Activation Of Estrogen Receptor Alpha Research Articles

LINC00173 silence and estrone supply suppress ER+ breast cancer by estrogen receptor α degradation and LITAF activation.

Persistent activation of estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in driving the progression of estrogen receptor positive (ER+) breast cancer (BC). In the current study, LINC00173, a long non-coding RNA, was found to bind both ERα and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFα) factor (LITAF), then cooperatively to inhibit ERα protein degradation by impeding the nuclear export of ERα. Concurrently, LITAF was found to attenuate TNFα transcription after binding to LINC00173, and this attenuating transcriptional effect was quite significant under lipopolysaccharide stimulation. Distinct functional disparities between estrogen subtypes emerge, with estradiol synergistically promoting ER+ BC cell growth with LINC00173, while estrone (E1) facilitated LITAF-transcriptional activation. In terms of therapeutic significance, silencing LINC00173 alongside moderate addition of E1 heightened TNFα and induced apoptosis, effectively inhibiting ER+ BC progression.

Abstract PO3-29-03: An Emerging Class of ER mutations Enhances ER Dimerization and Promotes ER Activity

Abstract Physiological activation of estrogen receptor alpha (ERα) requires the binding of estradiol (E2) to the ligand binding domain (LBD) of the receptor. This interaction triggers a repositioning of helix 12 (H12), facilitating the recruitment of coactivator proteins to the unoccupied coactivator binding groove. However, in the context of breast cancer, point mutations of ERα at H12 lead to its repositioning and self-activation in the absence of E2. Through MSK clinical sequencing breast cancer cohort (MSK-IMPACT), we assayed 8302 samples of ER+ breast cancer patients and identified 649 mutations within the LBD of ERα. 471 of these mutations (73%) were located at H11-H12 loops (D538, Y537, L536), while the remaining 27% were located near the dimer interface of ERα (V422, G442, F461, S463P, L469). Through in vitro biochemical and cell assays, we identified that these mutations promote dimer formation and hormone-independent transcriptional activity. Tumors expressing these mutations exhibited significantly accelerated growth compared to their wild-type controls. Unlike Y537S, cells harboring mutations at the dimer interface maintain their sensitivity to Selective Estrogen Receptor Degraders (SERDs), including Fulvestrant, recently FDA-approved Elacestrant, and Camizestrant, as well as Selective Estrogen Receptor Modulators (SERMs) including Tamoxifen and Raloxifene. By utilizing machine learning and computational structural analyses, we uncovered that these mutants, unlike Y537 repositions H12, act distinctly through conformational change across the ERα dimer interface. Further cell assays proved that the dimerization leads to increased stability of the receptor and facilitates its nuclear localization, which is required for ERα activation. Collectively, our finding unveiled a new class of ER mutations that enforce receptor dimerization and activation of the ER signaling pathway. The discovery opens up a new therapeutic interventional possibility, suggesting that targeting dimerization could emerge as a new strategy to combat these malignancies. Citation Format: Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John Katzenellenbogen, Kathryn Carlson, Benita S. Katzenellenbogen, Yang Shen, Sarat Chandarlapaty. An Emerging Class of ER mutations Enhances ER Dimerization and Promotes ER Activity [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-29-03.

Silver ciprofloxacin (CIPAG): a multitargeted metallodrug in the development of breast cancer therapy.

The anti-proliferative activity of the known metalloantibiotic {[Ag(CIPH)2]NO3∙0.75MeOH∙1.2H2O} (CIPAG) (CIPH = ciprofloxacin) against the human breast adenocarcinoma cancer cells MCF-7 (hormone dependent (HD)) and MDA-MB-231 (hormone independent (HI)) is evaluated. The in vitro toxicity and genotoxicity of the metalloantibiotic were estimated toward fetal lung fibroblast (MRC-5) cells. The molecular mechanism of the CIPAG activity against MCF-7 cells was clarified by the (i) cell morphology, (ii) cell cycle arrest, (iii) mitochondrial membrane permeabilization, and (iv) by the assessment of the possible differential effect of CIPAG on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) transcriptional activation, applying luciferase reporter gene assay. Moreover, the ex vivo mechanism of CIPAG was clarified by its binding affinity toward calf thymus (CT-DNA).

