GPR30 Research Articles

G-Protein Coupled Receptor 1 Is Involved in Tetrachlorobisphenol A-Induced Inflammatory Response in Jurkat Cells.

Estrogens can affect the immune inflammatory response through estrogen receptor alpha (ERα), but the specific role of estrogen member receptor G-protein coupled receptor 1 (GPER1) in this process remains unclear. In this study, we evaluated the effects of tetrachlorobisphenol A (TCBPA), which has estrogen activity, on immune inflammatory-related indicators of Jurkat cells, as well as investigated the role of GPER1 in these effects. The results showed that TCBPA at lower concentrations significantly promoted the viability of Jurkat cells, whereas higher concentrations decreased cell viability. TCBPA at concentrations ranging from 1 to 25 μM increased the intracellular reactive oxygen species (ROS) levels. Additionally, treatment with 10 μM TCBPA increased the protein expression of ERα and GPER1, elevated the phosphorylation of protein kinase B (p-Akt), and upregulated the mRNA levels of GPER1, Akt, and phosphoinositide 3-kinase (PI3K) genes. Treatment with 10 μM TCBPA also upregulated the protein or gene expression of pro-inflammatory cytokines, such as interleukins (IL1β, IL2, IL6, IL8, IL12α) and tumor necrosis factor alpha (TNFα) in Jurkat cells. Furthermore, pretreatment with a GPER1 inhibitor G15 significantly reduced the mRNA levels of Akt induced by 10 μM TCBPA. Moreover, the upregulation of mRNA expression of RelA (p65), TNFα, IL6, IL8, and IL12α induced by 10 μM TCBPA was also significantly attenuated after G15 pretreatment. These findings suggest that TCBPA upregulates the expression of genes related to inflammatory responses by activating the GPER1-mediated PI3K/Akt signaling pathway. This study provides new insights into the mechanism of TCBPA-induced inflammatory response.

Activation of GPER1 by G1 prevents PTSD-like behaviors in mice: Illustrating the mechanisms from BDNF/TrkB to mitochondria and synaptic connection.

G1 is a specific agonist of G protein-coupled estrogen receptor 1 (GPER1), which binds and activates GPER1 to exert various neurological functions. However, the preventive effect of G1 on post-traumatic stress disorder (PTSD) and its mechanisms are unclear. To evaluate the protective effect of G1 against synaptic and mitochondrial impairments and to investigate the mechanism of G1 to improve PTSD from brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling. This study initially detected GPER1 expression in the hippocampus of single prolonged stress (SPS) mice, utilizing both Western blot and immunofluorescence staining. Subsequently, the effects of G1 on PTSD-like behaviors, synaptic, and mitochondrial functions in SPS mice were investigated. Additionally, the involvement of BDNF/TrkB signaling involved in the protection was further confirmed using GPER1 antagonist and TrkB inhibitor, respectively. The expression of GPER1 was reduced in the hippocampus of SPS mice, and G1 treatment given for 14 consecutive days significantly improved PTSD-like behaviors in SPS mice compared with model group. Electrophysiological local field potential (LFP) results showed that G1 administration for 14 consecutive days could reverse the abnormal changes in the gamma oscillation in the CA1 region of SPS mice. Meanwhile, G1 administration for 14 consecutive days could significantly improve the abnormal expression of synaptic proteins, increase the expression of mitochondria-related proteins, increase the number of synapses in the hippocampus, and ameliorate the damage of hippocampal mitochondrial structure in SPS mice. In addition, G15 (GPER1 inhibitor) and ANA-12 (TrkB inhibitor) blocked the ameliorative effects of G1 on PTSD-like behaviors and aberrant expression of hippocampal synaptic and mitochondrial proteins in SPS mice and inhibited the reparative effects of G1 on structural damage to hippocampal mitochondria, respectively. G1 improved PTSD-like behaviors in SPS mice, possibly by increasing hippocampal GPER1 expression and promoting BDNF/TrkB signaling to repair synaptic and mitochondrial functional impairments. This study would provide critical mechanism for the prevention and treatment of PTSD.

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 1h following SAH. For mechanistic exploration, the selective inhibitor of adenosine monophosphate-activated protein kinase (AMPK), dorsomorphin, was administered via intracerebroventricular injection 30min 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 6h and peaked at 24h 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.

