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Divisions of
*
Rheumatology and
Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI 48109; and
Division of Chemistry II, Karolinska Institutet, MBB, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or TNF-
treatment induces
mPGES mRNA from very low levels at baseline to maximum levels at
24 h. IL-1-induced mPGES mRNA was inhibited by dexamethasone in
a dose-dependent fashion. Western blot analysis demonstrated that mPGES
protein was induced by IL-1, and maximum expression was sustained
for up to 72 h. There was a coordinated up-regulation of
cyclooxygenase-2 protein, although peak expression was earlier.
Differential Western blot analysis of the microsomal and the cytosolic
fractions revealed that the induced expression of mPGES protein was
limited to the microsomal fraction. The detected mPGES protein was
catalytically functional as indicated by a 3-fold increase of PGES
activity in synoviocytes following treatment with IL-1; this
increased synthase activity was limited to the microsomal fraction. In
summary, these data demonstrate an induction of mPGES in rheumatoid
synoviocytes by proinflammatory cytokines. This novel pathway may be a
target for therapeutic intervention for patients with
arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the stable PGs produced in rheumatoid synovia, PGE2 plays an important role. Injection of PGE2 recapitulates the cardinal signs of inflammation via vasodilation with plasma extravasation and sensitization of nociceptors (10). Furthermore, PGE2 stimulates production of matrix metalloproteinases (11) and angiogenesis (12) and inhibits apoptosis of T lymphocytes (13). PGE2 is specifically implicated in the symptoms of arthritis because neutralizing Abs against PGE2 are able to inhibit acute and chronic inflammation in the rat adjuvant arthritis model (14). Previous data point to a selective induction of PGE2 synthesis in inflammation (15, 16, 17, 18), suggesting that a PGE synthase (PGES) may be linked to the inducible pathway for PG synthesis.
Until recently, the ability to study regulation of terminal synthase enzymes was hampered by the fact that the PGES enzyme(s) had not been purified. However, two forms of the terminal synthase involved in production of PGE2 were recently cloned and characterized (19, 20, 21, 22). Similar to the more proximal PG synthetic enzymes, one isoform is constitutively expressed and unresponsive to proinflammatory stimuli, whereas the other isoform is inducible. The constitutive PGES is expressed in the cytosol (cPGES) under basal conditions in a wide variety of mammalian cell lines and rat tissues (21). Expression is generally unaltered by stimulation with bacterial LPS.
The inducible PGES is localized to the microsomal compartment and hence
termed mPGES (19, 22). The enzyme was identified as a
member of the membrane-associated proteins involved in eicosanoid and
glutathione metabolism family of enzymes originally called
microsomal glutathione S-transferase 1-like 1 (23). The
same sequence was also identified as a gene induced by p53
(24). Jakobsson et al. characterized this enzyme as a
human PGES that was dependent on glutathione for its activity and, more
important, inducible by IL-1 in human cells (19, 25).
Subsequently, Murakami et al. reported that induced PGES activity
previously identified in LPS-stimulated rat macrophages actually
originated from this enzyme (22). Rat mPGES exhibited a
high degree of sequence homology to the human mPGES, and a mouse
homologue was also cloned (22). Thorén and Jakobsson
described coordinate regulation of mPGES and COX-2 in a human lung
cancer cell line (25). Using cotransfection experiments,
functional coupling between constitutive cPGES and COX-1 and inducible
mPGES and COX-2 has also been demonstrated (21, 22).
The concept of a regulated PG production pathway including both COX-2 and PGES has important implications for understanding inflammation in arthritis. mPGES has not previously been studied in any clinically relevant primary cell type, including synovial cells. We hypothesized that mPGES would be regulated coordinately with COX-2 by proinflammatory cytokines in synoviocytes isolated from patients with rheumatoid arthritis (RA). This pathway could lead to the marked increase in PGE2 observed in inflammatory arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial tissue specimens were obtained from patients with RA at the time of total joint replacement surgery. Tissues were minced and digested for 4 h with 4 mg/ml collagenase (type I; Worthington Biochemical, Freehold, NJ) in DMEM at 37°C in 5% CO2. The dissociated cells were plated in 75-ml flasks containing DMEM supplemented with 10% human AB serum (BioWhittaker, Walkersville, MD), 10% FBS (Life Technologies, Rockville, MD), penicillin (100 U/ml), and streptomycin (100 µg/ml). When they reached 95% confluency, they were harvested with trypsin/EDTA (Life Technologies) and passed 1:3 into 175-ml flasks.
