Effect of Genistein As a Selective Estrogen Receptor Beta Agonist on the Expression of Calbindin-D9k in the Uterus of Immature Rats

Geun-Shik Lee*, Kyung-Chul Choi{dagger}, Hoe-Jin Kim* 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

Received June 17, 2004; accepted September 7, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genistein, a phytoestrogen possessing a high affinity for estrogen receptor ß (ERß), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERß on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERß agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ER{alpha} and ERß and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ER{alpha} protein, but not ERß and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERß ligand, may regulate CaBP-9k gene through ER{alpha} pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ER{alpha} in the uterus of immature rats, suggesting that ER{alpha} may be a key mediator in uterine CaBP-9k gene induction in immature rats.

Key Words: Calbindin-D9k; genistein; estrogen receptors; endocrine disruption.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Flavonoid compounds in foods originating from plants, including the isoflavone genistein, are known to have a wide spectrum of biological activities and have been linked to chronic disease prevention (Kuo, 1997Go; Middleton et al., 2000Go). Soya protein concentrates, routinely used as the primary source of protein in mammalian diets, contain several isoflavones, including genistein, diadzein, glycitein, and their conjugates (Brown and Setchell, 2001Go). These substances are readily absorbed (Chang et al., 2000Go; Degen et al., 2002Go; King, 1998Go) and act as pharmacological estrogens both in vitro and in vivo via estrogen receptors (ERs) (Farmakalidis et al., 1985Go; Farmakalidis and Murphy, 1984Go; Markiewicz et al., 1993Go; Song et al., 1999Go). A second form of ER, ERß, has a higher relative binding affinity to genistein in in vitro competitive binding assays than a classical form of ER, ER{alpha} (Kuiper et al., 1997Go).

The 9-kDa cytosolic calcium-binding protein, Calbindin-D9k (CaBP-9k), belongs to a family of intracellular proteins that have high affinities to calcium and two calcium-binding domains (Christakos et al., 1989Go; Kumar et al., 1989Go; Wasserman et al., 1982Go). This protein is expressed in several mammalian tissues such as intestine, uterus, kidney, and bone (Armbrecht et al., 1989Go; Delorme et al., 1983Go; Mathieu et al., 1989Go; Seifert et al., 1988Go). Functionally, intestinal CaBP-9k is involved in intestinal calcium absorption and is regulated at the transcriptional level 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). In addition, uterine CaBP-9k has been postulated to control myometrial activity related to intracellular calcium levels (Mathieu et al., 1989Go). In rats, the hormonal mechanism controlling the uterine CaBP-9k gene is relatively well understood. In the rat uterus, 17beta-estradiol (E2) upregulates and progesterone (P4) downregulates CaBP-9k gene expression during estrous cycle and early pregnancy (Krisinger et al., 1992Go, 1994Go; L'Horset et al., 1993Go, 1994Go). In our previous studies, we demonstrated that E2 and estrogenic compounds increased uterine CaBP-9k gene expression, suggesting that this gene could be a biomarker for the estrogenicity of chemicals (An et al., 2002Go, 2003aGo). In normal rats, CaPB-9k protein was limited mainly to the myometrium and partially to the endometrial stroma, but in pregnant rats, CaBP-9k gene could also be expressed in the uterine epithelium (Warembourg et al., 1987Go).

Genistein has been shown to have 20-fold higher binding affinity to ERß than ER{alpha} by a solid-phase binding assay (Kuiper et al., 1997Go, 1998Go). To determine which ER is involved in the induction of CaBP-9k gene, genistein was used as a potent ERß agonist to clarify its effect on uterine CaBP-9k regulation. At the same time, we tested the potency of CaBP-9k gene as a potential biomarker for the detection of phytoesterogen. Thus, the effect of genistein on uterine accumulation of CaBP-9k mRNA and protein in immature rats was analyzed by Northern blot and immunoblot analyses, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ER{alpha} and ERß and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals and genistein treatments. Immature female Sprague Dawley rats (18 days old) were obtained from Dae Han Biolink Co, Ltd (Eumsung, Chungbuk, Korea). Animal care and procedures were approved by the Chungbuk National University Ethics Committee. 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-hour light cycle). Animals were fed soy-free pellet food (Dyets Inc, Bethlehem, PA). To determine the effect of genistein at different doses, three groups of five rats (total n = 15) were injected with genistein (Sigma-Aldrich Corp, St. Louis, MO) subcutaneously (sc) at doses of 0.4, 4, or 40 mg/kg body weight (BW), daily for 3 days and euthanized 24 h after final injection. Genistein was dissolved in dimethyl sulfoxide (DMSO, Amresco Inc, Solon, OH) as a vehicle. The rats were injected with 40 µg/kg BW 17beta-estradiol (E2, n = 3) as a positive control or DMSO (n = 3) as a negative control daily for 3 days. To determine the effects of genistein over time, two groups of 18 rats (total n = 36) were injected sc with 40 mg/kg BW genistein or DMSO for 3 days, as indicated above. Three animals from each group were euthanized at 3, 6, 12, 24, 48, and 72 h after final injection. To determine the effect of ICI on E2 and genistein action, immature rats (n = 10) were injected sc daily for 3 days with ICI 182,780 (ICI) at 30 min prior to E2 or genistein injection and euthanized 24 h after final injection.

