ERα Knockout Mice Research Articles

The Bone Sparing Effects of 2-Methoxyestradiol Are Mediated via Estrogen Receptor-α in Male Mice.

2-Methoxyestradiol (2ME2), a metabolite of 17β-estradiol (E2), exerts bone sparing effects in animal models. We hypothesized that the underlying mechanism is back conversion of 2ME2 to E2, which subsequently acts via estrogen receptor (ER)α. We measured serum E2 levels in orchidectomized wild-type (WT) mice treated with 2ME2 66.6 μg/d or placebo. In placebo-treated animals, E2 was below the detection limit. In 2ME2-treated mice, the serum E2 level was 4.97 ± 0.68 pg/mL. This corresponds to the level found in diesterus in cycling female mice. Next, we investigated bone parameters in orchidectomized WT and ERα knockout mice treated with 2ME2 or placebo for 35 days. 2ME2 (6.66 μg/d) preserved trabecular and cortical bone in WT mice. Trabecular volumetric-bone mineral density was 64 ± 20%, and trabecular bone volume/total volume was 60 ± 20% higher in the metaphyseal region of the femur in the 2ME2 group, compared with placebo (P < .01). Both trabecular number and trabecular thickness were increased (P < .01). Cortical bone mineral content in the diaphyseal region of the femur was 31 ± 3% higher in the 2ME2 group, compared with placebo (P < .001). This was due to larger cortical area (P < .001). Three-point bending showed an increased bone strength in WT 2ME2-treated animals compared with placebo (maximum load [Fmax] +19±5% in the 2ME2 group, P < .05). Importantly, no bone parameter was affected by 2ME2 treatment in ERα knockout mice. In conclusion, 2ME2 treatment of orchidectomized mice results in increased serum E2. ERα mediates the bone sparing effects of 2ME2. The likely mediator of this effect is E2 resulting from back conversion of 2ME2.

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.

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.

Inhibition of p38 pathway-dependent MPTP-induced dopaminergic neurodegeneration in estrogen receptor alpha knockout mice

Approximately, 7–10 million people in the world suffer from Parkinson's disease (PD). Recently, increasing evidence has suggested the protective effect of estrogens against nigrostriatal dopaminergic damage in PD. In this study, we investigated whether estrogen affects 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral impairment in estrogen receptor alpha (ERα)-deficient mice. MPTP (15mg/kg, four times with 1.5-h interval)-induced dopaminergic neurodegeneration was evaluated in ERα wild-type (WT) and knockout (KO) mice. Larger dopamine depletion, behavioral impairments (Rotarod test, Pole test, and Gait test), activation of microglia and astrocytes, and neuroinflammation after MPTP injection were observed in ERα KO mice compared to those in WT mice. Immunostaining for tyrosine hydroxylase (TH) after MPTP injection showed fewer TH-positive neurons in ERα KO mice than WT mice. Levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC, metabolite of dopamine) were also lowered in ERα KO mice after MPTP injection. Interestingly, a higher immunoreactivity for monoamine oxidase (MAO) B was found in the substantia nigra and striatum of ERα KO mice after MPTP injection. We also found an increased activation of p38 kinase (which positively regulates MAO B expression) in ERα KO mice. In vitro estrogen treatment inhibited neuroinflammation in 1-methyl-4-phenyl pyridium (MPP+)-treated cultured astrocyte cells; however, these inhibitory effects were removed by p38 inhibitor. These results indicate that ERα might be important for dopaminergic neuronal survival through inhibition of p38 pathway.

Estrogen receptors alpha mediates postischemic inflammation in chronically estrogen-deprived mice

Estrogens are known to exert neuroprotective and immuneomodulatory effects after stroke. However, at present, little is known about the role of estrogens and its receptors in postischemic inflammation after menopause. Here, we provide important in vivo evidence of a distinct shift in microglial phenotypes in the model of postmenopause brain. Using a model-system for live imaging of microglial activation in the context of chronic estrogen- and ERα-deficiency associated with aging, we observed a marked deregulation of the TLR2 signals and/or microglial activation in ovariectomized and/or ERα knockout mice. Further analysis revealed a 5.7-fold increase in IL-6, a 4.7-fold increase in phospho-Stat3 levels suggesting an overactivation of JAK/STAT3 pathway and significantly larger infarction in ERα knockouts chronically deprived of estrogen. Taken together, our results suggest that in the experimental model of menopause and/or aging, ERα mediates innate immune responses and/or microglial activation, and ischemia-induced production of IL-6. Based on our results, we propose that the loss of functional ERα may lead to deregulation of postischemic inflammatory responses and increased vulnerability to ischemic injury in aging female brains.

