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B in HepG2 Cells
Women’s Health Research Institute, Wyeth-Ayerst Laboratories, Inc., Radnor, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Douglas C. Harnish, Women’s Health Research Institute, Wyeth-Ayerst Laboratories, Inc., 145 King of Prussia Road, Radnor, Pennsylvania 19087. E-mail: harnisd{at}war.wyeth.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B (NF-B) may play a major role in ligand-mediated
modification of diseases processes. In particular, the cardioprotective
effects of estrogen replacement therapy are thought to be due in part
to the ability of ligand-bound estrogen receptor (ER) to inhibit
NF-B function. In the current study 17ß-estradiol-bound ER
interfered with cytokine-induced activation of a NF-B reporter in
HepG2 cells. The estrogen metabolite, 17-ethinyl estradiol, and the
phytoestrogen, genistein, were also effective inhibitors of NF-B
activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were
inactive. This inhibition was reciprocal, as NF-B interfered with
the trans-activation properties of ER. Ligand-bound
ER did not inhibit NF-B binding to DNA, but it did decrease the
histone acetyltransferase activity required for NF-B transcriptional
activity. Coexpression of the transcription coactivator CREB binding
protein (CBP), but not steroid receptor coactivator 1a,
reversed the ER-mediated inhibition of NF-B activity. Mammalian
two-hybrid experiments also revealed that ligand-bound ER can
interact functionally with CBP-NF-B complexes. We suggest that CBP
targeting by ER results in the inhibition of NF-B and may occur
through formation of transcriptionally inert multimeric complexes that
are dependent upon the nature of the ER ligand. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent concepts on the etiology of atherogenesis suggest that
inflammation plays a major role (2) and that nuclear factor-B
(NF-B) may be involved in both early and late stages of the
inflammatory-proliferative process (3, 4). Activated NF-B and the
expression of a number of NF-B-dependent proinflammatory gene
products have been demonstrated in endothelial cells, macrophages, and
smooth muscle cells comprising the human atheroma. (5, 6, 7). Other
studies with C57BL/6 mice fed a high fat diet showed that this dietary
regimen induces NF-B activity and expression of acute phase response
genes, such as serum amyloid A and ceruloplasmin, in the liver of these
mice (8). These acute phase proteins are thought to remodel high
density lipoproteins to a form no longer effective in protecting low
density lipoproteins from oxidation and suggests that that NF-B
activation in the liver may also indirectly contribute to atherogenesis
(8).
Recent clinical data suggest that treatment of postmenopausal women
with estrogen replacement therapy reduces circulating levels of
adhesion molecules and other markers of endothelial dysfunction
(9, 10, 11). Consistent with this, it has been shown that ligand-activated
ER inhibits cytokine-induced cell adhesion molecule expression in
cultured endothelial cells by interfering with NF-B activation (12).
These findings have raised the possibility that at least part of the
cardioprotective effects of estrogen may be due to its ability to
interfere with NF-B-mediated gene activation in the vasculature and
the liver.
NF-B is composed of homodimeric and heterodimeric complexes of the
Rel family of proteins, p65 (Rel A), p50/105, c-Rel, p52/100, and Rel
B. These proteins share a 300-amino acid Rel homology domain
responsible for DNA binding, dimerization, and nuclear localization of
NF-B. This domain is also the target site for binding of the
inhibitory IB proteins (IB, IBß, IB
, Bcl-3, p105
and p100). Binding of IB to NF-B masks the NF-B nuclear
localization signal and sequesters NF-B in a nonactivated form in
the cytoplasm. Cell activation by a variety of extracellular signals,
such as UV irradiation, oxidative stress, cytokines, phorbol esters,
and lipopolysaccharide, induces a cascade of events that leads to
phosphorylation, ubiquitination, and proteolytic removal of IB. This
exposes the NF-B nuclear localization signal, and NF-B then
translocates to the nucleus, where it interacts with regulatory B
elements in gene promoters and enhancers.
Two types of estrogen receptors have been described (ER and ERß),
and both are present in most tissues, including arterial cells (13).
The liver, however, appears to express predominantly ER (14).
Similar to NF-B, ER is a signal (i.e. ligand)-activated
transcription factor that upon activation results in conformational
changes in the receptor followed by recruitment of various
transcription coactivators to form multiprotein complexes responsible
for transcription initiation. Ligands that confer different structural
arrangements to ER and exert tissue selective functions are referred to
as selective estrogen receptor modulators (SERMs). Several of the
coactivators [e.g. steroid receptor coactivator
(SRC)-1, CREB binding protein (CBP)/p300, and
p300/CBP-associated factor (pCAF)] are thought to contribute to
transcriptional activation by chromatin remodeling through their
intrinsic histone acetyltransferase (HAT) activity (reviewed in Ref.
