From the Departments of Biochemistry and Molecular
Biology and ¶ Pharmacology, Merck Frosst Centre for Therapeutic
Research, Kirkland, Quebec H9R 4P8, Canada
Received for publication, July 31, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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We have cloned and expressed the inducible form
of prostaglandin (PG) E synthase from rat and characterized its
regulation of expression in several tissues after in vivo
lipopoylsaccharide (LPS) challenge. The rat PGE synthase is 80%
identical to the human enzyme at the amino acid level and catalyzes the
conversion of PGH2 to PGE2 when overexpressed
in Chinese hamster ovary K1 (CHO-K1) cells. PGE synthase
activity was measured using [3H]PGH2 as
substrate and stannous chloride to terminate the reaction and convert
all unreacted unstable PGH2 to PGF2 Prostaglandin (PG)1
E2 is a major prostanoid derived from PGH2 that
can be generated by either degradation of PGH2 or by a reaction catalyzed by PGE synthase (1). PGH2 is formed by
the bis-oxygenation of arachidonic acid catalyzed by either isoform cyclooxygenase (COX)-1 or COX-2 and serves as the precursor to all
prostanoid products formed, including prostaglandins, prostacyclin, and
thromboxanes (2-4). Prostanoids have diverse biological functions including the maintenance of vascular and kidney homeostasis, relaxation and contraction of smooth muscle, regulation of
gastrointestinal secretion and motility, and induction of sleep, pain,
and inflammation (5). Within all these varied roles, data on COX-2
regulation of expression and the pharmacological effects of selective
COX-2 inhibitors have delineated that COX-2 is the major isoform
responsible for synthesis of inflammatory and pyretic prostanoids
(2-4, 6). In addition, studies with a specific monoclonal antibody to
PGE2 indicated that the major prostanoid that contributes
to inflammation is PGE2 (7). Therefore, it is compelling to
suggest that an inducible PGE synthase may provide a novel therapeutic
target for arthritis and pain downstream of COX-2 activity.
PGE synthase is a member of the membrane-associated proteins in
eicosanoid and glutathione metabolism (MAPEG) superfamily, which
consists of six human proteins with divergent functions (8). The
initial discovery of this family came to fruition through work on the
leukotriene pathway. Leukotrienes are derived from arachidonic acid
through the 5-lipoxygenase pathway (9) and act as potent mediators of
inflammation and bronchoconstriction (10). The initial discovery of the
MAPEG family was initiated upon the cloning of LTC4
synthase, which was found to be 31% identical to
5-lipoxygenase-activating protein (an arachidonate transfer protein
required for leukotriene biosynthesis) (11, 12). The search for new
members led to the discovery of three novel proteins (microsomal
glutathione transferases (MGSTs) 2 and 3 (13, 14) and PGE synthase) and
one preexisting enzyme (MGST1). Four members of this family can
conjugate glutathione to lipophilic substrates; however, one of these
enzymes, LTC4 synthase, conjugates glutathione specifically
to LTA4 to form the potent bronchoconstrictive leukotriene C4. The best-characterized member is MGST1, which is
involved in cellular detoxification of various xenobiotics (15). MGST2 and MGST3 can both conjugate glutathione to LTA4, whereas
only MGST2 can conjugate glutathione to the classical glutathione
transferase substrate 1-chloro-2,4-dinitrobenzene. Both MGST2 and MGST3
also possess a glutathione-dependent peroxidase activity
with hydroperoxy fatty acid substrates (14). The final member of this
family, PGE synthase, which has the highest sequence identity to
MGST1, could not conjugate glutathione to either LTA4 or
1-chloro-2,4-dinitrobenzene and, interestingly, was found to possess
PGE synthase activity (1).
The cDNA for human PGE synthase has recently been cloned, and the
enzyme has been shown to be inducible by IL-1 CHO-K1 cells were obtained from the American Type Culture
collection. Cell culture media, serum, antibiotics, and LipofectAMINE were purchased from Life Technologies, Inc. Oligonucleotides and a
polyclonal peptide antisera to human PGE synthase (1) were obtained
from Research Genetics (Huntsville, AL). Restriction enzymes, Pwo
polymerase, ligase, and Complete protease mixture were obtained from
Roche Molecular Biochemicals. PGH2,
[3H]PGH2, PGF2 Identification, Cloning, and Expression of Rat PGE
Synthase--
The human PGE synthase protein sequence was used to
perform a BLAST search of the GenBankTM expressed sequence
tag rodent data base. A rat expressed sequence tag was
identified with significant sequence identity to the human enzyme and
with the accession number AI136526. The expressed sequence tag clone
was obtained from Research Genetics and subcloned into the
EcoRI-NotI site of pcDNA 3.1 (Invitrogen).
The clone was sequenced using an Applied Biosystems 373A automated
sequencer and dye terminator reactions as described by the
manufacturer's instructions. The clone was full length and was
therefore transfected into CHO-K1 cells using LipofectAMINE 2000 (Life
Technologies, Inc.). Cells were harvested 24-48 h after transfection
and resuspended in 15 mM Tris-HCl, pH 8.0, 0.25 M sucrose, 0.1 mM EDTA, and 1 mM
glutathione. Resuspended cells were sonicated four times for 30 s
at 4 °C using a Cole Parmer 4710 Ultrasonic Homogenizer at 70% duty
cycle. Disrupted cells were subjected to centrifugation at 5,000 × g for 10 min, and the supernatant was further centrifuged at 100,000 × g for 1.5 h. The membrane pellet
obtained was resuspended in 10 mM potassium phosphate (pH
7.0), 20% glycerol, 0.1 mM EDTA, and 1 mM
glutathione. Both mock and human PGE synthase in pcDNA 3.1-transfected cells were prepared in a similar fashion. Protein concentrations were determined using the Coomassie protein assay (Pierce) as described by the manufacturer.