Investigating the effect of hesperetin on estrogen receptor alpha (ERα) expression, phosphorylation and activity in MCF-7 cells

PurposeThe most common malignancy among women worldwide is breast cancer. The estrogen receptor plays a vital role in this cancer. One of the most well-known mechanisms that affects the activity of this receptor is its phosphorylation by protein kinase pathways. Hesperetin, a flavonoid abundant in citrus species such as lemons, grapefruits, and oranges, is the aglycone form of hesperidin. It has undergone thorough evaluation for its potential anti-cancer properties, particularly in the context of breast cancer. Studies have shown that hesperetin has an effect on intracellular kinase pathways. The aim of this study was to investigate the effect of hesperetin on the expression, phosphorylation and activity of estrogen receptor alpha (ERα) in MCF-7 breast cancer cell line. Study design and methodsMCF-7 cells were cultured in RPMI-1640 phenol red-free medium supplemented with charcoal-stripped FBS and treated with hesperetin. The MTT method was used to evaluate cell survival. The levels of the ERα protein and its phosphorylated form (Ser118) were determined via western blotting. A luciferase reporter vector was used to evaluate ERE activity. ResultsThe results of this study indicated that hesperetin reduced the survival of MCF-7 cells in a dose-dependent manner. The expression and phosphorylation (at Ser118) of the ERα significantly increased and decreased, respectively, in the groups treated with hesperetin. Hesperetin increased the activity of the ERα in the absence of E2, although these differences were not statistically significant. Conversely, in the presence of E2, hesperetin caused a significant decrease in receptor activity. ConclusionBased on the results of this study, it can be concluded that hesperetin has a significant effect on ERα expression, phosphorylation and activity.

The Estrogen Receptor Alpha Regulates the Sex-dependent Expression and Pronociceptive Role of Bestrophin-1 in Neuropathic Rats

Bestrophin-1, a calcium-activated chloride channel (CaCC), is involved in neuropathic pain; however, it is unclear whether it has a dimorphic role in female and male neuropathic rats. This study investigated if 17β-estradiol and estrogen receptor alpha (ERα) activation regulate bestrophin-1 activity and expression in neuropathic rats. Neuropathic pain was induced by L5-spinal nerve transection (SNT). Intrathecal administration of CaCCinh-A01 (.1–1 µg), a CaCC blocker, reversed tactile allodynia induced by SNT in female but not male rats. In contrast, T16Ainh-A01, a selective anoctamin-1 blocker, had an equal antiallodynic effect in both sexes. SNT increased bestrophin-1 protein expression in injured L5 dorsal root ganglia (DRG) in female rats but decreased bestrophin-1 protein in L5 DRG in male rats. Ovariectomy prevented the antiallodynic effect of CaCCinh-A01, but 17β-estradiol replacement restored it. The effect of CaCCinh-A01 was prevented by intrathecal administration of MPP, a selective ERα antagonist, in rats with and without prior hormonal manipulation. In female rats with neuropathy, ovariectomy prevented the increase in bestrophin-1 and ERα protein expression, while 17β-estradiol replacement allowed for an increase in both proteins in L5 DRG. Furthermore, ERα antagonism (with MPP) prevented the increase in bestrophin-1 and ERα protein expression. Finally, ERα activation with PPT, an ERα selective activator, induced the antiallodynic effect of CaCCinh-A01 in neuropathic male rats and prevented the reduction in bestrophin-1 protein expression in L5 DRG. In summary, data suggest ERα activation is necessary for bestrophin-1’s pronociceptive action to maintain neuropathic pain in female rats. PerspectiveThe mechanisms involved in neuropathic pain differ between male and female animals. Our data suggest that ERα is necessary for expression and function of bestrophin-1 in neuropathic female but not male rats. Data support the idea that a therapeutic approach to relieving neuropathic pain must be based on patient's gender.

Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

Liver receptor homolog-1 (NR5A2) orchestrates hepatic inflammation and TNF-induced cell death

The nuclear receptor liver receptor homolog-1 (LRH-1) has been shown to promote apoptosis resistance in various tissues and disease contexts; however, its role in liver cell death remains unexplored. Hepatocyte-specific deletion of LRH-1 causes mild steatosis and inflammation but unexpectedly shields female mice from tumor necrosis factor (TNF)-induced hepatocyte apoptosis and associated hepatitis. LRH-1-deficient hepatocytes show markedly attenuated estrogen receptor alpha and elevated nuclear factor κB (NF-κB) activity, while LRH-1 overexpression inhibits NF-κB activity. This inhibition relies on direct physical interaction of LRH-1's ligand-binding domain and the Rel homology domain of NF-κB subunit RelA. Mechanistically, increased transcription of anti-apoptotic NF-κB target genes and the proteasomal degradation of pro-apoptotic BCL-2 interacting mediator of cell death prevent mitochondrial apoptosis and ultimately protect mice from TNF-induced liver damage. Collectively, our study emphasizes LRH-1 as a critical, sex-dependent regulator of cell death and inflammation in the healthy and diseased liver.

Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

Resveratrol and 3,3'-Diindolylmethane Differentially Regulate Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha Activity through Multiple Transcriptomic Targets in MCF-7 Human Breast Cancer Cells.

Inhibitory crosstalk between estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AHR) regulates 17β-estradiol (E2)-dependent breast cancer cell signaling. ERα and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Dietary ligands resveratrol (RES) and 3,3'diindolylmethane (DIM) also activate ERα while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and anti-inflammatory properties. Studies with genome-wide targets and AHR- and ERα-regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ERα as well as AHR coregulation in MCF-7 human breast cancer cells treated with DIM, RES, E2, or TCDD alone or E2+TCDD for 1 and 6 h, respectively. ERα bound sites after being DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after being DIM and E2+TCDD enriched for the ERE motif but not after TCDD. More than 90% of the differentially expressed genes closest to an AHR binding site after DIM or E2+TCDD also had an ERα site, and 60% of the coregulated genes between DIM and E2+TCDD were common. Collectively, our data show that RES and DIM differentially regulate multiple transcriptomic targets via ERα and ERα/AHR coactivity, respectively, which need to be considered to properly interpret their cellular and biological responses. These novel data also suggest that, when both receptors are activated, ERα dominates with preferential recruitment of AHR to ERα target genes.

Effects of long‐term oral treatment with a novel estrogen receptor beta agonist on memory, vasomotor, and anxiety outcomes in an EFAD mouse model of Alzheimer’s disease

AbstractBackgroundThe prevalence and severity of Alzheimer’s disease (AD) are increased by female sex, apolipoprotein E (APOE) genotype, and age, such that postmenopausal female carriers of the Apoe4 allele are at greater risk of developing AD than age‐matched males. Although estrogen therapy shows promise in preventing and reducing menopause‐ and AD‐related symptoms, activation of estrogen receptor alpha (ERa) is associated with increased risk for health concerns, such as breast cancer. Therefore, treatments more selective for estrogen receptor beta (ERb) may provide estrogen‐related health benefits without the risks of activating ERa. Long‐term or acute treatment of wild‐type mice with the novel ERb agonist, EGX358, improves memory and reduces drug‐induced vasomotor alterations. However, efficacy of this compound in an AD model is unknown.MethodFemale APOE‐TR+/+/5xFAD+/− (EFAD) mice expressed five familial AD and were homozygous for human APOE3 (E3FAD) or heterozygous for APOE3 and APOE4 (E3/4FAD). Mice were ovariectomized at 5 months of age and then treated orally with vehicle (1% DMSO) or EGX358 (10 mg/kg/day) via hydrogel for 8 weeks. Mice were weighed weekly and tested for spatial and object memory in the object placement (OP) and object recognition (OR) tasks, respectively. Anxiety‐like behaviors were also tested in the open field and elevated plus maze, and vasomotor symptoms were assessed via tail temperature following injection of the tachykinin receptor 3 agonist, senktide.ResultEGX358 significantly enhanced OR memory in both E3FAD and E3/4FAD mice. However, treatment did not affect OP memory or anxiety in the open field or elevated plus maze. Interestingly, there were significant genotype differences in open field, vasomotor response, and body weight such that E3/4FADs spent more time in the center of open field, had a greater increase in tail temperature following senktide injection, and gained more weight compared to E3FADs.ConclusionOur findings indicate a potential for ERβ‐specific estrogen treatment to improve memory for the identity of objects. Unexpected genotype differences indicate the need to further study how APOE genotype affects anxiety and metabolic outcomes in AD.

