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and Non-ERß Mechanism1
Departments of Biochemistry and Child Health (D.G., J.A.T., D.B.L.) and Animal Sciences (J.A.G., D.B.L.), University of Missouri, Columbia, Missouri 65211
Address all correspondence and requests for reprints to: Dr. Dennis B. Lubahn, Departments of Biochemistry and Child Health, 163A Animal Science Research Center, 920 East Campus Drive, University of Missouri, Columbia, Missouri 65211. E-mail: lubahnd{at}missouri.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-knockout (ERKO) mice were treated with Mxc or
estradiol-17ß (E2) to determine whether Mxc acts via
pathways that involve ER. In WT mice, both E2 and Mxc
stimulated increases in uterine LF and G6PD mRNA concentrations in a
dose-dependent manner. Competitive pretreatment with the pure
antiestrogen ICI 182,780 dramatically reduced
E2-stimulated increases in mRNA concentrations but had no
effect on Mxc-induced effects. Competitive pretreatment with
E2 had only a partially inhibitory effect on Mxc-induced
responses. In the ERKO mouse, E2 had little effect on
uterine LF or G6PD mRNA concentrations, whereas Mxc stimulated marked
increases in both LF and G6PD mRNAs. The Mxc-induced increases in LF
and G6PD mRNAs in the ERKO mouse were not suppressed by competitive
pretreatment with either E2 or ICI 182,780. Fold increases
in mRNA concentrations for both genes induced by Mxc were similar for
WT and ERKO mice. The results surprisingly indicate that a
xenoestrogen, Mxc, can increase LF and G6PD mRNA concentrations by a
mechanism that is not mediated through ER or ERß, and acts through
another pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mxc was long believed to act through the classic ER (ER) protein, a
ligand-activated transcription factor and a member of a large family of
evolutionarily conserved nuclear hormone receptors. However, the
discovery of an additional ER, ERß (8, 9), has made it necessary to
reevaluate estrogen action. Although the functional importance of ERß
vs. ER is not yet established, the tissue-specific
distribution of these two receptor forms (10) may imply tissue-specific
agonistic or antagonistic actions of estrogens. For example, it has
been shown that Mxc itself acts as an estrogen agonist at the level of
uterus and oviduct but as an antagonist in the ovary (11). Although it
has been shown that Mxc binds to both ER and ERß (10, 12), it is
not yet known through which receptor Mxc or its estrogenic
metabolite(s) acts. Mxc is converted in vivo by the liver to
2,2-bis(p-hydroxyphenyl)-1,1,1,-trichloroethane (HPTE). HPTE is thought
to be the principal active metabolite of Mxc because it has a higher
affinity for ER than Mxc (13) and shows potent in vitro
estrogenic activity (4).
The widespread presence in the environment of chemicals with the capacity to disrupt the functioning of the endocrine system has been extensively studied. These chemicals include pesticides and herbicides such as Mxc, DDT, chlorodecone (kepone), the polychlorinated biphenyls, and phenolic compounds; and they may act via many different mechanisms. One category of endocrine-disrupting chemicals are those that are able to bind to ERs (14) and have effects similar to those of endogenous estrogens. DDT, for example, now banned in the United States for pesticide use, has been shown to advance vaginal opening and increase ovarian and uterine weights in rats (15, 16). Kepone, another pesticide, also induces precocious vaginal opening in immature rats (17), and polychlorinated biphenyls too have been shown to induce precocious puberty and uterine growth in rats (18, 19). These chemicals can act in vivo via multiple mechanisms [for example, o,p’DDT, a structural analog of Mxc, binds to ERs, whereas p,p’DDE binds to androgen receptors (20)]. Though the action mechanisms of these estrogenic chemicals are not clear, these compounds have received a great deal of attention, in the past decade, as a possible cause of certain cancers and impaired reproduction in animals (21, 22, 23).
Kuiper et al. (10, 12) have shown that some natural
estrogens, including various xenobiotics and the estradiol metabolite
4-hydroxy estradiol (a catecholestrogen) may act through binding to
both ER and ERß. Using the ER knockout (ERKO) mice, which
show negligible or no classical responses to E2 (24), Das
et al. (25, 26) have shown that 4-hydroxy estradiol and the
xenoestrogen kepone have estrogenic actions mediated via a non-ER
and non-ERß pathway.
