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Am J Physiol Lung Cell Mol Physiol 290: L501-L508, 2006. First published November 18, 2005; doi:10.1152/ajplung.00420.2005
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Regulatory features of interleukin-1beta-mediated prostaglandin E2 synthesis in airway smooth muscle

Rodolfo M. Pascual,1 Elizabeth M. Carr,1 Michael C. Seeds,1 Manhong Guo,1 Reynold A. Panettieri, Jr.,2 Stephen P. Peters,1 and Raymond B. Penn1

1Department of Internal Medicine and Center for Human Genomics, Wake Forest University Health Sciences Center, Winston-Salem, North Carolina; and 2Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Submitted 3 October 2005 ; accepted in final form 21 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Exposure of airway smooth muscle (ASM) cells to the cytokine IL-1beta results in an induction of PGE2 synthesis that affects numerous cell functions. Current dogma posits induction of COX-2 protein as the critical, obligatory event in cytokine-induced PGE2 production, although PGE2 induction can be inhibited without a concomitant inhibition of COX-2. To explore other putative regulatory features we examined the role of phospholipase A2 (PLA2) and PGE synthase (PGES) enzymes in IL-1beta-induced PGE2 production. Treatment of human ASM cultures with IL-1beta caused a time-dependent induction of both cytosolic PLA2 (cPLA2) and microsomal PGES (mPGES) similar to that observed for COX-2. Regulation of COX-2 and mPGES induction was similar, being significantly reduced by inhibition of p42/p44 or p38, whereas cPLA2 induction was only minimally reduced by inhibition of p38 or PKC. COX-2 and mPGES induction was subject to feed-forward regulation by PKA, whereas cPLA2 induction was not. SB-202474, an SB-203580 analog lacking the ability to inhibit p38 but capable of inhibiting IL-1beta-induced PGE2 production, was effective in inhibiting mPGES but not COX-2 or cPLA2 induction. These data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-beta in human ASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. mPGES induction and, possibly, cPLA2 induction appear to cooperate with COX-2 to determine IL-1beta-mediated PGE2 production in human ASM cells.

cyclooxygenase-2; phospholipase A2; prostaglandin E2 synthase

RECENT STUDIES HAVE REVEALED the pleiotropic nature of airway smooth muscle (ASM). Beyond its ability to contract and regulate airway patency, ASM works in concert with other airway cells in an immunomodulatory capacity (11, 21). The resultant inflammatory milieu in turn exerts regulatory effects on ASM contractile state and synthetic functions.

The proinflammatory cytokine interleukin-1beta (IL-1beta) activates numerous pathways in ASM capable of modulating multiple ASM functions (1). One such pathway is that resulting in the production of autocrine PGE2 synthesis. A large induction of PGE2 synthesis in ASM elicited by chronic treatment with IL-1beta or combined IL-1beta and tumor necrosis factor-{alpha} (TNF-{alpha}) results in profound inhibition of agonist-induced contraction and cell proliferation, promotes desensitization or sensitization of various G protein-coupled receptors, and has differential effects on cytokine synthesis (2, 9, 10, 15, 24–26, 32) in multiple models of ASM function including primary cell cultures, ex vivo ASM strips, and in vivo models assessing pulmonary function [reviewed in Billington and Penn (4)].

Studies examining regulatory features of cytokine-induced PGE2 synthesis have posited induction of cyclooxygenase (COX)-2 protein as the critical, obligatory event in PGE2 production and the functional consequences thereof. COX-2-dependent PGE2 synthesis involves coordinated activation of phospholipase A2 (secretory or cytosolic), which provides arachidonic acid substrate to COX-2 and prostaglandin E2 synthases (PGES), which convert the COX-2 product PGH2 into PGE2. In ASM, inhibition of either induction of COX-2 protein or COX-2 activity causes a profound reduction in cytokine-induced PGE2 synthesis, demonstrating the essential role of COX-2 induction (2, 7, 13, 23, 25, 28, 31). To what extent cytokine-induced alterations in either phospholipase A2 (PLA2) or PGES activities contribute toward induced PGE2 levels is unclear, although the current dogma suggests that COX-2 activity is limiting in PGE2 synthesis. In other cell systems induction of cytosolic PLA2 (cPLA2) or microsomal PGES (mPGES) by cytokines has been observed, although the relevance of such induction is unclear (6, 19, 29).

