Prostaglandin E2-Transporting Pathway and Its Roles via EP2/EP4 in Cultured Human Dental Pulp.

INTRODUCTION: Prostaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. METHODS: Protein localization of microsomal PGE2 (mPGES)synthase, PGE2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. RESULTS: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). CONCLUSIONS: PGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

Studies in Zebrafish and Rat Models Support Dual Blockade of EP2 and EP4 (Prostaglandin E2 Receptors Type 2 and 4) for Renoprotection in Glomerular Hyperfiltration and Albuminuria.

BACKGROUND: Glomerular hyperfiltration (GH) is an important mechanism in the development of albuminuria in hypertension. Upregulation of COX2 (cyclooxygenase 2) and prostaglandin E2 (PGE2) was linked to podocyte damage in GH. We explored the potential renoprotective effects of either separate or combined pharmacological blockade of EP2 (PGE2 receptor type 2) and EP4 (PGE2 receptor type 4) in GH. METHODS: We conducted in vivo studies in a transgenic zebrafish model (Tg[fabp10a:gc-EGFP]) suitable for analysis of glomerular filtration barrier function and a genetic rat model with GH, albuminuria, and upregulation of PGE2. Similar pharmacological interventions and primary outcome analysis on albuminuria phenotype development were conducted in both model systems. RESULTS: Stimulation of zebrafish embryos with PGE2 induced an albuminuria-like phenotype, thus mimicking the suggested PGE2 effects on glomerular filtration barrier dysfunction. Both separate and combined blockade of EP2 and EP4 reduced albuminuria phenotypes in zebrafish and rat models. A significant correlation between albuminuria and podocyte damage in electron microscopy imaging was identified in the rat model. Dual blockade of both receptors showed a pronounced synergistic suppression of albuminuria. Importantly, this occurred without changes in arterial blood pressure, glomerular filtration rate, or tissue oxygenation in magnetic resonance imaging, while RNA sequencing analysis implicated a potential role of circadian clock genes. CONCLUSIONS: Our findings confirm a role of PGE2 in the development of albuminuria in GH and support the renoprotective potential of combined pharmacological blockade of EP2 and EP4 receptors. These data support further translational research to explore this therapeutic option and a possible role of circadian clock genes.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

Prostaglandin E receptor 2 mediates the inducible effects of prostaglandin E2 on expression of growth factors and enzymes in cattle endometrial epithelial cells and explants.

Prostaglandin E2 (PGE2 ) is able to induce the expression of several growth factors and enzymes in cattle endometria. However, the specific type of PGE2 receptors which mediates this effect is not fully clear. In this study, the role of prostaglandin E receptor 2 (PTGER2) in PGE2 -mediated induction of growth factors and enzymes expression in cattle endometrial explants and epithelial cells were investigated. PTGER2 was blocked by a PTGER2 antagonist, AH6809, before PGE2 treatment, then the mRNA and protein expression levels of several growth factors and enzymes were compared with that in PGE2 alone treatment group by real-time RT-PCR and Western blotting analysis in endometrial epithelial cells and explants. Results indicated that PGE2 significantly increased the mRNA and protein levels of these growth factors and enzymes, while the rates of increment in the expression of these growth factors and enzymes were inhibited by AH6809. In addition, a PTGER2 agonist, butaprost, significantly increased the expression levels of these growth factors and enzymes, and the effect could be blocked by AH6809. In conclusion, PTGER2 was found to be one dominant receptor mediating the inducible effects of PGE2 on the expression of these growth factors and enzymes in cattle endometrial explants and epithelial cells.

Effects of selective inhibition of prostaglandin E2 receptors EP2 and EP4 on the miRNA profile in endometriosis.

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 E2 (PGE2) plays an important role in the survival and growth of endometriotic lesions. MicroRNAs (miRNAs) are small, noncoding RNAs that control gene expressions through multiple mechanisms and have important roles in the pathogenesis of endometriosis. The objective of the present study is to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on miRNA profile in endometriosis. The novel results collectively indicate that inhibition of PGE2-EP2/EP4 signaling regulated several miRNA clusters associated with cell adhesion, migration, invasion, survival and growth in cell-specific and the chromosome-specific manner and reverses the epigenetic silencing of proapoptotic miRNAs 15a and 34c in the human endometriotic epithelial and stromal cells and experimental endometriotic lesions. Thus, selective inhibition of EP2/EP4 receptors could emerge as a potential nonsteroidal therapy for endometriosis.

PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment.

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 PGE2-EP2/EP4 signaling simultaneously promotes active inflammation by inducing expression of the NF-κB genes in myeloid cells and elicits immunosuppression by driving the mregDC (mature DC enriched in immunoregulatory molecules)-Treg (regulatory T cell) axis for Treg recruitment and activation in the tumor. Importantly, the EP2/EP4 expression level is strongly correlated with the gene signatures of both active inflammation and the mregDC-Treg axis and has significant prognosis value in various human cancers. Thus, PGE2-EP2/EP4 signaling functions as the key regulatory node linking active inflammation and immunosuppression in TME, which can be targeted by EP2 and EP4 antagonists for cancer therapeutics.

Reduced tumor growth in EP2 knockout mice is related to signaling pathways favoring an increased local anti­tumor immunity in the tumor stroma.

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 well­established how signaling pathways in tumor tissue relate to simultaneous signaling alterations in the surrounding tumor­stroma, at conditions of reduced disease progression due to decreased host inflammation. In the present study, wild­type tumor cells, producing high levels of prostaglandin E2 (MCG 101 cells, EP2+/+), were inoculated into EP2 knockout (EP2­/­) and EP2 wild­type (EP2+/+) mice. Solid tumors were dissected into tumor­ and tumor­stroma tissue compartments for RNA expression microarray screening, followed by metabolic pathway analyses. Immunohistochemistry was used to confirm adequate dissections of tissue compartments, and to assess cell proliferation (Ki­67), prostaglandin enzymes (cyclooxygenase 2) and immunity biomarkers (CD4 and CD8) at the protein level. Microarray analyses revealed statistically significant alterations in gene expression in the tumor­stroma compartment, while significantly less pathway alterations occurred in the tumor tissue compartment. The host knockout of EP2 receptors led to a significant downregulation of cell cycle regulatory factors in the tumor­stroma compartment, while interferon γ­related pathways, chemokine signaling pathways and anti­tumor chemokines [chemokine (C­X­C motif) ligand 9 and 10] were upregulated in the tumor compartment. Thus, such gene alterations were likely related to reduced tumor growth in EP2­deficient hosts. On the whole, pathway analyses of both tumor­ and tumor­stroma compartments suggested that absence of host EP2 receptor signaling reduces 'remodeling' of tumor microenvironments and increase local immunity, probably by decreased productions of stimulating growth factors, perhaps similar to well­recognized physiological observations in wound healing.

Intact prostaglandin signaling through EP2 and EP4 receptors in stromal progenitor cells is required for normal development of the renal cortex in mice.

Cyclooxygenase (Cox) inhibitors are known to have severe side effects during renal development. These consist of reduced renal function, underdeveloped subcapsular glomeruli, interstitial fibrosis, and thinner cortical tissue. Global genetic deletion of Cox-2 mimics the phenotype observed after application of Cox inhibitors. This study aimed to investigate which cell types express Cox-2 and prostaglandin E2 receptors 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 FoxD1+ stromal progenitors; EP1 and EP3, on the other hand, were expressed in tubules. Cox-2 mRNA was detected in medullary interstitial cells and macula densa cells. Functional investigations were performed with a cell-specific approach to delete Cox-2, EP2, and EP4 in FoxD1+ stromal progenitor cells. Our data show that Cox-2 expression in macula densa cells is sufficient to drive renal development. Deletion of EP2 or EP4 in FoxD1+ cells had no functional effect on renal development. Codeletion of EP2 and EP4 in FoxD1+ stromal cells, however, led to severe glomerular defects and a strong decline of glomerular filtration rate (1.316 ± 69.7 µL/min/100 g body wt in controls vs. 644.1 ± 64.58 µL/min/100 g body wt in FoxD1+/Cre EP2-/- EP4ff mice), similar to global deletion of Cox-2. Furthermore, EP2/EP4-deficient mice showed a significant increase in collagen production with a strong downregulation of renal renin expression. This study shows the distinct localization of EP receptors in mice. Functionally, we could identify EP2 and EP4 receptors in stromal FoxD1+ progenitor 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 is unclear 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 stromal progenitor cells may require intact prostaglandin E2 signaling through EP2 and EP4 receptors for normal renal development.

