Despite Transcriptional and Functional Coordination, Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 Largely Reside in Distinct Lipid Microdomains in WISH Epithelial Cells
Department of Obstetrics and Gynecology (Laboratory of Perinatal Research and Division of Maternal-Fetal Medicine) (WEA,DAK), Center for Biomedical Engineering (DAK), and Department of Physiology and Cell Biology (JMR), The Ohio State University, Columbus, Ohio
Correspondence to: Douglas A. Kniss, Laboratory of Perinatal Research, Department of Obstetrics and Gynecology, The Ohio State University, 5th Floor Means Hall, 1654 Upham Drive, Columbus, OH 43210. E-mail: kniss.1{at}osu.edu
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Summary |
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Key Words: inflammation cytokines prostaglandin E2 cyclooxygenase-2 microsomal prostaglandin E synthase-1 lipid microdomains epithelial cells
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Introduction |
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COX catalyzes the committing and rate-limiting step in PGE2 synthesis. COX-1 is constitutively expressed and contributes to immediate, low-amplitude PG release (Smith et al. 2000). The inducible COX-2 isoform is essential for high amplitude, delayed PG production, as is often seen under inflammatory conditions (Smith et al. 2000
). Several distinct gene products bearing PGES activity have been cloned and characterized. As with COX-2, the expression of microsomal PGES-1 (mPGES-1, a member of the membrane associated proteins involved in eicosanoid and glutathione metabolism superfamily) is induced in response to cytokines and other inflammatory stimuli (Jakobsson et al. 1999
; Forsberg et al. 2000
; Thoren and Jakobsson 2000
; Stichtenoth et al. 2001
). In contrast, cytosolic PGES (cPGES) and microsomal PGES-2 (mPGES-2) show only limited inducibility in response to inflammatory mediators (Stichtenoth et al. 2001
; Claveau et al. 2003
; Puxeddu et al. 2003
; Giannico et al. 2005
). It has further been demonstrated that these PGES isoforms contribute unequally to the generation of PGE2 under inflammatory conditions. Cotransfection studies suggest that mPGES-1 is functionally coupled with COX-2 during delayed PG release (Murakami et al. 2000
), whereas cPGES is preferentially association with COX-1 (Tanioka et al. 2000
; Han et al. 2002
). Microsomal PGES-2 appears to be COX nonselective, and current evidence suggests that the contribution of mPGES-2 to high-amplitude PGE2 release through COX-2 is limited (Watanabe et al. 1997
; Murakami et al. 2003
).
PGE2 is generated at sites of inflammation in substantial amounts. Because newly synthesized PGs are released soon after they are produced, cells must be functionally adapted to generate substantial amounts of PGE2 in a relatively short time. We have previously demonstrated that PGE2 production in response to pro-inflammatory cytokines (e.g., interleukin 1ß [IL-1ß] and tumor necrosis factor-) requires increased COX-2 expression in WISH epithelial cells (Albert et al. 1994
; Perkins and Kniss 1997b
). We hypothesized that cooperation between COX-2 and mPGES-1 might serve as an inducible pathway to facilitate this inflammatory cascade. In the present study, we used WISH cells to investigate the induced expression and subcellular localization of mPGES-1 in relation to COX-2. Furthermore, results from recent studies have indicated that a fraction of induced COX-2 may be localized to caveolar structures and may interact with caveolin-1 (Cav-1) (Liou et al. 2000
,2001
). As it has been speculated that PGE synthase isoforms could also be localized to these specific areas (Liou et al. 2001
), we tested whether mPGES-1 and COX-2 were coordinated in Cav-1containing lipid rafts.
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Materials and Methods |
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Northern Blot Analysis
Total RNA was extracted using TRIZOL (Invitrogen) and prepared for Northern blotting as previously described (Ackerman et al. 2004). Bound transcripts were identified using digoxigenin-labeled cDNA probes (Roche Diagnostics; Indianapolis, IN). A 1.8-kb cDNA fragment encoding human COX-2 (a kind gift from Dr. Timothy Hla, University of Connecticut, Farmington, CT), a 0.4-kb cDNA fragment corresponding to the coding region of human mPGES-1 (Cayman Chemical; Ann Arbor, MI), and a 0.6-kb cDNA fragment encoding human glyceraldehyde-3-phosphate dehydrogenase were used for detection. Chemiluminescent signals were detected using the VersaDoc Imaging System and analyzed using Quantity One software (Bio-Rad Laboratories; Hercules, CA).