In this study, we set out to characterize the effects and actions of
the widely-used estrogenic pesticide Mxc on the messenger RNA (mRNA)
concentrations of estrogen-responsive genes in the mouse uterus.
Lactoferrin (LF) and glucose-6-phosphate dehydrogenase (G6PD) are two
well-known estrogen-responsive genes (27, 28, 29, 30), and this study includes
both dose-response and time-course effects of Mxc on these genes. To
separate out effects that might be mediated via ER, from those
mediated by ERß and/or other receptors, we examined the effects of
Mxc on estrogen-inducible increases in mRNA concentration in the
ERKO mice and wild-type (WT) controls. Our results demonstrate that
Mxc can induce increases in estrogen-sensitive mRNA concentrations in a
manner similar to E2 but through a pathway that does not
involve either the classical ER or the recently discovered
ERß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and injection schedule
Animals were maintained and treated in accordance with
University of Missouri Animal Care and Use Committee guidelines. Adult
WT (+/+) or homozygous ERKO (-/-) sibling mice of the same mixed
genetic background (129/C57BL/6J) were ovariectomized and rested for 2
weeks before treatment. All treatments were given as two dorsal sc
0.1-ml injections, 6 h apart, of olive oil (vehicle control;
Sigma Chemical Co.), E2 (10 or 100
µg/kg BW), ICI (15 mg/kg), or Mxc (1.8, 3.75, 5.7, 7.5, 10.5, 15, 22,
30, 45, and 60 mg/kg BW), and animals were killed 12 h after the
final injection. In a separate group of mice, Mxc (15 mg/kg) was
injected sc together with E2 at doses of 10 and 100 µg/kg
BW, or with ICI at a dose of 15 mg/kg BW. In these animals,
E2 and ICI were injected 30 min before Mxc
injection.
Time-course studies (2, 6, 12, 18, and 24 h for Mxc; and 12, 18, and 24 h for estradiol) were carried with a single injection of Mxc (15 mg/kg) or estradiol (10 µg/kg). All compounds (0.1 ml/mouse) were injected dorsally sc in olive oil vehicle.
Isolation of RNA
Treated animals were euthanized, and the uterus was quickly
collected and snap frozen in liquid nitrogen. Total RNA was isolated
using Tri-Reagent (Sigma Chemical Co.). After isolation,
total RNA concentration was measured in a spectrophotometer.
Based on the optical density reading, all RNA samples were brought to a
concentration of 1 µg/µl and run out on a 1% agarose gel to
confirm the uniformity of the 18S and 28S RNA bands. The integrity and
quality of the purified RNA were also monitored by measurement of the
A260/280 ratio. Only RNA samples exhibiting a 260/280 ratio greater
than 1.6 and showing integrity of RNA by electrophoresis were used in
further experiments.
Reverse transcriptase (RT)-PCR
Complementary DNA (cDNA) was prepared for LF, G6PD, and RPL7,
using specific antisense primers (0.4 µM) in the presence
of 0.25 µl of avian myeloblastosis virus (AMV-RT), 2.5x
AMV-RT buffer, 0.25 mM MgCl2, and 1
mM deoxynucleotide triphosphates, in a total reaction vol
of 20 µl. RPL7 was used as a housekeeping gene to further
countercheck for uniform RNA loading and to monitor the efficiency of
RT reaction. One microgram of RNA was used as template in each
reaction. The RT reaction was carried out at 48 C for 1 h, and the
AMV-RT was then inactivated at 93 C for 3 min and brought to 14 C for
10 min. One microliter of sample cDNA template for each gene, including
RPL7, was then amplified by PCR in separate sets of reactions. A
negative control (reaction mix but no template) was run in each RT-PCR
reaction, both in RT and PCR reactions, to monitor for nonspecific
amplification.
For RT and PCR of mouse LF, the primers used were 5'-AGGAAAGCCCCCCTACAAAC-3' [nucleotide number (nt) 289–308, sense] and 5'-GGAACACAGCTCTTTGAGAAGAAC-3' (nt 564–541, antisense); GenBank accession no. D88510.
