Inhibitory Effect Of Prostaglandin E2 Research Articles

Pharmacological evidence that the inhibitory effects of prostaglandin E2 are mediated by the EP2 and EP4 receptors in human neutrophils.

Prostaglandin E2 (PGE2) is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis, and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of ROS production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Separation and Isolation of a New Hydroxylated Resveratrol Trimer Together with Other Stilbenoid Compounds from the Lianas of Gnetum microcarpum Blume and Their Inhibitory Effects of Prostaglandin E2

A new oligostilbene trimer, malaysianol F (1), together with ten known stilbenes (2–11), were successfully separated and purified from the acetone extract of the lianas of Gnetum microcarpum. Malaysianol D (2) was isolated for the first time in Gnetum plants. The tanninless crude extract (52.5 g) was fractionated using a vacuum liquid chromatography (VLC) technique to give five major fractions. Fraction 2 (4.68 g), 3 (4.79 g) and 4 (9.29 g) were all subjected to further isolation and purification using VLC, column chromatography (CC) and repetitive radial chromatography (RC) techniques with the best solvent system to yield malaysianol F (1) (6.2 mg), malaysianol D (2) (62.5 mg), malaysianol E (3) (2.4 mg), ε-viniferin (4) (10 mg), resveratrol (5) (6.5 mg), gnetol (6) (3.5 mg), gnetucleistol C (7) (12.2 mg), isorhapontigenin (8) (8 mg), cuspidan B (9) (3.2 mg), parvifolol D (10) (4.8 mg) and gnetifolin M (11) (2.5 mg). Their structures were determined on the basis of the analysis of spectral evidence by extensive NMR data analyses and comparison with the related published data. Several compounds were tested for anti-inflammatory activity. Their inhibitory effect on Prostaglandin E2 (PGE2) was tested using radioimmunoassay techniques. Compound 6 exhibited significant concentration-dependent inhibitory effects on PGE2 production with IC50 values of 1.84 µM comparable with the positive control, indomethacin (IC50 1.29 µM).

A novel immunochemotherapy based on targeting of cyclooxygenase and induction of immunogenic cell death

Cyclooxygenase (COX) plays a crucial role in the “inflammogenesis of cancer”, which leads to tumor progression, metastasis, and immunotherapy resistance. Therefore, reducing “inflammogenesis” by COX inhibition may be a key perspective for cancer therapy. However, the role of tumor-derived COX in the actions of COX inhibitors remains incompletely understood. In this study, applying “old drug new tricks” to repurpose 5-aminosalicylic acid (5-ASA), a COX inhibitor, we examined the effect of 5-ASA, alone or in combination with doxorubicin (DOX), in several cancer cell lines with different levels of COX expression. To facilitate the evaluation of the combination effect on tumors in vivo, a new micellar carrier based on PEG-b-PNHS polymer-conjugated 5-ASA (PASA) was developed to enhance codelivery of 5-ASA and DOX. Folate was also introduced to the polymer (folate-PEG-NH2-conjugated PASA (FASA)) to further improve delivery to tumors via targeting both tumor cells and tumor macrophages. An unprecedented high DOX loading capacity of 42.28% was achieved through various mechanisms of carrier/drug interactions. FASA was highly effective in targeting to and in inhibiting the growth of both 4T1.2 and CT26 tumors in BALB/c mice. However, FASA was more effective in CT26 tumor that has a high level of COX expression. Codelivery of DOX via PASA and FASA led to a further improvement in antitumor activity. Mechanistic studies suggest that inhibition of COX in vivo led to a more active tumor immune microenvironment. Interestingly, treatment with FASA led to upregulation of PD-1 on T cells, likely due to repressing the inhibitory effect of prostaglandin E2 (PGE2) on PD-1 expression on T cells. Combination of FASA/DOX with anti-PD-1 antibody led to a drastic improvement in the overall antitumor activity including regression of some established tumors at a suboptimal dose of FASA/DOX. Our data suggest that FASA/DOX may represent a new and effective immunochemotherapy for various types of cancers, particularly those cancers with high levels of COX expression.