Immunoblot Analysis
Cellular proteins were extracted in PBS containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and 10 µg/ml each of leupeptin, aprotinin, and antipain. Proteins (30 µg/lane) were resolved by SDS-PAGE and transferred to nitrocellulose. Nonspecific binding was blocked by incubation in Tris-buffered saline (pH 8.0) containing 0.1% Tween-20 (Sigma; St Louis, MO) and 5% nonfat dry milk. Membranes were then probed with polyclonal antibodies directed against mPGES-1 (160140; Cayman Chemical) or COX-2 (sc-1745; Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were exposed to horseradish peroxidase-conjugated secondary antibodies and immune complexes were revealed by enhanced chemiluminescence. As a control for loading precision, membranes were reprobed with a monoclonal antibody directed against glyceraldehyde-3-phosphate dehydrogenase (MAB374; Chemicon International, Temecula, CA). Immunoreactive proteins were visualized using the VersaDoc Imaging System and analyzed using Quantity One software (Bio-Rad).
Immunofluorescence
For whole-mount specimens, WISH cells were grown in monolayer culture on flame-sterilized glass cover slips. After stimulation with 10 ng/ml of recombinant human IL-1ß (R & D Systems; Minneapolis, MN) for 6 hr, cells were fixed for 1 hr in 4% paraformaldehyde PBS and made permeable by the addition of 0.2% Triton X-100 (Sigma) in PBS for 15 min at room temperature. Alternatively, cells were incubated in the presence of 0.05% saponin (Sigma) as a means for permeabilization (Goldenthal et al. 1985). Cells were blocked in 1% nonfat dry milk/5% normal goat serum in PBS before the addition of antibodies. For colocalization experiments, rabbit polyclonal antibodies directed against mPGES-1 (160140; Cayman Chemical) were applied simultaneously with mouse monoclonal COX-2 antibodies (clone 33; BD Biosciences, San Jose, CA). To control for specificity, antibodies were preincubated with a 10-fold excess of either blocking peptide (for mPGES-1, Cayman Chemical) or recombinant human COX-2 (Oxford Biomedical Research; Oxford, MI) before application. Additional negative controls were conducted in which primary antibodies were omitted. After stringent washing in PBS, the cover slips were exposed to Alexa Fluorconjugated secondary antibodies (Molecular Probes; Eugene, OR) and nuclei were counterstained with 5 µg/ml 4',6-diamidino-2-phenylindole (DAPI; Sigma). Specimens were then mounted on glass slides using the ProLong Antifade Kit (Molecular Probes) and visualized using standard epifluorescence (Nikon Instruments; Melville, NY) or confocal microscopy (Zeiss 510 META; Carl Zeiss Inc., Thornwood, NY). Images from whole-mount specimens were captured with MetaVue version 5.0 image analysis software (Universal Imaging Corp.; Downingtown, PA). Maximum fluorescence intensity measurements for structures labeled with anti-COX-2 and antimPGES-1 in each cell were obtained using the line scan function drawn through the long axis of the cell. Background fluorescence adjacent to each positive structure was subtracted from the peak fluorescence to obtain a fluorescence intensity measurement for each fluorochrome in each cell.
In tandem experiments, semithin cryosections were prepared for immunocytochemistry as previously described (Takizawa et al. 2003), with modifications. Briefly, confluent monolayer cultures grown in 175 cm2 flasks and stimulated for 6 hr with IL-1ß were fixed for 1 hr with 4% paraformaldehyde in 0.1 M sodium cacodylate (pH 7.4)/5% sucrose. Specimens were washed, collected by scraping, centrifuged briefly to pellet, and embedded in 10% gelatin made with 0.1 M sodium cacodylate (pH 7.4)/5% sucrose. Within the solidified gelatin, cell pellets were cut into small pieces, infiltrated with 2.3 M sucrose in 0.1 M sodium cacodylate (pH 7.4), and mounted on specimen pins. Specimens were then stored in liquid N2 until the time of sectioning. Semithin sections (
400 nm) were cut using a cryoultramicrotome, collected on droplets of 2 M sucrose containing 0.75% gelatin, and transferred to glass cover slips coated with 2% of 3-aminopropyltriethoxysilane (Sigma). Specimens were then exposed to antiCOX-2 and antimPGES-1 antibodies as before.
