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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Several distinct phospholipase
A2s (PLA2s) and two cyclooxygenases
(COXs) were transfected, alone or in combination, into human embryonic
kidney 293 cells, and their functional coupling during immediate and
delayed prostaglandin (PG)-biosynthetic responses was reconstituted.
Signaling PLA2s, i.e. cytosolic
PLA2 (cPLA2) (type IV) and two secretory
PLA2s (sPLA2), types IIA
(sPLA2-IIA) and V (sPLA2-V), promoted
arachidonic acid (AA) release from their respective transfectants after
stimulation with calcium ionophore or, when bradykinin receptor was
cotransfected, with bradykinin, which evoked the immediate response,
and interleukin-1 plus serum, which induced the delayed response.
Experiments on cells transfected with either COX alone revealed subtle
differences between the PG-biosynthetic properties of the two isozymes
in that COX-1 and COX-2 were favored over the other in the presence of
high and low exogenous AA concentrations, respectively. Moreover,
COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and
COX were coexpressed, AA released by cPLA2,
sPLA2-IIA and sPLA2-V was converted to
PGE2 by both COX-1 and COX-2 during the immediate response
and predominantly by COX-2 during the delayed response.
Ca2+-independent PLA2 (iPLA2) (type
VI), which plays a crucial role in phospholipid remodeling, failed to
couple with COX-2 during the delayed response, whereas it was linked to
ionophore-induced immediate PGE2 generation via COX-1 in
marked preference to COX-2. Finally, coculture of PLA2 and
COX transfectants revealed that extracellular sPLA2s-IIA
and -V, but neither intracellular cPLA2 nor
iPLA2, augmented PGE2 generation by neighboring
COX-expressing cells, implying that the heparin-binding
sPLA2s play a particular role as paracrine amplifiers of
the PG-biosynthetic response signal from one cell to another.
Phospholipase A2
(PLA2),1 which
regulates the release of arachidonic acid (AA) from membrane
phospholipids, and cyclooxygenase (COX), which converts AA to the
intermediate prostaglandin (PG) precursor PGH2, represent
the two crucial rate-limiting steps for the PG-biosynthetic pathway. To
date, more than 10 PLA2 (1, 2) and 2 COX (3) isozymes have
been identified in mammals. The existence of two kinetically distinct
PG-biosynthetic responses, the immediate and delayed phases, implies
the recruitment of different sets of biosynthetic enzymes to this
pathway. Although several works have suggested that preferential
coupling between these biosynthetic enzymes accounts for the
differential regulation of the immediate and delayed responses,
conflicting evidence has been yielded by different experimental systems
(4-11).
A rapidly expanding body of evidence suggests that the two COXs, the
constitutive COX-1 and inducible COX-2, play distinct roles in
regulating AA metabolism (3). Several investigators have proposed that
COX-1 and COX-2 respectively metabolize exogenous and endogenous
arachidonic acid (AA) to PGs (12, 13). This statement, however, is an
oversimplification, because in certain situations, COX-1 metabolizes
endogenous AA to PGs, e.g. thromboxane generation by
platelets (14) and macrophages (11) and PGD2 generation by
mast cells (4-6), and COX-2 also utilizes exogenous AA (11). More
generally, utilization of COX-1 is observed during the early phase of
PG biosynthesis occurring within several minutes of stimulation,
whereas COX-2-dependent PG generation proceeds over several
hours in parallel with the induction of COX-2 expression (4-6, 10, 11,
15). Although subtle differences in the subcellular distributions of
these two isozymes may be responsible for their separate functions, a
recent electron microscopic analysis showed that their locations in the
perinuclear and endoplasmic reticular membranes were indistinguishable
(16).
Whether distinct PLA2s are utilized selectively in the
different PG-biosynthetic phases and couple specifically with each COX
isozyme is controversial. PLA2s are subdivided into several classes, among which cytosolic PLA2 (cPLA2;
recently called cPLA2 Recently, in an attempt to elucidate the general functions of each
PLA2 in AA release, we analyzed the functional effects of
transfecting various PLA2 isozymes into mammalian cell
lines, such as human embryonic kidney 293 cells and Chinese hamster
ovary (CHO) cells. This approach enabled us to assess the overlapping and different functions of five distinct PLA2s, namely
cPLA2, sPLA2s (IIA, IIC, and V), and
iPLA2 (32). We demonstrated that cPLA2 and the
two heparin-binding sPLA2s, sPLA2-IIA and
sPLA2-V, act as "signaling" PLA2s that
promote stimulus-dependent AA release during both the
immediate and delayed responses, whereas iPLA2 mediates
spontaneous fatty acid release during culture, consistent with its
proposed role in "phospholipid remodeling" rather than signaling
(30, 31).
