p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes

We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-kappaB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and interferon (IFN)-gamma. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 microM) or the proteosome inhibitor MG-132 (1 microM). SB-203580 did not affect cytokine-stimulated IkappaBalpha degradation, NF-kappaB nuclear binding activity, or NF-kappaB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-kappaB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-kappaB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-kappaB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.     

Induction of cyclooxygenase-2 by heat shock protein 60 in macrophages and endothelial cells

The 60-kDa heat shock protein (HSP60), an endogenous ligand for the toll-like 4 receptor, is generated in response to inflammation, tissue injury, and/or stress and stimulates macrophages to produce cytotoxic and proinflammatory mediators including nitric oxide, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12. In the present studies we report that HSP60 is an effective inducer of cyclooxygenase-2 (COX-2) in macrophages, as well as endothelial cells. In both cell types, the synthesis of COX-2 was coordinate with induction of nitric oxide synthase (NOS)-2 and with nitric oxide production. With the use of promoter constructs in transient transfection assays, optimal expression of COX-2 in macrophages was found to require nuclear factor (NF)-kappaB, the cAMP-response element (CRE), and NF-IL-6, but not the E-box. Mobility shift assays revealed that HSP60 induced NF-kappaB and CRE binding activity, while CCAAT/enhancer binding protein (C/EBP), which binds to NF-IL-6, was constitutively active in the cells. Both c-Jun and CRE binding protein (CREB) bound to the CRE, while C/EBP-beta bound to NF-IL-6. These data indicate that NF-kappaB, C/EBP-beta, c-Jun, and CREB are important in HSP60-induced expression of COX-2. The c-Jun-NH(2)-terminal kinase (JNK), p44/42 mitogen-activated protein (MAP) kinase [extracellular signal-regulated kinase 1/2 (ERK1/2)], and p38 MAP kinase were rapidly activated by HSP60 in the macrophages. PD-98059, an inhibitor of phosphorylation of ERK1/2, caused a marked inhibition of HSP60-induced COX-2 and NOS-2 expression. Unexpectedly, SB-203580, a p38 kinase antagonist, was found to block HSP60-induced expression of COX-2, but not NOS-2. These data indicate that both ERK1/2 kinase and p38 kinase play a role in regulating HSP60-induced expression of COX-2.     

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.     

Platelet-activating factor induces matrix metalloproteinase-9 expression through Ca(2+)- or PI3K-dependent signaling pathway in a human vascular endothelial cell line

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of a variety of angiogenic factors, via the nuclear factor (NF)-kappaB activation. Recently, we reported that PAF upregulates MMP-9 expression in a NF-kappaB-dependent manner. In this study, we investigated the signaling pathway involved in PAF-induced MMP-9 expression in ECV304 cells. Our current data indicate that the Ca(2+)- or phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway is necessary for PAF-induced MMP-9 expression. Furthermore, PAF-induced NF-kappaB activation was blocked by selective inhibitors of Ca(2+), PI3K, or extracellular signal-regulated kinase (ERK). Our results suggest that PAF-induced MMP-9 expression, in a NF-kappaB-dependent manner, is regulated by Ca(2+), PI3K and ERK signaling pathways.     

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.     

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.     

A novel function of Goalpha: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells

Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goalpha as a mediator of opioid receptor-induced extracellular signal-regulated kinase activation in neural cells. The current study found that, in neuroblastoma x glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbetagamma subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goalpha1, but not Gialpha2, completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goalpha expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gialpha2 did not. By taking advantage of differences between human and mouse Goalpha gene sequences, we simultaneously knocked down endogenous Goalpha expression and expressed exogenous human Goalpha subunits. We found that both human Goalpha1 and Goalpha2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goalpha in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.     

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.     

Gangliosides activate cultured rat brain microglia

Microglia, brain resident macrophages, are activated in brain injuries and several neurodegenerative diseases. However, microglial activators that are produced in the brain are not yet defined. In this study, we showed that gangliosides, sialic acid-containing glycosphingolipids, could be a microglial activator. Gangliosides induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) and expression of cyclooxygenase-2 (COX-2). The effect of gangliosides on NO release increased dose-dependently in the range of 10-100 microgram/ml; however, the effect decreased at concentrations higher than 200 microgram/ml. Specific types of gangliosides showed differential effects on microglial activation. Similar to gangliosides, GT1b induced production of NO and TNF-alpha and expression of COX-2. However, GM1 and GD1a induced expression of COX-2 but had little effect on NO and TNF-alpha release. The effect of gangliosides and GT1b on NO release was reduced in the presence of neuraminidase, which removes sialic acid residues from gangliosides and GT1b. Gangliosides activated extracellular signal-regulated kinase significantly but activated c-jun N-terminal kinase/stress-activated protein kinase and p38 relatively weakly. The inhibition of extracellular signal-regulated kinase by PD98059 reduced NO release from both gangliosides- and GT1b-treated microglia whereas inhibition of p38 by SB203580 increased it rather slightly. Gangliosides activated NF-kappaB, and N-acetyl cystein, an inhibitor of NF-kappaB, reduced NO release. These results suggest that gangliosides could be a microglial activator that functions via activation of mitogen-activated protein kinase and NF-kappaB.     

Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells

Variations in the matrix metalloproteinase (MMP)-9 gene are related to the presence and severity of atherosclerosis. The aim of this study was to determine the signaling pathways of MMP-9 in endothelial cells subjected to low fluid shear stress. We found that low fluid shear stress significantly increased MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and phosphorylation of MAPK in cultured human umbilical vein endothelial cells (HUVECs). Inhibition of NF-kappaB resulted in remarkable downregulation of stress-induced MMP-9 expression. Pretreatment of HUVECs with inhibitors of p38 mitogen-activating protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) also led to significant suppression of stress-induced MMP-9 expression and NF-kappaB DNA-binding activity. Similarly, addition of integrins inhibitor to HUVECs suppressed the stress-induced MMP-9 expression, IkappaBalpha degradation, NF-kappaB DNA-binding activity and the phosphorylation of p38 MAPK, ERK1/2. Our findings demonstrated that the shear stress-induced MMP-9 expression involved integrins-p38 MAPK or ERK1/2-NF-kappaB signaling pathways.     

Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.