EP4 Agonist Research Articles

EP1 Receptor Antagonism as a Therapeutic Intervention Strategy in Renal Fibrosis

Renal fibrosis is a hallmark of Chronic Kidney Disease (CKD), which affects 10–16% of the world’s adult population. It has previously been shown that the cyclooxygenase/prostaglandin (COX/PG) system affects the progression of renal fibrosis. The downstream elements of this system, the eicosanoid prostaglandin (EP) receptors, EP1–4, have various physiological roles in health and disease. We therefore evaluated the effect of antagonizing one of these downstream receptors for prostaglandin, the EP1 receptor, on renal fibrosis.This was accomplished with a translational approach by utilizing three models of renal fibrosis; Transforming growth factor β (TGF‐β) treated Madin‐Darby Canine Kidney (MDCK) cells, mice that underwent seven days of unilateral ureteral obstruction (UUO), as well as TGF‐β treated Human Precision Cut Kidney Slices (HPCKS). We evaluated the extent of fibrosis in these models on the gene and protein level using QPCR as well as Western blotting and immunohistochemical analysis. Moreover, we determined the corresponding effects of the EP1 agonist 17‐phenyl trinor prostaglandin E2 (17‐phenyl PGE2) and the EP1 antagonist SC‐19220 on the intracellular Ca2+ concentration in MDCK cells measured by Flou‐4‐AM.We found, that as a whole, treatment with the EP1 receptor antagonist SC‐19220 successfully ameliorated renal fibrosis in all three models. Treatment with the EP1 receptor antagonist SC‐19220 (25mg/kg per day) diminished fibrosis in mice that underwent seven days of UUO. UUO‐induced protein expression of the fibrosis markers fibronectin (FN), α Smooth Muscle Actin (αSMA) and collagen measured by western blot, qPCR and immunohistochemical staining, was lowered by SC‐19220 treatment when compared to SHAM operated mice. Adding to this, treatment of HPCKS with SC‐19220 reduced TGF‐β‐mediated fibrosis as shown by decreased collagen 1A1, FN and αSMA gene expression as well as reduced staining intensity of Sirius Red and Masson’s Trichrome stains. In MDCK cells, fibrosis induced by TGF‐β was significantly reduced when treated with SC‐19220. This was evident by lower FN protein levels and possibly mediated by hampering of TGF‐β signalling. The specificity of SC‐19220 as an antagonist of the 17‐phenyl PGE2‐induced [Ca2+]i response was determined with standard concentration response experiments.Thus, this study shows that the EP1 receptor is a promising target for the treatment of renal fibrosis. However, further investigations into the mechanism underlying the anti‐fibrotic effect of EP1 receptor antagonism are necessary. It seems though, that intracellular calcium regulation is profoundly altered by the EP1 receptor and that this may alter fibrosis dynamics.Support or Funding InformationFunding for the project was provided by the Danish Research Council.

Distribution and Function of Prostaglandin E2Receptors in Mouse Uterus: Translational Value for Human Reproduction

Prostaglandin (PG) E analogues are used clinically to ripen the cervix and induce labour. However, selective receptor agonists may have potential to improve induction response rates or manage unwanted uterine hypercontractility in conditions such as dysmenorrhoea and preterm labour. To characterise their therapeutic value, PGE2 analogues were used to investigate the functional EP receptor population in isolated human uterus. Responsiveness in mouse tissues was also examined to validate its use as a pre-clinical model. Uterine samples were obtained from mice at dioestrus (n=12), term gestation (n=14) and labour (n=12) and from the lower uterus of women undergoing hysterectomy (n=12) or Caesarean section (n=18). Vehicle and agonist effects were assessed using superfusion and immersion techniques. PGE2 evoked predominant excitatory responses in mouse and relaxation in human tissues. Selective EP4 agonists inhibited tissue activity in both non-pregnant species, whilst the EP2 mimetic CP533536 also attenuated uterine contractions throughout gestation. The uterotonic effects of the EP3/1 agonist sulprostone were more pronounced than the EP1 agonist ONO-D1-004, corresponding to abundant EP3 receptor expression in all samples. The contractile phenotype in mouse compared to human uteri may relate to regional differences as well as high expression of EP3 receptor transcripts. Similarities in non-pregnant and gestational tissues across species suggest that EP3 may represent a valuable translational drug target for preventing uterine hypercontractility by employing a selective antagonist. SIGNIFICANCE STATEMENT: This research validates the use of non-pregnant mice for pre-clinical drug discovery of uterine EP receptor targets. To determine the utility of novel drugs and delivery systems at term pregnancy and labour, pharmacological agents interacting with EP3 receptors have clear translational value.

