ERα Isoform Research Articles

Ontogenic profile of estrogen receptor alpha and beta mRNA and protein expression in fetal rat brain

It has been shown that increased estrogen can down-regulate its receptor, but there is no data to determine if that mechanism acts in the fetal brain as a consequence of high maternal estrogen levels. The aim of this study was to explore the expression of estrogen receptor (ER) in the developing fetal brain at 16, 19 and 21 days gestation (dg). The results revealed that both ERalpha and ERbeta isoforms, and some of their variants, were present in rat fetal brain at the transcript level and at the protein level. PCR results showed that the amount of ERalpha and ERbeta mRNA did not change significantly between 16, 19 and 21 dg; however, changes in protein expression were apparent. Two bands were detected for ERalpha protein by immunoblotting: the expression of the 73 kDa band, relative to the expression of actin, decreased significantly between 16 and 21 dg, while expression of the 67 kDa band did not change. Multiple variants of ERbeta were detected, including wild type ERbeta, ERbeta2 and ERbetaDelta5; the amounts of all decreased significantly, relative to the amount of actin, between 16 and 21 dg. A decrease in protein expression of some of the ER variants without an equivalent decrease in the amount of mRNA suggests that high levels of estrogen might triggered posttranslational modifications of ER, including the ER ubiquitin-dependent proteolysis. Overall, these findings suggest that the fetal brain might be less responsive to estrogens during late pregnancy in the rat, thereby minimizing the known harmful effects of high levels of circulating maternal estrogens.

Estrogens modulate intracellular Ca2+ in human airway smooth muscle

Asthma is a disease characterized by airway hyperresponsiveness and inflammation. One‐third of pregnant women with pre‐existing disease experience a decrease in symptoms during the third trimester, when estrogen levels are highest. Previous studies have shown that estrogens relax vascular smooth muscle, however, little is known about estrogen effects on airway smooth muscle (ASM). We tested the hypothesis that estrogen modulates human ASM contractility. Western blot analysis and immunohistochemical studies showed the presence of both full‐length and truncated isoforms of ERα and β. Furthermore, ER isoforms were localized to caveolae. Caveolin‐1 siRNA downregulated ERα isoform in human ASM. Exposure to the pro‐inflammatory cytokine TNFα substantially increased ERα expression in ASM. In fura‐2 loaded human ASM cells, acute exposure to biologically‐active 17β‐estradiol decreased the intracellular Ca2+ response to contractile agonists such as histamine and bradykinin; this effect was blunted by the ER antagonist ICI 182, 780. Overall, these data suggest that estrogens may have a bronchodilatory effect. Accordingly, increased ER expression with airway inflammation may actually be protective during pregnancy in that bronchodilation resulting from higher estrogen levels (acting via ERs) may alleviate the increased ASM contractility of asthma.Supported by the Mayo Graduate School and NIH grant UL1RR024150‐01(Mayo Clinic CTSA awards to YSP and CMP)

Levels of Estrogen Receptors α and β in Frontal Cortex of Patients with Alzheimers Disease: Relationship to Mini-Mental State Examination Scores

Estrogen exerts beneficial effects on the brain throughout life. Studies demonstrate that estrogen is neuroprotective and that reduced brain estrogen activity may influence the clinical course of Alzheimer's disease (AD). Changes in levels of estrogen receptors have been detected in postmortem brain tissue of AD patients. Very little is known about the relationship between clinical stage and levels of estrogen receptors in postmortem brain. We hypothesized that estrogen receptor levels would be related to severity of cognitive impairment assessed proximate to death. Western blotting was used to quantify ER-alpha and ER-beta in nuclear, cytosolic, and crude membrane fractions of superior frontal cortex from 25 AD patients. Multiple linear regression analyses adjusted for age, sex, and education showed a significant linear relationship between Mini-Mental State Examination score (MMSE) and wild-type nuclear ER-alpha (â = 5.463, p = 0.03), but none between MMSE and wild-type nuclear ER-beta (â = 2.29, p = 0.36). We incidentally observed additional higher and lower molecular mass bands for ER-alpha in study subjects. Additional experiments performed on frontal cortex nuclear fractions prepared from subjects enrolled in a different study confirmed that these same bands are present in female and males with and without AD. Together our data show a relationship between wild-type ER-alpha and level of cognitive impairment in AD, and also suggest the possibility that variant isoforms of ER-alpha may be present in frontal cortex of patients with and without AD.

Dynamic regulation of estrogen receptor-α isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.