Impaired adenylate cyclase signaling in acute myocardial ischemia: Impact on effectiveness of P2Y12 receptor antagonists

IntroductionP2Y12 receptor antagonists reduce risk of thrombotic complications after stent implantation but increase bleeding risk. Activation of P2Y12 receptors by ADP causes Gi-protein-mediated inhibition of adenylate cyclase (AC), thus limiting platelet response to anti-aggregatory effect of prostacyclin (PGI2). However, P2Y12 blockade reverses this ADP-induced suppression of the platelet PGI2/AC signaling pathway. We previously demonstrated that impairment of this pathway predicts poor response to clopidogrel. ObjectivesTo identify clinical correlates of variability in PGI2/AC signaling, and to assess the impact of such variability on individual responses to the direct P2Y12 receptor antagonists ticagrelor (in vivo) and 2-methyl-thioadenosine-monophosphate (2MeSAMP) (in vitro). Patients/MethodsWe compared the inhibitory effects of prostaglandin E1 (PGE1) and the PGI2 analog Iloprost (Ilt) on platelet aggregation in whole blood samples from healthy control subjects (n = 17), and patients with stable angina pectoris (SAP; n = 35) or acute coronary syndromes (ACS; n = 23), with or without associated diabetes/hyperglycemia. ResultsCompared to control subjects, patients with ACS and – to a lesser extent – those with SAP, exhibited impaired responses to PGE1, accentuated in the presence of hyperglycemia. Efficacy of ticagrelor treatment, measured as change in platelet reactivity index, was directly related to pre-treatment PGE1 response, both at univariate and multivariate analysis. There was a strong correlation between extent of inhibition of platelet aggregation, whether by PGE1 or Ilt, and the anti-aggregatory effect of 2MeSAMP in vitro. ConclusionsThe integrity of PGI2/AC signaling, which is impaired in the presence of ACS and hyperglycemia, predetermines the anti-aggregatory efficiency of P2Y12 receptor antagonists.

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

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

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-β (TGF-β) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-β in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-β-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-β-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-β. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf.

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2.

Mucoadhesive chitosan/OA nanoparticles charged with celecoxib inhibit prostaglandin E2 LPS‐induced in U937 cell line

ABSTRACTThe present study shows the potential of new carriers of celecocib (CEL) for prostaglandin E2 (PGE2) inhibition in U937 cell line. Self‐assembled nanoparticles based on oleic acid‐modified chitosan were covered with hyaluronic acid (HA) obtaining systems with spherical shape and particle size close to 300 nm. CEL was encapsulated and the encapsulation efficiency (%EE) was dependent of the drug solubility in acid media, reaching %EE of 75.5% and 58.2% to strong and weak acid, respectively. The covering with HA increased the mucoadhesive properties and, the cellular binding and cellular uptake in U937 cells. Nanoparticles prepared in strong acid presented zeta potential (ζ) of 43.8 ± 0.4 mV, which become toxic and stimulate the PGE2 production in U937 cells, at the concentration 1 mg/mL. However, nanoparticles prepared in weaker acid presented ζ of 36.5 ± 1.21 mV, showing nontoxic effect and inhibitory effect of PGE2 from 80.8 pg/mL until 43.4 pg/mL. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45288.

Neutrophils Modulate Osteoblastic Niche for Hematopoietic Progenitors Via Prostaglandin E2 Production Triggered By Sympathetic Nervous System