Immunoblot Analysis--
Protein samples were resolved by
SDS-polyacrylamide gel electrophoresis using 4-20% gradient gels
supplied by Novex (Invitrogen) and transferred electrophoretically to
polyvinylidene difluoride membranes using a Novex immunoblot transfer
apparatus according to the manufacturer's instructions. Nonspecific
sites on polyvinylidene difluoride membranes were blocked with 5%
nonfat dry milk in PBST for 1 h at room temperature, followed by
two 5-min washes with PBST. The polyvinylidene difluoride membrane was
probed with a 1:500-1:5,000 dilution of PGE synthase antisera in 1%
milk/PBST for 1 h. A polyclonal peptide antisera raised to the
synthetic peptide as described by Jakobsson et al. (1) or an
affinity-purified antibody purchased from Cayman Chemical Co. raised to
residues 59-75 of human PGE synthase was utilized for immunoblot
analysis. The blot was washed four times with PBST for 15 min each and
then incubated with a 1:3,000 dilution of horseradish peroxidase-linked anti-rabbit IgG antibody (Amersham Pharmacia Biotech) in 1% milk/PBST for 1 h. The blot was washed four times with PBST for 15 min
each, and immunodetection was performed using Renaissance
Western blot Chemiluminescence Reagent (PerkinElmer Life Sciences)
according to the manufacturer's instructions. Detection and
quantitative analysis were performed using a Fuji Film LAS-1000
charge-coupled device and Image gauge software.
In Vivo Induction of PGE Synthase--
All procedures used in
the in vivo assays were approved by the Animal Care
Committee at the Merck Frosst Center for Therapeutic Research
(Kirkland, Quebec, Canada) according to guidelines established by the
Canadian Council on Animal Care.
Harlan Sprague-Dawley rats were injected with a single i.v. bolus of
0.12 mg/kg LPS or saline vehicle control, and 7 h after infusion,
the rats were sacrificed, and tissues were perfused with saline and
dissected. The adjuvant-induced arthritis model was performed with two
groups of four Harlan Sprague-Dawley rats each and an intradermal
injection of 0.5 mg of Mycobacterium butyricum in mineral
oil into the left hind foot pad as described previously (17). The
tissues were flash-frozen in liquid nitrogen and used for RNA
preparation. mRNA was isolated from the tissues using the kit
reagents of the Fast Track 2.0 mRNA Isolation Kit (Invitrogen). RNA
concentration was quantified by spectrophotometry. mRNA (0.1 µg)
was reverse-transcribed into cDNA with random hexamers using kit
reagents and following the manufacturer's recommended conditions (GeneAmp RNA PCR Kit; PerkinElmer Life Sciences). The RT
reaction was incubated in a thermal cycler (GeneAmp PCR System 9600, Perkin Elmer Cetus) at 62 °C for 1 h and 94 °C for 5 min and
then cooled to 4 °C. Half of the reverse-transcribed cDNA
product (10 µl) was amplified by PCR in a 100 µl reaction. The
reaction contained 0.2 µM deoxynucleotide triphosphates,
5 units of Taq polymerase (Roche Molecular Biochemicals),
and either 0.3 µM primers (PGE synthase) or 0.2 µM primers ( PGE Synthase Assay--
Microsomal membranes from
mock-transfected CHO-K1 cells or rat PGE synthase-transfected
CHO-K1 cells were diluted into 0.1 M potassium phosphate,
pH 7.0, and 2.5 mM reduced glutathione. The reaction
was initiated with 10 µM PGH2 and 0.2 µCi
of [3H]PGH2 (100 µCi/mmol) and terminated
with an equal volume of acetonitrile/H2O/acetic acid
(35%:65%:0.1%) containing 1 mg/ml stannous chloride. Samples were
analyzed by reverse phase HPLC using a Waters Nova-Pak C18 column
(3.9 × 150 mm, 4 µm particle size) and a Waters 625 HPLC system
with a Beckman 171 radioisotope detector. Radiolabeled standards,
[3H]PGE2 (Amersham Pharmacia Biotech),
[3H]PGF2 Identification and Expression of Rat PGE Synthase--
The
GenBankTM expressed sequence tag data base was searched
using the sequence of the previously identified human PGE synthase, and
a clone with high sequence identity from rats was identified. The clone
was obtained, isolated, and sequenced (Fig.
1). The amino acid sequence of rat PGE
synthase is 80% identical to that of the human PGE synthase. The
divergences occur mainly at the N- and C-terminal regions of the
proteins. The sequence was subcloned into a pcDNA 3.1 vector, and
the recombinant construct was transfected into CHO-K1 cells. Membrane
preparations of recombinant protein were prepared from CHO-K1 cells
transfected with either mock vector, rat PGE synthase, or human PGE
synthase. The samples were subjected to SDS-polyacrylamide gel
electrophoresis and analyzed by immunoblot with peptide antisera to
human PGE synthase. The rat PGE synthase-transfected cells contained an
immunoreactive band of 17 kDa that comigrates with human PGE synthase,
whereas the mock-transfected cells (Fig. 2) showed no detectable signal.
