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Biology and physiology of Calbindin-D9k in female reproductive tissues: Involvement of steroids and endocrine… Choi, Kyung-Chul; Leung, Peter C; Jeung, Eui-Bae Nov 16, 2005

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ralReproductive Biology and ssBioMed CentEndocrinologyOpen AcceReviewBiology and physiology of Calbindin-D9k in female reproductive tissues: Involvement of steroids and endocrine disruptorsKyung-Chul Choi1, Peter CK Leung1 and Eui-Bae Jeung*2Address: 1Department of Obstetrics and Gynecology, British Columbia Children's and Women's Hospital, Child and Family Research Institute, University of British Columbia, Vancouver, BC V6H 3V5 Canada and 2Laboratory 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 KoreaEmail: Kyung-Chul Choi - kchoi@cw.bc.ca; Peter CK Leung - peleung@interchange.ubc.ca; Eui-Bae Jeung* - ebjeung@chungbuk.ac.kr* Corresponding author    AbstractAlthough Calbindin-D9k (CaBP-9k), a cytosolic calcium binding protein which has calcium bindingsites, is expressed in various tissues, i.e., intestine, uterus, and placenta, potential roles of this geneand its protein are not clearly understood. Uterine CaBP-9k may be involved in controllingmyometrial activity related with intracellular calcium level and is not under the control of vitaminD despite the presence of vitamin D receptors. But, it is under the control of the sex steroidhormones, estrogen (E2) and progesterone (P4), in female reproductive systems including theuterus and placenta. Thus, in this review, we summarize recent research literature in regards tothe expression and regulation of CaBP-9k in mammals and introduce the research data of recentstudies by us and others.IntroductionA 9-kilodalton cytosolic calcium-binding protein termedas Calbindin-D9k (CaBP-9k) belongs to a family of intra-cellular proteins which have high affinities for calcium,and has two calcium binding domains [1]. The full-lengthcDNA encoding the human CaBP-9k has been clonedusing reverse transcription/PCR, which includes codingregion of 79 amino acids, 57 nucleotides 5'- and 159nucleotides 3'-non-coding region, and a poly(A) tail (total600 nucleotides in length) [2]. Further, our study revealedthat this gene spans about 5.5-kb and is localized on theX-chromosome, consists of three exons and carries fourAlu repeats [3]. In addition to its genomic structure, asequence of 50 nucleotides downstream from the pro-moter showed an extensive homology to the estrogenchange within this region in human causes the gene tolack expression in human uterus and placenta [3].It has been demonstrated that CaBP-9k is expressed indiverse mammalian tissues, i.e., intestine, uterus, kidney,and bone [4-7]. The functional role of CaBP-9k isinvolved in intestinal calcium absorption and its gene isregulated at the transcriptional or post-transcriptionallevel by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), ahormonal form of vitamin D [8,9]. This hormonal formcaused a parallel increase in CaBP-9k mRNA and intesti-nal absorption of calcium in rats [10]. In addition, uterineCaBP-9k may be involved in controlling myometrialactivity related with intracellular calcium level [6], but anexact role of CaBP-9k in the uterus is still under investiga-Published: 16 November 2005Reproductive Biology and Endocrinology 2005, 3:66 doi:10.1186/1477-7827-3-66Received: 02 September 2005Accepted: 16 November 2005This article is available from: http://www.rbej.com/content/3/1/66© 2005 Choi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)response element (ERE) at the same location within therat calbindin-D9k gene, suggesting that a two-nucleotidetion by us and a few of other research groups. Recently, wedemonstrated that uterine CaBP-9k is responsive to exog-Reproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66enous estrogen (E2) and can be a biomarker for environ-mental estrogenic chemicals, so called as "endocrinedisruptors" in rat models [11-15]. Thus, in this review, wesummarize recent research literature in regards to theexpression and regulation of CaBP-9k in mammals andintroduce updated research results by us and others.Uterine