ERα Deficiency Research Articles

WITHDRAWN: Estrogen regulates duodenal glucose absorption by affecting estrogen receptor-α on glucose transporters

The mechanisms of estrogen in glucose metabolism are well established; however, its role in glucose absorption remains unclear. In this study, we investigated the effects of estrogen on glucose absorption in humans, mice, and SCBN intestinal epithelial cells. We first observed a correlation between estrogen and blood glucose in young women and found that glucose tolerance was significantly less in the premenstrual phase than in the preovulatory phase. Similarly, with decreased serum estradiol levels in ovariectomized mice, estrogen receptors alpha (ERα) and beta (ERβ) in the duodenum were reduced, and weight and abdominal fat increased significantly. The expression of sodium/glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) and glucose absorption in the duodenum decreased significantly. Estrogen significantly upregulated SGLT1 and GLUT2 expression in SCBN cells. Silencing of ERα, but not ERβ, reversed this trend, suggesting that ERα may be key to estrogen-regulating glucose transporters. A mechanistic study revealed that downstream, estrogen regulates the protein kinase C (PKC) pathway. Overall, our findings indicate that estrogen promotes glucose absorption, and estrogen and ERα deficiency can inhibit SGLT1 and GLUT2 expression through the PKC signaling pathway, thereby reducing glucose absorption.

Estrogen receptor α-mediated signaling inhibits type I interferon response to promote breast carcinogenesis.

Estrogen receptor α (ERα)is an important driver and therapeutic target in ∼70% of breast cancers. How ERαdrives breast carcinogenesis is not fully understood. In this study, we show that ERαis a negative regulator of type I interferon (IFN) response. Activation of ERα by its natural ligand estradiol inhibits IFN-β-induced transcription of downstream IFN-stimulated genes (ISGs), whereas ERα deficiency or the stimulation with its antagonist fulvestrant has opposite effects. Mechanistically, ERα induces the expression of the histone 2A variant H2A.Z to restrict the engagement of the IFN-stimulated gene factor 3 (ISGF3) complex to the promoters of ISGs and also interacts with STAT2 to disrupt the assembly of the ISGF3 complex. These two events mutually lead to the inhibition of ISG transcription induced by type I IFNs. In a xenograft mouse model, fulvestrant enhances the ability of IFN-β to suppress ERα+ breast tumor growth. Consistently, clinical data analysis reveals that ERα+ breast cancer patients with higher levels of ISGs exhibit higher long-term survival rates. Taken together, our findings suggest that ERαinhibits type I IFN response via two distinct mechanisms to promote breast carcinogenesis.

Conditional knockout of oestrogen receptor alpha in CD11c+ cells impacts female survival and inflammatory cytokine profile in murine lupus.

Oestrogen and oestrogen receptor alpha (ERα) have been implicated in systemic lupus erythematosus pathogenesis. ERα signalling influences dendritic cell (DC) development and function, as well as inflammation and downstream immune responses. We previously reported that ERα modulates multiple Toll-like receptor-stimulated pathways in both conventional and plasmacytoid DCs in lupus-prone mice. For example, CD11chi MHCII+ cell numbers are reduced in mice with global ERα deficiency or when expressing a short variant of ERα. Herein, RNA-seq analysis of CD11chi cells from bone marrow of NZM2410 mice expressing WT ERα versus ERα short versus ERα null revealed differentially expressed complement genes, interferon-related genes and cytokine signalling (e.g., IL-17 and Th17 pathways). To better understand the role of ERα in CD11c+ cells, lupus prone NZM2410 mice with selective deletion of the Esr1 gene in CD11c+ cells were generated. Phenotype and survival of these mice were similar with the exception of Cre positive (CrePos) female mice. CrePos females, but not males, all died unexpectedly prior to 35 weeks. DC subsets were not significantly different between groups. Since ERα is necessary for robust development of DCs, this result suggests that DC fate was determined prior to CD11c expression and subsequent ERα deletion (i.e., proximally in DC ontogeny). Overall, findings point to a clear functional role for ERα in regulating cytokine signalling and inflammation, suggesting that further study into ERα-mediated regulatory mechanisms in DCs and other immune cell types is warranted.

