Absence Of E2 Research Articles

17β-estradiol inhibits Notch1 activation in murine macrophage cell line RAW 264.7.

Macrophages are major effectors in regulating immune response and inflammation. The pro-inflammatory phenotype (M1) is induced by the activation of the Toll-like receptor 4 (TLR4) on the macrophage surface, which recognizes lipopolysaccharide (LPS), a component of Gram-negative bacterial wall, and by the binding of interferon-gamma (IFNγ), a cytokine released by activated T lymphocytes, to its receptor (IFNGR). Among the pathways activated by LPS/IFNγ is the Notch pathway, which promotes the M1 phenotype. Conversely, 17β-estradiol (E2) has been shown to blunt LPS-mediated inflammatory response. While it has been shown that E2 regulates the activity of the Notch1 receptor in human endothelial cells, there is no evidence of estrogen-mediated regulation of Notch1 in macrophages. In this study, RAW 264.7 cells were stimulated with LPS/IFNγ in the presence or absence of E2 and/or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of γ-secretase, the enzyme involved in Notch activation. The effects of treatment on inducible nitric oxide synthase (iNOS), on components of the Notch pathway, and MAPK (mitogen-activated protein kinase) were assessed by quantitative PCR and Western blotting. We found that E2, through a mechanism involving the inhibition of p38 phosphorylation, reduces the activation of Notch1 induced by LPS/IFNγ. On the contrary, Notch1 exerts a negative control on the estrogen receptor α (ERα) since Notch1 inhibition increases the protein levels of this receptor. In conclusion, we report for the first time a Notch-ERα interaction in macrophages. Our data suggest that E2 may reduce LPS/IFNγ-mediated M1 pro-inflammatory phenotype in macrophages by inhibiting Notch1. This finding encourages further studies on Notch1 inhibitors as novel treatments for inflammation-related diseases.

Abstract Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats

Background: Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear. Aim: This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction. Methods: Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies. Results: Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p<0.05) body weight gain and fat mass; (2) higher (p<0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p<0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p<0.05) redox enzymes; (2) increased (p<0.05) ROS level and glutathione depletion (p<0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p<0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p<0.05) compared to findings in OVX+EtOH rats in the absence of E2. Conclusion: This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.

Abstract PO3-29-03: An Emerging Class of ER mutations Enhances ER Dimerization and Promotes ER Activity

Abstract Physiological activation of estrogen receptor alpha (ERα) requires the binding of estradiol (E2) to the ligand binding domain (LBD) of the receptor. This interaction triggers a repositioning of helix 12 (H12), facilitating the recruitment of coactivator proteins to the unoccupied coactivator binding groove. However, in the context of breast cancer, point mutations of ERα at H12 lead to its repositioning and self-activation in the absence of E2. Through MSK clinical sequencing breast cancer cohort (MSK-IMPACT), we assayed 8302 samples of ER+ breast cancer patients and identified 649 mutations within the LBD of ERα. 471 of these mutations (73%) were located at H11-H12 loops (D538, Y537, L536), while the remaining 27% were located near the dimer interface of ERα (V422, G442, F461, S463P, L469). Through in vitro biochemical and cell assays, we identified that these mutations promote dimer formation and hormone-independent transcriptional activity. Tumors expressing these mutations exhibited significantly accelerated growth compared to their wild-type controls. Unlike Y537S, cells harboring mutations at the dimer interface maintain their sensitivity to Selective Estrogen Receptor Degraders (SERDs), including Fulvestrant, recently FDA-approved Elacestrant, and Camizestrant, as well as Selective Estrogen Receptor Modulators (SERMs) including Tamoxifen and Raloxifene. By utilizing machine learning and computational structural analyses, we uncovered that these mutants, unlike Y537 repositions H12, act distinctly through conformational change across the ERα dimer interface. Further cell assays proved that the dimerization leads to increased stability of the receptor and facilitates its nuclear localization, which is required for ERα activation. Collectively, our finding unveiled a new class of ER mutations that enforce receptor dimerization and activation of the ER signaling pathway. The discovery opens up a new therapeutic interventional possibility, suggesting that targeting dimerization could emerge as a new strategy to combat these malignancies. Citation Format: Seema Irani, Wuwei Tan, Qing Li, Weiyi Toy, Catherine Jones, Mayur Gadiya, Antonio Marra, John Katzenellenbogen, Kathryn Carlson, Benita S. Katzenellenbogen, Yang Shen, Sarat Chandarlapaty. An Emerging Class of ER mutations Enhances ER Dimerization and Promotes ER Activity [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-29-03.

