Estrogen Receptor {alpha} Pathway Is Involved in the Regulation of Calbindin-D9k in the Uterus of Immature Rats

Geun-Shik Lee*, Hoe-Jin Kim*, Yong-Woo Jung*, Kyung-Chul Choi{dagger} and Eui-Bae Jeung*,1

* Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763 Republic of Korea, and {dagger} Department of Obstetrics and Gynecology, British Columbia Children's and Women's Hospital, British Columbia Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, BC, V6H 3V5 Canada

1 To whom correspondence should be addressed at Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea. Fax: +82-43-267-3150. E-mail: ebjeung{at}chungbuk.ac.kr.

Received November 8, 2004; accepted December 22, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been demonstrated in our previous studies that Calbindin-D9k (CaBP-9k) is a potent biomarker for screening estrogen-like chemicals in the rat model. Although treatments with 17beta-estradiol (E2) and endocrine disrupting compounds resulted in the up-regulation of uterine CaBP-9k, the mechanism of CaBP-9k induction by these compounds through two subtypes of estrogen receptors (ER{alpha} and ERß) is unclear. Thus, in the present study, immature rats were treated with propyl pyrazole triol (PPT, an ER{alpha}-selective ligand), diarylpropionitrile (DPN, an ERß-selective ligand), E2, or dimethyl sulfoxide (DMSO, a vehicle control) for three days in order to clarify which subtype of ER is involved in the uterine CaBP-9k induction. Following injection with these ER ligands, uterine CaBP-9k expression was analyzed by Northern blot and immunoblot assays. Uterine CaBP-9k expression is mainly mediated by PPT in a dose- and time-dependent manner in immature rats, whereas no significant alteration of the uterine CaBP-9k gene was observed after DPN treatment. In addition, an estrogenicity of PPT in inducing CaBP-9k expression was completely blocked by the anti-estrogen ICI 182,780, implying that uterine CaBP-9k is solely induced by ER{alpha}. A single treatment with PPT rapidly increased the protein levels of ER{alpha} and PR, an E2-mediated gene, in these tissues. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via the ER{alpha} pathway, but not ERß, in the uterus of immature rats.

Key Words: Calbindin-D9k; estrogen receptor; progesterone receptor; PPT; DPN.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 9-kDa cytosolic calcium-binding protein, Calbindin-D9k (CaBP-9k), belongs to a family of intracellular proteins that have high affinities for calcium and contain two calcium-binding domains (Christakos et al., 1989Go; Kumar et al., 1989Go; Wasserman et al., 1982Go). CaBP-9k is expressed in several mammalian tissues such as the intestine, uterus, kidney, and bone (Armbrecht et al., 1989Go; Delorme et al., 1983Go; Lee et al., 2003Go; Mathieu et al., 1989Go; Seifert et al., 1988Go). Functionally, intestinal CaBP-9k is involved in intestinal calcium absorption and is regulated at both the transcriptional and post-transcriptional level by 1,25-dihydroxyvitamin D3, the hormonal form of Vitamin D (Darwish and DeLuca, 1992Go; Roche et al., 1986Go; Wasserman and Fullmer, 1989Go). Although CaBP-9k is mainly expressed in the female reproductive tissues of various species, the exact role of this gene remains unknown. Uterine CaBP-9k has been postulated to control myometrial activity related to intracellular calcium levels. In addition, evidence has been presented for an absolute requirement for CaBP-9k and –28k during the early phase of embryo implantation; this suggests that regulation of calcium availability in the vicinity of the implanting embryo is critical for successful implantation (Luu et al., 2004Go; Mathieu et al., 1989Go). Again, placental CaBP-9k plays a role in calcium transfer from the mother to the fetus and is important in fetal growth. It appears that the CaBP-9k gene is not under the control of vitamin D in the uterus despite the presence of vitamin D receptors in this tissue; instead it is under the control of the sex steroid hormones. The hormonal mechanism controlling the uterine CaBP-9k gene is relatively well understood in rats. In the uterus of rats, 17beta-estradiol (E2) up-regulates and progesterone (P4) down-regulates CaBP-9k gene expression during estrous cycle and early pregnancy (Krisinger et al., 1992Go, 1994Go; L'Horset et al., 1993Go, 1994Go). The uterine CaBP-9k is mainly distributed in the myometrium and partially in endometrial stroma cells and luminal endometrium (An et al., 2003aGo; Bruns et al., 1988Go; Hong et al., 2004aGo; Warembourg et al., 1987Go). In addition, recent studies by us indicate that estrogenic compounds, i.e., octyl-phenol, nonyl-phenol, and bisphenol A, strongly induce uterine CaBP-9k mRNA and protein in the uterus of rodents. Therefore, this gene could be a potential biomarker for environmental estrogenic chemicals (An et al., 2002Go, 2003aGo,bGo; Hong et al., 2003bGo, 2004bGo).