Total RNA extraction and Northern blot analysis. The uteri were rapidly excised from euthanized rats and washed in cold sterile 0.9% NaCl. Total uterine RNA was prepared with TRIzol reagent (Invitrogen Life Technologies, Inc, Carlsbad, CA), and the concentration determined by measuring absorbance at 260 nm. Total RNA was denatured by heating at 85°C for 10 min. Ten micrograms of total RNA was electrophoresed on 1% formaldehyde denaturing agarose for 1 h at 110 V. 18S rRNA was used as an indicator of the quantity of total RNA. A Vacuum Blotter (Bio-Rad Laboratories, Inc, Hercules, CA) was used to transfer RNA to a nylon membrane (Amersham Pharmacia Biotech, Morgan, ON, Canada), according to the manufacturer's suggested instructions. RNA was UV cross-linked to the membrane by a Gene Cross-Linker (Bio-Rad Laboratories, Inc). The membranes were prehybridized in 50% formamide, 5x SSPE, 5x Denhardt's solution, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA for 2 h at 42°C. The radiolabeled CaBP-9k probe was prepared by using a Random Primer DNA Labeling Kit (TaKaRa Bio, Inc, Otsu, Shiga, Japan), according to the manufacturer's suggested instructions. 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 exposed to X-ray film (Eastman Kodak, Co, Rochester, NY), and the films scanned and analyzed by Molecular Analysis Program version 1.5 (Gel Doc 1000, Bio-Rad Laboratories, Inc.). The assay was repeated three times.

Semiquantitative RT-PCR. RT-PCR was performed as previously described (Lee et al., 2003Go). Briefly, total RNA (1 µg) was reverse transcribed into first strand complementary DNA (cDNA) using M-MLV reverse transcriptase (iNtRON Bio Inc, Sungnam, Kyungki-Do, Korea) and random primer (9 mer, TaKaRa Bio). To determine the conditions for logarithmic phase PCR amplification for PR mRNA, aliquots (1 µl) were amplified using different numbers of cycles. The 1A gene (cytochrome oxidase subunit 1) was PCR-amplified to rule out the possibility of RNA degradation and was used to control for variation in mRNA concentrations in the RT reaction (Nephew et al., 2000Go). A linear relationship between PCR products and amplification cycles was observed for PR and 1A mRNAs. PR and 1A were quantified using 30 cycles and 25 cycles, respectively. The cDNA was amplified in a 20 µl PCR reaction containing 1 U Taq polymerase (iNtRON), 1.5 mM MgCl2, 2 mM deoxy-NTP, and 50 pmol specific primers. PCR reactions were denatured at 95°C for 60 seconds, annealed at 55°C for 60 seconds, and extended at 72°C for 90 seconds. The oligonucleotide sequences for PR were 5'-CACAG GAGTT TGTCA AGCTC-3' (sense) and 5'-GGGAT TGGAT GAACG TATTC-3' (antisense). The oligonucleotide sequences for 1A were 5'-CCAGG GTTTG GAATT ATTTC-3' (sense) and 5'-GAAGA TAAAC CCTAA GGCTC-3' (antisense). PCR products (10 µl) were fractionated on a 2% agarose gel, stained with ethidium bromide, and photographed under UV-illumination. The photograph was scanned and analyzed using Molecular Analysis Program version 1.5 (Gel Doc 1000, Bio-Rad Laboratories, Inc).