15). It has been recently shown that these coactivators associate with
different transcription factors, including members of the nuclear
receptor family such as ER and NF-B (16, 17, 18).
Recent experiments on the down-regulation of the IL-6 promoter by
estrogen indicated a functional interaction or cross-talk between
ligand-bound ER and NF-B (19, 20, 21). Similarly, antagonism of
NF-B activity has been observed with other nuclear receptors,
including glucocorticoid receptor (GR) (22), androgen receptor (23),
progesterone receptor (24), retinoid X receptor (25), and peroxisome
proliferator-activated receptor (26). The underlying mechanisms for
these interactions are not clearly understood. For GR, the mechanism of
NF-B repression appears to be dependent upon the cell type used and
can involve direct protein-protein interactions (20, 22), induction
of IB expression (27), or competition for limiting coactivators
(28).
This study was undertaken to further understand the mechanisms
behind ER and NF-B cross-talk and to begin to elucidate its
involvement in the cardioprotective actions of estrogens. The data show
that ligand-bound ER antagonizes cytokine-induced NF-B activity
in human hepatoma HepG2 cells without affecting NF-B translocation
into the nucleus or binding to DNA. This antagonism was reciprocal, in
that activated NF-B inhibited the activity of ligand-bound ER.
These functional interactions involved the coactivator CBP that is
common to both NF-B and ER. The data suggest a novel mechanism of
mutual inhibition of NF-B and ER through CBP sharing and may
involve the formation of a nonproductive, transcriptionally inactive
complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B·luciferase (LUC) and estrogen
response element (ERE)·LUC constructs were described previously (29),
as was the pcDNA3-ER expression vector (30). For the mammalian
two-hybrid experiments, the CBP-GAL4 (encompassing amino acids 1–253)
and ER·vasopressin-16 (VP16) constructs were subcloned by PCR
amplification. The pFR·LUC reporter and
p65-trans-activation domain (TAD) were purchased from
Stratagene (La Jolla, CA). The adenoviral vector
expressing the NF-B luciferase reporter was prepared by transferring
the NF-B·LUC reporter segment into an Ad5
E1a plasmid that
contained adenovirus sequences from map unit 0–17 with a deletion of
the E1a region between map unit 1.4–9.1. The Ad5E1a-NF-B·LUC
plasmid was linearized with NdeI and transfected along with
ClaI and a fragment of Ad5 virus with an E3 region deletion
(80–88 map units) into 293 cells. Viral plaques generated by
homologous recombination were isolated, amplified, and characterized by
restriction DNA analysis. Confirmatory tests indicated that the
recombinant virus contained the expected DNA fragments and was
replication defective. The virus was purified, amplified, and tested,
and one plaque was selected as a seed stock and titrated in 293 cells
by plaque assay.
Cell transfections
HepG2 cells were maintained in growth medium at 37 C in a 5%
CO2 incubator. The cells were seeded in deficient
growth medium [phenol red-free DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with heat-inactivated 10%
FBS, 1% Glutamax, 1% MEM nonessential amino acids, 100 U/ml
penicillin, and 100 µg/ml streptomycin] at 2.5 x
105 cells/well in a 12-well dish (Falcon,
Franklin Lakes, NJ) before transfection. The cells were transfected by
the calcium phosphate coprecipitation method as described previously
(31). Luciferase activity was determined by a chemiluminescent method
using a luciferase assay system (Promega Corp., Madison,
WI). ß-Galactosidase activity was determined using Galacto-Light
(Tropix, Inc., Bedford, MA). The data shown represent the mean ±
SEM from at least three independent experiments, each
performed in duplicate.
Hep89 cell adenoviral infections
HepG2 cells stably expressing ER (Hep89) (31) were infected
with the adenoviral vector expressing the NF-B luciferase reporter
with a multiplicity of infection of 100% for 2 h. The cells were
washed with deficient growth medium and allowed to recover overnight.
The next day the cells were cotreated with increasing concentrations of
17ß-estradiol (E2) and interleukin-1ß
(IL-1ß; 100 U/ml) overnight.
Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts were prepared as described previously
(32), and cells were lysed with hypotonic buffer [20 mM
HEPES (pH 7.9), 20 mM NaF, 1 mM
NaV3O4, 1 mM
Na4P2O7,
1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, and 20 µl/ml protease inhibitor cocktail
(Sigma, St. Louis, MO)] containing 0.2% Nonidet P-40 and
centrifuged. The nuclear pellets were resuspended in 150 µl high salt
buffer (hypotonic buffer with 420 mM NaCl and 20%
glycerol), rocked at 4 C for 30 min, and centrifuged (16,000 x
g for 20 min). The resulting nuclear extracts were incubated
with 32P-labeled DNA probes corresponding to the
major histocompatibility complex class I promoter NF-B-binding site,
a consensus SP1 binding site, or the vitellogenin ERE. An
affinity-purified human monoclonal ER antibody (Stress Gen) was used
for supershift experiments. Protein-DNA complexes were analyzed by
electrophoresis in low ionic strength polyacrylamide gels.
Western blot experiments
A portion of the whole cell extracts from the transfected cells
was run on a 4–20% SDS-PAGE, transferred to nitrocellulose, and
blotted using a rabbit anti-p65 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was performed
using the Renaissance Chemiluminescence Western Blotting Detection
System (NEN Life Science Products, Boston, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity (Hep89)
(31) were used to determine whether ligand-bound ER interferes with
cytokine-induced NF-B activity. Hep89 cells were infected with an
adenoviral NF-B luciferase reporter and treated with increasing
concentrations of E2 in the presence of IL-1ß.
In the absence of E2, NF-B luciferase
expression was induced approximately 14-fold. In the presence of
E2, however, luciferase expression decreased by
approximately 60% in a dose-dependent manner (Fig. 1A
). This response was receptor
dependent, as the ER antagonist, ICI 182,780 (ICI), effectively
reversed the E2-mediated inhibition of NF-B
luciferase expression (Fig. 1B), and no inhibition of NF-B reporter
activity occurred with the parental HepG2 cell line, which lacks
endogenous ER.
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ligands may also
influence NF-B transcriptional activity, dose-response experiments
were performed with a panel of natural and synthetic estrogens. As
shown in Table 1, the estrogen
metabolite, 17-ethinyl estradiol, and the phytoestrogen, genistein,
were effective inhibitors of cytokine-induced NF-B transcriptional
activity. In contrast, none of the SERMs, tamoxifen,
4-hydroxytamoxifen, or raloxifene, inhibited NF-B activity.
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interferes with
IL-1ß-induced NF-B activity in HepG2 cells. In addition, these
results indicate that the ability of ER to interfere with NF-B
activity is determined by the chemical nature of the ligand and suggest
that ligand-specific conformations of the ER play a major role in
this cross-talk pathway.
Reciprocal inhibition of ER-mediated transcriptional activity by
NF-B subunits
Although ER bound by certain ligands interfered with
cytokine-induced NF-B activity, it was not clear whether ER
remained transcriptionally active in this context. To address this,
HepG2 cells were cotransfected with an ER expression vector and a
luciferase reporter driven by an ERE (ERE·LUC) and were treated with
E2 and IL-1ß. IL-1ß inhibited the
E2-mediated transcriptional activation of
ERE·LUC by approximately 75% (Fig. 2A). This inhibitory effect was a direct
effect of activated NF-B, as cotransfection with p65 and c-Rel
expression vectors repressed ERE·LUC activity to levels similar to
those observed with IL-1ß. Interestingly, p50 expression did not
significantly inhibit ER activity (Fig. 2A). Reciprocal inhibition
of p65 transcriptional activity by ligand-bound ER was also observed
(Fig. 2B). These results suggest that p65 and c-Rel activation by
IL-1ß are probably involved in inhibition of the transcriptional
activity of ligand-bound ER.
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B DNA binding is not inhibited by
E2 treatmentB activation is its translocation to the
nucleus and binding to B elements in the promoters of its target
genes. To determine whether the inhibition of NF-B transcriptional
activity by ligand-bound ER was due to interference with NF-B
binding to DNA, EMSA experiments were performed. Hep89 cells were
treated with 10-7 M
E2, and nuclear extracts were prepared at various
time points after IL-1ß stimulation. As shown in Fig. 3A, NF-B binding was induced 15 min
after exposure to IL-1ß and returned to basal levels by 1 h,
with a subsequent reinduction by 2 h, consistent with the biphasic
stimulation of NF-B in HepG2 cells (33). Supershift analysis
demonstrated that the slower migrating, upper complex was composed of
p65/p50 heterodimers, whereas the faster migrating, lower complex
contained p50 homodimers (indicated by arrows and data not
shown). The induction of NF-B binding in response to IL-1ß
treatment was specific, as protein binding to an SP1 oligonucleotide
probe was not altered by IL-1ß treatment (Fig. 3B). Exposure of the
cells to E2, however, did not influence NF-B
binding at any time point (Fig. 3A), even after supplementation of
E2 in the binding reaction (Fig. 3C), despite the
presence of ER in these extracts (Fig. 3D).