Identification of ACOT13 and PTGER2 as novel candidate genes of autosomal dominant polycystic kidney disease through whole exome sequencing.

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.

Polymorphisms rs2745557 in PTGS2 and rs2075797 in PTGER2 are associated with the risk of chronic obstructive pulmonary disease development in a Tunisian cohort.

We hypothesized that polymorphisms of genes involved in the prostaglandin pathway could be associated with COPD. In this study we explored the involvement of genetic polymorphisms in PTGS2, PTGER2 and PTGER4 genes in the development and severity of COPD and their effects on plasma concentrations of inflammatory/oxidative stress markers. We identified genotypes of PTGS2, PTGER2 and PTGER4 SNPs in a Tunisian cohort including COPD patients (n = 138) and control subjects (n = 216) using PCR-RFLP and PCR TaqMan. Pulmonary function (FEV1 and FVC) were assessed by plethsmography. PGE2, PGD2 and cytokine plasma (IL-6, IL-18, TNF-α, TGF-ß) concentrations were measured using ELISA and colorimetric standard methods were used to determine oxidative stress concentrations. Genotype frequencies of rs2745557 in PTGS2 and rs2075797 in PTGER2 were different between COPD cases and controls. There was no correlation between these polymorphisms and lung function parameters. For rs2745557, the A allele frequency was higher in COPD cases than in controls. For rs2075797, carriers of the GG genotype were more frequent in the COPD group than in controls. Only rs2745557 in PTGS2 had an effect on PGD2 and cytokine plasma concentrations. PGD2 was significantly decreased in COPD patients with the GA or AA genotypes. In contrast, IL-18 and NO plasma concentrations were increased in COPD rs2745557 A allele carriers as compared to homozygous GG subjects. Our findings suggest that rs2745557 in PTGS2 and rs2075797 in PTGER2 are associated with COPD development but not with its severity.

Restoring metabolism of myeloid cells reverses cognitive decline in ageing.

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1-3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer's disease4-6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

Peripheral Myeloid Cell EP2 Activation Contributes to the Deleterious Consequences of Status Epilepticus.

A 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 IL-6 after pilocarpine SE was nearly abolished in EP2 conditional 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. 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.

Expression of trophoblast derived prostaglandin E2 receptor 2 (EP2) is reduced in patients with recurrent miscarriage and EP2 regulates cell proliferation and expression of inflammatory cytokines.

BACKGROUD: Prostaglandin E2 (PGE2), an inflammatory mediator, modulates cytokines, regulates immune responses in reproductive processes and stimulates inflammatory reactions via the prostaglandin E2 receptor 2 (EP2). However, the regulatory effects of EP2 signaling on trophoblasts and its role in unexplained recurrent miscarriage (uRM) remains unclear. PATIENTS AND METHODS: A total of 19 placentas from patients with a history of more than two consecutive pregnancy losses of unknown cause (uRM group) and placentas of 19 healthy patients following a legal termination of their pregnancy were used for PGE2 receptor (EP1, EP2 and EP4) expression analyses via immunohistochemistry. Double immunofluorescence was also used to identify EP2 expressing cells in the decidua. Finally, HTR-8/SVneo cells were used to clarify the role of EP2 in in vitro experiments. RESULTS: The expression of EP2 and EP4 was found to be reduced in the syncytiotrophoblast and decidua of uRM patients. A selective EP2 receptor antagonist (PF-04,418,948) reduced the proliferation and secretion of ß-hCG, inhibited interleukin -6 (IL-6) and interleukin-8 (IL-8) and up-regulated the production of the tumor necrosis factor-α (TNF-α) and plasminogen activator inhibitor type 1 (PAI-1) in HTR-8/SVneo cells in vitro. CONCLUSION: PGE2-EP2 signaling pathway may represent a novel therapy option for uRM. The involvement of EP2 in uRM acts perhaps via inflammatory cytokines and indicates that the PGE2-EP2 signaling pathway might represent an unexplored etiology for uRM.

PGE2 accounts for bidirectional changes in alveolar macrophage self-renewal with aging and smoking.

