concomitant activation of extracellular signal-regulated kinase and induction of cox-2 stimulates...

Prostaglandins & other Lipid Mediators 81 (2006) 126–135

Concomitant activation of extracellular signal-regulated kinaseand induction of COX-2 stimulates maximum prostaglandin

E2 synthesis in human airway epithelial cells

Nenad Petrovic a, Darryl A. Knight a, John S. Bomalaski b,Philip J. Thompson a, Neil L.A. Misso a,∗

a Lung Institute of Western Australia and Centre for Asthma, Allergy and Respiratory Research,The University of Western Australia, Perth, Australia

b Department of Medicine, MCP Hahnemann Medical School, Philadelphia, USA

Received 15 June 2006; received in revised form 1 August 2006; accepted 30 August 2006Available online 2 October 2006

Abstract

The intracellular regulation and kinetics of prostaglandin (PG)E2 synthesis in human airway epithelial (NCI-H292) cells wasinvestigated. Interleukin (IL)-1�, tumor necrosis factor (TNF)-� and lipopolysaccharide (LPS) all induced PGE2 synthesis (p < 0.001)and transient (5–15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) andcalcium ionophore, A23187 further enhanced PGE2 synthesis (p < 0.001) and caused phosphorylation of ERK that was sustainedfor up to 16 h. COX-2 protein expression and PGE2 synthesis were increased following exposure to combinations of stimuli thatincreased intracellular Ca2+, and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE2

synthesis in response to all stimuli. Sustained, maximum PGE2 synthesis was observed when cells were stimulated such that ERKphosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathwaysfor PGE2 synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2expression, so as to tightly regulate the kinetics and amount of PGE2 produced by airway epithelial cells in response to lunginflammation.© 2006 Elsevier Inc. All rights reserved.

Keywords: Prostaglandin E2; Cyclooxygenase-2; Extracellular signal regulated kinase; Phospholipase A2; Airway epithelium; Inflammation

1. Introduction

Aberrant airway epithelium function and interactions with the underlying mesenchymal layer play an importantrole in the persistent airway inflammation, smooth muscle dysfunction and airway remodeling that are characteristicof asthma [1]. In addition to providing a protective barrier against environmental insults, the airway epithelium alsoproduces a number of cytokines, chemokines, growth factors and lipid mediators that influence the recruitment andfunction of mesenchymal and inflammatory cells [1,2]. The major lipid mediator produced by airway epithelium is

∗ Corresponding author at: Lung Institute of Western Australia (Inc.), Ground Floor, E Block, Sir Charles Gairdner Hospital, Verdun Street,Nedlands, WA 6009, Australia. Tel.: +61 8 9346 3198; fax: +61 8 9346 4159.

E-mail address: [email protected] (N.L.A. Misso).

1098-8823/$ – see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.prostaglandins.2006.08.006

mailto:[email protected]

dx.doi.org/10.1016/j.prostaglandins.2006.08.006

N. Petrovic et al. / Prostaglandins & other Lipid Mediators 81 (2006) 126–135 127

prostaglandin (PG)E2, which inhibits fibroblast chemotaxis and collagen production [3,4], smooth muscle proliferation[5], T lymphocyte proliferation and function [6], as well as eosinophil apoptosis [7,8]. In addition PGE2 promoteswound closure in airway epithelium [9], and protects against allergen- and methacholine-induced bronchoconstrictionand airway inflammation [10,11]. Therefore the regulation of PGE2 production by airway epithelial cells and its rolein normal physiology and homeostasis, as well as in inflammation and immune responses, have potentially importantimplications for inflammatory lung diseases such as asthma [6,12–14].

