|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 51, 40028-40035, December 22, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the School of Pharmaceutical Sciences, Kitasato University,
5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Received for publication, April 14, 2000, and in revised form, September 27, 2000
Antigenic cross-linking of the high affinity IgE
receptors on mast cells induced the synthesis of prostaglandin
D2 (PGD2). The production of
PGD2 in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in
the control line of cells (S1 cells). The reduction in the formation of
PGD2 in L9 cells was reversed upon inhibition of PHGPx
activity by buthionine sulfoximine. Experiments with inhibitors
demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme
responsible for the production of PGD2 upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to
prostaglandin H2 (PGH2) was strongly inhibited
in L9 cells, whereas the rate of conversion of PGH2 to
PGD2 was the same in L9 cells and S1 cells, indicating that
PGHS was inactivated in L9 cells. The PGHS activity in L9 cells
was about half that in S1 cells. However, PGHS activity in L9 cells
increased to the level in S1 cells upon the addition of the
hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of
3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2
in nucleus and endoplasmic reticulum via reductions in levels of the
hydroperoxides that are required for full activation of PGHS.
Therefore, it appears that PHGPx might function as a modulator of the
production of prostanoids, in addition to its role as an antioxidant enzyme.
Reactive oxygen species
(ROS),1 such as the
superoxide anion, hydrogen peroxide, and hydroxy radicals, are
constantly formed in aerobic cells as a result of mitochondrial
respiration and reactions catalyzed by enzymes such as NADH/NADPH
oxidase, xanthine oxidase, monooxidases, and cyclooxygenase (1-4).
Thus, cellular DNA, proteins, and membrane lipids are continually
exposed to ROS and suffer from damage, such as DNA cleavage, protein
denaturation, and lipid peroxidation. The ROS-mediated damage to
intracellular molecules is limited by cellular antioxidant enzymes,
such as phospholipid hydroperoxide glutathione peroxidase (PHGPx),
classical glutathione peroxidase (cGPx), superoxide dismutase, and
catalase (5).
Prostaglandins are important mediators both in host defense mechanisms
and in inflammation. Their production is stimulated by low levels of
ROS via the activation of prostaglandin H synthase (PGHS) and/or
phospholipase A2 in intact cells (6-9). Prostaglandin D2 (PGD2) is one of the major products formed
in mast cells from arachidonic acid by PGHS in response to
pharmacological or physiological stimulation, such as treatment with a
calcium ionophore, oxidative stress, or cross-linking of IgE receptors
(10-14). The biosynthesis of PGD2 is initiated by the
release of arachidonic acid from membrane phospholipids. The liberated
arachidonic acid is oxidized to an unstable hydroperoxide intermediate,
prostaglandin G2 (PGG2), by cyclooxygenase reaction, and subsequent
reduction of C-15 hydroperoxide moiety of PGG2 yields
PGH2. These sequential reactions are catalyzed by PGHS, the
enzyme that is the primary enzymatic regulator of the biosynthesis of
prostanoids (15). The cyclooxygenase activity of PGHS requires reaction
of PGHS peroxidase with hydroperoxide to generate a tyrosyl radical
species, which is the active form of PGHS (16-18). Thus, it has been
proposed that the cellular synthesis of prostaglandin is regulated in
part by intracellular levels of hydroperoxide (26). Intracellular
levels of hydroperoxides can be regulated by enzymes such as
glutathione peroxidases (GPx).
Two types of GPx, cGPx and PHGPx, are distributed in various tissues
and cells. cGPx is present predominantly in the cytosol and some is
located in mitochondria (19). cGPx catalyzes the reduction of fatty
acid hydroperoxides and hydrogen peroxide using glutathione as a
cofactor. PHGPx is present in cytosolic, nuclear, and mitochondrial
fractions (19-21) and is the only known antioxidative enzyme that can
directly reduce peroxidized phospholipids (22), fatty acids (23), and
cholesterol (24) in membranes and lipoproteins. Although direct
evidence for the involvement of GPx in reduction of the hydroperoxide,
which is required for activation of the cyclooxygenase activity of
PGHS, is still lacking, it has been shown that elevation of glutathione
levels inhibits the production of prostaglandin in peritoneal
macrophages (25). Furthermore, the selenoorganic compound ebselen,
which reduces intracellular peroxides, decreases PGHS activity in
NIH3T3 fibroblasts (26). These results suggest that GPx might regulate
the activity of PGHS in the cellular level. Therefore, we wondered
whether cGPx or PHGPx might be responsible for the regulation of the
formation of prostaglandins. We are interested in the possible role of
PHGPx in the regulation of prostanoid synthesis since PHGPx can
interact with biomembrane, in which the synthesis of prostaglandins occurs.
We previously established stable transformants of rat basophile
leukemia 2H3 (RBL-2H3) cells, in which a short 20-kDa
(non-mitochondrial type) or a long 23-kDa form (mitochondrial
type) of PHGPx was overexpressed (19, 27). The amount of PHGPx in L9
cells, which overexpressed the short 20-kDa form of PHGPx, was
significantly elevated in the cytosol, microsomal, and nuclear
fractions as compared with that in mock-transfected cells (S1 cells),
but the amounts of PHGPx in mitochondrial fractions from L9 and S1
cells were similar. The amount of PHGPx in the mitochondrial fraction of M15 cells, which overexpressed the long 23-kDa form of PHGPx, was
twice that in the same fraction from L9 cells. These three lines of
cells provide a useful model system with which to attempt to clarify
the ability of PHGPx to modulate the synthesis of prostaglandin. Cells
that overexpressed non-mitochondrial PHGPx were resistant to cell death
due to oxidative damage (27). Moreover, production of leukotrienes,
catalyzed by 5-lipoxygenase, was markedly inhibited as a result of
decreased levels of intracellular hydroperoxides (21). By contrast, in
cells that overexpressed mitochondrial PHGPx, the generation of
intracellular hydroperoxides induced by mitochondrial damage was
suppressed, and the cells were more resistant than cells that
overexpressed non-mitochondrial PHGPx to cell death caused by direct
damage to mitochondria, to oxidative stress (19), and also to a
mediator of apoptosis that induced the liberation of cytochrome
c (28).
In the present study, we used RBL-2H3 cells that overexpressed either
non-mitochondrial or mitochondrial PHGPx to examine the functional
roles of two types of PHGPx in the formation of prostaglandins. RBL-2H3
cells produce PGD2 predominantly in response to stimulation
by antigenic cross-linking of the high affinity immunoglobulin E (IgE)
receptors or to calcium ionophores (29, 30). We show here that
non-mitochondrial PHGPx significantly suppressed the production of
PGD2 via prevention of increases in levels of
hydroperoxides in the nucleus and endoplasmic reticulum.
Reagents--
Antibodies, raised in rabbit, against PGHS-1 from
rat were a kind gift from Dr. M. Murakami (Showa University, Tokyo,
Japan). PGHS-2-specific mouse monoclonal antibodies,
5,6-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA) and
PGD2-MOX Enzyme Immunoassay Kit were obtained from
Funakoshi Co., Ltd. (Tokyo, Japan). [1-14C]Arachidonic
acid (2.22 GBq/mmol) was purchased from PerkinElmer Life Sciences.
Dinitrophenyl-human serum albumin (DNP-hSA), BSO, and A23187 were
obtained from Sigma. IgE against dinitrophenyl (IgE-DNP) was obtained
from Seikagaku Co., Ltd. (Tokyo, Japan). PGH2 (>98%) was
obtained from Wako Pure Chemical (Tokyo, Japan).
