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Originally published In Press as doi:10.1074/jbc.M412075200 on February 18, 2005

J. Biol. Chem., Vol. 280, Issue 17, 16579-16585, April 29, 2005
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Redirection of Eicosanoid Metabolism in mPGES-1-deficient Macrophages*

Catherine E. Trebino, James D. Eskra, Timothy S. Wachtmann, Jose R. Perez, Thomas J. Carty, and Laurent P. Audoly{ddagger}

From the Inflammation, Pfizer Global Research and Development, Groton Laboratories, Pfizer Inc., Groton, Connecticut 06340

Received for publication, October 25, 2004 , and in revised form, February 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1–/– animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2) > thromboxane B2 (TxB2) > 6-keto prostaglandin F1{alpha} (PGF1{alpha}), prostaglandin F2{alpha} (PGF2{alpha}), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1–/– macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2 > 6-keto PGF1{alpha} and PGF2{alpha} > PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 µM [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1–/– macrophages compared with controls resulted in TxB2 and 6-keto PGF1{alpha} becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1–/– samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1–/– cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of prostaglandin metabolism is at the center of current anti-inflammatory therapies. Non-steroidal anti-inflammatory drugs and aspirin-related drugs block the activity of cyclooxygenases and their ability to convert arachidonic acid (AA)1 into PGH2. PGH2 can be subsequently metabolized by terminal prostaglandin synthases to the corresponding biologically active PGs, namely, PGI2, thromboxane A2, PGD2, PGF2{alpha}, and PGE2. A combination of pharmacological, genetic, and neutralizing antibody approaches demonstrates the importance of PGE2 in inflammation (1–6). In many respects, disruption of PGE2-dependent signaling in animal models of inflammation can be as effective as treatment with non-steroidal anti-inflammatory drugs or Cox-2 inhibitors (1, 3). The conversion of PGH2 to PGE2 may therefore represent a pivotal step in the propagation of inflammatory stimuli.

Several prostaglandin E synthases, the enzymes that convert PGH2 into PGE2, have been isolated and identified to date (7–11). Five mammalian PGE synthase isoforms have now been cloned, and these isoforms are referred to as microsomal PGE synthase (mPGES)-1, mPGES-2, GSTM2-2, GSTM3-3, and cPGES/p23 (12–15). mPGES-1 has emerged as an important modulator of inflammation in vivo (3, 16). mPGES-1 expression has been detected in multiple disease states both in animal models and in the clinic (17–19), and it is down-regulated in vitro by classical anti-inflammatory drugs such as dexamethasone (20, 21). In the absence of specific mPGES-1 inhibitors, antisense and transgenic approaches have provided further evidence supporting a role for mPGES-1 in inflammation (3, 17, 22). Importantly, mPGES-1–/– mice in a chronic model of inflammation, collagen-induced arthritis, are mostly resistant to the development of joint arthritis (3). The marked reduction in chronic inflammation is accompanied at the cellular level by a >95% reduction in LPS-induced PGE2 release from mPGES-1–/– peritoneal macrophages (3, 23, 24).

PGH2 serves as a common substrate for multiple prostaglandin synthases, and it is therefore conceivable that the modulation of one arm of the pathway may impact substrate availability for other terminal prostaglandin synthases. For example, prostaglandin I synthase (PGIS)-deficient mice lose a significant in vivo portion of their prostacyclin production ability, and this is accompanied by marked increases in systemic PGE2 and TxB2 production (25). It is difficult to conclude, especially in view of the developmental vascular disorders observed in PGIS-deficient animals, that these results were not the reflection of complex physiological adaptation secondary to genetic compensation. However, these studies do provide preliminary indication that in the absence of a major terminal prostanoid synthase, divergence of flow to other prostanoids can occur. Our studies were designed to test this hypothesis in mPGES-1-deficient macrophages.

