R(+)-methanandamide-induced cyclooxygenase-2 expression in H4 human neuroglioma cells: possible involvement of membrane lipid rafts

Cannabinoids induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells via a pathway independent of cannabinoid- or vanilloid receptor activation. The underlying mechanism was recently shown to involve increased synthesis of ceramide, which in turn leads to activation of p38 and p42/44 mitogen-activated protein kinases (MAPKs). The present study investigates a possible contribution of membrane lipid rafts to cannabinoid-induced COX-2 expression. To address this issue, we tested the influence of methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, on COX-2 expression by the endocannabinoid analogue R(+)-methanandamide (R(+)-MA). Incubation of H4 cells with MCD was associated with a loss of lipid raft integrity and a substantial inhibition of R(+)-MA-induced COX-2 expression and subsequent formation of prostaglandin E2. Moreover, MCD was shown to suppress signal transduction steps upstream to COX-2 induction by R(+)-MA. Accordingly, the cholesterol depletor suppressed R(+)-MA-induced formation of ceramide as well as phosphorylation of p38 and p42/44 MAPKs. Together, our results suggest that R(+)-MA induces COX-2 expression in human neuroglioma cells via a pathway linked to lipid raft microdomains.     

BK-induced COX-2 expression via PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB in astrocytes

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.     

Prostaglandin E2 induces cyclooxygenase-2 expression in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 mitogen-activated protein kinases

Prostaglandins (PGs) have been implicated in lowering intraocular pressure (IOP). A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing impaired COX-2 expression in the non-pigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. The present study investigates the effect of the major COX-2 product, PGE(2), on the expression of its synthesizing enzyme in human NPE cells (ODM-2). PGE(2) led to an increase of COX-2 mRNA and protein expression, whereas the expression of COX-1 remained unchanged. Upregulation of COX-2 expression by PGE(2) was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, and was abrogated by inhibitors of both pathways. Moreover, PGE(2)-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II, whereas the protein kinase A inhibitor H-89 was inactive in this respect. Induction of COX-2 expression was also elicited by butaprost (EP(2) receptor agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) receptor agonist), but not by EP(1)/EP(3) receptor agonists (17-phenyl-omega-trinor PGE(2), sulprostone). Consistent with these findings, the EP(1)/EP(2) receptor antagonist, AH-6809, and the selective EP(4) receptor antagonist, ONO-AE3-208, significantly reduced PGE(2)-induced COX-2 expression. Collectively, our results demonstrate that PGE(2) at physiologically relevant concentrations induces COX-2 expression in human NPE cells via activation of EP(2)- and EP(4) receptors and phosphorylation of p38 and p42/44 MAPKs. Positive feedback regulation of COX-2 may contribute to the production of outflow-facilitating PGs and consequently to regulation of IOP.     

Cyclooxygenase is regulated by ET-1 and MAPKs in peripheral lung microvascular smooth muscle cells

We examined the hypothesis that the potent vasoconstrictor endothelin (ET)-1 regulates both its own production and production of the vasodilator prostaglandins PGE(2) and prostacyclin in sheep peripheral lung vascular smooth muscle cells (PLVSMC). Confluent layers of PLVSMC were exposed to 10 nM ET-1; expression of the prepro (pp)-ET-1, cyclooxygenase (COX)-1, and COX-2 genes was examined by RT-PCR and Western analysis. Intracellular levels of ET-1 were measured by ELISA with and without addition of the protein synthesis inhibitor brefeldin A (50 microg/ml). Prostaglandin levels were measured by gas chromatography-mass spectrometry. Through use of ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively), the contribution of the ET receptors to COX-1 and -2 expression and ppET-1 gene expression was examined. The contribution of phosphorylated p38 and p44/42 MAPK on COX-1 and COX-2 expression was also examined with MAPK inhibitors (p38, SB-203580 and p44/42, PD-98056). ET-1 resulted in transient increases in ppET-1, COX-1, and COX-2 gene and protein expression and release of 6-keto-PGF(1alpha) and PGE(2) (P < 0.05). Both internalization of ET-1 and synthesis of new peptide contributed to an increase in intracellular ET-1 (P < 0.05). Although increased ppET-1 was regulated by both ET(A) and ET(B), COX-2 expression was upregulated only by ET(A); COX-1 expression was unaffected by either antagonist. ET-1 treatment resulted in transient phosphorylation of p38 and p44/42 MAPK; inhibitors of these MAPKs suppressed expression of COX-2 but not COX-1. Our data indicate that local production of ET-1 regulates COX-2 by activation of the ET(A) receptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Inducible nitric oxide synthase upregulates cyclooxygenase-2 in mouse cholangiocytes promoting cell growth

