Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines

Background and purpose:

Prostaglandin (PG) E₂ and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE₂ signals principally through prostanoid receptors of the EP₂ and/or EP₄ subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP₂ and EP₄ receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE₂-induced IL-8 production.

Experimental approach:

To identify the causative receptor, we knocked-down and over-expressed EP₂ and EP₄ receptor subtypes in colonic epithelial cells and studied the effect of several selective EP₂/EP₄ receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE₂ and Bmax values for the EP₂ and EP₄ receptor on colonic epithelial cells were determined by radioligand-binding assays with [³H]PGE₂.

Key results:

PGE₂ had the highest affinity for the EP₄ receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP₄ receptor agonist, ONO-AE1-329, and abolished by silencing the EP₄ receptor gene by using siRNA techniques, a selective EP₄ receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.

Conclusions and implications:

These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE₂ acting via the EP₄ receptor subtype.     

Tanshinones inhibit mast cell degranulation by interfering with IgE receptor-mediated tyrosine phosphorylation of PLCgamma2 and MAPK

Recently we have isolated four active components from Tanshen (the root of Salvia miltiorrhiza Bunge, Labiatae) responsible for the anti-allergic activities. In this study, the molecular mechanism of action of tanshinones for the inhibition of mast cell degranulation was investigated by testing their effects on the signaling components of the high affinity IgE receptor FcepsilonRI. Activation of FcepsilonRI produced immediate tyrosine phosphorylation of Syk, mitogen-activated protein kinase extracellular signal-regulated kinase, ERK1/ERK2 (p44, p42), and phospholipase Cgamma2 (PLCgamma2). 5,16-Dihydrotanshinone-I possessed the strongest inhibitory effects on mast cell degranulation and markedly reduced FcepsilonRI-mediated tyrosine phosphorylation of ERK and PLCgamma2. This suggests that tanshinones possibly exert their anti-allergic activities by affecting FcepsilonRI-mediated tyrosine phosphorylation of ERK and PLCgamma2. Abbreviations. FcepsilonRI:high affinity IgE receptor ERK:extracellular signal regulated kinase PLC: phospholipase C     

Excavation of lead compounds that inhibit mast cell degranulation by combinatorial chemistry and activity-guided

An allergic reaction ensues after antigen binds to mast cell or basophil high affinity IgE receptor, Fc epsilonRI, resulting in degranulation of various inflammatory mediators that produce various allergic symptoms. In this study, i) we isolated the active component for the inhibition of mast cell degranulation from the extract of leaves of Castanea crenata and identified it as quercetin; ii) we established the total synthesis procedure of quercetin; iii) using quercetin as positive control, we excavated some lead compounds that possess inhibitory activities for mast cell degranulation by screening the chemical libraries of 1,3-oxazolidine derivatives prepared by solid phase combinatorial chemistry. Some of 1,3-oxazolidine compounds possessing acetyl and 3',4'-dichlorophenyl group displayed strong inhibitory activities on Fc epsilonRI-mediated mast cell degranulation, suggesting that they can be used as lead compounds for the development of anti-allergic agents.     

Prostaglandin E2 inhibits the phospholipase D pathway stimulated by formyl-methionyl-leucyl-phenylalanine in human neutrophils. Involvement of EP2 receptors and phosphatidylinositol 3-kinase gamma

Prostaglandin E(2) (PGE(2)), originally discovered as a pro-inflammatory mediator, also inhibits several chemoattractant-elicited neutrophil functions, including adhesion, secretion of cytotoxic enzymes, production of superoxide anions, and chemotaxis. In this study, we have examined the effects of PGE(2) and prostaglandin E (EP) receptor-selective agonists/antagonists on several steps of the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation pathway in human neutrophils to elucidate the PGE(2) inhibitory mechanism. PGE(2) and EP(2) receptor agonists inhibited the stimulation of the activity of PLD induced by fMLP in a concentration-dependent manner. The fMLP-stimulated translocation to membranes of protein kinase C alpha, Rho, and Arf GTPases was diminished in the presence of PGE(2) or EP(2) agonists. Moreover, PGE(2) and EP(2) agonists decreased the activation of phosphatidylinositol 3-kinase gamma (PI3Kgamma) and Tec kinases as well as the tyrosine phosphorylation of proteins stimulated by fMLP. These data provide strong evidence that 1) the inhibitory effects of PGE(2) on the fMLP-induced PLD activation pathway were mediated via EP(2) receptors and that 2) the suppression of PI3Kgamma activity was the crucial step in the EP(2)-mediated inhibition of the fMLP-induced signaling cascade.     