Macrophages are key determinants of disease processes involved in the initiation, progression, and resolution of inflammation. These cells process external inflammatory cues and in turn propagate physiological responses via the release of inflammatory mediators such as PGE2, PGD2, PGI2, PGF2{alpha}, and TxA2 (26–28). The orchestrated synthesis of these prostaglandins is an important aspect of inflammation because each of these mediators displays unique and sometimes opposing cellular functions (29, 30). Therefore, macrophages provide a cellular model for characterizing prostaglandin metabolism. We posited that the absence of mPGES-1 expression in macrophages would be accompanied by marked "PGH2 shunting" to prostaglandins other than PGE2. To test this hypothesis, murine macrophages isolated from wild type and littermate mPGES-1-deficient animals were exposed to diverse pharmacological inhibitors and/or pro-inflammatory stimuli. Detailed expression analyses of mPGES-1+/+ and mPGES-1–/– cells revealed no overt difference in levels of prostaglandin metabolic enzymes. Furthermore, there was no detectable retrograde effect of mPGES-1 deletion on leukotriene levels. However, in the absence of mPGES-1 expression, our results suggest that PGH2 metabolism can be diverted to biologically active prostaglandins other than PGE2, suggesting that the mPGES-1 enzyme is a primary release mechanism of PGH2 accumulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Macrophages—mPGES-1–/– and control mPGES-1+/+ mice were generated as previously described (3). All animals used in our studies were on an inbred DBA/1lacJ genetic background. All experiments were performed under protocols approved by the Pfizer Global Research and Development Institutional Animal Care and Use Committees. Mice received intraperitoneal injection with 0.5 ml of 10% thioglycollate media on day 0. On day 4, peritoneal lavages were then collected and processed as previously described (2). No significant difference in the number of cells recovered from peritoneal exudates was noted between genotypes.

Cellular Prostaglandin Formation Assays—Four different types of cellular experiments were performed with the peritoneal macrophages isolated from mPGES-1–/– and mPGES-1+/+ mice. At the completion of each experiment, supernatants were isolated and stored at –80 °C until assayed. Media controls for each condition reflected the highest possible amount of solvent used to prepare the corresponding stimulating agent (LPS: PBS, arachidonic acid: ethanol, dazoxiben: Me2SO).

For the prostaglandin measurement time course experiments, macrophages (105 cells/well in a 96-well plate format) were incubated with 1 µg/ml LPS (Escherichia coli 0111:B4) for the corresponding incubation period, and supernatants were then removed and discarded. Cells were then washed three times with PBS and incubated for 1 additional hour in the presence of fresh cell culture media. For the prostaglandin accumulation experiments, macrophages (107 cells/dish in a 100-mm dish) were incubated without or with 1 µg/ml LPS (E. coli 0111:B4) for 16 h. For the AA post-stimulation experiments, macrophages (105 cells/well in a 96 well-plate format) were pre-treated with LPS for 16 h, followed by incubation with 10 µM arachidonic acid for increasing periods of time (up to 60 min). For the thromboxane synthase (TxAS) inhibitor studies (dazoxiben), cells (105 cells/well in a 96 well-plate format) were pre-treated with LPS (16-h incubation), followed by a 15-min pre-incubation with increasing concentrations of dazoxiben and subsequent treatment with 10 µM AA for 10 min.

Microsomal Preparation and Western Blot Analyses—Macrophages growing in a 10-cm culture dish (107 cells/dish) were processed, and immunodetections of microsomal proteins were performed as described previously (22). The following antibodies were used for immunodetection of prostaglandin synthetic enzymes: mPGES-1, rabbit polyclonal antibody (1:500; Cayman Chemicals, Ann Arbor, MI); Cox-2, rabbit polyclonal antibody (1:3000; Abcam, Cambridge, UK); PGIS, mouse monoclonal antibody (1:30; Oxford Biomedical Research, Oxford, MI); and TxAS, rabbit polyclonal antibody (1:500; Cayman Chemicals).