Cell Culture--
We used the previously established control
line of cells (S1 cells), L9 and L28 cells, which overexpressed
non-mitochondrial PHGPx (27), and M15 cells, which overexpressed
mitochondrial PHGPx (19). These lines of rat basophile leukemia
(RBL-2H3) cells were maintained at 37 °C in a humidified atmosphere
of 95% air and 5% CO2 in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum
(FCS), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. S1-Se(+) cells were grown
in DMEM supplemented with 250 nM sodium selenite, 5% FCS,
2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin for 2 weeks. Aliquots of 1 × 106
cells/ml were transferred to fresh medium when cells had reached semi-confluence.
Quantitation of PGD2 by Enzyme Immunosorbent
Assay--
RBL-2H3 cells were seeded at a density of 4 × 105 cells/ml into 6-cm diameter dishes and cultured for 2 days. After washing with phosphate-buffered saline (PBS), cells were
primed with IgE-DNP at a concentration of 1 µg/106 cells
in 2 ml of DMEM that contained 5% FCS for 2 h at 37 °C. After three washes with PBS, receptor cross-linking was effected by incubation with 10 µg/ml DNP-hSA for 1 h at 37 °C. The
incubation medium were collected and centrifuged at 10,000 × g for 5 min at 4 °C to pellet small numbers of dislodged
cells. Supernatants were prepared for quantitation of PGD2
with the PGD2-MOX Enzyme Immunoassay Kit.
Cells were treated with A23187 as described previously (13, 21).
RBL-2H3 cells (5 × 106 cells) were incubated in PBS
that contained 1 mM CaCl2 and 0.5 mM MgSO4 for 10 min at 37 °C and then
treated with 5 µM A23187 for 5 min. After centrifugation
at 10,000 × g for 5 min at 4 °C, supernatants were
prepared for enzyme immunosorbent assay.
Immunoblotting Analysis--
RBL-2H3 cells (5 × 106 cells) were activated by cross-linking their IgE
receptors, as described above, by sequential treatment with IgE-DNP and
DNP-hSA. Cells were incubated for 2 h with IgE-DNP (1 µg/106 cells), washed, and treated with DNP-hSA (10 µg/ml) for 1 h. The cells were harvested, washed with PBS, and
sonicated in PBS with a sonicator (20 s, 50 watts, model 5202; Ohtaka
Works, Tokyo). Proteins (30 µg) from sonicated cells were separated
by SDS-PAGE on a 10% polyacrylamide gel, as described by Laemmli (31),
and they were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane filter (Atto Instruments, Tokyo) at 2 mA/cm2 for 1 h in 100 mM Tris, 192 mM glycine, and 5% (v/v) methanol in a protein transfer
system (Atto Instruments), as described previously (13). The PVDF
membrane with the blotted proteins was blocked for 2 h by
incubation with 3% (w/v) defatted milk in 10 mM Tris-HCl
(pH 7.4) that contained 150 mM NaCl and 0.1% Tween 20 (TBS-T). The membrane was then incubated for 1.5 h with rabbit
polyclonal antibodies against PGHS-1 or mouse monoclonal antibodies
against PGHS-2 that had been diluted with TBS-T to an appropriate
concentration. After the PVDF membrane had been washed twice with
TBS-T, it was incubated with horseradish peroxidase-conjugated goat
antibodies against rabbit IgG or against mouse IgG (Zymed Laboratories Inc., South San Francisco, CA). The binding of
antibodies to antigens on the PVDF membrane was detected with an
enhanced chemiluminescence Western blotting analysis system (Amersham
Pharmacia Biotech).
Subcellular Fractionation of Cells--
RBL-2H3 cells (3 × 107 cells) were activated by cross-linking their IgE
receptors by sequential treatment with IgE-DNP and DNP-hSA. Cells were
washed twice with PBS and harvested. Cells were centrifuged at 700 × g for 5 min at room temperature. The obtained pellets
were suspended in 2 ml of sucrose buffer (0.25 M sucrose, 1 mM EDTA, 3 mM imidazole, and 0.1% (v/v)
ethanol that contained 10 µM leupeptin, 10 µM pepstatin A, 10 µM antipain, 10 µM chymostatin, and 100 µM
phenylmethylsulfonyl fluoride, pH 7.2) and homogenized in a
Teflon/glass Potter-Elvehjem homogenizer. Nuclear and microsomal
fractions were obtained by differential centrifugation according to the
previous method (19). Proteins (30 µg) from these fractions were used
for immunoblotting analysis.
The Formation of PGD2 from
[1-14C]Arachidonic Acid--
RBL-2H3 cells (2.5 × 106 cells) were incubated with 0.3 µM
[1-14C]arachidonic acid (3.7 kBq; 2.22 GBq/mmol) for
24 h at 37 °C. Labeled cells were washed three times with PBS
that contained 1 mg/ml fatty acid-free bovine serum albumin. The cells
were primed with IgE-DNP at a concentration of 1 µg/106
cells, and then receptors were cross-linked by the addition of DNP-hSA.
After incubation, all cells were placed on ice, and the incubation
medium was separated by centrifugation. Three volumes of ethyl acetate
(pH 3.0), supplemented with 0.1 ml of 0.1 M HCl, were added
to the incubation medium for extraction of metabolites. Each extract
was evaporated to dryness under reduced pressure, and the residue was
dissolved in a small amount of a mixture of chloroform and methanol
(2:1, v/v) and applied to a TLC plate (Silica Gel 60 F254;
Merck, Darmstadt, Germany). The plate was developed with organic layer
of a mixture of ethyl acetate, 2,2,4-trimethylpentane, acetic acid, and
H2O (110:50:20:100, v/v), as described by Green and
Samuelsson (32). The rate of production of each metabolite was
calculated from results of scanning densitometry after autoradiography with a Bio-Imaging analyzer (BAS2000; Fuji Film, Tokyo).
The Formation of PGD2 from Exogenously Added
Substrates--
RBL-2H3 cells (5 × 106 cells) were
incubated with IgE-DNP for 2 h and then treated with DNP-hSA for
1 h. After removal of the medium, cells were washed twice with PBS
and incubated in PBS that contained 1 mM CaCl2,
0.5 mM MgSO4, and 20 µM
[1-14C]arachidonic acid (3.7 kBq; 170.7 MBq/mmol) for 30 min. The supernatant was collected and supplemented with 3 volumes of
ethyl acetate (pH 3.0). The metabolites of labeled arachidonic acid
were extracted in the ethyl acetate layer. The extract was evaporated
to dryness under reduced pressure, and the residue was dissolved in a
small amount of a mixture of chloroform and methanol (2:1, v/v) and applied to a TLC plate. The plate was developed as described by Green
and Samuelsson (32). The rate of production of each metabolite was
calculated from results of scanning densitometry after autoradiography with the Bio-Imaging analyzer. The rate of production of
PGD2 from exogenously added prostaglandin H2
was determined as follows. RBL-2H3 cells, which had been stimulated by
cross-linking of IgE receptors, were incubated in PBS that contained 1 mM CaCl2, 0.5 mM MgSO4,
and 10 µM prostaglandin H2 for 30 min. The
incubation medium was collected, and the level of PGD2 was
determined by enzyme immunosorbent assay.
