RESUMO
Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia-reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia-neuron interactions. We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry. OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression. OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2-EP2-cAMP-PKA pathway by modulating glia-neuron interaction.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Ciclo-Oxigenase 2 , Neuroglia , Fármacos Neuroprotetores , Células Ganglionares da Retina , Animais , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Fármacos Neuroprotetores/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , AMP Cíclico/metabolismo , Camundongos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/agonistas , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ratos Sprague-Dawley , Ratos , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Camundongos Endogâmicos C57BL , Masculino , N-Metilaspartato/farmacologia , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
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.
Assuntos
Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4 , Humanos , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Polpa Dentária/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais , Dinoprostona/farmacologia , Dinoprostona/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Células CultivadasRESUMO
It is known that prostaglandin E2 (PGE2) induces proliferation of epithelia in bovine endometrial explants, however, the detailed mechanism of regulation of PGE2 in inducing bovine endometrial epithelial cell (bEEC) proliferation is unclear. In this study, we determined whether proliferation of bEECs is promoted by PGE2-prostaglandin E receptor 2 (PTGER2) signaling activation through cell cycle regulation. The results demonstrated that bEECs proliferation was induced by treatment of PGE2 and PTGER2 agonist butaprost. These processes were down-regulated by PTGER2 antagonist AH6809 and CDK inhibitors (LEE011, CDK2 Inhibitor II and Ro 3306). PGE2 and butaprost induced cyclins (A, B1, D1, D3 and E2), cyclin-dependent kinases (CDKs, 1, 2, 4 and 6), and epidermal growth factor (EGF) expression were inhibited by AH6809 treatment in bEECs. Moreover, proliferating cell nuclear antigen (PCNA), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and PTGER2 expression in bEECs were up-regulated by PGE2 and butaprost treatment. Our data demonstrate that PGE2-PTGER2 signaling activation has a direct molecular association with cell cycle regulation and cell proliferation in bEECs. Collectively, these findings will improve our understanding of the roles for PGE2-PTGER2 signaling activation in the physiological and pharmacological processes of bovine endometrium.
Assuntos
Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dinoprostona/metabolismo , Endométrio/citologia , Células Epiteliais/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Aminopiridinas/farmacologia , Animais , Bovinos , Células Cultivadas , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/farmacologia , Feminino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Purinas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , XantonasRESUMO
Purpose: Cyclic adenosine monophosphate (cAMP) and peroxisome proliferator-activated receptor alpha (PPARα) levels mediate extracellular matrix (ECM) changes by altering the levels of hypoxia-inducible factor 1-alpha (HIF-1α) in various tissues. We aimed to determine, in the sclera of guinea pigs, whether a prostanoid receptor (EP2)-linked cAMP modulation affects PPARα and HIF-1α signaling during myopia. Methods: Three-week-old guinea pigs (n = 20 in each group), were monocularly injected with either an EP2 agonist (butaprost 1 µmol/L/10 µmol/L), an antagonist (AH6809 10 µmol/L/30 µmol/L) or a vehicle solution for two weeks during normal ocular growth. Separate sets of animals received these injections and underwent form deprivation (FD) simultaneously. Refraction and axial length (AL) were measured at two weeks, followed by scleral tissue isolation for quantitative PCR (qPCR) analysis (n = 10) and cAMP detection (n = 10) using a radioimmunoassay. Results: Butaprost induced myopia development during normal ocular growth, with proportional increases in AL and cAMP levels. FD did not augment the magnitude of myopia or cAMP elevations in these agonist-injected eyes. AH6809 suppressed cAMP increases and myopia progression during FD, but had no effect in a normal visual environment. Of the diverse set of 27 genes related to cAMP, PPARα and HIF-1α signaling and ECM remodeling, butaprost differentially regulated 15 of them during myopia development. AH6809 injections during FD negated such differential gene expressions. Conclusion: EP2 agonism increased cAMP and HIF-1α signaling subsequent to declines in PPARα and RXR mRNA levels, which in turn decreased scleral fibrosis and promoted myopia. EP2 antagonism instead inhibited each of these responses. Our data suggest that EP2 suppression may sustain scleral ECM structure and inhibit myopia development.
Assuntos
Alprostadil/análogos & derivados , Matriz Extracelular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miopia Degenerativa , PPAR alfa/metabolismo , Receptores de Prostaglandina E Subtipo EP2 , Xantonas/farmacologia , Alprostadil/farmacologia , Animais , AMP Cíclico/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Cobaias , Miopia Degenerativa/etiologia , Miopia Degenerativa/metabolismo , Miopia Degenerativa/prevenção & controle , Antagonistas de Prostaglandina/farmacologia , Prostaglandinas E Sintéticas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de SinaisRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.
