Estrogen Receptor Antagonist ICI Research Articles

Ulipristal acetate simultaneously provokes antiproliferative and proinflammatory responses in endometrial cancer cells

Ulipristal acetate (UPA), a selective progesterone receptor modulator, is used for the treatment of uterine fibroids and selectively inhibits the proliferation and inflammation of leiomyoma cells. As few studies have focused on the molecular biological mechanism of UPA in Ishikawa endometrial cancer cells, we aimed to identify the effects of UPA on these cells. Ishikawa cells were treated with different concentrations of UPA. Cell viability and colony formation assays were performed to assess the growth of cancer cells, whereas invasion and migration assays were used to measure cell motility and invasiveness. Western blotting, caspase 3/7 assay, TUNEL assay, and flow cytometry were performed to analyze apoptosis. Moreover, expression levels of the proinflammatory cytokines oncostatin M, its receptor, interleukin 6, and interleukin 8 were examined using quantitative real-time PCR. UPA decreased cell viability and growth, thereby inhibiting cell migration and invasion via induction of apoptosis. Contrary to expectation, stand-alone application of UPA increased the expression of the proinflammatory cytokines but concomitant use of UPA and the estrogen receptor antagonist ICI 182,720 decreased it. These data revealed a novel dual role of UPA: It could attenuate cell growth via activation of apoptosis while simultaneously provoking the activation of proinflammatory cytokines in endometrial cancer cells. These indicate that the combination of UPA and an estrogen receptor antagonist may be useful in suppressing the secretion of proinflammatory cytokines by UPA alone.

Estrogen regulates the expression of retinoic acid synthesis enzymes and binding proteins in mouse skin

Topical 17-beta-estradiol (E2) regulates the hair cycle, hair shaft differentiation, and sebum production. Vitamin A also regulates sebum production. Vitamin A metabolism proteins localized to the pilosebaceous unit (PSU; hair follicle and sebaceous gland); and were regulated by E2 in other tissues. This study tests the hypothesis that E2 also regulates vitamin A metabolism in the PSU. First, aromatase and estrogen receptors localized to similar sites as retinoid metabolism proteins during mid-anagen. Next, female and male wax stripped C57BL/6J mice were topically treated with E2, the estrogen receptor antagonist ICI 182,780 (ICI), letrozole, E2 plus letrozole, or vehicle control (acetone) during mid-anagen. E2 or one of its inhibitors regulated most of the vitamin A metabolism genes and proteins examined in a sex-dependent manner. Most components were higher in females and reduced with ICI in females. ICI reductions occurred in the premedulla, sebaceous gland, and epidermis. Reduced E2 also reduced RA receptors in the sebaceous gland and bulge in females. However, reduced E2 increased the number of retinal dehydrogenase 2 positive hair follicle associated dermal dendritic cells in males. These results suggest that estrogen regulates vitamin A metabolism in the skin. Interactions between E2 and vitamin A have implications in acne treatment, hair loss, and skin immunity.

17β-Estradiol alleviates intervertebral disc degeneration by inhibiting NF-κB signal pathway

AimTo investigate the effect of 17β-Estradiol (E2) on intervertebral disc degeneration (IVDD) and the related mechanism. Materials and methodsImmunohistochemistry was used to detect the expression of estrogen receptor β (ERβ) within intervertebral discs of humans and rats. After that, rat IVDD model was established by needle puncture and bilateral ovariectomy. Then, the serum E2 level was detected by enzyme linked immunosorbent assay, and the degree of IVDD was evaluated by X-ray, magnetic resonance imaging, hematoxylin and eosin staining, and Safranin O-Fast Green staining. Finally, we used immunohistochemistry and immunofluorescence staining to determine the effect of E2 on nuclear factor kappa-B (NF-κB) signal pathway both in vivo and in vitro. Key findingsWe identified that IVDD was associated with lower levels of ERβ and ERβ levels were inversely correlated with IVDD. The histological staining and radiological results showed that E2 supplement could alleviate IVDD progression. Additionally, immunohistochemistry staining demonstrated that E2 could inhibit nucleus pulposus cell (NPC) apoptosis, matrix metalloproteinases (MMPs) synthesis, and degradation of extracellular matrix (ECM) by inhibiting the activation of NF-κB signal pathway. Furthermore, immunofluorescence staining showed that the above effects of E2 on the NF-κB signal pathway could be blocked by the estrogen receptor antagonist ICI182780 in vitro. Finally, inhibition of NF-κB signal pathway by BAY11-7082 could reduce MMPs synthesis and ECM degradation of NPCs. SignificanceCollectively, these findings indicated that E2 could effectively ameliorate IVDD by inhibiting NPC apoptosis via inhibition of NF-κB signal pathway.

