ERβ Agonist Research Articles

Prediction of compounds with antiosteoporosis activity in Chrysophyllum cainito L. leaves through in silico approach.

Estrogen deficiency causes various health problems in postmenopausal women, including osteoporosis. Phytoestrogen emerged as a potential alternative of estrogen with minimum side effects. The aims of this study were to analyze the metabolite profiling results of various extract of Chyrsophyllum cainito L. leaves, which contain phytoestrogen, through in silico study against 3OLS protein, an X-ray protein of ERβ, so it can predict the types of the phytoestrogen contents which have antiosteoporosis property. In silico analysis was carried out for the compounds from the metabolite profiling data of C.cainito leaves from our previous study. The structure compounds from metabolite profiling results of various extract of C.cainito leaves were prepared with Avogadro 1.0.1 software, molecular docking was done using PyRx 0.8 software, and Biovia Discovery Studio Visualizer 2016 software was used to visualize the structure of compounds against 3OLS protein. The physicochemical characteristics of the compounds were analyzed using the SwissADME web tool. From in silico studies, it was known that there were total 11 compounds in C.cainito leaves that predicted as phytoestrogens which have ERβ agonist properties against 3OLS protein. The ERβ agonist was a compound that has parameters similar to 17β-estradiol in its interaction with 3OLS protein, which has a pharmacophore distance of 10.862Å, and binding to amino acids His 475 and Glu 305 or Arg 346at receptor-ligand docking simulation. C.cainito leaves contain 11 compounds that are predicted to be phytoestrogens with ERβ agonist properties, which is responsible for antiosteoporosis activity.

Estrogen Receptor β Contributes to Both Hypertension and Hypothalamic Plasticity in a Mouse Model of Peri-Menopause.

Hypertension susceptibility in women increases at the transition to menopause, termed perimenopause, a state characterized by erratic estrogen fluctuation and extended hormone cycles. Elucidating the role of estrogen signaling in the emergence of hypertension during perimenopause has been hindered by animal models that are confounded by abrupt estrogen cessation or effects of aging. In the present study, accelerated ovarian failure (AOF) in estrogen receptor β (ERβ) reporter mice was induced by 4-vinylcyclohexene diepoxide in young mice to model early-stage ovarian failure (peri-AOF) characteristic of peri-menopause. It was found that administering ERβ agonists suppressed elevated blood pressure in a model of neurogenic hypertension induced by angiotensin II (AngII) in peri-AOF, but not in age-matched male mice. It was also found that ERβ agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERβ neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERβ in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERβ signaling in the PVN plays an important role in blood pressure regulation in female mice and contributes to hypertension susceptibility in females at an early stage of ovarian failure comparable to human perimenopause.

Estrogen Receptor β Is Involved in Acquired Resistance to EGFR-tyrosine Kinase Inhibitors in Lung Cancer.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has posed serious clinical problems in the treatment of lung adenocarcinoma (LADC) patients harboring relevant EGFR mutations. In this study, we explored the role of estrogen receptor β (ERβ) in the development of acquired resistance to EGFR-TKIs in human LADC. First, the role of ERβ in erlotinib resistance of LADC cell lines (PC9/ER) was examined. Then, the immunolocalization of ERβ in 28 LADC patient samples treated with EGFR-TKIs was investigated. Cytoplasmic ERβ was upregulated in erlotinib resistant cell lines. EGFR-TKIs sensitivity increased with ERβ inhibition in PC9/ER cells. ERK1/2 and AKT activities were both markedly increased by specific ERβ agonists even under erlotinib treatment of PC9/ER cells. Cytoplasmic ERβ immunoreactivity was significantly associated with clinical response to EGFR-TKIs. Cytoplasmic ERβ in LADC cells was involved in the development of resistance to EGFR-TKIs.