Investigating the effect of hesperetin on estrogen receptor alpha (ERα) expression, phosphorylation and activity in MCF-7 cells

PurposeThe most common malignancy among women worldwide is breast cancer. The estrogen receptor plays a vital role in this cancer. One of the most well-known mechanisms that affects the activity of this receptor is its phosphorylation by protein kinase pathways. Hesperetin, a flavonoid abundant in citrus species such as lemons, grapefruits, and oranges, is the aglycone form of hesperidin. It has undergone thorough evaluation for its potential anti-cancer properties, particularly in the context of breast cancer. Studies have shown that hesperetin has an effect on intracellular kinase pathways. The aim of this study was to investigate the effect of hesperetin on the expression, phosphorylation and activity of estrogen receptor alpha (ERα) in MCF-7 breast cancer cell line. Study design and methodsMCF-7 cells were cultured in RPMI-1640 phenol red-free medium supplemented with charcoal-stripped FBS and treated with hesperetin. The MTT method was used to evaluate cell survival. The levels of the ERα protein and its phosphorylated form (Ser118) were determined via western blotting. A luciferase reporter vector was used to evaluate ERE activity. ResultsThe results of this study indicated that hesperetin reduced the survival of MCF-7 cells in a dose-dependent manner. The expression and phosphorylation (at Ser118) of the ERα significantly increased and decreased, respectively, in the groups treated with hesperetin. Hesperetin increased the activity of the ERα in the absence of E2, although these differences were not statistically significant. Conversely, in the presence of E2, hesperetin caused a significant decrease in receptor activity. ConclusionBased on the results of this study, it can be concluded that hesperetin has a significant effect on ERα expression, phosphorylation and activity.

Dynamic regulation of BDNF gene expression by estradiol and lncRNA HOTAIR

Brain derived neurotrophic factor (BDNF) is a major neurotransmitter that controls growth and maintenance of neurons and its misregulation is linked to neurodegeneration and human diseases. Estradiol (E2) is well-known to regulate the process of differentiation and plasticity of hippocampal neurons. Here we examined the mechanisms of BDNF gene regulation under basal conditions and under stimuli such as E2. Our results demonstrated that BDNF expression is induced by E2 in vitro in HT22 cells (hippocampal neuronal cells) and in vivo (in ovariectomized mouse brain under E2-treatment). Using chromatin immunoprecipitation assay, we demonstrated that estrogen receptors (ERα, ERβ) were enriched at the BDNF promoter in presence of E2. Additionally, ER-coregulators (e.g., CBP/p300, MLL3), histone acetylation, H3K4-trimethylation, and RNA polymerase II levels were also elevated at the BDNF promoter in an E2-dependent manner. Additionally, under the basal conditions (in the absence of E2), the long noncoding RNA HOTAIR and its interacting partners PRC2 and LSD1 complexes binds to the promoter of BDNF and represses its expression. HOTAIR knockdown -relieves the repression resulting in elevation of BDNF expression. Further, levels of HOTAIR-interacting partners, EZH2 and LSD1 were reduced at the BDNF promoter upon HOTAIR-knockdown revealing that HOTAIR plays a regulatory role in BDNF gene expression by modulating promoter histone modifications. Additionally, we showed that E2 induced-BDNF expression is mediated by the displacement of silencing factors, EZH2 and LSD1 at BDNF promoter and subsequent recruitment of active transcription machinery. These results reveal the mechanisms of BDNF gene regulation under the basal condition and in presence of a positive regulator such as E2 in neuronal cells.

A novel photoelectrochemical aptasensor based on nitrogen vacancies g-C3N5/TiO2 heterostructure and endonuclease assisted recycling amplification for ultrasensitive analysis of 17β-estradiol

Herein, we developed nitrogen vacancies g-C3N5/TiO2 heterostructure modified electrode for sensitive photoelectrochemical (PEC) sensing of 17β-estradiol (E2) based on target-triggered Endonuclease assisted recycling and horseradish peroxidase (HRP)-induced biocatalytic precipitation (BCP) cascade signal amplification strategies. The nitrogen vacancies g-C3N5 (NV-g-C3N5) was produced with thermal polycondensation method and employed as a photoactive material. Since the synergy effect between the TiO2 and NV-g-C3N5 can reduce the photo-induced electron-hole recombination and expedite the spatial charge separation, NV-g-C3N5/TiO2 heterostructure showed higher PEC activity. HRP-labeled Assistant DNA (HRP-AD) hybridized with Substrate DNA (SD) on the electrode in the absence of E2, and precipitation generated by HRP catalyzed could quench the photocurrent of the electrode. In the presence of E2, the target triggered Endonuclease assisted recycling reaction and achieved the conversion of the E2 to a massive output of template DNA (tDNA). Owing to tDNA strong affinity to the SD, HRP-AD was released from the electrode, gradually terminating the BCP reaction and restoring the photocurrent. The PEC aptasensor for E2 detection exhibited a broad linear range from 0.01 pM to 100 pM, with a detection limit of 0.0087 pM, and could be used to detect E2 in bioanalysis of environmental samples.