ERa-36 instead of ERa mediates the stimulatory effects of estrogen on the expression of viral oncogenes HPV E6/E7 and the malignant phenotypes in cervical cancer cells

High risk human papillomavirus (HPV) is the main causative factor of cervical cancer. In addition, estrogen and its receptors are also involved in the development of carcinogenesis. The canonical estrogen receptor ERα is frequently deficient while its variant ERα-36 is highly expressed in cervical cancer cells. The biological significance for this receptor transition from ERα to ERα-36 remains unclear. In the present study, the results of RT-PCR and Western blot demonstrated that ERα and ERα-36 function antagonistically on the expression of the viral oncogenes HPV E6 and E7. At mRNA and protein levels, ERα inhibited HPV E6/E7 expression whereas ERα-36 stimulated HPV E6/E7 expression. Overexpression of ERα-36 promoted cell proliferation while reintroduction of ERα into cervical cancer cells did not significantly affect cell proliferation which is in line with the different effects of . ERα-36 and ERα on the expression of cell cycle regulator, namely p53, p21 and cyclin D1. Furthermore, ERα suppressed whereas ERα-36 promoted the migration and invasion of cervical cancer cells, which should be related to the oppositive roles of ERα and ERα-36 in the Wnt/β-catenin/MRTF-A signaling pathway which is activated by HPV E7. Results of this study suggest that ERα functions as a tumor suppressor whereas ERα-36 is an oncoprotein in cervical cancer cells. ERα deficiency together with ERα-36 overexpression might enhance the expression of HPV E6/E7 genes and facilitate the development of cervical cancer. Targeting ERα-36 with selective antagonists should be a promising strategy for cervical cancer therapy.

Quercetin regulates ERα mediated differentiation of BMSCs through circular RNA.

Circular RNA (circRNA) participates in regulation of gene transcription, while estrogen receptor alpha (ERα) and quercetin (QUE) positively regulate bone formation, but little is known about the correlation among circRNA, ERα and QUE. In this experiment, we created an ERα-deficient rBMSC model treated with QUE and evaluated the effects of ERα or QUE on rBMSCs, then analyzed differentially-expressed circRNAs by RNA-Seq and bioinformatics. The results showed that ERα deficiency constrained osteogenic differentiation and stimulated adipocytic differentiation of rBMSCs, while QUE abrogated those effects. We identified 136 differentially-expressed circRNAs in the Lv-shERα group and 120 differentially-expressed circRNAs in the Lv-shERα+QUE group. Thirty-two circRNAs retroregulated by ERα and QUE were involved in Rap1 and Wnt signaling, and four of them together sponged miR-326-5p, the target genes of which are osteogenic and adipogenic differentiation factors. Further study showed that over-expressed miR-326-5p could stimulate osteogenic differentiation, while attenuating adipogenic differentiation of rBMSCs. Therefore, we concluded that ERα and QUE might regulate the differentiation of rBMSCs through the circRNA-miR-326-5p-mRNA axis.

295-OR: Estrogen Receptor a Controls Energy Homeostasis in White and Brown Adipose Tissue by Regulating Polg1 Expression and Mitochondrial Remodeling

