siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.     

Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development

The hypothesis of fetal origins of adult disease posits that early developmental exposures involve epigenetic modifications, such as DNA methylation, that influence adult disease susceptibility. In utero or neonatal exposure to bisphenol A (BPA), a high-production-volume chemical used in the manufacture of polycarbonate plastic, is associated with higher body weight, increased breast and prostate cancer, and altered reproductive function. This study shows that maternal exposure to this endocrine-active compound shifted the coat color distribution of viable yellow agouti (Avy) mouse offspring toward yellow by decreasing CpG (cytosine-guanine dinucleotide) methylation in an intracisternal A particle retrotransposon upstream of the Agouti gene. CpG methylation also was decreased at another metastable locus, the CDK5 activator-binding protein (CabpIAP). DNA methylation at the Avy locus was similar in tissues from the three germ layers, providing evidence that epigenetic patterning during early stem cell development is sensitive to BPA exposure. Moreover, maternal dietary supplementation, with either methyl donors like folic acid or the phytoestrogen genistein, negated the DNA hypomethylating effect of BPA. Thus, we present compelling evidence that early developmental exposure to BPA can change offspring phenotype by stably altering the epigenome, an effect that can be counteracted by maternal dietary supplements.     

Bisphenol A and Hormone-Associated Cancers: Current Progress and Perspectives

Bisphenol A (BPA), a carbon-based synthetic compound, exhibits hormone-like properties and is present ubiquitously in the environment and in human tissues due to its widespread use and biological accumulation. BPA can mimic estrogen to interact with estrogen receptors α and β, leading to changes in cell proliferation, apoptosis, or migration and thereby, contributing to cancer development and progression. At the genetic level, BPA has been shown to be involved in multiple oncogenic signaling pathways, such as the STAT3, MAPK, and PI3K/AKT pathways. Moreover, BPA may also interact with other steroid receptors (such as androgen receptor) and plays a role in prostate cancer development. This review summarizes the current literature regarding human exposure to BPA, the endocrine-disrupting effects of BPA, and the role of BPA in hormone-associated cancers of the breast, ovary, and prostate.     

Estrogen receptor (ER) β expression in oligodendrocytes is required for attenuation of clinical disease by an ERβ ligand