In a previous analysis of cytokine-promoted COX-2 induction we observed discordance between regulation of COX-2 and PGE2 induction (25). These findings suggest that the activity of another element(s) in the pathway mediating PGE2 synthesis might be regulated by cytokines and could be selectively inhibited as a means of inhibiting PGE2 synthesis. Here we examine regulatory features of cPLA2, mPGES, COX-2, and PGE2 induction by IL-1beta in human ASM (HASM) cells. We find that like COX-2, cPLA2 and mPGES are induced by IL-1beta treatment of ASM and provide evidence that regulation of multiple elements contributes to cytokine-induced PGE2 synthesis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Materials. Arachidonic acid-[5,6,8,9,11,12,14,15-3H](N) and 1-palmitoyl-2-[14C]arachidonyl phosphatidylcholine were purchased from Perkin Elmer (Boston, MA). The Biotrak 125I-PGE2 RIA kit was purchased from Amersham (Arlington Heights, IL). Human anti-COX-2 antibody,human anti-COX-1, and human anti-mPGES antibodies were purchased from Cayman Chemical (Ann Arbor, MI). Human anti-cPLA2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SB-203580, SB-202474, bisindolylmaleimide I (Bis I), and U0126 were purchased from Calbiochem (San Diego, CA). IL-1beta and TNF-{alpha} were purchased from Roche (Indianapolis, IN). Solid-phase extraction cartridges were purchased from Agilent Technologies (Palo Alto, CA). Fluorescent antibodies were purchased from Rockland (Gilbersville, PA) or Molecular Probes (Eugene, OR). Odyssey blocking buffer and the Li-Cor imaging system were purchased from Li-Cor (Lincoln, NE). All other reagents were purchased from Sigma (St. Louis, MO) or from previously identified sources (20).

HASM cell culture. HASM cultures were established as described by Panettieri et al. (22) from human tracheae obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Characterization of this cell line with regard to immunofluorescence of smooth muscle actin and agonist-induced changes in cytosolic calcium has been previously reported (22). Cells of passages 2–5 were plated at a density of 1 x 104 cells/cm2 on the appropriate dishes or coverslips as described below in fetal bovine serum-supplemented medium (20). Seven days later, we growth-arrested cells by washing them with phosphate-buffered saline (PBS) and refeeding them with serum-free Ham's F-12 medium for 48 h as described previously (20).

Assay of PGE2 accumulation. HASM cultures growth-arrested for 48 h were treated with vehicle or indicated inhibitors for 30 min before being treated with either 20 IU/ml IL-1beta, 10 ng/ml TNF-{alpha}, or both for 18 h. PGE2 from the harvested culture media was purified using C-18 columns as described previously (12) and subsequently quantified with the Biotrak RIA kit as per the manufacturer's instructions.

Analysis of COX-1, COX-2, mPGES, cPGES, and cPLA2 expression. HASM cultures were grown in six-well plates and treated as described for PGE2 assays. After 18 h of treatment with cytokines, the plates were placed on ice, the media were then aspirated from the well, the well was washed once with cold PBS, and 200 µl of 1% sodium dodecyl sulfate (SDS) sample buffer (20) were applied directly to the well. Cell lysates were harvested by scraping, boiled for 5 min in 1% SDS sample buffer, and sonicated. Samples were electrophoresed on either a 4–20% gradient SDS polyacrylamide gel (to observe mPGES) or 10% SDS polyacrylamide gel, transferred to nitrocellulose membranes, and subsequently analyzed by immunoblotting for COX-2 or COX-1 expression using a 1:1,000 dilution of primary antibody, for cPLA2 expression using a 1:500 dilution of primary antibody, for mPGES and cPGES antibodies using a 1:1,000 dilution of primary antibody, and either Alexa 688 or IRDye 800 secondary antibodies conjugated with infrared fluorophores. Bands were visualized and directly quantified with the Odyssey Infrared Imaging System (Li-Cor) and, where appropriate, normalized to the value obtained for the vehicle-treated condition or to the IL-1beta-treated condition when basal protein expression was undetectable.