Cells were used between the third and seventh passage. After medium
change to DMEM supplemented with 2% FBS, 1% glutamine, penicillin
(100 U/ml), and streptomycin (100 µg/ml), cultures were treated as
indicated in the figure legends using human recombinant IL-1 and
TNF- (Genzyme, Cambridge, MA, or R&D Systems, Minneapolis, MN) or
dexamethasone (Sigma, St. Louis, MO).
Determination of eicosanoid profile
Cells were labeled overnight with
3H-labeled arachidonic acid (AA) (1 µCi, sp.
act. 76–100 µCi/mmol; DuPont/New England Nuclear, Boston, MA).
Unincorporated label was removed by washing. Some cells were examined
under unstimulated conditions. Some cells were treated with
acetylsalicylic acid (ASA; aspirin) (1 mM) for 30 min to
irreversibly inhibit constitutive COX-1, washed extensively to remove
the ASA, then treated with IL-1 (1 ng/ml) for 6 h to induce
COX-2. All cells were then treated with the calcium ionophore A23187
(2.5 µM) 30 min before harvesting to stimulate activity of
PLA2.
Radiolabeled AA and eicosanoid metabolites were extracted from the medium using a Sep-Pak cartridge (Waters, Milford, MA). Lipid extracts were subjected to reversed-phase HPLC as previously described (26). Radioactivity was determined online using a Radiomatic Flo-One Beta Detector (Packard, Downers Grove, IL). PG products were identified by comparison with the elution of known standards.
To determine whether PGE2 production was
dependent on COX-2, synoviocytes were treated in the presence or
absence of IL-1 (1 ng/ml) or TNF- (10 ng/ml) with or without
aspirin (1 mM) or the selective COX-2 inhibitor NS-398 (3 µM) added
simultaneously. Medium was collected after 16 h and analyzed for
PGE2 by enzyme-linked immunoassay (Cayman
Chemicals, Ann Arbor, MI). Cells were lysed, and total protein was
determined by the Bradford method (Bio-Rad, Hercules, CA).
PGE2 levels were normalized to protein
concentration.
RNA preparation and analysis
Total RNA was prepared from synoviocytes using Tri-Reagent (Molecular Research Center, Cincinnatti, OH). For Northern blot analysis, 20 µg of total RNA was subjected to electrophoresis through a 1.5% agarose-4% formaldehyde gel and transferred onto a nylon membrane (Nytran; Schleicher & Schuell, Keene, NH). To generate a probe for mPGES, the human mPGES coding sequence of 792 bp (19) was amplified from the pT7T3D-Pac vector, purified, and 32P-labeled by the random prime method (Random Primed DNA Labeling Kit; Boehringer Mannheim, Mannheim, Germany). A probe for G3PDH was synthesized by random prime method from a cDNA fragment of human G3PDH generated by PCR using primers as described below. Membranes were hybridized in 50% formamide at 42°C for 24 h with 9 x 106 cpm/ml cDNA probe. Washing was performed twice for 15 min in 2x SSC, 0.1% SDS at room temperature and twice for 15 min in 0.1x SSC, 0.1% SDS at 65°C. The membranes were exposed to Kodak XAR film for 48 h at -80°C (Kodak, Rochester, NY).
The cDNA was prepared by reverse transcription of 5 µg total RNA in 50 µl containing 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 0.4 mM dNTPs, 2 µM random hexamer primers (Life Technologies), 0.1 U/µl RNase inhibitor (Life Technologies), and 8 U/µl Moloney murine leukemia virus reverse transcriptase (Life Technologies). Reaction mixtures were incubated at 22°C for 10 min, at 42°C for 30 min, and at 95°C for 5 min. Complementary DNA was diluted 10-fold and used for PCR.