BPDCN MYB Fusions Bind Cell Cycle Gene Promoters, Reactivate Self-Renewal in Progenitor Cells and Induce Myeloid/Dendritic Leukemia

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare acute leukemia with poor prognosis arising from the plasmacytoid dendritic cell (pDC) lineage. MYB is a hematopoietic transcription factor overexpressed in many leukemias. Genomic rearrangements of MYB were identified in a significant fraction of BPDCNs, including in cases without any other clear driver mutations, and result in its fusion to one of several partner genes (Suzuki et al., Leukemia 2017). These fusions are rare or absent in other acute leukemias and the mechanisms by which they contribute to BPDCN oncogenesis are not understood. To examine the impact of Myb fusions on hematopoietic differentiation and leukemogenesis, we used mouse bone marrow progenitors immortalized by estradiol (E2)-dependent activation of HoxB8 and culture with Flt3 ligand (HoxB8-FL cells). We used HoxB8-FL cells deficient in Cdkn2a, since CDKN2A deletion is present in up to ~67% of BPDCNs (Lucioni et al., Blood 2011) but is rare in other myeloid leukemias. HoxB8-FL cells differentiate into myeloid, lymphoid and dendritic cells in vitro upon withdrawal of E2, or in vivo upon transplantation. They therefore represent lymphoid-primed multipotent progenitor (LMPP)-like cells that are unable to self-renew in the absence of E2. We co-expressed a dTom reporter with V5-tagged Myb constructs in HoxB8-FL cells: wild-type full-length Myb (Myb-FL), truncated Myb (Myb-TR) or Myb-PLEKHO1. MYB-PLEKHO1 is the most frequent fusion in BPDCN patients, while truncated Myb is analogous to recurrent BPDCN MYB fusions in which the partner is out-of-frame. Empty vector was used as a control. HoxB8-FL cells expressing Myb-FL, Myb-TR or Myb-PLEKHO1 displayed arrested differentiation upon withdrawal of E2 in vitro (Figure 1). While all empty vector-transduced cells had upregulated CD11b and/or CD11c at day 7 post-E2 withdrawal, Myb overexpression caused an accumulation of CD11b-CD11c- undifferentiated cells. In vivo, a similar effect was observed at day 7 post-transplantation: dTom+ CD11b-CD11c-CD19-B220- undifferentiated cells were still present in the bone marrow of Myb-FL, Myb-TR and Myb-PLEKHO1 recipient mice, while in empty vector recipients all dTom+ cells expressed at least one of these differentiation markers. Surprisingly, we observed long-term persistence of dTom+ cells in the peripheral blood of Myb-TR and Myb-PLEKHO1, but not Myb-FL, recipients. This developed into lethal malignancy in 100% of Myb-TR and Myb-PLEKHO1 recipients by 28 weeks (Figure 2). Malignant cells showed blast-like morphology of acute leukemia and expressed markers of a myeloid/dendritic progenitor (Kit+CD11b+Cx3cr1+). dTom+ disease was never observed in Myb-FL or empty vector recipients (log-rank P<0.001). Together, while expression of either full-length Myb or Myb fusions impaired myeloid/dendritic differentiation, only Myb rearrangements associated with BPDCN generated a fully penetrant leukemia. To investigate chromatin-level differences between physiologically expressed wild-type MYB and MYB fusions, we performed Cut&Run in K562 cells with V5-tagged MYB-FL, MYB-TR or MYB-PLEKHO1 knocked in to the endogenous MYB locus using CRISPR/Cas9 and homology-directed repair. We observed increased binding of MYB-TR and MYB-PLEKHO1 relative to MYB-FL at promoters of cell cycle genes that function at the G2/M checkpoint, including CDC20, CDCA3 and CKS2. These binding sites contained canonical MYB binding motifs, indicating that MYB fusions increase direct DNA binding at chromatin involved in control of specific cell cycle genes. Cut&Run for V5-Myb-PLEKHO1 in mouse leukemias revealed similar direct DNA binding at cell cycle gene promoters. Moreover, expression of G2/M cell cycle genes was enriched in patient BPDCNs relative to normal pDCs. Our data suggest that BPDCN MYB fusions exert distinct oncogenic effects to overexpression of wild-type MYB. In addition to arresting differentiation, BPDCN MYB fusions can reactivate self-renewal in progenitor cells, which may occur through increased direct DNA binding at cell cycle gene promoters. This may explain the simple genetics of MYB-rearranged BPDCN, which lack other recurrent myeloid-type mutations, if the fusions are sufficient to generate leukemia via these dual roles. These data also indicate that therapeutic strategies targeting MYB and/or specific cell cycle checkpoints may be active in BPDCN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of 2-methoxyestradiol on hydrogen peroxide induced neuronal cell death and tau hyperphosphorylation