Estrogen activates endothelial exocytosis

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Estrogen receptor β and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate

Knockout of ERβ in the mouse leads to nuclear expression of epidermal growth factor receptor (EGFR) in the prostate. To examine whether ERβ plays a similar role in the human prostate, we used four cohorts of men: 1) a Swedish cohort of normal prostates and PCa (prostate cancer) of different Gleason grades; 2) men with benign prostatic hyperplasia (BPH) treated with the 5α-reductase inhibitor, finasteride, and finasteride together with the ERβ agonists, soy isoflavones; 3) men with PCa above Gleason grade 4 (GG4), treated with ADT (androgen deprivation therapy) and abiraterone (AA), the blocker of androgen synthesis for different durations; and 4) men with GG4 PCa on ADT or ADT with the AR (androgen receptor) blocker, enzalutamide, for 4 mo to 6 mo. In men with BPH, finasteride treatment induced EGFR nuclear expression, but, when finasteride was combined with isoflavones, EGFR remained on the cell membrane. In GG4 patients, blocking of AR for 4 mo to 6 mo resulted in loss of ERβ and PTEN expression and increase in patients with nuclear EGFR from 10 to 40%. In the men with GG4 PCa, blocking of adrenal synthesis of testosterone for 2 mo to 7 mo had the beneficial effect of increasing ERβ expression, but, on treatment longer than 8 mo, ERβ was lost and EGFR moved to the nucleus. Since nuclear EGFR is a predictor of poor outcome in PCa, addition of ERβ agonists together with abiraterone should be considered as a treatment that might sustain expression of ERβ and offer some benefit to patients.

Selective ER-β agonists alleviate neuronal deficits in insulin-resistant estrogen-deficient rats

Objective The present study aimed to determine the effect of estrogen receptor (ER) agonists on depression and memory impairment in insulin-resistant ovariectomized (OVX) rats. Methods Rats underwent bilateral ovariectomy, and low-dose streptozotocin (STZ) and a high-fat diet (58% fat, 25% protein, and 17% carbohydrates as a percentage of kilocalories) were administered to induce an estrogen-deficient insulin-resistant state. After 1 week of STZ administration, rats were treated with 17β-estradiol (17βE2) and selective ER-α (propylpyrazoletriol) and ER-β (diarylpropionitrile) agonists (10 μg/kg subcutaneously). Memory was evaluated using the Morris water maze and depression using the forced swim test. Results Treatment with selective ER-β agonist and 17βE2 but not with selective ER-α agonist significantly modulated the neurobehavioral deficits in insulin-resistant OVX rats. These neurobehavioral parameters were further correlated with brain-derived neurotrophic factor (BDNF) levels and acetylcholinesterase (AChE) activity. Selective ER-β agonist and 17βE2 significantly modulated BDNF levels and AChE activity in insulin-resistant OVX rats. Significant increases in estradiol and uterine weight were observed in 17βE2-treated rats, but selective ER agonists did not show any effect. Conclusion ER-β agonist can be an effective strategy for the mitigation of memory loss and depression in an estrogen-deficient insulin-resistant state without all of the deleterious feminizing effects that occur with the use of 17βE2.

Long-term oral administration of a novel estrogen receptor beta agonist enhances memory and alleviates drug-induced vasodilation in young ovariectomized mice

Development of estrogen therapies targeting the β (ERβ) but not α (ERα) estrogen receptor is critically needed for the treatment of negative menopausal symptoms, as ERα activation increases health risks like cancer. Here, we determined the effects of long-term oral treatment with EGX358, a novel highly selective ERβ agonist, on memory, vasodilation, and affect in young ovariectomized mice. Mice were orally gavaged daily for 9 weeks with vehicle, 17β-estradiol (E2), the ERβ agonist diarylpropionitrile (DPN), or EGX358 at doses that enhance memory when delivered acutely. Tail skin temperature was recorded as a proxy for vasodilation following injection of vehicle or senktide, a tachykinin receptor 3 agonist used to model hot flashes. Anxiety-like behavior was assessed in the open field (OF) and elevated plus maze (EPM), and depression-like behavior was measured in the tail suspension (TST) and forced swim tests (FST). Finally, memory was assessed in object recognition (OR) and object placement (OP) tasks. E2, DPN, and EGX358 reduced senktide-mediated increases in tail skin temperature compared to vehicle. All three treatments also enhanced memory in the OR and OP tasks, whereas vehicle did not. Although E2 increased time spent in the center of the OF, no other treatment effects were observed in the OF, EPM, TST, or FST. These data suggest that long-term ERβ activation can reduce hot flash-like symptoms and enhance spatial and object recognition memories in ovariectomized mice. Thus, the highly selective ERβ agonist EGX358 may be a promising avenue for reducing menopause-related hot flashes and memory dysfunction.

