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Regulation of Delayed Prostaglandin Production in Activated
P388D1 Macrophages by Group IV Cytosolic and Group V Secretory
Phospholipase A2s*
(Received for publication, May 21, 1998, and in revised form, February 26, 1999)
Hiroyuki Shinohara, Marı́a A. Balboa, Christina A. Johnson, Jesús Balsinde, and
Edward A. Dennis‡
From the Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California at
San Diego, La Jolla, California 92093-0601
Group V secretory phospholipase A2 (sPLA2) rather
than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin
E2 (PGE2) production in the macrophage-like cell line
P388D1. When a new clone of these cells, P388D1/MAB,
selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6–24 h) in response to lipopolysaccharide alone occurred in parallel with the
induction of Group V sPLA2 and cyclooxygenase-2 (COX2). No changes in the level of cytosolic phospholipase A2
(cPLA2) or COX-1 were observed, and Group IIA sPLA2
was not detectable. Use of a potent and selective sPLA2
inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that
delayed PGE2 was largely dependent on the induction of
Group V sPLA2. Also, COX-2, not COX-1, was found to
mediate delayed PGE2 production because the response
was completely blocked by the specific COX-2 inhibitor
NS-398. Delayed PGE2 production and Group V sPLA2
expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca21-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid
release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response
as well. Collectively, the results suggest a model
whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2
production via COX-2.
Arachidonic acid (AA)1 mobilization and the generation of
prostaglandins by major immunoinflammatory cells such as
macrophages and mast cells usually occur in two phases. The
immediate phase, which takes minutes and is elicited by Ca21mobilizing agonists such as platelet-activating factor (PAF), is
characterized by a burst of AA liberation. In some cells such as
P388D1 macrophages (1, 2) and MMC-34 mast cells (3), this
burst is mainly produced by a secretory phospholipase A2
(sPLA2) but is strikingly regulated by the cytosolic Group IV
phospholipase A2 (cPLA2).
The delayed phase of prostaglandin production is accompanied by the continuous supply of AA over long incubation periods spanning several hours. There is some discrepancy about
the identity of the PLA2 isoform(s) involved in the delayed
phase. Despite this phase being independent of a Ca21 increase, the cPLA2 has often been suggested to be critically
involved (3–5). However, other studies have suggested the
quantitatively more important role of the sPLA2, an enzyme
that is dramatically induced during long term incubation of the
cells with a variety of stimuli (4–6). There is, however, agreement that COX-2, another enzyme whose expression is augmented dramatically after long term stimulation, is absolutely
required for long term PGE2 production, irrespective of the
constitutive presence of COX-1 (7–9).
Using a new clone of the P388D1 macrophage-like cells
termed P388D1/MAB, we provide herein evidence for the involvement of Group V sPLA2 in delayed PGE2 production.
Furthermore, our results suggest that Group V sPLA2 expression is dependent upon the activation of Group IV cPLA2.
EXPERIMENTAL PROCEDURES
Materials—Mouse P388D1 macrophage-like cells were obtained from
the American Type Culture Collection (Rockville, MD). Iscove’s modified Dulbecco’s medium (endotoxin ,0.05 ng/ml) was from Whittaker
Bioproducts (Walkersville, MD). Fetal bovine serum was from Hyclone
Laboratories (Logan, UT). Nonessential amino acids were from Irvine
Scientific (Santa Ana, CA). [5,6,8,9,11,12,14,15-3H]Arachidonic acid
(specific activity, 100 Ci/mmol) was from NEN Life Science Products,
and 1-palmitoyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine (specific
activity, 54 mCi/mmol) was from Amersham Pharmacia Biotech. PAF,
LPS (Escherichia coli 0111:B4), and actinomycin D were from Sigma.
Methylarachidonyl fluorophosphonate (MAFP) and NS-398 were from
Biomol (Plymouth Meeting, PA). Antibodies against murine COX isoforms were generously provided by Dr. W. L. Smith (Department of
Biochemistry, Michigan State University, East Lansing, MI). The antibody against Group IV cPLA2 was generously provided by Dr. Ruth
Kramer (Lilly). The sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), was generously provided by Dr. Edward Mihelich (Lilly). cDNA probes for Groups V and
IIA sPLA2s were synthesized as described previously (11). cDNA probes
for murine glyceraldehyde-3-phosphate dehydrogenase were from Cayman (Ann Arbor, MI).
