N-(2-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Induces Apoptosis and Cell Cycle Arrest in Breast Cancer Cells, Decreasing GPER Expression.

In this work, we performed anti-proliferative assays for the compound N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) on breast cancer (BC) cells (MCF-7, SKBR3, and triple-negative BC (TNBC) MDA-MB-231 cells) to explore its pharmacological mechanism regarding the type of cell death associated with G protein-coupled estrogen receptor (GPER) expression. The results show that HO-AAVPA induces cell apoptosis at 5 h or 48 h in either estrogen-dependent (MCF-7) or -independent BC cells (SKBR3 and MDA-MB-231). At 5 h, the apoptosis rate for MCF-7 cells was 68.4% and that for MDA-MB-231 cells was 56.1%; at 48 h, that for SKBR3 was 61.6%, that for MCF-7 cells was 54.9%, and that for MDA-MB-231 (TNBC) was 43.1%. HO-AAVPA increased the S phase in MCF-7 cells and reduced the G2/M phase in MCF-7 and MDA-MB-231 cells. GPER expression decreased more than VPA in the presence of HO-AAVPA. In conclusion, the effects of HO-AAVPA on cell apoptosis could be modulated by epigenetic effects through a decrease in GPER expression.

(-)-Epicatechin metabolites as a GPER ligands: a theoretical perspective.

Diet habits and nutrition quality significantly impact health and disease. Here is delve into the intricate relationship between diet habits, nutrition quality, and their direct impact on health and homeostasis. Focusing on (-)-Epicatechin, a natural flavanol found in various foods like green tea and cocoa, known for its positive effects on cardiovascular health and diabetes prevention. The investigation encompasses the absorption, metabolism, and distribution of (-)-Epicatechin in the human body, revealing a diverse array of metabolites in the circulatory system. Notably, (-)-Epicatechin demonstrates an ability to activate nitric oxide synthase (eNOS) through the G protein-coupled estrogen receptor (GPER). While the precise role of GPER and its interaction with classical estrogen receptors (ERs) remains under scrutiny, the study employs computational methods, including density functional theory, molecular docking, and molecular dynamics simulations, to assess the physicochemical properties and binding affinities of key (-)-Epicatechin metabolites with GPER. DFT analysis revealed distinct physicochemical properties among metabolites, influencing their reactivity and stability. Rigid and flexible molecular docking demonstrated varying binding affinities, with some metabolites surpassing (-)-Epicatechin. Molecular dynamics simulations highlighted potential binding pose variations, while MMGBSA analysis provided insights into the energetics of GPER-metabolite interactions. The outcomes elucidate distinct interactions, providing insights into potential molecular mechanisms underlying the effects of (-)-Epicatechin across varied biological contexts.

17ß-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17ß-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

Secoisolariciresinol diglucoside attenuates neuroinflammation and cognitive impairment in female Alzheimer's disease mice via modulating gut microbiota metabolism and GPER/CREB/BDNF pathway.

BACKGROUND: Gender is a significant risk factor for late-onset Alzheimer's disease (AD), often attributed to the decline of estrogen. The plant estrogen secoisolariciresinol diglucoside (SDG) has demonstrated anti-inflammatory and neuroprotective effects. However, the protective effects and mechanisms of SDG in female AD remain unclear. METHODS: Ten-month-old female APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with SDG to assess its potential ameliorative effects on cognitive impairments in a female AD model through a series of behavioral and biochemical experiments. Serum levels of gut microbial metabolites enterodiol (END) and enterolactone (ENL) were quantified using HPLC-MS. Correlation analysis and broad-spectrum antibiotic cocktail (ABx) treatment were employed to demonstrate the involvement of END and ENL in SDG's cognitive improvement effects in female APP/PS1 mice. Additionally, an acute neuroinflammation model was constructed in three-month-old C57BL/6J mice treated with lipopolysaccharide (LPS) and subjected to i.c.v. injection of G15, an inhibitor of G protein-coupled estrogen receptor (GPER), to investigate the mediating role of the estrogen receptor GPER in the cognitive benefits conferred by SDG. RESULTS: SDG administration resulted in significant improvements in spatial, recognition, and working memory in female APP/PS1 mice. Neuroprotective effects were observed, including enhanced expression of CREB/BDNF and PSD-95, reduced ß-amyloid (Aß) deposition, and decreased levels of TNF-α, IL-6, and IL-10. SDG also altered gut microbiota composition, increasing serum levels of END and ENL. Correlation analysis indicated significant associations between END, ENL, cognitive performance, hippocampal Aß-related protein mRNA expression, and cortical neuroinflammatory cytokine levels. The removal of gut microbiota inhibited END and ENL production and eliminated the neuroprotective effects of SDG. Furthermore, GPER was found to mediate the inhibitory effects of SDG on neuroinflammatory responses. CONCLUSION: These findings suggest that SDG promotes the production of gut microbial metabolites END and ENL, which inhibit cerebral ß-amyloid deposition, activate GPER to enhance CREB/BDNF signaling pathways, and suppress neuroinflammatory responses. Consequently, SDG exerts neuroprotective effects and ameliorates cognitive impairments associated with AD in female mice.

