The physiological and biochemical basis of potency thresholds modeled using human estrogen receptor alpha: implications for identifying endocrine disruptors.

The endocrine system functions by interactions between ligands and receptors. Ligands exhibit potency for binding to and interacting with receptors. Potency is the product of affinity and efficacy. Potency and physiological concentration determine the ability of a ligand to produce physiological effects. The kinetic behavior of ligand-receptor interactions conforms to the laws of mass action. The laws of mass action define the relationship between the affinity of a ligand and the fraction of cognate receptors that it occupies at any physiological concentration. We previously identified the minimum ligand potency required to produce clinically observable estrogenic agonist effects via the human estrogen receptor-alpha (ERα). By examining data on botanical estrogens and dietary supplements, we demonstrated that ERα ligands with potency lower than one one-thousandth that of the primary endogenous hormone 17ß-estradiol (E2) do not produce clinically observable estrogenic effects. This allowed us to propose a Human-Relevant Potency Threshold (HRPT) for ERα ligands of 1 × 10-4 relative to E2. Here, we test the hypothesis that the HRPT for ERα arises from the receptor occupancy by the normal metabolic milieu of endogenous ERα ligands. The metabolic milieu comprises precursors to hormones, metabolites of hormones, and other normal products of metabolism. We have calculated fractional receptor occupancies for ERα ligands with potencies below and above the previously established HRPT when normal circulating levels of some endogenous ERα ligands and E2 were also present. Fractional receptor occupancy calculations showed that individual ERα ligands with potencies more than tenfold higher than the HRPT can compete for occupancy at ERα against individual components of the endogenous metabolic milieu and against mixtures of those components at concentrations found naturally in human blood. Ligands with potencies less than tenfold higher than the HRPT were unable to compete successfully for ERα. These results show that the HRPT for ERα agonism (10-4 relative to E2) proposed previously is quite conservative and should be considered strong evidence against the potential for disruption of the estrogenic pathway. For chemicals with potency 10-3 of E2, the potential for estrogenic endocrine disruption must be considered equivocal and subject to the presence of corroborative evidence. Most importantly, this work demonstrates that the endogenous metabolic milieu is responsible for the observed ERα agonist HRPT, that this HRPT applies also to ERα antagonists, and it provides a compelling mechanistic explanation for the HRPT that is grounded in basic principles of molecular kinetics using well characterized properties and concentrations of endogenous components of normal metabolism.

Low dose of propyl-pyrazole-triol, an agonist of estrogen receptor alpha, administration stimulates the Coolidge effect in fadrozole-treated male rats.

Estrogen receptor (ER) α is involved in male sexual function. Here, we aim to investigate how ERα activation influences sexual satiety and the Coolidge effect (i.e., when a rat, that has reached sexual satiety, experiences an increased arousal after exposure to a novel sexual partner) in estrogen-deprived male rats. Male rats (8 per group) were treated daily for 29 days with either saline (Control group) or fadrozole dissolved in saline (1 mg/kg/day) 1 h before mating. On Days 13 and 29, rats treated with fadrozole received either no additional treatment (fadrozole group) or a single injection of propyl-pyrazole-triol (ERα-agonist group, dissolved in sesame oil, 1 mg/kg). Rats mated until reaching sexual satiety on Days 13 and 29. In these sessions, the Control group displayed higher frequency of intromission and ejaculation than the other groups. The ERα-agonist group mounted more frequently but reached sexual satiety sooner than the Control group. On Day 29, when exposed to a new sexual partner, the fadrozole-treated rats were less likely to display intromission than the other groups, or ejaculation than the Control group, or mounting than the ERα-agonist group. The Control group showed more ejaculatory behavior and shorter ejaculation latency than the other groups. Body weights, testosterone levels, estradiol levels, and ERα-immunoreactive cell counts in brain regions for sexual behavior were comparable between groups after 29 days of treatments. Our data suggest that estrogen helps regulate sexual satiety and the Coolidge effect in male rats.

Estrogen signaling in the dorsal raphe regulates binge-like drinking in mice.