Assay of PGHS Activity--
RBL-2H3 cells (5 × 106 cells) were activated by cross-linking their IgE
receptors by sequential treatment with IgE-DNP and DNP-hSA. Then cells
(5 × 106 cells) were washed three times with PBS and
harvested with a cell scraper. The cells were suspended in sucrose
buffer (0.25 M sucrose, 1 mM EDTA, 3 mM imidazole, and 0.1% (v/v) ethanol that contained 10 µM leupeptin, 10 µM pepstatin A, and 100 µM phenylmethylsulfonyl fluoride, pH 7.2) and homogenized
in a Teflon/glass Potter-Elvehjem homogenizer. The enzymatic reaction
was performed in 100 µl of 0.1 M Tris-HCl (pH 8.0) that
contained 5 mM tryptophan, 20 µM hematin, 1 mM p-chloromercuriphenylsulfonic acid, and 30 µg of protein in a cell homogenate at 24 °C for 2 min after
addition of 40 µM [1-14C]arachidonic acid
(0.654 GBq/mmol), unless otherwise stated. The reaction was terminated
by addition of a mixture of diethyl ether, methanol, and 1 M citric acid (30:4:1, v/v), with centrifugation at
1,000 × g for 5 min at 4 °C. The diethyl ether
layer was applied to a TLC plate. The plate was developed with a
mixture of diethyl ether, methanol, and acetic acid (90:2:0.1, v/v).
The products were identified by comparison with chromatographic
standards. Enzyme activities were calculated from results of scanning
densitometry after autoradiography with the Bio-Imaging analyzer.
Assay of PHGPx Activity--
Cells (1.5 × 107
cells) were sonicated in 1 ml of 10 mM Tris-HCl buffer (pH
7.4) containing 5 mg/ml leupeptin and 17 mg/ml phenylmethylsulfonyl
fluoride. The homogenate was centrifuged at 10,000 × g
for 10 min at 4 °C, and the supernatant was used for assay. PHGPx
activity was determined by using phosphatidylcholine hydroperoxide
(PCOOH) as the substrate according to our previous paper (21).
Flow Cytometric Analysis of Intracellular Peroxides--
We used
an oxidant-sensitive fluorescent probe,
5,6-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA), to assess
levels of intracellular peroxides. After sequential treatment with
IgE-DNP and DNP-hSA, cells were washed with PBS and incubated with 5 µM DCFH-DA in PBS for 15 min. DCFH-DA was deacylated to
the non-fluorescent compound 2',7'-dichlorofluorescein (DCFH) within
the cells, and DCFH was oxidized to the fluorescent compound
2',7'-dichlorofluorescein by peroxides. The fluorescent intensity of
dichlorofluorescein in cells was analyzed with a flow cytometer
(EPICS® Elite Flow Cytometer; Coulter, Hialeah, FL).
Quantitation of Proteins--
Concentrations of proteins were
determined with Protein Assay Reagent (Bio-Rad) with bovine serum
albumin as the standard.
Production of Prostaglandin D2 in PHGPx-overexpressing
RBL-2H3 Cells--
The production of prostaglandins was examined in
mock-transfected cells (S1 cells) and in cells that overexpressed
non-mitochondrial PHGPx (L9 cells) by autoradiography after prior
labeling with radioactive arachidonic acid (Fig.
1A). S1 cells predominantly produced prostaglandin D2 (PGD2) upon
activation with IgE-DNP, whereas production of other types of
prostaglandin was not observed. Levels of PGD2 were
determined by enzyme immunosorbent assays after treatment of cells with
IgE-DNP (Fig. 1B). Quiescent RBL-2H3 cells continuously
produced PGD2 to yield a level of approximately 100 pg/106 cells. The level of PGD2 in S1 cells
activated by IgE-DNP was approximately three times that in
non-stimulated cells. The production of PGD2 was markedly
inhibited by the overexpression of non-mitochondrial PHGPx (in L9 and
L28 cells) (Fig. 1, A and B). Kinetic analysis of
IgE-DNP-activated S1 cells revealed that the level of PGD2 reached a maximum within 1 h, and then the level of
PGD2 declined gradually (Fig.
2). By contrast, synthesis of
PGD2 in L9 cells was suppressed during culture for 18 h with IgE-DNP. These results indicated that non-mitochondrial PHGPx
might be involved in regulation of the formation of PGD2 by
RBL-2H3 cells in response to IgE-DNP.
We examined the effects of buthionine sulfoximine (BSO) on the
production of PGD2 in order to determine whether or not
suppression of the production of PGD2 was due to the
overexpression of PHGPx (Table I). BSO
reduces the activity of GPx by lowering the level of glutathione in
cells. BSO decreases the level of glutathione to about 2% of that in
non-treated RBL-2H3 cells under our present conditions (13). The
production of PGD2 in S1 cells was moderately enhanced by
treatment with BSO. However, treatment with BSO caused a marked
increase in the production of PGD2 in L9 cells in response to activation by IgE-DNP. The suppression of formation of
PGD2 in L9 cells was abolished upon inhibition of PHGPx
activity, and the level of PGD2 recovered to 92% that in
S1 cells. These results indicated that the reduction in the rate of
production of PGD2 was indeed due to the induction of PHGPx
activity in RBL-2H3 cells.
S1 cells were cultured in DMEM containing 250 nM sodium
selenite for 2 weeks (S1-Se(+) cells). The
activity of endogenous PHGPx in S1-Se(+) cells
was significantly increased by the incubation with selenite. Specific
activities of PHGPx in cell lysate of S1 and
S1-Se(+) cells were 0.11 ± 0.02 and
0.38 ± 0.05 nmol PCOOH/min/mg, respectively. The production of
PGD2 in S1-Se(+) cells after
stimulation with IgE-DNP was 119.4 ± 50.1 pg
PGD2/106 cells, one-third of that in S1 cells.
Effects of Inhibitors of PGHS on Production of PGD2 in
RBL-2H3 Cells--
We attempted to identify the PGHS isozyme
responsible for production of PGD2 in IgE-DNP-stimulated
RBL-2H3 cells using inhibitors of PGHS (Fig.
3). The production of PGD2 in
response to IgE-DNP was abolished by treatment with NS-398, a
PGHS-2-specific inhibitor (Fig. 3A). Inhibition of
production of PGD2 was also observed after treatment with
aspirin (acetyl salicylate), which is a common inhibitor of both PGHS-1
and PGHS-2. No significant difference in the release of arachidonic
acid was observed between IgE-DNP-stimulated cells and cells treated
with the individual inhibitors (Fig. 3B).
Expression of PGHS-1 and PGHS-2 was monitored by immunoblotting
analysis with PGHS-1-specific polyclonal antibodies and PGHS-2-specific monoclonal antibodies, respectively (Fig.
4A). Both forms of PGHS in L9
cells were expressed at the same respective levels as those in S1
cells. Although expression of PGHS-1 was unchanged both in S1 and L9
cells after stimulation by IgE-DNP, the expression of PGHS-2 was
apparently enhanced in both S1 and L9 cells within 1 h by the
stimulation with IgE-DNP. There was no difference in the extent of the
increase in the level of expression between S1 and L9 cells. Expression
of PGHS-2 in response to activation by IgE-DNP was not sensitive to
depletion of intracellular glutathione by prior treatment with BSO
(Fig. 4B). These results indicated that inducible PGHS-2 was
responsible for the production of PGD2 in RBL-2H3 cells
that had been activated by cross-linking of IgE receptors. Furthermore,
we examined the subcellular localization of PGHS-1 and PGHS-2. PGHS-1
and PGHS-2 were in both microsomal and nuclear fractions. PGHS-2 was
predominantly localized in nuclear fraction as compared with microsomal
fraction, whereas expression of PGHS-1 in nuclear fraction was similar
to that in microsomal fraction (Fig. 4C).