Estrogen inhibition of norepinephrine responsiveness is initiated at the plasma membrane of GnRH-producing GT1-7 cells

The modulatory action of estradiol (E2) on the GnRH network can be exerted indirectly on presynaptic neurons or directly on estrogen receptors (ERs) located within GnRH hypothalamic neurons. Using the GnRH-producing GT1-7 cell line, we have investigated whether E2 is able to modify the response of these cells to norepinephrine (NE) stimulation. A 48-h exposure of GT1-7 cells to 10 nM E2 reduced NE-induced cAMP accumulation. However, 15-min exposure was enough to induce this inhibitory action, provided that a hormone-free period of 48 h after steroid treatment was allowed. Furthermore, this effect was mimicked by E2 coupled to (E-BSA), indicating that it may be exerted through a membrane-mediated mechanism. In addition, competition experiments using E-BSA coupled to fluorescein isothiocyanate (FITC) revealed the presence of cell membrane-binding sites for E2. Binding of E-BSA coupled to FITC was blocked by preincubation of cells with either E2, antiestrogen ICI 182 780, or tamoxifen. Moreover, fluorescence staining of non-permeabilized cells with antibodies against receptors alpha and beta confirmed the presence of both receptor subtypes at the cell membrane. To determine the nature of the ER involved in this response, specific agonists for ERalpha 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT) and ERbeta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) were used. Since PPT, but not DPN, reproduced the effect of E2, it is suggested that estrogen-induced modulatory action on NE responsiveness was mediated by the ERalpha isoform. Taken together, these results indicate that E2 modulates the adrenergic sensitivity of GT1-7 cells by a mechanism compatible with the activation of membrane-associated ERs.

Estrogenic and antiestrogenic activities of 2,4-diphenylfuran-based ligands of estrogen receptors α and β

The estrogen receptor (ER) exists in two isoforms ERα and ERβ with a different distribution in the body and different functions which are not clearly identified yet. Thus, it is desirable to have both agonists and antagonists with selectivity for one or the other ER isoform available. In a previous study we showed that 2,5-diphenylfurans can be converted into pure antiestrogens with preference for ERα. When the arrangement of the phenyl rings was altered to a 2,4-substitution, the α-selectivity was lost as demonstrated by comparative assays using recombinant human ERα and ERβ. 3,5-Dialkyl-2,4-bis(4-hydroxyphenylfurans) were shown to act as agonists with preference for ERβ. Replacement of one of the alkyl groups by the [(pentylsulfanyl)propyl]aminohexyl side chain afforded estrogen antagonists without receptor selectivity. These derivatives were characterized as pure antiestrogens in transcription and proliferation assays in ER+ MCF-7 breast cancer cells. The most potent antagonists displayed IC 50 values of ca. 20 nM (fulvestrant 4 nM). The data showed that the 2,4-arrangement of the phenyl rings in the furan structure increases the binding affinity for ERβ in comparison to the isomeric 2,5-diphenylfurans but does not lead to a pure antagonist with selectivity for ERβ.

Association of estrogen receptor-α and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment

IntroductionMedroxyprogesterone acetate (MPA) induces estrogen receptor (ER)-positive and progesterone receptor (PR)-positive ductal invasive mammary carcinomas in BALB/c mice. We sought to reproduce this MPA cancer model in C57BL/6 mice because of their widespread use in genetic engineering. Within this experimental setting, we studied the carcinogenic effects of MPA, the morphologic changes in mammary glands that are induced by MPA and progesterone, and the levels of ER and PR expression in MPA-treated and progesterone-treated mammary glands. Finally, we evaluated whether the differences found between BALB/c and C57BL/6 mouse strains were due to intrinsic differences in epithelial cells.MethodsThe carcinogenic effect of MPA was evaluated in C57BL/6 mice using protocols proven to be carcinogenic in BALB/c mice. In addition, BALB/c and C57BL/6 females were treated with progesterone or MPA for 1 or 2 months, and mammary glands were excised for histologic studies and for immunohistochemical and Western blot evaluation of ER and PR. Hormone levels were determined by radioimmunoassay. Isolated mammary epithelial cells were transplanted into cleared fat pads of 21-day-old female Swiss nu/nu mice or control congenic animals.ResultsMPA failed to induce mammary carcinomas or significant morphologic changes in the mammary glands of C57BL/6 mice. The expression of ER-α and PR isoform A in virgin mice was surprisingly much higher in BALB/c than in C57BL/6 mammary glands, and both receptors were downregulated in progestin-treated BALB/c mice (P < 0.05). PR isoform B levels were low in virgin control mice and increased after progestin treatment in both strains. ER-β expression followed a similar trend. No differences in hormone levels were found between strains. Surprisingly, the transplantation of the epithelial mammary gland cells of both strains into the cleared fat pads of Swiss (nu/nu) mice abolished the mammary gland morphologic differences and the ER and PR differences between strains.ConclusionC57BL/6 mammary glands are resistant to MPA-induced carcinogenesis and to hormone action. MPA and progesterone have different effects on mammary glands. Low ER-α and PR-A levels in untreated mammary glands may be associated with a low-risk breast cancer profile. Although we cannot at this time rule out the participation of other, untested factors, our findings implicate the stroma as playing a crucial role in the strain-specific differential hormone receptor expression and hormone responsiveness.

