| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Laboratories of Molecular Carcinogenesis (D.M.K., R.P.D.) and Reproductive and Developmental Toxicology (S.C.H., K.S.K.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Dr. Richard P. DiAugustine, National Institute of Environmental Health Sciences, P.O. Box 12233, Mail Drop D4–04, Research Triangle Park, North Carolina 27709. E-mail: diaugus2{at}niehs.nih.gov
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) and elicit estrogenic responses also mimic E2 by
activating the uterine IGF-I signaling pathway. Ovariectomized adult
female mice were treated with both environmental and clinically
relevant chemicals previously reported to display estrogenic and/or
antiestrogenic properties, and their uteri were evaluated for an
activated IGF-I signaling pathway. Diethylstilbestrol,
4-hydroxytamoxifen, the raloxifene analog LY353381,
2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE),
bisphenol A, and genistein were shown to mimic E2 in the
uterus by increasing the level of IGF-I messenger RNA, inducing IGF-I
receptor (IGF-IR) tyrosine phosphorylation, stimulating the formation
of IGF-IR signaling complexes, and increasing both proliferating cell
nuclear antigen expression and the number of mitotic cells in the
epithelium. The dose of chemical necessary to activate IGF-I signaling
varied, with the order of potency: E2 =
diethylstilbestrol > LY353381 > 4-hydroxytamoxifen >
genistein > HPTE > bisphenol A. Administration of the chemicals
to ER knockout mice did not activate IGF-IR, indicating that ER
is required for activation of uterine IGF-IR by these diverse
chemicals. This study demonstrates that several chemicals shown
previously to display estrogenic activities also mimic E2
by activating uterine IGF-I signaling. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To identify chemicals that are potential estrogens and to understand the mechanisms through which such chemicals may act, a variety of in vitro assays have been established. These in vitro assays include ER binding assays, cell proliferation assays, and transcription assays using estrogen-responsive reporter genes (Ref. 19 and references therein). With the wealth of in vitro screening assays available to test the estrogenic potential of environmental chemicals and other exogenous chemicals, researchers can use the information gathered from in vitro assays to examine specific chemicals for estrogenic actions in vivo. In vivo assays include the animal studies mentioned previously as well as uterotropic assays (20, 21), steroidogenic enzyme assays (22), and the detection of estrogen-regulated gene products in estrogen target cells (23, 24, 25).
It is generally accepted that E2 exerts its effects in target organs by binding to ER and modulating the expression of various proteins such as growth factors (Ref. 26 and references therein). In the uterus, one such growth factor is insulin-like growth factor I (IGF-I). Studies have shown that administration of E2 to ovariectomized adult female mice and rats results in an increase in uterine IGF-I transcripts (27, 28). Subsequent studies have provided further evidence for IGF-I being a mediator of the actions of E2 in the uterus. A survey of tyrosine kinase receptors in the mouse uterus showed that IGF-I receptor (IGF-IR), but not the receptors for fibroblast growth factor, platelet-derived growth factor, or epidermal growth factor, is activated after administration of E2 to ovariectomized adult female mice (29). In addition, E2 induced the formation of a uterine signaling complex composed of IGF-IR, insulin receptor substrate-1 (IRS-1), and p85, the regulatory subunit of phosphatidylinositol-3 kinase (29, 30). Although the biological end point of this signaling cascade has not been resolved fully, a recent study has indicated that IGF-I is required for E2-induced mitosis in the uterine epithelium (31). The study showed that the appearance of E2-induced mitotic figures was reduced in the uteri of IGF-I-null mice compared with their wild-type counterparts, indicating a role for IGF-I in G2 progression of uterine epithelial cells.
Based on the above information, which argues a role for IGF-I in
mediating some of the actions of E2 in the
uterus, the present study was designed to examine whether chemicals
that have been reported previously to display estrogenic properties
also mimic E2 by activating the IGF-I signaling
pathway. Representatives from different classes of reported ER
agonists/antagonists, both clinical and environmental, are shown herein
to increase uterine IGF-I messenger RNA (mRNA) levels, activate uterine
IGF-IR signaling, and induce proliferation of the uterine epithelium.
In addition, ER knockout mice (ERKO), in which both alleles of
the ER gene have been disrupted, resulting in a female reproductive
tract that is refractory to the mitogenic effects of
E2 as previously described (32, 33), were
included in this study as a model system for examining the uterine
effects of exogenous estrogens in the absence of ER. It is
demonstrated that all chemicals tested required ER in the uterus to
elicit the responses that were evaluated. The chemicals chosen for
examination were DES, 4-hydroxytamoxifen (4OH-tamoxifen), genistein,
2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE, the
estrogenic metabolite of the pesticide methoxychlor) (34), bisphenol A
(a chemical found in plastics) (35, 36), and LY 353381 (a
benzothiophene compound similar to raloxifene that has been reported to
have little stimulatory effect on the rat endometrium) (37).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
knockout mice
and their wild-type counterparts (C57/BL6 background) were
ovariectomized at 76–84 days of age and treated sc with hormone or
chemical at 14–28 days postovariectomy. For consistency all compounds
were administered in 100 µl sesame oil. For long
R3-IGF-I injections, mice were treated by three
consecutive injections of 100 µl PBS or three consecutive injections
of 200 µg each of long R3-IGF-I (I) at 5-min
intervals.
Chemicals
E2, DES, bisphenol A, genistein, and
4OH-tamoxifen were purchased from Sigma (St. Louis, MO).
HPTE was a gift from Wendy N. Jefferson (NIEHS), and LY 353381 was a
gift from Eli Lilly & Co. (Indianapolis, IN). Long
R3-IGF-I was purchased from Diagnostics Systems Laboratories, Inc. (Webster, TX).
Immunoprecipitation of IGF-IR
Mice were injected with chemicals and killed 6 h after
treatment. Immediately after death, uteri were removed and homogenized
at 4C in 600 µl solubilization buffer (20 mM HEPES, 2
mM EDTA, 2 mM EGTA, and 1% Triton X-100)
containing protease and phosphatase inhibitors (20 µg/ml aprotinin,
20 µg/ml leupeptin, 4 µg/ml
-phenylmethylsulfonylfluoride, 1 mM
Na3VO4, 20 mM
NaF, and 0.05 mM
Na2MoO4). Homogenates (200
µl) were then subjected to immunoprecipitation with 5 µg antimouse
IGF-IR polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with an equal volume of 2 x
immunoprecipitation buffer [100 mM Tris (pH 8.5), 300
mM NaCl, 10 mM EDTA, and 1% Triton X-100] for
1 h at 4 C. Antigen-antibody complexes were captured with protein
A-Sepharose for 3 h and subjected to three sequential washes
(first: 0.5% Triton X-100, 1 mM EDTA, and 500
mM NaCl in 50 mM Tris, pH 8.5; second: 0.5%
Triton X-100, 1 mM EDTA, and 150 mM NaCl in 50
mM Tris, pH 8.5; third: 0.1% Triton X-100 in 10
mM Tris, pH 8.5). Precipitated antigen was eluted from the
protein A-Sepharose by resuspending the pellets in Laemmli sample
buffer and boiling for 5 min.
Immunoblots
Immunoprecipitated proteins were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked in either
Tris-buffered saline with 5% BSA or PBS with 3% nonfat dry milk.
Membranes were then probed with antiphosphotyrosine (PY20, ICN Biomedicals, Inc., Aurora, OH), anti-IRS-1 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-p85 (Upstate Biotechnology, Inc.), or anti-IGF-IR antibodies. Enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech,
Piscataway, NY) was used for detection according to the manufacturer’s
specifications. Immunoblots are representative of three to five
independent experiments with two individual animals per dose.
RNA isolation and ribonuclease protection assays (RPAs)
Mice were injected with chemicals, and 6 h after treatment
uteri were removed and immediately snap-frozen in liquid
N2. Uteri were then pulverized, and total RNA was
isolated with TRIzol (Life Technologies, Inc., Grand
Island, NY) according to the manufacturer’s instructions. RNA
integrity was qualitatively assessed by agarose gel electrophoresis and
was quantified by measuring the absorbance at 260 nm. RNA from each
uterus was hybridized to 32P-labeled RNA probes,
which are complementary to nucleotides 73–487 of the reported mouse
IGF-I mRNA sequence (GenBank accession no. X04482), and which recognize
both the A and B forms of the IGF-I mRNA. To equate loading among
samples RNA was also hybridized to 32P-labeled
RNA probes complementary to mouse cyclophilin mRNA. Hybridization of
the B and A forms of IGF-I mRNA to the IGF-I probe results in protected
fragments of 317 and 265 nucleotides (nt), respectively; hybridization
to cyclophilin probe results in a protected fragment of 103 nt. RPAs
were performed using the RPA III Kit (Ambion, Inc.,
Austin, TX) according to the manufacturer’s instructions. Briefly, 10
µg total RNA were coprecipitated with 1 x
105 cpm of each probe, resuspended in
hybridization buffer, and incubated at 42 C overnight. Samples were
then digested with RNase A and T1 for 30 min at 37 C. After digestion,
samples were precipitated and resuspended in gel loading buffer.
Samples were electrophoresed on 6% polyacrylamide/7 M urea
gels, and after electrophoresis, the gels were vacuum dried, and film
was exposed to the gels at -70 C. Gels were also quantitatively
analyzed by phosphorimage analysis. ANOVA procedures were used to
assess the significance of differences among replicates and differences
among treatment groups. The variance-stabilizing logarithmic
transformation was used before statistical analysis. Pairwise
comparisons of each group to the vehicle control group were made using
Dunnett’s test (38). RPA results are representative of four
independent experiments for a total of at least eight individual
animals per chemical treatment.
Proliferating cell nuclear antigen (PCNA) immunohistochemistry and
visualization of mitotic cells
For analysis of PCNA expression, uteri were removed 18 h
after injection of estradiol or chemicals. Uteri were immediately fixed
in cold 10% buffered formalin. Tissues were cut into 5-µm sections,
mounted on SuperFrost Plus slides (Fisher Scientific,
Norcross, GA), and air-dried. Immunohistochemical staining of PCNA was
performed with adaptations to the method previously described (39). The
primary antibody used was antimouse PCNA 19A2 (Beckman Coulter, Inc., Hialeah, FL), and the secondary antibody used was
biotinylated antimouse IgM (Vector Laboratories, Inc.,
Burlingame, CA). ExtrAvidin peroxidase conjugate and diaminobenzidene
(Sigma) were added for detection. Each section is
representative of an individual animal from one of at least three
independent experiments with two animals per treatment. For
visualization of mitotic cells, mice were injected sc with
E2 or chemicals 22 h before removal of
uteri. Two hours before removal of uteri, mice were injected ip with
0.1 mg Colcemid (Sigma, St. Louis, MO) in 50 µl
H2O. Uteri were immediately fixed in cold 10%
buffered formalin. Tissues were cut in 5-µm sections and stained with
hematoxylin and eosin. Each section is representative of an individual
animal from one of three independent experiments with at least two
animals per treatment.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As shown in Fig. 1A
, all chemicals
administered to the mice induced tyrosine phosphorylation of uterine
IGF-IR; however, the dose of chemical necessary to observe IGF-IR-pY
varied depending on the chemical. Based on the doses of chemical used,
E2 and DES were equipotent in activating IGF-IR.
Both compounds stimulated IGF-IR tyrosine phosphorylation at doses as
low as 3 µg/kg. Previous studies have also shown that DES is as
potent as E2 or is a more potent ER agonist than
E2 depending on the assay for estrogenicity used
(Ref. 19 and references therein). 4OH-tamoxifen induced IGF-IR
tyrosine phosphorylation at 300 µg/kg, which is in agreement with
other studies indicating that 4OH-tamoxifen is indeed an ER agonist in
both human and rodent uterus (14, 15, 16) and is approximately 100-fold
less potent than E2 (Ref. 19 and references
therein). Interestingly, LY 353381, the benzothiophene developed as a
raloxifene analog, activated IGF-IR when administered at 300 µg/kg.
Among the compounds evaluated, genistein was the next most potent
chemical, activating IGF-IR at a concentration of 15,000 µg/kg or
higher. It should be noted that the level of response with genistein
varied. While all other chemicals generated consistent dose-response
curves each time the experiment was performed, some mice required
higher doses of genistein than others for uterine IGF-IR activation to
be observed. Finally, HPTE and bisphenol A also induced IGF-IR-pY, but
only at doses of 30,000 and 45,000 µg/kg, respectively. None of the
chemicals evaluated affected the levels of uterine IGF-IR, as
determined by Western blot analysis of the immunoprecipitated uterine
IGF-IR (data not shown).
|
). In all cases where a chemical
induced IGF-IR-pY, a complex containing IGF-IR, IRS-1, and p85 was also
formed, which further confirms that IGF-IR uses an
IRS-1/phosphatidylinositol 3-kinase pathway to transmit its
signal in response to an estrogenic stimulus in the uterus. Doses of
chemicals, including low doses of E2, that did
not induce IGF-IR-pY also did not induce the formation of an
IGF-IR/IRS-1/p85 complex (data not shown).
Increase in uterine IGF-I mRNA by ER modulators and environmental
chemicals
Based on previous studies that demonstrated an increase in uterine
IGF-I mRNA in response to E2 (27, 28), it was
hypothesized that the chemical-induced IGF-IR activation occurred as
the result of an increase in locally synthesized IGF-I. To evaluate
whether the compounds that induced IGF-IR activation also increased
IGF-I gene expression, RPA was used to measure IGF-I mRNA expression in
the uteri of mice treated with the chemicals. Doses of chemicals that
activated IGF-IR signaling (Fig. 1, A and B) were administered to mice,
and Fig. 2A is a representative RPA,
demonstrating that all chemicals that activated IGF-IR signaling also
increased the levels of uterine IGF-I mRNA. IGF-IB mRNA levels were
elevated within each uterine sample in response to every chemical
tested. IGF-IA mRNA levels were also significantly increased above
control levels, except in the samples from LY-treated animals. However,
the magnitude of increase in IGF-IA mRNA levels was lower than the
increase in IGF-IB mRNA levels. The dose of each chemical administered
was different depending on the chemical used; therefore, samples cannot
be directly compared among the chemical treatments. Nevertheless,
quantification of uterine IGF-I mRNA levels was performed by
phosphorimage analysis and is presented graphically to demonstrate that
the chemicals increased the levels of IGF-I mRNA compared with the
levels expressed in the uteri of vehicle-treated mice (Fig. 2B).
|
in estradiol- and chemical-induced IGF-IR
signaling, rather
than through an alternative mechanism of activation, ovariectomized
ERKO mice and ovariectomized wild-type counterparts were
administered the same doses of the various chemicals that were shown
earlier to activate uterine IGF-IR signaling and increase uterine
IGF-IR mRNA levels. As described in Materials and Methods,
uterine IGF-IR was analyzed by immunoblot analysis for tyrosine
phosphorylation. Figure 3A illustrates
that in the ERKO mice, IGF-IR was not activated by
E2 or any of the other chemicals tested.
|
ERKO mouse uterus, both wild-type and ERKO mice
were administered long R3-IGF-I. Long
R3-IGF-I was used because it exhibits decreased
binding to IGF-binding proteins compared with the native ligand (40),
thereby increasing tissue bioavailability. As shown in Fig. 3B, long
R3-IGF-I activates IGF-IR in the ERKO mouse
uterus, demonstrating that uterine IGF-IR in the ERKO mouse retains
its responsiveness to activation by IGF-I. Therefore, the failure of
E2 and the other chemicals to activate IGF-IR in
the ERKO uterus was due to the absence of ER, rather than to
IGF-I-insensitive uterine IGF-IR.
Increase in uterine epithelial PCNA expression and number of
mitotic epithelial cells by ER modulators and environmental
chemicals
The experiments described above showed that activation of
IGF-I signaling can be used as a marker for estrogen action in the
uterus. It was important to examine next whether the same doses of
chemicals that activated IGF-I signaling also mimicked other aspects of
estrogen action, namely proliferation of uterine epithelial cells.
Ovariectomized mice were administered doses of chemicals that were
shown previously to activate IGF-I signaling as well as doses of
chemicals that did not activate IGF-I signaling (
Figs. 1–3). Uteri
were examined immunohistochemically for either PCNA expression or the
presence of mitotic cells. Negligible amounts of PCNA expression and
mitotic cells were identified in uteri of vehicle-treated
ovariectomized mice (Figs. 4 and 5). In contrast, 18 h after
treatment with chemicals at doses that activated uterine IGF-I
signaling, increased PCNA immunoreactivity was observed in the uterine
luminal epithelium of wild-type mice (Fig. 4), indicating that the
uterine epithelial cells were actively proliferating in response to the
chemicals. PCNA expression was not observed in the uterine epithelium
when mice were treated with doses of chemicals that did not activate
IGF-I signaling (data not shown). Additionally, 24 h after
treatment, doses of chemicals that activated uterine IGF-I signaling
and increased uterine PCNA expression also increased the number of
observable mitotic cells in the uterine epithelium (Fig. 5). Again,
because doses differed among the chemicals evaluated, this study did
not quantitatively compare the number of mitotic cells that could be
observed in response to the chemicals. Rather, this study demonstrated
that compared with uteri from vehicle-treated mice, there was an
overall increase in mitoses in uterine epithelial cells from mice
treated with the chemicals. As in the case of uterine PCNA expression,
doses of chemicals that neither activated uterine IGF-I signaling nor
increased uterine PCNA expression failed to increase the number of
mitotic cells in the uterine epithelium (data not shown). Furthermore,
as expected, when E2, 4OH-tamoxifen, and
genistein were administered to ERKO mice, the amount of PCNA
expressed in the uterine epithelium did not increase compared with the
levels expressed in vehicle-treated mice (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies from this laboratory have shown that E2 activates IGF-I signaling in the mouse uterus (29, 30), indicating that IGF-I may play a role in mediating the mitogenic effects of E2 in the uterus. In support of this idea is the recent study using IGF-I nullizygous mice that demonstrated that IGF-I is required for G2 progression in E2-induced uterine epithelial cell mitosis (31). Studies have also shown that IGFs stimulate endometrial cancer cell growth in culture (41), and that E2 increases the sensitivity of endometrial cancer cells to IGFs by elevating IGF-IR and decreasing the levels of IGF-binding proteins (42). It will be important to further evaluate environmental and synthetic estrogens/SERMs for their effects on these events to better predict whether estrogenic environmental chemicals present a true risk factor in the development of endometrial diseases. Furthermore, it is essential to understand whether the proliferative effects that are exerted on endometrial cancer cells by environmental chemicals require activation of IGF-I signaling or whether these are distinct and independent events.
Unlike many other E2-regulated genes, the IGF-I gene apparently is not regulated through the classical mechanism that involves the direct binding of an E2-ER complex to an estrogen response element in the regulatory regions of target genes. Rather, E2 regulation of IGF-I gene transcription was shown to be mediated through an activating protein-1 (AP-1) site in the IGF-I gene promoter (43). It should be noted that the study described the estrogen-dependent activation of the IGF-I gene promoter in a HepG2 cell system, and the same mechanism of activation of the IGF-I gene in uterine cells has not been shown. Importantly, the study also demonstrated that the DNA-binding domain of the ER was required for E2 activation through the AP-1 site. Tamoxifen activation of the AP-1 pathway in vitro, including in endometrial cancer cells, has been reported previously and has also been shown to require the ER DNA-binding domain (44).
Other studies have established further that estrogens and antiestrogens
differentially activate reporter genes through AP-1 sites depending on
the ligand and whether the ER examined is ER or ERß (45, 46). It
was shown that antiestrogens/SERMs such as tamoxifen and raloxifene
activate transcription through an AP-1 site when acting through ERß.
The reports of ERß expression in the uterus are conflicting, and its
expression patterns appear to be species specific (47, 48, 49, 50); however,
the data herein indicate that in the mouse uterus, ERß is not
expressed at levels that can elicit an estrogenic response, evidenced
by the failure of the ERKO uterus to respond estrogenically to any
of the chemicals tested, particularly 4OH-tamoxifen or LY353381. If
ERß contributes to the estrogenic effects of various ER antagonists
and SERMS in the uterus, activation of the IGF-I gene and subsequent
IGF-I signaling events would be expected. Assuming the IGF-I gene is
regulated by E2 in the uterus through an AP-1
element, the absence of 4OH-tamoxifen activation of IGF-IR signaling in
the uterus of ERKO mice confirms the requirement of ER for
tamoxifen activation of an AP-1-responsive event in uterine cells. That
exposure of ERKO mice to the other chemicals tested did not result
in IGF-IR activation demonstrates that ER is absolutely required for
activation of uterine IGF-I signaling by many structurally diverse
estrogens.
