Estradiol Binding Sites Research Articles

Genistein Ameliorated Vascular Endothelial Growth Factor-A (VEGF-A) and Estrogen Receptor-Alpha (ER-α) in Endometriosis Mice Model, In Vivo and In Silico.

Endometriosis (EM) is a gynecological disorder that causes morbidity in women and is characterized by endometrial tissue in the uterus cavity. This study investigated the mechanism of genistein in the VEGF-A and ER-α expression through in vivo and in silico approaches. An in vivo study was conducted by thirty-six mice that were divided into six groups including control, EM, and EM treatment with genistein with the doses of 1.3, 1.95, 2.6, and 3.25 mg/day for 14 days. Peritoneal tissues with lesions were collected and analyzed by immunohistochemistry to measure the VEGF-A and ER-α expression. The data were analyzed using a statistical approach using one-way ANOVA followed by Tukey HSD test with a significant value p < 0.05. In silico study was conducted for investigating the inhibition mechanism of genistein in VEGF-A and ER-α protein. Genistein significantly reduced the VEGF-A and ER-α expression with the optimum dose of 3.25 mg/day. Molecular docking showed that genistein inhibited VEGF-A in several active site residues of VEGF-A, also blocked the ER-α protein in estradiol binding sites. This study concluded that genistein prevented endometriosis by performing the antiangiogenic activity and showed a similar function to estradiol.

Anti‐cancer Effects of Melatonin, Estrogen, and Progesterone Hormone Therapy in MCF‐7 and MDA‐MB‐231 Breast Cancer Cells

A novel melatonin, estrogen (E2), and progesterone (P4) hormone therapy (MEPT) was developed as a safe bio‐identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer (BC) risk and mortality of all types of BC in postmenopausal women. MEPT delayed tumor onset and reduced tumor incidence in Neu female mice (Dodda et al., 2019; Front Oncol). For other BCs, its actions are currently unknown. In this study, MEPT’s anti‐cancer actions (cell viability, migration) were tested in MCF‐7 (human ER+/PR+) and MDA‐MB‐231 (human TNBC with constitutively active ras mutation) using equivalent concentrations of melatonin (1nM), E2 (50pM), and P4 (10nM) observed in serum after an oral dose that produced anti‐tumor actions in neu mice (Dodda et al 2019; Front Oncol). Potential mechanisms underlying MEPT’s anti‐cancer actions were assessed by western blot (pERK1/2, pERK5, NF‐kB, RUNX2, β1‐INTEGRIN, RANKL, and ELF‐5) using MEK1/2 (PD98059) and MEK5 (Bix02189) inhibitors and radioligand binding using 2‐[125I]‐iodomelatonin or [3H]estradiol binding for melatonin and estrogen receptor expression, respectively. For MCF‐7 cells, BIX02189 combined with MEPT blocked MEPT‐mediated inhibition of migration and MEPT‐mediated ELF5 induction in MCF‐7 cells (vs. vehicle) and decreased pERK5 (vs. Bix02189) suggesting that MEK5, through pERK1/2 and ELF5, were involved in the anti‐cancer actions of MEPT in MCF‐7 cells. For MDA‐MB‐231 cells, although PD98059 and Bix02189 alone inhibited viability and migration, when added in combination with MEPT, no further blockade or enhancement occurred suggesting that MEK1/2 and MEK5 were not involved in MEPT’s anti‐cancer actions in MDA‐MB‐231 cells. MEPT’s differential actions on MCF‐7 and MDA‐MB‐231 cells were most likely attributed to unique melatonin/melatonin receptor actions within each cell. Both MCF‐7 and MDA‐MB‐231 cells express high‐affinity melatonin receptors (MCF‐7: Kd = 13 pM; Bmax = 20.55 fmol/mg protein vs. MDA‐231: Kd = 1.7 nM; Bmax = 60.09 fmol/mg protein) but only in MCF‐7 cells were [3H]‐estradiol binding sites detected (MCF‐7: Kd = 2.2 nM; Bmax = 8.6 pmol/mg protein) and stimulatory actions of E2 or P4 on MCF‐7 cell proliferation observed. These findings demonstrate novel anti‐cancer actions of MEPT in ER+ BC and TNBC cells through distinct mechanisms. Future studies assessing MEPT’s actions against ER+ BC and TNBC in vivo are warranted.Support or Funding InformationThis study was funded by the Marie‐Clement Rodier, C.S.Sp. Endowed Chair.