Eicosanoid Analysis (Non-radioactive Measurements)—Non-radioactive media were assayed for PGE2, 6-keto PGF1{alpha} (a stable metabolite of PGI2), TxB2 (a stable metabolite of TxA2), PGF2{alpha}, and PGD2 by EIA according to the manufacturer's protocol (Cayman Chemicals). The lower limit of detection by EIA was 15 pg/ml.

Radiolabeled Eicosanoid Analysis—Mouse peritoneal macrophages were added to 24-well culture dishes at a concentration of 106 cells/well in 1 ml of Dulbecco's modified Eagle's medium + 1% fetal bovine serum. After attaching for 4 h, 1 µg/ml LPS was added, and the cells were incubated for 20 h. Cells were washed first with Dulbecco's modified Eagle's medium (no fetal bovine serum), and 0.5 ml of Dulbecco's modified Eagle's medium containing 10 µM arachidonic acid and 1 µCi of [3H]arachidonic acid was subsequently added for a period of 5 min at 37 °C. The media were removed and found to contain 33% of the added radioactivity.

Solid phase extraction of the media was then performed. Acetonitrile (500 µl) containing a mixture of eicosanoid standards: 1 µg each of 6-keto PGF1{alpha}, TxB2, PGF2{alpha}, PGE2, and PGD2; 50 ng each of LTD4, LTC4, LTB4, and HHT; and 100 ng of indomethacin was added to 250 µl of media in a 1.5-ml microcentrifuge tube. After centrifugation at 8000 x g for 2 min, the supernatant was transferred to a 13 x 100-mm borosilicate glass tube. After dilution with 3.5 ml of 5 mM phosphoric acid in water, the supernatant was then passed through a 100-mg Bond Elut C18 column (Varian Inc., Palo Alto, CA) previously wetted with 4 ml of methanol followed by 2 x 1 ml of acetonitrile and 1 ml of water. The tube and column were then washed with 1 ml of acetonitrile/water/phosphoric acid/triethylamine (10:90:0.036:0.036, v/v/v/v) and eluted with 0.5 ml of solvent acetonitrile/water/phosphoric acid/triethylamine (70:30:0.036:0.036, v/v/v/v) followed by 0.5 ml of acetonitrile/water/phosphoric acid/triethylamine (10:90:0.036:0.036, v/v/v/v), resulting in a final 1.0-ml eluate in acetonitrile/water/phosphoric acid/triethylamine (35:65:0.036:0.036, v/v/v/v).

HPLC separation was then performed using a Waters Symmetry C8 HPLC column (3.0 x 0.46 cm) (Waters, Milford, MA). The entire 1 ml of solid phase extraction eluate was injected using a Gilson ASPEC autosampler programmed to dilute and mix the sample with 4.1 ml of water containing 5 mM phosphoric acid and injecting 5.0 ml using a 5.0-ml sample loop into the column equilibrated with acetonitrile/water/phosphoric acid/triethylamine (10:90:0.036:0.036, v/v/v/v) at a flow rate of 1.5 ml/min. A linear gradient to acetonitrile/water/phosphoric acid/triethylamine (60:40:0.036:0.036, v/v/v/v) over 20 min at a flow rate of 1.5 ml/min was used to elute the eicosanoids. The eluate was monitored with 2 PerkinElmer Life Sciences Series 200 absorbance detectors set at 195 and 280 nm. The percentage recovery of the spiked standards showed 94 ± 3% recovery of prostanoid standards and 76 ± 5% recovery of the HHT standards. Fractions (0.15 min) were collected from 10.00 to 25.00 min. For radiolabeled product profiles, 150 of 225 µl of each fraction was counted in 4.0 ml of Beckman Redisafe scintillation fluid, and the mixture was counted using a Beckman LS6500 scintillation counter.

Materials—Unless indicated otherwise, all reagents were obtained from Sigma-Aldrich.