Obesity is heightened during aging. Although the role of the estrogen receptor (ER)α to prevent obesity is supported by our previous work, ERα actions in adipocytes remain inadequately understood. Gene expression analysis from UKTwins Study, METSIM Study, and the study of the UCLA Hybrid Mouse Diversity Panel show that ESR1/Esr1 is inversely associated with adiposity and markers of metabolic health in both humans and mice. To determine the mechanistic role(s) of ERα in fat, we generated 2 adipose tissue-specific knockout mouse lines by selectively deleting Esr1 from white and brown adipose tissue (FERKO and ERαKOBAT, respectively). In white adipose tissue, Esr1 controls oxidative metabolism by reducing the E3 ubiquitin ligase Parkin-mediated mitochondrial degradation. Indeed, we report that adipose tissue-selective Parkin knockout mice have reduced body fat and elevated adipocyte mtDNA content. In brown adipose tissue, Esr1 is a requisite for mitochondrial remodeling and cold-induced thermogenesis. Although the role of mitochondrial metabolism differs between white and brown fat, the unique mitochondrial phenotypes observed in white and brown Esr1-deficient adipocytes, appear to be the consequence of reduced expression of the mtDNA polymerase, Polg1. We provide direct evidence that ERα binds the Polg1 promoter to control its expression, and that selective phenotypes of ERα deficiency are recapitulated by Polg1 deletion. Collectively our findings show that ERα has strong heritability in adipose tissue and support strategies targeting cell-specific actions of ERα to combat diseases associated with metabolic dysfunction. Disclosure Z. Zhou: None. A.L. Hevener: None. Funding National Institutes of Health (U54DK120342)

ERα Signaling Increased IL-17A Production in Th17 Cells by Upregulating IL-23R Expression, Mitochondrial Respiration, and Proliferation.

Women have increased prevalence of Th17-mediated autoimmune diseases, including lupus and multiple sclerosis, and severe asthma. While estradiol and progesterone increased IL-17A production in Th17 cells by inhibiting Let7f miRNA expression and increasing IL-23 receptor (IL-23R) expression, it remained unclear how estrogen signaling through the canonical nuclear receptors, estrogen receptor α (ERα) and/or ERβ, regulated this pathway. We hypothesized that estrogen signaling through ERα increased IL-23R expression and IL-17A production from Th17 cells. To test this hypothesis, naïve T cells from WT female, WT male, Esr1−/− and Esr2−/− female mice were differentiated into Th17 cells. IL-17A production and IL-23R expression were significantly increased in Th17 cells from WT female mice compared to Th17 cells from WT male mice. Deletion of ERα (Esr1−/−), but not ERβ (Esr2−/−), significantly decreased IL-17A production and IL-23R expression in Th17 cells by limiting IL-23R expression in a Let-7f dependent manner. ERα deficiency also decreased Th17 cell proliferation as well as decreased T cell metabolism as measured by ATP-linked oxygen consumption rate and proton leakage. Further, we found that Cox20 expression, a protein involved in mitochondrial respiration through assembly of cytochrome c oxidase in the electron transport chain, was increased in Th17 cells from WT female mice compared to Th17 cells from WT male and Esr1−/− female mice. Inhibition of Cox20 decreased IL-17 production in Th17 cells from WT female mice. Combined these studies showed that ERα signaling increased IL-17A production in Th17 cells by upregulating IL-23R expression and promoting mitochondrial respiration and proliferation.

Endocrine resistant breast cancer cells with loss of ERα expression retain proliferative ability by reducing caspase7-mediated HDAC3 cleavage.

Endocrine therapy is the most commonly used approach for the treatment of estrogen receptor (ERα)-positive breast cancer. The cure rate of patients with ERα-positive breast cancer is, however, limited due to the occurrence of endocrine resistance. Loss of ERα is one major mechanism for the occurrence of endocrine resistance. Recent studies have shown that pan-HDAC inhibitors may be effective in reversing endocrine resistance. However, the molecular mechanism underlying this reversal has remained largely unknown. Here we aimed to unravel this mechanism. Endocrine resistant breast cancer cell lines were established through exposure to tamoxifen. mRNA expression was assessed by qRT-PCR and protein expression by Western blotting. The effect of HDAC3 inhibition on the viability of endocrine resistant breast cancer cells was evaluated using CCK-8 and colony forming assays. Immunohistochemistry was used to detect protein expression in primary breast cancer tissues. We found that in endocrine resistant breast cancer cells loss of ERα led to HDAC3 stabilization via decreased ERα-mediated caspase7 expression, resulting in reduced caspase7-mediated HDAC3 cleavage. We also found that the ERα-caspase7-HDAC3 axis determined the global H3K9 and H4K16 acetylation status, which was positively correlated with ERα expression. Finally, we found that inhibition of HDAC3 significantly decreased the viability of endocrine resistant breast cancer cells exhibiting ERα deficiency. The ERα-caspase7-HDAC3 axis was subsequently verified in primary endocrine resistant breast cancer samples. From our data we conclude that the ERα-caspase7-HDAC3 axis may play a role in promoting the proliferation of endocrine resistant breast cancer cells. HDAC3 may serve as a therapeutic target for (a subset of) endocrine resistant breast cancers exhibiting ERα loss.