EP4 activation ameliorates liver ischemia/reperfusion injury via ERK1/2‑GSK3β‑dependent MPTP inhibition.

Prostaglandin E receptor subtype 4 (EP4) is widely distributed in the heart, but its role in hepatic ischemia/reperfusion (I/R), particularly in mitochondrial permeability transition pore (MPTP) modulation, is yet to be elucidated. In the present study, an EP4 agonist (CAY10598) was used in a rat model to evaluate the effects of EP4 activation on liver I/R and the mechanisms underlying this. I/R insult upregulated hepatic EP4 expression during early reperfusion. In addition, subcutaneous CAY10598 injection prior to the onset of reperfusion significantly increased hepatocyte cAMP concentrations and decreased serum ALT and AST levels and necrotic and apoptotic cell percentages, after 6 h of reperfusion. Moreover, CAY10598 protected mitochondrial morphology, markedly inhibited mitochondrial permeability transition pore (MPTP) opening and decreased liver reactive oxygen species levels. This occurred via activation of the ERK1/2-GSK3β pathway rather than the janus kinase (JAK)2-signal transducers and activators of transcription (STAT)3 pathway, and resulted in prevention of mitochondria-associated cell injury. The MPTP opener carboxyatractyloside (CATR) and the ERK1/2 inhibitor PD98059 also partially reversed the protective effects of CAY10598 on the liver and mitochondria. The current findings indicate that EP4 activation induces ERK1/2-GSK3β signaling and subsequent MPTP inhibition to provide hepatoprotection, and these observations are informative for developing new molecular targets and preventative therapies for I/R in a clinical setting.

The positive effect of selective prostaglandin E2 receptor EP2 and EP4 blockade on cystogenesis in vitro is counteracted by increased kidney inflammation in vivo

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major cause of end-stage kidney disease in man. The central role of cyclic adenosine monophosphate (cAMP) in ADPKD pathogenesis has been confirmed by numerous studies including positive clinical trial data. Here, we investigated the potential role of another major regulator of renal cAMP, prostaglandin E2 (PGE2), in modifying disease progression in ADPKD models using selective receptor modulators to all four PGE2 receptor subtypes (EP1-4). In 3D-culture model systems utilizing dog (MDCK) and patient-derived (UCL93, OX161-C1) kidney cell lines, PGE2 strikingly promoted cystogenesis and inhibited tubulogenesis by stimulating proliferation while reducing apoptosis. The effect of PGE2 on tubulogenesis and cystogenesis in 3D-culture was mimicked or abolished by selective EP2 and EP4 agonists or antagonists but not those specific to EP1 or EP3. In a Pkd1 mouse model (Pkd1nl/nl), kidney PGE2 and COX-2 expression were increased by two-fold at the peak of disease (week four). However, Pkd1nl/nl mice treated with selective EP2 (PF-04418948) or EP4 (ONO-AE3-208) antagonists from birth for three weeks had more severe cystic disease and fibrosis associated with increased cell proliferation and macrophage infiltration. A similar effect was observed for the EP4 antagonist ONO-AE3-208 in a second Pkd1 model (Pax8rtTA-TetO-Cre-Pkd1f/f). Thus, despite the positive effects of slowing cyst growth invitro, the more complex effects of inhibiting EP2 or EP4 invivo resulted in a worse outcome, possibly related to unexpected pro-inflammatory effects.