Prostaglandin EP receptors involved in modulating gastrointestinal mucosal integrity

Endogenous prostaglandins (PGs) play an important role in modulating the mucosal integrity and various functions of the gastrointestinal tract, and E type PGs are most effective in these actions. PGE? protected against acid-reflux esophagitis and prevented the development of gastric damage induced by ethanol or indomethacin, the effects mimicked by EP1 agonists and attenuated by an EP1 antagonist. Adaptive cytoprotection induced by mild irritants was also attenuated by the EP1 antagonist. On the other hand, the acid-induced duodenal damage was prevented by EP3/EP4 agonists and worsened by EP3/EP4 antagonists. Similarly, the protective effect of PGE? on indomethacin-induced small intestinal damage or DSS-induced colitis was mimicked by EP3/EP4 agonists or EP4 agonists, respectively. The mechanisms underlying these actions of PGE? are related to inhibition of stomach contraction (EP1), stimulation of duodenal HCO?? secretion (EP3/EP4), inhibition of small intestinal contraction (EP4), and stimulation of mucus secretion (EP3/EP4) or down-regulation of cytokine secretion in the colon (EP4), respectively. PGE? also showed a healing-promoting effect on gastric ulcers and intestinal lesions through the activation of EP4 receptors, the effect associated with stimulation of angiogenesis via an increase in VEGF expression. These findings should aid the development of new strategies for treatment of gastrointestinal diseases.     

Microfilament-associated degranulation of sensitized guinea-pig lung mast cells

When sensitized guinea-pig lung mast cells were exposed to antigen, granules were pushed out on to the cell surface. Subsequently, thin filaments, some extending as long as 15 microns, were projected radially with the extruded granules. The latter became swollen in the extracellular medium and the elongated filaments became shorter, until, within 7-8 min, the granules were reincorporated into the cytoplasm. The time course of morphological changes corresponded approximately to that of changes in the intracellular Ca2+ concentrations. The filaments connecting the extruded granules to the cell surface were stained with rhodamine-phalloidin, indicating that they consisted mainly of actin.     