Assuntos
Dinoprostona/toxicidade , Neurônios Motores/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Acetilcisteína/farmacologia , Animais , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , L-Lactato Desidrogenase/metabolismo , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP3/metabolismoRESUMO
Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 µmol/L, latanoprost free acid (LAT-A) at 0.1 µmol/L, or prostaglandin F2α (PGF2α), at 0.1 µmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 µmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.
Assuntos
Células 3T3-L1/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Glicina/análogos & derivados , Latanoprosta/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Células 3T3-L1/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Diferenciação Celular/efeitos dos fármacos , Cricetulus , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Glicina/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Camundongos , Prostaglandinas F Sintéticas/farmacologia , Radioimunoensaio/métodosRESUMO
Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3',5'-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.
Assuntos
AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Fibroblastos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo , Fibrose Pulmonar/tratamento farmacológico , Receptores de Prostaglandina E Subtipo EP2/agonistas , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Isoenzimas , Pulmão/enzimologia , Pulmão/patologia , Diester Fosfórico Hidrolases/genética , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Sistemas do Segundo Mensageiro , Regulação para CimaRESUMO
AIM: Renal 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. METHODS: We 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. RESULTS: Butaprost (50 µM) reduced TGF-ß-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-ß. The mechanism of action of butaprost appeared to be a direct effect on TGF-ß/Smad signalling, which was independent of the cAMP/PKA pathway. CONCLUSION: In 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.
Assuntos
Alprostadil/análogos & derivados , Fibrose/tratamento farmacológico , Nefropatias/metabolismo , Rim/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2/agonistas , Idoso , Alprostadil/farmacologia , Animais , Linhagem Celular , Cães , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Rim/patologia , Nefropatias/patologia , Antígeno MART-1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Tecidos , Obstrução Ureteral , Agentes Urológicos/farmacologiaRESUMO
We have reported the usefulness of chondrocyte sheets on articular cartilage repair in animal experiments. Here, we investigated the regenerative effects of EP2 signalling with or without chondrocyte sheets. Forty-five rabbits were used, with six rabbits in each of the six groups and nine rabbits for chondrocytes and synovial cells harvesting to fabricate triple-layered chondrocyte sheets: osteochondral defect only (control, Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheets (Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist and chondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4 weeks after surgery. The rabbits were sacrificed at 12 weeks, and the repaired tissues were evaluated for histology. The weight distribution ratio and International Cartilage Repair Society grading were as follows: Group A: 40.5% ± 0.2%, 14.8 ± 0.5; Group B: 43.4% ± 0.7%, 25.4 ± 0.8; Group C: 38.7% ± 0.7%, 13.7 ± 0.3; Group D: 48.6% ± 0.6%, 40.2 ± 0.5; Group E: 49.1% ± 0.3%, 40.5 ± 0.4; and Group F; 46.8% ± 0.4%, 38.7 ± 0.5. Significant differences in histology and pain alleviation were observed between groups except between Groups A and C, between Groups D and E, and between Groups D and F. These findings show that the intra-articular administration of an EP2 agonist achieved pain alleviation and tissue repair. However, no synergistic effect with chondrocyte sheets was observed.
Assuntos
Cartilagem Articular/lesões , Condrócitos/transplante , Traumatismos do Joelho/terapia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Transdução de Sinais , Aloenxertos , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Traumatismos do Joelho/metabolismo , Traumatismos do Joelho/patologia , Modelos Animais , Piridazinas/farmacologia , Coelhos , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Prostaglandin E2 (PGE2) is responsible for inflammatory symptoms. However, PGE2 also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE2 receptors, EP1-EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R-EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells. Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE2, alongside selective agonists of EP1-EP4 receptors, were assessed on LPS-induced TNF-α, IL-1ß and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs. PGE2 and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE2 on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE2. This indicates that PGE2 suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.
