Epstein-Barr virus Zta-induced immunomodulators from nasopharyngeal carcinoma cells upregulate interleukin-10 production from monocytes

During lytic infection with Epstein-Barr virus (EBV), several viral lytic proteins function to evade immune recognition or to actively suppress immune cells. An EBV lytic transactivator, Zta, induces an immunosuppressive cytokine interleukin 10 (IL-10) in B cells, but whether it regulates IL-10 in the context of epithelial cells is unclear. In this study, we tested nasopharyngeal carcinoma (NPC) cell lines and found that Zta did not induce IL-10 in these epithelial cells. Interestingly, the conditioned medium of Zta-expressing NPC cells enhanced IL-10 production from monocytes. We further revealed that the IL-10-inducing effect involved at least two immunomodulators that were upregulated by Zta and secreted from NPC cells: granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostaglandin E(2) (PGE(2)). Zta was recruited to and activated the GM-CSF promoter, thus upregulating GM-CSF expression. Zta also activated the promoter of cyclooxygenase-2 (COX-2), and Zta-induced COX-2 increased downstream PGE(2) production. Cotreatment with GM-CSF and PGE(2) synergistically induced IL-10 production from monocytes. The IL-10-inducing effect of the Zta-conditioned medium was reduced when GM-CSF or the COX-2/PGE(2) pathway was blocked. The conditioned medium of NPC cells with EBV lytic infection showed a similar increase of GM-CSF and PGE(2) levels as well as the IL-10-inducing effect on monocytes, and knockdown of Zta abolished all the effects. Therefore, through Zta-induced immunomodulators, EBV lytic infection in NPC cells can direct bystander monocytes to produce IL-10, which may be a novel way of EBV to promote local immunosuppression.     

Analysis of isoform specific ERK signaling on the effects of interleukin-1β on COX-2 expression and PGE2 production in human chondrocytes

The MAPK/ERK pathway is involved in IL-1β-induced cyclooxygenase (COX-2) expression and prostaglandin E2 (PGE2) production; two factors that play important roles in OA pathogenesis. In the present study, we find that IL-1β induced COX-2 expression and PGE2 production in human chondrocytes via a process that required the activation of the MAPK/ERK pathway. To evaluate the respective roles and relationship of ERK1 and ERK2 on IL-1β induced COX-2 expression and PGE2 production, small interfering RNA was used to knockdown ERK1, ERK2 or both in human chondrocytes. COX-2 expression and PGE2 production were significantly suppressed to a similar degree by the silencing of ERK1 or ERK2 alone. Moreover, the combined knockdown displayed a synergistic effect. Simultaneously, Western blotting indicated that the knockdown of ERK1 or ERK2 down regulated phospho-ERK1 and ERK1 or phospho-ERK2 and ERK2 levels, respectively. No significant compensatory mechanism through the upregulation of the other phospho-ERK and ERK isoform was observed. The combined silencing suppressed both phospho-ERK1/2 and ERK1/2. In conclusion, each ERK isoform similarly influenced IL-1β-mediated COX-2 expression and PGE2 production in human chondrocytes, and ERK1 and ERK2 displayed synergistic effects. Although, inhibition of both ERK1 and ERK2 would be a more effective, each ERK isoform may sufficiently regulate these effects in human chondrocytes. ERK1 or ERK2 may be potential therapeutic target for the inflammatory process of OA.     

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.     

Role of cyclooxygenase-2 in cytokine-induced beta-cell dysfunction and damage by isolated rat and human islets

Type I diabetes mellitus is an autoimmune disease characterized by the selective destruction of the insulin-secreting beta-cell found in pancreatic islets of Langerhans. Cytokines such as interleukin-1 (IL-1), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mediate beta-cell dysfunction and islet degeneration, in part, through the induction of the inducible isoform of nitric-oxide synthase and the production of nitric oxide by beta-cells. Cytokines also stimulate the expression of the inducible isoform of cyclooxygenase, COX-2, and the production of prostaglandin E(2) (PGE(2)) by rat and human islets; however, the role of increased COX-2 expression and PGE(2) production in mediating cytokine-induced inhibition of islet metabolic function and viability has been incompletely characterized. In this study, we have shown that treatment of rat islets with IL-1beta or human islets with a cytokine mixture containing IL-1beta + IFN-gamma +/- TNF-alpha stimulates COX-2 expression and PGE(2) formation in a time-dependent manner. Co-incubation of rat and human islets with selective COX-2 inhibitors SC-58236 and Celecoxib, respectively, attenuated cytokine-induced PGE(2) formation. However, these inhibitors failed to prevent cytokine-mediated inhibition of insulin secretion or islet degeneration. These findings indicate that selective inhibition of COX-2 activity does not protect rat and human islets from cytokine-induced beta-cell dysfunction and islet degeneration and, furthermore, that islet production of PGE(2) does not mediate these inhibitory and destructive effects.     