cPLA2 activity assay. Assay of cPLA2 activity was performed as described previously (33). In brief, cells were plated in 150-mm dishes and treated as described above. Cytosolic protein fractions were prepared in assay buffer: 100 mM KCl, 10 mM HEPES, 1 mM EDTA, and 1 mM EGTA containing 100 µM leupeptin, 50 µM pepstatin A, 2 mM sodium vanadate, and 10 mM diisopropyl fluorophosphates. The final assay mixture also contained 400 pM of sonicated micelles of 1-palmitoyl-2-[14C]arachidonyl-phosphatidylcholine, 5 mM CaCl2, 5 mM dithiothreitol (DTT), 200 µg BSA, and 40 µg of cytosolic protein in a final volume of 525 µl and was warmed to 37°C in a shaking bath to initiate reactions. Reactions were stopped by solvent extraction; lipids were resolved by TLC, visualized, scraped, and counted by liquid scintillation. The percent hydrolysis was calculated as described in Ref. 33.

Arachidonic acid release assay. Cells were cultured in 24-well dishes and serum starved for 24 h. We then loaded cells overnight with [3H]arachidonic acid ([3H]AA) by changing the media to serum-free media containing 0.5 µCi/ml [3H]AA and 5 mM CaCl2. Cells were washed 5x with serum-free media containing 0.5 mg/ml BSA/5 mM CaCl2 to remove unbound [3H]AA and treated with inhibitors and cytokines in the same media. Subsequent [3H]AA released into culture supernatants was assessed by liquid scintillation counting. Generation of ASM cultures with stable expression of green fluorescent protein (GFP) or protein kinase A inhibitor (PKI)-GFP was as described in Guo et al. (8).

Data presentation and statistical analysis. Data points from individual assays represent the mean values from duplicate or triplicate measurements. Statistical analysis of data was performed with GraphPad Prism (San Diego, CA) software. Except where noted, data are presented as means ± SE. To minimize interexperimental variability in subsequent experiments that examined the effect of various inhibitory agents, group values were normalized to values for the IL-1beta-vehicle condition. Statistically significant differences among groups were assessed either by ANOVA with Fisher’s post hoc analysis or by t-test for paired samples with P < 0.05 sufficient to reject the null hypothesis.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
IL-1beta-mediated PGE2 production in HASM is regulated by PKC, MAPKs, and corticosteroids. Initial studies characterized regulatory features of IL-1beta-mediated PGE2 production in HASM. In growth-arrested HASM cells, basal PGE2 production over 18 h was low (Fig. 1). Consistent with our previous results (25), 18-h treatment with IL-1beta resulted in a large (~25-fold) induction of PGE2 production over basal levels. Pretreatment with inhibitors of p42/p44 MAPK (U0126), p38 MAPK (SB-203580), or conventional PKC isoforms (Bis I) all significantly inhibited (~70–90%) this induction. Pretreatment with dexamethasone essentially eliminated IL-1beta-mediated PGE2 production. Somewhat surprisingly, pretreatment with SB-202474, an analog of SB-203580 that lacks the ability to inhibit p38 (16), also caused a profound inhibition of the induction of PGE2.


Figure 1
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  		Fig. 1. Regulation of IL-1beta-mediated PGE2 production in human airway smooth muscle (HASM). Confluent, growth-arrested cells grown in 12-well plates were pretreated with vehicle (Veh, 0.1% DMSO), 10 µM U0126, 1 µM SB-203580, 1 µM SB-202474, 3 µM bisindolylmaleimide (Bis) I, or 10 nM dexamethasone (Dexa) for 30 min followed by 20 IU/ml IL-1beta treatment for 18 h. PGE2 in cell culture supernatants was isolated and quantified by RIA. Data are presented as percentage of vehicle-pretreated IL-1beta value. Mean basal (vehicle-stimulated) PGE2 production was 13 pg/well (well volume = 1 ml), and for IL-1beta-treated cells 566 pg/well. Data represent mean ± SE values from 6 independent experiments. Differences between IL-1beta and IL-1beta with inhibitors were analyzed by repeated-measures ANOVA. *P < 0.05, IL-1beta + inhibitor vs. IL-1beta + vehicle.