Functional pharmacology of human prostanoid EP2 and EP4 receptors

Prostanoid EP(2) and EP(4) receptor-mediated responses are difficult to distinguish pharmacologically because of the lack of potent, selective antagonists. We describe systematic agonist fingerprints for recombinant human prostanoid EP(2) and EP(4) receptors expressed in CHO and HEK293 cells, respectively. The rank orders of potency of endogenous prostaglandins were: prostanoid EP(2) receptors: prostaglandin E(2)>prostaglandin D(2)=prostaglandin F(2alpha)>prostaglandin I(2); prostanoid EP(4) receptors: prostaglandin E(2)>prostaglandin I(2)>prostaglandin D(2)=prostaglandin F(2alpha). Butaprost free acid (9-oxo-11alpha,16R-dihydroxy-17-cyclobutyl-prost-13E-en-1-oic acid) behaved as a highly selective partial agonist at prostanoid EP(2) receptors while butaprost methyl ester elicited small, low potency responses. The prostanoid EP(1) and EP(3) receptor agonists misoprostol (9-oxo-11alpha,16-dihydroxy-16-methyl-prost-13E-en-1-oic acid, methyl ester), sulprostone (N-(methylsulphonyl)-9-oxo-11alpha,15R-dihydroxy-16-phenoxy-17,18,19,20-tetranor-prosta-5Z,13E-dien-1-amide), and GR63799X ([1R-[1alpha(Z),2beta(R*),3alpha]-(-)-4-benzoylamino)phenyl-7-[3-hydroxy-3-phenoxy-propoxy)-5-oxocyclopentyl]-4-heptenoate), and the prostanoid DP receptor agonist BW245C ((4S)-(3-[(3R,S)-3-cyclohexyl-3-hydropropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid), activated both prostanoid EP(2) and EP(4) receptors. Prostaglandin I(2), iloprost (6,9alpha-methylene-11alpha,15S-dihydroxy-16-methyl-prosta-5E,13E-dien-18-yn-1-oic acid, trometamol salt) and cicaprost (5-[(E)-(1S, 5S, 6S, 7R)-7-hydroxy-6-[(3S, 4S)-3-hydroxy-4-methylnona-1,6-diinyl]-bicyclo[3.3.0]octan-3-yliden]-3-oxapentanoic acid; ZK96480) were full agonists at prostanoid EP(4) receptors. Key differentiating agonists are: butaprost FA, 16,16-dimethyl-prostaglandin E(2), 19-(R)-hydroxy prostaglandin E(2), misoprostol, BW245C, prostaglandin F(2alpha) and prostaglandin D(2).     

Mesenteric mast cell degranulation in acute T-2 toxin poisoning.

T-2 toxin-induced alterations in rat mesenteric mast cell granulation were measured by cytophotometric analyses of the metachromatic reaction of mast cell granules with azure B. Hypogranulation (diminution of metachromatic material) was observed 8 h following injections of T-2 toxin (0.5-1.5 LD50, i.p.). These data suggest that mast cell activation occurs during acute T-2 intoxication and raise the possibility that mast cell mediators may contribute to toxin-induced cardiovascular collapse.     

UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation

UDP-glucose (UDPG), a glycosyl donor in the biosynthesis of carbohydrates, is an endogenous agonist of the G protein-coupled P2Y₁₄ receptor. RBL-2H3 mast cells endogenously express a P2Y₁₄ receptor at which UDPG mediates degranulation as indicated by β-hexosaminidase (HEX) release. Both UDPG and a more potent, selective 2-thio-modified UDPG analog, MRS2690 (diphosphoric acid 1-α-d-glucopyranosyl ester 2-[(2-thio)uridin-5″-yl] ester), caused a substantial calcium transient in RBL-2H3 cells, which was blocked by pertussis toxin, indicating the presence of the G_i-coupled P2Y₁₄ receptor, supported also by quantitative detection of abundant mRNA. Expression of the closely related P2Y₆ receptor was over 100 times lower than the P2Y₁₄ receptor, and the P2Y₆ agonist 3-phenacyl-UDP was inactive in RBL-2H3 cells. P2Y₁₄ receptor agonists also induced [³⁵S]GTPγS binding to RBL-2H3 cell membranes, and phosphorylation of ERK1/2, P38 and JNK. UDPG and MRS2690 concentration-dependently enhanced HEX release with EC₅₀ values of 1150 ± 320 and 103 ± 18 nM, respectively. The enhancement was completely blocked by pertussis toxin and significantly diminished by P2Y₁₄ receptor-specific siRNA. Thus, mast cells express an endogenous P2Y₁₄ receptor, which mediates G_i-dependent degranulation and is therefore a potential novel therapeutic target for allergic conditions.     

Microsomal prostaglandin E synthase-1 contributes to ischaemic excitotoxicity through prostaglandin E2 EP3 receptors

Background and purpose:

Although microsomal prostaglandin E synthase (mPGES)-1 is known to contribute to stroke injury, the underlying mechanisms remain poorly understood. This study examines the hypothesis that EP₃ receptors contribute to stroke injury as downstream effectors of mPGES-1 neurotoxicity through Rho kinase activation.