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does not interfere with the
IL-1ß-mediated NF-B activation, translocation into the nucleus, or
binding to DNA and suggest that ER may target some common component
necessary for NF-B transcriptional activity.
ER limits HAT activity required for maximal function of
NF-B
ER interference with coactivator recruitment to NF-B
could reduce the available coactivator histone acetyl-transferase (HAT)
activity required for NF-B function. To assess whether the loss of
histone acetylation may be involved in the ER-mediated repression of
NF-B activity, cytokine activation of the NF-B reporter in HepG2
cells was monitored in the presence of the deacetylase inhibitor
trichostatin A (TSA), which causes increased histone acetylation.
Although the nucleosome organization occurring with transiently
transfected DNA may be altered compared with that using stably
incorporated DNA (34), TSA can facilitate transcription on transiently
transfected reporters, indicating that transfected DNA can be organized
in nucleoprotein structures sensitive to histone acetylation (35, 36, 37).
As shown in Fig. 4, in the absence of
E2, TSA did not further stimulate NF-B
reporter activity, but did inhibit IL-1ß-induced NF-B activity at
the highest concentration, suggesting that further increases in histone
acetylase activity may result in transcriptional squelching effects.
Nevertheless, even at this high concentration TSA partially reversed
the E2-mediated suppression of NF-B activity.
These data indicate that ligand-bound ER limits the HAT activity
required for maximal NF-B activity and suggest that ER competes
with NF-B for coactivators with intrinsic HAT activity.
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B
inhibitionB activity by ligand-bound ER, vectors
expressing the coactivators CBP or SRC-1a were included in the
cotransfection experiments. More specifically, HepG2 cells were
cotransfected with the NF-B.LUC reporter, a constant amount of ER
expression vector, and increasing concentrations of either CBP or
SRC-1a. In the absence of E2, CBP further
stimulated IL-1ß-induced NF-B trans-activation,
whereas SRC-1a had no effect (see Fig. 5A). As previously shown (38), SRC-1a
potentiated ER-mediated trans-activation of a C3
luciferase reporter (data not shown). In the presence of
E2, CBP, but not SRC-1a, reversed the
E2-mediated repression of IL-1ß-induced NF-B
reporter activity. Similar results were observed with Hep89 cells (data
not shown). The ability of CBP to reverse the ER-mediated repression
appeared to be distinct from its trans-activating functions,
as it occurred at a concentration that did not significantly stimulate
NF-B reporter activity alone. In addition, the CBP-mediated reversal
plateaued, suggesting that additional coactivators may be required.
Western blot analysis showed that the ability of CBP to reverse the
ER-mediated repression did not result from inductions of p65 protein
levels (Fig. 5B). CBP coexpression also reversed the ER-mediated
repression of NF-B reporter activity directed by a vector expressing
the p65 subunit of NF-B (Fig. 5C). These results are consistent with
a model in which ligand-bound ER limits the availability of the
coactivator CBP, thus resulting in reduced NF-B function.
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with the p65-CBP
complex limits CBP available to
NF-B, mammalian two-hybrid studies were conducted in HepG2 cells.
Specifically, we determined whether the CBP interaction with p65 was
altered in the presence of ligand-bound ER. As both p65 and ER
have been shown to interact with the NH2-terminal
domain of CBP (17, 39), a vector expressing an
NH2-terminal fragment of CBP (amino acids 1–253)
fused to the DNA-binding protein GAL4 (CBP-GAL4), was cotransfected
with a vector expressing the p65·TAD (amino acids 364–550; p65-TAD)
or a vector expressing the ER ligand-binding domain fused to the
transcriptional activator VP16 (ER-VP16). As shown in Fig. 6, the CBP construct was capable of
trans-activation alone, as observed previously (17).
