ERβ Knockout Research Articles

Estrogen Protects the Female Heart from Ischemia/Reperfusion Injury through Manganese Superoxide Dismutase Phosphorylation by Mitochondrial p38β at Threonine 79 and Serine 106

A collective body of evidence indicates that estrogen protects the heart from myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism remains incompletely understood. We have previously delineated a novel mechanism of how 17β-estradiol (E2) protects cultured neonatal rat cardiomyocytes from hypoxia/reoxygenation (H/R) by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide dismutase (MnSOD) activity via p38β, leading to the suppression of reactive oxygen species (ROS) and apoptosis. Here we investigate these cytoprotective actions of E2 in vivo. Left coronary artery ligation and reperfusion was used to produce I/R injury in ovariectomized (OVX) female mice and in estrogen receptor (ER) null female mice. E2 treatment in OVX mice reduced the left ventricular infarct size accompanied by increased activity of mitochondrial p38β and MnSOD. I/R-induced infarct size in ERα knockout (ERKO), ERβ knockout (BERKO) and ERα and β double knockout (DERKO) female mice was larger than that in wild type (WT) mice, with little difference among ERKO, BERKO, and DERKO. Loss of both ERα and ERβ led to reduced activity of mitochondrial p38β and MnSOD at baseline and after I/R. The physical interaction between mitochondrial p38β and MnSOD in the heart was detected by co-immunoprecipitation (co-IP). Threonine 79 (T79) and serine 106 (S106) of MnSOD were identified to be phosphorylated by p38β in kinase assays. Overexpression of WT MnSOD in cardiomyocytes reduced ROS generation during H/R, while point mutation of T79 and S106 of MnSOD to alanine abolished its antioxidative function. We conclude that the protective effects of E2 and ER against cardiac I/R injury involve the regulation of MnSOD via posttranslational modification of the dismutase by p38β.

Estrogen Stimulation of Kiss1 Expression in the Medial Amygdala Involves Estrogen Receptor-α But Not Estrogen Receptor-β.

The neuropeptide kisspeptin, encoded by Kiss1, regulates reproduction by stimulating GnRH secretion. Neurons synthesizing kisspeptin are predominantly located in the hypothalamic anteroventral periventricular (AVPV) and arcuate nuclei, but smaller kisspeptin neuronal populations also reside in extrahypothalamic brain regions, such as the medial amygdala (MeA). In adult rodents, estradiol (E2) increases Kiss1 expression in the MeA, as in the AVPV. However, unlike AVPV and arcuate nuclei kisspeptin neurons, little else is currently known about the development, regulation, and function of MeA Kiss1 neurons. We first assessed the developmental onset of MeA Kiss1 expression in males and found that MeA Kiss1 expression is absent at juvenile ages but significantly increases during the late pubertal period, around postnatal day 35, coincident with increases in circulating sex steroids. We next tested whether developmental MeA Kiss1 expression could be induced early by E2 exposure prior to puberty. We found that juvenile mice given short-term E2 had greatly increased MeA Kiss1 expression at postnatal day 18. Although MeA Kiss1 neurons are known to be E2 up-regulated, the specific estrogen receptor (ER) pathway(s) mediating this stimulation are unknown. Using adult ERα knockout and ERβ knockout mice, we next determined that ERα, but not ERβ, is required for maximal E2-induced MeA Kiss1 expression in both sexes. These results delineate both the developmental time course of MeA Kiss1 expression and the specific ER signaling pathway required for E2-induced up-regulation of Kiss1 in this extrahypothalamic brain region. These findings will help drive future studies ascertaining the potential functions of this understudied kisspeptin population.

Estrogen receptor beta reduces colon cancer metastasis through a novel miR-205 - PROX1 mechanism.

Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ERβ) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ERβ represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ERβ and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ERβ upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3′UTR. Through the generation of intestine-specific ERβ knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo. Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo. In conclusion, ERβ in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3′UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ERβ-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.

Klotho/fibroblast growth factor 23- and PTH-independent estrogen receptor-α-mediated direct downregulation of NaPi-IIa by estrogen in the mouse kidney.

Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however, the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERβ) and the Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERβ or Klotho and their wild type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERβ knockout (KO) mice caused a significant reduction in food intake along with increased renal phosphate wasting. The latter resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were absent in ERα KO mice. Estrogen treatment of Klotho KO mice or parathyroid hormone (PTH)-depleted thyroparathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably coexpressing both ERα and ERβ caused a significant downregulation of NaPi-IIa protein when transiently transfected with a plasmid containing full-length or open-reading frame (ORF) 3'-untranslated region (UTR) but not 5'-UTR ORF of mouse NaPi-IIa transcript. In conclusion, estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3'-UTR of its mRNA and is independent of Klotho/fibroblast growth factor 23 and PTH signaling pathways.

Estrogen receptor β controls MMP-19 expression in mouse ovaries during ovulation.

Estrogen receptor beta (ERβ/ESR2) has a central role in mouse ovaries, as ERβ knockout (BERKO) mice are subfertile due to an increase in fibrosis around the maturing follicle and a decrease in blood supply. This has a consequence that these follicles rarely rupture to release the mature oocyte. Matrix metalloproteinases (MMPs) are modulators of the extracellular matrix, and the expression of one specific MMP, MMP-19, is normally increased in granulosa cells during their maturation until ovulation. In this study, we demonstrate that MMP-19 levels are downregulated in BERKO mouse ovaries. Using human MCF-7 cells that overexpress ERβ, we could identify MMP-19 to be a transcriptional target of ligand-bound activated ERβ acting on a specificity protein-1 binding site. These data provide a molecular explanation for the observed follicle rupture defect that contributes to the subfertility of female BERKO mice.

Revisiting the neural role of estrogen receptor beta in male sexual behavior by conditional mutagenesis

Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) β in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERβ in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERβ deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males.These data show that neural ERβ does not play a crucial role in the organization and activation of the neural circuitry underlying male sexual behavior. These discrepancies with the phenotype of global ERβ knockout models are discussed.

ERβ regulation of NF-kB activation in prostate cancer is mediated by HIF-1.

We examined the regulation of NF-κB in prostate cancer by estrogen receptor β (ERβ) based on the inverse correlation between p65 and ERβ expression that exists in prostate carcinomas and reports that ERβ can inhibit NF-κB activation, although the mechanism is not known. We demonstrate that ERβ functions as a gate-keeper for NF-κB p65 signaling by repressing its expression and nuclear translocation. ERβ regulation of NF-κB signaling is mediated by HIF-1. Loss of ERβ or hypoxia stabilizes HIF-1α, which we found to be a direct driver of IKKβ transcription through a hypoxia response element present in the promoter of the IKKβ gene. The increase of IKKβ expression in ERβ-ablated cells correlates with an increase in phospho-IκBα and concomitant p65 nuclear translocation. An inverse correlation between the expression of ERβ and IKKβ/p65 was also observed in the prostates of ERβ knockout (BERKO) mice, Gleason grade 5 prostate tumors and analysis of prostate cancer databases. These findings provide a novel mechanism for how ERβ prevents NF-κB activation and raise the exciting possibility that loss of ERβ expression is linked to chronic inflammation in the prostate, which contributes to the development of high-grade prostate cancer.

Neonatal Estrogen Receptor β Is Important in the Permanent Inhibition of Epithelial Cell Proliferation in the Mouse Uterus.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERβ inhibits cell proliferation. ERβ mRNA is expressed in neonatal uteri and vaginae; however, its functions in neonatal tissues have not been ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ERβ, and its roles in cell proliferation in neonatal uteri and vaginae of ERβ knockout (βERKO) mice. ERβ mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice and decreased with age. In uterine and vaginal epithelia of 2-day-old βERKO mice, cell proliferation was greater than that in wild-type animals and in uterine epithelia of 90- and 365-day-old βERKO mice. In addition, p27 protein, known as a cyclin-dependent kinase inhibitor, was decreased in the uteri of 90- and 365-day-old βERKO mice. Inhibition of neonatal ERs by ICI 182780 (an ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90-day-old mice but not in the vaginal epithelium. These results suggest that neonatal ERβ is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ERβ function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

