From the Department of Nuclear Receptors, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Estrogen replacement therapy increases plasma
concentrations of high density lipoprotein and its major protein
constituent, apolipoprotein AI (apoAI). Studies with animal model
systems, however, suggest opposite effects. In HepG2 cells stably
expressing estrogen receptor (ER
), 17
-estradiol (E2) potently
inhibited apoAI mRNA steady state levels. ApoAI promoter deletion
mapping experiments indicated that ER
plus E2 inhibited apoAI
activity through the liver-specific enhancer. Although the ER
DNA
binding domain was essential but not sufficient for apoAI enhancer
inhibition, ER
binding to the apoAI enhancer could not be detected
by electrophoretic mobility shift assays. Western blotting and
cotransfection assays showed that ER
plus E2 did not influence the
abundance or the activity of the hepatocyte-enriched factors HNF-3
and HNF-4, two transcription factors essential for apoAI enhancer
function. Expression of the ER
coactivator RIP140 dramatically
repressed apoAI enhancer function in cotransfection experiments,
suggesting that RIP140 may also function as a coactivator on the apoAI
enhancer. Moreover, estrogen regulation of apoAI enhancer activity was
dependent upon the balance between ER
and RIP140 levels. At low
ratios of RIP140 to ER
, E2 repressed apoAI enhancer activity,
whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ER
and E2 but also upon the
intracellular balance of ER
and coactivators utilized by ER
and
the apoAI enhancer.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apolipoprotein AI (apoAI)1 is the major protein constituent of plasma high density lipoproteins (HDLs), a class of lipoproteins thought to play a major role in protection against atherosclerosis (reviewed in Ref. 1). Because plasma HDL levels are correlated with plasma apoAI and liver apoAI mRNA levels (2), it is thought that factors affecting apoAI gene expression play an important role in atherosclerosis susceptibility. Although a number of pharmacological, dietary, and physiological factors affect apoAI and HDL plasma levels (3-8), the underlying molecular mechanisms remain obscure. For example, numerous observational studies and a recent randomized trial have shown that estrogen replacement therapy increases apoAI and HDL plasma levels (9-11). However, the underlying molecular mechanism(s) remains controversial. Both increased production of apoAI (12, 13) and reduced HDL catabolism (14) have been suggested as potential mechanisms.
Work with animals has further complicated the issue. In cynomolgus monkeys, ethinyl estradiol or conjugated equine estrogen markedly reduces apoAI and HDL plasma levels (15, 16). Similarly, ovariectomy increases hepatic apoAI mRNA levels in rats, further supporting the concept that estrogen may repress apoAI gene expression (17). Moreover, the ethinyl estradiol-induced increases in apoAI mRNA levels in these animals appears to occur via indirect dietary effects due to hormone treatment (17). Further, estrogen-induced increases in apoAI transcription rates in rats are dependent on the strains used (6).
Liver-specific expression of the apoAI gene is conferred by a powerful
hepatocyte-specific enhancer located in the nucleotide region 220 to
110 upstream of the apoAI transcriptional start site (18). The
activity of the enhancer depends on synergistic interactions between
transcription factors bound to three distinct sites: A (
214 to
192), B (
169 to
146), and C (
134 to
119) within the enhancer
(18, 19). Sites A and C bind various members of the nuclear receptor
superfamily including the hepatocyte nuclear factor 4, HNF-4 (20-22),
retinoid X receptor
(23), and apolipoprotein AI regulatory
protein-1 (24, 25). Site B binds members of the hepatocyte nuclear
factor 3 family, HNF-3 (19, 26). Synergy between these factors during
enhancer activation appears to involve interactions with
uncharacterized transcription auxiliary factors (18, 26). Recent
evidence suggests that one or more of these factors are regulated by
estrogen in heterologous nonhepatic cells (26).
The actions of estrogen are mediated primarily by the estrogen
receptors (ERs) (27) and
(28, 29); however, only ER
is
expressed in the liver (28, 29). Estrogen signal transduction involves
high affinity binding to intracellular ERs, ligand-induced conformational changes of ERs leading to the recruitment of
transcriptional auxiliary factors, binding of ERs to estrogen response
elements (EREs) in gene promoters, and regulation of transcriptional
activity in conjunction with other transcription factors bound to their cognate sites in the promoter. Recent efforts to characterize ER
transcription auxiliary factors have led to the identification of a
growing number of coactivators such as Trip/Sug1 (30, 31), ERAP140 and
ERAP160 (32), RIP140 (33), TIF1 (34), and SRC-1 (35, 36). Although all
of these proteins bind ER
in a ligand-dependent fashion,
the mechanisms by which they modulate ER signaling and "cross-talk"
with other signal transduction pathways is not understood. Recent
findings indicate that the related coactivators p300 (37) and CBP (38)
are also involved in ER
function and serve as a signal integrator
for several hormone-dependent and hormone-independent signal transduction pathways (Refs. 36, 39, and 40 and reviewed in Ref.