Assuntos
Dinoprostona/efeitos adversos , Monócitos/citologia , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP4/genética , Fator de Necrose Tumoral alfa/metabolismo , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/efeitos adversos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Células THP-1RESUMO
Prostaglandin (PG)E2 is an arachidonic acid-derived lipid mediator that plays an important role in inflammation and immunity. In this study, we demonstrate that PGE2 suppresses basal and 1,25-dihydroxy vitamin D3 (VD3)-induced expression of hCAP18/LL-37 via E prostanoid (EP)2 and EP4 receptors. In humans, VD3 up-regulates vitamin D receptor (VDR) expression and promotes transcription of the cathelicidin hCAP18/LL-37 gene, whereas PGE2 counteracts this effect. We find that PGE2 induces the cAMP/PKA-signaling pathway and enhances the expression of the inhibitory transcription factor cAMP-responsive modulator/inducible cAMP early repressor, which prevents VDR expression and induction of hCAP18/LL-37 in human macrophages. The negative regulation by PGE2 was evident in M1- and M2-polarized human macrophages, although PGE2 displayed more profound inhibitory effects in M2 cells. PGE2 impaired VD3-induced expression of cathelicidin and concomitant activation of autophagy during Mycobacterium tuberculosis (Mtb) infection and facilitated intracellular Mtb growth in human macrophages. An EP4 agonist also significantly promoted Mtb survival in human macrophages. Our results indicate that PGE2 inhibits hCAP18/LL-37 expression, especially VD3-induced cathelicidin and autophagy, which may reduce host defense against Mtb. Accordingly, antagonists of EP4 may constitute a novel adjunctive therapy in Mtb infection.-Wan, M., Tang, X., Rekha, R. S., Muvva, S. S. V. J. R., Brighenti, S., Agerberth, B., Haeggström, J. Z. Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Dinoprostona/farmacologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Tuberculose/metabolismo , Autofagia/efeitos dos fármacos , Calcitriol/farmacologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Receptores de Calcitriol/biossíntese , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tuberculose/patologia , Tuberculose/terapia , CatelicidinasRESUMO
Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2α (PGF2α) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2α (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 µM H-89 (a PKA inhibitor) and 3 µM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 µM chelerythrine chloride (a PKC inhibitor) and 3 µM U0126 negated OVGP1 downregulation by PGF2α. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2α through the PTGFR-Ca2+-PKC pathway.
Assuntos
Dinoprosta/metabolismo , Dinoprostona/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Oviductos/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina/agonistas , Matadouros , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Bovinos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/agonistas , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Oviductos/citologia , Oviductos/efeitos dos fármacos , Prostaglandinas F Sintéticas/farmacologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismoRESUMO
Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.
Assuntos
Dinoprostona/farmacologia , Dinoprostona/fisiologia , Neurônios Motores/citologia , Neuritos/fisiologia , Crescimento Neuronal/efeitos dos fármacos , Crescimento Neuronal/genética , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , AMP Cíclico/fisiologia , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Camundongos , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP3/agonistas , Transdução de Sinais/fisiologiaRESUMO
The domestic animal endometrium undergoes regular periods of regeneration and degeneration during cycles of oestrus and dioestrus. If blastocyst implantation occurs in the uterus, the endometrium will prepare for pregnancy by changing its pattern of development to build a connection with the embryo to nourish it. Prostaglandin E2 (PGE2) secretion synchronized with endometrial growth in these processes and could be involved in endometrial growth. One of the PGE2 receptors (PTGER2) is present in endometrium and its increased expression accompanies with endometrial growth in above processes. However, the association between PTGER2 and endometrial growth remains unclear. Endometrial growth factors and cell proliferation is the foundation for endometrial growth. Therefore, in this study, the response of growth factors and cell proliferation essential for endometrial growth to PTGER2 activation were investigated in bovine endometrium. The results indicated that mRNA and protein expression of connective tissue growth factor (CTGF), fibroblast growth factor-2 (FGF-2), interleukin-8 (IL-8), transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor A (VEGFA) were up-regulated after PTGER2 activation by corresponding agonist butaprost (P < 0.05), and proliferation of endometrial epithelia and fibroblasts were induced in response to increased levels of proliferating cell nuclear antigen (PCNA), cytokeratin-18 (CK-18) and fibroblast-specific protein 1 (FSP-1) expression in bovine endometrial explants in vitro (P < 0.05).
Assuntos
Alprostadil/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Endométrio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Receptores de Prostaglandina E Subtipo EP2/agonistas , Alprostadil/farmacologia , Animais , Bovinos , Endométrio/citologia , FemininoRESUMO
Chronic obstructive pulmonary disease (COPD) is often associated with co-morbidities. Metabolic disorders like hyperlipidemia and diabetes occur also in underweight COPD patients, although the mechanism is uncertain. Subcutaneous adipose tissue (SAT) plays an important role in energy homeostasis, since restricted capacity to increase fat cell number with increase in fat cell size occurring instead, is associated with lipotoxicity and metabolic disorders. The aim of this study is to show the protective role of SAT for the metabolic disorders in pulmonary emphysema of a murine model. We found ectopic fat accumulation and impaired glucose homeostasis with wasting of SAT in a murine model of elastase-induced pulmonary emphysema (EIE mice) reared on a high-fat diet. ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, improved angiogenesis and subsequently adipogenesis, and finally improved ectopic fat accumulation and glucose homeostasis with restoration of the capacity for storage of surplus energy in SAT. These results suggest that metabolic disorders like hyperlipidemia and diabetes occured in underweight COPD is partially due to the less capacity for storage of surplus energy in SAT, though the precise mechanism is uncertained. Our data pave the way for the development of therapeutic interventions for metabolic disorders in emphysema patients, e.g., use of pro-angiogenic agents targeting the capacity for storage of surplus energy in the subcutaneous adipose tissue.