Prostaglandin E2 Enzyme Immunoassay
PGE2 content of culture media was quantified using a commercially available enzyme immunoassay (EIA) kit, according to the instructions of the manufacturer (Cayman Chemical). All treatments were conducted in the presence of 5 µM exogenous arachidonic acid (Cayman Chemical). The intra- and interassay coefficients of variation were <10%. Cells were lysed using 1 N NaOH and analyzed for protein content. PGE2 content was then normalized to total protein for each sample.
Preparation of Detergent-resistant Membrane Fractions
Detergent-resistant membrane (DRM) fractions were prepared by flotation on a sucrose step gradient as described previously (Liou et al. 2001), with modifications. After treatment with IL-1ß (10 ng/ml) or vehicle for 6 hr, confluent WISH cells were washed in ice-cold PBS and collected by scraping and centrifugation at 400 x g for 5 min at 4C. Cell pellets were lysed in ice-cold 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA buffer (TNE) containing 1% Triton X-100, 1 mM PMSF, and 10 µg/ml each of leupeptin, aprotinin, and antipain. Homogenization was achieved using 10 strokes of a loose-fitting Dounce homogenizer. The 0.6 ml homogenate was adjusted to 40% sucrose with the addition of 1.4 ml of 56% sucrose prepared in TNE buffer. This was placed in the bottom of an ultracentrifuge tube and overlaid with equal volumes of TNE buffer containing 30% and 5% sucrose, respectively. After centrifugation at 250,000 x g for 20 hr at 4C, nine 600-µl fractions from top to bottom were collected. Equivalent volumes from these fractions were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed for COX-2, mPGES-1, and Cav-1 content by immunoblotting. The chicken anti-peptide Cav-1 antibody was described previously (Lyden et al. 2002
).
Statistical Analysis
EIA data were expressed as PGE2 produced (ng/mg of protein) and represent the means ± SEM for quadruplicate determinations, which were repeated twice. The data were assessed by one-way ANOVA followed by the Tukey-Kramer multiple comparisons post hoc test. Spearman rank correlation analysis was used to assess the relation between COX-2associated fluorescence intensity and that of mPGES-1 in a representative population of cells. Simple linear regression was used to model the relationship between these two variables. A p value of less than 0.05 was considered significant.
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Results |
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Inhibition of mPGES-1 Attenuates Cytokine-induced PGE2 Production
To investigate the contribution of COX-2 and mPGES-1 to cytokine-induced PGE2 production, cells were stimulated with IL-1ß (10 ng/ml) in the presence or absence of indomethacin (a nonselective COX inhibitor; Cayman Chemical), NS-398 (a COX-2 selective inhibitor; Cayman Chemical), or MK-886 (an inhibitor of mPGES-1 that also inhibits other membrane associated proteins involved in eicosanoid and glutathione metabolism proteins such as leukotriene C4 synthase [LTC4S] and 5-lipoxygenase activating protein [FLAP]; BIOMOL Research Laboratories, Plymouth Meeting, PA) (Mancini et al. 2001). Challenge with IL-1ß caused a 7-fold increase in PGE2 production relative to control cells (Figure 2, p<0.0001). Consistent with our previous work (Perkins and Kniss 1997a
), synthesis of PGE2 was completely attenuated when stimulated with IL-1ß in the presence of 1 µM of indomethacin or 1 µM of NS-398 (both p<0.0001). MK-886 attenuated IL-1ßinduced PGE2 synthesis in a dose-dependent manner. At doses that inhibit mPGES-1 activity in vitro by
70% (6.25 µM) or
85% (12.5 µM) (Mancini et al. 2001
), MK-886 reduced PGE2 production by 40% (p<0.01) and 49% (p<0.001), respectively. At higher doses (2550 µM), which completely inhibit mPGES-1 in vitro (Mancini et al. 2001
; Kamei et al. 2003
), IL-1ß-induced PGE2 production was reduced to that observed in control cells (both p<0.0001). Treatment with vehicle alone (ethanol) did not affect IL-1ßinduced PGE2 production (not shown). No morphological evidence of cellular toxicity was evident at the doses of MK-886 tested. These results suggest that, despite the constitutive presence of cPGES and mPGES-2 in these cells (Ackerman and Kniss, unpublished data), cytokine-induced PGE2 production proceeds predominantly, although not exclusively, through a COX-2/mPGES-1 pathway.