41). Additional pathways of liganded ER action involve recruitment of
ER
to gene promoters lacking EREs via protein-protein interactions
with promoter-bound transcription factors (42-45) and activation of
the mitogen-activated protein kinase pathway (46, 47).
This report shows that ER and 17
-estradiol repress apoAI promoter
activity in human hepatoma HepG2 cells. This effect appears to be due
to ER
partitioning of coactivators required for apoAI enhancer
function in liver cells. The data suggest that RIP140 may play an
important role in apoAI enhancer function and ER
-mediated repression. We propose that estrogen effects on apoAI gene expression vary in direction and magnitude depending upon the balance of coactivators shared by ER
and the apoAI enhancer.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid Constructions--
The 2500AI.LUC.CIII/AIV construct
was created by transferring a 3.0-kilobase HindIII 5' apoAI
DNA fragment and a 7-kilobase BamHI 3' apoAI fragment from
the previously reported construct
2500AI.CAT[CIII/AIV] (21) into
their respective sites in pGL2-Basic (Promega). The
2500AI.LUC
construct was created by transferring only the 3.0-kilobase
HindIII fragment into pGL2-Basic. The
256AI.LUC,
220/
110AI.LUC, and
41/+397.LUC constructs were created by
transferring their respective HindIII fragments from
256AI.CAT,
222[
110/
41]AI.CAT, and
41AI.CAT, respectively
(18), into pGL2-Basic. Construct
220/
110ABC.LUC was generated by
transferring an 110-base pair BamHI fragment from
222[
110/41]AI.CAT (18) into
41.LUC (19). The apoAI
enhancer-type mutants were created by transferring the approximately
110-base pair BamHI fragments from the corresponding chloramphenicol acetyltransferase (CAT) constructs (18) into
41.LUC
(19). Construct TK.LUC was generated by cloning a
105/+10 NheI/HindIII TK promoter fragment into
pGL2-Basic. Construct
220/
110ABC/TK.LUC was generated by
transferring a 110-base pair BamHI DNA fragment from
222[
110/41]AI.CAT (18) into TK.LUC. The A.LUC, B.LUC, and
ERE.LUC constructs were described previously (19, 48).
Stable Cell Line Creation--
HepG2 cells stably expressing
ER (Hep89) were created by transfecting the pcDNA3-ER
expression vector into HepG2 cells by electroporation using the BTX
Electro Cell Manipulator 600 according to the manufacturer's
recommended settings. Stably expressing cells were selected by
resistance to G418 (400 µg/ml). Distinct, well isolated colonies were
picked using Bellco cloning cylinders (6 × 8 mm) and assessed for
the presence of ER
.
Cell Transfections--
Plasmid DNAs were purified on Qiagen
columns and transfected into HepG2 cells by the calcium phosphate
coprecipitation method as described previously (26). The cells were
seeded in deficient growth media (phenol red-free Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with
heat-inactivated 10% fetal bovine serum, 1% Glutamax, 1% minimum
essential medium nonessential amino acids, 100 units/ml penicillin and
100 µg/ml streptomycin) at 2.5 × 105 cells/well in
a 12-well dish (Falcon) before transfection. Different amounts of the
expression vectors pMT2-ER, pMT2-HNF-4 (52), pCMV.HNF-3
(53),
pEFRIP (33), or RSV-CBP-HA (38) were cotransfected as indicated.
Luciferase and
-galactosidase activity was determined as described
previously (26). The data shown represent the mean ± S.E. from at
least three independent experiments, each in duplicate. Statistical
analysis of the data was carried out using the Dunnett's method (54)
to compare treated versus control samples.