In this study, we extended our previous study in order to investigate
particular functional cooperation between PLA2 and COX enzymes during different phases of PG biosynthesis by cotransfecting each PLA2 and COX into 293 cells. Reconstitution of the
immediate and delayed PGE2 biosynthetic responses of these
transfectants confirmed that distinct functional coupling between
different PLA2 and COX enzymes occurs during each phase.
Materials Establishment of PLA2 and COX
Transfectants--
Transformants of 293 cells that stably expressed
cPLA2, sPLA2-IIA, sPLA2-V,
sPLA2-IIC, and iPLA2 were established as
described previously (32). COX-1 and COX-2 cDNAs were transfected
into 293 cells using LipofectAMINE PLUS, according to the
manufacturer's instructions. Briefly, 1 µg of plasmid was mixed with
5 µl of LipofectAMINE PLUS in 200 µl of Opti-MEM medium for 30 min
and then added to cells that had attained 40-60% confluence in 6-well plates (Iwaki) containing 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium comprising
RPMI 1640 containing 10% (v/v) fetal calf serum (FCS). After overnight
culture, the medium was replaced again with 2 ml of fresh medium, and
culture was continued at 37 °C in a CO2 incubator
flushed with 5% CO2 in humidified air. For transient expression analysis, the cells were harvested 3 days after transfection and used immediately. In order to establish stable transfectants, cells
transfected with each cDNA were cloned by limiting dilution in
96-well plates in culture medium supplemented with 800 µg/ml G418
(Life Technologies). After culture for 2-4 weeks, wells containing a
single colony were chosen, and the expression of each COX was assessed
by immunoblotting. The established clones were expanded and used for
the experiments as described below.
In order to establish PLA2/COX double transformants, 293 transformants expressing each COX were subjected to a second
transfection with each PLA2 cDNA, which had been
subcloned into pcDNA3.1/Zeo (+) (Invitrogen). Three days after
transfection, the cells were used for the experiments or seeded into
96-well plates to be cloned by culture in the presence of 50 µg/ml
zeocin (Invitrogen) in order to establish stable transformants
overexpressing both PLA2 and COX. The expression of each
PLA2 was examined by RNA blotting, and in the case of
sPLA2s, by measuring PLA2 activities released into the supernatants. A similar strategy was employed to produce PLA2/BKR double transformants: after the second
transfection with cPLA2 or sPLA2-IIA cDNA
in pcDNA3.1/Zeo (+), G418-resistant 293 cells stably expressing rat
B2-type BKR were selected with zeocin.
Measurement of sPLA2 Activity--
Cells in 1 ml of
culture medium were seeded, at a density of 5 × 104
cells/ml, into each well of 24-well plates. After culture for 4 days,
the supernatants were collected, and the cells were incubated for a
further 15 min at 37 °C with 1 ml of culture medium containing 1 M NaCl. This allowed cell surface-associated
sPLA2s to be recovered quantitatively from the medium, as
described previously (32). The PLA2 activity was assayed by
measuring the amounts of free radiolabeled fatty acids released from
the substrates
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine
(NEN Life Science Products) and
1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine
(Amersham Pharmacia Biotech) for sPLA2-V and
sPLA2-IIA, respectively. Each reaction mixture consisted of
an aliquot of the required sample, 100 mM Tris-HCl, pH 6.0 (for sPLA2-V) or 7.4 (for sPLA2-IIA), 4 mM CaCl2, and 2 µM substrate.
After incubation for 10-30 min at 37 °C, the [14C]fatty acids released were extracted by the method of
Dole and Meinertz (40), and the radioactivity was counted.