Changes in the content of estrogen α and progesterone receptors during differentiation of mouse embryonic stem cells to dopamine neurons

Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinson's disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor α (ER-α) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-α expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-α. These results show a differential expression pattern of ER-α and PR isoforms during neuronal differentiation of ESC.

The complete nuclear estrogen receptor family in the rainbow trout: Discovery of the novel ERα2 and both ERβ isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERα (NR3A1) and ERβ (NR3A2). The ERβ subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERα have not been demonstrated in any fish species. In the rainbow trout ( Oncorhynchus mykiss), the only ER described is an isoform of the ERα subtype (i.e. ERα1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERα isoform, ERα2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERα1. Both the rainbow trout ERβ subtypes, ERβ1 [NR3A2a] and ERβ2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERαs (∼ 40% similarity). ERβ1 and ERβ2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERα subtype clade, with the closest relative to rainbow trout ERα2 being rainbow trout ERα1, suggesting a recent, unique duplication event to create these two isoforms. For the ERβ subtype there are two distinct subclades, one represented by the ERβ1 isoform and the other by the ERβ2 isoform. The rainbow trout ERβ1 and ERβ2 are not closely associated with each other, but instead fall into their respective ERβ subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms ( ERα1, ERα2, ERβ1, and ERβ2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.

Downregulation of beta-catenin and transdifferentiation of human osteoblasts to adipocytes under estrogen deficiency

Background and aim. Postmenopausal osteoporosis, caused by estrogen deficiency, is characterized by the structural deterioration of bone accompanied by an increase in bone marrow adipocytes. Transgenic animal models have shown that there is a reciprocal relationship between osteoblastogenesis and adipogenesis in vivo. The present study investigated whether the estrogen and the canonical Wnt signaling pathways are linked together and regulate the phenotype and function, differentiation and proliferation of human osteoblasts using an in vitro estrogen-deficiency model.Methods. Human osteoblasts (hFOB 1.19) and fulvestrant, an estrogen receptor blocker, were used to mimic estrogen deficiency in vitro. Osteogenic and adipogenic differentiation was measured by using specific stains and microscopy, as well as by measuring the expression of bone cell-specific markers with reverse transcription polymerase chain reaction. Expression of estrogen receptor-α (ERα) and β-catenin was detected in Western blots and by immunoprecipitation.Results. The cells expressed the 46-kDa and the 77-kDa ERα isoforms and β-catenin. Fulvestrant reduced expression of ERα and β-catenin. β-Catenin was co-immunoprecipitated with ERα, indicating that these two proteins form a new signaling complex and transcription factor. In addition, it induced intracellular lipid droplet accumulation and downregulation of bone cell markers, indicating adipocyte differentiation.

Regulation of human estrogen receptor α-mediated gene transactivation in Saccharomyces cerevisiae by human coactivator and corepressor proteins

Human estrogen receptor α (ERα)-mediated transcription activation was evaluated in the yeast Saccharomyces cerevisiae using both the native ERα and a G400V variant. A previous study [1] demonstrated that coexpression of human SRC-1, a potent stimulator of ERα function in mammalian cells, potentiated ERα-mediated gene expression in yeast over five-fold in an E 2-dependent manner. In the present study, two additional human coactivator proteins were shown to potentiate ERα-mediated gene expression in yeast. SRC2 potentiated transactivation two- to three-fold while SRC3 potentiated transactivation five- to eight-fold. Both human coactivators potentiated both the native ERα and the G400V variant in an E 2-dependent manner. The effect of a human corepressor protein was also evaluated in yeast. Repressor of estrogen receptor activity (REA) did not affect E 2-induced transactivation by ERα (either isoform). However, in a strain that coexpressed human SRC1, REA reduced E 2-induced transactivation to that observed with ERα alone. Furthermore, repression of SRC1 potentiation was specific for the native ERα since REA had no effect on SRC1 potentiation of the G400V variant. Additionally, REA repression was specific for SRC1 since potentiation of ERα (either isoform) transactivation by SRC2 and SRC3 was unaffected by coexpression of REA. These results support previous observations in mammalian cells that REA does not prevent ERα from binding to DNA but does inhibit potentiation of ERα-mediated transactivation by SRC1. The results in the present study further characterize REA-mediated repression, and demonstrate the utility of this yeast system for dissecting molecular mechanisms involved in regulating gene transactivation by human ERα.