Western blot analysis. Protein was extracted with Proprep (iNtRON) according to the manufacturer's instructions. Cytosolic protein (50 µg per lane) was resolved by SDS/PAGE (15% acrylamide) and transferred to nitrocellulose using a Semi-dry Transfer Cell (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. The membranes were blocked with phosphate-buffered saline containing 0.05% (w/v) Tween-20 and 5% (w/v) dry milk overnight. The blocked membranes were incubated sequentially with primary and secondary antibodies dissolved in 1% (w/v) bovine serum albumin for 1 h at room temperature. The rabbit antibody to rat CaBP-9k (Swant, Bellinzona, Switzerland), ER{alpha}, ERß, and ß-actin (Santa Cruz Biotech, Santa Cruz, CA) was used for detection process. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotech). The membranes were added with Immunoblot Lighting Chemiluminescence reagent (Perkin-Elmer Life Sciences, Boston, MA) according to the manufacturer's protocol and exposed to X-ray film (Eastman Kodak, Rochester, NY). The films were scanned and analyzed by Molecular Analysis Program version 1.5 (Gel Doc 1000).

Data analysis. Data were analyzed by the nonparametric one-way analysis of variance using the Kruskal Wallis test, followed by Dunnett's test for multiple comparisons to vehicle. Each data value was converted to rank for statistical analysis. All statistical analyses were performed with SPSS for Windows (SPSS Inc, Chicago, IL). p < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Genistein on CaBP-9k Gene Regulation
The expression of genistein-induced uterine CaBP-9k mRNA and protein was assessed by using different doses of genistein and was monitored over time by Northern blot and immunoblot analyses, respectively. As seen in Figure 1, treatment with genistein (40 mg/kg BW per day) increased CaBP-9k protein three-fold in the uterus of immature rats, whereas no significant difference was observed at 0.4 and 4 mg/kg BW per day of genistein. A single dose of E2 (40 µg/kg BW per day) significantly increased CaBP-9k protein (four-fold vs. vehicle, Fig. 1). In a time-dependent experiment, the expression of CaBP-9k mRNA was examined in this tissue after treatment with genistein (40 mg/kg BW per day) for 3 days. After final injection, total RNA and protein were prepared as described in the Materials and Methods. The level of CaBP-9k mRNA significantly increased until 12 h (maximal 10-fold vs. vehicle at 6 h as shown in Fig. 2A), then decreased to control level at 24 h after final injection with genistein. In addition, CaBP-9k protein increased at 3 h (eight-fold vs. vehicle), sustained until 24 h (five-fold vs. vehicle), and dropped to control level at 72 h after final injection, while its protein level was not altered by DMSO, a vehicle control during these time points (Fig. 2B).



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FIG. 1. Dose-dependent effects of genistein on CaBP-9k protein expression. Immature rats (n = 5 per group) were treated with genistein at doses of 0.4, 4 and 40 mg/kg BW each day for 3 days and euthanized 24 h after final injection. Control groups (n = 3 per group) received E2 as a positive control or DMSO as a negative control for 3 days, as described in the Materials and Methods. CaBP-9k protein expression was examined by immunoblot analysis as described in the Materials and Methods. Data were analyzed by the nonparametric Kruskal-Wallis test, followed by Dunnett's test for multiple comparisons. The values represent means ± SD. a, p < 0.05 versus vehicle.

 


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FIG. 2. Time-dependent effects of genistein on uterine CaBP-9k gene expression. Immature rats were treated with genistein at doses of 40 mg/kg BW (n = 3 per group) and DMSO (n = 3 per group) each day for 3 days and euthanized at 3, 6, 12, 24, 48, and 72 h after final injection. CaBP-9k mRNA expression was analyzed by Northern hybridization (A), and protein expression was analyzed by immunoblot analysis (B) as described in the Materials and Methods. The values represent means ± SD. a, p < 0.05 versus vehicle for each time point.

 
Effect of Estrogen Antagonist on Genistein-Induced CaBP-9k Expression
The promoter region of rat CaBP-9k gene has a putative estrogen response element (ERE), and this gene is regulated through a classical ER-ERE pathway (Darwish et al., 1991Go; L'Horset et al., 1994Go). To clarify the mechanism by which genistein induced the expression of CaBP-9k mRNA and protein in the uterus of immature rats, ICI 182,780 (ICI), a selective estrogen antagonist, was pretreated at 30 min before genistein. Treatment with genistein or E2 significantly increased CaBP-9k protein in the uterus of immature rats, and ICI completely blocked genistein-induced CaBP-9k protein (Fig. 3). In addition, pretreatment of immature rats with ICI before E2 significantly suppressed E2-induced CaBP-9k protein as well in this tissue.