Estrogens promote binge alcohol drinking and contribute to sex differences in alcohol use disorder. However, the mechanisms are largely unknown. This study aims to test if estrogens act on 5-hydroxytryptamine neurons in the dorsal raphe nucleus (5-HTDRN) to promote binge drinking. We found that female mice drank more alcohol than male mice in chronic drinking in the dark (DID) tests. This sex difference was associated with distinct alterations in mRNA expression of estrogen receptor α (ERα) and 5-HT-related genes in the DRN, suggesting a potential role of estrogen/ERs/5-HT signaling. In supporting this view, 5-HTDRN neurons from naïve male mice had lower baseline firing activity but higher sensitivity to alcohol-induced excitation compared to 5-HTDRN neurons from naïve female mice. Notably, this higher sensitivity was blunted by 17ß-estradiol treatment in males, indicating an estrogen-dependent mechanism. We further showed that both ERα and ERß are expressed in 5-HTDRN neurons, whereas ERα agonist depolarizes and ERß agonist hyperpolarizes 5-HTDRN neurons. Notably, both treatments blocked the stimulatory effects of alcohol on 5-HTDRN neurons in males, even though they have antagonistic effects on the activity dynamics. These results suggest that ERs' inhibitory effects on ethanol-induced burst firing of 5-HTDRN neurons may contribute to higher levels of binge drinking in females. Consistently, chemogenetic activation of ERα- or ERß-expressing neurons in the DRN reduced binge alcohol drinking. These results support a model in which estrogens act on ERα/ß to prevent alcohol-induced activation of 5-HTDRN neurons, which in return leads to higher binge alcohol drinking.

Loureirin C, from Chinese Dragon's Blood (Dracaena cochinchinensis S.C. Chen), is a novel selective estrogen receptor α modulator with anti-Alzheimer's disease effects.

As the incidence of Alzheimer's disease (AD) continues to rise in recent years, there are few therapeutic drugs for AD treatment with limited efficacy. AD occurs twice as often in women as that in men, partially due to the low estrogen level in women after menopause. Phytoestrogens (PEs), similar to endogenous estrogens in chemical structure with neuroprotection and fewer side effects, have good development and application prospects in AD-treatment. Loureirin C is an active ingredient isolated from Chinese Dragon's Blood (CDB) with a similar structure to 17ß-E2. In our study, we found that loureirin C targeted to ERα and had partial-agonistic activity using molecular docking prediction and dual-luciferase reporter assay. However, it is still unclear whether loureirin C has estrogenic effects in body, and whether exerts anti-AD effect through ERα. In this paper, the ERα selective inhibitor MPP or ERα specific small interfering RNA (siERα) mediated gene silencing technology were used. Besides,E-SCREEN method were used to evaluate the estrogen effects of loureirin C in vivo and in vitro. MTT assay, Western blot, real-time PCR technology and behaver tests was used to investigate the neuroprotective effect, cognitive function and the underlying mechanism. We found that loureirin C possessed estrogenic activity, had neuroprotective effects in AD cells and improved cognitive impairment in AD mice via ERα. Loureirin C may be a potential candidate for AD.

No detectable changes in anxiety-related and locomotor behaviors in adult ovariectomized female rats exposed to estradiol, the ERß agonist DPN or the ERα agonist PPT.

The sex steroid hormone 17ß-estradiol (estradiol) and its Estrogen Receptors (ERs) have been linked to modulation of anxiety-related and locomotor behaviors in female rodents. Research suggests that estradiol mitigates anxiety-related behaviors through activating Estrogen Receptor (ER)ß and increases locomotor behaviors through ERα. The influence of ERs on these behaviors cannot always be detected. Here we discuss two experiments in which we tested the hypothesis that anxiety-related behaviors would decrease after ERß activation and locomotor behaviors would increase after ERα activation, and also assessed the persistence of these behavioral effects by varying the timing of behavioral testing. Two cohorts of adult female ovariectomized rats were exposed to estradiol, the ERß agonist DPN, the ERα agonist PPT, or oil for four consecutive days. Body mass was assessed throughout as a positive control. In both cohorts, open field behaviors were assessed on the first day of exposure. In one cohort (Experiment 1), open field, light/dark box, and elevated plus maze behaviors were assessed on the final day of injections. In the second cohort (Experiment 2), these behaviors were assessed 24 h after the final exposure. As expected, significant differences in body mass were detected in response to estradiol and PPT exposure, validating the estradiol and ER manipulation. No significant differences were observed in anxiety-related or locomotor behaviors across treatment groups, indicating that the efficacy of these agonists as therapeutic agents may be limited. We review these results in the context of previous literature, emphasizing relevant variables that may obscure ER-related actions on behavior.