Cell Culture and Labeling Conditions—P388D1 cells were maintained at 37 °C in a humidified atmosphere at 90% air and 10% CO2 in
Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and nonessential amino acids. P388D1 cells were plated at
106/well, allowed to adhere overnight, and used for experiments the
following day. All experiments were conducted in serum-free Iscove’s
* This work was supported by Grants HD 26,171 and GM 20,501 from
the National Institutes of Health. This work was presented at the
Keystone Symposium on Signal Transduction and Lipid Second Messengers held in Taos, New Mexico on March 1–6, 1998. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 619-534-3055;
Fax: 619-534-7390.
1 The abbreviations used are: AA, arachidonic acid; PAF, plateletactivating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV
cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX,
cyclooxygenase (prostaglandin H2 synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca
21-independent phospholipase A2.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 18, Issue of April 30, pp. 12263–12268, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 12263
modified Dulbecco’s medium. When required, radiolabeling of the
P388D1 cells with [
3H]AA was achieved by including 0.5 mCi/ml [3H]AA
during the overnight adherence period (20 h). Labeled AA that had not
been incorporated into cellular lipids was removed by washing the cells
four times with serum-free medium containing 1 mg/ml albumin.
Measurement of PGE2 Production and Extracellular [
3H]AA Release—The cells were placed in serum-free medium for 30 min before
the addition of LPS for different periods of time. Afterward, the supernatants were removed and cleared of detached cells by centrifugation,
and PGE2 was quantitated using a specific radioimmunoassay (PersPective Biosystems, Framingham, MA). For [3H]AA release experiments, cells labeled with [3H]AA were used, and the incubations were
performed in the presence of 0.5 mg/ml bovine serum albumin. The
supernatants were removed, cleared of detached cells by centrifugation,
and assayed for radioactivity by liquid scintillation counting. The
standard LPS/PAF stimulation protocol for immediate responses has
been described previously (1). Briefly, the cells were incubated for 1 h
with 200 ng/ml LPS followed by a 10-min incubation with 100 nM PAF.
Western Blot Analyses—The cells were overlaid with a buffer consisting of 10 mM Hepes, 0.5% Triton X-100, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 20 mM leupeptin, 20 mM aprotinin, pH
7.5. Samples from cell extracts (10 mg for cPLA2, 200 mg for COX) were
separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide
gel) and transferred to Immobilon-P (Millipore). For cPLA2 mobility
shift studies, 24-cm acrylamide gels were run. Nonspecific binding was
blocked by incubating the membranes with 5% nonfat milk in phosphate-buffered saline for 1 h. Membranes were then incubated with
anti-cPLA2, anti-COX-1, or anti-COX-2 antisera and treated with
horseradish peroxidase-conjugated protein A (Amersham Pharmacia
Biotech). Bands were detected by enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech).
Northern Blot Analyses—Total RNA was isolated from unstimulated
or LPS-stimulated cells by the TriZOL reagent method (Life Technologies, Inc.), exactly as indicated by the manufacturer. Fifteen mg of RNA
were electrophoresed in a 1% formaldehyde/agarose gel and transferred
to nylon filters (Hybond, Amersham Pharmacia Biotech) in 103 SSC
buffer. Hybridizations were performed in QuickHyb solution (Stratagene) following the manufacturer’s instructions. 32P-Labeled probes
for Group IIA or glyceraldehyde-3-phosphate dehydrogenase were coincubated with the filters for 1 h at 66 °C followed by three washes with
23 SSC containing 0.1% SDS at room temperature for 30 min. A final
wash was carried out at 60 °C for 30 min with 13 SSC containing 0.1%
SDS. For Group V sPLA2, hybridizations were performed in ExpressHyb solution (CLONTECH) following the manufacturer’s instructions. The 32P-labeled probes were co-incubated with the filters for 1 h
at 66 °C followed by two washes with 23 SSC containing 0.05% SDS for
15 min: the first at room temperature and the second at 40 °C. Afterward the filters were washed twice more with 0.13 SSC containing
0.1% SDS for 15 min at room temperature. Bands were visualized by
autoradiography.
