From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
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ABSTRACT |
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Activation of rat fibroblastic 3Y1 cells with
interleukin-1 (IL-1
) and tumor necrosis factor
(TNF
)
induced delayed prostaglandin (PG) E2 generation over
6-48 h, which occurred in parallel with de novo induction
of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the
constitutive expression of type IV cytosolic PLA2
(cPLA2) and COX-1. Types V and IIC sPLA2s were
barely detectable in these cells. Studies using an anti-type IIA
sPLA2 antibody, sPLA2 inhibitors, and a type
IIA sPLA2-specific antisense oligonucleotide revealed that
IL-1
/TNF
-induced delayed PGE2 generation by these
cells was largely dependent on inducible type IIA sPLA2,
which was functionally linked to inducible COX-2. Delayed
PGE2 generation was also suppressed markedly by the
cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA
sPLA2, but not COX-2, expression. AACOCF3
inhibited the initial phase of cytokine-stimulated arachidonic acid
release, and supplementing AACOCF3-treated cells with
exogenous arachidonic acid partially restored type IIA
sPLA2 expression. These results suggest that certain
metabolites produced by the cPLA2-dependent
pathway are crucial for the subsequent induction of type IIA
sPLA2 expression and attendant delayed PGE2 generation. Some lipoxygenase-derived products might be involved in
this event, since IL-1
/TNF
-induced type IIA sPLA2
induction and PGE2 generation were reduced markedly by
lipoxygenase, but not COX, inhibitors. In contrast, Ca2+
ionophore-stimulated immediate PGE2 generation was
regulated predominantly by the constitutive enzymes cPLA2
and COX-1, even when type IIA sPLA2 and COX-2 were
maximally induced after IL-1
/TNF
treatment, revealing functional
segregation of the constitutive and inducible PG biosynthetic
enzymes.
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INTRODUCTION |
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Two kinetically different prostaglandin (PG)1-generating pathways from endogenous arachidonic acid, the immediate and delayed phases, imply the recruitment of different sets of biosynthetic enzymes, expression and activation of which are tightly regulated by distinct transmembrane signalings (1). The immediate phase of PG biosynthesis, occurring within several minutes of stimulation, is elicited by agonists that mobilize intracellular Ca2+ and characterized by a burst release of arachidonic acid initiated by phospholipase A2 (PLA2) and subsequent conversion to bioactive PGs by the sequential actions of cyclooxygenase (COX) and terminal PG synthases. The delayed phase of PG biosynthesis is accompanied by the continuous supply of arachidonic acid and its conversion to PGs, often PGE2, over long culture periods following growth or proinflammatory stimuli.
Segregated utilization of the two COX isoforms in these two distinct
phases has been demonstrated in several systems (2-4). COX-1 is
constitutively expressed in most cells and tissues and is generally
thought to serve certain physiologic housekeeping functions, whereas
COX-2 is dramatically induced in response to a wide variety of stimuli,
and is thought to contribute to the generation of PGs at certain stages
of cell proliferation and differentiation and at sites of inflammation
(5). The absolute requirement for COX-2 in the delayed PG generation,
irrespective of the constitutive presence of COX-1, has been shown by
studies using COX-2-selective inhibitors, antisense oligonucleotides, and knockout mice (2-4, 6). COX-1 is involved in immediate PG
generation, such as thromboxane A2 generation by activated platelets (7) and the IgE-dependent immediate phase of
PGD2 generation by mast cells (2, 4). In contrast,
interleukin (IL)-1-primed, platelet-derived growth factor-initiated
immediate PGE2 generation by mouse calvaria cells depends
on IL-1
-induced COX-2 in preference to constitutive COX-1 (8).
Several explanations of the functional segregation of the two COX
isoforms have been proposed, such as their different subcellular
localizations (9) and their different substrate concentration
requirements (10). In fact, the finding that the utilization of
arachidonic acid by COX-2 is favored over COX-1 at low peroxide
concentrations (10) appears consistent with the involvement of COX-1 in
the burst of events that follow immediate activation and of COX-2 in
the delayed phase, in which only a limited amount of arachidonic acid
would be supplied continuously. Moreover, preferential coupling between
particular PLA2s, COXs, and terminal PG synthases have been
suggested by several recent studies (11-13).