AimsAlzheimer's Disease (AD) is characterized by progressive cognitive impairment, and memory loss. It has been shown that depletion of estrogens renders women vulnerable to AD with menopause women presenting higher risk for AD development than men. However, women under hormone replacement therapy (HRT) with 17β-estradiol (E2) show lower risk for AD, implying that E2 may be protective. It has been shown that E2 exerts its effects through the estrogen receptor (ER) but also via its biologically active metabolites, 2-hydroxyestradiol (2OH), and 2-methoxyestradiol (2ME). We hypothesized that the neuroprotective effects of E2 are partly attributed to its metabolites. Materials and methodsSH-SY5Y neuronal cells were subjected oxidative stress (OS) cell death by hydrogen peroxide (H2O2), in the presence or absence of E2, 2ME and 2OH. Viability was assessed by trypan blue and thiazolyl blue tetrazolium bromide assays, intracellular OS with the Dichlorodihydrofluorescein Diacetate (DCFDA) assay, and Bax, p53 and PUMA quantified by RT-PCR. Tau hyperphosphorylation was studied by western blot. Key findingsE2 and its metabolites 2OH and 2ME protect from cell death as assessed by the viability assays. Their effect was partly attributed to their antioxidant properties evidenced by the reduction of intracellular OS. Treatment with 2ME resulted in a reduction of Bax, but not p53 or PUMA in cells challenged with OS. Finally, 2ME was able to inhibit tau hyperphosphorylation as well. SignificanceE2 protects neuron cells partly through its metabolites. Further studies are needed to fully delineate the mechanism for this protection.

Exploring the effect of estrogen on Candida albicans hyphal cell wall glycans and ergosterol synthesis.

Increased levels of 17-β estradiol (E2) due to pregnancy in young women or to hormonal replacement therapy in postmenopausal women have long been associated with an increased risk of yeast infections. Nevertheless, the effect underlying the role of E2 in Candida albicans infections is not well understood. To address this issue, functional, transcriptomic, and metabolomic analyses were performed on C. albicans cells subjected to temperature and serum induction in the presence or absence of E2. Increased filament formation was observed in E2 treated cells. Surprisingly, cells treated with a combination of E2 and serum showed decreased filament formation. Furthermore, the transcriptomic analysis revealed that serum and E2 treatment is associated with downregulated expression of genes involved in filamentation, including HWP1, ECE1, IHD1, MEP1, SOD5, and ALS3, in comparison with cells treated with serum or estrogen alone. Moreover, glucose transporter genes HGT20 and GCV2 were downregulated in cells receiving both serum and E2. Functional pathway enrichment analysis of the differentially expressed genes (DEGs) suggested major involvement of E2 signaling in several metabolic pathways and the biosynthesis of secondary metabolites. The metabolomic analysis determined differential secretion of 36 metabolites based on the different treatments’ conditions, including structural carbohydrates and fatty acids important for hyphal cell wall formation such as arabinonic acid, organicsugar acids, oleic acid, octadecanoic acid, 2-keto-D-gluconic acid, palmitic acid, and steriacstearic acid with an intriguing negative correlation between D-turanose and ergosterol under E2 treatment. In conclusion, these findings suggest that E2 signaling impacts the expression of several genes and the secretion of several metabolites that help regulate C. albicans morphogenesis and virulence.

17β-Estradiol-Induced Conformational Changes of Human Microsomal Triglyceride Transfer Protein: A Computational Molecular Modelling Study.

