GPR30 Research Articles

Activation of GPR81 Aggravates Remote Organ Injury During Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion injury (HIRI) is a serious situation with high morbidity and mortality, which is usually accompanied with hyperlactatemia due to impaired lactate clearance in liver. G-protein-coupled receptor 81 (GPR81) has recently been identified as the bioactive receptor of lactate. GPR81 is profoundly involved in the modulation of metabolism and inflammation, but its significance in HIRI remains unclear. The present study investigated the potential roles of GPR81 in HIRI by using the GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA). The results indicated that treatment with CHBA had no obvious effects on HIRI-induced histologic abnormalities and elevation of serum aspartate aminotransferase, alanine aminotransferase. However, CHBA significantly upregulated the serum level of tumor necrosis factor alpha and interleukin-6 in mice with HIRI. Administration of CHBA also exacerbated HIRI-induced histologic lesions in lung, increased the level of myeloperoxidase in lung tissue and the protein concentration in bronchoalveolar lavage fluid. In addition, the serum levels of brain natriuretic peptide and creatinine also increased in CHBA-treated mice. The results indicate that activation of GPR81 might aggravate HIRI-induced remote organ injury and result in serious outcomes.

Puerarin attenuates hepatic steatosis via G-protein-coupled estrogen receptor-mediated calcium and SIRT1 signaling pathways.

Puerarin, the major bioactive ingredient isolated from the root of Pueraria lobata (Willd.), attenuates body weight gain and reduces lipid levels in high-fat diet-induced obese mice; however, the underlying mechanism responsible for regulating lipid metabolism remains unclear. This study investigated the molecular mechanism(s) underlying the role of puerarin in regulating lipogenesis and lipolysis in human HepG2 cells. In this study, puerarin strongly inhibited the expression of fatty acid synthase (FASN) and sterol regulatory element binding protein 1c (SREBP-1c). Moreover, puerarin significantly induced the expression of adipose triglyceride lipase (ATGL), which is responsible for triacylglycerol hydrolase activity in cells. Puerarin enhanced 5' AMP-activated protein kinase (AMPK) activity, which is a central regulator of hepatic lipid metabolism. Furthermore, this AMPK activation could be mediated by sirtuin 1 (SIRT1) and calcium signaling pathways involved in G protein-coupled estrogen receptor (GPER) signaling. GPER blockage significantly reversed the effect of puerarin on lipid accumulation and the related signaling pathways. Docking studies showed that puerarin could bind in the GPER in a similar manner as GPER agonist G1. Our results suggest that puerarin can improve hepatic steatosis by activating GPER; it's signaling cascade sequentially induced calcium and SIRT1 signaling pathways. Thus, puerarin may be a potential therapeutic agent for the treatment of non-alcoholic fatty liver disease.

Molecular docking and identification of G-protein-coupled receptor 120 (GPR120) agonists as SARS COVID-19 MPro inhibitors

COVID-19 has become a pandemic, and any new drug for treating the disease could save millions of lives. Several drugs already in use for other diseases and medical conditions are repurposed for treating COVID-19 in an attempt to find treatment for the disease without spending research time on ADME TOX and other studies on side effects. In this exercise, the drugs repurposed are from antiviral, antibiotics, antiviral for HIV and HCV, anti-cancer, natural medicines, etc. Possible repurposing anti-diabetic GPR-120 agonists used as for SAR-CoV-2 is attempted in the study by carrying out docking of 68 GPR-120 agonists. Ten of these compounds were found to have docking scores −8.3 to −8.0, and the best docking score was observed for an arylsulfonamide and a biarylpropanoic acid belonging to GPR120 agonists previously evaluated for the treatment of type II diabetes. These GPR120 agonists could serve as start point for novel inhibitors for the discovery of drugs to treat COVID-19.