Perspective: Prospects for Nutraceutical Support of Intestinal Barrier Function

Impairment of intestinal barrier function is linked to certain pathologies and to aging, and can be a cause of bacterial infections, systemic and hepatic inflammation, food allergies, and autoimmune disorders. The formation and maintenance of intestinal tight junctions is supported by glucagon-like peptide-2 (GLP-2), which via insulin-like growth factor I activity boosts phosphoinositide 3-kinase/Akt/mammalian target of rapamycin complex 1 (PI3K/Akt/mTORC1) signaling in enterocytes. 5'-AMP-activated protein kinase (AMPK) activity as well as estrogen receptor-β (ERβ) activity are also protective in this regard. Conversely, activation of mitogen-activated protein kinases (MAPKs) and cellular Src (c-Src) under inflammatory conditions can induce dissociation of tight junctions. Hence, nutraceuticals that promote GLP-2 secretion from L cells-effective pre/probiotics, glycine, and glutamine-as well as diets rich in soluble fiber or resistant starch, can support intestinal barrier function. AMPK activators-notably berberine and the butyric acid produced by health-promoting microflora-are also beneficial in this regard, as are soy isoflavones, which function as selective agonists for ERβ. The adverse impact of MAPK and c-Src overactivation on the intestinal barrier can be combatted with various antioxidant measures, including phycocyanobilin, phase 2-inducer nutraceuticals, and N-acetylcysteine. These considerations suggest that rationally designed functional foods or complex supplementation programs could have clinical potential for supporting and restoring healthful intestinal barrier function.

A Multi-Omics Study Revealing the Metabolic Effects of Estrogen in Liver Cancer Cells HepG2.

Hepatocellular carcinoma (HCC) that is triggered by metabolic defects is one of the most malignant liver cancers. A much higher incidence of HCC among men than women suggests the protective roles of estrogen in HCC development and progression. To begin to understand the mechanisms involving estrogenic metabolic effects, we compared cell number, viability, cytotoxicity, and apoptosis among HCC-derived HepG2 cells that were treated with different concentrations of 2-deoxy-d-glucose (2-DG) that blocks glucose metabolism, oxamate that inhibits lactate dehydrogenase and glycolysis, or oligomycin that blocks ATP synthesis and mitochondrial oxidative phosphorylation. We confirmed that HepG2 cells primarily utilized glycolysis followed by lactate fermentation, instead of mitochondrial oxidative phosphorylation, for cell growth. We hypothesized that estrogen altered energy metabolism via its receptors to carry out its anticancer effects in HepG2 cells. We treated cells with 17β-estradiol (E2), 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) an estrogen receptor (ER) α (ERα) agonist, or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), an ERβ agonist. We then used transcriptomic and metabolomic analyses and identified differentially expressed genes and unique metabolite fingerprints that are produced by each treatment. We further performed integrated multi-omics analysis, and identified key genes and metabolites in the gene–metabolite interaction contributed by E2 and ER agonists. This integrated transcriptomic and metabolomic study suggested that estrogen acts on estrogen receptors to suppress liver cancer cell growth via altering metabolism. This is the first exploratory study that comprehensively investigated estrogen and its receptors, and their roles in regulating gene expression, metabolites, metabolic pathways, and gene–metabolite interaction in HCC cells using bioinformatic tools. Overall, this study provides potential therapeutic targets for future HCC treatment.

