Interleukin-1beta-induced mucin production in human airway epithelium is mediated by cyclooxygenase-2, prostaglandin E2 receptors, and cyclic AMP-protein kinase A signaling

We reported recently that interleukin (IL)-1beta exposure resulted in a prolonged increase in MUC5AC mucin production in normal, well differentiated, human tracheobronchial epithelial (NHTBE) cell cultures, without significantly increasing MUC5AC mRNA (Am J Physiol 286:L320-L330, 2004). The goal of the present study was to elucidate the signaling pathways involved in IL-1beta-induced MUC5AC production. We found that IL-1beta increased cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin (PG) E(2) production and that the COX-2 inhibitor celecoxib suppressed IL-1beta-induced MUC5AC production. Addition of exogenous PGE(2) to NHTBE cultures also increased MUC5AC production and IL-1beta-induced Muc5ac hypersecretion in tracheas from wild-type but not from COX-2-/- mice. NHTBE cells expressed all four E-prostanoid (EP) receptor subtypes and misoprostol, an EP2 and EP4 agonist, increased MUC5AC production, whereas sulprostone, an EP1 and EP3 agonist, did not. Furthermore, specific protein kinase A (PKA) inhibitors blocked IL-1beta and PGE(2)-induced MUC5AC production. However, neither inhibition of epidermal growth factor receptor (EGFR) activation with the tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline HCl (AG-1478) or EGFR blocking antibody nor inhibition of extracellular signal-regulated kinase/P-38 mitogen activated protein kinases with specific inhibitors blocked IL-1beta stimulation of MUC5AC mucin production. We also observed that tumor necrosis factor (TNF)-alpha, platelet activating factor (PAF), and lipopolysaccharide (LPS) induced COX-2 and increased MUC5AC production that was blocked by celecoxib, suggesting a common signaling pathway of inflammatory mediator-induced MUC5AC production in NHTBE cells. We conclude that the induction of MUC5AC by IL-1beta, TNF-alpha, PAF, and LPS involves COX-2- generated PGE(2), activation of EP2 and/or EP4 receptor(s), and cAMP-PKA-mediated signaling.     

Regulation of cyclooxygenase-2 expression by phospholipase D in human amnion-derived WISH cells

Prostaglandins (PGs) are known to play a key role in the initiation of labor, but the mechanisms regulating their synthesis in amnion are largely unknown. In this study, the regulatory mechanisms for PGE(2) production during phospholipase D (PLD) and p38-dependent activation of WISH cells were investigated. We found that the stimulation of WISH cells with interleukin (IL)-1 beta elicited dose-dependent synthesis of cyclooxygenase-2 (COX-2) mRNA, protein, and their products, PGE(2). Moreover, the treatment of [(3)H]myristate-labeled cells in the presence of 1-butanol caused the dose-dependent formation of [(3)H]phosphatidylbutanol (PBt), a product specific to PLD activity. Pretreating the cells with 1-butanol and Ro 31-8220 inhibited the IL-1 beta-induced COX-2 expression, but 3-butanol did not affect this response. In addition, evidence that PLD was involved in the stimulation of COX-2 expression was provided by the observations that COX-2 expression was stimulated by the dioctanoyl phosphatidic acid (PA) and that the prevention of PA dephosphorylation by 1-propranolol potentiated COX-2 expression by IL-1 beta. Moreover, IL-1 beta stimulation of the cells caused the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and IL-1 beta-induced COX-2 expression was inhibited by the pretreatment of WISH cells with a p38 inhibitor, in contrast ERK upstream inhibitor had no effect. Furthermore, Ro 31-8220 inhibited IL-1 beta-induced p38 phosphorylation but not ERK phosphorylation. The results of this study indicate that in human amnion cells, IL-1 beta might activate PLD through an upstream protein kinase C to elicit p38 and finally induce COX-2 expression.     