Maternal Zinc Diet Impairs Learning and Memory in Offspring Rats through the CREB/BDNF Pathway

BACKGROUND: Zinc released into the synaptic cleft able to modulate various signaling pathways, including brain derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB). Zinc binding to its receptor, G-protein coupled receptor 39 (GPR39), may trigger biochemical pathways leading to cAMP response element binding protein (CREB)-dependent gene transcription that subsequently promotes BDNF upregulation. Therefore, zinc dyshomeostasis should be considered as a condition that induces disruption of CREB/BDNF signaling. This study was conducted to examine the effect of maternal zinc diet on hippocampal expression levels of CREB and BDNF in offspring.METHODS: One-day pregnant rats were randomly divided into five groups: zinc-deficient (D), zinc-restricted (R), zinc-adequate (A), zinc-supplemented (S), and excess zinc-supplemented (ES). The groups had different zinc diets during pregnancy and lactation. The behavioral function of the offspring was tested with Y-maze at the 43th postnatal. Hippocampus was isolated, BDNF was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and CREB was examined using sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Spatial working memory measurement demonstrated that D and ES group exhibited a significantly lower spontaneous alternation than other groups. The qRT-PCR and ELISA analysis revealed the hippocampal expression level of BDNF and CREB decreased in groups D and ES, but tended to increase in groups R and S, until the highest expression peak was found in group A.CONCLUSION: High and low intake of zinc induces lower expression of BDNF and CREB in hippocampus, which further impairs learning and memory. Our findings suggest the signaling pathway of CREB/BDNF is involved in zinc dyshomeostasis-induced cognitive impairments.KEYWORDS: hippocampus, BDNF, CREB, TrkB, GPR39, zinc, diet, LTP

Gill transcriptomes analysis of Takifugu obscurus, Takifugu rubripes and their hybrid offspring in freshwater and seawater

Osmoregulation is essential for aquatic organisms to balance their internal fluids and ions and adapt to environmental changes. The gills are a crucial organ for maintaining osmotic balance in fish by regulating the uptake and excretion of ions. Changes in the salinity of the aquatic environment can lead to alterations in the expression of genes within gill cells, which helps to maintain the internal salinity balance and allows the fish to adapt to different salinity levels. In this study, we used transcriptomic data of the gill from the euryhaline Takifugu obscurus and the stenohaline Takifugu rubripes, as well as their hybrid offspring (Tor1 and Tor2), to investigate how the gill contributes to osmoregulation in response to freshwater and seawater. Our study revealed 8 commonly differentially expressed genes among these four fish species. Analysis of the T. obscurus transcriptome data has demonstrated that SLC1a5, a member of the solute carrier (SLC) family, and Gper1, a G-protein coupled estrogen receptor 1-like, are enriched in many biological processes and might be important for its osmoregulation in freshwater. Furthermore, PDE8B, Lrrc9 and Cplx4 might be important osmoregulatory genes that are inherited by offspring in T. obscurus. Moreover, upon encountering changes in water salinity, T. rubripes and the hybrid offspring Tor1 reveal a marked concentration of differentially expressed genes in metabolic pathways within their gills, whereas T. obscurus and the hybrid offspring Tor2 show a more even distribution of DEGs across various signaling pathways. Finally, the Pearson correlation analysis revealed that the T. rubripes displayed a significant correlation with Tor1, while the T. obscurus was significantly related to Tor2. Overall, this study provides a theoretical foundation for understanding the salinity adaptation mechanisms in hybrid offspring of Takifugu, and holds significant implications for guiding the breeding of economically valuable Takifugu strains with improved traits.

Estrogen receptor signaling and targets: Bones, breasts and brain (Review).