In silico exploration of hydroxylated polychlorinated biphenyls as estrogen receptor β ligands by 3D-QSAR, molecular docking and molecular dynamics simulations

Hydroxylated polychlorinated biphenyls (HO-PCBs), as the major metabolites of PCBs, have been reported to act as estrogen receptor β (ERβ) agonists. However, the chemical–biological interactions governing their activities toward ERβ have not been elucidated. Therefore, three dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulations, to the best of our knowledge, for the first time were performed to explore the correlation between the structures and activities. The best 3D-QSAR model presented higher predictive ability (R 2 cv=0.543, R 2 pred=0.5793/R 2 cv=0.543, R 2 pred=0.6795) based on comparative molecular field analysis (CoMFA) and comparative similarity indices analysis (CoMSIA), respectively. At the same time, the derived contour maps indicated the important structural features required for improving the activity. Furthermore, molecular docking studies and MD simulations predicted the binding mode and the interactions between the ligand and the receptor. All the results would lead to a better understanding of the specific mechanism of HO-PCBs on estrogen receptor β (ERβ). Communicated by Ramaswamy H. Sarma

Abstract PS18-39: Estrogen receptor beta agonists for triple negative breast cancer

Abstract Background:Triple negative breast cancer (TNBC) represents about 10-15% of all breast cancers. Given the lack of targeted therapies, chemotherapy and immunotherapy are the only treatment options. The five-year survival rate of TNBC patients is approximately 15% less than other forms of breast cancer. Therefore, there is an urgent need to develop novel effective therapeutics for TNBC.Material and Methods:RNA isolated from human immortalized mammary epithelial MCF10A, and various TNBC cell lines was DNase treated and quantitative RT-PCR (qRT-PCR) performed using gene-specific primers spanning exon-exon junctions that include introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. Immuno-blot analyses were performed on lysates prepared from cells in log phase of growth. Proteins were detected by Enhanced Chemi-Luminescence (ECL) method. Viability of cells treated with ERβ specific agonists was determined by using CellTiter-Glo® 2.0 assay. MB231 cells were treated with ERβ agonists following transfection with pcDNA3 (vector) or Flag-tagged ESR2 plasmids. Western blot analyses using specific antibodies and qRT-PCR were performed with the protein extracts and RNA isolated from the cells, respectively. Results:Our experiments demonstrate that ESR2 (ERβ) is differentially expressed and its level of expression is about 3 to 22-fold higher in TNBC cell lines tested than MCF10A. We have shown that viability of estrogen receptor (ER) positive breast cancer cell lines can be significantly inhibited by selective activation of ERβ. A highly selective ERβ agonist has been invented in the Drug Development Institute at the Ohio State University. Treatment of TNBC lines with these ERβ agonists exhibited a dramatic reduction of cell viability. While comparable IC50 values were observed with our in house and commercially available compounds, TNBC had 2-3-fold lesser IC50 values than ER-positive cell lines. Increased expression of FOXO3 and c-Myc1 and decreased expression of c-Myc2 isoform was noted in ERβ overexpressing MB231 cells upon treatment with ERβ agonists.Conclusion:Our results demonstrate that treatment with highly selective ERβ agonists could be an effective therapeutic strategy for TNBC patients. To improve and develop novel therapeutics for TNBC understanding the mechanism of action of these compounds will be crucial. Citation Format: Jharna Datta, Natalie Willingham, Jasmine Manouchehri, Joel David, Mirisha Sheth, Bhuvaneswari Ramaswamy, Ramesh Ganju, Mathew A Cherian. Estrogen receptor beta agonists for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-39.

Activation of estrogen receptor beta signaling reduces stemness of glioma stem cells.