expression of CaBP-9kIt has been demonstrated that CaBP-9k is mainlyexpressed in the endometrial stroma and myometrium ofthe uterus in non-pregnant rats [16,17], whereas this geneis translocated into the epithelium of the uterus in preg-nant rats [18]. However, it has been shown that the CaBP-9k is only expressed in the luminal and glandular epithe-lium of the endometrium, not in the myometrium and inthe stromal cells of the endometrium in non-pregnantcows [19]. In contrast to the regulation of CaBP-9k in theintestine, CaBP-9k gene is not under the control of vita-min D in the uterus despite the presence of vitamin Dreceptors in this tissue. This gene appears to be under thecontrol of the sex steroid hormones [17,20,21].There is a strong body of evidence that CaBP-9k is regu-the expression of CaBP-9k mRNA up to 300-fold and itsmRNA was shown to fluctuate in the uterus of rats duringestrous cycle, where serum E2 level was also under fluctu-ation [22]. Although the expression of CaBP-9k mRNA isnot detectable at diestrus when E2 level is at the lowest,this mRNA increases at proestrus and reaches the highestlevel at estrus in response to the rise in plasma E2 andthen decreases at metestrus in the uterus of rats [20,22]. Inaddition, E2-dependent regulation of CaBP-9k gene wasdemonstrated. i.e., CaBP-9k synthesis decreased drasti-cally in the uterus of ovariectomized rats, whereas it wasgreatly enhanced by low physiological doses of E2 in adose-dependent manner by CaBP-radioimmunoassay(RIA) [17]. This E2-dependent regulation of CaBP-9k genewas also approved in the uterus of mature ovariectomizedand immature rats by slot and Northern blot analysis[23,24]. In estrogen-primed ovariectomized rats, proges-terone (P4) inhibited E2-induced CaBP-9k gene expres-sion, which was completely abolished by co-administration of RU486, a P4 antagonist [20]. In thepregnant rats, P4 was shown to be responsible for down-regulation of CaBP-9k gene in the uterus during earlypregnancy [25]. In the ovariectomized (OVX) gilts, E2treatment induced an increase in CaBP-9k mRNA level,whereas P4 administration to ovariectomized pigsdecreased CaBP-9k mRNA levels [26]. Recently, we dem-onstrated that CaBP-9k mRNA and protein are domi-nantly expressed during luteal phase, indicating that P4may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus during luteal phase, whichis unlike the condition in the rat uterus (Fig. 1) [27]. Inthis study, the porcine CaBP-9k may be dominantlyexpressed in the epithelium and glandular structure of theporcine uterus during luteal phase, suggesting that CaBP-9k gene may also be differentially regulated during thiscycle presumably by steroid hormones, especially up-reg-ulated P4 level in this tissue [27]. In addition, a positiverole of P4 on the expression of CaBP-9k has been demon-strated in the bovine uterus, which indicates that theexpression of CaBP-9k was greatest during P4-dominatedluteal phase of the bovine estrous cycle [19].In contrast to the uterus of rats, the expression of CaBP-9kgene is not under strict E2 regulation in the uterus of mice.The uterus of mice has been demonstrated to expressCaBP-9k and its level increases in this tissue during earlypregnancy and implantation [28,29]. CaBP-9k mRNA isexpressed in the endometrial epithelia, both luminal andglandular, in the uterus at the time of implantation, andin the luminal, but not in the glandular, epithelia on earlypregnancy (day 5 of pregnancy). P4 enhanced CaBP-9kmRNA expression in the uterus, whereas E2 did not in theoophorectomized adult mice [28]. A higher expression ofPorcine CaBP-9k mRNA expression in the endometrium and whole uterus during an estrus cycleFigur  1Porcine CaBP-9k mRNA expression in the endometrium and whole uterus during an estrus cycle. To investigate a role of CaBP-9k in the tissue compartment of the pig uterus, the expression levels of CaBP-9k mRNA (A) and protein (B) were analyzed by Northern blot and immunoblot analyses. [Reproduced with permission from Yun S-M, Choi KC, Kim IH, An BS, Lee GS, Hong EJ, Son JH, Oh GT, Jeung E-B 2004 Calbindin-D9k mRNA expression and regulation during estrus cycle in the pig uterus. Mol Reprod Dev 67: 251\endash 