Estrogens are involved in a number of physiological functions, including in the development of the brain, growth, reproduction and metabolism. The biological actions of estrogens are achieved by binding to estrogen receptors (ERs) in numerous types of tissues. ERα and ERβ belong to the nuclear receptor superfamily and the G‑protein coupled ER1 (GPER1) is a membrane receptor. The primary biologically active estrogen, 17β‑estradiol demonstrates a high affinity for ERs. Mechanistically, estrogens bind to the ERs in the nucleus, and the complex then dimerize and bind to estrogen response elements (EREs) located in the promoter regions of the target genes. This is referred to as the genomic mechanism of ERs' function. Furthermore, ERs can also act through kinases and other molecular interactions leading to specific gene expression and functions, referred to as the non‑genomic mechanism. While ERα and ERβ exert their functions via both genomic and non‑genomic pathways, GPER1 exerts its function primarily via the non‑genomic pathways. Any aberrations in ER signaling can lead to one of a number of diseases such as disorders of growth and puberty, fertility and reproduction abnormalities, cancer, metabolic diseases or osteoporosis. In the present review, a focus is placed on three target tissues of estrogens, namely the bones, the breasts and the brain, as paradigms of the multiple facets of the ERs. The increasing prevalence of breast cancer, particularly hormone receptor‑positive breast cancer, is a challenge for the development of novel antihormonal therapies other than tamoxifen and aromatase inhibitors, to minimize toxicity from the long treatment regimens in patients with breast cancer. A complete understanding of the mechanism of action of ERs in bones may highlight options for novel targeted treatments for osteoporosis. Likewise, the aging of the brain and related diseases, such as dementia and depression, are associated with a lack of estrogen, particularly in women following menopause. Furthermore, gender dysphoria, a discordance between experienced gender and biological sex, is commonly hypothesized to emerge due to discrepancies in cerebral and genital sexual differentiation. The exact role of ERs in gender dysphoria requires further research.

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

BackgroundSex-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.ResultsASNS 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.ConclusionsOur 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.

Gut microbiota-derived acetate attenuates lung injury induced by influenza infection via protecting airway tight junctions

BackgroundGut microbiota (GM) have been implicated as important regulators of gastrointestinal symptom which is commonly occurred along with respiratory influenza A virus (IAV) infection, suggesting the involvement of the gut-to-lung axis in a host’s response to IAV. IAV primarily destroys airway epithelium tight junctions (TJs) and consequently causes acute respiratory disease syndrome. It is known that GM and their metabolism produce an anti-influenza effect, but their role in IAV-induced airway epithelial integrity remains unknown.MethodsA mouse model of IAV infection was established. GM were analyzed using 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) levels were measured. GM depletion and fecal microbiota transplantation (FMT) were conducted to validate the role of GM in IAV infection. A pair-feeding experiment was conducted to reveal whether IAV-induced GM dysbiosis is attributed to impaired food intake. Furthermore, human bronchial epithelial (HBE) cells were cocultured with IAV in the presence or absence of acetate. TJs function was analyzed by paracellular permeability and transepithelial electronic resistance (TEER). The mechanism of how acetate affects TJs integrity was evaluated in HBE cells transfected with G protein-coupled receptor 43 (GPR43) short hairpin RNA (shRNA).ResultsIAV-infected mice exhibited lower relative abundance of acetate-producing bacteria (Bacteroides, Bifidobacterium, and Akkermansia) and decreased acetate levels in gut and serum. These changes were partly caused by a decrease in food consumption (due to anorexia). GM depletion exacerbated and FMT restored IAV-induced lung inflammatory injury. IAV infection suppressed expressions of TJs (occludin, ZO-1) leading to disrupted airway epithelial barrier function as evidenced by decreased TEER and increased permeability. Acetate pretreatment activated GPR43, partially restored IAV-induced airway epithelial barrier function, and reduced inflammatory cytokines levels (TNF-α, IL-6, and IL-1β). Such protective effects of acetate were absent in HBE cells transfected with GPR43 shRNA. Acetate and GPR43 improved TJs in an AMP-activated protein kinase (AMPK)-dependent manner.ConclusionCollectively, our results demonstrated that GM protected airway TJs by modulating GPR43-AMPK signaling in IAV-induced lung injury. Therefore, improving GM dysbiosis may be a potential therapeutic target for patients with IAV infection.

Atrazine promotes cholangiocarcinoma cell proliferation and migration via GPER-mediated PI3K/Akt/NF-κB pathway

