IL-1beta inhibits intestinal smooth muscle proliferation in an organ culture system: involvement of COX-2 and iNOS induction in muscularis resident macrophages

Intestinal inflammation causes hyperplasia of smooth muscle that leads to thickening of the smooth muscle layer, resulting in dysmotility. IL-1beta is a proinflammatory cytokine that plays a central role in intestinal inflammation. In this study, to evaluate the effect of IL-1beta on proliferation of ileal smooth muscle cells in vivo, we utilized an organ culture system. When rat ileal smooth muscle tissue was cultured under serum-free conditions for 3 days, most smooth muscle cells maintained their arrangement and kept their contractile phenotype. When 10% FBS was added, an increased number of smooth muscle cells per unit area was observed. Moreover, immunohistochemical staining for PCNA demonstrated that FBS induced proliferation of smooth muscle cells. IL-1beta inhibited the proliferative effect of FBS. Furthermore, IL-1beta upregulated inducible nitric oxide (NO) synthase and cyclooxygenase-2 mRNA and protein and thus stimulated NO and PGE(2) productions. Moreover, exogenously applied NO and PGE(2) inhibited the increase of bromodeoxyuridine-positive cells stimulated with FBS. Immunostaining revealed that the majority of cyclooxygenase-2 and inducible NO synthase was located in the dense network of macrophages resident in the muscularis, which were immunoreactive to ED2. Based on these findings, IL-1beta acts as an anti-proliferative mediator, which acts indirectly through the production of PGE(2) and NO from resident macrophage within ileal smooth muscle tissue.     

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.     

Differential effects of prostaglandin E2 on contractions of airway smooth muscle

In an in vitro muscle bath, the active tension generated by strips of canine tracheal smooth muscle responding to cumulative additions of either histamine (10(-8) to 10(-3) M) or acetylcholine (10(-9) to 10(-3) M) was measured in the absence and presence of prostaglandin E2 (PGE2) (10(-6) to 10(-5) M). When contractile responses of equal magnitude were compared, the contractions elicited by acetylcholine were resistant to the inhibitory effects of PGE2, relative to comparable contractions elicited by histamine. To assess the role of adenylate cyclase in determining the different responses to histamine and acetylcholine in the presence of PGE2, we assayed adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and found that acetylcholine, but not histamine, decreased PGE2-stimulated adenylate cyclase activity by 48 +/- 2% (mean +/- SE; n = 5). However, in other experiments, we found that even large pharmacological increases in tissue adenosine 3',5'-cyclic monophosphate (cAMP) content only partially inhibited muscarinic tone. Also, exogenously applied analogues of cyclic AMP inhibited contractions induced by histamine more effectively than comparable contractions induced by acetylcholine. We concluded that acetylcholine decreased adenylate cyclase activity in membranes prepared from canine tracheal smooth muscle and that this effect may have contributed to, but did not completely account for, the relative resistance of muscarinic contractions to the inhibitory effects of PGE2.     

Regulatory features of interleukin-1beta-mediated prostaglandin E2 synthesis in airway smooth muscle

Exposure of airway smooth muscle (ASM) cells to the cytokine IL-1beta results in an induction of PGE2 synthesis that affects numerous cell functions. Current dogma posits induction of COX-2 protein as the critical, obligatory event in cytokine-induced PGE2 production, although PGE2 induction can be inhibited without a concomitant inhibition of COX-2. To explore other putative regulatory features we examined the role of phospholipase A2 (PLA2) and PGE synthase (PGES) enzymes in IL-1beta-induced PGE2 production. Treatment of human ASM cultures with IL-1beta caused a time-dependent induction of both cytosolic PLA2 (cPLA2) and microsomal PGES (mPGES) similar to that observed for COX-2. Regulation of COX-2 and mPGES induction was similar, being significantly reduced by inhibition of p42/p44 or p38, whereas cPLA2 induction was only minimally reduced by inhibition of p38 or PKC. COX-2 and mPGES induction was subject to feed-forward regulation by PKA, whereas cPLA2 induction was not. SB-202474, an SB-203580 analog lacking the ability to inhibit p38 but capable of inhibiting IL-1beta-induced PGE2 production, was effective in inhibiting mPGES but not COX-2 or cPLA2 induction. These data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-beta in human ASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. mPGES induction and, possibly, cPLA2 induction appear to cooperate with COX-2 to determine IL-1beta-mediated PGE2 production in human ASM cells.     

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

Interleukin-1 is cytoprotective, antisecretory, stimulates PGE2 synthesis by the stomach, and retards gastric emptying

Human recombinant interleukin 1 beta (IL-1) administered intraperitoneally to rats produced the following gastric effects: 1. It was cytoprotective, preventing gastric mucosal necrosis produced by oral administration of one ml of absolute ethanol to fasted animals. The ED50 was 1200 units/kg (110 ng per animal). IL-1 was 125 times more potent than prostaglandin E2 (on a weight basis), and 6,000 times more potent (on a molar basis). 2. The cytoprotective effect of IL-1 was blocked by indomethacin (inhibitor of prostaglandin synthesis) and by IRAP (a specific interleukin-1 receptor antagonist protein). IRAP did not inhibit cytoprotection induced by PGE2. 3. IL-1 prevented the formation of gastric erosions induced by aspirin. 4. IL-1 inhibited gastric secretion (volume, acid concentration and output), in the pylorus-ligated rat, with an ED50 of 300 units/kg (3.2 ng per animal). 5. Indomethacin and IRAP blocked the antisecretory effect of IL-1. 6. IL-1 retarded gastric emptying, an effect blocked by IRAP, but not by indomethacin. 7. IL-1 increased synthesis of prostaglandin E2 by the gastric mucosa by 111%. IL-1 is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory, and delay gastric emptying. It appears to act mostly by stimulating the synthesis of prostaglandins by the stomach. These studies suggest that the stomach possesses IL-1 receptors. These are probably located on parietal cells (that produce acid), on prostaglandin-producing cells, on smooth muscle cells (responsible for gastric emptying), and on as yet unidentified cells involved in gastric cytoprotection. Both IL-1 and IRAP, being natural substances, may play a physiological role in the maintenance of gastric mucosal integrity, and in the regulation of acid secretion and gastric motility.     

