Prostaglandin E(2) receptors, EP2 and EP4, differentially modulate TNF-alpha and IL-6 production induced by lipopolysaccharide in mouse peritoneal neutrophils

The expression and function of prostaglandin (PG) E(2) receptors were examined in mouse neutrophils exudated into the peritoneal cavity by casein treatment. Expressions of the EP2 and EP4 receptors were detected in neutrophils by Northern blot, but those of EP1 and EP3 receptors were not detected by RT-PCR. EP2-selective agonist, ONO-AE1-259, and EP4-selective agonist, ONO-AE1-329, stimulated cAMP formation in the cells. PGE(2) affected the TNF-alpha and IL-6 production in lipopolysaccharide (LPS)-treated neutrophils; it suppressed the TNF-alpha production and enhanced the IL-6 production. The PGE(2) effects were mimicked by dibutyryl cAMP. This is the first study of the enhancement of IL-6 production by cAMP-elevating reagents in neutrophils. Using neutrophils from EP2- and EP4-deficient mice in combination with EP2- and EP4-selective agonists, it was found that the augmentation of IL-6 was mediated mainly by the EP2 receptor and the suppression of TNF-alpha by the EP4 receptor and partially by the EP2 receptor. These findings indicate that casein-induced peritoneal neutrophils express Gs-coupled PGE(2) receptors, EP2 and EP4, which might differentially regulate the LPS-induced production of TNF-alpha and IL-6.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.     

Prostaglandin E(2) inhibits IL-18-induced ICAM-1 and B7.2 expression through EP2/EP4 receptors in human peripheral blood mononuclear cells

Costimulatory molecules play important roles in immune responses. In the present study we investigated the effects of PGE(2) on the expression of ICAM-1, B7.1, and B7.2 on monocytes in IL-18-stimulated PBMC using FACS analysis. Addition of PGE(2) to PBMC inhibited ICAM-1 and B7.2 expression elicited by IL-18 in a concentration-dependent manner. We examined the involvement of four subtypes of PGE(2) receptors, EP1, EP2, EP3, and EP4, in the modulatory effect of PGE(2) on ICAM-1 and B7.2 expression elicited by IL-18, using subtype-specific agonists. ONO-AE1-259-01 (EP2R agonist) inhibited IL-18-elicited ICAM-1 and B7.2 expression in a concentration-dependent manner with a potency slightly less than that of PGE(2), while ONO-AE1-329 (EP4R agonist) was much less potent than PGE(2). The EP2/EP4R agonist 11-deoxy-PGE(1) mimicked the effect of PGE(2) with the same potency. ONO-D1-004 (EP1R agonist) and ONO-AE-248 (EP3R agonist) showed no effect on IL-18-elicited ICAM-1 or B7.2 expression. These results indicated that EP2 and EP4Rs were involved in the action of PGE(2). Dibutyryl cAMP and forskolin down-regulated ICAM-1 and B7.2 expression in IL-18-stimulated monocytes. As EP2 and EP4Rs are coupled to adenylate cyclase, we suggest that PGE(2) down-regulates IL-18-induced ICAM-1 and B7.2 expression in monocytes via EP2 and EP4Rs by cAMP-dependent signaling pathways. The fact that anti-B7.2 as well as anti-ICAM-1 Ab inhibited IL-18-induced cytokine production implies that PGE(2) may modulate the immune response through regulation of the expression of particular adhesion molecules on monocytes via EP2 and EP4Rs.     

