Innately expressed estrogen-related receptors in the skeletal muscle are indispensable for exercise fitness.

Transcriptional determinants in the skeletal muscle that govern exercise capacity, while poorly defined, could provide molecular insights into how exercise improves fitness. Here, we have elucidated the role of nuclear receptors, estrogen-related receptor alpha and gamma (ERRα/γ) in regulating myofibrillar composition, contractility, and exercise capacity in skeletal muscle. We used muscle-specific single or double (DKO) ERRα/γ knockout mice to investigate the effect of ERRα/γ deletion on muscle and exercise parameters. Individual knockout of ERRα/γ did not have a significant impact on the skeletal muscle. On the other hand, DKO mice exhibit pale muscles compared to wild-type (WT) littermates. RNA-seq analysis revealed a predominant decrease in expression of genes linked to mitochondrial and oxidative metabolism in DKO versus WT muscles. DKO muscles exhibit marked repression of oxidative enzymatic capacity, as well as mitochondrial number and size compared to WT muscles. Mitochondrial function is also impaired in single myofibers isolated from DKO versus WT muscles. In addition, mutant muscles exhibit reduced angiogenic gene expression and decreased capillarity. Consequently, DKO mice have a significantly reduced exercise capacity, further reflected in poor fatigue resistance of DKO mice in in vivo contraction assays. These results show that ERRα and ERRγ together are a critical link between muscle aerobic capacity and exercise tolerance. The ERRα/γ mutant mice could be valuable for understanding the long-term impact of impaired mitochondria and vascular supply on the pathogenesis of muscle-linked disorders.

Benzotriazole Ultraviolet Stabilizers Promote Breast Cancer Cell Proliferation via Activating Estrogen-Related Receptors α and γ at Human-Relevant Levels.

Benzotriazole ultraviolet stabilizers (BUVSs) are ubiquitous emerging pollutants that have been reported to show estrogenic disruption effects through interaction with the classic estrogen receptors (ERs) in the fashion of low activity. The present study aims at revealing the potential disruption mechanism via estrogen-related receptors α and γ (ERRα and ERRγ) pathways. By the competitive binding assay, we first found that BUVSs bond to ERRγ ligand binding domain (ERRγ-LBD) with Kd ranging from 0.66 to 19.27 µM. According to the results of reporter gene assays, the transcriptional activities of ERRα and ERRγ were promoted by most tested BUVSs with the lowest observed effective concentrations (LOEC) from 10 to 100 nM, which are in the range of human exposure levels. At 1 µM, most tested BUVSs showed higher agonistic activity toward ERRγ than ERRα. The most effective two BUVSs promoted the MCF-7 proliferation dependent on ERRα and ERRγ with a LOEC of 100 nM. The molecular dynamics simulation showed that most studied BUVSs had lower binding free energy with ERRγ than with ERRα. The structure-activity relationship analysis revealed that molecular polarizability, electron-donating ability, ionization potential, and softness were the main structural factors impacting the binding of BUVSs with ERRγ. Overall, our results provide novel insights into the estrogenic disruption effects of BUVSs.

Estrogen-related receptors: novel potential regulators of osteoarthritis pathogenesis.

Osteoarthritis (OA) is a chronic inflammatory disease that is associated with articular cartilage destruction, subchondral bone alterations, synovitis, and even joint deformity and the loss of joint function. Although current basic research on the pathogenesis of OA has made remarkable progress, our understanding of this disease still needs to be further improved. Recent studies have shown that the estrogen-related receptor (ERR) family members ERRα and ERRγ may play significant roles in the pathogenesis of OA. In this review, we refer to the latest research on ERRs and the pathogenesis of OA, elucidate the structure and physiopathological functions of the ERR orphan nuclear receptor family, and systematically examine the relationship between ERRs and OA at the molecular level. Moreover, we also discuss and predict the capacity of ERRs as potential targets in the clinical treatment of OA.

Cardiac-Specific Overexpression of ERRγ in Mice Induces Severe Heart Dysfunction and Early Lethality.