Statistical Analysis—The values are expressed as the mean ± S.E. Group comparisons were performed by analysis of variance with a Bonferroni post-test unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Production of PGE2 in Macrophages—Incubation of wild type peritoneal macrophages with LPS caused a substantial increase in the release of PGE2 into the cellular supernatant starting after 4 h, lasting over the course of 48 h, and reaching a plateau at the 24 h time point (Fig. 1). In marked contrast, mPGES-1–/– cells showed no significant increase in PGE2 formation at all time points.



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  		FIG. 1.
Impact of mPGES-1 genetic deletion on PGE2 production in murine peritoneal macrophages. Peritoneal macrophages were isolated from mPGES-1+/+ and mPGES-1–/– mice and incubated with 1 µg/ml LPS in 96-well plates (105 cells/well) for the indicated periods of time. Media were isolated and assayed for PGE2 by EIA as described under "Experimental Procedures." Data shown are the mean ± S.E. from duplicate determinations with n = 3/time point/experiment.

 
Evidence of Prostaglandin Shunting in mPGES-1–/– Macrophages after Prolonged LPS Challenge—Based on the time course of PGE2 formation, we chose to further profile prostaglandin formation 16 h after LPS incubation. Cellular supernatants were isolated and assayed for PGE2, 6-keto PGF1{alpha} (a stable metabolite of PGI2), TxB2 (a stable metabolite of TxA2), PGF2{alpha}, and PGD2 production. No significant difference was detected between mPGES-1+/+ and mPGES-1–/– samples from cells treated with vehicle only (Fig. 2).



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  		FIG. 2.
Redirection of prostaglandin production in mPGES-1–/– macrophages. Peritoneal macrophages were isolated from mPGES-1+/+ and mPGES-1–/– mice and incubated with 1 µg/ml LPS overnight in 96-well plates (105 cells/well) as described under "Experimental Procedures." Media were isolated and assayed for prostaglandins by EIA. Data shown are the mean ± S.E. from triplicate determinations with n = 3/group/experiment. **, p < 0.01; ***, p < 0.001.

 
In contrast, incubation with LPS caused a significant increase in all prostanoids measured in the supernatants isolated from wild type cells. Consistent with our time course experiments, PGE2 levels were reduced by ~95% in media isolated from LPS-treated mPGES-1–/– cells. Furthermore, TxB2 became the most prominent prostaglandin formed in mPGES-1–/– samples. The relative order of prostaglandin abundance in mPGES-1–/– samples was as follows: TxB2 > 6-keto PGF1{alpha} > PGF2{alpha} > PGD2 > PGE2. In contrast, this ranking in mPGES-1+/+ samples was as follows: PGE2 > TxB2 > 6-keto PGF1{alpha} > PGF2{alpha} > PGD2. We detected an ~2-fold increase in the levels of TxB2, 6-keto PGF1{alpha}, PGF2{alpha}, and PGD2 in mPGES-1–/– supernatants relative to mPGES-1+/+ samples (Table I). To characterize the molecular mechanisms responsible for this altered pattern of expression, we focused on the expression of the terminal prostaglandin synthases and cyclooxygenases isoforms responsible for the formation of the most abundant prostaglandin.


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  		TABLE I
Prostaglandin profiles from mPGES-1+/+ and mPGES-1-/- macrophages incubated overnight with LPS

Data shown are the mean of three independent experiments. 90% of prostanoid production was recovered in the mPGES-1–/– samples compared with the mPGES-1+/+ samples by monitoring PGE2, 6-keto PGF1{alpha}, TxB2, PGD2, and PGF2{alpha} levels (total production: 22.5 ng/ml, mPGES-1+/+ samples; 20.2 ng/ml, –/–mPGES-1 samples). PG, prostaglandin.