Endothelin-1-induced prostaglandin E2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion

Cyclooxygenase (COX)-1- and COX-2-derived prostaglandins are implicated in the development and progression of several malignancies. We have recently demonstrated that treatment of ovarian carcinoma cells with endothelin-1 (ET-1) induces expression of both COX-1 and COX-2, which contributes to vascular endothelial growth factor (VEGF) production. In this study, we show that in HEY and OVCA 433 ovarian carcinoma cells, ET-1, through the binding with ETA receptor (ETAR), induces prostaglandin E2 (PGE2) production, as the more represented PG types, and increases the expression of PGE2 receptor type 2 (EP2) and type 4 (EP4). The use of pharmacological EP agonists and antagonists indicates that ET-1 and PGE2 stimulate VEGF production principally through EP2 and EP4 receptors. At the mechanistic level, we prove that the induction of PGE2 and VEGF by ET-1 involves Src-mediated epidermal growth factor receptor transactivation. Finally, we demonstrate that ETAR-mediated activation of PGE2-dependent signaling participates in the regulation of the invasive behavior of ovarian carcinoma cells by activating tumor-associated matrix metalloproteinase. These results implicate EP2 and EP4 receptors in the induction of VEGF expression and cell invasiveness by ET-1 and provide a mechanism by which ETAR/ET-1 can promote and interact with PGE2-dependent machinery to amplify its proangiogenic and invasive phenotype in ovarian carcinoma cells. Pharmacological blockade of ETAR can therefore represent an additional strategy to control PGE2 signaling, which has been associated with ovarian carcinoma progression.     

The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages

The expression and regulation of the PGE receptors, EP(2) and EP(4), both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP(4) but not EP(2) was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP(4) was down-regulated by LPS but not by medium change. PGE(2) increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP(4) agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP(2) agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-alpha and IL-12 production were equipotent with PGE(2) at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE(2) or dibutyryl cAMP alone, but not butaprost, reduced EP(4) expression, and indomethacin reversed the LPS-induced down-regulation of EP(4), indicating that the down-regulation of EP(4) is mediated by LPS-induced PG synthesis and EP(4) activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP(4) expression was found in the cells treated with PGE(2) alone, but not in LPS-treated cells. In contrast, up-regulation of EP(2) expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP(4) is involved mainly in the inhibition of cytokine release, and that the gene expression of EP(2) and EP(4) is differentially regulated during macrophage activation.     

Mechanisms of prostaglandin E2-induced interleukin-6 release in astrocytes: possible involvement of EP4-like receptors, p38 mitogen-activated protein kinase and protein kinase C.

The expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin-6 (IL-6) have all been suggested to propagate neuropathology in different brain disorders such as HIV-dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2-stimulated IL-6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2-induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2-induced IL-6 synthesis. This indicates that the cAMP pathway is not part of PGE2-induced signal transduction cascade leading to IL-6 release. The EP3/EP1-receptor agonist sulprostone failed to induce IL-6 release, suggesting an involvement of EP4-like receptors. PGE2-activated p38 mitogen-activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2-induced IL-6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL-6 via an EP4-like receptor by the activation of PKC and p38 MAPK via an EP4-like receptor independently of cAMP.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

Endothelin-1 stimulates human monocytes in vitro to release TNF-alpha , IL-1beta and IL-6

Increased plasma- and tissue levels of endothelin-1 (ET-1) during inflammatory diseases, have suggested a role of ET-1 in the pathophysiology of inflammatory reactions. The authors have studied the effect of ET-1 on cytokine release from monocytes and monocyte-derived macrophages. ET-1 increased secretion of TNF-alpha, IL-1beta and IL-6 in a dose- and time-dependent manner. Optimal ET-1 concentration ranged from 0.01 to 1 nM. The maximal response was a 200 to 400% increase in cytokine release. A time-course study revealed that the pattern of cytokines induced by ET-1 was different in monocytes and macrophages, although an early increase in TNF-alpha was observed in both monocyte and macrophage supernatants. In conclusion, ET-1 stimulates monocytes and macrophages to release cytokines thereby demonstrating a potential role for ET-1 in regulation of inflammatory responses.     

Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and NF-kappa B

In this study, we investigated the signaling pathway involved in IL-6 production caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused concentration- and time-dependent increases in IL-6, PGE(2), and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE(2) (EP2) antagonist (AH6809), a PGE(4) (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Furthermore, PGE(2), an EP2 agonist (butaprost), an EP2/PGE(3) (EP3)/EP4 agonist (misoprostol), and misoprostol in the presence of AH6809 all induced IL-6 production, whereas an EP1/EP3 agonist (sulprostone) did not. PGN caused time-dependent activations of IkappaB kinase alphabeta (IKKdbeta) and p65 phosphorylation at Ser(276), and these effects were inhibited by NS398 and KT5720. Both PGE(2) and 8-bromo-cAMP also caused IKKdbeta kinase alphabeta phosphorylation. PGN resulted in two waves of the formation of NF-kappaB-specific DNA-protein complexes. The first wave of NF-kappaB activation occurred at 10-60 min of treatment, whereas the later wave occurred at 2-12 h of treatment. The PGN-induced increase in kappaB luciferase activity was inhibited by NS398, AH6809, AH23848, KT5720, a protein kinase C inhibitor (Ro31-8220), and a p38 MAPK inhibitor (SB203580). These results suggest that PGN-induced IL-6 production involves COX-2-generated PGE(2), activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdbeta, kinase alphabeta, p65 phosphorylation, and NF-kappaB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.     

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.     

In vitro cytokine release by activated murine peritoneal macrophages: role of prostaglandins in the differential regulation of tumor necrosis factor alpha, interleukin 1, and interleukin 6

Distinct subpopulations of macrophages or differently activated macrophages display various functions in immune reactions. Some of their activities depend on specific sets of factors (i.e., cytokines and eicosanoids) produced by activated macrophages. We have studied the ability of murine (CBA/ca) peritoneal macrophages to selectively release tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and IL-6. We have found that the priming of cells (Mo) with different stimulants (thioglycolate vs. LPS) induces the release of particular cytokines by reactivated macrophages. The increased release of TNF-alpha correlates with lower levels of IL-1 and IL-6. We have also found that prostaglandin E2 (PGE2) and prostacyclin (PGI2) have opposing effects on the production of two of these cytokines. The release of TNF-alpha is inhibited by prostaglandins, whereas increased levels of PGE2 and PGI2 correlate with higher levels of IL-6.     

Interleukin-1 beta-induced expression of the prostaglandin E-receptor subtype EP3 in U373 astrocytoma cells depends on protein kinase C and nuclear factor-kappaB

Both interleukin-1beta (IL-1beta) and prostaglandins (PGs) are important mediators of physiological and pathophysiological processes in the brain. PGE2 exerts its effects by binding to four different types of PGE2 receptors named EP1-EP4. EP3 has found to be expressed in neurons, whereas expression of EP3 in glial cells has not been reported in the brain yet. Here we describe IL-1beta-induced EP3 receptor expression in human astrocytoma cells, primary astrocytes of rat and human origin and in rat brain. Using western blot, we found a marked up-regulation of EP3 receptor synthesis in human and rat primary glial cells. Intracerebroventricular administration of IL-1beta stimulated EP3 receptor synthesis in rat hippocampus. The analysis of involved signal transduction pathways by pathway-specific inhibitors revealed an essential role of protein kinase C and nuclear factor-kappaB in astrocytic IL-1beta-induced EP3 synthesis. Our data suggest that PGE2 signaling in the brain may be altered after IL-1beta release due to up-regulation of EP3 receptors. This might play an important role in acute and chronic conditions such as cerebral ischemia, traumatic brain injury, HIV-encephalitis, Alzheimer's disease and prion diseases in which a marked up-regulation of IL-1beta is followed by a prolonged increase of PGE2 levels in the brain.     

Induction of macrophage TNF alpha, IL-1, IL-6, and PGE2 production by DTH-initiating factors