Suppression of PGHS Activity by Overexpression of PHGPx--
The
production of PGD2 was examined after addition of exogenous
arachidonic acid or prostaglandin H2 (PGH2) to
the incubation medium in an attempt to identify the step in the
synthesis of PGD2 in L9 cells where inhibition occurred
(Fig. 5). The rate of formation of
PGD2 from exogenously added
[1-14C]arachidonic acid was determined (Fig.
5A). Formation of PGD2 from arachidonic acid was
enhanced in S1 cells after the challenge with IgE-DNP, but no
enhancement of the production of PGD2 was observed in L9
cells under the same conditions. Prior treatment with BSO allowed L9
cells to produce as much PGD2 as S1 cells. By contrast,
conversion of exogenously added PGH2 to PGD2,
catalyzed by PGD2 synthase, was not enhanced in S1 or in L9
cells after stimulation with IgE-DNP, and no difference in terms of the
extent of the conversion was observed between S1 and L9 cells (Fig.
5B). These results indicated that the activity of
PGD2 synthase was similar in L9 cells and S1 cells.
Moreover, since PGHS-2 activity was susceptible to the overexpression
of PHGPx, the insufficient activation of PGHS-2 resulted in the
suppression of production of PGD2 in L9 cells. To clarify
the effects of overexpression of PHGPx on PGHS activity, we measured
the activity of PGHS in a cell-free system (Table
II). Cell lysates prepared from quiescent cells and from IgE-DNP-stimulated cells were incubated with radioactive arachidonic acid, and the amount of radioactive PGH2
produced was determined. In lysates of quiescent cell, we detected no
significant difference in terms of PGHS activity between S1 cells and
L9 cells. Stimulation by IgE-DNP caused an increase in PGHS activity in the lysate prepared from S1 cells, but no such activation of PGHS was
observed in the lysate prepared from IgE-DNP-stimulated L9 cells. We
next examined the effects of hydroperoxides on the activity of PGHS in
an attempt to estimate whether inhibition of the activity of PGHS might
have been due to insufficient levels of hydroperoxides in
PHGPx-overexpressing cells (Fig. 6). We
determined PGHS activity in lysates, prepared from IgE-DNP-stimulated
cells, in the presence of various concentrations of the fatty acid
hydroperoxide 15-HpETE or of 3-chloroperoxybenzoic acid (3-CPBA), both
of which have been used as activators (33, 34). Concentrations of
reduced form of GSH in cell lysate prepared from S1 and L9 cells were 2.06 ± 0.39 and 1.81 ± 0.69 nmol/mg, respectively. However,
when cell lysate was added to the assay system of PGHS activity, which contained p-chloromercuriphenylsulfonic acid, the reduced
form of GSH and other non-protein thiol compounds was not detected (data not shown). These results indicated that the level of GSH in
lysate would not influence to the activity of PGHS by the peroxides. The cell lysates supplemented with 15-HpETE or 3-CPBA revealed differences in the dependence on hydroperoxides between L9 cells and S1
cells. In the lysate of S1 cells, the PGHS activity increased rapidly
with increase in the concentration of 15-HpETE and then it decreased
above 0.05 µM 15-HpETE. By contrast, in the lysate of L9
cells, PGHS was activated as the level of 15-HpETE was raised, with
maximum activity at 5 µM (Fig. 6B). These
results indicated that L9 cells required 100 times more 15-HpETE than
did S1 cells for maximum activation of PGHS. In lysate supplemented
with 3-CPBA, the observed PGHS activity increased with increases in the
concentration of 3-CPBA, indicating that the peroxide activated PGHS.
The observed activity leveled off above 0.5 and 50 µM
3-CPBA in S1 and L9 cells, respectively.
The levels of peroxides were determined in S1 and L9 cells by flow
cytometric analysis using the oxidant-sensitive fluorescent dye
2',7'-dichlorofluorescein diacetate (Fig.
7). After stimulating with IgE-DNP,
fluorescent intensity in S1 cells was larger than that in similarly
stimulated L9 cells. These results indicated that the levels of
peroxides in L9 cells were lower than those in S1 cells after
stimulation with IgE-DNP. The intensity of fluorescence after
stimulation with antigen of sensitized L9 cells that had been treated
with 3-CPBA was the same as that in S1 cells after stimulation with
IgE-DNP. These results indicated that the exposure of L9 cells to
peroxide 3-CPBA had increased the level of intracellular peroxides.
Effects of the Overexpression of Mitochondrial PHGPx on the
Production of PGD2--
PHGPx is selectively overexpressed
in the mitochondria of RBL-2H3 cells (M15 cells), as a result of
transfection with a plasmid that encodes mitochondrial-type PHGPx (19).
We monitored the production of PGD2 in M15 cells in order
to clarify the influence of mitochondrial PHGPx on the production of
PGD2 (Fig. 8). By contrast to
that in L9 cells, the formation of PGD2 was only slightly suppressed in M15 cells that had been activated with IgE-DNP (compare Figs. 1B and 8A). A23187 strongly induced the
production of PGD2 in S1 cells (Fig. 8B),
whereas production of PGD2 was considerably suppressed in
L9 cells but not in M15 cells. The overexpression of non-mitochondrial
PHGPx inhibited the production of PGD2 in response to
A23187 more strongly than did that of mitochondrial PHGPx. These
results indicated that PHGPx in mitochondria did not participate in
regulation of the synthesis of PGD2.
Effects of PHGPx on Production of Leukotriene C--
RBL-2H3 cells
have potent 5-lipoxygenase activity and they release significant
amounts of leukotriene C (LTC4) upon activation by A23187.
We previously monitored the production of LTC4 by L9 cells
after a challenge with A23187 and found that the production of
LTC4 was dramatically suppressed in L9 cells. However, the production of LTC4 by M15 cells was similar to that by S1
cells (Fig. 9). These results indicated
that mitochondrial PHGPx was involved neither in the regulation of
prostaglandin synthesis nor in the regulation of leukotriene
synthesis.
In the present study, we examined the effect of overexpression of
non-mitochondrial PHGPx in RBL-2H3 cells, which generated PGD2 predominantly, together with small amounts of other
prostanoid products such as PGE2,
6-keto-PGF1 There is a considerable evidence that the cyclooxygenase activity of
PGHS requires reaction of the peroxidase with hydroperoxide (16, 39).
Half-maximal activity was found to require about 2 nM
hydroperoxide in the case of PGHS-2 and 21 nM in the case of PGHS-1 (40). Peroxidase reduces hydroperoxides by two electrons to
generate the corresponding alcohol and a higher oxidation state, which
is consistent with the Fe(IV) protoporphyrin IX radical, from the
resting state of heme, Fe(III) protoporphyrin IX. A secondary oxidized
species, a tyrosyl radical generated from the Fe(IV) protoporphyrin IX
radical, is envisioned as the active species that removes
hydrogen from arachidonic acid to initiate the cyclooxygenase reaction
(17, 18). Therefore, we examined the effects of hydroperoxides on the
production of prostaglandins in an attempt to determine whether
inhibition of the activity of PGHS was due to insufficient levels of
hydroperoxides in PHGPx-overexpressing cells. The low level activity of
PGHS in L9 cells after IgE-DNP stimulation was increased upon treatment
of cells with the peroxides 3-CPBA, 15-HpETE (Fig. 6), and 12-HpETE
(data not shown). The maximum activity of PGHS was observed at 0.05 and
5 µM 15-HpETE in S1 cells and L9 cells, respectively, and
the specific activity was almost the same in the two cell lines.