The mobilization of hematopoietic stem/progenitor cells (HSC/HPCs) from the bone marrow (BM) to peripheral blood by granulocyte colony-stimulating factor (G-CSF) is an essential method in clinic. We have shown that the suppression of osteoblastic niche by β-adrenergic signal is critical for this phenomenon (Cell 2006; Cell Stem Cell 2013). Because G-CSF administration causes fever and back pain and these symptoms are ameliorated by non-steroidal anti-inflammatory drugs, we investigated the role of prostaglandin E2 (PGE2) in the BM microenvironment during G-CSF treatment. First, HPC (CFU-C) mobilization by G-CSF (125μg/kg/dose, every 12 hours, 8 divided doses) was significantly augmented in microsomal PGE syntase-1-deficient (mPGES-1-/-) mice (42% increase, n=16, p<0.05), and strongly inhibited by exogenous administration of PGE2 (6mg/kg/day for 2 weeks) to wild-type (WT) mice (52%, n=8-9 p<0.05). These data suggest that G-CSF induces mPGES-1-mediated PGE2 production, which suppresses the HPC mobilization. In the early phase (at 4 doses) of G-CSF administration, mPGES-1 mRNA in BM cells was upregulated (34% increase, n=5, p<0.05). Furthermore, WT mice reconstituted with mPGES-1-/- BM showed higher HPC mobilization than control mice reconstituted with mPGES-1+/+ BM (2.2-fold increase, n=4, p<0.05), which indicated that blood cells might be responsible for additional PGE2 synthesis. To address this, we examined the PGE2 production by ELISA from various lineage cell lines, such as neutrophil precursor 32D, macrophage RAW264.7, B cell Ba/F3, and T cell EL4. No increase was observed by G-CSF in any cell lines; whereas, isoproterenol induced PGE2 production significantly only in 32D culture supernatant (2.4-fold increase compared to vehicle treatment, n=4, p<0.05) accompanied with drastic increase of mPGES-1 mRNA in the cells and norepinephrine showed a similar effect. Primary neutrophils sorted from the BM also demonstrated prompt PGE2 production by isoproterenol (3-fold increase compared to vehicle treatment, n=4, p<0.05) but not by G-CSF. These data suggest that G-CSF-triggered high sympathetic tone stimulates the BM neutrophils to lead PGE2 production. We next assessed the exact roles of PGE2 in HPC mobilization. The inhibitory effect of PGE2 on HPC mobilization was completely abrogated in PGE2 receptor EP4-deficient (EP4-/-) mice, and the chimeric model generated by the reciprocal BM transplantation revealed that it was EP4 in microenvironment, but not in hematopoietic cells, that was critical for this effect. Since PGE2 did not change the CXCL12 behavior, we speculated that PGE2 increased another anchor in the niche, osteopontin (OPN). Immunofluorescence staining demonstrated upregulation of OPN by PGE2 and/or G-CSF in the endosteum, which was abolished in EP4-/- mice. Indeed, the inhibitory effect of PGE2 on HPC mobilization was canceled partially in OPN-/- mice and almost completely in anti-OPN antibody-treated WT mice. PGE2 also inhibited AMD3100-induced HPC mobilization, and this effect was canceled by anti-OPN antibody, which confirmed that PGE2-mediated niche modulation was independent of CXCL12 axis. To assess the induction of OPN by PGE2 directly, we fractionated non-hematopoietic (CD45-CD31-Ter119-) cells isolated from adult femur into three populations, i.e. Sca-1+ALCAM- immature mesenchymal cells, Sca-1-ALCAM- preosteoblasts that favorably support HPCs, and Sca-1-ALCAM+ mature osteoblasts that are most potent to maintain quiescent HSCs in vitro. PGE2 upregulated the OPN protein 2-fold in Sca-1+ALCAM- immature mesenchymal cells in cultures and more dramatically (6-fold) in Sca-1-ALCAM- preosteoblasts as assessed by flow cytometry. In sharp contrast, no OPN induction was observed in Sca-1-ALCAM+ mature osteoblasts. PGE2 failed to induce OPN in all three fractions from EP4-/- mice. In contrast to HPCs, the mobilization of long-term (6 months) repopulating HSCs was not altered in mPGES-1-/- and in PGE2-treated WT mice. This was consistent with the OPN induction profile in fractionated osteoblasts. These results suggest that PGE2 selectively regulates the osteoblastic niche for hematopoietic progenitors, but not for stem cells, by the induction of OPN via EP4 receptor. Collectively, we propose the inter-communication between the mature hematopoietic cells and the niche for their immature progenitors governed by the sympathetic nervous system. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory Effect of Prostaglandin E2 on the Migration of Nasal Fibroblasts

