GPR30 Research Articles

Structure of G protein-coupled receptor GPR1 bound to full-length chemerin adipokine reveals a chemokine-like reverse binding mode.

Chemerin is an adipokine with chemotactic activity to a subset of leukocytes. Chemerin binds to 3 G protein-coupled receptors, including chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C chemokine receptor-like 2 (CCRL2). Here, we report that GPR1 is capable of Gi signaling when stimulated with full-length chemerin or its C-terminal nonapeptide (C9, YFPGQFAFS). We present high-resolution cryo-EM structures of Gi-coupled GPR1 bound to full-length chemerin and to the C9 peptide, respectively. C9 insertion into the transmembrane (TM) binding pocket is both necessary and sufficient for GPR1 signaling, whereas the full-length chemerin uses its bulky N-terminal core for interaction with a β-strand located at the N-terminus of GPR1. This interaction involves multiple β-strands of full-length chemerin, forming a β-sheet that serves as a "lid" for the TM binding pocket and is energetically expensive to remove as indicated by molecular dynamics simulations with free energy landscape analysis. Combining results from functional assays, our structural model explains why C9 is an activating peptide at GPR1 and how the full-length chemerin uses a "two-site" model for enhanced interaction with GPR1.

Structural Basis for Chemerin Recognition and Signaling Through Its Receptors

Chemerin is a chemotactic adipokine that participates in a multitude of physiological processes, including adipogenesis, leukocyte chemotaxis, and neuroinflammation. Chemerin exerts biological functions through binding to one or more of its G protein-coupled receptors (GPCRs), namely chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and CC-motif receptor-like 2 (CCRL2). Of these receptors, CMKLR1 and GPR1 have been confirmed as signaling receptors of chemerin, whereas CCRL2 serves as a chemerin-binding protein without transmembrane signaling. High-resolution structures of two chemerin receptors are now available thanks to recent advancements in structure biology. This review focuses on the structural perspectives of the chemerin receptors with an emphasis on the structure–activity correlation, including key components of the two receptors for ligand recognition and conformational changes induced by chemerin and its derivative peptides for G protein activation. There are also comparisons between the two chemerin receptors and selected GPCRs with peptide ligands for better appreciation of the shared and distinct features of the chemerin receptors in ligand recognition and transmembrane signaling, and in the evolution of this subclass of GPCRs.

High glucose combined with lipopolysaccharide stimulation inhibits cell proliferation and migration of human HaCaT keratinocytes by impacting redox homeostasis and activating the polyol pathway.

High glucose level and chronic inflammation are characteristic features of diabetic cutaneous wounds. Keratinocytes make up the epidermis and play an important role in skin repair. However, metabolomic changes of keratinocytes in chronic diabetic ulcers have not been fully studied. This study used high levels of glucose combined with lipopolysaccharide to treat human HaCaT keratinocytes. Untargeted metabolomic combined with colorimetric assays were used to explore the changes of keratinocyte metabolites and related metabolic pathways caused by high glucose and lipopolysaccharide. Results demonstrated that high glucose combined with lipopolysaccharide treatment increased intracellular reactive oxygen species and impaired proliferation and migration of keratinocytes. Untargeted metabolomics analysis identified a total of 273 differential metabolites. Redox metabolism associated metabolites were largely altered. Reduced nicotinamide adenine dinucleotide, gamma-glutamylcysteine, superoxide dismutase activity and SOD2 gene expression were significantly upregulated while nicotinamide adenine dinucleotide, glutathione, glutathione peroxidase, several types of lysophosphatidylcholine, lysophosphatidylinositol, and GPR55 gene expression were downregulated. Alterations of glutathione and nicotinamide adenine dinucleotide were verified by colorimetric assays. For the first time, high glucose and LPS were observed to boost the levels of fructose, aldose reductase and sorbitol dehydrogenase of the polyol pathway in HaCaT cells. Further treatment of HaCaT with fructose leading to inhibition of cell proliferation and migration. Our data suggest high glucose combined with lipopolysaccharide significantly altered redox homeostasis associated metabolites and activate the polyol pathway in keratinocytes to impact cell proliferation and migration, providing new strategies for the treatment of chronic diabetic ulcers.