Glioblastoma (GBM) is the most common and deadliest tumor of the central nervous system. GBM has poor prognosis and glioma stem cells (GSCs) are implicated in tumor initiation and therapy resistance. Estrogen receptor β (ERβ) is expressed in GBM and exhibit tumor suppressive function. However, the role of ERβ in GSCs and the therapeutic potential of ERβ agonists on GSCs remain largely unknown. Here, we examined whether ERβ modulates GSCs stemness and tested the utility of two ERβ selective agonists (LY500307 and Liquiritigenin) to reduce the stemness of GSCs. The efficacy of ERβ agonists was examined on GSCs isolated from established and patient derived GBMs. Our results suggested that knockout of ERβ increased the proportion of CD133+ and SSEA+ positive GSCs and overexpression of ERβ reduced the proportion of GSCs in GBM cells. Overexpression of ERβ or treatment with ERβ agonists significantly inhibited the GSCs cell viability, neurosphere formation, self-renewal ability, induced the apoptosis and reduced expression of stemness markers in GSCs. RNA sequencing analysis revealed that ERβ agonist modulate pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that ERβ overexpression or agonist treatment reduced glutamate receptor signaling pathway and induced apoptotic pathways. In orthotopic models, ERβ overexpression or ERβ agonists treatment significantly reduced the GSCs mediated tumor growth and improved the mice overall survival. Immunohistochemical studies demonstrated that ERβ overexpression decreased SOX2 and GRM3 expression and increased expression of GFAP in tumors. These results suggest that ERβ activation could be a promising therapeutic strategy to eradicate GSCs.

Silibinin treatment protects human skin cells from UVB injury through upregulation of estrogen receptors

Ultraviolet B (UVB) from the sunlight is a major environmental cause for human skin damages, inducing cell death, inflammation, senescence and even carcinogenesis. The natural flavonoid silibinin, clinically used as liver protectant, has protective effects against UVB-caused skin injury in vivo and in vitro. Silibinin is often classified as a phytoestrogen, because it modulates the activation of estrogen receptors (ERs). However, whether silibinin's estrogenic effect contributes to the skin protection against UVB injury remains to be elucidated. The issue was explored in this study by using the human foreskin dermal fibroblasts (HFF) and human non-malignant immortalized keratinocytes (HaCaT). In HFF, pre-treatment with silibinin rescued UVB-irradiated cells from apoptosis. Interestingly, silibinin increased the whole cellular and nuclear levels of ERα and ERβ in UVB-irradiated cells. Activation of ERs by treatment with estradiol elevated the cell survival and reduced apoptosis in UVB-treated cells. ERα agonist increased cell survival, while its antagonist decreased it. ERβ agonist also increased cell survival, but the antagonist had no effect on cell survival. Transfection of the cells with the small interfering RNAs (si-RNAs) to ERα or ERβ diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERβ were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERβ by specific antagonists and si-RNAs, respectively, reduced cell survival in UVB-treated HaCaT cells regardless of silibinin treatment. Taken together, it is summarized that silibinin up-regulates both ERα and ERβ pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage.

Histone deacetylase inhibitors enhance estrogen receptor beta expression and augment agonist-mediated tumor suppression in glioblastoma.

BackgroundGlioblastomas (GBMs) are the most lethal primary brain tumors. Estrogen receptor β (ESR2/ERβ) function as a tumor suppressor in GBM, however, ERβ expression is commonly suppressed during glioma progression. In this study, we examined whether drugs that reverse epigenetic modifications will enhance ERβ expression and augment ERβ agonist-mediated tumor suppression.MethodsWe tested the utility of epigenetic drugs which act as an inhibitor of histone deacetylases (HDACs), histone methylases, and BET enzymes. Mechanistic studies utilized RT-qPCR, chromatin immunoprecipitation (ChIP), and western blotting. Cell viability, apoptosis, colony formation, and invasion were measured using in vitro assays. An orthotopic GBM model was used to test the efficacy of in vivo.ResultsOf all inhibitors tested, HDACi (panobinostat and romidepsin) showed the potential to increase the expression of ERβ in GBM cells. Treatment with HDACi uniquely upregulated ERβ isoform 1 expression that functions as a tumor suppressor but not ERβ isoform 5 that drives oncogenic functions. Further, combination therapy of HDACi with the ERβ agonist, LY500307, potently reduced cell viability, invasion, colony formation, and enhanced apoptosis. Mechanistic studies showed that HDACi induced ERβ is functional, as it enhanced ERβ reporter activities and ERβ target genes expression. ChIP analysis confirmed alterations in the histone acetylation at the ERβ and its target gene promoters. In orthotopic GBM model, combination therapy of panobinostat and LY500307 enhanced survival of tumor-bearing mice.ConclusionsOur results suggest that the combination therapy of HDACi and LY500307 provides therapeutic utility in overcoming the suppression of ERβ expression that commonly occurs in GBM progression.