The zinc-finger transcriptional factor Slug transcriptionally downregulates ER\u03b1 by recruiting lysine-specific demethylase 1 in human breast cancer

Estrogen receptor α (ERα) is related with epithelial–mesenchymal transition, invasion and metastasis, and serves as an important therapeutic predictor and prognostic factor in breast cancer patients. The triple negative breast cancer (TNBC) is characterized by loss of hormone receptors and human epidermal growth factor receptor 2 (Her2), and lacks effective targeted therapy with poor prognosis. Unfortunately, the molecular mechanisms of ERα deficiency, which becomes hormone independent and results in resistance to endocrine therapy, remain to be elucidated in breast cancer. In this study, we observed an inverse correlation between Slug, a zinc-finger transcriptional repressor, and ERα expression in both human breast cancer tissues and cell lines. In ERα-negative breast cancer patients, high Slug messenger RNA expression showed obviously shorter relapse-free survival. We found that Slug binds to the E-box located in the promoter of estrogen receptor 1 gene (ESR1) to suppress its expression. More specifically, Slug recruits lysine-specific demethylase 1 (LSD1) to the E-box and thereby inhibits ERα expression by demethylating H3K4me2, which is evidenced by the interaction between Slug and LSD1. Moreover, the amount of H3K4me2 binding to the E-box was significantly increased after LSD1 knockdown in MDA-MB-231 cells. Functionally, the ability to proliferate, invade and metastasize was significantly suppressed after knockdown of either Slug or LSD1 alone, or both simultaneously. Taken together, these results suggest that Slug transcriptionally inhibits ERα expression by recruiting LSD1 to the ESR1 promoter in breast cancers. Thus, targeted inhibition of Slug and LSD1 may restore ERα and lead to resensitization to hormone therapy, providing a novel therapeutic strategy for ERα-negative breast cancer patients, especially for TNBC.

ZEB1 induces ER-\u03b1 promoter hypermethylation and confers antiestrogen resistance in breast cancer

Antiestrogen resistance is a major obstacle to endocrine therapy for breast cancers. Although reduced estrogen receptor-α (ER-α) expression is a known contributing factor to antiestrogen resistance, the mechanisms of ER-α downregulation in antiestrogen resistance are not fully understood. Here, we report that ectopic zinc-finger E-box binding homeobox 1 (ZEB1) is associated with ER-α deficiency in breast cancer cells and thus confers antiestrogen resistance. Mechanistically, ZEB1 represses ER-α transcription by forming a ZEB1/DNA methyltransferase (DNMT)3B/histone deacetylase (HDAC)1 complex on the ER-α promoter, leading to DNA hypermethylation and the silencing of ER-α. Thus, ectopic ZEB1 downregulates ER-α expression and subsequently attenuates cell growth inhibition by antiestrogens, such as tamoxifen and fulvestrant. Notably, the depletion of ZEB1 by RNA interference causes ER-α promoter demethylation, restores ER-α expression, and increases the responsiveness of breast cancer cells to antiestrogen treatment. By studying specimens from a large cohort of subjects with breast cancer, we found a strong inverse correlation between ZEB1 and ER-α protein expression. Moreover, breast tumors that highly express ZEB1 exhibit ER-α promoter hypermethylation. Using a nude mouse xenograft model, we further confirmed that the downregulation of ZEB1 expression restores the responsiveness of breast cancer cells to antiestrogen therapy in vivo. Therefore, our findings suggest that ZEB1 is a crucial determinant of resistance to antiestrogen therapies in breast cancer.

