<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> induces prostaglandin E<Subscript>2</Subscript> synthesis in macrophages via signal pathways for calcium-dependent arachidonic acid production and PKC-dependent induction of cyclooxygenase-2

In this study, the intracellular signaling pathway of PGE₂ synthesis in macrophages (RAW264.7) induced by Toxoplasma gondii was investigated. The T. gondii-induced PGE₂ production in macrophages increased in a time-dependent manner, as PGE₂ induction began at 4 h, peaked at 12 h, and then plateaued at a high level. COX-2 mRNA in macrophages was detectable as early as 4 h after treatment; the maximal expression was observed at 8 h. The earliest induction of COX-2 protein occurred at 4 h and peaked at 16 h; meanwhile, COX-1 mRNA level and protein production remained unchanged throughout. Indomethacin and nimesulide inhibited tachyzoite-induced PGE₂ production and COX-2 mRNA expression in macrophages but they had no significant effect on COX-2 protein expression. EGTA, TFP and BAPTA/AM inhibited both arachidonic acid (AA) and PGE₂ production without effecting COX-2 protein expression, but verapamil inhibited neither AA nor PGE₂ production. H7 was found to inhibit PGE₂ production, and COX-2 mRNA expression and protein expression by tachyzoite or LPS stimulated macrophages in a dose-dependent manner. Our results demonstrate that T. gondii induces PGE₂ biosynthesis in RAW264.7 macrophages by regulating AA production through a calcium-dependent pathway and induction of COX-2 expression by a PKC-dependent pathway.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

Enhancement of activated β1-integrin expression by prostaglandin E2 via EP receptors in isolated human coronary arterial endothelial cells: implication for the treatment of Kawasaki disease

Objective:  Plasma prostaglandin E₂ (PGE₂) levels are markedly elevated in acute Kawasaki disease (KD). We evaluated the function of the EP receptors in the expression of activated β₁-integrin stimulated by PGE₂ in human coronary arterial endothelial cells (HCAEC). Methods:  We determined the mRNA expression of the PGE₂ receptors, EP receptors (EP_1-4) in HCAEC by RT-PCR and protein expression by Western blotting. We evaluated the function of the EP receptors in the expression of activated β₁-integrin stimulated by PGE₂ in HCAEC, using antagonists and agonists of the EP receptors, by flow cytometry. Results:  RT-PCR revealed mRNAs for all four EP receptors in HCAEC. Western blotting demonstrated EP₁, EP₂ and EP₃ expression in HCAEC. The EP₂ and EP₃ agonists enhanced the expression of activated β₁-integrin in HCAEC. The potency of the EP₂ agonist was significantly greater than that of the EP₃ agonist. Pretreatment with the EP₁, EP₂ and EP₃ antagonists inhibited the expression of activated β₁-integrin induced by PGE₂ in HCAEC. The potency of the EP₂ antagonist was significantly greater than that of the EP₁ and EP₃ antagonists. Conclusions:  Our results suggest that PGE₂ mainly induces the activation of β₁-integrins via the EP₂ receptor in HCAEC. Our results further suggest that the EP₂ antagonist modulates the inflammatory response during KD vasculitis. Received 13 August 2008; returned for revision 15 September 2008; returned for final revision 28 October 2008; accepted by G. Wallace 24 November 2008     

Effects of platelet release products on neutrophilic activity in human whole blood