Induction of COX-2 and PGE(2) biosynthesis by IL-1beta is mediated by PKC and mitogen-activated protein kinases in murine astrocytes

Interleukin-1 (IL-1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E(2) (PGE(2)) production after IL-1beta stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL-1beta or the phorbol ester, PMA significantly increased PGE(2) secretion. The stimulatory action of IL-1beta on PGE(2) production was totally abolished by NS-398, a specific inhibitor of cyclo-oxygenase-2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL-1beta induced the expression of COX-2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL-1 beta treatment also induced the expression of COX-2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H-7, bisindolylmaleimide and calphostin C) inhibited IL-1beta stimulation of PGE(2). In addition, PKC-depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL-1. The ablation of the effects of PMA and IL-1beta on PGE(2) production, likely results from down-regulation of phorbol ester sensitive-PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC-alpha from cytosol to membrane following treatment with IL-1beta. In addition, IL-1beta treatment led to activation of extracellular signal-regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL-1beta-induced PGE(2) release. ERK1/2 activation by IL-1beta was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE(2), likely by regulating the induction of cyclo-oxygenase-2, in IL-1beta-stimulated astroglial cells.     

Inhibition of acrolein-stimulated MUC5AC expression by Platycodon grandiflorum root-derived saponin in A549 cells

Mucin overproduction is a hallmark of chronic airway diseases such as chronic obstructive pulmonary disease. In this study, we investigated the inhibition of acrolein-induced expression of mucin 5, subtypes A and C (MUC5AC) by Changkil saponin (CKS) in A549 cells. Acrolein, a known toxin in tobacco smoke and an endogenous mediator of oxidative stress, increases the expression of airway MUC5AC, a major component of airway mucus. CKS, a Platycodon grandiflorum root-derived saponin, inhibited acrolein-induced MUC5AC expression and activity, through the suppression of NF-κB activation. CKS also repressed acrolein-induced phosphorylation of ERK1/2, JNK1/2, and p38MAPK, which are upstream signaling molecules that control MUC5AC expression. In addition, the MAPK inhibitors PD98059 (ERK1/2), SP600125 (JNK1/2), and SB203580 (p38 MAPK), and a PKC delta inhibitor (rottlerin; PKCδ) inhibited acrolein-induced MUC5AC expression and activity. CKS repressed acrolein-induced phosphorylation of PKCδ. Moreover, a reactive oxygen species (ROS) inhibitor, N-acetylcysteine, inhibited acrolein-induced MUC5AC expression and activity through the suppression of PKCδ and MAPK activation, and CKS repressed acrolein-induced ROS production. These results suggest that CKS suppresses acrolein-induced MUC5AC expression by inhibiting the activation of NF-κB via ROS-PKCδ-MAPK signaling.     

The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1beta-dependent PGE(2) release via mechanistically distinct processes

1. In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E(2) in response to pro-inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL-1beta-dependent release of PGE(2). 2. Following IL-1beta treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. 3. PD09059, UO126 and SB203580 prevented IL-1beta-induced PGE(2) release at doses that correlated closely with published IC(50) values. Small or partial effects at the relevant doses were observed on induction of cyclo-oxygenase (COX) activity or COX-2 protein suggesting that the primary effects were at the level of arachidonate availability. 4. Neither PD098059 nor SB203580 showed any effect on IL-1beta-induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX-2. 5. In contrast, UO126 was highly effective at inhibiting IL-1beta-dependent arachidonate release, implicating MEK2 in the activation of the PLA(2) that is involved in IL-1beta-dependent PGE(2) release. 6. We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti-inflammatory benefits.     