Ulipristal Acetate Simultaneously Provokes Antiproliferative and Proinflammatory Responses in Endometrial Cancer Cells

Ulipristal acetate (UPA), a selective progesterone receptor modulator, is used for the treatment of uterine fibroids and selectively inhibits the proliferation and inflammation of leiomyoma cells. As few studies have focused on the molecular biological mechanism of UPA in Ishikawa endometrial cancer cells, we aimed to identify the effects of UPA on these cells. Ishikawa cells were treated with different concentrations of UPA. Cell viability and colony formation assays were performed to assess the growth of cancer cells, whereas invasion and migration assays were used to measure cell motility and invasiveness. Western blotting, caspase 3/7 assay, TUNEL assay, and flow cytometry were performed to analyze apoptosis. Moreover, expression levels of the proinflammatory cytokines oncostatin M, its receptor, interleukin 6, and interleukin 8 were examined using quantitative real-time PCR. UPA decreased cell viability and growth, thereby inhibiting cell migration and invasion via induction of apoptosis. Contrary to expectation, stand-alone application of UPA increased the expression of the proinflammatory cytokines but concomitant use of UPA and the estrogen receptor antagonist ICI 182,720 decreased it. Our data revealed a novel dual role of UPA: It could attenuate cell growth via activation of apoptosis while simultaneously provoking the activation of proinflammatory cytokine s in endometrial cancer cells. These indicate that the combination of UPA and an estrogen receptor antagonist may be useful in suppressing the secretion of proinflammatory cytokines by UPA alone.

ZEB2 regulates endocrine therapy sensitivity and metastasis in luminal a breast cancer cells through a non-canonical mechanism.

The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17β-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17β-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.

Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4\u2009+\u2009T lymphocytes following hemorrhagic shock

The aim is to investigate that 17β-estradiol (E2)/estrogen receptors (ERs) activation normalizes splenic CD4 + T lymphocytes proliferation and cytokine production through inhibition of endoplasmic reticulum stress (ERS) following hemorrhage. The results showed that hemorrhagic shock (hemorrhage through femoral artery, 38–42 mmHg for 90 min followed by resuscitation of 30 min and subsequent observation period of 180 min) decreased the CD4+ T lymphocytes proliferation and cytokine production after isolation and incubation with Concanavalin A (5 μg/mL) for 48 h, induced the splenic injury with evidences of missed contours of the white pulp, irregular cellular structure, and typical inflammatory cell infiltration, upregulated the expressions of ERS biomarkers 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6). Either E2, ER-α agonist propyl pyrazole triol (PPT) or ERS inhibitor 4-Phenylbutyric acid administration normalized these parameters, while ER-β agonist diarylpropionitrile administration had no effect. In contrast, administrations of either ERs antagonist ICI 182,780 or G15 abolished the salutary effects of E2. Likewise, ERS inducer tunicamycin induced an adverse effect similarly to that of hemorrhagic shock in sham rats, and aggravated shock-induced effects, also abolished the beneficial effects of E2 and PPT, respectively. Together, the data suggest that E2 produces salutary effects on CD4+ T lymphocytes function, and these effects are mediated by ER-α and GPR30, but not ER-β, and associated with the attenuation of hemorrhagic shock-induced ERS.

Estrogenic activity of ethyl gallate and its potential use in hormone replacement therapy

We aimed to compare the estrogenic activities of compounds isolated from Moutan Cortex Radicis (MRC, Paeonia suffruticosa Andrews) and identify their potential use in hormone replacement therapy. We quantified seven marker components (gallic acid, oxypaeoniflorin, paeoniflorin, ethyl gallate, benzoic acid, benzoylpaeoniflorin, and paeonol) in MRC using a high-performance liquid chromatography simultaneous analysis assay. To investigate the estrogenic activity of MRC and the seven marker components, an E-screen assay was conducted using the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Among them, ethyl gallate caused cell proliferation in a concentration-dependent manner at concentrations above 25 µM and was clearly suppressed by combination treatment with the ER antagonist ICI 182,780. Therefore, ethyl gallate may be a compound of MRC that can increase the estrogenic effect in ER-positive MCF-7 cells.

Optimization of adipogenic differentiation conditions for canine adipose-derived stem cells.

BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.