Furthermore, 3-CPBA activated PGHS, and enzymatic activity reached a
plateau value at 0.5 and 50 µM 3-CPBA in S1 cells and L9
cells, respectively. L9 cells needed 100 times more 15-HpETE or 3-CPBA
than did S1 cells for maximum PGHS activity, indicating that the
intracellular level of hydroperoxides was significantly depressed
in PHGPx-overexpressing cells. A low level of hydroperoxides in
activated cells was also demonstrated in L9 cells, as compared with S1
cells, when intracellular hydroperoxides were estimated by flow
cytometric analysis (21). We also observed stimulation with IgE-DNP to
increase in levels of intracellular peroxides with flow cytometric
analysis (data not shown). The increase in the levels of intracellular
peroxides induced by IgE-DNP was suppressed by overexpression of
non-mitochondrial PHGPx. This effect by PHGPx was abolished by the
addition of 3-CPBA (Fig. 7). Our results indicated that
non-mitochondrial PHGPx was primarily responsible for the reduction of
lipid hydroperoxides and thereby caused suppression of the
activity of PGHS in RBL-2H3 cells.
We can assume that levels of intracellular hydroperoxides are
controlled by two kinds of intracellular isozymes of GPx. In previous
studies, we determined the specific activities and levels of PHGPx and
cGPx in S1 and L9 cells (19, 21). The levels of cGPx in S1 and L9 cells
were similar, but the ratio of cGPx content to PHGPx content was 4.6 in
S1 cells and only 1.1 in L9 cells (19).
PGHS-1 and PGHS-2 localize in the nuclear envelope and endoplasmic
reticulum (41), and these isozymes are distributed in the inner and
outer nuclear membranes (42). PGHS-2 localized in the nuclear fraction
more than the microsomal fraction. This result suggests that the
synthesis of PGH2 upon stimulation by IgE-DNP predominantly
occurs at the nuclear envelope, to which cytosolic phospholipase
A2 and 5-lipoxygenase have been translocated from the
cytosol (43, 44), although PGHS in endoplasmic reticulum partially
provides PGH2 in stimulated cells. Mitochondria are a major
physiological source of ROS, which can be generated during mitochondrial respiration. The ROS produced in mitochondria can cause
peroxidation of membrane lipids and, possibly, induce the liberation of
hydroperoxides, such as fatty acid hydroperoxides and hydrogen
peroxide. PHGPx is specifically localized in nuclear and mitochondrial
fractions of rat testis (45). In a previous study, we demonstrated that
overexpression of mitochondrial PHGPx, but not of non-mitochondrial
PHGPx, suppressed apoptotic cell death that occurs via the
mitochondrial death pathway. M15 cells were resistant to the induction
of apoptosis by deprivation of glucose or by treatment with etoposide,
staurosporine, UV irradiation, actinomycin D, or cycloheximide (28).
However, overexpression of PHGPx in the mitochondrial fraction induced
slight suppression of the production of PGD2 in RBL-2H3
cells that had been stimulated by either IgE-DNP (Fig. 8A)
or A23187 (Fig. 8B). The amount of PHGPx in the nuclear
fraction from L9 cells was six and three times higher than that from S1
and M15 cells, respectively, but the amounts of PHGPx in the
mitochondrial fractions from L9 and from S1 cells were similar.
However, the amount of PHGPx in the mitochondrial fraction from M15
cells was twice that from S1 and L9 cells, and the amounts of PHGPx in
the microsomal fractions from L9 and M15 cells were approximately the
same level (19). Mitochondrial PHGPx that has a targeting signal
sequence should be exclusively imported into mitochondria. Therefore,
it is supposed that PHGPx in nuclear, microsomal, and cytosolic
fractions of M15 cells would be partially degraded and lose the ability
to penetrate into mitochondria and to possess full activity of PHGPx. These results indicate that inactivation of PGHS-2 was due to high
level expression of PHGPx in the nucleus and endoplasmic reticulum.
Subcellular distribution of non-mitochondrial PHGPx in nuclear,
microsome, and cytosol of L9 cells was almost same as that in S1 cells
(19). The activity of endogenous PHGPx in S1-Se(+) cells that cultured in
selenite-supplemented medium was three times higher than that in S1
cells, and production of PGD2 after stimulation with
IgE-DNP in S1-Se(+) cells was one-third of that
in S1 cells. These data suggest that the endogenous PHGPx could
modulate the production of PGD2.
5-Lipoxygenase catalyzes the formation of leukotrienes from arachidonic
acid at the nuclear envelope (44, 46). This conversion requires
expression of 5-lipoxygenase-activating protein (FLAP), which is
exclusively localized to the nuclear envelope and probably delivers
arachidonic acid to 5-lipoxygenase (47). The overexpression of
non-mitochondrial PHGPx suppressed the production of LTC4
in RBL-2H3 cells that had been stimulated with A23187 (21), but
overexpression of mitochondrial PHGPx had no effect on the rate of
production of LTC4 (Fig. 9). Thus, PHGPx in the nucleus, but not in mitochondria, might be critical for modulation of the level
of hydroperoxides required for the activation of 5-lipoxygenase.
Our results suggest that PHGPx in the nucleus might play an important
role in regulation of the metabolism of arachidonic acid, the synthesis
of leukotrienes, and the synthesis of prostanoids via the reduction of
lipid hydroperoxides around nucleus. A recent report indicates that
PGJ, derived from PGD2, reduces mitochondrial activity and
induces apoptosis (48). Thus, metabolites of arachidonic acid generated
by PGHS appear to have biological activities in addition to their
functions as chemical mediators. The present study suggests that
non-mitochondrial PHGPx might be involved in the regulation of cellular
functions and signal transduction via modulation of the production of prostaglandins.
We thank Junko Matsubara, Tomomi
Hoshino, and Reiko Nagakawa for their expert technical assistance.
*
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.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M003191200
The abbreviations used are:
ROS, reactive oxygen
species;
PHGPx, phospholipid hydroperoxide glutathione peroxidase;
cGPx, classical glutathione peroxidase;
PG, prostaglandin;
PGHS, prostaglandin H synthase;
PLA2, phospholipase
A2;
RBL, rat basophile leukemia cells;
Ig, immunoglobulin;
LT, leukotriene;
DNP, dinitrophenyl;
hSA, human serum albumin;
PBS, phosphate-buffered saline;
DMEM, Dulbecco's modified Eagle's medium;
FCS, fetal calf serum;
PVDF, polyvinylidene difluoride;
TBS-T, Tris-buffered saline plus Tween 20;
TLC, thin-layer chromatography;
BSO, buthionine sulfoximine;
HpETE, hydroperoxyeicosatetraenoic acid;
3-CPBA, 3-chloroperoxybenzoic acid;
DCFH-DA, 5,6-carboxy-2',7'-dichlorofluorescein diacetate;
PCOOH, phosphatidylcholine hydroperoxide;
PAGE, polyacrylamide gel
electrophoresis.