Hormonal Modulation of Dendritic Cells Differentiation, Maturation and Function: Implications for the Initiation and Progress of Systemic Autoimmunity.

Hormonal homeostasis is crucial for keeping a competent and healthy immune function. Several hormones can modulate the function of various immune cells such as dendritic cells (DCs) by influencing the initiation of the immune response and the maintenance of peripheral tolerance to self-antigens. Hormones, such as estrogens, prolactin, progesterone and glucocorticoids may profoundly affect DCs differentiation, maturation and function leading to either a pro-inflammatory or an anti-inflammatory (or tolerogenic) phenotype. If not properly regulated, these processes can contribute to the pathogenesis of autoimmune disease. An unbalanced hormonal status may affect the production of pro-inflammatory cytokines, the expression of activating/inhibitory receptors and co-stimulatory molecules on conventional and plasmacytoid DCs (pDCs), conferring susceptibility to develop autoimmunity. Estrogen receptor (ER)-α signaling in conventional DCs can promote IFN-α and IL-6 production and induce the expression of CD40, CD86 and MHCII molecules. Furthermore, estrogen modulates the pDCs response to Toll-like receptor ligands enhancing T cell priming. During lupus pathogenesis, ER-α deficiency decreased the expression of MHC II on pDCs from the spleen. In contrast, estradiol administration to lupus-prone female mice increased the expression of co-stimulatory molecules, enhanced the immunogenicity and produced large amounts of IL-6, IL-12 and TNF-α by bone marrow-derived DCs. These data suggest that estradiol/ER signaling may play an active role during lupus pathology. Similarly, understanding hormonal modulation of DCs may favor the design of new therapeutic strategies based on autologous tolerogenic DCs transfer, especially in sex-biased systemic autoimmune diseases. In this review, we discuss recent data relative to the role of different hormones (estrogen, prolactin, progesterone and glucocorticoids) in DC function during systemic autoimmune pathogenesis.

Regulation of Metabolism by Estrogen Receptor Alpha

Postmenopausal estrogen deficiency increases the risk for metabolic syndrome, obesity, and type 2 diabetes. Estrogen treatment improves metabolic function in postmenopausal women, and estrogen receptor α (ERα) is thought to be the primary player in these protective metabolic effects. However, the mechanisms behind ERα regulation of metabolic function have not been elucidated. The purpose of this study was to investigate the impact of ERα loss on whole body and skeletal muscle oxidative capacity in a rat model. 14 wk‐old female Wildtype (WT) and ERα knockout (ERα−/−) rats were exercised to exhaustion and one week later a glucose tolerance test was performed. At sacrifice, body weight and adipose tissue depot weights (subcutaneous, retroperitoneal and periuterine) were measured. Adipocyte cell size and mitochondrial protein content were assessed by immunohistochemistry and western blot, respectively. Intramuscular triglyceride storage was also assessed. ERα deficient rats had increased body weight and subcutaneous adipose tissue mass compared with WT rats. Although there was no increase in retroperitoneal adipose tissue mass, adipocyte size was increased in ERα deficient rats. HOMA‐IR values were also increased in ERα−/− animals indicating increased insulin resistance. ERα deficiency also decreased exercise time to exhaustion when compared with WT rats. Mitochondrial proteins Complex IV and peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) were significantly decreased and intramuscular triglyceride storage was increased in skeletal muscles from ERα deficient rats. Our data demonstrate the potential regulatory role of ERα to enhance oxidative capacity and improve energy metabolism both systemically and in skeletal muscle. Pharmacological activation of ERα could be an effective strategy for the prevention and treatment of metabolic dysfunction. Selective estrogen receptor modulators currently used as treatment for various postmenopausal symptoms may have the potential to restore metabolic function in postmenopausal women and improve quality of life.Support or Funding InformationThis research was supported in part by an NIH grant (R01 AG031575) and the IRHRM Pilot grant.