256]CaBP-9kFollicular phase Luteal phaseEndo Whole Endo Wholeβ-actinCaBP-9kFollicular phase Luteal phaseActinABPage 2 of 8(page number not for citation purposes)lated by sex steroid hormones in the uterus of rats. Treat-ment of 21-day-old rats with E2 resulted in an increase inCaBP-9k mRNA was observed in the uterus of mice at die-strus and metestrus, whereas only basal level of its expres-Reproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66sion at proestrus and estrus, and E2 alone did not induceuterine CaBP-9k mRNA in this study [29]. Taken together,these results suggest the complex hormonal regulation ofCaBP-9k in the uterus of different species. To date, therewas no evidence that CaBP-9k gene may be regulated byE2 in female reproductive tract of mice. In the recentstudy, we demonstrated that RU486, a P4 antagonist,induced a significant decrease in CaBP-9k mRNA expres-sion, whereas tamoxifen and ICI 182,780, an E2 antago-nists, had no effect on CaBP-9k mRNA expression,suggesting that P4, not E2, is a key regulator of CaBP-9kmRNA expression during late pregnancy and lactation inthe uterus of mice [30].The mechanism involved in the regulation of uterineCaBP-9k gene by steroids is relatively well understood inrats. In the uterus of rats, estrogen is known to up-regulateand progesterone down-regulate the expression of CaBP-9k gene during estrous cycle and early pregnancy[20,22,24,25]. The expression level of CaBP-9k mRNAfluctuates during the estrous cycle, but shows very differ-ent expression pattern in the uterus of between rats andmice as abovementioned [29]. The mechanism of distinctof CaBP-9k appears to be mediated through an imperfectestrogen-responsive-like element (ERE) identified in theintron A of mouse CaBP-9k gene [8,24]. The regulation ofthis gene is known to be mediated by an E2 response ele-ment located at the first intron of rat CaBP-9k gene [31].Recently, cloning of intron A of the mouse CaBP-9k genehave revealed single-base difference in the ERE comparedto that of the rat [32]. This may partially explain theobserved difference in the hormonal regulation of CaBP-9k gene in the uterus of between rats and mice. In addi-tion, a distinct regulation of porcine CaBP-9k gene in theuterus is explained by no presence of a functional EREwithin intron A region [26]. However, we can not rule outthe possibility of involvement of other unknown cell-, tis-sue-, and species-specific factors in the CaBP-9k geneexpression. This idea is supported by the finding that E2regulation of CaBP-9k gene in rats was only uterine-spe-cific and this does not occur in the intestine [17].Although the putative ERE failed to bind to the estrogenreceptor (ER) from the mouse uterus, we isolated mousegenomic clones of the CaBP-9k gene and analyzed theirexpression in the mouse uterus [33]. In addition, wefound a promoter region of CaBP-9k gene containing theputative progesterone response element (PRE) and itsexpression was stimulated by P4, suggesting that themouse uterine CaBP-9k gene is expressed under the con-trol of a PRE [33]. In the recent study, we demonstratedthat P4 and PR may be a dominant factor in the regulationof CaBP-9k and E2 and ERα can also influence the expres-sion of CaBP-9k gene via an indirect pathway in the uterusof immature mice [34]. Currently, endocrine disruptor-induced expression of CaBP-9k mRNA and protein wasreversed or abolished by pretreatment with RU486 or ICI182,780, suggesting that these synthetic chemicals mayhave both progestogenic and estrogenic properties by act-ing through PR or ER in the induction of uterine CaBP-9kmRNA and protein in the uterus of immature mice [35].Uterine induction of CaBP-9k as a biomarker for endocrine disruptionEndocrine disruptors (EDs) are environmental chemicalsthat interfere with physiological systems, adversely affect-ing hormone balance (endocrine system) or disruptingnormal function in the organs that hormones regulate ormodulate, for example, female and male reproductive sys-tem [36]. Representative example of suspected environ-mental estrogenic EDs includes the drugs which havebeen specifically designed to treat hormone imbalance inhumans. These estrogenic compounds, including