The primers used for mouse G6PD were 5'-CTCCTGCAGATGTTGTGTCT-3' (nt 842–861, sense) and 5'-TCATTGGGCTGCATACGGA-3' (nt 1245–1227, antisense); GenBank accession no. Z11911.
The primers for mouse RPL7 were 5'-TCAATGGAGTAAGCCCAAAG-3' (nt 383–402, sense) and 5'-CAAGAGACCGAGCAATCAAG-3' (nt 628–609, antisense); GenBank accession no. M29016.
For each gene, PCR was done in the presence of specific sense and antisense primers (0.4 µM), 0.1 mM MgCl2, 0.4 mM deoxynucleotide triphosphates, 0.25 µl Fisher-Taq DNA polymerase (Fisher Scientific, St. Louis, MO), and 2x Fisher-Taq polymerase buffer in a total reaction vol of 50 µl. The thermal cycling condition for LF and RPL7 was 30 cycles at 94 C for 30 sec, 55 C for 30 sec, 68 C for 50 sec, with a preincubation at 94 C for 3 min and final incubation at 68 C for 7 min. For G6PD, the thermal cycling conditions were slightly different at 30 cycles at 94 C for 30 sec, 55 C for 40 sec, and 68 C for 1 min. Pre- and postincubation temperatures were the same as above. Uniformity of RNA loading for each sample was confirmed by electrophoresis of the RPL7 cDNA, and then LF and G6PD cDNA samples were coamplified with six different concentrations of competitor, as described below.
Competitive RT-PCR of LF
Competitive RT-PCR was the method chosen to quantify the changes
of RNA message because of the very limited amount of RNA obtainable
from ERKO mouse uteri. It is essentially the same procedure as that
employed by Das et al. (25). The competitor template
contains the same primer template sequence as the mouse target cDNA.
This competitor template was a gift from Drs. S. K. Das and
S. K. Dey and was generated by introducing a nonspecific DNA
fragment into a mouse target cDNA clone. A 185-bp blunt-ended fragment
(SspI), obtained from a pGEM7Zf(+) vector, was inserted into
the LF cDNA at the StuI site. This DNA template was used as
the competitor for competitive PCR of LF cDNA templates derived from
the RT of uterine RNAs. One tenth of the total RT product was
coamplified with 10-fold increasing amounts of the competitive template
(1 fg–100 pg) by PCR for 30 cycles, with the mixture of sense and
antisense oligonucleotides. The final sizes of the competitor template
and target cDNA were 460 bp and 275 bp, respectively. The PCR
amplification conditions were the same as for RT-PCR.
Competitive RT-PCR of G6PD
The competitor template contains the same primer-annealing site
as mouse target cDNA. A PCR product (350 bp) of the G6PD cDNA,
generated by using the mouse G6PD primers described above, was
subcloned into the PGEM-T Easy Vector. A 200-bp foreign piece of
blunt-ended (ClaI) DNA was inserted (blunt-end ligation)
within this PCR product in the KpnI restriction site and was
used as a competitor template for quantitative PCR. The amplification
conditions were the same as for RT-PCR, and the final sizes of the
competitor template and target cDNA were 550 bp and 350 bp,
respectively. For G6PD, one fifth of the RT product was coamplified
with 10-fold increasing amounts of the competitive template (1 fg–100
pg) by PCR for 30 cycles with the mixture of sense and antisense
oligonucleotides. One fifth of the RT product was used in PCR
amplification of G6PD instead of the one tenth used for LF, because
G6PD mRNA was expressed at lower levels than LF mRNA.
Amplified products were separated on 2% agarose (Agarose Low EEO, Fisher Scientific) gels and stained with ethidium bromide. Gels from different assays were scanned, and optical density units (peak area) for each sample and competitor were determined by using Gptools, version 3.0 (BioPhotonics Corp., Ann Arbor, MI). The ratio of band intensities of the competitor and target cDNA was calculated for each sample and plotted against the amounts of competitor. For each sample, a separate standard curve was prepared to determine the amount of mRNA for each specific gene. The amount of target cDNA was determined from the logarithm plot at the zero equivalence point, which represents 10% of the total (because only one tenth of the total reaction was used) for LF and 20% for G6PD total (because one fifth of the total reaction was used).
