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1.
Kaneki H Takasugi I Fujieda M Kiriu M Mizuochi S Ide H 《Journal of cellular biochemistry》1999,73(1):36-48
The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period. 相似文献
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Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy
PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats. 相似文献
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The purpose of this study was to investigate the effect of corticotropin-releasing hormone (CRH) on the expression of the prostaglandin (PG) E(2) EP1 receptor subtype and PGE(2) production in amnion WISH cells (AWC). AWC cultures were incubated with CRH. Culture fluid was collected for PGE(2) measurement, and the cells were collected and analyzed for EP1 protein and mRNA. Immunohistochemical localization of the EP1 receptor was also performed. Incubation of AWC with CRH resulted in a dose-dependent increase (r = 0.97) in the level of EP1 receptor protein (P < 0.001). Coincubation of AWC with CRH and indomethacin resulted in the decreased production of PGE(2) while having no effect on EP1 receptor expression. A significant but not dose-dependent increase in EP1 mRNA expression was also observed (P < 0.01). Immunohistochemical evaluation verified cell membrane localization of the receptor in both stimulated and unstimulated cells and confirmed the increased expression of EP1 receptor in response to CRH. Incubation of AWC with CRH also resulted in increased culture fluid PGE(2) levels (P < 0.01). These results suggest that the role CRH plays in the initiation of labor may also involve the promotion of elevated PGE(2) levels and increased expression of the EP1 receptor in amnion. 相似文献
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Prostaglandin E2 (PGE2) is an important mediator of diverse biologic functions in many tissues and binds with high affinity to four cell surface, seven-transmembrane domain, G protein-coupled receptors (EP1-EP4). The EP4 receptor subtype has a long intracellular carboxy-terminal region and is functionally coupled to adenylate cyclase, resulting in elevated intracellular cyclic adenosine 5' monophosphate (cAMP) levels upon activation. To further study EP4 receptor subtype function, a canine kidney cDNA library was screened and three clones were isolated and sequenced. The longest clone was 3,103 bp and contained a single open reading frame of 1,476 bp, potentially encoding a protein of 492 amino acids with a predicted molecular weight of 53.4 kDa. Sequence analysis of this open reading frame reveals 89% identity to the human EP4 protein coding region at the nucleotide level and 90% identity when the putative canine and human protein sequences are compared. Northern blot analysis showed relatively high levels of canine EP4 expression in heart, lung and kidney, while Southern blot analysis of canine genomic DNA suggests the presence of a single copy gene. Following transfection of canine EP4 into CHO-KI cells, Scatchard analysis revealed a dissociation constant of 24 nM for PGE, while competition binding studies using 3H-PGE2 as ligand demonstrated specific displacement by PGE2 prostaglandin E, (PGE1), and prostaglandin A3 (PGA3). Treatment with PGE2 also resulted in increased levels of cAMP in transfected, but not in parental, CHO-KI cells. In contrast, butaprost, an EP2 selective ligand, and sulprostone, an EP1/EP3 selective ligand, did not bind to this receptor at the maximal concentration used (320 nM). To further investigate secondary signaling, the canine EP4 cDNA was truncated to produce an 1,117 bp fragment encoding a 356 amino acid protein lacking the intracellular carboxy-terminus. When transfected, this truncated cDNA produced a protein with a dissociation constant of 11 nM for PGE2 and a binding and cAMP accumulation profile similar to that of the full-length protein. Both full-length and truncated canine EP4 underwent short term PGE2-induced desensitization as shown by a lack of continuing cAMP accumulation after the initial PGE2 stimulation, suggesting no involvement of the C-terminal intracellular tail. This result is in contrast to that reported for the human EP4 receptor, where residues within the C-terminal intracellular tail were shown to mediate short term, ligand induced desensitization. 相似文献
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Suppression of prostaglandin E2 receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth 总被引:3,自引:0,他引:3