Proper cardiac function depends on the coordinated expression of multiple gene networks related to fuel utilization and mitochondrial ATP production, heart contraction, and ion transport. Key transcriptional regulators that regulate these gene networks have been identified. Among them, estrogen-related receptors (ERRs) have emerged as crucial modulators of cardiac function by regulating cellular metabolism and contraction machinery. Consistent with this role, lack of ERRα or ERRγ results in cardiac derangements that lead to functional maladaptation in response to increased workload. Interestingly, metabolic inflexibility associated with diabetic cardiomyopathy has been recently associated with increased mitochondrial fatty acid oxidation and expression of ERRγ, suggesting that sustained expression of this nuclear receptor could result in a cardiac pathogenic outcome. Here, we describe the generation of mice with cardiac-specific overexpression of ERRγ, which die at young ages due to heart failure. ERRγ transgenic mice show signs of dilated cardiomyopathy associated with cardiomyocyte hypertrophy, increased cell death, and fibrosis. Our results suggest that ERRγ could play a role in mediating cardiac pathogenic responses.

Modulation of estrogen-related receptors subtype selectivity: Conversion of an ERRß/γ selective agonist to ERRα/ß/γ pan agonists.

Estrogen Related Receptors (ERRs) are key regulators of energy homeostasis and play important role in the etiology of metabolic disorders, skeletal muscle related disorders, and neurodegenerative diseases. Among the three ERR isoforms, ERRα emerged as a potential drug target for metabolic and neurodegenerative diseases. Although ERRß/γ selective agonist chemical tools have been identified, there are no chemical tools that effectively target ERRα agonism. We successfully engineered high affinity ERRα agonism into a chemical scaffold that displays selective ERRß/γ agonist activity (GSK4716), providing novel ERRα/ß/γ pan agonists that can be used as tools to probe the physiological roles of these nuclear receptors. We identified the structural requirements to enhance selectivity toward ERRα. Molecular modeling shows that our novel modulators have favorable binding modes in the LBP of ERRα and can induce conformational changes where Phe328 that originally occupies the pocket is dislocated to accommodate the ligands in a rather small cavity. The best agonists up-regulated the expression of target genes PGC-1α and PGC-1ß, which are necessary to achieve maximal mitochondrial biogenesis. Moreover, they increased the mRNA levels of PDK4, which play an important role in energy homeostasis.

Novel discoveries in acid-base regulation and osmoregulation: A review of selected hormonal actions in zebrafish and medaka.

Maintenance of internal ionic and acid-base homeostasis is critical for survival in all biological systems. Similar to mammals, aquatic fishes have developed sophisticated homeostatic mechanisms to mitigate metabolic or environmental disruptions in ionic and acid-base status of systemic body fluids via hormone-controlled transport of ions or acid equivalents. The present review summarizes newly discovered actions of several hormones in zebrafish (Danio rerio) and medaka (Oryzias latipes) that have greatly contributed to our overall understanding of ionic/acid-base regulation. For example, isotocin and cortisol were reported to enhance transport of various ions by stimulating the proliferation and/or differentiation of ionocyte progenitors. Meanwhile, stanniocalcin-1, a well-documented hypocalcemic hormone, was found to suppress ionocyte differentiation and thus downregulate secretion of H+ and uptake of Na+ and Cl-. Estrogen-related receptor and calcitonin gene-related peptide also regulate the differentiation of certain types of ionocytes to either stimulate or suppress H+ secretion and Cl- uptake. On the other hand, endothelin and insulin-like growth factor 1 activate the respective secretion of H+ and Na+/Cl through fast actions. These new findings enhance our understanding of how hormones regulate fish ionic and acid-base regulation while further providing new insights into vertebrate evolution, mammalian endocrinology and human disease-related therapeutics.

The estrogen-related receptors in metabolism and cancer: newer insights.

The eukaryotic nuclear receptors (NRs) super-family of transcriptional factors include the estrogen-related receptors (ERRs) that have diverse roles in control of cellular energy balance, general metabolism, growth and development, immunity etc. Mouse knock-out models of specific ERR isoforms (ERRα, ERRß and ERRγ) exhibit defects in several phenotypic traits. Newer findings indicate important roles of ERRs in the regulation of brown adipocyte tissue mitochondrial oxidative functions as well as metabolic control in association with hypoxia-inducible factors during cellular hypoxic state. Genes involved in cardiac metabolism is also influenced by ERRα and ERRγ in association with the co-activators PGC-1α and PGC-1ß. On the other hand, ERRs have crucial involvement at the interface of metabolism and diseases such as cancer. Recent findings have implicated ERRα in the progression of tumor and malignancy of the breast, prostate, colon, endometrium etc. In this article, new insights into the regulatory role of ERRs in metabolism and cancer shall be reviewed.