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FIG. 3. Effect of ICI on genistein-induced uterine CaBP-9k protein expression. Immature rats were injected SC with ICI at 30 min prior to genistein (40 mg/kg BW per day) or E2, and euthanized 24 h after final injection. The level of CaBP-9k protein was analyzed by immunoblot analysis as described in the Materials and Methods. The values represent means ± SD. a, p < 0.05 versus vehicle; b, p < 0.05 versus genistein or E2 treatment only.

 
Effect of Genistein on ER{alpha} and ERß Proteins
To assess an involvement of ER in the regulation of CaBP-9k gene, the proteins of ER{alpha} and ERß were analyzed in a time-dependent manner by immunoblot blot analysis. Treatment with genistein increased ER{alpha} protein at 3 and 6 h in the uterus of immature rats as indicated in Figure 4. ER{alpha} protein reached its maximal level (three-fold vs. vehicle) 3 h after final injection and returned to control level at 12 h (Fig. 4). However, there was no significant alteration in ERß protein when CaBP-9k was highly induced by genistein in this tissue.



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FIG. 4. Effect of genistein on ER{alpha} and ERß protein expression. Immature rats were given 40 mg/kg BW genistein (n = 6 per group) or DMSO (n = 6 per group) daily for 3 days and euthanized at 3, 6, 12, and 24 h after final injection. ER{alpha} and ERß protein expression was examined by immunoblot analysis as described in the Materials and Methods. The values represent means ± SD. a, p < 0.05 versus vehicle for each time point.

 
Effect of genistein on PR mRNA
The expression of PR mRNA was further analyzed in a time-dependent manner by RT-PCR because PR is one of E2-induced genes in female reproductive tissues. This study showed that PR mRNA increased at 3 h following genistein and decreased to control level at 6 h in the uterus of immature rats (Fig. 5).



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FIG. 5. Effect of genistein on PR mRNA expression. Immature rats were given 40 mg/kg BW genistein or DMSO each day for 3 days and euthanized at 3, 6, 12, and 24 h after final injection. PR mRNA expression was examined by RT-PCR analysis as described in the Materials and Methods. The values represent means ± SD. a, p < 0.05 versus vehicle for each time point.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CaBP-9k is a cytosolic calcium-binding protein expressed mainly in female rat reproductive organs; however, the exact role of CaBP-9k in these organs has not been clearly described. In this study, we evaluated the effect of genistein, a selective ERß agonist, on the expression of CaBP-9k mRNA and protein in the uterus of immature rats. The expression of CaBP-9k gene is highly modulated by estrogen in the rat uterus throughout estrous cycle and during the perinatal period (Blin et al., 1995Go; Krisinger et al., 1993Go, 1994Go). This gene, which was regulated by E2 and P4, was located in the uterine luminal epithelium of pregnant rats and also found in endometrial stromal cells and uterine smooth muscle (Bruns et al., 1988Go). The uterine expression of CaBP-9k mRNA and protein in rats was rapidly and strongly induced by E2 and estrogenic compounds (octyl-phenol, nonylphenol, and bisphenol A), possibly through ER. Presumably, these compounds activated ER directly (An et al., 2002Go, 2003aGo,bGo; Hong et al., 2003Go). Genistein and other flavonoids are also agonists for ER and modulators of ER responsive genes (Kuiper et al., 1998Go; Makela et al., 1994Go; Miksicek, 1993Go; Picherit et al., 2000Go). Using an oligonucleotide array to determine the expression levels of approximately 7,000 rat genes and over 1,000 expressed sequence tags (ESTs), CaBP-9k was the most altered uterine gene after exposure to E2 or estrogenic compounds, BPA and genistein (Naciff et al., 2002Go). In this study, a high dose (40 mg/kg BW per day) of genistein significantly increased CaBP-9k mRNA and protein in the uterus of immature rats, indicating that genistein acts to induce CaBP-9k like E2 in this tissue. After a 3-day treatment with genistein, five- to eight-fold increase in CaBP-9k mRNA was maintained for 12 h then declined steadily to the control level over next 24 h. These results suggest that genistein, one of the phytoestrogens, could be regarded as a potent biomarker for phytoestrogens and xenoestrogens in terms of uterine CaBP-9k induction.

Estrogenic compounds have transcriptional activities at a number of estrogen-responsive promoters containing diverse response elements where ER upregulates 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 which binds to ER (Darwish et al., 1991Go; L'Horset et al., 1994Go). In addition, it was reported that CaBP-9k ERE cooperates with ER, in cis, to induce this estrogenic response in vivo (L'Horset et al., 1994Go). To confirm that effect of genistein is derived from an involvement of ER in the upregulation of CaBP-9k mRNA and protein, we preinjected immature rats with ICI, a pure antagonist of E2, at 30 min prior to E2 or genistein administration. ICI was reported to dramatically reduce E2-induced CaBP-9k mRNA expression (Blin et al., 1995Go; Krisinger et al., 1992Go). Pretreatment with ICI completely blocked genistein- or E2-induced CaBP-9k protein level in the uterus of immature rats, indicating that ER solely mediates this effect of genistein.