Atrazine (ATZ), an herbicide widely distributed on a global scale, possess a potential risk for the development of various cancers upon environmental exposure. However, the effect and molecular mechanism of ATZ in cholangiocarcinoma (CCA), is still unclear. This study aimed to investigate the effect of ATZ on the proliferation and migration of CCA cell in vitro. Immortalized human cholangiocytes (MMNK-1) and three CCA cell lines (KKU-055, KKU-100 and KKU-213B) were treated with 0.01 to 100 μM of ATZ and 17β-estradiol (E2). The results showed that, similar to E2, low doses (0.01 to 1 μM) of ATZ promoted the proliferation of all CCA and MMNK-1 cells. ATZ exposure increased non-genomic G protein-coupled estrogen receptor (GPER) expression in the cell membrane and cytoplasm of KKU-213B and KKU-055 cells via G2/M cell cycle accumulation. This, in turn, promoted the proliferation and migration of CCA cells. ATZ exposure induced the upregulation of GPER and increased expression levels of PI3K, p-PI3K, Akt, p-Akt, NF-κB and PCNA. In contrast, following ATZ treatment, the GPER antagonist G15 significantly downregulated the GPER/PI3K/Akt/NF-κB pathway. These results suggest that ATZ promotes CCA cell proliferation and migration through the GPER/PI3K/Akt/NF-κB pathway. This information can enhance public health awareness regarding ATZ contamination to prevent the relative risk of CCA.

GPCR Function in Autophagy Control: A Systematic Approach of Chemical Intervention

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

Modulation of neuroinflammation and oxidative stress by targeting GPR55 - new approaches in the treatment of psychiatric disorders.

Pharmacological treatment of psychiatric disorders remains challenging in clinical, pharmacological, and scientific practice. Even if many different substances are established for treating different psychiatric conditions, subgroups of patients show only small or no response to the treatment. The neuroinflammatory hypothesis of the genesis of psychiatric disorders might explain underlying mechanisms in these non-responders. For that reason, recent research focus on neuroinflammatory processes and oxidative stress as possible causes of psychiatric disorders. G-protein coupled receptors (GPCRs) form the biggest superfamily of membrane-bound receptors and are already well known as pharmacological targets in various diseases. The G-protein coupled receptor 55 (GPR55), a receptor considered part of the endocannabinoid system, reveals promising modulation of neuroinflammatory and oxidative processes. Different agonists and antagonists reduce pro-inflammatory cytokine release, enhance the synthesis of anti-inflammatory mediators, and protect cells from oxidative damage. For this reason, GPR55 ligands might be promising compounds in treating subgroups of patients suffering from psychiatric disorders related to neuroinflammation or oxidative stress. New approaches in drug design might lead to new compounds targeting different pathomechanisms of those disorders in just one molecule.

Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells.

Chronic inflammation is driven by proinflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and chemokines, such as c-c motif chemokine ligand 2 (CCL2), CCL3, C-X-C motif chemokine ligand 2 (CXCL2), and CXCL10. Inflammatory processes of the central nervous system (CNS) play an important role in the pathogenesis of various neurological and psychiatric disorders like Alzheimer's disease, Parkinson's disease, and depression. Therefore, identifying novel anti-inflammatory drugs may be beneficial for treating disorders with a neuroinflammatory background. The G-protein-coupled receptor 55 (GPR55) gained interest due to its role in inflammatory processes and possible involvement in different disorders. This study aims to identify the anti-inflammatory effects of the coumarin-based compound KIT C, acting as an antagonist with inverse agonistic activity at GPR55, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells in comparison to the commercial GPR55 agonist O-1602 and antagonist ML-193. All compounds significantly suppressed IL-6, TNF-α, CCL2, CCL3, CXCL2, and CXCL10 expression and release in LPS-treated BV2 microglial cells. The anti-inflammatory effects of the compounds are partially explained by modulation of the phosphorylation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK (ERK 1/2), protein kinase C (PKC) pathways, and the transcription factor nuclear factor (NF)-κB, respectively. Due to its potent anti-inflammatory properties, KIT C is a promising compound for further research and potential use in inflammatory-related disorders.

GPR30 alleviated subarachnoid hemorrhage-induced blood-brain barrier dysfunction by activating the PI3K/Akt and Nrf2/HO-1 pathways.

