* Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763 Republic of Korea, and 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
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ABSTRACT |
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Key Words: Calbindin-D9k; estrogen receptor; progesterone receptor; PPT; DPN.
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INTRODUCTION |
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The classical estrogen receptor (now referred to as ER) 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., 1996
; Mosselman et al., 1996
). 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
is expressed in the epithelial cells, glandular epithelial cells, and stromal cells; however, ERß is only detected in glandular epithelial cells (Hiroi et al., 1999
; Pelletier et al., 2000
). Judging from the localization of ERs and CaBP-9k, it is anticipated that ER
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
agonist) and diarylpropionitrile (DPN, an ERß agonist). PPT is a potent ER
agonist that has 410-fold higher binding affinity for ER
than ERß, and it has been demonstrated to have almost no binding affinity to ERß (Kraichely et al., 2000
; Stauffer et al., 2000
). However, DPN is a potency-selective agonist for ERß, and 70 times more selective for ERß than ER
(Meyers et al., 2001
). 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
and progesterone receptor (PR) was determined following single PPT treatment in the same tissues.
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MATERIALS AND METHODS |
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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., 2003; Harris et al., 2002
; Tena-Sempere et al., 2004
) 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 (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.
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RESULTS |
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DISCUSSION |
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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., 2002, 2003a
). 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., 2004
). 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., 2003a
; Lemmen et al., 2004
). 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., 2004
, 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., 1995; Krisinger et al., 1993
, 1994
; Naciff et al., 2002
). 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., 1988
). In the present study, it was investigated the effects of PPT, an ER
-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
may have the same effect on uterine CaBP-9k induction in immature rats, supporting the view that ER
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
after the saturation of this agonist into ERß isoform and act as a very low potent agonist for ER
(Harrington et al., 2003
). 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., 1990). 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., 1990
). 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., 2002, 2003a
; Anderson et al., 1999
; Blin et al., 1995
). 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., 1991
; L'Horset et al., 1994
). 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., 1995
; Krisinger et al., 1992
). In this study, a pre-treatment with ICI completely blocked uterine CaBP-9k stimulation by PPT or E2. This indicates that ER
is solely concerned with PPT-induced uterine CaBP-9k expression, because PPT lacks any ERß binding affinity (Kraichely et al., 2000
; Stauffer et al., 2000
).
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., 1988; Mathieu et al., 1989
). The P4-induced antagonistic effect is reversed by RU486, an anti-progestin, showing the primary action of PR (L'Horset et al., 1993
). ER is considered as a critical factor to modulate the uterine CaBP-9k gene in rats (Krisinger et al., 1993
, 1994
). There are two subtypes of ERs in reproductive tissues, ER
and ERß, which differ in tissue distribution. In the rat uterus, ER
is dominantly expressed and is regulated by cycling hormone levels during normal estrous cycle (Brenner et al., 1979
; Wang et al., 2000
). However, the uterine ERß isoform is expressed constantly through the cycle. Although ERß shares many functional characteristics with ER
, a molecular mechanism controlling this gene is distinct from that of ER
(Frasor et al., 2003
; Kuiper et al., 1996
, 1997
). Overall, the effect of ER
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., 2003
). To clarify a coordination of ERs involving uterine CaBP-9k gene modulation following ER
activation, we investigated the effect of combined PPT and DPN on the uterine CaBP-9k expression.
In normal estrous cycle of rats, ER is predominantly expressed in the uterus at proestrus just before the uterine CaBP-9k induction by endogenous E2 secretion (Krisinger et al., 1993
). Thus, uterine expression of ER
is tightly linked to that of CaBP-9k in this tissue. In this study, we measured ER
protein expression followed by a single injection with PPT. As expected, uterine ER
was increased before CaBP-9k induction, suggesting that an increased ER
protein may stimulate uterine CaBP-9k gene expression. PR is one of the E2-responsive genes and an indicator of ER
-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., 2002
).
In summary, we demonstrated that uterine CaBP-9k gene expression is mainly mediated by PPT, an ER-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
. Taken together, these results indicate that uterine CaBP-9k is induced by E2 and endocrine disrupting chemicals via ER
pathway, but not ERß, in the uterus of immature rats.
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ACKNOWLEDGMENTS |
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