The classical estrogen receptor (now referred to as ER{alpha}) was thought to be the only form of nuclear receptor able to bind estrogen and mediate its hormonal effects in target tissues. However, the first cloning of a second form of estrogen receptor by Kuiper et al., now referred to as ERß, has caused a re-examination of the estrogen signaling system (Kuiper et al., 1996Go; Mosselman et al., 1996Go). Although the CaBP-9k gene expression is tightly regulated by estrogens in the rat uterus, it is unclear how ER agonists induce the CaBP-9k expression in this tissue and which type of ER is involved. In the uterus, ER{alpha} is expressed in the epithelial cells, glandular epithelial cells, and stromal cells; however, ERß is only detected in glandular epithelial cells (Hiroi et al., 1999Go; Pelletier et al., 2000Go). Judging from the localization of ERs and CaBP-9k, it is anticipated that ER{alpha} mediates uterine CaBP-9k regulation. To determine the involvement of specific ER subtypes in the induction of uterine CaBP-9k, the present study was performed with two novel ER subtype-selective ligands, propyl pyrazole triol (PPT, an ER{alpha} agonist) and diarylpropionitrile (DPN, an ERß agonist). PPT is a potent ER{alpha} agonist that has 410-fold higher binding affinity for ER{alpha} than ERß, and it has been demonstrated to have almost no binding affinity to ERß (Kraichely et al., 2000Go; Stauffer et al., 2000Go). However, DPN is a potency-selective agonist for ERß, and 70 times more selective for ERß than ER{alpha} (Meyers et al., 2001Go). Using these novel ER- selective chemicals, we examined the effects of PPT and DPN on uterine CaBP-9k mRNA and protein accumulation in the uteri of immature rats. Northern blot and immunoblot analyses were used to elucidate the mechanism of induction of CaBP-9k by estrogens through ERs in the uterus of immature rats. Further, the expression of uterine ER{alpha} and progesterone receptor (PR) was determined following single PPT treatment in the same tissues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Dimethyl sulfoxide (DMSO) was purchased from Amresco, Inc. (Solon, OH) to dissolve reagents. Propyl pyrazole triol (PPT, Cat. #1426, an ER{alpha} -specific agonist), diarylpropionitrile (DPN, Cat. #1496, an ERß-specific agonist), and ICI 182,780 (ICI, Cat. #1047, an E2 antagonist) were obtained from Tocris (Eslisvill, MO), whereas 17beta-estradiol (E2, Cat. #E-8875) was purchased from the Sigma-Aldrich Corp. (St. Louis, MO).

