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Regional distribution of the prostaglandin E2 receptor EP1 in the rat brain: accumulation in Purkinje cells of the cerebellum  [PDF]
E. Candelario-Jalil,H. Slawik,I. Ridelis,A. Waschbisch,R. S. Akundi,M. Hull,B. L. Fiebich
Quantitative Biology , 2007,
Abstract: Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3, and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem, and cerebellum) using reverse transcription- polymerase chain reaction, Western blotting, and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found very strong expression of EP1 in cerebellum, as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed very high immunolabeling of their perikaryon and dendrites, as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region-specific response to PGE2.
Microglial inhibition of neuroprotection by antagonists of the EP1 prostaglandin E2 receptor
Noel G Carlson, Monica A Rojas, John-David Black, Jonathan W Redd, John Hille, Kenneth E Hill, John W Rose
Journal of Neuroinflammation , 2009, DOI: 10.1186/1742-2094-6-5
Abstract: Primary neuronal cultures systems with or without non-neuronal cells were used to examine how the neuroprotective properties of EP1 antagonists were influenced by non-neuronal cells. The influence of astrocytes or microglia were individually tested in excitotoxicity assays using a co-culture system with these cells grown on permeable transwell inserts above the neuronal-enriched cultures. The influence of microglia on PGE2 synthesis and EP1 receptor expression was examined.EP1 antagonists were neuroprotective in neuronal-enriched cultures (> 90% neurons) but not in mixed cultures (30% neurons plus other non-neuronal cells). Co-cultures of microglia on permeable transwell inserts above neuronal-enriched cultures blocked neuroprotection by EP1 antagonists. Incubation of microglia with neuronal-enriched cultures for 48 hours prior to NMDA challenge was sufficient to block neuroprotection by EP1 antagonists. The loss of neuroprotection by EP1 antagonists was accompanied by a decrease of neuronal EP1 expression in the nucleus in cultures with microglia present.These findings demonstrate microglial modulation of neuronal excitotoxicity through interaction with the EP1 receptor and may have important implications in vivo where microglia are associated with neuronal injury.Cyclooxygenase-2 (COX-2), the enzyme that catalyzes the rate limiting step in the synthesis of prostanoids, contributes to neuronal death. Inhibitors of COX, termed non-steroidal anti-inflammatory drugs (NSAIDs) [1], can protect neurons following an assault with toxic stimuli that promote excitotoxic death; both in vitro [2,3] and in vivo [4-7]. COX-2 knockout mice are also less susceptible to excitotoxicity following exposure to the glutamate receptor agonist N-methyl D-aspartate (NMDA) [8]. Therefore, a loss of COX-2 activity either by inhibition of the enzyme or loss of expression is associated with increased neuronal viability. Conversely, increased COX-2 activity appears to augment neuronal death. Th
Cyclooxygenase-2 enhances α2β1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells
Ju-Fang Liu, Yi-Chin Fong, Chih-Shiang Chang, Chun-Yin Huang, Hsien-Te Chen, Wei-Hung Yang, Chin-Jung Hsu, Long-Bin Jeng, Chih-Yi Chen, Chih-Hsin Tang
Molecular Cancer , 2010, DOI: 10.1186/1476-4598-9-43
Abstract: We found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and α2β1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCβ, PKCα, c-Src and NF-κB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-κB cascades.Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the EP1/PLC/PKCα/c-Src/NF-κB signal transduction pathway.Chondrosarcoma is the second most common malignancy of bone and it has a poor response to chemotherapy or radiation treatment currently-used, making the management of chondrosarcomas a complicated challenge [1]. Clinically, surgical resection remains the primary mode of therapy for chondrosarcoma. In the absence of an effective adjuvant therapy, this mesenchymal malignancy has a poor prognosis and therefore, it is important to explore novel and adequate remedies [2]. Since chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and metastasize distantly [2], an approach that decreases its ability to invade and metastasize may facilitate the development of effective adjuvant therapy.Cyclooxygenases (COXs) are the rate-limiting enzymes that ca
TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells, Which Is Involved in Fibrotic Response to TGF-β1  [PDF]
Erina Takai, Mitsutoshi Tsukimoto, Shuji Kojima
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076346
Abstract: Transforming growth factor-?1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.