The blood-brain barrier (BBB) plays a critical role in the development and outcome of subarachnoid hemorrhage (SAH). This study focuses on the potential mechanism by which G-protein-coupled estrogen receptor 30 (GPR30) affects the BBB after SAH. A rat SAH model was established using an intravascular perforation approach. G1 (GPR30 agonist) was administered to investigate the mechanism of BBB damage after SAH. Brain water content, Western blotting, Evans blue leakage, and immunofluorescence staining were performed. Brain microvascular endothelial cells were induced by hemin to establish SAH model in vitro. By adding LY294002 [a phosphatidylinositol 3-kinase (PI3K) blocker] and zinc protoporphyrin IX (ZnPP IX) [a heme oxygenase 1 (HO-1) antagonist], the mechanism of improving BBB integrity through the activation of GPR30 was studied. In vivo, GPR30 activation improved BBB disruption, as evidenced by decreased cerebral edema, downregulated albumin expression, and reduced extravasation of Evans blue and IgG after G1 administration in SAH rats. Moreover, SAH downregulated the levels of tight junction (TJ) proteins, whereas treatment with G1 reversed the effect of SAH. The protective effect of G1 on BBB integrity in vitro was consistent with that in vivo, as evidenced by G1 reducing the impact of hemin on transendothelial electrical resistance (TEER) value, dextran diffusivity, and TJ protein levels in brain microvascular endothelial cells. In addition, G1 activated the PI3K/ protein kinase B (Akt) and nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 pathways both in vivo and in vitro. Furthermore, the administration of LY294002 and ZnPP IX partially reversed the protective effect of G1 on BBB integrity in hemin-stimulated cells. We demonstrated that the activation of GPR30, at least partly through the PI3K/Akt and Nrf2/HO-1 pathways, alleviated BBB damage both in vivo and in vitro. This study introduced a novel therapeutic approach for protecting the BBB after SAH.NEW & NOTEWORTHY The PI3K/Akt and Nrf2/HO-1 pathways might be potential mechanisms by which GPR30 protected the integrity of the BBB in SAH models. Therefore, treatment of SAH with GPR30 activator might be a promising therapeutic strategy.

Estradiol contributes to sex differences in resilience to sepsis-induced metabolic dysregulation and dysfunction in the heart via GPER-1-mediated PPARδ/NLRP3 signaling

Background and aimClinically, septic males tend to have higher mortality rates, but it is unclear if this is due to sex differences in cardiac dysfunction, possibly influenced by hormonal variations. Cardiac dysfunction significantly contributes to sepsis-related mortality, primarily influenced by metabolic imbalances. Peroxisome proliferator-activated receptor delta (PPARδ) is a key player in cardiac metabolism and its activation has been demonstrated to favor sepsis outcomes. While estradiol (E2) is abundant and beneficial in females, its impact on PPARδ-mediated metabolism in the heart with regards to sex during sepsis remains unknown. Methods and resultsHere, we unveil that while sepsis diminishes PPARδ nuclear translocation and induces metabolic dysregulation, oxidative stress, apoptosis and dysfunction in the heart thereby enhancing mortality, these effects are notably more pronounced in males than females. Mechanistic experiments employing ovariectomized(OVX) mice, E2 administration, and G protein-coupled estrogen receptor 1(GPER-1) knockout (KO) mice revealed that under lipopolysaccharide (LPS)-induced sepsis, E2 acting via GPER-1 enhances cardiac electrical activity and function, promotes PPARδ nuclear translocation, and subsequently ameliorates cardiac metabolism while mitigating oxidative stress and apoptosis in females. Furthermore, PPARδ specific activation using GW501516 in female GPER-1−/− mice reduced oxidative stress, ultimately decreasing NLRP3 expression in the heart. Remarkably, targeted GPER-1 activation using G1 in males mirrors these benefits, improving cardiac electrical activity and function, and ultimately enhancing survival rates during LPS challenge. By employing NLRP3 KO mice, we demonstrated that the targeted GPER-1 activation mitigated injury, enhanced metabolism, and reduced apoptosis in the heart of male mice via the downregulation of NLRP3. ConclusionOur findings collectively illuminate the sex-specific cardiac mechanisms influencing sepsis mortality, offering insights into physiological and pathological dimensions. From a pharmacological standpoint, this study introduces specific GPER-1 activation as a promising therapeutic intervention for males under septic conditions. These discoveries advance our understanding of the sex differences in sepsis-induced cardiac dysfunction and also present a novel avenue for targeted interventions with potential translational impact.

