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Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder. Half of the patients would slowly progress to end-stage renal disease. However, the potential target for ADPKD treatment is still lacking. Methods Four ADPKD patients and two healthy family members were included in this study. The peripheral blood samples were obtained and tested by the whole exome sequencing (WES). The autosomal mutations in ADPKD patients were retained as candidate sites. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein–protein interaction network (PPI) analyses were performed by clusterProfiler R package. A dataset containing 18 ADPKD patients and three normal samples were downloaded from the Gene Expression Omnibus (GEO) database and analyzed using the limma R package. Results A total of six mutant genes were identified based on the dominant genetic pattern and most of them had not been reported to be associated with ADPKD. Furthermore, 19 harmful genes were selected according to the harmfulness of mutation. GO and KEGG enrichment analyses showed that the processes of single-organism cellular process, response to stimulus, plasma membrane, cell periphery, and anion binding as well as cyclic adenosine monophosphate (cAMP) signaling pathway and pathways in cancer were significantly enriched. Through integrating PPI and gene expression analyses, acyl-CoA thioesterase 13 (ACOT13), which has not been reported to be related to ADPKD, and prostaglandin E receptor 2 (PTGER2) were identified as potential genes associated with ADPKD. Conclusions Through combination of WES, gene expression, and PPI network analyses, we identified ACOT13 and PTGER2 as potential ADPKD-related genes.

Background Alzheimer’s disease (AD) causes substantial medical and societal burden with no therapies ameliorating cognitive deficits. Centralized pathologies involving amyloids, neurofibrillary tangles, and neuroinflammatory pathways are being investigated to identify disease-modifying targets for AD. Cyclooxygenase-2 (COX-2) is one of the potential neuroinflammatory agents involved in AD progression. However, chronic use of COX-2 inhibitors in patients produced adverse cardiovascular effects. We asked whether inhibition of EP2 receptors, downstream of the COX-2 signaling pathway, can ameliorate neuroinflammation in AD brains in presence or absence of a secondary inflammatory stimuli. Methods We treated 5xFAD mice and their non-transgenic (nTg) littermates in presence or absence of lipopolysaccharide (LPS) with an EP2 antagonist (TG11-77.HCl). In cohort 1, nTg (no-hit) or 5xFAD (single-hit—genetic) mice were treated with vehicle or TG11-77.HCl for 12 weeks. In cohort 2, nTg (single-hit—environmental) and 5xFAD mice (two-hit) were administered LPS (0.5 mg/kg/week) and treated with vehicle or TG11-77.HCl for 8 weeks. Results Complete blood count analysis showed that LPS induced anemia of inflammation in both groups in cohort 2. There was no adverse effect of LPS or EP2 antagonist on body weight throughout the treatment. In the neocortex isolated from the two-hit cohort of females, but not males, the elevated mRNA levels of proinflammatory mediators (IL-1β, TNF, IL-6, CCL2, EP2), glial markers (IBA1, GFAP, CD11b, S110B), and glial proteins were significantly reduced by EP2 antagonist treatment. Intriguingly, the EP2 antagonist had no effect on either of the single-hit cohorts. There was a modest increase in amyloid–plaque deposition upon EP2 antagonist treatment in the two-hit female brains, but not in the single-hit genetic female cohort. Conclusion These results reveal a potential neuroinflammatory role for EP2 in the two-hit 5xFAD mouse model. A selective EP2 antagonist reduces inflammation only in female AD mice subjected to a second inflammatory insult.

The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.

PURPOSE: PGF(2α) analogs are commonly used to treat glaucoma and are associated with higher rates of meibomian gland dysfunction (MGD). The purpose of this study was to evaluate the physiological effects of PGF(2α) and PGE(2) on immortalized human meibomian gland epithelial cells (HMGECs). METHODS: HMGECs were immunostained for the four PGE(2) receptors (EP1, EP2, EP3, EP4) and the one PGF(2α) receptor (FP) and imaged. Rosiglitazone-differentiated HMGECs were exposed to PGF(2α) and PGE(2) (10(−9) to 10(−6) M) for three hours. Cell viability was assessed by an ATP-based luminescent assay, and lipid extracts were analyzed for cholesteryl esters (CEs), wax esters (WEs), and triacylglycerols (TAGs) by ESI-MSMS(ALL) in positive ion mode by a Triple TOF 5600 Mass Spectrometer using SCIEX LipidView 1.3. RESULTS: HMGECs express three PGE(2) receptors (EP1, EP2, EP4) and the one PGF(2α) receptor (FP). Neither PGE(2) nor PGF(2α) showed signs of cytotoxicity at any of the concentrations tested. WEs were not detected from any of the samples, but CEs and TAGs both exhibited a diverse and dynamic profile. PGE(2) suppressed select CEs (CE 22:1, CE 26:0, CE 28:1, CE 30:1). PGF(2α) dose dependently increased several CEs (CE 20:2, CE 20:1, CE 22:1, CE 24:0) yet decreased others. Both prostaglandins led to nonspecific TAG remodeling. CONCLUSION: PGE(2) and PGF(2α) have minimal effect on HMGEC viability. PGF(2α) influences lipid expression greater than PGE(2) and may do so by interfering with meibocyte differentiation. This work may provide insight into the mechanism of MGD development in glaucoma patients treated with PGF(2α) analogs.

