycerol, 1 mM dithiothreitol,
and 2.5 µg poly(dI-dC) (Pharmacia Biotech, Uppsala,
Sweden). The radiolabeled oligonucleotide (105
cpm) was added to the mixture after preincubation for 15 min at 4 C,
and then the total reaction mixture was incubated for 20 min at room
temperature. For competition assay, 50-fold excess amounts of
appropriate unlabeled oligonucleotide were added to the binding
reaction mixture. For supershift assay, appropriate antibody was
incubated with nuclear extract in 1 x binding buffer for 20 min
at room temperature before the addition of
32P-labeled probe. After electrophoresis, the
gels were dried and analyzed by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Untreated MCF-7 cells failed to produce
MCP-1 in the medium. MCP-1 was detectable at 24 h after treatment
with IL-1. The concentration of MCP-1 in the medium continued to
increase during the first 72 h after IL-1 treatment.
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-induced MCP-1 mRNA expression in MCF-7
cells by Northern blot analysis (Fig. 1
, B and C). The expression of
MCP-1 mRNA was clearly detected 4 h after IL-1 stimulation.
MCP-1 mRNA was not detected in unstimulated MCF-7 cells at any time
point (data not shown).
Inhibition of IL-1-induced MCP-1 expression by
E2 and XEs
To examine the effects of E2 on MCP-1
expression in MCF-7 cells, cells were cultured with IL-1 alone or in
combination with E2 (Fig. 2A). Increasing the concentration of the
added E2 resulted in a dose-dependent decrease in
the MCP-1 concentration in the culture medium of IL--stimulated
cells. These concentrations of E2 represent the
physiological range of circulating E2 levels.
Ethanol at 0.1% had no effect on the MCP-1 concentration.
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alone or in combination with
these compounds (Fig. 2B). The addition of either BPA or NP resulted in
a decrease in the MCP-1 concentration in a dose-dependent manner;
however, the effects of these agents were approximately 1,000- or
10,000-fold less potent than that of E2. The
combined treatment of MCF-7 cells with a mixture of BPA and NP resulted
in additive effects. These inhibitory effects of
E2 or XEs were antagonized by a pure
antiestrogen, ICI 182,780, implying that the negative effects of these
compounds are mediated by the ER.
Regulation of MCP-1 mRNA expression by E2
and XEs
To determine whether the expression of MCP-1 mRNA could be
down-regulated by E2 or XEs, MCF-7 cells were
cultured for 24 h with IL-1 in the presence or absence of
E2 or XEs. MCP-1 mRNA was not detected in
unstimulated cells (lane 1). The addition of E2
(lanes 3–6) or XEs (lanes 7–10) to MCF-7 cells resulted in inhibition
of IL-1-induced MCP-1 mRNA expression in a dose-response manner
similar to the effects on MCP-1, suggesting that the effects were at
the pretranslational level (Fig. 3).
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induces human MCP-1 expression via NF-B sites of human
MCP-1 geneB binding sites, termed A1 and A2, have been identified
in the enhancer region of the human MCP-1 gene (32, 33). To investigate
the role of this region in the expression of MCP-1 mRNA, MCF-7 cells
were transfected with pGLM-ENH containing the enhancer region of the
human MCP-1 gene (Fig. 4A). Treatment of
MCF-7 cells with IL-1 increased luciferase activity significantly.
Next, to clarify the role of the A1 and A2 sites, the sequences of the
A1 and the A2 sites were mutated, and then these constructs were
transfected into MCF-7 cells. The enhancer activity disappeared almost
completely by the mutation in either the A1 or A2 sequence, indicating
that the enhancer activity was mediated by both A1 and A2 sites (Fig. 4A).
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, both E2 and XEs partially
but significantly inhibited the IL-1-induced luciferase
activity.
Induction of the nuclear protein-MCP-1 A1 probe
complexes by IL-1 and inhibition by E2 or
XEs
To confirm that the increased MCP-1 mRNA expression by IL-1 was
due to the binding of NF-B to the enhancer region, EMSA was
performed with the A1 probe using the nuclear extracts of
IL-1-stimulated MCF-7 cells. As shown in Fig. 5A, one distinct DNA-nuclear protein complex
formation was found 4 h after IL-1 stimulation (lane 5,
indicated by an arrow). The formation of the complex was
inhibited with an excess amount of the unlabeled A1 probe (Fig. 5B, lane 3), but not with the mutated MA1 probe (Fig. 5B, lane 4),
indicating that this complex was specific.