Effects of moxibustion in improving synovitis in adjuvant arthritis rats by regulating synovial GPR43 and peripheral blood Th17/Treg balance

To observe the effects of moxibustion on G protein-coupled receptor 43 (GPR43) and helper T cell 17 (Th17)/regulatory T cell (Treg) balance in rats with adjuvant arthritis (AA), so as to investigate the partial mechanism of moxibustion therapy on rheumatoid arthritis (RA). A total of 24 Wistar rats were randomly divided into a normal group, a model group and a moxibustion group, with 8 rats in each group. The AA rat model was replicated using wind, cold and moisture environmental factors + Freund's complete adjuvant (CFA) injection method. In the moxibustion group, moxibustion was performed on bilateral "Zusanli" (ST36) and "Shenshu" (BL23) for 20 min/time, once daily, for 21 d. The changes of joint swelling degree (JSD) and arthritis index (AI) were observed in each group of rats. Transmission electron microscopy and HE staining were used to examine changes in the cellular structure of the ankle joint synovial tissue in each group. Western blot analysis was employed to detect the expression levels of GPR43 in the synovial tissue of the ankle joints. Flow cytometry was used to measure the percentages of Treg and Th17 in peripheral blood. ELISA was used to determine the contents of interleukin-10 (IL-10) and transforming growth factor-β 1 (TGF-β1) in serum. Compared with the normal group, the JSD and AI in the model group increased before and after treatment (P<0.01), with significant synovial pathological and ultrastruct ural injury observed. Additionally, compared with the normal group, the expression of GPR43 in the synovial tissue decreased (P<0.01), the Treg percentage in peripheral blood decreased (P<0.01) and the Th17 percentage increased (P<0.01), serum IL-10 and TGF-β1 contents decreased in the model group (P<0.01). Compared with the model group, the moxibustion group exhibited a significant reduction in JSD and AI after treatment (P<0.01), the degree of pathological and ultrastruct ural damage in the synovium decreased, the expression of GPR43 in the synovial tissue increased (P<0.05), the Treg percentage in peripheral blood increased (P<0.01) and the Th17 percentage decreased (P<0.01), serum IL-10 and TGF-β1 contents increased (P<0.01). The relative expression of GPR43 in synovial tissue was positively correlated with the percentage of Treg in peripheral blood (r=0.967, P<0.01). Moxibustion can significantly improve the inflammatory response in the synovial membrane of AA rats. The mechanism may be related to moxibustion restoring the Th17/Treg balance through regulating GPR43.

Omega-3 polyunsaturated fatty acids alleviate hyperuricemic nephropathy by inhibiting renal pyroptosis through GPR120

Hyperuricemic nephropathy (HN) is characterized by increased serum uric acid levels that incite renal inflammation. While omega-3 polyunsaturated fatty acids (PUFAs) are known for their anti-inflammatory properties, their impact on HN remains unclear. This study explored the effects of omega-3 PUFAs, specifically docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on HN. Using a mouse model induced by adenine and potassium oxonate, we treated HN mice with DHA, EPA, or both for four weeks. The results showed that omega-3 PUFAs significantly reduced serum uric acid levels and improved kidney function, with DHA, EPA, and their combination showing similar efficacy. Transcriptome sequencing and further analysis revealed that these fatty acids alleviate renal pyroptosis by reducing key markers such as NOD-like receptor pyrin containing 3 (NLRP3), cleaved gasdermin-D, caspase-1, and interleukin-1β. To further investigate the underlying mechanism, we focused on G-protein coupled receptor 120 (GPR120), a receptor activated by DHA. The use of a GPR120 antagonist (AH7614) partially blocked DHA’s effects, while the agonist (TUG891) mimicked its anti-pyroptotic actions. Co-immunoprecipitation assays showed that DHA activates GPR120, leading to its internalization and interaction with β-arrestin2, ultimately inhibiting NLRP3 inflammasome formation and reducing inflammation. Overall, omega-3 PUFAs, particularly through GPR120 activation, appear to protect against renal inflammation in HN by modulating the NLRP3/caspase-1/GSDMD pathway.

