ToxSci Advance Access originally published online on March 21, 2006
Toxicological Sciences 2006 91(2):419-430; doi:10.1093/toxsci/kfj167
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Differential Expression Patterns of Wnt and ß-Catenin/TCF Target Genes in the Uterus of Immature Female Rats Exposed to 17
-Ethynyl Estradiol
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* Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Kamisu, Ibaraki 314-0255, Japan; Science of Bioresource Production, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan; Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan; and Department of Bioproduction, Faculty of Agriculture, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan
1 To whom correspondence should be addressed at Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Kamisu, Ibaraki 314-0255, Japan. Fax: +81-479-46-5097. E-mail: katayama{at}ankaken.co.jp.
Received December 15, 2005; accepted March 13, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To characterize the effects of an estrogen receptor (ER) agonist on the gene expressions in the uterus, immature female rats were administered once orally with 17
-ethynyl estradiol (EE, 3 µg/kg), a potent ER agonist. We focused on four categories of sex steroid hormone receptor genes: well-known estrogen target genes, Wnt genes, and ß-catenin/T-cell factor (TCF) target genes. ER, ERß, progesterone receptor, and androgen receptor mRNAs were all downregulated at 24 and/or 48 h after EE administration. Complement C3 and insulin-like growth factor 1 mRNAs were markedly induced after EE administration. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA status varied until 12 h after EE administration, all of them were simultaneously downregulated at 24 and 48 h. The remarkable downregulation of Wnt7a mRNA in response to EE was considered to be important to understand the various uterine phenomena affected by ER agonists. In the ß-catenin/TCF target genes, the downregulation of anti-Mullerian hormone type 2 receptor and bone morphogenetic protein 4 mRNA after EE administration appeared to be closely related to the downregulation of Wnt7a. The upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration was considered to have been affected by the upregulation of Wnt4. These results indicate that an ER agonist influences not only the mRNA expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes (Wnt4, Wnt5a, Wnt7a) and ß-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli. Key Words: estrogen receptor agonist; uterus; Wnt genes; ß-catenin/TCF target genes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The evidence that several synthetic compounds released into the environment may cause developmental and reproductive abnormalities in wildlife by disrupting normal endocrine functions has increased the concern about potential adverse human health effects from such endocrine disruptors (Colborn et al., 1993
; Kavlock et al., 1996). Among the compounds considered as endocrine disruptors, many of them have estrogenic activity (Kanno et al., 2001, 2003; Owens and Ashby, 2002). However, there are still many unresolved aspects about the molecular mechanism in the process to induce abnormal differentiation of the female reproductive tract exposed to estrogenic compounds.
Estrogen is a steroid hormone that plays a pivotal role in the regulation of mammalian reproduction and acts by regulating the transcription of specific genes through the specific nuclear receptors, estrogen receptor alpha (ER) and estrogen receptor beta (ERß) (DeMayo et al., 2002). Therefore, changes in the expression of estrogen target genes are considered to be a useful index for evaluating the estrogenic activity of synthetic compounds. However, it is difficult to predict the full range of effects of estrogenic compounds from changes of the expression level of only the well-known estrogen target genes. Therefore, we considered whether there was any possible candidate with a further broad range of effects among the genes responding to estrogenic compounds.
Wnt genes encode a large family of secreted cysteine-rich proteins that play key roles as intercellular signaling molecules in embryonic development (Wodarz and Nusse, 1998). In the Wnt family, Wnt4, Wnt5a, and Wnt7a play important roles in the female reproductive system (Heikkila et al., 2001). Wnt4 expression is crucial for the formation of Mullerian ducts, and thus both male and female Wnt4-deficient mice completely lack Mullerian ducts (Vainio et al., 1999). Wnt5a-deficient mice die at birth due to a failure to complete anteroposterior body axis development (Yamaguchi et al., 1999). Mericskay et al. (2004) demonstrated that Wnt5a is required to appropriately establish the development of the posterior region of the female reproductive tract. Furthermore, although the oviduct, uterine, and cervical compartments of the female reproductive tract developed in the absence of Wnt5a, the mutant uterus failed to form glands that are essential for adult function (Mericskay et al., 2004). Wnt7a is expressed in the luminal epithelial cells of the fetal Mullerian tracts and is maintained at high levels in the adult uterine luminal epithelium (Miller et al., 1998b). Wnt7a-deficient mice are viable, but Wnt7a mutant males are infertile due to the consequences of ectopic Mullerian ducts. Mutant females are sterile because of the abnormal development of the oviduct and uterus, both of which are Mullerian duct derivatives (Miller and Sassoon, 1998; Parr and McMahon, 1998). The abnormal female reproductive tract caused by the deficiency of Wnt7a closely resembles the abnormalities in female humans and mice prenatally exposed to diethylstilbestrol (Miller et al., 1998a).