Objectives:  To investigate the effects of Platelet release products (PRPr) and their phagocytosis activators including ATP, ADP, and macromolecular activators of phagocytosis (MAPPs) on phagocytosis and oxidative burst activity by neutrophils in human whole blood. Methods:  A whole blood-based flow cytometric assay was used to assess neutrophilic activity. Escherichia coli were used as the target organisms. Results:  Phagocytosis and oxidative burst by neutrophils were markedly increased after treatment with PRPr or MAPPs  + ATP + ADP. The phagocytosis activation was more prominent in the first minute, and displayed a maximum enhancement of 4-fold. The maximum augmentation of oxidative burst was 5-fold which occurred at 5 mins. A striking finding was that the effect of MAPPs was evident from 5 mins onwards and increased with further incubation in both the phagocytosis and oxidative burst assays. Conclusion:  These data suggest that the neutrophilic activity enhanced by PRPr depends more on ATP and ADP during the early phase and on MAPPs during the later phase of the phagocytic process. The present study reveals an important role for PRPr and their phagocytosis activators in the enhancement of neutrophilic activity in human whole blood. Received 30 September 2008; returned for revision 28 November 2008; received from final revision 30 October 2008; accepted by Graham R. Wallace 9 December 2008     

The COX-2 inhibitor, rofecoxib, ameliorates dextran sulphate sodium induced colitis in mice

Objective: We have evaluated the efficacy of the selective cyclo-oxygenase (COX)-2 inhibitor, rofecoxib, for the prevention of experimental colitis.Material and methods: To induce colitis BALB/c mice received 5% dextran sulphate sodium (DSS) in their drinking water continuously for 7 days. Rofecoxib (2.5–10 mg/kg body weight, p.o.) was administered throughout the treatment period with DSS. Colitis was quantified by a clinical damage score, colon length, weight loss, stool consistency and rectal bleeding. Inflammatory response was assessed by neutrophil infiltration, determined by histology and myeloperoxidase (MPO) activity. Interleukin (IL)-1, prostaglandin (PG)E₂ and PGD₂ levels in colon mucosa and the immunohistochemical expression of COX-1 and –2 were also studied.Results: The COX-2 inhibitor ameliorated severe colitis, reduced the degree of inflammation through reduction of neutrophil infiltration and IL-1 levels. PGE₂, and PGD₂ synthesis were significantly reduced in DSS-treated groups. Indeed, treatment with rofecoxib diminished the lost of COX-1 caused by DSS in the crypt epithelium whereas expression of COX-2 remained unaffected.Conclusions: Rofecoxib is protective in acute DSS – induced colitis, probably by reducing neutrophil infiltration, inhibiting up-regulation of IL-1 and returning to normal COX-1 expression in the inflamed colonic mucosa.Received 19 April 2004; returned for revision 17 June 2004; accepted by I. Ahnfelt-Rønne 23 November 2004     

META060 inhibits multiple kinases in the NF-κB pathway and suppresses LPS – mediated inflammation in vitro and ex vivo

Objective:  We investigated whether a novel candidate META060 targeted the inflammatory signal transduction without affecting constitutive COX-2 enzymatic activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We also investigated its bioavailability in humans and its anti-inflammatory effect ex vivo. Methods:  We measured prostaglandin E₂, nitric oxide, TNFα and IL-6 by ELISA, COX-2 protein by Western blot, NF-κB nuclear binding by electrophoretic mobility shift assays, and NF-κB activation by luciferase assay. Kinase inhibitions were measured by cell-free assays. Bioavailability was tested in 4 human subjects consuming 940 mg META060. LPS-activated TNFα and IL-6 were measured in peripheral blood mononuclear cells (PBMC) isolated from 1 subject up to 6 hours post administration. Results:  META060 dose-dependently inhibited prostaglandin E₂ and nitric oxide formation, COX-2 abundance, and NF-κB activation. In cell-free assays, META060 inhibited multiple kinases in the NF-κB signaling pathway, including BTK, PI3K, and GSK3. META060 was detected in the plasma of the subjects; isolated PBMC were resistant to LPS-stimulated TNFα and IL-6 production. Conclusion:  Without inhibiting COX-2 enzyme, META060 reduces the inflammation by inhibiting multiple kinases involved in NF-κB pathway, and may have potential as a safe anti-inflammatory therapeutic. Received 28 July 2008; returned for revision 30 October 2008; received from final revision 4 November 2008; accepted by J. Di Battista 13 November 2008     