Mapping of estradiol binding sites through receptor micro-autoradiography in the endometrial stroma of early pregnant mice.

Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.

Crystallographic Analysis and Mimicking of Estradiol Binding: Pedersen et al. Respond

Previous studies have addressed the biological effects of brominated flame retardants (Birnbaum and Staskal 2004; Koike et al. 2013; Mariussen and Fonnum 2003; Ogunbayo et al. 2008), including a 2-year bioassay study performed by the National Toxicology Program (NTP), which demonstrated that tetrabromobisphenol A (TBBPA) can induce aggressive uterine tumors in rats (NTP 2013). As pointed out by Osimitz et al., TBBPA has been shown to bind poorly to the estrogen receptor, providing the impetus to study other pathways such as disruption of steroid transport and metabolism. Other groups have demonstrated the ability of TBBPA and flame retardant metabolites to inhibit estrogen sulfotransferase (SULT1E1), with IC50 (median inhibitory concentration) values near the Km for estradiol (Hamers et al. 2008; Kester et al. 2002; Zhang et al. 1998). Our work (Gosavi et al. 2013) was focused solely on understanding the structural mechanism by which these compounds bind to and inhibit SULT1E1’s ability to metabolize estradiol. The results of our work demonstrate that TBBPA and the 3-OH metabolite of BDE-47, although structurally different, bind in a similar manner at the estradiol binding site. This work suggests that these compounds could have an additive effect on the inhibition of this enzyme. We wholeheartedly agree with Osimitz et al. that the results of our work warrant future studies addressing the potential additive effect of these compounds on steroid metabolism in target tissues.

Multiple Sites of Type II Site Ligand (Luteolin and BMHPC) Regulation of Gene Expression in PC-3 Cells

Type II [3H]estradiol binding site ligands including luteolin (a naturally occurring bioflavonoid) and synthetic compounds such as 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)cyclohexanone (BMHPC) inhibit normal and malignant prostate cell (PC-3, LNCaP, DU-145) proliferation in vitro and in vivo. Type II sites represent a binding domain on histone H4 possibly involved in an epigenetic mechanism for controlling gene transcription. Treatment of PC-3 human prostate cancer cells with luteolin or BMHPC modulated the expression of a number of genes in the epidermal growth factor receptor signaling pathway (EGFRSP) and cell cycle pathway (CCP). Pronounced stimulation (400-2000% of control) of c-FOS and p21 RNA expression was observed, suggesting that these were primary sites of action. Both compounds also caused irreversible G2/M arrest (p&lt;0.001). siRNA's for c-FOS or p21 reduced the RNA expression of their respective targets by 85-95%, with minimal effects on cell proliferation. Furthermore, neither siRNA alone (single knockdown), or in combination (double knockdown), blocked luteolin or BMHPC inhibition of PC-3 cell proliferation. Thus, although c-FOS and p21 are known to modulate the expression of genes in the ESGRSP (EGFR, SOS, GRB2, JNK1, MKK4, RasGAP) and CCP (CCNA2, CCNE2, CDC25A, CDKN1A, CDKN1B, p27, PLK1) involved in the regulation of cell proliferation by luteolin and BMHPC, the c-FOS and p21 siRNA knockdown studies reported here suggest that c-FOS and p21 may be secondary bystanders in the overall response to these ligands in the regulation of PC-3 cell proliferation.