Estrogen Receptor α Deficiency Modulates TLR Ligand-Mediated PDC-TREM Expression in Plasmacytoid Dendritic Cells in Lupus-Prone Mice.

Female lupus-prone NZM2410 estrogen receptor α (ERα)-deficient mice are protected from renal disease and have prolonged survival compared with wild-type littermates; however, the mechanism of protection is unknown. Plasmacytoid dendritic cells (pDCs) and type I IFN drive lupus pathogenesis. Estrogen acting via ERα enhances both pDC development and IFN production. The objectives for this study were to determine if ERα modulates pDC function and IFN activity in predisease NZM2410 mice as a possible protective mechanism of ERα deficiency in lupus-prone mice. We measured the effect of ERα deficiency on spleen pDC frequency, number, maturation, and activation state. ERα deficiency reduced type I IFN activity and the frequency of MHC class II(+) pDCs in the spleen without altering overall pDC frequency, number, or maturation state. Additionally, ERα-deficient NZM2410 mice had a significantly decreased frequency of pDCs expressing PDC-TREM, a modulator of TLR-mediated IFN production. After in vitro TLR9 stimulation, ERα deficiency significantly reduced the expression of PDC-TREM on pDCs from both NZM2410 and C57BL/6 mice. Thus, we have identified a significant effect of ERα deficiency on pDCs in predisease NZM2410 mice, which may represent a mechanism by which ERα deficiency protects NZM2410 mice from lupuslike disease.

Protective Hematopoietic Effect of Estrogens in a Mouse Model of Thrombosis: Respective Roles of Nuclear Versus Membrane Estrogen Receptor α.

We recently reported that chronic 17β-estradiol (E2) treatment in mice decreases platelet responsiveness, prolongs the tail-bleeding time and protects against acute thromboembolism via the hematopoietic estrogen receptor alpha (ERα), and independently of ERβ. Here, we have explored the respective roles of membrane vs nuclear actions of ERα in this process, using: 1) the selective activator of membrane ERα: estrogen dendrimer conjugate, and 2) mouse models with mutations in ERα. The selective targeting of activation function 2 of ERα provides a model of nuclear ERα loss-of-function, whereas mutation of the ERα palmitoylation site leads to a model of membrane ERα deficiency. The combination of pharmacological and genetic approaches including hematopoietic chimera mice demonstrated that absence of either membrane or nuclear ERα activation in bone marrow does not prevent the prolongation of the tail-bleeding time, suggesting a redundancy of these two functions for this E2 effect. In addition, although hematopoietic membrane ERα is neither sufficient nor necessary to protect E2-treated mice from collagen/epinephrine-induced thromboembolism, the protection against death-induced thromboembolism is significantly reduced in the absence of hematopoietic nuclear ERα activation. Overall, this study emphasizes that hematopoietic cells (likely megakaryocytes and possibly immune cells) constitute an important target in the antithrombotic effects of estrogens, and delineate for the first time in vivo the respective roles of membrane vs nuclear ERα effects, with a prominent role of the latter.

Estrogen receptor alpha signaling promotes Sle1-induced loss of tolerance and immune cell activation and is responsible for sex bias in B6.Sle1 congenic mice

Sex bias in lupus incidence is thought to be due, in part, to the ability of estrogens to promote loss of tolerance. Previously, we showed that estrogens promote lupus via estrogen receptor α (ERα). C57BL/6 (B6) mice carrying the Sle1 lupus susceptibility locus (B6.Sle1) display loss of tolerance and develop anti-nuclear antibodies and immune cell hyperactivation. The incidence of loss of tolerance in B6.Sle1 females is greater than in males. Here, we show that a deficiency of either estrogens or ERα attenuates loss of tolerance and autoantibody development in B6.Sle1 females. Furthermore, we demonstrate that immune cell activation in B6.Sle1 mice shows sex bias and that ERα deficiency diminishes this phenotype in B6.Sle1 females. Thus, estrogens, acting via ERα, control sex bias in the Sle1 phenotype. Furthermore, we show that ERα may impact the Sle1 phenotype by modulating the expression of Pbx1, one of genes that underlies the Sle1 locus.