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different, antigen-specific, Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This cell clone, WP-3.27, releases an antigen-specific factor (OX-F) that sensitizes mast cells such that specific antigen challenge will induce serotonin release which mediates the early phase of DTH. In normal mice contact sensitized with picryl chloride (PCl), a similar polyclonal factor (PCl-F) has a similar activity and is also known to bind to macrophages. Thus, we measured macrophage production of TNF alpha, IL-1, IL-6, and PGE2 in response to the hapten affinity-purified DTH-initiating factors OX-F and PCl-F. Both factors induced significant release of each cytokine and PGE2. The production of TNF alpha, IL-1, and IL-6 was measured by bioassays. Northern blot analysis showed rapid accumulation of cytokine mRNA (2-4 hr), while maximal production of PGE2 occurred at approximately 8 hr. These macrophage activating properties of OX-F and PCl-F were not due to contamination with LPS as determined by the low levels of LPS present in OX-F and PCl-F and by the failure of polymyxin B to inhibit factor-induced PGE2 and TNF alpha production. Also, macrophage activation was shown not to be due to the action of several lymphokines known to be produced by WP3.27. Separation of OX-F and PCl-F by preparative isoelectric focusing showed a similar pattern: there were two major peaks of PGE2-inducing activity observed for both factors (for PCl-F at pI of 2-3 and 5.0, and for OX-F at pI of 3.5-4 and 5.0), but not for a sham factor produced by WEHI-3 cells. The ability of DTH-initiating factors to rapidly induce macrophage cytokine release and PGE2 synthesis 4-6 hr later may suggest a role for these mediators during the respective early vascular and late cellular phases of inflammation in DTH.     

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.     

Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1beta of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP(2), EP(3), and EP(4) receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1beta promoted the elaboration of G-CSF, which was augmented by PGE(2). Cicaprost (IP receptor agonist) was approximately equiactive with PGE(2), whereas PGD(2), PGF(2alpha), and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP(1) receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE(2), the EP receptor agonists 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3) selective), 17-phenyl-omega-trinor PGE(2) (EP(1) selective), ONO-AE1-259, and butaprost (both EP(2) selective) were full agonists at enhancing G-CSF release. AH 6809 (10 microM) and L-161,982 (2 microM), which can be used in HASMC as selective EP(2) and EP(4) receptor antagonists, respectively, failed to displace to the right the PGE(2) concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE(2)-induced G-CSF release. Augmentation of G-CSF release by PGE(2) was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE(2) facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP(2) and EP(4) prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.     

Prostaglandin E2 augments IL-10 signaling and function

In inflamed joints of rheumatoid arthritis, PGE(2) is highly expressed, and IL-10 and IL-6 are also abundant. PGE(2) is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE(2) on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE(2) significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE(2) suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE(2)-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE(2)-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE(2) selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE(2) may modulate immune responses by alteration of cytokine signaling in THP-1 cells.     

EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.     

Prostaglandin E2 aggravates gastric mucosal injury induced by histamine in rats through EP1 receptors

We demonstrated that prostaglandin (PG) E2 aggravates gastric mucosal injury caused by histamine in rats, and investigated using various EP agonists which EP receptor subtype is involved in this phenomenon. Rats were used after 18 hr fasting. Histamine (80 mg/kg) dissolved in 10% gelatin, was given s.c., either alone or in combination with i.v. administration of PGE2 or various EP agonists such as 17-phenyl PGE2 (EP1), butaprost (EP2), sulprostone (EP1/EP3), ONO-NT012 (EP3) and ONO-AE1-329 (EP4). The animals were killed 4 hr later, and the mucosa was examined for lesions. The mucosal permeability was determined using Evans blue (1%). Histamine alone induced few lesions in the gastric mucosa within 4 hr. PGE2 dose-dependently worsened the lesions induced by histamine, the response being inhibited by tripelennamine but not cimetidine. The effect of PGE2 was mimicked by 17-phenyl PGE2 and sulprostone, but not other EP agonists, including EP2, EP3, and EP3/EP4 agonists. The mucosal vascular permeability was slightly increased by histamine, and this response was markedly enhanced by co-administration of 17-phenyl PGE2 as well as PGE2. The mucosal ulcerogenic and vascular permeability responses induced by histamine plus PGE2 were both suppressed by pretreatment with ONO-AE829, the EP1 antagonist. These results suggest that PGE2 aggravates histamine-induced gastric mucosal injury in rats. This action of PGE2 is mediated by EP1 receptors and functionally associated with potentiation of the increased vascular permeability caused by histamine through stimulation of H1-receptors.