Phospholipase A2 Assay—Aliquots (50–100 ml) of supernatants from
LPS-treated cells were assayed for PLA2 activity as follows. The assay
mixture (500 ml) consisted of 100 mM 1-palmitoyl-2-[14C]palmitoyl-snglycero-3-phosphocholine substrate (2000 cpm/nmol), 10 mM CaCl2, 100
mM KCl, 25 mM Tris-HCl, pH 8.5. Reactions proceeded at 40 °C for 30
min, after which [14C]palmitate release was determined by a modified
Dole procedure (10).
Antisense Inhibition Studies in P388D1 Cells—Transient transfection of P388D1 cells with antisense oligonucleotide, ASGV-2, or its sense
counterpart, SGV-2, plus LipofectAMINE was carried out as described
(11). Briefly, P388D1 cells were transfected with oligonucleotide (125
nM) in the presence of 5 mg/ml LipofectAMINE (Life Technologies, Inc.)
under serum-free conditions for 8 h prior to treating the cells with or
without 100 ng/ml LPS for 10 h after transfection (11). Antisense
oligonucleotide ASGV-2 (59-GGA CUU GAG UUC UAG CAA GCC-39) is
complementary to nucleotides 64–84 of the mouse Group V PLA2 gene.
SGV-2 (59-GGC UUG CUA GAA CUC AAG UCC-39) is the sense complement of ASGV-2.
For Group VI iPLA2 antisense experiments, a protocol identical to
that reported elsewhere was followed (12).
Data Presentation—Assays were carried out in duplicate or triplicate. Each set of experiments was repeated three times with similar
results. Unless otherwise indicated, the data presented are from representative experiments.
RESULTS
AA Release in a Novel P388D1 Cell Clone (MAB)—Stimulation of murine P388D1 macrophages with nanomolar amounts
of the receptor agonist PAF results in very little AA mobilization. However, preincubation of the cells with LPS prior to
stimulation with PAF increases the release of AA by these cells
well above unstimulated levels, the relative magnitude of the
response being dependent on the cell batch (13, 14). We have
now selected by limit dilution a clone of the P388D1 cells
termed MAB, which shows a remarkably higher [3H]AA release
response to LPS/PAF when compared with the ATCC batch of
P388D1 cells from which the MAB clone was obtained (Fig. 1).
More interestingly, in addition to an immediate response to
LPS/PAF (Fig. 2A), cells from the MAB clone also exhibited a
delayed [3H]AA release response, spanning several hours, to
LPS alone (Fig. 2A). The dose response of the effect of LPS on
long term [3H]AA release is shown in Fig. 2B. The maximal
effect was observed at a LPS dose as low as 10 ng/ml.
Prostaglandin Production by P388D1/MAB Cells—Fig. 2C
shows the time course of PGE2 production by LPS in these cells
as measured by radioimmunoassay, which corresponded well
with the [3H]AA mobilization response. Thus, LPS-induced
PGE2 barely increased above controls within the first 3 h of
treatment, rising afterward, and reaching a plateau after 12 h.
The effect of LPS on the protein levels of the two COX
isoenzymes these cells express (2) was assessed by immunoblot.
Expression of COX-1 did not change along the time course of
LPS activation (data not shown), whereas COX-2 levels noticeably increased with maximal expression between 12 and 18 h
(Fig. 3A). Interestingly, LPS-induced COX-2 expression almost
parallels PGE2 generation (cf. Figs. 2C and 3A), suggesting
that COX-2 is the enzyme responsible for LPS-induced PGE2
synthesis. Indeed, the COX-2-specific inhibitor NS-398 (15)
completely blocked LPS-induced PGE2 production (Fig. 3B).