The rat PGE synthase membrane preparation was tested for enzymatic
activity using [3H]PGH2 as substrate and
separation of the reaction products by reverse phase HPLC. The assay
was comprised of 10 µg/ml membrane protein in 100 mM
potassium phosphate, pH 7.0, and 2.5 mM glutathione and
initiated with 10 µM PGH2 and 0.2 µCi of
[3H]PGH2 (100 µCi/µmol). The reaction was
terminated with 1 mg/ml stannous chloride in HPLC running buffer to
quantitatively convert the remaining PGH2 substrate to
PGF2 Induction of Rat PGE Synthase with in Vivo LPS
Challenge--
Prostaglandin E2 levels have been shown to
be elevated upon LPS challenge in rat brain with a concomitant
induction of cyclooxygenase-2 (18). We designed a study to compare the
inducibility of PGE synthase, COX-2, and IL-1
Prostaglandins play an important role in the maintenance of the
integrity of the gastrointestinal mucosa. We have therefore analyzed
tissues of the GI tract from these vehicle- and LPS-treated rats (Fig.
5). In vehicle-treated animals, PGE synthase mRNA was detectable in
the stomach, but low or undetectable signals were seen in colon, ileum,
and jejunum. Upon LPS stimulation, significant up-regulation of PGE
synthase was only detected in the colon. An extra sample from
myeloproliferative tissue, the thymus, was included, and a slight
induction of PGE synthase was detected. COX-2 mRNA levels were
detected in the colon, ileum, jejunum, and stomach but were similar in
both vehicle- and LPS-treated animal tissues, whereas up-regulation in
the thymus was detected for COX-2 upon LPS challenge. IL-1 Induction of PGE Synthase in the Rat Adjuvant Arthritis
Model--
The rat adjuvant arthritis model is used extensively as a
pharmacological model of clinical arthritis and has a major
prostaglandin component. Rats injected with adjuvant will begin
demonstrating edema and hyperalgesia within several days of induction
of disease, and this process is diminished in the presence of
cyclooxygenase-2 inhibitors such as rofecoxib (6). We have utilized
this model to detect PGE synthase, and 5 days after adjuvant treatment,
a significant increase in the inducible PGE synthase was detected (Fig.
6). As seen in Fig. 6A, no PGE
synthase is detected in the naïve (vehicle-treated) rat
paw.
Quantitative Analysis of PGE Synthase Expression--
We have also
quantitated the RNA induction of PGE synthase and COX-2 in the tissues
from LPS-treated rats and adjuvant-treated paws. Normalizing for
Inhibition of PGE Synthase--
As described earlier, PGE synthase
is a member of the MAPEG family, which also contains
5-lipoxygenase-activating protein (FLAP) and LTC4 synthase.
We analyzed several inhibitors of FLAP such as MK-886 and the reaction
product of LTC4 synthase, and we found that
LTC4 and MK-886 inhibit rat PGE synthase with
IC50 values of 1.2 and 3.2 µM, respectively
(Fig. 8). In contrast, the cyclooxygenase
inhibitors indomethacin and acetaminophen were inactive up to
concentrations of 100 and 1000 µM, respectively, as
inhibitors of rat PGE synthase.
Induction of prostaglandin synthesis through the induction of the
COX-2 enzyme is widely accepted as the mechanism responsible for
prostanoid mediated pain, fever, and inflammation (2-6). The major
prostanoids implicated in these pathophysiological conditions are
PGI2 and PGE2. PGI2 has been
implicated because the knockout of the prostacyclin receptor
results in decreased paw swelling in the carageenan-induced acute
inflammatory model in mice (20). A monoclonal antibody to
PGE2 also demonstrates efficacy in a carageenan rat paw
model of inflammation (7). The role of the prostaglandin E
receptor is more difficult to define because there are four identified
receptors for PGE2 (5). One approach to assess the role of
PGE2 in various models would be to evaluate the regulation
of the enzyme directly involved in its synthesis, PGE synthase. We have
now cloned the rat enzyme and examined its induction along with COX-2
in the rat upon LPS challenge. Using a new assay for PGE synthase
activity in which the remaining PGH2 at the end of the
reaction is converted to PGF2 The coinduction of PGE synthase with COX-2 and IL-1 It is well established that nonsteroidal anti-inflammatory drugs
cause GI lesions, and GI ulceration is a major clinical side effect of
nonsteroidal anti-inflammatory drugs that nonselectively inhibit COX-1
and COX-2 (27-29). These GI side effects have been attributed to
prostanoids derived from COX-1, which cause alterations in mucosal
blood flow, and changes in mucous secretion and bicarbonate and tumor
necrosis factor PGE synthase is a member of the MAPEG family, which includes FLAP and
LTC4 synthase. Comparison of the hydropathy plots of these
three proteins demonstrates an identical putative membrane topography
for all three of these family members, although the sequence identity
of PGE synthase with FLAP and LTC4 synthase is less than
20% at the amino acid level. MK-886, which is a potent inhibitor of
leukotriene biosynthesis (IC50 = 100 nM (34)), was also found to inhibit PGE synthase with a moderate potency (IC50 = 3.2 µM). MK-886 is also a weak
inhibitor of LTC4 synthase with an IC50 of 11 µM (35). Interestingly as depicted in Fig. 9, the region of FLAP that is essential
for binding compounds such as MK-886 (36, 37) is highly conserved in
both LTC4 synthase and PGE synthase. In fact, the negative
charge of the aspartate or a glutamate at position 62 of FLAP is
essential for binding MK-886 analogues. MK-886 appears to inhibit
leukotriene biosynthesis by binding to an arachidonate binding site on
FLAP (38). The presence of a consensus amino acid sequence and
sensitivity to indole inhibitors of the MK-886 series for FLAP,
LTC4 synthase, and PGE synthase suggest that this region
might also be involved in the binding of eicosanoids for each of these
proteins. The motif ERXXXAXXNXXD/E
could represent a consensus sequence for interaction with arachidonic
acid and/or several of its oxygenation products.
