Interferon-gamma and interleukin 4 inhibit interleukin 1beta-induced delayed prostaglandin E(2)generation through suppression of cyclooxygenase-2 expression in human fibroblasts

Interleukin (IL-)1 stimulates prostaglandin E(2)(PGE(2)) generation in fibroblasts, and preferential couplings between particular phospholipase A(2)(PLA(2)) and cyclooxygenase (COX) isozymes are implicated with IL-1-induced delayed PGE(2)generation. The regulatory effects of interferon (IFN)-gamma and IL-4 on IL-1beta-induced COX, PLA(2)isoforms expression and terminal delayed PGE(2)generation were examined in three types of human fibroblasts. These human fibroblasts constitutively expressed cytosolic PLA(2)(cPLA(2)) and COX-1 enzymes, and exhibited delayed PGE(2)generation in response to IL-1beta. IL-1beta also stimulated expression of cPLA(2)and COX-2 only, while constitutive and IL-1beta-induced type IIA and type V secretory PLA(2)s (sPLA(2)s) expression could not be detected. A COX-2 inhibitor and cPLA(2)inhibitor markedly suppressed the IL-1beta-induced delayed PGE(2)generation, while a type IIA sPLA(2)inhibitor failed to affect it. IFN-gamma and IL-4 dramatically inhibited the IL-1beta-induced delayed PGE(2)generation; these cytokines apparently suppressed IL-1beta-stimulated COX-2 expression and only weakly suppressed cPLA(2)expression in response to IL-1beta. These results indicate that IL-1beta-induced delayed PGE(2)generation in these human fibroblasts mainly depends on de novo induction of COX-2 and cPLA(2), irrespective of the constitutive presence of COX-1, and that IFN-gamma and IL-4 inhibit IL-1beta-induced delayed PGE(2)generation by suppressing, predominantly, COX-2 expression.     

Activation of cytosolic phospholipase A2alpha through nitric oxide-induced S-nitrosylation. Involvement of inducible nitric-oxide synthase and cyclooxygenase-2

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)alpha protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)alpha activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.     

Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.     

EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.     

Cyclooxygenase-1 and -2 enzymes differentially regulate the brain upstream NF-kappa B pathway and downstream enzymes involved in prostaglandin biosynthesis

We have recently reported that cyclooxygenase (COX)-2-deficiency affects brain upstream and downstream enzymes in the arachidonic acid (AA) metabolic pathway to prostaglandin E2 (PGE2), as well as enzyme activity, protein and mRNA levels of the reciprocal isozyme, COX-1. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and - 2) in wild type and COX-1(-/-) mice. We found that brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1(-/-) mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the NF-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. The mechanism of NF-kappaB activation in the COX-1(-/-) mice involved the up-regulation of protein expression of the p50 and p65 subunits of NF-kappaB, as well as the increased protein levels of phosphorylated IkappaBalpha and of phosphorylated IKKalpha/beta. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2, to overcome defects in brain AA cascade.     

COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.     

A bifunctional role for group IIA secreted phospholipase A2 in human rheumatoid fibroblast-like synoviocyte arachidonic acid metabolism

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA(2)-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA(2)-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE(2) production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA(2)-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A(2)-α (cPLA(2)-α) inhibitors abrogate TNF/sPLA(2)-IIA-mediated PGE(2) production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA(2)-α. Nonetheless, exogenous sPLA(2)-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE(2) production. sPLA(2)-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA(2)-IIA but not cPLA(2)-α. Exogenous H48Q alone, like sPLA(2)-IIA, increases COX-2 protein levels without inducing PGE(2) production. Unlike TNF, sPLA(2)-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA(2)-IIA regulates AA flux through the cPLA(2)-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA(2)-IIA.     

Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal

Atherosclerosis is a disorder of lipid metabolism as well as a chronic inflammatory disease. Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses, mediates a variety of biological actions involved in vascular pathophysiology. We have previously shown that COX-2 gene expression is dramatically induced by a lipid-derived endogenous electrophile, 4-hydroxy-2-nonenal (HNE) (Kumagai, T., Matsukawa, N., Kaneko, Y., Kusumi, Y., Mitsumata, M., and Uchida, K. (2004) J. Biol. Chem. 279, 48389-48396). In the present study, based on the finding that HNE induced COX-2 expression only in the serum-containing media, we characterized a serum component essential for the HNE-induced COX-2 induction and found that low density lipoprotein (LDL) that had been denatured by freeze-thawing or oxidized LDL might be involved in the COX-2 induction. Moreover, we characterized the cellular events triggered by the combined stimulus of HNE and oxidized LDL and established that COX-2 induction is regulated by two sets of signaling mechanisms, one for the up-regulation of the scavenger receptor CD36 by HNE and one for the CD36-mediated COX induction by oxidized LDL. These findings represent a demonstration of a link between lipoprotein modification and activation of the inflammatory potential of macrophages.     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.     

Adenovirus type 5 exerts multiple effects on the expression and activity of cytosolic phospholipase A2, cyclooxygenase-2, and prostaglandin synthesis

In this study, we examine how infection of murine and human fibroblasts by adenovirus (Ad) serotype 5 (Ad5) affects the expression and activity of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of PGs. Our experiments showed that infection with Ad5 is accompanied by the rapid activation of cPLA2 and the cPLA2-dependent release of [3H]arachidonic acid ([3H]AA). Increased expression of COX-2 was also observed after Ad infection, as was production of PGE2 and PGI2. Later, however, as the infection progressed, release of [3H]AA and production of PGs stopped. Late-stage Ad5-infected cells also did not release [3H]AA or PGs following treatment with a panel of biologically diverse agents. Experiments with UV-inactivated virus confirmed that Ad infection is accompanied by the activation of a host-dependent response that is later inhibited by the virus. Investigations of the mechanism of suppression of the PG pathway by Ad5 did not reveal major effects on the expression or activity of cPLA2 or COX-2. We did note a change in the intracellular position of cPLA2 and found that cPLA2 did not translocate normally in infected cells, raising the possibility that Ad5 interferes with the PG pathway by interfering with the intracellular movement of cPLA2. Taken together, these data reveal dynamic interactions between Ad5 and the lipid mediator pathways of the host and highlight a novel mechanism by which Ad5 evades the host immune response. In addition, our results offer insight into the inflammatory response induced by many Ad vectors lacking early region gene products.     

alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.     

Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-alpha-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-alpha-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-alpha. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-alpha-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-alpha are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-alpha-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.     

Human group V phospholipase A2 induces group IVA phospholipase A2-independent cysteinyl leukotriene synthesis in human eosinophils

We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA2 activation.     

IL1β-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1β in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1β, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1β. Further analysis indicated that the major COX-2 product, prostaglandin E(2), directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1β. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1β in the fibroblasts.