Byakangelicol,isolated from Angelica dahurica,inhibits both the activity and induction of cyclooxygenase-2 in human pulmonary epithelial cells

We examined the inhibitory mechanism of byakangelicol, isolated from Angelica dahurica, on interleukin-1beta (IL-1beta)-induced cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human pulmonary epithelial cell line (A549). Byakangelicol (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 expression and PGE2 release. The selective COX-2 inhibitor, NS-398 (0.01-1 microM), and byakangelicol (10-50 microM) both concentration-dependently inhibited the activity of the COX-2 enzyme. Byakangelicol, at a concentration up to 200 microM, did not affect the activity and expression of COX-1 enzyme. IL-1beta-induced p44/42 mitogen-activated protein kinase (MAPK) activation was inhibited by the MAPK/extracellular signal-regulated protein kinase (MEK) inhibitor, PD 98059 (30 microM), while byakangelicol (50 microM) had no effect. Treatment of cells with byakangelicol (50 microM) or pyrrolidine dithiocarbamate (PDTC; 50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol, translocation of p65 NF-kappaB from the cytosol to the nucleus and the NF-kappaB-specific DNA-protein complex formation. Taken together, we have demonstrated that byakangelicol inhibits IL-1beta-induced PGE2 release in A549 cells; this inhibition may be mediated by suppression of COX-2 expression and the activity of COX-2 enzyme. The inhibitory mechanism of byakangelicol on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. Therefore, byakangelicol may have therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs-AP-1 and IKKs-IκB-NF-κB signaling pathways

Naringenin, the aglycone of naringin, has been reported to attenuate MUC5AC secretion by inhibiting activity of nuclear factor kappa B (NF-κB) via EGFR-PI3K-Akt/ERK MAPKinase signaling pathways. However, previous studies demonstrated that the MUC5AC promoter was located in two different regions: an activator protein-1 (AP-1) binding site and a NF-κB binding site. The current study comprehensively determined the involvement of MAPKs/AP-1 and IKKs/IκB/NF-κB in epidermal growth factor (EGF)-induced A549 cells, and sought to ascertain the signaling pathways of naringin imparted in suppression of EGF-induced MUC5AC secretion. The results showed that naringin of 100μM not only significantly decreased EGF-induced overexpressions of both MUC5AC mucin and mRNA in A549 cells, but also suppressed the phosphorylation of EGF receptor, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK), as well as nucleus NF-κB p65 and AP-1. Moreover, any of three MAPKs inhibitors (PD98059, SB203580, and SP600125) significantly inhibited EGF-induced MUC5AC secretion. And as compared to MG132, the inhibitor κB (IκB) phosphorylation inhibitor of SN50 was more effective in reducing EGF-induced MUC5AC secretion because of suppression of nucleus AP-1. Meanwhile, as compared to naringin, both SP600125 and azithromycin were less effective in suppressing EGF-induced secretion of MUC5AC because of the unchanged nucleus NF-κB p65. These results indicated that naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs/AP-1 and IKKs/IκB/NF-κB signaling pathways.     

Mitogen-activated protein kinase signalling pathways in IL-1 beta-dependent rat airway smooth muscle proliferation

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen-activated protein kinase (MAPK) pathway in IL-1beta induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen-activated and extracellular regulated kinase kinase, MEK-1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c-jun N-terminal kinase, JNK, inhibitor) on IL-1beta-induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL-1beta in a dose-dependent manner. p38, JNK and ERK MAPKs were activated by IL-1beta in a time-dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 microM) had no effect on IL-1beta-induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose-dependently inhibited IL-1beta-dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL-1beta-dependent proliferation of rat ASM.     

Involvement of protein kinase C-gamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