Objectives: Fibroblast migration is crucial for normal wound repair after sinonasal surgery. Prostaglandin E2 (PGE2) is a potent inhibitor of fibroblast functions including chemotaxis, proliferation, and matrix production. The purpose of this study was to determine whether PGE2 affects the migration of nasal fibroblasts and to investigate the mechanism of action of PGE2 on nasal fibroblasts. Methods: Primary cultures of nasal fibroblasts were established from inferior turbinate samples. Fibroblast migration was evaluated with scratch assays. Reverse transcription-polymerase chain reaction was performed for E-prostanoid (EP)1, EP2, EP3 and EP4 receptors. EP receptor-selective agonists and antagonists were used to evaluate receptor functions. Stimulatory G (Gs) proteins were activated to evaluate mechanisms. Intracellular cyclic adenosine monophosphate (cAMP) levels were measured by enzyme-linked immunosorbent assay, and fibroblast cytoskeletal structures were visualized with immunocytochemistry. Results: PGE2 significantly reduced the migration of nasal fibroblasts. Agonists selective for the EP2 and EP4 receptors significantly reduced the nasal fibroblast migration. Antagonists of the EP2 and EP4 receptors inhibited the effect of PGE2 on nasal fibroblast migration. Activation of Gs protein and adenyl cyclase reduced nasal fibroblast migration. Conclusions: PGE2 inhibited the migration of nasal fibroblasts via the EP2 and EP4 receptors, and this inhibition was mediated by cAMP elevation. Targeting specific EP receptors could offer therapeutic opportunities for conditions such as delayed wound healing after nasal surgery.

Fish oil attenuates methylmalonate-induced seizures

Methylmalonic acidemias are inherited metabolic disorders characterized by methylmalonate (MMA) accumulation and neurological dysfunction, including seizures. Dietary fatty acids are known as an important energy source and reduce seizure activity in selected acute animal models. This study investigated whether chronic treatment with fish oil or with oleic acid attenuates MMA-induced seizures and whether maintenance of Na(+),K(+)-ATPase activity was involved in such an effect. Adult male Wistar rats were given fish oil (85 mg/kg), oleic acid (85 mg/kg) or vehicle (0.42% aqueous Cremophor EL™, 4 mL/kg/body weight/day), p.o., for 75 days. On the 73th day a cannula was implanted in the right lateral ventricle with electrodes over the parietal cortex for EEG recording. On the 76th day the animals were injected with NaCl (2.5 μmol/2.5 μL, i.c.v.), or with MMA (2.5 μmol/2.5 μL, i.c.v.), and seizure activity was measured by electroencephagraphic (EEG) recording with concomitant behavior monitoring. The effect of prostaglandin E2 (PGE2) on Na(+),K(+)-ATPase activity of slices of cerebral cortex from NaCl-injected animals was determined. Fish oil increased the latency to MMA-induced tonic-clonic seizures, reduced the mean amplitude of ictal EEG recordings, and prevented PGE2-induced decrease of Na(+),K(+)-ATPase activity in cortical slices in vitro. Oleic acid decreased mean amplitude of ictal EEG recordings. The results support that fish oil decreases MMA-induced seizures. The decreased sensitivity of Na(+),K(+)-ATPase to the inhibitory effect of PGE2 in fish oil-treated animals may be related to the currently reported anticonvulsant activity.

Differential effects of prostaglandin E2 and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes

Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E(2) (PGE(2) ) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. Cells from healthy human gingiva or skin were treated with PGE(2) and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. In hGFs, PGE(2) (1 μm) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE(2) -induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal-regulated kinase (p-ERK), PGE(2) reduced it (by 70-80%) and EMD restored it when present with PGE(2). Prostaglandin E(2) stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE(2) was milder than with hGFs. When present together, EMD abolished the PGE(2) -induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE(2) had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP(2) receptor mRNA than hGFs, while hGKs contained none. Prostaglandin E(2) inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE(2) can be explained by the levels of EP(2) expression.

Regulation of receptor activator of nuclear factor kappaB ligand and osteoprotegerin expression by prostaglandin E2 in osteoblastic-like cells