 
COX-2, cPLA2, and mPGES protein expression are induced by IL-1beta and exhibit differential regulation. As previously demonstrated by our group and others chronic treatment with IL-1beta resulted in a large induction of COX-2 protein expression in HASM, whereas levels of COX-1 were unchanged (Fig. 2). Consistent with our analysis of the regulation of PGE2 induction and the essential role of COX-2, pretreatment with U0126, SB-203580, or dexamethasone significantly inhibited IL-1beta-induced COX-2. Interestingly, neither SB-202474 nor Bis I, both effective in inhibiting IL-1beta-mediated PGE2 production, had a significant effect on the induction of COX-2. This finding suggested a component/feature of the PGE2 synthetic machinery whose regulation differs from that of COX-2 induction.


Figure 2
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  		Fig. 2. IL-1beta-mediated cyclooxygenase (COX) expression in HASM. Cells were pretreated with the indicated inhibitors for 30 min then treated with 20 IU/ml IL-1beta for 18 h. Cell lysates were harvested and subject to immunoblotting with secondary antibodies conjugated to infrared fluorophores. Band intensity reflecting COX-2 expression was directly quantified using the Odyssey Imaging System as described in METHODS. Representative blots for COX-2 (A and B) and COX-1 (C) are shown. Data from 6 independent experiments are presented as mean ± SE values in D. *P < 0.05, IL-1beta + inhibitor vs. IL-1beta + vehicle.

 
We therefore considered the contribution of altered PLA2 and PGES activities to IL-1beta-mediated PGE2 production. Examination of cPLA2 protein expression following chronic IL-1beta treatment revealed a significant (>4-fold) induction of cPLA2 protein expression (Fig. 3). Regulation of this induction was radically different from that observed for the induction of COX-2. Inhibition of p42/p44 had no effect on cPLA2 induction. A small but significant inhibitory effect (~25%) by Bis I and SB-203580 was observed. Pretreatment with SB-202474 had no significant effect, and dexamethasone pretreatment essentially eliminated cPLA2 induction.


Figure 3
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  		Fig. 3. IL-1beta-mediated cytosolic phospholipase A2 (cPLA2) expression in HASM. Cells were pretreated with the indicated inhibitors for 30 min then treated with 20 IU/ml IL-1beta for 18 h. Cell lysates were harvested and subject to immunoblotting with secondary antibodies conjugated to infrared fluorophores. Band intensity reflecting cPLA2 expression was directly quantified with the Odyssey Imaging System as described in METHODS. A representative blot is shown in B. Data from 6 independent experiments are presented as mean ± SE values in C. *P < 0.05, IL-1beta + inhibitor vs. IL-1beta + vehicle.

 
To determine whether the induction of cPLA2 protein translated into an increase in cellular PLA2 activity, we examined PLA2 activity in both intact cells and cell-free assays. Interestingly, we found that IL-1beta did not affect whole cell arachidonic acid release over the course of 18-h treatment (Fig. 4). Although pretreatment with U0126 and Bis I inhibited arachidonic acid release in both vehicle (not shown)- and IL-1beta-treated cells, none of the other inhibitors affected arachidonic acid release. Because both secretory PLA2 (sPLA2) and cPLA2 isoforms can contribute to cellular arachidonic acid release and because studies examining cPLA2 knockout mice suggest that COX-dependent PGE2 synthesis requires cPLA2 (5), we examined the regulation of cPLA2 in an in vitro assay. PLA2 activity in cellular lysates (in which sPLA2 activity was neutralized) was significantly increased by 18-h treatment of cells by IL-1beta (Fig. 5). Inhibition of p38 or PKC had a small but nonsignificant effect, whereas dexamethasone pretreatment significantly inhibited this induction of activity. Thus, unlike global PLA2 activity (Fig. 4), which appears dominated by sPLA2 activity, cPLA2 enzymatic activity is induced and regulated in a manner similar to the observed induction of cPLA2 protein.