Prostaglandin synthesis is regulated by phospholipase A2 (PLA2), constitutive and inducible cyclooxygenases(COX-1 and COX-2), and cell-specific prostaglandin synthases such as microsomal PGE synthase [12,15]. Previousstudies have also implicated one or more of the three principal mitogen-activated protein kinases (MAPK), extracellularsignal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38-MAPK in the regulation of COX-2 expressionand prostaglandin synthesis in different cell types [16–18]. MAPK is also involved in the regulation of prostaglandinsynthesis at the level of PLA2 and it is well established that activation of cPLA2 requires phosphorylation of serine505

by ERK [19].In human airway epithelial cells, COX-2 is expressed even in non-inflammatory conditions [20], but inflammatory

stimuli induce a marked up-regulation of COX-2 expression that appears to involve ERK, and possibly p38-MAPKand JNK as well [16,21–23]. However, it is not clear how phosphorylation of ERK is coordinated with COX-2induction to stimulate maximum PGE2 synthesis following an inflammatory insult to airway epithelial cells. Phospho-rylation of ERK is usually transient, lasting up to 30 min, and this is probably sufficient to bring about the sustainedphosphorylation and activation of cPLA2 [19]. However, maximum induction of COX-2 requires gene transcrip-tion and protein synthesis and may take up to 24 h [16,21,22]. It has been hypothesized that constitutive COX-1catalyses the immediate synthesis of prostaglandins, which occurs within minutes of stimulation and parallels thetransient phosphorylation of ERK [24]. However, the relationship between ERK phosphorylation and the induction ofCOX-2 necessary for delayed, maximum prostaglandin biosynthesis that may continue for several hours after stim-ulation is unclear. Furthermore the relationship between ERK activation, COX-2 expression and maximization ofPGE2 synthesis may vary with the nature and complexity of the specific inflammatory stimulus received by airwayepithelial cells.

Therefore the aims of this study were to investigate the intracellular regulation and kinetics of PGE2 synthesis inhuman airway epithelial (NCI-H292) cells exposed to different inflammatory stimuli and combinations of stimuli. Inaddition, we investigated interactions between the different signal transduction pathways, and the association of ERKactivation with COX-2 expression and maximization of PGE2 synthesis in response to different inflammatory stimuli.

2. Materials and methods

2.1. Materials

Recombinant human TNF-� and IL-1� were purchased from Roche Diagnostics (Sydney, Australia). Phorbol-12-myristate-13 acetate (PMA), calcium ionophore A23187, the protein kinase C (PKC) inhibitor, Ro-32-0432, and theERK inhibitor, PD98059, were purchased from Calbiochem-Novabiochem (Sydney, Australia). The PLA2 inhibitors,arachidonyl trifluoromethylketone (AACOCF3), methyl arachidonyl fluorophosphate (MAFP), oleyloxyethyl phos-phorylcholine (OOEPC) and bromoenol lactone (BEL) were obtained from Cayman Chemical (Ann Arbor, MI, USA).Lipopolysaccharide (LPS, E. coli serotype 0127:B8) and all other chemicals were obtained from Sigma Chemical Co.(Sydney, Australia).

2.2. Cell culture and treatment with inflammatory stimuli and inhibitors

The human airway epithelium NCI-H292 cell line was obtained from American Type Culture Collection (ATCC,Rockville, MD, USA) [25], and cultured in RPMI-1640 medium, supplemented with 10% FCS and gentamicin(50 �g/ml), at 37 ◦C in a humidified 5% CO2 atmosphere. NCI-H292 cells were grown in 24-well tissue cultureplates, and when confluent, were treated with inflammatory stimuli, which were added to the medium at the follow-ing concentrations: TNF-�, 25 ng/ml; IL-1�, 5 ng/ml; LPS, 50 �g/ml; A23187, 0.95 �M; PMA, 30 nM. In inhibitionexperiments, cells were pre-incubated for 60 min with Ro-32-0432 (5 �M), PD98059 (50 �M), AACOCF3 (40 �M),MAFP (10 �M), OOEPC (20 �M) or BEL (20 �M). Stimuli for PGE2 synthesis were then added to the medium for

128 N. Petrovic et al. / Prostaglandins & other Lipid Mediators 81 (2006) 126–135

the required incubation period, after which the cells in each well were disaggregated with trypsin-versene solution andviable cells counted by trypan blue exclusion.

2.3. PGE2 assay

A competitive enzyme immunoassay (EIA) was used according to the manufacturer’s instructions (Cayman Chem-ical). PGE2 concentrations in cell culture supernatants were expressed as ng of PGE2 per 105 viable cells.