Involvement of Phospholipid Hydroperoxide Glutathione Peroxidase
in the Modulation of Prostaglandin D2 Synthesis*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Production of prostaglandin D2 in
the control line of cells and in cells that overexpressed
non-mitochondrial PHGPx. A, autoradiogram of prostanoid
products. Control cells (S1) and cells that overexpressed
non-mitochondrial PHGPx (L9) were labeled with
[1-14C]arachidonic acid for 24 h and then
activated by cross-linking their IgE receptors, by sequential treatment
with IgE-DNP and DNP-hSA. Labeled cells were primed with IgE-DNP at a
concentration of 1 µg/106 cells. Two hours later, cells
were washed, and then receptor cross-linking was effected by incubation
with 10 µg/ml DNP-hSA for 1 h at 37 °C in culture medium.
Culture media were collected and supplemented with ethyl acetate-HCl
(pH 3.0) for extraction of labeled metabolites. Extracts were
concentrated and applied to a TLC plate. After the plate had been
developed, the radioactivity of products was determined by
autoradiography with a Bio-Imaging analyzer. The products are indicated
on the left. AA, arachidonic acid. B,
rates of production of PGD2 in cells that overexpressed
non-mitochondrial PHGPx (L9 and L28 cells). S1, L9, and L28 cells were
activated by cross-linking the IgE receptors as described in
A. Culture media were collected, and PGD2 was
quantitated with enzyme immunosorbent assay kit. Open bars
represent non-activated cells, and hatched bars represent
cells activated by cross-linking of IgE receptors. Values given are
means ± S.D. of results from three independent experiments.
View larger version (15K):
[in a new window]
Fig. 2.
Time course of the generation of
prostaglandin D2 by a control line of cells and by cells
that overexpressed non-mitochondrial PHGPx. Control cells (S1) and
L9 cells were activated by cross-linking their IgE receptors and by
sequential treatment with IgE-DNP and DNP-hSA. S1 cells (open
circles) and L9 cells (closed circles) were incubated
for 2 h with IgE-DNP (1 µg/106 cells), washed, and
treated with DNP-hSA (10 µg/ml) for the indicated periods. Culture
media were collected, and PGD2 was quantitated. For
details, see legend to Fig. 1.
Effects of buthionine sulfoximine on the production of prostaglandin
D2 by a control line of cells and by cells that
overexpressed non-mitochondrial PHGPx
) 0.5 mM buthionine
sulfoximine (BSO). Then they were incubated with IgE-DNP (1 µg/106 cells) for 2 h, washed, and treated with DNP-hSA
for 1 h. Amounts of PGD2 released into the supernatant
were quantitated. See legend to Fig. 1 for details.
View larger version (28K):
[in a new window]
Fig. 3.
Dependence of the production of prostaglandin
D2 on a specific isozyme of PGHS in cells with cross-linked
IgE receptors. Control cells (S1) were labeled with
[1-14C]arachidonic acid for 24 h and activated by
cross-linking their IgE receptors, as described in the legend to Fig.
1. 1st column, labeled cells were cultured with no
additions. 2nd column, labeled cells were incubated with
IgE-DNP (1 µg/106 cells) for 2 h, washed, and then
incubated with DNP-hSA for 1 h to activate the cells. 3rd
column, labeled cells were incubated for 2 h with IgE-DNP
and for 1 h with 100 µM aspirin, washed, and then they
were treated for 1 h with DNP-hSA plus aspirin (aspirin irreversibly
inactivates PGHS-1 and PGHS-2). 4th column, labeled cells
were incubated for 2 h with IgE-DNP and for 15 min with 1 µM NS-398, washed, and then they were treated for 1 h with DNP-hSA plus NS-398 (NS-398 blocks PGHS-2-dependent
production of PGD2). Culture media were collected and mixed
with ethyl acetate-HCl (pH 3.0) for extraction of labeled metabolites.
Extracts were concentrated and applied to a TLC plate. After the plate
had been developed, rates of production of PGD2
(A) and release of arachidonic acid (B) were
calculated from results of scanning densitometry after autoradiography
with the Bio-Imaging analyzer.
View larger version (13K):
[in a new window]
Fig. 4.
Cellular levels of PGHS-1 and PGHS-2 in a
control line of cells and in cells that overexpressed non-mitochondrial
PHGPx. A, cellular levels of PGHS-1 and PGHS-2 in
RBL-2H3 cells stimulated by cross-linking of IgE receptors. Control
cells (S1) and L9 cells were incubated for 2 h with
IgE-DNP (1 µg/106 cells), washed, and treated with
DNP-hSA. One hour later, cells were collected and sonicated. The
proteins in sonicates were subjected to SDS-PAGE, and then proteins
were blotted onto a PVDF membrane. Immunoblotting was performed using
polyclonal antibodies against PGHS-1 and monoclonal antibodies against
PGHS-2 as described in the text. B, effects of BSO on
cellular levels of PGHS-2 in RBL-2H3 cells. S1 cells were pretreated
with 0.5 mM BSO for 18 h and then incubated with
IgE-DNP for 2 h. After washing, cells were treated with DNP-hSA
for 1 h. Cell lysates were subjected to SDS-PAGE and then the
proteins were blotted onto a PVDF membrane. Immunoblotting was
performed with monoclonal antibodies against PGHS-2 as described in the
text. C, localization of PGHS-1 and PGHS-2 in RBL-2H3 cells.
After S1 cells were stimulated by cross-linking of IgE receptors,
nuclear and microsomal fractions were prepared as described in the
text. The proteins in fractions were subjected to SDS-PAGE and then
proteins were blotted onto a PVDF membrane. Immunoblotting was
performed as described in A. The cell fractions are
indicated on the top. Nuc, nuclear fraction; Mic,
microsomal fraction.
View larger version (22K):
[in a new window]
Fig. 5.
Production of prostaglandin D2
from exogenously added substrates in a control line of cells and in
cells that overexpressed non-mitochondrial PHGPx. A,
production of PGD2 from exogenously added arachidonic acid.
Control cells (S1, open bars) and L9 cells (hatched
bars) were incubated with IgE-DNP for 2 h, washed, and then
treated with DNP-hSA for 1 h. After removal of the medium, cells
were incubated in phosphate-buffered saline that contained 1 mM CaCl2, 0.5 mM MgSO4,
and 3.7 kBq of [1-14C]arachidonic acid (170.7 MBq/mmol)
for 30 min. Supernatants were collected, and mixed with 3 volumes of
ethyl acetate (pH 3.0). The metabolites of labeled arachidonic acid,
which were extracted in the ethyl acetate layer, were separated by TLC,
and rates of production of metabolites were calculated from results of
scanning densitometry after autoradiography with the Bio-Imaging
analyzer. B, production of PGD2 from exogenously
added prostaglandin H2. Control cells (S1, open
bars) and L9 cells (hatched bars), which had been
stimulated by cross-linking of IgE receptors, were incubated in
phosphate-buffered saline that contained 1 mM
CaCl2, 0.5 mM MgSO4, and 10 µM prostaglandin H2 for 30 min. Incubation
media were collected, and PGD2 was quantitated. See legend
to Fig. 1 for details.
PGHS activity in a control line of cells and in cells that
overexpressed non-mitochondrial PHGPx
View larger version (15K):
[in a new window]
Fig. 6.
Effects of peroxides on PGHS activity in
IgE-DNP-stimulated cells. Control cells (S1, open
circles) and L9 cells (closed circles) were treated
with IgE-DNP for 2 h and then with DNP-hSA for 1 h. Cells
were washed with PBS, harvested with a cell scraper, and homogenized.
The PGHS activity in the homogenates was determined in the presence of
increasing concentrations of 3-CPBA (A) and 15-HpETE
(B).
View larger version (13K):
[in a new window]
Fig. 7.