Interleukin-1-mediated PGE2 production and sphingomyelin metabolism. Evidence for the regulation of cyclooxygenase gene expression by sphingosine and ceramide.

We recently demonstrated that sphingosine enhances interleukin-1 beta (IL-1)-mediated prostaglandin E2 (PGE2) production in human dermal fibroblasts (Ballou, L. R., Barker, S. C., Postlethwaite, A. E., and Kang, A. H. (1990) J. Immunol. 145, 4245-4251). Because sphingosine and ceramide are interconvertable, we extended previous studies by treating cells with C2-ceramide (C2-cer), a membrane-soluble analogue of ceramide, and found that C2-cer stimulates IL-1-mediated PGE2 production to the same degree as sphingosine. In an effort to elucidate the mechanistic basis by which sphingosine and C2-cer affect PGE2 production, we examined the effect of these molecules on the expression of genes encoding cyclooxygenase (EC 1.14.99.1, Cox) and phospholipase A2 (EC 3.1.1.4, PLA2), the rate-limiting enzymes in PGE2 biosynthesis. We found that sphingosine and C2-cer treatment resulted in an 8-fold induction of Cox mRNA within 1-2 h which declined thereafter; concomitant changes in Cox protein were also observed. In contrast, expression of phospholipase A2 remained unaltered. We also found that IL-1-mediated PGE2 production was dramatically enhanced in cells treated simultaneously with sphingomyelinase which led us to directly test the effect of IL-1 on sphingomyelin turnover. IL-1 treatment induced the hydrolysis of a significant fraction of prelabeled sphingomyelin which was accompanied by increased levels of intracellular ceramide. Taken together, our results suggest that enhanced Cox expression may account for the observed enhancement of IL-1-mediated PGE2 production by sphingosine and C2-cer. These data also suggest that endogenous sphingomyelin metabolites, generated in response to IL-1, may play an important role in IL-1 signal transduction.     

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.     

Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells

Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.     

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.     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.     

Cytokines regulate beta-2-adrenergic receptor responsiveness in airway smooth muscle via multiple PKA- and EP2 receptor-dependent mechanisms

Beta2AR desensitization in airway smooth muscle (ASM) mediated by airway inflammation has been proposed to contribute to asthma pathogenesis and diminished efficacy of beta-agonist therapy. Mechanistic insight into this phenomenon is largely conceptual and lacks direct empirical evidence. Here, we employ molecular and genetic strategies to reveal mechanisms mediating cytokine effects on ASM beta2AR responsiveness. Ectopic expression of inhibitory peptide (PKI-GFP) or a mutant regulatory subunit of PKA (RevAB-GFP) effectively inhibited intracellular PKA activity in cultured human ASM cells and enhanced beta2AR responsiveness by mitigating both agonist-specific (beta-agonist-mediated) desensitization and cytokine (IL-1beta and TNF-alpha)-induced heterologous desensitization via actions on multiple targets. In the absence of cytokine treatment, PKA inhibition increased beta2AR-mediated signaling by increasing both beta2AR-G protein coupling and intrinsic adenylyl cyclase activity. PKI-GFP and RevAB-GFP expression also conferred resistance to cytokine-promoted beta2AR-G protein uncoupling and disrupted feed-forward mechanisms of PKA activation by attenuating the induction of COX-2 and PGE2. Cytokine treatment of tracheal ring preparations from wild-type mice resulted in a profound loss of beta-agonist-mediated relaxation of methacholine-contracted rings, whereas rings from EP2 receptor knockout mice were largely resistant to cytokine-mediated beta2AR desensitization. These findings identify EP2 receptor- and PKA-dependent mechanisms as the principal effectors of cytokine-mediated beta2AR desensitization in ASM.     

Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo

Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.     

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.     

Prostaglandin receptors: advances in the study of EP3 receptor signaling

Prostaglandin (PG) E(2) produces a broad range of physiological and pharmacological actions in diverse tissues through specific receptors on plasma membranes for maintenance of local homeostasis in the body. PGE receptors are divided into four subtypes, EP1, EP2, EP3, and EP4, which have been identified and cloned. These EP receptors are members of the G-protein coupled receptor family. Among these subtypes, the EP3 receptor is unique in its ability to couple to multiple G proteins. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via G(i) activation, and in Ca(2+)-mobilization through G(beta)(gamma) from G(i). Along with G(i) activation, the EP3 receptor can stimulate cAMP production via G(s) activation. Recent evidence indicates that the EP3 receptor can augment G(s)-coupled receptor-stimulated adenylyl cyclase activity, and can also be coupled to the G(13) protein, resulting in activation of the small G protein Rho followed by morphological changes in neuronal cells. This article focuses on recent studies on the novel pathways of EP3 receptor signaling.     

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.