ER Ligands Research Articles

0053: The estrogen receptor alpha C451 palmitoylation site is absolutely required for vascular membrane-initiated action of estrogens in mice

Estrogen Receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signalling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss-of-function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild-type. However, E2 vascular actions such as rapid dilatation, the acceleration of endothelial repair and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ER (ERα-AF2°) provided selective loss-of-function of nuclear ERα actions. In ERα-AF2°, the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2° whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor crosstalk in vivo is modest in the uterus. Thus, this work is the first to genetically segregate membrane versus nuclear actions of a steroid hormone receptor and to demonstrate their in vivo tissue-specific roles.

Estrogen receptor ligands ameliorate fatty liver through a nonclassical estrogen receptor/Liver X receptor pathway in mice

Liver X receptor (LXR) activation stimulates triglyceride (TG) accumulation in the liver. Several lines of evidence indicate that estradiol-17β (E2) reduces TG levels in the liver; however, the molecular mechanism underlying the E2 effect remains unclear. Here, we show that administration of E2 attenuated sterol regulatory element-binding protein (SREBP)-1 expression and TG accumulation induced by LXR activation in mouse liver. In estrogen receptor alpha (ERα) knockout (KO) and liver-specific ERα KO mice, E2 did not affect SREBP-1 expression or TG levels. Molecular analysis revealed that ERα is recruited to the SREBP-1c promoter through direct binding to LXR and inhibits coactivator recruitment to LXR in an E2-dependent manner. Our findings demonstrate the existence of a novel liver-dependent mechanism controlling TG accumulation through the nonclassical ER/LXR pathway. To confirm that a nonclassical ER/LXR pathway regulates ERα-dependent inhibition of LXR activation, we screened ERα ligands that were able to repress LXR activation without enhancing ERα transcriptional activity, and, as a result, we identified the phytoestrogen, phloretin. In mice, phloretin showed no estrogenic activity; however, it did reduce SREBP-1 expression and TG levels in liver of mice fed a high-fat diet to an extent similar to that of E2. We propose that ER ligands reduce TG levels in the liver by inhibiting LXR activation through a nonclassical pathway. Our results also indicate that the effects of ER on TG accumulation can be distinguished from its estrogenic effects by a specific ER ligand.

In Search of Novel Drug Target Sites on Estrogen Receptors Using RNA Aptamers

Estrogen receptor α (ERα) is a well-validated drug target for a majority of breast cancers. But the target sites on this receptor are far from exhaustively defined. Almost all ER antagonists in clinical use function by binding to the ligand-binding pocket to occlude agonist access. Resistance to this type of drugs may develop over time, not caused by the change of ERα itself, but by changes in ER associated proteins. This observation is fueling the development of reagents that downregulate ER activity through novel binding sites. However, it is challenging to find general ER antagonists that act independently from other known ER ligands. In this report, we describe the utility of RNA aptamers in the search for new drug target sites on ERα. We have identified three high affinity aptamers and characterized one of them in detail. This aptamer interacted with ERα in a way not affected by the presence or absence of either the steroidal ligands or the estrogen response DNA elements, and effectively inhibited ER-mediated transcriptional activation in a breast cancer cell line. Serving as a novel drug lead, it may also be used to guide the rational chemical synthesis of small molecule drugs or to perform screens of small molecule libraries for those that are able to displace the aptamer from its binding site.

Identification of Compounds by High-Content Screening That Induce Cytoplasmic to Nuclear Localization of a Fluorescent Estrogen Receptor α Chimera and Exhibit Agonist or Antagonist Activity In Vitro

We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z′ values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein–tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.

Synthesis of 11β-ether-17α-ethinyl-3,17β-estradiols with strong ER antagonist activities

Abstract We have previously found that several families of nonpolar short chain 11 β -ethers and esters of estradiol are selective estrogen receptor modulators (SERMs). Surprisingly, the transformation from potent estrogen to anti-estrogen occurs when the 11 β -side chain is increased slightly in length from four to five non-hydrogen atoms. To generate strong antagonists for preclinical development, we have synthesized other similar ER ligands with 11 β -ethers and with an additional ethinyl group at the 17 α -position in order to slow metabolism of the steroidal moiety. Here we report the synthesis and biological activity of two such compounds (11 β - i -PrO-propyl and 11 β - t -BuO-propyl ethers) with extremely strong antagonist activities.