In the Wnt signaling pathway, at least three kinds of the Wnt/ß-catenin pathway, the Wnt/c-Jun N-terminal kinase pathway (Wnt/JNK pathway), and the Wnt/Ca2+ pathway are identified (Kuhl et al., 2000; Veeman et al., 2003; Willert and Nusse, 1998). The Wnt/ß-catenin pathway regulates the transcription of various target genes via stabilized ß-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) family members. If a certain estrogenic compound influences the expressions of Wnt genes and/or ß-catenin/TCF target genes, then the compound may cause altered development of the female reproductive tract. However, only limited information is available whether estrogenic compounds influence the regulation of such gene expression.
The objective of this study was to investigate the effects of ER agonist on the expression of Wnt genes and ß-catenin/TCF target genes. In this study, we used 17-ethynyl estradiol (EE) as an ER agonist. EE is the estrogenic chemical used medically in oral contraceptive. It is estimated that each year about 3% of women in the United States and Europe who use oral contraceptives become pregnant accidentally, primarily because of missed pills (Thayer et al., 2001; Timms et al., 2005). Oral contraceptive pills may be taken for many months until the unplanned and unexpected pregnancy is discovered (Li et al., 1995; Timms et al., 2005). Therefore, there is concern about the effect of EE on the reproductive tract including uterus because it has the possibility of becoming an unexpected source of exposure of human fetuses to estrogenic compounds. We report here that EE influences the mRNA expression of Wnt genes (Wnt4, Wnt5a, Wnt7a) and ß-catenin/TCF target genes in the uterus of immature rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
EE (purity: 99%) and corn oil were obtained from Sigma-Aldrich Co. (St. Louis, MO). Ethanol was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Corn oil containing 1% ethanol was prepared as the vehicle solution.
Animals.
Thirteen-day-old, female Crj:CD(SD) IGS rats were obtained from Charles River Japan, Inc. (Kanagawa, Japan) with lactating maternal animals. After arrival, immature and maternal animals were acclimated for 5 days. Weaning and group assignment were performed on the day before administration (at 18 days old) to minimize the stress associated with weaning. Sixty animals were assigned to 12 experimental groups to give similar intergroup variations in body weight among the resulting groups. Animals were housed in polycarbonate cages (380 x 330 x 175 mm [width x depth x height], CLEA Japan, Inc., Tokyo, Japan). During the quarantine period, animals were accommodated in cages in groups of 10 immature animals and one maternal animal per cage. After group assignment, immature animals were accommodated at five animals per cage. The animal room was maintained at a temperature of 19.0–25.0°C, with a relative humidity of 35.0–75.0%, and at a 12 h light/dark cycle. The animals were allowed free access to a pellet diet for experimental animals (MF, Oriental Yeast, Co., Ltd., Tokyo, Japan) and sterilized water. The animals were cared for in accordance with "The Guidelines for Animal Experimentation" for our laboratory, Mitsubishi Chemical Safety Institute Ltd.
Study design.