Estrogen receptor β exon 3-deleted mouse: The importance of non-ERE pathways in ERβ signaling

In 1998, an estrogen receptor β (ERβ) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERβ mutant mouse was generated by deleting ERβ exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ERβ. In the present study, we created an ERβ exon 3-deleted mouse (ERβ-Δex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ERβ protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ERβ antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ERβ-Δex3 protein. Moreover, with nuclear extracts from ERβ-Δex3 prostates, there was an ERβ-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ERβ-Δex3 mouse. We conclude that very little of ERβ transcriptional activity depends on binding to classical estrogen response elements (EREs).

MP31-19 ESTROGEN RECEPTOR-ALPHA IS NECESSARY FOR HORMONAL INDUCTION OF PROSTATE GROWTH IN MALE MICE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2015MP31-19 ESTROGEN RECEPTOR-ALPHA IS NECESSARY FOR HORMONAL INDUCTION OF PROSTATE GROWTH IN MALE MICE Tristan Nicholson, Jalissa Wynder, and William Ricke Tristan NicholsonTristan Nicholson More articles by this author , Jalissa WynderJalissa Wynder More articles by this author , and William RickeWilliam Ricke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1374AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgens and estrogens act in synergy to promote prostatic growth, and there is an increasing focus on the role of estrogens in benign prostatic hyperplasia (BPH). We have previously shown that adult male mice treated with a combination of testosterone and estradiol (T+E2) develop features of BPH, including bladder hypertrophy, prostate growth and bladder outlet obstruction. Previous work has shown that bladder enlargement in this model depends on intact estrogen receptor-alpha (ERα), but not estrogen receptor-beta (ERβ). Therefore, we determined the contribution of these estrogen receptors to prostate growth in male mice treated with T+E2. METHODS ERα and ERβ knockout (KO) male mice were generated on a C57bl/6 background and respective wild-type littermates served as controls. Adult (6-8 weeks) mice underwent surgical implantation of 25 mg T and 2.5 mg E2 slow release hormone pellets for 4 months and were compared to untreated mice. Following euthanasia, urogenital tracts were excised and prostate and bladder mass was determined with a precision balance. Statistical analysis consisted of one-way ANOVA with the Bonferroni post-hoc comparison. RESULTS Hormonal (T+E2) treatment of wild-type mice caused a 30% increase in prostate mass relative to untreated control mice (P < 0.05), which was associated with bladder hypertrophy (P < 0.05). The combination of T+E2 induced similar increased prostate (P < 0.001) and bladder mass in ERβKO mice (P < 0.001) relative to untreated mice. However, in ERαKO mice, T+E2 treatment did not cause increased prostate or bladder mass relative to untreated control ERαKO mice. CONCLUSIONS Hormonally induced prostate growth was associated with bladder hypertrophy in male mice treated with T+E2. The present results support the conclusion that ERα, but not ERβ, is necessary for hormonal induction of prostate growth in male mice treated with T+E2. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e363 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tristan Nicholson More articles by this author Jalissa Wynder More articles by this author William Ricke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Roles of estrogen in the formation of intracranial aneurysms in ovariectomized female mice.

Epidemiological studies have indicated that postmenopausal women have a higher incidence of intracranial aneurysms than men in the same age group. To investigate whether estrogen or estrogen receptors (ERs) mediate protective effects against the formation of intracranial aneurysms. Intracranial aneurysms were induced in mice by combining a single injection of elastase into the cerebrospinal fluid with deoxycorticosterone acetate salt hypertension. The mice were treated with estrogen (17β-estradiol), an ERα agonist (propyl pyrazole triol), and an ERβ agonist (diarylpropionitrile) with and without a nitric oxide synthase inhibitor. The ovariectomized female mice had a significantly higher incidence of aneurysms than the male mice, which was consistent with findings in previous epidemiological studies. In ovariectomized female mice, an ERβ agonist, but not an ERα agonist or 17β-estradiol, significantly reduced the incidence of aneurysms. The protective effect of the ERβ agonist was absent in the ovariectomized ERβ knockout mice. The protective effect of the ERβ agonist was negated by treatment with a nitric oxide synthase inhibitor. The effects of sex, menopause, and estrogen treatment observed in this animal study were consistent with previous epidemiological findings. Stimulation of estrogen receptor-β was protective against the formation of intracranial aneurysms in ovariectomized female mice.