Estrogenic activities of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside and physcion in MCF-7 cells

Polygonum multiflorum (PM) is a perennial herbal medicine that inhibits osteopenia of osteoporosis caused by estrogen deficiency and shows estrogen-like activity. Components such as 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), physcion, rhein, emodin, and chrysophanol are contained in PM, and TSG and physcion of the stilbene and anthraquinone series are dominant in PM. Among them, TSG and physcion are known to have various biological activities, but the estrogen activity is unknown. In this study, we investigated the estrogenic activities of TSG and physcion in MCF-7 cells (a breast cancer cell line). Cell proliferation and cytotoxicity were measured using BrdU assay and cell counting kit-8. The cell cycle of MCF-7 cells and expression of estrogen receptor alpha (ERα) were analyzed using FACS. Both TSG and physcion dose-dependently stimulated cell proliferation of MCF-7 cells in the concentration range 1 to 100 μM. Furthermore, this proliferation was inhibited by cotreatment with the estrogen receptor antagonist ICI 182,780. Treatment with TSG (10 to 100 μM) or physcion (1 to 100 μM) was found to increase the proportion of cells in the S phase significantly, and this increase was also inhibited by ICI 182,780 cotreatment. In addition, the nuclear expression of ERα in MCF-7 cells was significantly upregulated by TSG (1 to 100 μM) or physcion (10 to 100 μM) but these upregulations were also inhibited by ICI 182,780 cotreatment. These observations indicate both TSG and physcion and induce estrogen-like activity via ERα.

Issue Cover (December 2020)

Journal of NeurochemistryVolume 155, Issue 5 Issue CoverFree Access Issue Cover (December 2020) First published: 25 November 2020 https://doi.org/10.1111/jnc.14771 Read the full article: ‘Donepezil-induced oligodendrocyte differentiation is mediated through estrogen receptors’ by O. Imamura, M. Arai, M. Dateki, K. Oishi, K. Takishima, (J. Neurochem. 2020, vol. 155 (5), pp. 494–507) on doi:10.1111/jnc.14927 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract Front cover: We previously demonstrated that donepezil stimulates the differentiation of neural stem cells into oligodendrocytes and neurons, albeit at the expense of astrogenesis. In this study, we aimed at elucidating the molecular pathways contributing to donepezil-induced differentiation using mouse induced pluripotent stem cell-derived neural stem cells. The signaling pathway reporter assay revealed that donepezil increased the transcriptional activity of estrogen response element under differentiating conditions. Moreover, estrogen receptors α (ERα) and β (ERβ) were highly expressed in myelin basic protein (MBP)-positive mature oligodendrocytes. The estrogen receptor (ER) antagonist ICI 182,780 abrogated the number of MBP-positive oligodendrocytes induced by donepezil but exerted no effect on their differentiation into Tuj1-positive neurons and GFAP-positive astrocytes. These findings indicate that the donepezil-stimulated oligodendrocyte is mediated through the ER signaling pathway. Image content: Cover image shows a micrographic image of the neurospheres derived from mouse induced pluripotent stem cells. Read the full article ‘Donepezil-induced oligodendrocyte differentiation is mediated through estrogen receptors’ by O. Imamura, M. Arai, M. Dateki, K. Oishi, K. Takishima, (J. Neurochem. 2020, vol. 155 (5), pp. 494–507) on doi:10.1111/jnc.14927 Volume155, Issue5December 2020 RelatedInformation

Total saponin extract, ginsenoside Rb1, and compound K alleviate peripheral and central neuropathic pain through estrogen receptors on rats.

In this study, we investigated whether total saponin extract (TSE), ginsenoside Rb1, and Rb1 metabolite compound K, which are isolated from red ginseng, have antinociceptive effects on peripheral and central neuropathic pain (PNP and CNP, respectively). PNP and CNP were induced by tail nerve injury (TNI) at S1 and by contusive spinal cord injury (SCI) at T9 in male Sprague-Dawley rats, respectively. Two weeks after TNI or 4 weeks after SCI, pain-induced rats were orally administered vehicle, TSE (50 mg/kg), Rb1 (12.5 mg/kg), compound K (7 mg/kg), or gabapentin (GBP, 60 mg/kg), and the antinociceptive effects were examined by von Frey filament, cold/warm water, and hot plate analyses. Allodynia and hyperalgesia were significantly alleviated by TSE, Rb1, and GBP 1 hr after drug administration. The immunohistochemistry and real-time RT-PCR results showed that the activation of microglia/astrocytes and the expression of inflammatory mediators such as Il-1β, Il-6, iNOS, and Cox-2 were also significantly inhibited in L4-L5 spinal cord of CNP-induced rats 1 hr after drug administration. Furthermore, the antinociceptive effects of TSE and Rb1 were reversed by treatment with the estrogen receptor (ER) antagonist ICI182780. In particular, compound K also significantly alleviated both PNP and CNP. Therefore, our results indicate that TSE, Rb1, and compound K have potential antinociceptive effects against neuropathic pain that might be mediated through the ER.