G-protein–coupled estrogen receptor 30 regulation of signaling downstream of protein kinase Cε mediates sex dimorphism in hyaluronan-induced antihyperalgesia

Abstract High molecular weight hyaluronan (HMWH) inhibits hyperalgesia induced by diverse pronociceptive inflammatory mediators and their second messengers, in rats of both sexes. However, the hyperalgesia induced by ligands at 3 pattern recognition receptors, lipopolysaccharide (a toll-like receptor 4 agonist), lipoteichoic acid (a toll-like receptor 2/6 agonist), and nigericin (a NOD-like receptor family, pyrin domain containing 3 activator), and oxaliplatin and paclitaxel chemotherapy–induced peripheral neuropathy are only attenuated in males. After gonadectomy or intrathecal administration of an antisense to G-protein–coupled estrogen receptor 30 (GPER) mRNA, HMWH produces antihyperalgesia in females. In nociceptors cultured from rats that had been treated with oxaliplatin, HMWH reverses nociceptor sensitization from male and GPER antisense–treated female, but not from gonad intact females. G-protein–coupled estrogen receptor–dependent sex dimorphism for HMWH-induced antihyperalgesia was also observed for the prolongation of prostaglandin E2 (PGE2)-induced hyperalgesia in primed nociceptors. While in primed rats, HMWH inhibits early, protein kinase A-dependent hyperalgesia, 30 minutes post PGE2 injection, in both sexes; measured 4 hours post-PGE2, HMWH inhibits the protein kinase Cε (PKCε)-dependent prolongation of PGE2 hyperalgesia only in males and GPER antisense–treated females. In females, hyperalgesia induced by PKCε agonist, ψεRACK, in control but not in primed nociceptors, was inhibited by HMWH. Inhibitors of 2 GPER second messengers, extracellular-regulated kinase 1/2 and nonreceptor tyrosine kinase, also unmasked HMWH antihyperalgesia in females with oxaliplatin chemotherapy–induced peripheral neuropathy, a condition in which nociceptors are primed as well as sensitized. Our results support GPER-dependent sex dimorphism in HMWH-induced antihyperalgesia for pain induced by pattern recognition receptor agonists, and chronic inflammatory and neuropathic pain, mediated by changes in signaling downstream of PKCε in primed nociceptors.

GPR30 Agonist G1 Mitigates Sepsis-Induced Cardiac Dysfunction by Inhibiting ACE2/c-FOS-Mediated Necroptosis in Female Mice.

Sepsis is a severe inflammatory syndrome with high mortality and morbidity. Sepsis-induced myocardial dysfunction (SIMD) is a common cause of death in sepsis. The female sex is less susceptible to sepsis-related organ dysfunction, although the underlying mechanism of this sex difference remains unclear. This study explored the role of estrogen receptor G protein-coupled estrogen receptor 30 (GPR30) in septic cardiac dysfunction. Results from the present study indicated that GPR30 activation by the G1 agonist protected female mouse hearts against SIMD exposed to lipopolysaccharides. However, this beneficial effect was absent in female ACE2-knockout mice, as demonstrated by poorer cardiac contractility, myocardial injury, and necroptosis. We also demonstrated that the Stat6 transcription factor induced ace2 transcription by enhancing its promoter activity under GPR30 activation in septic hearts. The adenovirus-mediated inhibition of ACE2 targeting c-FOS expression reversed the deterioration, restored cardiac function, and improved survival in female ACE2-knockout mice. These results demonstrate the essential role of GPR30/STAT6/ACE2/c-FOS-mediated necroptosis in G1-mediated protection and provide novel insight into the pathogenesis of sepsis-related organ damage.

A computational and machine learning approach to identify GPR40-targeting agonists for neurodegenerative disease treatment.