Opsonizing activities of IgG,IgM antibodies and the C3b inactivator-cleaved third component of complement in macrophage phagocytosis

Phagocytosis of SRBC by guinea-pig peritoneal macrophages is enhanced by opsonizing IgG antibody alone. IgM antibody requires the presence of bound C3. Treatment of C3b coated SRBC with purified C3b inactivator (yielding EA_IgM C1423d) does not reduce attachment to, and phagocytosis by, peritoneal macrophages. This finding suggests the existence of a C3d receptor on peritoneal macrophages. EC43b intermediates which have been produced by removing IgM antibody by mercaptoethanol treatment and by subsequent removal of C1 and C2, are phagocytosed despite the absence of IgM antibody. Furthermore, treatment of EC43b with C3b inactivator does not change phagocytosis. Thus, IgM antibody does not appear to be a necessary prerequisite for the stimulation of phagocytosis, C3b or C3d alone being sufficient.     

Induction of heme oxygenase-1 attenuates lipopolysaccharide-induced cyclooxygenase-2 expression in mouse brain endothelial cells

Background  

Prostaglandin E₂ (PGE₂), an arachidonic acid metabolite converted by cyclooxygenase-2 (COX-2), plays important roles in the regulation of endothelial functions in response to bacterial infection. The enzymatic activity of COX-2 can be down-regulated by heme oxygenase-1 (HO-1) induction. However, the mechanisms underlying HO-1 modulating COX-2 protein expression are not known.     

The effect of BisGMA on cyclooxygenase-2 expression,PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE₂ production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 μm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE₂ production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE₂ production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE₂ production.     

Evidence that 13–14 di-hydro, 15-keto prostaglandin D2-induced airway eosinophilia in guinea-pigs is independent of interleukin-5

Objective and design:  Prostaglandin D₂ (PGD₂) has been shown to cause eosinophil, basophil and Th2 cell chemotaxis in vitro and in vivo through an action on the prostaglandin CRTH2 receptor. In the present study, the dependence of PGD₂-induced eosinophil accumulation in vivo on interleukin-5 (IL-5) blood eosinophilia was investigated. Materials:  Guinea-pigs were exposed to aerosols of 13,14dihydro 15-keto PGD₂ (DK-PGD₂) or platelet activating factor (PAF) and the eosinophil content of the bronchoalveolar lavage fluid or blood determined. In some experiments, DK-PGD₂ was administered systemically and eosinophilia measured. Results:  Aerosols of DK-PGD₂ caused eosinophil accumulation in the lungs 24h after exposure. DK-PGD₂ (10 μg.ml^-1) -induced airway eosinophilia was inhibited when animals were treated with the CRTH2 receptor antagonist ramatroban. 1–4h after exposure to DK-PGD₂ a significant decrease in blood eosinophil count was measured. The anti-IL-5 antibody TRFK-5 had no inhibitory effect of DK-PGD₂-induced airway eosinophilia, but abolished airway eosinophilia induced by exposure of guinea-pigs to aerosols of PAF. Intracardiac injection of DK-PGD₂ induced a dose-related increase in blood eosinophil numbers. Conclusions:  It is concluded that, in the guinea-pig, DKPGD₂-induced airway eosinophilia is mediated by an action on prostaglandin CRTH2 receptors and that this response appears to be independent of IL-5. Received 28 May 2008; returned for revision 9 June 2008; received from final revision 17 June 2008; accepted by M. Parnham 18 June 2008     

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in PGE₂ production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated fromPorphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) andEschericia coli (E. coli) by the phenol-water procedure. The three LPS preparations produced PGE₂ up to 48 h in a time-dependent manner in human gingival fibroblasts.P. gingivalis-LPS was the most potent stimulator of PGE₂ production and, to a lesser extent,A. actinomycetemcomitans- andE. coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE₂ production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE₂ production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h afterP. gingivalis-LPS stimulation, while expression of COX-1 protein was not affected byP. gingivalis-LPS. In order to investigate the regulation of PGE₂ production,P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE₂ production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE₂ not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE₂ may be involved in the pathogenesis of periodontal diseases.     