The PCR was performed in 50 µl containing 5 µl cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 100 µM dNTPs, 0.05 U/µl Taq polymerase (Life Technologies), and MgCl2 in appropriate concentrations (1 mM for mPGES and prostacylin synthase (PGIS), 2 mM for COX-2 and G3PDH, and 2.5 mM for cPGES). The oligonucleotide primers used were as follows: for mPGES, sense 5'-ATGCCTGCCCACAGCCTG-3', and antisense, 5'-TCACAGGTGGCGGGCCGC-3'; for cPGES, sense, 5'-ATGCAGCCTGCTTCTGCA-3', and antisense 5'-TTACTCCAGATCTGGCAT-3'; for COX-2, sense, 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3', and antisense, 5'-AGATCATCTCTGCCTGAGTATCTT-3'; for PGIS, sense, 5'-GGAGCAAATGGCTGGAGAGTTAC-3', and antisense, 5'-ATCCGTCAGGGTTCAGGAATCG-3'; and for G3PDH, sense, 5'-CCACCCATGGCAAATTCCATGGCA-3', and antisense, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. Cycling conditions were as follows: denaturing at 94°C for 2 min; annealing at 70°C (mPGES and PGIS), 65°C (COX-2), 60°C (G3PDH), or 50°C (cPGES) for 1 min; extension at 72°C for 1 min; 25 cycles for G3PDH and 30 cycles for mPGES, COX-2, cPGES, and PGIS. All PCR conditions were adjusted to assure that product formation was on the linear portion of a cycle curve.
Immunoblot analysis
Synoviocytes were trypsinized, pelleted, washed in ice-cold phosphate-buffered saline, and resuspended in lysis buffer (10 mM HEPES (pH 7.9) at 4°C, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitors). Protein concentration of the lysates was determined using the Bradford method (Bio-Rad), and 40 µg of protein was separated by electrophoresis in a denaturing sodium dodecyl sulfate polyacrylamide gel and transferred to a nylon membrane (Polyvinylidene Difluoride-Plus; Micron Separations, Westborough, MA). Rabbit polyclonal antiserum against human PGES and PGES electrophoresis standard were prepared as described previously (19).
For immunoblot anaysis of cytosolic and microsomal fractions, frozen cell pellets were resuspended in 1.0 ml homogenization buffer consisting of 0.1 M potassium phosphate buffer (pH 7.4), 1x Complete Protease Inhibitor mixture (Boehringer Mannheim), and 0.25 M sucrose. Cells were sonicated three times for 20 s and subjected to differential centrifugation at 1,000 x g for 10 min, 10,000 x g for 15 min, and 170,000 x g for 1 h at 4°C. The cytosolic fractions were centrifuged once more at 170,000 x g. Fractions were resuspended in 100 µl homogenization buffer, and protein concentrations were determined (Bio-Rad). Electrophoresis and immunoblotting were performed as previously described (25).
PGES activity assay
To determine PGES activity in whole cells, primary RA
synoviocytes were cultured in six-well plates in complete medium. After
they reached confluence, cells were washed with 2 ml PBS, and the
medium was changed to DMEM supplemented with 2% FBS and 1 mM ASA to
inhibit enzymatic activity of endogenous COXs. Cells were treated with
IL-1 for 24 or 72 h, washed with 2 ml PBS, and medium was
exchanged. In some experiments, cells were pelleted and frozen in
liquid N2 for fractionation.
For analysis of PGES activity in intact synoviocytes, plates were placed on ice and 7.05 µg PGH2 (Cayman Chemicals) was added. The reaction was stopped by adding 100 µl of 400 mM FeCl2-4 mM citric acid at the times indicated in the figure legends. Incubation of cells with acetone for 5 min served as negative control. Measurement of PGE2 was performed by enzyme-linked immunoassay (Cayman Chemicals).