Cellular Metabolism of Estradiol in Models for Select Molecular Subtypes of Clinical Breast Cancer

The mitogenic ovarian steroid hormone 17β-estradiol (E2) is associated with progression of Estrogen Receptor Positive (ER+) breast cancer, and ER represents a major therapeutic target for endocrine therapy. In addition to the ER dependent signal transduction, cellular oxidative metabolism of the ER ligand E2 generates several metabolites with pleotropic growth modulatory effects on breast cancer cells, providing valuable leads for novel therapeutic approach. The molecular classification of clinical breast cancer has defined cancer subtypes based on differential expression of the genes for hormones and growth factor receptors, thereby facilitating subtype targeted therapeutic interventions. However, de novo or acquired resistance to conventional endocrine and targeted small molecule based treatment limits the therapeutic efficacy and promotes therapy resistant disease progression. These aspects emphasize the need for identification of new efficacious non-toxic lead compounds. The present review summarizes critical experiments conducted to i) develop and optimize human tissue derived cell culture models for select molecular subtypes of clinical breast cancer, ii) determine the status of homeostatic growth control, cellular metabolism of 17β-estradiol (E2) and cancer risk in the developed models, and iii) evaluate the therapeutic efficacy and identify possible mechanisms of action of select herbal extracts/phyto-chemical. Additionally, this review discusses the evidence for the role of E2 metabolism in breast carcinogenesis and therapy. The data generated from the cell culture experiments demonstrate that the models for select molecular sub-types exhibit aberrant hyperproliferation, altered cellular metabolism of E2 and enhanced cancer risk. Select mechanistically distinct herbal extracts and natural phyto-chemical at their respective maximum cytostatic concentrations modulate cellular metabolism of E2 favoring generation of anti-proliferative metabolites and inhibit anchorage independent growth, thus reducing cancer risk. Collectively, these data validate the present mechanism based cell culture approach to identify and prioritize novel efficacious lead compounds for subtype targeted therapy of clinical breast cancer.

Erbium(III) in Aqueous Solution: An Ab Initio Molecular Dynamics Study

Structural and dynamical properties of the erbium(III) ion in water have been obtained by means of ab initio quantum mechanical charge field molecular dynamics (QMCF-MD) simulations for the ground state and an excited state. The quality of the simulations has been monitored by recording UV/vis and Raman spectra of dilute solutions of ErCl3 and Er(NO3)3 in water and by comparison with EXAFS data from literature. Slight deviations between these data can be mainly attributed to relativistic effects, which are not sufficiently considered by the methodological framework. In both simulations, a mixture of coordination numbers eight and nine and a ligand exchange on the picosecond range are observed. The strength of the Er-ligand bond is considerably lower than that of trivalent transition metal ions but higher than that for La(III) and Ce(III) in aqueous solution. The main difference between ground state and excited state is the ligand exchange rate of the first shell. The second hydration shell is stable in both cases but with significantly different properties.

A Titratable Two-Step Transcriptional Amplification Strategy for Targeted Gene Therapy Based on Ligand-Induced Intramolecular Folding of a Mutant Human Estrogen Receptor

The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium. We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ER(G521T)) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression. Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given. The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.

Benzofuran-Based Estrogen Receptor &amp;#945; Modulators as Anti-Cancer Therapeutics: In Silico and Experimental Studies

In the search for new estrogen receptor alpha (ERα) modulators, a trial molecular screening was conducted and 5,6-dihydroxybenzofuran was identified as a possible drug target for ERα. The target molecular modelling molecule 1 and a series of 5,6-dihydroxybenzofurans have been synthesized and evaluated for their anti-proliferation activities against MCF-7 and MDA-MB-231 cells. From the SAR studies, potential functional groups have been identified, the two hydroxyl groups at C-5 and C-6 and the phenyl ring at C-2, which showed considerable cytotoxicity in MCF-7 breast cancer cells. In addition, the apoptotic abilities of the compounds have been measured in both MCF-7 ER(+) and MDA-MB-231 ER(-) breast cancer cells. The results demonstrated that our compounds inhibit MCF-7 breast cancer cells via ER(+). These preliminary results provide valuable information towards the identification of important functional groups present on 5,6-dihydroxybenzofuran, which could be a promising scaffold for designing novel ER ligands.