Northern Analysis-- HepG2 and Hep89 cells were seeded in deficient growth media and treated over 72 h in the presence or absence of 1 µM E2. Total RNA was isolated (Biotecx Labs), subjected to electrophoresis, and hybridized with 32P-labeled apoAI PstI cDNA fragment (55) or 32P-labeled human glyceraldehyde-3-phosphate dehydrogenase cDNA (Stratagene). The relative intensities of the hybridized signals were quantitated by phosphoimaging (Molecular Dynamics).
Electrophoretic Mobility Shift Assays--
Protein-DNA complexes
were analyzed by incubation of bacterially expressed HNF-4, HepG2
nuclear extracts (26), or baculovirus-expressed human ER (Panvera)
with 32P-labeled DNA probes corresponding to either the
110-base pair apoAI enhancer or the vitellogenin ERE followed by
electrophoresis in low ionic strength polyacrylamide gels as described
previously (26).
Western Blot Experiments--
Nuclear extracts were prepared as
described previously (26). Protein concentrations were determined by
the BCA method. Proteins were transferred from a 4, 20%
SDS-polyacrylamide gel to nitrocellulose and blotted using
affinity-purified human monoclonal ER antibody (Stress Gen), rabbit
anti-HNF-3 antibody (gift of R. Costa), or rabbit anti-HNF4 serum
(from F. Sladek) as the primary antibodies followed by
peroxidase-conjugated goat anti-rabbit IgG antibody (Zymed
Laboratories Inc.). Detection was performed using the Enhanced Chemiluminescence Western blotting Detection System (Amersham Pharmacia
Biotech).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Estrogen Represses apoAI mRNA Levels in HepG2 Cells Stably
Expressing ER--
Human hepatoma HepG2 cells retain many
liver-specific functions; however, they no longer express ER
.
Therefore, HepG2 cells stably expressing ER
were created to monitor
the regulation of the apoAI gene by estrogen. The resulting cell line,
Hep89, expressed ER
by Western blot (Fig.
1A). Further, the activity of
a synthetic vitellogenin estrogen response element luciferase reporter
(ERE.LUC) was stimulated 25-fold by 100 nM 17
-estradiol
(E2) in Hep89 cells, whereas no E2-dependent promoter
activity was observed in the parental HepG2 cells (Fig. 1B).
Northern blotting demonstrated a 2-3-fold decrease in apoAI mRNA
steady state levels in Hep89 cells after a 72-h treatment with 1 µM E2, whereas apoAI mRNA levels remained unchanged
in HepG2 cells (Fig. 1, C and D). Therefore, apoAI mRNA steady state levels are regulated by E2 in a
receptor-dependent manner.
|
Estrogen Represses apoAI Promoter Activity in HepG2 Cells--
The
ER effects on apoAI mRNA levels could be due to changes in
mRNA stability or apoAI transcription. To determine the potential of ER
to regulate apoAI gene transcription, a luciferase reporter under the control of both 5'- and 3'-flanking regulatory sequences of
the human apoAI gene (reporter-2500 AI.LUC.CIII/AIV, Ref. 21) was
cotransfected with an ER
expression vector into HepG2 cells. After
transfection, the cells were treated with estrogen agonists and
antagonists, and the reporter activity in cell extracts was determined.
Dose-response experiments with E2 resulted in a 75% maximal repression
of apoAI promoter activity with an EC50 value of
approximately 12 nM (Fig.
2A). The repression was both
ligand- and receptor-dependent, since it did not occur with
either ER
or E2 alone (Fig. 2B). The estrogen receptor
antagonist ICI 182,780 (1 µM) also repressed promoter
activity but to a lesser extent (30% repression). A 10-fold molar
excess of ICI (1 µM) over E2 (100 nM)
effectively competed the E2-mediated repression to the level seen with
ICI alone. ERE.LUC reporter activity was also regulated by ER
in a
ligand-dependent fashion, except that in contrast to the
apoAI promoter, ICI acted as a pure antagonist for ERE activation. The
mechanism of ER
repression on the apoAI promoter may be distinct
from those involved in ER
activation of an ERE.
|
Mapping of the apoAI Promoter Estrogen Response Element--
A
collection of apoAI promoter reporters (18) was used to delineate
elements involved in ER-induced repression of the apoAI promoter.
Deletion of the entire 3'-flanking region of the apoAI gene (reporter
2500 AI.LUC) or deletion of both the 3' region and approximately 2.25 kilobases of the 5' region (reporter
256AI.LUC) did not affect ER
and E2-induced repression (Fig.