Coexpression of ER-VP16 with the CBP-GAL4 vector resulted in a
ligand-dependent 2.7-fold increase in transcriptional activity compared
with that of ER-VP16 alone, whereas coexpression with the p65-TAD
expression vector resulted in a 3.1-fold activation. Interestingly,
when the ER·VP16 expression vector was included in the CBP-GAL4- and
p65-TAD-expressing cells a ligand-dependent synergistic activation
occurred (
12-fold). These results suggest that ligand-bound ER
interacts with p65-bound CBP and implies that the loss of NF-B
functional activity may not occur through ER partitioning of CBP but,
rather, through formation of an ER-, CBP-, NF-B-inactive
complex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B may be involved in both early and late stages of
the inflammatory-proliferative process of atherogenesis, and the
negative cross-talk between ER and NF-B may be a fundamental
mechanism behind estrogen’s cardioprotection. Recently, fibrates,
peroxisome proliferator-activated receptor ligands, have been
demonstrated to decrease COX-2 expression via inhibition of NF-B
activity, suggesting that targeted inhibition of NF-B-related
pathways may be a viable therapeutic intervention for atherosclerosis
(26).
Although cross-talk between ER and NF-B has been previously
observed, the diversity of the mechanisms involved have become apparent
only recently. Thus, direct interactions of ER with NF-B (20, 40), inhibition of NF-B DNA binding (21, 41), and ER-mediated
stabilization of IB (42) have been suggested as potential
mechanisms. In addition, the effectiveness of these cross-talk systems
appears to be determined by cell-specific contexts (43, 44).
Exploration of the mechanism by which ligand-activated ER interferes
with IL-1ß-stimulated NF-B activity in HepG2 cells revealed that
despite the reduced functionality of NF-B, there was very little, if
any, inhibition of its nuclear translocation or DNA-binding activity.
Consistent with this, ligand-activated ER inhibited the
transcriptional activity of the NF-B subunit p65, which activates
transcription in a cytokine-independent fashion. Similar
observations have been reported with other NF-B inhibitors,
including certain flavonoids (45) and high density lipoproteins (46).
This implies that there must be events following binding of NF-B to
DNA, but before transcriptional activation, that are susceptible to
inhibition by ligand-bound ER. The precise nature of these events
and their possible diversity in different cell types is not clearly
understood.
Our findings that supplementation of CBP, but not SRC-1a, was able to
reverse the E2-mediated repression of NF-B
activity adds another mechanism by which ER can exert its action. CBP
does not fully restore IL-1ß-induced NF-B activity, but appears to
plateau, suggesting the involvement of additional coactivators. The
observation that the deacetylase inhibitor TSA partially reversed the
ER-mediated inhibition of NF-B is consistent with the idea that
antagonistic signals from ER and NF-B influence HAT activity
levels associated with these multiprotein complexes and may contribute
to their reduced transcription activation properties. It was recently
shown that the HAT activity associated with NF-B originates from
pCAF, a cofactor that is associated with CBP, but not by CBP alone
(47). It is, therefore, tempting to speculate that as a consequence of
the CBP, ER, and NF-B interaction, the pCAF interaction with CBP
is weakened, resulting in pCAF loss and thus loss of the HAT activity
required for nucleosome restructuring and transcriptional
activation.
The inability of the cofactor SRC-1a to reverse the ER-mediated
inhibition of NF-B is consistent with similar experiments in HeLa
cells (44), but in contrast to its involvement in GR-mediated
inhibition of NF-B (28). As SRC-1a binds to the p50 subunit of
NF-B (48), and the p50 subunit does not interfere with ERE
activation by ligand-activated ER, it is possible that ER
interferes primarily with the p65 subunit of NF-B, whereas GR
influences mainly the p50 subunit. Collectively, these findings raise
the possibility that multiple pathways contribute to the cross-talk
between nuclear receptors and NF-B.
The current studies show that ligand-bound ER inhibits
IL-1ß-stimulated activation of NF-B by an apparently novel
mechanism that involves binding of ligand-activated ER to
NF-B/coactivator CBP complexes, leading to formation of
transcriptionally inactive, higher order complexes. This is based on
the mutual inhibition of ER and NF-B and the two-hybrid
functional assays that indicate cooperative interactions among CBP,
ER, and NF-B. Clearly, direct physical evidence for the existence
of this multiprotein complex will provide additional support for this
mechanism, and attempts to show this by immunoprecipitation are
currently underway.
The striking synergism between ligand-bound ER·VP16 and p65
TAD in activation of the GAL4 DNA-bound CBP, although strongly
suggesting a direct or indirect interaction of these proteins, is at
odds with the repressive effects of ligand-bound ER on cytokine
induction of the NF-B reporter. Although we currently we do not have
a clear explanation for this paradox, there are several possible
contributing factors. First, it has been previously reported that
binding of transcription factors to their cognate sites induces
conformational changes in the transcription factors (49) as well as the
coactivators upon docking to transcription factors (50). Therefore, it
is possible that the conformational change in a coactivator differs
depending on whether the transcription factor is or is not DNA bound.