Investigation of estrogen-like effects of parabens on human neutrophils

This study investigated the estrogen-like effects and mechanism of action most commonly used parabens: methyl- (MeP), ethyl- (EtP), propyl- (PrP) and butylparaben (BuP) in human neutrophils.Neutrophils were isolated from 50 blood donors, pre-incubated with antagonists of estrogen receptor α (ERα), ERβ and G-protein coupled estrogen receptor 1 (GPER), then incubated with MeP, EtP, PrP, BuP and 17β-estradiol (E2; 10 nM). Cytotoxic effect was evaluated by MTT test. Neutrophils apoptosis, necrosis and NETs formation were assessed in flow cytometry and confocal microscopy. The ability of the neutrophils for chemotaxis, phagocytosis, NADPH oxidase activity and generation of superoxide anion was assessed in Boyden's chamber, Park's method with latex, the NBT test, and reduction of cytochrome C, respectively. The total nitric oxide concentration was measured in neutrophils supernatants by the Griess reaction. The expression of cathepsin G, neutrophil elastase, proteinase 3, ERα, ERβ and GPER was assessed in Western blot method.In our research, parabens did not cause a cytotoxic effect on human neutrophils nor affect their lifespan. Parabens exposure did not change neutrophils functions (chemotaxis, phagocytosis, NETs formation and oxygen-dependent killing mechanism) and expression of estrogen receptors.Our results suggest that parabens do not cause estrogen receptor-mediated neutrophils-related effects at concentrations measured in the plasma of individuals using products preserved with parabens.

The G-Protein-Coupled Estrogen Receptor Agonist Prevents Cardiac Lipid Accumulation by Stimulating Cardiac Peroxisome Proliferator-Activated Receptor α: A Preclinical Study in Ovariectomized-Diabetic Rat Model.

Type 2 diabetes mellitus (T2DM) is associated with cardiometabolic changes, and menopause exacerbates these conditions, leading to a greater risk of cardiovascular diseases (CVDs). The G protein-coupled estrogen receptor (GPER), which mediates the rapid effects of estrogen, has beneficial cardiac effects in both T2DM and menopause, but its mechanism of action is not well understood. This study aimed to determine whether G1 as a selective GPER-agonist has beneficial effects on cardiac lipid metabolism in ovariectomized rats with T2DM. Female Wistar rats were divided into 5 groups (n = 7 in each group): Sham-control (Sh-Ctl), T2DM, ovariectomized-T2DM (OVX-T2DM), OVX-T2DM-G1 (GPER-agonist), and OVX-T2DM-vehicle (OVX-T2DM-Veh). After stabilization of T2DM, G1 (200 μg/Kg) was administrated for 6 weeks. Then, the levels of free fatty acids (FFAs), CD36, peroxisome proliferator-activated receptor α (PPARα), and lipid accumulation in the cardiac tissue were determined. Compared with the Sh-Ctl group, cardiac FFAs (P < 0.001), CD36 (P < 0.05), and lipid accumulation (P < 0.001) increased, and cardiac PPARα (P < 0.01) decreased in T2DM animals; ovariectomy intensified these changes. Also, cardiac FFAs, PPARα, and lipid accumulation (P < 0.05) significantly decreased in the OVX-T2DM-G1 group compared to the OVX-T2DM-Veh group. However, cardiac CD36 levels did not change. G1 as a selective GPER-agonist affects lipid metabolism in T2DM animals. It also plays a vital role in improving cardiac metabolism during postmenopausal diabetic conditions.

G protein-coupled estrogen receptor (GPER/GPR30) levels in pelvic floor muscles and its association with estrogen status in female rabbits

Objective: To assess the relative expression of the G-protein coupled estrogen receptor (GPER) in the bulbospongiosus (Bsm) and pubococcygeus (Pcm) muscles in control, ovariectomized (OVX), and OVX with estradiol benzoate supplementation (OVX + EB) rabbits.Methods: We used tissues from C, 1-month OVX, and OVX plus 15-day EB implanted (OVX + EB) groups. The GPER expression was evaluated by Western blot and immunohistochemistry for both Bsm and Pcm. Results: Both muscles showed a GPER immunoreactivity in blood vessels, inside myofibers next to myonuclei, and in polymorphonuclear cells. Four-week ovariectomy did not modify the GPER expression in the Bsm and Pcm, but two-week estradiol benzoate increased it in the latter muscle alone.Conclusions: We demonstrated that the Bsm and Pcm of female rabbits express GPER. High serum estradiol levels elevate GPER relative expression in the Pcm alone. The present study supports the remarkable estrogen sensitivity of the Pcm.