The G protein-coupled receptor 40 (GPR40) is known to exert a significant influence on neurogenesis and neurodevelopment within the central nervous system of both humans and rodents. Research findings indicate that the activation of GPR40 by an agonist has been observed to promote the proliferation and viability of hypothalamus cells in the human body. The objective of the present study is to discover new agonist compounds for the GPR40 protein through the utilization of machine learning and pharmacophore-based screening techniques, in conjunction with other computational methodologies such as docking, molecular dynamics simulations, free energy calculations, and investigations of the free energy landscape. In the course of our investigation, we successfully identified five unreported agonist compounds that exhibit robust docking score, displayed stability in ligand RMSD and consistent hydrogen bonding with the receptor in the MD trajectories. Free energy calculations were observed to be higher than control molecule. The measured binding affinities of compounds namely 1, 3, 4, 6 and 10 were -13.9, -13.5, -13.4, -12.9, and -12.1 Kcal/mol, respectively. The identified molecular agonist that has been found can be assessed in terms of its therapeutic efficacy in the treatment of neurological diseases.

GPR34 is a metabolic immune checkpoint for ILC1-mediated antitumor immunity.

Type 1 innate lymphoid cells (ILC1s) are a class of tissue-resident cells with antitumor activity, suggesting its possible role in solid tumor immune surveillance, but it is not clear whether manipulating ILC1s can induce potent antitumor immune responses. Here, we found that G-protein-coupled receptor 34 (GPR34), a receptor for lysophosphatidylserine (LysoPS), was highly expressed on ILC1s but not on conventional natural killer cells in the tumor microenvironment. LysoPS was enriched in the tumor microenvironment and could inhibit ILC1 activation via GPR34. Genetic deletion of LysoPS synthase Abhd16a expression in tumors or Gpr34 expression in ILC1s or antagonizing GPR34 enhanced ILC1 antitumor activity. In individuals with cancer, ABHD16A expression in tumors or GPR34 expression in ILC1s was inversely correlated with the antitumor activity of ILC1s or ILC1-like cells. Thus, our results demonstrate that manipulating ILC1s can induce potent antitumor immunity, and GPR34 is a metabolic immune checkpoint that can be targeted to develop ILC1-based immunotherapy.

Endothelin receptor B is required for the blood pressure-lowering effect of G protein-coupled estrogen receptor 1 in ovariectomized rats.

Activation of 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 (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. Animals were then subjected to ovariectomy and simultaneously implanted with minipumps to deliver either the GPER1 agonist G1 or its corresponding vehicle. Two weeks post surgery, we initiated treatment of rats with the ETB antagonist A-192621. Animals were maintained on a normal-salt diet and then challenged with a high-salt diet for an additional 5 days. Assessment of mean arterial blood pressure revealed an increase in vehicle-treated, but not G1-treated, rats in response to ovariectomy. A-192621 increased blood pressure in normal-salt diet-fed vehicle- and G1-treated rats. G1 improved the circadian blood pressure rhythms that 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 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.NEW & NOTEWORTHY Systemic G protein-coupled estrogen receptor 1 (GPER1) activation in rats prevents the increase in blood pressure evoked by ovariectomy. Blockade of endothelin receptor B negates the blood pressure-lowering impact of GPER1 in ovariectomized rats. Endothelin receptor B plays an important role in mediating the blood pressure-lowering action of GPER1 activation in female rats.

MNAM enhances Blautia abundance and modulates Th17/Treg balance to alleviate diabetes in T2DM mice