Female mice lacking ERβ display excitatory/inhibitory synaptic imbalance to drive the pathogenesis of temporal lobe epilepsy.

Epilepsy is a highly prevalent and drug-refractory neurological disorder characterized by spontaneous recurrent seizures. Estrogen is identified to be proconvulsant and lowers the seizure threshold of female epilepsy. Estrogen receptor β (ERβ) has been proposed to mediate neuroprotection in epilepsy, although the underlying mechanism remains unknown.Rationale: In this study, we investigated the role of ERβ in the epileptogenesis of female temporal lobe epilepsy (TLE).Methods: Immunohistochemistry, immunofluorescence, western blots, Golgi staining, 1H MRS and whole-cell patch-clamp were used to evaluate ERβ expression, pathological changes, and synaptic excitation /inhibition (E/I) balance in female TLE patients and ovariectomized (OVX) chronic epileptic mice. Electroencephalogram (EEG) recordings were recorded to evaluate the epileptic susceptibility in OVX WT and ERβ-/- mice. And high-throughput RNA-sequence was performed to identify differential expression genes (DEGs) which can elucidate the potential mechanism of ERβ regulating the seizure susceptibility.Results: ERβ expression was decreased in the brains of female TLE patients and OVX chronic epileptic mice. ERβ deletion enhanced seizure susceptibility and exacerbated the imbalance of synaptic E/I in hippocampal CA1 area of OVX epileptic mice. In line with these observations, RNA-sequence data further identified glutamine ligase (GLUL) as the target of ERβ involved in regulating synaptic E/I in CA1. Furthermore, ERβ agonist WAY-200070 markedly suppressed epileptic phenotypes and normalized GLUL expression in CA1 region of kainic acid (KA) induced OVX chronic epileptic model.Conclusions: Our data provide novel insight into the pathogenesis of female TLE, and indicate ERβ provides a new therapeutic strategy for female TLE patients.

N-myc downstream-regulated gene 2 promotes the protein stability of estrogen receptor beta via inhibition of ubiquitin-protein ligase E3A to suppress colorectal cancer.

N-myc downstream-regulated gene 2 (NDRG2) and estrogen receptor beta (ERβ) both play key roles in cellular differentiation in colorectal cancer (CRC). Previous studies have demonstrated that ERβ co-locates with and directly transactivates NDRG2. However, the effect of NDRG2 on ERβ and its underlying mechanism remain largely unknown. Our aim of the study is to explore the effect of NDRG2 on ERβ and their contributions to progression of CRC. The Cancer Genome Atlas (TCGA) database was first utilized to validate the clinical significance of ERβ and NDRG2 in CRC. MTT and scratch migration assays were carried out to verify the role of ERβ and NDRG2 in CRC cells. Western blotting and polymerase chain reaction were performed to analyze the effect of NDRG2 on ERβ, and an immunoprecipitation assay was conducted to explore the protein-protein interaction. ERβ and NDRG2 were both found to be significantly down-regulated in tumor tissues from the TCGA-CRC database. NDRG2 was also observed to enhance the protein stability of ERβ while could not change messenger RNA (mRNA) level of ESR2 (encoding ERβ). A positive relationship was found to exist between the two proteins in CRC cells, with NDRG2 prolonging the half-life of ERβ and improving its nuclear translocation. Through detecting expression of ERβ downstream genes (such as TP53 and JNK) and performing related function experiment, we demonstrated that NDRG2 could promote transcriptional activation of ERβ target genes and enhance the function of tumor suppressors when the ERβ agonist diarylpropionitrile (DPN). The immunoprecipitation assay showed that NDRG2 could affect the complex components of ubiquitin-protein ligase E3A (UBE3A, known as E6AP) and ERβ, reducing the ubiquitin-mediated proteasome degradation of ERβ. In the current study, we found that NDRG2 could bind with UBE3A to hinder the binding of UBE3A with ERβ. Moreover, a positive feedback loop was discovered between NDRG2 and ERβ, which provides a novel insight and therapeutic target for CRC.