Emerging Roles of Estrogen-Related Receptors in the Brain: Potential Interactions with Estrogen Signaling.

In addition to their well-known role in the female reproductive system, estrogens can act in the brain to regulate a wide range of behaviors and physiological functions in both sexes. Over the past few decades, genetically modified animal models have greatly increased our knowledge about the roles of estrogen receptor (ER) signaling in the brain in behavioral and physiological regulations. However, less attention has been paid to the estrogen-related receptors (ERRs), the members of orphan nuclear receptors whose sequences are homologous to ERs but lack estrogen-binding ability. While endogenous ligands of ERRs remain to be determined, they seemingly share transcriptional targets with ERs and their expression can be directly regulated by ERs through the estrogen-response element embedded within the regulatory region of the genes encoding ERRs. Despite the broad expression of ERRs in the brain, we have just begun to understand the fundamental roles they play at molecular, cellular, and circuit levels. Here, we review recent research advancement in understanding the roles of ERs and ERRs in the brain, with particular emphasis on ERRs, and discuss possible cross-talk between ERs and ERRs in behavioral and physiological regulations.

Chlorinated biphenyls effect on estrogen-related receptor expression, steroid secretion, mitochondria ultrastructure but not on mitochondrial membrane potential in Leydig cells.

To characterize polychlorinated biphenyls (PCBs) action on Leydig cells, PCBs congeners, low-chlorinated (delor 103; d103) and high-chlorinated ones (delor 106; d106) were selected. The cells were treated according to PCBs dose (d103 or d106 0.2 ng/ml in low doses:, or 2 ng/ml in high doses) and type (d103 + d106 in low doses or 103 + 106 in high doses). After 24 h treatment with PCBs, a distinct increase in estrogen-related receptors (ERRs type α, ß and γ) expression was revealed. However, the dose- and type-dependent PCBs effect was mostly exerted on ERRα expression. A similar increase in ERRs expression was demonstrated by estradiol but not testosterone, which was without an effect on ERRs. PCBs caused no decrease in the membrane potential status of Leydig cells (either in dose or type schedule) but had severe effects on the mitochondria number and structure. Moreover, PCBs markedly increased calcium (Ca2+) concentration and sex steroid secretion (both androgens and estrogens were elevated). These findings suggest a similar estrogenic action of PCBs congeners (d103 and d106) on Leydig cell function. We report dose- and type-specific effects of PCBs only on Leydig cell ERRs expression. Both delors showed common effects on the mitochondria ultrastructural and functional status. Based on our results, ERRα seems to be the most sensitive to hormonal modulation. The increases in Ca2+ and sex steroid secretion may be due to the activation of ERRs by PCBs binding and/or direct effect of PCBs on ERRs mRNA/protein expression. Nevertheless, to confirm the existence of possible relationships between ERRs signaling (including PCBs as ligands) and mitochondria function in Leydig cells, further intensive studies are needed.

Identification of novel inverse agonists of estrogen-related receptors ERRγ and ERRß.

Estrogen-related receptors (ERRs, α, ß, and γ) are orphan nuclear receptors most closely related in sequence to estrogen receptors (ERα and ERß). Much attention has been paid recently to the functions of ERRs for their potential roles as new therapeutic targets implicated in the etiology of metabolic disorders. While no endogenous ligand has been identified for any of the ERR isoforms to date, the potential for using synthetic small molecules to modulate their activity has been demonstrated. In the present study, a series of novel inverse agonists of ERRγ and ERRß were synthesized using regio- and stereo-specific direct substitution of triarylethylenes. These compounds were evaluated for their ability to modulate the activities of ERRs. The rational directed substitution approach and extensive SAR studies resulted in the discovery of compound 4a (DY40) as the most potent ERRγ inverse agonist described to date with mixed ERRγ/ERRß functional activities, which potently suppressed the transcriptional functions of ERRγ with IC50=0.01µM in a cell-based reporter gene assay and antagonized ERRγ with a potency approximately 60 times greater than its analog Z-4-OHT (Z-4-hydroxytamoxifen). In addition, compound 3h (DY181) was identified as the most potent synthetic inverse agonist for the ERRß that exhibited excellent selectivity over ERRα/γ in functional assays. This selectivity was also supported by computational docking models that suggest DY181 forms more extensive hydrogen bound network with ERRß which should result in higher binding affinity on ERRß over ERRγ.