Estrogen receptor β: the guardian of the endometrium.

The endometrium is the primary target organ for the 'female' sex steroid hormone estrogen, which exerts effects in the endometrium via two main classical estrogen receptor (ER) isoforms, ERα and ERβ. The main function of the endometrium, embryo implantation, appears unperturbed in ERβ knockout mice, which has led researchers to disregard other potentially important functional roles that ERβ may have in endometrium. This review focuses on ERβ in the human endometrium and its protective role from the undesired effects of ERα. We conducted a systematic search using PubMed and Ovid for publications between January 1996 and February 2014. All studies that examined ERβ expression or function in non-pregnant endometrium or cells derived from the endometrium were considered, including human and animal studies. Studies of the basic function of ERβ isoforms in restraining ERα-mediated cell-specific trophic/mitotic responses to estrogen in other tissues has allowed appreciation of the important potential role of ERβ in the regulation of cell fate in the human endometrium. Our current understanding of ERβ expression and function in endometrium is, however, incomplete. ERβ is dynamically expressed in healthy premenstrual endometrium, persists in post-menopausal atrophic endometrium and may play an important role in endometrial disease. All endometrial cell types express ERβ and aberrations in ERβ expression have been reported in almost all benign and malignant endometrial proliferative disease. The collective evidence suggests that ERβ has an important role in normal endometrial function and also in most, if not all, benign and malignant endometrial diseases. However, the conduct of studies of endometrial ERβ expression needs to be standardized: agreement is needed regarding the most appropriate control tissue for endometrial cancer studies as well as development of standardized methods for the quantification of ERβ immunohistochemical data, similar to those scoring systems employed for other hormonally regulated tissues such as breast cancer, since these data may have direct clinical implications in guiding therapy.

Abstract 2118: Differential efficiency in the deletion of estrogen receptor β DNA binding domain generates distinct phenotypes in the mouse ventral prostate and ovary

Abstract The anti-proliferative properties of estrogen receptor β (ERβ) and its ability to regulate growth and differentiation of the prostate epithelium make it interesting as a therapeutic target in prostate cancer. In 1998, the first ERβ knockout (ERβ-/-) mouse was created by inserting a neocassette into the ERβ gene to interrupt the gene at the DNA binding domain (DBD). In this original knockout, aging ERβ-/- mice develop prostate hyperplasia and upon estradiol treatment, they also develop prostatic intraepithelial neoplasia lesions, which are believed to be premalignant. These mice were also characterized by a reduction in the ability to ovulate and disruption of neuronal migration during fetal development. In 2008, another ERβ mutant mouse was generated by inserting LoxP sites around the third exon of the gene. Upon deletion of exon 3, the female mice were infertile, due to the inability of the female mice to ovulate but there were no other defects in either males or females. Thus it was concluded that, ERβ has very limited physiological function and the phenotype of the original ERβ-/- mouse deemed to be due to the presence of the neocassette which remained in the gene. In the present study, a new ERβ knockout mouse was generated by inserting Lox P sites around the third exon and this exon was deleted by crossing this mouse with either a CMV-Cre or a Rosa-Cre deleter mouse. The only abnormality found in the new mutant ERβ mouse was the inability of the female mice to ovulate. Sequencing of the ovary mRNA showed that the knockout strategy worked i.e., exon 3 was deleted and a shift in the reading frame was generated, resulting in the creation of two in frame stop codons. Nevertheless, in this new mutant mouse, there were no detectable defects in the prostate, the lungs or the brain. We demonstrated that although two stop codons should have prevented translation of the protein, ventral prostates, lungs and skeletal muscle of these mice produce a full length ERβ protein. This protein was detected in the nuclei of prostate epithelial cells by immunohistochemistry and western blot assays, using a set of four anti-ERβ antibodies directed against different epitopes. In contrast to the original knockout mouse, the expression of Ki67 and androgen receptor in the prostate epithelium was unchanged in the new ERβ-/- mouse. Moreover, DNA binding of prostate nuclear extracts from these mice to Estrogen Responsive Elements and AP-1 responsive elements was inhibited by ERβ-specific antibodies. We conclude that differential efficiency in the deletion of ERβ DBD generates divergent phenotypes in the mouse ventral prostate and ovary and this discrepancy may be mediated by tissue variations in the efficiency of the Cre recombinase system. Citation Format: Laure Maneix, Per Antonson, Sabrina S. Rochel-Maia, Hyun-Jin Kim, Margaret Warner, Jan-Ake Gustafsson. Differential efficiency in the deletion of estrogen receptor β DNA binding domain generates distinct phenotypes in the mouse ventral prostate and ovary. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2118. doi:10.1158/1538-7445.AM2014-2118

Modification of female and male social behaviors in estrogen receptor beta knockout mice by neonatal maternal separation.

Maternal separation (MS) is an animal model mimicking the effects of early life stress on the development of emotional and social behaviors. Recent studies revealed that MS stress increased social anxiety levels in female mice and reduced peri-pubertal aggression in male mice. Estrogen receptor (ER) β plays a pivotal role in the regulation of stress responses and anxiety-related and social behaviors. Behavioral studies using ERβ knockout (βERKO) mice reported increased social investigation and decreased social anxiety in βERKO females, and elevated aggression levels in βERKO males compared to wild-type (WT) mice. In the present study, using βERKO and WT mice, we examined whether ERβ contributes to MS effects on anxiety and social behaviors. βERKO and WT mice were separated from their dam daily (4 h) from postnatal day 1–14 and control groups were left undisturbed. First, MS and ERβ gene deletion individually increased anxiety-related behaviors in the open field test, but only in female mice. Anxiety levels were not further modified in βERKO female mice subjected to MS stress. Second, βERKO female mice showed higher levels of social investigation compared with WT in the social investigation test and long-term social preference test. However, MS greatly reduced social investigation duration and elevated number of stretched approaches in WT and βERKO females in the social investigation test, suggesting elevated levels of social anxiety in both genotypes. Third, peri-pubertal and adult βERKO male mice were more aggressive than WT mice as indicated by heightened aggression duration. On the other hand, MS significantly decreased aggression duration in both genotypes, but only in peri-pubertal male mice. Altogether, these results suggest that βERKO mice are sensitive to the adverse effects of MS stress on subsequent female and male social behaviors, which could then have overrode the ERβ effects on female social anxiety and male aggression.

Oestradiol-induced synapse formation in the female hippocampus: roles of oestrogen receptor subtypes.

During the oestrus cycle, varying spine synapse density correlates positively with varying local synthesis of oestradiol in the hippocampus. In this context, the roles of the oestrogen receptor (ER) subtypes ERα and β are not fully understood. In the present study, we used neonatal hippocampal slice cultures from female rats because these cultures synthesise oestradiol and express both receptor subtypes, and inhibition of oestradiol synthesis in these cultures results in spine synapse loss. Using electron microscopy, we tested the effects on spine synapse density in response to agonists of both ERα and ERβ. Application of agonists to the cultures had no effect. After inhibition of oestradiol synthesis, however, agonists of ERα induced spine synapse formation, whereas ERβ agonists led to a reduction in spine synapse density in the CA1 region of these cultures. Consistently, up-regulation of ERβ in the hippocampus of adult female aromatase-deficient mice is paralleled by hippocampus-specific spine synapse loss in this mutant. Finally, we found an increase in spine synapses in the adult female ERβ knockout mouse, but no effect in the adult female ERα knockout mouse. Our data suggest antagonistic roles of ERβ and ERα in spine synapse formation in the female hippocampus, which may contribute to oestrus cyclicity of spine synapse density in the hippocampus.