Therefore, LPS-delayed PGE2 generation depends exclusively
on COX-2, irrespective of the continued presence of COX-1.
cPLA2 Involvement in LPS-induced Long Term Responses—
Expression of the Group IV cPLA2 protein in P388D1/MAB cells
was constitutive and did not change after exposure to LPS. To
address the possible involvement of cPLA2 in LPS-induced AA
mobilization in P388D1/MAB cells, experiments were conducted with MAFP, an inhibitor that has previously been
shown to block the immediate, cPLA2-dependent [
3H]AA release in LPS/PAF-treated macrophages (1). As shown in Fig. 4,
MAFP strongly blocked the LPS-induced long term [3H]AA
release response. MAFP has recently been observed to inhibit
the Group VI iPLA2 in addition to the cPLA2 (10). Therefore, it
FIG. 1. AA release in a new P388D1 cell clone, MAB. [
3H]AA
release in LPS/PAF-treated (closed bars) or untreated (open bars) was
assayed in cells from the ATCC or the MAB clone as indicated. The cells
were incubated with 200 ng/ml LPS for 1 h followed by a 10-min
incubation with 100 nM PAF.
Long Term Prostaglandin Production in Macrophages12264
could be possible that part of the MAFP effects reported herein
resulted from inhibition of the iPLA2 in addition to any effect
on the cPLA2. We have recently described the inhibition of
iPLA2 expression in P388D1 cells by antisense RNA oligonucleotides (12). Using this technique, we have been able to
significantly inhibit iPLA2 expression, assayed both by protein
content by immunoblot and activity by a specific in vitro assay
(12). Antisense RNA inhibition of the iPLA2 under identical
conditions as those shown previously (12) showed no reduction
in AA release in response to LPS (not shown). Therefore these
data make it likely that the above reported effects of MAFP on
the response are because of the inhibition of the cPLA2.
Role of sPLA2—LPS-induced long term [
3H]AA release was
also noticeably blocked by the selective sPLA2 inhibitor
LY311727 (17), indicating that in addition to the cPLA2, a
sPLA2 is also involved in the process (Fig. 4). PGE2 production
by LPS was also inhibited by LY311727 by about 90%. Although originally described as a selective Group II sPLA2 inhibitor (17), we have recently shown that this compound is also
a potent Group V sPLA2 inhibitor (18).
Our previous work (11) has demonstrated that P388D1 cells
express measurable message levels for Group V sPLA2, both
under unstimulated and LPS/PAF-stimulated conditions. However, message levels for Groups IIA sPLA2 or IIC sPLA2 were
undetectable even by reverse transcriptase-polymerase chain
reaction (11). As shown in Fig. 5A, an antisense oligonucleotide
specific for Group V sPLA2 (ASGV-2) strongly blocked PGE2
FIG. 2. LPS-stimulated long term [3H]AA metabolism in
P388D1 macrophages. A, time course of [
3H]AA release in response to
LPS/PAF (the latter was added 1 h after the former, and the incubations proceeded for the time indicated) (closed triangles), LPS alone
(closed circles), or neither (open circles). B, dose response of the LPS
effect (20-h incubation). C, the time course of PGE2 production by cells
treated with (closed circles) or without (open circles) 100 ng/ml LPS.
FIG. 3. LPS-stimulated long term PGE2 production. A, the effect
of 100 ng/ml LPS on COX-2 protein levels at the indicated times (h) as
measured by immunoblot. B, the effect of NS-398 on LPS-induced PGE2
production. The cells were treated with (closed bars) or without (open
bars) 5 mM NS-398 for 20 min before the addition of LPS for 18 h.
FIG. 4. Effect of MAFP and LY311727 on LPS-induced AA release. The cells were treated with 25 mM MAFP (closed triangles), 25 mM
LY311727 (closed squares), or neither (closed circles) for 20 min before
the addition of LPS. Open circles denote control incubations, i.e. those
that received neither LPS nor inhibitors. The inhibitors alone did not
change the control release.
Long Term Prostaglandin Production in Macrophages 12265
production in LPS-treated cells, whereas its sense control
(SGV-2) had no effect. Moreover, mRNA analyses by Northern
blot at long times of stimulation with LPS confirmed the presence of mRNA for Group V sPLA2 (Fig. 5, B and C) but not for
Group IIA sPLA2 (data not shown). The signal for Group V
sPLA2 markedly increased after LPS stimulation, reaching a
peak at approximately 10 h.
