Effects Of ERβ Research Articles

Overexpression of estrogen receptor β inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation

BackgroundEstrogen receptor β (ERβ) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERβ. Here, we explore the effect of overexpressed ERβ on human HSCs and the role of ERβ in pharmacological action of calycosin.MethodsLX-2 cells were transfected with lentivirus to overexpress ERβ. In the presence or absence of overexpressed ERβ, the effects of ERβ and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-β1-induced LX-2 cells and the role of ERβ in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERβ or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERβ on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot.ResultsERβ overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERβ or calycosin alone inhibited the TGF-β1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERβ and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERβ overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed.ConclusionsERβ mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect.Graphical

Effects of ERβ and ERα on OVX-induced changes in adiposity and insulin resistance.

Loss of ovarian hormones leads to increased adiposity and insulin resistance (IR), increasing the risk for cardiovascular and metabolic diseases. The purpose of this study was to investigate whether the molecular mechanism behind the adverse systemic and adipose tissue-specific metabolic effects of ovariectomy requires loss of signaling through estrogen receptor alpha (ERα) or estrogen receptor β (ERβ). We examined ovariectomized (OVX) and ovary-intactwild-type (WT), ERα-null (αKO), and ERβ-null (βKO) female mice (age ~49 weeks; n = 7-12/group). All mice were fed a phytoestrogen-free diet (<15 mg/kg) and either remained ovary-intact (INT) or were OVX and followed for 12 weeks. Body composition, energy expenditure, glucose tolerance, and adipose tissue gene and protein expression were analyzed. INT αKO were ~25% fatter with reduced energy expenditure compared to age-matched INT WT controls and βKO mice (all P < 0.001). Following OVX, αKO mice did not increase adiposity or experience a further increase in IR, unlike WT and βKO, suggesting that loss of signaling through ERα mediates OVX-induced metabolic dysfunction. In fact, OVX in αKO mice (i.e., signaling through ERβ in the absence of ERα) resulted in reduced adiposity, adipocyte size, and IR (P < 0.05 for all). βKO mice responded adversely to OVX in terms of increased adiposity and development of IR. Together, these findings challenge the paradigm that ERα mediates metabolic protection over ERβ in all settings. These findings lead us to suggest that, following ovarian hormone loss, ERβ may mediate protective metabolic benefits.

Abstract P6-05-02: Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits its anti-cancer effects, in part, via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-02.

Estrogen and mechanical loading-related regulation of estrogen receptor-β and apoptosis in tendinopathy.

Female-dominant tendinopathies are musculoskeletal disorders caused by repetitive hand posture and motion; they are considered overuse syndromes. Both external mechanical stress and changes in hormone levels might affect disease progression. We have previously reported that estrogen receptor-β (ER)-β expression was associated with the pathogenesis of de Quervain's disease. To study the underlying mechanisms, a cyclic stretching culture system was applied to tendon tissue from ovariectomized (OVX) rats. Furthermore, a collagenase I-induced rat tendinopathy model was established to examine the association of ER-β with disease progression. Our results showed that ER-β expression and the number of apoptotic cells were higher and associated with disease severity in rats with tendinopathy. Mechanical stress altered the morphology of primary tenocytes and collagen fiber alignment in tendons, and up-regulated the expression of matrix metalloproteinase-9, ER-β, and interleukin-1β, as well as induced apoptosis in tenocytes and tendon tissue from OVX rats. This is the first report on the effects of ER-β and mechanical stress in tendinopathy. We hope these findings contribute to new pharmacological therapies targeting ER-β signaling pathways to treat tendon-related diseases.