The enzymatic properties of type IV 85-kDa cytosolic PLA2 (cPLA2), i.e. arachidonate selectivity, Ca2+-dependent translocation, and phosphorylation-dependent activation via kinases belonging to the mitogen-activated protein kinase family (14, 15), are in agreement with its role in immediate PG biosynthesis, which is usually accompanied by rapid and transient cytoplasmic Ca2+ mobilization. Under such conditions, cPLA2 translocates to the perinuclear envelope and endoplasmic reticular membranes (16, 17), where downstream COXs, 5-lipoxygenase (LOX), 5-LOX-activating protein, and some terminal PG and leukotriene synthases colocalize (9, 18-20). The functional association of cPLA2 with constitutive COX-1 resulting in immediate PG biosynthesis has been demonstrated in several studies (21, 22). cPLA2 has been also implicated in the inducible COX-2-dependent delayed PG generation lasting for hours (12, 23, 24) and COX-2-dependent immediate PGE2 generation (8) that are initiated and primed, respectively, by inflammatory stimuli.
Secretory PLA2 (sPLA2) enzymes comprise a growing family of distinct enzymes with similar molecular masses of about 14 kDa. To date, five mammalian sPLA2s, designated types I, IIA, IIC, V, and X, have been identified (1, 25-27). Type I sPLA2 is abundantly expressed in the pancreas, where it functions as a digestive enzyme for dietary phospholipids. It is also expressed in several nondigestive organs, where it may act as a regulator of cellular functions via specific sPLA2 receptor (28, 29). Type IIA sPLA2 was originally isolated from inflamed sites and inflammatory cells, is induced by proinflammatory stimuli, and is thought to play some roles in cellular inflammatory responses (1, 30). Type IIA sPLA2 has been shown to augment the delayed phase of PG generation by several cells in response to proinflammatory cytokines (31-34), and further support for these biological actions has been supplied by several animal studies (35-37). Recently, two groups have demonstrated that type V sPLA2, the transcript of which was originally found to be expressed in the heart and lung (38), is crucial for stimulus-initiated PG biosynthesis by mouse macrophages (39) and mast cells (40). Type IIC sPLA2 is abundantly expressed in rodent testes, whereas it is a nonfunctional pseudogene in human (41). The overall structures of types IIA, IIC, and V sPLA2s, the genes of which are tightly linked on human chromosome 1, are more closely related to one another than to that of type I sPLA2, the gene of which maps to human chromosome 12 (26). More recently, a novel group X sPLA2 has been cloned; it is distributed in immune tissues, exhibits some features characteristic of both types I and IIA sPLA2s, and maps to human chromosome 16 (27). Thus, the sPLA2 family is growing rapidly; therefore, it has been proposed that some of the previously described functions of sPLA2s, particularly type IIA sPLA2, need to be reevaluated, since studies based upon enzyme activities and using inhibitors or antibodies against type IIA sPLA2 may not discriminate these sPLA2s (26).
Here, we report the regulation of PGE2 biosynthesis in rat fibroblastic 3Y1 cells, which have the capacity to express high levels of type IIA sPLA2, cPLA2 and the two COXs and to exhibit both immediate and delayed PGE2 generation in response to appropriate stimuli. Pharmacologic, immunochemical, and genetic studies have provided evidence that both cPLA2 and type IIA sPLA2 are required for COX-2-dependent delayed, whereas cPLA2, but not type IIA sPLA2, is utilized for COX-1-dependent immediate, PGE2 biosynthesis by 3Y1 cells. Of particular interest is that functional cPLA2 may be crucial for the subsequent type IIA sPLA2 induction and attendant COX-2-dependent delayed PGE2 generation. Thus, this study (i) reconfirmed the involvement of type IIA sPLA2 in biological responses, (ii) demonstrated significant cross-talk between the two Ca2+-dependent PLA2s where one enzyme is required for the induction of the other, and (iii) revealed segregated coupling of discrete PLA2 and COX enzymes in the different phases of PG biosynthesis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Mouse IL-1 was purchased from Genzyme. Human
TNF
was provided by Dr. H. Ishimaru (Asahi Chemical Industry).