before
high pressure liquid chromatography analysis. We assessed the induction of PGE synthase in tissues from Harlan Sprague-Dawley rats after LPS-induced pyresis in vivo. Rat PGE synthase was
up-regulated at the mRNA level in lung, colon, brain, heart,
testis, spleen, and seminal vesicles. Cyclooxygenase (COX)-2 and
interleukin 1
were also up-regulated in these tissues, although to
different extents than PGE synthase. PGE synthase and COX-2 were also
up-regulated to the greatest extent in a rat model of adjuvant-induced
arthritis. The RNA induction of PGE synthase in lung and the
adjuvant-treated paw correlated with a 3.8- and 16-fold induction of
protein seen in these tissues by immunoblot analysis. Because PGE
synthase is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family, of which leukotriene (LT) C4 synthase and 5-lipoxygenase-activating
protein are also members, we tested the effect of LTC4 and
the 5-lipoxygenase-activating protein inhibitor MK-886 on PGE synthase
activity. LTC4 and MK-886 were found to inhibit the
activity with IC50 values of 1.2 and 3.2 µM,
respectively. The results demonstrate that PGE synthase is up-regulated
in vivo after LPS or adjuvant administration and suggest
that this is a key enzyme involved in the formation of PGE2
in COX-2-mediated inflammatory and pyretic responses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in the lung carcinoma-derived A549 cell line (1). The corresponding rat sequence
has been cloned and shown to be up-regulated during
-amyloid treatment of rat brain (16). In the present study, we present the
cloning, expression, and demonstration of activity for rat PGE
synthase. In addition, to investigate the role that PGE synthase might
play in an inflammatory process, we have determined the inducibility
and tissue distribution of PGE synthase RNA in comparison with COX-2 in
LPS-induced pyresis in rats and adjuvant-treated rat paws. We also
present the first induction of PGE synthase at the protein level in the
latter two models of inflammation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
PGE2, PGD2, LTC4, and a partially
purified PGE synthase antibody were purchased from Cayman Chemical Co. (Ann Arbor, MI). Glutathione and stannous chloride were obtained from
Sigma and BDH, Inc., respectively. [32P]dCTP was
obtained from PerkinElmer Life Sciences, and the random prime kit for
generating radiolabeled cDNA was obtained from Amersham Pharmacia Biotech.
-actin, COX-2, and IL-1
). The PCR reaction was incubated at 94 °C for 5 min and then amplified for 25-50 cycles using the reaction conditions as follows: 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 1 min. After
amplification, the reactions were incubated at 72 °C for 7 min and then cooled to 4 °C. Synthetic DNA amplimers for PGE
synthase were as follows: sense, 5'-ATGACTTCCCTGGGTTTGGTGATGGAG-3'; and
antisense, 5'-ACAGATGGTGGGCCACTTCCCAGA-3'. Synthetic primers for
-actin were ordered from CLONTECH. Synthetic DNA
amplimers for COX-2 were as follows: sense,
5'-GACGATCAAGATAGTGATCGAA-3'; and antisense,
5'-AAGCGTTTGCGGTACTCATTG-3'. Synthetic DNA amplimers for IL-1
were
as follows: sense, 5'-GCACCTTCTTTTCCTTCATC-3'; and antisense,
5'-CTGATGTACCAGTTGGGGAA-3'. Reverse transcription-PCR products were
analyzed by 1% (w/v) agarose gel electrophoresis. PGE synthase RT-PCR
products were transferred to nitrocellulose membrane and analyzed by
Southern blot. The full-length PGE synthase cDNA was labeled with
[
-32P]dCTP using the T7QuickPrime Kit
(Amersham Pharmacia Biotech). Hybridization was performed in 5× SSC,
5× Denhardt's solution, 0.1% SDS, and 0.1 mg/ml denatured salmon
sperm DNA for 18 h at 42 °C. Blots were washed to a final
stringency of 0.5× SSC, 0.5% SDS at 65 °C followed by
autoradiography at
80 °C. cDNA from PGE synthase- or
COX-2-transformed bacteria was used as a template, with PGE synthase
and COX-2 primers respectively, and served as a positive control.
Comparative analysis between the amount of PCR product and the amount
of initial template demonstrated that PCR amplification was in the
linear range at the conditions utilized for each amplification.
Detection and quantitative analysis were performed using a Fuji Film
LAS-1000 charge-coupled device and Image gauge software.
(PerkinElmer Life Sciences), and
[3H]PGD2 (PerkinElmer Life Sciences) were
utilized for determining the separation by reverse phase HPLC and for
quantitation of product formation of PGE2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence and the predicted
translation of rat PGE synthase. The open reading frame of rat PGE
synthase is presented (GenBankTM accession number
AF280967). The cDNA sequence presented was subcloned into pcDNA
3.1 for expression in CHO-K1 cells.
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Fig. 2.
