Expression of estrogen receptors,androgen receptor and steroid receptor coactivator-3 is negatively correlated to the differentiation of astrocytic tumors

Astrocytic tumors are the most common primary brain tumors. It has been reported that androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERβ) and their coactivator SRC-1 and SRC-3 are involved in the regulation of the growth and development of many tumors, but their expression profiles and significances in the astrocytic tumors remain largely unknown. In this study, the expression of AR, ERs, and SRCs, and the possible roles of them in astrocytic neoplasm were evaluated and compared to normal brain tissues by nickel-intensified immunohistochemistry with tissue microarrays. The results showed that there were no age- or gender-differences regarding to the levels of these receptors or coactivators in astrocytic or normal brain tissues. In the high-grade astrocytic tissue, the levels of AR, ERs and SRC-3 were significantly decreased when compared to the low-grade astrocytic tissues, but the levels of SRC-1 remain unchanged. Correlation analysis revealed that the levels of AR, ERs and SRC-3 were negatively correlated to tumor differentiation, and the levels of SRC-3 were positively correlated to that of ERα. Furthermore, the decreased levels of SRC-3 were associated with an increase of ERβ in astrocytic tumors when compared to that of normal brain tissues. These above results indicate a combination of decreased expression of ERs, AR and SRC-3 but not SRC-1 may be involved in the tumorigenesis of gliomas, ERα/SRC-3 axis may play central role in the regulation these tumors.     

Structure–activity relationships of bisphenol A analogs at estrogen receptors (ERs): Discovery of an ERα-selective antagonist

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERβ subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERβ and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.     

The role of androgen receptors in the masculinization of brain and behavior: what we've learned from the testicular feminization mutation

Many studies demonstrate that exposure to testicular steroids such as testosterone early in life masculinizes the developing brain, leading to permanent changes in behavior. Traditionally, masculinization of the rodent brain is believed to depend on estrogen receptors (ERs) and not androgen receptors (ARs). According to the aromatization hypothesis, circulating testosterone from the testes is converted locally in the brain by aromatase to estrogens, which then activate ERs to masculinize the brain. However, an emerging body of evidence indicates that the aromatization hypothesis cannot fully account for sex differences in brain morphology and behavior, and that androgens acting on ARs also play a role. The testicular feminization mutation (Tfm) in rodents, which produces a nonfunctional AR protein, provides an excellent model to probe the role of ARs in the development of brain and behavior. Tfm rodent models indicate that ARs are normally involved in the masculinization of many sexually dimorphic brain regions and a variety of behaviors, including sexual behaviors, stress response and cognitive processing. We review the role of ARs in the development of the brain and behavior, with an emphasis on what has been learned from Tfm rodents as well as from related mutations in humans causing complete androgen insensitivity.     

Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro-in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E(2) treatment where we observed a trigger in ERβ2 expression upon E(2) exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.     

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.     

Expression of estrogen receptors in human corpus cavernosum and male urethra.

Estrogen, largely produced in testis and adrenal gland, may play important roles in male reproduction. Most of the effects of estrogens are mediated by binding of estrogen to one or both of the two estrogen receptor (ER) subtypes alpha and beta. Recently, they have been described in testis, prostate, and efferent ducts, mostly in rodents. The goal of this study was to prove the evidence of ERs in human corpus cavernosum and male urethra, exploring the protein expression of these receptors by immunohistochemistry. Corpus cavernosum and corpus spongiosum smooth muscle was immunoreactive for the androgen receptor (AR), ER alpha, and strongly for ER beta. Endothelial cells were negative for AR, sporadically positive for ER alpha, and positive for ER beta. Urethral epithelium showed strong nuclear expression of AR, predominantly in the basal cell layer, and nuclear expression of ER alpha in the intermediate cells. ER beta was highly expressed in almost all urethral nuclei and, much more weakly, in cytoplasm. Progesterone receptor (PGR) was negative in all cases and all tissues. These results represent the first report that ER alpha and particularly ER beta are regularly expressed in human penile tissue.     

Cell-specific distributions of estrogen receptor alpha (ERα) and androgen receptor (AR) in anterior pituitary glands from adult cockerels as revealed by immunohistochemistry

Estrogens and androgens play important roles in regulating the hormone-secreting functions of the pituitary gland by binding to their corresponding receptors. However, the expression of estrogen receptors (ERs) and the androgen receptor (AR) and the cell types containing ERs and AR in the anterior pituitary gland of adult chickens have not been well-studied. In this study, the distribution of ERα, AR and their corresponding cell types in the anterior pituitary gland of adult cockerels was detected by immunohistochemistry. The results showed that ERα was expressed in 68.63 % of luteinizing hormone (LH) producing cells but was not found in thyrotropes, lactotropes, somatotropes, corticotropes and folliculo-stellate (FS) cells. Pituitary hormone and AR double labeling results showed that about 37 % of LH cells and 50 % of thyroid-stimulating hormone (TSH) producing cells expressed AR, respectively. In contrast, less than 1 % of the somatotropes had an AR positive signal and AR signals were not detected in lactotropes, corticotropes or FS cells. In addition, there were only a few AR and ERα dual-labeled cells observed. These novel results provide evidence for a cell-specific distribution of ERα and AR in the anterior pituitary from adult cockerels by immunohistochemistry. The different distributions of ERα and AR in the LH cells suggest that the feedback-regulating mechanisms of estrogen and androgen on the pituitary hormones secretion are different. The functions and related mechanisms still need to be elucidated further.     