Figure 4
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  		Fig. 4. IL-1beta-mediated arachidonic acid (AA) release in HASM. Confluent growth-arrested cells grown in 24-well plates were loaded with [3H]tritiated AA overnight then washed extensively as described in METHODS. Loaded cells were then pretreated with vehicle (0.1% DMSO), 10 µM U0126, 1 µM SB-203580, 1 µM SB-202474, 3 µM Bis I, or 10 nM Dexa for 30 min and then treated with 20 IU/ml IL-1beta for 18 h. AA release into cell culture supernatants was assessed by liquid scintillation counting. Data from 5 independent experiments are presented as mean ± SE values. *P < 0.05, IL-1beta + inhibitor vs. IL -1beta + vehicle.

 

Figure 5
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  		Fig. 5. IL-1beta-mediated in vitro cPLA2 activity. Confluent growth-arrested cells grown in 100-mm dishes were pretreated with vehicle (0.1% DMSO), 10 µM U0126, 1 µM SB-203580, 1 µM SB-202474, 3 µM Bis I, or 10 nM Dexa for 30 min and then treated with 20 IU/ml IL-1beta for 18 h. Cytosolic fractions were then prepared, and enzyme activity was assessed as described in METHODS. Data from 4 independent experiments are presented as mean ± SE values. *P < 0.05, IL-1beta + inhibitor vs. IL-1beta + vehicle.

 
As with both COX-2 and cPLA2, mPGES protein expression was upregulated (~4-fold) by chronic IL-1beta treatment (Fig. 6, A and B). Conversely, no change in cPGES expression after IL-1beta pretreatment was observed (Fig. 6C). The mPGES induction was sensitive to each of the inhibitory agents examined. Pretreatment with U0126 or SB-203580 significantly reduced (~30 and 50%, respectively) the induction of mPGES, whereas dexamethasone eliminated it. Bis I pretreatment caused a small mPGES induction (~=25%) that was not statistically significant (P = 0.2). Regulation of mPGES induction was unique in that SB-202474 had a significant inhibitory effect, suggesting that inhibition of mPGES expression/activity could contribute to the inhibitory effect of SB-202474 on IL-1beta-mediated PGE2 production.


Figure 6
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  		Fig. 6. IL-1beta-mediated microsomal PGE synthase (mPGES) expression in HASM. Cells were pretreated with the indicated inhibitors for 30 min then treated with 20 IU/ml IL-1beta for 18 h. Cell lysates were harvested and subject to immunoblotting with secondary antibodies conjugated to infrared fluorophores. Band intensity reflecting mPGES expression was directly quantified using the Odyssey Imaging System as described in METHODS. Representative blots are provided depicting the time-dependent induction of mPGES (A) and regulation by the indicated inhibitors (B). C: representative blot of cPGES expression and regulation. Data from 5 independent experiments examining the effect of inhibitors on induced mPGES expression are presented as mean ± SE values in D. *P < 0.05, IL-1beta + inhibitor vs. IL-1beta + vehicle.

 
PKA differentially regulates COX-2, cPLA2, and mPGES. We have recently established that PKA participates in a feed-forward mechanism that augments IL-1beta-induced COX-2 and PGE2 synthesis in HASM (8). To examine the regulatory role of PKA in the induction of cPLA2 and mPGES, HASM cells selected for stable expression of GFP or a GFP chimera of the PKA inhibitory peptide PKI (PKI-GFP) [characterized in Guo et al. (8)] were treated 18 h with vehicle, IL-1beta, or IL-1beta + TNF-{alpha}, and protein expression of COX-2, cPLA2, and mPGES was assessed. As previously shown, the induction of COX-2 was mitigated in cells expressing PKI-GFP, and this was associated with a reduction in PGE2 production (Fig. 7, A, B, E). Conversely, the induction of cPLA2 was minimally affected by expression of PKI-GFP (Fig. 7, A and D), suggesting that regulation of cPLA2 gene transcription is not PKA dependent and unaffected by autocrine PGE2 production. As with COX-2, induction of mPGES was reduced (~35%) in lines expressing PKI-GFP (Fig. 7, A and C), suggesting that mPGES upregulation is similarly subject to feed-forward regulation mediated by PGE2 production and associated PKA activation.