Alveolar macrophages (AMs) are resident immune cells of the lung that are critical for host defense. AMs are capable of proliferative renewal, yet their numbers are known to decrease with aging and increase with cigarette smoking. The mechanism by which AM proliferation is physiologically restrained, and whether dysregulation of this brake contributes to altered AM numbers in pathologic circumstances, however, remains unknown. Mice of advanced age exhibited diminished basal AM numbers and contained elevated PGE2 levels in their bronchoalveolar lavage fluid (BALF) as compared with young mice. Exogenous PGE2 inhibited AM proliferation in an E prostanoid receptor 2 (EP2)-cyclic AMP-dependent manner. Furthermore, EP2 knockout (EP2 KO) mice exhibited elevated basal AM numbers, and their AMs resisted the ability of PGE2 and aged BALF to inhibit proliferation. In contrast, increased numbers of AMs in mice exposed to cigarette smoking were associated with reduced PGE2 levels in BALF and were further exaggerated in EP2 KO mice. Collectively, our findings demonstrate that PGE2 functions as a tunable brake on AM numbers under physiologic and pathophysiological conditions.

Prostaglandin E2 receptor subtypes 1 and 2 play a role in TGF-ß1-induced renal fibrosis by regulating endoplasmic reticulum stress.

OBJECTIVE: This study aimed to investigate the effects of prostaglandin E2 receptor subtypes 1 (EP1) and 2 (EP2) on endoplasmic reticulum (ER) stress induced by TGF-ß1 in mouse mesangial cells (MCs) and to explore its potential mechanisms. MATERIALS AND METHODS: Mouse mesangial cells were isolated and cultured. EP-siRNAs were transfected into mesangial cells for silencing EP1 and EP2. Mesangial cell proliferation was assessed by the CCK-8 method. Expression of PGE2 was measured by enzyme-linked immunosorbent assay (ELISA). GRP78, TRPC1, ERK1/2, and phospho-ERK1/2 levels were examined by Western blot. RESULTS: TGF-ß1 induced mesangial cell proliferation and increased PGE2 secretion. Besides, TGF-ß1 significantly upregulated GRP78 and TRPC1 expression at the protein level. Phospho-ERK1/2 protein amounts were also increased (p<0.05). Compared with the TGF-ß1 group, cell proliferation in the EP1-siRNA+TGF-ß1 group was reduced, while GRP78, TRPC1, and ERK1/2 protein amounts were downregulated (p<0.05). EP1 agonist significantly enhanced above changes and their activities (p<0.05). EP1 antagonist significantly attenuated the above changes (p<0.05). Compared with TGF-ß1 group, cell proliferation in EP2-siRNA+TGF-ß1 group was increased, while GRP78, TRPC1, and ERK1/2 protein amounts were increased (p<0.05). EP2 agonist significantly attenuated the above changes (p<0.05). CONCLUSIONS: EP1 receptor may increase TGF-ß1-induced cell damage by increasing the activities of GRP78, TRPC1, and ERK1/2 via ER stress. Meanwhile, the EP2 receptor may reduce TGF-ß1-induced cell damage by suppressing GRP78, TRPC1, and ERK1/2 activities, also via ER stress. EP1 inhibition and EP2 stimulation may be a therapeutic option for delaying renal fibrosis.

Concerted EP2 and EP4 Receptor Signaling Stimulates Autocrine Prostaglandin E2 Activation in Human Podocytes.

Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman's space. Elevated Prostaglandin E2 (PGE2) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE2, 15-keto-PGE2, and 13,14-dihydro-15-keto-PGE2 levels. hPC were treated with PGE2 with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE2 led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE2. PTGER4 was downregulated after PGE2 stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE2 and 15-keto-PGE2 levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE2 pathway in hPC linked to concerted EP2 and EP4 signaling.

Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions.

Understanding the signaling pathways that regulate proliferation and differentiation of muscle progenitors is essential for successful cell transplantation for treatment of Duchenne muscular dystrophy. Here, we report that a γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine tertial butyl ester), which inhibits the release of NICD (Notch intercellular domain), promotes the fusion of human muscle progenitors in vitro and improves their engraftment in the tibialis anterior muscle of immune-deficient mice. Gene expression analysis revealed that DAPT severely down-regulates PTGER2, which encodes prostaglandin (PG) E2 receptor 2 (EP2), in human muscle progenitors in the differentiation condition. Functional analysis suggested that Notch signaling inhibits differentiation and promotes self-renewal of human muscle progenitors via PGE2/EP2 signaling in a cAMP/PKA-independent manner.

Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression.

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.

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.

Role of the PGE2 receptor subtypes EP1, EP2, and EP3 in repetitive traumatic brain injury.

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.