Ceramide-induced enhancement of secretory phospholipase A2 expression via generation of reactive oxygen species in tumor necrosis factor-alpha-stimulated mesangial cells

Since prostanoids such as prostaglandin E2 play a pivotal role in modulating renal function, we investigated the involvement of ceramide in expression of secretory phospholipase A2 (sPLA2) and cyclooxygenase-2 (COX-2) in tumor necrosis factor-alpha (TNF-alpha)-stimulated mesangial cells. TNF-alpha stimulation increased ceramide generation in parallel with a decrease in sphingomyelin. Pretreatment with exogenous sphingomyelinase (SMase) dose-dependently enhanced TNF-alpha-stimulated increases in COX-2 protein and sPLA) activity. SMase also augmented TNF-alpha-mediated nuclear factor kappaB (NF-kappaB) activation. N-acetylcysteine (NAC), an antioxidant, completely inhibited the SMase-induced increase in sPLA2 activity, whereas NAC inhibited partially the activity stimulated with TNF-alpha alone. Under the conditions, NAC completely inhibited reactive oxygen species (ROS) production induced by SMase followed by TNF-alpha. These results suggest that ceramide elicits up-regulation of NF-kappaB through ROS production, which, in turn, leads to stimulation of COX-2 and sPLA2 expression. Therefore, ceramide may be implicated in the pathogenesis of renal abnormalities.     

Enhanced expression of cyclooxygenase-2 and prostaglandin E2 in response to endotoxin after trauma is dependent on MAPK and NF-kappaB mechanisms

Macrophage prostaglandin E2 (PGE2) production is important in cellular immune suppression and in affecting the potential development of sepsis after trauma. We hypothesized that macrophage PGE2 production after trauma is regulated by mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB). Mice were subjected to trauma and splenic macrophages isolated 7 days later. Macrophages from traumatized mice showed increased cyclooxygenase-2 (COX-2) mRNA, protein expression, and PGE2 production compared with controls. Increased phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase was observed in macrophages from traumatized mice. Pharmacologic inhibition of MAPK blocked trauma-induced COX-2 expression, and PGE2 production. Trauma macrophages showed increased IkappaBalpha phosphorylation and NF-kappaB binding to DNA. Inhibiting IkappaBalpha blocked trauma-induced NF-kappaB activity, COX-2 expression and PGE2 production. This suggests that trauma-induced PGE2 production is mediated through MAPK and NF-kappaB activation and offers potential for modifying the macrophages' responses following injury.     

Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors

The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C(2)-ceramide) on LPS-induced production of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10-50 microM, C(2)-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE(2) formation. By contrast, a structural analog of C(2)-ceramide that does not elicit functional activity, C(2)-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB and AP-1 activation. The immunocomplex kinase assay indicated that IkappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IkappaBalpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-kappaB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-kappaB and AP-1, which might result from ceramide's inhibition of LPS-stimulated IkappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.     

PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.     

Green tea proanthocyanidins inhibit cyclooxygenase-2 expression in LPS-activated mouse macrophages: molecular mechanisms and structure-activity relationship

The inhibitory effects of green tea proanthocyanidins on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) release were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Prodelphinidin B2 3,3' di-O-gallate (PDGG) caused a dose-dependent inhibition of COX-2 at both mRNA and protein levels with the attendant release of PGE(2). Molecular evidence revealed that PDGG inhibited the degradation of Ikappa-B, nuclear translocation of p65 and CCAAT/enhancer-binding protein (C/EBP)delta, and phosphorylation of c-Jun, but not CRE-binding protein (CREB), which regulate COX-2 expression. Moreover, PDGG suppressed the activations of mitogen-activated protein kinase (MAPK) including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. The results demonstrated that PDGG suppressed COX-2 expression via blocking MAPK-mediated activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and C/EBPdelta. Furthermore, studies on structure-activity relationship using five kinds of proanthocyanidins revealed that the galloyl moiety of proanthocyanidins appeared important to their inhibitory actions. Thus, our findings provide the first molecular basis that green tea proanthocyanidins with the galloyl moiety might have anti-inflammatory properties through blocking MAPK-mediated COX-2 expression.     

Ceramide pathways modulate ethanol-induced cell death in astrocytes

We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.     

Differentiation status-dependent regulation of cyclooxygenase-2 expression and prostaglandin E2 production by epidermal growth factor via mitogen-activated protein kinase in articular chondrocytes

Although large amounts of epidermal growth factor (EGF) are found in the synovial fluids of arthritic cartilage, the role of EGF in arthritis is not clearly understood. This study investigated the effect of EGF on differentiation and on inflammatory responses such as cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in articular chondrocytes. EGF caused a loss of differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. EGF also induced COX-2 expression and PGE(2) production. EGF-induced dedifferentiation was caused by EGF receptor-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) but not p38 kinase, whereas the activation of both ERK1/2 and p38 kinase was necessary for COX-2 expression and PGE(2) production. Neither the inhibition of COX-2 expression and PGE(2) production nor the addition of exogenous PGE(2) affected EGF-induced dedifferentiation. However, COX-2 expression and PGE(2) production were significantly enhanced in chondrocytes that were dedifferentiated by serial subculture, and EGF also potentiated COX-2 expression and PGE(2) production, although these cells were less sensitive to EGF. Dedifferentiation-induced COX-2 expression and PGE(2) production were mediated by ERK1/2 and p38 kinase signaling. Our results indicate that EGF in articular chondrocytes stimulates COX-2 expression and PGE(2) production via ERK and p38 kinase signaling in association with differentiation status.     