Data analysis and statistics
The concentration of mRNA for each gene was calculated as
fg/µg total RNA. Statistical analysis of the data was performed by
ANOVA, followed by LSM t test, using a SAS computer program
(SAS system, version V- 6.12, TS 020). Significance was accepted at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After performing the RT reaction, each sample was amplified with its
respective competitor template and quantified as described (details in
Materials and Methods). Fig. 1A
shows a representative diagram of
coamplification of competitor and target cDNA for LF and G6PD genes in
both WT and ERKO animals. Figure 1B shows an example of the
logarithmic plots for control and induced samples (oil- and
estradiol-treated WT animals) that are used to calculate mRNA
concentrations.
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). For LF, a slow increase in mRNA
concentrations starts from around 3-fold at 12 h, rising to about
6-fold at 18 h. The maximum (14-fold) response within the
timeframe studied for LF was seen at 24 h. In contrast, G6PD gave
a peak response (17-fold) at 12 h and a decline in response (down
to 4.8-fold) at 18 h. A further stimulation (6-fold) started at
24 h, suggesting its stimulation to be bimodal.
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KO mice (Fig. 2B
). A
7-fold increase was found at 18 h, which was the time of maximum
response for LF in this timeframe, followed by a slight decline (down
to 5-fold) at 24 h. No differences in responses were observed
between WT and ERKO mice in this time-course study.
For G6PD, no response was observed at 2 h, either in WT or ERKO
mice (Fig. 2C). A slight increase (2-fold) was observed at 6 h in
WT mice and also (3.8-fold) in ERKO animals. At 12 and 18 h, WT
mice did not show any further increase beyond that at 6 h (about
3-fold), but a 6-fold increase was found at 24 h. However, ERKO
mice showed a further increase (to about 6-fold) at 12 h and
18 h and gave a peak response (9-fold) at 24 h.
For further studies, 18 h was chosen for the end point, because this appeared optimal for both genes within the timeframe studied.
Effects of different doses of Mxc on uterine LF mRNA
concentrations
Mxc (3.75, 5.7, 7.5, 10.5, 15, 22, 30, 45, and 60 mg/kg) induced
increases in LF mRNA concentrations in a dose-dependent manner in WT
mice, up to 30 mg/kg (Fig. 3A). Little
effect was seen before the 5.7-mg/kg dose, after which message
concentrations rose to a peak at 30 mg/kg and then declined. The
30-mg/kg dose seemed to be a maximally effective dose in WT mice. In WT
animals, a sharper decline in message was seen, starting from 45 mg/kg;
and a gradual fall was seen at the 60-mg/kg dose. The scenario in the
ERKO mouse was quite different. Although the 1.8-mg/kg dose was
ineffective, the next lower doses (3.75, 5.7, and 7.5 mg/kg) gave
similar magnitude responses (about 4.5-fold increase), forming a short
plateau. After this plateau, a further stimulation was seen at the
10-mg/kg dose (7-fold), with a sharper increase (up to 10.5-fold) at 15
mg/kg; message concentrations declined after this dose, down to
4.8-fold at 60 mg/kg.
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). Although
the lower (1.8- and 3.75-mg/kg) doses seemed ineffective in WT animals,
injection with Mxc at the higher doses induced increases in G6PD mRNA
concentrations in WT animals in a dose-dependent manner, rising to a
maximum-fold increase at 15 mg/kg. After this point, a decrease in mRNA
concentrations was seen, although fold increase did not drop further
and remained at around 7-fold at the higher doses (22, 30, and 60
mg/kg).
ERKO mice again showed a small plateau response at the lower doses
(3.5-fold at 3.75 mg/kg, and 5-fold at 5.7 and 7.5 mg/kg). A further
increase in message concentration was seen at 10.5 mg/kg (5.9-fold),
and a sharp increase at 15 mg (12-fold) was observed that was
maintained at 22 mg (11-fold). Nevertheless, as seen for LF, a fall in
G6PD mRNA concentration was seen at 30 mg (4.6-fold), 45 mg (4-fold),
and 60 mg (3-fold).