Ligand-binding dynamics rewire cellular signaling via estrogen receptor-α

Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands displayed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways, and reveals a novel role of the DBD in allosteric control of ERα-mediated signaling.

ChemInform Abstract: Synthesis and Evaluation of 4‐(Substituted styryl/alkenyl)‐3,5‐bis(4‐hydroxyphenyl)‐isoxazoles as Ligands for the Estrogen Receptor.

Abstract4‐(Substituted styryl/alkenyl)‐3,5‐bis(4‐hydroxyphenyl)‐isoxazoles of type (III) and (VII) are prepared and evaluated as ligands for the estrogen receptor‐alpha (ERα) The 4‐(4‐hydroxy‐styryl) derivative (VII) displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα‐LBD tolerates the presence of the added vinyl group at the 4‐position of the isoxazole ring.

Molecular Mechanisms of Selective Estrogen Receptor Modulator Activity in Human Breast Cancer Cells: Identification of Novel Nuclear Cofactors of Antiestrogen–ERα Complexes by Interaction Proteomics

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.

Selective estrogen receptor modulators (SERMs) for uterine leiomyomas

Uterine fibroids are benign tumours that arise from individual smooth muscle cells of the uterus. Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that act as estrogens in some tissues while blocking estrogen activity in others. There have been many clinical studies of various SERMs for uterine fibroids. However, their effectiveness is controversial. To evaluate the effectiveness and safety of selective estrogen receptor modulators in women with uterine fibroids. We searched the Cochrane Menstrual Disorders and Subfertility Group Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, PubMed, EMBASE, the Register of Chinese trials developed by the Chinese Cochrane Centre, and the Chinese Med Database (Chinese Biomedical Disc), VIP, and China National Knowledge Infrastructure. We handsearched a number of journals and searched reference lists, and searched databases of ongoing trials and the Internet. The searches were conducted in March and April 2012. We included randomised controlled studies of selective estrogen receptor modulators versus other forms of medical therapy, placebo or no treatment in women of reproductive age (18 to 45 years old) with confirmed uterine fibroids. Two review authors independently extracted data and assessed trial quality. As the studies identified were not sufficiently similar, we did not do a meta-analysis but summarised the data in a narrative format. Three studies involving 215 participants were included, the trial size varied from 25 to 100 women. The SERM in all cases was raloxifene. In one study women in both arms received gonadotrophin releasing hormone (GnRH) analogue. Comparison interventions included no treatment and placebo. Two of the three included studies found a significant benefit from raloxifene, but the third study found no benefit at three or six-month follow-up. The overall quality of the evidence was low or very low. All three studies mentioned adverse reactions but data were limited. There is no consistent evidence from the limited number of studies that SERMs reduce the size of fibroids or improve clinical outcomes. Further studies are required to establish evidence of benefit of SERMs in treating women with uterine fibroids. This updated review did not find any new study for inclusion.

New polyoxometalate-based mononuclear lanthanide complexes with slow relaxation of magnetization

A series of new polyoxometalate(POM)-based mononuclear lanthanide(III)–bi pyridine-N-oxide complexes, [Ln(bpyno)4][PMo12O40]·2H2O (Ln=Dy (1), Tb (2), Er (3), Ho (4); bpyno=4,4′-dimethyl-2,2′-bipyridyl-N-N′-dioxide), have been synthesized by the reactions between LnPMo12O40 salts (Ln=Dy (1), Tb (2), Er (3), Ho (4)) and bpyno ligand. All compounds were characterized by elemental analyses, TG analyses, IR, powder X-ray diffraction and single-crystal X-ray diffraction. In 1–4, each mononuclear Ln center is coordinated by eight O atoms derived from four bpyno ligands, exhibiting an approximate square antiprismatic coordination geometry. Furthermore, the [Ln(bpyno)4]3+ units are well separated by the α-Keggin-type polyoxoanions. Magnetic measurements indicate that compound 1 exhibits frequency dependent ac magnetic susceptibilities indicative of slow relaxation of the magnetization.

Inhibition of Neu-induced mammary carcinogenesis in transgenic mice expressing ERΔ3, a dominant negative estrogen receptor α variant.