Experimental approach:

We used a glutamate-induced excitotoxicity model in cultured rat and mouse hippocampal slices and a mouse middle cerebral artery occlusion–reperfusion model. Effects of an EP₃ receptor antagonist on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice.

Key results:

In cultures of rat hippocampal slices, the mRNAs of EP_1–4 receptors were constitutively expressed and only the EP₃ receptor antagonist ONO-AE3-240 attenuated and only the EP₃ receptor agonist ONO-AE-248 augmented glutamate-induced excitotoxicity in CA1 neurons. Hippocampal slices from mPGES-1 KO mice showed less excitotoxicity than those from WT mice and the EP₃ receptor antagonist did not attenuate the excitotoxicity. In transient focal ischaemia models, injection (i.p.) of an EP₃ antagonist reduced infarction, oedema and neurological dysfunction in WT mice, but not in mPGES-1 KO mice, which showed less injury than WT mice. EP₃ receptor agonist-induced augmentation of excitotoxicity in vitro was ameliorated by the Rho kinase inhibitor Y-27632 and Pertussis toxin. The Rho kinase inhibitor HA-1077 also ameliorated stroke injury in vivo.

Conclusion and implications:

Activity of mPGES-1 exacerbated stroke injury through EP₃ receptors and activation of Rho kinase and/or G_i. Thus, mPGES-1 and EP₃ receptors may be valuable therapeutic targets for treatment of human stroke.This article is commented on by Andreasson, pp. 844–846 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00715.x     

Inhibition of rat mast cell degranulation by verapamil

Calcium antagonists, e.g. verapamil, prevent exercise-induced asthma. This protective effect may proceed from inhibition of contraction of bronchial smooth muscle, release of mediators by primary effector cells, e.g. mast cells, or both. Therefore, we studied the inhibitory effect of increasing concentrations of verapamil on both in vitro antigen-induced degranulation and ionophore A23187-induced release of labelled serotonin by rat peritoneal mast cells. There was a dose-dependent inhibition by verapamil of both ovalbumin-induced degranulation of mast cells passively sensitized by incubation with mice IgE-rich serum and ionophore-induced release of tritiated serotonin by mast cells previously incubated with (3H)-5HT; the 50% inhibiting concentration was 1.4 X 10(-4) mol I-1 and 5.2 X 10(-5) mol I-1, respectively. An attractive explanation of our results is that verapamil inhibits the antigen-induced release of mediators by mast cells through its calcium antagonist effect. Our results also suggest that the preventing effect of calcium antagonists on asthma may be multi-factorial since other authors have clearly shown that these drugs inhibit contraction of guinea-pig tracheal smooth muscle in vitro.     

Inhibition of IgE-mediated mast cell degranulation by sulphasalazine

The effect of sulphasalazine on two mast cell populations and human peripheral leukocytes is reported. Sulphasalazine inhibited histamine release from mouse and rat mast cells, but it caused a potentiation of secretion in human peripheral leukocytes. The drug alone did not induce histamine release when administered without an anaphylactic stimulus. The results are discussed in terms of a possible mode of action of sulphasalazine in the treatment of inflammatory bowel disease.     

Regulation of M2-type pyruvate kinase mediated by the high-affinity IgE receptors is required for mast cell degranulation