Experimental animals and treatments.
Immature female Sprague-Dawley rats (14 days old) were obtained from Dae Han Biolink Co, Ltd. (Eumsung, Chungbuk, Korea) and 137 rats in total were used in the present experiments. All animals were housed in polycarbonate cages, and used after acclimation to an environmentally controlled room (temperature: 23 ± 2°C, relative humidity: 50 ± 10%, frequent ventilation and 12 h light cycle). They were fed with soy-free pellet food (Dyets, Inc., Bethlehem, PA). Sixty rats were divided into four groups (n = 15 per group), and each group was subcutaneously (sc) treated with DMSO as a vehicle, 40 µg/kg body weight (BW) E2, 10 mg/kg BW PPT or DPN (Frasor et al., 2003Go; Harris et al., 2002Go; Tena-Sempere et al., 2004Go) for three days. The rats were euthanized at 3, 6, 12, 24, and 48 h after final injection. In a dose-dependent experiment, four groups of rats (total n = 32) were given sc injection with PPT or DPN at the dose of 0.01, 0.1, 1, and 10 mg/kg BW for three days and euthanized. Three rats were given E2 (40 µg/kg BW) as a positive control and another three rats were given DMSO as a vehicle control. In a time-dependent experiment, the rats (total n = 24) were given a single sc injection with PPT (1 mg/kg BW), and euthanized at 0, 3, 6, 12, 24, 48, 72, and 96 h after injection. For the effect of antagonism, nine rats were injected sc with three doses of ICI (0.1, 1, and 10 mg/kg BW) 30 min before injection with 0.1 mg/kg BW PPT for three days, and two groups were a positive (n = 3, 0.1mg/kg BW PPT) or negative (n = 3, DMSO) control. All experimental procedures and animal use were approved by the Ethics Committee of the Chungbuk National University.

Total RNA extraction and Northern blot analysis.
Rats were euthanized and uteri were rapidly excised and washed in cold sterile NaCl (0.9%). Total RNA was prepared from the uteri with TRIzol reagent (Invitrogen, Co., Carlsbad, CA), and the concentration of RNA was determined by the absorbance at 260 nm. Total RNA was denatured by heating at 85°C for 10 min. Ten micrograms of total RNA were electrophoresed on 1% formaldehyde denaturing agarose gels for 1 h at 110 volts, and 18S rRNA served as an indicator of quantity of total RNA. The RNA was then transferred from agarose gel to nylon membrane with Vacuum blotter (Bio-Rad, Hercules, CA) according to manufacturer's suggested procedure. RNA was UV cross-linked to the membrane using a Gene Cross-Linker (Bio-Rad). The membranes were prehybridized in 50% formamide, 5x SSPE, 5x Denhardt's, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA for 2 h at 42°C. The radioactive labeled CaBP-9k probe was prepared from the Random Primer DNA Labeling Kit (TaKaRa Bio., Inc., Otsu, Shiga, Japan) according to the manufacturer's suggested procedure. The [32P]dCTP labeled CaBP-9k probe was added to the hybridization solution and incubated overnight at 42°C. The membranes were washed three times at 42°C in 2x SSC, 0.1% SDS, at 54°C in 1x SSC, 0.1% SDS and at 68°C in 0.1x SSC, 0.1% SDS. The membranes were then exposed to x-ray films (Eastman Kodak, Co., Rochester, NY). The films were scanned and analyzed by Molecular Analysis Program version 1.5 (Gel Doc 1000, Bio-Rad). The assay was repeated three times.

Western blot analysis.
Protein was extracted with Proprep (iNtRON Bio., Inc., Sungnam, Kyungki-Do, Korea) according to the supplier's instructions. Thirty µg of cytosolic protein per lane was resolved by SDS/PAGE (12% acrylamide) and transferred to a nitrocellulose membrane by a Trans-Blot Cell (Bio-Rad), according to the manufacturer's protocol. The membranes were then blocked with phosphate-buffered saline containing 0.05% Tween-20 and 5% dry milk and left overnight; they were then incubated sequentially with primary and secondary antibodies dissolved in 1% bovine serum albumin (BSA) for 1 h at room temperature. The antibodies to rat CaBP-9k (diluted 1:3000, Cat. #CB9, Swant, Bellinzona, Switzerland), ER{alpha} (1: 3000, Cat. #sc-8002, Santa Cruz Biotech, Santa Cruz, CA), PR (1:2000, Cat. #sc-538, Santa Cruz Biotech) and ß-actin (1:1000, Cat. #sc-10731, Santa Cruz Biotech) were employed. A horseradish peroxidase conjugated anti-rabbit IgG (1:3000, Santa Cruz Biotech) was used as a secondary antibody. The membranes were visualized using Lighting Chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's manual and exposed to x-ray film (Eastman Kodak). The films were scanned and analyzed by molecular analysis program version 1.5 (Gel Doc 1000).