3A). A reporter containing
only the apoAI hepatocyte-specific enhancer driving the expression of
the apoAI basal promoter (reporter
220/
110AI.LUC) was also
repressed approximately 60% by ER
plus E2. The activity of the
apoAI enhancer in a heterologous reporter containing the thymidine
kinase promoter was also repressed by ER
plus E2, whereas the
thymidine kinase basal promoter reporter activity remained unaffected
(Fig. 3B), demonstrating that ER
regulation occurs directly on the apoAI enhancer.
|
The ER DNA Binding Domain and Transcription Activation Functions
1 or 2 Are Necessary for apoAI Enhancer Repression--
To further
probe the mechanism by which ER
and E2 repressed the apoAI enhancer,
vectors expressing ER
with a deletion in the N-terminal
transcription activation function (AF1) or point mutations in the
C-terminal transcription activation function (AF2) or in the DBD were
cotransfected with either the
220/
110 ABC.LUC or ERE.LUC reporter
into HepG2 cells (Fig. 4). Deletion of
AF1 (mutant X-DBD-AF2) had no effect on apoAI enhancer repression but
inhibited ERE activation by 80%. In contrast, inactivation of AF2
(mutant AF1-DBD-X) diminished both apoAI repression and ERE activation.
Inactivation of both AF1 and AF2 completely abolished both apoAI
repression and ERE activation. Finally, point mutations within the DNA
binding domain that converts its binding selectivity from an ERE to a
glucocorticoid response element (mutant AF1-X-AF2) eliminated
repression of the apoAI enhancer. As shown previously (51), this ER
DNA binding domain mutant activated a reporter driven by a
glucocorticoid response element (data not shown).
|
ER Does Not Bind to the apoAI Enhancer--
The requirement of
an intact DNA binding domain for ER
repression of the apoAI enhancer
suggested that ER
could bind directly to the enhancer and interfere
with the synergistic interactions between bound transcription factors.
We therefore determined if partially purified ER
binds to the
220/
110 apoAI enhancer fragment by electrophoretic mobility shift
assays. Under these conditions, ER
did not bind to the apoAI
enhancer but bound efficiently to the vitellogenin ERE as expected
(Fig. 5, A and B).
Control experiments showed that HNF-4 bound strongly to the apoAI
enhancer as expected (20, 25) (Fig. 5A) and weakly to the
vitellogenin ERE (Fig. 5B). Since HNF-4 does not activate
the ERE.LUC reporter in HepG2 cell transfections (data not shown), this
binding interaction is unlikely to occur in the intact cell. The
presence of additional cellular proteins did not overcome the inability
of ER
to bind to the apoAI enhancer, as demonstrated in experiments
comparing extracts from HepG2 cells and Hep89 cells (Fig.
5C) or when HepG2 extracts were supplemented with ER
(Fig. 5D). In both instances, no ER
binding to the
enhancer could be detected, although binding to the ERE was observed.
Thus even under conditions where nonspecific binding of HNF-4 to a
noncognate sequence could be detected, no direct binding of ER
to
the apoAI enhancer was observed.
|
ER and E2 Do Not Influence HNF-4 or HNF-3
Abundance or
Function--
HNF-4, which binds to sites A and C in the apoAI
enhancer (20, 22, 25), and HNF-3
, which binds to site B (19), are two fundamental transcription factors involved in apoAI enhancer function. Ligand-activated ER
could repress apoAI enhancer activity by reducing the abundance or by inhibiting the activation properties of
endogenous HNF-4, HNF-3
, or both (see for example Refs. 42 and 56).
To examine the first possibility, the effects of E2 on the relative
abundance of HNF-4 and HNF-3
in Hep89 cells was determined by
Western blotting analysis. As shown in Fig.
6, A and B, neither
HNF-4 nor HNF-3
protein levels were altered after treatment with
E2.
|
RIP140 Is a Coactivator for the apoAI Enhancer--
Although
optimal transcriptional activity of the apoAI enhancer depends on both
DNA-bound transcription factors and non-DNA-bound coactivators (18,
26), there is no information regarding the identity of these
coactivators. We tested two well characterized ER coactivators, CBP
(38) and RIP140 (33) for their possible involvement in apoAI enhancer
activity. Specifically, the apoAI enhancer reporter (
220/
110
ABC.LUC) was cotransfected with increasing amounts of either RIP140 or
CBP expression vectors. CBP did not alter apoAI enhancer activity. In
contrast, RIP140 repressed activity in a dose-dependent
fashion (Fig. 7), reminiscent of its
squelching affects on ER
activation of ERE reporters (33). RIP140
did not affect the function of an SV40 enhancer driven reporter (data not shown), demonstrating that the RIP140 effect was specific to the
apoAI enhancer.