Differential Regulation of Ovarian Anti-Müllerian Hormone (AMH) by Estradiol through α- and β-Estrogen Receptors

Anti-müllerian hormone (AMH) is a member of the TGF-β family, which limits follicle maturation. Recently serum AMH has been recognized as a useful diagnostic and prognostic tool in human reproductive endocrinology. The aim of this study was to investigate the regulation of human ovarian AMH by estradiol and FSH. AMH mRNA were quantified by real time RT-PCR in human granulosa cells (GC). AMH transcription was studied in KK1 GC cotransfected with estrogen receptors (ER)-β or ERα, and normal human AMH promoter-luciferase construct (hAMH-luc) or mutated AMH promoter reporter constructs. Binding sites for estradiol (estrogen response element half-site) and steroidogenic factor 1 were disrupted by targeted mutagenesis. The level of ER in GC was determined by quantitative RT-PCR and Western blotting. In KK1 cells, estradiol up-regulated and inhibited hAMH-luc in the presence of ERα and ERβ respectively. Disruption of estrogen response element half-site and/or steroidogenic factor 1 binding sites did not modify ERβ-mediated effect of estradiol on hAMH-luc, whereas it affected that conveyed by ERα. The FSH enhancement of hAMH-luc was abolished by estradiol in cells overexpressing ERβ. When both ER were transfected, estradiol inhibited hAMH-luc or had no effect. Estradiol repressed AMH mRNAs in human GC, which express a little more ERα than ERβ mRNA. Our results show that AMH expression can be differentially regulated by estradiol depending on the ER and suggest that its decrease in GC of growing follicles, which mainly express ERβ, and during controlled ovarian hyperstimulation is due to the effect of estradiol.

Characterization of the Estradiol-Binding Site Structure of Human Pancreas-Specific Protein Disulfide Isomerase: Indispensable Role of the Hydrogen Bond between His278 and the Estradiol 3-Hydroxyl Group

Estradiol (E(2)), a female sex hormone, has important biological functions. Human pancreas-specific protein disulfide isomerase (PDIp), a protein folding catalyst, was recently found to be able to bind E(2). Here we report the characterization of its E(2)-binding site by using biochemical methods coupled with molecular modeling tools. Analysis of various truncated PDIp proteins showed that the b-b' fragment contains an intact E(2)-binding site that has the same binding affinity as the full-length PDIp protein, with apparent K(d) values of approximately 170 nM. Computational modeling and docking analyses revealed that the E(2)-binding site in the b-b' fragment is located in a hydrophobic pocket composed mainly of the b' domain and partially of the b domain. The hydrogen bond, formed between the 3-hydroxyl group of E(2) (donor) and PDIp's His278 (acceptor), is indispensable for its binding. By contrast, the 17β-hydroxyl group of E(2) is of negligible importance for E(2) binding. This binding model was jointly confirmed by a series of experiments, such as selective mutation of the binding site amino acid residues and selective modification of the ligand structures.

Regulation of cell cycle and RNA transcription genes identified by microarray analysis of PC-3 human prostate cancer cells treated with luteolin

Prostate cancer is the second leading cause of cancer-related deaths in men in the United States. Our previous studies have shown that ligands for the nuclear type II [ 3H]estradiol binding site such as luteolin significantly inhibit prostate cancer cells in vitro and in vivo; however, the role of these ligands in cell growth and proliferation is poorly understood. In order to further elucidate the molecular mechanism through which luteolin exerts its effects on PC-3 cells, cRNA microarray analyses was performed on 38,500 genes to determine the genes altered by luteolin treatment. The expression of 3331 genes was changed greater than 1.2-fold after luteolin treatment. Analysis of the altered genes identified two pathways that were significantly affected by luteolin. The Cell Cycle Pathway contained 22 down-regulated genes (including polo-like kinase 1, cyclin A2, cyclin E2 and proliferation cell nuclear antigen) and one up-regulated gene (cyclin-dependent kinase inhibitor 1B). In addition, 13 genes were down-regulated by luteolin in the RNA Transcription Pathway. Real-time polymerase chain reactions and western blots verified the observations from the microarray. In addition, two synthetic, chemically distinct type II ligands, ZN-2 and BMHPC, mimicked the effects of luteolin on gene expression at the mRNA and protein level in PC-3 cells. Finally, chromatin immunoprecipitation assays indicated that luteolin exerts its effects on genes by altering the acetylation state of promoter-associated histones. Taken together, the data suggest that type II ligands inhibit cell growth and proliferation through epigenetic control of key genes involved in cell cycle progression and RNA transcription.