Data analysis.
Data were analyzed by nonparametric one way analysis of variance using the Kruskal Wallis test, followed by Dunnett's test for multiple comparisons to vehicle. The value of data was converted to ranks for these tests. All statistical analyses were performed with SPSS for Windows & E (SPSS Inco, Chicago, IL). p < 0.01 or lower was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of ER{alpha}- and ERß-Specific Ligands on Uterine CaBP-9k Gene Expression
To determine which ER subtype is involved in the induction of uterine CaBP-9k gene, its mRNA and protein levels were analyzed in a time-dependent manner following sc injections with DMSO as a vehicle, 40 µg/kg E2, 10 mg/kg PPT (a ER{alpha}-specific ligand), and DPN (a ERß-specific ligand) for three days. The expression of CaBP-9k mRNA was induced through 3 to 48 h after a final treatment with PPT (10 mg/kg BW), whereas no significant induction of CaBP-9k mRNA was observed by a DPN-treatment at 3 to 48 h (Fig. 1). Treatment with DPN appeared to induce CaBP-9k mRNA, but it was not significant as shown in Figure 1. As expected, a maximal induction of CaBP-9k mRNA after final E2 injection was observed at 6 h and E2-induced CaBP-9k mRNA was retracted to the vehicle level at 48 h (Fig. 1). To confirm a translational level of CaBP-9k by these ligands, the levels of CaBP-9k protein were further examined following treatment with E2, PPT, and DPN for 12 h using a specific antibody to CaBP-9k protein in the uterus of immature rats. The parallel significant induction of CaBP-9k protein was observed in E2 and PPT treated groups, and an increase in CaBP-9k protein was 2.5-fold higher by PPT rather than by E2 (Fig. 2). DPN was shown to have a minor effect on the induction of CaBP-9k, but this was not significant compared to the vehicle, as demonstrated in Figure 2. These results suggest that the induction of uterine CaBP-9k by E2 or estrogenic chemicals may occur mainly through ER{alpha} signaling pathway, but not through ERß.



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FIG. 1. Time-dependent effect of ER{alpha}- and ERß- selective ligands on uterine CaBP-9k mRNA expression following three days of injections. Sixty immature rats were treated with PPT and DPN (10 mg/kg BW), E2 (40 µg/kg BW) as a positive control, and DMSO as a negative control for three days and euthanized at 3, 6, 12, 24, and 48 h after final injection. In sequential, CaBP-9k mRNA expression was examined using Northern hybridization analysis as described in Materials and Methods. Data were analyzed by nonparametric way of Kruskal Wallis test, followed by Dunnett's test for multiple comparisons to vehicle. The values represent means ± SD. a, p < 0.01 vs. vehicle.

 


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FIG. 2. Effect of ERs selective ligands on the induction of uterine CaBP-9k protein. Sixty immature rats were treated sc with PPT and DPN (10 mg/kg BW), E2 (40 µg/kg BW) as a positive control, and DMSO as a negative control for three days and euthanized at 12 h after final injection. The expression of CaBP-9k protein was analyzed by Western blot analysis as described in Materials and Methods. The values represent means ± SD. a, p < 0.01 vs. vehicle.