octyl-phenol (OP), nonylphenol (NP), bisphenol A (BPA), andmethoxychlor (MXC), can also be transferred through theplacenta to the fetus and through breast milk to infants[11,13,37]. Screening methods to detect endocrine dis-Induction of uterine CaBP-9k protein by estrogenic com-pounds, i.e., OP, NP and BPA was assessed in a dose-depend-ent man r at 24 h after final injection by immun blot a alysiFig re 2Induction of uterine CaBP-9k protein by estrogenic com-pounds, i.e., OP, NP and BPA was assessed in a dose-depend-ent manner at 24 h after final injection by immunoblot analysis. Data are presented as the mean ± SD. a; significantly different compared with vehicle at P < 0.05. [Reproduced with permission from An B-S, Choi KC, Kang SK, Hwang WS, Jeung EB 2003 Novel Calbindin-D9k protein as a useful biomarker for environmental estrogenic compounds in the uterus of immature rats. Reprod Toxicol 17: 311–319]Page 3 of 8(page number not for citation purposes)regulation of CaBP-9k gene between the rat and mouse isnot clear at the moment. The effect of E2 on the regulationrupters have been evaluated by many groups, i.e. thereceptor binding assay, reporter gene assay, and immatureReproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66rat uterotrophic assay. The reporter gene assay has manybenefits as a promising prescreening procedure, becausethis assay could be performed as a high throughputscreening process to detect an endocrine disruptor fromthousands of chemicals and no use of experimental ani-mals is required [38]. To screen estrogenic chemicals inthe induction of endocrine disruption, genetically sensi-tive animal models, mice and rats, should be developed.Efforts to identify the mechanisms of endocrine disrup-tion by estrogenic chemicals need to be supported foroptimal test methods for thousands of potential chemi-cals in reproductive development and function [39]. Thus,we have recently established a sensitive method to detectCaBP-9k mRNA and protein using immature rats, whichcan be used as a biomarker for endocrine disruptors, thus,we introduce our current results in regard to estrogeniceffect of endocrine disruptors in the uterus of immaturerats [12]. Among the assays for the estrogenic activity ofchemicals, an assay to detect an endogenous gene expres-sion that measures estrogen-induced changes either incultured cells or in selected tissues from exposed animalshas been proposed and is widely being used. In our previ-ous study, we demonstrated that phthalate esters exhibit aweak estrogenic activity in vitro assay at high concentra-tions. Although phthalates resulted in an increase in MCF-7 cell proliferation by estrogenic effect, they could notmetabolized to inactive forms in vivo system. These resultssuggest that a conflict may exist in estrogenic effect by var-ious phthalates between in vitro and in vivo models relatedto the expression of CaBP-9k [40].The expression levels of CaBP-9k mRNA and protein arestrongly up-regulated by estrogenic compounds (OP, NPand BPA) and E2 itself in the uterus of immature rats (Fig.2), indicating that CaBP-9k can be a useful biomarker fordetection of the estrogenicity of putative estrogenic com-pounds. Thus, regarding to risk assessment, we proposethat CaBP-9k mRNA and protein assay in the immaturerat uterus can be a very sensitive and powerful tool toidentify compounds with estrogenic activity when used incombination with classical assays [11,23]. Treatments ofdams with OP, NP and BPA resulted in an increase ofCaBP-9k mRNA and protein in maternal and fetal uteri(Fig. 3) of immature rats [13]. These results demonstratethat maternally injected estrogenic compounds resultedin an increase of CaBP-9k mRNA and/or protein in thematernal tissues (uterus and placenta) and fetal uterusduring late pregnancy, suggesting that placenta may notbe a reliable barrier against these estrogenic compoundsfor fetal health [13]. The uterus is a highly estrogen-responsive tissue, which can be measured throughEffect of endocrine disruptors on the induction of CaBP-9k mRNA in e natal ute sFigure 4Effect of endocrine disruptors on the induction of CaBP-9k mRNA in neonatal uterus. RT-PCR/Southern blot analysis