Distribution of Cannabinoid Receptors in Keratinocytes of Healthy Dogs and Dogs With Atopic Dermatitis

It is commonly accepted that some form of skin barrier dysfunction is present in canine atopic dermatitis (AD), one of the most common cutaneous pruritic inflammatory diseases of dogs. The impaired skin barrier function facilitates the penetration of allergens and subsequently stronger sensitization responses. The role of the endocannabinoid system (ECS) in the physiology and pathology of the skin is becoming increasingly established. It has been demonstrated that cannabinoid receptors are expressed in healthy and diseased skin and, based on current knowledge, it could be stated that cannabinoids are important mediators in the skin. The present study has been designed to immunohistochemically investigate the expression of the cannabinoid receptors type 1 (CB1R) and 2 (CB2R) and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors alpha (PPARα), and serotoninergic receptor 1a (5-HT1aR) in keratinocytes of healthy dogs and of dogs with AD. Samples of skin tissues were collected from 7 healthy controls (CTRL-dogs) and from 8 dogs with AD (AD-dogs). The tissue samples were processed using an immunofluorescence assay with commercially available antibodies, and the immunolabelling of the receptors studied was quantitatively evaluated. The keratinocytes of the CTRL- and the AD-dogs showed immunoreactivity for all the receptors investigated with a significant upregulation of CB2R, TRPA1, and 5-HT1aR in the epidermis of the AD-dogs. The presence of cannabinoid and cannabinoid-related receptors in healthy keratinocytes suggested the possible role of the ECS in canine epidermal homeostasis while their overexpression in the inflamed tissues of the AD-dogs suggested the involvement of the ECS in the pathogenesis of this disease, having a possible role in the related skin inflammation and itching. Based on the present findings, the ECS could be considered a potential therapeutic target for dogs with AD.

GPR39 Deficiency Impairs Memory and Alters Oxylipins and Inflammatory Cytokines Without Affecting Cerebral Blood Flow in a High-Fat Diet Mouse Model of Cognitive Impairment.

Vascular cognitive impairment (VCI) is the second most common cause of dementia. There is no treatment for VCI, in part due to a lack of understanding of the underlying mechanisms. The G-protein coupled receptor 39 (GPR39) is regulated by arachidonic acid (AA)-derived oxylipins that have been implicated in VCI. Furthermore, GPR39 is increased in microglia of post mortem human brains with VCI. Carriers of homozygous GPR39 SNPs have a higher burden of white matter hyperintensity, an MRI marker of VCI. We tested the hypothesis that GPR39 plays a protective role against high-fat diet (HFD)-induced cognitive impairment, in part mediated via oxylipins actions on cerebral blood flow (CBF) and neuroinflammation. Homozygous (KO) and heterozygous (Het) GPR39 knockout mice and wild-type (WT) littermates with and without HFD for 8 months were tested for cognitive performance using the novel object recognition (NOR) and the Morris water maze (MWM) tests, followed by CBF measurements using MRI. Brain tissue and plasma oxylipins were quantified with high-performance liquid chromatography coupled to mass spectrometry. Cytokines and chemokines were measured using a multiplex assay. KO mice, regardless of diet, swam further away from platform location in the MWM compared to WT and Het mice. In the NOR test, there were no effects of genotype or diet. Brain and plasma AA-derived oxylipins formed by 11- and 15-lipoxygenase (LOX), cyclooxygenase (COX) and non-enzymatically were increased by HFD and GPR39 deletion. Interleukin-10 (IL-10) was lower in KO mice on HFD than standard diet (STD), whereas IL-4, interferon γ-induced protein-10 (IP-10) and monocyte chemotactic protein-3 (MCP-3) were altered by diet in both WT and KO, but were not affected by genotype. Resting CBF was reduced in WT and KO mice on HFD, with no change in vasoreactivity. The deletion of GPR39 did not change CBF compared to WT mice on either STD or HFD. We conclude that GPR39 plays a role in spatial memory retention and protects against HFD-induced cognitive impairment in part by modulating inflammation and AA-derived oxylipins. The results indicate that GPR39 and oxylipin pathways play a role and may serve as therapeutic targets in VCI.

