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.     

Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.     

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.     

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.     

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.     

Novel regulatory mechanisms of CD40-induced prostanoid synthesis by IL-4 and IL-10 in human monocytes

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE(2), a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE(2) via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE(2) production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-kappaB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-kappaB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-kappaB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.     

Bisgma Stimulates Prostaglandin E2 Production in Macrophages via Cyclooxygenase-2, Cytosolic Phospholipase A2, and Mitogen-Activated Protein Kinases Family

Background

Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation via macrophage activation. Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages.

Methodology/Principal Findings

Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway (MEK1/2, ERK1/2, and Elk), p38 pathway (MEK3/6, p38, and MAPKAPK2), and JNK pathway (MEK4, JNK, and c-Jun) in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05).

Conclusions

These results suggest that BisGMA induced-PGE2 production may be via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

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.     

Induction of COX-2 protein expression by vanadate in A549 human lung carcinoma cell line through EGF receptor and p38 MAPK-mediated pathway

Vanadate is a transition metal widely distributed in the environment. It has been reported that vanadate associated with air pollution particles can modify DNA synthesis, causing cell growth arrest, and apoptosis. Moreover, vanadium exposure was also found to cause the synthesis of inflammatory cytokines, such as interleukin-1, tumor necrosis factor-alpha, and prostaglandin E(2). Here, we found that exposure of A549 human lung carcinoma cells to vanadate led to extracellular signal-regulated kinase, c-Jun NH(2)-terminal protein kinases (JNKs), p38 mitogen-activated protein kinase (p38) activation, and COX-2 protein expression in a dose-dependent manner. SB203580, a p38 MAPK inhibitor, but not PD098059 and SP600125, specific inhibitor of MKK1 and selective inhibitor of JNK, respectively, suppressed COX-2 expression. Furthermore, the epithelial growth factor (EGF) receptor specific inhibitor (PD153035) reduced vanadate-induced COX-2 expression. However, scavenging of vanadate-induced reactive oxygen species by catalase, a specific H(2)O(2) inhibitor, or DPI, an NADPH oxidase inhibitor, resulted in no inhibition on COX-2 expression. Together, we suggested that EGF receptor and p38 MAPK signaling pathway may be involved in vanadate-induced COX-2 protein expression in A549 human lung carcinoma cell line.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells

The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal-transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

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.