for CaBP-9k mRNA on day 6 of lactation was performed as pre-viously described. The values represent means ± SD (n = 5). a, P < 0.05 vs. vehicle (VE). [Reproduced with permission from Hong E-J, Choi KC, Jung YW, Leung PCK, Jeung E-B 2004 Transfer of maternally injected endocrine disruptors through breast milk during lactation induces neonatal Calbin-din-D9k in the rat model. Reprod Toxicol 18: 661–668]Induction of CaBP-9k mRNA expression in fetal uterus by estrogenic compoundsFig r  3Induction of CaBP-9k mRNA expression in fetal uterus by estrogenic compounds. The values represent means ± SD. a, P < 0.05 vs. vehicle. [Reproduced with permission from Hong EJ, Choi KC, Jeung E-B 2003 Maternal-fetal transfer of endocrine disruptors in the induction of Calbindin-D9k mRNA and protein during pregnancy in rat model. Mol Cell Endocrinol 212: 63–72]Page 4 of 8(page number not for citation purposes)induce CaBP-9k expression in vivo system following oraltreatments, assuming that these phthalates are easilychanges in CaBP-9k expression. In addition, we investi-gated the potential for estrogenic compounds, OP, NP,Reproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66BPA, diethylstilbestrol (DES) and E2 to be transferredthrough breast milk from the dam to the neonate duringlactation via measuring the induction of CaBP-9k in uter-ine tissue [14]. These results indicate that these com-pounds have an estrogenic effect on the maternal uterusduring the lactation period, as shown by the induction ofboth CaBP-9k mRNA and protein. There was a significantincrease in CaBP-9k mRNA in neonatal uterus when thedams were treated with high doses of estrogenic com-pounds, but protein levels of CaBP-9k were undetectable(Fig. 4). Taken together, these findings suggest that mater-nally injected estrogenic compounds may be transferredto neonates through breast milk and thus, affect uterinefunction, as shown by the induction of CaBP-9k geneexpression in neonatal uterus [14]. In addition, werecently examined the effect of OP, NP and BPA on theexpression of CaBP-9k following maternal exposures dur-ing late pregnancy in maternal and fetal uterus [15,41].The expression of CaBP-9k mRNA was also induced fol-lowing treatment with a high dose (600 mg/kg BW) of OPtransferred from mother exposed to fetuses during latepregnancy and persisted in 5 day of lactation (Fig. 5). Inparallel with mRNA level, the expression level of CaBP-9kprotein was significantly induced by treatment with a highsures to OP, NP and BPA during late pregnancy increasedthe expression levels of CaBP-9k mRNA and protein inmaternal or neonatal uteri, suggesting that the absorptionand distribution of environmental estrogenic compoundsin maternal and neonatal uteri are extremely rapid, andthese chemicals can easily pass though placenta duringpregnancy to affect functions of neonatal reproductive tis-sues [15,41]. In addition, a novel in vivo model was intro-duced to detect both estrogenic and progestogenicactivities of EDs in the induction of CaBP-9k mRNA andprotein in the uterus of immature mice [35].Genistein, a phytoestrogen, has been shown to have rela-tively 20-fold higher binding affinity to the ERβ than ERby a solid-phase binding assay [42]. To determine whichER is involved in the induction of CaBP-9k gene, weemployed genistein as a potent ERβ agonist to clarify itseffect on uterine CaBP-9k regulation [43]. Both CaBP-9kmRNA and protein levels were significantly induced bygenistein in the uterus of immature rats. It is of interestthat the pre-treatment of immature rats with ICI 182,780α.Localization of CaBP-9k protein by immunohistochemical-staining in maternal uterusFigure 6Localization of CaBP-9k protein by immunohistochemical-staining in maternal uterus. Immuno-reactivity of CaBP-9k protein expression following treatment with OP and NP was investigated in endometrium and smooth myometrial fibers dose-dependently. Especially, these proteins that are widely spaced through the stromal cells in endometrium s, stroma cells; le, Luminal epithelial cell; ge, glandular epithelial cell. Magnification × 100. [Reproduced with permission from Hong EJ, Choi KC, Jeung EB 2004 Induction of Calbindin-D9k mRNA