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Spatial Segregation of Induced COX-2 and mPGES-1 Within Cells
Having established the intercellular expression pattern for COX-2 and mPGES-1, we next evaluated the degree of intracellular colocalization of these proteins. In whole-mount specimens permeabilized with Triton X-100 and visualized by standard epifluorescence microscopy, examination of cells showing coordinate increases in both COX-2 and mPGES-1 immunoreactivity revealed apparent intracellular colocalization, most prominent in the perinuclear area (arrows in Figure 4C). Equivalent results were obtained when viewed by confocal microscopy (Figure 4D). Given that Triton X-100 might cause artifactual disruption of intracellular architecture, particularly when examining membrane-associated antigens (Goldenthal et al. 1985), we sought to confirm our initial observations in the presence of a second permeabilization agent (saponin). However, in the presence of saponin, the degree of apparent intracellular colocalization of COX-2 and mPGES-1 was less pronounced compared with specimens permeabilized with Triton X-100 when assessed either by standard epifluorescence (Figure 4G) or confocal microscopy (Figure 4H). Notably, in the presence of saponin, the pattern of mPGES-1like immunoreactivity was altered. Specifically, perinuclear mPGES-1 immunostaining was reduced compared with that seen after Triton X-100 treatment, whereas punctate cytoplasmic staining was increased (Figure 4F).
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Segregation of mPGES-1 and COX-2 within Intracellular Membrane Lipid Microdomains
According to the contemporary view of membrane organization, it is widely recognized that certain proteins may reside in, or be excluded from, specialized membrane microdomains (such as lipid rafts) within the context of the cellular environment (Munro 2003; Cohen et al. 2004
). In this model, cholesterol- or sphingolipid-enriched membrane microdomains may develop tightly packed, liquid-ordered phases that are distinct from the fluid-phase properties of the membrane in general (Munro 2003
; Zurzolo et al. 2003
). Experiments in our laboratory have indicated that WISH cells contain lipid microdomain-enriched caveolae-like structures, and express the lipid raft-associated caveolar protein, Cav-1 (Ackerman, Robinson, and Kniss, unpublished observations). Furthermore, recent studies have indicated that a proportion of catalytically active COX-2 may be localized to caveolae-like structures, and may interact with Cav-1 (Liou et al. 2000
, 2001
). Given the mutual exclusivity of mPGES-1 and COX-2 immunostaining within intracellular membranes, we hypothesized that segregation of COX-2, but not mPGES-1, to lipid microdomains might provide an explanation for these observations.
The relative enrichment in tightly packed cholesterol and sphingolipids imparts a resistance of lipid microdomains to extraction in cold Triton X-100 (Munro 2003; Cohen et al. 2004
). As a consequence of their low buoyant densities in sucrose gradients, lipid microdomain-enriched DRM fractions can be separated biochemically from other cellular components. To assess localization of COX-2 and mPGES-1 to such microdomains, DRMs from IL-1ßtreated WISH cells were prepared as described previously (Liou et al. 2000
, 2001
). As in previous studies, immunoreactive Cav-1 was associated with detergent-insoluble fractions (Figure 5A, fractions 26). The majority (56%) of immunoreactive Cav-1 was located within fractions 2 and 3 (containing
5% sucrose), whereas 43% was located within fractions 46 (containing
30% sucrose) (Figures 5A and 5B). Unexpectedly, 96% of immunoreactive COX-2 was located within the detergent-soluble fractions 79 (containing
40% sucrose) (Figures 5A and 5B). Equivalent results were obtained when COX-2 was induced using phorbol 12-myristate 13-acetate (data not shown), indicating that this observation was not restricted to the mode of induction.