Bisphenol F blocks Leydig cell maturation and steroidogenesis in pubertal male rats through suppressing androgen receptor signaling and activating G-protein coupled estrogen receptor 1 (GPER1) signaling

Bisphenol F (BPF) is a new analog of bisphenol A (BPA). BPA has deleterious effects on the male reproductive system, but the effect of BPF has not been studied in detail. In this study we focus on the effect of BPF on Leydig cell maturation. Male Sprague-Dawley rats were gavaged with 0, 1, 10, or 100 mg/kg BPF from postnatal days 35–56. BPF significantly reduced serum testosterone levels and sperm count in cauda epididymis at dose ≥1 mg/kg. It significantly down-regulated the expression of steroidogenic enzymes, while increasing FSHR and SOX9 levels at 10 and 100 mg/kg. Further studies showed that BPF reduced NR3C4 expression in Leydig and Sertoli cells without affecting its levels in peritubular myoid cells. BPF markedly increased GPER1 in Leydig cells at 100 mg/kg, and it significantly reduced SIRT1 and PGC1α levels in the testes at 100 mg/kg. BPF significantly inhibited testosterone production by immature Leydig cells at 50 μM after 24 h of treatment, which was completely reversed by NR3C4 agonist 7α-methyl-19-nortestosterone and partially reversed by GPER1 antagonist G15 not by ESR1 antagonist ICI 182,780. In conclusion, BPF negatively affects Leydig cell maturation in pubertal male rats through NR3C4 antagonism and GPER1 agonism.

GPR87 promotes tumor cell invasion and mediates the immunogenomic landscape of lung adenocarcinoma

The purpose of this study is to examine the association between G protein-coupled receptor 87 (GPR87) and lung adenocarcinoma (LUAD) metastasis and immune infiltration. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets extract clinical data. According to the TCGA database, increased GPR87 expression predicts poor overall survival, progression-free interval, and disease-specific survival in LUAD patients. The meta-analysis also reveals a significant association between high GPR87 expression and poor overall survival. Moreover, functional experiments demonstrate that GPR87 silencing reduces LUAD cell invasion and migration. Immunoblotting shows that GPR87 knockdown decreased Vimentin and N-cadherin expression and increased E-cadherin expression in LUAD cells. GPR87 expression in LUAD is positively correlated with immune infiltration. In addition, GPR87 expression is associated with immune and chemotherapy resistance in LUAD patients. Our findings indicate that GPR87 promotes tumor progression and is correlated with immune infiltration, suggesting GPR87 as a possible biomarker for prognosis prediction in LUAD.

Differential contribution of estrogen receptors to the intestinal therapeutic effects of 17β-estradiol in a murine model of Parkinson’s disease

Beneficial effects of estrogens have been reported in Parkinson's disease (PD) for many years. We previously reported their neuroprotective and anti-inflammatory potentials in the enteric nervous system of the intestine, a region possibly affected during the early stages of the disease according to Braak’s hypothesis. Three different estrogen receptors have been characterized to date: the estrogen receptor alpha (ERα), the estrogen receptor beta (ERβ) and the G protein coupled estrogen receptor 1 (GPER1). The aim of the present study was to decipher the individual contribution of each estrogen receptor to the therapeutic properties of 17β-estradiol (E2) in the myenteric plexus of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Different agonists, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT; ERα), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERβ), G1 (GPER1), and antagonists, ICI 182,780 (ERα and ERβ), G15 (GPER1), were used to analyze the involvement of each receptor. We confirmed that G1 protects dopamine (DA) neurons to a similar extent as E2. An anti-inflammatory effect on proinflammatory macrophages and cultured human monocytes was also demonstrated with E2 and G1. The effects of PPT and DPN were less potent than G1 with only a partial neuroprotection of DA neurons by PPT and a partial reduction of interleukin (IL)− 1β production in monocytes by PPT and DPN. Overall, the present results indicate that the positive outcomes of estrogens are mainly through activation of GPER1. Therefore, this suggests that targeting GPER1 could be a promising approach for future estrogen-based hormone therapies during early PD.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17β-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Effects of Estrogen on Proliferation and Apoptosis of Osteoblasts through Regulating GPER/AKT Pathway.