Estrogen Receptor-α Knockout Mice.

Tissue specific knockout mice are valuable tools to study gene function in vivo. The method uses the Cre/loxP system in which loxP sites are cloned into the genome surrounding one or more exons of a gene and the targeted exon(s) are deleted when the Cre enzyme is expressed. Mouse lines that are prepared for the generation of knockout ERα mice have been developed independently by many research groups and the number of available transgenic mouse lines that express Cre under tissue specific promoters is large. Here, we describe how tissue specific ERα knockout mice are generated.

Identification of proteins highly expressed in uterine fluid from mice with hydrometra

Estrogen receptor alpha (ERα) is an important regulator of the estrous cycle and mice with global ERα deletion, as well as some conditional knockout mouse lines, have an interruption in the estrous cycle. In this study we observed that conditional ERα knockout mice where the Cre gene is regulated by the rat insulin promoter (RIP), RIP-Cre/ERαKO mice, have a 3.7-fold increase in serum 17β-estradiol levels, blocked estrous cycle, and develop a fluid-filled uterus (hydrometra). Using a proteomics approach, we identified three proteins, lactoferrin, complement C3 and chitinase 3-like protein 1 (CHI3L1), as highly expressed proteins in hydrometra fluid. The mRNA levels of the corresponding genes were more than 50-fold higher in RIP-Cre/ERαKO uterus compared to controls. High expression of CHI3L1 in the uterine fluid was not reflected as elevated levels in the serum. The high expression of lactoferrin, complement C3 and CHI3L1 in the uterine fluid, in association with elevated estrogen levels, prompted us to address if the expression of these genes is related to reproduction. However, gonadotropin treatment of mice reduced the uterine expression of these genes in a model of in vitro fertilization. Our findings identify lactoferrin, complement C3 and CHI3L1 as highly expressed proteins in hydrometra fluid in association with chronically elevated serum estradiol levels.

Abstract 2876: Supplemental estrogen protects against obesity induced mammary gland inflammation in mice

Abstract Obesity is a risk factor for the development of several malignancies including hormone receptor-positive breast cancer in postmenopausal women and has been associated with an increased risk of recurrence and reduced survival. A link between obesity, breast inflammation and hormone receptor-positive breast cancer was unknown. Obesity causes subclinical inflammation in visceral and subcutaneous white adipose tissue (WAT), characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures (CLS). Estrogen synthesis is catalyzed by aromatase. Previously, we demonstrated CLS and elevated levels of pro-inflammatory mediators and aromatase in the mammary glands (MG) of obese mice and breast tissue of obese women. Recent clinical data suggests that supplemental estrogen given as monotherapy may reduce the risk of breast cancer. Here we tested the hypothesis that supplemental estrogen could prevent or reverse WAT inflammation (WATi) and related molecular changes in the MG of mice. In a prevention study, female C57BL/6J mice were ovariectomized (OVX) to simulate the postmenopausal state. The OVX mice received subcutaneous 17β estradiol (E2) or placebo prior to being initiated on a high fat diet (HFD). E2 supplementation protected mice against HFD-induced weight gain at least, in part, by suppressing caloric consumption. Suppression of weight gain was associated with a marked decrease in MG WATi (CLS/cm2) and a corresponding reduction in adipocyte diameter. Furthermore, expression of pro-inflammatory mediators (Cox-2, TNF-α, IL-1β) and aromatase were also reduced in the MG of mice that received supplemental E2. Next, to determine whether E2 supplementation can also reverse WATi, obese OVX mice were treated with either E2 or placebo and then continued on HFD. E2 supplementation led to physiologically relevant concentrations of 17β estradiol and caused weight loss along with a reversal in MG inflammation (CLS/cm2). Caloric consumption, adipocyte hypertrophy and MG expression of pro-inflammatory mediators and aromatase were also reduced with E2 treatment. Finally, we determined whether the protective effects of E2 were mediated by estrogen receptor-α (ERα), which has been suggested to play a key role in metabolic processes. Interestingly, ovary intact ERα knockout mice gained more weight and consumed more calories than HFD fed ovary intact wild-type mice. These changes were paralleled by adipocyte hypertrophy, WATi and elevated levels of pro-inflammatory mediators and aromatase in the MG. Collectively, our findings indicate that estrogen via ERα protects against weight gain, CLS formation and associated increases in pro-inflammatory mediators and aromatase in the MG. Whether similar effects occur in women warrants further investigation. Citation Format: Priya Bhardwaj, Baoheng Du, Xi Kathy Zhou, Erika Sue, Dilip Giri, Michael D. Harbus, Domenick J. Falcone, Clifford A. Hudis, Kotha Subbaramaiah, Andrew J. Dannenberg. Supplemental estrogen protects against obesity induced mammary gland inflammation in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2876. doi:10.1158/1538-7445.AM2015-2876