Pathogenesis of aspirin-exacerbated respiratory disease

The underlying respiratory disease is activated by unknown mechanism and results in an intense infiltration of mast cells and eosinophils into the entire respiratory mucosa. These cells synthesize leukotrienes (LTs) at a very high rate and mast cells also release histamine and tryptase and synthesize PGD₂ a vasodilator and bronchoconstrictor. Furthermore, AERD patients under synthesize from arachidonic acid (AA) a peculiar product called lipoxins, which opposes inflammation generated by leukotrienes. Finally, cysLT₁ receptors are over expressed and highly responsive to LTE₄, further augmenting the underlying inflammatory disease. This inflammatory condition is partly inhibited by synthesis of PGE₂ through COX-1. PGE₂ partially inhibits 5-lipogygenase conversion of AA to LTA₄ and blocks release of histamine and tryptase from mast cells. When COX-1 is inhibited by ASA or NSAIDs, PGE₂ synthesis stops and an enormous release of histamine and synthesis of LTs occurs. The upper respiratory reaction is mediated by both histamine and LTs but the bronchospastic reaction is mediated by LTs. The systemic effects of flush, gastric pain and hives are mediated by histamine. Aspirin desensitization can not be explained by disappearance of LT synthesis since urine LTE₄ levels are still elevated at acute ASA desensitization. However, mast cell products such as histamine, tryptase and PGD₂ are no longer released or synthesized at acute desensitization. It is more likely that a diminution in number or function of cysLT receptors accounts for the diminished inflammatory response found in ASA desensitization.     

Clinical and Immunomodulatory Effects of Celecoxib Plus Interferon-Alpha in Metastatic Renal Cell Carcinoma Patients with COX-2 Tumor Immunostaining

Introduction  

Cycloxygenase-2 (COX-2) is an enzyme involved in prostaglandin E2 (PGE₂) synthesis associated with higher renal cell carcinoma stage. COX-2 inhibition enhances interferon (IFN-α) anti-tumor immune effects in pre-clinical models. A phase II trial of celecoxib and IFN-α in a targeted population of metastatic renal cell carcinoma patients with maximal COX-2 expression was conducted.     

Kakkalide and Its Metabolite Irisolidone Ameliorate Carrageenan-Induced Inflammation in Mice by Inhibiting NF-κB Pathway

The anti-inflammatory activities of kakkalide, a major constituent of the flower of Pueraria thunbergiana, and irisolidone, a metabolite of kakkalide produced by intestinal microflora, against carrageenan-induced inflammation in air pouches on the backs of mice and in lipopolysaccharide (LPS)-stimulated peritoneal macrophages were investigated. Kakkalide and irisolidone down-regulated the gene expression of cytokines [tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β)] and cyclooxygenase-2 (COX-2) and the production of pro-inflammatory cytokines, TNF-α and IL-1β, and inflammatory mediators, NO and prostaglandin E₂ (PGE₂), in LPS-stimulated peritoneal macrophages. These agents also inhibited the phosphorylation of IκB-α and the nuclear translocation of nuclear factor-kappa B (NF-κB). Orally administered kakkalide and irisolidone significantly reduced carrageenan-induced inflammatory markers, leukocyte number, and protein amount in the exudates of the air pouch. These constituents also inhibited PGE₂ production and COX-2 inducible nitric oxide synthase, IL-1β, and TNF-α expression. These agents also inhibited NF-κB activation. The anti-inflammatory effects of irisolidone were more potent than those of kakkalide. Based on these findings, kakkalide and irisolidone may inhibit inflammatory reactions via NF-κB pathway, and irisolidone, a metabolite of kakkalide, may more potently inhibit these inflammatory reactions.     