Reduction in ERRα is associated with lichen sclerosus and vulvar squamous cell carcinoma.

OBJECTIVE: ERRs (estrogen-related receptors) regulate energy metabolism, the cell cycle and inflammatory processes in both normal and cancer cells. Chronic inflammation induced by lichen sclerosus (LS) or human papilloma virus (HPV) precedes vulvar squamous cell carcinoma (vulvar SCC). We investigated the expression of ERRα, ERRß and ERRγ in normal vulvar skin, LS as well as LS-dependent and LS-independent/HPV-related vulvar SCC. METHODS: A total of 203 samples were analyzed for ERRα, ERRß and ERRγ by using immunohistochemistry. These included 37 normal vulvar skin samples, 110 LS samples, 6 vulvar intraepithelial neoplasia (VIN) samples and 50 vulvar SCC samples. RESULTS: A substantial reduction in or disappearance of ERRα was detected in all vulvar SCC samples. A total of 79% of childhood-onset LS and 51% of adulthood-onset LS lesions showed decreases in ERRα staining. A gradual reduction in ERRα cytoplasmic staining was observed from healthy vulvar skin to precursor lesions and further to SCC. Nuclear ERRα staining was observed in 8/33 (24%) LS-dependent and 10/17 (59%) LS-independent SCC samples. CONCLUSIONS: ERRα, a key regulator of cell energy metabolism, may play a role in the pathogenesis of both LS and vulvar SCC.

Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming.

Treatment of Duchenne muscular dystrophy (DMD) by replacing mutant dystrophin or restoring dystrophin-associated glycoprotein complex (DAG) has been clinically challenging. Instead, identifying and targeting muscle pathways deregulated in DMD will provide new therapeutic avenues. We report that the expression of nuclear receptor estrogen-related receptor-γ (ERRγ), and its metabolic and angiogenic targets are down-regulated (50-85%) in skeletal muscles of mdx mice (DMD model) vs. wild-type mice. Corelatively, oxidative myofibers, muscle vasculature, and exercise tolerance (33%) are decreased in mdx vs. wild-type mice. Overexpressing ERRγ selectively in the dystrophic muscles of the mdx mice restored metabolic and angiogenic gene expression compared with control mdx mice. Further, ERRγ enhanced muscle oxidative myofibers, vasculature, and blood flow (by 33-66%) and improved exercise tolerance (by 75%) in the dystrophic mice. Restoring muscle ERRγ pathway ameliorated muscle damage and also prevented DMD hallmarks of postexercise muscle damage, hypoxia, and fatigue in mdx mice. Notably, ERRγ did not restore sarcolemmal DAG complex, which is thus dispensable for antidystrophic effects of ERRγ. In summary, ERRγ-dependent metabolic and angiogenic gene program is defective in DMD, and we demonstrate that its restoration is a potential strategy for treating muscular dystrophy.

Perspectives of drug-based neuroprotection targeting mitochondria.

Mitochondrial dysfunction has been reported in most neurodegenerative diseases. These anomalies include bioenergetic defect, respiratory chain-induced oxidative stress, defects of mitochondrial dynamics, increase sensitivity to apoptosis, and accumulation of damaged mitochondria with instable mitochondrial DNA. Significant progress has been made in our understanding of the pathophysiology of inherited mitochondrial disorders but most have no effective therapies. The development of new metabolic treatments will be useful not only for rare mitochondrial disorders but also for the wide spectrum of common age-related neurodegenerative diseases shown to be associated with mitochondrial dysfunction. A better understanding of the mitochondrial regulating pathways raised several promising perspectives of neuroprotection. This review focuses on the pharmacological approaches to modulate mitochondrial biogenesis, the removal of damaged mitochondria through mitophagy, scavenging free radicals and also dietary measures such as ketogenic diet.

Identification and characterization of GSK-9089 metabolites through high resolution-mass spectrometry based in vitro and in vivo rat biological sample analysis.