RNA Blotting--
Approximately equal amounts (~10 µg) of
the total RNAs obtained from transfected cells 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 probed with the respective cDNA probes that had been
labeled with [32P]dCTP (Amersham Pharmacia Biotech) by
random priming (Takara Shuzo). All hybridizations were carried out as
described previously (10).
SDS-Polyacrylamide Gel Electrophoresis/Immunoblotting--
Cell
lysates (105 cell equivalents) were subjected to
SDS-polyacrylamide gel electrophoresis (7.5% (w/v) for
cPLA2 and 10% for COX-1 and COX-2) under reducing
conditions. In order to analyze sPLA2-IIA, the cells were
treated for 15 min with culture medium containing 1 M NaCl,
and aliquots of the resulting supernatants were subjected to 15%
SDS-polyacrylamide gel electrophoresis under nonreducing 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 probed with the respective antibodies and visualized
using the ECL Western blot analysis system (Amersham Pharmacia
Biotech), as described previously (10).
Cell Activation--
Cells (5 × 104 in 1 ml of
culture medium) were seeded into each well of 24-well plates. In order
to assess AA release, 0.1 µCi/ml [3H]AA (Amersham
Pharmacia Biotech) was added to the cells in each well on day 3, when
they had nearly reached confluence, and culture was continued for
another day. After three washes with fresh medium, 250 µl of RPMI
1640 medium with or without 10 µM A23187, 10 µM BK, or 1 ng/ml IL-1 PGE2 Generation by Transfectants Expressing Either COX
Alone--
Human COX-1 and COX-2 cDNAs, subcloned into a mammalian
expression vector with a neomycin-resistant marker, were individually transfected into 293 and CHO cells to establish stable transformants. Expression of COX-1 and COX-2 proteins was barely detectable in parental 293 cells but was detected clearly in the respective transformants (Fig. 1A). In
order to assess immediate and delayed PGE2 generation from
endogenous AA by each COX, we stimulated the transformants with 10 µM A23187 for 30 min and 1 ng/ml IL-1 in the presence of
10% FCS for 4 h, respectively (Fig. 1B). Whereas treatment of parental 293 cells with A23187 resulted in minimal PGE2 generation, consistent with undetectable expression
levels of both COX isozymes (Fig. 1A), replicate cells
expressing COX-1 and COX-2 produced approximately 15 and 35 times more
PGE2 in response to A23187 than the control cells (Fig.
1B, top). When COX-2-expressing cells were stimulated with
IL-1/FCS, delayed PGE2 generation increased markedly,
reaching approximately 14 times the amount generated by the control
cells (Fig. 1B, bottom). In contrast, COX-1-expressing cells
produced only minimal amounts of PGE2 that did not differ
significantly from the amounts produced by the control cells.
To investigate the functional differences between COX-1 and COX-2
further, we measured the COX activities by supplying the transfectants
with exogenous AA (Fig. 1C). In our assay system, COX-1
transfectants produced PGE2 when the exogenous AA
concentration exceeded 10 µM, reaching nearly 100 ng of
PGE2 in the presence of 50 µM AA, whereas
conversion to PGE2 was undetectable when the AA
concentration was below 10 µM. COX-2-expressing cells
produced substantial amounts of PGE2 even when the AA
concentration was below 10 µM. Approximately 20 ng of
PGE2 was produced by COX-2-expressing cells treated with 50 µM AA, only
We observed that some PGE2 was produced by cells expressing
COX-2, but not COX-1, even in the absence of exogenous AA or any extracellular stimulus (Fig. 1, C and E),
indicating that COX-2 could utilize endogenous AA in this situation.
Interestingly, COX-2-expressing cells not only spontaneously produced
more PGE2 but also released more AA than the control cells
(Fig. 1E). This increased AA release induced by COX-2
transfection was significantly suppressed by the cPLA2
inhibitor MAFP but not by the iPLA2 inhibitor bromoenol
lactone, the COX-2 inhibitor NS-398 (Fig. 1E), or the sPLA2 inhibitor LY311727 (data not shown), whereas
PGE2 generation by COX-2 transfectants was suppressed by
MAFP and NS-398 but not by bromoenol lactone. Immunoblot analysis
revealed a trace amount of cPLA2 (Fig. 1F) but
none of other PLA2s examined in this study (data not shown)
in parental 293 cells. These observations suggest that COX-2 can
activate AA release mediated by the MAFP-sensitive cPLA2
through a mechanism independent of COX activity.