Rabbit antiserum to human cPLA2 was provided by J. D. Clark (Genetics Institute), mouse cPLA2 cDNA by M. Tsujimoto (RIKEN Institute), rabbit antiserum to mouse COX-1 by W. L. Smith (Michigan State University), mouse COX-2 cDNA and the
COX-2 inhibitor NS-398 (42) by J. Trzaskos (Merck DuPont), rat type V
sPLA2 cDNA by J. A. Tischfield (Indiana University
School of Medicine), and mouse
-actin cDNA by J. P. Arm
(Harvard Medical School). Mouse COX-1 cDNA, rabbit antiserum to
COX-2, arachidonic acid, and the PGE2 enzyme immunoassay
kit were purchased from Cayman Chemical. The LOX inhibitors, including nordihydroguaiaretic acid (NDGA; general LOX inhibitor), AA-861 (5-LOX
inhibitor), cinnamyl-3,4-dihydroxy-
-cyanocinnamate (12-LOX inhibitor) and 5,8,11,14-eicosatetraynoic acid (15-LOX inhibitor) (43),
were purchased from BIOMOL Research Laboratories. Rat type IIA
sPLA2 cDNA (44) and rabbit polyclonal antibody against rat type IIA sPLA2 (45) were prepared as described
previously. The cDNA probes for rat cPLA2 and rat type
IIC sPLA2 were obtained by the reverse
transcriptase-polymerase chain reaction (RT-PCR) using RNA extracted
from 3Y1 cells (34) and rat testis (41), respectively, as described
previously. The antisense and sense oligonucleotides and RT-PCR primers
were obtained from Greiner Japan. The type IIA sPLA2
inhibitor thielocin A1 (35, 36) was donated by Dr. T. Yoshida (Shionogi
Pharmaceutical). A23187 and the cPLA2 inhibitor
arachidonoyl trifluoromethyl ketone (AACOCF3) (47) were
purchased from Calbiochem. Aspirin, dexamethasone, and heparin were
purchased from Sigma. CellFectin reagent, Opti-MEM medium, and TRIzol
reagent were from Life Technologies, Inc. The IL-3-dependent mouse bone marrow-derived mast cell line
MC-MKM, which originated from BALB/cJ mice (48), was cultured in
enriched medium supplemented with 50% (v/v) WEHI-3B-conditioned medium as a source of IL-3 and 100 ng/ml recombinant mouse c-kit
ligand, which had been expressed using a baculovirus system (2).
Activation of 3Y1 Cells--
The 3Y1 cells were a gift from Dr.
Y. Uehara (National Institute of Infectious Disease, Tokyo, Japan) and
maintained in culture medium composed of Dulbecco's modified Eagle's
medium (DMEM; Nissui Pharmaceutical) supplemented with 10% (v/v) fetal
calf serum (FCS), penicillin/streptomycin (100 units/ml and 100 µg/ml, respectively) (Flow Laboratories) and 2 mM
glutamine (Life Technologies, Inc.). The media of 3Y1 cells that had
attained 60-80% confluence in six-well plates (Iwaki) were replaced
with 2 ml of DMEM supplemented with 2% FCS. After culture for 24 h, 1 ng/ml IL-1, 100 units/ml human TNF
, or both were added to
the cultures to assess the delayed response. Replicated cells were
activated with 1 µM A23187 for 10 min in DMEM
supplemented with 2% FCS to assess the immediate response. The
supernatants were taken for PGE2 enzyme immunoassay. For
immunoblot analysis and the PLA2 assay (see below), the
cells were trypsinized, washed once with 10 mM phosphate
buffer, pH 7.4, containing 150 mM NaCl
(phosphate-buffered saline), resuspended in 10 mM Tris-HCl
(pH 7.4) containing 1 mM EDTA and 250 mM
sucrose at 1 × 107 cells/ml, and disrupted by 1-min
pulse sonication (50% work cycle, setting 4) with a Branson
Sonifier (Branson Sonic Power Co.). As for the RNA blot analysis,
TRIzol was directly added to the cell monolayer.
RNA Blotting--
All the procedures were performed as described
elsewhere (34). Briefly, equal amounts (10 µg) of total RNA, purified
using TRIzol reagent, were applied to each lane of 1.2% (w/v)
formaldehyde-agarose gels, electrophoresed, and transferred to
Immobilon-N membranes (Millipore). The resulting blots were then
sequentially probed with cPLA2, type IIA sPLA2,
COX-2, and -actin cDNA probes that had been labeled with
[32P]dCTP (Amersham Life Science) by random priming
(Takara Biomedicals). All hybridizations were carried out at 42 °C
overnight in a solution comprising 50% (v/v) formamide, 0.75 M NaCl, 75 mM sodium citrate, 0.1% (w/v) SDS,
1 mM EDTA, 10 mM sodium phosphate, pH 6.8, 5 × Denhardt's solution (Sigma), 10% (w/v) dextran sulfate
(Sigma), and 100 µg/ml salmon sperm DNA (Sigma). The membranes were
washed three times at room temperature with 150 mM NaCl, 15 mM sodium citrate, 1 mM EDTA, 0.1% SDS, and 10 mM sodium phosphate, pH 6.8, for 5 min each, followed by
two washes at 55 °C with 30 mM NaCl, 3 mM
sodium citrate, 1 mM EDTA, 0.1% SDS, and 10 mM
sodium phosphate, pH 6.8, for 15 min each. The blots were visualized by
autoradiography with Kodak X-Omat AR films and double intensifying
screens at
80 °C.