In the wake of health disasters associated with the chronic use of cyclooxygenase-2 (COX-2) inhibitor drugs, it has been widely proposed that modulation of downstream prostanoid synthases or receptors might provide more specificity than simply shutting down the entire COX cascade for anti-inflammatory benefits. The pathogenic actions of COX-2 have long been thought attributable to the prostaglandin E2 (PGE(2)) signaling through its Gα(s)-coupled EP2 receptor subtype; however, the truly selective EP2 antagonists did not emerge until 2011. These small molecules provide game-changing tools to better understand the EP2 receptor in inflammation-associated conditions. Their applications in preclinical models also reshape our knowledge of PGE(2)/EP2 signaling as a node of inflammation in health and disease. As we celebrate the 10-year anniversary of this breakthrough, the exploration of their potential as drug candidates for next-generation anti-inflammatory therapies has just begun. The first decade of EP2 antagonists passes, while their future looks brighter than ever.

Increased fluid-flow shear stress (FFSS) contributes to hyperfiltration-induced podocyte and glomerular injury resulting in progression of chronic kidney disease (CKD). We reported that increased FFSS in vitro and in vivo upregulates PGE2 receptor EP2 (but not EP4 expression), COX2-PGE

Prostaglandins are important signaling lipids with prostaglandin EPharmacological treatment was performed in transgenic zebrafish embryos using 500 μM 15-keto-PGEHere, we show that 15-keto-PGEAltogether, our results reveal 15-keto-PGE

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E

Aims Prostaglandin E2 (PGE2) is involved in the development of cardiac hypertrophy. However, whether PGE2 regulates the chronic kidney disease‐associated cardiac hypertrophy and the tentative mechanism remains to be elucidated. Methods and results We explored the effect of PGE2 receptor inhibitors on cardiac hypertrophy in vitro and in a 5/6 nephrectomy (5/6NT) rat model using quantitative reverse transcription polymerase chain reaction, western blotting, enzyme‐linked immunosorbent assay, immunohistochemical staining, and immunofluorescence staining assays. The result showed that EP2 and EP4 receptors were both up‐regulated in the PGE2‐treated cardiomyocyte cells. PGE2 treatment enhanced active β‐catenin (non‐phosphorylated) signalling through mediating EP2 and EP4 receptors. Interestingly, inhibition of EP2 receptor suppressed PGE2‐induced cardiomyocyte hypertrophy and cardiac fibrosis‐related proteins in vitro. In the 5/6NT rat model, the increased secretion PGE2 was identified in the 5/6NT rat model for 2 weeks (P = 0.0251). EP2 receptor inhibitor administration significantly improved the cardiac function and fibrosis in 5/6NT rats. Conclusions Our study demonstrated that inhibition of EP2 receptor could improve PGE2‐induced cardiac hypertrophy in 5/6NT rats. The exploration of these mechanisms may contribute to the optimization of therapy in chronic kidney disease accompanied cardiac hypertrophy in clinic.

Purpose: To investigate the intraocular pressure (IOP)-lowering effects of omidenepag isopropyl (OMDI), a potent and highly selective prostanoid EP2 receptor agonist, as a potential first-line ocul...

Cancer cells can effectively suppress the natural immune response in humans, and prostaglandin E

Inflammatory signaling through prostaglandin E2 receptor subtype 2 (EP2) is associated with malignant tumor growth in both experimental models and cancer patients. Thus, the absence of EP2 receptors in host tissues appears to reduce tumor growth and systemic inflammation by inducing major alterations in gene expression levels across tumor tissue compartments. However, it is not yet well‑established how signaling pathways in tumor tissue relate to simultaneous signaling alterations in the surrounding tumor‑stroma, at conditions of reduced disease progression due to decreased host inflammation. In the present study, wild‑type tumor cells, producing high levels of prostaglandin E2 (MCG 101 cells, EP2

Cyclooxygenase-2 (COX-2) activity protects against oxidative stress and apoptosis early in experimental kidney injury. The present study was designed to test the hypothesis that COX-2 activity attenuates fibrosis and preserves microvasculature in injured kidney. The murine unilateral ureteral-obstruction (UUO) model of kidney fibrosis was employed and compared with human nephrectomy tissue with and without chronic hydronephrosis.Fibrosis and angiogenic markers were quantified in kidney tissue from wild-type and COX-2COX-2 immunosignal was observed in interstitial fibroblasts at baseline and after UUO. Fibronectin, collagen I, III, alpha-smooth muscle actin, and fibroblast specific protein-1 mRNAs increased significantly more after UUO in COX-2COX-2 protects in the early phase against obstruction-induced fibrosis and maintains angiogenic factors. Increased PGE