Effect of estradiol 17ß and ICI on Mxc-induced increases in LF
mRNA concentrations
This study was carried out to check whether competitive
pretreatment with estrogen or antiestrogen would inhibit the
Mxc-induced stimulation of LF (Fig. 4A)
and G6PD (Fig. 4B) mRNAs. Pretreatment with E2 at 10
µg/kg did not inhibit, but did reduce, the magnitude of the
Mxc-induced increases in LF mRNA concentrations in both WT and ERKO
mice (Fig. 4A). The fold increase was reduced from 10- to 7-fold in WT
mice and from 10- to 8-fold in ERKO animals. E2 alone,
at doses of 10 and 100 µg/kg, gave 14.6-fold and 16.4-fold increases,
respectively, in WT mice; whereas no responses were observed in ERKO
mice other than a very slight (3-fold) stimulation at 10 µg/kg.
Pretreatment with E2 at 100 µg/kg considerably reduced
the Mxc-induced LF response (from 10- to 2.7-fold increase) in WT mice.
The ERKO mice showed magnitudes of response almost similar to those
of Mxc, both with and without pretreatment with either dose of
E2, although the lower dose of E2 seemed to
inhibit the Mxc-induced effect (from 10.5- to 8-fold). Treatment with
the antiestrogen ICI alone, at a dose of 15 mg/kg, had no effect on LF
mRNA concentrations. Pretreatment with ICI had no effect on the
Mxc-induced response in WT or ERKO mice, but the same dose did
inhibit the response to estradiol in WT mice.
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). In contrast, this
dose of E2 did not inhibit the Mxc-induced increases in
G6PD mRNA concentrations in the ERKO mouse and, instead, had an
additive effect. However, the higher, 100-µg/kg E2 dose led to a
partial reduction of the Mxc-induced response in both subject groups
(from 10- to 5-fold in the WT, and 11- to 6-fold in the ERKO). The
response pattern of G6PD message to ICI was similar to that for LF. ICI
did not have any effect when injected alone, inhibited the
E2-induced response seen in WT mice, but did not inhibit
Mxc-induced G6PD response in WT or ERKO mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by an imperfect
palindromic ERE in the 5'-flanking region of the LF gene (28); less is
known about G6PD activation. To determine whether uterine responses to
Mxc were also mediated through ER, we compared the effects of these
agents on uterine LF and G6PD mRNA concentrations in ovariectomized
ERKO and WT mice by using quantitative RT-PCR.
We show here that, in the WT mouse, Mxc (like E2)
stimulated increases in uterine LF and G6PD mRNA in a saturable,
dose-dependent manner and that E2-stimulated increases were
dramatically reduced by competitive pretreatment with the pure
antiestrogen ICI. In contrast, the increases in mRNA concentrations
induced by Mxc were not inhibited by this antiestrogen. Competitive
pretreatment with E2, at 10 µg/kg, had only a partially
inhibitory effect on the Mxc-induced responses, and the antiestrogen
alone did not influence the concentrations of uterine LF or G6PD mRNA.
Collectively, these results indicate that, under normal conditions, the
WT uterus responds to both Mxc and E2, in terms of
increased LF and G6PD mRNA concentrations. Moreover, the lack of ICI
inhibition of Mxc-induced uterine LF and G6PD mRNA accumulation, and
their only-partial inhibition by E2, suggested that Mxc can
act through an additional independent signaling pathway not involving
ER or ERß. We confirmed these results by using ovariectomized
ERKO mice.