 
Effect of ER-Selective Ligands on Uterine CaBP-9k Gene Expression
The immature rats were treated with increasing doses of PPT and DPN (0.01, 0.1, 1, and 10 mg/kg BW) in a dose-dependent manner to investigate whether an accumulation of CaBP-9k gene by these ligands is dose-dependent or not. The expression of CaBP-9k mRNA was significantly induced by the treatments with PPT (1 and 10 mg/kg BW), and a 4- or 5-fold increase over E2 was observed by PPT treatments, respectively (Fig. 3A). But no significant alteration was observed in the mRNA level at 0.01 and 0.1 mg/kg BW PPT treatments, even though treatment with 0.1 mg/kg PPT appears to increase CaBP-9k mRNA as seen in Figure 3A. In parallel with its mRNA levels, the protein levels of CaBP-9k were significantly enhanced by the treatments with PPT at 0.1, 1, and 10 mg/kg BW (Fig. 3B). These data indicate that uterine CaBP-9k mRNA and protein are regulated in a dose-dependent manner by PPT. However, all doses of DPN did not increase or alter the protein level of uterine CaBP-9k (Fig. 3C).



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FIG. 3. Dose-dependent effect of PPT or DPN on uterine CaBP-9k mRNA and protein expressions. Total 38 immature rats were treated sc with PPT or DPN at the doses of 0.01, 0.1, 1, and 10 mg/kg BW, E2 as a positive control (n = 3), and DMSO as a negative control (n = 3) for three days. In sequential, PPT-induced CaBP-9k mRNA (A) and protein levels (B), and DPN-induced protein levels (C) were examined by Northern hybridization and Western blot analysis, respectively, described in Materials and Methods. Data were analyzed by nonparametric way of Kruskal Wallis test, followed by Dunnett's test for multiple comparisons to vehicle. The values represent means ± SD. a, p < 0.01 vs. vehicle.

 
For a time-course study in the regulation of CaBP-9k by PPT (an ER{alpha} selective ligand), the uteri were collected at 0, 3, 6, 12, 24, 48, 72, and 96 h after a single treatment with PPT (1 mg/kg BW). A significant increase in CaBP-9k mRNA was observed from 6 to 24 h, and a maximal accumulation of CaBP-9k mRNA was detected at 12 h after PPT injection (Fig. 4A). After this, the highest mRNA level fell steadily until 72 h, and it was scarcely detectable at 72 and 96 h after PPT treatment (Fig. 4A). It is of interest that CaBP-9k protein levels gradually increased in a time-dependent fashion and was maximally accumulated at 72 h following PPT treatment, as seen in Figure 4B. In addition, the effect of PPT on the uterine CaBP-9k protein level started to decrease at 96 h after a single PPT injection, so that the protein levels showed a slower time course than their mRNA levels after PPT (Fig. 4B).



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FIG. 4. Time-dependent effects of PPT on uterine CaBP-9k mRNA and protein expression. Immature rats (total n = 24) were singly treated sc with PPT at the dose of 1 mg/kg BW day and euthanized at 0, 3, 6, 12, 24, 48, 72, and 96 h after injection. Uterine CaBP-9k mRNA (A) and protein (B) expression were analyzed by Northern hybridization and immunoblot analysis, respectively, described in Materials and Methods. The values represent means ± SD. a, p < 0.01 vs. 0 h groups.

 
Effect of Estrogen Antagonist on ER{alpha}-Induced CaBP-9k Expression
To clarify the mechanism of PPT in the induction of CaBP-9k gene in the uterus of immature rats, ICI 182,780 (ICI), a selective estrogen antagonist on both ERs, was employed in the present study. As shown in Figure 5, treatments with PPT (0.1 mg/kg BW) induced a significant increase of uterine CaBP-9k protein as expected, and a pre-treatment with low and moderate doses of ICI (0.1 and 1 mg/kg BW) prior to PPT injection did not block PPT-induced CaBP-9k protein. However, a high-dose of ICI (10 mg/kg BW) completely blocked PPT-induced CaBP-9k protein (0.1 mg/kg BW) as indicated in Figure 5. These results imply that the effect of PPT on uterine CaBP-9k is exclusively derived via an ER{alpha}-dependent mechanism.