 
Induction Kinetics of Prostaglandin Synthetic Enzymes—To begin to address the mechanisms that could have led to this diversion of prostaglandin flow, we profiled the induction kinetics of Cox-2, mPGES-1, PGIS, and TxAS by Western blot. All four proteins were detected at t = 0 h. Genetic ablation of the mPGES-1 gene resulted in a significant down-regulation of detectable mPGES-1 protein expression (Fig. 3A). Importantly, the absence of detectable mPGES-1 expression did not result in significant overt modulation of either Cox-2, PGIS, or TxAS protein levels as determined by densitometry (Fig. 3B; data not shown for PGIS and TxAS). Treatment of macrophages with either vehicle or LPS did not have a significant impact on TxAS or PGIS protein levels in either mPGES-1+/+ or mPGES-1–/– samples. In contrast, expression of mPGES-1 and Cox-2 in mPGES-1+/+ samples was up-regulated several fold above baseline levels at multiple time points following LPS incubation (Fig. 3B).



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  		FIG. 3.
Western blot analysis of prostaglandin synthetic enzymes. Peritoneal macrophages were plated in 10-cm dishes (107 cells/dish) in the absence or presence of 1 µg/ml LPS for the indicated periods of time. Cells were rinsed three times with PBS, and microsomes were isolated and subjected to Western blot analysis as described under "Experimental Procedures." A, specific antibodies against mPGES-1, Cox-2, PGIS, and TxAS were then utilized to probe for the presence of specific proteins followed by an enhanced chemiluminescence detection method to monitor the expression time course of each of these proteins in murine macrophages. B, Western blots were scanned to quantitatively compare mPGES-1 and Cox-2 induction kinetics. Results shown are from a representative experiment performed in duplicate.

 
The pattern of Cox-2 protein expression did not differ significantly between mPGES-1+/+ and mPGES-1–/– cells, consistent with the absence of detectable genetic compensation at the protein expression level (Fig. 3B). Whereas both mPGES-1 and Cox-2 are modulated in response to an inflammatory stimulus, their relative patterns of expression differ markedly. Peak expression of Cox-2 protein occurs at 8 h after LPS challenge and returns to baseline by 24 h. In contrast, mPGES-1 protein expression follows a biphasic pattern, reaching an intermediate plateau at 8 h and a sustained peak at 24 h. The 48 h time point typifies the distinct pattern of expression of these two proteins: at this time point, mPGES-1 is maximally up-regulated (relative to the time course of this experiment), and Cox-2 levels have returned back to baseline.

Kinetics of Prostaglandin Formation during an Acute Challenge—LPS challenge experiments suggest that murine macrophages divert their prostaglandin production to PGI2 and TxA2 (and, to a lesser extent, PGD2 and PGF2{alpha}) in the absence of mPGES-1 expression. To further examine the role of mPGES-1 in acute inflammatory settings, LPS-stimulated cells were exposed to 10 µM AA from 30 s to 60 min. The first 10 min of AA challenge were associated with the highest rate of prostanoid production (Fig. 4). Consistent with our previous results, PGE2 production in mPGES-1–/– samples was significantly reduced compared with wild type samples (Fig. 4A). Both mPGES-1–/– and mPGES-1+/+ samples produced significant quantities of TxB2 and 6-keto PGF1{alpha} (Fig. 4, B and C) within the first 10 min of AA challenge. Greater amounts of both of these prostaglandins were detected in mPGES-1–/– samples, but both reached maximum amounts 10 min post-challenge.



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  		FIG. 4.
AA-stimulated prostaglandin production in LPS-pre-treated macrophages. Macrophages isolated from mPGES-1+/+ and mPGES-1–/– cells were incubated with 1 µg/ml LPS in 96-well plates at a density of 105 cells/well overnight and washed three times with PBS, followed by an incubation with AA for the indicated periods of time as described under "Experimental Procedures." PGE2, 6-keto PGF1{alpha}, and TxB2 levels were determined by EIA. Data shown are the mean ± S.E. from triplicate determinations with n = 3/group/experiment.