RT-PCR--
Specific primers for the PCR, based on the sequences
of sPLA2s reported previously (38, 41, 45), were
synthesized. The type IIA sPLA2 primers used were 5-ATG
AAG GTC CTC CTC CTG CTA G-3
and 5
-TCA GCA TTT GGG CTT CTT CC-3
(45),
type IIC sPLA2 primers were 5
-ATG GAC CTC CTG GTC TCC TCA
GG-3
and 5
-CTA GCA ATG AGT TTG TCC CTG C-3
(41), and type V
sPLA2 primers were 5
-CAG GGG GCT TGC TAG AAC TCA A-3
and
5
-AAG AGG GTT GTA AGT CCA GAG G-3
(38). The RT-PCR was carried out
using a RNA PCR kit (avian myeloblastosis virus) version-2 (Takara
Biomedicals), according to the manufacturer's instructions, using 1 µg of total RNA from IL-1
/TNF
-treated 3Y1 or mast cells as a
template. Equal amounts of each RT product were amplified by PCR with
ex Taq polymerase (Takara Biomedicals) for 30 cycles
consisting of 30 s each at 94 °C, 55 °C and 72 °C. The
amplified cDNA fragments were resolved electrophoretically on 1.5%
(w/v) agarose gels and visualized by ethidium bromide. The fragments
were then transferred onto Immobilon-N membranes and probed with
[32P]dCTP-labeled cDNAs for types IIA, IIC, and V
sPLA2s. Hybridization and subsequent washing were carried
out as described above for RNA blot analysis.
Immunoblotting-- All the procedures were performed as described previously (34). Briefly, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing (for type IIA sPLA2) and reducing (for cPLA2, COX-1, and COX-2) conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were washed once with 10 mM Tris-HCl (pH 7.2) containing 150 mM NaCl and 0.1% (v/v) Tween 20 (TBS-T) and then blocked for 1 h in TBS-T containing 3% (w/v) skim milk. After washing the membranes with TBS-T, antibodies against cPLA2, type IIA sPLA2, COX-1, and COX-2 were added at a dilution of 1:5,000 in TBS-T and incubated for 2 h. Following three washes with TBS-T, the membranes were treated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.; diluted 1:7,000) in TBS-T. After six washes with TBS-T, the protein bands were visualized using an ECL Western blot analysis system (Amersham Life Science).
Measurement of PLA2 Activity-- The PLA2 activities in the resulting lysates were measured after incubation with 100 mM Tris-HCl, pH 9.0, 4 mM CaCl2, and 2 µM 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Life Science) or 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products), as the substrates, for 30 min at 37 °C (34).
Transfection of Antisense Oligonucleotide into 3Y1
Cells--
The antisense (5-TAG CAA CAG GAG GAC CTT CAT-3
) and sense
(5
-ATG AAG GTC CTC CTG TTG CTA-3
) oligonucleotides for rat type IIA
sPLA2, corresponding to the translation initiation site,
100 nM each, were incubated individually with CellFectin
reagent (Life Technologies, Inc.) in 200 µl of Opti-MEM medium (Life
Technologies, Inc.) for 15 min at room temperature and then added to
cells that had attained 60-80% confluence in 6-well plates and been
supplemented with 800 µl of Opti-MEM. After incubation for 6 h
at 37 °C, the medium was replaced with 2 ml of DMEM supplemented
with 2% FCS in the continued presence of 100 nM
oligonucleotide. After culture for 24 h, 1 ng/ml IL-1
and 100 units/ml TNF
were added to the culture to activate the cells.