Estrogen receptor β exhibited anti-tumor effects on osteosarcoma cells by regulating integrin, IAP, NF-kB/BCL-2 and PI3K/Akt signal pathway

This study aimed to investigate the effects of Estrogen receptor β (ERβ) on osteosarcoma cells, and explore the regulatory mechanisms involved in this process. Osteosarcoma U2-OS cells consisted four groups, and treated by E2, E2 + LY294002 (ERβ agonists), E2 + ERβ siRNA, E2 + ERβ siRNA + LY294002, respectively. Cell counting kit 8 (CCK-8) assay was performed to detect the cell viability of U2-OS cells in each group. The effects of ERβ on the migration and invasion ability of U2-OS cells were examined by wound healing assay and transwell cell culture chamber, respectively. The expression of Inhibitor of apoptosis protein (IAP) and integrin α5 in U2-OS cells of each group was detected by quantitative RT-PCR, and the expression of phosphorylated p65 (p-p65), p-AKT and Bcl-2 was detected by western blotting. The cell viability, migration and invasion ability of U2-OS cells were significantly increased by ERβ siRNA, but inhibited by ERβ agonists LY294002 (p < 0.05). ERβ siRNA significantly downregulated Integrin α5 and unregulated IAP in U2-OS cells (p < 0.05). The expression of p-p65, p-AKT and Bcl-2 was significantly reduced by LY294002, but increased by ERβ siRNA (p < 0.05). In conclusion, ERβ exhibited obvious anti-tumor effects on osteosarcoma cells by regulating integrin, IAP, NF-kBBCL-2 and PI3K/Akt signal pathway.

Abstract 4401: Role of estrogen signaling in colon microenvironment during obesity

Abstract Recent epidemiological studies highlight the strong association between high body mass index (BMI) and enhanced risk of colon cancer. This association appears stronger in men than in women, indicating a possible protective role of estrogens in this context. The biological action of the estrogens is mediated by specific transcription factors, the estrogen receptors (ERs). ERβ is the main ER in the intestine; and it is expressed in both epithelial cells and immune cells. Anti-inflammatory and anti-tumorigenic effects of ERβ in colitis-associated neoplasia have been demonstrated. Several rodent models for obesity show an impaired intestinal epithelial barrier function and an increase of pro-inflammatory markers in the colon. Moreover, in murine colitis models, high fat diet (HFD) exacerbates colonic inflammation. However, the specific molecular mechanisms linking obesity to increased colon cancer development, where estrogen signaling may be involved, is not clear. We hypothesize that ERβ-selective agonists can oppose the HFD-mediated inflammatory pro-tumorigenic colonic signalling in a gender-specific manner during obesity, and thereby protect against colorectal cancer. Our study is focused on the initiation of colon inflammation during obesity and aims to investigate the impact of ERβ activation in this context. To address this question, male and female mice were fed a HFD for 26 weeks and treated with a ERβ-selective agonist for 3 weeks prior sacrifice. Stool pellets were regularly collected. Animals were sacrificed; colon samples and caecal contents were collected for further analysis. We show that the expression pattern of inflammatory genes in colon is modified by the ERβ-selective agonist in HFD fed mice. Moreover the ERβ-selective agonist appears to induce gender-specific changes of the microbiota of HFD-fed mice. Our results show that an ERβ-selective agonist can modulate the colon microenvironment during obesity in mice and will provide a better comprehension of the molecular mechanisms leading to obesity-associated colonic inflammation. Citation Format: Amena Archer, Linnea Pettersson, Marcela González-Granillo, Christina Savva, Marion Korach-André, Cecilia Williams. Role of estrogen signaling in colon microenvironment during obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2017-4401

Abstract P5-04-04: Activation of ERβ in triple negative breast cancer results in cell cycle arrest