 
To further profile the impact of mPGES-1 deletion on eicosanoid metabolism, radiolabeling studies were performed using a 15-min [3H]AA incubation preceded by overnight treatment with LPS. The elution profile clearly showed the reduction in PGE2 production accompanied by elevations in other prostaglandins, 6-keto PGF1{alpha}, TxB2, and PGF2{alpha} (Fig. 5, A and B). Leukotriene levels were not overtly altered in mPGES-1–/– samples. Our results suggest that a compound that co-elutes with HHT was increased in mPGES-1–/– samples by ~5-fold relative to wild type samples. These results clearly indicate that the absence of mPGES-1 can be accompanied by metabolic shunting toward prostaglandins other than PGE2, and possibly HHT, at the cellular level.



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  		FIG. 5.
Prostanoid profile in AA-stimulated LPS-pretreated macrophages. Macrophages incubated with LPS were washed three times with PBS and incubated with [3H]AA for 10 min. Radiolabeled metabolites were separated by HPLC and identified by co-elution with unlabeled authentic standards as described under "Experimental Procedures." A, profile from supernatant isolated from mPGES-1+/+ cells. B, profile from supernatant isolated from mPGES-1–/– cells.

 
Shunting of Prostaglandin Formation after Pharmacological Inhibition of a Terminal Prostaglandin Synthase—To establish the relevance of our findings following genetic manipulation of the prostaglandin pathway, we tested the effects of a pharmacological inhibitor on prostaglandin shunting. Wild type and mPGES-1–/– macrophages treated with increasing concentrations of the thromboxane synthase inhibitor, dazoxiben, were exposed to 10 µM arachidonic acid following overnight incubation with LPS. The IC50 values were similar for cells of either genotype, although the absolute TxB2 levels were higher in mPGES-1–/– samples compared with mPGES-1+/+ samples (Fig. 6A). TxAS inhibition caused a corresponding increase in PGE2 levels (Fig. 6B). The inflection points for the TxB2 and PGE2 curves were 1 and 0.1 µM, respectively. These results suggest that either pharmacological inhibition or genetic manipulation can produce diversion of prostaglandin metabolism in our experimental system.



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  		FIG. 6.
Pharmacologically induced prostaglandin shunting. LPS-pretreated macrophages were incubated with increasing concentrations of a thromboxane synthase inhibitor (dazoxiben) for 15 min at the indicated concentrations and exposed to 10 µM AA for 10 min as described under "Experimental Procedures." PGE2 and TxB2 levels were determined by EIA. A, dazoxiben is equally potent in blocking TxB2 release from mPGES-1+/+ and mPGES-1–/– cells. B, redirection of prostaglandin production to PGE2 following the inhibition of TxB2 production in mPGES-1+/+ cells. Data shown are the mean ± S.E. from duplicate determinations with n = 3/group/experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Stimulation of murine macrophages with LPS or a combination of LPS and AA causes a marked increased in prostaglandins released into the extracellular medium, with PGE2 being the most abundant prostaglandin. The coordinated induction of Cox-2 and mPGES-1 expression occurred concomitantly with the cumulative release of PGE2. This also accompanied the specific increase in microsomal PGE synthase (mPGES-1) activity (3). The up-regulation of mPGES-1 expression is in part responsible for the release of PGE2, based on the observation that mPGES-1-deficient macrophages displayed a >95% reduction in PGE2 release. These results suggest that mPGES-1 activity is a key factor in LPS-induced PGE2 production.

We recently reported an absence of overt physiological defects in macrophages isolated from mPGES-1-deficient mice based on morphology, {beta}-hexasaminidase and interleukin-6 release (3). In the present study, we have extended the characterization of these cells by comparing the expression profiles of Cox-2, PGIS, and TxAS between wild type and mPGES-1–/– cells, and we detected no significant differences in a detailed time course analysis following LPS treatment. We focused on the prostacyclin and thromboxane pathways because of their relative abundance over other prostaglandins in our experiments and their putative role in cellular inflammation (29, 31).