Arachidonic Acid Release--
3Y1 cells grown in 24-well plates
were preincubated overnight with [3H]arachidonic acid
(100 Ci/nmol; Amersham Life Science) that was diluted 1:500 with fresh
culture medium. After three washes with fresh medium, 250 µl of fresh
culture medium with or without IL-1 and TNF
was added to each
well and the amount of free [3H]arachidonic acid released
into each supernatant during culture was measured. The amount of
arachidonic acid released was calculated as a percentage, using the
formula [S/(S + P)] × 100, where
S and P are the radioactivities of equal portions
of supernatant and cell pellet, respectively.
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RESULTS |
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Cytokine-induced COX-2-dependent Delayed
PGE2 Generation--
When rat fibroblastic 3Y1 cells were
cultured with 1 ng/ml IL-1 and 100 units/ml TNF
, PGE2
generation started to increase after culture for 6 h, increased
markedly by 12 h, and reached its maximum to a plateau level after
24-48 h (Fig. 1A). The
concentrations of the cytokines used shown in Fig. 1 and in subsequent
experiments were based upon the results of dose-response experiments,
in which the maximal response was observed when >1 ng/ml IL-1
and
>100 units/ml TNF
were combined (data not shown). Each cytokine
alone elicited a similar, but weaker, effect than the combination of the two (data not shown).
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Expression of PLA2--
Expression of the 85-kDa
cPLA2 protein and mRNA was constitutive and did not
alter significantly during culture with IL-1/TNF
for 48 h
(Fig. 2A). Expression of the 14-kDa type IIA
sPLA2 protein was minimal before stimulation and increased
dramatically after culture with IL-1
/TNF
for 6-12 h, reaching
its maximum and plateau level after 12-48 h (Fig. 2A).
Increased expression of type IIA sPLA2 protein was
accompanied by that of its 0.9-kilobase mRNA, which was barely
detectable during the initial 3 h, then increased to reach a peak
at 24 h (approximately 60-fold increase over the control level),
and tended to decline gradually thereafter (Fig. 2A).
PLA2 activity associated with 3Y1 cells increased markedly over 6-24 h of culture with IL-1
/TNF
(Fig. 1B), in
parallel with type IIA sPLA2 protein expression (Fig.
2A). The increased PLA2 activity was sensitive
to dithiothreitol, stable after acid extraction, and neutralized by an
antibody raised against type IIA sPLA2, whereas the basal
PLA2 activity was resistant to dithiothreitol, acid-labile,
did not react with the anti-type IIA sPLA2 antibody, and
was inhibited by the cPLA2 inhibitor AACOCF3
(data not shown).
Involvement of Type IIA sPLA2 in Delayed
PGE2 Generation--
To clarify the involvement of type
IIA sPLA2 in IL-1/TNF
-induced PGE2
biosynthesis, we carried out several approaches using agents that
inhibit sPLA2 activity, prevent type IIA sPLA2
association with plasma membranes, or reduce type IIA sPLA2
expression specifically. First, we examined the effects of the
neutralizing anti-rat type IIA sPLA2 antibody (45) and the
sPLA2-specific inhibitor thielocin A1 (46) on
PGE2 generation. The addition of 10 µg/ml anti-rat type
IIA sPLA2 antibody to 3Y1 cells stimulated for 48 h
with IL-1
/TNF
attenuated PGE2 generation almost to
the basal level (Fig. 3A). Similar reduction of
PGE2 generation was observed when the cells were stimulated
with IL-1
/TNF
in the presence of 10 µg/ml thielocin A1 (Fig.