Endometriosis is an estrogen-dependent, progesterone-resistant, chronic inflammatory gynecological disease of reproductive-age women. Two major clinical symptoms of endometriosis are chronic pelvic pain and infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent disease recurrence. Prostaglandin E

Prostaglandin E

Cognitive comorbidities can substantially reduce quality of life in people with epilepsy. Inflammation is a component of all chronic diseases including epilepsy, as well as acute events like status epilepticus (SE). Neuroinflammation is the consequence of several broad signaling cascades including cyclooxygenase-2 (COX-2)-associated pathways. Activation of the EP2 receptor for prostaglandin E2 appears responsible for blood-brain barrier leakage and much of the inflammatory reaction, neuronal injury and cognitive deficit that follows seizure-provoked COX-2 induction in brain. Here we show that brief exposure of mice to TG11-77, a potent, selective, orally available and brain permeant EP2 antagonist, eliminates the profound cognitive deficit in Y-maze performance after SE and reduces delayed mortality and microgliosis, with a minimum effective i.p. dose (as free base) of 8.8 mg/kg. All in vitro studies required to submit an investigational new drug (IND) application for TG11-77 have been completed, and non-GLP dose range-finding toxicology in the rat identified no overt, organ or histopathology signs of toxicity after 7 days of oral administration at 1000 mg/kg/day. Plasma exposure in the rat was dose-linear between 15 and 1000 mg/kg dosing. TG11-77 thus appears poised to continue development towards the initial clinical test of the hypothesis that EP2 receptor modulation after SE can provide the first preventive treatment for one of the chief comorbidities of epilepsy.

The cellular mechanism by which epoxy fatty acids (EpFA) improves disease status is not well characterized. Previous studies suggest the involvement of cellular receptors and cyclic AMP (cAMP). Herein, the action of EpFAs derived from linoleic acid (LA), arachidonic acid (ARA), and docosahexaenoic acid on cAMP levels was studied in multiple cell types to elucidate relationships between EpFAs, receptors and cells' origin. cAMP levels were enhanced in HEK293 and LLC-PK1 cells by EpFAs from LA and ARA. Using selective antagonists, the EpFA effects on cAMP levels appear dependent on the prostaglandin E

EP2 and EP4 prostanoid receptors have long been considered to have similar roles, since they are known to couple with Gαs-protein and activate cAMP-mediated signaling pathways. In this study, we re-evaluated the results of cAMP assays with or without phosphodiesterase (PDE) inhibitor pretreatment. Here, we show that in the absence of PDE inhibitor pretreatment, prostaglandin E2 causes accumulation of cAMP in EP2 receptors, whereas markedly low levels of cAMP accumulated in EP4 receptors. By applying the Black/Leff operational model calculation, we found that EP2 receptors have a biased ability to intrinsically activate the Gαs-protein-mediated pathway, whereas EP4 receptors have strong biased activity for the Gαi-protein-mediated pathway. Thus, EP2 and EP4 receptors may not be similar Gαs-coupled receptors but instead substantially different receptors with distinct roles.

Chronic inflammatory bowel disease (IBD), which is characterized by prolonged inflammation of the gastrointestinal tract is associated with an increased risk of colorectal cancer. Recent studies revealed that the pathology of IBD is caused by hyperactivated immune responses mediated by differentiated CD4

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl− channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone (a Food and Drug Administration-approved drug known to target prostaglandin receptors) and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary-cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current ( ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to the levels comparable to forskolin (Fsk). Pretreatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pretreated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) cotreatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ∼60% compared with the treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that cotreatment with lubiprostone can further restore modulator-rescued CFTR.