This experiment was carried out to study the effects of estrogen on the proliferation and apoptosis of osteoblasts through regulating the G protein-coupled estrogen receptor (GPER)/protein kinase B (AKT) pathway. For this aim, osteoblasts were cultured in vitro and divided into control group, estrogen group and inhibitor group after passage. The osteoblasts in the control group were cultured normally, estrogen intervention was made in the estrogen group and G15 inhibitor intervention was made in the inhibitor group. After intervention for 24 h, osteoblasts were collected for detection. The positive expression of GPER and the double-positive expression of Tom20/Lamp2 were detected via immunofluorescence assay. The protein expressions of GPER, AKT and phosphorylated (p)-AKT were detected via Western blotting. The mRNA expression of GPER was detected via qPCR. Moreover, the autophagosomes were observed under a transmission electron microscope, and the apoptosis and cell proliferation were detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and cell counting kit-8 (CCK8) assay, respectively. Results of the immunofluorescence assay revealed that the positive expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05), while the double-positive expression of Tom20/Lamp2 in the estrogen group was lower than that in control group and inhibitor group (p<0.05). According to the results of Western blotting, the relative protein expression of AKT had no differences among the three groups (p>0.05), while the relative protein expressions of GPER and p-AKT in the estrogen group were higher than those in the control group and inhibitor group (p<0.05). The results of qPCR showed that the relative mRNA expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05). There were a small number of autophagosomes in osteoblasts in the control group and inhibitor group, while the number of autophagosomes in osteoblasts was smaller in the estrogen group. Besides, the estrogen group had a remarkably lower apoptosis rate of osteoblasts than the control group and inhibitor group and a remarkably higher proliferation rate than the control group and inhibitor group. Then estrogen can inhibit the mitochondrial autophagy of osteoblasts by regulating the GPER/AKT pathway, thereby inhibiting apoptosis and promoting cell proliferation.

P-337 Melatonin exerts a therapeutic effect on endometriosis by suppressing estrogen receptors mediated angiogenesis pathway

Abstract Study question What are the underlying mechanisms of melatonin to treat endometriosis? Summary answer Melatonin inhibited estrogen receptors/HIF1α/VEGF signaling pathway and limited the endometriotic lesions growth and angiogenesis. What is known already Currently, alternative pharmaceutical for endometriosis is needed urgently due to lack of satisfactory treatment option. The role of melatonin in inhibiting progression of endometriosis has attracted widespread attention in recent years. Several experimental animal studies showed melatonin decreased endometriotic lesion volume, albeit the exact mechanism is still unknown. As an estrogen-dependent disease, estrogen can not only stimulate the proliferation and growth of endometriotic lesions, but also stabilize HIF-1α and thus promote HIF-1α–induced VEGF via binding to estrogen receptors. Herein, estrogen receptors might be a therapeutic target to block the lesion growth and angiogenesis, subsequently inhibit the progression of endometriosis. Study design, size, duration We investigated the role of melatonin in endometriosis in vitro and in vivo. Endometriotic epithelial (12Z) and stromal (Hs 832(C).T) cell lines were cultured and treated with melatonin for 24 hours. Subcutaneous endometriotic model was surgically established in C57BL/6 mice, and randomly assigned to receive vehicle or melatonin for 21days. All experiments were performed 3-8 times for comparisons. Participants/materials, setting, methods The effect of melatonin on endometriotic cells was determined by MTT cell viability assay. Endometriotic lesion size and body weight were measured every 2-3 days after transplantation. RNA and protein expressions of estrogen receptors, HIF1α and VEGF were detected by qPCR and Western blotting and/or immunostaining in in vitro and in vivo experiments. In vivo imaging by Cellvizio was used to examine microvascular network of the developing endometriotic lesions. Main results and the role of chance Melatonin exerted inhibitory effects on cell growth in a dose and time-dependent manner in 12Z and Hs 832(C).T cell lines, and reduced the volume and weight of the ectopic endometriotic lesions without major change in body weight and behaviour. In addition, melatonin significantly decreased the percentage of area (48.41 vs 33.13%), average length (546.7 vs 240.9um) and total number of junctions (53.30 vs 26.68) of microvessels in the lesions. Melatonin also specially and significantly downregulated estrogen receptor β (ERβ) and G protein-coupled estrogen receptor 30 (GPR30) at RNA and protein levels while estrogen receptor α (ERα) was not significantly changed in both 12Z and Hs 832(C).T cells and endometriotic lesions. Furthermore, the expressions of downstream HIF1α and VEGF were significantly decreased after melatonin treatment. Limitations, reasons for caution Melatonin has been reported to employ a wide range of biological effects. Therefore, its suppressive effects on estrogen receptors/HIF1α/VEGF signaling pathway might be part of the pharmacological mechanisms to treat endometriosis. Further studies are also needed to explore the therapeutic mechanisms of melatonin from other perspectives. Wider implications of the findings Our findings add mechanistic insight in support of the use of melatonin as an adjuvant therapy in the management of endometriosis. Melatonin shows the potential to act as a novel alternative therapeutic drug to treat endometriosis with fewer side effects. Trial registration number NA