Immunoblot analysis of PGE synthase expressed
transiently in CHO cells. Rat and human PGE synthase
subcloned into pcDNA3.1 and vector alone were separately
transfected into CHO-K1 cells. Twenty-four h after transfection, cells
were harvested, and 100,000 × g membrane fractions
were prepared. Protein samples (5 µg/lane) were separated by
SDS-polyacrylamide gel electrophoresis on 4-20% gradient gels (Novex)
and subjected to immunoblotting with a polyclonal peptide antisera (1)
after electrophoretic transfer to polyvinylidene difluoride membrane.
Detection was performed using enhanced chemiluminescence (PerkinElmer
Life Sciences). The position of migration of molecular mass standards
(Life Technologies, Inc.) is depicted.
. In assays with mock preparations,
PGF2
was the predominant product, with low amounts of
PGE2 and PGD2. In contrast, rat PGE synthase
extracts formed primarily PGE2, with concomitant decreases
in PGF2
and PGD2. These results demonstrate
that this cloned sequence encodes for a protein with PGE synthase
activity. A time course of product formation was performed using the
same conditions mentioned above. The conversion of PGH2 to
PGE2 with rat PGE synthase was rapid, and the reaction
reached a plateau after 1 min, mainly due to substrate depletion (Fig.
3). Product accumulation obtained under
these conditions was maximal at 1 µmol PGE2/mg
protein/min. The time course of the reaction is similar to that
reported for human PGE synthase, with product formation for the rat
enzyme being 3.6-fold higher than the activity reported for the human microsomal enzyme preparation. The mock-transfected cells show a small
amount of PGH2 conversion to PGE2, which only
increases slightly with incubation time and corresponds mainly to a
nonenzymatic degradation of PGH2.
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Fig. 3.
Time course of product formation.
Membrane preparations of mock-transfected or rat PGE
synthase-transfected CHO cells were incubated with
[3H]PGH2 for various time periods (0-5 min).
The reaction was terminated with the addition of 1 mg/ml stannous
chloride in 35% acetonitrile and 0.1% acetic acid. The reaction
products were subjected to reverse phase HPLC as described under
"Materials and Methods." The retention times of the prostaglandin
products were verified using authentic
[3H]PGE2,
[3H]PGF2 , and
[3H]PGD2 standards.
in Harlan
Sprague-Dawley rats after LPS treatment. Harlan Sprague-Dawley rats
were treated with 0.12 mg/kg LPS by a single i.v. bolus, and 7 h
after challenge, various tissues were collected for analyses by
quantitative PCR. This dose of LPS causes a significant elevation of
body temperature of rats (from 36.4 ± 0.3 °C to 38.5 ± 0.2 °C). Characterization of eight major tissues from vehicle- or
LPS-treated rats by RT-PCR followed by Southern blot analysis
demonstrated that lung, brain, heart, spleen, and seminal vesicles
contain increased mRNA levels of PGE
synthase as compared with the vehicle-treated control animals (Figs. 4
and 5). RT-PCR was also performed for
COX-2 and IL-1
, and the results were compared with those for PGE
synthase in these tissues. COX-2 was also up-regulated in lung, brain,
and heart tissues, as seen for PGE synthase, with a weak and not
clearly detectable COX-2 induction in spleen and seminal vesicle. COX-2 was also present in non-LPS-treated rat brain, and this observation is
consistent with constitutive expression of COX-2 in this tissue (19).
IL-1
was significantly up-regulated in lung, spleen, and seminal
vesicle, and a slight induction was also detected in brain and heart.
The lung seems to be very responsive to LPS administration, with a
significant induction of PGE synthase, COX-2, and IL-1
mRNA.
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Fig. 4.
Induction of rat PGE synthase after in
vivo LPS challenge. Harlan Sprague-Dawley rats were
challenged with either saline vehicle control ( LPS) or a single i.v.
bolus of 0.12 mg/kg LPS (+LPS) and sacrificed 7 h after challenge.
Tissues were removed after saline perfusion, and mRNA templates
were isolated. RT-PCR amplification was performed, and the samples were
electrophoresed and transferred to nitrocellulose. The membrane was
probed for PGE synthase using a 32P-labeled cDNA
encoding the open reading frame of rat PGE synthase.
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Fig. 5.
Induction of PGE synthase, COX-2, and
IL-1 in LPS-challenged rat tissues.
Messenger RNA was prepared from tissues of control- or LPS-treated rats
as described in Fig. 4. RT-PCR was performed for PGE synthase, COX-2,
and IL-1
during the linear phase of amplification.
-Actin was
used as the control to compare non-LPS-treated and LPS-treated
rat tissues. The signal obtained is from RT-PCR samples separated on
1% agarose gels and stained with 0.5% ethidium bromide.
was
detectable in all LPS-treated tissues.
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Fig. 6.
Induction of PGE synthase in rat
adjuvant-induced arthritis. Paws from adjuvant-treated rats were
isolated and fresh frozen for analysis by RT-PCR. RT-PCR was performed
for PGE synthase, COX-2, and -actin (control) in both naïve
and adjuvant-treated paws (A). The signal obtained is from
RT-PCR samples separated on 1% agarose gels and stained with 0.5%
ethidium bromide. The edema of the naïve versus the
injected paw is presented for n = 4 rats at day 0 and
day 5 and is expressed as paw volume (B).