In the ERKO mouse, we show that E2 was ineffective at
stimulating marked increases in uterine LF or G6PD mRNA concentrations,
agreeing with work by Das et al. (25). Mxc, however, did
stimulate both LF and G6PD mRNA, in a saturable and dose-dependent
manner, indicative of a receptor-mediated mechanism of action. This
increase in uterine LF and G6PD mRNA concentrations, induced by Mxc,
was not suppressed by competitive pretreatment with E2 or
ICI in the manner seen in WT animals, thus confirming that Mxc can work
through a non-ER and non-ERß mechanism. It is interesting to note
that both LF and G6PD mRNA concentrations were stimulated equally in WT
and ERKO mice, suggesting that the predominant pathway for Mxc
action on these mRNAs is not via ER. Again, the lack of inhibition
by ICI or E2 of Mxc-induced effects indicates that ER
and ERß are minimally involved in mediating the effects of Mxc on LF
and G6PD mRNA concentrations. In support of this, it should be noted
that the concentrations of ERß are remarkably low, in comparison with
ER, in the WT and ERKO mouse uterus (31).
Taken together, these uterine responses to Mxc in the mouse clearly
establish the presence of a pathway that is not mediated via the
classical ER or ERß but, instead, through an additional signaling
pathway. However, although the saturable dose responses are indicative
of a receptor-mediated mechanism, this pathway may not necessarily
involve an additional ER (such as an ER
). It is possible, for
example, that Mxc effects are mediated via a membrane receptor or
another nuclear receptor.
Although the molecular pathway(s) by which Mxc alters these
estrogen-sensitive uterine mRNA concentrations has yet to be
characterized, several possibilities can be ruled out. First, although
ERKO mice lack full-length ER, they may still have alternatively
spliced forms of ER. The existence of alternatively spliced forms of
ER that contain the ER ligand-binding domain has recently been
documented, either with sequence changes upstream of the exon 5/6
boundary in rat pituitary or lacking exon 5 in rat brain and human
smooth muscle cells (32, 33, 34). Second, it is possible that effects of
estrogenic ligands in ERKO mice could be mediated by different ER
subtypes, such as ERß or its alternatively spliced forms (35, 36, 37).
However, it is known that E2 and antiestrogens bind to both
ER and ERß ligand-binding domains (12). Our observations
demonstrate that E2 has little or no effect on ERKO
uterine LF and G6PD mRNA concentrations, and that neither
E2 nor ICI markedly inhibits Mxc-induced increases in
uterine LF and G6PD mRNAs in ERKO mice. This lack of inhibition
suggests that the effects of this xenoestrogen on the uterine LF and
G6PD genes are not mediated via the ligand-binding domains of ER or
ERß. A third factor to consider is that the LF gene has been shown to
have two promoter regions (38), and it is possible that Mxc may exert
its effects on LF and G6PD through promoter regions on these genes that
differ from those used by E2.
One important factor to consider is that mRNA concentrations, at any given point in time, represent a balance between synthesis and degradation. Estrogen has been shown to regulate the stability of specific mRNAs (39), and the possibility remains that xenoestrogens may regulate mRNA concentrations through altering expression or via effects on stabilization or destabilization of mRNAs.
The relative pharmacokinetics of Mxc as a pro-drug and E2
and ICI as drugs might be considered to present technical difficulties
for this study, especially in competition experiments. However, we do
not believe that this is likely to be a problem, because our
dose-response times (12 h after last dose) are short, and the
inhibitory effects of ICI on estradiol are evident in the WT controls
throughout the time course of our experiments. From the controls, it is
clear that ICI is still present and would be capable of inhibiting Mxc
if it were working through either an ER or ERß ligand-binding
domain. In addition, it has been shown that ICI is a comparatively
long-acting drug (days longer than tamoxifen), with sustained
antiestrogenic effects from a single injection in oil that last at
least 3 weeks (39A ). Therefore, even if it takes hours, or even a day,
for Mxc to be metabolized into an active form, the antiestrogenic
effect of ICI would still exist when the active form of Mxc was
generated. From this, it is clear that ICI is still present and capable
of inhibiting the pro-drug Mxc or its metabolites if it were working
through either a ER or ERß ligand-binding domain.