3A). The induced sPLA2 activity was virtually abrogated, whereas the cPLA2 activity was not affected
appreciably, by these agents at the concentrations we used (data not
shown). Furthermore, neither the anti-type IIA sPLA2
antibody nor thielocin A1 inhibited recombinant types IIC and V
sPLA2s, revealing their strict specificity to type IIA
sPLA2.2
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Role of cPLA2 in Type IIA sPLA2 Expression
and Delayed PGE2 Generation--
Culture of 3Y1 cells with
IL-1/TNF
for 48 h in the presence of AACOCF3
reduced PGE2 generation considerably almost to the basal
level (Fig. 5A). This
observation suggests that the AACOCF3-sensitive cPLA2 (47) is also involved in delayed PGE2
generation by 3Y1 cells, consistent with previous studies demonstrating
the involvement of cPLA2 in the delayed responses of some
cells (12, 23, 24). Interestingly, we found that AACOCF3
markedly suppressed IL-1
/TNF
-induced expression of type IIA
sPLA2 without affecting that of COX-2 (Fig. 5, B
and C). The inhibitory effect of AACOCF3 on type
IIA sPLA2 expression was evident at the mRNA level
(Fig. 5C). The addition of excess exogenous arachidonic acid
to AACOCF3-treated, IL-1
/TNF
-stimulated cells
restored type IIA sPLA2 expression to some extent (Fig. 5,
B and C), although it did not exhibit this effect
when added to unstimulated cells (data not shown). Restoration of type
IIA sPLA2 expression was only minimal when exogenous
arachidonic acid was supplied to AACOCF3-treated cells
24 h after starting the culture (Fig. 5C). As shown in
Fig. 5D, IL-1
/TNF
stimulation evoked rapid
[3H]arachidonic acid release by
[3H]arachidonic acid-prelabeled cells, and this release
was abrogated by AACOCF3. These results suggest that
activation of cPLA2 occurring during the early phase of
cell activation following cytokine treatment is crucial for the
subsequent induction of type IIA sPLA2 expression in 3Y1
cells.
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Involvement of cPLA2 and COX-1 in Ionophore-stimulated
Immediate PGE2 Generation--
When 3Y1 cells were
stimulated with 1 µM A23187, immediate PGE2
generation was elicited within 10 min, with PGE2
concentrations in the supernatants reaching approximately 20 ng/ml
(Table I). A23187-initiated immediate
PGE2 generation by cells pretreated for 12-48 h with
IL-1/TNF
, in which both type IIA sPLA2 and COX-2
proteins had been maximally induced (Fig. 2A), did not
differ significantly from that by control cells (Table I). Immediate PGE2 generation was inhibited by AACOCF3 almost
completely, but not by NS-398 (Table I). We conclude, therefore, that
A23187-induced immediate PGE2 generation by 3Y1 cells is
mediated by cPLA2 and COX-1, even when enough type IIA
sPLA2 and COX-2 to mediate IL-1
/TNF
-induced delayed
PGE2 generation are present in these cells.
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DISCUSSION |
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Several recent studies have shown that cPLA2 appears to be one of the most important PLA2 isozymes involved in regulating immediate eicosanoid generation, which is triggered by an increase in the cytoplasmic Ca2+ concentration (21, 22, 57-60). cPLA2 has also been implicated in delayed PG generation lasting for hours (12, 23, 24), although the mechanisms that activate cPLA2 under such Ca2+-less conditions remain to be elucidated. Several pieces of evidence have shown that sPLA2 enzymes also regulate certain phases of eicosanoid biosynthesis. Delayed PG generation initiated by proinflammatory stimuli is often accompanied by marked induction of type IIA sPLA2 expression and secretion, in which a relationship between type IIA sPLA2 function and cytokine-stimulated delayed PG generation has been suggested using type IIA sPLA2 inhibitors or anti-type IIA sPLA2 antibodies (31-33, 50, 51). We recently showed that cell surface-associated type IIA sPLA2 introduced forcibly into cells augmented the cytokine-induced, COX-2-dependent delayed PGE2 generation and proposed that cPLA2 and type IIA sPLA2 initiate and enhance delayed eicosanoid biosynthesis, respectively (24, 34). More recently, several investigators demonstrated that type V sPLA2, rather than type IIA sPLA2, plays an important role in relatively rapid eicosanoid biosynthesis in some murine cells expressing type V, but not IIA, sPLA2 (39, 40). These findings, together with the identification of types IIC (41) and X (27) sPLA2s, have provided new insights into the redundant functions of sPLA2 family members, which may explain why some inbred mouse strains in which the type IIA sPLA2 gene is naturally disrupted (61, 62) remain relatively normal throughout their lives.
In the present study, we examined the distinct roles of
PLA2 enzymes and their functional coupling with downstream
COX enzymes during particular phases of PG biosynthesis by the rat
fibroblastic cell line 3Y1. Several lines of evidence suggest that
sPLA2 type IIA plays an important role in
COX-2-dependent delayed PGE2 generation. First,
IL-1/TNF
-elicited delayed PGE2 generation was
accompanied by concordant induction of type IIA sPLA2 and
COX-2, with little change in the constitutive expression of
cPLA2 and COX-1 and no detectable level of types IIC and V
sPLA2 expression. Second, PGE2 generation,
which was abolished by the COX-2 inhibitor NS-398, was suppressed
markedly by a neutralizing antibody against type IIA sPLA2,
by the sPLA2 inhibitor thielocin A1, by heparin, which inhibits type IIA sPLA2-mediated PG biosynthesis by
preventing it from associating with cell surfaces (32-34), and by
dexamethasone, which suppressed type IIA sPLA2 expression.
