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

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Abstract
Genistein, a phytoestrogen possessing a high affinity for estrogen receptor β (ERβ), 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 ERβ on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERβ 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 ERα and ERβ 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 ERα protein, but not ERβ and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERβ ligand, may regulate CaBP-9k gene through ERα pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERα in the uterus of immature rats, suggesting that ERα may be a key mediator in uterine CaBP-9k gene induction in immature rats.

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