Estrogen receptor alpha deficiency protects against development of cognitive impairment in murine lupus.

BackgroundOne of the more profound features of systemic lupus erythematosus (SLE) is that females have a 9:1 prevalence of this disease over males. Up to 80% of SLE patients have cognitive defects or affective disorders. The mechanism of CNS injury responsible for cognitive impairment is unknown. We previously showed that ERα deficiency significantly reduced renal disease and increased survival in lupus-prone mice. We hypothesized that ERα deficiency would be similarly protective in the brain, and that ERα may play a role in modulating blood-brain barrier (BBB) integrity and/or neuroinflammation in lupus-prone mice.MethodsMRL/lpr ERα+/+ and ERαKO mice (n = 46) were ovariectomized, received 17β-estradiol pellets, and underwent radial arm water maze (WRAM) and novel object recognition (NOR) testing starting at eight weeks of age. Mice were sacrificed and brains were hemisected and processed for either immunohistochemistry, or hippocampus and parietal cortex dissection for Western blotting.ResultsMRL/lpr ERαKO mice (n = 21) performed significantly better in WRAM testing than wild-type MRL/lpr mice (n = 25). There was a significant reduction in reference memory errors (P <0.007), working memory errors (P <0.05), and start arm errors (P <0.02) in ERαKO mice. There were significant differences in NOR testing, particularly total exploration time, with ERα deficiency normalizing behavior. No significant differences were seen in markers of tight junction, astrogliosis, or microgliosis in the hippocampus or cortex by Western blot, however, there was a significant reduction in numbers of Iba1+ activated microglia in the hippocampus of ERαKO mice, as evidenced by immunohistochemietry (IHC).ConclusionERα deficiency provides significant protection against cognitive deficits in MRL/lpr mice as early as eight weeks of age. Additionally, the significant reduction in Iba1+ activated microglia in the MRL/lpr ERαKO mice was consistent with reduced inflammation, and may represent a biological mechanism for the cognitive improvement observed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0171-x) contains supplementary material, which is available to authorized users.

Estrogen Receptor Alpha Modulates Mesangial Cell Responses to Toll-Like Receptor Ligands

The female predominance in lupus is incompletely understood. The mechanisms for this difference are multifactorial involving the sex chromosomes, the hormones, and their receptors. We, and others, demonstrated that estrogen receptor alpha (ERα)–deficient female mice developed significantly less lupus-like renal disease. This protective effect of ERα deficiency occurred despite no impact on glomerular immune complex deposition. We hypothesized that decreased renal disease in ERα-deficient mice was due to a dampened renal response to inflammatory stimuli. Given the role of Toll-like receptors (TLRs) in lupus, we assessed whether there was an interaction between TLR responses and ERα. Herein, we show that TLR3, 4, and 7 ligands all enhanced mesangial cell (MC) ERα expression, whereas neither estrogen, nor ERα, impacted TLR3, 4, or 7 expression in MCs. The lack of ERα markedly decreased MC production of interleukin 6 and monocyte chemoattractant protein 1 (MCP-1) following addition of TLR3, 4, and 7 ligands. In MCs, TLR ligands induced ERα phosphorylation and nuclear localization. TLR3-induced nuclear factor κB nuclear translocation in MCs was not significantly affected by estrogen or ERα. Finally, we demonstrate that female MCs express more TLR3 and respond to TLR ligands with a significantly increased production of interleukin-6 compared with male MCs. These results identify a significant impact/interaction of ERα in TLR-mediated inflammatory responses in MCs.