Evaluation of PGES activity in microsomal fractions was performed as previously described (25). In these experiments, PGE2 was measured by reversed-phase HPLC with UV detection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in primary
synoviocytes
Under basal conditions, RA synoviocytes produced low levels of PGs
from endogenous AA released by treatment with calcium ionophore, mostly
PGI2 (Fig. 1
). To
determine changes in the profile of stable PGs after induction of
COX-2, we treated synoviocytes with aspirin (1 mM) to inhibit
constitutive COX-1. Aspirin was removed, and cells were treated for
6 h with IL-1, which we have previously shown to be sufficient
to induce COX-2 protein (9). Levels of
PGI2 were increased, but there was a
proportionally greater induction of PGE2
(Fig. 1).
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or TNF-. These experiments were
performed three times using cells from three different RA patients.
Complete inhibition of PGE2 in conditioned medium
was seen in the aspirin- and NS-398-treated cells, demonstrating that
substrate for PGE2 production was generated by a
COX-2-dependent pathway. mPGES mRNA expression is induced by pro-inflammatory cytokines
A specific increase in PGE2 could be
explained by induction of a specific PGE synthase. To evaluate that
possibility, we examined expression of the inducible mPGES. Treatment
with IL-1 led to a marked increase of mPGES mRNA from very low
levels at baseline to maximum levels at 24 h, as shown by Northern
blot analysis (Fig. 2A). Levels were
increased by 4 h after treatment with IL-1, and high level mRNA
expression was sustained for up to 72 h after treatment. TNF-
treatment for 24 h also resulted in an enhanced mPGES mRNA signal,
but to a lesser degree than with IL-1 (Fig. 2B).
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and dexamethasone
As previously documented, COX-2 mRNA levels were up-regulated by
IL-1 (Fig. 3). IL-1-induced COX-2
and mPGES transcripts, and mRNA was inhibited by dexamethasone in a
dose-dependent fashion. cPGES is expressed under basal conditions, and
expression is not altered by IL-1 or glucocorticoids. PGIS
expression is also constitutive and unaffected by either treatment.
Expression of G3PDH is shown for comparison.
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mPGES and COX-2 protein expression followed the same pattern as
shown above for mRNA (Fig. 4). There was
an expected sharp increase in COX-2 protein after IL-1 treatment.
Peak expression of COX-2 was more rapid than for mPGES, and levels were
beginning to decline by 24 h after treatment (Fig. 4).
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).
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induces mPGES activity
The increase of mPGES protein was associated with a 3-fold
increase of PGES activity in intact primary synovioctyes (Fig. 6), thus indicating that the detected
mPGES protein is a catalytically functional PGES enzyme. Synovial cells
were cultured in the presence of aspirin to achieve irreversible
inhibition of both constitutive COX-1 and any COX-2 induced by IL-1.
Aspirin acetylation of COX prevents the entry of AA to the catalytic
site of the enzyme. Because endogenous PGH2
synthesis is blocked, we measure PGES activity by conversion of
exogenous PGH2 to PGE2.
Nonenzymatic degradation of PGH2 to
PGE2 is minimal in our experimental conditions,
as shown in the control wells without cells. In accordance with
localization of mPGES protein to the microsomes, we observed a
time-dependent increase in PGES activity in microsomal fractions of
IL-1-treated synoviocytes (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar to the COX enzymes, it appears that the PGES enzymes function
as a pair of isoforms (21, 22). The experiments presented
here demonstrate that the mPGES is the inducible isoform responsible
for specific up-regulation of PGE2 in response to
inflammatory stimuli in synovial tissues. In addition to increased mRNA
and protein expression, we show that endogenous PGES activity is
increased in intact cells after treatment with IL-1. Although both
cPGES and mPGES mRNA are present in synoviocytes, only mPGES expression
is regulated. Induction of PGES activity by IL-1 suggests the mPGES
is responsible. The observation that PGES activity is increased in the
microsomal fraction further implicates mPGES rather than cPGES as
mediating increased PGE2 production. Moreover, we
demonstrated that mPGES expression occurs in a pattern generally
similar to COX-2 with expression increased by proinflammatory cytokines
and blocked by corticosteroids. These observations suggest a functional
linkage between COX-2 and mPGES. This idea is supported by the finding
that specific COX-2 inhibitors reduce production of
PGE2 more than other stable PGs
(27). Finally, our results provide an additional target by
which glucocorticoids exert anti-inflammatory effects, because
dexamethasone completely suppressed the induction of mPGES by
IL-1.