In the experiments with either the NF-B or the ERE reporters, the
corresponding transcription factors were DNA bound, whereas in the
experiments with the GAL4 reporter neither of these transcription
factors was DNA bound. In addition, depending upon the conformation
change inputs, this could lead to transcription factor synergy or
antagonism. In the case of NF-B and ligand-bound ER in the
functional studies, it suggests that interaction with CBP leads to
input of antagonistic trans-activation signals. This
antagonism could involve CBP conformational changes that are not
compatible with recruitment of additional auxiliary factors necessary
for p65 and CBP nucleoprotein assembly (51). Second, in contrast with
the NF-B and ERE reporter experiments in which the intact CBP was
used, in the experiments with the GAL4 reporter, only a portion of CBP
was used. Thus, any contribution of the deleted portion of CBP would
have been lost in these assays. Nevertheless, whatever the explanations
for these contradictory findings, it is clear that these results point
to a functional interaction among ER, CBP, and NF-B.
It is also important to emphasize that this functional
interaction is clearly dependent upon the chemical character of the
ER ligand. Binding of ligand to ER is thought to induce
ligand-specific conformational changes with different functionalities
in different cell types and tissues. Both 17-ethinyl estradiol and
genistein resulted in synergistic activation in the mammalian
two-hybrid assays, whereas tamoxifen and raloxifene did not promote
this activity (data not shown), consistent with the functional data.
Thus, although all of these compounds have been reported to be
effective in lowering cholesterol (52, 53, 54), they appear to be
drastically different in their abilities to inhibit NF-B in HepG2
cells. This strongly suggests that only specific conformations of ER
induced by specific ligands can participate in the formation of this
multiprotein complex and NF-B inhibition. This has important
implications for the use of SERMs as therapeutics, because it raises
the possibility of pathway-selective estrogens as therapeutics in
addition to the current tissue-selective estrogens concept. It will,
therefore, be of great interest to determine whether the differences in
NF-B inhibitory properties of the above compounds and other SERMs
correlate with differences in different functional end points in
vivo. In particular, due to the recent interest in the use of
SERMs as cardiovascular therapeutics and the fundamental role of
NF-B in inflammatory processes associated with cardiovascular
disease, it will be important to determine the activity of clinically
used SERMs in their function as NF-B inhibitors.
In summary, we provided experimental evidence in support of a novel
mechanism by which ligand-activated ER inhibits cytokine-induced
NF-B activity in human liver cells through CBP sharing and may
result in the formation of a transcriptionally inactive multiprotein
complex. The dependence of this interaction on the chemical nature of
the ER ligand raises the possibility of the discovery of ER
agonists with antiinflammatory cardioprotective effects without the
common side-effects observed with estrogens.
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Acknowledgments |
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B luciferase reporter. |
Footnotes |
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Received February 18, 2000.
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K. J. Higgins, S. Liu, M. Abdelrahim, K. Vanderlaag, X. Liu, W. Porter, R. Metz, and S. Safe Vascular Endothelial Growth Factor Receptor-2 Expression Is Down-Regulated by 17{beta}-Estradiol in MCF-7 Breast Cancer Cells by Estrogen Receptor {alpha}/Sp Proteins Mol. Endocrinol., February 1, 2008; 22(2): 388 - 402. [Abstract] [Full Text] [PDF]
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 R. A. Ansari and J. Gandy Determining the Transrepression Activity of Xenoestrogen on Nuclear Factor-{kappa}B in Cos-1 Cells by Estrogen Receptor-{alpha} International Journal of Toxicology, September 1, 2007; 26(5): 441 - 449. [Abstract] [Full Text] [PDF]
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 R. H. Straub The Complex Role of Estrogens in Inflammation Endocr. Rev., August 1, 2007; 28(5): 521 - 574. [Abstract] [Full Text] [PDF]