and protein by maternal exposure to alkylphenols during late pregnancy in maternal and postnatal uteri of rats. Biol Reprod 71: 669–675]200 400 600Vehicle E2 DESControlOctylphenolNonylphenollessssssssgegelesEffect of OP and NP on the induction of CaBP-9k mRNA in neonatal uterusFigure 5Effect of OP and NP on the induction of CaBP-9k mRNA in neonatal uterus. RT-PCR/Southern blot assay was performed during lactation period in neonatal uterus. The values repre-sent means ± SD. a, P < 0.05 vs. vehicle. [Reproduced with permission from Hong EJ, Choi KC, Jeung EB 2004 Induction of Calbindin-D9k mRNA and protein by maternal exposure to alkylphenols during late pregnancy in maternal and postnatal uteri of rats. Biol Reprod 71: 669–675]Page 5 of 8(page number not for citation purposes)dose of OP and NP in the maternal uterus by immunohis-tochemistry (Fig. 6). In conclusion, the maternal expo- (ICI), followed by genistein or E2, completely blockedReproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66genistein- and E2-induced CaBP-9k protein in this tissueas demonstrated in Fig. 7. In addition, genistein was dem-onstrated to induce ERα protein, but not ERβ or PRmRNA, an E2-responsive gene, in this tissue. These resultsimply that genistein, an ERβ ligand, may regulate CaBP-9kgene through ERα pathway and ERα may be a key media-tor in the induction of uterine CaBP-9k gene in immaturerats [43]. To support an involvement of ERα-dependentpathway by EDs, we demonstrated that uterine CaBP-9kgene expression is mainly mediated by propyl pyrazoletriol (PPT), an ERα-selective ligand, in a dose- and time-dependent manner, in the uterus of immature rats [44]. Incontrast, no significant alteration in the uterine CaBP-9kgene was observed after diarylpropionitrile (DPN), anERβ-selective ligand. In addition, an estrogenicity of PPTin inducing CaBP-9k expression was completely blockedindicate that uterine CaBP-9k is induced by E2 and endo-crine disrupting chemicals via ERα pathway, but not ERβ,in the uterus of immature rats [44].Placental expression of CaBP-9kTransport of Ca2+ through placenta is responsible fordeveloping fetus, and CaBP-9k appears to play an impor-tant role in the regulation of Ca2+ from the mother to thefetus during pregnancy. However, the role of CaBP-9k isunknown to date in placenta during pregnancy. A recentstudy demonstrated that CaBP-9k transcript is present incytotrophoblast cells and syncytiotrophoblasts of humanterm placenta, with a lower expression in cytotrophoblastcells as compared to syncytiotrophoblasts [45]. In addi-tion, CaBP-9k protein was present in cytotrophoblast andsyncytiotrophoblast placental tissue sections as well as incultured cells, indicating that CaBP-9k is unequivocallyexpressed by trophoblast cells from human term placenta[45]. The expression of CaBP-9k gene has been investi-gated in the placenta of other species [26,46-48]. The highlevel of CaBP-9k has been localized to epithelial cells ofthe yolk sac and endodermal cells of the placenta [16].The expression of CaBP-9k mRNA was not detectable byNorthern blot analysis, while this transcript was detectedin porcine myometrium and placenta by RT-PCR [26]. Aspreviously described, CaBP-9k mRNA has been also local-ized in the trophoblasts in various species. It is hypothe-sized that CaBP-9k plays a role in calcium transfer andfetal growth by parallel gestational changes in placentalCaBP-9k which reflects the fetal accumulation of calcium.An increased level of CaBP-9k gene in the caruncular epi-thelium during the last trimester is in response to theincreasing need for calcium to supply the fetal skeletonwith mineralization, suggesting that CaBP9k may play arole in transporting calcium across the placenta in cattle[48]. In the placenta of mice, the distinct regulation ofCaBP-9k has been demonstrated in the placenta com-pared to other tissues such as intestine and kidney, indi-cating that the expression of this gene is not dependent onVitamin D receptor [49]. Recently we demonstrated theexpression of CaBP-9k for the first time in mouse placentaand extra-embryonic membrane separately, and CaBP-9kmRNA may be regulated by sex steroid hormones (E2 andP4) and their