2.4. Western immunoblot analysis of ERK and COX expression

Cell lysates were analysed by Western blotting as described previously [26]. Briefly, cells were lysed (RIPA buffer,Roche Diagnostics) and after centrifugation (11,000 × g, 20 min, 4 ◦C), the supernatants (20 �g of total protein) wereseparated on gradient (4–16%) SDS polyacrylamide gels and transferred to PVDF membranes. Monoclonal antibodies(mAb) to the phosphorylated form of ERK (phospho-p44/42 ERK1/2 mAb, Cell Signaling Technology, Beverly, MA,USA) or to COX-1 or COX-2 (Cayman Chemical) were used as primary antibodies. Protein bands were visualizedusing appropriate secondary antibodies with detection by enhanced chemiluminescence (Amersham).

2.5. Statistical analyses

Data are presented as the mean (±S.D.) of six replicates. Results were confirmed in at least two separate experiments.Mean data were compared using one-way analysis of variance with the Bonferroni multiple comparison test. A p value<0.05 was considered significant.

3. Results

3.1. Effects of inflammatory stimuli on PGE2 synthesis

Treatment of NCI-H292 cells with IL-1� or TNF-� for 4 h resulted in 2- and 1.5-fold increases in PGE2 synthesis,while LPS alone and in combination with IL-1� and TNF-� induced a >6-fold increase in PGE2 (p < 0.001, Fig. 1).The combination of LPS with IL-1� and TNF-� was used to mimic the complex inflammatory milieu to which cellsmight be exposed in vivo, and similar combinations have previously been shown to induce COX-2 and nitric oxidesynthase in human airway epithelial cells [27,28].

Fig. 1. Effects of inflammatory mediators on PGE2 synthesis. Confluent NCI-H292 cells were incubated for 4 h without stimulation (control) orwith IL-1� (5 ng/ml), TNF-� (25 ng/ml), or LPS (50 �g/ml) singly or in combination. PGE2 released into the culture medium was measured byenzyme immunoassay. Data are mean ± S.D. of six replicates. ***P < 0.001 compared with control.


Table 1Effects of phospholipase A2 inhibitors on PGE2 production by airway epithelial cells

Stimulus Inhibitor PGE2 (ng/105 cells) % inhibition

LPS – 1.41 ± 0.22LPS AACOCF3 0.22 ± 0.08* 84.4LPS MAFP 0.31 ± 0.3* 78.0LPS OOEPC 1.34 ± 0.2 5.0LPS BEL 1.32 ± 0.2 6.4LPS/TNF�/IL-1� – 2.54 ± 0.39LPS/TNF�/IL-1� AACOCF3 0.32 ± 0.29* 87.4LPS/TNF�/IL-1� MAFP 0.35 ± 0.17* 86.2LPS/TNF�/IL-1� OOEPC 2.49 ± 0.32 2.0LPS/TNF�/IL-1� BEL 2.45 ± 0.31 3.5

Confluent NCI-H292 cells were pre-incubated without inhibitor or with AACOCF3 (40 �M), MAFP (10 �M), OOEPC (20 �M) or BEL (20 �M) for60 min. Cells were then stimulated for 4 h with LPS alone (50 �g/ml) or a combination of IL-1� (5 ng/ml), TNF-� (25 ng/ml) and LPS (50 �g/ml).PGE2 released into the culture medium was measured by enzyme immunoassay. Data are mean ± S.D. of six replicates. *p < 0.001 compared withno inhibitor.

3.2. Effects of PLA2 inhibitors on PGE2 synthesis

PGE2 synthesis stimulated by LPS alone or a combination of IL-1�, TNF-� and LPS, was potently inhibited by theselective cPLA2 inhibitors AACOCF3 and MAFP (p < 0.001, Table 1), whereas selective inhibitors of secretory PLA2(OOEPC) or calcium-independent PLA2 (BEL) had little effect.