Human microsomal triglyceride transfer protein (hMTP) plays an essential role in the assembly of apoB-containing lipoproteins, and has become an important drug target for the treatment of several disease states, such as abetalipoproteinemia, fat malabsorption and familial hypercholesterolemia. hMTP is a heterodimer composed of a larger hMTPα subunit and a smaller hMTPβ subunit (namely, protein disulfide isomerase, hPDI). hPDI can interact with 17β-estradiol (E2), an endogenous female sex hormone. It has been reported that E2 can significantly reduce the blood levels of low-density lipoprotein, cholesterol and triglyceride, and modulate liver lipid metabolism in vivo. However, some of the estrogen’s actions on lipid metabolism are not associated with estrogen receptors (ER), and the exact mechanism underlying estrogen’s ER-independent lipid-modulating action is still not clear at present. In this study, the potential influence of E2 on the stability of the hMTP complex is investigated by jointly using multiple molecular dynamics analyses based on available experimental structures. The molecular dynamics analyses indicate that the hMTP complex in the presence of E2 has reduced interface contacts and surface areas. A steered molecular dynamics analysis shows that the forces required to separate the two subunits (namely, hPDI and hMTPα subunit) of the hMTP complex in the absence of E2 are significantly higher than the forces required to separate the complex in which its hPDI is already bound with E2. E2 makes the interface between hMTPα and hPDI subunits more flexible and less stable. The results of this study suggest that E2-induced conformational changes of the hMTP complex might be a novel mechanism partly accounting for the ER-independent lipid-modulating effect of E2.

Binding affinity and synergy between agonists and peptides of the estrogen receptor‐beta (ERß) needed for arain‐selective SERB Activity

The estrogen receptor (ER) exist predominantly in two isoforms (ERα and ERß). Both are primarily activated by the naturally occurring hormone 17ß-Estradiol (E2) which regulates a variety of physiological processes. One of ER's major role is acting as key neuroprotective modulator for hippocampal plasticity in the brain. The ERß isoform is predominantly found in the brain, which makes it a promising drug target for memory consolidation. Selectivity for ERß over ERα is crucial to the development of drug leads, since ERα–induced cell proliferation results in risk of cancer. We have developed a selective and potent ERß agonist with in vivo efficacy for improving cognitive function within mice. A prior study determined that selectivity for ER is related to the ligand's ability to induce the conformational change necessary for the activation of transcription. Selective estrogen receptor modulators (SERMs) like raloxifene & tamoxifen are used to treat breast cancer by selectively targeting ERα in mammary tissue. This is done through the synergistic binding of coactivator proteins to the ER in the presence of an agonist. Similarly, it is predicted that selective estrogen beta agonists (SERBAs) have the ability to be tissue specific in the presence of specific coactivators. Through the use of computational modeling, a splice variant of SRC1 (steroid receptor coactivator-1), SRC1-4, was identified to be the most favorable brain-relevant peptide for ERß. This study determines the binding affinity of agonist for ERß and ERα in the presence and absence of SRC1-4. In addition we dive into the extent to which binding synergy between agonist and coactivator exist, which could lead to selective activity in the brain. The synergy associated with the conformational change is measured by ΔΔGsynergy based on binding affinity for both agonist and coactivator. The greater the synergy between the agonist and coactivator, the more promising the lead compound will be as a SERBA. Preliminary studies have been done for the binding of SRC1-4 using TR-FRET assays where the concentration of coactivator was varied. Fluorescence polarization (FP) studies were also utilized to determine a Kd. In these studies, FL-SRC1-4 remained constant while ERß-LBD concentrations varied in the presence of 1 μM (saturating) E2. The two methods are compared and contrasted. The same FP assay was completed again in the absence of E2 (no agonoism). This provides a binding affinity for SRC1-4 to the receptor when an agonist is not present. Computational modeling studies were performed to compare ER's affinity for SRC1-4 peptides over other coactivator peptides. 3 unlabeled peptides (SRC1-1, SRC1-3 and PGC1a) were used in a competitive FP binding assay with FL-SRC1-4. The data was consistent with prior data, indicating SRC1-4 has the highest affinity for ERß. The agonistic-synergy effect will be calculated through the measurement of E2 affinity for ERß in the presence and absence of SRC1-4. The comparison of ΔΔG(synergy) for ERß vs. ERα will ultimately provide a measurement of selectivity and synergy that is biologically relevant for SERB activity. Hanson, A. M. et al. A-C estrogens as potent and selective estrogen receptor-beta agonists (SERBAs) to enhance memory consolidation under low-estrogen conditions. J. Med. Chem.61, 4720–4738 (2018).

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice

BackgroundPrevious studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. MethodsICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10−7 to 10−4 M) in vitro and in vivo. ResultsERα and ERβ expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10−4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10−7 to 10−5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10−4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). ConclusionExposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Nonlinear relationship between chromatin accessibility and estradiol-regulated gene expression.