PGIS and TxAS are the two key terminal prostaglandin synthases responsible for the formation of their respective prostaglandins. There was no obvious pattern of protein induction for either PGIS or TxAS, despite a marked increase in wild type control cells of the corresponding prostaglandin end products. Furthermore, >75% of total prostaglandins was recovered in the form of 6-keto PGF1{alpha} or TxB2, consistent with an enzymatic reservoir afforded by other terminal prostaglandin synthases during times when PGH2 and a specific prostaglandin synthase may not be metabolically coupled in an efficient manner. Both 6-keto PGF1{alpha} and TxB2 levels were increased by a similar magnitude in the mPGES-1–/– samples, consistent with the uniform distribution of PGH2 metabolism to either of these pathways. Based on these observations, we propose that the redirection of PGH2 metabolism is not a direct reflection of genetic adaptation but rather a phenotypic adaptation in the absence of a major metabolic pathway.

Pharmacological or genetic disruption of a specific terminal prostaglandin synthase can modulate the production of remaining prostaglandins. Prostanoid tissue analysis in PGIS mice revealed that PGE2 and TxB2 levels were significantly elevated in PGIS–/– animals (25). Transfection of adenovirus encoding hematopoietic prostaglandin D synthase into fibroblasts caused an increase in PGD2 coupled with a decrease in PGE2 released into the extracellular environment (32). In our experimental system, the thromboxane synthase inhibitor, dazoxiben, decreased TxB2 and increased PGE2 in a dose-dependent manner consistent with shunting as previously reported by other researchers (33–35). We also noted that between baseline and the 10 µM dazoxiben-treated groups, the maximal difference in PGE2 levels was higher than that for TxB2 levels. This observation, combined with a 10-fold difference in inflection points between the PGE2 and TxB2 curves, suggests that part of the increase in PGE2 production possibly reflects a mechanism other than a simple intracellular divergence of PGH2 to prostaglandin E synthase(s). Interestingly, there was no increase in 6-keto PGF1{alpha} levels in wild type or knock-out cells incubated with up to 10 µM dazoxiben,2 suggesting that the selective blockade of a terminal prostaglandin synthase does not necessarily result in the non-discriminatory shunting of PGH2 to the remaining prostaglandin pathways.

5-Lipoxygenase-deficient mice display both decreases in LTC4 and increases in PGE2 following stimulation with Ca2+ ionophore, suggesting that at the level of arachidonic acid metabolism, shunting to any eicosanoid subclass is possible in principle (36). We examined the possibility that the absence of mPGES-1 expression could be accompanied by an increase in leukotriene secretion. No such evidence was obtained in our experiments. However, increases in 6-keto PGF1{alpha}, TxB2, and PGF2{alpha} were observed consistently in our diverse experimental approaches. Furthermore, significant increases in a compound that co-elutes with HHT were detected possibly as a result of PGH2 breakdown, which can occur through a number of enzymatic (via TxAS or cytochrome P450s) or non-enzymatic pathways (37, 38).

The coordination of prostaglandin metabolism is emerging as an important hypothesis in the regulation of inflammatory processes (30). A recent study of human atherosclerotic plaques suggests the balance between lipocailin-type prostaglandin D synthase and mPGES-1 expression may be a distinguishing feature of asymptomatic and symptomatic plaques (39). These observations suggest that the disruption of "prostaglandin homeostasis" may thus serve as a biomarker for certain pathophysiological conditions. The balance of prostaglandins could also impact the phenotype of terminal prostaglandin synthase-deficient mice. Administration of PGD2 causes an anti-inflammatory effect in a rat model of carrageenan-induced pleurisy (30). Genetic disruption of mPGES-1-dependent PGE2 production using genetically modified mice causes a significant diminution in arthritis (3). These observations raise the possibility that redirection to PGD2 may be an added benefit as a result of PGH2 redirection.