Post-stroke periodic estrogen receptor-beta agonist improves cognition in aged female rats.

Women have a higher risk of having an ischemic stroke and increased cognitive decline after stroke as compared to men. The female sex hormone 17ß-estradiol (E2) is a potent neuro- and cognitive-protective agent. Periodic E2 or estrogen receptor subtype-beta (ER-ß) agonist pre-treatments every 48 h before an ischemic episode ameliorated ischemic brain damage in young ovariectomized or reproductively senescent (RS) aged female rats. The current study aims to investigate the efficacy of post-stroke ER-ß agonist treatments in reducing ischemic brain damage and cognitive deficits in RS female rats. Retired breeder (9-10 months) Sprague-Dawley female rats were considered RS after remaining in constant diestrus phase for more than a month. The RS rats were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min and treated with either ER-ß agonist (beta 2, 3-bis(4-hydroxyphenyl) propionitrile; DPN; 1 mg/kg; s.c.) or DMSO vehicle at 4.5 h after induction of tMCAO. Subsequently, rats were treated with either ER-ß agonist or DMSO vehicle every 48 h for ten injections. Forty-eight hours after the last treatment, animals were tested for contextual fear conditioning to measure post-stroke cognitive outcome. Neurobehavioral testing, infarct volume quantification, and hippocampal neuronal survival were employed to determine severity of stroke. Periodic post-stroke ER-ß agonist treatment reduced infarct volume, improved recovery of cognitive capacity by increasing freezing in contextual fear conditioning, and decreased hippocampal neuronal death in RS female rats. These data suggest that periodic post-stroke ER-ß agonist treatment to reduce stroke severity and improve post-stroke cognitive outcome in menopausal women has potential for future clinical investigation.

Effects of estrogen receptor ß or G protein-coupled receptor 30 activation on anxiety-like behaviors in relation to GABAergic transmission in stress-ovariectomized rats.

For mental disorders such as anxiety and depression, stress and stressful events are considered as precipitating causes that may be enhanced by estrogen variability. This condition is proven by the higher vulnerability of women than men. Despite the complexity of underlying mechanisms, the gamma-aminobutyric acid (GABA) system piques interest as its receptor contains multiple psychoactive modulatory sites including neurosteroids. Moreover, according to clinical and experimental reports, GABA-associated genes can be altered by stress and hormonal status. Therefore, this study investigated the effects of estrogen receptor ß (ERß) or G protein-coupled receptor 30 (GPR30) activation on anxiety/depression-like behaviors and the alterations in the GABA-associated gene of ovariectomized rats under chronic mild stress (CMS). Mild stressors were focused on because they represent a realistic simulation of daily life stress. In this study, ovariectomized rats were treated with vehicle, estradiol (E2), diarylpropionitrile (DPN; ERß agonist) or G1 (GPR30 agonist) and exposed to 4-week CMS. The results showed that E2, DPN, and G1 treatments reduced anxiety-like behaviors without affecting depression-like behaviors. Concurrently, the GABA level and most GABA- and neurosteroid-associated mRNAs were altered by E2. Similar mRNA profiles were observed in DPN- and E2-administrations but not in G1 treatment. Collectively, these data suggest that estrogen exerts an anxiolytic-like action through either ERß and/or GPR30 activation, and the modulatory effects of estrogen on GABAergic system are likely to be modulated through ERß. The findings of this study therefore further provide insights into the roles of estrogen and daily mild stressors in GABA-related activity and behavioral responses, especially anxiety.

Estrogen receptor α activation modulates the gut microbiome and type 2 diabetes risk factors.