Estrogen receptors in human bladder cells regulate innate cytokine responses to differentially modulate uropathogenic E. coli colonization

The bladder epithelial cells elicit robust innate immune responses against urinary tract infections (UTIs) for preventing the bacterial colonization. Physiological fluctuations in circulating estrogen levels in women increase the susceptibility to UTI pathogenesis, often resulting in adverse health outcomes. Dr adhesin bearing Escherichia coli (Dr E. coli) cause recurrent UTIs in menopausal women and acute pyelonephritis in pregnant women. Dr E. coli bind to epithelial cells via host innate immune receptor CD55, under hormonal influence. The role of estrogens or estrogen receptors (ERs) in regulating the innate immune responses in the bladder are poorly understood. In the current study, we investigated the role of ERα, ERβ and GPR30 in modulating the innate immune responses against Dr E. coli induced UTI using human bladder epithelial carcinoma 5637 cells (HBEC). Both ERα and ERβ agonist treatment in bladder cells induced a protection against Dr E. coli invasion via upregulation of TNFα and downregulation of CD55 and IL10, and these effects were reversed by action of ERα and ERβ antagoinsts. In contrast, the agonist-mediated activation of GPR30 led to an increased bacterial colonization due to suppression of innate immune factors in the bladder cells, and these effects were reversed by the antagonist-mediated suppression of GPR30. Further, siRNA-mediated ERα knockdown in the bladder cells reversed the protection against bacterial invasion observed in the ERα positive bladder cells, by modulating the gene expression of TNFα, CD55 and IL10, thus confirming the protective role of ERα. We demonstrate for the first time a protective role of nuclear ERs, ERα and ERβ but not of membrane ER, GPR30 against Dr E. coli invasion in HBEC 5637 cells. These findings have many clinical implications and suggest that ERs may serve as potential drug targets towards developing novel therapeutics for regulating local innate immunity and treating UTIs.

Estrogen receptor β regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3

Loss of the tumor suppressor, PTEN, is one of the most common findings in prostate cancer (PCa). This loss leads to overactive Akt signaling, which is correlated with increased metastasis and androgen independence. However, another tumor suppressor, inositol-polyphosphate 4-phosphatase type II (INPP4B), can partially compensate for the loss of PTEN. INPP4B is up-regulated by androgens, and this suggests that androgen-deprivation therapy (ADT) would lead to hyperactivity of AKT. However, in the present study, we found that in PCa, samples from men treated with ADT, ERβ, and INPP4B expression were maintained in some samples. To investigate the role of ERβ1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERβ1. In these cells, INPP4B was induced by ERβ ligands, and this induction was accompanied by inhibition of Akt activity and reduction in cell migration. These findings reveal that, in the absence of androgens, ERβ1 induces INPP4B to dampen AKT signaling. Since the endogenous ERβ ligand, 3β-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERβ1 on AKT signaling, an ERβ agonist should be added along with ADT.

17β-Estradiol Protects Neural Stem/Progenitor Cells Against Ketamine-Induced Injury Through Estrogen Receptor β Pathway.