EP1 receptor within the ventrolateral periaqueductal grey controls thermonociception and rostral ventromedial medulla cell activity in healthy and neuropathic rat
Enza Palazzo, Francesca Guida, Luisa Gatta, Livio Luongo, Serena Boccella, Giulia Bellini, Ida Marabese, Vito de Novellis, Francesca Rossi, Sabatino Maione
Molecular Pain , 2011, DOI: 10.1186/1744-8069-7-82
Abstract: It has been well established that prostaglandin E2 (PGE2) sensitizes peripheral nociceptors through the activation of prostaglandin EP receptors present on the peripheral terminals of sensory neurons, leading to a reduction in pain threshold and increased responsiveness [1]. As well as a peripheral role, spinal prostaglandins (PGs) contribute to dorsal horn sensitization in persistent pain states in the spinal cord [2,3]. Nevertheless, little attention has been given to PG action at supraspinal level and in particular within the antinociceptive descending pathway, consisting of periaqueductal grey (PAG), rostral ventromedial medulla (RVM) and spinal dorsal horn components. PAG-induced control of nociception is produced concomitantly with the modulation of neuron activity within the RVM: ON-cells, which are activated and OFF-cells, which are inhibited by cutaneous nociceptive stimuli [4]. Unlike ON and OFF cells, another class of neurons; the neutral cells, are instead unaffected by noxious stimuli.Both isoforms of cyclooxygenases (COXs), COX-1 and COX-2, PGE2 and the prostaglandin EP3 receptor have been identified within the PAG [5-7]. Intra-PAG microinjection of a COX1-2 inhibitor, lysine-acetylsalcylate, reduced nociceptive processing [8,9]. The involvement of PAG PGs in tonic facilitatory control on spinal nociception [10] and that of PGE2 in the genesis of hyperalgesia and spontaneous pain at spinal dorsal horn level [11] has already been recognized. On this subject, EP receptor subtype antagonist may potentially behave like an analgesic at this level. Indeed, in one of our previous studies, we demonstrated that intra-PAG microinjections of EP1-4 receptor subtype antagonists prevented formalin and misoprostol-induced hyperalgesia in mice, demonstrating a key role of PGs in a facilitating nociceptive response throughout EP receptors at PAG level [12]. As far as chronic pain is concerned, insights into the role of EP1 receptor are still scant, although it has been
Prostanoid receptor EP1 and Cox-2 in injured human nerves and a rat model of nerve injury: a time-course study
Pascal F Durrenberger, Paul Facer, Maria A Casula, Yiangos Yiangou, Roy A Gray, Iain P Chessell, Nicola C Day, Sue D Collins, Sharon Bingham, Alex W Wilson, David Elliot, Rolfe Birch, Praveen Anand
BMC Neurology , 2006, DOI: 10.1186/1471-2377-6-1
Abstract: Tissue sections were immunostained with specific antibodies to EP1, Cox-2, CD68 (human macrophage marker) or OX42 (rat microglial marker), and neurofilaments (NF), prior to image analysis, from the following: human brachial plexus nerves (21 to 196 days post-injury), painful neuromas (9 days to 12 years post-injury), avulsion injured DRG, control nerves and DRG, and rat CCI model tissues. EP1 and NF-immunoreactive nerve fibres were quantified by image analysis.EP1:NF ratio was significantly increased in human brachial plexus nerve fibres, both proximal and distal to injury, in comparison with uninjured nerves. Sensory neurones in injured human DRG showed a significant acute increase of EP1-IR intensity. While there was a rapid increase in EP1-fibres and CD-68 positive macrophages, Cox-2 increase was apparent later, but was persistent in human painful neuromas for years. A similar time-course of changes was found in the rat CCI model with the above markers, both in the injured nerves and ipsilateral dorsal spinal cord.Different stages of infiltration and activation of macrophages may be observed in the peripheral and central nervous system following peripheral nerve injury. EP1 receptor level increase in sensory neurones, and macrophage infiltration, appears to precede increased Cox-2 expression by macrophages. However, other methods for detecting Cox-2 levels and activity are required. EP1 antagonists may show therapeutic effects in acute and chronic neuropathic pain, in addition to inflammatory pain.Tissue damage induces an inflammatory response including the production of prostaglandins (PGs) such as PGE2, which activate the EP1 receptor expressed by sensory fibres. PGs produced in the spinal cord may also play an important role in the development of hypersensitivity following peripheral nerve injury [1]; PGs generated by Cox-2 in the spinal cord have been shown to contribute to the maintenance of hyperalgesia [2].The enzymes involved in the production of PGs are
PPAR Agonist Rosiglitazone Suppresses Renal mPGES-1/PGE2 Pathway in db/db Mice  [PDF]
Ying Sun,Zhanjun Jia,Gang Liu,Li Zhou,Mi Liu,Baoxue Yang,Tianxin Yang
PPAR Research , 2013, DOI: 10.1155/2013/612971