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FIG. 5. Effect of ICI 182,780 on PPT-induced uterine CaBP-9k protein. Immature rats were injected sc with increasing doses of ICI 182,780 (ICI; 0.1, 1, and 10 mg/kg BW) prior to 30 min before PPT treatment (0.1 mg/kg BW) for three days and euthanized at 12 h after final injection. Uterine CaBP-9k protein was analyzed by Western blot analysis as described in Materials and Methods. The values represent means ± SD. a, p < 0.01 vs. vehicle; b, p < 0.01 vs. PPT treatment only.

 
Effect of PPT on Uterine ER{alpha} and PR Expression
To assess an involvement of ER{alpha} pathway in the regulation of CaBP-9k gene, the protein levels of ER{alpha} and PR (an ER regulated gene) were further analyzed in a time-dependent manner by Western blot analysis. A single treatment with PPT rapidly increased ER{alpha} protein at 3 h, but this protein returned to endogenous level at 12 h in the immature rat uteri, as indicated in Figure 6. In addition, PR, an E2-mediated gene, was highly increased by PPT at 6 and 12 h and dropped to background level at 24 h (Fig. 6). These results indicate that uterine CaBP-9k expression was relatively delayed compared to the expressions of ER and PR.



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FIG. 6. Time-dependent effect of PPT on uterine ER{alpha} and PR protein expression. Immature rats (total n = 24) were treated with PPT at the dose of 1 mg/kg BW and euthanized at 0, 3, 6, 12, 24, 48, 72, and 96 h after final injection. Uterine ER{alpha} and PR protein were analyzed by immunoblot analysis described in Materials and Methods. The values represent means ± SD. a, p < 0.01 vs. 0 h ER{alpha} expression; b, p < 0.01 vs. 0 h PR expression.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although CaBP-9k is mainly expressed in female reproductive tissues, i.e., uterus and placenta of various species, the role of CaBP-9k remains unknown. Uterine CaBP-9k has been thought to be involved in controlling myometrial activity related to intracellular calcium level, and placental CaBP-9k plays a role in calcium transfer from the mother to the fetus. In the uterus of rats, E2 is known to up-regulate and P4 down-regulate the expression of the CaBP-9k gene during estrous cycle and pregnancy. We demonstrated that CaBP-9k mRNA may be regulated by sex steroid hormones (E2 and P4) and their receptors, through a complex pathway, in these tissues (An et al., 2003aGo,bGo, 2004Go; Yun et al., 2004Go). The elucidation of other factors that regulate CaBP-9k mRNA will provide further insight into the understanding of regulation of CaBP-9k in these tissues, and of its roles in the control of reproductive functions. The uterus is a highly estrogen-responsive tissue, and its responses can be measured through changes in CaBP-9k expression.

We demonstrated that the expression levels of CaBP-9k mRNA and protein are controlled by estrogenic chemicals, octyl-phenol, nonyl-phenol, and bisphenol A, in the uterus of immature rats (An et al., 2002Go, 2003aGo). Genistein, a phytoestrogen which has the higher affinity to ERß, induced uterine CaBP-9k mRNA and protein levels in the uterus of immature rats (Lee et al., 2004Go). In addition, maternally injected estrogenic compounds resulted in an increase of CaBP-9k mRNA and/or protein in the fetal uterus during late pregnancy, suggesting that placenta may not be a reliable barrier against these estrogenic compounds, and they could be detrimental to fetal health as shown recently in a transgenic mouse model (Hong et al., 2003aGo; Lemmen et al., 2004Go). It is of interest that maternally injected estrogenic compounds may be transferred to neonates through breast milk and thus, affect uterine function, as shown by the induction of CaBP-9k gene in the neonatal uterus (Hong et al., 2004Go, in press). These results, which we have established and developed, suggest that the expression of uterine CaBP-9k mRNA and/or protein is an excellent biomarker to detect estrogenic compounds in immature rats.