EP3 signaling in dendritic cells promotes liver repair by inducing IL-13-mediated macrophage differentiation in mice.

Macrophage plasticity is essential for liver wound healing; however, the mechanisms underlying macrophage phenotype switching are largely unknown. Dendritic cells (DCs) are critical initiators of innate immune responses; as such, they orchestrate inflammation following hepatic injury. Here, we subjected EP3-deficient (Ptger3-/- ) and wild-type (WT) mice to hepatic ischemia-reperfusion (I/R) and demonstrate that signaling via the prostaglandin E (PGE) receptor EP3 in DCs regulates macrophage plasticity during liver repair. Compared with WT mice, Ptger3-/- mice showed delayed liver repair accompanied by reduced expression of hepatic growth factors and accumulation of Ly6Clow reparative macrophages and monocyte-derived DCs (moDCs). MoDCs were recruited to the boundary between damaged and undamaged liver tissue in an EP3-dependent manner. Adoptive transfer of moDCs from Ptger3-/- mice resulted in impaired repair, along with increased numbers of Ly6Chigh inflammatory macrophages. Bone marrow macrophages (BMMs) up-regulated expression of genes related to a reparative macrophage phenotype when co-cultured with moDCs; this phenomenon was dependent on EP3 signaling. In the presence of an EP3 agonist, interleukin (IL)-13 derived from moDCs drove BMMs to increase expression of genes characteristic of a reparative macrophage phenotype. The results suggest that EP3 signaling in moDCs facilitates liver repair by inducing IL-13-mediated switching of macrophage phenotype from pro-inflammatory to pro-reparative.

Prostaglandin E2 and Its Receptor EP2 Modulate Macrophage Activation and Fusion in Vitro.

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.

EP4 signaling facilitates mucosal repair after DSS-induced colitis by enhancement of lymphangiogenesis

Lymphatic development in the intestine is related to inflammatory bowel diseases. Prostaglandin E2 (PGE2)/EP4 prevents from the development of experimental colitis. We previously reported that EP4 facilitates wound healing and wound-associated lymphangiogenesis. In the present study, we examined whether EP4 is involved in tissue repair from colitis by enhancement of lymphangiongenesis. Experimental colitis was induced in male C57/Bl6 mice by the administration of dextran sulphate sodium (DSS) via drinking water for 5 days followed by free water drinking. Mice were received EP4 selective agonist, EP4 selective antagonist, or vehicle from Day 5 through Day 14. At Day 14, mice post-treated with EP4 antagonist were susceptible to signs of colitis, increased colonic tissue destruction and infiltration of macrophages. The increased lymphatic vessel density (LVD) was increased, but percentage of lymphatic vessel area (LVA%) was reduced. These were associated with decreased levels of lymphatic markers and pro-lymphangiogenic factors including VEGF-C and VEGF-D. By contrast, post-treatment with EP4 agonist improved signs of DSS-colitis, suppressed macrophage infiltration and cytokines. This was associated with further enhancement of lymphatic factors and lymphangiogenesis as evidenced by higher LVD and LVA%, and fluorescence intensity in the mesenteric lymph nodes. These results suggest that EP4 activation is helpful for attenuation of DSS-induced colitis by enhancement of drainage function through enlarged lymphatic vessels.

Prostaglandin E2 receptor EP4 regulates cell migration through Orai1

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.

In Vivo Bone Effects of a Novel Bisphosphonate-EP4a Conjugate Drug (C3) for Reversing Osteoporotic Bone Loss in an Ovariectomized Rat Model.

