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.     

p38 MAP kinase regulates IL-1 beta responses in cultured airway smooth muscle cells

We have previously reported that interleukin (IL)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.     

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.     

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.     

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.     

Sphingosine-1-phosphate enhances IL-1{beta}-induced COX-2 expression in mouse intestinal subepithelial myofibroblasts

Intestinal subepithelial myofibroblasts (SEMFs) is a specific population of cells involved in intestinal inflammation and carcinogenesis via an elaborate network of cytokines, chemokines and other inflammatory factors, including PGE(2). Sphingosine-1-phosphate (S1P) has been implicated as an important mediator of inflammation and cancer and in certain cell types increases cyclooxygenase-2 (COX-2) expression. In the present study, we aimed to assess involvement of S1P in COX-2 expression by SEMFs. Primary SEMFs were obtained from C57BL/6J mouse and their identity was verified by fluorescent staining of specific marker proteins. Expression of S1P receptors 1, 2, 3 and sphingosine kinases 1 and 2 in SEMFs were determined by RT-PCR analysis. COX-2 expression and PGE(2) production were assayed by Western blotting and ELISA, respectively. COX-2 mRNA stability was assayed by Northern blotting. S1P produced dose-dependent increase in COX-2 expression, resulting in increased PGE(2) release from SEMFs. Using specific inhibitors, we show that actions of p38, ERK, IKK, and PKC were involved in S1P-induced COX-2 expression. On the other hand, p38 and PKC had lesser roles in IL-1beta-induced COX-2 expression. Inhibition of sphingosine kinase to block S1P production did not affect IL-1beta-induced COX-2 expression, but S1P amplified IL-1beta-induced p38 activation and COX-2 expression. PKC inhibition blocked S1P amplified COX-2 expression. S1P addition increased COX-2 mRNA stability. In SEMFs, S1P amplifies IL-1beta-induced COX-2 expression through increased mRNA stability. These observations point to involvement of S1P in activation of SEMFs that may contribute to intestinal inflammation and carcinogenesis.     

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.     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Osteoprotegerin levels increased by interleukin-1beta in human periodontal ligament cells are suppressed through prostaglandin E(2) synthesized de novo

Osteoprotegerin (OPG) is a novel tumor necrosis factor receptor superfamily that inhibits osteoclast differentiation, activity, and survival. Interleukin-1beta (IL-1beta) increases OPG expression. IL-1beta also increases prostaglandin E(2) (PGE(2)) production and stimulates bone resorption. In the present study, we examined the involvement of PGE(2) in IL-1beta-induced increases in OPG levels in human periodontal ligament cells (HPL cells) in an effort to clarify apparently conflicting IL-1beta actions on bone resorption and understand IL-1beta-induced increases in secretion of OPG and PGE(2) in HPL cells. 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a mRNA synthesis inhibitor, partly inhibited the increase in OPG mRNA levels induced by IL-1beta. Cycloheximide, a protein synthesis inhibitor, enhanced the stimulatory effect of IL-1beta. Etodolac, a selective cyclooxygenase-2 inhibitor, suppressed the increase in PGE(2) levels. Furthermore, etodolac reinforced the promotion of OPG expression by IL-1beta at the mRNA and protein levels. PGE(2) added to cultures of HPL cells decreased OPG mRNA levels in a dose- and time- dependent manner. These findings suggest that the increase in OPG levels induced by IL-1beta in HPL cells is suppressed through PGE(2) synthesized de novo.     

IL-1 beta and prostaglandins regulate integrin mRNA expression.

The purpose of this study was to examine the effects of IL-1 beta on integrin expression in MG-63 human osteosarcoma cells. Human recombinant IL-1 beta (rIL-1 beta) produced significant increases in both alpha 2- and alpha 5-subunit mRNA levels, as well as a smaller increase in alpha v-subunit mRNA. In contrast, IL-1 beta decreased alpha 4-subunit mRNA levels by approximately 30% relative to untreated controls. These findings suggest that human IL-1 beta differentially regulates expression of integrins. When cultures were treated with both IL-1 beta and the cyclooxygenase inhibitor, indomethacin, the expression of alpha 2-, alpha 5-, and alpha v-subunit mRNA levels were dramatically increased relative to untreated controls; co-treatment with 0.5 mM prostaglandin E2 (PGE2) partially reversed this effect. Indomethacin alone did not affect integrin mRNA levels. Treatment with IL-1 beta or IL-1 beta + indomethacin also induced significant changes in MG-63 morphology (i.e., increased cell elongation) and increased the ability of cells to contract collagen gels. PGE2 reversed the above effects on cell morphology and gel contraction. These findings indicate that (a) IL-1 beta differentially regulates the expression of integrins and (b) that PGE2, which is induced by IL-1 beta, may provide a negative feedback loop which counteracts the stimulatory effect of IL-1 beta on integrin gene expression. It is suggested that products of inflammation may affect cell behavior by differentially regulating the expression of various integrins.     

Interleukin-1beta-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS,and NF-kappaB pathways in canine tracheal smooth muscle cells

Interleukin-beta (IL-1beta) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1beta-enhanced cyclooxygenase (COX)-2 expression associated with PGE(2) synthesis in tracheal smooth muscle cells (TSMCs). IL-1beta markedly increased COX-2 expression and PGE(2) formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE(2) formation in response to IL-1beta were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1beta-induced activation of NF-kappaB correlated with the degradation of IkappaB-alpha in TSMCs. IL-1beta-induced NF-kappaB activation, COX-2 expression, and PGE(2) synthesis were inhibited by the dominant negative mutants of NIK and IKK-alpha, but not by IKK-beta. IL-1beta-induced COX-2 expression and PGE(2) synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1beta-induced NF-kappaB activation, indicating that activation of p42/44 and p38 MAPK and NF-kappaB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE(2) from IL-1beta-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-kappaB signalling pathways in canine TSMCs. IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, tyrosine kinase, and PI3-K in these cells.     

Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.