Endothelin receptor B is Required for the Blood Pressure lowering Effect of G Protein-coupled Estrogen Receptor 1 in Ovariectomized Rats.

Activation of the G protein-coupled estrogen receptor 1 (GPER1) elicits antihypertensive actions in different animal models. The endothelin-1 signaling system plays a fundamental role in blood pressure regulation. Lack of functional endothelin receptor B receptors (ETB) evokes hypertension and salt sensitivity. GPER1 and ETB interact to promote urinary sodium excretion in female rats. We hypothesized that activation of GPER1 protects against hypertension and salt sensitivity induced by ETB antagonism in female rats. Female Sprague Dawley rats were implanted with radiotelemetry. Then, animals were subjected to ovariectomy and simultaneously implanted with minipumps to deliver either the GPER1 agonist, G1, or its corresponding vehicle (Veh). Two weeks post-surgery, we initiated treatment of rats with the ETB antagonist, A-192621. Animals were maintained on a normal salt (NS) diet then challenged with a high salt (HS) diet for an additional 5 days. Assessment of mean arterial blood pressure revealed an increase in Veh-treated, but not G1-treated, rats in response to ovariectomy. A-192621 increased blood pressure in NS-fed Veh and G1-treated rats. G1 improved the circadian blood pressure rhythms which were disrupted in A-192621-treated ovariectomized rats. Thus, although systemic GPER1 activation did not protect ovariectomized rats from hypertension and salt sensitivity induced by ETB antagonism, it maintained the circadian blood pressure rhythms. Functional ETB is required to elicit the antihypertensive actions of GPER1. Additional studies are needed to improve our understanding of the interaction between G protein-coupled receptors in regulating circadian blood pressure rhythm.

G protein-coupled estrogen receptor expression in postnatal developing mouse retina.

Introduction: Estrogen has emerged as a multifaceted signaling molecule in the retina, playing an important role in neural development and providing neuroprotection in adults. It interacts with two receptor types: classical estrogen receptors (ERs) alpha and beta, and G protein-coupled estrogen receptor (Gper). Gper differs from classical ERs in structure, localization, and signaling. Here we provide the first report of the temporal and spatial properties of Gper transcript and protein expression in the developing and mature mouse retina. Methods: We applied qRT-PCR to determine Gper transcript expression in wild type mouse retina from P0-P21. Immunohistochemistry and Western blot were used to determine Gper protein expression and localization at the same time points. Results: Gper expression showed a 6-fold increase during postnatal development, peaking at P14. Relative total Gper expression exhibited a significant decrease during retinal development, although variations emerged in the timing of changes among different forms of the protein. Gper immunoreactivity was seen in retinal ganglion cells (RGCs) throughout development and also in somas in the position of horizontal cells at early time points. Immunoreactivity was observed in the cytoplasm and Golgi at all time points, in the nucleus at early time points, and in RGC axons as the retina matured. Discussion: In conclusion, our study illuminates the spatial and temporal expression patterns of Gper in the developing mouse retina and provides a vital foundation for further investigations into the role of Gper in retinal development and degeneration.