Third, the antisense oligonucleotide directed to the translation
initiation site for type IIA sPLA2 suppressed type IIA
sPLA2 expression and reduced delayed PGE2
generation concomitantly. Finally, as mentioned below, cPLA2 or 12-/15-LOX inhibitors blocked type IIA
sPLA2 expression, leading to reduced delayed
PGE2 generation.
Intriguingly, the cPLA2 inhibitor AACOCF3,
which suppressed IL-1/TNF
-induced PGE2 generation by
3Y1 cells at concentrations comparable to those generally used to
assess cPLA2 function in cells (21, 57-59), markedly
reduced IL-1
/TNF
-induced type IIA sPLA2 expression
without affecting COX-2 expression. The need for deacylation of
arachidonate substrate was overridden by evaluating AACOCF3
in the presence of exogenous arachidonic acid, which partially prevented inhibition of type IIA sPLA2 expression by
AACOCF3. Moreover, AACOCF3 blocked the
[3H]arachidonic acid release from
IL-1
/TNF
-stimulated cells that occurred immediately after adding
these cytokines. Similar inhibition of [3H]arachidonic
acid release was observed with another cPLA2 inhibitor, methylarachidonyl fluorophosphate (12, 63) (data not shown). These
results suggest that arachidonic acid released by cPLA2 at
the early stage of cytokine stimulation is crucial for the subsequent
type IIA sPLA2 induction and that the inhibitory effect of
the cPLA2 inhibitor on delayed PGE2 generation
may be, at least in part, due to suppression of type IIA
sPLA2 expression in 3Y1 cells. Our results are not only in
line with several reports that inhibitors or antisense oligonucleotides
for cPLA2 suppressed the delayed phase of PG biosynthesis
in several cell types (12, 23, 24), but also consistent with the
recently proposed hypothesis that prior activation of cPLA2
is necessary for sPLA2 to act (24, 40, 63). That the
counteracting effect of exogenous arachidonic acid on inhibition of
type IIA sPLA2 expression by AACOCF3 was only
partial, even when present in excess, predicts the need for some
additional PLA2 metabolites, such as lysophospholipids or their derivatives, in the regulation of type IIA sPLA2
induction. Indeed, we recently found that lysophosphatidylcholine
exhibited significant type IIA sPLA2-inducing
activity.3 It should be noted
that this kind of cross-talk between cPLA2 and type IIA
sPLA2 may not be always applicable to other systems, since
type IIA sPLA2-independent delayed PGE2
generation does occur in several cells, including those derived from
type IIA sPLA2-deficient mice (11, 24, 40). In these cases,
some other sPLA2s, such as type V sPLA2 (39,
40), may compensate for type IIA sPLA2, or
cPLA2 alone is sufficient to remote delayed PG generation
(24).
IL-1/TNF
-induced type IIA sPLA2 expression and
PGE2 generation were also suppressed by inhibitors for 12- or 15-LOXs, but not by those for COX or 5-LOX, suggesting that certain
arachidonate metabolites produced via the cPLA2 and 12- or
15-LOX pathway are required for type IIA sPLA2 induction.
Although in our preliminary experiments the addition of
12-hydroxyeicosatetraenoic acid to 3Y1 cells restored
sPLA2-IIA expression to some extent,3 further
studies should be awaited to determine the precise roles of the
particular 12-/15-LOX metabolites in type IIA sPLA2
expression. The importance of some LOX pathway products in the
homeostasis of mammalian cells has been suggested by several studies.
For instance, Honn and co-workers (64) recently reported that
12-/15-LOX metabolites were essential regulators of cell survival and
apoptosis. Rao et al. (43) have shown that 12- and
15-hydroperoxyeicosatetraenoic acids induced the expression of
c-fos and c-jun that form the transcription
factor AP-1 in vascular smooth muscle cells. Some growth factors
require arachidonic acid release and metabolism through the LOX pathway
for induction of growth in certain cell types (65, 66). The
proliferator-activated receptors, which were originally identified as a
group of transcription factors that regulate gene expression of enzymes
associated with lipid homeostasis (67), have been shown to be the
nuclear receptors for particular arachidonate metabolites, such as
J-series PGs (68) and leukotriene B4 (69).