Protein tyrosine kinase p56lck is required for ceramide-induced but not tumor necrosis factor-induced activation of NF-kappa B, AP-1, JNK, and apoptosis

Ceramide has been implicated as an intermediate in the signal transduction of several cytokines including tumor necrosis factor (TNF). Both ceramide and TNF activate a wide variety of cellular responses, including NF-kappaB, AP-1, JNK, and apoptosis. Whether ceramide transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56(lck) in ceramide- and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, isogeneic Lck-deficient T cells. Treatment with ceramide activated NF-kappaB, degraded IkappaBalpha, and induced NF-kappaB-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56(lck) kinase. These effects were specific to ceramide, as activation of NF-kappaB by phorbol 12-myristate 13-acetate, lipopolysaccharide, H(2)O(2), and TNF was minimally affected. p56(lck) was also found to be required for ceramide-induced but not TNF-induced AP-1 activation. Similarly, ceramide activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. Ceramide also induced cytotoxicity and activated caspases and reactive oxygen intermediates in Jurkat cells but not in JCaM1 cells. Ceramide activated p56(lck) activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56(lck) tyrosine kinase reversed the ceramide-induced NF-kappaB activation and cytotoxicity. Overall our results demonstrate that p56(lck) plays a critical role in the activation of NF-kappaB, AP-1, JNK, and apoptosis by ceramide but has minimal or no role in activation of these responses by TNF.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Cyclooxygenase-2-derived E prostaglandins down-regulate matrix metalloproteinase-1 expression in fibroblast-like synoviocytes via inhibition of extracellular signal-regulated kinase activation

We examined the regulation of matrix metalloproteinase (MMP) production by mitogen-activated protein kinases and cyclooxygenases (COXs) in fibroblast-like synoviocytes (FLSCs). IL-1beta and TNF-alpha stimulated FLSC extracellular signal-regulated kinase (ERK) activation as well as MMP-1 and -13 release. Pharmacologic inhibitors of ERK inhibited MMP-1, but not MMP-13 expression. Whereas millimolar salicylates inhibited both ERK and MMP-1, nonsalicylate COX and selective COX-2 inhibitors enhanced stimulated MMP-1 release. Addition of exogenous PGE(1) or PGE(2) inhibited MMP-1, reversed the effects of COX inhibitors, and inhibited ERK activation, suggesting that COX-2 activity tonically inhibits MMP-1 production via ERK inhibition by E PGs. Exposure of FLSCs to nonselective COX and selective COX-2 inhibitors in the absence of stimulation resulted in up-regulation of MMP-1 expression in an ERK-dependent manner. Moreover, COX inhibition sufficient to reduce PGE levels increased ERK activity. Our data indicate that: 1) ERK activation mediates MMP-1 but not MMP-13 release from FLSCs, 2) COX-2-derived E PGs inhibit MMP-1 release from FLSCs via inhibition of ERK, and 3) COX inhibitors, by attenuating PGE inhibition of ERK, enhance the release of MMP-1 by FLSC.     

Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step

Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2 levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2 levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-kappaB decreased rotavirus infection by at least 40%. PGE2 counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-kappaB inhibitors. Conclusively, COXs and PGE2 are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-kappaB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway

TNF-alpha induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-alpha-induced COX-2 expression. TNF-alpha also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-alpha-induced response. The 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-kappaB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-alpha, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-kappaB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-alpha-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-gamma2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-alpha, NF-kappaB-inducing kinase, or I-kappaB (inhibitory protein that dissociates from NF-kappaB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-alpha might activate phospholipase C-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and protein tyrosine kinase, resulting in the activation of NF-kappaB-inducing kinase and IKK1/2, and NF-kappaB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.     

Streptococcus pneumoniae induced p38 MAPK- and NF-kappaB-dependent COX-2 expression in human lung epithelium

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.     

Methylglyoxal mediates vascular inflammation via JNK and p38 in human endothelial cells

Methylglyoxal (MGO) is a reactive metabolite of glucose. Since the plasma concentration of MGO is increased in diabetic patients, MGO is implicated in diabetes-associated vascular endothelial cells (ECs) injury, which might be responsible for atherosclerosis. In the present study, we examined effects of treatment of human umbilical vein ECs with MGO on EC morphology and inflammatory responses. MGO (24 h) induced cytotoxic morphological changes in a concentration-dependent manner (0-420 microM). MGO induced mRNA and protein expression of cyclooxygenase (COX)-2 in a concentration (0-420 microM)- and time (6-24 h)-dependent manner. COX-2 induction was associated with increased PGE(2) release. Acute treatment with MGO (20 min) induced concentration-dependent (0-420 microM) activation of JNK and p38 MAP kinase but not ERK or NF-kappaB. Both the JNK inhibitor SP600125 and the p38 inhibitor SB203580 prevented the MGO induction of COX-2. However, inhibiting JNK and p38 or COX-2 was ineffective to the morphological damage by MGO (420 microM, 24 h). EUK134, a synthetic combined superoxide dismutase/catalase mimetic, had no effect on MGO-induced COX-2. Present results indicated that MGO mediates JNK- and p38-dependent EC inflammatory responses, which might be independent of oxidative stress. On the other hand, MGO-induced morphological cell damage seems unlikely to be associated with COX-2-PGE(2).