Flow cytometric analysis of intracellular
peroxides in a control line of cells and in cells that overexpressed
non-mitochondrial PHGPx. S1 cells (fine line) and L9
cells (bold line) were incubated for 2 h with IgE-DNP
(1 µg/106 cells), washed, and treated with DNP-hSA (10 µg/ml) for 30 min. L9 cells sensitized with IgE-DNP were incubated
with 50 µM 3-CPBA for 10 min at 37 °C and then treated
with DNP-hSA (10 µg/ml) for 30 min (dotted line).
Stimulated cells were incubated with 5 µM DCFH-DA for 15 min. The intensity of fluorescence from DCFH of cells was quantified by
flow cytometry and is plotted on a logarithmic scale, in arbitrary
units, against the number of cells.
View larger version (31K):
[in a new window]
Fig. 8.
Production of prostaglandin D2 in
a control line of cells and in cells that overexpressed mitochondrial
PHGPx. A, production of PGD2 in RBL-2H3
cells stimulated by cross-linking of IgE receptors. Control cells
(S1) and cells that overexpressed mitochondrial PHGPx
(M15) were either untreated (open bars) or
treated (hatched bars) with IgE-DNP for 2 h and then
with DNP-hSA for 1 h. The amounts of PGD2 released
into the media were determined. See legend to Fig. 1 for details.
B, production of PGD2 in RBL-2H3 cells
stimulated with 5 µM A23187 for 5 min at 37 °C.
Control cells (S1), L9 cells, and M15 cells were either untreated
(open bars) or treated (hatched bars) with
A23187, and the amounts of PGD2 in media were determined as
above.
View larger version (30K):
[in a new window]
Fig. 9.
Production of leukotriene C4 in a
control line of cells and in cells that overexpressed mitochondrial
PHGPx. Control cells (S1) and M15 cells were either
untreated (open bars) or treated (hatched bars)
with 5 µM A23187 for 5 min at 37 °C. The amounts of
LTC4 in media were determined by an enzyme immunosorbent
assay. Data are expressed as means ± S.D. of results from three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and thromboxane B2 in response to IgE-DNP or A23187 (30). The overexpression of non-mitochondrial PHGPx in RBL-2H3 cells caused the dramatic suppression of the production of PGD2 in response to stimulation by IgE
antigen (Fig. 1). Two isoforms of PGHS are known. PGHS-1 is expressed
constitutively in nearly all mammalian cells. PGHS-2, although normally
absent from most cells, is synthesized in many types of cell upon
treatment with a variety of cytokines, growth factors, and tumor
promoters (35). The RBL-2H3 cells used in the present experiments
expressed PGHS-1 and PGHS-2 constitutively. No apparent differences
were observed in terms of the levels of expression of PGHS-1 and
induced PGHS-2 between the control line of cells (S1 cells) and cells that overexpressed non-mitochondrial PHGPx, namely L9 cells (Fig. 4A). It is known that aspirin (acetyl salicylate) inhibits
both PGHS-1 and PGHS-2 by acetylation of specific serine residues, Ser-530 in human PGHS-1 and Ser-516 in human PGHS-2 (36, 37). Our
finding that the production of PGD2 was completely
suppressed both by aspirin and by an inhibitor of PGHS-2, NS-398, which
reacts selectively with the active site of PGHS-2 but not with that of PGHS-1 (38), indicated that PGHS-2 was responsible for the production of PGD2 in RBL-2H3 cells upon stimulation by IgE-DNP (Fig.
3). In RBL-2H3 cells, PGH2 is converted to PGD2
by the hematopoietic form of PGD2 synthase, which requires
glutathione as a coenzyme for its activity. Exogenously added
PGH2 was transformed to PGD2 at almost the same
rate in both S1 and L9 cells (Fig. 5B), but the production
of PGD2 from exogenously added arachidonic acid was
suppressed in L9 cells (Fig. 5A). Moreover, the activity of PGHS was suppressed in L9 cells after stimulation with IgE-DNP (Table
II). These results indicated that inhibition of the synthesis of
PGD2 was not due to inhibition of the isomerization of
PGH2 to PGD2 but, rather, was due to inhibition
of the production of PGH2 from arachidonic acid by PGHS-2.
A change in phospholipase A2 activity was not involved in
the suppression of PGD2 production, since, under our
conditions, the rates of release of arachidonic acid by S1 cells and L9
cells were the same (data not shown).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel./Fax:
81-3-3444-4943; E-mail: nakagaway@pharm.kitasato-u.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Cerutti, P. A.
(1985)
Science
227,
375-381
2.
Fridovich, I.
(1978)
Science
201,
875-880
3.
Jolly, S. R.,
Kane, W. J.,
Bailie, M. B.,
Abrams, G. D.,
and Lucchesi, B. R.
(1984)
Circ. Res.
54,
277-285
4.
Guarnieri, C.,
Muscari, C.,
and Calderera, C. M.
(1992)
in
Free Radicals and Aging
(Emerit, I.
, and Chance, B., eds)
, pp. 73-77, Birkhauser Verlag, Basel, Switzerland
5.
Hayes, J. D.,
and McLellan, L. I.
(1999)
Free Radical Res.
31,
273-300
6.
Harlan, J. M.,
and Callahan, K. S.
(1984)
J. Clin. Invest.
74,
442-448
7.
Marshall, P. J.,
and Lands, W. E.
(1986)
J. Lab. Clin. Med.
108,
525-534
8.
Sporn, P. H.,
Peters, G. M.,
and Simon, R. H.
(1988)
Am. Rev. Respir. Dis.
137,
49-56
9.
Taylor, L.,
Menconi, M. J.,
and Polgar, P.
(1983)
J. Biol. Chem.
258,
6855-6857
10.
Lewis, R. A.,
Soter, N. A.,
Diamond, P. T.,
Austen, K. F.,
Oates, J. A.,
and Roberts, L.
(1982)
J. Immunol.
129,
1627-1631
11.
Holgate, S. T.,
Burns, G. B.,
Robinson, C.,
and Church, M. K.
(1984)
J. Immunol.
133,
2138-2144
12.
Peters, S. P.,
MacGlashan, D. J.,
Schulman, E. S.,
Schleimer, R. P.,
Hayes, E. C.,
Rokach, J.,
Adkinson, N. J.,
and Lichtenstein, L. M.
(1984)
J. Immunol.
132,
1972-1979
13.
Sakamoto, H.,
Kitahara, J.,
and Nakagawa, Y.
(1999)
J. Biochem. (Tokyo)
125,
90-95
14.
Kawata, R.,
Reddy, S. T.,
Wolner, B.,
and Herschman, H. R.
(1995)
J. Immunol.
155,
818-825
15.
Vane, J.
(1994)
Nature
367,
215-216
16.
Wei, C.,
Kulmacz, R. J.,
and Tsai, A. L.
(1995)
Biochemistry
34,
8499-8512
17.
Tsai, A. L.,
Wu, G.,
Palmer, G.,
Bambai, B.,
Koehn, J. A.,
Marshall, P. J.,
and Kulmacz, R. J.
(1999)
J. Biol. Chem.
274,
21695-21700
18.
Xiao, G.,
Tsai, A. L.,
Palmer, G.,
Boyar, W. C.,
Marshall, P. J.,
and Kulmacz, R. J.
(1997)
Biochemistry
36,
1836-1845
19.
Arai, M.,
Imai, H.,
Koumura, T.,
Yoshida, M.,
Emoto, K.,
Umeda, M.,
Chiba, N.,
and Nakagawa, Y.
(1999)
J. Biol. Chem.
274,
4924-4933
20.