Cyclooxygenase (Cox) inhibitors are known to have severe side effects during renal development. These consist of reduced re-nal function, underdeveloped subcapsular glomeruli, interstitialfibrosis, and thinner cortical tissue. Global genetic deletion ofCox-2 mimics the phenotype observed after application of Cox inhibitors. This study aimed to investigate which cell typesexpress Cox-2 and prostaglandin E2receptors and what functions are mediated through this pathway during renal development.Expression of EP2 and EP4 mRNA was detected by RNAscope mainly in descendants of FoxD1þstromal progenitors; EP1 andEP3, on the other hand, were expressed in tubules. Cox-2 mRNA was detected in medullary interstitial cells and macula densacells. Functional investigations were performed with a cell-specific approach to delete Cox-2, EP2, and EP4 in FoxD1þstromalprogenitor cells. Our data show that Cox-2 expression in macula densa cells is sufficient to drive renal development. Deletion ofEP2 or EP4 in FoxD1þcells had no functional effect on renal development. Codeletion of EP2 and EP4 in FoxD1þstromal cells,however, led to severe glomerular defects and a strong decline of glomerularfiltration rate (1.316 ± 69.7mL/min/100 g body wt incontrols vs. 644.1 ± 64.58mL/min/100 g body wt in FoxD1þ/CreEP2⁻/⁻EP4ffmice), similar to global deletion of Cox-2.Furthermore, EP2/EP4-deficient mice showed a significant increase in collagen production with a strong downregulation of renalrenin expression. This study shows the distinct localization of EP receptors in mice. Functionally, we could identify EP2 and EP4receptors in stromal FoxD1þprogenitor cells as essential receptor subtypes for normal renal development. NEW & NOTEWORTHY Cyclooxygenase-2 (Cox-2) produces prostaglandins that are essential for normal renal development. It isunclear in which cells Cox-2 and the receptors for prostaglandin E2(EP receptors) are expressed during late nephrogenesis.This study identified the expression sites for EP subtypes and Cox-2 in neonatal mouse kidneys. Furthermore, it shows that stro-mal progenitor cells may require intact prostaglandin E2signaling through EP2 and EP4 receptors for normal renal development.

The ultrafiltrate flow over the major processes and cell body generates fluid flow shear stress (FFSS) on podocytes. Hyperfiltration-associated increase in FFSS can lead to podocyte injury and detachment. Previously, we showed that FFSS-induced upregulation of the cyclooxygenase 2 (COX2)-PGE2-prostaglandin E receptor 2 (EP2) axis in podocytes activates Akt-glycogen synthase kinase-3β-β-catenin and MAPK/ERK signaling in response to FFSS. Integrative MultiOmics Pathway Resolution (IMPRes) is a new bioinformatic tool that enables simultaneous time-series analysis of more than two groups to identify pathways and molecular connections. In the present study, we used previously characterized COX2 [prostaglandin-endoperoxide synthase 2 ( Ptgs2)], EP2 ( Ptger2), and β1-catenin ( Ctnnb1) as “seed genes” from an array data set of four groups analyzed over a time course. The 3 seed genes shared 7 pathways and 50 genes of 14 pathways and 89 genes identified by IMPRes. A composite of signaling pathways highlighted the temporal molecular connections during mechanotransduction signaling in FFSS-treated podocytes. We investigated the “proteoglycans in cancer” and “galactose metabolism” pathways predicted by IMPRes. A custom-designed PCR array validated 60.7% of the genes predicted by IMPRes analysis, including genes for the above-named pathways. Further validation using Western blot analysis showed increased expression of phosho-Erbb2, phospho-mammalian target of rapamycin (mTOR), CD44, and hexokinase II (Hk2); decreased total Erbb2, galactose mutarotase (Galm), and β-1,4-galactosyltransferase 1 (B4galt1); and unchanged total mTOR and AKT3. These findings corroborate our previously reported results. This study demonstrates the potential of the IMPRes method to identify novel pathways. Identifying the “proteoglycans in cancer” and “galactose metabolism” pathways has generated a lead to study the significance of FFSS-induced glycocalyx remodeling and possible detachment of podocytes from the glomerular matrix.

Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 μmol/L, latanoprost free acid (LAT-A) at 0.1 μmol/L, or prostaglandin F2α (PGF2α), at 0.1 μmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 μmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.

The foreign body response (FBR) has impaired progress of new implantable medical devices through its hallmark of chronic inflammation and foreign body giant cell (FBGC) formation leading to fibrous encapsulation. Macrophages are known to drive the FBR, but efforts to control macrophage polarization remain challenging. The goal for this study was to investigate whether prostaglandin E2 (PGE2), and specifically its receptors EP2 and/or EP4, attenuate classically activated (i.e., inflammatory) macrophages and macrophage fusion into FBGCs in vitro. Lipopolysaccharide (LPS)-stimulated macrophages exhibited a dose-dependent decrease in gene expression and protein production of tumor necrosis factor alpha (TNF-α) when treated with PGE2. This attenuation was primarily by the EP4 receptor, as the addition of the EP2 antagonist PF 04418948 to PGE2-treated LPS-stimulated cells did not recover TNF-α production while the EP4 antagonist ONO AE3 208 did. However, direct stimulation of EP2 with the agonist butaprost to LPS-stimulated macrophages resulted in a ∼60% decrease in TNF-α secretion after 4 h and corresponded with an increase in gene expression for Cebpb and Il10, suggesting a polarization shift toward alternative activation through EP2 alone. Further, fusion of macrophages into FBGCs induced by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited by PGE2 via EP2 signaling and by an EP2 agonist, but not an EP4 agonist. The attenuation by PGE2 was confirmed to be primarily by the EP2 receptor. Mrc1, Dcstamp, and Retlna expressions increased upon IL-4/GM-CSF stimulation, but only Retnla expression with the EP2 agonist returned to levels that were not different from controls. This study identified that PGE2 attenuates classically activated macrophages and macrophage fusion through distinct EP receptors, while targeting EP2 is able to attenuate both. In summary, this study identified EP2 as a potential therapeutic target for reducing the FBR to biomaterials.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.