Chromatin accessibility is central to basal and inducible gene expression. Through ATAC-seq experiments in estrogen receptor-positive (ER+) breast cancer cell line MCF-7 and integration with multi-omics data, we found estradiol (E2) induced chromatin accessibility changes in a small number of breast cancer-relevant E2-regulated genes. As expected, open chromatin regions associated with E2-inducible gene expression showed enrichment of estrogen response element (ERE) and those associated with E2-repressible gene expression were enriched for ERE, PBX1, and PBX3. While a significant number of open chromatin regions showed pioneer factor FOXA1 occupancy in the absence of E2, E2-treatment further enhanced FOXA1 occupancy suggesting that ER-E2 enhances chromatin occupancy of FOXA1 to a subset of E2-regulated genes. Surprisingly, promoters of 80% and enhancers of 60% of E2-inducible genes displayed closed chromatin configuration both in the absence and presence of E2. Integration of ATAC-seq data with ERα ChIP-seq data revealed that ~40% ERα binding sites in the genome are found in chromatin regions that are not accessible as per ATAC-seq. Such ERα binding regions were enriched for binding sites of multiple nuclear receptors including ER, ESRRB, ERRγ, COUP-TFII (NR2F2), RARα, EAR2 as well as traditional pioneer factors FOXA1 and GATA3. Similar data were also obtained when ERα ChIP-seq data were integrated with MNase-seq and DNase-seq data sets. In summation, our results reveal complex mechanisms of ER-E2 interaction with nucleosomes. Notably, "closed chromatin" configuration as defined by ATAC-seq or by other techniques is not necessarily associated with lack of gene expression and technical limitations may preclude ATAC-seq to demonstrate accessibility of chromatin regions that are bound by ERα.

Hormonal modulation of ESR1 mutant metastasis.

Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer (MBC), and confer clinical resistance to aromatase inhibitors (AIs). Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estrogen withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of estrogen; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a 9 gene expression signature which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone (DHT) significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.

Genome-wide estrogen receptor-α binding and action in human endometrial stromal cells

To investigate the gene targets of estradiol (E2)-estrogen receptor-α (ESR1) in human endometrial stromal cells. Basic science. University research center. Premenopausal women with or without endometriosis. Primary cultures of human endometrial stromal cells from healthy endometrium, with or without small-interfering RNA (siRNA) knockdown of ESR1 expression, were treated with E2 or vehicle control. Genome-wide RNA expression by RNA sequencing was compared in endometrial stromal cells with or without siRNA knockdown of ESR1 in the presence or absence of E2. Genome-wide recruitment of ESR1 to chromatin was assessed by chromatin immunoprecipitation sequencing. Gene expression by real-time qualitative polymerase chain reaction of a potential E2-ESR1 target gene was determined in endometrial stromal cells and endometriotic stromal cells. We identified several important pathways that are dependent on E2-ESR1 signaling in endometrial stromal cells, including progesterone signaling, cell-matrix adhesion, and cytoskeleton rearrangement, as well as paracrine signaling by members of the fibroblast growth factor family. We detected a total of 709 ESR1 target sites on chromatin. By integrating data on genome-wide transcriptomic changes and E2-ESR1 binding sites, we identified inositol polyphosphate phosphatase type II (INPP4B) as a candidate E2-mediated suppressor of proliferation in healthy endometrial cells. INPP4B was downregulated in endometriosis-derived stromal cells. E2-ESR1 activates genes involved in human endometrial stromal cell cycle regulation, progesterone response, and production of stromal growth factors. Understanding the direct role of estrogen on the endometrial stroma and identifying downstream targets of E2-ESR1 can inform the development of targeted therapies for endometriosis and diminished endometrial receptivity.

17β-Estradiol Promotes Proinflammatory and Procoagulatory Phenotype of Innate Immune Cells in the Presence of Antiphospholipid Antibodies.

Antiphospholipid syndrome (APS) is the most common cause of acquired thrombophilia and recurrent spontaneous miscarriages associated with extended persistence of antiphospholipid antibodies (aPL). How circulating aPL and high-17β-estradiol (E2) environment contribute to the pregnancy complications in APS is poorly defined. Therefore, we aimed to analyse whether E2 could be responsible for the immune cell hyperactivation in aPL- positive (lupus anticoagulant, anti-cardiolipin, anti-β2-glycoprotein) in women. For this, peripheral blood mononuclear cells (PBMCs) from 14 aPL- positive and 13 aPL- negative women were cultured in the presence or absence of E2, LPS or E2+LPS and cell immunophenotype and cytokine release were analysed. In the aPL+ group, E2 presence markedly increased the percentage of NK cells positive for CD69 (p < 0.05), monocytes positive for tissue factor (TF, CD142) (p < 0.05), and B cells expressing PD-L1 (p < 0.05), as well as the elevated production of IL-1β comparing to aPL- women (p < 0.01). Regardless of aPL positivity, E2 augmented the procoagulatory response elicited by LPS in monocytes. Our findings show the ability of E2 to promote proinflammatory and procoagulatory phenotype of innate immune cells in individuals with aPL positivity. Our data highlights the significant impact of female hormones on the activation of immune cells in the presence of aPL.