We used sexually immature female rats in which significant ovarian estrogen synthesis and regulation by the hypothalamic-pituitary-gonadal axis had not been initiated. Rats were used for the experiment at 19 days of age. Animals were administered once by oral gavage with either the vehicle or 3 µg/kg EE. The dose of EE was selected at a dose known to cause hypertrophy of the uterus in a 3-day uterotrophic assay (Kanno et al., 2001). Initial body weight before the administration and final body weight before the necropsy were measured using an electronic balance (PM3000, Mettler Toledo K.K., Tokyo, Japan). The administration volume was 10 ml/kg and was adjusted individually based on the initial body weight before administration. Five animals were included in each treatment group. Animals were euthanized by CO2 asphyxiation at 1, 3, 6, 12, 24, and 48 h after administration. The uterus was removed from the body, placed on gauze, and cut at several sites to discharge gently the fluid in the uterus. The absolute "blotted uterine weight" (weight of the uterus excluded the inner fluid) was measured using an electronic balance (Model AE260, Mettler Toledo K.K.). In addition, the relative weight of the uterus from each animal was calculated by dividing the absolute blotted uterine weight by final body weight before the necropsy. The uterus was submerged in the RNA preservative reagent RNAlater (Ambion, Inc., Austin, TX), kept at 4°C overnight, and then was stored at –20°C until processing for RNA isolation.
Isolation of total RNA.
The uterus was homogenized in the dissolving and absorption liquid containing 1% 2-mercaptoethanol (TOYOBO Co., Ltd., Osaka, Japan) for 300 s at –20°C using an automatic sample preparation system (Twist Crusher HMX-2000, TOYOBO Co., Ltd.). Total RNA was isolated using MagExtractor-RNA- (TOYOBO Co., Ltd.) and an automatic nucleic acid extraction system (MagExtractor System MFX-2000, TOYOBO Co., Ltd.) according to the manufacturer's recommended protocol and was subsequently DNase treated with RNase-free DNase I (TAKARA BIO INC., Shiga, Japan) for 30 min at 37°C in the presence of RNase OUT (Invitrogen Corporation, Carlsbad, CA). The amount of total RNA was determined using the RiboGreen RNA Quantitation kit (Molecular Probes, Inc., Eugene, OR) or spectrophotometer (DU-7400, Beckman Coulter, Inc., Fullerton, CA). The absence of genomic DNA contamination in the total RNA samples was confirmed by real-time polymerase chain reaction (PCR) for each RNA sample without reverse transcriptase using TaqMan Rodent GAPDH Control Reagents VIC (Applied Biosystems, Foster City, CA), according to the manufacturer's recommended protocol.
Real-time reverse transcription–PCR.
One-step real-time reverse transcription (RT)–PCR was performed to determine changes in gene expression using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Total RNA (0.5–20 ng) isolated from each uterus was added to a reaction mixture containing forward primer, reverse primer, TaqMan probe, and TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems) in a final volume of 50 µl according to the manufacturer's instruction. Rat-specific primers and TaqMan probes were designed for the genes of interest (Table 1) using Primer Express software (Applied Biosystems). The mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was determined using primer and probe sets in the TaqMan Rodent GAPDH Control Reagents VIC (Applied Biosystems). TaqMan probes were labeled with a fluorescent reporter dye (FAM or VIC) at the 5' end and a quencher dye (TAMRA) at the 3' end. Thermal cycling conditions were as follows: 1 cycle of 30 min at 48°C for reverse transcription, 1 cycle of 10 min at 95°C for activation of DNA polymerase, 40 cycles of 15 s at 95°C for denaturation, and 1 min at 60°C for annealing/extension. The expression levels of target gene and 18S rRNA in each sample were calculated based on the standard curve generated with the rat total RNA for the uterus (UNITECH Co., Ltd., Chiba, Japan) or ovary (Ambion, Inc.). The expression level of target gene was then normalized by the expression level of 18S rRNA using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems) to control the quantity of the isolated RNA. Real-time RT-PCR analyses were performed in duplicate on all five animals in each treatment group.
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Statistical analysis.
The data of each group were expressed as the means ± SDs. Fold changes for uterine weights and gene expression data were expressed as the ratio of the mean value of the group treated with EE (3 µg/kg) to the mean value of the time-matched vehicle group. Differences of body weights, uterine weights, and gene expression data in the EE treatment group from those in the time-matched vehicle group were analyzed for statistical significance. F-test was applied to analyze the homogeneity of the variance. When the variance was homogeneous, Student's t-test was performed. When the variance was not homogeneous, Aspin-Welch' t-test was performed. The statistical analysis was performed with SAS Proprietary Software Release 8.2 (SAS Institute, Inc., Cary, NC). Significance was determined by a two-tailed significance level of 5%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of EE on the Body Weight in Immature Female Rats
No statistically significant differences were noted in body weights between any of the 3-µg/kg EE groups and the time-matched vehicle groups (Table 2). Since there were no abnormal clinical signs observed in any groups, 3 µg/kg of EE was considered not to produce serious toxicity in the animals.