Estrogen modulates cardiac growth through an estrogen receptor α-dependent mechanism in healthy ovariectomized mice

The modulation of cardiac growth by estrogen in healthy mice is not completely understood. The aim was to investigate the effects of estrogen on cardiac growth in healthy mice lacking either estrogen receptor (ER) α or β. Wild-type (WT), ERα knockout (ERKO) and ERβ knockout (BERKO) 2-month-old mice were ovariectomized and randomly assigned to groups receiving an estradiol (E2)-containing or soy-free (control, CON) diet (n=5–7/group). After three months of E2 administration, WT and BERKO mice had significantly lower body weight, higher relative uterus and heart weight than CON mice, while there was no major E2 effect in ERKO mice. Furthermore, there was a higher concentration of E2-responsive genes Igf1 and Myocd in WT and BERKO but not in ERKO mice. Together, these findings indicate that the estrogenic regulation of cardiac growth in healthy mice is primarily mediated through ERα and not ERβ.

Suppression of ERβ signaling via ERβ knockout or antagonist protects against bladder cancer development

Epidemiological studies showed that women have a lower bladder cancer (BCa) incidence, yet higher muscle-invasive rates than men, suggesting that estrogen and the estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), may play critical roles in BCa progression. Using in vitro cell lines and an in vivo carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced mouse BCa model, we found that ERβ plays a positive role in promoting BCa progression. Knockdown of ERβ with ERβ-shRNA in ERβ-positive human BCa J82, 647v and T24 cell lines led to suppressed cell growth and invasion. Mice lacking ERβ have less cancer incidence with reduced expression of the proliferation marker Ki67 in BBN-induced BCa. Consistently, our results show that non-malignant urothelial cells with ERβ knockdown are more resistant to carcinogen-induced malignant transformation. Mechanism dissection found that targeting ERβ suppressed the expression of minichromosome maintenance complex component 5 (MCM5), a DNA replication licensing factor that is involved in tumor cell growth. Restoring MCM5 expression can partially reverse ERβ knockdown-mediated growth reduction. Supportively, treating cells with the ERβ-specific antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), reduced BCa cell growth and invasion, as well as MCM5 expression. Furthermore, we provide the first evidence that BCa burden and mortality can be controlled by PHTPP treatment in the carcinogen-induced BCa model. Together, these results demonstrate that ERβ could play positive roles in promoting BCa progression via MCM5 regulation. Targeting ERβ through ERβ-shRNA, PHTPP or via downstream targets, such as MCM5, could serve as potential therapeutic approaches to battle BCa.

ERα regulates lipid metabolism in bone through ATGL and perilipin

A decrease in bone mineral density during menopause is accompanied by an increase in adipocytes in the bone marrow space. Ovariectomy also leads to accumulation of fat in the bone marrow. Herein we show increased lipid accumulation in bone marrow from estrogen receptor alpha (ERα) knockout (ERαKO) mice compared to wild-type (WT) mice or estrogen receptor beta (ERβ) knockout (ERβKO) mice. Similarly, bone marrow cells from ERαKO mice differentiated to adipocytes in culture also have increased lipid accumulation compared to cells from WT mice or ERβKO mice. Analysis of individual adipocytes shows that WT mice have fewer, but larger, lipid droplets per cell than adipocytes from ERαKO or ERβKO animals. Furthermore, higher levels of adipose triglyceride lipase (ATGL) protein in WT adipocytes correlate with increased lipolysis and fewer lipid droplets per cell and treatment with 17β-estradiol (E2) potentiates this response. In contrast, cells from ERαKO mice display higher perilipin protein levels, promoting lipogenesis. Together these results demonstrate that E2 signals via ERα to regulate lipid droplet size and total lipid accumulation in the bone marrow space in vivo.

Abstract LB-256: Estrogen and its receptors alpha and beta in different stages of breast carcinogenesis.