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B/Rel subunit in this complex, a supershift assay
was performed with a specific antibody against each NF-B/Rel
protein. As shown in Fig. 5C, an antibody against c-Rel had no effect
on band formation with the A1 probe (lane 4). Addition of an anti-p50
supershifted (lane 2) and addition of an antibody against p65 disrupted
the formation of this complex (lane 3), indicating that the band
consists of p50/p65.
To examine whether E2 or XEs had any effect on
the nuclear factors bound to the A1 binding site that were required for
MCP-1 gene activation, we performed EMSA with the A1 probe. Results
show that nuclear proteins from the cells treated with
E2 (Fig. 6A) or XEs
(Fig. 6B) formed decreased levels of the p50/p65 complex in a
dose-dependent manner (indicated by an arrow).
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-induced nuclear protein-MCP-1-A2 probe-stimulated MCF-7 cells. As shown in Fig. 7A, four distinct DNA-nuclear protein
complexes (C1, C2, C3, and C4) were detected by EMSA. After IL-1
treatment, the amount of C3 increased, then declined thereafter (lanes
1–5). The band intensity of C2 was slightly increased after IL-1
treatment. The formations of C2 and C3 were inhibited with a 50-fold
molar excess amount of the unlabeled A2 probe, but not with the MA2
probe carrying three nucleotide substitutions, indicating that these
complexes were specific (Fig. 7B). In contrast, both C1 and C4 were
inhibited with an excess amount of both unlabeled A2 and MA2 probes,
indicating that these complexes were nonspecific (Fig. 7B).
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B/Rel subunit in each complex, supershift assay
was performed with a specific antibody against each NF-B/Rel
protein. Addition of anti-p50 affected both C2 and C3 bands (Fig. 7C, lane 2). C3 was disrupted with anti-p65 (lane 3), and C2 was disrupted
with anti-c-Rel (lane 4). These results indicated that C2 and C3 were
p50/c-Rel and p50/p65, respectively.
We next examined whether E2 and XEs were able to
alter the binding of nuclear proteins to the A2 probe. Nuclear proteins
from the cells treated with E2 (Fig. 8A) or XEs (Fig. 8B) diminished p50/p65
complex formation dose dependently. The band intensity of p50/c-Rel, to
a lesser extent, appeared diminished after the treatment with
E2 or XEs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation, MCP-1 protein in the culture medium continued to increase
during the first 72 h, whereas mRNA content decreased before
72 h. This time delay of MCP-1 protein production in the medium
may probably due to the time required for processing of the MCP-1
protein in the cytoplasm. E2 was able to inhibit
IL-1-induced MCP-1 expression in a dose-dependent manner over a
physiological concentration range
(10-12–10-9
M). The observation that a pure antiestrogen ICI182,780
antagonized the E2-dependent inhibition of MCP-1
suggests that negative regulation of MCP-1 expression by
E2 is mediated through ER.
XEs are nonsteroidal, human-produced chemicals that can enter the body
by ingestion or adsorption. Previous studies (5, 6) evaluated the
estrogenic activity of XEs. Kuiper et al. performed
competition binding assays with ER or ERß protein and a transient
gene expression assay using cells in which an acute estrogenic response
was created by cotransfection with recombinant human ER or ERß
cDNA in the presence of an estrogen-dependent reporter plasmid (6).
They revealed that both BPA and NP have an affinity 1,000- to
10,000-fold lower than that of E2 for both ER
subtypes (6). In our study the estrogenic potency of BPA or NP to
reduce MCP-1 expression was 3–4 orders of magnitude lower than that of
E2, comparable to these studies (5, 6). The
decrease in p50/p65 binding to A1 probe in cells treated with XEs is
not apparent compared to that of E2 treatment.
Thus, XEs may repress MCP-1 expression in a different manner by
E2 treatment. Indeed, a recent study suggested
that BPA exhibits a distinct mechanism of action at the ER (36).
Although the information concerning the levels of exposure to XEs in
humans is limited, further in vivo study is necessary to
understand whether XEs affect chemokine production and ultimately
immune system function. BPA may be an order of magnitude more potent
in vivo than in vitro (37).
Regulatory elements in the promoter regions of the MCP-1 gene have been
identified by several groups (21, 22, 32, 33, 38, 39, 40, 41, 42). Ueda et
al. investigated the mechanisms involved in the expression of
human MCP-1 gene transcription in different types of cells (32, 33).