Organizational Effects of Estrogens and Androgens on Estrogen and Androgen Receptor Expression in Pituitary and Adrenal Glands in Adult Male and Female Rats.

Sex steroid hormones, such as androgens and estrogens, are known to exert organizational action at perinatal periods and activational effects during adulthood on the brain and peripheral tissues. These organizational effects are essential for the establishment of biological axes responsible for regulating behaviors, such as reproduction, stress, and emotional responses. Estradiol (E2), testosterone, and their metabolites exert their biological action through genomic and non-genomic mechanisms, bounding to canonical receptors, such as estrogen receptor (ER)α, ERβ, and androgen receptor (AR) or membrane receptors, such as the G protein-coupled estrogen receptor (GPER), respectively. Expression of ERs and AR was found to be different between males and females both in the brain and peripheral tissues, suggesting a sex-dependent regulation of their expression and function. Therefore, studying the ERs and AR distribution and expression levels is key to understand the central and peripheral role of sex steroids in the establishment of sex-specific behaviors in males and females. We investigated the organizational effects of estrogens and androgens in the pituitary and adrenal glands of adult male and female rats. For this, selective blockade of AR with flutamide or 5α-reductase with finasteride or aromatase with letrozole during the first 5 days of life has been performed in male and female pups and then quantification of ERs and AR expression in both glands has been carried out in adulthood. Data show that inhibition of dihydrotestosterone (DHT) and E2 production during the first five postnatal days mainly decreases the ER expression in male to female values and AR expression in female to male levels in the pituitary gland and increases AR expression in female to male levels in the adrenal gland. In contrast, blocking the action of androgens differentially modulates the ERs in males and females and decreases AR in both males and females in both glands. Altogether, the results suggest that neonatal modifications of the androgen and estrogen pathways can potentially lead to permanent modifications of the neuroendocrine functions of the pituitary and adrenal glands in the adulthood of both sexes.

Positive interaction between GPER and β-alanine in the dorsal root ganglion uncovers potential mechanisms: mediating continuous neuronal sensitization and neuroinflammation responses in neuropathic pain

BackgroundThe pathogenesis of neuropathic pain and the reasons for the prolonged unhealing remain unknown. Increasing evidence suggests that sex oestrogen differences play a role in pain sensitivity, but few studies have focused on the oestrogen receptor which may be an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors on the nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain.MethodsWe intrathecally (i.t.) administered a class of oestrogen receptor antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we assessed genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitization and neuroinflammatory responses in neuropathic pain. The excitability of DRG neurons was detected using the patch-clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, RT-PCR and behavioural testing were used to assess the expression, cellular distribution, and actions of the main receptor and its related signalling molecules.ResultsIncreasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-β (ERβ), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitization. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by simultaneously downregulating iNOS, IL-1β and IL-6 expression and restoring GABAα2 expression. Additionally, the positive interaction between GPER and β-alanine and subsequent β-alanine accumulation enhances pain sensation and promotes chronic pain development.ConclusionGPER activation in the DRG induces a positive association between β-alanine with iNOS, IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG neurons and microglia may prevent neuropathic pain development.