Activation of prostanoid EP2 receptor exacerbates neuroinflammatory and neurodegenerative pathology in central nervous system diseases such as epilepsy, Alzheimer's disease, and cerebral aneurysms. A selective and brain-permeable EP2 antagonist will be useful to attenuate the inflammatory consequences of EP2 activation and to reduce the severity of these chronic diseases. We recently developed a brain-permeable EP2 antagonist 1 (TG6-10-1), which displayed anti-inflammatory and neuroprotective actions in rodent models of status epilepticus. However, this compound exhibited moderate selectivity to EP2, a short plasma half-life in rodents (1.7 h) and low aqueous solubility (27 μM), limiting its use in animal models of chronic disease. With lead-optimization studies, we have developed several novel EP2 antagonists with improved water solubility, brain penetration, high EP2 potency, and selectivity. These novel inhibitors suppress inflammatory gene expression induced by EP2 receptor activation in a microglial cell line, reinforcing the use of EP2 antagonists as anti-inflammatory agents.

The EP4 prostanoid receptors are one of four receptor subtypes for prostaglandin E2 (PGE2). Therefore, EP4 may play an important role in cancer progression. However, little information is available regarding their function per se, including migration and the cellular signaling pathway of EP4 in oral cancer. First, we found that mRNA and protein expression of EP4 was abundantly expressed in human‐derived tongue squamous cell carcinoma cell lines HSC‐3 and OSC‐19. The EP4 agonist (ONO‐AE1‐437) significantly promoted cell migration in HSC‐3 cells. In contrast, knockdown of EP4 reduced cell migration. Furthermore, we confirmed that knockdown of EP4 suppressed metastasis of oral cancer cells in the lungs of mice in vivo. Therefore, we focused on the mechanism of migration/metastasis in EP4 signaling. Interestingly, EP4 agonist significantly induced intracellular Ca2+ elevation not in only oral cancer cells but also in other cells, including normal cells. Furthermore, we found that EP4 activated PI3K and induced Ca2+ influx through Orai1 without activation of store depletion and stromal interaction molecule 1 (STIM1). Immunoprecipitation showed that EP4 formed complexes with Orai1 and TRPC1, but not with STIM. Moreover, the EP4 agonist ONO‐AE1‐437 phosphorylated ERK and activated MMP‐2 and MMP‐9. Knockdown of Orai1 negated EP4 agonist‐induced ERK phosphorylation. Taken together, our data suggested that EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration. Migration is regulated by EP4/PI3K/Orai1 signaling in oral cancer., EP4 activated PI3K and then induced Ca2+ influx from the extracellular space through Orai1, resulting in ERK phosphorylation and promoting cell migration.

Purpose: Omidenepag isopropyl (OMDI) is a prodrug of OMD, a selective, nonprostaglandin, prostanoid EP2 receptor agonist. This phase I study aimed to investigate the pharmacokinetic properties, safety, and intraocular pressure (IOP)-lowering efficacy of OMDI. Methods: Fourteen healthy male volunteers (7 Japanese and 7 Caucasian) 20–35 years of age received 1 drop of OMDI 0.0025% at 9:00 h in both eyes for 7 days. Blood samples were taken predose and up to 8 h postdose on days 1, 3, and 7. The plasma concentration of OMD was determined using high-performance liquid chromatography–tandem mass spectrometry. Pharmacokinetic parameters measured included the maximum plasma concentration (Cmax) and the half-life (t½) of OMD. IOP, adverse events (AEs), ophthalmic examinations, vital signs, and laboratory values were assessed. Results: Cmax for all subjects was reached after 10–15 min and decreased with a t½ of ∼30 min. Ad hoc statistical analyses found significant differences in some pharmacokinetic parameters between Japanese and Caucasian subjects, likely due to differences in body weight. These differences reduced over 7 days of dosing and were not thought to be clinically meaningful. There was no OMD accumulation after 7 days of repeated dosing. Mean IOP was reduced by ∼4–5 mmHg between baseline and 2 h postdose, remaining stable from day 3 onward. All AEs were mild and considered treatment related. Conclusions: Pharmacokinetic parameters of OMD were similar between Japanese and Caucasian subjects. There was no accumulation of OMD after 7 days of dosing. OMDI was well tolerated and demonstrated clinically significant IOP reductions.