PLA2 activity measurements in the supernatants of LPSstimulated cells revealed a time-dependent increase in activity
(Fig. 6), which correlated well with the time course of Group V
sPLA2 mRNA induction (cf. Figs. 5B and 6). Extracellular PLA2
activity was decreased if the experiments were conducted in
the presence of the RNA synthesis inhibitor actinomycin D
(Fig. 7A). This increased extracellular activity was found to
correspond to that of Group V sPLA2 by the following criteria:
(i) it was completely inhibited by the sPLA2 inhibitor LY311727
(Fig. 7B) and (ii) it was decreased in supernatants from cells
treated with an antisense RNA oligonucleotide specific for
Group V sPLA2, ASGV-2 (11) (Fig. 7C).
Role of cPLA2 in sPLA2 Activation—Our previous studies
have indicated that the immediate AA release triggered by
LPS/PAF in these cells involves the sequential action of both
cPLA2 and sPLA2, with the activity of the latter being dependent on previous activation of the former (1, 2). Thus we sought
to investigate if a similar cross-talk exists between the two
enzymes during long term stimulation conditions. We found
that no increased PLA2 activity beyond what was observed in
the basal state could be found in supernatants from cells
treated with MAFP (Fig. 6). In addition, the cPLA2 inhibitor
markedly decreased the LPS-induced expression of Group V
sPLA2 mRNA (Fig. 8).
DISCUSSION
A striking hallmark of the immunoinflammatory response is
the generation of oxygenated derivatives of AA such as the
prostaglandins. The response of major prostaglandin-secreting
cells such as macrophages and mast cells to proinflammatory
stimuli is generally biphasic (4). The first phase is completed
within minutes after the addition of the stimulus, whereas the
second phase usually takes several hours (4). Using the murine
macrophage-like cell line P388D1, we have been studying the
molecular mechanisms responsible for AA mobilization and
prostaglandin production in response to LPS/PAF. When
primed by LPS, these cells will respond to Ca21-mobilizing
stimuli such as PAF by generating a rapid burst of free AA,
part of which is converted to prostaglandins such as PGE2.
Strikingly, this process is completed within a few minutes after
the addition of PAF (19). No free AA or prostaglandins are
produced after the immediate phase is completed, not even
after several hours of cell exposure to LPS/PAF (13). Such a
behavior, which is abnormal for a macrophage cell, has prevented us from studying the molecular mechanisms responsible for delayed prostaglandin production in macrophages. In an
attempt to overcome this problem, we subcloned the P388D1
cells by limit dilution, and selecting on the basis of high responsivity to LPS/PAF, we obtained a clone termed MAB,
FIG. 5. Group V sPLA2 involvement in AA release. A, the effect of
a specific Group V antisense oligonucleotide (AGV-2) or its sense control
(SGV-2) on PGE2 production in cells treated without (open bars) or with
(closed bars) 100 ng/ml LPS for 10 h. None denotes incubations that
received no oligonucleotide. B, the effect of LPS on Group V sPLA2
message levels as determined by Northern blot. Total RNA from cells
incubated with (1) or without (2) 100 ng/ml LPS for the indicated
periods of time was isolated and analyzed by Northern blot with probes
specific for Group V sPLA2 or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). C, densitometric analysis of the Group V sPLA2 signals
normalized for the glyceraldehyde-3-phosphate dehydrogenase signal
in each lane.
FIG. 6. Effect of MAFP on the appearance of sPLA2 activity in
the supernatants of P388D1 cells and the effect of MAFP. The
cells, pretreated with (closed triangles) or without (closed circles) 25 mM
MAFP for 20 min, were challenged with (closed symbols) or without
(open circles) 100 ng/ml LPS for the indicated times. Afterward, supernatants were collected and assayed for PLA2 activity. The amount of
PLA2 activity detected in supernatants of cells not treated with LPS
(open circles) was not changed whether the cells were pretreated or not
with MAFP.
Long Term Prostaglandin Production in Macrophages12266
which shows enhanced sensitivity to LPS/PAF in the immediate phase (min) and exhibits a delayed response (h) to LPS
alone.