Casein kinase 1 controls the shuttling of epidermal growth factor receptor and estrogen receptor in endometrial carcinoma induced by breast cancer hormonal therapy: Relevance of GPER1/Src

Casein kinase 1 plays a crucial role in carcinogenesis. 4-Hydroxytamoxifen (4-OHT), which is widely used to treat breast cancer, often leads to the development of endometrial carcinoma with poor prognosis, particularly among women who receiving long-term treatment. This study was performed to elucidate whether specific inhibition of casein kinase 1 (CK1) controls 4-OHT-mediated Ishikawa cell carcinogenesis. 4-OHT significantly stimulated the activity of estrogen receptor alpha (ERα) and nuclear translocation and expression of epidermal growth factor receptor (EGFR) from the plasma membrane to perinuclear or nuclear regions, as well as the activities of G-protein-coupled estrogen receptor 1 (GPER1) and Src in Ishikawa cells. However, inhibition of EGFR by Gefitinib blocked all these events, and inhibition of GPER1 or Src produced a partial block. GPER1 and Src controlled Ishikawa cell carcinogenesis in different manners: GPER1 accelerated EGFR mobility without affecting ERα activity, while Src activated ERα and EGFR without any change in GPER1 expression. EGFR and GPER1 performed reciprocal regulation in endometrial cell carcinogenesis via direct interaction in 4-OHT-treated Ishikawa cells, implying a possible key role of GPER1 in these events. Inhibition of CK1 by CKI-7 and IC261, however, impeded all changes beginning with EGFR translocation and activity in 4-OHT-treated Ishikawa cells. These findings indicate that inhibition of CK1 could control 4-OHT-mediated activation and translocation of ER/EGFR and GPER1/Src expression, inhibiting 4-OHT-triggered endometrial carcinogenesis. Therefore, targeting of CK1 by CKI-7 and IC261 could be a prospective adjuvant therapy for breast cancer patients taking tamoxifen.

Glyphosate mimics 17β-estradiol effects promoting estrogen receptor alpha activity in breast cancer cells

Glyphosate, the active ingredient in several broad-spectrum herbicide formulations, has been validated and widely used throughout the world. Recent reports have questioned its safety, showing that glyphosate may act as an endocrine disruptor by promoting estrogenic activity. However, the molecular mechanism involved in this phenomenon remains unclear. Therefore, here we aimed to elucidate the mechanism by which glyphosate induces estrogenic activity using estrogen-sensitive breast cancer cell line models. Our results show that glyphosate mimics the cell effects of 17β-estradiol (E2), promoting estrogen receptor α (ERα) phosphorylation, its degradation, and transcriptional activity at high concentrations. The molecular mechanism seems involved in the ERα ligand-binding domain (LBD). Molecular simulations suggest a plausible interaction between glyphosate and the LBD through a coordinated complex involving divalent cations such as Zn (II). In addition, glyphosate exposure alters the level of Cyclin-dependent kinase 7 that contribute to ERα phosphorylation. Finally, glyphosate increases cell proliferation rate and levels of cell cycle regulators, accompanied by an increase in anchorage-independent growth capacity. These findings suggest that glyphosate at high concentrations, induces estrogen-like effects through an ERα ligand binding site-dependent mechanism, leading to cellular responses resulting from a complex interplay of genomic and non-genomic events.