ABSTRACTPathological bone loss is a regular feature of postmenopausal osteoporosis, and the microstructural changes along with the bone loss make the individual prone to getting hip, spine, and wrist fractures. We have developed a new conjugate drug named C3, which has a synthetic, stable EP4 agonist (EP4a) covalently linked to an inactive alendronate (ALN) that binds to bone and allows physiological remodeling. After losing bone for 12 weeks, seven groups of rats were treated for 8 weeks via tail‐vein injection. The groups were: C3 conjugate at low and high doses, vehicle‐treated ovariectomy (OVX) and sham, C1 (a similar conjugate, but with active ALN at high dose), inactive ALN alone, and a mixture of unconjugated ALN and EP4a to evaluate the conjugation effects. Bone turnover was determined by dynamic and static histomorphometry; μCT was employed to determine bone microarchitecture; and bone mechanical properties were evaluated via biomechanical testing. Treatment with C3 significantly increased trabecular bone volume and vertebral BMD versus OVX controls. There was also significant improvement in the vertebral load‐bearing abilities and stimulation of bone formation in femurs after C3 treatment. This preclinical research revealed that C3 resulted in significant anabolic effects on trabecular bone, and EP4a and ALN conjugation components are vital to conjugate anabolic efficacy. A combined therapy using an EP4 selective agonist anabolic agent linked to an inactive ALN is presented here that produces significant anabolic effects, allows bone remodeling, and has the potential for treating postmenopausal osteoporosis or other diseases where bone strengthening would be beneficial. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Novel Microglia Cell Line Expressing the Human EP2 Receptor.

Recently, EP2 signaling pathways were shown to regulate the classical activation and death of microglia in rat primary microglial culture. The study of microglial cells has been challenging because they are time-consuming to isolate in culture, they are demanding in their growth requirements, and they have a limited lifespan. To circumvent these difficulties, we created a murine BV2 microglial cell line stably expressing human EP2 receptors (BV2-hEP2) and further explored EP2 modulation of microglial functions. The BV2-hEP2 cells displayed cAMP elevation when exposed to the selective EP2 receptor agonists (ONO-AE1-259-1 and CP544326), and this response was competitively inhibited by TG4-155, a selective EP2 antagonist (Schild KB = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to the case of rat primary microglia, BV2-hEP2 microglia treated with lipopolysaccharide (LPS) (100 ng/mL) displayed rapid and robust induction of the inflammatory mediators COX-2, IL-1β, TNFα, and IL-6. EP2 activation depressed TNFα induction but exacerbated that of the other inflammatory mediators. Like primary microglia, classically activated BV2 microglia phagocytose fluorescent-labeled latex microspheres. The presence of EP2, but not its activation by agonists, in BV2-hEP2 microglia reduced phagocytosis and proliferation by 65% and 32%, respectively, compared to BV2 microglia. Thus, BV2-hEP2 is the first microglial cell line that retains the EP2 modulation of immune regulation and phagocytic ability of native microglia. Suppression of phagocytosis by the EP2 protein appears unrelated to classical EP2 signaling pathways, which has implications for therapeutic development of EP2 antagonists.

Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gβγ Subunits.

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C terminus of β-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Further characterization of a novel EP2 and EP3 receptor dual agonist, ONO-8055, on lower urinary tract function in normal and lumbar canal stenosis rats.

To further explore the effects of a novel EP2 and EP3 dual agonist, ONO-8055, on detrusor contractility, we investigated the responses of bladder strips from sham and lumbar canal stenosis (LCS) rats to this agonist, its effects on lower urinary tract function in normal rats, and mRNA expression of EP2 and EP3 receptors in the sham and LCS rats. The responses of bladder strips from sham and LCS rats to ONO-8055 were measured. The effects of ONO-8055 on LUT function of normal rats were investigated with awake cystometry and intraurethral perfusion pressure (Pura) measurements. The relative mRNA of bladder and urethral tissue of the sham and LCS rats was quantified using specific probes for EP1, EP2, EP3, and EP4 genes. Compared with the vehicle, the muscle tensions of both the sham and LCS rats were significantly increased after adding this agonist. On awake cystometry of normal rats, bladder capacity and Pura were decreased in the ONO-8055 groups, but a statistically significant difference in mean changes was demonstrated only between the vehicle group and the group receiving the highest dose. Compared with the sham rats, mRNA expressions of the four EP receptors in the lower urinary tract of the LCS rats did not show a statistically significant difference. This agonist did not augment bladder contractility or urethral relaxation in normal rats.