Novel estrogen receptor alpha promoter polymorphism increases ventricular hypertrophic response to hypertension

Given the strong genetic contribution to blood pressure and left ventricular hypertrophy (LVH), and the influence of estrogen on these parameters, we hypothesized that polymorphisms in the estrogen receptor alpha (ERα) promoter may influence LVH. Three novel polymorphisms were identified upstream of the ERα alternatively spliced exon 1E, within sequence which demonstrated significant promoter activity in vitro. Demonstration of ERα E isoform expression in human ventricle by RT-PCR supported a possible functional role for the 1E novel polymorphisms in estrogen signaling in the heart. Indeed, G > A (−721 E) was significantly associated with LVH after controlling for systolic blood pressure and sex in a healthy population ( n = 74), contributing to 23% of interventricular septum (IVS) width variance ( p < 0.001) and 9.4% of left ventricular mass index (LVMI) variance ( p = 0.035). In a separate hypertensive cohort, male carriers of the A allele ( n = 8) had a 17% increase in IVS (95% CI: 6–28%) and a 19% increase in LVMI (3–34%) compared to GG homozygotes ( n = 84). We conclude that a novel polymorphism in the promoter of a cardiac mRNA splice isoform of ERα is associated with LVH.

Estrogen receptor α and its splice variants in the hippocampus in aging and Alzheimer's disease

Clinical and experimental studies show that estrogens can have beneficial effects on hippocampus-dependent cognitive functions that may be mediated by estrogen receptor (ER)α. We investigated whether menopause and Alzheimer's disease (AD) cause changes in this ER subtype. Immunocytochemical staining of ERα, aromatase and Golgi complex (GC) was performed on paraffin embedded hippocampal tissue from women of the pre-, peri- and postmenopausal age. Canonical ERα mRNA amplicons, ERα splice variants (del.2, del.4, del.7, MB1) and aromatase transcripts were measured by Q-PCR in frozen hippocampal samples of AD and matched control cases. Nuclear ERα, aromatase and the GC enhanced during aging in women indicating availability of locally synthesized estrogens that may up-regulate ERα by which neuronal metabolism can be augmented in the hippocampus after the menopause. In AD cases canonical and alternatively spliced ERα mRNA, and aromatase gene expression were down-regulated suggesting a deficit in local estrogen levels and diminished signalling through ERα. The major ERα splice variants in the hippocampus were found to be MB1 and del.4. A novel ERα isoform TADDI was isolated and sequenced from two female patients. It lacks 31 bp at the junction between exons 3 and 4 with an insertion of 13 nucleotides from the middle of the exon 2.

Heart estrogen receptor alpha: Distinct membrane and nuclear distribution patterns and regulation by estrogen

Estrogen receptor α (ERα) is present in the heart consistent with estrogen-induced modulation of cardiac function by genomic and non-genomic mechanisms, and with estrogen-mediated cardioprotective effects. We show that, in heart from adult male rats, ERα is detected mainly as two distinct isoforms: (i) a ∼66 kDa isoform with the expected mass of the classical full-length ERα and (ii) an additional isoform of ∼45 kDa. Differential centrifugation separated the 66 kDa isoform into the cytosolic fraction; while the 45 kDa isoform was enriched in the membrane fraction. High-resolution confocal studies show that ERα is distributed in the nucleus, cytosol, and various membranes including the plasmalemma. Notoriously, ERα labeling was very prominent in T-tubular membranes defined by α-actinin staining and the intercalated disks. In the T-tubules, ERα degree of association to α-actinin depends on the distribution pattern of the receptor along the T-tubules; association is high when ERα pattern is “continuous,” while it is low when the receptor has a discontinuous “granular” distribution. Nuclear ERα has a distinct trabecular distribution and it is excluded from the heterochromatin, consistent with an active transcription factor. Treatment with estrogen (∼4 h) produced an overall decrease in both nuclear and non-nuclear ERα levels and made more evident discrete ERα nuclear puncta uncovering cellular mechanism(s) of short term action of estrogen in the heart. The results indicate that the levels of the cardiac ERα isoforms are downregulated by estrogen and are differentially distributed: the full-length ERα is mainly compartmentalized in the cytosol and nucleus, while the 45 kDa isoform is mainly present in membrane structures. The membrane localization of ERα may support the rapid effects of estrogens on heart function.