Ovarian cancer is the leading cause of death among gynecological malignancies. Cyclooxygenase 2 is widely expressed in various cancer cells and participates in the occurrence and development of tumors by regulating a variety of downstream signaling pathways. However, the function and molecular mechanisms of cyclooxygenase 2 remain unclear in ovarian cancer. Here, we demonstrated that cyclooxygenase 2 was highly expressed in ovarian cancer and the expression level was highly correlated with ovarian tumor grades. Further, ovarian cancer cells with high expression of cyclooxygenase 2 exhibit enhanced proliferation and invasion abilities. Specifically, cyclooxygenase 2 promoted the release of prostaglandin E2 upregulated the phosphorylation levels of phospho-nuclear factor-kappa B p65. Celecoxib, AH6809, and BAY11-7082 all can inhibit the promoting effect of cyclooxygenase 2 on SKOV3 and OVCAR3 cell proliferation and invasion. Besides, celecoxib inhibited SKOV3 cell growth in the xenograft tumor model. These data suggest that high expression of cyclooxygenase 2 promotes the proliferation and invasion of ovarian cancer cells through the prostaglandin E2/nuclear factor-kappa B signaling pathway. Cyclooxygenase 2 may be a potential therapeutic target for the treatment of ovarian cancer.

Aims The goal was to explore the signaling pathways of PGE2 to investigate therapeutic effects against secondary injuries following TBI. Methods Young (4.9 ± 1.0 months) and aged (20.4 ± 1.4 months) male wild type (WT) C57BL/6 and PGE2 EP1, 2, and 3 receptor knockout mice were selected to either receive sham or repetitive concussive head injury. Immunohistochemistry protocols with Iba1 and GFAP were performed to evaluate microgliosis and astrogliosis in the hippocampus, two critical components of neuroinflammation. Passive avoidance test measured memory function associated with the hippocampus. Results No differences in hippocampal microgliosis were found when aged EP2-/- and EP3-/- mice were compared with aged WT mice. However, the aged EP1-/- mice had 69.2 ± 7.5% less hippocampal microgliosis in the contralateral hemisphere compared with WT aged mice. Compared with aged EP2-/- and EP3-/- , EP1-/- aged mice had 78.9 ± 5.1% and 74.7 ± 6.2% less hippocampal microgliosis in the contralateral hemisphere. Within the EP1-/- mice, aged mice had 90.7 ± 2.7% and 81.1 ± 5.6% less hippocampal microgliosis compared with EP1-/- young mice in the contralateral and ipsilateral hemispheres, respectively. No differences were noted in all groups for astrogliosis. There was a significant difference in latency time within EP1-/- , EP2-/- , and EP3-/- on day 1 and day 2 in aged and young mice. Conclusion These findings demonstrate that the PGE2 EP receptors may be potential therapeutic targets to treat repetitive concussions and other acute brain injuries.

This study aimed to re-examine the receptor subtype that mediates the fever-producing effects of prostaglandin E

Cyclooxygenase metabolizes dihomo-γ-linolenic acid and arachidonic acid to form prostaglandin (PG) E, including PGE1 and PGE2, respectively. Although PGE2 is well known to play an important role in the development and maintenance of hyperalgesia and allodynia, the role of PGE1 in pain is unknown. We confirm whether PGE1 induced pain using orofacial pain behavioral test in mice and determine the target molecule of PGE1 in TG neurons with whole-cell patch-clamp and immunohistochemistry. Intradermal injection of PGE1 to the whisker pads of mice induced a reduced threshold, enhancing the excitability of HCN channel-expressing trigeminal ganglion (TG) neurons. The HCN channel-generated inward current (Ih) was increased by 135.3 ± 4.8% at 100 nM of PGE1 in small- or medium-sized TG, and the action of PGE1 on Ih showed a concentration-dependent effect, with a median effective dose (ED50) of 29.3 nM. Adenylyl cyclase inhibitor (MDL12330A), 8-bromo-cAMP, and the EP2 receptor antagonist AH6809 inhibited PGE1-induced Ih. Additionally, PGE1-induced mechanical allodynia was blocked by CsCl and AH6809. PGE1 plays a role in mechanical allodynia through HCN2 channel facilitation via the EP2 receptor in nociceptive neurons, suggesting a potential therapeutic target in that PGE1 could be involved in pain as endogenous substances under inflammatory conditions.