Although COX-2 and mPGES expression vary similarly in response to the
same stimuli, there are differences in the specific timing for
induction. In keeping with the differences in the time course of COX-2
and mPGES expression after stimulation with IL-1, the promoter of
the mPGES gene lacks many of the elements usually associated with
cytokine-inducible genes (20). Examination of a 651-bp
promoter region revealed the presence of GC boxes, barbie boxes, and an
aryl hydrocarbon regulatory element but the mPGES promoter lacked a
TATAA box and other known transcriptional elements. In addition to
transcriptional stimulation by IL-1, promoter reporter constructs
demonstrated enhanced transcription in response to
2,3,7,8-tetrachlorodibenzo-p-dioxin, known to stimulate
transcription via aryl hydrocarbon regulatory elements. Transcription
was inhibited by phenobarbital, presumably acting via the barbie boxes
(20). COX-2 expression is regulated by posttranscriptional
mechanisms (28, 29). The 3' region of mPGES lacks the
AUUUA instability sequences found in the COX-2 gene, suggesting another
possible difference in regulatory mechanisms of the two enzymes
(20).
Similar to our findings, cytokine induction of mPGES in human A549 cells and rat macrophages was suppressed by dexamethasone (22, 25). Moreover, a functional linkage between mPGES and COX-2 was proven in transfected human embryonic kidney 293 cells, which stably expressed both enzymes. In those experiments, functional linkage could not be explained solely on the basis of subcellular localization because mPGES, COX-1, and COX-2 were all expressed in a perinuclear distribution. The basis for functional linkage remains unclear at the current time. However, mPGES is a member of a superfamily of enzymes involved in eicosanoid metabolism which includes 5-lipoxygenase-activating protein (FLAP). FLAP is involved in the transfer of AA to 5-lipoxygenase to achieve efficient leukotriene production (30). It is concievable that mPGES could facilitate interaction with COX-2 by similar mechanisms. It has been noted that transfection of FLAP into certain cell lines increases PGE2 synthesis and COX-2 expression (31).
Our findings have considerable clinical relevance for understanding and treating inflammatory arthritis. Although specific COX-2 inhibitors have decreased gastrointestinal toxicity compared with traditional nonsteroidal anti-inflammatory drugs, it is clear that there are adverse effects due to inhibition of COX-2 (2). For example, specific inhibition of COX-2 inhibits production of renal and systemic PGI2 (32, 33). Consistent with an important physiologic role for COX-2 in the kidney, specific COX-2 inhibition can lead to altered excretion of sodium, edema, and elevated blood pressure (34). In addition, specific inhibition of COX-2 alters the balance between platelet-derived thromboxane A2 and endothelial PGI2, leading to altered vascular tone and perhaps to an increased risk of thrombosis (35, 36). Thus, a more selective modulation of the prostanoid pathway is desirable. It is currently unclear how much of the inflammation in human arthritis is mediated by PGE2; indeed, mice with a targeted deletion of the prostacyclin receptor have reduced pain and inflammation (37). Nevertheless, these data suggest a novel pathway that may be a target for therapeutic intervention for patients with arthritis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leslie J. Crofford, Division of Rheumatology, University of Michigan, Room 5510E, Medical Science Research Building I, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0680. E-mail address: crofford{at}umich.edu
3 Abbreviations used in this paper: PLA2, phospholipase A2; COX, cyclooxygenase; PGES, PGE synthase; cPGES, cytosolic PGES; mPGES, inducible microsomal PGES; RA, rheumatoid arthritis; AA, arachidonic acid; ASA, acetylsalicyclic acid; PGIS, prostacyclin synthase; FLAP, 5-lipoxygenase-activating protein.
Received for publication December 28, 2000. Accepted for publication April 20, 2001.