Estradiol and exercise can decrease risk factors associated with type 2 diabetes (T2D) including total body weight gain and abdominal fat gain. Estradiol functions through estrogen receptor (ER) α and ERß. Some studies suggest that activation of ERα may provide protection against T2D. Female Wistar rats were ovariectomized and fed a high-fat diet for 10 weeks and divided into the following 5 experimental groups: (1) no treatment (control), (2) exercise, (3) estradiol, (4) propylpyrazoletriyl (a selective ERα agonist), and (5) diarylpropionitrile (a selective ERß agonist). ERα activation decreased the abundance of Firmicutes, and ERα and ERß activation increased the abundance of Bacteroidetes. ERα activation decreased food consumption, and ERα and ERß activation increased voluntary activity. Exercise was the only treatment to decrease the blood glucose and serum insulin levels. ERα activation, but not ERß, increased hepatic protein expression of ACC and FAS and decreased hepatic protein expression of LPL. ERα activation also decreased hepatic mRNA expression of PPARα and PPARγ. This study elucidates the functions of estradiol by assessing specific activation of ERα and ERß. As obesity increases the abundance of Firmicutes and decreases the abundance of Bacteroidetes, our study shows that ERα activation can restore the gut microbiome to non-obese abundances. This study further provides novel insights into ERα's role in hepatic fat metabolism via regulation of ACC, FAS, LPL, PPARα, and PPARγ.

2',3',4'-Trihydroxychalcone changes estrogen receptor α regulation of genes and breast cancer cell proliferation by a reprogramming mechanism.

BACKGROUND: Menopausal hormone therapy (MHT) is recommended for only five years to treat vasomotor symptoms and vulvovaginal atrophy because of safety concerns with long-term treatment. We investigated the ability of 2',3',4'-trihydroxychalcone (2',3',4'-THC) to modulate estrogen receptor (ER)-mediated responses in order to find drug candidates that could potentially prevent the adverse effects of long-term MHT treatment. METHODS: Transfection assays, real time-polymerase chain reaction, and microarrays were used to evaluate the effects of 2',3',4'-THC on gene regulation. Radioligand binding studies were used to determine if 2',3',4'-THC binds to ERα. Cell proliferation was examined in MCF-7 breast cancer cells by using growth curves and flow cytometry. Western blots were used to determine if 2',3',4'-THC alters the E2 activation of the MAPK pathway and degradation of ERα. Chromatin immunoprecipitation was used to measure ERα binding to genes. RESULTS: The 2',3',4'-THC/E2 combination produced a synergistic activation with ERα on reporter and endogenous genes in human U2OS osteosarcoma cells. Microarrays identified 824 genes that we termed reprogrammed genes because they were not regulated in U2OS-ERα cells unless they were treated with 2',3',4'-THC and E2 at the same time. 2',3',4'-THC blocked the proliferation of MCF-7 cells by preventing the E2-induced activation of MAPK and c-MYC transcription. The antiproliferative mechanism of 2',3',4'-THC differs from selective estrogen receptor modulators (SERMs) because 2',3',4'-THC did not bind to the E2 binding site in ERα like SERMs. CONCLUSION: Our study suggests that 2',3',4'-THC may represent a new class of ERα modulators that do not act as a direct agonists or antagonists. We consider 2',3',4'-THC to be a reprogramming compound, since it alters the activity of ERα on gene regulation and cell proliferation without competing with E2 for binding to ERα. The addition of a reprogramming drug to estrogens in MHT may offer a new strategy to overcome the adverse proliferative effects of estrogen in MHT by reprogramming ERα as opposed to an antagonist mechanism that involves blocking the binding of estrogen to ERα.

Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Elucidation of Agonist and Antagonist Dynamic Binding Patterns in ER-α by Integration of Molecular Docking, Molecular Dynamics Simulations and Quantum Mechanical Calculations.

Estrogen receptor alpha (ERα) is a ligand-dependent transcriptional factor in the nuclear receptor superfamily. Many structures of ERα bound with agonists and antagonists have been determined. However, the dynamic binding patterns of agonists and antagonists in the binding site of ERα remains unclear. Therefore, we performed molecular docking, molecular dynamics (MD) simulations, and quantum mechanical calculations to elucidate agonist and antagonist dynamic binding patterns in ERα. 17ß-estradiol (E2) and 4-hydroxytamoxifen (OHT) were docked in the ligand binding pockets of the agonist and antagonist bound ERα. The best complex conformations from molecular docking were subjected to 100 nanosecond MD simulations. Hierarchical clustering was conducted to group the structures in the trajectory from MD simulations. The representative structure from each cluster was selected to calculate the binding interaction energy value for elucidation of the dynamic binding patterns of agonists and antagonists in the binding site of ERα. The binding interaction energy analysis revealed that OHT binds ERα more tightly in the antagonist conformer, while E2 prefers the agonist conformer. The results may help identify ERα antagonists as drug candidates and facilitate risk assessment of chemicals through ER-mediated responses.