Progestin type affects the increase of heparanase level and procoagulant activity mediated by the estrogen receptor.

Does progestin have an effect on heparanase level and procoagulant activity? Progestin increases the heparanase level and procoagulant activity via the estrogen receptor and the magnitude of the effect depends on the progestin type. Users of combined oral contraceptives (COCs) containing third- and fourth-generation progestins have a higher risk of venous thrombosis compared to those employing second-generation progestins. Heparanase protein is capable of degrading heparan sulfate (HS) chains and enhancing activation of the coagulation system. We have previously demonstrated that estrogen enhances the expression and procoagulant activity of heparanase. Estrogen and progestin receptor positive breast carcinoma cell lines: EMT6, T47D and MCF-7 were compared to the MDA-231 breast carcinoma cell line devoid of these receptors. This observational study incorporated 45 users of third-generation COCs progestins, 21 users of fourth-generation COCs progestins and 28 individuals not using hormonal therapy and not pregnant per history. Second-generation progestin-levonorgestrel, third-generation progestin-desogastrel (DSG), an estrogen receptor antagonist-ICI 182.780 and a progestin receptor antagonist-mifepristone, were added to cell lines. Heparanase level and procoagulant activity, HS levels, tissue factor (TF) activity and factor Xa levels were evaluated in the plasma of the study group. Levonorgestrel and DSG increased heparanase levels in the cells and medium. The effect of DSG was more prominent and additive to that of estrogen. The effect was inhibited by ICI 182.780. In the plasma of COC users, heparanase procoagulant activity, HS levels, TF activity and factor Xa levels were significantly higher compared to controls. In COC pills containing the same dose of estrogen, the procoagulant effect of drospirenone was significantly stronger than that of DSG and gestodene. The limitations of the study include a small number of participants in each study group, although the results are statistically significant and evaluated by several different coagulation parameters. The study demonstrates a new mechanism through which progestin affects coagulation system activation and shows that this effect is progestin type-dependent. Development of a progestin derivative with an attenuated effect on heparanase procoagulant activity may reduce thrombotic risk. No external funding was sought for this study. Y.N. is named in a European patent application No. IL201200027 filed on 18 January 2012. Other authors have no conflict of interest to declare. N/A.

Expression characteristics and regulatory mechanism of Apela gene in liver of chicken (Gallus gallus).

Apela, a novel endogenous peptide ligand for the G-protein-coupled apelin receptor, was first discovered and identified in human embryonic stem cells in 2013. Apela has showed some biological functions in promoting angiogenesis and inducing vasodilatation of mammals by binding apelin receptor, but little is known about its expression characteristics and regulatory mechanism in chicken. In the present study, the coding sequences of Apela in chicken was cloned. The evolution history and potential function of Apela were analyzed. Subsequently, the spatiotemporal expression characteristics of chicken Apela were investigated. Furthermore, the regulatory mechanism of Apela mRNA responsing to estrogen was explored by in vitro and in vivo experiments. The results showed that the length of the CDs of Apela mRNA was 165 bp and encoded a protein consisting of 54 amino acids residues with a transmembrane domain in chicken. The Apela was derived from the same ancestor of Apelin, and abundantly expressed in liver, kidney and pancreas tissues. The expression levels of Apela in the liver of hens were significantly higher at the peak-laying stage than that at the pre-laying stage (p ≤ 0.05). The Apela mRNA levels were significantly up-regulated in primary hepatocytes treated with 17β-estradiol (p ≤ 0.05), and could be effectively inhibited by estrogen receptor antagonists MPP, ICI 182780 and tamoxifen. It indicated that chicken Apela expression was regulated by estrogen via estrogen receptor α (ERα). In individual levels, both the contents of TG, TC and VLDL-c in serum, and the expression of ApoVLDLII and Apela in liver markedly up-regulated by 17β-estradiol induction at 1mg/kg and 2mg/kg concentrations (p ≤ 0.05). This study lays a foundation for further research on Apela involving in hepatic lipid metabolism.