Although the expression of the CaBP-9k gene is tightly regulated by E2 or other ER agonists in the uterus of rats, it is not clear how ER agonists induce the expression of CaBP-9k in this tissue, and what mechanism is involved in this induction. The expression of the CaBP-9k gene is highly modulated by E2 in the rat uterus throughout estrous cycle and during the perinatal periods (Blin et al., 1995Go; Krisinger et al., 1993Go, 1994Go; Naciff et al., 2002Go). In addition, E2 and P4 regulate cellular CaBP-9k expression patterns in endometrial stromal cells, uterine smooth muscle and uterine luminal epithelium in pregnant rats (Bruns et al., 1988Go). In the present study, it was investigated the effects of PPT, an ER{alpha}-specific ligand, and DPN, an ERß-specific ligand, on the expressions of CaBP-9k mRNA and protein in the uterus of immature rats to elucidate which ER subtype is involved in the induction of uterine CaBP-9k. Treatments with increasing doses of PPT resulted in an induction of uterine CaBP-9k mRNA and protein, but DPN did not induce any significant change of CaBP-9k in this tissue. As expected, treatment with E2 induced the CaBP-9k gene in a time- and concentration-dependent fashion. Based on the previous and present studies, an activated ER{alpha} may have the same effect on uterine CaBP-9k induction in immature rats, supporting the view that ER{alpha} could be the major ER regulating uterine CaBP-9k gene expression in this tissue. However, a minor and insignificant increase in CaBP-9k mRNA and protein by the high dose of DPN (10 mg/kg) also occurred. This can be partially explained because a high concentration of DPN (10 mg/kg BW) may bind to ER{alpha} after the saturation of this agonist into ERß isoform and act as a very low potent agonist for ER{alpha} (Harrington et al., 2003Go). In an agreement with this, a lower dose of DPN (0.01, 0.1, 1 mg/kg BW) did not alter uterine CaBP-9k gene expression in rats.

Uterine CaBP-9k mRNA and protein is undetectable in mature ovariectomized and immature rats (L'Horset et al., 1990Go). A single dose of E2 results in a detectable level of CaBP-9k mRNA at 1 h, a significant increase at 3 h after injection, and a maximal CaBP-9k mRNA level at 6 to 12 h post injection (L'Horset et al., 1990Go). In this study, a significant increase in CaBP-9k mRNA by PPT was observed at 6 h and a maximal CaBP-9k expression was detected at 12 h. When compared to the previous findings, a time-sequence CaBP-9k transcription by PPT was similar to that produced by E2. In a time-course study of its related protein, a significant increase in CaBP-9k protein started at 12 h and the peak was reached at 72 h; this suggests that the translation of CaBP-9k RNA to protein, in general, took 60 h. The effect of PPT on the expressions of CaBP-9k mRNA and protein was further examined in a dose-dependent manner. The increasing doses of PPT resulted in an induction of both CaBP-9k mRNA and protein in the uterus of immature rats. At the dose of 0.1 mg/kg PPT, there is a time-difference between CaBP-9k mRNA and protein production because of the time needed for accumulation of sufficient protein.