This study investigated the therapeutic effects of N1-Methylnicotinamide (MNAM), a metabolic derivative, on T2DM mice induced by a high-fat diet and streptozotocin (STZ), focusing on its impact on the gut microbiome and immune modulation. MNAM significantly reduced hyperglycemia and enhanced insulin secretion, effects that were dependent on the presence of gut microbiota. It also mitigated STZ-induced weight loss and improved islet cell morphology, reducing islet cell mortality and increasing insulin (INS) levels. Flow cytometry analysis showed a decrease in T helper 17 cells (Th17) and an increase in Treg cells after MNAM treatment, corresponding to the upregulation of Treg markers [interleukin (IL)-10, forkhead box P3 (FOXP3)] and downregulation of Th17 markers [IL17A, RAR-related orphan receptor gamma (RORγt)]. Additionally, MNAM raised anti-inflammatory IL-10 levels while reducing pro-inflammatory cytokines [IL-17α, tumor necrosis factor (TNF-α), IL-6]. Microbiome analysis revealed decreased diversity and increased Blautia abundance post-MNAM administration. Treatment with Blautia not only reversed diabetes indicators but also modulated the Th17/Treg balance and reduced inflammation, with its metabolite sodium acetate mimicking these effects through the G protein-coupled receptor 43 (GPR43) pathway. These findings suggest that MNAM’s mitigation of diabetes operates through modulation of the gut microbiota and immune regulation, highlighting Blautia and its metabolite as potential therapeutic agents and providing a theoretical foundation for novel treatment strategies in T2DM.

The Expression of Cannabinoid and Cannabinoid-Related Receptors on the Gustatory Cells of the Piglet Tongue.

The gustatory system is responsible for detecting and evaluating the palatability of the various chemicals present in food and beverages. Taste bud cells, located primarily on the tongue, communicate with the gustatory sensory neurons by means of neurochemical signals, transmitting taste information to the brain. It has also been found that the endocannabinoid system (ECS) may modulate food intake and palatability, and that taste bud cells express cannabinoid receptors. The purpose of this study was to investigate the expression of cannabinoid and cannabinoid-related receptors in the gustatory cells of the papillae vallatae and foliatae of ten piglets. Specific antibodies against the cannabinoid receptors (CB1R and CB2R), G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) were applied on cryosections of lingual tissue; the lingual tissue was also processed using Western blot analysis. Cannabinoid and cannabinoid-related receptors were found to be expressed in the taste bud cells and the surrounding epithelial cells. The extra-papillary epithelium also showed strong immunolabeling for these receptors. The results showed that these receptors were present in both the taste bud cells and the extra-gustatory epithelial cells, indicating their potential role in taste perception and chemesthesis. These findings contributed to understanding the complex interactions between cannabinoids and the gustatory system, highlighting the role of the ECS within taste perception and its potential use in animal production in order to enhance food intake.

The G-Protein-Coupled Estrogen Receptor Agonist G-1 Mediates Antitumor Effects by Activating Apoptosis Pathways and Regulating Migration and Invasion in Cervical Cancer Cells.

Estrogens and HPV are necessary for cervical cancer (CC) development. The levels of the G protein-coupled estrogen receptor (GPER) increase as CC progresses, and HPV oncoproteins promote GPER expression. The role of this receptor is controversial due to its anti- and pro-tumor effects. This study aimed to determine the effect of GPER activation, using its agonist G-1, on the transcriptome, cell migration, and invasion in SiHa cells and non-tumorigenic keratinocytes transduced with the HPV16 E6 or E7 oncogenes. Transcriptome analysis was performed to identify G-1-enriched pathways in SiHa cells. We evaluated cell migration, invasion, and the expression of associated proteins in SiHa, HaCaT-16E6, and HaCaT-16E7 cells using various assays. Transcriptome analysis revealed pathways associated with proliferation/apoptosis (TNF-α signaling, UV radiation response, mitotic spindle formation, G2/M cell cycle, UPR, and IL-6/JAK/STAT), cellular metabolism (oxidative phosphorylation), and cell migration (angiogenesis, EMT, and TGF-α signaling) in SiHa cells. Key differentially expressed genes included PTGS2 (pro/antitumor), FOSL1, TNFRSF9, IL1B, DIO2, and PHLDA1 (antitumor), along with under-expressed genes with pro-tumor effects that may inhibit proliferation. Additionally, DKK1 overexpression suggested inhibition of cell migration. G-1 increased vimentin expression in SiHa cells and reduced it in HaCaT-16E6 and HaCaT-16E7 cells. However, G-1 did not affect α-SMA expression or cell migration in any of the cell lines but increased invasion in HaCaT-16E7 cells. GPER is a promising prognostic marker due to its ability to activate apoptosis and inhibit proliferation without promoting migration/invasion in CC cells. G-1 could potentially be a tool in the treatment of this neoplasia.