Asparagine synthetase and G-protein coupled estrogen receptor are critical responders to nutrient supply in KRAS mutant colorectal cancer.

Survival differences exist in colorectal cancer (CRC) patients by sex and disease stage. However, the potential molecular mechanism(s) are not well understood. Here we show that asparagine synthetase (ASNS) and G protein-coupled estrogen receptor-1 (GPER1) are critical sensors of nutrient depletion and linked to poorer outcomes for females with CRC. Using a 3D spheroid model of isogenic SW48 KRAS wild-type (WT) and G12A mutant (MT) cells grown under a restricted nutrient supply, we found that glutamine depletion inhibited cell growth in both cell lines, whereas ASNS and GPER1 expression were upregulated in KRAS MT versus WT. Estradiol decreased growth in KRAS WT but had no effect on MT cells. Selective GPER1 and ASNS inhibitors suppressed cell proliferation with increased caspase-3 activity of MT cells under glutamine depletion condition particularly in the presence of estradiol. In a clinical colon cancer cohort from The Cancer Genome Atlas, both high GPER1 and ASNS expression were associated with poorer overall survival for females only in advanced stage tumors. These results suggest KRAS MT cells have mechanisms in place that respond to decreased nutrient supply, typically observed in advanced tumors, by increasing the expression of ASNS and GPER1 to drive cell growth. Furthermore, KRAS MT cells are resistant to the protective effects of estradiol under nutrient deplete conditions. The findings indicate that GPER1 and ASNS expression, along with the interaction between nutrient supply and KRAS mutations shed additional light on the mechanisms underlying sex differences in metabolism and growth in CRC, and have clinical implications in the precision management of KRAS mutant CRC.

Advances in immune regulation of the G protein-coupled estrogen receptor.

Estrogen and related receptors have been shown to have a significant impact on human development, reproduction, metabolism and immune regulation and to play a critical role in tumor development and treatment. Traditionally, the nuclear estrogen receptors (nERs) ERα and ERß have been thought to be involved in mediating the estrogenic effects. However, our group and others have previously demonstrated that the G protein-coupled estrogen receptor (GPER) is the third independent ER, and estrogen signaling mediated by GPER is known to play an important role in normal physiology and a variety of abnormal diseases. Interestingly, recent studies have progressively revealed GPER involvement in the maintenance of the normal immune system, abnormal immune diseases, and inflammatory lesions, which may be of significant clinical value primarily in the immunotherapy of tumors. In this article, we review current advances in GPER-related immunomodulators and provide a theoretical basis and potential clinical targets to ameliorate immune-related diseases and immunotherapy for tumors.

The G-Protein-Coupled Estrogen Receptor Selective Agonist G-1 Attenuates Cell Viability and Migration in High-Grade Serous Ovarian Cancer Cell Lines.

The G-protein-coupled estrogen receptor (GPER; G-protein-coupled estrogen receptor 30, also known as GPR30) is a novel estrogen receptor and has emerged as a promising target for ovarian cancer. GPER, a seven-transmembrane receptor, suppresses cellular viability and migration in studied ovarian cancer cells. However, its impact on the fallopian tube, which is the potential origin of high-grade serous (HGSC) ovarian cancer, has not been addressed. This study was conducted to evaluate the relationship of GPER, ovarian cancer subtypes, i.e., high-grade serous cell lines (OV90 and OVCAR420), as well as the cell type that is the potential origin of HGSC ovarian cancer (i.e., the fallopian tube cell line FT190). The selective ligand assessed here is the agonist G-1, which was utilized in an in vitro study to characterize its effects on cellular viability and migration. As a result, this study has addressed the effect of a specific GPER agonist on cell viability, providing a better understanding of the effects of this compound on our diverse group of studied cell lines. Strikingly, attenuated cell proliferation and migration behaviors were observed in the presence of G-1. Thus, our in vitro study reveals the impact of the origin of HGSC ovarian cancers and highlights the GPER agonist G-1 as a potential therapy for ovarian cancer.

The G Protein Estrogen Receptor (GPER) is involved in the resistance to the CDK4/6 inhibitor palbociclib in breast cancer.