Estrogen receptor alpha modulates toll-like receptor signaling in murine lupus

Systemic lupus erythematosus (SLE) is a disease that disproportionately affects females. Despite significant research effort, the mechanisms underlying the female predominance in this disease are largely unknown. Previously, we showed that estrogen receptor alpha knockout (ERαKO) lupus prone female mice had significantly less pathologic renal disease and proteinuria, and significantly prolonged survival. Since autoantibody levels and number and percentage of B/T cells were not significantly impacted by ERα genotype, we hypothesized that the primary benefit of ERα deficiency in lupus nephritis was via modulation of the innate immune response. Using BMDCs and spleen cells/B cells from female wild-type or ERαKO mice, we found that ERαKO-derived cells have a significantly reduced inflammatory response after stimulation with TLR agonists. Our results indicate that the inflammatory response to TLR ligands is significantly impacted by the presence of ERα despite the absence of estradiol, and may partially explain the protective effect of ERα deficiency in lupus-prone animals.

Abstract 1631: Protection of colon carcinogenesis by estrogen receptor-beta may involve regulation of angiogenesis and invasion specific signaling molecules

Abstract Estrogen receptor-beta (ERα) has been suggested to exert anti-tumorigenic effects in the colon. However, its mechanism of action in colon cancer remains unclear. Previously, we demonstrated that azoxymethane (AOM) treated ERα knockout (≥ERKO) mice showed a significantly higher incidence of colonic precancerous lesions compared to AOM-treated WT mice. These results were associated with a decrease in apoptosis and loss of crypt organization in the colonic epithelium of αERKO mice. In the present study, we investigated whether ERα deficiency would also correlate with a higher incidence of colonic tumors. For this, αERKO and WT mice were injected subcutaneously with AOM (10mg/kg) once a week, for six weeks. Forty weeks after the beginning of the study, mice were sacrificed and colonic sections were collected for histopathology, RNA, and protein expression analysis. Evaluation of hematoxylin and eosin sections from each group showed that αERKO mice developed higher number of colonic adenomas (1.40 ± 0.40) than WT mice (0.33 ± 0.21) (p &amp;lt; 0.05). Furthermore, Mouse Cancer PathwayFinder Superarray revealed a greater than two-fold increase of angiogenesis and invasion specific signaling molecules in ERα-deficient tumors compared to WT ones. These findings highlight the importance of ERα against colon carcinogenesis progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1631. doi:1538-7445.AM2012-1631

Type I interferon and pro-inflammatory cytokine synthesis induced by Toll-like receptor signaling in dendritic cells is regulated by estrogen receptor alpha (57.7)

Abstract Estrogen receptors (ER) regulate target gene expression by forming complexes with chromatin-modifying coregulators and transcription factors. Indeed, ER alter the activity of NF-κB and C/EBPβ, which are crucial regulators of cytokine gene expression. To test the hypothesis that ER alpha (ERα) signaling regulates pro-inflammatory cytokine and type I interferon production by CD11c+ dendritic cells (DC), we developed a novel line of mice, bearing CD11c-Cre and a conditional allele of ERα, in which ERα deficiency is restricted to CD11c+ cells. Isolated ERα+ or ERα-/- splenic DC were stimulated by Toll-like receptor (TLR) ligands in steroid-deficient medium with physiological levels (1 nM) of estradiol, followed by assessment of cytokine RNA and protein synthesis. ERα deficiency significantly reduced the synthesis of Ifna and Il6 RNA and protein by plasmacytoid DC triggered via TLR7 with influenza virus or via TLR9 with unmethylated CpG-rich DNA. In conventional DC stimulated via TLR9, ERα deficiency led to reduced levels of Il1a, Csf2, Ifnb, Nos2 and Ifng RNA and IL-1α and GM-CSF protein. Taken together, these data show that cell autonomous ERα signaling in DC promotes type I interferon and pro-inflammatory cytokine synthesis induced by pathogen-specific nucleic acids. Thus, estradiol-bound ERα may interact with transcription factors activated by TLR 7 and 9 signaling to increase transcription of cytokine genes, thereby regulating the ability of DC to promote pathogen immunity.