Sodium octanoate mediates GPR84-dependent and independent protection against sepsis-induced myocardial dysfunction

IntroductionThis study aims to evaluate the therapeutic effects of sodium octanoate (SO), a medium-chain fatty acid salt, on SIMD in a murine model and to explore its underlying mechanisms. MethodsMale mice were subjected to sepsis models through two methods: intraperitoneal injection of lipopolysaccharide (LPS) and cecal ligation and punction (CLP). Mice received interval doses of SO every 2 hours or 4 hours for a total of six times or three times after LPS treatment. The relationship between SO and G protein-coupled receptor 84 (GPR84) was evaluated through GEO data analysis and molecular docking studies. DBA/2 mice were used to study the role of the GPR84 protein in the SO-mediated protection. Energy metabolomics was utilized to comprehensively assess the impact of SO on the levels of cardiac energy metabolic products in septic mice. histone modification identification techniques were used to further identify the specific sites of histone modification in the hearts of SO-treated septic mice. ResultsSO treatment significantly improved myocardial contractile function, restored the oxidative stress imbalance and enhanced the myocardium's resistance to oxidative injury. SO significantly promotes the expression of GPR84. The loss of GPR84 function markedly attenuates the protective effects of SO. SO enhanced myocardial energy metabolism by promoting the synthesis of acetyl-CoA and upregulating genes involved in fatty acid β-oxidation which were abolished by medium-chain acyl-CoA dehydrogenase (MCAD) knockdown. SO induced histone acetylation, particularly at H3K123 and H3K80. ConclusionOur study demonstrates that SO exerts protective effects against SIMD through both GPR84-mediated anti-inflammatory and antioxidant actions and GPR84-independent enhancement of myocardial energy metabolism, possibly mediated by MCAD.

GPR40 agonist ameliorates neurodegeneration and motor impairment by regulating NLRP3 inflammasome in Parkinson’s disease animal models

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and accumulation of intracellular α-synuclein (ɑ-syn) aggregates known as Lewy bodies and Lewy neurites. Levels of polyunsaturated fatty acids (PUFAs) have previously been shown to be reduced in the SN of PD patients. G protein-coupled receptor 40 (GPR40) serves as a receptor for PUFAs, playing a role in neurodevelopment and neurogenesis. Additionally, GPR40 has been implicated in several neuropathological conditions, such as apoptosis and inflammation, suggesting its potential as a therapeutic target in PD.In this study, we investigated the neuroprotective effects of the GPR40 agonist, TUG469 in PD models. Our results demonstrated that TUG469 reduces the neurotoxicity induced by 6-OHDA in SH-SY5Y cells. In 6-OHDA-induced PD model mice, TUG469 treatment improved motor impairment, preserved dopaminergic fibers and cell bodies in the striatum (ST) or SN, and attenuated 6-OHDA-induced microgliosis and astrogliosis in the brain. Furthermore, in a PD model involving the injection of mouse ɑ-syn fibrils into the brain (mPFFs-PD model), TUG469 treatment reduced the levels of pSer129 ɑ-syn, and decreased microgliosis and astrogliosis. Our investigation also revealed that TUG469 modulates inflammasome activation, apoptosis, and autophagy in the 6-OHDA-PD model, as evidenced by the results of RNA-seq and western blotting analyses.In summary, our findings highlight the neuroprotective effects of GPR40 agonists on dopaminergic neurons and their potential as therapeutic agents for PD. These results underscore the importance of targeting GPR40 in PD treatment, particularly in mitigating neuroinflammation and preserving neuronal integrity.