Estrogen receptor alpha is required for macrophage iron sequestration and homeostasis in female mice (INM1P.438)

Abstract Sexual dimorphisms in immunity are known to confer protection against infections at the cost of heightened risk for autoimmunity in women. The female sex hormone, estradiol, can modulate both pro/anti-inflammatory macrophage signals and the mechanisms of action remain unclear. Iron sequestration by macrophages is fundamentally important to limit bacterial infection, and since estradiol is thought to influence iron metabolism in females, we asked whether ERα impacts the innate immune response by regulating macrophage iron metabolism. To address this question, we crossed floxed Esr1 (fl/fl control) and LysM-Cre transgenic animals to generate myeloid-specific ERα knockout (MACER) mice. We studied the effect of ERα deletion on iron homeostasis upon endotoxin treatment in female MACER and fl/fl conrols. MACER mice had increased serum iron (92%; p=0.01) compared to fl/fl under basal conditions. As expected, endotoxin treatment reduced serum iron levels in fl/fl controls (41%, p=.008), however there was no change in serum iron in MACER mice. Consistent with impaired macrophage iron homeostasis, expression of genes associated with iron absorption (TfR, Slc11a2, Cybrd1), efflux (Fpn), and storage (Ftl) were significantly altered in female MACER mice compared to fl/fl controls. Together, these data suggest that ERα regulates macrophage iron metabolism in females, is required for systemic iron homeostasis, and may play a previously unrecognized role in female innate immunity.

MP68-13 ESTROGEN RECEPTOR ALPHA PREVENTS BLADDER CANCER VIA INPP4B INHIBITED AKT PATHWAY IN VITRO AND IN VIVO

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2015MP68-13 ESTROGEN RECEPTOR ALPHA PREVENTS BLADDER CANCER VIA INPP4B INHIBITED AKT PATHWAY IN VITRO AND IN VIVO Chiuan-Ren Yeh, Iawen Hsu, Hiroshi Miyamoto, Xue-Ru Wu, Chawnshang Chang, Elizabeth Guancial, Edward M. Messing, and Shuyuan Yeh Chiuan-Ren YehChiuan-Ren Yeh More articles by this author , Iawen HsuIawen Hsu More articles by this author , Hiroshi MiyamotoHiroshi Miyamoto More articles by this author , Xue-Ru WuXue-Ru Wu More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Elizabeth GuancialElizabeth Guancial More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2475AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERa in BCa development remains largely unknown. METHODS To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERaKO) and used the pre- carcinogen BBN to induce BCa. Immunoblotting and Immunohistochemistry (IHC) were used to detect phospho-AKT, INPP4B, ERα and p27. Malignant transformation assay and Q-PCR, lentivial shRNA were applied for functional characterization. RESULTS Earlier reports indicated that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERa could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. CONCLUSIONS Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e862 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chiuan-Ren Yeh More articles by this author Iawen Hsu More articles by this author Hiroshi Miyamoto More articles by this author Xue-Ru Wu More articles by this author Chawnshang Chang More articles by this author Elizabeth Guancial More articles by this author Edward M. Messing More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Estrogen Receptor (ER)α-regulated Lipocalin 2 Expression in Adipose Tissue Links Obesity with Breast Cancer Progression

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity.

Estradiol decreases taurine level by reducing cysteine sulfinic acid decarboxylase via the estrogen receptor-α in female mice liver.