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Effects of EE on the Uterine Weight in Immature Female Rats
Uterine weights significantly increased between 6 and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 1, Table 3). An increase of the relative uterine weight in the 3-µg/kg EE group reached a peak at 24 h (1.99-fold) after administration.
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Effects of EE on the Expression of Reference Genes in the Uterus of Immature Female Rats
Reference genes, which are often referred to as housekeeping genes, are frequently used to normalize mRNA levels between different samples. However, the expression level of these genes may vary in different tissues, different cell types, and different disease stages, so the selection of the reference genes is critical for the interpretation of the expression data. Therefore, to determine a stable endogenous reference gene in the uterus after treatment with ER agonist, time-course changes were measured in the expressions of commonly used reference genes, Gapdh mRNA and 18S rRNA. Firstly, the expression of Gapdh mRNA and 18S rRNA were normalized with an amount of total RNA. The expression of Gapdh mRNA normalized with total RNA significantly increased between 3 and 12 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 2B). However, no statistically significant differences were noted in the expression of 18S rRNA normalized with total RNA between any of the 3-µg/kg EE groups and the time-matched vehicle groups (Fig. 2A). Therefore, 18S rRNA was judged to be suitable as a stable endogenous reference gene to normalize the target mRNA expression in the uterus after treatment with ER agonist.
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The expression of Gapdh mRNA normalized with 18S rRNA significantly increased between 3 and 24 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 2C). An increase of the Gapdh mRNA in the 3-µg/kg EE group reached a peak at 12 h (9.64-fold) after administration.
Effects of EE on the Expression of Sex Steroid Hormone Receptor Genes in the Uterus of Immature Female Rats
ER, ERß, progesterone receptor (PR), and androgen receptor (AR) mRNAs were selected to evaluate the effect of EE on the expression of sex steroid hormone receptor genes (Diel et al., 2000; Waters et al., 2001).
The expression level of ER mRNA was about 100 times higher than that of ERß mRNA in the uterus of immature rats. The expression of ER mRNA significantly decreased from 24 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups and kept decreasing until 48 h (0.57-fold) (Fig. 3A, Table 3).
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The expression of ERß mRNA significantly increased at 1 h (2.01-fold) after treatment with 3 µg/kg EE, but thereafter it decreased more than those of the time-matched vehicle groups (the lowest: 0.25-fold at 6 h) (Fig. 3B, Table 3).
The expression of PR mRNA significantly decreased from 12 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups and kept decreasing until 48 h (the lowest: 0.40-fold at 24 h) (Fig. 3C, Table 3).
The expression of AR mRNA significantly increased at 3 h (1.93-fold) after treatment with 3 µg/kg EE as compared with that in the time-matched vehicle group but significantly decreased more than that of the time-matched vehicle group at 48 h (0.50-fold) (Fig. 3D, Table 3).
Since ER, ERß, PR, and AR are transcription factors to exert physiological functions specific to each ligand (Mangelsdorf et al., 1995; Tasset et al., 1990), it is suggested that estrogenic compounds may affect the expressions of their downstream target genes.
Effects of EE on the Expression of Well-Known Estrogen Target Genes in the Uterus of Immature Female Rats
Complement C3 (Diel et al., 2000; Sundstrom et al., 1989) and insulin-like growth factor 1 (Igf1: Klotz et al., 2000) mRNAs were selected to evaluate the effect of EE on the expression of well-known estrogen target genes.
The expression of complement C3 mRNA significantly increased between 6 and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 4A). An increase of the complement C3 mRNA in the 3-µg/kg EE group reached a peak at 24 h (43.90-fold) after administration (Table 3). Since complement C3 mRNA was markedly induced after EE administration, this gene was considered as a useful biomarker for evaluating the effects of estrogenic compounds in the uterus.