Roveni, A.,
Maiorino, M.,
and Ursini, F.
(1994)
Methods Enzymol.
143,
307-313
21.
Imai, H.,
Narashima, K.,
Arai, M.,
Sakamoto, H.,
Chiba, N.,
and Nakagawa, Y.
(1998)
J. Biol. Chem.
273,
1990-1997
22.
Ursini, F.,
Maiorino, M.,
and Gregolin, C.
(1985)
Biochim. Biophys. Acta
839,
62-70
23.
Schnurr, K.,
Belkner, J.,
Ursini, F.,
Schewe, T.,
and Kuhn, H.
(1996)
J. Biol. Chem.
271,
4653-4658
24.
Thomas, J. P.,
Maiorino, M.,
Ursini, F.,
and Girotti, A. W.
(1990)
J. Biol. Chem.
265,
454-461
25.
Margalit, A.,
Hauser, S. D.,
Zweifel, B. S.,
Anderson, M. A.,
and Isakson, P. C.
(1998)
Am. J. Physiol.
274,
R294-R302
26.
Shitashige, M.,
Morita, I.,
and Murota, S.
(1998)
Biochim. Biophys. Acta
1389,
57-66
27.
Imai, H.,
Sumi, D.,
Sakamoto, H.,
Hanamoto, A.,
Arai, M.,
Chiba, N.,
and Nakagawa, Y.
(1996)
Biochem. Biophys. Res. Commun.
222,
432-438
28.
Nomura, K.,
Imai, H.,
Koumura, T.,
Arai, M.,
and Nakagawa, Y.
(1999)
J. Biol. Chem.
274,
29294-29302
29.
Peden, D. B.,
and Dailey, L.
(1995)
Am. J. Physiol.
268,
L902-L910
30.
Westcott, J. Y.,
Wenzel, S. E.,
and Dreskin, S. C.
(1996)
Biochim. Biophys. Acta
1303,
74-81
31.
Laemmli, U. K.
(1970)
Nature
227,
680-685
32.
Green, K.,
and Samuelsson, B.
(1964)
J. Lipid Res.
5,
117-120
33.
Lu, G.,
Tsai, A. L.,
Van, W. H.,
and Kulmacz, R. J.
(1999)
J. Biol. Chem.
274,
16162-16167
34.
Chen, W.,
Pawelek, T. R.,
and Kulmacz, R. J.
(1999)
J. Biol. Chem.
274,
20301-20306
35.
Herschman, H. R.
(1996)
Biochim. Biophys. Acta
1299,
125-140
36.
Lecomte, M.,
Laneuville, O.,
Ji, C.,
DeWitt, D. L.,
and Smith, W. L.
(1994)
J. Biol. Chem.
269,
13207-13215
37.
Wennogle, L. P.,
Liang, H.,
Quintavalla, J. C.,
Bowen, B. R.,
Wasvary, J.,
Miller, D. B.,
Allentoff, A.,
Boyer, W.,
Kelly, M.,
and Marshall, P.
(1995)
FEBS Lett.
371,
315-320
38.
Futaki, N.,
Takahashi, S.,
Yokoyama, M.,
Arai, I.,
Higuchi, S.,
and Otomo, S.
(1994)
Prostaglandins
47,
55-59
39.
Smith, W. L.,
and Marnett, L. J.
(1991)
Biochim. Biophys. Acta
1083,
1-17
40.
Kulmacz, R. J.,
and Wang, L. H.
(1995)
J. Biol. Chem.
270,
24019-24023
41.
Morita, I.,
Schindler, M.,
Regier, M. K.,
Otto, J. C.,
Hori, T.,
DeWitt, D. L.,
and Smith, W. L.
(1995)
J. Biol. Chem.
270,
10902-10908
42.
Spencer, A. G.,
Woods, J. W.,
Arakawa, T.,
Singer, I. I.,
and Smith, W. L.
(1998)
J. Biol. Chem.
273,
9886-9893
43.
Glover, S.,
Bayburt, T.,
Jonas, M.,
Chi, E.,
and Gelb, M. H.
(1995)
J. Biol. Chem.
270,
15359-15367
44.
Malaviya, R.,
Malaviya, R.,
and Jakschik, B. A.
(1993)
J. Biol. Chem.
268,
4939-4944
45.
Godeas, C.,
Tramer, F.,
Micali, F.,
Roveri, A.,
Maiorino, M.,
Nisii, C.,
Sandri, G.,
and Panfili, E.
(1996)
Biochem. Mol. Med.
59,
118-124
46.
Brock, T. G.,
McNish, R. W.,
and Peters, G. M.
(1995)
J. Biol. Chem.
270,
21652-21658
47.
Dixon, R. A.,
Diehl, R. E.,
Opas, E.,
Rands, E.,
Vickers, P. J.,
Evans, J. F.,
Gillard, J. W.,
and Miller, D. K.
(1990)
Nature
343,
282-284
48.
Keelan, J. A.,
Sato, T. A.,
Marvin, K. W.,
Lander, J.,
Gilmour, R. S.,
and Mitchell, M. D.
(1999)
Biochem. Biophys. Res. Commun.
262,
579-585
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. J. Swindle, J. W. Coleman, F. R. DeLeo, and D. D. Metcalfe Fc{epsilon}RI- and Fc{gamma} Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent J. Immunol., November 15, 2007; 179(10): 7059 - 7071. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Imai, M. Saito, N. Kirai, J. Hasegawa, K. Konishi, H. Hattori, M. Nishimura, S. Naito, and Y. Nakagawa Identification of the Positive Regulatory and Distinct Core Regions of Promoters, and Transcriptional Regulation in Three Types of Mouse Phospholipid Hydroperoxide Glutathione Peroxidase J. Biochem., October 1, 2006; 140(4): 573 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Liu, D. Cao, S. F. Oh, C. N. Serhan, and R. J. Kulmacz Divergent cyclooxygenase responses to fatty acid structure and peroxide level in fish and mammalian prostaglandin H synthases FASEB J, June 1, 2006; 20(8): 1097 - 1108. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Ran, H. Liang, M. Gu, W. Qi, C. A. Walter, L. J. Roberts II, B. Herman, A. Richardson, and H. Van Remmen Transgenic Mice Overexpressing Glutathione Peroxidase 4 Are Protected against Oxidative Stress-induced Apoptosis J. Biol. Chem., December 31, 2004; 279(53): 55137 - 55146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Rouzer, P. J. Kingsley, H. Wang, H. Zhang, J. D. Morrow, S. K. Dey, and L. J. Marnett Cyclooxygenase-1-dependent Prostaglandin Synthesis Modulates Tumor Necrosis Factor-{alpha} Secretion in Lipopolysaccharide-challenged Murine Resident Peritoneal Macrophages J. Biol. Chem., August 13, 2004; 279(33): 34256 - 34268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bambai, C. E. Rogge, B. Stec, and R. J. Kulmacz Role of Asn-382 and Thr-383 in Activation and Inactivation of Human Prostaglandin H Synthase Cyclooxygenase Catalysis J. Biol. Chem., February 6, 2004; 279(6): 4084 - 4092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakamoto, T. Tosaki, and Y. Nakagawa Overexpression of Phospholipid Hydroperoxide Glutathione Peroxidase Modulates Acetyl-CoA, 1-O-Alkyl-2-lyso-sn-glycero-3-phosphocholine Acetyltransferase Activity J. Biol. Chem., December 20, 2002; 277(52): 50431 - 50438. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |