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Endocrinology Vol. 141, No. 9 3430-3439
Copyright © 2000 by The Endocrine Society

ARTICLES

Activation of a Uterine Insulin-Like Growth Factor I Signaling Pathway by Clinical and Environmental Estrogens: Requirement of Estrogen Receptor-{alpha}

Diane M. Klotz, Sylvia Curtis Hewitt, Kenneth S. Korach and Richard P. Diaugustine

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
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent data indicate that insulin-like growth factor I (IGF-I) may have a function in mediating the mitogenic effects of 17ß-estradiol (E2) in the uterus and in regulating the growth of uterine neoplasms. This study was designed to determine whether synthetic and plant-derived chemicals that interact with estrogen receptor-{alpha} (ER{alpha}) 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{alpha} knockout mice did not activate IGF-IR, indicating that ER{alpha} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STRUCTURALLY DIVERSE synthetic and plant-derived chemicals have been shown to mimic 17ß-estradiol (E2) in both in vitro and in vivo experimental systems by interacting with estrogen receptor (ER) and eliciting responses generally attributed to E2, leading to concern for their potentials to disrupt the endocrine systems of animals and humans (Refs. 1, 2 and references therein). The ability of exogenous chemicals to interfere with female reproductive tract functions by mimicking the endogenous steroid hormone E2 is perhaps one of the most widely studied properties of endocrine-disrupting chemicals to date. Both laboratory and field studies have indicated that wildlife populations, including birds, fish, and alligators, may suffer adverse reproductive effects after exposure to environmental chemicals such as DDT, polychlorinated biphenyls, and nonylphenol (3, 4, 5, 6). Research has also focused on understanding the estrogenic actions of phytochemicals such as genistein, a compound that has been shown both in vitro and in vivo to display estrogenic properties (7, 8, 9). In addition to the hormonal effects of environmental chemicals, studies have demonstrated the endocrine-disrupting effects of clinically related exogenous estrogens. Studies of the synthetic estrogen diethylstilbestrol (DES) have shown that in utero exposure to this chemical interferes with the proper development of both the male and female reproductive tracts, as well as increases the incidence of reproductive tract neoplasms (10, 11, 12, 13). Further emphasis has been placed on designing selective ER modulators (SERMs) that can be used clinically as antiestrogens in the treatment or prevention of breast cancer without increasing the risk of endometrial cancer through their partial estrogen agonist activity. For example, tamoxifen was developed for clinical use as an antiestrogen; however, it is documented to have estrogenic effects in both rodent and human uterus (14, 15, 16). On the other hand, raloxifene, a newer clinical SERM, is reported to lack any uterotropic activity (17, 18).

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{alpha} knockout mice ({alpha}ERKO), in which both alleles of the ER{alpha} 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{alpha}. It is demonstrated that all chemicals tested required ER{alpha} 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures involving animals were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the NIEHS animal care and use committee. Animals were housed in the NIEHS animal facility under a 12-h light, 12-h dark cycle and fed food and water ad libitum. CD-1 mice (Charles River Laboratories, Inc., Raleigh, NC) or ER{alpha} 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 {alpha}-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of uterine IGF-IR signaling by ER modulators and environmental chemicals
Previous studies showed that administration of E2 to ovariectomized adult mice results in tyrosine phosphorylation of uterine IGF-IR (IGF-IR-pY), and the formation of an IGF-IR/IRS-1/p85 complex, which established the IGF-I signaling pathway as a component of E2 action in the uterus. To determine whether select environmental chemicals also mimic E2 by activating uterine IGF-IR signaling, ovariectomized adult female CD-1 mice were administered various doses of chemicals or E2, and immunoblot analysis was performed on uterine IGF-IR immunoprecipitates as described in Materials and Methods. Where data were available, mice were administered chemicals at doses above and below the minimal dose of the chemical that had been reported previously to elicit a uterotropic response (20, 36).

As shown in Fig. 1AGo, 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).



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  		Figure 1. Tyrosine phosphorylation of uterine IGF-IR and formation of uterine IGF-IR signaling complexes in response to ER modulators and environmental chemicals. Mice were administered 100 µl sesame oil (V), 30 µg/kg E2, or various doses of chemicals. Uterine IGF-IR was analyzed as described in Materials and Methods. Each lane represents an individual animal. A, Tyrosine-phosphorylated uterine IGF-IR, visualized by a diffuse band at approximately 105 kDa, is indicated by solid arrows. Tyrosine-phosphorylated IRS-1 associated with uterine IGF-IR after treatment is indicated by open arrows. A dose response for E2-induced tyrosine phosphorylation of IGF-IR is included at the bottom for comparison; doses 1–5 of E2 administered are as follows: 1) 0.003 µg/kg, 2) 0.03 µg/kg, 3) 0.3 µg/kg, 4) 3 µg/kg, and 5) 30 µg/kg. OHT, 4OH-tamoxifen; GEN, genistein; BPA, bisphenol A; LY, LY 353381. B, IRS-1 and p85 associated with uterine IGF-IR after treatment with the same doses of chemicals that induced IGF-IR tyrosine phosphorylation in A. The upper panel for each chemical represents IRS-1, and the lower panel represents p85. C, Increasing doses of each chemical administered to the mice.

 
In addition to stimulating the tyrosine phosphorylation of IGF-IR, the administration of the chemicals resulted in the formation of uterine IGF-IR/IRS-1/p85 complexes (Fig. 1BGo). 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. 1Go, A and B) were administered to mice, and Fig. 2AGo 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. 2BGo).



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  		Figure 2. Increase in uterine IGF-I mRNA by ER modulators and environmental chemicals. A, Ovariectomized adult female CD-1 mice were administered 100 µl sesame oil (VEH), 30 µg/kg E2, 30 µg/kg DES, 3000 µg/kg 4OH-tamoxifen (OHT), 15,000 µg/kg genistein (GEN), 30,000 µg/kg HPTE, 30,000 µg/kg bisphenol A (BPA), or 3000 µg/kg LY 353381 (LY). Total uterine RNA was analyzed for the presence of IGF-I transcripts as described in Materials and Methods. The protected fragments representing IGF-IA mRNA (IGF-IA), IGF-IB mRNA (IGF-IB), and cyclophilin mRNA (CYC) species are indicated. IGF-I and cyclophilin probes are indicated with arrows as 1 and 2, respectively. Each lane represents an individual animal. B, IGF-IA and IGF-IB mRNA levels represented graphically as the fold induction above the vehicle-treated control value ± SEM. Levels of cyclophilin transcripts were used for normalization among samples. *, P <= 0.005 compared with vehicle treatment.

 
Requirement of ER{alpha} in estradiol- and chemical-induced IGF-IR signaling
To demonstrate that the activation of uterine IGF-IR signaling in response to the various chemicals was mediated through ER{alpha}, rather than through an alternative mechanism of activation, ovariectomized {alpha}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 3AGo illustrates that in the {alpha}ERKO mice, IGF-IR was not activated by E2 or any of the other chemicals tested.



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  		Figure 3. Requirement of ER{alpha} in estrogen- and environmental chemical-induced IGF-IR signaling. {alpha}ERKO mice were administered A) 100 µl sesame oil (VEH), 30 µg/kg E2, 30 µg/kg DES, 3000 µg/kg 4OH-tamoxifen (OHT), 15,000 µg/kg genistein (GEN), 30,000 µg/kg HPTE, 30,000 µg/kg bisphenol A (BPA), or 3000 µg/kg LY 353381 (LY) or B) 100 µl sesame oil (OIL), 30 µg/kg E2, PBS, and long R3-IGF-I (I) as described in Materials and Methods. Uterine IGF-IR was analyzed as described in Materials and Methods. The open arrow indicates the expected size of tyrosine-phosphorylated IGF-IR. Each lane represents an individual animal.

 
To establish that activation of the IGF-I signaling pathway by IGF-I was intact in the {alpha}ERKO mouse uterus, both wild-type and {alpha}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. 3BGo, long R3-IGF-I activates IGF-IR in the {alpha}ERKO mouse uterus, demonstrating that uterine IGF-IR in the {alpha}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 {alpha}ERKO uterus was due to the absence of ER{alpha}, 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. 1–3GoGoGo). 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. 4Go and 5Go). 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. 4Go), 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. 5Go). 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 {alpha}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).



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  		Figure 4. Increase in uterine epithelial PCNA expression by ER modulators and environmental chemicals. Mice were administered 100 µl sesame oil (VEH), 30 µg/kg E2, 30 µg/kg DES, 3000 µg/kg 4OH-tamoxifen (OHT), 15,000 µg/kg genistein (GEN), 30,000 µg/kg HPTE, 30,000 µg/kg bisphenol A (BPA), or 3000 µg/kg LY 353381 (LY). Uteri were analyzed for PCNA expression as described in Materials and Methods. PCNA immunoreactivity is visualized as brown staining. The control section (CTRL) was analyzed with the omission of PCNA antibody to demonstrate the lack of nonspecific staining. Magnification, x200.

 


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  		Figure 5. Increase in uterine epithelial mitotic figures by ER modulators and environmental chemicals. Ovariectomized adult mice were administered 100 µl sesame oil (VEH), 30 µg/kg E2, 30 µg/kg DES, 3000 µg/kg 4OH-tamoxifen (OHT), 15,000 µg/kg genistein (GEN), 30,000 µg/kg HPTE, 30,000 µg/kg bisphenol A (BPA), or 3000 µg/kg LY 353381 (LY). Uteri were analyzed for mitotic figures as described in Materials and Methods. Individual or groups of mitotic figures are indicated with arrows. Magnification of uteri from treated animals, x200. Magnification of the uterus from a vehicle-treated animal, x100 (to show a larger area of the uterine epithelium).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented herein show that the clinically used estrogens/antiestrogens, phytochemicals, and synthetic environmental chemicals examined, which had been reported to display estrogenic properties, also mimic E2 by activating the full complement of IGF-I signaling events previously shown to be stimulated by E2 in the mouse uterus. These events include an increase in uterine IGF-I mRNA, tyrosine phosphorylation of IGF-IR, and the formation of an IGF-IR/IRS-1/p85 complex. Additionally, the chemicals evaluated increased both the expression of PCNA and the number of mitotic cells in the uterine epithelium.

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{alpha} 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 {alpha}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 {alpha}ERKO mice confirms the requirement of ER{alpha} for tamoxifen activation of an AP-1-responsive event in uterine cells. That exposure of {alpha}ERKO mice to the other chemicals tested did not result in IGF-IR activation demonstrates that ER{alpha} 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{alpha}-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{alpha}-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{alpha}-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
 
The authors thank Wendy Jefferson (NIEHS) for providing advice on the PCNA staining, Dr. Joseph Haseman (NIEHS) for assistance in performing and interpreting the statistical analyses, and Drs. Barbara Davis (NIEHS), Sandra Dunn (NIEHS), and Blake Neubauer (Eli Lilly & Co.) for careful and thoughtful review of the manuscript.

Received May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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