Abstract: Evidence had shown the detrimental effect of prostaglandin (PG) E2 in diabetic nephropathy (DN) of STZ-induced type-1 diabetes but its role in the development of DN of type-2 diabetes remains uncertain. The present study was undertaken to investigate the regulation of PGE2 synthetic pathway and the interaction between peroxisome proliferator-activated receptor (PPAR)γ and PGE2 synthesis in the kidneys of db/db mice. Strikingly, urinary PGE2 was remarkably elevated in db/db mice paralleled with the increased protein expressions of COX-2 and mPGES-1. In contrast, the protein expressions of COX-1, mPGES-2, cPGES, and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) were not altered. Following 1-week rosiglitazone (Rosi) therapy, urinary PGE2, but not other prostanoids, was reduced by 57% in parallel with significant reduction of mPGES-1 protein and EP4 mRNA expressions. By immunohistochemistry, mPGES-1 was significantly induced in the glomeruli of db/db mice, which was almost entirely abolished by Rosi. In line with the reduction of glomerular mPGES-1, the glomerular injury score showed a tendency of improvement after 1 week of Rosi therapy. Collectively, the present study demonstrated an inhibitory effect of PPARγ activation on renal mPGES-1/PGE2/EP4 pathway in type-2 diabetes and suggested that mPGES-1 may potentially serve as a therapeutic target for treating type-2 diabetes-associated DN. 1. Introduction The abnormality of renal prostaglandins in diabetic kidney was thought to be an important factor in mediating the glomerular injury, tubular interstitial fibrosis, and fluid imbalance [1–4]. Among five prostanoids of PGE2, PGD2, PGI2, PGF2α, and thromboxane (TX) A2, PGI2 and TXA2 are important players in regulation of renal hemodynamics [5–7]; PGE2 serves as an important regulator of glomerular integrity, SS hemodynamics, and tubular fluid reabsorption [8–10], while the PGD2 and PGF2α in kidney are less studied and their functions are poorly understood. The renal PGE2 receptor (EP1-EP4) expression profile is altered in type-1 diabetic mice [11]. Makino et al. demonstrated that EP1-selective antagonist prevented the progression of nephropathy in streptozotocin (STZ) diabetic rats [12]. Most recently, Mohamed et al. reported that EP4 agonist exacerbated kidney injury in STZ-induced type-1 diabetes in mice [13]. These reports strongly suggested a detrimental role of PGE2 in kidney of STZ-induced type-1 diabetes via EP1 and/or EP4 receptors. However, no prior studies examine the role of PGE synthase in the kidney injury of type-2 diabetes. In clinic, type-2
Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells
Tveteraas Ingun,Müller Kristin,Aasrum Monica,?deg?rd John
Journal of Experimental & Clinical Cancer Research , 2012, DOI: 10.1186/1756-9966-31-72
Abstract: Background It is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells. Methods The rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay. Results In the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family. Conclusion The results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).
Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins
Tomoko Moriyama, Tomohiro Higashi, Kazuya Togashi, Tohko Iida, Eri Segi, Yukihiko Sugimoto, Tomoko Tominaga, Shuh Narumiya, Makoto Tominaga
Molecular Pain , 2005, DOI: 10.1186/1744-8069-1-3
Abstract: Tissue damage and inflammation produce an array of chemical mediators such as ATP, bradykinin, prostanoids, protons, cytokines and peptides including substance P that can excite or sensitize nociceptors to elicit pain at the site of injury. Among them prostanoids were shown to influence inflammation, and their administration was found to reproduce the major signs of inflammation including augmented pain [1]. Prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are the products of arachidonic acid metabolism through the cyclooxygenase pathway. In addition to numerous other physiological actions in vivo, previous studies have indicated important roles for PGE2 in nociception and inflammation [2,3]. PGE2 is generated in most cells in response to mechanical, thermal or chemical injury and inflammatory insult, resulting in sensitization or direct activation of nearby sensory nerve endings. Analgesic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are attributed predominantly to inhibition of prostaglandin synthesis. Prostaglandins act upon a family of pharmacologically distinct prostanoid receptors including EP1, EP2, EP3, EP4 and IP that activate several different G protein-coupled signaling pathways [2,4,5]. Primary sensory neurons in dorsal root ganglion (DRG) are known to express mRNAs encoding several prostanoid receptor subtypes, IP, EP1, EP3 and EP4 [6,7]. The role of IP in inflammation has been clearly shown by the analysis of IP-deficient mice, although the underlying cellular mechanisms still remain to be elucidated [8]. In contrast, the potential involvement of EP receptors other than IP in inflammation and pain generation has not been well studied, although some earlier studies have suggested that prostanoids contribute to the development of pain through EP receptors [9,10].The capsaicin receptor TRPV1 is a non-selective cation channel expressed predominantly in unmyelinated C-fibers [11]. TRPV1 is activated not only by capsaicin, but also by protons
An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening
Annirudha J Chillar, Parastoo Karimi, Kathy Tang, Ke-He Ruan
BMC Complementary and Alternative Medicine , 2011, DOI: 10.1186/1472-6882-11-11
Abstract: We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney). PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened.After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy.Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE1 and PGE2.The subtype receptors of prostaglandin E (PGE) isomer-1 (PGE1) and isomer-2 (PGE2) (termed EP1, EP2, EP3 and EP4) are widely distributed and have been extensively studied for their involvement in a variety of cancers and stem cell differentiation. Expression of EP1 is frequently seen in human breast cancers and colon tumor cells. Nuclear expression of EP1 in human breast cancers correlates w
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