Estrogen related receptors (ERRs) agonist GSK-9089 (DY-131) reported to pose a potential in increasing exercise endurance. High resolution mass spectrometry (HRMS) based analysis has utmost importance in the detection, identification, or characterization of a molecule including its metabolites in human body. In this study, in vitro metabolism profile of GSK-9089 was investigated after incubation with liver microsomes and S9 fractions. Additionally, in vivo metabolites of the molecule were identified in plasma, urine, and faeces samples of rats. Structures of all the potential metabolites were revealed by employing an in silico tool and HRMS based analysis through data-dependent and data-independent mining strategies. Nine unknown metabolites of GSK-9089 have been identified which were found to be present in a trace amount in in vivo matrices. Most of the in vitro and in vivo phase I metabolites of the molecule were formed after imine bond hydrolysis followed by deamidation, oxidation, and N-oxidation. The molecule underwent phase II metabolism to generate more polar metabolites mainly through glucuronide, sulfate conjugation biotransformation reactions. The in vitro and in vivo metabolites of GSK-9089 could be useful to identify the abuse of this ERRs agonist in the future.

Role of Estrogen-Related Receptor γ and PGC-1α/SIRT3 Pathway in Early Brain Injury After Subarachnoid Hemorrhage.

Estrogen-related receptors (ERRs) were shown to play an important role in the regulation of free radical-mediated pathology. This study aimed to investigate the neuroprotective effect of ERRγ activation against early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the potential underlying mechanisms. In a rat model of SAH, the time course of ERRs and SIRT3 and the effects of ERRγ activation were investigated. ERRγ agonist DY131, selective inhibitor GSK5182, or SIRT3 selective inhibitor 3-TYP were administered intracerebroventricularly (icv) in the rat model of SAH. The use of 3-TYP was for validating SIRT3 as the downstream signaling of ERRγ activation. Post-SAH assessments included SAH grade, neurological score, Western blot, Nissl staining, and immunofluorescence staining in rats. In an vitro study, the ERRγ agonist DY131 and ERRγ siRNA were administered to primary cortical neurons stimulated by Hb, after which cell viability and neuronal deaths were accessed. Lastly, the brain ERRγ levels and neuronal death were accessed in SAH patients. We found that brain ERRγ expressions were significantly increased, but the expression of SIRT3 dramatically decreased after SAH in rats. In the brains of SAH rats, ERRγ was expressed primarily in neurons, astrocytes, and microglia. The activation of ERRγ with DY131 significantly improved the short-term and long-term neurological deficits, accompanied by reductions in oxidative stress and neuronal apoptosis at 24 h after SAH in rats. DY131 treatment significantly increased the expressions of PGC-1α, SIRT3, and Bcl-2 while downregulating the expressions of 4-HNE and Bax. ERRγ antagonist GSK5182 and SIRT3 inhibitor 3-TYP abolished the neuroprotective effects of ERRγ activation in the SAH rats. An in vitro study showed that Hb stimulation significantly increased intracellular oxidative stress in primary cortical neurons, and DY131 reduced such elevations. Primary cortical neurons transfected with the ERRγ siRNA exhibited notable apoptosis and abolished the protective effect of DY131. The examination of SAH patients' brain samples revealed increases in ERRγ expressions and neuronal apoptosis marker CC3. We concluded that ERRγ activation with DY131 ameliorated oxidative stress and neuronal apoptosis after the experimental SAH. The effects were, at least in part, through the ERRγ/PGC-1α/SIRT3 signaling pathway. ERRγ may serve as a novel therapeutic target to ameliorate EBI after SAH.

Chemerin and Chemokine-like Receptor 1 Expression in Ovarian Cancer Associates with Proteins Involved in Estrogen Signaling.

Chemerin, a pleiotropic adipokine coded by the RARRES2 gene, has been reported to affect the pathophysiology of various cancer entities. To further approach the role of this adipokine in ovarian cancer (OC), intratumoral protein levels of chemerin and its receptor chemokine-like receptor 1 (CMKLR1) were examined by immunohistochemistry analyzing tissue microarrays with tumor samples from 208 OC patients. Since chemerin has been reported to affect the female reproductive system, associations with proteins involved in steroid hormone signaling were analyzed. Additionally, correlations with ovarian cancer markers, cancer-related proteins, and survival of OC patients were examined. A positive correlation of chemerin and CMKLR1 protein levels in OC (Spearman's rho = 0.6, p < 0.0001) was observed. Chemerin staining intensity was strongly associated with the expression of progesterone receptor (PR) (Spearman´s rho = 0.79, p < 0.0001). Both chemerin and CMKLR1 proteins positively correlated with estrogen receptor ß (ERß) and estrogen-related receptors. Neither chemerin nor the CMKLR1 protein level was associated with the survival of OC patients. At the mRNA level, in silico analysis revealed low RARRES2 and high CMKLR1 expression associated with longer overall survival. The results of our correlation analyses suggested the previously reported interaction of chemerin and estrogen signaling to be present in OC tissue. Further studies are needed to elucidate to which extent this interaction might affect OC development and progression.