Aromatase and estrogen receptors in male reproduction

Aromatase is a terminal enzyme which transforms irreversibly androgens into estrogens and it is present in the endoplasmic reticulum of numerous tissues. We have demonstrated that mature rat germ cells express a functional aromatase with a production of estrogens equivalent to that of Leydig cells. In humans in addition to Leydig cells, we have shown the presence of aromatase in ejaculated spermatozoa and in immature germ cells. In most tissues, high affinity estrogen receptors, ERα and/or ERβ, mediate the role of estrogens. Indeed, in human spermatozoa, we have successfully amplified ERβ mRNA but the protein was not detectable. Using ERα antibody we have detected two proteins in human immature germ cells: one at the expected size 66 kDa and another at 46 kDa likely corresponding to the ERα isoform lacking exon 1. In spermatozoa only the 46 kDa isoform was present, and we suggest that it may be located on the membrane. In addition, in men genetically deficient in aromatase, it is reported that alterations of spermatogenesis occur both in terms of the number and motility of spermatozoa. All together, these observations suggest that endogenous estrogens are important in male reproduction.

Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.

Acteoside and martynoside exhibit estrogenic/antiestrogenic properties

Acteoside and martynoside are plant phenylpropanoid glycosides exhibiting anticancer, cytotoxic and antimetastatic activities. We investigated their potential to activate estrogen receptor isoforms ERα and ERβ in HeLa cells transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERα or ERβ expression vector. Their estrogenic/antiestrogenic effects were also assessed in breast cancer cells (MCF7), endometrial cancer cells (Ishikawa) and osteoblasts (KS483), by measuring IGFBP3 levels, cell viability and number of mineralized nodules, respectively, seeking for a natural selective estrogen receptor modulator (SERM). Acteoside and martynoside antagonized both ERα and ERβ ( p < 0.001), whereas they reversed the effect of E 2 mainly via ERα ( p < 0.001). Martynoside was a potent antiestrogen in MCF-7 cells, increasing, like ICI182780, IGFBP3 levels via the ER-pathway. In osteoblasts, martynoside induced nodule mineralization, which was abolished by ICI182780, implicating an ER-mediated mechanism. Furthermore, its antiproliferative effect on endometrial cells suggests that martynoside may be an important natural SERM. Acteoside was an antiestrogen in breast cancer cells and osteoblasts, without any effect on endometrial cells. Our study suggests that the nature is rich in selective ERα and ERβ ligands, the discovery of which may lead to the development of novel neutraceutical agents.

The phytoestrogen biochaninA weakens acute cardiac allograft rejection without affecting the reproductive system

Background Since the two estrogen receptor isoforms ERα and ERβ have been discovered it is unclear by which receptor immunomodulating or feminizing effects are mediated. In this study, the effects of the two selective ERα- and ERβ-agonists ethinylestradiol and biochaninA, respectively, on acute cardiac allograft rejection, uterus growth, vascular adhesion molecule and MHC-II expression were investigated and verified using in vitro cell culture. Methods Heterotopic Lewis to ovarectomized F344 cardiac transplantations were performed. The study groups received supplemental biochaninA or ethinylestradiol, the control group received no treatment. Grafts and uteri were harvested on the fifth postoperative day and blood was taken for hormone plasma level quantifications. Purified Lewis aortic endothelial cell cultures were pre-treated with biochaninA or ethinylestradiol and stimulated with TNF-α or IFN-γ for quantification of ICAM-1/VCAM-1 and MHC-II expression. Endothelium–lymphocyte adhesion assays were performed using purified F344 lymphocytes. Results Both biochaninA and ethinylestradiol treatment significantly reduced graft mononuclear infiltration of CD8+ and ED1+cells and markedly reduced ISHLT grading compared to untreated controls. Either agent significantly inhibited lymphocyte adhesion, endothelial VCAM-1 upregulation during graft rejection and during TNF-α-stimulation in vitro, whereas no effect was observed for ICAM-1 upregulation. BiochaninA but not ethinylestradiol significantly reduced endothelial MHC-II upregulation in vivo and in vitro. Only ethinylestradiol treatment strongly affected uterus growth in ovarectomized recipients. Conclusions Only the treatment with the phytoestrogen biochaninA reduced endothelial MHC-II expression in vivo and in vitro and weakened allograft rejection without affecting the reproductive system. Supplemental phytoestrogens may therefore provide further benefits in the clinical setting.