Treatment of experimental autoimmune encephalomyelitis (EAE) mice with the estrogen receptor (ER) β ligand diarylpropionitrile (DPN) has been shown to have neuroprotective effects via stimulation of endogenous myelination. The direct cellular mechanisms underlying the effects of this ERβ ligand on the central nervous system are uncertain because different cell types in both the peripheral immune system and central nervous system express ERs. ERβ is the target molecule of DPN because DPN treatment fails to decrease EAE clinical symptoms in global ERβ-null mice. Here we investigated the potential role of ERβ expression in cells of oligodendrocyte (OL) lineage in ERβ ligand-mediated neuroprotection. To this end, we selectively deleted ERβ in OLs using the well-characterized Cre-loxP system for conditional gene knockout (CKO) in mice. The effects of this ERβ CKO on ERβ ligand-mediated neuroprotective effects in chronic EAE mice were investigated. ERβ CKO in OLs prevented DPN-induced decrease in EAE clinical disease. DPN treatment during EAE did not attenuate demyelination, only partially improved axon conduction, and did not activate the phosphatidylinositol 3-kinase/serine-threonine-specific protein kinase/mammalian target of rapamycin signaling pathway in ERβ CKO mice. However, DPN treatment significantly increased brain-derived neurotrophic factor levels in ERβ CKO mice. These findings demonstrate that signaling through ERβ in OLs is essential for the beneficial myelination effects of the ERβ ligand DPN in chronic EAE mice. Further, these findings have important implications for neuroprotective therapies that directly target OL survival and myelination.Multiple sclerosis (MS) is an inflammatory, demyelinating neurodegenerative disease characterized by physical, and often cognitive, deficits that can progress to severe debilitation. Although current MS treatments exist in the form of immunomodulatory or immunosuppressive agents, these treatments fail to halt disease progression and are not directly neuroprotective.Building on a wealth of research supporting a role for estrogens in neuroprotection, we have demonstrated that treatment of experimental autoimmune encephalomyelitis (EAE) mice with the estrogen receptor (ER) β ligand 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) attenuates clinical disease, neurodegeneration, and axon demyelination and improves axon conduction (1–4). Notably, these effects were observed with both prophylactic and therapeutic treatment regimens, and they occurred in the presence of peripheral cytokine production and central nervous system (CNS) inflammation. Evidence of direct neuroprotection by an ERβ ligand is welcomed, because it circumvents ERα-mediated adverse effects of synthetic estrogens [i.e., increased breast and uterine endometrial growth in females and feminizing effects in males (5)].Because ERs are present in various cell types in the peripheral immune system and CNS, including cells of oligodendrocyte (OL) lineage, it is difficult to assess which cell type(s) mediate ERβ ligand-conferred neuroprotection (6). ERβ is the target molecule of DPN: DPN treatment fails to decrease EAE clinical symptoms in global ERβ-null mice (4). Although informative, such studies do not elucidate the cell type(s) on which DPN acts. We have recently demonstrated that therapeutic treatment with DPN increases mature OL numbers, remyelination-induced callosal conduction, and phosphorylated ERβ levels in OLs, and that it activates the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway implicated in OL survival, differentiation, and axon myelination (1, 7, 8). Taken together with the pronounced, functional improvement in endogenous myelination observed in DPN-treated chronic EAE mice, a direct effect on ERβ in OLs may mediate ERβ ligand-induced myelination/remyelination improvement and neuroprotection. To test this hypothesis, ERβ was selectively deleted from OLs using the well-characterized Cre-loxP recombination system for conditional gene knockout (CKO) in mice (9–12). The Olig2,ERβ CKO mice that resulted from crossing ERβ^flox/flox and Olig2-tva-Cre mice were viable and displayed normal behavior and gross CNS anatomy; hence, the effect of ERβ CKO in OLs on ERβ ligand-induced neuroprotection in chronic EAE mice was investigated. ERβ CKO in OLs prevented DPN-induced attenuation of clinical disease and demyelination and failed to activate the PI3K/Akt/mTOR signaling pathway. However, DPN treatment in Olig2,ERβ CKO mice partially improved axon conduction and reduced axonal loss and, as in WT mice, significantly increased BDNF levels. These findings reveal a direct action of the ERβ ligand DPN on ERβ in OLs in DPN-induced myelination/remyelination effects within a chronic mouse model of MS.     

Differential colocalization of estrogen receptor β (ERβ) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: An immunocytochemical study

Evidence exists for the localization of the newly identified estrogen receptor β (ERβ) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERα. Presently, we investigate whether ERβ-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague–Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17β (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERβ antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERβ-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERβ-ir within magnocellular areas was noticeably less intense. OT-/ERβ-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains (≈50% of OT and 25% of ERβ-labeled cells between bregma −1.78 and −2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERβ-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERβ-ir. In the SON, most nuclear ERβ-ir colocalized with AVP-ir, whereas few OT-/ERβ-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERβ-mediated mechanism, in a tissue-specific manner.     

High ERα36 Expression Level and Membrane Location Predict Poor Prognosis in Renal Cell Carcinoma

Estrogen receptor alpha 36 (ERα36), a truncated variant of ERα, is located in cytoplasm and membrane that is different from other nuclear receptors of ERα family. ERα36 is involved in progression and treatment resistance of a variety of carcinomas. However, the clinical and prognostic significance of ERα36 in renal tumors have not been fully elucidated.Here, renal tumor tissues from 125 patients were collected and immunohistochemical stained with ERα36 antibody. ERα36 expression level and location in these cases were analyzed for their correlations with clinical characteristics. The differential diagnosis value was also assessed for benign and malignant renal tumors, as well as its prognostic value.The results showed that membrane ERα36 expression was rarely detected in benign tumors but predominantly observed in malignant renal tumors. Kaplan–Meier analysis indicated that significant correlations of high ERα36 level and ERα36 membrane expression were correlated with both poor disease-free survival and overall survival. Univariate and multivariate analysis confirmed that both ERα36 high expression and membrane location can serve as unfavorable prognostic indicators for renal cell carcinoma.It is thus concluded that membrane ERα36 expression is valuable for differential diagnosis of malignant renal tumors from benign ones. Both ERα36 high expression and membrane location indicate poor prognosis in renal cell carcinoma.     