Prostaglandin E(2) (PGE(2)), a major cyclooxygenase (COX-2) metabolite, plays important roles in tumor biology and its functions are mediated through one or more of its receptors EP1, EP2, EP3, and EP4. We have shown that the matrix glycoprotein fibronectin stimulates lung carcinoma cell proliferation via induction of COX-2 expression with subsequent PGE(2) protein biosynthesis. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibited this effect and induced cellular apoptosis. Here, we explore the role of the PGE(2) receptor EP2 in this process and whether the inhibition observed with PPARgamma ligands is related to effects on this receptor. We found that human non-small cell lung carcinoma cell lines (H1838 and H2106) express EP2 receptors, and that the inhibition of cell growth by PPARgamma ligands (GW1929, PGJ2, ciglitazone, troglitazone, and rosiglitazone [also known as BRL49653]) was associated with a significant decrease in EP2 mRNA and protein levels. The inhibitory effects of BRL49653 and ciglitazone, but not PGJ2, were reversed by a specific PPARgamma antagonist GW9662, suggesting the involvement of PPARgamma-dependent and -independent mechanisms. PPARgamma ligand treatment was associated with phosphorylation of extracellular regulated kinase (Erk), and inhibition of EP2 receptor expression by PPARgamma ligands was prevented by PD98095, an inhibitor of the MEK-1/Erk pathway. Butaprost, an EP2 agonist, like exogenous PGE(2) (dmPGE(2)), increased lung carcinoma cell growth, however, GW1929 and troglitazone blocked their effects. Our studies reveal a novel role for EP2 in mediating the proliferative effects of PGE(2) on lung carcinoma cells. PPARgamma ligands inhibit human lung carcinoma cell growth by decreasing the expression of EP2 receptors through Erk signaling and PPARgamma-dependent and -independent pathways. 相似文献
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Kimberly O. Cameron Bruce A. Lefker Hua Z. Ke Mei Li Michael P. Zawistoski Christina M. Tjoa Ann S. Wright Shari L. DeNinno Vishwas M. Paralkar Thomas A. Owen Li Yu David D. Thompson 《Bioorganic & medicinal chemistry letters》2009,19(7):2075-2078
Sulfonamides, exemplified by 3a, were identified as highly selective EP2 agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP2 agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing. 相似文献
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Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice 总被引:19,自引:0,他引:19
Sonoshita M Takaku K Sasaki N Sugimoto Y Ushikubi F Narumiya S Oshima M Taketo MM 《Nature medicine》2001,7(9):1048-1051
Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma. 相似文献
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Lyons C Beharry K Akmal Y Attenello F Nageotte MP 《Prostaglandins & other lipid mediators》2003,70(3-4):317-329
We examined and compared the in vitro effects of misoprostol (synthetic prostaglandin E1 (PGE1) analogue) on prostaglandin E2 (PGE2) secretion and EP3 receptor mRNA expression in the pregnant rat myometrium and cervix at 19 days gestation. Myometrial and cervical tissue samples were exposed to media with or without misoprostol (50 or 100 pg/ml) and incubated for 15 and 30 min, and 1, 3, 6, 12, and 24 h. Media and tissue samples were collected for quantification of PGE2 and mRNA expression of rEP3alpha and rEP3beta receptor, respectively. PGE2 secretion increased (P < or = 0.05) in the myometrium exposed to 50 and 100 pg/ml misoprostol. Cervical PGE2 secretion increased following exposure to the 100 pg/ml dose only. In the myometrium, 50 and 100 pg/ml misoprostol induced elevations in rEP3alpha and rEP3beta receptor mRNA expression. rEP3alpha and rEP3beta receptor mRNA expression in the cervix was not different from controls. These data demonstrate that the EP3 receptor is differentially expressed in the myometrium and cervix in response to misoprostol. This may account for the ability of misoprostol to stimulate the myometrium when administered for cervical ripening. 相似文献
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Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells. 相似文献
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Li CC Shen HL Lii CK Liu KL Yang JJ Chen HW 《Biochemical and biophysical research communications》2005,327(2):424-430
Cytochrome P-450 is an important bioactivation-detoxification system in vivo. Its expression is regulated by foreign chemicals and dietary factors, and lipids have been found to regulate its gene expression. We showed previously that prostaglandin E(2) (PGE(2)), a fatty acid metabolite, down-regulates cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital. The objective of the present study was to determine whether PGE(2) type 2 receptor (EP(2))-which is coupled to Gs-protein when bound by PGE(2), leading to cAMP production-is involved in this down-regulation. We also determined the possible roles of EP(2) downstream pathways in this down-regulation. We used a primary rat hepatocyte culture model in which EP(2) was shown to be present to study this question. The intracellular cAMP concentration in primary rat hepatocytes was significantly higher after treatment with 1microM PGE(2) than after treatment with 0, 0.01, or 0.1microM PGE(2). Butaprost, an EP(2) agonist, down-regulated CYP 2B1 expression in a dose-dependent manner. SQ22536, an adenylate cyclase inhibitor, reversed the down-regulation by PGE(2) as did H-89, a protein kinase A inhibitor. These results suggest that EP(2) and the downstream pathways of cAMP and protein kinase A are involved in the down-regulation of CYP 2B1 expression by PGE(2) in the presence of phenobarbital. 相似文献
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Tabata H Tanaka S Sugimoto Y Kanki H Kaneko S Ichikawa A 《Biochemical and biophysical research communications》2002,298(3):398-402