Low Levels of Estrogen Receptor β Protein Predict Resistance to Tamoxifen Therapy in Breast Cancer

Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor alpha (ER-alpha) in tumors is used clinically to predict the likelihood of response to hormonal therapies. The clinical value of the second recently identified ER isoform, called ER-beta, is less clear, and there is currently conflicting data concerning its potential role as a prognostic or predictive factor. To assess whether ER-beta expression is associated with clinical outcome, protein levels were measured by immunoblot analysis of a retrospective bank of tumor cell lysates from 305 axillary node-positive patients. A total of 119 received no adjuvant therapy, and 186 were treated with tamoxifen only. The median follow-up time was 65 months. Univariate and multivariate Cox regression modeling was done to assess the prognostic and predictive significance of ER-beta expression. Expression of ER-beta protein did not correlate significantly with any other clinical variables, including ER and progesterone levels (as measured ligand binding assay), tumor size, age, or axillary nodal status. In the untreated population, those patients whose tumors who expressed both receptor isoforms exhibited the most favorable outcome as compared with those patients who had lost ER-alpha expression. However, there was no association between ER-beta levels alone and either disease-free or overall survival in the untreated patient population. In contrast, in both univariate and multivariate analyses, high levels of ER-beta predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. These findings provide evidence that ER-beta may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-beta may influence tumor progression in ways different from those mediated by the ER-alpha isoform.

Biological Role of Pituitary Estrogen Receptors ERα and ERβ on Progesterone Receptor Expression and Action and on Gonadotropin and Prolactin Secretion in the Rat

Estrogen (E) is a key regulator of the synthesis and secretion of pituitary reproductive hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL)]. Until recently, it was thought that all biological actions of E at the pituitary were manifested through a single E receptor (R). The pituitary, like many other reproductive tissues, expresses two isoforms of ER, α and β, both activated by E. The relative contribution of α and β forms in E regulatory actions is largely unknown. To this end, 2-week-old ovariectomized (OVX) rats were injected over 3 days with 25 µg estradiol benzoate (EB), 1.5 mg of propylpyrazole triol (PPT), a selective ERα agonist, 1.5 mg of the selective ERβ agonist diarylpropionitrile (DPN) or a combination of PPT and DPN. Controls were injected with 0.2 ml oil. At 10:00 h on the day after treatment, trunk blood was collected to determine serum concentration of LH, FSH and PRL, and pituitaries were processed for RT-PCR analysis of total (A+B) progesterone receptor (PR) mRNA, immunocytochemistry of PR and incubation. Pituitaries from each of the five groups were incubated in DMEM, with or without 20 nM of the antiprogestin at the receptor ZK299, for 3 h with: 10^–8M 17β-estradiol, 10^–6M PPT, 10^–6M DPN, PPT+DPN or medium alone, respectively, to determine LH, FSH and PRL secretion, and, when challenged with two pulses of 15 min 1 h apart of 10^–8M gonadotropin-releasing hormone (GnRH) (GnRH self-priming). EB, PPT and PPT+DPN treatments increased PR mRNA and the number and intensity of nuclei immunoreactive (IR) for PR in gonadotropes, and reduced the number of gonadectomy cells. Like E, PPT alone or in combination with DPN stimulated PRL secretion, increased basal and GnRH-stimulated LH and FSH secretion and induced GnRH self-priming in the absence of ZK299 in the incubation medium. DPN alone had only a significant E-like effect on gonadectomy cells and IR-PR, but not on GnRH self-priming. In addition, while DPN lacked an agonistic action on peripheral tissue and serum pituitary reproductive hormones concentration, EB, PPT and PPT+DPN induced similar uterine ballooning and vaginal cornification, and increased and decreased, respectively, serum concentrations of PRL and gonadotropins. Overall, these results indicate that most of these E actions on the pituitary are exerted through the ERα isoform. The finding that activation of ERβ with its selective DPN agonist had an estrogenic effect on IR-PR nuclei, but not on GnRH self-priming, a characteristic ERα-mediated effect of E, suggests that the biological action of E at the pituitary may involve both isoforms of ER.