The binding of Sp1 to the proximal GC box located between bp -64 and
-59 was critical for maintenance of the basal transcription of this
gene (33). In the distal enhancer region, 2.6 kb upstream from the
transcription initiation site, they identified two closely located
NF-B binding sites that were important for the tissue- and
stimulus-specific transcription of the human MCP-1 gene (32, 33).
Hence, human MCP-1 gene activity is regulated through protein
interactions within at least two distinct regions of its 5'-flanking
region: a proximal promoter region and a distal enhancer region. In
MCF-7 cells, two NF-B sites in the distal MCP-1 enhancer conferred
IL-1 responsiveness. EMSA revealed that IL-1 induced NF-B
binding to both A1 and A2 probes. Using luciferase assays in
transiently transfected MCF-7 cells, we showed that the up-regulation
by IL-1 was inhibited by E2 or XEs. NF-B
binding to both A1 and A2 sites were decreased by using nuclear
extracts treated with E2 or XEs, indicating that
NF-B sites in the enhancer region are targets of estrogen inhibition
of IL-1-induced MCP-1 expression in MCF-7 cells. However, the
effects of E2 and XEs on the expression of MCP-1
seem to be much more dramatic than the effects of these agents on the
promoters used in our luciferase assay, suggesting that the inhibitory
effects of E2 or XEs on MCP-1 expression may not
be completely explained by preventing NF-B binding to the MCP-1
gene. It cannot be excluded that estrogens, in addition to the
suppression of NF-B-dependent activity, modulate MCP-1 mRNA levels
by influencing the activity of other transcription factors important
for MCP-1 induction, such as AP-1 (22), or at the posttranscriptional
level. A full analysis of promoters of the MCP-1 gene will be required
to identify the other cis-elements for the inhibitory
effects by estrogens.
Our results indicated that E2 or XE inhibition of
IL-1-induced MCP-1 expression in MCF-7 cells is mediated partly
through NF-B sites of the human MCP-1 gene. Although the precise
molecular mechanism of the inhibitory effects of MCP-1 by
E2 or XEs is not clear, several possibilities may
be considered. First, a direct interaction between ER and NF-B could
account for the repression. This association may introduce
conformational changes in both proteins that lead to the inability to
bind DNA or result in the formation of inactive complexes on the DNA by
preventing the interaction with essential cofactors or the basal
transcriptional machinery. Second, estrogens may act by modifying the
expression of factors binding to the DNA or by altering nuclear
translocation of NF-B. ER and NF-B might function in competition
for a common transcriptional cofactor(s). Third, the ligand-activated
ER may impair the cooperative interactions between the SP-1 and NF-B
family factors, thus altering the capacity of NF-B to bind to its
site and/or to activate transcription.
MCP-1 is considered to be a "slow kinetics" subset of immediate
early genes (21, 41). Slow kinetics immediate early genes display a 60-
to 90-min lag period before initiation of transcription in response to
serum or growth factor stimulation. A recent study shows that PDGF
induced JE/MCP-1 transcription at 15 min after stimulation, which
returned to near the baseline level within 1 h in rat aortic
smooth muscle cells (42). Thus, the time course of MCP-1 expression in
MCF-7 cells may be rather slow compared with that in other cell types,
and the induction may be indirect. Although the reason for this slow
response to IL-1 is unclear, the differences appear to depend on the
presence of cell type-specific cooperating factors or signaling
pathways. Further study will be necessary to clarify the molecular
mechanism of cell type- and stimulus-specific responses.
In summary, our data demonstrate that E2 and XEs
inhibit IL-1-induced MCP-1 expression in MCF-7 cells and indicate
that this inhibition is mediated partly by the inhibition of NF-B
binding to the human MCP-1 gene. The results presented here give
detailed insight into how E2 and XEs inhibit
MCP-1 expression at the transcriptional level. Chemokines are a rapidly
expanding family of cytokines that are chemoattractants and activators
for specific types of leukocytes (43, 44). MCP-1 may also play a role
in reproductive processes such as ovulation and parturition (45, 46).
Whether XEs can affect other kinds of chemokine production and,
ultimately, immune or reproductive system function in vivo
remains to be established.
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Acknowledgments |
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Footnotes |
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Received May 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1. Eur J Immunol 27:1091–1097[Medline]
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induces
expression of monocyte chemoattractant JE via fos and
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Biol Chem 268:9526–9532B and a 90-kDa
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