The estrogen receptor α (ERα) splicing variant with an in-frame deletion of exon 3 (ERΔ3) is frequently expressed in the normal breast, but its influence on tumorigenesis has not been explored. In vitro, ERΔ3 has dominant negative activity, suggesting it may suppress estrogen stimulation in the breast. ERΔ3 may inhibit classical signaling on estrogen response element (ERE)-regulated genes as well as activate non-classical pathways at Sp1 and AP-1 sites. Transgenic mice were developed that express mouse ERΔ3 in all tissues examined, including the mammary gland. To investigate if ERΔ3 expression affects tumorigenesis, ERΔ3 mice were crossbred with MMTV-Neu mice. Mammary tumor onset was significantly delayed in ERΔ3/Neu versus MMTV-Neu females and metastatic incidence and burden was significantly reduced. Consequently, ERΔ3 expression suppressed tumor development and metastasis in this aggressive model of HER2/Neu-positive breast cancer. To determine if ER ligands with anticancer activity may augment ERΔ3 protection, the bitransgenic mice were treated with tamoxifen and soy isoflavones starting at age 2 months. Soy protein with isoflavones (181 mg/1,800 kcal) did not affect tumor development in MMTV-Neu or ERΔ3/Neu mice; however, metastatic progression was not inhibited in soy-treated ERΔ3/Neu mice, as it was in untreated ERΔ3/Neu mice. In contrast, tamoxifen (20 mg/1,800 kcal) significantly enhanced tumor prevention in ERΔ3/Neu versus MMTV-Neu mice (98% vs. 81% tumor free). The results in ERΔ3/Neu mice demonstrate that ERΔ3 influences estrogen-dependent mammary carcinogenesis and, thus, may be protective in women expressing ERΔ3 in the breast. However, exposure to different estrogens may augment or block its beneficial effects.

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.

Primary study of a novel reporter gene/probe system human ER ligand binding domain/radiolabeled estradiol

Objective To evaluate the feasibility of a new reporter gene/probe system,namely human ER ligand binding domain (hERL)/radionuclide labeled estradiol,and to provide basis for its monitoring gene and cell therapy from in vitro cellular uptake study and in vivo imaging experiment.Methods Recombinant plasmid pDC316- hERL -internal ribosome entry site-VEGF165 ( pDC316-hERL-IRES-VEGF165,or EIV) and recombinant Ad-EIV were constructed,which carried a reporter gene (hERL) and a therapeutic gene (VEGF165 ) through IRES.Adenovirus was used as a vector.MSCs were obtained from tibias and femurs of rat,and cultured normally.Ad-EIV and EIV coated with lipofectamine 2000 (Lipo-EIV) were transfected into MSCs.RT-PCR and Western blot were performed to detect the expression of hERL and VEGF165 from mRNA and protein level.The cellular uptake values of 125I labeled estradiol ( 125I-E2 ) were measured in Ad-EIV,Lipo-EIV and non-transfected MSCs at different incubation time ( 1,3,6,9,12 and 24 h).Ad-EIV transfected MSCs were injected into the left upper limb of rats,and non-transfected MSCs into the right upper limb as self-control.Micro PET/CT images were obtained after 1 d of transfection.Ttest and Pearson linear correlation analysis were used to analyze the data.Results After transfected with Ad-EIV,mRNA and protein expressions of hERL and VEGF165 in MSCs were increased with adenovirus multiplicity of infection ( MOI),and positive correlation could be seen ( r2 =0.953 and 0.966,both P ＜0.05).The expressions of mRNA and protein in Ad-EIV group were higher than those of Lipo-EIV transfected MSCs.Time-dependent accumulation of 125I-E2 was observed in the Ad-EIV group and Lipo-EIV group,and the highest uptake rates occurred at 24 h,with peak values of ( 10.94 ± 0.30) ％ and (8.93 ± O.18)％,respectively.Higher cellular uptakes could be seen at all time points in the Ad-EIV group than those of the Lipo-EIV group (t =4.132-16.168,all P ＜0.05).Moreover,the cellular uptakes with two vectors were significantly higher than those of non-transfected control cells at all the tested time points ( t =15.489- 26.560 for Ad-EIV and t =10.523 - 24.204 for Lipo-EIV,respectively,all P ＜ 0.05 ).Compared with the right upper limb,significantly higher radioactivity could be seen in the left side where Ad5-EIV transfected MSCs were injected.Conclusions Reporter gene hERL could be transfected into the cells via adenovirus and liposome as vectors.Its successful expression in cells could be detected and imaged by radiolabled estradiol.The reporter gene/probe system hERL/radiolabled estradiol might be used as a novel reporter gene/probe system for monitoring gene and cell therapy. Key words: Genes, reporter; Estrogen receptor ligand binding domain; Radionuclide imaging; Estradiol; Isotope labeling; Rats