Estrogen receptor–β activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNFα mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133⁺ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.     

Bisphenol A Exposure in Exclusively Breastfed Infants and Lactating Women: An Observational Cross-sectional Study

Objective:Bisphenol A (BPA) is a known endocrine disruptor and free BPA will interact with estrogen. BPA is also fat soluble and will therefore contaminate breast milk. The European Food Safety Authority has set a limit for temporary tolerable daily intake of 4 μg/kg body weight/day in breastfeeding infants. The aim of this study was to measure human milk BPA concentrations in Turkish women and thus exclusively breastfed infants’ exposure to BPA.Methods:Healthy, postnatal, exclusively breastfeeding women were recruited and breast milk samples were collected. Free BPA concentration was analyzed in the milk samples using competitive enzyme-linked immunosorbent assay. Participants’ demographic characteristics and nutritional habits were investigated through face-to-face interviews using a detailed questionnaire.Results:Eighty women participated. Median milk free BPA level was 0.63 μg/L. There was no statistically significant association between maternal body mass index, birth type, parity, infant birth week, infant birth weight, and human milk BPA concentration. Nevertheless, there was a significant association between human milk BPA level and consumption of fast-food and carbonated drinks (p=0.022 and p=0.018, respectively). Exclusively breastfed infants’ mean BPA exposure was 0.0099±0.0079 μg/kg bw/day. There was a moderate negative significant correlation between infant BPA exposure and infant current body weight (r=0.327, p=0.003).Conclusion:BPA exposure in exclusively breastfed infants was within accepted limits and the current dietary exposure level of infants in this cohort was safe.     

Therapeutic Value of Estrogen Receptor α in Hepatocellular Carcinoma Based on Molecular Mechanisms

The incidence of hepatocellular carcinoma (HCC) is significantly lower in women than men, implying that estrogen receptors (ERs) may play an important role in this sex dimorphism. Recently, considerable progress has been made in expanding our understanding of the mechanisms of ERs in HCC. As one of the most important ERs, ERα functions as a tumor suppressor in the progression of HCC through various pathways, such as STAT3 signaling pathways, lipid metabolism-related signaling pathways, and non-coding RNAs. However, the function of ERα was reduced with the changes of some molecules in the liver, which may develop further into HCC and make it difficult to achieve an effective hormone treatment effect. Intriguingly, there are signs that individualized hormone therapy according to the activity of ERα will overcome this challenge. Based on these observations, it is particularly imperative to reassess and extend the function of ERα. In this review, we mainly elucidated molecular mechanisms associated with ERα in HCC and investigated the individualized hormone therapy based on these mechanisms, with the aim of providing new insights for HCC treatment.     

Cell lineage tracing links ERα loss in Erbb2-positive breast cancers to the arising of a highly aggressive breast cancer subtype