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 R. Dai, R. A. Phillips, and S. A. Ahmed Despite Inhibition of Nuclear Localization of NF-{kappa}B p65, c-Rel, and RelB, 17-beta Estradiol Up-Regulates NF-{kappa}B Signaling in Mouse Splenocytes: The Potential Role of Bcl-3 J. Immunol., August 1, 2007; 179(3): 1776 - 1783. [Abstract] [Full Text] [PDF]
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 J M W Gee, V E Shaw, S E Hiscox, R A McClelland, N K Rushmere, and R I Nicholson Deciphering antihormone-induced compensatory mechanisms in breast cancer and their therapeutic implications Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S77 - S88. [Abstract] [Full Text] [PDF]
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 H.-C. Shih, C.-L. Lin, T.-Y. Lee, W.-S. Lee, and C. Hsu 17{beta}-Estradiol Inhibits Subarachnoid Hemorrhage-Induced Inducible Nitric Oxide Synthase Gene Expression by Interfering With the Nuclear Factor {kappa}B Transactivation Stroke, December 1, 2006; 37(12): 3025 - 3031. [Abstract] [Full Text] [PDF]
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 E. R. LaVallie, P. S. Chockalingam, L. A. Collins-Racie, B. A. Freeman, C. C. Keohan, M. Leitges, A. J. Dorner, E. A. Morris, M. K. Majumdar, and M. Arai Protein Kinase C{zeta} Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-{kappa}B by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes J. Biol. Chem., August 25, 2006; 281(34): 24124 - 24137. [Abstract] [Full Text] [PDF]
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 J. K. Kundu, Y. K. Shin, S. H. Kim, and Y.-J. Surh Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-{kappa}B in mouse skin by blocking I{kappa}B kinase activity Carcinogenesis, July 1, 2006; 27(7): 1465 - 1474. [Abstract] [Full Text] [PDF]
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 S. Olivier, P. Close, E. Castermans, L. de Leval, S. Tabruyn, A. Chariot, M. Malaise, M.-P. Merville, V. Bours, and N. Franchimont Raloxifene-Induced Myeloma Cell Apoptosis: A Study of Nuclear Factor-{kappa}B Inhibition and Gene Expression Signature Mol. Pharmacol., May 1, 2006; 69(5): 1615 - 1623. [Abstract] [Full Text] [PDF]
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B. S. Komm, Y. P. Kharode, P. V. N. Bodine, H. A. Harris, C. P. Miller, and C. R. Lyttle Bazedoxifene Acetate: A Selective Estrogen Receptor Modulator with Improved Selectivity Endocrinology, September 1, 2005; 146(9): 3999 - 4008. [Abstract] [Full Text] [PDF]
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Y Zhou, S Eppenberger-Castori, U Eppenberger, and C C Benz The NF{kappa}B pathway and endocrine-resistant breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S37 - S46. [Abstract] [Full Text] [PDF]
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G. Soucy, G. Boivin, F. Labrie, and S. Rivest Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain J. Immunol., May 15, 2005; 174(10): 6391 - 6398. [Abstract] [Full Text] [PDF]
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 C. C. Chadwick, S. Chippari, E. Matelan, L. Borges-Marcucci, A. M. Eckert, J. C. Keith Jr., L. M. Albert, Y. Leathurby, H. A. Harris, R. A. Bhat, et al. Identification of pathway-selective estrogen receptor ligands that inhibit NF-{kappa}B transcriptional activity PNAS, February 15, 2005; 102(7): 2543 - 2548. [Abstract] [Full Text] [PDF]
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 M. E. Jung, M. B. Gatch, and J. W. Simpkins Estrogen Neuroprotection Against the Neurotoxic Effects of Ethanol Withdrawal: Potential Mechanisms Experimental Biology and Medicine, January 1, 2005; 230(1): 8 - 22. [Abstract] [Full Text] [PDF]
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K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho Estrogen Regulation of Growth Hormone Action Endocr. Rev., October 1, 2004; 25(5): 693 - 721. [Abstract] [Full Text] [PDF]
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 D. C. Harnish, L. M. Albert, Y. Leathurby, A. M. Eckert, A. Ciarletta, M. Kasaian, and J. C. Keith Jr. Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G118 - G125. [Abstract] [Full Text] [PDF]
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 H. Liu, E.-S. Lee, C. Gajdos, S. T. Pearce, B. Chen, C. Osipo, J. Loweth, K. McKian, A. De Los Reyes, L. Wing, et al. Apoptotic Action of 17{beta}-Estradiol in Raloxifene-Resistant MCF-7 Cells In Vitro and In Vivo J Natl Cancer Inst, November 5, 2003; 95(21): 1586 - 1597. [Abstract] [Full Text] [PDF]