Assuntos
Dinoprostona/análogos & derivados , Doenças Metabólicas/complicações , Doenças Metabólicas/tratamento farmacológico , Enfisema Pulmonar/complicações , Receptores de Prostaglandina E Subtipo EP2/agonistas , Gordura Subcutânea/efeitos dos fármacos , Animais , Dieta Hiperlipídica/efeitos adversos , Dinoprostona/farmacologia , Dinoprostona/uso terapêutico , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Masculino , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Gordura Subcutânea/patologiaRESUMO
BACKGROUND: Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS. METHODS: Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS. RESULTS: PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect. CONCLUSIONS: These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Animais Recém-Nascidos , Córtex Cerebral/citologia , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/fisiologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Prostaglandin E2 is a potent endogenous molecule that binds to four different G-protein-coupled receptors: EP1-4. Each of these receptors is a valuable drug target, with distinct tissue localisation and signalling pathways. We review the structural features of EP modulators required for subtype-selective activity, as well as the structural requirements for improved pharmacokinetic parameters. Novel EP receptor subtype selective agonists and antagonists appear to be valuable drug candidates in the therapy of many pathophysiological states, including ulcerative colitis, glaucoma, bone healing, B cell lymphoma, neurological diseases, among others, which have been studied in vitro, in vivo and in early phase clinical trials.
Assuntos
Preparações Farmacêuticas/química , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4 , Animais , Dinoprostona/química , Desenho de Fármacos , Humanos , Ligantes , Estrutura Molecular , Terapia de Alvo Molecular , Ligação Proteica , Receptores de Prostaglandina E Subtipo EP1/agonistas , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP3/agonistas , Receptores de Prostaglandina E Subtipo EP3/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidoresRESUMO
Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling in AQP2 membrane targeting in Madin-Darby canine kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost), and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of the EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively nonphosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs-coupled receptors can also signal through an alternative pathway that increases AQP2 membrane targeting.
Assuntos
Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renais Coletores/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais/fisiologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Desamino Arginina Vasopressina/farmacologia , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Cães , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Pirrolidinonas/farmacologia , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Vasopressinas/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
Prostaglandin E2 (PGE2), a major metabolite of arachidonic acid produced by cyclooxygenase pathways, exerts its bioactive responses by activating four E-prostanoid receptor subtypes, EP1, EP2, EP3, and EP4. PGE2 enables modulating N-methyl-D-aspartate (NMDA) receptor-mediated responses. However, the effect of E-prostanoid receptor agonists on large-conductance Ca(2+)-activated K(+) (BK) channels, which are functionally coupled with NMDA receptors, remains unclear. Here, we showed that EP2 receptor-mediated signaling pathways increased NMDA-induced outward currents (I NMDA-OUT), which are associated with the BK channel activation. Patch-clamp recordings from the acutely dissociated mouse cortical neurons revealed that an EP2 receptor agonist activated I NMDA-OUT, whereas an EP3 receptor agonist reduced it. Agonists of EP1 or EP4 receptors showed no significant effects on I NMDA-OUT. A direct perfusion of 3,5'-cyclic adenosine monophosphate (cAMP) through the patch pipette facilitated I NMDA-OUT, which was abolished by the presence of protein kinase A (PKA) inhibitor. Furthermore, facilitation of I NMDA-OUT caused by an EP2 receptor agonist was significantly suppressed by PKA inhibitor. Finally, the activation of BK channels through EP2 receptors facilitated the recovery phase of NMDA-induced dendritic beading in the primary cultured cortical neurons. These results suggest that a direct activation of BK channels by EP2 receptor-mediated signaling pathways plays neuroprotective roles in cortical neurons.
Assuntos
Dinoprostona/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Carbazóis/farmacologia , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Éteres Metílicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Pirróis/farmacologia , Receptores de Prostaglandina E Subtipo EP1/agonistas , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP3/agonistas , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/metabolismoRESUMO
The modification of the novel G protein-biased EP2 agonist 1 has been investigated to improve its G protein activity and develop a better understanding of its structure-functional selectivity relationship (SFSR). The optimization of the substituents on the phenyl ring of 1, followed by the inversion of the hydroxyl group on the cyclopentane moiety led to compound 9, which showed a 100-fold increase in its G protein activity compared with 1 without any increase in ß-arrestin recruitment. Furthermore, SFSR studies revealed that the combination of meta and para substituents on the phenyl moiety was crucial to the functional selectivity.