David W. Singleton

XENOESTROGEN EXPOSURE AND MECHANISMS OF ENDOCRINE DISRUPTION

Environmental xenoestrogens can be divided into natural compounds (e.g. from plants or fungi), and synthetically derived agents including certain drugs, pesticides and industrial by-products. Dietary exposure comes mainly from plant-derived phytoestrogens, which are thought to have a number of beneficial actions. However, high levels of exogenous estrogens including several well-known synthetic agents are associated with harmful effects. Chemicals like xenoestrogens, which can mimic endogenous hormones or interfere with endocrine processes, are collectively called endocrine disruptors. Adverse effects by endocrine disrupting chemicals (particularly xenoestrogens) include a number of developmental anomalies in wildlife and humans. Critical periods of urogenital tract and nervous system development in-utero and during early post-natal life are especially sensitive to hormonal disruption. Furthermore, damage during this vulnerable time is generally permanent, whereas in adulthood, ill effects may sometimes be alleviated if the causative agent is removed. The most commonly studied mechanism in which xenoestrogens exert their effects is through binding and activation of estrogen receptors a and similar to endogenous hormone. However, endocrine disruptors can often affect more than one hormone (sometimes in opposite directions), or different components of the same endocrine pathway, therefore making it difficult to predict effects on human health. In addition, xenoestrogens have the potential to exert tissue specific and nongenomic actions, which are sensitive to relatively low estrogen concentrations. The true risk to humans is a controversial issue; to date, little evidence exists for clear-cut relationships between xenoestrogen exposure and major human health concerns. However, because of the complexity of their mechanism and potential for adverse effects, much interest remains in learning how xenoestrogens affect normal estrogen signaling.

The F-domain of estrogen receptor-alpha inhibits ligand induced receptor dimerization.

The role of the carboxyl terminal F-domain of estrogen receptor (ERalpha) is uncertain, but evidence suggests that this region may impart internal restraint on ER dimerization in the presence of 17beta-estradiol (E2). To identify the C-terminal residues affecting human ERalpha activation, we created a series of deletions and examined E2 induced receptor dimerization and transactivation. Deletion of the final 24 C-terminal amino acids of the F-domain (Delta7b) yielded a fivefold increase in dimerization, when compared to wild type (wt) ERalpha in the presence of 2nM E2, utilizing a yeast two-hybrid assay. This increase in dimerization is similar to that observed when the entire F-domain was deleted. Measurement of mutant:mutant homodimer formation yielded similar increases compared to mutant:wt interactions. Interestingly, a point mutation at the C-terminus (mut 3) showed increases in dimerization comparable to that of Delta7b in the presence of nanomolar amounts of E2. However, at sub-nanomolar levels of E2, mut 3 behaved similarly to wt ERalpha, whereas Delta7b maintained striking increases in dimerization. Determination of E2 binding affinity (Kd) constants revealed only marginal differences for wt and F-domain mutants, suggesting that the F-domain affects dimerization directly. We also observed enhanced interaction of F domain mutants with p160 family coactivator SRC1. Finally, transcriptional regulation of estrogen responsive reporters, 2XERE-LacZ and 3XERE-Luc in yeast and mammalian cells, respectively, reflected the increased propensity for dimerization by F domain mutants. Together, these data indicate that the C-terminal amino acids of ERalpha are critical for attenuation of E2 induced receptor dimerization and transcriptional activity.

Bisphenol-A and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells

Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17beta-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ERalpha-HA) human breast cancer cells derived from the MCF-7 cell line. The ERalpha-HA cells (stably over-expressing ERalpha) were exposed to E2 (10(-8)M) or BPA (10(-6)M), for 3h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ERalpha-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4-12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott-Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ERalpha expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth.

Phosphorylation of estrogen receptor alpha, serine residue 305 enhances activity

Upon ligand binding the estrogen receptor alters its conformation, dimerizes, binds to estrogen response elements (EREs), recruits cofactors and initiates the formation of a transcriptional complex. In addition to estradiol binding, hormone receptor activity is modulated by phosphorylation at several key residues. Previous studies have shown that p21-activated kinase-1 (Pak1) and cyclic-AMP dependent protein kinase (PKA) can phosphorylate ERalpha at serine residue 305. However, the effects of serine 305 phosphorylation on ERalpha activity have not been fully characterized. To study these effects, ERalpha S305E and S305A mutants were created to mimic constitutively phosphorylated or un-phosphorylated states, respectively. Using yeast two-hybrid assays we showed that dimerization of ERalpha S305E was still ligand dependent. However, the capability of dimerization in the presence of estradiol was significantly higher in S305E compared to wild-type ERalpha. Transactivation assays demonstrated that phospho-mimetic ERalpha S305E is active in the absence of ligand. Chromatin immunoprecipitation (ChIP) analysis shows a change of in vivo DNA binding in which S305E mutant binds to ERalpha DNA target sequences and exhibits increased residency in the absence of ligand. We also observed increased cell growth in cells stably transfected with S305E ERalpha. Thus, we suggest that phosphorylation of S305 does not trigger ERalpha dimerization but increases binding to target gene promoters, which can lead to increased cell growth in the absence of estradiol. This implies a shift from hormone-induced activation of ERalpha to activation through phosphorylation, which could confer resistance to hormone based therapies for breast cancer.

The Genomic Response of a Human Uterine Endometrial Adenocarcinoma Cell Line to 17α-Ethynyl Estradiol

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p </= 0.0001). The annotation available for the genes affected indicates that EE exposure results in changes in multiple molecular pathways affecting various biological processes, particularly associated with development, morphogenesis, organogenesis, cell proliferation, cell organization, and biogenesis. All of these processes are also affected by estrogen exposure in the uterus of the rat. Comparison of the response to EE in both the rat uterus and the Ishikawa cells showed that 71 genes are regulated in a similar manner in vivo as well as in vitro. Further, some of the genes that show a robust response to estrogen exposure in Ishikawa cells are well known to be estrogen responsive, in various in vivo studies, such as PGR, MMP7, IGFBP3, IGFBP5, SOX4, MYC, EGR1, FOS, CKB, and CCND2, among others. These results indicate that transcript profiling can serve as a viable tool to select reliable in vitro systems to evaluate potential estrogenic activities of target chemicals and to identify genes that are relevant for the estrogen response.

Mutation of Serines 104, 106, and 118 Inhibits Dimerization of the Human Estrogen Receptor in Yeast

Ligand‐dependent dimerization and phosphorylation participate in regulating transcriptional activation of the estrogen receptor‐α (ER). We investigated the role of serines 104, 106, and 118 located in the activation function‐1 (AF‐1) domain of ER in ligand‐induced receptor dimerization. These serines, previously documented as important sites for transactivation, were mutated to alanine, and yeast genetic systems were used to determine their effect on receptor dimerization and transcriptional activity. The serine to alanine mutants resulted in 50–80% decreased dimerization in response to 17β‐estradiol, while having modest effects on ER‐mediated transactivation. We further demonstrated that ER expressed in yeast became hyperphosphorylated in the presence of estradiol, most likely at a site(s) different than the serines under investigation. Ligand‐induced phosphorylation was inhibited by U0126 indicating that the ER was phosphorylated via the MAPK pathway. Taken together, these data indicate that serines 104, 106, and 118 are important for ligand‐dependent ER dimerization, and that MAP kinase mediated phosphorylation may be important for ER function, in yeast model systems.

DNA Homologous Recombination Factor SFR1 Physically and Functionally Interacts with Estrogen Receptor Alpha

Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER’s ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER’s transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.