-actin expression, the lung and the adjuvant-treated paw had the
most significant induction of PGE synthase with 7- and 20-fold
increases, respectively, as compared with tissues from vehicle-treated
animals (Fig. 7A). COX-2 was
also elevated 2.6-fold in lung and 6.5-fold in the adjuvant-treated paw
(Fig. 7A). The remaining tissues that contained up-regulated
PGE synthase (Fig. 5) RNA (testes, spleen, seminal vesicles, colon, and
thymus) showed a 2- to 3-fold induction over non-LPS-exposed tissues, and the brain and heart contained a slightly higher increase of PGE
synthase (5- to 6-fold). COX-2 mRNA was induced 2- to 5.8-fold in
brain, heart, testes, spleen, and seminal vesicles, with the highest
induction (as a ratio of
-actin expression) observed in heart and
brain. Because RNA induction may not always correlate with protein
expression, we analyzed PGE synthase protein expression by immunoblot.
Protein expression was examined in lung tissue from LPS-treated animals
and adjuvant-treated paws (Fig. 7B) because these tissues
contained the highest induction and levels of PGE synthase mRNA
(Figs. 6 and 7A). The most significant protein induction detected was in the rat adjuvant-treated paw with a 16-fold increase of
PGE synthase protein as compared with the naïve paw. This is in
concordance with the 20-fold increase in mRNA (Fig. 7A) obtained from rat paws treated in a similar fashion. A 3.8-fold induction of PGE synthase protein was also detected in lung tissue (Fig. 7B) from LPS-treated animals, which is within 2-fold
of the RNA induction (7-fold) obtained from similar tissues (Fig. 7A). This is the first reported evidence of PGE synthase
protein in two major models of inflammation, LPS-induced pyresis and
adjuvant-induced arthritis.
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Fig. 7.
Quantitative analysis of PGE synthase
induction and protein expression in lung and in adjuvant-treated
paw. Amplification of RNA in the described tissues was performed
using RT-PCR. Various cycles of PCR were performed to quantitate
(A) the signal during a linear part of the PCR amplification
curve. The results for PGE synthase (n = 2) and COX-2
(n = 1) induction in lung are presented and demonstrate
a 7- and 2.6-fold induction, respectively. The induction in the
adjuvant-treated paw is 20-fold for PGE synthase (n = 2) and 6.5-fold for COX-2 (n = 2). These similar
tissues were analyzed for protein expression as shown (B).
Equal amounts of protein were electrophoresed on 10-20%
SDS-polyacrylamide gel electrophoresis, transferred
electrophoretically, and analyzed by immunoblot using an
affinity-purified PGE synthase polyclonal antibody (see "Materials
and Methods"). The increase in PGE synthase protein expression is
presented. PGE synthase standards are from a membrane preparation of
transfected CHO cells.
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Fig. 8.
Inhibition of PGE synthase by
LTC4 and MK-886. Rat PGE synthase (0.01 mg/ml) in 100 mM phosphate buffer, pH 7.0, was incubated with either
Me2SO or ethanol vehicle or various concentrations of
LTC4 (A) or MK-886 (B) at room
temperature for 15 min. The reaction was then initiated with 10 µM PGH2 and 0.2 µCi of
[3H]PGH2 for 2 min. The reaction was
terminated with the addition of 1 mg/ml stannous chloride in 35%
acetonitrile and 0.1% acetic acid. The reaction products were
subjected to reverse phase HPLC as described under "Materials and
Methods." The results are an average of duplicates and are plotted as
the percentage inhibition of PGE2 formation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
with stannous chloride,
rat PGE synthase was found to be active when expressed in CHO cells.
The rat PGE synthase is 80% identical to the human enzyme.
demonstrates
that in vivo this enzyme is up-regulated under
proinflammatory conditions such as LPS-induced pyresis and
adjuvant-induced arthritis. Interestingly, the highest induction of rat
PGE synthase was seen in the lung and in the adjuvant-treated paw.
Aerosilized PGE2 has been demonstrated to enhance
respiratory function when administered before antigen challenge (21),
and this increased PGE synthase may be a method of maintaining
significant oxygenation in the lungs for tissues undergoing damage
after LPS provocation. The significant induction of PGE synthase in
adjuvant-induced arthritis is consistent with the important role that
prostaglandins play in this inflammatory model (22, 23). Most striking
is the induction of PGE synthase protein in this model, which
correlates well with the mRNA induction. The protein
detection in the paw and lung tissues and the similarity of induction
as compared with RNA levels suggest concordance of PGE synthase
mRNA expression with protein expression in these tissues. During
preparation of the manuscript for this article, another study
confirming the LPS-stimulated induction of PGE synthase has been
published (24). This recent study has also demonstrated preferential
coupling of inducible PGE synthase with COX-2 as opposed to COX-1. This further strengthens the role of PGE synthase as a therapeutic target
for inflammation. The kidney is also an important target tissue for
prostaglandins, and they play an important role in regulating renal
hemodynamics. We have not detected either COX-2 or PGE synthase in the
kidney, but this is not unexpected because COX-2 is localized mainly to
one specific region, the macula densa (25, 26). In situ
hybridization would be required for more precise determinations of
mRNA induction of COX-2 and PGE synthase in the kidney and other
tissues that express these enzymes in localized regions.
production. The importance of prostaglandins as
cytoprotectants in the GI tract has been described previously (30). PGE
synthase is constitutively expressed in the stomach, and its role in
cytoprotection and its relationship to COX-1 and COX-2 in GI tissues
remain to be investigated. Also, because COX-2 has been implicated to
be a mediator of colonic tumors (31), the induction of PGE synthase in
this tissue and its link to COX-2 provide impetus to examine its
expression in colon tumors as compared with normal colonic epithelium
(32). The constitutive mRNA expression of COX-2 in several GI
tissues is in contrast to the undetectable level of COX-2 protein (33)
in these tissues, and this may result from induction during
manipulation of tissues, or it may suggest tight translational control
of protein expression.