Abstract P157: Acute GPER1 Activation Reduces Blood Pressure during the Inactive Period in Aged Female Mice

Non-dipping of blood pressure (BP) during the nighttime is an independent risk factor for hypertension. Importantly, BP dipping is diminished in postmenopausal women, suggesting that the female sex hormones may contribute to the circadian rhythm of BP. Estradiol, activates the G protein-coupled estrogen receptor 1 (GPER1), which elicits protective actions within the cardiovascular system. However, little is known about the GPER1 effect on circadian BP regulation in aged females. Taken together, we hypothesized that systemic GPER1 activation lowers BP and regulates circadian rhythm of BP in aged females. To test the hypothesis, 23-24 months-old C57BL/6J female mice (n=6) were implanted with radio-telemeters for BP recording. After 21 days of postsurgical recovery, BP was recorded. Mice were then given vehicle intraperitoneally daily for 3 consecutive days, followed by the selective GPER1 agonist (LNS8801, 400 µg/kg/day) for an additional 3 days. Data were collected over 24-hour periods according to the 12-hour active period and the 12-hour inactive period. GPER1 activation lowered 24-hour mean arterial pressure (MAP) compared to vehicle control values (113±3 vs. 117±3 mmHg; p=0.01). Similar to MAP, 24-hour systolic blood pressure (SBP) and diastolic blood pressure (DBP) were lower in LNS8801-treated mice compared to corresponding vehicle-treated mice. However, there was not a significant difference in 24-hour heart rate (HR). Interestingly, MAP during the inactive period was lower in LNS8801-treated mice compared to corresponding vehicle-treated mice (104±2 vs. 110±3, respectively, p=0.02). Whereas, GPER1 activation did not elicit significant changes in MAP during the active period. Similarly, LNS8801 reduced DBP during the inactive period (88±4 vs. 96±4 mmHg; p=0.04). We extended the study in aged female mice (14–16 months) with genetic deletion of GPER1 (GPER1 KO; n=6) and wildtype littermates (WT; n=5). Genetic deletion of GPER1 increased 24-hour MAP, SBP, and DBP, whereas HR was not significantly different between genotypes. Noteworthy, GPER1 KO increased MAP during the active period (126±2 vs. 114±1 mmHg; p=0.009) and during the inactive period, compared to WT (110±3 vs. 101±1 mmHg; p=0.01). The results indicate that GPER1 activity lowers BP. Interestingly, LNS8801 acutely improves BP dipping during the inactive period. Overall, GPER1 activation elicits BP-lowering effects in aged female mice, possibly by regulating the circadian BP rhythm.

The activation of the G-protein-coupled estrogen receptor promotes the aggressiveness of MDA-MB231 cells by targeting the IRE1α/TXNIP pathway

Background and purpose: This study investigated modulating the G protein-coupled estrogen receptor (GPER) on the IRElα/TXNIP pathway and its role in drug resistance in MDA-MB231 cells. Experimental approach: To determine the optimal concentrations of G1 and 4-hydroxytamoxifen (TAM), GPER expression and ERK1/2 phosphorylation were analyzed using qRT-PCR and western blotting, respectively. Cells were treated with individual concentrations of G1 (1000 nM), G15 (1000 nM), and TAM (2000 nM), as well as combinations of these treatments (G1 + G15, TAM + G15, and G1 + TAM) for 24 and 48 h. The expression levels of GPER, IRE1α, miR-17-5p, TXNIP, ABCB1, and ABCC1 genes and TXNIP protein expression were evaluated. Finally, apoptosis and cell migration were examined using flow cytometry and the wound-healing assay, respectively. Findings/Results: Activating GPER with its specific agonist G1 and TAM significantly increased IRE1α levels in MDA-MB231 cells. IRE1α through splicing XBP1 led to unfolded protein response. In addition, decreased TXNIP gene and protein expression reduced apoptosis, increased migration, and upregulated the genes associated with drug resistance. Conclusion and implication: Our investigation revealed that blocking the GPER/IRE1α/TXNIP pathway in MDA-MB231 cells could enhance treatment efficacy and improve chemotherapy responsiveness. The distinct unfolded protein response observed in MDA-MB231 cells may stem from the unique characteristics of these cells, which lack receptors for estrogen, progesterone, and HER2/neu hormones, possessing only the GPER receptor (ER-/PR-/HER2-/GPER+). This study introduced a new pathway in TNBC cells, indicating that targeting GPER could be crucial in comprehensive therapeutic strategies in TNBC cells.