Using the MAB clone, we have characterized the LPS-induced delayed prostaglandin production in terms of the role
played by distinct PLA2 enzymes and their coupling with downstream COX enzymes during LPS signaling. Our previous work
on the immediate response of the cells to LPS/PAF highlighted
the very important role played by the novel Group V sPLA2 as
the provider of most of the free AA directed to PGE2 biosynthesis (11). Herein, several lines of evidence suggest that Group
V sPLA2 also behaves as a major provider of AA for the delayed
phase of PGE2 production in LPS-treated cells. First, delayed
[3H]AA release and PGE2 production correspond with the induction of Group V sPLA2 mRNA and enhanced secretion of a
sPLA2-like activity to the supernatants, with no change in the
constitutive levels of cPLA2 and no detectable induction of
Group IIA sPLA2. Second, delayed PGE2 production is strongly
blunted by LY311727, a selective sPLA2 inhibitor. Third, an
antisense oligonucleotide specific for Group V sPLA2 (11) suppresses Group V sPLA2 activity and inhibits delayed PGE2
production. Our conclusions in this regard fully agree with
recent works by Kudo and co-workers (20, 21) that were published while this manuscript was under review. By using transfection techniques, Kudo and co-workers (20, 21) have also
documented the importance of Group V sPLA2 in delayed AA
release and PGE2 production.
Our data have also implicated the cPLA2 as an important
step in LPS signaling by enabling the subsequent action of the
sPLA2. Thus the cPLA2 inhibitor MAFP (1) markedly blocked
both long term [3H]AA release and Group V sPLA2 mRNA
induction. Collectively, these results suggest an intriguing
cross-talk between the cPLA2 and the Group V sPLA2 for the
delayed phase of prostaglandin production in macrophages.
This is a very interesting concept because cross-talk appears to
exist as well between these two enzymes during the immediate
phase of prostaglandin production (1, 2). In the immediate
phase, cPLA2 activation generates a rapid and early burst of
free AA inside the cell that enables sPLA2 activation by an as
yet unidentified mechanism (1, 2). In the delayed phase, cPLA2
activation influences sPLA2 apparently by regulating sPLA2
mRNA levels.
Cross-talk between cPLA2 and sPLA2 in the immediate
phase of prostaglandin production was also found to take place
in mast cells (3) when the same protocol originally used in
macrophages (1) was employed. Furthermore, a recent study by
Kuwata et al. (22) about fibroblasts suggests that cross-talk
between cPLA2 and sPLA2 in the delayed phase could also
constitute a general mechanism of activation. Using a different
cPLA2 inhibitor, arachidonyl trifluoromethyl ketone, Kuwata
et al. (22) found that cPLA2 inhibition blocked sPLA2 expression in fibroblasts, leading to reduced PGE2 generation. The
FIG. 7. Effect of different treatments on the appearance of
PLA2 activity in supernatants from LPS-treated cells. A, the
effect of actinomycin D is shown. PLA2 activity in the supernatants
from cells treated with LPS plus the indicated concentrations of actinomycin D for 18 h is indicated. Control denotes incubations carried out
without either LPS or actinomycin D. B, blockage by LY311727 of the
PLA2 activity of supernatants from LPS-treated or untreated cells. An
aliquot of the culture medium of cells treated without (Control) or with
LPS for 18 h was incubated with or without 25 mM LY311727 for 20 min
at 40 °C and then assayed for PLA2 activity. C, the effect of a specific
Group V antisense oligonucleotide (AGV-2) or its sense control (SGV-2)
on PLA2 activity in the supernatants from LPS-treated or untreated
cells.
FIG. 8. The effect of 25 mM MAFP on Group V sPLA2 expression
from LPS-treated (100 ng/ml, 18 h) or untreated cells.
Long Term Prostaglandin Production in Macrophages 12267
study by Kuwata et al. (22) is interesting not only because it
supports the possible universality of cross-talk between cPLA2
and sPLA2 but because the sPLA2 expressed by rat fibroblasts
is a Group IIA enzyme, not Group V. This lends further support
to the emerging notion that Group IIA and Group V sPLA2 may
be functionally redundant (23). In addition, Kuwata et al. (22)
were able to show that overcoming cPLA2 inhibition by exogenous AA partially restored the Group IIA sPLA2 expression.