G-protein coupled estrogen receptor 1, amyloid-β, and tau tangles in older adults

Accumulation of amyloid-β (Aβ) and tau tangles are hallmarks of Alzheimer’s disease. Aβ is extracellular while tau tangles are typically intracellular, and it is unknown how these two proteinopathies are connected. Here, we use data of 1206 elders and test that RNA expression levels of GPER1, a transmembrane protein, modify the association of Aβ with tau tangles. GPER1 RNA expression is related to more tau tangles (p = 0.001). Moreover, GPER1 expression modifies the association of immunohistochemistry-derived Aβ load with tau tangles (p = 0.044). Similarly, GPER1 expression modifies the association between Aβ proteoforms and tau tangles: total Aβ protein (p = 0.030) and Aβ38 peptide (p = 0.002). Using single nuclei RNA-seq indicates that GPER1 RNA expression in astrocytes modifies the relation of Aβ load with tau tangles (p = 0.002), but not GPER1 in excitatory neurons or endothelial cells. We conclude that GPER1 may be a link between Aβ and tau tangles driven mainly by astrocytic GPER1 expression.

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

BackgroundEstetrol (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 ERβ, 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.MethodsThe 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.ResultsAfter 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.ConclusionsOverall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.

The Expression of Adipogenic Marker Is Significantly Increased in Estrogen-Treated Lipedema Adipocytes Differentiated from Adipose Stem Cells In Vitro.

Lipedema is a chronic, idiopathic, and painful disease characterized by an excess of adipose tissue in the extremities. The goal of this study is to characterize the gene expression of estrogen receptors (ERα and ERβ), G protein-coupled estrogen receptor (GPER), and ER-metabolizing enzymes: hydroxysteroid 17-beta dehydrogenase (HSD17B1, 7, B12), cytochrome P450 (CYP19A1), hormone-sensitive lipase (LIPE), enzyme steroid sulfatase (STS), and estrogen sulfotransferase (SULT1E1), which are markers in Body Mass Index (BMI) and age-matched non-lipedema (healthy) and lipedema ASCs and spheroids. Flow cytometry and cellular proliferation assays, RT-PCR, and Western Blot techniques were used to determine the expression of ERs and estrogen-metabolizing enzymes. In 2D monolayer culture, estrogen increased the proliferation and the expression of the mesenchymal marker, CD73, in hormone-depleted (HD) healthy ASCs compared to lipedema ASCs. The expression of ERβ was significantly increased in HD lipedema ASCs and spheroids compared to corresponding healthy cells. In contrast, ERα and GPER gene expression was significantly decreased in estrogen-treated lipedema spheroids. CYP19A1 and LIPE gene expressions were significantly increased in estrogen-treated healthy ASCs and spheroids, respectively, while estrogen upregulated the expression of PPAR-ϒ2 and ERα in estrogen-treated lipedema-differentiated adipocytes and spheroids. These results indicate that estrogen may play a role in adipose tissue dysregulation in lipedema.

17-β-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-β-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.

Abstract PO4-24-07: The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer

Abstract Background: The G-protein coupled estrogen receptor (GPER) is a noncanonical estrogen receptor (ER) that is estimated to be present in roughly 50-60% of all breast cancer (BC) subtypes. Previous studies have suggested that the presence of GPER during primary disease aids in disease progression, metastasis, as well as chemotherapy resistance due to the agonistic activity of traditional chemotherapies for ER+ BC, tamoxifen and fulvestrant. It is also well established that the extracellular matrix (ECM), specifically collagen, contributes to the overall risk and progression of BC in patients. Importantly, fibroblasts are one of the primary cell types responsible for ECM deposition in the tumor microenvironment. We hypothesize that the modulation of cancer-associated fibroblasts (CAFs) via estrogen signaling through GPER regulates the deposition and structure of key ECM proteins, such as collagen, to aid in disease progression. Methods: CAFs, isolated from human BC surgical resections, were plated at 100% confluency, and incubated with either 10nM β-estradiol (E2), 50nM G1 (a GPER specific agonist) or a vehicle control and 50ug/mL ascorbic acid daily for 14 days to induce matrix deposition. Phenol-free, high glucose DMEM with 10% charcoal stripped FBS was used as media to reduce FBS sourced estrogen. Following the 14-day incubation, cell proliferation was quantified prior to extracting the cells to isolate the CAF-derived matrix (CDM). The organization and composition of ECM was analyzed in CDMs through immunofluorescence (IF) or western blot, respectively. An unpaired t-test was conducted on biological triplicates and between vehicle control and either G1 or E2. Significance was determined as p&amp;lt; 0.05. Results: Culturing CAFs with E2 resulted in a 10% increase in cell proliferation (n=3, p&amp;lt; 0.05) and a 1.6-fold (n=3, p&amp;lt; 0.05) increase in total ECM deposition by western blot. Key ECM proteins such as collagen, fibronectin, and periostin had a 1.8, 1.3, and 1.3-fold increase, respectively (n=3, p&amp;lt; 0.05), in protein deposition with E2 treatment when normalized to cell number. Collagen fiber analysis of IF stained CDM for collagen resulted in a coefficient of alignment with E2 of 0.76 compared to 0.51 without (n=3 for E2 and n=6 for vehicle, p&amp;lt; 0.05). To confirm E2 was acting through GPER, CDMs were subsequently generated with exposure to 50nM G1, a specific GPER agonist. Collagen fiber analysis of the G1 exposed CDMs resulted in a coefficient of alignment with G1 of 0.71 compared to 0.51 without (n=3 for G1 and n=6 for vehicle, p&amp;lt; 0.05). Conclusions: This study demonstrates that estrogen signaling through GPER modulates the deposition of fibronectin, periostin, and collagen as well as the organization of collagen architecture. These results highlight the need to further investigate how the interplay between the ECM, GPER activity, and fibroblasts impacts breast cancer progression. Citation Format: Shelby Fertal, Brian Burkel, Fern Murdoch, Kathy O'Leary, Linda Schuler, Suzanne Ponik. The Impact of GPER Modulation on Cancer Associated Fibroblasts on Extracellular Matrix Formation in Breast Cancer [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 PO4-24-07.