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 K. Watashi, M. Hijikata, A. Tagawa, T. Doi, H. Marusawa, and K. Shimotohno Modulation of Retinoid Signaling by a Cytoplasmic Viral Protein via Sequestration of Sp110b, a Potent Transcriptional Corepressor of Retinoic Acid Receptor, from the Nucleus Mol. Cell. Biol., November 1, 2003; 23(21): 7498 - 7509. [Abstract] [Full Text] [PDF]
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J. Frasor, J. M. Danes, B. Komm, K. C. N. Chang, C. R. Lyttle, and B. S. Katzenellenbogen Profiling of Estrogen Up- and Down-Regulated Gene Expression in Human Breast Cancer Cells: Insights into Gene Networks and Pathways Underlying Estrogenic Control of Proliferation and Cell Phenotype Endocrinology, October 1, 2003; 144(10): 4562 - 4574. [Abstract] [Full Text] [PDF]
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K. Lai, D. C. Harnish, and M. J. Evans Estrogen Receptor {alpha} Regulates Expression of the Orphan Receptor Small Heterodimer Partner J. Biol. Chem., September 19, 2003; 278(38): 36418 - 36429. [Abstract] [Full Text] [PDF]
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 B. Martin-McNulty, D. M. Tham, V. da Cunha, J. J. Ho, D. W. Wilson, J. C. Rutledge, G. G. Deng, R. Vergona, M. E. Sullivan, and Y.-X. Wang 17{beta}-Estradiol Attenuates Development of Angiotensin II-Induced Aortic Abdominal Aneurysm in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1627 - 1632. [Abstract] [Full Text] [PDF]
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K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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 K. M. M. Kelley, B. G. Rowan, and M. Ratnam Modulation of the Folate Receptor {alpha} Gene by the Estrogen Receptor: Mechanism and Implications in Tumor Targeting Cancer Res., June 1, 2003; 63(11): 2820 - 2828. [Abstract] [Full Text] [PDF]
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M. R. Adams, D. L. Golden, T. C. Register, M. S. Anthony, J. B. Hodgin, N. Maeda, and J.K. Williams The Atheroprotective Effect of Dietary Soy Isoflavones in Apolipoprotein E-/- Mice Requires the Presence of Estrogen Receptor-{alpha} Arterioscler. Thromb. Vasc. Biol., November 1, 2002; 22(11): 1859 - 1864. [Abstract] [Full Text] [PDF]
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P. Kannan-Thulasiraman and D. J. Shapiro Modulators of Inflammation Use Nuclear Factor-kappa B and Activator Protein-1 Sites to Induce the Caspase-1 and Granzyme B Inhibitor, Proteinase Inhibitor 9 J. Biol. Chem., October 18, 2002; 277(43): 41230 - 41239. [Abstract] [Full Text] [PDF]
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M. J. Evans, H. A. Harris, C. P. Miller, S. K. Karathanasis, and S. J. Adelman Estrogen Receptors {alpha} and {beta} Have Similar Activities in Multiple Endothelial Cell Pathways Endocrinology, October 1, 2002; 143(10): 3785 - 3795. [Abstract] [Full Text] [PDF]
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 K. K. Koh Effects of estrogen on the vascular wall: vasomotor function and inflammation Cardiovasc Res, September 1, 2002; 55(4): 714 - 726. [Full Text] [PDF]
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M. J. Evans, K. Lai, L. J. Shaw, D. C. Harnish, and C. C. Chadwick Estrogen Receptor {alpha} Inhibits IL-1{beta} Induction of Gene Expression in the Mouse Liver Endocrinology, July 1, 2002; 143(7): 2559 - 2570. [Abstract] [Full Text] [PDF]
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R.-C. Wu, J. Qin, Y. Hashimoto, J. Wong, J. Xu, S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator Activity by I{kappa}B Kinase Mol. Cell. Biol., May 15, 2002; 22(10): 3549 - 3561. [Abstract] [Full Text] [PDF]
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J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
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 R. V. Sharma, M. V. Gurjar, and R. C. Bhalla Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappa B activation in VSM cells from female rats J Appl Physiol, November 1, 2001; 91(5): 2400 - 2406. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, E. M. Resnick, V. Y. Lin, and M. A. Shupnik Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways Endocrinology, August 1, 2001; 142(8): 3361 - 3368. [Abstract] [Full Text] [PDF]
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 M. J. Evans, A. Eckert, K. Lai, S. J. Adelman, and D. C. Harnish Reciprocal Antagonism Between Estrogen Receptor and NF-{kappa}B Activity In Vivo Circ. Res., October 26, 2001; 89(9): 823 - 830. [Abstract] [Full Text] [PDF]
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