Sodium butyrate modulates blood pressure and gut microbiota in maternal tryptophan-free diet-induced hypertension rat offspring

Maternal nutrition, gut microbiome composition, and metabolites derived from gut microbiota are closely related to the development of hypertension in offspring. A plethora of metabolites generated from diverse tryptophan metabolic pathways show both beneficial and harmful effects. Butyrate, one of the short-chain fatty acids (SCFAs), has shown vasodilation effects. We examined whether sodium butyrate administration in pregnancy and lactation can prevent hypertension induced by a maternal tryptophan-free diet in adult progeny and explored the protective mechanisms. Pregnant Sprague–Dawley rats received normal chow (CN), tryptophan-free diet (TF), sodium butyrate 400 mg/kg/d in drinking water (CNSB), or TF diet plus sodium butyrate (TFSB) in pregnancy and lactation. Male offspring were sacrificed at the age of 16 weeks (n=8 per group). Compared with normal chow, offspring exposed to the maternal tryptophan-free diet had markedly increased blood pressure, associated with activation of the renin–angiotensin system (RAS). Treatment with sodium butyrate rescued maternal TF-exposed offspring from hypertension. The protective effect of sodium butyrate is related to alterations to microbiome composition, increased renal expression of SCFA receptor G protein-coupled receptor 41 (GPR41) and GPR109A, and restoration of RAS balance. In summary, these results suggest that sodium butyrate protects against maternal TF-induced offspring hypertension, likely by modulating gut microbiota, its derived metabolites, and the RAS.

Abstract 1852: G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma cells

Abstract Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. Nearly 50% of the patients develop hepatic metastasis, and when this occurs the 1-year survival rate is 12 months. While the primary tumor can be treated with radiotherapy and surgery, current treatments for metastatic disease have shown limited clinical activity in UM patients and there is an urgent need for new effective therapies. Recent reports have shown that male patients have more metastases than female patients, and the time from the diagnosis until development of metastases was shorter, suggesting a potential role of female hormones in disease progression. The G-protein-coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/β and its activation with a specific agonist has tumor-suppressive roles in several cancers. Here, we demonstrate that the novel clinical GPER agonist, LNS8801 (Linnaeus Therapeutics), has potent anticancer activities in UM cells including inhibition of cell migration and cell proliferation. LNS8801 treatment induced a G2/M cell cycle arrest due to disruption of tubulin polymerization and defective mitotic spindle formation. There was a time-dependent increase of mitotic proteins, such as phosphorylated aurora-A, aurora-B and histone-3, while c-Myc and phospho-Rb were downregulated. The drug treatment also induced significant cell death, as detected by caspase 3/7 activation and PARP cleavage. Finally, LNS8801 inhibited tumor growth in a UM xenograft model in vivo. In conclusion, our studies suggest that GPER agonists may be a novel treatment for uveal melanoma, and clinical trials with the GPER agonist LNS8801 are warranted and currently underway. Citation Format: Grazia Ambrosini, Christopher A. Natale, Gary K. Schwartz. G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1852.