Figure 7
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  		Fig. 7. Effects of PKA inhibition on induction of COX-2, mPGES, and cPLA2 by cytokines. HASM lines stably expressing either green fluorescent protein (GFP) or protein kinase A inhibitor (PKI)-GFP were generated as described in Guo et al. (8), growth-arrested, and treated 18 h with vehicle, 20 IU/ml IL-1beta, or 20 IU/ml IL-1beta plus 10 ng/ml TNF-{alpha} (I+T). A: representative blot demonstrating cytokine-induced changes in COX-2, mPGES, and cPLA2 expression, and levels of the intracellular PKA substrate vasodilator-associated phosphoprotein (VASP) is presented. B–D: mean ± SE values of COX-2, mPGES, and cPLA2 expression under the indicated conditions, quantified from 5–6 different experiments each using a different set of clonal lines. E: mean ± SE values (n = 6) of PGE2 measured in culture supernatant in cells treated under the indicated conditions. *P < 0.05, PKI-GFP vs. GFP condition, IL-1beta-stimulated condition; **P < 0.05, PKI-GFP vs. GFP condition, IL-1beta + TNF-{alpha}-stimulated condition.

 
Collectively, these data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-1beta in HASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. Differential regulation of COX-2 and mPGES induction may explain the role of PKC, and possibly the effects of SB-202474 on IL-1beta-induced PGE2, and suggests a role for mPGES induction in promoting the full effect of IL-1beta on autocrine PGE2 production.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The present study demonstrates that in human ASM, IL-1beta-mediated cPLA2, COX-2, and mPGES protein induction are regulated by distinct mechanisms. Whereas IL-1beta effects the coordinate induction of all three enzymes, albeit to different extents, the profile of signaling pathways mediating the induction of each enzyme is distinct. These findings represent the first report of cytokine-mediated induction of cPLA2 protein expression and enzymatic activity in ASM. Novel mechanistic insight is also provided by demonstration that regulation of IL-1beta-mediated cPLA2 induction differs from that for COX-2 or mPGES. IL-1beta-mediated cPLA2 induction was abrogated by steroid pretreatment, modestly sensitive to PKC and p38 MAPK inhibition, but resistant to the inhibition of p42/p44 MAPK or PKA. In contrast, IL-1beta-mediated COX-2 induction was highly sensitive to p42/p44 MAPK, PKA, and p38 inhibition, whereas PKC inhibition had a minimal effect. Similar to what has been reported previously in ASM, IL-1beta-mediated COX-2 induction was highly sensitive to steroid pretreatment (3, 24, 32).

Also we demonstrate for the first time a cytokine-mediated induction of mPGES protein expression in ASM. Interestingly, IL-1beta-mediated mPGES induction was sensitive to inhibition of all of the pathways tested including p38, p42/p44, PKA, and classical PKC; however, the inhibition in each case was only modest. Thus the profile of signaling pathways regulating IL-1beta-mediated mPGES induction was qualitatively similar to that of COX-2. However, in contrast to what was observed for cPLA2 and COX-2, IL-1beta-mediated mPGES induction was sensitive to pretreatment with the imidazole compound SB-202474. This finding may explain, in part, the observed effect of SB-202474 on IL-1beta-mediated PGE2 production. Notably this finding suggests that induction of mPGES contributes significantly to the observed PGE2 production.