|
The Role of Coactivator Partitioning in ER-mediated Repression
of the apoAI Enhancer--
Transcriptional interference among nuclear
receptors is due, at least in part, to partitioning of limited amounts
of shared transcriptional coactivators (36, 39). It is therefore
possible that ER
and the apoAI enhancer share common cofactors and
that partitioning of these cofactors to ER
results in repression of the enhancer. To test this possibility, HepG2 cells were cotransfected with the
220/
110 ABC.LUC reporter, a constant amount of ER
expression vector, and increasing amounts of RIP140 expression vector.
In the absence of E2, increasing amounts of RIP140 expression vector
repressed apoAI enhancer activity as observed above (Fig. 8A). However, in the presence
of E2, low amounts of RIP140 expression vector (50 ng) reversed ER
and E2-induced repression from 70% to approximately 35%. In fact, the
reduced enhancer activity obtained at high RIP140 levels was actually
stimulated 3-fold by ER
in an E2-dependent fashion (Fig.
8B). RIP140 does not appear to repress ER
production as
determined by transient transfections in which different
promoter/enhancers (i.e. CMV enhancer or adenovirus major
late promoter) used to drive expression of ER
gave similar results
(data not shown). Together, these data indicate that ER
can affect
apoAI gene expression via transcription coactivators and that RIP140 or
an endogenous RIP140-like protein may be involved in apoAI enhancer
regulation by ER
and E2.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The apoAI liver-specific enhancer plays a central role in integration of diverse physiological and environmental signals affecting apoAI gene expression. Our working hypothesis is that signal-induced transient multiprotein complexes containing both DNA binding factors and factors not directly bound to DNA assemble onto the apoAI enhancer and regulate one or more steps in transcription initiation.
In this study, estrogenic signals influenced apoAI expression at the
mRNA level by repressing apoAI gene transcription. More specifically, ER and E2 potently repressed apoAI liver-specific enhancer activity in a ligand- and receptor-dependent
manner. Although gel shift experiments did not provide evidence for
ER
binding to the enhancer, the DBD was essential for apoAI
repression. In addition, the ER
transcription activation functions
AF1 and AF2, although not sufficient by themselves, were individually essential for repression when associated with the DBD. Experiments with
ER
mutants showed that the combination of the DBD and AF2 domain is
as effective in enhancer repression as the wild type ER
, whereas the
combination of the DBD and AF1 domain is only 50% as effective as
full-length ER
. All ER
mutants tested were capable of ligand
binding (51, 57, 58), and apoAI enhancer repression by them was
strictly ligand-dependent. Thus, it appears that a
ligand-induced change in the receptor cooperates with the DBD and
either AF1 or AF2 to impart an apoAI enhancer repressing activity.
How does ER and E2 repress apoAI enhancer function? Previous studies
showed that maximal activity of the apoAI enhancer depends on
synergistic interactions between transcription factors bound to
enhancer sites A, B, and C (18). Because ER
does not bind to the
enhancer under our gel-shifting conditions, repression mechanisms
involving transcription interference by quenching (59) seem unlikely.
ER
may repress the apoAI enhancer by inhibiting the activity of
transcription factors required for enhancer function (see for example
Refs. 42 and 56). For example, we have recently observed that
adenovirus E1A inhibits apoAI enhancer activity by selective
inactivation of HNF-3, the factor that binds to site B.2 To test this possibility,
two liver-specific transcription factors, HNF-4 and HNF-3
, involved
in apoAI enhancer function were assayed. The results showed that
neither HNF-3
nor HNF-4 activities or protein levels were influenced
by ER
plus E2, suggesting that ER
represses the apoAI enhancer at
some other level. Although the possibility that ER
activates a
repressing transcription factor that binds to the enhancer and inhibits
its function cannot be unequivocally excluded, the observation that
ER
could mediate apoAI enhancer repression independent of individual
cis-elements mutations within the enhancer suggests that repression
occurs at a level secondary to DNA binding.
