Environmental Estrogens Stimulate Gene Transcription in the Prolactin Promoter Danae Fitzmaurice and Winnifred Bryant Ph. D. Department of Biology University of Wisconsin, Eau Claire INTRODUCTION 17 β-estradiol (E 2 ) is produced primarily by the ovaries and governs gene expression in a number of target tissues, including the brain and pituitary. In the pituitary particularly, the roles of estrogen extends to stimulating reproductive hormone secretion. Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor that mediates the physiological effects of E 2. Within a target cell, occupancy of ERs by E 2 results in receptor activation and nuclear localization. Subsequent binding to an estrogen response element (ERE) in a target gene allows recruitment of various coregulator proteins which enhance or suppress gene expression. The actions of endogenous E 2 can be mimicked by environmental estrogens that are produced in plants (phytoestrogens) or produced commercially as synthetic compounds (xenoestrogens). These studies examine the ability of E2 and environmental estrogens to alter transcriptional activity a pituitary cell line MATERIALS AND METHODS Cells GH 3 cells (rat pituitary cell line expressing endogenous ER  ) were cultured in DMEM supplemented with growth serum and maintained at 37 o C in 95%O 2 /5%CO 2. Gene Expression in Mammalian Cells All cell lines were transiently transfected using GeneJuice transfection reagent. To determine dose response relationships, 250ng pGL3 reporter (ERE gene fused to firefly luciferase gene) were transfected into GH 3 cells and treated with increasing concentrations of the xenoestrogen bisphenol A and the phytoestrogen daidzein. Alternately, cells were transfected with 100 ng prolactin promoter and treated with increasing concentrations of the xenoestrogen bisphenol A and the phytoestrogen. To demonstrate that the effects of environmental estrogens were receptor-mediated, treatments were also administered in the presence of ICI , a pure E 2 antagonist. To examine the role of the transcription factors, Pit-1, cells were transfected with prolactin promoter and Pit- 1, then treated with environmental estrogens. For each set of experiments, period of treatment was 24 hours. Cells were then collected in 200 ul 1X Promega lysis buffer. 50ul luciferin was added to an equal volume of lysate and luciferase activity assessed using a Turner Biosystems 20/20n luminometer. Data are expressed as arbitrary light units (ALU) per 50 ul lysate or as ALU/mg protein. Statistical Analysis. Transfections were performed in triplicate. Data were analyzed by one way ANOVA and a Bonferroni post- hoc test using GraphPad Prism software. Figure 1. Environmental estrogens stimulate transcription in a dose related manner. GH 3 cells were transfected with the 250 ng pGL3 model promoter (Panel A) or 250 ng of the physiologically complex PRL promoter (Panel B). Cells were treated for 24 hours with BPA (solid grey bars) or D (shadowed bars) at the doses indicated in the figure. Cells were collected, lysed and subjected to luciferase assay. Data are expressed as arbitrary light units per microgram of protein. Bars represent mean ± SEM for 4 experiments. For purposes of comparison, response of transfected cells to E 2 is also shown (black bar).*, significantly different from vehicle treated control, P<0.05. †, significant difference in respective responses of EEs, P< C Figure 2. ERs mediate EE-induced transcription of a model and complex promoter. GH 3 cells were transfected with the 250 ng pGL3 model promoter (Panel A) or 250 ng of the physiologically complex PRL promoter (Panel B). Cells were treated for 24 hours with stimulatory doses of E 2, G, BPA, D, and K in the absence (white bars) or presence (gray bars) of ICI 182, 780, a pure estrogen antagonist. Cells were collected, lysed, and subjected to luciferase assay. Data are expressed as arbitrary light units per microgram of protein. Bars represent mean ± SEM for 4 experiments. *, significantly different from vehicle treated control (black bar), P<0.05. Figure 3. The nuclear transcription factor Pit-1 enhances EE-induced transcriptional responses in the PRL promoter. GH 3 cells were transfected with the 250 ng of the PRL promoter (white bars). Additionally, some cells were cotransfected with Pit-1 (black bars). Cells were treated for 24 hours with stimulatory doses of E 2, BPA or D. Cells were collected, lysed and subjected to luciferase assay. Data are expressed as arbitrary light units per microgram of protein. Bars represent mean ± SEM for 4 experiments. For purposes of comparison, response of transfected cells to E 2 is also shown (black bar). Bars represent mean ± SEM for 4 experiments.*, significantly different from vehicle treated control, P<0.05. †, significant difference in transcriptional responses in presence of Pit-1, P<0.05 SUMMARY AND CONCLUSIONS Transcriptional activity in both promoter constructs were stimulated by EEs in a dose related fashion. These stimulatory effects are mediated via the ER, since ICI (which competitively inhibits the binding of estrogen to the ER) is observed to abolish these effects. Relative to the potency E 2, BPA, G and D proved to be more effective at stimulating transcription in the PRL promoter. In our experiments, the cotransfection of Pit-1 resulted in a moderate increase in EE-induced transcription. Thus, occupancy of the ER by EEs may elicit responses in the PRL promoter that require Pit-1 for full transcriptional activity. Collectively, these data indicate that EEs indeed behave similarly as E 2 in the stimulation of the prolactin promoter with regard to mechanism but not magnitude. These data in these studies are significant because they demonstrate the effects of EEs on a pituitary specific gene promoter that is not regulated by a palindromic ERE, but multiple promoter elements. PRL is of physiological significance for its ability to initiate and maintain lactation in mammals, but the prolactin gene, however, is not the only E 2 sensitive gene expressed in the pituitary. Indeed, E 2 regulation gene expression and hormone release in the anterior pituitary is evidenced by the ubiquitous expression of ERs in the APG (Shupnik, 2002). Thus, the ability of xenoestrogens and phytoestrogens to stimulate complex promoter activity in pituitary cells may present some particular implications for overall health, but reproductive health in particular (Adeyoa-Osiguwa et al., 2003). With detailed information on specific nuclear mechanisms of EE action, we may be better able to assess risk of incidental or deliberate exposure to such compounds. REFERENCES Shupnik, M.A. (2002). Oestrogen receptors, receptor variants and oestrogen actions in the hypothalamic-pituitary axis. Journal of Neuroendocrinology, 14, Adeoya-Osiguwa, et al. (2003). 17  -Estradiol and environmental estrogens significantly affect mammalian sperm function. Human Reproduction, 18: Korb et al. Environmental Estrogens Stimulate Gene Transcription in the Prolactin Promoter. International Journal of Biology 2: ACKNOWLEGMENTS These studies were supported by ORSP-UWEC * * * * * From: NEJM, 346 (5):