It has been suggested (40) that one way xenobiotics disrupt endocrine
function is by interfering with the ability of natural ligands to bind
receptors and/or binding proteins, perhaps at multiple levels of
activation. Mxc is known to be active in vivo, acting as a
proestrogen, which requires metabolism for estrogenic effectiveness
(41). Mxc’s metabolites have been less extensively studied than Mxc
itself, but Gaido et al. (42) have reported that HPTE,
thought to be the active in vivo metabolite, acts as an
agonist for ER but as an antagonist for ERß. Katzenellenbogen
et al. (43) have also reported finding ER ligands that are
full agonists via ER but antagonists via ERß, as well as one gene,
quinone reductase, whose activity is up-regulated by antiestrogens
acting through ER and ERß. This mixed agonist/antagonist function
has also been reported for other compounds. The antiestrogen
hydroxytamoxifen has mixed agonist/antagonist activity through ER,
depending on the tissue and gene (44, 45), and can exert synergistic
effects when combined with E2 (46).
We had expected that, because both Mxc and E2 are thought
to act through ER, their effects would be additive, but this was not
seen. It is possible that, in the WT uterus (that is, in the presence
of ER), Mxc may act as a mixed agonist/antagonist to E2
action by partial binding with ER, as well as acting through another
pathway. These interactions may, in some way, account for the
unexpected decrease in Mxc activity we observed in WT animals when
E2 was added. It is also possible that the receptor used by
Mxc is an ER repressor.
The additive effect of Mxc, with low doses of E2, in the
ERKO mouse, was also unexpected. Although the error bar for these
samples was unusually large, the decrease in Mxc-induced increases in
G6PD mRNA levels in the presence of E2 (100 µg/kg) was
not statistically different from Mxc alone. Clearly, this in
vivo system is complex, and it involves potential interactions of
ER, ERß, and ER-ERß heterodimers, as well as interactions
between these ERs and the additional receptor used by Mxc. Further,
there is likely to be some degree of cross-talk between ER/ERß and
the Mxc-signaling pathway. It is also possible that there are
activators or repressors that are important in the control of ER
function whose normal actions are altered by the lack of ER in the
ERKO mouse uterus.
One interesting feature was the differing time course of Mxc-induced
responses for G6PD and LF mRNA. Mxc stimulated both LF and G6PD mRNA
concentrations in a dose-dependent fashion, but the increase in LF
message concentrations was slower than that of G6PD (increases in LF
mRNA concentrations were not seen until 12 h, whereas increases in
G6PD mRNA concentrations were seen at around 6 h). This is similar
to the findings of Curtis et al. (47), examining the effects
of DES and its metabolites, and suggests that Mxc acts on these two
genes through two different mechanisms, one more rapid than the other.
Another fact that suggests independence of the two pathways is that,
for G6PD (but not LF) mRNA concentrations, the lower dose of
E2 (10 µg/kg) seemed to be additive with Mxc, in the
ERKO mouse. Additive or synergistic actions of E2 are
not unknown and have been reported elsewhere, with testosterone in
prostate growth (48), with kepone in uterus (49), and with isomers of
DDT in MCF-7 cells (50). Finally, in these experiments, there is also a
clear difference in how LF and G6PD are regulated in the presence of E2
and Mxc together. Understanding of the molecular cause of these
differences awaits a detailed comparative analysis of the LF and G6PD
promoter regions in in vitro transcriptional reporter
assays.
In summary, our findings in ERKO mice demonstrate that a
xenoestrogen can up-regulate the expression of two estrogen-responsive
genes in the uterus, via one or more pathways that do not seem to
involve ER or ERß. Currently, there is much interest in
xenoestrogens and other environmental endocrine disrupters because of
their potential adverse effects on human and animal health. Further
characterization of this novel pathway will enhance our understanding,
not only of xenoestrogen action but also of diverse steroid hormone and
endocrine disrupter actions in target organs.