Dual activation of estrogen receptor alpha and glucocorticoid receptor upregulate CRTh2-mediated type 2 inflammation; mechanism driving asthma severity in women?

Type 2-high asthma is characterized by elevated levels of circulating Th2 cells and eosinophils, cells that express chemoattractant-homologous receptor expressed on Th2 cells (CRTh2). Severe asthma is more common in women than men; however, the underlying mechanism(s) remain elusive. Here we examined whether the relationship between severe asthma and type 2 inflammation differs by sex and if estrogen influences Th2 cell response to glucocorticoid (GC). Type 2 inflammation and the proportion of blood Th2 cells (CD4+ CRTh2+ ) were assessed in whole blood from subjects with asthma (n=66). The effects of GC and estrogen receptor alpha (ERα) agonist on in vitro differentiated Th2 cells were examined. Expression of CRTh2, type 2 cytokines and degree of apoptosis (Annexin V+ , 7-AAD) were determined by flow cytometry, qRT-PCR, western blot and ELISA. In severe asthma, the proportion of circulating Th2 cells and hospitalizations were higher in women than men. Women with severe asthma also had more Th2 cells and serum IL-13 than women with mild/moderate asthma. Th2 cells, eosinophils and CRTh2 mRNA correlated with clinical characteristics associated with asthma control in women but not men. In vitro, GC and ERα agonist treated Th2 cells exhibited less apoptosis, more CRTh2 as well as IL-5 and IL-13 following CRTh2 activation than Th2 cells treated with GC alone. Women with severe asthma had higher levels of circulating Th2 cells than men, which may be due to estrogen modifying the effects of GC, enhancing Th2 cell survival and type 2 cytokine production.

Ameliorating Effect of the Edible Mushroom Hericium erinaceus on Depressive-Like Behavior in Ovariectomized Rats.

Estrogen deficiency during menopause causes a variety of neurological symptoms, including depression. The edible Lion's Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (HE), is a medicinal mushroom that has the potential for a neuroprotective effect and ameliorating neurological diseases, such as depression, anxiety, and neurodegenerative diseases. HE contains phytoestrogens, including daidzein and genistein. However, the ameliorating effect of HE on menopausal symptoms is not well understood. Here we investigated the impact of methanol extract of the HE fruiting body on depressive-like behavior in postmenopausal model rats. The activation of estrogen receptor alpha (ERα) causes body weight loss and uterine weight gain. Body weight gain and uterine weight loss by estrogen deficiency in ovariectomized (OVX) rats were reversed with 17β-estradiol (E2) but not with HE. Thus, the phytoestrogens in HE may hardly activate ERα. Estrogen receptor beta (ERβ) is expressed in the brain, and activation of ERβ ameliorates menopausal depressive symptoms. Notably, depressive-like behavior in OVX rats evaluated in forced swim test was reduced by administration of not only E2 but also HE for 92 d. Long-term activation of ERα increases the risk of breast and uterine cancers. HE, therefore, may be effective in treating menopausal depression without the risk of carcinogenesis caused by ERα activation.

Hypothalamic Estrogen Signaling and Adipose Tissue Metabolism in Energy Homeostasis.