3.3. Effects of PMA and calcium ionophore (A23187) on PGE2 synthesis

To investigate the role of protein kinase C and intracellular Ca2+ in PGE2 synthesis, cells were treated withPMA or A23187, both in the presence and absence of IL-1�, TNF-� or LPS. Both PMA and A23187 increasedPGE2 synthesis by unstimulated cells (8- and 18-fold, respectively, p < 0.001) (Table 2). Furthermore, both agentsgreatly enhanced PGE2 synthesis when used in combination with IL-1�, TNF-� or LPS (p < 0.001). The most pro-nounced increases in PGE2 synthesis were detected when cells were treated with PMA or A23187 and stimulatedwith LPS, which resulted in 47- and 100-fold increases in PGE2 synthesis, respectively, compared with untreatedcells.

Table 2Effects of PMA and A23187 on PGE2 synthesis in airway epithelial cells

Stimulus Treatment PGE2 (ng/105 cells)

None – 0.23 ± 0.05PMA 1.55 ± 0.34*

A23187 2.72 ± 0.6*

IL-1� – 0.46 ± 0.1PMA 4.06 ± 0.9*

A23187 8.26 ± 1.83*

TNF� – 0.35 ± 0.08PMA 2.88 ± 0.64*

A23187 6.76 ± 1.50*

LPS – 1.41 ± 0.31PMA 10.76 ± 2.38*

A23187 24.79 ± 5.49*

NCI-H292 cells were incubated for 4 h with PMA (30 nM) or A23187 (0.95 �M) without a stimulus (control), or together with IL-1� (5 ng/ml),TNF-� (25 ng/ml) or LPS (50 �g/ml). PGE2 release was measured by enzyme immunoassay. Data are mean ± S.D. of six replicates. *p < 0.001compared to cells not treated with PMA or A23187.


Fig. 2. Effects of PKC and ERK inhibitors on PGE2 synthesis. Confluent NCI-H292 cells were pre-incubated for 60 min with the PKC inhibitor,Ro-32-0432 (5 �M, �), the MEK inhibitor, PD98059 (50 �M, ) or without inhibitor (�). Cells were then treated with no stimulus (control) orstimulated for 4 h with IL-1� (5 ng/ml), TNF-� (25 ng/ml) or LPS (50 �g/ml). PGE2 release was measured by enzyme immunoassay. Inserts showthe effects of Ro-32-0432 (�) and PD98059 ( ) on PGE2 synthesis induced by A23187 (0.95 �M) (insert A) or PMA (30 nM) (insert B). Data aremean ± S.D. of six replicates. ***p < 0.001, **p < 0.01 compared with no inhibitor.

3.4. Effects of PKC and ERK inhibitors on PGE2 synthesis

The role of PKC activation in PGE2 synthesis induced by IL-1�, TNF-� or LPS was investigated with the selectivecell-permeable PKC inhibitor, Ro-32-0432 (Fig. 2). The effects of Ro-32-0432 varied with the inflammatory stimulus.IL-1�-induced PGE2 synthesis was almost completely inhibited by Ro-32-0432 (92% inhibition, p < 0.001, Fig. 2),while inhibition of LPS- and TNF�-induced PGE2 synthesis was partial, but still significant (p < 0.001). In contrast,A23187-mediated PGE2 synthesis was unaffected by the PKC inhibitor (Fig. 2, insert A). The concentration of inhibitorused (5 �M) was sufficient to completely block PKC activation, since PMA-mediated PGE2 synthesis was completelyabolished in the presence of Ro-32-0432 (Fig. 2, insert B).

The ERK inhibitor, PD98059, at the concentration of 50 �M used previously [29], almost completely blocked PGE2synthesis, irrespective of whether it was induced by IL-1�, TNF-�, LPS, A23187 or PMA (p < 0.001, Fig. 2). Since ithas been reported that PD98059 directly inhibited COX-2 activity in intact human platelets [30], control experimentswere performed in which NCI-H292 cells were incubated with PD98059 in the presence of exogenous arachidonicacid. No inhibition of PGE2 synthesis was observed in these experiments (data not shown), indicating that in thesecells PD98059 was acting upstream of COX-2 rather than directly inhibiting COX-2 activity.