Quercetin promotes human epidermal stem cell proliferation through the estrogen receptor/<bold><roman>β</roman></bold>-catenin/c-Myc/cyclin A2 signaling pathway

Skin epidermal stem cells (EpSCs) play an important role in wound healing. Quercetin is a phytoestrogen reported to accelerate skin wound healing, but its effect on EpSCs is unknown. In this study, we investigated the effect of quercetin on human EpSC proliferation and explored the underlying mechanisms. We found that quercetin at 0.1~1μM significantly promoted EpSC proliferation and increased the number of cells in S phase. The pro-proliferative effect of quercetin on EpSCs was confirmed in cultured human skin tissue. Mechanistic studies showed that quercetin significantly upregulated the expressions of β-catenin, c-Myc, and cyclins A2 and E1. Inhibitor for β-catenin or c-Myc significantly inhibited quercetin-induced EpSC proliferation. The β-catenin inhibitor XAV-939 suppressed quercetin-induced expressions of β-catenin, c-Myc, and cyclins A2 and E1. The c-Myc inhibitor 10058-F4 inhibited the upregulation of c-Myc and cyclin A2 by quercetin. Pretreatment of EpSCs with estrogen receptor (ER) antagonist ICI182780, but not the G protein-coupled ER1 antagonist G15, reversed quercetin-induced cell proliferation and upregulation of β-catenin, c-Myc, and cyclin A2. Collectively, these results indicate that quercetin promotes EpSC proliferation through ER-mediated activation of β-catenin/c-Myc/cyclinA2 signaling pathway and ER-independent upregulation of cyclin E1 and that quercetin may accelerate skin wound healing through promoting EpSC proliferation. As EpSCs are used not only in clinic to treat skin wounds but also as seed cells in skin tissue engineering, quercetin is a useful reagent to expand EpSCs for basic research, skin wound treatment, and skin tissue engineering.

Determination of the Estrogenic Activity of Coffee Extract Solutions Prepared from Capsule Coffee Using the BG1Luc4E2 Assay

Abstract Objectives Estrogenic chemicals (ECs) possess estrogenic activity (EA) which can have harmful effects on the reproductive system. Coffee is known to have estrogenic potency due to its natural phytoestrogens, but coffee prepared from plastic capsules (capsule coffee) may increase exposure to ECs and consequently increase EA, potentially increasing risk to reproductive health. The objective of this study was to determine the EA of capsule coffee extract solutions in vitro. Methods Six varieties of capsule coffee and two varieties of coffee prepared from whole beans using a stainless-steel French press were first brewed then concentrated and extracted. The BG1Luc4E2 assay was then conducted to determine the EA of the coffee extracts. The normalized EA (% RME2) of the coffee extracts was determined as the relative estrogenic potency compared to the maximum normalized EA of the positive control 17β-estradiol (set to 100 % RME2). EA was determined if at least one data point on the concentration-response curve was above 15% RME2 and confirmed via inhibition with the estrogen receptor antagonist ICI 182,780. The correlation between the EA estimated by the BG1Luc4E2 assay and the estrogenic potential (EEQ) was determined with Spearman's rank correlation coefficient. Results All eight coffee extract solutions tested positive for EA and results were confirmed by their complete inhibition with ICI. The level of EA for the six capsule coffee extracts ranged from 48 to 56 % RME2, while the level of EA for the two coffee varieties prepared from whole beans were 40 and 42 % RME2. There was a significant correlation between EA and EEQ was (ρ = 0.8857, P = 0.0333). Conclusions These results indicate that the EAs of capsule coffees were higher than that of coffee prepared from a plastic-free method and that the EA measured in an in vitro model was correlated with the calculated estrogenic potential of the coffee extract's EC contents. Future studies are warranted in in vivo models as well as in humans as tests of estrogenic potency in vitro do not necessarily predict the effects in living organisms. Funding Sources This study was supported by the National Institutes of Health.