Estrogenic compounds have transcriptional activities at a number of estrogen-responsive promoters containing diverse response elements where ER up-regulates gene expression via mechanisms involving direct or indirect DNA-binding transcription factors (An et al., 2002Go, 2003aGo; Anderson et al., 1999Go; Blin et al., 1995Go). The rat CaBP-9k gene contains an imperfect ERE on the boundary between exon I and intron A, and activated ER binds to the ERE to induce this estrogenic response in vivo (Darwish et al., 1991Go; L'Horset et al., 1994Go). To confirm that the effect of PPT involves ER in the up-regulation of CaBP-9k mRNA and protein, we performed a pre-treatment with ICI 30 min prior to PPT. In the previous studies, ICI dramatically reversed E2-induced CaBP-9k mRNA and protein levels (Blin et al., 1995Go; Krisinger et al., 1992Go). In this study, a pre-treatment with ICI completely blocked uterine CaBP-9k stimulation by PPT or E2. This indicates that ER{alpha} is solely concerned with PPT-induced uterine CaBP-9k expression, because PPT lacks any ERß binding affinity (Kraichely et al., 2000Go; Stauffer et al., 2000Go).

It has been shown that E2 is a major factor for controlling CaBP-9k gene expression in vivo, and P4 antagonizes E2-induced CaBP-9k gene expression (Bruns et al., 1988Go; Mathieu et al., 1989Go). The P4-induced antagonistic effect is reversed by RU486, an anti-progestin, showing the primary action of PR (L'Horset et al., 1993Go). ER is considered as a critical factor to modulate the uterine CaBP-9k gene in rats (Krisinger et al., 1993Go, 1994Go). There are two subtypes of ERs in reproductive tissues, ER{alpha} and ERß, which differ in tissue distribution. In the rat uterus, ER{alpha} is dominantly expressed and is regulated by cycling hormone levels during normal estrous cycle (Brenner et al., 1979Go; Wang et al., 2000Go). However, the uterine ERß isoform is expressed constantly through the cycle. Although ERß shares many functional characteristics with ER{alpha}, a molecular mechanism controlling this gene is distinct from that of ER{alpha} (Frasor et al., 2003Go; Kuiper et al., 1996Go, 1997Go). Overall, the effect of ER{alpha} was of greater magnitude; it had marked estrogenic effects on uterine weight gain, luminal epithelial cell proliferation and uterine estrogen-response genes. However, in other studies, ERß appeared to play a modulatory role in uterine functions (Frasor et al., 2003Go). To clarify a coordination of ERs involving uterine CaBP-9k gene modulation following ER{alpha} activation, we investigated the effect of combined PPT and DPN on the uterine CaBP-9k expression.

In normal estrous cycle of rats, ER{alpha} is predominantly expressed in the uterus at proestrus just before the uterine CaBP-9k induction by endogenous E2 secretion (Krisinger et al., 1993Go). Thus, uterine expression of ER{alpha} is tightly linked to that of CaBP-9k in this tissue. In this study, we measured ER{alpha} protein expression followed by a single injection with PPT. As expected, uterine ER{alpha} was increased before CaBP-9k induction, suggesting that an increased ER{alpha} protein may stimulate uterine CaBP-9k gene expression. PR is one of the E2-responsive genes and an indicator of ER{alpha}-mediated transcription in the uterus. PR protein was enhanced after PPT treatment in the uterus of immature rats after 12 h, and it was induced via an ER-AP1 pathway (Petz et al., 2002Go).

In summary, we demonstrated that uterine CaBP-9k gene expression is mainly mediated by PPT, an ER{alpha}-selective ligand, in a dose- and time-dependent manner, in the uterus of immature rats. In contrast, no significant alteration in the uterine CaBP-9k gene was observed after DPN, an ERß-selective ligand. In addition, an estrogenicity of PPT in inducing CaBP-9k expression was completely blocked by ICI 182,780; this suggests that uterine CaBP-9k is solely enhanced though ER{alpha}. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via ER{alpha} pathway, but not ERß, in the uterus of immature rats.


    ACKNOWLEDGMENTS
 
This work was supported by grant no. R01-2002-000-00015-0 from the Basic Research Program of the Korea Science & Engineering Foundation. We are grateful to Dr. Anthony M. Perks at the British Columbia Research Institute for Children's and Women's Health, University of British Columbia for editing the manuscript.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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