receptors through complex pathway in thesetissues [50].Concluding RemarksAlthough CaBP-9k is mainly expressed in female repro-ductive tissues, i.e., uterus and placenta of various species,the role of CaBP-9k remains unknown. It can be hypoth-esized that uterine CaBP-9k may be involved in control-ling myometrial activity related with intracellular calciumlevel and placental CaBP-9k plays a role in calcium trans-Effect of ICI on genistein-induced uterine CaBP-9k protein expressionFigure 7Effect 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. The values rep-resent means ± SD. a, P < 0.05 vs. vehicle; b, P < 0.05 vs. gen-istein or E2 treatment only [Reproduced with permission from Lee GS, Choi KC, Kim HJ, Jeung EB 2004 Effect of gen-istein on the expression of Calbindin-D9k as a potential estro-genic compound in the uterus of immature rats through estrogen receptors. Toxicol Sci 82: 451–457]Page 6 of 8(page number not for citation purposes)by ICI 182,780; which suggests that uterine CaBP-9k issolely enhanced though ERα. Taken together, these resultsfer from the mother to the fetus for fetal growth. It appearsthat CaBP-9k gene is not under the control of vitamin DReproductive Biology and Endocrinology 2005, 3:66 http://www.rbej.com/content/3/1/66in the uterus despite the presence of vitamin D receptorsin this tissue; instead it is under the control of the sex ster-oid hormones. The hormonal mechanism controllinguterine CaBP-9k gene is relatively well understood in therat. In the uterus of rats, estrogen is known to up-regulateand progesterone down-regulate the expression of CaBP-9k gene during estrous cycle and pregnancy. However, therecent studies demonstrated that CaBP-9k is mainly regu-lated by progesterone, not estrogen in the uterus of micebecause of lack of responsiveness caused by a single-basedifference in the ERE of mouse CaBP-9k gene compared tothat of rats. Until now, a few studies demonstrated theexpression and regulation of CaBP-9k gene in the placentaof various species. It appears that CaBP-9k mRNA may beregulated by sex steroid hormones (E2 and P4) and theirreceptors through complex pathway in these tissues. Weassumed that ERα may be a key mediator in uterine CaBP-9k gene induction in immature rats. The elucidation ofother factors that regulate CaBP-9k mRNA will furtherprovide insight into the understanding of regulation ofCaBP-9k in these tissues, and its roles in the control ofreproductive functions.The uterus is a highly estrogen-responsive tissue, whichcan be measured through changes in CaBP-9k expression.We demonstrated that the expression levels of CaBP-9kmRNA and protein are induced by estrogenic chemicals,so called "endocrine disruptors", in the uterus of imma-ture rats. In addition, maternally injected estrogenic com-pounds resulted in an increase of CaBP-9k mRNA and/orprotein in the fetal uterus during late pregnancy, suggest-ing that placenta may not be a reliable barrier againstthese estrogenic compounds for fetal health. It is of inter-est that maternally injected estrogenic compounds may betransferred to neonates through breast milk and thusaffect uterine function, as shown by the induction ofCaBP-9k gene expression in neonatal uterus. The expres-sion of CaBP-9k mRNA and/or protein is an excellentbiomarker to detect an estrogenic chemical in the uterusof immature rats which we developed and established.Availability of this gene using immature rats will providean insight of risk assessment for estrogenic and pro-gestogenic chemicals in our environment.AcknowledgementsThis work was supported by the research grant of the Chungbuk National University in 2005. The authors would like to thank Mr. Geun-Shik LEE (College of Veterinary Medicine, Chungbuk National University) for editing the figures.References1. Christakos S, Gabrielides C, Rhoten WB: Vitamin D-dependentcalcium binding proteins: chemistry, distribution, functionalconsiderations, and molecular biology.  Endocr Rev 1989,10:3-26.3. Jeung EB, Leung PC, Krisinger J: The human calbindin-D9k gene.Complete structure and implications on steroid hormoneregulation.  J Mol Biol 1994, 235:1231-1238.4. 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