Oestradiol and testosterone binding sites in mice tibiae and their relationship with bone growth.

High affinity oestradiol and testosterone binding sites were found in tibiae cytosol from entire male and female of different ages. Scatchard assay allowed to estimate a Kd of 2.7 X 10(-9) M for oestradiol binding sites indicating that the 3H-oestradiol binding was of high affinity. Oestradiol and testosterone binding sites abundance in mice tibiae are subject to change with age. It is not easy to establish a direct correlation between these changes and the values reported here on bone growth in weight and length, however seems possible to point a negative relationship between bone lengthening and oestradiol binding site levels in female, as well a positive relationship with testosterone in both sexes. The presence of oestradiol and testosterone binding sites in epiphyses and not in the diaphyses reinforces the hypothesis that both are playing some role in bone growth.

Two Types of Nuclear Oestradiol Binding Sites in Human Myometrium and Leiomyoma during the Menstrual Cycle1)

Two types of oestradiol binding sites were identified in human myometrium and myoma by saturation analysis. Nuclear type I receptors bind Oe with high affinity (KD = 1.4 nM) and low capacity (0.1-2.0 pmole/mg DNA) in a competitive fashion (Hill coeff. = 1.0), while nuclear type II sites bind the hormone with reduced affinity (KD: approximately 20 nM in myometrium and KD: approximately 40 nM in myoma), but high capacity (1-15 pmole/mg DNA) by positive cooperation (Hill coeff.: 4-5 in myometrium and 2-3 in myoma). Binding properties of type II sites in myoma are similar to those found in endometrium in previous studies of our laboratory, rather than those found in normal myometrium. The concentration of both nuclear Oe binding sites varied with the menstrual cycle. In myometrium, maximal concentration of nuclear type I and type II sites occurred between 10-14 days of cycle, when blood Oe level is the highest. In myoma the concentration of type I receptors was highest during the late follicular phase, but type II sites were uniformly high during the first 14 days of the cycle. In the luteal phase, receptor concentrations were low and apparently unaltered. It is possible that these changes in Oe binding capability of leiomyoma play a role in the pathomechanism.

Nuclear type II [ 3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [ 3H]estradiol binding to nuclear type II sites, but did not compete for [ 3H]estradiol binding to recombinant ERα or ERβ. ZN-1 and ZN-2 inhibited the proliferation of ERα and ERβ positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [ 3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19–30, 2005).

Reconstitution of the type II [ 3H]estradiol binding site with recombinant histone H4

Previously, we identified the rat uterine nuclear type II [ 3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [ 3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [ 3H]estradiol with a low affinity ( K d ∼20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient ∼4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [ 3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the “nucleosomal ligand-binding domain” for methyl- p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.

Homology-Modeled Ligand-Binding Domains of Zebrafish Estrogen Receptors α, β1, and β2: From in Silico to in Vivo Studies of Estrogen Interactions in Danio rerio as a Model System

Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) alpha, beta(1), and beta(2). Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfERalpha and found to bind in the same manner as in human ERalpha, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: New insights based on studies with carboxy-biochanin A

Estradiol-17β (E 2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca 2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E 2 on DNA synthesis, intracellular Ca 2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E 2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca 2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERα and suppressed ERβ mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

A two-site model for antiestrogen action

Evidence is presented for a unified model for the reaction of antiestrogens with estrogen receptors that explains much of the unusual pharmacology of these clinically important agents. Agonist activity results from occupancy of the estradiol-binding (primary) site in the receptor and antagonism from the additional interaction with a secondary locus not recognized by hormone. In the case of type I antiestrogens, such as tamoxifen, this is weaker than primary site binding, so these substances are agonists at low concentrations and antagonists at higher levels. With type II antiestrogens, affinities for both sites are comparable, so one never has the agonist situation (only primary site occupied), and these agents are pure antagonists. Reproductive tissues of the mouse and guinea pig contain a small macromolecule that binds hydroxytamoxifen, but not estradiol, keeping the free antiestrogen concentration below that required for secondary binding. Thus, in these species, tamoxifen is a pure agonist.