3.5. Time course of PGE2 synthesis

IL-1�, TNF-�, or LPS, singly and in combination (IL-1�/TNF-� and LPS/IL-1�/TNF-�) all induced rapid increasesin PGE2 synthesis, with maximum concentrations of up to 2.7 ng/105 cells being detected 1 h after stimulation (Fig. 3).In contrast, treatment of cells with PMA, A23187 or a combination of LPS, PMA and A23187 resulted in steadyincreases in PGE2 concentrations that reached a plateau after approximately 4 h. Furthermore, treatment of cells witha combination of LPS, PMA and A23187 resulted in greatly enhanced maximum PGE2 synthesis (40 ng/105 cells),which was 130-fold greater than that observed for untreated cells.

3.6. Time course of ERK activation

In order to investigate the correlation between ERK activation and PGE2 synthesis, the time course of ERK phos-phorylation in response to different stimuli was assessed (Fig. 4). ERK phosphorylation at time 0 was similar for allexperiments, providing a reasonably constant basis on which to assess the extent of ERK phosphorylation at subsequenttime points with the different stimuli. In cells treated with IL-1�, TNF-� or LPS (or combinations thereof) ERK phos-phorylation was transient, reaching a maximum at 5 or 15 min. In contrast, ERK phosphorylation was sustained for 1 h incells treated with PMA, and for up to 16 h following treatment with A23187 or a combination of A23187, PMA and LPS.


Fig. 3. Time course of PGE2 synthesis induced by different stimuli. Confluent NCI-H292 cells were incubated for the indicated times with IL-1�

(5 ng/ml), TNF-� (25 ng/ml), LPS (50 �g/ml), A23187 (0.95 �M) or PMA (30 nM), alone or in combinations, and PGE2 synthesis was measuredby enzyme immunoassay. Treatments are indicated by the following symbols: control (�, dashed line); IL-1� (�); TNF� (�); IL-1� + TNF� (�);LPS (�); LPS + IL-1� + TNF� (�); PMA (©); A23187 (�) and LPS + PMA + A23187 (�). Data are mean ± S.D. of six replicates.

3.7. Time course of COX-2 protein expression

The time course of COX-2 protein expression was substantially different to that of ERK activation with increases inCOX-2 protein occurring later than ERK phosphorylation. Increases in COX-2 protein were observed after 15 min oftreatment with TNF-�, IL-1�/TNF-� and IL-1�/TNF-�/LPS (Fig. 5). However with most stimuli, substantial increases

Fig. 4. Time course of ERK phosphorylation. Confluent NCI-H292 cells were incubated for the times indicated with IL-1� (5 ng/ml), TNF-� (25 ng/ml), LPS (50 �g/ml), A23187 (0.95 �M) or PMA (30 nM), alone and in combination. Cell lysates were analysed for the presence ofphosphorylated ERK by Western blotting using phospho-p44/42 ERK1/2 mAb and appropriate secondary antibody with ECL detection.


Fig. 5. Time course of COX-2 protein expression. Confluent NCI-H292 cells were treated for the times indicated with IL-1� (5 ng/ml), TNF-�(25 ng/ml), LPS (50 �g/ml), A23187 (0.95 �M) or PMA (30 nM), alone and in combination. Cell lysates were analysed for COX-2 and COX-1protein expression by Western blotting using COX-2 and COX-1 mAb and appropriate secondary antibody with ECL detection.

in COX-2 protein expression were only observed after 4 h and reached a maximum only after 16 h. Expression ofCOX-2 protein was greater when cells were treated with combinations of stimuli, with the highest levels being detectedfollowing treatment with A23187, PMA and LPS. Expression of COX-1 protein was not changed significantly by anyof the treatments (Fig. 5). Constitutively expressed COX-1 protein also provided a reference for the effects of loadingat different time points.