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Acknowledgments |
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Footnotes |
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Received October 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 deficient
mice reveal a distinct estrogen signaling pathway. Proc Natl Acad Sci
USA 94:12786–12791 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 and ß. Program of the
80th Annual Meeting of The Endocrine Society, New Orleans,
LA,,1998 (Abstract OR 14-2), p 71
and ß. Biochem
Biophys Res Commun 236:140–145[CrossRef][Medline]
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C. L. Bevan, D. M. Porter, C. R. Schumann, E. Y. Bryleva, T. J. Hendershot, H. Liu, M. J. Howard, and L. P. Henderson The Endocrine-Disrupting Compound, Nonylphenol, Inhibits Neurotrophin-Dependent Neurite Outgrowth Endocrinology, September 1, 2006; 147(9): 4192 - 4204. [Abstract] [Full Text] [PDF]
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 D. Tomic, M. S. Frech, J. K. Babus, R. K. Gupta, P. A. Furth, R. D. Koos, and J. A. Flaws Methoxychlor Induces Atresia of Antral Follicles in ER{alpha}-Overexpressing Mice Toxicol. Sci., September 1, 2006; 93(1): 196 - 204. [Abstract] [Full Text] [PDF]
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 S. Musatov, W. Chen, D. W. Pfaff, M. G. Kaplitt, and S. Ogawa RNAi-mediated silencing of estrogen receptor {alpha} in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors PNAS, July 5, 2006; 103(27): 10456 - 10460. [Abstract] [Full Text] [PDF]
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X. Fei, H. Chung, and H. S. Taylor Methoxychlor Disrupts Uterine Hoxa10 Gene Expression Endocrinology, August 1, 2005; 146(8): 3445 - 3451. [Abstract] [Full Text] [PDF]
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D. A. Symonds, D. Tomic, K. P. Miller, and J. A. Flaws Methoxychlor Induces Proliferation of the Mouse Ovarian Surface Epithelium Toxicol. Sci., February 1, 2005; 83(2): 355 - 362. [Abstract] [Full Text] [PDF]
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 S. K. Dey, H. Lim, S. K. Das, J. Reese, B. C. Paria, T. Daikoku, and H. Wang Molecular Cues to Implantation Endocr. Rev., June 1, 2004; 25(3): 341 - 373. [Abstract] [Full Text] [PDF]
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 D. E. Frigo, Y. Tang, B. S. Beckman, A. B. Scandurro, J. Alam, M. E. Burow, and J. A. McLachlan Mechanism of AP-1-mediated gene expression by select organochlorines through the p38 MAPK pathway Carcinogenesis, February 1, 2004; 25(2): 249 - 261. [Abstract] [Full Text] [PDF]
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P. J. Shughrue, G. R. Askew, T. L. Dellovade, and I. Merchenthaler Estrogen-Binding Sites and Their Functional Capacity in Estrogen Receptor Double Knockout Mouse Brain Endocrinology, May 1, 2002; 143(5): 1643 - 1650. [Abstract] [Full Text] [PDF]
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K. M. Waters, S. Safe, and K. W. Gaido Differential Gene Expression in Response to Methoxychlor and Estradiol through ER{alpha}, ER{beta}, and AR in Reproductive Tissues of Female Mice Toxicol. Sci., September 1, 2001; 63(1): 47 - 56. [Abstract] [Full Text] [PDF]
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 W. Shang, I. Konidari, and D. W. Schomberg 2-Methoxyestradiol, an Endogenous Estradiol Metabolite, Differentially Inhibits Granulosa and Endothelial Cell Mitosis: A Potential Follicular Antiangiogenic Regulator Biol Reprod, August 1, 2001; 65(2): 622 - 627. [Abstract] [Full Text] [PDF]
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N. B. Schwartz Perspective: Reproductive Endocrinology and Human Health in the 20th Century--A Personal Retrospective Endocrinology, June 1, 2001; 142(6): 2163 - 2166. [Full Text] [PDF]
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O. M. Conneely Perspective: Female Steroid Hormone Action Endocrinology, June 1, 2001; 142(6): 2194 - 2199. [Full Text] [PDF]
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O. Vidal, M. K. Lindberg, K. Hollberg, D. J. Baylink, G. Andersson, D. B. Lubahn, S. Mohan, J.-A. Gustafsson, and C. Ohlsson Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice PNAS, May 9, 2000; 97(10): 5474 - 5479. [Abstract] [Full Text] [PDF]
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L. C. Hodges, J. S. Bergerson, D. S. Hunter, and C. L. Walker Estrogenic Effects of Organochlorine Pesticides on Uterine Leiomyoma Cells in Vitro Toxicol. Sci., April 1, 2000; 54(2): 355 - 364. [Abstract] [Full Text] [PDF]
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