We previously reported that the prostaglandin E(2) (PGE(2)) receptor subtype EP(1) is coupled to intracellular Ca(2+) mobilization in CHO cells, which is dependent on extracellular Ca(2+) in a pertussis toxin-insensitive manner [H. Katoh, et al., Biochim. Biophys. Acta 1244 (1995) 41-48]. However, it remains unknown about the signal transduction involved in this response. To investigate the mechanism regulating Ca(2+) mobilization mediated by EP(1) receptors in detail, we performed a series of experiments using the Xenopus laevis oocyte expression system and found that endogenous G(q) and/or G(11), and not G(i1) is involved in the Ca(2+) mobilization induced by PGE(2). We further investigated the receptor-activated Ca(2+) channel (RACC)-related response by introducing mRNA for mouse transient receptor potential 5 (TRP5), a possible candidate for the RACC, and found effective coupling between them. These results suggest that the EP(1) receptors induce Ca(2+) mobilization via G(q) and/or G(11) and Ca(2+) influx via TRP. 相似文献
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This study was conducted to investigate the mechanism of interleukin-1beta (IL-1beta)-induced IL-6 production in human osteoblasts (MG-63 cells). Stimulation with IL-1beta resulted in the production of IL-6 and prostaglandin E(2) (PGE(2)). IL-6 production gradually increased and peaked 96 h after stimulation. IL-6 mRNA was detected between 4 and 72 h after IL-1beta stimulation. The patterns of PGE(2) production and the expression of cyclooxygenase-2 (COX-2) mRNA were biphasic after stimulation. Actinomycin D, cycloheximide, indomethacin, and NS-398 (COX-2 inhibitor) suppressed the production of IL-6 and PGE(2). Anti-PGE(2) antibody markedly reduced the production of IL-6. In addition, stimulation with 17-phenyl-PGE(2), a PGE receptor-1 (EP-1 receptor) agonist, led to the expression of IL-6 mRNA after pretreatment with IL-1beta. These findings indicate that IL-1beta-induced IL-6 production in MG-63 cells involves the following sequence of steps: IL-1beta-induced COX-2 activation, PGE(2) production, and EP-1 receptor signaling prior to IL-6 production. 相似文献
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The prostaglandin E(2) (PGE(2)) G protein-coupled receptor (GPCR), EP2, plays important roles in mouse skin tumor development (Chun, K. S., Lao, H. C., Trempus, C. S., Okada, M., and Langenbach, R. (2009) Carcinogenesis 30, 1620-1627). Because keratinocyte proliferation is essential for skin tumor development, EP2-mediated signaling pathways that contribute to keratinocyte proliferation were investigated. A single topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication via activation of epidermal growth factor receptor (EGFR) and PKA signaling. Because GPCR-mediated activation of EGFR can involve the formation of a GPCR-β-arrestin-Src signaling complex, the possibility of a β-arrestin1-Src complex contributing to EP2-mediated signaling in keratinocytes was investigated. Butaprost induced β-arrestin1-Src complex formation and increased both Src and EGFR activation. A role for β-arrestin1 in EP2-mediated Src and EGFR activation was demonstrated by the observation that β-arrestin1 deficiency significantly reduced Src and EGFR activation. In agreement with a β-arrestin1-Src complex contributing to EGFR activation, Src and EGFR inhibition (PP2 and AG1478, respectively) indicated that Src was upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/2, and STAT3, and both β-arrestin1 deficiency and EGFR inhibition (AG1478 or gefitinib) decreased their activation. In addition to β-arrestin1-dependent EGFR activation, butaprost increased PKA activation, as measured by phospho-GSK3β (p-GSK3β) and p-cAMP-response element-binding protein formation. PKA inhibition (H89 or R(P)-adenosine-3',5'-cyclic monophosphorothioate (R(P)-cAMPS)) decreased butaprost-induced cAMP-response element-binding protein and ERK activation but did not affect EGFR activation, whereas β-arrestin1 deficiency decreased EGFR activation but did not affect butaprost-induced PKA activation, thus indicating that they were independent EP2-mediated pathways. Therefore, the results indicate that EP2 contributed to mouse keratinocyte proliferation by G protein-independent, β-arrestin1-dependent activation of EGFR and G protein-dependent activation of PKA. 相似文献
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J R Sheller D Mitchell B Meyrick J Oates R Breyer 《Journal of applied physiology》2000,88(6):2214-2218
PGE(2) is an important cyclooxygenase product that modulates airway inflammatory and smooth muscle responses. Signal transduction is mediated by four EP receptor subtypes that cause distinct effects on cell metabolism. To determine the role of EP(2) receptor activation, we produced a mouse lacking the EP(2) receptor by targeted gene disruption. The effect of aerosolized PGE(2) and other agonists was measured using barometric plethysmography and by measurements of lung resistance in mechanically ventilated mice. Inhalation of PGE(2) inhibited methacholine responses in wild-type but not in mice lacking the EP(2) receptor [EP(2)(-/-)]. After airway constriction was induced by methacholine aerosol, PGE(2) reduced the airway constriction enhanced pause in wild-type mice (from 0.88 +/- 0.15 to 0.55 +/- 0.06) but increased it in EP(2)(-/-) mice (from 0.73 +/- 0. 08 to 1.27 +/- 0.19). Similar results were obtained in mechanically ventilated mice. These data indicate that the EP(2) receptor mediates the bronchodilation effect of PGE(2). 相似文献