View larger version (19K):
[in a new window]
Fig. 9.
Alignment of a putative substrate and
inhibitor binding site of FLAP, LTC4 synthase, and PGE
synthase. The amino acid region 50-62 for human FLAP is an
essential region for binding leukotriene inhibitors such as
MK-886. The identity in this region is compared with LTC4
synthase and PGE synthase. Gray shading represents identical
residues, and black shading represents conserved
residues.
The EP3 knockout has elegantly demonstrated that this
receptor, along with PGE2, is the major mechanism of LPS-
and IL-1-induced pyresis (39). The induction of PGE synthase in the
brain during LPS-induced pyresis and in the paw in adjuvant-induced
arthritis suggests that this enzyme may have an important function in
the initiation of pyresis, pain, and inflammation. The development of
selective inhibitors of PGE synthase and a mouse deletion of this gene
will provide substantial input in the role of PGE2 as compared with other prostanoids in the initiation of inflammatory responses.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P. O. Box 1005, Pointe-Claire/Dorval, Quebec H9R 4P8, Canada. Tel.: 514-428-3167; Fax: 514-428-4930; E-mail: joseph_mancini@merck.com.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M006865200
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ABBREVIATIONS |
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The abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; FLAP, 5-lipoxygenase-activating protein; GI, gastrointestinal; LT, leukotriene; LPS, lipopolysaccharide; HPLC, high pressure liquid chromatography; IL, interleukin; MGST, microsomal glutathione transferase; PBST, Dulbecco's phoshate-buffered saline, 0.05% Tween 20; RT, reverse transcription; PCR, polymerase chain reaction; CHO, Chinese hamster ovary.
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REFERENCES |
---|
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---|
1. |
Jakobsson, P. J.,
Thoren, S.,
Morgenstern, R.,
and Samuelsson, B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7220-7225 |
2. | Smith, W. L., and Marnett, L. J. (1991) Biochim. Biophys. Acta 1083, 1-17[Medline] [Order article via Infotrieve] |
3. | Vane, J. R., and Botting, R. M. (1995) Inflamm. Res. 44, 1-10[Medline] [Order article via Infotrieve] |
4. | Herschman, H. R. (1996) Biochim. Biophys. Acta 1299, 125-140[Medline] [Order article via Infotrieve] |
5. |
Narumiya, S.,
Sugimoto, Y.,
and Ushikubi, F.
(1999)
Physiol. Rev.
79,
1193-1226 |
6. |
Chan, C. C.,
Boyce, S.,
Brideau, C.,
Charleson, S.,
Cromlish, W.,
Ethier, D.,
Evans, J.,
Ford-Hutchinson, A. W.,
Forrest, M. J.,
Gauthier, J. Y.,
Gordon, R.,
Gresser, M.,
Guay, J.,
Kargman, S.,
Kennedy, B.,
Leblanc, Y.,
Leger, S.,
Mancini, J.,
O'Neill, G. P.,
Ouellet, M.,
Patrick, D.,
Percival, M. D.,
Perrier, H.,
Prasit, P.,
Rodger, I.,
Tagari, P.,
Therien, M.,
Vickers, P.,
Visco, D.,
Wang, Z.,
Webb, J.,
Wong, E.,
Xu, L.-J.,
Young, R. N.,
Zamboni, R.,
and Riendeau, D.
(1999)
J. Pharmacol. Exp. Ther.
290,
551-560 |
7. | Portanova, J. P., Zhang, Y., Anderson, G. D., Hauser, S. D., Masferrer, J. L., Seibert, K., Gregory, S. A., and Isakson, P. C. (1996) J. Exp. Med. 184, 883-891[Abstract] |
8. | Jakobsson, P. J., Morgenstern, R., Mancini, J., Ford-Hutchinson, A., and Persson, B. (1999) Protein Sci. 8, 689-692[Abstract] |
9. | Samuelsson, B. (1983) Science 220, 568-575[Medline] [Order article via Infotrieve] |
10. |
Leff, J. A.,
Busse, W. W.,
Pearlman, D.,
Bronsky, E. A.,
Kemp, J.,
Hendeles, L.,
Dockhorn, R.,
Kundu, S.,
Zhang, J.,
Seidenberg, B. C.,
and Reiss, T. F.
(1998)
N. Engl. J. Med.
339,
147-152 |
11. |
Welsch, D. J.,
Creely, D. P.,
Hauser, S. D.,
Mathis, K. J.,
Krivi, G. G.,
and Isakson, P. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9745-9749 |
12. | Mancini, J. A., Abramovitz, M., Cox, M. E., Wong, E., Charleson, S., Perrier, H., Wang, Z., Prasit, P., and Vickers, P. J. (1993) FEBS Lett. 318, 277-281[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Jakobsson, P. J.,
Mancini, J. A.,
and Ford-Hutchinson, A. W.
(1996)
J. Biol. Chem.
271,
22203-22210 |
14. |
Jakobsson, P. J.,
Mancini, J. A.,
Riendeau, D.,
and Ford-Hutchinson, A. W.
(1997)
J. Biol. Chem.
272,
22934-22939 |
15. | Morgenstern, R., Guthenberg, C., and Depierre, J. W. (1982) Eur. J. Biochem. 128, 243-248[Abstract] |
16. | Satoh, K., Nagano, Y., Shimomura, C., Suzuki, N., Saeki, Y., and Yokota, H. (2000) Neurosci. Lett. 283, 221-223[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Fletcher, D. S.,
Widmer, W. R.,
Luell, S.,
Christen, A.,
Orevillo, C.,
Shah, S.,
and Visco, D.