During the completion of this work, Boulet et al. (23) and Kamei et al. (24) described a prostaglandin metabolic profile in mPGES-1-deficient macrophages. These investigators did not detect significant increases in 6-keto PGF1{alpha} and TxB2 levels in mPGES-1–/– supernatants compared with mPGES-1+/+ samples isolated from thioglycollate-elicited macrophages stimulated with LPS. In contrast, significant increases in both 6-keto PGF1{alpha} and TxB2 were measured in our experimental system. Our studies were performed on an inbred DBA/1 genetic background, whereas the other studies were completed on a mixed 129xC57/Bl6 genetic background. Multiple reports have described how differences in genetic background can impact phenotypes vis-à-vis eicosanoid-related phenotypes (40–42). To begin to understand these differences, it is interesting to note that 129 and C57/Bl6 mice, in contrast to DBA/1 or Balb/C mice, carry a mutation in the spla2 gene translating into the absence of sPLA2 IIA protein expression (41, 43). sPLA2 IIA has been implicated in Cox-2-dependent PGE2 production, and this may contribute in part to differences in prostanoid profiles (44). Notably, significant differences in PGE2 production and levels of prostaglandin-synthesizing enzymes have been previously reported between Balb/C and C57/Bl6 mice (40). It is therefore not surprising that a similar genetic deletion on two distinct genetic backgrounds can potentially result in different phenotypes.

Several questions that extend beyond the scope of this study remain to be answered. What other PGH2-dependent products are modulated, besides TxA2, PGI2, PGF2{alpha}, and PGD2, when mPGES-1 expression is reduced? Several groups have reported that levuglandin E2-protein adducts can be formed from PGH2 (45–47). The biological role and formation of this molecule during states of decreased mPGES-1 expression has yet to be elucidated, especially in view of the absence of overt baseline phenotypes in mPGES-1-deficient mice over 1 year of age.2 How prevalent is the redirection of PGH2 metabolism extracellularly? Redirection of PGH2 metabolism can conceivably occur both intracellularly and/or extracellularly. Transcellular (extracellular) PGH2 metabolism can occur between platelet and endothelial cells (33). This raises the question that shunting patterns may differ from one cell type to another, resulting in different shunting patterns at the macroscopic level from one organ to the next (23).

In summary, our experiments demonstrate that the absence or pharmacological inhibition of a PGH2 metabolic enzyme results in an altered prostanoid metabolic profile in murine macrophages. Expression analysis of key prostanoid metabolic enzymes does not support the idea that the phenotype of mPGES-1-deficient macrophages is due in part to alterations in the expression profile of prostaglandin metabolic enzymes. However, our data demonstrate that mPGES-1-dependent PGH2 metabolism represents a major metabolic outlet for PGH2, but other terminal prostaglandin synthases have the capacity to absorb the excess PGH2 created by this unusual situation. Importantly, non-enzymatic formation of PGE2 is not a significant occurrence. The combination of genetic and pharmacological approaches in these studies further supports the importance of mPGES-1 in PGE2 synthesis and provides insights into the contribution of mPGES-1 to PGH2 metabolism.


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

{ddagger} To whom correspondence should be addressed. Present address: Dept. of Pharmacology, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada. Tel.: 514-428-2864; Fax: 514-428-3921; E-mail: laurent_audoly{at}merck.com.

1 The abbreviations used are: AA, arachidonic acid; mPGES, microsomal prostaglandin E synthase; PGE2, prostaglandin E2; PGH2, prostaglandin H2; PGF1{alpha}, prostaglandin F1{alpha}; PGF2{alpha}, prostaglandin F2{alpha}; PGD2, prostaglandin D2; PGI2, prostaglandin I2; TxB2, thromboxane B2; TxA2, thromboxane A2; LPS, lipopolysaccharide; HPLC, high pressure liquid chromatography; Cox, cyclooxygenase; TxAS, thromboxane synthase; EIA, enzyme immunoassay; PGIS, prostaglandin I synthase; PBS, phosphate-buffered saline; HHT, 12-hydroxyheptadecatrienoic acid. Back

2 C. E. Trebino, personal communication. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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