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K. M. A. O'Reilly, R. P. Phipps, T. H. Thatcher, B. A. Graf, J. Van Kirk, and P. J. Sime Crystalline and amorphous silica differentially regulate the cyclooxygenase-prostaglandin pathway in pulmonary fibroblasts: implications for pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1010 - L1016. [Abstract] [Full Text] [PDF]
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C. E. Trebino, J. D. Eskra, T. S. Wachtmann, J. R. Perez, T. J. Carty, and L. P. Audoly Redirection of Eicosanoid Metabolism in mPGES-1-deficient Macrophages J. Biol. Chem., April 29, 2005; 280(17): 16579 - 16585. [Abstract] [Full Text] [PDF]
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D. Kamei, K. Yamakawa, Y. Takegoshi, M. Mikami-Nakanishi, Y. Nakatani, S. Oh-ishi, H. Yasui, Y. Azuma, N. Hirasawa, K. Ohuchi, et al. Reduced Pain Hypersensitivity and Inflammation in Mice Lacking Microsomal Prostaglandin E Synthase-1 J. Biol. Chem., August 6, 2004; 279(32): 33684 - 33695. [Abstract] [Full Text] [PDF]
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J. Guay, K. Bateman, R. Gordon, J. Mancini, and D. Riendeau Carrageenan-induced Paw Edema in Rat Elicits a Predominant Prostaglandin E2 (PGE2) Response in the Central Nervous System Associated with the Induction of Microsomal PGE2 Synthase-1 J. Biol. Chem., June 4, 2004; 279(23): 24866 - 24872. [Abstract] [Full Text] [PDF]
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L. Boulet, M. Ouellet, K. P. Bateman, D. Ethier, M. D. Percival, D. Riendeau, J. A. Mancini, and N. Methot Deletion of Microsomal Prostaglandin E2 (PGE2) Synthase-1 Reduces Inducible and Basal PGE2 Production and Alters the Gastric Prostanoid Profile J. Biol. Chem., May 28, 2004; 279(22): 23229 - 23237. [Abstract] [Full Text] [PDF]
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S. Cheng, H. Afif, J. Martel-Pelletier, J.-P. Pelletier, X. Li, K. Farrajota, M. Lavigne, and H. Fahmi Activation of Peroxisome Proliferator-activated Receptor {gamma} Inhibits Interleukin-1{beta}-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1 J. Biol. Chem., May 21, 2004; 279(21): 22057 - 22065. [Abstract] [Full Text] [PDF]
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K. Subbaramaiah, K. Yoshimatsu, E. Scherl, K. M. Das, K. D. Glazier, D. Golijanin, R. A. Soslow, T. Tanabe, H. Naraba, and A. J. Dannenberg Microsomal Prostaglandin E Synthase-1 Is Overexpressed in Inflammatory Bowel Disease: EVIDENCE FOR INVOLVEMENT OF THE TRANSCRIPTION FACTOR Egr-1 J. Biol. Chem., March 26, 2004; 279(13): 12647 - 12658. [Abstract] [Full Text] [PDF]
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T J Jang, S K Min, J D Bae, K H Jung, J I Lee, J R Kim, and W S Ahn Expression of cyclooxygenase 2, microsomal prostaglandin E synthase 1, and EP receptors is increased in rat oesophageal squamous cell dysplasia and Barrett's metaplasia induced by duodenal contents reflux Gut, January 1, 2004; 53(1): 27 - 33. [Abstract] [Full Text] [PDF]
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K. Sun, R. Ma, X. Cui, B. Campos, R. Webster, D. Brockman, and L. Myatt Glucocorticoids Induce Cytosolic Phospholipase A2 and Prostaglandin H Synthase Type 2 But Not Microsomal Prostaglandin E Synthase (PGES) and Cytosolic PGES Expression in Cultured Primary Human Amnion Cells J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5564 - 5571. [Abstract] [Full Text] [PDF]
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M. Murakami, K. Nakashima, D. Kamei, S. Masuda, Y. Ishikawa, T. Ishii, Y. Ohmiya, K. Watanabe, and I. Kudo Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2 J. Biol. Chem., September 26, 2003; 278(39): 37937 - 37947. [Abstract] [Full Text] [PDF]
