ToxSci Advance Access published online on September 29, 2004
Toxicological Sciences, doi:10.1093/toxsci/kfh296
Toxicological Sciences © Society of Toxicology 2004; all rights reserved
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1 Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763 Republic of Korea
* To whom correspondence should be addressed. E-mail: ebjeung{at}chungbuk.ac.kr.
Genistein, a phytoestrogen possessing a high affinity for estrogen receptor
Accepted September 7, 2004
Endocrine Toxicology
Effect of Genistein as a Selective Estrogen Receptor Beta Agonist on the Expression of Calbindin-D9k in the Uterus of Immature Rats
2 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
Abstract 
(ER), 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 ER on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ER agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for 3 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
and ER 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 pre-treatment 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 protein, but not ER and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ER ligand, may regulate CaBP-9k gene through ER pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ER in the uterus of immature rats, suggesting that ER may be a key mediator in uterine CaBP-9k gene induction in immature rats.
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