BACKGROUND: The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients. METHODS: BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored. RESULTS: Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs. CONCLUSIONS: Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

Growth characteristics of HCT116 xenografts lacking asparagine synthetase vary according to sex.

BACKGROUND: Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase (ASNS) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS-/- and HCT 116 ASNS+/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice. RESULTS: ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor (GPER1) in tumors from male and female mice with HCT116 ASNS-/- xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth. CONCLUSIONS: Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS/GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients.

Selective Activation of G Protein-Coupled Estrogen Receptor 1 (GPER1) Reduces ER Stress and Pyroptosis via AMPK Signaling Pathway in Experimental Subarachnoid Hemorrhage.

Neuroinflammation is a critical pathogenic event following hemorrhagic stroke. Endoplasmic reticulum (ER) stress-induced apoptosis and nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3(NLRP3)-associated pyroptosis can contribute to the escalation of neuroinflammatory responses, leading to increased brain damage. G protein-coupled estrogen receptor 1(GPER1), as the most extensively characterized brain-derived estrogen, was reported to trigger neuroprotective effects. However, the anti-apoptotic and anti-pyroptotic effect of GPER1 activation and the underlying mechanism has not been fully elucidated. We established the experimental SAH model by intravascular perforation. The GPER1 selective agonist G1 was intravenously administered 1 h following SAH. For mechanistic exploration, the selective inhibitor of adenosine monophosphate-activated protein kinase (AMPK), dorsomorphin, was administered via intracerebroventricular injection 30 min prior to SAH induction. Post-SAH assessments included SAH grade, the short-term and long-term neurological outcomes, brain edema, cerebral blood flow, transmission electron microscopy (TEM), western blot (WB), ELISA, TUNEL staining, Fluoro-Jade C staining (FJC), and immunofluorescence staining. The expression of GPER1 was observed to elevate at 6 h and peaked at 24 h subsequent to SAH, predominantly co-localized with neurons. Post-treatment with G1 markedly ameliorated both the short-term and long-term neurological deficits of SAH mouse, as well as inhibiting the expression of neuronal ER stress-associated apoptotic proteins (i.e., CHOP, GRP78, Caspase-12, Cleaved Caspase-3, Bax, Bcl2) and pyroptosis-associated proteins (i.e., NLRP3, ASC, Cleaved Caspase-1). Additionally, our research revealed that inhibition of AMPK with dorsomorphin attenuated the neuroprotective effects of G1. This was accompanied by modifications in the molecular pathways associated with ER stress-induced apoptosis and pyroptosis. These data herein elucidated that GPER1 exerted neuroprotective effects by mitigating neuroinflammation in an AMPK-dependent manner, which modulates neuronal ER stress-associated apoptosis and pyroptosis. Boosting the anti-apoptotic and anti-pyroptotic effect by activating GPER1 may be an efficient treatment strategy for SAH patients.

G protein-coupled estrogen receptor agonist G-1 decreases ADAM10 levels and NLRP3-inflammasome component activation in response to Staphylococcus aureus alpha-hemolysin.

The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.

New Mechanism for the Apoptosis of Human Neuroblastoma Cells by the Interaction between Fluorene-9-Bisphenol and the G Protein-Coupled Estrogen Receptor 1.

Fluorene-9-bisphenol (BHPF) is an emerging contaminant. Presently, there is no report on its interaction with G protein-coupled estrogen receptor 1 (GPER). By using an integrated toxicity research scenario that combined theoretical study with experimental methods, BHPF was found to inhibit the GPER-mediated effect via direct receptor binding. Molecular dynamics simulations found that Trp2726.48 and Glu2756.51 be the key amino acids of BHPF binding with GPER. Moreover, the calculation indicated that BHPF was a suspected GPER inhibitor, which neither can activate GPER nor is able to form water channels of GPER. The role of two residues was successfully verified by following gene knockout and site-directed mutagenesis assays. Further in vitro assays showed that BHPF could attenuate the increase in intracellular concentration of free Ca2+ induced by G1-activated GPER. Besides, BHPF showed an enhanced cytotoxicity compared with G15, indicating that BHPF might be a more potent GPER inhibitor than G15. In addition, a statistically significant effect on the mRNA level of GPER was observed for BHPF. In brief, the present study proposes that BHPF be a GPER inhibitor, and its GPER molecular recognition mechanism has been revealed, which is of great significance for the health risk and assessment of BHPF.