It has been demonstrated that E2 is a major factor controlling CaBP-9k gene expression in vivo, and P4 antagonized E2-induced CaBP-9k expression (Krisinger et al., 1994Go). RU486, a P4 antagonist, interfered with the effect of P4 on E2-induced CaBP-9k gene expression (Krisinger et al., 1994Go; L'Horset et al., 1993Go). ER is a critical factor modulating uterine CaBP-9k gene in rats (Krisinger et al., 1993Go, 1994Go). There are two subtypes of ERs in reproductive tissues of rats, ER{alpha} and ERß, which differ in tissue distribution. In rat uterus, ER{alpha} is dominantly expressed, but the affinity of genistein has a substantially 20-fold higher affinity to ERß than ER{alpha} (Kuiper et al., 1997Go, 1998Go). Pharmacologic concentrations of genistein can modulate ER{alpha}, ERß, and other uterine steroid receptor genes in prostate and other rat tissues (Cotroneo et al., 2001Go; Fritz et al., 2002Go; Wang et al., 2003Go). To clarify which ER subtype is involved in uterine CaBP-9k gene modulation, we investigated uterine expression of ER{alpha} and ERß protein following genistein treatment. Our results demonstrated that genistein significantly increased ER{alpha} protein at 3 and 6 h after final injection when CaBP-9k gene was highly expressed. The level of ER{alpha} protein returned to control level between 6 and 12 h, suggesting a possible role of ER{alpha} in the regulation of CaBP-9k gene by genistein.

ERß appeared not to be involved in CaBP-9k regulation, because genistein did not modulate ERß protein. During estrous cycle, E2 tightly regulated ER{alpha} (Wang et al., 2000Go), and the estrous cycle tightly regulated CaBP-9k expression in the rat uterus (Krisinger et al., 1992Go), suggesting that uterine ER{alpha} may contribute to CaBP-9k regulation in this tissue. The expression pattern of uterine CaBP-9k after genistein treatment indicates a lag period in CaBP-9k protein production following an increase of ER{alpha} protein. Pretreatment with ICI, an antiestrogen, before genistein or E2, completely blocked ER{alpha} mRNA expression in the uterus of immature rats (data not shown). In addition, we measured the protein levels of ER{alpha} and ER{alpha}, and PR mRNA, one of the estrogen-responsive genes and an indicator of ER-mediated transcription in the uterus (Kraus and Katzenellenbogen, 1993Go; Manni et al., 1981Go). Treatment with genistein induced ER{alpha} protein and PR mRNA in the uterus of immature rats: thus, it can be assumed that genistein stimulated ER{alpha} and PR expression via AP1 site (Petz et al., 2002Go). These results confirm that genistein may be involved in the regulation of CaBP-9k gene via ER{alpha} pathway because no difference was observed in ERß protein by genistein. However, we cannot rule out possibility of involvement of genistein via ERß in the regulation of CaBP-9k in this tissue.

In summary, we demonstrated for the first time that CaBP-9k gene expression was induced by genistein, a high-ERß-affinity chemical, in the uterus of immature rats. An estrogenicity of genistein was completely blocked by ICI, indicating that uterine CaBP-9k is solely regulated through ERs. ER{alpha} was highly expressed by genistein just before uterine CaBP-9k was highly expressed in this tissue, suggesting that genistein-induced ER{alpha} may take part in the regulation of CaBP-9k gene in the uterus of immature rats. Genistein, despite its higher affinity to ERß, may have an estrogenic property to modulate uterine CaBP-9k gene expression in this tissue via a classical ER{alpha} pathway. Thus, genistein, one of the phytoestrogens, induced uterine CaBP-9k in immature rats, confirming this gene is a potent biomarker for phytoestrogens.


    ACKNOWLEDGMENTS
 
This work was supported by grant No. R01-2002–000–00015–0 from the Basic Research Program of the Korea Science & Engineering Foundation and Korea Research Foundation Grant (KRF 2003–041-E00238). We are grateful to Dr. Barb Conway at the British Columbia Research Institute for Children's and Women's Health, University of British Columbia for a critical review of the manuscript.


    NOTES
 

1 To whom correspondence should be addressed at Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, 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.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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