With respect to the clinical relevance of this study, two recent publications reported no increase in endometrial cancer in women on raloxifene therapy, although the women were examined after only 3 yr of treatment (51, 52). Even in the absence of increased cancer risk, increases in IGF-I mRNA and IGF-IR signaling in response to antiestrogens such as 4OH-tamoxifen and raloxifene may be important in gynecological diseases that affect compartments of the uterus other than the endometrium. Studies have shown that IGF-I promotes leiomyoma cell growth in vitro (53). Although IGF-I autocrine loops have been suggested for leiomyoma cells, paracrine stimulation of leiomyomas by IGF-I from other uterine cells may contribute to their growth. In vitro studies have also shown that both tamoxifen and raloxifene can inhibit estrogen-induced growth of a leiomyoma-derived cell line (54); however, the data herein demonstrate that the raloxifene analog LY 353381 and 4OH-tamoxifen increased the levels of uterine IGF-I mRNA and activated uterine IGF-IR signaling. Increases in uterine IGF-I by tamoxifen or other SERMS in vivo could potentially counteract or antagonize the growth inhibitory actions of tamoxifen on the leiomyoma cells in vitro. Furthermore, whereas tamoxifen or raloxifene may inhibit estrogen-induced leiomyoma cell growth when administered with E2, in other cases where no estrogen is present, such as in postmenopausal women, tamoxifen or raloxifene might display their weaker estrogenic effects and, therefore, increase the growth of leiomyomas over that in untreated control cases. In support of this hypothesis is a study that examined uterine samples from postmenopausal women who had been treated with tamoxifen. Uterine IGF-I expression was determined to be up-regulated in specimens from tamoxifen-treated patients compared with that in control specimens (55).
The raloxifene analog LY353381, in addition to activating uterine IGF-I
signaling, increased the expression of PCNA and the number of mitotic
cells in the uterine epithelium. This is also in contrast to reports
that raloxifene does not display estrogenic properties in the human
uterus (17, 18), and LY 353381 does not do so in the rat uterus (37).
Although the rat study reports an overall lack of estrogenic activity
of LY 353381 in the uterus, certain doses did induce significant
increases in uterine epithelial cell height and uterine weight. The
studies presented herein were performed using a mouse model system, and
species or model system differences may play a role in the
contradictory results among studies. Additionally, in the clinical
setting, raloxifene is administered orally, and in the studies reported
herein the LY353381 compound was directly injected into the mice.
Differences based on the route of administration may, therefore, also
account for the estrogenic activity of LY353381 observed here, but not
in other studies with raloxifene. It is also worth restating that the
LY353381 compound is a raloxifene analog, and although belonging to the
same class of compounds, it is not structurally identical to
raloxifene; therefore, slight structural differences may also affect
the estrogenic activity of LY353381 compared with the lack of activity
reportedly observed with raloxifene. However, one study has shown that
raloxifene can activate ER-dependent transcription from an AP-1 site
(45). Activation of IGF-I signaling by LY353381, therefore, is not
unexpected considering that uterine IGF-I gene expression appears to be
regulated by E2 through an AP-1 site, and in the
studies presented here LY353381 appears to function estrogenically in
an ER-dependent manner.
LY353381 was the only compound that did not significantly increase the levels of both IGF-I mRNAs. Whether IGF-IA and -B mRNAs are regulated differently by the LY compound remains to be determined. Most likely, however, the lack of a significant increase in IGF-IA mRNA in response to the LY compound is due to greater interanimal variation for LY exposure than for the other chemicals. Although genistein significantly increased the levels of both IGF-I mRNAs, it was noted that there was variability in the lowest dose of genistein that increased IGF-IR tyrosine phosphorylation. This may be due to variable uptake and/or metabolism of the genistein in different mice, compared with more consistent and equal uptake and/or metabolism (if applicable) of the other chemicals in different mice. Alternatively, the reported tyrosine kinase inhibitory activity of genistein (56) may also play a role in elevating the concentration of genistein necessary to induce IGF-IR-pY.