Abstract Background: Triple negative breast cancer (TNBC), which comprises approximately 20% of breast cancer diagnoses, lacks estrogen receptor alpha, progesterone receptor and Her-2 expression. However, we have identified that 30% of TNBC patients express estrogen receptor beta (ERβ), a nuclear hormone receptor and potential therapeutic target. Here we examine the effects of ERβ in triple negative breast cancer cell lines. Methods: Cell lines that stably express ERβ were used to perform microarray analyses following five days of estrogen treatment. Ingenuity pathway analysis was conducted on differentially expressed genes to determine alterations in biological pathways. The effects of ERβ on cell cycle progression and apoptosis was determined. Cell cycle-related expression changes were confirmed with RT-qPCR and western blotting. The impact of siRNA mediated gene silencing of CDK1 on TNBC cell proliferation was assessed as was the ability of ERβ to elicit anti-proliferative effects in the setting of CDK1 knockdown. Results: We have shown that estrogen or ERβ-specific agonist treatment causes decreased proliferation of ERβ+ TNBC cells. This inhibitory effect is not due to programmed cell death but rather a G1/S phase cell cycle arrest as indicated by flow cytometry experiments. Microarray data and ingenuity pathway analysis revealed a number of down regulated genes involved in cell-cycle progression. Specifically, estrogen treatment of ERβ positive TNBC cells was shown to result in suppression of cyclin-dependent kinase 1 (CDK1) and Cyclin B, effects that were confirmed following ERβ-specific agonist treatment at both the mRNA and protein levels via RT-qPCR and western blotting, respectively. Knockdown of CDK1 in ERβ+ TNBC cells using siRNA resulted in decreased proliferation and diminished the anti-proliferative effects observed following estrogen or ERβ-specific agonist treatment. Conclusions: Our data demonstrate that estrogen and ERβ-specific agonists cause cell cycle arrest in ERβ positive TNBC. These effects are due to ERβ-mediated suppression of multiple genes involved in cell cycle progression including CDK1 and Cyclin B. Following knockdown of CDK1, estrogen or ERβ-specific agonist treatment displayed minimal impact on cell proliferation. Therefore, ERβ's effects on proliferation may primarily be mediated by blockade of CDK1 and Cyclin B. Regardless of ERβ, our data suggest that inhibition of CDK1 activity may have therapeutic benefit in a subset of TNBC patients, an area of study that has yet to be explored. Citation Format: Reese JM, Bruinsma ES, Monroe DG, Goetz MP, Hawse JR. Activation of ERβ in triple negative breast cancer results in cell cycle arrest [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-04-04.

Abstract P6-03-06: Therapeutic targeting of ERβ in triple negative breast cancer

Abstract Background: While the biological functions and clinical importance of ERα are well understood in breast cancer, much less is known about its most closely related family member, ERβ, particularly in the setting of triple negative disease. Additionally, the ability to therapeutically target ERβ in triple negative breast cancer (TNBC) has not been fully explored. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in a cohort of 71 TNBCs. To further define the biological functions of ERβ in TNBC, novel ERβ expressing triple negative cell lines (MDA-MB-231 and Hs578T) were developed and comprehensively characterized at the level of global gene expression profiling, modulation of important biological pathways, cellular proliferation and response to targeted therapies. Results: In TNBCs from 71 patients, nuclear and cytoplasmic ERβ was detected at moderate to high levels in 24% and 32% of cases respectively. This moderate to high expression of both nuclear and cytoplasmic ERβ was associated with higher levels of Ki67. Of the 17 tumors expressing ERβ, 13 (76%) were negative for androgen receptor expression. In the triple negative MDA-MB-231-ERβ and Hs578T-ERβ cell lines, expression of ERβ led to inhibition of proliferation in response to both estrogen and multiple ERβ specific agonists. Microarray analysis and RT-PCR profiling of these cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors, while suppressing the expression of many interleukins. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells inhibited the proliferation rates and blocked TGFβ signaling in non-ERβ expressing TNBC cells, effects that were completely reversed following depletion of cystatins from the conditioned media. Conclusions: ERβ is expressed in a substantial proportion of TNBCs, and most do not express the androgen receptor. In TNBC, where targeted therapies are lacking, our data suggest that estrogen or ERβ specific agonists would be expected to elicit anti-tumor effects when ERβ is expressed. These tumor suppressive effects of ERβ appear to be mediated in part through the actions of cystatins, and their inhibition of TGFβ signaling, suggesting that this family of secreted proteins may represent novel biomarkers for monitoring ERβ specific drug responsiveness and/or patient outcomes. Citation Format: Jordan M Reese, Malayannan Subramaniam, Vera J Suman, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse. Therapeutic targeting of ERβ in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-06.