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C. E. Trebino, J. L. Stock, C. P. Gibbons, B. M. Naiman, T. S. Wachtmann, J. P. Umland, K. Pandher, J.-M. Lapointe, S. Saha, M. L. Roach, et al. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase PNAS, July 22, 2003; 100(15): 9044 - 9049. [Abstract] [Full Text] [PDF]
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S. Thoren, R. Weinander, S. Saha, C. Jegerschold, P. L. Pettersson, B. Samuelsson, H. Hebert, M. Hamberg, R. Morgenstern, and P.-J. Jakobsson Human Microsomal Prostaglandin E Synthase-1: PURIFICATION, FUNCTIONAL CHARACTERIZATION, AND PROJECTION STRUCTURE DETERMINATION J. Biol. Chem., June 13, 2003; 278(25): 22199 - 22209. [Abstract] [Full Text] [PDF]
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D. Kamei, M. Murakami, Y. Nakatani, Y. Ishikawa, T. Ishii, and I. Kudo Potential Role of Microsomal Prostaglandin E Synthase-1 in Tumorigenesis J. Biol. Chem., May 23, 2003; 278(21): 19396 - 19405. [Abstract] [Full Text] [PDF]
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J. W. Meadows, A. L. W. Eis, D. E. Brockman, and L. Myatt Expression and Localization of Prostaglandin E Synthase Isoforms in Human Fetal Membranes in Term and Preterm Labor J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 433 - 439. [Abstract] [Full Text] [PDF]
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A. I. Ivanov, R. S. Pero, A. C. Scheck, and A. A. Romanovsky Prostaglandin E2-synthesizing enzymes in fever: differential transcriptional regulation Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2002; 283(5): R1104 - R1117. [Abstract] [Full Text] [PDF]
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R. Han and T. J. Smith Cytoplasmic Prostaglandin E2 Synthase Is Dominantly Expressed in Cultured KAT-50 Thyrocytes, Cells That Express Constitutive Prostaglandin-endoperoxide H Synthase-2. BASIS FOR LOW PROSTAGLANDIN E2 PRODUCTION J. Biol. Chem., September 20, 2002; 277(39): 36897 - 36903. [Abstract] [Full Text] [PDF]
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H. Naraba, C. Yokoyama, N. Tago, M. Murakami, I. Kudo, M. Fueki, S. Oh-ishi, and T. Tanabe Transcriptional Regulation of the Membrane-associated Prostaglandin E2 Synthase Gene. ESSENTIAL ROLE OF THE TRANSCRIPTION FACTOR Egr-1 J. Biol. Chem., August 2, 2002; 277(32): 28601 - 28608. [Abstract] [Full Text] [PDF]
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M. Lazarus, C. J. Munday, N. Eguchi, S. Matsumoto, G. J. Killian, B. K. Kubata, and Y. Urade Immunohistochemical Localization of Microsomal PGE Synthase-1 and Cyclooxygenases in Male Mouse Reproductive Organs Endocrinology, June 1, 2002; 143(6): 2410 - 2419. [Abstract] [Full Text] [PDF]
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A. N. McEvoy, E. A. Murphy, T. Ponnio, O. M. Conneely, B. Bresnihan, O. FitzGerald, and E. P. Murphy Activation of Nuclear Orphan Receptor NURR1 Transcription by NF-{kappa}B and Cyclic Adenosine 5'-Monophosphate Response Element-Binding Protein in Rheumatoid Arthritis Synovial Tissue J. Immunol., March 15, 2002; 168(6): 2979 - 2987. [Abstract] [Full Text] [PDF]
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K. Yoshimatsu, D. Golijanin, P. B. Paty, R. A. Soslow, P.-J. Jakobsson, R. A. DeLellis, K. Subbaramaiah, and A. J. Dannenberg Inducible Microsomal Prostaglandin E Synthase Is Overexpressed in Colorectal Adenomas and Cancer Clin. Cancer Res., December 1, 2001; 7(12): 3971 - 3976. [Abstract] [Full Text] [PDF]
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)
were exposed to the calcium ionophore A23187 (2.5 µM). These cells
synthesize low levels of PGs, particularly PGI2, as
measured by 6-keto PGF1
), all PGs were more abundant. The
data are from four separate labeling experiments using cell lines from
two different patients with RA. The fold increase in PGE2
is greater than that of other PGs in all cell lines
(p < 0.05 by paired Student t
test).