Clearly the regulation of IL-1beta-mediated PGE2 production in human ASM is a complex process. Existing dogma holds that IL-1beta-mediated COX-2 induction in ASM is the key regulatory step that ultimately determines PGE2 production. Previous analyses of the regulatory features of IL-1beta-mediated PGE2 production in ASM have focused on the role of COX-2 induction (3, 7, 15, 23, 24, 31). Studies in other cell types have suggested but have not directly shown that PLA2 is upregulated by IL-1beta treatment. Saunders et al. (27) reported that in A549 pulmonary epithelial cells IL-1beta treatment for 24 h mediated COX-2 induction but that the subsequent rate of PGE2 release was very low. Treatment of the IL-1beta-primed cells with bradykinin or the calcium ionophore A-23187 resulted in large increases in PGE2 production. The authors proposed that IL-1beta-mediated PLA2 was stimulated by these agents to release arachidonic acid that was subsequently utilized by COX-2/mPGES to make PGE2. Newton et al. (18), also using A549 cells, demonstrated that inhibition of p38 and p42/p44 resulted in minimal inhibition of IL-1beta-mediated COX-2 induction but a disproportionate reduction in PGE2 production. Because p38 and p42/p44 inhibition also diminished IL-1beta-mediated arachidonic acid release, they asserted a key role for PLA2-mediated arachidonic acid release in subsequent PGE2 production. This group had also previously shown that in A549 cells IL-1beta treatment resulted in the upregulation of cPLA2 mRNA and that this effect was steroid sensitive (19). These results cannot be generalized to ASM because p38 and p42/p44 signaling appears to be required for maximal IL-1beta-mediated COX-2 induction in ASM (13, 14, 25) but plays a minor role in A549 cells.

Clarifying the regulation of PLA2 and PGES enzymes and their role in PGE2 generation in ASM.

There are a large number of phospholipases that are capable of releasing arachidonic acid from membrane phospholipid and hence potentially capable of contributing to PGE2 generation in ASM. Indeed the fact that IL-1beta does not change overall arachidonic acid release (Fig. 4), even though cPLA2 expression is clearly increased, suggests that other phospholipase activity exists that does not necessarily contribute to the observed induction of PGE2. That in vitro PLA2 activity increases proportionately with cPLA2 expression and because this assay is performed in the presence of DTT (a potent inhibitor of sPLA2 enzymatic activity) supports the notion that IL-1beta-mediated PLA2 activity is due to cPLA2 and not sPLA2. Most studies to date that have examined PLA2-COX enzyme coupling suggest that cPLA2 is the PLA2 that is functionally coupled to COX-2 (17). Indeed, the targeted deletion of cPLA2 in mice results in an animal that is not capable of producing PGE2 in response to cytokine stimulation (5, 30). We examined the effect of IL-1beta treatment on the constitutively expressed cPGES and found that cPGES is constitutively expressed and not regulated by IL-1beta. This result suggests that cPGES does not contribute in a substantial way towards IL-1beta-mediated PGE2 production.

We previously examined IL-1beta-mediated COX-2 induction and PGE2 production in HASM and identified conditions under which regulation of PGE2 induction appears dissociated from the regulation of COX-2 induction (25). For example, the imidazole SB-202474 reduced IL-1beta-mediated PGE2 production by >80%, whereas its effect on IL-1beta-mediated COX-2 induction was minimal. Similarly, MEK1/2 inhibitor U0126 abrogated PGE2 generation even though its inhibition of IL-1beta-mediated COX-2 induction was considerably less. Moreover, cotreatment of human ASM with IL-1beta + TNF-{alpha} or IL-1beta + EGF resulted in an additive effect on COX-2 induction but more than additive increases in PGE2 production. Interestingly, COX-2 induction under these conditions was resistant to the inhibition of p38 or p42/p44 even though the inhibition of PGE2 remained nearly complete. These results strongly suggested that the regulation of cytokine-mediated PGE2 production was not totally dependent on the level of COX-2 but that other regulatory elements were involved.