Abstract P447: Impact of Altered Receptor Expression On the Cardiovascular Response to Estrogen

Premenopausal women benefit from estrogen's protective effects, including lower arterial stiffness than men of the same age. Despite estrogen's protective role, menopausal hormone therapy showed no cardiovascular benefits in the Women's Health Initiative. One theory is that the response to menopausal hormone therapy is modulated by the presence of pre-existing cardiovascular disease. Estrogen signals through nuclear estrogen receptors ERα and ERβ, and the membrane-bound G protein-coupled estrogen receptor (GPER). Our lab has shown that GPER induces protective cardiovascular effects including decreased blood pressure, protection from renal damage, and attenuation of arterial remodeling. In this study, we hypothesized that GPER deletion would attenuate the protective vascular effects of estrogen after menopause. Female C57Bl/6J wildtype (wt) and global GPER knockout (ko) mice underwent ovariectomy (OVX) at 46 weeks of age and concurrently administered estrogen (E) or vehicle (Veh). Angiotensin II (Ang II; 700 ng/kg/day) infusion was used to induce hypertension either four weeks prior (Ang-E) or at the time of OVX (E-Ang) to evaluate the impact of cardiovascular diseases preceding or coinciding with menopause. Blood pressure was measured by tail-cuff plethysmography and arterial stiffness was assessed by pulse wave velocity (PWV). Statistical analysis was performed using one-way ANOVA. Ang II induced a similar level of hypertension in wt and ko mice and was not impacted by E or timing. In wt mice, PWV was significantly higher in Veh-treated compared with E-treated hypertensive animals, independent of timing (P&lt;0.001). In contrast, PWV was not impacted by E in GPER ko mice (P=0.99). In conclusion, we found that E did not affect the hypertensive response to Ang II in both wt and GPER ko mice. GPER deletion reduced estrogen’s protection from arterial stiffening. Whether Ang II preceded or coincided with menopause did not alter the effects of E. These studies are important for determining whether cardiovascular status should be a factor when considering menopausal hormone therapy.

17β-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 17β-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.

Activation of GPR55 alleviates neuropathic pain and chronic inflammation.

Neuropathic pain (NP) significantly impacts the quality of life due to its prolonged duration and lack of effective treatment. Recent findings suggest that targeting neuroinflammation is a promising approach for treating NP. G protein-coupled receptor 55 (GPR55), a member of the GPCR family, plays an important role in neuroinflammatory regulation. CID16020046, a GPR55 agonist, possesses promising anti-neuroinflammatory effects. Herein, the therapeutic effect of CID16020046 on NP was investigated in an NP rat model. The NP model was established using the unilateral sciatic nerve chronic constriction injury (CCI) assay. Both sham and CCI rats were intraperitoneally administered with 20mg/kg CID16020046. NP was assessed using paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). First, we showed that GPR55 was downregulated in the spinal dorsal horn of CCI rats. After CCI rats were treated with CID16020046, the values of PWT and PWL were increased, indicating their effect on pain relief. The treated rats had attenuated release of inflammatory cytokines in the spinal cord, decreased spinal malondialdehyde (MDA) levels, and increased spinal glutathione peroxidase (GSH-PX) activity. Additionally, the increased levels of phosphorylated nuclear factor (NF)-κB p65 in CCI rats were significantly alleviated by CID16020046 treatment. Mechanistically, we showed that CID16020046 significantly suppressed the activation of the Janus kinase (JAK2)/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in the spinal cord of CCI-treated rats. However, Colivelin TFA (a STAT3 agonist) abolished the effect of CID16020046 on JAK2/STAT3 activation. In conclusion, our data demonstrate that the activation of GPR55 by CID16020046 alleviates NP and neuroinflammation in CCI rats by mediating the JAK2/STAT3 pathway.