Background. To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. Methods. The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). Results. The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

The objective of the current study was to examine the drug-induced effects of the EP2 agonist, omidenapag (OMD), on human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs). The drug-induced effects on 2D monolayers and 3D spheroids were characterized by examining the ultrastructures by scanning electron microscope (SEM), transendothelial electrical resistance (TEER) measurements, and fluorescein isothiocyanate (FITC)-dextran permeability. The physical properties of 3D spheroids with respect to size and stiffness were also examined. In addition, the gene expressions of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4, and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9, and 14, aquaporin1 (AQP1), and several endoplasmic reticulum (ER) stress-related factors were evaluated. In the 2D HCSFs, OMD induced (1) a significant increase in ECM deposits, as evidenced by SEM, the mRNA expression of COL4 and FN, and (2) a decrease in TEER values and a concentration-dependent increase in FITC-dextran permeability. In the case of 3D spheroids, OMD had no effect on size but a substantial increase in stiffness was observed. Furthermore, such OMD-induced effects on stiffness were dramatically modulated by the osmotic pressure of the system. In contrast to the above 2D cultures, among the ECM molecules and the modulators of 3D spheroids, namely, TIMPS and MMPs, the down-regulation of COL1, TIMP1 and 2 and the up-regulation of MMP9 were observed. Interestingly, such diversity in terms of OMD-induced gene expressions between 2D and 3D cultures was also recognized in AQP1 (2D; no significant change, 3D; significant up-regulation) and ER stress-related genes. The findings presented herein suggest that the EP2 agonist, OMD, alters the physical stiffness of 3D spheroids obtained from human corneal stroma fibroblasts and this alteration is dependent on the osmotic pressures. 2D and 3D cell cultures may be useful for evaluating the drug induced effects of OMD toward human corneal stroma.

Peripheral Myeloid Cell EP2 Activation Contributes to the Deleterious Consequences of Status Epilepticus Varvel NH, Espinosa-Garcia C, Hunter-Chang S, Chen Di, Biegel A, Hsieh A, Blackmer-Raynolds L, Ganesh T, Dingledine R. J Neurosci. 2021;41(5):1105-1117. doi: 10.1523/JNEUROSCI.2040-20.2020A multidimensional inflammatory response ensues after status epilepticus (SE), driven partly by cyclooxygenase-2-mediated activation of prostaglandin EP2 receptors. The inflammatory response is typified by astrocytosis, microgliosis, erosion of the blood–brain barrier (BBB), formation of inflammatory cytokines, and brain infiltration of blood-borne monocytes. Our previous studies have shown that inhibition of monocyte brain invasion or systemic administration of an EP2 receptor antagonist relieves multiple deleterious consequences of SE. Here, we identify those effects of EP2 antagonism that are reproduced by conditional ablation of EP2 receptors in immune myeloid cells and show that systemic EP2 antagonism blocks monocyte brain entry in male mice. The induction of hippocampal interleukin-6 after pilocarpine SE was nearly abolished in EP2 conditional knockout (KO) mice. Serum albumin levels in the cortex, a measure of BBB breakdown, were significantly higher after SE in EP2-sufficient mice but not in EP2 conditional KOs. The EP2 deficiency in innate immune cells accelerated the recovery from sickness behaviors following SE. Surprisingly, neurodegeneration was not alleviated in myeloid conditional KOs. Systemic EP2 antagonism prevented monocyte brain infiltration and provided broader rescue of SE-induced effects than myeloid EP2 ablation, including neuroprotection and broader suppression of inflammatory mediators. Reporter expression indicated that the cellular target of CD11b-driven Cre was circulating myeloid cells but, unexpectedly, not microglia. These findings indicate that activation of EP2 receptors on immune myeloid cells drives substantial deficits in behavior and disrupts the BBB after SE. The benefits of systemic EP2 antagonism can be attributed, in part, to blocking brain recruitment of blood-borne monocytes. SIGNIFICANCE STATEMENT Unabated seizures reduce quality of life, promote the development of epilepsy, and can be fatal. We previously identified activation of prostaglandin EP2 receptors as a driver of undesirable consequences of seizures. However, the relevant EP2-expressing cell types remain unclear. Here we identify peripheral innate immune cells as a driver of the EP2-related negative consequences of seizures. Removal of EP2 from peripheral immune cells was beneficial, abolishing production of a key inflammatory cytokine, accelerating weight regain, and limiting behavioral deficits. These findings provide evidence that EP2 engagement on peripheral immune and brain endothelia contributes to the deleterious effects of SE, and will assist in the development of beneficial therapies to enhance quality of life in individuals who suffer prolonged seizures.

Inflammaging is associated with aging-associated cognitive loss and neurodegeneration. Chronic nonsteroidal anti-inflammatory drug (NSAID) use has been reported to reduce the incidence of Alzheimer's disease (AD), presumably by inhibiting inflammation, although NSAIDs appear to not be good candidates for anti-AD therapeutics given disappointing clinical trial results. Prostaglandin E2 (PGE2) acts downstream of NSAID target COX-2, a cyclooxygenase, to activate several G-protein coupled receptors (GPCRs) including EP2, which is now reported to reduce glycolysis and oxidative phosphorylation during aging by increasing glycogen synthesis and polarizing myeloid cells toward the M1 proinflammatory phenotype. Inhibiting EP2 using small molecule drugs polarizes macrophages toward the anti-inflammatory phenotype, restores youthful metabolism and mitochondrial morphology as well as youthful hippocampus-based memory capability. EP2 may be a better target than COXs for the development of drugs that improve age-associated mild cognitive impairment and possibly even for the development of drugs to treat dementias.