An alternative explanation for the ER- and E2-induced repression of
the apoAI enhancer is that a coactivator common to the enhancer and
ER
is partitioned to ER
, leading to transcriptional interference
with the enhancer. HNF-4, which binds to site A, is a member of the
nuclear receptor superfamily and could share coactivators with ER
similar to other nuclear receptors (reviewed in Ref. 41). For example,
partitioning of p300/CBP to ER
was recently shown to be the major
mechanism of transcriptional interference between ER
and the
progesterone receptor (39) or the transcription factor AP-1 (36). The
involvement of coactivators in nuclear receptor function and their
importance in apoAI enhancer function prompted us to test the
possibility that CBP or RIP140 is involved in apoAI enhancer function.
The results showed that expression of CBP does not affect apoAI
enhancer activity. However, similar experiments with RIP140 showed that
this cofactor repressed apoAI enhancer activity, reminiscent of the
RIP140 squelching effects on ER
activation (33). In the presence of
E2, low RIP140 expression vector levels reversed the ER
-mediated
repression of apoAI enhancer activity. Therefore, RIP140-like factors
may be one class of non-DNA binding coactivators involved in apoAI
enhancer function that are partitioned to ER
.
The observation that two different activation functions in ER
(i.e. AF1 and AF2) can in conjunction with the DBD repress apoAI enhancer activity independently of each other raises the possibility that ER
partitions an additional cofactor(s) involved in
apoAI enhancer function. Consistent with this, RIP140 interaction with
ER
requires a functional AF2 domain (60) that is inactive in the
ER
mutant AF1-DBD-X that represses enhancer activity. Therefore a
model whereby, in addition to RIP140, ER
shares other cofactors with
the apoAI enhancer and RIP140 binding to ER
alters its conformation,
so that some, but not all, of these factors are released and used by
the enhancer is possible.
How could the disparities regarding ER regulation of apoAI and HDL
plasma levels between different cell and animal systems (see the
Introduction) be reconciled? It is clear from our previous work that
the multiprotein complexes assembled onto the apoAI enhancer are
transient, and their protein composition is influenced by the
prevailing developmental, physiological, and environmental factors
affecting apoAI gene expression. For example, although apoAI enhancer
activity does not depend on retinoids, prior repression of enhancer
activity by the nuclear receptor apolipoprotein AI regulatory protein-1
converts the enhancer into a retinoid-responsive element (61). A
similar phenomenon may be occurring with E2 in which the state of the
apoAI enhancer due to intracellular coactivator levels determines the
mode of ER
regulation. When cofactors shared by the apoAI enhancer
and ER
are present in limiting amounts compared with ER
, their
partitioning by ER
will result in enhancer inhibition. This latter
case appears to be operating in the Hep89 cell system. In contrast,
when these cofactors are in excess compared with ER
and apoAI
enhancer activity is partially repressed due to endogenous squelching,
estrogen-activated ER
will alleviate repression by partitioning
cofactors in excess and result in enhancer stimulation. Consistent with
this, the low level enhancer activity obtained at high RIP140
expression vector levels was stimulated 3-fold by ER
in a
ligand-dependent fashion. In addition, differences in ER
affinity for these coactivators, induced by different ligands, may
explain the opposite effects of different estogen agonists and
antagonists on apoAI plasma levels (62, 63). Therefore, we propose that
the effects of estrogen on apoAI gene transcription will depend upon
the balance between cofactors shared by the apoAI enhancer and ER
and may explain the disparities observed for apoAI gene regulation by E2 obtained using different cell and animal systems.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Goodman, R. Costa, F. Sladek, M. Parker, and P. Chambon for providing the expression vectors
for CBP, HNF-3, HNF-4, RIP140, and ER, respectively, and Dr. E. Allegretto for providing the various mutant ER
expression vectors.
We are also grateful to E. Ferris for the cell culture work.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Nuclear
Receptors, Wyeth-Ayerst Research, 145 King of Prussia Rd., Radnor, PA
19087. Tel.: 610-341-2670; Fax: 610-989-4588; E-mail:
karaths{at}war.wyeth.com.
1
The abbreviations used are: apoAI,
apolipoprotein AI; HDL, high density lipoprotein; HNF, hepatocyte
nuclear factor; ER, estrogen receptor; ERE, estrogen response element;
CBEB, cAMP-response element binding protein; CBP, CREB-binding protein;
DBD, DNA binding domain; E2, 17-estradiol; CMV,
cytomegalovirus.
2 E. Kilbourne and S. K. Karathanasis, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|