Effects of TGF-beta, TNF-alpha, IL-beta and IL-6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, prostaglandin E2 production and bone resorption in mouse calvarial bone cells

Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E(2) (PGE(2)) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and, to a lesser extent, TNF-alpha, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1beta and TNF-alpha resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE(2) biosynthesis compared to cells treated with IL-1beta or TNF-alpha or IL-6 alone. In contrast, the presence of TGF-beta reduced COX-2 mRNA level, PGE(2) biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or a combination thereof. However, neither IL-1beta, TNF-alpha, IL-6 nor a combination of IL-1beta, TNF-alpha, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE(2) production induced by IL-1beta, TNF-alpha and IL-6 or a combination of IL-1beta, TNF-alpha, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-beta, TNF-alpha, IL-6 on PGE(2) production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss.     

Prostaglandin E(2) stimulates prostatic intraepithelial neoplasia cell growth through activation of the interleukin-6/GP130/STAT-3 signaling pathway.

Cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) secretion are increased in prostatic intraepithelial neoplasia (PIN) and prostate cancer. PGE(2) biosynthesis by cyclooxygenase (COX)-2 plays a pivotal role in inflammation and carcinogenesis. One of the critical proinflammatory cytokines in the prostate is interleukin-6 (IL-6). We hypothesized that increased expression of COX-2, with resultant increased levels of PGE(2) in human PIN cells, activates the IL-6 signaling pathway. We demonstrate an autocrine upregulation of PGE(2) mediated by IL-6 in a human PIN cell line. We further demonstrate that PGE(2) stimulates soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, Stat-3 protein phosphorylation, and DNA binding activity. These events, induced by PGE(2), lead to increased PIN cell growth. Treatment of PIN cells with a selective COX-2 inhibitor decreases cell growth. Finally, PGE(2)-stimulated PIN cell growth was abrogated by the addition of IL-6 neutralizing antibodies. These data provide mechanistic evidence that increased expression of COX-2/PGE(2) contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway.     

Viscum album exerts anti-inflammatory effect by selectively inhibiting cytokine-induced expression of cyclooxygenase-2

Viscum album (VA) preparations are extensively used as complementary therapy in cancer and are shown to exert anti-tumor activities which involve the cytotoxic properties, induction of apoptosis, inhibition of angiogenesis and several other immunomodulatory mechanisms. In addition to their application in cancer therapy, VA preparations have also been successfully utilized in the treatment of several inflammatory pathologies. Owing to the intricate association of inflammation and cancer and in view of the fact that several anti-tumor phytotherapeutics also exert a potent anti-inflammatory effect, we hypothesized that VA exerts an anti-inflammatory effect that is responsible for its therapeutic benefit. Since, inflammatory cytokine-induced cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) play a critical role in the pathogenesis of inflammatory diseases, we investigated the anti-inflammatory effect of VA on regulation of cyclo-oxygenase expression and PGE2 biosynthesis by using human lung adenocarcinoma cells (A549 cells) as a model. A549 cells were stimulated with IL-1β and treated with VA preparation (VA Qu Spez) for 18 hours. PGE2 was analysed in the culture supernatants by enzyme immunoassay. Expression of COX-2 and COX-1 proteins was analyzed by immunoblotting and the expression of COX-2 mRNA was assessed by semi-quantitative RT-PCR. We found that VA Qu Spez inhibit the secretion of IL-1β-induced PGE2 in a dose-dependent manner. Further, we also show that this inhibitory action was associated with a reduced expression of COX-2 without modulating the COX-1 expression. Together these results demonstrate a novel anti-inflammatory mechanism of action of VA preparations wherein VA exerts an anti-inflammatory effect by inhibiting cytokine-induced PGE2 via selective inhibition of COX-2.     

Nucleobindin co-localizes and associates with cyclooxygenase (COX)-2 in human neutrophils

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca(2+)-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2) biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.     

Heregulin-alpha and heregulin-beta expression is linked to a COX-2-PGE2 pathway in human gastric fibroblasts

We have previously shown heregulin (HRG)-alpha expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE(2) or interleukin (IL)-1beta, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG-alpha and HRG-beta mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE(2), or IL-1beta, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE(2). As with erbB3 phophorylation, IL-1beta stimulated both HRG-alpha and HRG-beta mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1beta-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE(2) restored this inhibitory effect, suggesting the activation of an IL-1beta-COX-2-PGE(2) pathway that culminates in the release of HRG from fibroblasts. HRG-alpha and HRG-beta mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.     

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.     

Role of p38 in nitric oxide synthase and cyclooxygenase expression, and nitric oxide and PGE2 synthesis in human gingival fibroblasts stimulated with lipopolysaccharides

Periodontal disease, a gingival inflammatory disease caused by gram-negative bacteria, is the main cause of tooth loss. Lipopolysaccharides (LPS) present in bacterial cell walls induce human gingival fibroblasts' production of pro-inflammatory cytotoxins such as IL-1beta and TNFalpha. The goal of this study was to determine p38 role in the expression of inducible nitric oxide synthase enzyme (i-NOS) and cyclooxygenase (COX-2), as well as in PGE(2) and nitric oxide synthesis in human gingival fibroblasts challenged with LPS. We found that lipopolysaccharides induced a rapid and significant increase in p38 phosphorylation. After interruption of p38 transduction pathway by pre-treatment with inhibitor SB203580, no response to stimulation with LPS was observed; i-NOS expression and nitric oxide synthesis was completely blocked. However, p38 inhibition only partially blocked COX-2 expression and PGE2 synthesis. We conclude that p38 is critically involved in i-NOS induction, and that it participates in COX-2 expression and in PGE2 synthesis.     