Selective estrogen receptor‐β activation stimulates skeletal muscle growth and regeneration

There is increasing evidence suggesting that estrogens augment skeletal muscle regeneration processes after injury. To study the contribution of estrogen receptors α and β (ERα and ERβ) during muscle regeneration, skeletal muscles of ovariectomized (OVX) rats, as well as ERα- and ERβ-knockout (αErko and βErko) mice, were injured with a myotoxin (notexin). OVX rats were simultaneously treated with the ER-selective ligands genistein, ERα agonist 16α-LE2 (alpha), ERβ agonist 8β-VE2 (beta), or 17β-estradiol (E(2)). OVX rats showed significantly elevated serum creatine kinase (CK) activity after muscle injury compared to intact sham-treated animals. Treatment with ER ligands significantly reduced CK activity. TNF-α, IL-10, and MCP-1 expression served to characterize immune responses. Treatment with all ER ligands, but particularly E(2) and beta, reduced TNF-α, but elevated MCP-1 and IL-10 expression. PCNA and MyoD expression served to define satellite cell activation and proliferation and were found to be up-regulated by beta and E(2). To further study muscle regeneration responses, expression of the embryonic myosin heavy chain (MHC) was analyzed. Beta and E(2) but not alpha increased embryonic MHC expression compared to OVX. The absence of ERβ in βErko mice negatively affected CK activity levels and expression of satellite cell and muscle regeneration markers (MHC embryonic, MyoD, Pax7) compared with αErko and wild-type mice. In a classic Hershberger assay using male rats, beta stimulated muscle growth, accompanied by a strong induction of IGF-1 expression. Our data provide evidence that ERβ signaling is involved in the regulation of skeletal muscle growth and regeneration by stimulating anabolic pathways, activating satellite cells and modulating immune responses.

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

IntroductionThe multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells.MethodsMolecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells.ResultsMIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments.ConclusionsOur findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist.

Efficacy of pharmacological estrogen receptor antagonists in blocking activation of zebrafish estrogen receptors

A variety of pharmacological agonists, antagonists and selective estrogen receptor modulators (SERM) have been used to better understand the role of specific receptors in various physiological processes. Despite similar structure and function, less is known about the effect of agonists and antagonists on teleost estrogen receptors and the results of these studies have indicated wide variation among species. The goal of this study was to determine the ability of two human SERMs to modulate activation of three zebrafish estrogen receptor isoforms. Full length cDNA of zebrafish estrogen receptor 1 (esr1), estrogen receptor 2a (esr2a) and estrogen receptor 2b (esr2b) were cloned into expression vectors and transfected into cells that do not endogenously express any estrogen receptor along with an estrogen responsive luciferase vector. Cells transfected with any of the zebrafish estrogen receptors individually and then exposed to 17β-estradiol (E 2) or 17α-ethinylestradiol (EE 2) exhibited a dose dependent increase in luciferase activity. None of the pharmacological antagonists, ICI 182, 780, methyl-piperidino-pyrazole (MPP) or pyrazolo [1,5-a] pyrimidine (PHTPP), were able to independently transactivate luciferase expression with any of the zebrafish estrogen receptors. Of the three ER antagonists, only ICI 182, 780 was able to block EE 2 induced luciferase activity, although a 10 to 100-fold excess of ICI 182, 780 was necessary with all receptors. Neither MPP nor PHTPP were able to block EE 2 induced luciferase activity with any isoform of zebrafish estrogen receptor. These results indicate that the difference between human ER and zebrafish ER ligand binding is not conserved enough for the SERMs MPP or PHTPP to elicit similar effects in zebrafish as those manifested in humans.