Cyclic AMP levels and their regulation by prostaglandins in peritoneal macrophages of rats and humans

Basal levels of cyclic AMP and their alterations following stimulation by prostaglandins have been examined in rat peritoneal macrophages and in such cells of humans with renal disease on continuous ambulatory peritoneal dialysis (CAPD). Resident cells of rats contained more cyclic AMP than elicited macrophages, but the responsiveness to either PGE₂ or DC-PGI₂ (a stable synthetic analogue of PGI₂) was higher with elicited than with resident cells. However, both kinds of peritoneal macrophages of rats were, in terms of cyclic AMP elevation, more sensitive to stimulation by PGE₂ than by DC-PGI₂. The CAPD macrophages of humans were elicited cells. The unstimulated levels of cyclic AMP in these human macrophages were much higher than those in elicited rat cells. Furthermore the human macrophages proved more sensitive to stimulation by DC-PGI₂ than by PGE₂. The reversed sensitivity, in comparison with rat cells, reflects the utterly poor effects of PGE₂ in the human macrophages.The distinction in responsiveness to PGE₂ and DC-PGI₂ of the rat macrophages is compatible with the earlier reported distribution of and affinity to receptor binding sites of these PGs in the rat cells. The findings with the human macrophages suggest, however, that in these cells either the distribution of specific binding sites or the affinity of the two PGs to such sites might be substantially different from that of rats.     

Up-Regulation of the Inflammatory Response by Ovariectomy in Collagen-Induced Arthritis. Effects of Tin Protoporphyrin IX

We have studied the influence of ovariectomy on the inflammatory response and bone metabolism on CIA as a model of postmenopausal arthritis as well as the effects of tin protoporphyrin IX (SnPP), a heme oxygenase inhibitor. Ovariectomy in non-arthritic mice produced increased serum PGD₂ levels and up-regulated the expression of COX-2, h-PGDS, l-PGDS, and HO-1 in the joints. In CIA, ovariectomy potentiated the inflammatory response with higher levels of serum IL-6 and MMP-3, local PGD₂ and MMP-3 as well as trabecular bone erosion. In OVX-CIA, SnPP decreased the serum levels of IL-6, MMP-3, and PGD₂; down-regulated TNFα, COX-2, hPGDS, PGD₂, PGE₂, and MMP-3 in joint tissues; and also decreased focal bone loss in the inflamed joint. Ovariectomy up-regulates inflammatory mediators in non-arthritic and in arthritic animals. In the OVX-CIA model, SnPP exerts anti-inflammatory effects which are not associated with the prevention of systemic bone loss.     

In vitro effects of Helicobacter pylori-induced infection in gastric epithelial AGS cells on microglia-mediated toxicity in neuroblastoma SH-SY5Y cells

Objective:  To investigate the in vitro effects of H. pylori-conditioned medium (HCM) from gastric epithelial AGS cell cultures on microglia and neuronal cells. Material:  H. pylori, human gastric epithelial AGS cells, microglia-like BV-2 cells and human neuroblastoma SH-SY5Y cells. Treatment:  Treated AGS cells with H. pylori at ratios from 1:100 to 1:900 for 24 h. Cultured BV-2 cells and SH-SY5Y cells were treated with HCM from AGS cell cultures. Methods:  Cell viability was measured by a quantitative colorimetric assay with MTT. Nitric oxide (NO) was determined by using Griess reagent. IL-8 was measured by an enzyme-linked immunosorbent assay. Protein expressions were revealed by western blot analysis. Results:  H. pylori increased IL-8, NO, COX-2 and gp91^phox in AGS cell cultures. When BV-2 cells were cocultured with AGS cells, HCM increased COX-2, gp91^phox, iNOS and NO of BV-2 cells. HCM also enhanced the degradation of IκBα in BV-2 cells. HCM up-regulated expression of nNOS, COX-2, and gp91^phox of SH-SY5Y cells co-cultured with BV-2 cells. Particularly, the decrease of cell viability of SH-SY5Y induced by HCM was dependent on the presence of BV-2 cells. Conclusions:  H. pylori-induced infection induces microglia-mediated inflammation and neurotoxicity. The present results suggest that microglia play a critical role in HCM-induced toxicity of neuronal SH-SY5Y cells. Received 15 April 2008; returned for revision 10 May 2008; received from final revision 14 September 2008; accepted by M. Katori 18 September 2008     

Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes

We previously reported that hydrogen sulfide (H₂S) preconditioning (SP) produces cardioprotection in isolated rat cardiomyocytes. The present study was designed to determine the involvement of cyclooxygenase-2 (COX-2) in the SP-induced delayed cardioprotection. Isolated cardiac myocytes were treated with NaHS (100 μM, a H₂S donor) for 30 min and then cultured for 20 h followed by ischemia/reperfusion insults. SP significantly increased cell viability, percentage of rod-shaped cells, and myocyte contractility after 10 min of reperfusion. Given 30 min before and during lethal ischemia, two selective COX-2 inhibitors, NS-398 and celebrex, abrogated SP-induced cardioprotective effects. Moreover, SP upregulated the expression of COX-2 and increased PGE₂ production in the cardiac myocytes. These effects were significantly attenuated by glibenclamide, an ATP-sensitive K⁺ channel (K_ATP) blocker, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting that activation of both K_ATP and PKC is required for the stimulation of COX-2. Additionally, NG-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, failed to regulate COX-2 protein expression but inhibited SP-enhanced COX-2 activity and PGE₂ production. In conclusion, we provided the first evidence that SP may produce delayed cardioprotection via K_ATP/PKC dependent induction of COX-2 expression and via nitric oxide-induced COX-2 activation.     

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE₂, PGF_2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8 h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE₂ and beraprost, but not PGF_2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE₂ and beraprost but not by PGF_2α. The distinct effect of PGE₂ and beraprost from PGF_2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE₂ and PGI₂ stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.     

Signal transduction pathways involving p38 MAPK, JNK, NFκB and AP-1 influences the response of chondrocytes cultured in agarose constructs to IL-1β and dynamic compression

Objective and Design:  To examine whether inhibitors of the MAPK pathways will influence the response of bovine chondrocytes cultured in agarose constructs to IL-1β and dynamic compression. Methods:  Dose-response studies were conducted under IL-1β conditions with either SB203580, SP600125, PDTC or curcumin. In separate experiments, constructs were treated with IL-1β and an appropriate concentration of inhibitor and subjected to 15% dynamic compression. Nitrite and PGE₂ release, ³⁵SO₄ and [³H]-thymidine incorporation were subsequently measured using biochemical assays. Results:  All inhibitors reduced the IL-1β induced nitrite and PGE₂ release in a dose-dependent manner. The inhibition of [³H]-thymidine incorporation by IL-1β was partially reversed with SB203580, SP600125 or curcumin, but not PDTC. In most cases, the inhibitors reduced ³⁵SO₄ incorporation with IL-1β. For the mechanical loading studies, the inhibitors reduced the compression-induced inhibition of nitrite and PGE₂ release and restored [³H]-thymidine and ³⁵SO₄ incorporation. Conclusions:  The MAPK, AP-1 and NF-κB signalling pathways are involved in the upregulation of NO and PGE₂ release by IL-1β. Dynamic compression stimulates cell proliferation and proteoglycan synthesis in the presence of IL-1β and/or inhibitors of the MAPKs and NFκB and AP-1 signalling pathways. This experimental approach could provide valuable information for the biophysical/pharmacological treatment of OA. Received 11 July 2007; returned for revision 27 September 2007; received from final revision 15 January 2008; accepted by J. Di Battista 15 January 2008