Anthocyanins Activate Membrane Estrogen Receptors With Nanomolar Potencies to Elicit a Nongenomic Vascular Response Via NO Production.

Background The vascular pharmacodynamics of anthocyanins is only partially understood. To examine whether the anthocyanin-induced vasorelaxation is related to membrane estrogen receptor activity, the role of ERα or GPER antagonism was ascertained on anthocyanins or 17-ß estradiol-(E2) induced vasodilatations and NO production. Methods and Results The rat arterial mesenteric bed was perfused with either anthocyanins or corresponding 3-O-glycosides, or E2, to examine rapid concentration-dependent vasorelaxations. The luminally accessible fraction of NO in mesenteric perfusates before and after anthocyanins or E2 administration was quantified. Likewise, NO-DAF signal detected NO production in primary endothelial cells cultures incubated with anthocyanins or E2 in the absence and presence of ERα (ICI 182,780) or GPER (G-36) selective antagonists. Anthocyanins or corresponding glycosides elicited, within minutes, vasodilation with nanomolar potencies; half maximal anthocyanin response reached 50% to 60% efficacy, in contrast to acetylcholine. The vasorelaxation is of rapid onset and exclusively endothelium-dependent; NOS inhibition annulled the vasorelaxation. The delphinidin vascular response was not modified by 100 nmol/L atropine but significantly attenuated by joint application of ICI plus G-36 (52±4.6 versus 8.5±1.5%), revealing the role of membrane estrogen receptors. Moreover, the anthocyanin or E2-induced NO production was antagonized up to 70% by these antagonists. NO-DAF signal elicited by anthocyanins was annulled by NOS inhibition or by ICI plus G-36 addition. Conclusions The biomedical effect of anthocyanins or 3-O-glycosylates derivatives contained in naturally purple-colored foods or berries is due to increased NO production, and not to the phytochemical's antioxidant potential, highlighting the nutraceutical role of natural products in cardiovascular diseases.

Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

The physiological role of estrogen receptor functional domains.

Estrogen receptor (ER) is a member of the nuclear receptor superfamily whose members share conserved domain structures, including a DNA-binding domain (DBD) and ligand-binding domain (LBD). Estrogenic chemicals work as ligands for activation or repression of ER-mediated transcriptional activity derived from two transactivation domains: AF-1 and AF-2. AF-2 is localized in the LBD, and helix 12 of the LBD is essential for controlling AF-2 functionality. The positioning of helix 12 defines the ER alpha (ERα) ligand properties as agonists or antagonists. In contrast, it is still less well defined as to the ligand-dependent regulation of N-terminal AF-1 activity. It has been thought that the action of selective estrogen receptor modulators (SERMs) is mediated by the regulation of a tissue specific AF-1 activity rather than AF-2 activity. However, it is still unclear how SERMs regulate AF-1 activity in a tissue-selective manner. This review presents some recent observations toward information of ERα mediated SERM actions related to the ERα domain functionality, focusing on the following topics. (1) The F-domain, which is connected to helix 12, controls 4-hydroxytamoxifen (4OHT) mediated AF-1 activation associated with the receptor dimerization activity. (2) The zinc-finger property of the DBD for genomic sequence recognition. (3) The novel estrogen responsive genomic DNA element, which contains multiple long-spaced direct-repeats without a palindromic ERE sequence, is differentially recognized by 4OHT and E2 ligand bound ERα transactivation complexes.

PARP7 and Mono-ADP-Ribosylation Negatively Regulate Estrogen Receptor α Signaling in Human Breast Cancer Cells.

ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17ß-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17ß-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17ß-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor's ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) ß (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERß)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERß)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 µM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

Estrogenic Effects of Extracts and Isolated Compounds from Belowground and Aerial Parts of Spartina anglica.

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.

ERα-agonist and ERß-antagonist bifunctional next-generation bisphenols with no halogens: BPAP, BPB, and BPZ.