In contrast to cytokine-induced delayed PGE2 generation,
which requires both type IIA sPLA2 and cPLA2
that are linked cooperatively to COX-2 but not to COX-1,
ionophore-stimulated immediate PGE2 generation is regulated
predominantly by constitutive cPLA2 and COX-1, even when
type IIA sPLA2 and COX-2 are maximally induced. Dissociation of endogenous type IIA sPLA2 from the
immediate response is consistent with several previous observations
(34, 70, 71), although there are some exceptions, particularly when
excess exogenous type IIA enzyme is added to agonist-primed cells, in which it elicits the immediate response (72-74). In this regard, the
role of type IIA sPLA2 in 3Y1 cells appears to differ from that of type V sPLA2 in certain mast cells, in which type V
enzyme is reported to be crucial for immediate, rather than delayed, PGD2 generation (40). The failure of COX-2 to mediate the
immediate response in 3Y1 cells is in line with the observation that
IgE/antigen-mediated immediate PGD2 generation by mouse
bone marrow-derived mast cells was entirely dependent upon COX-1, even
when COX-2 is present in these cells following cytokine priming (2,
75). In contrast, we recently found that IL-1-primed mouse calvarial
osteoblasts (8) and lipopolysaccharide-primed rat peritoneal
macrophages2 elicited immediate PGE2 generation
in response to secondary Ca2+-mobilizing stimuli that was
dependent upon COX-2 rather than COX-1. Therefore, it is likely that
utilization of COX isoforms in the immediate response, relative to the
absolute dependence of the delayed response on COX-2 (2-4, 6), appears
to be cell type- or stimulus-specific and may reflect the presence of
some particular machineries, such as different intracellular
localizations (9), separate intracellular transport of arachidonic
acid, the presence of presumptive COX isoform-selective co-regulatory factors (76), and selective coupling with the downstream terminal PG
synthases (13).
In summary, we have reconfirmed that inducible type IIA sPLA2 is essential for optimal delayed PGE2 biosynthetic pathway, in which it is functionally linked to inducible COX-2, in 3Y1 cells. Furthermore, our results suggest that cPLA2 contributes to delayed PGE2 generation at least partly through the induction of type IIA sPLA2 via the 12-/15-LOX pathway. These results are in line with our recent findings that cPLA2 and COX-2 are functionally linked in cells derived from type IIA sPLA2-deficient mice and forced introduction of type IIA sPLA2 into these cells augmented the delayed PGE2 biosynthetic response (24). In contrast, immediate PGE2 generation is mediated predominantly by the constitutive enzymes cPLA2 and COX-1. These observations reveal the particular routes for functional cross-talk between and segregation of the constitutive and inducible enzymes in the different phases of PG biosynthetic pathway.
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ACKNOWLEDGEMENTS |
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We thank S. Shimbara, K. Tada, and T. Ando
for their technical assistance; Drs. J. D. Clark, W. L. Smith, J. Trzaskos, J. A. Tischfield, J. P. Arm, and T. Yoshida for providing cDNAs, antibodies, and inhibitors; and Drs.
Y. Uehara and H. Ishimaru for providing 3Y1 cells and recombinant
TNF, respectively.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency.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. Tel.: 81-3-3784-8196;
Fax: 81-3-3784-8245; E-mail: kudo{at}pharm.showa-u.ac.jp.
1
The abbreviations used are: PG, prostaglandin;
PLA2, phospholipase A2; sPLA2,
secretory PLA2; cPLA2, cytosolic
PLA2; COX, cyclooxygenase; LOX, lipoxygenase; IL,
interleukin; TNF, tumor necrosis factor
; TBS-T, Tris-buffered
saline containing 0.05% Tween 20; FCS, fetal calf serum; DMEM,
Dulbecco's modified Eagle medium, RT-PCR, reverse
transcriptase-polymerase chain reaction; PCR, polymerase chain
reaction; AACOCF3, arachidonoyl trifluoromethyl ketone;
NDGA, nordihydroguaiaretic acid.
2 H. Naraba, M. Murakami, H. Matsumoto, S. Shimbara, I. Kudo, A. Ueno, and S. Oh-ishi, submitted for publication.
3 H. Kuwata, M. Murakami, Y. Nakatani, and I. Kudo, unpublished observation.
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