BACKGROUND AND PURPOSE: M2-type pyruvate kinase (M2PK) was found to interact directly with the 'ITAM' region of the gamma chain of the high-affinity IgE receptor (FcvarepsilonRI). Our hypothesis was that mast cell degranulation might require the FcvarepsilonRI-mediated inhibition of M2PK activity. EXPERIMENTAL APPROACH: In rat basophilic leukaemia (RBL-2H3) cells, the effects of directly inhibiting M2PK or preventing the FcvarepsilonRI-mediated inhibition of M2PK (disinhibition) on degranulation was measured by hexosaminidase release. Effects of blocking the FcvarepsilonRI-mediated inhibition of M2PK was also assessed in vivo in a mouse model of allergen-induced airway hyper-responsiveness. KEY RESULTS: Activation of FcvarepsilonRI in RBL-2H3 cells caused the rapid phosphorylation of tyrosine residues in M2PK, associated with a decrease in M2PK enzymatic activity. There was an inverse correlation between M2PK activity and mast cell degranulation. FcvarepsilonRI-mediated inhibition of M2PK involved Src kinase, phosphatidylinositol 3-kinase, PKC and calcium. Direct inhibition of M2PK potentiated FcvarepsilonRI-mediated degranulation and prevention of the FcvarepsilonRI-mediated inhibition of M2PK attenuated mast cell degranulation. Transfection of RBL-2H3 cells with M1PK which prevents FcvarepsilonRI-induced inhibition of M2PK, markedly reduced their degranulation and exogenous M1PK (i.p.) inhibited ovalbumin-induced airway hyper-responsiveness in vivo. CONCLUSIONS AND IMPLICATIONS: We have identified a new control point and a novel biochemical pathway in the process of mast cell degranulation. Our study suggests that the FcvarepsilonRI-mediated inhibition of M2PK is a crucial step in responses to allergens. Moreover, the manipulation of glycolytic processes and intermediates could provide novel strategies for the treatment of allergic diseases.     

PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes

Background and Purpose

Different protease-activated receptors (PARs) activated by thrombin are involved in cardiovascular disease, via up-regulation of inflammatory proteins including COX-2. However, the mechanisms underlying thrombin-regulated COX-2 expression in human cardiomyocytes remain unclear.

Experimental Approach

Human cardiomyocytes were used in the study. Thrombin-induced COX-2 protein and mRNA expression, and signalling pathways were determined by Western blot, real-time PCR and COX-2 promoter luciferase reporter assays, and pharmacological inhibitors or siRNAs. PGE₂ generation and cell proliferation were also determined.

Key Results

Thrombin-induced COX-2 protein and mRNA expression, promoter activity and PGE₂ release was attenuated by the PAR1 antagonist ({"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797) or the inhibitors of proteinase activity (PPACK), MEK1/2 (U0126), p38 MAPK (SB202190) or JNK1/2 (SP600125), and transfection with small interfering RNA (siRNA) of PAR1, p38, p42 or JNK2. These results suggested that PAR1-dependent MAPKs participate in thrombin-induced COX-2 expression in human cardiomyocytes. Moreover, thrombin stimulated phosphorylation of MAPKs, which was attenuated by PPACK and {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797. Furthermore, thrombin-induced COX-2 expression was blocked by the inhibitors of AP-1 (tanshinone IIA) and NF-κB (helenalin). Moreover, thrombin-stimulated phosphorylation of c-Jun/AP-1 and p65/NF-κB was attenuated by tanshinone IIA and helenalin, respectively, suggesting that thrombin induces COX-2 expression via PAR1/MAPKs/AP-1 or the NF-κB pathway. Functionally, thrombin increased human cardiomyocyte proliferation through the COX-2/PGE₂ system linking to EP₂ receptors, as determined by proliferating cell nuclear antigen and cyclin D1 expression.

Conclusions and Implications

These findings demonstrate that MAPKs-mediated activation of AP-1/NF-κB pathways is, at least in part, required for COX-2/PGE₂/EP₂-triggered cell proliferation in human cardiomyocytes.     

聚山梨酯-80致RBL-2H3肥大细胞脱颗粒的研究

目的:研究聚山梨酯-80对RBL-2H3肥大细胞脱颗粒释放组胺的影响.方法:培养大鼠来源的RBL-2H3肥大细胞,取不同厂家来源的聚山梨酯-80与RBL-2H3细胞共培养60 min,用荧光分光光度法定量检测RBL-2H3细胞释放的组胺量,计算组胺释放率.结果:不同厂家来源的聚山梨酯-80与RBL-2H3细胞作用60 min后,细胞的组胺释放率与空白对照组相比均显著增加,在一定浓度范围内,组胺的释放随聚山梨酯-80浓度的增加而增加.结论:聚山梨酯-80可导致RBL-2H3肥大细胞脱颗粒,并存在着明显的量效关系,为研究聚山梨酯80致过敏反应机制提供了一定的依据.     