Selenium restored mitophagic flux to alleviate cadmium-induced hepatotoxicity by inhibiting excessive GPER1-mediated mitophagy activation.

Cadmium (Cd) is a common environmental pollutant, while selenium (Se) can ameliorate heavy metal toxicity. Consequently, this study aimed to investigate the protective effects of Se against Cd-induced hepatocyte injury and its underlying mechanisms. To achieve this, we utilized the Dongdagou-Xinglong cohort, BRL3A cell models, and a rat model exposed to Cd and/or Se. The results showed that Se counteracted liver function injury and the decrease in GPER1 levels caused by environmental Cd exposure, and various methods confirmed that Se could protect against Cd-induced hepatotoxicity both in vivo and in vitro. Mechanistically, Cd caused excessive mitophagy activation, evidenced by the colocalization of LC3B, PINK1, Parkin, P62, and TOMM20. Transfection of BRL3A cells with mt-keima adenovirus indicated that Cd inhibited autophagosome-lysosome fusion, thereby impeding mitophagic flux. Importantly, G1, a specific agonist of GPER1, mitigated Cd-induced mitophagy overactivation and hepatocyte toxicity, whereas G15 exacerbates these effects. Notably, Se supplementation attenuated Cd-induced GPER1 protein reduction and excessive mitophagy activation while facilitating autophagosome-lysosome fusion, thereby restoring mitophagic flux. In conclusion, this study proposed a novel mechanism whereby Se alleviated GPER1-mediated mitophagy and promoted autophagosome-lysosome fusion, thus restoring Cd-induced mitophagic flux damage, and preventing hepatocyte injury.

Epigenetic Modulation of GPER Expression in Gastric and Colonic Smooth Muscle of Male and Female Non-Obese Diabetic (NOD) Mice: Insights into H3K4me3 and H3K27ac Modifications.

Type 1 diabetes (T1D) affects gastrointestinal (GI) motility, favoring gastroparesis, constipation, and fecal incontinence, which are more prevalent in women. The mechanisms are unknown. Given the G-protein-coupled estrogen receptor's (GPER) role in GI motility, we investigated sex-related diabetes-induced epigenetic changes in GPER. We assessed GPER mRNA and protein expression levels using qPCR and Western blot analyses, and quantified the changes in nuclear DNA methyltransferases and histone modifications (H3K4me3, H3Ac, and H3K27Ac) by ELISA kits. Targeted bisulfite and chromatin immunoprecipitation assays were used to evaluate DNA methylation and histone modifications around the GPER promoter by chromatin immunoprecipitation assays in gastric and colonic smooth muscle tissues of male and female control (CTR) and non-obese diabetic (NOD) mice. GPER expression was downregulated in NOD, with sex-dependent variations. In the gastric smooth muscle, not in colonic smooth muscle, downregulation coincided with differences in methylation ratios between regions 1 and 2 of the GPER promoter of NOD. DNA methylation was higher in NOD male colonic smooth muscle than in NOD females. H3K4me3 and H3ac enrichment decreased in NOD gastric smooth muscle. H3K4me3 levels diminished in the colonic smooth muscle of NOD. H3K27ac levels were unaffected, but enrichment decreased in NOD male gastric smooth muscle; however, it increased in the NOD male colonic smooth muscle and decreased in the female NOD colonic smooth muscle. Male NOD colonic smooth muscle exhibited decreased H3K27ac levels, not female, whereas female NOD colonic smooth muscle demonstrated diminished enrichment of H3ac at the GPER promoter, contrary to male NOD. Sex-specific epigenetic mechanisms contribute to T1D-mediated suppression of GPER expression in the GI tract. These insights advance our understanding of T1D complications and suggest promising avenues for targeted therapeutic interventions.

Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells.

BACKGROUND: Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERß, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells. METHODS: The potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients. RESULTS: After the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients. CONCLUSIONS: Overall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

17-ß-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity.

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-ß-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2',7'-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.