Antiglaucoma EP2 Agonists: A Long Road That Led Somewhere.

For >2 decades, EP2 agonists have been the subject of antiglaucoma research and development by scientists in industry and academia around the world. The road has led to the recent approval of the first drug of this class. This article reviews the development of EP2 agonists from conception to clinical approval, discussing pharmacology, structure, biodistribution, therapeutics, and drug delivery. An extensive list of source references is provided for the reader's benefit.

Prostaglandin E2 (PGE2)–EP2 signaling negatively regulates murine atopic dermatitis–like skin inflammation by suppressing thymic stromal lymphopoietin expression

Atopic dermatitis (AD) is a common and chronic inflammatory skin disease of type 2 immunity. Keratinocyte-derived cytokines, including thymic stromal lymphopoietin (TSLP) and IL-33, are considered to induce the development of AD. Production of prostanoids, a family of lipid mediators, is increased in AD lesions. However, their physiologic functions remain to be clarified. We sought to elucidate the functions of prostanoids in the development of AD. The roles of prostanoids were investigated in a mouse model of AD induced by repeated application of hapten and PAM212, a keratinocyte cell line. Application of indomethacin, which blocks prostanoid synthesis, leads to enhanced TSLP and IL-33 production in the skin, increased serum IgE levels, and exacerbation of skin inflammation in this AD model. The skin inflammation was attenuated in TSLP receptor-deficient mice but not in IL-33-deficient mice, and the indomethacin-enhanced type 2 immune responses were abolished in TSLP receptor-deficient mice. Indomethacin increased protease-activated receptor 2-mediated TSLP production in keratinocytes invitro, and prostaglandin E2 reversed the increase in TSLP levels through its receptor, the prostaglandin E2 receptor (EP2), by downregulating surface expression of protease-activated receptor 2. Administration of an EP2 agonist canceled indomethacin-enhanced TSLP production and type 2 immune responses in the skin, whereas an EP2 antagonist caused an enhancement of TSLP production and type 2 immune responses in the skin. Prostaglandin E2-EP2 signaling negatively regulates murine AD-like skin inflammation by suppressing TSLP expression.

Prostaglandin E2 and an EP4 receptor agonist inhibit LPS-Induced monocyte chemotactic protein 5 production and secretion in mouse cardiac fibroblasts via Akt and NF-κB signaling

BackgroundProstaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We have previously reported that mice lacking the EP4 receptor in the cardiomyocytes develop heart failure with a phenotype of dilated cardiomyopathy. Also, these mice have increased levels of chemokines, like MCP-5, in their left ventricles. We have recently reported that overexpression of the EP4 receptor could improve cardiac function in the myocardial infarction model. Furthermore, we showed that overexpression of EP4 had an anti-inflammatory effect in the whole left ventricle. It has also been shown that PGE2 can antagonize lipopolysaccharide-induced secretion of chemokines/cytokines in various cell types. We therefore hypothesized that PGE2 inhibits lipopolysaccharide (LPS)-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. Methods and resultsOur hypothesis was tested using isolated mouse adult ventricular fibroblasts (AVF) treated with LPS. Pre-treatment of the cells with PGE2 and the EP4 agonist CAY10598 resulted in reductions of the pro-inflammatory response induced by LPS. Specifically, we observed reductions in MCP-5 secretion. Western blot analysis showed reductions in phosphorylated Akt and IκBα indicating reduced NF-κB activation. The anti-inflammatory effects of PGE2 and EP4 agonist signaling appeared to be independent of cAMP, p-44/42, or p38 pathways. ConclusionExogenous treatment of PGE2 and the EP4 receptor agonist blocked the pro-inflammatory actions of LPS. Mechanistically, this was mediated via reduced Akt phosphorylation and inhibition of NF-κB.

Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body

Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1–4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive.We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath.PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction.The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux.

Activation of the prostaglandin E2 EP2 receptor attenuates renal fibrosis in unilateral ureteral obstructed mice and human kidney slices.

AimRenal fibrosis plays a pivotal role in the development and progression of chronic kidney disease, which affects 10% of the adult population. Previously, it has been demonstrated that the cyclooxygenase‐2 (COX‐2)/prostaglandin (PG) system influences the progression of renal injury. Here, we evaluated the impact of butaprost, a selective EP2 receptor agonist, on renal fibrosis in several models of kidney injury, including human tissue slices.MethodsWe studied the anti‐fibrotic efficacy of butaprost using Madin‐Darby Canine Kidney (MDCK) cells, mice that underwent unilateral ureteral obstruction and human precision‐cut kidney slices. Fibrogenesis was evaluated on a gene and protein level by qPCR and Western blotting.ResultsButaprost (50 μM) reduced TGF‐β‐induced fibronectin (FN) expression, Smad2 phosphorylation and epithelial‐mesenchymal transition in MDCK cells. In addition, treatment with 4 mg/kg/day butaprost attenuated the development of fibrosis in mice that underwent unilateral ureteral obstruction surgery, as illustrated by a reduction in the gene and protein expression of α‐smooth muscle actin, FN and collagen 1A1. More importantly, a similar anti‐fibrotic effect of butaprost was observed in human precision‐cut kidney slices exposed to TGF‐β. The mechanism of action of butaprost appeared to be a direct effect on TGF‐β/Smad signalling, which was independent of the cAMP/PKA pathway.ConclusionIn conclusion, this study demonstrates that stimulation of the EP2 receptor effectively mitigates renal fibrogenesis in various fibrosis models. These findings warrant further research into the clinical application of butaprost, or other EP2 agonists, for the inhibition of renal fibrosis.

Expression of prostaglandin E2 receptor 3 in the eyelid epidermis of patients with Stevens-Johnson syndrome/toxic epidermal necrolysis

Background/aimsIn a previous genome-wide association study of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) patients we reported the association between SJS/TEN and the prostaglandin E receptor 3 (PTGER3) gene, and that its...

Involvement of EP2 and EP4 Receptors in Eosinophilic Esophagitis: A Pilot Study

BackgroundThe prostaglandin D2 receptor DP2 has been implicated in eosinophil infiltration and the development of eosinophilic esophagitis (EoE).Aims and MethodsIn this study, we investigated an involvement of PGE2 (EP1–EP4) and PGD2 (DP1) receptors in EoE by measuring their expression in peripheral blood eosinophils and esophageal mucosal biopsies of EoE patients and by performing migration and adhesion assays with eosinophils from healthy donors.ResultsExpression of EP2 and EP4, but not EP1 and EP3, was decreased in blood eosinophils of patients with EoE vs. control subjects. Adhesion of eosinophils to esophageal epithelial cells was decreased by EP2 receptor agonist butaprost and EP4 agonist ONO-AE1-329, whereas DP1 agonist BW245C increased adhesion. In chemotaxis assays with supernatant from human esophageal epithelial cells, only ONO-AE1-329 but not butaprost or BW245C inhibited the migration of eosinophils. Expression of EP and DP receptors in epithelial cells and eosinophils was detected in sections of esophageal biopsies from EoE patients by immunohistochemistry. qPCR of biopsies from EoE patients revealed that gene expression of EP4 and DP1 was the highest among PGE2 and PGD2 receptors. Esophageal epithelial cells in culture showed high gene expression for EP2 and EP4. Activation of EP2 and EP4 receptors decreased barrier integrity of esophageal epithelial cells in impedance assays.ConclusionsActivation of EP2 and EP4 receptors may inhibit eosinophil recruitment to the esophageal mucosa. However, their activation could negatively affect esophageal barrier integrity suggesting that eosinophilic rather than epithelial EP2 and EP4 have a protective role in EoE.