Prostaglandin D2 release from human skin mast cells in response to ionophore A23187

1. Cells were dispersed from human foreskin by proteolytic digestion and enriched or depleted in mast cell content by density gradient flotation on discontinuous gradients of Percoll. 2. Cells were harvested at six interfaces on the density gradient. Mast cell purity ranged from 0.6-85.0%, compared to 5.5% in the unfractionated cells. 3. Challenge of the cells with the calcium ionophore A23187 resulted in release of both histamine and prostaglandin D2 (PGD2). In fractions depleted of mast cells, histamine release and net PGD2 generation were low, but increasing amounts of these mediators were released as mast cell purity was increased up to 59%. 4. Overall, there was a significant correlation between the net generation of PGD2 and histamine (r = 0.9234, P less than 0.001) and also between PGD2 release and mast cell number (r = 0.7475, P less than 0.001). 5. These data provide the first direct evidence of the capacity of the human cutaneous mast cell to synthesize and release PGD2.     

Prostaglandins of the E series inhibit monoamine release via EP3 receptors: proof with the competitive EP3 receptor antagonist L-826,266

Prostaglandin E₂ (PGE₂) and its analogue sulprostone inhibit noradrenaline and serotonin release in rodent tissues. We examined whether the receptor involved is blocked by the EP₃ antagonist L-826,266, whether such receptors also occur on central cholinergic neurones and retinal dopaminergic cells, whether PGE₂ is produced by the degradation of the endocannabinoid virodhamine and whether EP₃ receptor activation stimulates ³⁵S-GTPγS binding. Transmitter release was studied as electrically evoked tritium overflow in superfused tissues preincubated with ³H-noradrenaline (which in the guinea pig retina labels dopaminergic cells), ³H-serotonin or ³H-choline. ³⁵S-GTPγS binding, a measure of G protein activation, was studied in mouse and guinea pig hippocampal membranes. L-826,266 antagonised the effect of sulprostone on noradrenaline release in the rat cortex, yielding a Schild plot-based pA₂ value of 7.56. Apparent pA₂ values in mouse cortex and rat vas deferens (noradrenaline release) and rat cortex (serotonin release) were 7.55, 7.87 and 7.67, respectively. PGE₂ did not affect acetylcholine release in rat brain and dopamine release in guinea pig retina. In seven mice tissues, noradrenaline release was inhibited by sulprostone but not affected by virodhamine. ³⁵S-GTPγS binding was not altered by sulprostone but stimulated by the cannabinoid agonist WIN 55,212-2. Prostaglandins of the E series inhibit monoamine release via EP₃ receptors at which L-826,266 is a competitive antagonist. EP₃ receptors that inhibit transmitter release are not present on central cholinergic neurones and retinal dopaminergic cells. Virodhamine is not converted to PGE₂. An EP₃ receptor model based on ³⁵S-GTPγS binding could not be identified.     

Prostaglandin E2 synthases

Prostaglandin E2 (PGE2) is widely distributed in various tissues, and exhibits various biologically important activities. PGE2 synthase (PGES) catalyzes conversion of COX-derived PGH2 to PGE2. It now appears that there are at least three distinct types of PGES in mammals. We identified two distinct glutathione-dependent PGESs. Cytosolic PGES (cPGES), known as p23, is constitutively and ubiquitously expressed and predominantly converts COX-1-derived PGH2 to PGE2. We find that the regulation of cPGES/p23 activity in cells depends on its association with hsp90. Microsomal PGES-1 (mPGES-1), identical to MGST1-L1, is an inducible perinuclear enzyme that is functionally linked with COX-2 in marked preference to COX-1. COX-2 and mPGES-1 are essential components for delayed PGE2 synthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer. Most recently, glutathione-nonspecific mPGES-2, homologous to glutaredoxin and thioredoxin, was identified. These PGESs seem to be a potential novel target for drug development.