GPER1 Activation Upregulates Renal Purinergic P2Y2 Receptor Expression and Lowers Blood Pressure in Ovariectomized Rats Fed High Salt Diet

Renal purinergic P2Y2 receptor promotes urinary Na+ excretion via inhibiting epithelial Na+ channel (ENaC) activity. Recent evidence implicates that ovarian hormones regulate P2Y2/ENaC natriuretic pathway, however the underlying mechanism remains unclear. Estradiol activates the G protein-coupled estrogen receptor 1 (GPER1), which promotes natriuresis in females. Taken together, we hypothesized that GPER1 activation regulates renal P2Y2 receptor expression and maintains blood pressure in ovariectomized (OVX) rats. To test our hypothesis, we determined the effect of pharmacologic activation GPER1 on (i) blood pressure and (ii) mRNA expression of renal P2Y2 receptor. In particular, female (16–20 weeks of age) Sprague Dawley rats were implanted with telemeters. After recording baseline blood pressure, rats were ovariectomized and simultaneously implanted with an osmotic minipump to deliver either G1, selective GPER1 agonist (OVX+G1), or Vehicle (OVX+Veh) for 3 weeks. Rats were fed a normal salt (NS, 0.49% NaCl) throughout the study and challenged with a high salt (HS, 4% NaCl) during the last week. Finally, animals were euthanized, renal cortex and medulla were assessed for P2Y2 and αENaC mRNA expression. We found that OVX+Veh rats fed a HS diet had greater mean arterial pressure (MAP) when compared to their baseline values prior to ovariectomy (113 ± 1 vs. 105 ± 1 mmHg; p &lt; 0.01; n = 6). Interestingly, OVX+G1 rats fed a HS diet did not elicit significant changes in MAP when compared to their baseline values (106 ± 3 vs. 104 ± 2 mmHg; p = 0.25; n = 6). Moreover, renal cortical P2Y2 mRNA expression in OVX+G1 rats was higher compared to corresponding vehicle-treated rats (1.95 ± 0.21 vs. 1.00 ± 0.17 fold change from OVX+Veh; p &lt; 0.01; n = 6-7/group). Renal outer and inner medullary P2Y2 mRNA expression elicited similar trends; however, it did not reach statistical significance. No differences were evident in the renal αENaC mRNA expression in response to G1 treatment. We also extended the study in female mice to determine the effect of genetic deletion of GPER1 on renal P2Y2 receptor mRNA expression. We found that P2Y2 mRNA expression in kidneys obtained from GPER1 KO mice was markedly lower when compared to wildtype litter mates. However, no differences were detected in the mRNA expression of αENaC in response to GPER1 deletion. The current results indicate that GPER1 activity upregulates the renal P2Y2 expression which may contribute to preventing the increase in blood pressure in HS-fed OVX animals, possibly by promoting natriuresis. Additional studies are required to improve our understanding for GPER1/P2Y2/ENaC signaling pathway in the postmenopausal female population. NIH/NIDDK R00DK119413 to EYG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.