Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J2

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7beta-hydroxy-epiandrosterone in rats with colitis

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Regulation of prostaglandin E synthases: effects of siRNA-mediated inhibition of microsomal prostaglandin E synthase-1

Prostaglandin E2 (PGE2) is a key mediator involved in several inflammatory conditions. In this study, we investigated the expression and regulation of the terminal PGE2 synthesizing enzyme prostaglandin E synthases (mPGES-1, mPGES-2 and cPGES) in gingival fibroblasts stimulated with pro-inflammatory cytokines. We used siRNA knockdown of mPGES-1 to elucidate the impact of mPGES-1 inhibition on mPGES-2 and cPGES expression, as well as on PGE2 production. The cytokines TNFalpha and IL-1beta increased protein expression and activity of mPGES-1, accompanied by increased COX-2 expression and PGE2 production. The isoenzymes mPGES-2 and cPGES, constitutively expressed at mRNA and protein levels, were unaffected by the pro-inflammatory cytokines. We show for the first time that treatment with mPGES-1 siRNA down-regulated the cytokine-induced mPGES-1 protein expression and activity. Interestingly, mPGES-1 siRNA did not affect the cytokine-stimulated PGE2 production, whereas PGF(2alpha) levels were enhanced. Neither mPGES-2 nor cPGES expression was affected by siRNA silencing of mPGES-1. Dexamethasone and MK-886 both inhibited the cytokine-induced mPGES-1 expression while mPGES-2 and cPGES expression remained unaffected. In conclusion, mPGES-1 siRNA down-regulates mPGES-1 expression, and neither mPGES-2 nor cPGES substituted for mPGES-1 in a knockdown setting in gingival fibroblasts. Moreover, mPGES-1 siRNA did not affect PGE2 levels, whereas PGF(2alpha) increased, suggesting a compensatory pathway of PGE2 synthesis when mPGES-1 is knocked down.     

Cyclooxygenase-2, Not Microsomal Prostaglandin E Synthase-1, Is the Mechanism for Interleukin-1β-induced Prostaglandin E2 Production and Inhibition of Insulin Secretion in Pancreatic Islets

Arachidonic acid is converted to prostaglandin E(2) (PGE(2)) by a sequential enzymatic reaction performed by two isoenzyme groups, cyclooxygenases (COX-1 and COX-2) and terminal prostaglandin E synthases (cPGES, mPGES-1, and mPGES-2). mPGES-1 is widely considered to be the final enzyme regulating COX-2-dependent PGE(2) synthesis. These generalizations have been based in most part on experiments utilizing gene expression analyses of cell lines and tumor tissue. To assess the relevance of these generalizations to a native mammalian tissue, we used isolated human and rodent pancreatic islets to examine interleukin (IL)-1β-induced PGE(2) production, because PGE(2) has been shown to mediate IL-1β inhibition of islet function. Rat islets constitutively expressed mRNAs of COX-1, COX-2, cPGES, and mPGES-1. As expected, IL-1β increased mRNA levels for COX-2 and mPGES-1, but not for COX-1 or cPGES. Basal protein levels of COX-1, cPGES, and mPGES-2 were readily detected in whole cell extracts but were not regulated by IL-1β. IL-1β increased protein levels of COX-2, but unexpectedly mPGES-1 protein levels were low and unaffected. In microsomal extracts, mPGES-1 protein was barely detectable in rat islets but clearly present in human islets; however, in neither case did IL-1β increase mPGES-1 protein levels. To further assess the importance of mPGES-1 to IL-1β regulation of an islet physiologic response, glucose-stimulated insulin secretion was examined in isolated islets of WT and mPGES-1-deficient mice. IL-1β inhibited glucose-stimulated insulin secretion equally in both WT and mPGES-1(-/-) islets, indicating that COX-2, not mPGES-1, mediates IL-1β-induced PGE(2) production and subsequent inhibition of insulin secretion.     

Legionella pneumophila-induced PKCalpha-, MAPK-, and NF-kappaB-dependent COX-2 expression in human lung epithelium

Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE(2) synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E(2) (PGE(2)) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE(2) production in pulmonary epithelial cells. Legionella induced the release of PGE(2) in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA(2) activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE(2) release in A549 cells. L. pneumophila induced the degradation of IkappaBalpha and activated NF-kappaB. Inhibition of IKK blocked L. pneumophila-induced PGE(2) release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCalpha was observed in infected cells. PKCalpha and p38 and p42/44 MAP kinase inhibitors reduced PGE(2) release and COX-2 expression. In summary, PKCalpha and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE(2) release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.     

Platelet-derived growth factor-induced arachidonic acid release for enhancement of prostaglandin E(2) synthesis in human gingival fibroblasts pretreated with interleukin-1beta

Platelet-derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E(2) (PGE(2)) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time- and dose-dependent manner, and simultaneously induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), but less provoked PGE(2) release and cyclooxygenase-2 (COX-2) mRNA expression. When [Ca(2+)](i) was increased by Ca(2+)-mobilizing reagents, arachidonic acid release was increased. The PDGF-induced arachidonic acid release and increase in [Ca(2+)](i) were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre-stimulated with interleukin-1beta (IL-1beta), PDGF clearly increased PGE(2) release. The PDGF-induced PGE(2) release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL-1beta, arachidonic acid strongly enhanced PGE(2) release and COX-2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca(2+)](i) via tyrosine kinase activation, and which contributes to PGE(2) production via COX-2 expression in HGF primed with IL-1beta.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

Cyclooxygenase-1 and -2 enzymes differentially regulate the brain upstream NF-kappa B pathway and downstream enzymes involved in prostaglandin biosynthesis

We have recently reported that cyclooxygenase (COX)-2-deficiency affects brain upstream and downstream enzymes in the arachidonic acid (AA) metabolic pathway to prostaglandin E2 (PGE2), as well as enzyme activity, protein and mRNA levels of the reciprocal isozyme, COX-1. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and - 2) in wild type and COX-1(-/-) mice. We found that brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1(-/-) mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the NF-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. The mechanism of NF-kappaB activation in the COX-1(-/-) mice involved the up-regulation of protein expression of the p50 and p65 subunits of NF-kappaB, as well as the increased protein levels of phosphorylated IkappaBalpha and of phosphorylated IKKalpha/beta. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2, to overcome defects in brain AA cascade.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.     

The regulation of PGE(2) biosynthesis in MG-63 osteosarcoma cells by IL-1 and FGF is cell density-dependent

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1beta (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE(2) production compared to controls. While treatment with FGF-1 alone did not affect PGE(2) biosynthesis, it enhanced the formation of PGE(2) by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE(2) biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA(2) (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA(2) protein (>3-fold) and PGHS-2 protein (>1. 5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA(2) expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA(2) expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE(2) biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE(2) induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE(2) biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell-cell or cell-extracellular matrix interactions.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calcium

Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582-1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production.     

Differential modulation of COX-2 expression in A549 airway epithelial cells by structurally distinct PPAR(gamma) agonists: evidence for disparate functional effects which are independent of NF-(kappa)B and PPAR(gamma)

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.