The signaling pathway of protein kinase C (PKC) is known to play a role in mediating the action of various cytokines. Here we examined the signal transduction pathway of PKC activation and the role of PKC isoforms in interleukin-1beta (IL-1beta)-mediated cyclooxygenase-2 (COX-2) expression in human pulmonary epithelial cell line (A549). The tyrosine kinase inhibitors (genistein and tyrphostin AG126) and phosphatidylcholine-phospholipase C inhibitor (D-609) prevented IL-1beta-induced prostaglandin E(2) (PGE(2)) release and COX-2 expression, whereas U-73122 (a phosphatidylinositol-phospholipase C inhibitor) and propranolol (a phosphatidate phosphohydrolase inhibitor) had no effect. The PKC inhibitors (Go 6976 and Ro 31-8220) and NF-kappaB inhibitor, pyrrolidine dithiocarbamate, also attenuated IL-1beta-induced PGE(2) release and COX-2 expression. Western blot analysis using PKC isoenzyme-specific antibodies indicated that A549 cells expressed PKC-alpha, -gamma, -iota, -lambda, -zeta, and -micro. IL-1beta caused the translocation of PKC-gamma but not other isoforms from cytosol to the membrane fraction. Moreover, the translocation of PKC-gamma was inhibited by genistein or D-609, but not by U-73122. IL-1beta caused the translocation of p65 NF-kappaB from cytosol to the nucleus as well as the degradation of IkappaB-alpha in cytosol. Furthermore, the translocation of p65 NF-kappaB was inhibited by genistein, Go 6976, Ro 31-8220, or pyrrolidine dithiocarbamate. These results indicate that in human pulmonary epithelial cells, IL-1beta might activate phosphatidylcholine-phospholipase C through an upstream tyrosine phosphorylation to elicit PKC activation, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE(2) release. Of the PKC isoforms present in A549 cells, only activation of PKC-gamma is involved in regulating IL-1beta-induced responses.     

2-methoxycinnamaldehyde reduces IL-1beta-induced prostaglandin production in rat cerebral endothelial cells

Prostaglandin E2 (PGE2) works as a common final mediator of the febrile. Guizhi-Tang, one of the most famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions, was previously reported to reduce the production of PGE 2 in rats. 2-Methoxycinnamaldehyde is a principle compound isolated from Guizhi-Tang. The aim of the present study was to investigate the effects of 2-methoxycinnamaldehyde on PGE2 production of rat cerebral endothelial cells (CECs). 2-Methoxycinnamaldehyde dose-dependently inhibited interleukin (IL)-1beta-induced PGE2 production in CECs with IC50 values of 174 microM. IL-1beta stimulation increased the protein, activity and mRNA expression of cyclooxygenase (COX)-2 but not COX-1. 2-Methoxycinnamaldehyde reduced IL-1beta-induced protein and activity of COX-2, but did not influence the COX-2 mRNA expression. Our results show that prostaglandin production in CECs during stimulated conditions is sensitive to inhibition by 2-methoxycinnamaldehyde and suggest that 2-methoxycinnamaldehyde may reduce COX-2 protein level and activity but not COX-2 mRNA.     

15-Deoxy-delta12,14-prostaglandin-J2 up-regulates cyclooxygenase-2 but inhibits prostaglandin-E2 production through a thiol antioxidant-sensitive mechanism

15-Deoxy-delta12,14-prostaglandin-J(2) (15d-PGJ(2)) has potent anti-inflammatory effects including the inhibition of interleukin-1beta (IL-1beta)-induced expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) production in several cell types. 15d-PGJ(2) contains an alpha,beta-unsaturated electrophilic ketone and several evidences suggest that thiol reducing agents prevent or revert the cellular effects of 15d-PGJ(2). The present study was devoted to analyze the effect of 15d-PGJ(2) on COX-2 expression in cultured human mesangial cells (HMC). 15d-PGJ(2) induced an increase in the reduced glutathione (GSH) content and up-regulated COX-2 protein expression, but not COX-1, in a manner which was unaffected by selective peroxisome proliferator-activated receptor gamma (PPARgamma) blockade nor mimicked by ciglitazone, a PPARgamma agonist. N-acetylcysteine (NAC), a thiol reducing agent, but not reactive oxygen species scavengers, prevented 15d-PGJ(2)-induced COX-2 up-regulation. Depletion of GSH by buthionine sulfoximine, which diminishes thiol antioxidant activity, cooperated with 15d-PGJ(2) to accumulate COX-2. Therefore, 15d-PGJ(2) up-regulated COX-2 through a thiol antioxidant-sensitive mechanism. Interestingly, NAC did not inhibit the COX-2 expression induced by the electrophilic alpha,beta-unsaturated compound PGA(2). Up-regulation of COX-2 by 15d-PGJ(2) did not result in increased PGE(2) production. Furthermore, preincubation with 15d-PGJ(2) inhibited IL-1beta-induced PGE(2) production although IL-1beta-induced COX-2 expression remained unaffected by the treatment with 15d-PGJ(2). On the contrary, PGA(2) elicited an increase in PGE(2) production and it acted synergistically with IL-1beta to enhance PGE(2) production. These results indicate for the first time that 15d-PGJ(2) inhibits PGE(2) production independently of its effect on COX-2 expression.     