As demonstrated for bisphenol AF (BPAF), the electrostatic halogen bond based on the London dispersion force of halogen atoms was found to be a major driving force of their bifunctional ERα-agonist and ERß-antagonist activities. Because similar electronic effects are anticipated for hydrocarbon groups (alkyl or aryl groups), we hypothesized that bisphenol compounds consisting of such groups also work bifunctionally. In the present study, we examined bisphenol AP (BPAP), B (BPB), and Z (BPZ). After recognizing their considerably strong receptor binding affinities, we evaluated the abilities of BPAP, BPB, and BPZ to activate ERα and ERß in a luciferase reporter gene assay. These bisphenols were fully active for ERα but completely inactive for ERß. When we examined their inhibitory activities for 17ß-estradiol in ERß by two different qualitative and quantitative analytical methods, we found that those bisphenols worked as definite antagonists. Consequently, they were established as bifunctional ERα-agonists and ERß-antagonists. The present structure-activity analyses revealed that the dispersion force works not only on the halogens but also on the hydrocarbon groups, and that it is a major driving force of bifunctional ERα-agonist and ERß-antagonist activities.

Estrogen Receptors Alpha and Beta Mediate Synaptic Transmission in the PFC and Hippocampus of Mice.

Distinct from ovarian estradiol, the steroid hormone 17ß-estradiol (E2) is produced in the brain and is involved in numerous functions, particularly acting as a neurosteroid. However, the physiological role of E2 and the mechanism of its effects are not well known. In hippocampal slices, 17ß-estradiol has been found to cause a modest increase in fast glutamatergic transmission; because some of these effects are rapid and acute, they might be mediated by membrane-associated receptors via nongenomic action. Moreover, activation of membrane estrogen receptors can rapidly modulate neuron function in a sex-specific manner. To further investigate the neurological role of E2, we examined the effect of E2, as an estrogen receptor (ER) agonist, on synaptic transmission in slices of the prefrontal cortex (PFC) and hippocampus in both male and female mice. Whole-cell recordings of spontaneous excitatory postsynaptic currents (sEPSC) in the PFC showed that E2 acts as a neuromodulator in glutamatergic transmission in the PFC in both sexes, but often in a cell-specific manner. The sEPSC amplitude and/or frequency responded to E2 in three ways, namely by significantly increasing, decreasing or having no response. Additional experiments using an agonist selective for ERß, diarylpropionitrile (DPN) showed that in males the sEPSC and spontaneous inhibitory postsynaptic currents sIPSC responses were similar to their E2 responses, but in females the estrogen receptor ß (ERß) agonist DPN did not influence excitatory transmission in the PFC. In contrast, in the hippocampus of both sexes E2 potentiated the gluatmatergic synaptic transmission in a subset of hippocampal cells. These data indicate that activation of E2 targeting probably a estrogen subtypes or different downstream signaling affect synaptic transmission in the brain PFC and hippocampus between males versus females mice.

Bisphenol-C is the strongest bifunctional ERα-agonist and ERß-antagonist due to magnified halogen bonding.

We reported that bisphenol AF (BPAF) works as an agonist for estrogen receptor (ER) ERα but as an antagonist for ERß. Similar results were observed for bisphenol E analogs (BPE-X) such as BPE-F, BPE-Cl, and BPE-Br, each consisting of a series of a tri-halogenated methyl group CX3 in the central alkyl moiety. It was demonstrated that the electrostatic halogen bond based on the dispersion force of halogen atoms is a major driving force in the activities of bifunctional ERα-agonist and ERß-antagonist. Since the chlorine atoms present in bisphenol C (BPC) exist in a π-π conjugated system due to the presence of an adjacent C = C double bond, we intended to prove that BPC is also a bifunctional ERα-agonist and ERß-antagonist exhibiting greatly enhanced agonist/antagonist activities. BPC was evaluated for its ability to activate ERα and ERß in the luciferase reporter gene assay using HeLa cells. With high receptor-binding ability to both ERs, BPC was found to be fully active for ERα but inactive for ERß. BPC's definite antagonist activity in ERß was revealed by its inhibitory activity against 17ß-estradiol. Thus, BPC is a bifunctional ERα-agonist and ERß-antagonist. These agonist/antagonist activities were discovered to be extremely high among series of halogen-containing bisphenol compounds. This comparative structure-activity study revealed that the ascending order of ERα-agonist and ERß-antagonist activities was BPE-F ≪ BPE-Cl ≲ BPAF < BPE-Br ≪ BPC. The highly intensified receptor interaction of BPC is attributable to the presence of an n-π-π-n conjugation system mediated through the >C = CCl2 double bond.