Primary tumors are drivers of pre-metastatic niche formation, but the coordination by the secondary organ toward metastatic dissemination is underappreciated. Here, by single-cell RNA sequencing and immunofluorescence, we identified a population of cyclooxygenase 2 (COX-2)-expressing adventitial fibroblasts that remodeled the lung immune microenvironment. At steady state, fibroblasts in the lungs produced prostaglandin E2 (PGE2), which drove dysfunctional dendritic cells (DCs) and suppressive monocytes. This lung-intrinsic stromal program was propagated by tumor-associated inflammation, particularly the pro-inflammatory cytokine interleukin-1β, supporting a pre-metastatic niche. Genetic ablation of Ptgs2 (encoding COX-2) in fibroblasts was sufficient to reverse the immune-suppressive phenotypes of lung-resident myeloid cells, resulting in heightened immune activation and diminished lung metastasis in multiple breast cancer models. Moreover, the anti-metastatic activity of DC-based therapy and PD-1 blockade was improved by fibroblast-specific Ptgs2 deletion or dual inhibition of PGE2 receptors EP2 and EP4. Collectively, lung-resident fibroblasts reshape the local immune landscape to facilitate breast cancer metastasis.

Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing effector T‐cell functions. The actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we employed pharmacological and genetic approaches to examine whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. We found that TGF‐β‐induced Foxp3 expression and iTreg cell differentiation in vitro is markedly inhibited by PGE2, which was mediated by the receptors EP2 and EP4. Mechanistically, PGE2‐EP2/EP4 signalling interrupts TGF‐β signalling during iTreg differentiation. Moreover, EP4 deficiency in T cells impaired iTreg cell differentiation in vivo. Thus, our results demonstrate that PGE2 negatively regulates iTreg cell differentiation through a direct action on T cells, highlighting the potential for selectively targeting the PGE2‐EP2/EP4 pathway to control T cell‐mediated inflammation., We have found that the development of inducible Treg cells is inhibited by PGE2 through its receptors EP2/EP4 and direct down‐regulation of TGF‐β signalling. Our results suggest a role for the PGE2‐EP2/EP4 pathway in T cell‐mediated inflammation.

Emerging evidence suggests a mechanistic role for myeloid-derived suppressor cells (MDSCs) in lung diseases like asthma. Previously, we showed that adoptive transfer of MDSCs dampens lung inflammation in murine models of asthma through cyclooxygenase-2 and arginase-1 pathways. Here, we further dissected this mechanism by studying the role and therapeutic relevance of the downstream mediator prostaglandin E2 receptor 4 (EP4) in a murine model of asthma. We adoptively transferred MDSCs generated using an EP4 agonist in a murine model of asthma and studied the consequences on airway inflammation. Furthermore, pegylated human arginase-1 was used to model MDSC effector activities. We demonstrate that the selective EP4 agonist L-902,688 increased the number and suppressive activity of MDSCs through arginase-1 and nitric oxide synthase-2. These results showed that adoptive transfer of EP4-primed MDSCs, EP4 agonism alone or arginase-1 administration ameliorated lung inflammatory responses and histopathological changes in asthmatic mice. Collectively, our results provide evidence that MDSCs dampen airway inflammation in murine asthma through a mechanism involving EP4.

Prostaglandin E

Active inflammation generally promotes immune activation. However, in the tumor microenvironment (TME), active inflammation occurs in parallel with immunosuppression, and both contribute to tumor growth. Why inflammation does not lead to immune activation in TME remains unclear. In this study, using the immune checkpoint inhibitor-insensitive mouse cancer model and single-cell RNA sequencing, we show that PGE

Hypothalamic feeding circuits have been identified as having innate synaptic plasticity, mediating adaption to the changing metabolic milieu by controlling responses to feeding and obesity. However, less is known about the regulatory principles underlying the dynamic changes in agouti-related protein (AgRP) perikarya, a region crucial for gating of neural excitation and, hence, feeding. Here we show that AgRP neurons activated by food deprivation, ghrelin administration, or chemogenetics decreased their own inhibitory tone while triggering mitochondrial adaptations in neighboring astrocytes. We found that it was the inhibitory neurotransmitter GABA released by AgRP neurons that evoked this astrocytic response; this in turn resulted in increased glial ensheetment of AgRP perikarya by glial processes and increased excitability of AgRP neurons. We also identified astrocyte-derived prostaglandin E(2), which directly activated — via EP2 receptors — AgRP neurons. Taken together, these observations unmasked a feed-forward, self-exciting loop in AgRP neuronal control mediated by astrocytes, a mechanism directly relevant for hunger, feeding, and overfeeding.