Ent-pimara-8(14), 15-dien-19-oic acid isolated from the roots of Aralia cordata inhibits induction of inflammatory mediators by blocking NF-kappaB activation and mitogen-activated protein kinase pathways

Macrophages play central roles in the innate immune system. The roots of Aralia cordata are widely used in Oriental medicine as a remedy for arthritis. During our program to screen medicinal plants for potential anti-inflammatory compounds, ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) was isolated from the roots of A. cordata. We examined the effect of PA on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. PA was found to significantly inhibit the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and interleukin-6 (IL-6), as well as the expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and IL-6. Furthermore, we examined whether mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are involved in LPS-induced RAW 264.7 cells. We found that a p38 inhibitor (SB203580) and an ERK 1/2 inhibitor (PD98059) significantly affected LPS-induced IL-6 production. In contrast, a JNK 1/2 inhibitor (SP600125) and PI3K inhibitor (wortmannin or LY294002) did not block the induction of IL-6 production by LPS. The LPS-induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) was inhibited by PA, but not the phosphorylation of JNK 1/2 and AKT (Ser473). Moreover, PA suppressed I kappaB alpha degradation, NF-kappaB activation and luciferase activity. These results suggest that PA isolated from A. cordata has a potential regulatory effect on inflammatory iNOS, COX-2 and IL-6 expression through blockade of the phosphorylation of MAPKs following I kappaB alpha degradation and NF-kappaB activation.     

Kaempferol Regulates the Expression of Airway MUC5AC Mucin Gene via IκBα-NF-κB p65 and p38-p44/42-Sp1 Signaling Pathways

In the present study, kaempferol, a flavonoidal natural compound found in Polygonati Rhizoma, was investigated for its potential effect on the gene expression and production of airway MUC5AC mucin. A human respiratory epithelial NCI-H292 cells was pretreated with kaempferol for 30 min and stimulated with epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA), for the following 24 h. The effect on PMA-induced nuclear factor kappa B (NF-κB) signaling pathway or EGF-induced mitogen-activated protein kinase (MAPK) signaling pathway was investigated. Kaempferol suppressed the production and gene expression of MUC5AC mucins, induced by PMA through the inhibition of degradation of inhibitory kappa Bα (IκBα), and NF-κB p65 nuclear translocation. Also, kaempferol inhibited EGF-induced gene expression and production of MUC5AC mucin through regulating the phosphorylation of EGFR, phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2 (p44/42), and the nuclear expression of specificity protein-1 (Sp1). These results suggest kaempferol regulates the gene expression and production of mucin through regulation of NF-κB and MAPK signaling pathways, in human airway epithelial cells.     

IL-1β对人鼻黏膜上皮细胞黏蛋白MUC5AC mRNA的表达的影响

目的探讨IL-1β对鼻黏膜上皮细胞黏蛋白MUC5ACmRNA表达的影响。方法在培养的第二代人鼻黏膜上皮细胞加入IL-1β(10ng/ml)刺激24h后,采用荧光定量巢式RT-PCR检测人鼻黏膜上皮细胞中MUC5ACmRNA的定量表达。结果在培养的鼻黏膜上皮细胞上检测到MUC5ACmRNA的表达,IL-1β刺激组MUC5ACmRNA定量表达[(4.60±1.89)108拷贝/μg]均高于对照组[(2.50±1.40)107拷贝/μg],差异具有统计学意义(P<0.05﹚。结论 IL-1β诱导鼻黏膜上皮细胞中MUC5ACmRNA的表达增高,提示IL-1β可能具有上调鼻黏膜上皮细胞黏蛋白mRNA的表达的作用。