Abstract 329: Understanding the role of GPR30 in the estrogen non-genomic signaling in inflammatory breast cancer

Abstract Inflammatory Breast Cancer (IBC) is rare and the most aggressive subtype of all breast cancers, reporting rapid progression, poor prognosis, and a unique clinical diagnosis (blockage of lymph vessels by tumor emboli). Approximately, 40% of IBC cases are classified as triple-negative breast cancer (TNBC), meaning they test negative for hormone receptors (estrogen and progesterone) and the amplification of the epidermal growth factor receptor 2 (HER2), which leads to no effective targeted therapies availability. However, 17β-estrogen (E2) has been found to enhance the migration and invasion phenotypes of TN-IBC cells and it has been associated with the presence of a membrane G-protein coupled estrogen receptor (GPR30) and a novel cytoplasmic estrogen receptor (ERα36 isoform) that can undergo an estrogen non-genomic (rapid) cell’s response. Recent studies have also reported a crosstalk between these estrogen non-genomic receptors and the epidermal growth factor receptor (EGFR) signaling pathway in the presence of E2, inducing phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and promoting oncogenic phenotypes (migration &amp; invasion) in TN-IBC cells. However, the exact role of GPR30 and ERα36 in the non-genomic signaling pathway remains elusive in TN-IBC. We hypothesis that GPR30 and ERα36 are necessary to undergo de estrogen non-genomic signaling and promote oncogenic phenotypes in TN-IBC SUM149 cells after E2 treatment. Methods: We have measured migratory activity and tumor emboli formation of SUM149 cells (by wound-healing and 3D-cell proliferation assay), and phosphorylation levels of ERK1/2 and AKT kinases (by western-blot) after treatments in time-points with E2 alone and in combination with G15 (GPR30 antagonist drug). We are also working to identify protein partners of GPR30 (by Ip and Mass-spec) after E2 treatment, including physical interaction with ERα36, in TN-IBC and other types of breast cancers. Results: G15 treatment in Sum149 cells significantly reduced E2-induced ERK1/2 phosphorylation, indicating that GPR30 is necessary for the estrogen non-genomic signaling pathway and has direct involvement with the EGFR signaling cascade. Data acquired from these experiments will contribute to a better understanding of TN-IBC’s molecular structure and create new opportunities to develop novel targeted therapies. Citation Format: Xavier S. Bittman Soto, Adrian J. Rivera Lopez, Esther A. Peterson. Understanding the role of GPR30 in the estrogen non-genomic signaling in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 329.

Modulatory Effects of Estradiol and Its Mixtures with Ligands of GPER and PPAR on MAPK and PI3K/Akt Signaling Pathways and Tumorigenic Factors in Mouse Testis Explants and Mouse Tumor Leydig Cells.

The present study was designed to evaluate how estradiol alone or in combination with G protein-coupled estrogen receptor (GPER) agonists and GPER and peroxisome proliferator-activated receptor (PPAR) antagonists alter the expression of tumor growth factor β (TGF-β), cyclooxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1α), and vascular endothelial growth factor (VEGF) in mouse testis explants and MA-10 mouse tumor Leydig cells. In order to define the hormone-associated signaling pathway, the expression of MAPK and PI3K/Akt was also examined. Tissue explants and cells were treated with estradiol as well as GPER agonist (ICI 182,780), GPER antagonist (G-15), PPARα antagonist (GW6471), and PPARγ antagonist (T00709072) in various combinations. First, we showed that in testis explants GPER and PPARα expressions were activated by the GPER agonist and estradiol (either alone or in mixtures), whereas PPARγ expression was activated only by GPER agonist. Second, increased TGF-β expression and decreased COX-2 expression were found in all experimental groups of testicular explants and MA-10 cells, except for up-regulated COX-2 expression in estradiol-treated cells, compared to respective controls. Third, estradiol treatment led to elevated expression of HIF-1α and VEGF, while their lower levels versus control were noted in the remaining groups of explants. Finally, we demonstrated the up-regulation of MAPK and PI3Kp85/Akt expressions in estradiol-treated groups of both ex vivo and in vitro models, whereas estradiol in mixtures with compounds of agonistic or antagonistic properties either up-regulated or down-regulated signaling kinase expression levels. Our results suggest that a balanced estrogen level and its action together with proper GPER and PPAR signaling play a key role in the maintenance of testis homeostasis. Moreover, changes in TGF-β and COX-2 expressions (that disrupted estrogen pathway) as well as disturbed GPER-PPAR signaling observed after estradiol treatment may be involved in testicular tumorigenesis.