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The expression of Igf1 mRNA significantly increased between 3 and 12 h (the peak: 9.73-fold at 3 h) after treatment with 3 µg/kg EE (Fig. 4B, Table 3). However, thereafter, the expression of Igf1 mRNA kept decreasing and significantly decreased more than that of the time-matched vehicle group at 48 h (0.52-fold) (Table 3).
Time courses of the expression of these genes (particularly the maximal response time) were greatly different individually. Consequently, it is considered important to closely investigate the time course of gene expressions for evaluating the estrogen-like effects of chemicals based on changes in mRNA expression level.
Effects of EE on the Expression of Wnt Genes in the Uterus of Immature Female Rats
Wnt4, Wnt5a, and Wnt7a mRNAs were selected to evaluate the effect of EE on the expression of Wnt genes (Carta and Sassoon, 2004; Hou et al., 2004; Mericskay et al., 2004; Miller et al., 1998b).
The expression of Wnt4 mRNA increased between 3 and 12 h after treatment with 3 µg/kg EE, as compared with those in the time-matched vehicle groups, and significant differences were noted at 6 h (1.71-fold) and 12 h (1.95-fold) after administration (Fig. 5A, Table 3). However, thereafter, the expression of Wnt4 mRNA kept decreasing, and significant differences were noted at 24 h (0.51-fold) and 48 h (0.43-fold) after administration.
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The expression of Wnt5a mRNA significantly increased at 3 h (2.12-fold) after treatment with 3 µg/kg EE as compared with that in the time-matched vehicle group (Fig. 5B, Table 3). However, thereafter, the expression of Wnt5a mRNA kept decreasing, and significant differences were noted at 24 h (0.45-fold) and 48 h (0.39-fold) after administration.
The expression of Wnt7a mRNA significantly decreased from 1 h after treatment with 3 µg/kg EE (0.70-fold) as compared with those in the time-matched vehicle groups (Fig. 5C, Table 3). No statistically significant difference was noted in the expression of Wnt7a mRNA at 3 h after treatment with 3 µg/kg EE as compared with that in the time-matched vehicle group. However, the mean Wnt7a mRNA level was similar to that observed at 1 h (0.70-fold) after administration. Thereafter, the expression of Wnt7a mRNA significantly decreased between 6 and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups. The decrease of Wnt7a mRNA in the 3-µg/kg EE group fell to the lowest at 24 h (0.15-fold) after administration.
Effects of EE on the Expression of ß-Catenin/TCF Target Genes in the Uterus of Immature Female Rats
Anti-Mullerian hormone type 2 receptor (Amhr2, also known as Mullerian inhibiting substance type II receptor: Hossain and Saunders, 2003), bone morphogenetic protein 4 (Bmp4: Kim et al., 2002; Schwartz et al., 2003), fibroblast growth factor 9 (Fgf9: Imai et al., 2002; Schwartz et al., 2003), cyclin D1 (Schwartz et al., 2003; Shtutman et al., 1999), follistatin (Willert et al., 2002), fibronectin (Gradl et al., 1999), and matrix metalloproteinase 7 (Mmp7: Brabletz et al., 1999; Schwartz et al., 2003) mRNAs were selected to evaluate the effect of EE on the expression of ß-catenin/TCF target genes.
The expression of Amhr2 mRNA significantly decreased from 6 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups, and they kept decreasing until 48 h after administration (Fig. 6A, Table 3). Significant differences were noted between 6 and 24 h (the lowest: 0.14-fold at 6 h) after administration.
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The expression of Bmp4 mRNA significantly decreased between 6 and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 6B, Table 3). A decrease of the Bmp4 mRNA in the 3-µg/kg EE group fell to the lowest at 12 h (0.13-fold) after administration.
The expression of Fgf9 mRNA significantly decreased at 24 and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 6C, Table 3). A decrease of the Fgf9 mRNA in the 3-µg/kg EE group fell to the lowest at 24 h (0.40-fold) after administration.
The expression of cyclin D1 mRNA significantly increased between 3 and 12 h (the peak: 1.89-fold at 3 h) after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 7A, Table 3). However, thereafter, the expression of cyclin D1 mRNA kept decreasing and significantly decreased more than that of the time-matched vehicle group at 24 h (0.45-fold) after administration.