HER2-positive (HER2⁺) breast cancers (BrCs) contain approximately equal numbers of ERα⁺HER2⁺ and ERα⁻HER2⁺ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα⁺HER2⁺ BrCs could lose ERα to become ERα⁻HER2⁺ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERα^CreRFP-T mice that produce an RFP-marked ERα⁺ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα⁺RFP⁺Erbb2⁺ and ERα⁻RFP⁻Erbb2⁺ MGECs. Early hyperplasia developed mostly from ERα⁺RFP⁺Erbb2⁺ cells and ERα⁻RFP⁻Erbb2⁺ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα⁺RFP⁺Erbb2⁺ cells, 15% fast-proliferating ERα⁻RFP⁺Erbb2⁺ cells derived from ERα⁺RFP⁺Erbb2⁺ cells, and 20% fast-proliferating ERα⁻RFP⁻Erbb2⁺ cells. The advanced tumors had mostly ERα⁻RFP⁺Erbb2⁺ and ERα⁻RFP⁻Erbb2⁺ cells and only a very small population of ERα⁺RFP⁺Erbb2⁺ cells. In ERα⁻RFP⁺Erbb2⁺ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα⁻RFP⁺Erbb2⁺ cells, a few ERα⁻RFP⁻Erbb2⁺ cells, and no ERα⁺RFP⁺Erbb2⁺ cells. The high metastatic capacity of ERα⁻RFP⁺Erbb2⁺ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα⁺RFP⁺Erbb2⁺ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα⁻RFP⁺Erbb2⁺ cells. The ERα⁻Erbb2⁺ BrCs with an ERα⁺ origin are more aggressive than those ERα⁻Erbb2⁺ BrCs with an ERα⁻ origin, and thus, they should be distinguished and treated differently in the future.

The mammary gland (MG) epithelium contains both ERα⁺ and ERα⁻ luminal epithelial cells (1). Breast cancers (BrCs) may arise from either ERα⁺ or ERα⁻ MG epithelial cells (MGECs). BrCs are heterogeneous and can be roughly grouped into ERα⁺, HER2⁺, and triple negative BrCs. About 70% of BrCs belong to the ERα⁺ group, which is associated with a relatively good prognosis, and about 20% fall into the HER2⁺ group with a much worse prognosis. About 50% of HER2⁺ BrCs express ERα (2), so HER2⁺ BrCs are accordingly designated as ERα⁺HER2⁺ and ERα⁻HER2⁺ BrCs. In ERα⁺HER2⁺ cancers, the cross-talk between ERα and HER2 signaling pathways and loss of ERα partially account for resistance to endocrine therapy (3, 4). ERα⁺HER2⁺ cancers exhibit a wide range of disease relapse time, metastatic potential, and responsiveness to anti-HER2 treatment, while ERα⁻HER2⁺ cancers generally are more malignant with earlier relapse, stronger metastatic potential, and much worse prognosis (5–13). ERα and HER2 expression vary during tumor progression, and patients can have both ERα⁺ primary tumors as well as ERα⁻ metastases (3, 14, 15). In some patients with recurrent BrCs, the ratios of ERα⁺ to ERα⁻ cancer cells may be reduced (16). These observations suggest that some ERα⁺HER2⁺ BrCs may progress to ERα⁻HER2⁺ BrCs; yet, direct evidence is missing since cell lineage tracing in people is unethical.To elucidate the relationships among the HER2⁺ BrC subtypes, we set out to answer three unresolved biomedical questions: First, do ERα⁺HER2⁺ cancers lose ERα to become the more aggressive ERα⁻HER2⁺ cancers? Second, do ERα⁻HER2⁺ BrCs originate directly from ERα⁻ MGECs or indirectly from ERα⁺HER2⁺ cancer cells? Third, are ERα⁻HER2⁺ cancers derived from preexisting ERα⁺HER2⁺ cancers and do ERα⁻HER2⁺ cancers that stem from ERα⁻ MGECs have different cell proliferation rates and metastatic capabilities? To answer these important questions, we developed a trigenic mouse model that allows for in vivo tracing of the ERα⁺ and ERα⁻ MGEC lineages during tumorigenesis and metastasis. In this model, breast carcinogenesis was induced by Erbb2 (the rodent homolog of HER2) expression in both ERα⁺ and ERα⁻ MGECs in adulthood, and all tumor cells arising from the ERα⁺ MGEC lineage were traced with red fluorescent protein (RFP) expression during the entire process of cancer initiation, progression, and metastasis. ERα expression history, cell proliferation rate, and metastatic capability were compared and characterized among different subtypes of BrC cells.     

Generation and reproductive phenotypes of mice lacking estrogen receptor β

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.