Abstract Excessive exposure to Estrogen has long been indicated to increase breast cancer risk. Although the anti-estrogen endocrine therapy by selective estrogen receptor modulator (SERM) is widely used to treat the ERα-positive breast cancers, there are several mysteries in the field: 1) Some ERα positive breast cancers do not respond to tamoxifen; 2) Some ERα negative breast cancers also respond to tamoxifen; 3) Strikingly, physiological estrogen can cause dramatic tumor regression of long-term tamoxifen-resistant ERα-positive breast cancer; 4) When ERα is overexpressed in genetically engineered mice, mammary tumors form but they do not progress to invasive cancer, which indicates that the activation of ERα alone does not fully explain the action of estrogen. To uncover these mysteries, we focus on the following two main points: 1) Role of ERβ, the second estrogen receptor, which is the more abundant ER in the normal breast. Estradiol, the main type of estrogen, binds with ERα and ERβ with the similar affinity, but there are quite differences for some SERMs. 2) Differential function of two ERs in each stage of breast carcinogenesis. Breast cancer is a heterogeneous disease and also is a chronically developed disease, which occurs through a series of steps. The tumor cells in different development steps have distinct molecular profiles and clinical behaviors. First we used a large set of well-documented surgical excised human ductal breast cancer samples and carefully examined the tumor histology and the expression of ERα and ERβ by IHC staining. Most of these samples contained normal areas as well as areas of hyperplasia, ductal carcinoma in situ (DCIS) and invasive ductal cancer (IDC). We compared ERα and ERβ expression in different cancer stages within each woman. Our results suggest that ERβ loss is an early event in breast carcinogenesis, ERα has dual role in cancer proliferation and invasion, and ERβ/ERα ratio seems to be one of the determinants for the breast cancer initiation and progression to malignancy. Then we use inducible ERα transgenic mouse model to verify the findings in the human samples study. This ERα transgenic mouse develops DCIS, but not IDC. We use estradiol as well as several specific ligands for ERα and ERβ to treat this mouse and also generate a unique animal model by crossing ERα transgenic mouse with ERβ knockout mice to address the differential role of ERα and ERβ in breast cancer initiation and progression. The clinical application of our findings will be discussed. Citation Format: Bo Huang, Yoko Omoto, Hirotaka Iwase, Hiroko Yamashita, Tatsuya Toyama, Margaret Warner, Jan-Åke Gustafsson. Estrogen and its receptors alpha and beta in different stages of breast carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-256. doi:10.1158/1538-7445.AM2013-LB-256

The Role of Estrogen Receptor β in Transplacental Cancer Prevention by Indole-3-Carbinol

In the present study, the efficacy of indole-3-carbinol (I3C), a key bioactive component of cruciferous vegetables, for prevention of cancer in offspring exposed in utero to the environmental carcinogen dibenzo[def,p]chrysene (DBC) was evaluated using an estrogen receptor β (ERβ) knockout mouse model. I3C was provided either through the maternal diet coincident with carcinogen exposure during pregnancy or directly to offspring postinitiation with DBC. I3C was effective at reducing T-cell acute lymphoblastic lymphoma/leukemia (T-ALL)-related mortality in offspring only if provided via the maternal diet, although a gender difference in the role of ERβ in mediating this response was evident. In female offspring, chemoprevention of T-ALL by maternal dietary I3C required expression of ERβ; survival in Esr2 wild-type and heterozygous female offspring was more than 90% compared with 66% in Esr2 null females. Alternatively, ERβ status did not significantly impact the transplacental chemoprevention by I3C in males. The possible role of ERβ in mediating lung carcinogenesis or chemoprevention by I3C was similarly complicated. Lung tumor incidence was unaltered by either dietary intervention, whereas lung tumor multiplicity was substantially reduced in Esr2 null females on the control diet and marginally lower in Esr2 null males exposed to I3C via the maternal diet compared with their wild-type and heterozygous counterparts. These findings suggest that I3C may act via ERβ to prevent or suppress DBC-initiated transplacental carcinogenesis but that the involvement of this receptor seems to differ depending on the cancer type and gender of the offspring.