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Discussion |
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A fascinating finding in our current work was the unexpected observation of cell-to-cell variability in induced expression of mPGES-1 and COX-2. Despite a modest positive correlation, coordinate increases in immunoreactive mPGES-1 and COX-2 occurred in only 23% of cells (Figures 3A and 3D). Such an observation cannot be appreciated using methods in which cell lysates are pooled (such as with Northern or Western blotting). Although common signaling cascades and transcription factors (such as nuclear factor-kappa B) contribute to upregulation of both enzymes (Catley et al. 2003), our data suggest that the overall regulatory control for each of these genes is distinct, which is consistent with a number of recent reports (reviewed in Murakami and Kudo 2004
). Our data additionally suggest that individual cells may contribute unequally to overall cytokine-induced PGE2 release, such that the most substantial PG production may occur in a limited number of cells. That this may be relevant in vivo is supported by our recent report of heterogeneous expression of COX-2 protein in human amnion epithelial cells from placental membranes in the setting of term human labor (Dunn-Albanese et al. 2004
).
The role of intracellular compartmentalization between COX isoforms and terminal PGE isomerases is not well-delineated, but has profound functional consequences. It has been suggested that the enzymes involved in the liberation and conversion of arachidonic acid to PGE2 (including COX-1, COX-2, phospholipase A2 isoforms, and certain terminal PG isomerases) might colocalize to the same subcellular organelles, which might facilitate coordination of their metabolic activities (Naraba et al. 1998; Murakami and Kudo 2004
). Our data indicate that, in WISH epithelial cells, COX-2 and mPGES-1 reside primarily within intracellular membranes, most probably within the endoplasmic reticulum (ER) and along the contiguous outer nuclear envelope. This has been corroborated by the finding of immunoreactive mPGES-1 and COX-2 in microsomal, but not cytosolic, fractions prepared from these cells (data not shown), which is consistent with previous reports (Jakobsson et al. 1999
; Murakami et al. 2000
). However, our data also indicate that discrete lipid microdomains may physically separate the two enzymes. In whole-cell immunocytochemical preparations permeabilized with Triton X-100, we found cytokine-induced COX-2 and mPGES-1 to be particularly concentrated (and apparently colocalized) within the perinuclear area (Figures 4C and 4D). Using saponin as alternate permeabilization agent, however, we noted that the degree of overlap between immunofluorescently stained COX-2 and mPGES-1 was significantly reduced (Figures 4G and 4H). Similar reagent-specific inconsistencies in antigen distribution have been noted previously (Goldenthal et al. 1985
), suggesting that standard permeabilization methods may be inadequate to characterize accurately the intracellular arrangement of some membrane-associated proteins. Using a higher resolution methodology employing semithin physical sections (which circumvents the need for postfixation detergent treatment), we discovered that COX-2/mPGES-1 were largely noncolocalized, even in the perinuclear area (Figures 4K and 4L). This indicates that the extent of overlap observed by conventional analysis might be overestimated, suggesting that a majority of COX-2 and mPGES-1 are not in immediate spatial proximity.
In further support of a topological separation between COX-2 and mPGES-1, we found that the majority (68%) of immunoreactive mPGES-1 partitioned with Cav-1enriched DRMs in which COX-2 was excluded (Figures 5A and 5B). Consequently, most intracellular COX-2 and mPGES-1 appear to be biochemically segregated, even when residing in the same subcellular compartment. The finding of COX-2 (together with other ER-associated membrane proteins, not shown) in soluble fractions constitutes an important negative control in these experiments, because it excludes incomplete solubilization of bulk membranes as a trivial explanation for the finding of mPGES-1 in DRMs (Schuck et al. 2003). These results are in contrast to those described by Liou et al. (2001)
, who found that in human foreskin fibroblasts, a significant proportion of induced COX-2 was present in DRM fractions concomitantly with Cav-1. This suggests that COX-2/Cav-1 interactions may be cell typespecific.
Given that mPGES-1 partitioned into DRM fractions, it is tempting to speculate that this enzyme might reside in lipid rafts within the cell. According to the lipid raft model, cholesterol- and sphingolipid-enriched membrane microdomains may develop tightly packed, liquid-ordered phases that are distinct from the fluid-phase properties of the membrane in general (Munro 2003; Zurzolo et al. 2003
). Transmembrane proteins can reside in, or be excluded from, such lipid rafts depending on their physical characteristics (Munro 2003
). A widely used method to delineate protein content of lipid rafts is to extract membranes in cold Triton X-100, in which proteins associated with DRM fractions are not solubilized (Munro 2003
; Schuck et al. 2003
). As a consequence of their low buoyant densities, DRM fractions can be physically separated detergent soluble fractions by isopycnic centrifugation and analyzed for protein content by immunoblotting.