These results suggest that the AA mobilized by cPLA2 is responsible for cross-talk between cPLA2 and sPLA2 (22). This is
again reminiscent of what happens in the immediate phase of
activation, wherein the cPLA2-derived AA is also responsible
for cross-talk between cPLA2 and sPLA2, albeit by different
mechanisms (1, 2). Unfortunately, inhibition by MAFP of
Group V sPLA2 expression and activity could not be reversed in
our macrophage studies with LPS alone by supplementing the
medium with exogenous AA (up to 100 mM). This was not
unexpected because P388D1 cells manifest an extraordinarily
high capacity to import free AA from exogenous sources and
incorporate it into membranes (19, 24, 25), which is much
higher than that of most other cells (26). Thus, the half-life of
the free AA in the cell would be too short to adequately mimic
the low but continued production of AA-derived cPLA2 upon
long term LPS exposure.
A model has recently emerged suggesting differential actions
of COX-1 and COX-2 by virtue of differential coupling to distinct PLA2s (2, 3, 6, 8, 20, 21, 27). Thus, depending on whether
cPLA2 or sPLA2 is the provider of free AA, either COX-1 or
COX-2 would be responsible for PGE2 release. However, which
PLA2 form couples to which COX isoform appears to depend
strongly on cell type. We have recently demonstrated that the
immediate, PAF receptor-mediated phase of PGE2 production
in LPS-primed macrophages involves sPLA2 coupling to COX-2
(2). The current results support a similar kind of coupling for
the delayed PGE2 production in LPS-treated cells. Identical
coupling has been suggested by Arm and co-workers (6) for the
delayed phase of PGE2 generation in mast cells. These results
raise another interesting concept regarding the regulation of
PGE2 during both phases of activation. As is the case for AA
release (Fig. 2A), we have observed that the amount of PGE2
generated during the Ca21-dependent short term stimulation
is comparable to the amount produced in the late phase. It
follows from this comparison that although the effector enzymes involved in the response are the same (i.e. cPLA2, sPLA2,
COX-2), the regulatory mechanisms differ. Thus, in the short
phase at low levels of COX-2, it appears that the dramatic burst
in AA release is what determines the amount of PGE2 produced. In contrast, in the delayed phase at comparably lower
AA availability, it appears that both the induction of large
amounts of COX-2 protein and of the AA provider, Group V
sPLA2, determine the amount of PGE2 produced.
It is important to note, however, that our results have not
excluded that a minor fraction of the long term PGE2 produced
in response to LPS could arise from the AA generated by the
cPLA2. Should this be the case, some cPLA2/COX-2 coupling
may exist as well, similar to what has been suggested by Reddy
and Herschman (3) for delayed PGD2 production in mast cells
and by Murakami et al. (5) in cells derived from Group IIAdeficient mice. The striking feature of the current work is that
although COX-1 is present in active form in the P388D1 cells
(2), it appears to be spared from the process of long term PGE2
production. This finding remains unexplained but has recently
been recognized in other cell types as well (6, 8, 22). Recent
work by Spencer et al. (16) showed no differences in the distribution of COX-1 versus COX-2 among subcellular fractions in a
variety of cells. Thus subcellular compartmentalization may
not be the cause for COX-1 not being utilized during LPS
signaling. Other putative explanations may include the existence of COX-selective regulatory components, selective coupling to terminal PG synthases, or kinetic differences in AA
utilization by the two isoforms.
In summary, we have established a subclone of P388D1 cells,
MAB, that displays long term responsiveness to LPS in terms
of PGE2 generation. We have confirmed (11) that these cells
express Group V sPLA2, not Group IIA sPLA2, and found that
(i) Group V sPLA2 is a key enzyme in long term AA mobilization as well and (ii) Group V sPLA2 is functionally coupled to
COX-2. Furthermore, our results have suggested that cPLA2
plays a key role in long term AA mobilization, at least partly by
regulating the expression of Group V sPLA2.
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