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The expression of follistatin mRNA significantly increased between 1 and 6 h (the peak: 4.18-fold at 6 h) after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 7B, Table 3). However, thereafter, the expression of follistatin mRNA significantly decreased more than those of the time-matched vehicle groups between 12 and 48 h (the lowest: 0.32-fold at 24 h).
The expression of fibronectin mRNA significantly increased at 1 and 3 h (the peak: 3.06-fold at 3 h) after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 7C, Table 3). However, thereafter, the expression of fibronectin mRNA decreased remarkably, and no statistically significant difference was noted from 6 to 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups.
The expression of Mmp7 mRNA increased remarkably from 3 to 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups (Fig. 7D, Table 3). A statistically significant difference was noted in the expression of Mmp7 mRNA at 6, 12, and 48 h after treatment with 3 µg/kg EE as compared with those in the time-matched vehicle groups, respectively. An increase of the Mmp7 mRNA in the 3-µg/kg EE group reached a peak at 6 h (41.93-fold) after administration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to estrogenic compounds during critical in utero and early postnatal stages of development is the particular concern because many feedback mechanisms functioning in the adult are absent and adverse effects may be noted at doses lower than those observed in the adult (Colborn et al., 1993
; Crisp et al., 1998). Furthermore, it may lead to permanent alterations in the development of reproductive organs and other tissues with ER (Bigsby et al., 1999). Therefore, it is important to understand whether estrogenic compounds interfere with the expression of morphogenesis-related gene in the immature uterus.
We demonstrated that EE altered the mRNA expression of Wnt genes and ß-catenin/TCF target genes by various time-course patterns in the uterus of immature female rats. Wnt7a is the key target gene assumed to be responsible for the effects of diethylstilbestrol (Mericskay et al., 2005; Miller et al., 1998a; Sassoon, 1999). In this study, Wnt7a mRNA was drastically downregulated after EE administration, decreasing to 15% of the time-matched control level at 24 h. Although the time courses of Wnt4, Wnt5a, and Wnt7a mRNA varied until 12 h after EE administration, all of them were downregulated at 24 and 48 h. Since these genes are intimately involved with development and differentiation of organs (Heikkila et al., 2001; Wodarz and Nusse, 1998), simultaneous downregulation of Wnt4, Wnt5a, and Wnt7a mRNA by EE may cause altered development of the female reproductive tract, indicating that estrogenic compounds affect the Wnt signaling pathway regulated by Wnt4, Wnt5a, and Wnt7a.
The Wnt/ß-catenin pathway is the most understood Wnt signaling pathway. In the absence of Wnt signals, free ß-catenin is phosphorylated by glycogen synthase kinase-3ß in the cytosol. Adenomatous polyposis coli and Axin are part of the large multiprotein complex that facilitates this phosphorylation process (Rubinfeld et al., 1993). Phosphorylated ß-catenin is ubiquitinated and ultimately degraded by the proteasome (Orford et al., 1997). In the presence of a Wnt signal, this phosphorylation of ß-catenin is blocked, and the free cytosolic ß-catenin is translocated to the nucleus and heterodimerized with one of the TCF/LEF family members and can regulate the transcription of ß-catenin/TCF target genes (Giles et al., 2003).
