* Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361763 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
Received June 17, 2004; accepted September 7, 2004
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ABSTRACT |
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Key Words: Calbindin-D9k; genistein; estrogen receptors; endocrine disruption.
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INTRODUCTION |
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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., 1989; Kumar et al., 1989
; Wasserman et al., 1982
). This protein is expressed in several mammalian tissues such as intestine, uterus, kidney, and bone (Armbrecht et al., 1989
; Delorme et al., 1983
; Mathieu et al., 1989
; Seifert et al., 1988
). 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, 1992
; Roche et al., 1986
; Wasserman and Fullmer, 1989
). In addition, uterine CaBP-9k has been postulated to control myometrial activity related to intracellular calcium levels (Mathieu et al., 1989
). 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., 1992
, 1994
; L'Horset et al., 1993
, 1994
). 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., 2002
, 2003a
). 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., 1987
).
Genistein has been shown to have 20-fold higher binding affinity to ERß than ER by a solid-phase binding assay (Kuiper et al., 1997
, 1998
). 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
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.
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MATERIALS AND METHODS |
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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., 2003). 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., 2000
). 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, 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.
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RESULTS |
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DISCUSSION |
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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., 2002, 2003a
; Anderson et al., 1999
; Blin et al., 1995
). The rat CaBP-9k gene contains an imperfect ERE which binds to ER (Darwish et al., 1991
; L'Horset et al., 1994
). 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., 1994
). 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., 1995
; Krisinger et al., 1992
). 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., 1994). RU486, a P4 antagonist, interfered with the effect of P4 on E2-induced CaBP-9k gene expression (Krisinger et al., 1994
; L'Horset et al., 1993
). ER is a critical factor modulating uterine CaBP-9k gene in rats (Krisinger et al., 1993
, 1994
). There are two subtypes of ERs in reproductive tissues of rats, ER
and ERß, which differ in tissue distribution. In rat uterus, ER
is dominantly expressed, but the affinity of genistein has a substantially 20-fold higher affinity to ERß than ER
(Kuiper et al., 1997
, 1998
). Pharmacologic concentrations of genistein can modulate ER
, ERß, and other uterine steroid receptor genes in prostate and other rat tissues (Cotroneo et al., 2001
; Fritz et al., 2002
; Wang et al., 2003
). To clarify which ER subtype is involved in uterine CaBP-9k gene modulation, we investigated uterine expression of ER
and ERß protein following genistein treatment. Our results demonstrated that genistein significantly increased ER
protein at 3 and 6 h after final injection when CaBP-9k gene was highly expressed. The level of ER
protein returned to control level between 6 and 12 h, suggesting a possible role of ER
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 (Wang et al., 2000
), and the estrous cycle tightly regulated CaBP-9k expression in the rat uterus (Krisinger et al., 1992
), suggesting that uterine ER
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
protein. Pretreatment with ICI, an antiestrogen, before genistein or E2, completely blocked ER
mRNA expression in the uterus of immature rats (data not shown). In addition, we measured the protein levels of ER
and ER
, and PR mRNA, one of the estrogen-responsive genes and an indicator of ER-mediated transcription in the uterus (Kraus and Katzenellenbogen, 1993
; Manni et al., 1981
). Treatment with genistein induced ER
protein and PR mRNA in the uterus of immature rats: thus, it can be assumed that genistein stimulated ER
and PR expression via AP1 site (Petz et al., 2002
). These results confirm that genistein may be involved in the regulation of CaBP-9k gene via ER
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 was highly expressed by genistein just before uterine CaBP-9k was highly expressed in this tissue, suggesting that genistein-induced ER
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
pathway. Thus, genistein, one of the phytoestrogens, induced uterine CaBP-9k in immature rats, confirming this gene is a potent biomarker for phytoestrogens.
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ACKNOWLEDGMENTS |
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NOTES |
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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, 361763, Republic of Korea. Fax: +82-43-267-3150. E-mail: ebjeung{at}chungbuk.ac.kr.
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