Senescence and autophagy relation with the expressional status of non-canonical estrogen receptors in testes and adrenals of roe deer (Capreolus capreolus) during the pre-rut period.

The roe deer bucks represent a spontaneous model to study the synchronized testicular involution and recrudescence cycles. However, cellular processes and hormonal control of steroidogenic glands are scarcely known. For the present study testes and adrenal glands obtained from roe deer during the pre-rut season were used. We aimed to determine (i) senescence and autophagy involvement in testis atrophy (immunohistochemical analysis for tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A; p16 and microtubule-associated protein 1A/1B-light chain 3; LC3, respectively), (ii) the size of the adrenal cortex and medulla (morphometric analysis), (iii) G-protein coupled estrogen receptor (GPER) and estrogen-related receptors (ERRs; type α, ß, and Y) distribution and expression (qRT-PCR and immunohistochemical analyses) and (iv) serum testosterone and estradiol levels (immunoassay ELISA). This study revealed pre-rut characteristics of testis structure with the presence of both senescence and autophagy-positive cells and higher involvement of senescence, especially in spermatogenic cells (P < 0.05). In the adrenal cortex, groups of cells exhibiting shrinkage were observed. The presence of ERRs in cells of the seminiferous epithelium and interstitial Leydig cells and GPER presence distinctly in Leydig cells was revealed. In adrenals, these receptors were localized in groups of normal-looking cells and those with shrinkage. Morphometric analysis showed differences in cortex width which was smaller (P < 0.05) than that of the medulla. A weak immunohistochemical signal was observed for ERRß when compared to ERRα and ERRγ. The mRNA expression level of ERRα and ERRγ was lower (P < 0.001 and P < 0.05, respectively) while ERRß was higher (P < 0.001) in adrenals when compared to testes. mRNA GPER expression was similar in both glands. In the pre-rut season, the testosterone level was 4.89 ng/ml while the estradiol level was 0.234 ng/ml. We postulate that: (i) senescence and autophagy may be involved in both reinitiation of testis function and/or induction of abnormal processes, (ii) hormonal modulation of testis inactivity may affect adrenal cortex causing cell shrinkage, (iii) ERRs and GPER localization in spermatogenic cells and interstitial cells, as well as cortex cells, may maintain and control the morpho-functional status of both glands, and (iv) androgens and estrogens (via ERRs and GPER) drive cellular processes in the testis and adrenal pre-rut physiology.

Estrogen-Related Receptors Gene Expression and Copy Number Alteration Association With the Clinicopathologic Characteristics of Breast Cancer.

Purpose: It has been suggested that dysregulation of transcription factors expression or activity plays significant roles in breast cancer (BC) severity and poor prognosis. Therefore, our study aims to thoroughly evaluate the estrogen-related receptor isoforms (ESRRs) expression and copy number alteration (CNA) status and their association with clinicopathologic characteristics in BC. Methods: A METABRIC dataset consist of 2509 BC patients' samples was obtained from the cBioPortal public domain. The gene expression, putative CNA, and relevant tumor information of ESRRs were retrieved. ESRRs messenger RNA (mRNA) expression in BC cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE). Association and correlation analysis of ESRRs expression with BC clinicopathologic characteristics and molecular subtype were performed. Kaplan-Meier survival analysis was conducted to evaluate the prognostic value of ESRRs expression on patient survival. Results: ESRRα expression correlated negatively with patients' age and overall survival, whereas positively correlated with tumor size, the number of positive lymph nodes, and Nottingham prognostic index (NPI). Conversely, ESRRγ expression was positively correlated with patients' age and negatively correlated with NPI. ESRRα and ESRRγ expression were significantly associated with tumor grade, expression of hormone receptors, human epidermal growth factor receptor 2 (HER2), and molecular subtype, whereas ESRRß was only associated with tumor stage. A significant and distinct association of each of ESRRs CNA with various clinicopathologic and prognostic factors was also observed. Kaplan-Meier survival analysis demonstrated no significant difference for survival curves among BC patients with high or low expression of ESRRα, ß, or γ. On stratification, high ESRRα expression significantly reduced survival among premenopausal patients, patients with grade I/II, and early-stage disease. In BC cell lines, only ESRRα expression was significantly higher in HER2-positive cells. No significant association was observed between ESRRß expression and any of the clinicopathologic characteristics examined. Conclusions: In this clinical dataset, ESRRα and ESRRγ mRNA expression and CNA show a significant correlation and association with distinct clinicopathologic and prognostic parameters known to influence treatment outcomes; however, ESRRß failed to show a robust role in BC pathogenesis. ESRRα and ESRRγ can be employed as therapeutic targets in BC-targeted therapy. However, the role of ESRRß in BC pathogenesis remains unclear.