Cysteine sulfinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO) are two rate-limiting enzymes in taurine de novo synthesis, and their expressions are associated with estrogen concentration. The present study was designed to determine the relationship between 17β-estradiol (E₂) and taurine in female mice liver. We initially observed the mice had lower levels of CSAD, CDO, and taurine during estrus than diestrus. We then, respectively, treated the ovariectomized mice, the cultured hepatocytes, and Hep G2 cells with different doses of E₂, and the CSAD and CDO expressions and taurine levels were analyzed. The results showed that E₂ decreased taurine level in the serum and the cultured cells by inhibiting CSAD and CDO expressions. Furthermore, we identified the molecular receptor types through which E₂ plays its role in regulating taurine synthesis, and our results showed that estrogen receptor-α (ERα) expression was much higher than estrogen receptor-β (ERβ) in the liver and hepatocytes, and the inhibiting effects of E₂ on CSAD, CDO, and taurine level were partially abrogated in the ICI-182,780-pretreated liver and hepatocytes, and in ERα knockout mice. These results indicate that estradiol decreases taurine content by reducing taurine biosynthetic enzyme expression in mice liver.

Memory Impairment in Estrogen Receptor α Knockout Mice Through Accumulation of Amyloid-β Peptides.

Estrogen has been known to reduce the development of Alzheimer’s disease (AD). However, exact mechanisms are not clear. We investigated whether estrogen can increase amyloid-beta (Aβ) degradation and affects Aβ-induced memory impairment in an estrogen deficiency model. Estrogen receptor alpha (ERα) knockout mice and wild-type mice were intracerebroventricular (ICV) infused with Aβ (300 pmol) for 2 weeks. Cognitive function was then assessed by the Morris water maze test and passive avoidance test. In addition, Western blot analysis, immunostaining, immunofluorescence staining, ELISA, and enzyme activity assays were used to examine the degree of Aβ deposition in the brains of ERα knockout mice. In our present study, Aβ was accumulated more in the ERα knockout mice brain and greatly worsened memory impairment and glial activation as well as neurogenic inflammation. These results suggest that estrogen may protect memory impairment by stimulating the degradation of Aβ and down-regulate neurogenic inflammation as well as amyloidogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-014-8853-z) contains supplementary material, which is available to authorized users.

Estrogen receptor alpha prevents bladder cancer via INPP4B inhibited akt pathway in vitro and in vivo.

Clinical reports show males have a higher bladder cancer (BCa) incidence than females. The sexual difference of BCa occurrence suggests that estrogen and its receptors may affect BCa development. Estrogen receptor alpha (ERα) is the classic receptor to convey estrogen signaling, however, the function of ERα in BCa development remains largely unknown. To understand the in vivo role of ERα in BCa development, we generated total and urothelial specific ERα knockout mice (ERαKO) and used the pre- carcinogen BBN to induce BCa. Earlier reports showed that ERα promotes breast and ovarian cancers in females. Surprisingly and of clinical importance, our results showed that ERα inhibits BCa development and loss of the ERα gene results in an earlier onset and higher incidence of BBN-induced in vivo mouse BCa. Supportively, carcinogen induced malignant transformation ability was reduced in ERα expressing urothelial cells as compared to ERα negative cells. Mechanism studies suggest that ERα could control the expression of INPP4B to reduce AKT activity and consequently reduce BCa cell growth. In addition, IHC staining of clinical sample analyses show that INPP4B expression, in correlation with reduced ERα, is significantly reduced in human BCa specimens. Together, this is the first report using the in vivo cre-loxP gene knockout mouse model to characterize ERα roles in BCa development. Our studies provide multiple in vitro cell studies and in vivo animal model data as well as human BCa tissue analyses to prove ERα plays a protective role in BCa initiation and growth at least partly via modulating the INPP4B/Akt pathway.

Estrogen receptor α promotes non-amyloidogenic processing of platelet amyloid precursor protein via the MAPK/ERK pathway

Deposition of amyloid β peptide (Aβ), a proteolytic product of amyloid precursor protein (APP), in senile plaques and in the walls of cerebral blood vessels is a hallmark of Alzheimer's disease (AD). Platelets contain high levels of APP and Aβ and may contribute to amyloid deposits seen in AD. However, the biochemical mechanism(s) involved in the regulation of platelet APP metabolism are largely unknown. The estrogen receptor α (ERα) is found to be expressed in platelets. It has not been elucidated whether ERα-mediated non-genomic signaling intervenes with platelet APP processing. Using ERα knock-out (α-ERKO) mice and wild type (WT) littermates, the present study demonstrated that ERα-specific agonist propylpyrazole triol (PPT) promoted non-amyloidogenic processing of platelet APP via the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) pathway. The underlying basis involves direct association of activated ERK with a disintegrin and metalloprotease domain 17 (ADAM17, an α-secretase candidate) and ERK-dependent threonine phosphorylation of ADAM17. These results suggest that selective modulation of ERα in peripheral target tissues may serve as an anti-amyloidogenic strategy for AD and other amyloidogenic diseases.