17β-Estradiol and/or Estrogen Receptor β Attenuate the Autophagic and Apoptotic Effects Induced by Prolonged Hypoxia Through HIF-1α-Mediated BNIP3 and IGFBP-3 Signaling Blockage

Background/Aims: The risk of heart disease is higher in males than in females. However, this advantage of females declines with increasing age, presumably a consequence of decreased estrogen secretion and malfunctioning of the estrogen receptor. We previously demonstrated that 17β-estradiol (E2) prevents cardiomyocyte hypertrophy, autophagy and apoptosis via estrogen receptor α (ERα), but the effects of ERβ on myocardial injury remained elusive. The present paper thus, investigated the cardioprotective effects of estrogen (E2) and ERβ against hypoxia-induced cell death. Methods: Transient transfection of Tet-On ERβ gene construct was used to overexpress ERβ in hypoxia-treated H9c2 cardiomyoblast cells. Results: Our data revealed that IGF1R, Akt phosphorylation and Bcl-2 expression are enhanced by ERβ in H9c2 cells. Moreover, ERβ overexpression reduced accumulation of hypoxia-related proteins, autophagy-related proteins and mitochondria-apoptotic proteins and enhanced the protein levels of Bcl-2, pAkt and Bad under hypoxic condition. In neonatal rat ventricular myocytes (NRVMs), we observed that hypoxia induced cell apoptosis as measured by TUNEL staining, and E2 and/or ERβ could totally abolish hypoxia-induced apoptosis. The suppressive effects of E2 and/or ERβ in hypoxia-treated NRVMs were totally reversed by ER antagonist, ICI. Taken together, E2 and/or ERβ exert the protective effect through repressed hypoxia-inducible HIF-1α, BNIP3 and IGFBP-3 levels to restrain the hypoxia-induced autophagy and apoptosis effects in H9c2 cardiomyoblast cells. Conclusion: The results suggest that females probably could tolerate better prolonged hypoxia condition than males, and E2/ERβ treatment could be a potential therapy to prevent hypoxia-induced heart damage.”

Inhibitory effects of ERβ on proliferation, invasion, and tumor formation of MCF-7 breast cancer cells-prognostication for the use of ERβ-selective therapy

Context: Estrogen is well-known as an important factor in the physiological functions and pathological processes of breast. Estrogen receptor β (ERβ) is expressed in the majority of breast cancers at lower levels compared with the normal breast tissue.Objective: The effect of ERβ on the characteristics of breast tumor cells and its prognostication for the use of ERβ-selective therapy were investigated here for the first time.Materials and methods: ERβ was overexpressed in ERα positive MCF-7 breast cancer cells by gene transfection. The proliferation, motility, and xenografts growth of MCF-7 cells were investigated by MTT assays, wound-healing assay and animal study.Results: Results demonstrated that ERβ-GFP localized in both the cytoplasm and the nucleus in the presence of 17β-estradiol (E2), with stronger fluorescence-signal intensity in the nucleus, 2.8-times higher than that in the cytoplasm. The ERβ overexpressed MCF-7 cells resulted in a 38.7% decreased growth rate and motility in vitro. Furthermore, ERβ overexpression enhanced the antiproliferative effects of phytoestrogen, antiestrogen, and histone deacetylase inhibitor. Exogenous ERβ expression reduced tumor volume by 99% at 27 days postadministration, indicated that overexpression of ERβ led to retardation of tumor formation and growth in immunodeficient mice.Discussion and conclusion: This study provided a relatively new evidence to support that ERβ is an important modulator of proliferation and motility of breast cancer cells, and implied for the first time a possibility for the use of novel ERβ-selective therapies in breast cancer treatment.