Molecular dynamics of estrogen-related receptors and their regulatory proteins: roles in transcriptional control for endocrine and metabolic signaling.

Estrogen-related receptor (ERR) is a member of the nuclear receptor (NR) superfamily and has three subtypes α, ß, and γ. Despite their strong homology with estrogen receptor (ER) α, ERRs cannot accommodate endogenous hormones. However, they are able to regulate gene expression without ligand binding. ERRα and ERRγ orchestrate the expression of genes involved in bioenergetic pathways, while ERRß controls placental development and stem cell maintenance. Evidence from recent studies, including clinical research, has also demonstrated close associations of ERRs with the pathophysiology of hormone-related cancers and metabolic disorders including type 2 diabetes mellitus. This review summarizes the basic knowledge and recent advances in ERRs and their associated proteins, focusing on the subcellular dynamics involved in transcriptional regulation. Fluorescent protein labeling enabled monitoring of ERRs in living cells and revealed previously unrecognized characteristics. Using this technique, we demonstrated a role of ERRß in controlling estrogen signaling by regulating the subnuclear dynamics of ligand-activated ERα. Visualization of ERRs and related proteins and subsequent analyses also revealed a function of ERRγ in promoting liver lactate metabolism in association with LRPGC1, a recently identified lactic acid-responsive protein. These findings suggest that ERRs activate unique transregulation mechanisms in response to extracellular stimuli such as hormones and metabolic signals, implying an adaptive system behind the cellular homeostatic regulation by orphan NRs. Control of subcellular ERR dynamics will contribute toward the development of therapeutic approaches to treat various diseases including hormone-related cancers and metabolic disorders associated with abnormal ERR signaling pathways.

The potential endocrine disruption mechanism of anthelmintic drug niclosamide by activating estrogen receptors and estrogen-related receptors.

Niclosamide (NIC), a helminthic drug used widely for controlling schistosomiasis, can reportedly disrupt the endocrine system. However, its underlying mechanisms are still unclear. In this study, we revealed the potential endocrine disruption mechanism of NIC by activating estrogen receptors (ERs) and estrogen-related receptors (ERRs). The binding potency of NIC with ERα, ERß and ERRγ were determined by fluorescence competitive binding assays, which shows an IC50 (the concentration of NIC needed to displace 50 % of the probe from the receptor) of 90 ± 4.1, 10 ± 1.7 nM and 0.59 ± 0.07 nM respectively. The IC50 for ERRγ is the lowest one among the three detected receptors, which is three orders of magnitude lower than the known agonist GSK4716.The transcriptional activities of NIC on ERs and ERRs were detected by MVLN cells (stably transfected with ERs reporter gene) and HeLa cells (transiently transfected with ERRs reporter gene)-based luciferase reporter gene assay. The lowest observable effective concentration (LOEC) ranked as follows: ERRγ (0.5 nM) < ERRα (10 nM) < ERs (100 nM). The maximum observed induction rate for ERRγ (294 %) was higher than that for ERRα (191 %). The maximum observed induction rate of NIC for ERs was 30 % relative to 17ß-estradiol. In addition, we simulated the interactions of NIC with ERs and ERRs by molecular docking. NIC could dock into the ligand binding pockets of ERs and ERRs and form hydrogen bonds with different amino acids. The binding energy ranked as follows: ERRγ (-8.90 kcal/mol) < ERß (-7.57 kcal/mol) < ERRα (-7.15 kcal/mol) < ERα (-6.53 kcal/mol), which implied that NIC bound to ERRγ with higher binding affinity than the other receptors. Overall, we clarify that ERRγ might be the dominant target for NIC in cells rather than ERRα and ERs. We reveal potential novel mechanisms for the endocrine disruption effects of NIC by activating both ERRs and ERs at environmentally-related nanomolar levels.