Prostaglandin E2 Inhibits LPS‐Induced Monocyte Chemoattractant Protein 5 in Adult Cardiac Fibroblasts Through its EP4 Receptor via Reduced Akt and NF‐κB Signaling

Prostaglandin E2 (PGE2) signals through 4 G‐protein coupled receptor sub‐types (EP1–EP4) to elicit a variety of pathophysiological effects. We previously reported that mice lacking the EP4 receptor in cardiomyocytes (EP4 KO) develop heart failure with a phenotype of dilated cardiomyopathy. These mice also have increased levels of monocyte chemoattractant protein‐5 (MCP‐5), in their left ventricles. We recently published that overexpression of the EP4 receptor improves cardiac function in a model of myocardial infarction; associated with anti‐inflammatory effects. Moreover, conditioned media from EP4 KO cardiomyocyte cultures increased macrophage migration in vitro, which was attenuated with a MCP‐5 neutralizing antibody. In a variety of cell types, PGE2 antagonizes lipopolysaccharide (LPS)‐induced secretion of chemokines/cytokines. We therefore hypothesized that PGE2 inhibits LPS‐induced MCP‐5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. Our hypothesis was tested using 16–20‐week old mouse adult ventricular fibroblasts treated with LPS (10 μg/ml). Media and cells were harvested after 1, 2, 4, and 24 hrs of LPS treatment. MCP‐5 secretion was determined by ELISA. LPS treatment for 4 hrs stimulated MCP‐5 secretion 2.3‐fold compared to vehicle (p<0.05) and this stimulation persisted for at least 24 hrs (p < 0.001). Pre‐treatment with PGE2 or an EP4 agonist significantly reduced LPS‐stimulated MCP‐5 by 24 hours (p< 0.01). Both the LPS‐stimulation of MCP‐5 and its inhibition by PGE2‐EP4 takes 4 hours to be detectable, suggesting that PGE2 via EP4 signaling may regulate MCP‐5 transcription. To test this, we examined MCP‐5 mRNA levels after 1, 2, 4, and 24 hours post LPS stimulation. After 1 hour of LPS stimulation, MCP‐5 mRNA significantly increased compared to vehicle (4.49 ± 0.71 vs. 1.00, respectively; p < 0.005). Treatment with the EP4 agonist significantly blunted the response to LPS (2.09 ± 0.82 vs. 4.49 ± 0.71 in LPS‐treated cells; p < 0.005). PGE2 treatment also attenuated MCP‐5 mRNA after LPS stimulation, although this failed to reach statistical significance (2.68 ± 1.04 vs. 4.49 ± 0.71 in LPS‐treated cells; p=0.186). PGE2 and the EP4 agonist only reduced LPS‐stimulated MCP‐5 mRNA at the 1 hr time point, suggesting that EP4 receptor activation leads to downstream signaling that quickly downregulates MCP‐5. Western blot showed PGE2‐EP4 reduced LPS stimulated phosphorylation of Akt and IκBα, indicating reduced NF‐κB activation. Furthermore, blocking NF‐κB with Cardamonin (10 μM) reduced LPS‐stimulated MCP‐5 after 24 hours of treatment (1.71 ± 0.41 vs. 9.11 ± 3.09 in LPS treated cells; p<0.005). Our data also show that the inhibitory effects of PGE2 or the EP4 agonist are independent of cAMP, JNK, p‐44/42, or p38 pathways. Therapeutically, our study may help shed light on the mechanism(s) regulating chemokine production in cardiac fibroblasts.Support or Funding InformationPH: NIH grant [5P01HL028982] (sub‐project 2). TB: NIH T32 Detroit Cardiovascular Training Grant (5T32HL12082205)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.