Estrogen Attenuates Phenotypic Switch of Human Arteriolar Smooth Muscle Cells: Role of p53 and pRb

Cardiovascular diseases (CVD) remains the leading cause of mortality worldwide. Vascular inflammation is a major contributor to the onset and the pathogenesis of this disease. In response to inflammatory stimuli, vascular smooth muscle cells (VSMCs) switch from a contractile to a dedifferentiated synthetic phenotype, characterized by decreased expression of differentiation markers and increased proliferative and migratory abilities. By virtue of its ability to attenuate these processes, estrogen (E2) is known to play a vasculoprotective role. While the vast majority of studies investigating the role of estrogen on phenotypic modulation were conducted on VSMCs isolated from large vessels, the effect of estrogen on VSMCs extracted from microvessels (microVSMCs) remains largely obscure. Thus, we sought assess the effect of estrogen on phenotypic switch of microVSMCs, and characterize the underlying molecular mechanism.We were able to extract microVSMCs from human dermal arterioles by enzymatic sprouting method. The purity of the cells was identified by their morphology and by immunostaining of two VSMC markers: smooth muscle alpha‐actin and calponin. In addition, we previously assessed the concentration‐dependent effect of estrogen on the VSMC response, with 10−10 M being the optimal physiologic concentration. Results showed that E2 (10−10 M) inhibited FBS‐induced cell proliferation and migration. This estrogen‐induced inhibition of was accompanied by decreased activation of mitogenic extracellular signal–regulated kinase (ERK1/2) and focal adhesion kinase (FAK), involved in cell proliferation and migration, respectively. Furthermore, E2 (10−10 M) increased 5′ AMP‐activated protein kinase (AMPK) phosphorylation of. In addition, E2 blocked the cells in G0/G1 phase of cell cycle. These estrogenic effects were not inhibited by ICI 280,780 (5 μM, estrogen receptor (ERα/β) antagonist). Surprisingly, ICI 280,780 mimicked estrogen, acting as an ER agonist. Furthermore, estrogen‐induced cell cycle arrest was concomitant with increased expression of p53 and decreased phosphorylation of retinoblastoma protein (pRb). The regulation of the aforementioned tumor suppressor proteins, p53 and pRb, was not mirrored by apoptosis evident by the absence of E2‐induced caspase‐3 cleavage. Alternatively, SA‐β‐gal staining showed that E2 induced senescence of VSMC. Moreover, E2 increased the expression of contractile differentiation markers: calponin and caldesmon.Taken together, this is the first study to report the effect of estrogen on phenotypic switch of VSMCs extracted from human arterioles. Our results indicate that estrogen attenuated VSMC proliferation by inducing VSMC senescence, via an ERα/β‐independent p53/pRb‐mediated mechanism. These results potentially implicate estrogen membrane receptor, GPR30, as the mediator of rapid estrogenic response in microVSMCs. In addition, due to the potential of estrogen to favorably modulate VSMC phenotype, our results may explain the lower incidence of CVDs in estrogen‐replete premenopausal females.Support or Funding InformationMPP # 320133 to A.E. and CNRS to M.F.Effect of estrogen on VSMC proliferationCells were treated with 10% FBS in the presence or absence of E2 (10‐10 M) for 1,3,5,7, and 9 days. E2 was added 30 minutes before and during treatment with FBS.Figure 1Effect of estrogen on cell senescence in VSMCs. Cells were treated with FBS, in the presence or absence of E2 (10‐10 M) or ICI 280,780 (5 μM). E2 or ICI 280,780 were added 30 minutes before and during treatment of FBS. Photography image (magnification 5 X) of SA‐β‐gal stained cells were obtained.Figure 2

Estrogen inhibits vascular calcification in rats via hypoxia-induced factor-1α signaling.

ObjectiveCalcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway.MethodsEight-week-old healthy female Sprague–Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17β-estradiol) and bone morphogenetic protein 2 siRNA intervention.ResultsThe estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification (p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro (p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein (p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 (p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells (p < 0.01).ConclusionEstrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.