To investigate the regulation of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression by prostaglandin E2 (PGE2) in osteoblastic-like cells and involved signaling pathways. Rat UMR106 osteoblast-like cells were cultured and treated with various dose of PGE2 or regulators of different signaling pathways such as protein kinase A, protein kinase C, ERK-MAPK and calcium/calmodulin pathways for different period of time,the cells were then harvested at indicated time points, total RNA were isolated and RANKL/OPG mRNA expression were studied by real-time PCR. PGE2, Forskolin and db-cAMP increased RANKL mRNA by 2.8 times (P = 0.002), 2.2 times (P = 0.006) and 2.1 times (P = 0.005) respectively, and inhibited OPG mRNA expression by 12% (P < 0.01), 85% (P = 0.005) and 70% (P = 0.013) respectively, while RANKL and OPG mRNA expression were down-regulated by A23187 by 58% (P = 0.002) and 53% (P = 0.017) respectively. As for inhibitory experiments, the stimulatory effects of PGE2 on RANKL mRNA expression could be inhibited only by KT-5720 by 53% (P < 0.01), while the other inhibitors did not have any effect at all. The inhibitory effects of PGE2 on OPG mRNA expression were partially blocked by KT-5720 by 47% (P = 0.01), verapamil by 38% (P = 0.029) and W7 by 43% (P < 0.01) respectively, while KN-62, chelerythrine and PD98059 had no effects. PGE2 can up-regulate the expression of RANKL but down-regulate the expression of OPG. The up-regulation of RANKL mRNA expression by PGE2 was mediated through the activation of PKA signaling pathway while PGE2 induced down-regulation of OPG mRNA expression was predominantly mediated via PKA as well as the Ca2+ /Calmodulin signaling pathways.

Effect of leukotriene B4 and prostaglandin E2 on the adhesion of lymphocytes to endothelial cells

The arachidonic acid metabolites leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) may play an important role in inflammation. It is not known whether these mediators influence the binding of lymphocytes to endothelial cells, a process which is important in the extravasation of lymphocytes in inflammatory states. In the present investigation, the effect of LTB4 and PGE2 on the binding interaction between lymphocytes and endothelial cells was examined using a centrifugation cell binding assay. Although LTB4 elicited an aggregation response on human polymorphonuclear leucocytes (PMNL) and enhanced their binding to endothelial cells it had no effect on lymphocyte binding. By contrast, PGE2 caused a dose-dependent inhibition of lymphocyte binding to endothelial cells. The inhibitory effect of PGE2 had a rapid onset but was exhibited only when PGE2 was present continuously during the cell binding assay. Although the mechanism by which PGE2 acts is not clear, it may provide a negative feedback mechanism in regulating the influx of lymphocytes into inflammatory sites.

Cooperation of adenosine and prostaglandin E2 (PGE2) in amplification of cAMP-PKA signaling and immunosuppression.

We hypothesize that adenosine and PGE2 could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. To test this hypothesis, the effect of adenosine and PGE2 on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. PGE2 and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE2 and adenosine receptors we found that cooperation of PGE2 and adenosine in their inhibitory effects are mediated via EP2 and A2A receptors, respectively. LAK cells have 35-fold higher expression of EP2 than A2A. Combined PGE2 and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE2. In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE2 and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE2 are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE2 could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.

Melatonin synthesized by Jurkat human leukemic T cell line is implicated in IL‐2 production

Human lymphocytes have recently been described as an important physiological source of melatonin (N-acetyl-5-methoxytryptamine), which could be involved in the regulation of the human immune system. On the other hand, stimulation of IL-2 production by exogenous melatonin has been shown in the Jurkat human lymphocytic cell line. Furthermore, both melatonin membrane and nuclear receptors are present in these cells. In this study, we show that the necessary machinery to synthesize melatonin is present and active in resting and stimulated Jurkat cells. Accordingly, we have found that cells synthesize and release melatonin in both conditions. Therefore, we investigated whether endogenous melatonin produced by Jurkat cells was involved in the regulation of IL-2 production. When melatonin membrane and nuclear receptors were blocked using specific antagonists, luzindole and CGP 55644, respectively, we found that IL-2 production decreased, and this drop was reverted by exogenous melatonin. Additionally, PHA activation of Jurkat cells changed the profile of melatonin nuclear receptor mRNA expression. A previous study showed that exogenous melatonin is able to counteract the decrease in IL-2 production caused by prostaglandin E2 (PGE2) in human lymphocytes via its membrane receptor. In our model, when we blocked the melatonin membrane receptor with luzindole, the inhibitory effect of PGE2 on IL-2 production was higher. Therefore, we have demonstrated the physiological role of endogenous melatonin in this cell line. These findings indicate that endogenous melatonin synthesized by human T cells would contribute to regulation of its own IL-2 production, acting as an intracrine, autocrine, and/or paracrine substance.

Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10(-8) M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.

Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells

Exposure to pathogens induces dendritic cells to release inflammatory cytokines and chemokines. The inflammatory response is controlled by endogenous agents such as anti-inflammatory cytokines, glucocorticoids, anti-inflammatory neuropeptides, and lipid mediators. This study is the first report on the inhibition by prostaglandin E2 (PGE2) of TNF release from bone marrow-derived dendritic cells stimulated with lipopolysaccharide (LPS), a TLR4 ligand, or peptidoglycan, a TLR2 ligand. The inhibition of TNF occurs at both mRNA and protein level. The inhibitory effect of PGE2 is mediated by the EP2 and EP4 receptors, and involves both PKA signaling and mediation by DC-derived IL-10. Intraperitoneal administration of PGE2 together with LPS results in a reduction in serum TNF and intracellular TNF in peritoneal exudate cells, compared to LPS alone. In addition, administration of PGE2 in vivo reduces the numbers of CD11c+ DCc that accumulate in the peritoneal cavity in response to LPS. The various implications of the PGE2-induced reduction in TNF are discussed.

Prostaglandin E1 at clinically relevant concentrations inhibits aggregation of platelets under synergic interaction with endothelial cells.

The inhibitory effect of prostaglandin E1 (PGE1) on platelet aggregation is considered an important characteristic of this agent. However, the concentration of PGE1 to inhibit aggregation in vitro is higher than those of clinical use (1 ng/ml). To clarify whether PGE1 at clinically relevant concentrations inhibits aggregation under synergic action with endothelial cell-derived factors (nitric oxide and prostacyclin), we evaluated the minimum effective concentration of PGE1 to enhance the anti-aggregating activity of endothelial cells. Inhibitory effects of PGE1 and/or the incubation buffer from cultured porcine aortic endothelial (PAE) cells on human platelet aggregation induced by 2 micro g/ml collagen were examined by turbidimetry. PGE1 concentration-dependently (>3 ng/ml) inhibited aggregation: the incubation buffer from PAE cells stimulated by bradykinin also inhibited aggregation. Bradykinin concentration-dependently increased the anti-aggregating activity of the PAE incubation buffer. The half-maximum effective concentration of bradykinin to inhibit aggregation (95.4+/-22.3 nM) was significantly decreased to 10.3+/-2.5 nM by 0.1 ng/ml PGE1 and to 0.9+/-0.5 nM by 1 ng/ml PGE1, respectively. These indicated that PGE1 (=0.1 ng/ml) inhibits aggregation through synergism with endothelial cells. The synergic effect of PGE1 and the anti-aggregating activity of the PAE cells preincubated with 10 micro M indomethacin for 30 min was more potent than that of these cells preincubated with 1 mM NG-nitro-L-arginine methyl ester. This suggested that the interaction of PGE1 with endothelial cell-derived nitric oxide is more powerful than that with endothelial cell-derived prostacyclin. Prostaglandin E1 (=0.1 ng/ml) inhibited platelet aggregation under synergic interaction with endothelial cells.

Regulation of von Willebrand Factor Binding to the Platelet Glycoprotein Ib-IX by a Membrane Skeleton-dependent Inside-out Signal

The platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX (GPIb-IX), mediates initial platelet adhesion and activation. We show here that the receptor function of GPIb-IX is regulated intracellularly via its link to the filamin-associated membrane skeleton. Deletion of the filamin binding site in GPIb(alpha) markedly enhances ristocetin- (or botrocetin)-induced vWF binding and allows GPIb-IX-expressing cells to adhere to immobilized vWF under both static and flow conditions. Cytochalasin D (CD) that depolymerizes actin also enhances vWF binding to wild type GPIb-IX. Thus, vWF binding to GPIb-IX is negatively regulated by the filamin-associated membrane skeleton. In contrast to native vWF, binding of the isolated recombinant vWF A1 domain to wild type and filamin binding-deficient mutants of GPIb-IX is comparable, suggesting that the membrane skeleton-associated GPIb-IX is in a state that prevents access to the A1 domain in macromolecular vWF. In platelets, there is a balance of membrane skeleton-associated and free forms of GPIb-IX. Treatment of platelets with CD increases the free form and enhances vWF binding. CD also reverses the inhibitory effects of prostaglandin E1 on vWF binding to GPIb-IX. Thus, GPIb-IX-dependent platelet adhesion is doubly controlled by vWF conformation and a membrane skeleton-dependent inside-out signal.