Ketamine inhibits neural stem/progenitor cell (NSPC) proliferation and disrupts normal neurogenesis in the developing brain. 17β-Estradiol alleviates neurogenesis damage and enhances behavioral performance after ketamine administration. However, the receptor pathway of 17β-estradiol that protects NSPCs from ketamine-induced injury remains unknown. In the present study, we investigated the role of estrogen receptor α (ER-α) and estrogen receptor β (ER-β) in 17β-estradiol’s protection against ketamine-exposed NSPCs and explored its potential mechanism. The primary cultured NSPCs were identified by immunofluorescence and then treated with ketamine and varying doses of ER-α agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) or ER-β agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) for 24 h. NSPC proliferation was analyzed by 5-bromo-2-deoxyuridine incorporation test. The expression of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) was quantified by western blotting. It was found that treatment with different concentrations of PPT did not alter the inhibition of ketamine on NSPC proliferation. However, treatment with DPN attenuated the inhibition of ketamine on NSPC proliferation at 24 h after their exposure (P < 0.05). Furthermore, treatment with DPN increased p-GSK-3β expression in NSPCs exposed to ketamine. These findings indicated that ER-β mediates probably the protective effects of 17β-estradiol on ketamine-damaged NSPC proliferation and GSK-3β is involved in this process

Formononetin attenuates atopic dermatitis by upregulating A20 expression via activation of G protein-coupled estrogen receptor

Ethnopharmacological relevanceAtopic dermatitis (AD) is a complex skin disease with highly heterogeneous inflammation, which ranks among the largest component of the nonfatal diseases worldwide. The medications currently used to treat AD primarily include antihistamines, vitamin D and anti-inflammatory drugs, etc. But, the usage of these drugs is usually accompanied by various side-effects. Formononetin (FMN), a natural active ingredient of Astragalus membranaceus (Fisch.) Bunge, decreases the AD relapse rate, reduces recurring severity incidence and resists the inflammation in the initial stage of AD. However, the underlying mechanism of FMN on repressing the development of AD is still unknown. Aim of the studyTo investigate the potential mechanism of FMN on relieving the initial responses of AD and elucidate its possible therapeutic targets in vivo and in vitro. Materials and methodsA fluorescein isothiocyanate (FITC)-induced mouse model of the initial stage of AD was established in vivo. Human keratinocytes (HaCaT) cells were co-stimulated with tumor necrosis factor alpha (TNF-α) and polyinosinic-polycytidylic acid (Poly(I:C)) in vitro. The production of thymic stromal lymphopoietin (TSLP) and immunoglobulin E (IgE) were detected by enzyme-linked immunosorbnent assay (ELISA). The protein expression was measured through immunohistochemistry and western blotting. The mRNA expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The impact of TNF-α-induced protein 3 (TNFAIP3/A20) was reflected using its small interfering RNA (siRNA). The role of G protein-coupled estrogen receptor (GPER) was explored using its agonist (G1), antagonist (G15) or siRNA (siGPER) in vitro. ResultsWe found that FMN upregulated the expression of A20 protein and mRNA in the initial stage of AD model, especially in the epithelial region of ear tissue, and inhibited the production of TSLP simultaneously. Consistently, FMN significantly upregulated A20 protein and its mRNA expression while reduced TSLP protein and its mRNA expression in vitro, and this effect could be antagonized by A20 siRNA (siA20). Moreover, compared with PPT (ERα agonist) and DPN (ERβ agonist), G1 could significantly increase the expression of A20. In addition, compared with MPP (ERα antagonist) and PHTPP (ERβ antagonist), G15 could markedly reduce the expression of A20. Furthermore, the effects of FMN on A20 were interfered by siGPER and G15 in vitro and in vivo. ConclusionsThese results demonstrated that FMN attenuated AD by upregulating A20 expression via activation of GPER. This new strategy might have effective therapeutic potential for AD and other inflammatory disorders.