Abstract P6-05-05: The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen

Abstract Background: The premise that estrogen promotes the growth of all estrogen receptor positive (ER) + breast cancers has led to anti-estrogen therapies as the standard of care for all ER+ tumors. Paradoxically, estrogen (E2) has demonstrated a therapeutic effect in the adjuvant, palliative and prevention of breast cancers in some women suggesting that there is a subset of ER+ breast cancers that may be growth-suppressed by E2. Our companion window-of-opportunity trial (abstract submitted) found E2 is growth suppressive for some post-menopausal women with high ER expressing tumors. We utilized an in vitro cell model to investigate whether differential growth responses to E2 could be due to transcriptional differences mediated by the level of the ER. Method: We generated a stable MCF-7 transfectant (MCF7-ER) using a fluorescently labelled lentiviral plasmid with a doxycycline inducible ER and compared the response to E2 against an MCF-7 mock transfectant (MCF7-EM). We assessed changes in proliferation (S-phase fraction), gene regulation (RNA-Seq) and differential ER-DNA binding (chromatin immunoprecipitation (ChIP-Seq)). To determine if high levels of ER were causing changes in the underlying chromatin configuration, we compared our ChIP-Seq profiles against previously mapped DNA loop anchor regions obtained by chromatin interaction analysis by paired end-tag sequencing (ChIA-PET) on the MCF-7 parental cell line treated with E2 (Fullwood M et al. Nature 2009). Changes in DNA loop formation were assessed by fluorescence in situ hybridization (FISH) and chromatin conformation capture (3C). Results: We confirmed previously published findings that increased ER expression in the presence of E2 leads to significant decreases in S-phase fraction (P = 0.003) with impaired transition at the G1/S and G2/M checkpoints. RNA-Seq studies defined 342 basally-upregulated genes in the MCF7-ER cells that became down-regulated after E2 treatment indicative of inverse regulation compared to the effect of E2 in the MCF7-EM mock transfectants. In E2-deprived conditions, there was an increased number of ER-DNA binding peaks in the high ER expressing cells (2,776 in MCF7-ER vs. 1,906 in MCF7-EM), suggesting unliganded ER may mediate basal gene transcription. Only 17 (5%) of the basally upregulated genes had ER binding at a proximal or distant anchor associated with previously mapped ER-DNA loops. This suggests that high ER expressing cells may develop a novel chromatin configuration that is distinct from those of parental MCF-7 cells. FISH analysis confirmed a significant increase in loop formation at the promoter region of the TFF1 gene in the MCF7-ER cells compared to MCF7-EM mock transfectants (P = 0) in the absence of E2. This DNA loop was maintained, though slightly decreased, after E2 treatment. Conclusions: These results support a theoretical model where increased ER expression may enable a novel chromatin configuration which mediates a basal level of gene expression that switches to a growth-suppressive transcriptional response upon E2 treatment. Further elucidation of an anti-proliferative DNA configuration signature using MCF7-ER cells may generate a novel set of interactions that could serve as a strategy to predict a patient’s response to estrogens and endocrine therapies. Citation Format: Lacey Haddon, Xiuying Hu, Hosna Jabbari, Judith Hugh. The estrogen switch: Estrogen receptor alpha levels determine the proliferative response to estrogen [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 P6-05-05.

Ovariectomy Activates Chronic Low-Grade Inflammation Mediated by Memory T Cells, Which Promotes Osteoporosis in Mice.

The loss of estrogen (E2 ) initiates a rapid phase of bone loss leading to osteoporosis in one-half of postmenopausal women, but the mechanism is not fully understood. Here, we show for the first time how loss of E2 activates low-grade inflammation to promote the acute phase of bone catabolic activity in ovariectomized (OVX) mice. E2 regulates the abundance of dendritic cells (DCs) that express IL-7 and IL-15 by inducing the Fas ligand (FasL) and apoptosis of the DC. In the absence of E2 , DCs become long-lived, leading to increased IL-7 and IL-15. We find that IL-7 and IL-15 together, but not alone, induced antigen-independent production of IL-17A and TNFα in a subset of memory T cells (TMEM ). OVX of mice with T-cell-specific ablation of IL15RA showed no IL-17A and TNFα expression, and no increase in bone resorption or bone loss, confirming the role of IL-15 in activating the TMEM and the need for inflammation. Our results provide a new mechanism by which E2 regulates the immune system, and how menopause leads to osteoporosis. The low-grade inflammation is likely to cause or contribute to other comorbidities observed postmenopause. © 2020 American Society for Bone and Mineral Research.