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 signaling prevents diet-induced hepatic insulin resistance in male mice with obesity

The development of insulin resistance in the liver is a key event that drives dyslipidemia and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor-α (ERα). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ERα signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ERα-knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared with their wild-type floxed (fl/fl) littermates during hyperinsulinemic euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of Akt-Ser(473) and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin-stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ERα in the liver helps prevent whole body and hepatic insulin resistance associated with HFD feeding in males. Augmenting hepatic estrogen signaling through ERα may lessen the impact of obesity on diabetes and cardiovascular risk in males.

Estrogen receptor ligands ameliorate fatty liver through a nonclassical estrogen receptor/Liver X receptor pathway in mice

Liver X receptor (LXR) activation stimulates triglyceride (TG) accumulation in the liver. Several lines of evidence indicate that estradiol-17β (E2) reduces TG levels in the liver; however, the molecular mechanism underlying the E2 effect remains unclear. Here, we show that administration of E2 attenuated sterol regulatory element-binding protein (SREBP)-1 expression and TG accumulation induced by LXR activation in mouse liver. In estrogen receptor alpha (ERα) knockout (KO) and liver-specific ERα KO mice, E2 did not affect SREBP-1 expression or TG levels. Molecular analysis revealed that ERα is recruited to the SREBP-1c promoter through direct binding to LXR and inhibits coactivator recruitment to LXR in an E2-dependent manner. Our findings demonstrate the existence of a novel liver-dependent mechanism controlling TG accumulation through the nonclassical ER/LXR pathway. To confirm that a nonclassical ER/LXR pathway regulates ERα-dependent inhibition of LXR activation, we screened ERα ligands that were able to repress LXR activation without enhancing ERα transcriptional activity, and, as a result, we identified the phytoestrogen, phloretin. In mice, phloretin showed no estrogenic activity; however, it did reduce SREBP-1 expression and TG levels in liver of mice fed a high-fat diet to an extent similar to that of E2. We propose that ER ligands reduce TG levels in the liver by inhibiting LXR activation through a nonclassical pathway. Our results also indicate that the effects of ER on TG accumulation can be distinguished from its estrogenic effects by a specific ER ligand.

The Islet Estrogen Receptor-α Is Induced by Hyperglycemia and Protects Against Oxidative Stress-Induced Insulin-Deficient Diabetes

The female steroid, 17β-estradiol (E2), is important for pancreatic β-cell function and acts via at least three estrogen receptors (ER), ERα, ERβ, and the G-protein coupled ER (GPER). Using a pancreas-specific ERα knockout mouse generated using the Cre-lox-P system and a Pdx1-Cre transgenic line (PERαKO−/−), we previously reported that islet ERα suppresses islet glucolipotoxicity and prevents β-cell dysfunction induced by high fat feeding. We also showed that E2 acts via ERα to prevent β-cell apoptosis in vivo. However, the contribution of the islet ERα to β-cell survival in vivo, without the contribution of ERα in other tissues is still unclear. Using the PERαKO−/− mouse, we show that ERα mRNA expression is only decreased by 20% in the arcuate nucleus of the hypothalamus, without a parallel decrease in the VMH, making it a reliable model of pancreas-specific ERα elimination. Following exposure to alloxan-induced oxidative stress in vivo, female and male PERαKO−/− mice exhibited a predisposition to β-cell destruction and insulin deficient diabetes. In male PERαKO−/− mice, exposure to E2 partially prevented alloxan-induced β-cell destruction and diabetes. ERα mRNA expression was induced by hyperglycemia in vivo in islets from young mice as well as in cultured rat islets. The induction of ERα mRNA by hyperglycemia was retained in insulin receptor-deficient β-cells, demonstrating independence from direct insulin regulation. These findings suggest that induction of ERα expression acts to naturally protect β-cells against oxidative injury.

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.