Estrogen receptor-regulated SOCS3 modulation via JAK2/STAT3 pathway is involved in BPF-induced M1 polarization of macrophages

As an alternative to bisphenol A (BPA), bisphenol F (BPF) has been increasingly used in manufacturing various consumer products. Exposured to BPF may lead to imbalanced immune homeostasis, yet the underlying mechanisms have not been fully elucidated. The present study was aimed to investigate the effects of BPF on macrophages and the underlying mechanism in regard to its association with estrogen receptor (ER), janus kinase 2/signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (JAK2/STAT3/SOCS3) pathway. In this study, after treatment of RAW264.7 macrophages with BPF (0, 5, 10, 20 μM), the macrophage M1 polarization was promoted, and the gene expression of M1 functional markers and pro-inflammatory cytokines was upregulated, which suggested the involvement of a vicious circle associated with chronic inflammation. Moreover, BPF facilitated SOCS3 expression in the cells in a dose-dependent manner, via activation of the JAK2/STAT3 signaling pathway, which may promote the transcription of many pro-inflammatory factors. Additionally, the above effects of BPF were blocked by either JAK2/STAT3 inhibitor AG490 (10 μM) or ER antagonist ICI 182,780 (10 μM). Taken together, the results of this study indicate that BPF promotes macrophage polarization toward pro-inflammatory M1 subtype, through activation of the ER-JAK2/STAT3/SOCS3 signaling pathway. Our finding may provide a new insight into the link between bisphenol exposure and immune dysfunction.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

STK3/4 Expression Is Regulated in Uterine Endometrial Cells during the Estrous Cycle.

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Bidirectional Synaptic Plasticity Is Driven by Sex Neurosteroids Targeting Estrogen and Androgen Receptors in Hippocampal CA1 Pyramidal Neurons.

Neuroactive estrogenic and androgenic steroids influence synaptic transmission, finely modulating synaptic plasticity in several brain regions including the hippocampus. While estrogens facilitate long-term potentiation (LTP), androgens are involved in the induction of long-term depression (LTD) and depotentiation (DP) of synaptic transmission. To examine sex neurosteroid-dependent LTP and LTD in single cells, patch-clamp recordings from hippocampal CA1 pyramidal neurons of male rats and selective antagonists for estrogen receptors (ERs) and androgen (AR) receptors were used. LTP induced by high-frequency stimulation (HFS) depended on activation of ERs since it was prevented by the ER antagonist ICI 182,780 in most of the neurons. Application of the selective antagonists for ERα (MPP) or ERβ (PHTPP) caused a reduction of the LTP amplitude, while these antagonists in combination, prevented LTP completely. LTP was never affected by blocking AR with the specific antagonist flutamide. Conversely, LTD and DP, elicited by low-frequency stimulation (LFS), were impeded by flutamide, but not by ICI 182,780, in most neurons. In few cells, LTD was even reverted to LTP by flutamide. Moreover, the combined application of both ER and AR antagonists completely prevented both LTP and LTD/DP in the same neuron. The current study demonstrates that the activation of ERs is necessary for inducing LTP in hippocampal pyramidal neurons, whereas the activation of ARs is required for LTD and DP. Moreover, both estrogen- and androgen-dependent LTP and LTD can be expressed in the same pyramidal neurons, suggesting that the activation of sex neurosteroids signaling pathways is responsible for bidirectional synaptic plasticity.

Estradiol inhibits fMLP-induced neutrophil migration and superoxide production by upregulating MKP-2 and dephosphorylating ERK

Estrogen has been reported to inhibit neutrophil infiltration related inflammation and suppress neutrophils migration in vitro, but the underlying mechanism is not fully understood. By using HL-60 differentiated neutrophil-like cells (dHL-60) and human neutrophils, we examined the effect of 17-β estradiol (E2) on cell migration and superoxide production in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and explored the mechanisms involved. We found that fMLP significantly induced dHL-60 cell and neutrophil migration and superoxide production, which was inhibited by ERK inhibitor PD98059. E2 significantly inhibited fMLP-induced dHL-60 cell and neutrophil migration and superoxide production at both physiological and pharmacological concentrations. Mechanistic studies showed that pretreatment of these cells with E2 rapidly elevated the protein level of mitogen-activated protein kinase phosphatase 2 (MKP-2) and inhibited fMLP-induced ERK phosphorylation. Pretreatment of these cells with estrogen receptor (ER) antagonist ICI 182780 reversed the inhibition of fMP-induced cell migration and superoxide production, and the induction of MKP-2 expression and the suppression of fMP-induced ERK phosphorylation by E2. However, pretreatment of cells with G-protein coupled ER antagonist G15 had no such effect. Collectively, these results demonstrate that fMLP stimulates neutrophil chemotaxis and superoxide production through activating ERK, and indicate that ER-mediated upregulation of MKP-2 may dephosphorylate ERK and contribute to the inhibitory effect of E2 on neutrophil activation by fMLP. Our study reveals new mechanisms involved in the anti-inflammatory activity of estrogen.