3.8. Effect of concomitant ERK activation and COX-2 protein expression on PGE2 synthesis

When cells were incubated for 4 h with TNF-�/IL-1� and then stimulated for 15 min with LPS, high levels ofCOX-2 protein were expressed concomitantly with increased ERK phosphorylation (Fig. 6B), resulting in high lev-els of PGE2 synthesis (Fig. 6A). In contrast, PGE2 synthesis was significantly lower under conditions where ERK

Fig. 6. Effect of concomitant ERK activation and COX-2 expression on PGE2 synthesis. Confluent NCI-H292 cells were incubated with IL-1�

(5 ng/ml), TNF-� (25 ng/ml) and LPS (50 �g/ml) for 4 h, or with IL-1�/TNF-� for 4 h with or without further stimulation with LPS for 15 minas indicated. (A) PGE2 synthesis was measured by enzyme immunoassay (mean ± S.D. of six measurements). (B) Cell lysates were analysed forphosphorylated ERK and COX-2 protein by Western blotting using phospho-p44/42 ERK1/2 and COX-2 mAb, appropriate secondary antibodiesand ECL detection. ***p < 0.001 compared to all other treatments, **p < 0.01 compared to LPS (15 min).


was phosphorylated but COX-2 expression was low (stimulation with LPS for 15 min, p < 0.001), or where COX-2was highly expressed but the level of ERK phosphorylation was low (TNF�/IL-1� for 4 h, p < 0.001). Furthermore,concomitant ERK phosphorylation and COX-2 expression, obtained by treatment with TNF-�/IL-1� for 4 h followedby LPS for 15 min, resulted in 1.8-fold higher PGE2 synthesis compared with simultaneous treatment with all threemediators for 4 h (p < 0.01), which resulted in very high COX-2 expression without a concomitant increase in ERKphosphorylation (Fig. 6A and B).

4. Discussion

The intracellular regulation and kinetics of prostaglandin synthesis is complex. In this study we have attempted toelucidate the relationship of ERK phosphorylation and COX-2 expression to PGE2 synthesis following exposure ofhuman airway epithelial cells to different inflammatory stimuli. We have also assessed the kinetics of these pathways andshown that simultaneous ERK activation and COX-2 induction are necessary for maximum PGE2 synthesis. The resultsindicate that in human airway epithelial cells, ERK is the major kinase regulating PGE2 synthesis, and inhibition ofERK phosphorylation with PD98059 almost completely abrogated PGE2 synthesis in response to all stimuli. However,the signaling pathways leading to ERK activation and PGE2 synthesis varied for the different inflammatory stimuli.Thus, the effects of IL-1� appeared to be mediated exclusively by PKC, whereas PGE2 synthesis induced by TNF-�or LPS was only partially dependent on PKC. Increases in intracellular Ca2+ may also contribute to ERK activation asdemonstrated by the effect of the Ca2+ ionophore, A23187. Therefore in airway epithelial cells, inflammatory stimulimay activate ERK and induce PGE2 synthesis by at least three separate signaling pathways: (a) by direct activation of theRas/Raf cascade via cytokine receptors, (b) through PKC-dependent mechanisms and (c) by increases in intracellularCa2+. Although some members of the PKC family are Ca2+-dependent, our results as well as those of others [31,32]suggest that in airway epithelium, Ca2+ does not play a major role in PKC-mediated PGE2 synthesis.

The involvement of more than one signaling pathway leading to ERK activation and prostaglandin synthesis hasbeen observed previously. Activation of ERK by PKC-dependent mechanisms has been reported in many cell types,including airway epithelial cells [33,34], and probably involves phosphorylation of an upstream Raf kinase [35,36].Synergism between PMA and A23187 has been reported for PGE2 synthesis in MDCK cells [37], while in airwaysmooth muscle cells, activation of ERK by endothelin requires Ca2+ influx as well as activation of Raf kinase [38].Separate PKC and Ca2+ mediated pathways for ERK activation have also been demonstrated in rabbit vascular smoothmuscle where Ca2+/calmodulin dependent kinase may mediate the effects of Ca2+ influx on ERK activation [39].

The role of ERK in phosphorylation and activation of cPLA2 is well documented in other cell types [19]. In this study,the observation that inhibition of cPLA2 and ERK, but not sPLA2 or iPLA2, completely abolished PGE2 synthesis,is consistent with a mechanism in airway epithelial cells, in which the transient phosphorylation of ERK mediatesthe activation of cPLA2 and the ‘switching on’ of immediate PGE2 synthesis of via COX-1. A second and equallyimportant role for ERK is likely to be its participation in the induction of COX-2 expression, as previously observedin airway epithelial cells [22,40] and other cell types [17,18,41]. However, an important finding in the present studywas that PGE2 synthesis was greatly enhanced by combinations of stimuli such as IL-1�/TNF�/LPS, to which airwayepithelial cells are likely to be exposed during in vivo lung inflammation. Under such conditions, ERK may be a keydownstream effector that regulates PGE2 synthesis in airway epithelial cells by activating I�B kinase and the bindingof NF-�B to the promoter on the COX-2 gene [15,22,31].

However, under conditions of maximum stimulation, some induction of COX-2 may also occur by mechanisms thatare independent of ERK. Both p38-MAPK and JNK have been implicated as regulators of NF-�B-dependent COX-2expression in other cell systems [18,42], and may also play a role in airway epithelial cell PGE2 synthesis under somecircumstances [43]. Furthermore, in some cells, PGE2 acts at EP2 or EP4 receptors to stimulate phosphorylation ofthe cAMP response element binding protein (CREB), and thereby the autocrine induction of COX-2 transcription[44,45]. It is not clear whether ERK is involved in this pathway, but it nevertheless represents a potentially importantmechanism for delayed amplification of PGE2 production following initial exposure of airway epithelial cells to aninflammatory stimulus.

As might be expected, there was a significant delay between ERK phosphorylation, which was essentially transient,and induction of COX-2, which requires gene transcription and protein synthesis, and therefore reached a maximumonly after several hours. In airway epithelial cells, activation of ERK by the physiological inflammatory stimuli, IL-1�,TNF�, or LPS reached a maximum within 5–15 min, consistent with its role in the activation of sub-maximal PGE2 syn-


thesis, which reached a peak 1 h after exposure to these stimuli. However, induction of COX-2 expression and maximumPGE2 synthesis with these physiological stimuli, even in combination, was only observed at around 16 h, after ERKactivation had declined. Furthermore we observed that PGE2 synthesis was greatly enhanced if multiple intracellularpathways, including PKC and intracellular Ca2+, were activated with the non-physiological stimuli, PMA and A23187,so that ERK phosphorylation was sustained for several hours and overlapped with increased COX-2 expression.

These observations prompted us to investigate whether the timing of exposure to physiological stimuli (IL-1�,TNF�, LPS) could be experimentally manipulated so as to achieve ERK activation that was concomitant with COX-2expression, or a secondary activation of ERK after COX-2 had been induced. The results shown in Fig. 6, demonstratethat under these circumstances PGE2 synthesis was greatly enhanced, by comparison with the situation in whichthere was either transient ERK activation or independent delayed COX-2 expression. While it is likely that in theseexperiments, PGE2 measured in the medium at 4 h reflects accumulation as a result of earlier signaling events, theresults quite clearly show that PGE2 synthesis was enhanced by inducing ERK phosphorylation (with LPS for 15 min)so that it was contemporaneous with high levels of COX-2 expression. A possible explanation for these findings is thatduring the initial phase of the inflammatory response, when ERK is only transiently activated, cPLA2 is activated andsub-maximal PGE2 synthesis results from the activity of constitutively expressed COX-1 [24]. In the later stages of theinflammatory response, greater, more prolonged PGE2 synthesis occurs when ERK activation is sustained and parallelsCOX-2 expression, possibly due to the involvement of additional inflammatory mediators, including PGE2 itself.

In conclusion, our findings suggest that in human airway epithelial cells, inflammatory stimuli induce prostaglandinsynthesis through multiple pathways, including Ras/Raf, Ca2+-independent PKC and increases in intracellular Ca2+.ERK plays a major role in the induction of COX-2 expression, and when ERK activation is concomitant with COX-2induction PGE2 synthesis is greatly enhanced. Thus, the effects of numerous inflammatory mediators acting via differentintracellular signaling pathways may not simply reflect redundancy in the induction of PGE2 synthesis. Rather, stimuliproduced at different stages of the inflammatory process may influence ERK activation and COX-2 induction such thatthere is tight regulation of the kinetics and magnitude of PGE2 production in response to inflammation in the lung.

Acknowledgement

This work was supported in part by a research grant from the Asthma Foundation of Western Australia.

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concomitant activation of extracellular signal-regulated kinase and induction of cox-2 stimulates...

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