Interaction of estrogen mimics, singly and in combination, with plasma sex steroid-binding proteins in rainbow trout ( Oncorhynchus mykiss)

The plasma sex steroid-binding protein (SBP) is believed to be involved in regulating circulating endogenous sex steroids as well as cellular signal transduction to nuclear steroid receptors in sex steroid-sensitive tissues. In this study, a variety of estrogen mimics (EMs), which may contribute to the endocrine disrupting effects observed in fish, were tested for the ability to interact with the rainbow trout plasma sex steroid-binding protein (rtSBP) either singly or in binary combinations. The EMs ethynylestradiol, diethylstilbestrol, 4-hydroxytamoxifen, genistein, zearalenone, 4- t-octylphenol, bisphenol A and o,p′-DDT were all able to displace 1,2,4,6,7-[ 3H]estradiol from the sex steroid-binding site at the rtSBP ( K d=2.1±0.5 nM, B max=2963±303 fmol estradiol/mg protein) in a dose-dependent and competitive manner. The plastizicer n-butyl benzyl phthalate only displayed weak binding affinity for the rtSBP, whereas the pesticide dieldrin was not able to compete for the high affinity estradiol-binding sites in plasma. None of the compounds tested was able to clearly promote the binding of the others when given in combination, indicating that synergy did not occur at the ligand-SBP binding level. The rtSBP binding affinity for EMs ranged over several orders of magnitude, but were consistently lower than those for the endogenous sex steroids (about 10 2–10 6 less potent). The results suggest that the presence of rtSBP may potentially modulate the bioavailability of EMs to estrogen receptors relative to each other and to the endogenous sex steroids themselves. Since the binding of the endogenous ligands is reversible, SBP-bound estrogens (or androgens) potentially may also become displaced by potent EMs.

Expression and cellular localization of naturally occurring beta estrogen receptors in uterine and mammary cell lines.

The protein ER-alpha has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-beta protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-alpha and -beta isoforms. Native ER-beta was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-beta as a tight approximately 50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-beta version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-beta immunolabeling was localized at membrane subfractions. ER-beta expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-alpha was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-beta could be expressed as functional estrogen binding proteins among a dominant population of ER-alpha sites in the cell lines under study.

Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata

Municipal effluents are an important source of estrogens to the aquatic environment. The purpose of this study was to examine the estrogenicity of municipal effluents to the indigenous freshwater mussel, Elliptio complanata. First, estradiol-binding sites in gonad homogenates were characterized to determine the binding affinity and specificity of estrogens. Mussels were exposed to increasing concentrations of a municipal effluent for 96 h at 15°C. In another experiment, mussels were placed in cages and submerged for 62 days at 1.5 km upstream and 5 km downstream of a municipal effluent plume in the St. Lawrence River. Mussels were harvested for assessment of vitellogenin-like proteins in the hemolymph and determination of total lipid, carbohydrate and protein in the gonad. The presence of specific estrogen-binding sites was found in both male and female gonads. Binding of estradiol to cytosol proteins reached saturation, yielding a dissociation constant of 0.4 nM. Vitellogenin (Vg) levels increased significantly in both the hemolymph and the gonad after exposure to the effluent. Moreover, females appeared to be more sensitive than males to producing Vg. Mussels exposed in situ to contaminated surface waters had higher levels of Vg at the downstream site, again, females had higher levels of Vg than did males. On the other hand, lipid and sugar levels in male gonads were significantly increased at the downstream site. Moreover, mussels at the downstream site had decreased shell growth length and increased total and soft tissue weights. We conclude that municipal effluents contain bio-available xenoestrogens at levels sufficient to elicit effects in freshwater mussels.

Hormone Binding by Protein Disulfide Isomerase, a High Capacity Hormone Reservoir of the Endoplasmic Reticulum

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.