To gain an appreciation of which regulatory elements may be involved we first examined the role of cPLA2 in IL-1beta-mediated PGE2 production. This isoform of PLA2 was chosen because although several PLA2 enzymes can release arachidonic acid from membrane phospholipids, the central role of cPLA2 in prostanoid production has been established by its targeted deletion in mice (5, 30). Moreover, cotransfection studies have suggested that cPLA2 preferentially couples to COX-2 with resultant prostanoid production (17). In HASM we observe that cPLA2 is constitutively expressed under basal conditions, and somewhat surprisingly we observed that chronic IL-1beta treatment resulted in a robust three- to fourfold induction of both cPLA2 expression and in vitro PLA2 activity. Interestingly, IL-1beta-mediated cPLA2 expression was not regulated by p42/p44 but was modestly sensitive to p38 and PKC inhibition. Thus the differential effects on PGE2 production and COX-2 expression by p38 and PKC can be explained in part by IL-1beta-mediated regulation of cPLA2. However, the effects of p38 and PKC inhibition on cPLA2 expression are only modest, and p42/p44 inhibition has no effect on IL-1beta-mediated cPLA2 expression. Interestingly, none of the inhibitors tested had any effect on IL-1beta-mediated PLA2 activity in vitro. However, because of the repertoire of PLA2 enzymes that can release arachidonic acid in vitro, it is likely that we cannot discriminate the regulation of cPLA2 using an in vitro assay.

We next turned our attention to the study of IL-1beta-mediated mPGES. This enzyme was of interest because several studies in different cell systems have demonstrated that IL-1beta mediates the coordinate induction of COX-2 and mPGES (6, 29). Consistent with these studies, we found that mPGES was also induced by IL-1beta in ASM cells and that under most conditions in which COX-2 induction was inhibited, mPGES induction was similarly inhibited. The notable exception was the significant inhibitory effect of SB-202474 on mPGES induction only. Because SB-202474 also significantly inhibits IL-1beta-mediated PGE2 induction, these results implicate the induction of mPGES as an important contribution to the induction of PGE2 by IL-1beta.

Because we had previously determined that COX-2 induction by both IL-1beta and combined IL-1beta and TNF-{alpha} treatment was augmented in a feed-forward manner by the PKA activity associated with COX-2/PGE2 induction, we examined whether PKA similarly regulates the induction of cPLA2 and mPGES. As previously demonstrated (8), PKI-GFP inhibited cytokine-induced PKA activity as evidenced by inhibition of PKA-mediated phosphorylation of the intracellular PKA substrate vasodilator-associated phosphoprotein, and this was associated with a reduction in cytokine-induced PGE2 production (Fig. 7, A and E). Interestingly, we again observed that regulation of mPGES induction was similar to that of COX-2, with cells expressing a PKI inhibitory peptide exhibiting a significantly lesser induction of both COX-2 and mPGES in response to cytokine treatment (Fig. 7, B and C). However, induction of cPLA2 was not affected by PKA inhibition (Fig. 7D), demonstrating another instance in which the regulation of this enzyme was distinct from that of COX-2 and mPGES.

In summary, the present study details the regulatory features of IL-beta-mediated COX-2, cPLA2, and mPGES induction in ASM cells and relates these features to the regulation of PGE2 induction. Future studies emphasizing imaging of subcellular compartmentalization of the induced enzymes will likely enhance our understanding of how the activity of these enzymes is coordinated to promote PGE2 synthesis.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-58506 and HL-67663.


   DISCLOSURES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
R. A. Panettieri has consultant arrangements with GlaxoSmithKline (GSK), Centocor, Epigenesis, Prolexys, and BioMarck; grants from GSK, Epigenesis, and Centocor; and serves on the Speaker's Bureaus of GSK, Merck, Centocor, Genentech, and Novartis. S. P. Peters has been involved in clinical trials supported by Abaris, AstraZeneca, Altana, Boehringer Ingelheim, Centocor, Genentech, GlaxoSmithKline, Novartis, Pfizer, and Wyeth. He has also performed consulting duties for Adelphi, AstraZeneca Pharmaceuticals, Discovery, Genentech, Novartis, Omnicare, RAND Corporation, and SanofiAventis. He has also participated in physician education programs (including speakers' bureaus) sponsored by AstraZeneca, Merck, Genentech, and Novartis.


   ACKNOWLEDGMENTS
 
The authors thank Uma Ghandi for assistance in generating the GFP and PKI-GFP lines.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. B. Penn, Wake Forest Univ. Health Sciences Center, Center for Human Genomics, Medical Center Blvd, Winston-Salem, NC 27157 (e-mail: rpenn{at}wfubmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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