Involvement of tyrosine kinases on cyclooxygenase expression and prostaglandin E2 production in human gingival fibroblasts stimulated with interleukin-1beta and epidermal growth factor

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tyrosine kinase on prostaglandin E2 (PGE2) production in human gingival fibroblasts stimulated by interleukin-1beta (IL-1beta) and/or epidermal growth factor (EGF). The cytokine IL-1beta and to a lesser extent EGF, enhanced COX-2 mRNA levels in gingival fibroblasts. Simultaneous treatment with EGF and IL-1beta resulted in enhanced COX-2 mRNA levels accompanied by a synergistic stimulation of PGE2 biosynthesis compared to the cells treated with IL-1beta or EGF alone. Neither IL-1beta EGF nor the combination of IL-1beta and EGF enhanced COX-1 mRNA levels in gingival fibroblasts. The tyrosine kinase inhibitors, Herbimycin A and PD 153035 hydrochloride, reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1beta or the combination of IL-1beta and EGF whereas COX-1 mRNA levels were not affected. Furthermore, the COX-2 specific inhibitor, NS-398, abolished the PGE2 production induced by IL-1beta, EGF, or the combination. These results indicate that the synergy between IL-1beta and EGF on PGE2 production is due to an enhanced gene expression of COX-2 and that tyrosine kinase(s) are involved in the signal transduction of COX-2 in gingival fibroblasts.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

TLR3-dependent induction of nitric oxide synthase in RAW 264.7 macrophage-like cells via a cytosolic phospholipase A2/cyclooxygenase-2 pathway

dsRNA is a by-product of viral replication capable of inducing an inflammatory response when recognized by phagocyte cells. In this study, we identify group IVA cytosolic phospholipase A2 (cPLA2alpha) as an effector of the antiviral response. Treatment of RAW 264.7 murine macrophage-like cells with the dsRNA analog polyinosinic:polycytidylic acid (poly-IC) promotes the release of free arachidonic acid that is subsequently converted into PGE2 by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA2alpha inhibitor pyrrophenone, pointing out to cPLA2alpha as the effector involved. In keeping with this observation, the cPLA2alpha phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA2alpha expression and activity by either small interfering RNA (siRNA) or pyrrophenone leads to inhibition of the expression of the inducible NO synthase (iNOS) gene. Moreover, COX-2-derived PGE2 production appears to participate in iNOS expression, because siRNA inhibition of COX-2 also leads to inhibition of iNOS, the latter of which is restored by exogenous addition of PGE2. Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE2 generation, and iNOS induction by poly-IC. Collectively, these findings suggest a model for macrophage activation in response to dsRNA, whereby engagement of TLR3 leads to cPLA2alpha-mediated arachidonic acid mobilization and COX-2-mediated PGE2 production, which cooperate to induce the expression of iNOS.     

Potential role of group X secretory phospholipase A(2) in cyclooxygenase-2-dependent PGE(2) formation during colon tumorigenesis

Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.     

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.     

Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts

We have previously shown that the cyclooxygenase (COX)-2/PGE2 pathway plays a key role in VEGF production in gastric fibroblasts. Recent studies have identified three PGE synthase (PGES) isozymes: cytosolic PGES (cPGES) and microsomal PGES (mPGES)-1 and -2, but little is known regarding the expression and roles of these enzymes in gastric fibroblasts. Thus we examined IL-1beta-stimulated mPGES-1 and cPGES mRNA and protein expression in gastric fibroblasts by quantitative PCR and Western blot analysis, respectively, and studied both their relationship to COX-1 and -2 and their roles in PGE2 and VEGF production in vitro. IL-1beta stimulated increases in both COX-2 and mPGES-1 mRNA and protein expression levels. However, COX-2 mRNA and protein expression were more rapidly induced than mPGES-1 mRNA and protein expression. Furthermore, MK-886, a nonselective mPGES-1 inhibitor, failed to inhibit IL-1beta-induced PGE2 release at the 8-h time point, while totally inhibiting PGE2 at the later stage. However, MK-886 did inhibit IL-1beta-stimulated PGES activity in vitro by 86.8%. N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (NS-398), a selective COX-2 inhibitor, totally inhibited PGE2 production at both the 8-h and 24-h time points, suggesting that COX-2-dependent PGE2 generation does not depend on mPGES-1 activity at the early stage. In contrast, NS-398 did not inhibit VEGF production at 8 h, and only partially at 24 h, whereas MK-886 totally inhibited VEGF production at each time point. These results suggest that IL-1beta-induced mPGES-1 protein expression preferentially coupled with COX-2 protein at late stages of PGE2 production and that IL-1beta-stimulated VEGF production was totally dependent on membrane-associated proteins involved in eicosanoid and glutathione metabolism (MAPEG) superfamily proteins, which includes mPGES-1, but was partially dependent on the COX-2/PGE2 pathway.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.