It is difficult to reconcile that mPGES-1 appears to localize to internal membranes while residing in largely in lipid microdomains. As traditionally conceived, lipid rafts are commonly present in the plasma membrane, but occur at low levels within most internal membranes (Munro 2003). However, given that the ER is the site of sterol production, and that high levels of sterols favor the development of liquid-ordered domains, it is conceivable that liquid-ordered domains may exist within ER membranes. Indeed, detergent-resistant ER lipid microdomains have been described, potentially associated with lipid body biogenesis (Hayashi and Su 2003
; Tauchi-Sato et al. 2002
). Hayashi and Su (2003)
noted that these ER-associated DRM microdomains exhibited lower buoyancy in Triton X-100 sucrose flotation compared with plasma membrane DRM fractions. Similar to their results, we observed that peak Cav-1 immunoreactivity (as a marker for plasma membrane rafts) occurred in a more buoyant fraction (fraction 3, Figures 5A and 5B) than that of mPGES-1 (fractions 46, Figures 5A and 5B), suggesting that mPGES-1 may partition into a similar ER-associated microdomains. We are attempting to define further these putative ER-associated lipid microdomains in terms of lipid composition, protein content, and sensitivity to cholesterol depletion, as well as through the use of more refined extraction methodologies. Although we are sensitive to the realization that there is controversy regarding extrapolating data derived from DRMs to preexisting structures within living cells (Munro 2003
; Zurzolo et al. 2003
), we believe our results are sufficiently compelling to warrant further investigation.
In light of our present findings, the mechanism for preferential coupling between COX-2 and mPGES-1 may be more dynamic and complex than would be anticipated based on a simple compartmentalization model. Our current fluorescence imaging and biochemical data suggest either that immediate proximity may not be required for COX-2/mPGES-1 functional coupling or that, if required, it may occur only transiently. The finding of coincident partitioning of a minority of mPGES-1 and essentially all of COX-2 into detergent-soluble membrane fractions suggests that transient mPGES-1/COX-2 interactions may occur within liquid-disordered internal membranes of a given cell. To date, however, no direct interactions between COX-2 and mPGES-1 have been documented. Additionally, we speculate that intracellular COX-2/mPGES-1 coupling may be but one of the mechanisms through which PGE2 biosynthesis occurs. Our current immunofluorescence data suggest that transcellular arachidonic acid metabolism might also occur in WISH, such that cells expressing high levels of COX-2 may donate metabolic intermediates (PGG2/H2) to neighboring cells enriched in mPGES-1. A precedent for such a scenario stems from observations that endothelial cell-derived PGH2 can be used as a substrate for thromboxane production by platelets (Karim et al. 1996; Camacho and Vila 2000
) or for PGI2 production by lymphocytes (Merhi-Soussi et al. 2000
).
In summary, our results suggest that synergistic activity between COX-2/mPGES-1 is the principal means through which cytokine-elicited PGE2 production proceeds within WISH epithelial cells. The mechanisms through which mPGES-1 and COX-2 may functionally couple remain incompletely defined. Our data suggest that the role of intracellular compartmentalization in COX-2/mPGES-1 interactions may be more complex than current models indicate. Future efforts will be directed at exploring these and other potential mechanisms for mPGES-1/COX-2 coupling.
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Acknowledgments |
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We gratefully acknowledge Dr. Toshihiro Takizawa, whose expert guidance was indispensable for the preparation of the semi-thin physical sections. We are indebted to Kathleen Wolken of the Campus Microscopy and Imaging Facility for her excellent technical assistance. We thank Drs. Eric J. Smart and William V. Everson of the University of Kentucky for critical review of this manuscript. Portions of this work were presented at the 50th Annual Meeting of the Society for Gynecologic Investigation, March 2630, 2003, Washington, DC, and the 10th Annual Meeting of the International Federation of Placenta Associations, September 2529, 2004, Asilomar, CA.
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Footnotes |
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