Design and synthesis of peptide-based chimeric molecules to induce degradation of the estrogen and androgen receptors

Peptide-based inducers of estrogen receptor (ER) α and androgen receptor (AR) degradations via the ubiquitin–proteasome system (UPS) were developed. The designated inducers were composed of two biologically active scaffolds: the helical peptide PERM3, which is an LXXLL-like mimic of the coactivator SRC-1, and various small molecules (MV1, LCL161, VH032, and POM) that bind to E3 ligases (IAPs, VHL, and cereblon, respectively), to induce ubiquitylation of nuclear receptors that bind to SRC-1. All of the synthesized chimeric E3 ligand-containing molecules induced the UPS-mediated degradation of ERα and AR. The PERM3 peptide was applicable for the development of the ERα and AR degraders using these E3 ligands.     

Estrogen receptor alpha interacts with 17β-hydroxysteroid dehydrogenase type 10 in mitochondria

Estrogen receptor alpha (ERα) is present in the nucleus, the cytosol and in mitochondria. The rat ERα ligand binding domain was employed as bait in a bacterial two-hybrid screening of a human heart cDNA library to detect novel protein-protein interaction partners of ERα in the heart. 17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10), which converts potent (17β-estradiol) to less potent estrogens (estrone), co-localized with 17β-HSD10 in the mitochondria of rat cardiac myocytes. GST pull-down experiments confirmed the interaction of ERα and 17β-HSD10. These findings suggest that the ERα estrogen receptor might be involved in regulating intracellular estrogen levels by modulating 17β-HSD10 activity.     

Homology-modeled ligand-binding domains of zebrafish estrogen receptors alpha, beta1, and beta2: from in silico to in vivo studies of estrogen interactions in Danio rerio as a model system

Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) alpha, beta(1), and beta(2). Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfERalpha and found to bind in the same manner as in human ERalpha, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.     

The protective role of estrogen and estrogen receptors in cardiovascular disease and the controversial use of estrogen therapy

Epidemiologic studies have previously suggested that premenopausal females have reduced incidence of cardiovascular disease (CVD) when compared to age-matched males, and the incidence and severity of CVD increases postmenopause. The lower incidence of cardiovascular disease in women during reproductive age is attributed at least in part to estrogen (E2). E2 binds to the traditional E2 receptors (ERs), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ), as well as the more recently identified G-protein-coupled ER (GPR30), and can exert both genomic and non-genomic actions. This review summarizes the protective role of E2 and its receptors in the cardiovascular system and discusses its underlying mechanisms with an emphasis on oxidative stress, fibrosis, angiogenesis, and vascular function. This review also presents the sexual dimorphic role of ERs in modulating E2 action in cardiovascular disease. The controversies surrounding the clinical use of exogenous E2 as a therapeutic agent for cardiovascular disease in women due to the possible risks of thrombotic events, cancers, and arrhythmia are also discussed. Endogenous local E2 biosynthesis from the conversion of testosterone to E2 via aromatase enzyme offers a novel therapeutic paradigm. Targeting specific ERs in the cardiovascular system may result in novel and possibly safer therapeutic options for cardiovascular protection.     

Epigenetic setting for long-term expression of estrogen receptor α and androgen receptor in cells

Epigenetic regulation of the nuclear estrogen and androgen receptors, ER and AR, constitutes the molecular basis for the long-lasting effects of sex steroids on gene expression in cells. The effects prevail at hundreds of gene loci in the proximity of estrogen- and androgen-responsive elements and many more such loci through intra- and even inter-chromosomal level regulation. Such a memory system should be active in a flexible manner during the early development of vertebrates, and later replaced to establish more stable marks on genomic DNA. In mammals, DNA methylation is utilized as a very stable mark for silencing of the ERα and AR isoform expression during cancer cell and normal brain development. The factors affecting the DNA methylation of the ERα and AR genes in cells include estrogen and androgen. Since testosterone induces brain masculinization through its aromatization to estradiol in a narrow time window of the perinatal stage in rodents, the autoregulation of estrogen receptors, especially the predominant form of ERα, at the level of DNA methylation to set up the “cell memory” affecting the sexually differentiated status of brain function has been attracting increasing attention. The alternative usage of the androgen-AR system for brain masculinization and estrogenic regulation of AR expression in some species imply that the DNA methylation pattern of the AR gene can be established by closely related but different systems for sex steroid-induced phenomena, including brain masculinization.     

Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.