If ER agonist interfered with the Wnt/ß-catenin pathway in the immature uterus, the mRNA expression of ß-catenin/TCF target genes should also alter in the uterus of immature rats. Expression patterns of ß-catenin/TCF target gene after EE administration were roughly classified into four patterns. Group 1, consisting of Amhr2, Bmp4, and Fgf9 mRNA, revealed downregulation in expression levels after EE administration. Amhr2 and Bmp4 mRNA were downregulated after EE administration, demonstrating time courses linked closely to Wnt7a mRNA. Fgf9 mRNA was downregulated during the period when Wnt7a mRNA was downregulated most remarkably. Consequently, it was suggested that mRNA expressions of Amhr2, Bmp4, and Fgf9 after EE administration might be affected by the downregulation of Wnt7a but not Wnt4 and Wnt5a. Group 2, consisting of cyclin D1 and follistatin mRNA, revealed upregulation at the early phase after EE administration followed by downregulation, as compared with those in the time-matched vehicle groups. Time-course expression patterns of these genes were similar to that of Wnt4 mRNA. Hou et al. (2004) reported that estrogen in an ER-independent manner rapidly upregulates the mRNA expressions of Wnt4 and Wnt5a in the uterus of ovariectomized wild-type or ER (–/–) mice. Terada et al. (2003) showed that the overexpression of Wnt4 and ß-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Yao et al. (2004) reported that follistatin is a downstream component of Wnt4 signaling and proposed that Wnt4 acts through follistatin to regulate vascular boundaries and maintain germ cell survival in the ovary. Therefore, it was suggested that the upregulation of cyclin D1 and follistatin mRNA at the early phase after EE administration might be affected by the upregulation of Wnt4. Group 3, consisting of fibronectin mRNA, revealed upregulation at the early phase after EE administration, followed by changes similar to the level of the time-matched vehicle group. Group 4, consisting of Mmp7 mRNA, revealed continuous upregulation after EE administration. Mmp7 is well known as the ß-catenin/TCF target gene described above; however, it is also well known as a gene that increases in response to estrogen (Naciff et al., 2003). In this study, Mmp7 mRNA was remarkably upregulated after EE administration regardless of the alteration of expression levels of Wnt4, Wnt5a, and Wnt7a. Therefore, it was suggested that the Mmp7 mRNA was not downregulated as the ß-catenin/TCF target gene but was remarkably upregulated as the estrogen target gene in the uterus of immature rats. According to these results, mRNA expression level of the ß-catenin/TCF target genes in the uterus after EE administration was considered dependent on the balance between estrogen and Wnt signaling pathway under the presence of multiple stimuli. In the ongoing experiment, to determine whether the gene expression changes caused by EE were ER-mediated reactions, immature female rats were treated once by oral gavage with vehicle, EE, or EE plus ICI 182,780 (ER antagonist). At the present moment, we confirmed that the mRNA expression of Wnt5a, Wnt7a, Amhr2, and Fgf9 was regulated by ER (data not shown).
Wnt5a is known as a ligand of the Wnt/Ca2+ pathway, and it plays a role in inhibiting the Wnt/ß-catenin pathway (Kuhl et al., 2000; Veeman et al., 2003). However, the signaling pathway of Wnt4 and Wnt7a is complex, and there are still many uncertain aspects of their molecular mechanism of action. For example, it has been reported that the signal of Wnt7a might be transmitted via both the ß-catenin–dependent pathway (Lyu and Joo, 2005; Shimizu et al., 1997) and the ß-catenin–independent pathway (Kengaku et al., 1998; Lyu and Joo, 2005). Lyu and Joo (2005) demonstrated that Wnt7a induced the accumulation of cytosolic ß-catenin and the activation of small GTPase Rac and ß-catenin in the SV40-immortalized human corneal epithelial cells. The regulation of ß-catenin–independent pathway by Wnt7a depended on the activation of Rac and c-Jun, suggesting that Wnt7a could activate the Wnt/JNK pathway. In this study, the precise mechanisms of action of ER agonist on the expression of Wnt genes and ß-catenin/TCF target genes remain to be elucidated. Therefore, further studies are needed to examine whether the expression of Wnt4, Wnt5a, and Wnt7a, which are altered by ER agonist, influences the expression of the ß-catenin/TCF target genes through ß-catenin-dependent pathway and/or ß-catenin–independent pathway.
It is rare that the cell is exposed only to the single extracellular or intracellular signal in the process of development and differentiation of organs. Actually, it is thought that the multiple signal transduction pathways are activated and/or inhibited at the same time in one cell, and they influence each other. In this study, we demonstrated that ER agonist influenced not only the expression of sex steroid hormone receptor genes and well-known estrogen target genes but also Wnt genes and ß-catenin/TCF target genes in the uterus of immature rats, indicating that their molecules are the potential players affected by estrogenic stimuli. Our findings in this study will provide important clues for evaluating the effects of estrogenic compounds in the immature uterus.
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ACKNOWLEDGMENTS |
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The authors thank Mr. T. Sato, Mr. T. Shinozuka, and Ms. K. Tsutsumi for their excellent technical assistance and Dr. G. J. Wishart, University of Abertay Dundee, United Kingdom, for kindly reviewing the manuscript.
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