Obesity has become a global epidemic, and it is a major risk factor for other metabolic disorders such as type 2 diabetes and cardiometabolic disease. Accumulating evidence indicates that there is sex-specific metabolic protection and disease susceptibility. For instance, in both clinical and experimental studies, males are more likely to develop obesity, insulin resistance, and diabetes. In line with this, males tend to have more visceral white adipose tissue (WAT) and less brown adipose tissue (BAT) thermogenic activity, both leading to an increased incidence of metabolic disorders. This female-specific fat distribution is partially mediated by sex hormone estrogens. Specifically, hypothalamic estrogen signaling plays a vital role in regulating WAT distribution, WAT beiging, and BAT thermogenesis. These regulatory effects on adipose tissue metabolism are primarily mediated by the activation of estrogen receptor alpha (ERα) in neurons, which interacts with hormones and adipokines such as leptin, ghrelin, and insulin. This review discusses the contribution of adipose tissue dysfunction to obesity and the role of hypothalamic estrogen signaling in preventing metabolic diseases with a particular focus on the VMH, the central regulator of energy expenditure and glucose homeostasis.

Molecular profiling of ginsenoside metabolites to identify estrogen receptor alpha activity.

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17β-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

UXT, a novel DNMT3b-binding protein, promotes breast cancer progression via negatively modulating lncRNA MEG3/p53 axis

Overexpressed ubiquitously expressed transcript (UXT) in breast tumors and derived cell lines modulated the transcriptional activity of estrogen receptor alpha. However, how UXT exerts its biological functions in the tumorigenicity of breast cancer remains largely unknown. Expressions of UXT and maternally expressed gene 3 (MEG3) were examined by qRT-PCR and Western blot. The capacity of cell proliferation, apoptosis, migration, and invasion was assessed using CCK-8, flow cytometry, and transwell assays. Methylation-specific PCR (MS-PCR) was employed to evaluate the methylation of the MEG3 imprinting control region. Co-immunoprecipitation was performed to verify the UXT/DNMT3b interaction. RNA immunoprecipitation (RIP) was subjected to assess the regulation of MEG3 on p53 activity. A xenograft tumor model was further conducted to certify the molecular mechanism. UXT was upregulated, while MEG3 was downregulated in breast cancer tissues and cell lines. UXT knockdown or MEG3 overexpression inhibited cell proliferation, promoted apoptosis, and weakened cell migration and invasion. Hypermethylation of the MEG3 imprinting control region was modulated by highly expressed DNMT3b. UXT inhibited MEG3 expression via recruiting DNMT3b to its imprinting control region. MEG3 positively regulated p53 activity. UXT negatively regulated the MEG3/p53 axis in a DNMT3b-dependent manner to promote tumor growth. UXT, a novel DNMT3b-binding protein, aggravates the progression of breast cancer through MEG3/p53 axis.

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner.

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

Hibiscus sabdariffa anthocyanins are potential modulators of estrogen receptor alpha activity with favourable toxicology: a computational analysis using molecular docking, ADME/Tox prediction, 2D/3D QSAR and molecular dynamics simulation

The estrogen hormone receptor (ER) mediated gene expression mainly regulate the development and physiology of the primary and secondary reproductive system alongside bone-forming, metabolism and behaviour. Over-expressed ER is associated with several pathological conditions and play a crucial role in breast cancer occurrence, progression and metastasis. Hibiscus sabdariffa L. or roselle is a rich source of naturally occurring polyphenolic compounds that reportedly have robust estrogenic activity. However, the estrogen-like ingredient of the plant remains ambiguous. This study has screened a library of already recorded and less-explored compounds of Hibiscus sabdariffa for their estrogen receptor binding affinity and safety using a suite of computational methods that include protein-ligand docking, ADME and Toxicity prediction, and 2D/3D QSAR. The study revealed that the estrogen-receptor binding potential of Pelargonidin, Delphinidin, Cyanidin, and Hibiscetin are more efficient than popular breast cancer drugs, Tamoxifen and Raloxifene. Besides, the compounds exhibited favourable toxicological parameters with potent bioactivity towards binding ER-α subunit. Thus, these compounds can serve as prototypes for designing novel Selective Estrogen Receptor Modulator molecules with a few structural modifications. This is the first report exploring the phytochemical basis of estrogenic activity of Hibiscus sabdariffa L. Communicated by Ramaswamy H. Sarma