(1998)
J. Pharmacol. Exp. Ther.
284,
714-721 |
18. |
Cao, C.,
Matsumura, K.,
Yamagata, K.,
and Watanabe, Y.
(1997)
Am. J. Physiol.
272,
R1712-R1725 |
19. | Yamagata, K., Andreasson, K. I., Kaufmann, W. E., Barnes, C. A., and Worley, P. F. (1993) Neuron 11, 371-386[Medline] [Order article via Infotrieve] |
20. | Murata, T., Ushikubi, F., Matsuoka, T., Hirata, M., Yamasaki, A., Sugimoto, Y., Ichikawa, A., Aze, Y., Tanaka, T., Yoshida, N., Ueno, A., Oh-ishi, S., and Narumiya, S. (1997) Nature 388, 678-682[CrossRef][Medline] [Order article via Infotrieve] |
21. | Pavord, I. D., Wong, C. S., Williams, J., and Tattersfield, A. E. (1993) Am. Rev. Respir. Dis. 148, 87-90[Medline] [Order article via Infotrieve] |
22. |
Philippe, L.,
Gegout-Pottie, P.,
Guingamp, C.,
Bordji, K.,
Terlain, B.,
Netter, P.,
and Gillet, P.
(1997)
Am. J. Physiol.
273,
R1550-R1556 |
23. | Mukherjee, A., Hale, V. G., Borga, O., and Stein, R. (1996) Inflamm. Res. 45, 531-540[Medline] [Order article via Infotrieve] |
24. |
Murakami, M.,
Naraba, H.,
Tanioka, T.,
Semmyo, N.,
Nakatani, Y.,
Kojima, F.,
Ikeda, T.,
Fueki, M.,
Ueno, A.,
Oh-Ishi, S.,
and Kudo, I.
(2000)
J. Biol. Chem.
275,
32783-32792 |
25. | Harris, R. C., McKanna, J. A., Akai, Y., Jacobson, H. R., Dubois, R. N., and Breyer, M. D. (1994) J. Clin. Invest. 94, 2504-2510[Medline] [Order article via Infotrieve] |
26. | Nantel, F., Meadows, E., Denis, D., Connolly, B., Metters, K. M., and Giaid, A. (1999) FEBS Lett. 457, 475-477[CrossRef][Medline] [Order article via Infotrieve] |
27. | Allison, M. C., Howatson, A. G., Torrance, C. J., Lee, F. D., and Russell, R. I. (1992) N. Engl. J. Med. 327, 749-754[Abstract] |
28. | Langman, M. J., Weil, J., Wainwright, P., Lawson, D. H., Rawlins, M. D., Logan, R. F., Murphy, M., Vessey, M. P., and Colin-Jones, D. G. (1994) Lancet 343, 1075-1078[Medline] [Order article via Infotrieve] |
29. | Traversa, G., Walker, A. M., Ippolito, F. M., Caffari, B., Capurso, L., Dezi, A., Koch, M., Maggini, M., Alegiani, S. S., and Raschetti, R. (1995) Epidemiology 6, 49-54[Medline] [Order article via Infotrieve] |
30. | Goldstein, J. L., Larson, L. R., and Yamashita, B. D. (1998) Am. J. Manag. Care 4, 687-697[Medline] [Order article via Infotrieve] |
31. | Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trzaskos, J. M., Evans, J. F., and Taketo, M. M. (1996) Cell 87, 803-809[Medline] [Order article via Infotrieve] |
32. | Kargman, S. L., O'Neill, G. P., Vickers, P. J., Evans, J. F., Mancini, J. A., and Jothy, S. (1995) Cancer Res. 55, 2556-2559[Abstract] |
33. | Kargman, S., Charleson, S., Cartwright, M., Frank, J., Riendeau, D., Mancini, J., Evans, J., and O'Neill, G. (1996) Gastroenterology 111, 445-454[Medline] [Order article via Infotrieve] |
34. |
Rouzer, C. A.,
Ford-Hutchinson, A. W.,
Morton, H. E.,
and Gillard, J. W.
(1990)
J. Biol. Chem.
265,
1436-1442 |
35. | Gupta, N., Nicholson, D. W., and Ford-Hutchinson, A. W. (1997) Can. J. Physiol. Pharmacol. 75, 1212-1219[CrossRef][Medline] [Order article via Infotrieve] |
36. | Vickers, P. J., Adam, M., Charleson, S., Coppolino, M. G., Evans, J. F., and Mancini, J. A. (1992) Mol. Pharmacol. 42, 94-102[Abstract] |
37. | Mancini, J. A., Coppolino, M. G., Klassen, J. H., Charleson, S., and Vickers, P. J. (1994) Life Sci. 54, PL137-PL142[Medline] [Order article via Infotrieve] |
38. |
Mancini, J. A.,
Waterman, H.,
and Riendeau, D.
(1998)
J. Biol. Chem.
273,
32842-32847 |
39. | Ushikubi, F., Segi, E., Sugimoto, Y., Murata, T., Matsuoka, T., Kobayashi, T., Hizaki, H., Tuboi, K., Katsuyama, M., Ichikawa, A., Tanaka, T., Yoshida, N., and Narumiya, S. (1998) Nature 395, 281-284[CrossRef][Medline] [Order article via Infotrieve] |