Both inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) have been implicated in the biliary tract carcinogenesis. However, it is not known whether these inflammatory mediators are induced by interdependent or parallel pathways. Because iNOS activity has been associated with diverse gene expression, the aim of this study was to determine whether iNOS induces COX-2. To address this objective, immortalized, but nonmalignant, murine cholangiocytes, 603B cells were employed for these studies. Both iNOS and COX-2 protein and mRNA were expressed in these cells. However, iNOS inhibition with either N-[3-(aminomethyl) benzyl]acetamidine or stable transfection with an iNOS antisense construct inhibited COX-2 mRNA and protein expression, an effect that was reversed by NO donors. COX-2 mRNA expression in 603B cells was reduced by pharmacological inhibitors of the p38 MAPK and JNK1/2 pathways. In contrast, neither inhibitors of the soluble guanylyl cyclase inhibitor/protein kinase G nor p42/44 MAPK pathways attenuated COX-2 mRNA expression. Finally, 603B cells grew at a rate threefold greater than 603B-iNOS antisense cells. The low growth rate of 603B-iNOS antisense cells could be restored to near that of the parent cell line with exogenous PGE(2.) In conclusion, iNOS induces COX-2 expression in cholangiocytes, which promotes cell growth. COX-2 induction may contribute to iNOS-associated carcinogenesis.     

Induction of cyclooxygenase-2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB pathways

Lipopolysaccharide (LPS) was found to induce inflammatory responses in the airways and exerted as a potent stimulus for PG synthesis. This study was to determine the mechanisms of LPS-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). LPS markedly increased the expression of COX-2 and release of PGE(2) in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Both the expression of COX-2 and the generation of PGE(2) in response to LPS were attenuated by a tyrosine kinase inhibitor genistein, a phosphatidylcholine-phospholipase C inhibitor D609, a phosphatidylinositol-phospholipase C inhibitor U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. Furthermore, LPS-induced NF-kappaB activation correlated with the degradation of IkappaB-alpha, COX-2 expression, and PGE(2) synthesis, was inhibited by transfection with dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. LPS-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK inhibitor), but these two inhibitors had no effect on LPS-induced NF-kappaB activation, indicating that NF-kappaB is activated by LPS independently of activation of p42/p44 MAPK and p38 MAPK pathways in TSMCs. Taken together, these findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from LPS-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways. LPS-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation

Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP(1-4)). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 micromol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.     

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.     

Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E(2) (PGE(2)). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE(2) formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE(2) accumulation. The same pathways as those obtained (2)in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

The CCK-2/gastrin splice variant receptor retaining intron 4 transactivates the COX-2 promoter in vitro

The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.     

Enterovirus 71 induces COX-2 expression via MAPKs,NF-κB,and AP-1 in SK–N–SH cells: Role of PGE2 in viral replication

The enterovirus 71 (EV71) causes severe neurological diseases that were mediated through cyclooxygenase-2 (COX-2) expression in brain. However, the mechanisms underlying EV71-initiated intracellular signaling pathways leading to COX-2 expression remain unknown in neurons. Here we report that exposure of SK–N–SH cells to EV71 increased COX-2 expression and PGE₂ generation in a time- and virus titer-dependent manner, revealed by Western blot, real-time PCR, and PGE₂ analyses. These EV71-induced responses were mediated through activation of p42/p44 MAPK, p38 MAPK, JNK, NF-κB, and AP-1, revealed by using selective pharmacological inhibitors or transfection with respective siRNAs. Consistently, EV71-stimulated translocation of NF-κB into the nucleus and degradation of IκBα in the cytosol was blocked by pretreatment with the selective inhibitors of MEK1/2 (U0126) and NF-κB (Bay11-7085), respectively, suggesting that MEK1/2-p42/p44 MAPK cascade linking to NF-κB was involved in COX-2 expression. In addition, EV71-induced AP-1 subunits (c-jun and c-fos mRNA) expression was also attenuated by pretreatment with a selective JNK inhibitor SP600125, suggesting that JNK cascade linking to AP-1 was involved in COX-2 expression induced by EV71. These findings suggested that up-regulation of COX-2 associated with the release of PGE₂ from EV71-infected SK–N–SH cells which was mediated through activation of p38 MAPK, JNK, p42/p44 MAPK, NF-κB, and AP-1 pathways.