In summary, these studies show that a myriad of estrogenic chemicals,
both those found in the environment as well as synthetic chemicals that
are of clinical interest, activate uterine IGF-I signaling in an
ER-dependent manner. From the literature it is evident that IGF-I
may affect several different aspects of uterine physiology, including
mitogenesis of the normal epithelium and proliferation of both
endometrial cancers and leiomyoma cells. Although it appears that in
the mouse uterus ERß does not play a role in mediating the activation
of IGF-I signaling by ER agonists or antagonists/SERMs, it may do so in
other species or in other tissues in which IGF-I signaling is subject
to regulation by estrogens. Therefore, it may be important to consider
including an IGF-I promoter-based assay in screens for
estrogens/antiestrogens when action in such tissues is of concern.
These studies also support an earlier suggestion (44) that analysis of
estrogen action and screening for therapeutic SERMs should consider
both classical and AP-1-directed responses.
|
Acknowledgments |
|---|
Received May 22, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621 and ß. Endocrinology 138:863–870This article has been cited by other articles:
 |
|
 |
|
  X. Di, L. Yu, A.B. Moore, L. Castro, X. Zheng, T. Hermon, and D. Dixon A low concentration of genistein induces estrogen receptor-alpha and insulin-like growth factor-I receptor interactions and proliferation in uterine leiomyoma cells Hum. Reprod., August 1, 2008; 23(8): 1873 - 1883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.G. Groothuis, H.H.N.M. Dassen, A. Romano, and C. Punyadeera Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human Hum. Reprod. Update, July 1, 2007; 13(4): 405 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Marini, L. Minutoli, F. Polito, A. Bitto, D. Altavilla, M. Atteritano, A. Gaudio, S. Mazzaferro, A. Frisina, N. Frisina, et al. Effects of the Phytoestrogen Genistein on Bone Metabolism in Osteopenic Postmenopausal Women: A Randomized Trial Ann Intern Med, June 19, 2007; 146(12): 839 - 847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Wierman Sex steroid effects at target tissues: mechanisms of action Advan Physiol Educ, March 1, 2007; 31(1): 26 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. V. Henley and K. S. Korach Endocrine-Disrupting Chemicals Use Distinct Mechanisms of Action to Modulate Endocrine System Function Endocrinology, June 1, 2006; 147(6): s25 - s32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Katayama, K. Ashizawa, T. Fukuhara, M. Hiroyasu, Y. Tsuzuki, H. Tatemoto, T. Nakada, and K. Nagai Differential Expression Patterns of Wnt and {beta}-Catenin/TCF Target Genes in the Uterus of Immature Female Rats Exposed to 17{alpha}-Ethynyl Estradiol Toxicol. Sci., June 1, 2006; 91(2): 419 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Toyofuku, T. Hara, T. Taguchi, Y. Katsura, K. Ohama, and Y. Kudo Cyclic and characteristic expression of phosphorylated Akt in human endometrium and decidual cells in vivo and in vitro Hum. Reprod., May 1, 2006; 21(5): 1122 - 1128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Ford Jr., S. G. Clark, E. M. Walters, M. B. Wheeler, and W. L. Hurley Estrogenic effects of genistein on reproductive tissues of ovariectomized gilts J Anim Sci, April 1, 2006; 84(4): 834 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Durrer, K. Maerkel, M. Schlumpf, and W. Lichtensteiger Estrogen Target Gene Regulation and Coactivator Expression in Rat Uterus after Developmental Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor Endocrinology, May 1, 2005; 146(5): 2130 - 2139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Fabian and B. F. Kimler Selective Estrogen-Receptor Modulators for Primary Prevention of Breast Cancer J. Clin. Oncol., March 10, 2005; 23(8): 1644 - 1655. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Curtis Hewitt, J. Collins, S. Grissom, B. Deroo, and K. S. Korach Global Uterine Genomics in Vivo: Microarray Evaluation of the Estrogen Receptor {alpha}-Growth Factor Cross-Talk Mechanism Mol. Endocrinol., March 1, 2005; 19(3): 657 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhang, T. McElrath, W. Tong, and J. W Pollard The molecular basis of tamoxifen induction of mouse uterine epithelial cell proliferation J. Endocrinol., January 1, 2005; 184(1): 129 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Guzeloglu Kayisli, U. A. Kayisli, G. Luleci, and A. Arici In Vivo and In Vitro Regulation of Akt Activation in Human Endometrial Cells Is Estrogen Dependent Biol Reprod, September 1, 2004; 71(3): 714 - 721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Richards, D. M. Klotz, M. P. Walker, and R. P. DiAugustine Mammary Gland Branching Morphogenesis Is Diminished in Mice with a Deficiency of Insulin-like Growth Factor-I (IGF-I), But Not in Mice with a Liver-Specific Deletion of IGF-I Endocrinology, July 1, 2004; 145(7): 3106 - 3110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ji, W. Chang, M. Centrella, and T. L. McCarthy Activation Domains of CCAAT Enhancer Binding Protein {delta}: Regions Required for Native Activity and Prostaglandin E2-Dependent Transactivation of Insulin-Like Growth Factor I Gene Expression in Rat Osteoblasts Mol. Endocrinol., September 1, 2003; 17(9): 1834 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. N. Jefferson, J. F. Couse, E. Padilla-Banks, K. S. Korach, and R. R. Newbold Neonatal Exposure to Genistein Induces Estrogen Receptor (ER){alpha} Expression and Multioocyte Follicles in the Maturing Mouse Ovary: Evidence for ER{beta}-Mediated and Nonestrogenic Actions Biol Reprod, October 1, 2002; 67(4): 1285 - 1296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sato, G. Wang, M. P. Hardy, T. Kurita, G. R. Cunha, and P. S. Cooke Role of Systemic and Local IGF-I in the Effects of Estrogen on Growth and Epithelial Proliferation of Mouse Uterus Endocrinology, July 1, 2002; 143(7): 2673 - 2679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yoshida, A. Takenaka, S.-I. Katsuda, Y. Kurokawa, and A. Maekawa Neonatal Exposure to p-tert-octylphenol Causes Abnormal Expression of Estrogen Receptor {alpha} and Subsequent Alteration of Cell Proliferating Activity in the Developing Donryu Rat Uterus Toxicol Pathol, April 1, 2002; 30(3): 357 - 364. [Abstract] [PDF]
|
|
|
|
 |
|
 D. M. Klotz, S. C. Hewitt, P. Ciana, M. Raviscioni, J. K. Lindzey, J. Foley, A. Maggi, R. P. DiAugustine, and K. S. Korach Requirement of Estrogen Receptor-alpha in Insulin-like Growth Factor-1 (IGF-1)-induced Uterine Responses and in Vivo Evidence for IGF-1/Estrogen Receptor Cross-talk J. Biol. Chem., March 1, 2002; 277(10): 8531 - 8537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Dardes, D. Bentrem, R. M. O'Regan, J. M. Schafer, and V. C. Jordan Effects of the New Selective Estrogen Receptor Modulator LY353381.HCl (Arzoxifene) on Human Endometrial Cancer Growth in Athymic Mice Clin. Cancer Res., December 1, 2001; 7(12): 4149 - 4155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Papaconstantinou, B. R. Fisher, T. H. Umbreit, P. L. Goering, N. T. Lappas, and K. M. Brown Effects of {beta}-Estradiol and Bisphenol A on Heat Shock Protein Levels and Localization in the Mouse Uterus Are Antagonized by the Antiestrogen ICI 182,780 Toxicol. Sci., October 1, 2001; 63(2): 173 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
B. Fournier, S. Gutzwiller, T. Dittmar, G. Matthias, P. Steenbergh, and P. Matthias Estrogen Receptor (ER)-alpha , but Not ER-beta , Mediates Regulation of the Insulin-like Growth Factor I Gene by Antiestrogens J. Biol. Chem., September 14, 2001; 276(38): 35444 - 35449. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
0.005 compared with vehicle treatment.