In order to verify that the observations described above was also
applicable to other cells, we carried out similar experiments using CHO
cells transfected with COX-1 and COX-2 (Fig. 1, F and G). CHO cells expressing COX-1 and COX-2 produced 16 and 25 times more PGE2, respectively, than the parental cells
during the immediate response to A23187 (Fig. 1G, left).
Significant and comparable increases in PGE2 generation by
parental and COX-1-expressing cells after stimulation with IL-1 plus
FCS was observed (Fig. 1G, middle). This increase was
suppressed almost completely by NS-398 (data not shown) (32),
indicating its dependence upon endogenous COX-2, trace amounts of which
were expressed in CHO cells (Fig. 1F). The COX-2
transfectants exhibited a 3-fold increase in delayed PGE2
generation compared with the control cells (Fig. 1G,
middle). The experiment using exogenous AA revealed that, as in
the experiments on 293 cells (Fig. 1, A-E), COX-2 was more active than COX-1 when the AA concentrations were below 10 µM, whereas COX-1 was active in the presence of high AA
concentrations (Fig. 1G, right). Moreover, significant
PGE2 generation by COX-2, but not COX-1, transfectants
occurred even without extracellular stimuli or exogenous AA (Fig.
1G). CHO cells expressed more endogenous cPLA2
than 293 cells (Fig. 1F), and the spontaneous
PGE2 generation by CHO COX-2 transfectants was inhibited by
MAFP (data not shown).
PGE2 Generation by Transfectants Expressing Both
cPLA2 and COX--
In order to assess the functional
coupling between particular PLA2 enzymes and each COX, we
attempted to establish PLA2/COX double transfectants. Fig.
2A depicts the expression
levels of cPLA2, COX-1 and COX-2 in cells transfected with
these cDNAs alone or in combination. Although the expression of
cPLA2 alone was accompanied by a marked increase in
A23187-induced AA release, as reported previously (32), no appreciable
PGE2 generation occurred (Fig. 2B) due to the
absence of constitutive expression of endogenous COXs (Fig.
2A). Although the expression of COX-1 alone increased
PGE2 generation only minimally, coexpression of cPLA2 and COX-1 led to a dramatic increase in
PGE2 generation (Fig. 2B). As described above,
the expression of COX-2 alone evoked AA release, which was accompanied
by a significant increase in PGE2 generation. When
cPLA2 and COX-2 were coexpressed, they increased AA release
and PGE2 generation in a synergistic manner (Fig.
2B). A23187-induced PGE2 generation by these
double transformants, which was dependent upon the coexistence of
cPLA2 and either COX, displayed the typical immediate
response pattern, reaching a peak 5-10 min after stimulation and a
plateau by 30 min (Fig. 2C).
As A23187 is a nonphysiologic stimulus, we wanted to establish whether
cPLA2 could couple with COX-1 and COX-2 in the immediate response following a more physiologic stimulus. In order to address this issue, we introduced BKR, a G-protein-linked receptor with seven
membrane-spanning regions, into 293 cells, cPLA2 cDNA
was then transfected into these cells, and
G-protein-dependent activation of cPLA2 was
assessed (Fig. 3, A and
B). Note that the expression levels of cPLA2 in
transfectants expressing cPLA2 alone (Fig. 2A)
and those expressing both cPLA2 and BKR (Fig.
3A) were comparable. After stimulation for 30 min with 10 µM BK, cells stably expressing both cPLA2 and
BKR released approximately 3 times more AA than the replicate cells
expressing BKR alone (Fig. 3B). Significant increases in
BK-dependent PGE2 generation after transfecting
either COX-1 or COX-2 into cPLA2/BKR, but not BKR alone,
transformants were observed (Fig. 3C), confirming that
cPLA2 can couple with both COX-1 and COX-2 in the immediate
phase elicited by a physiologic stimulus.
Expression of cPLA2 alone increased IL-1/FCS-induced
delayed AA release, which was accompanied by a modest increase in
PGE2 generation that was dependent upon endogenously
induced COX-2 (Fig. 2D), as demonstrated in our previous
study (32). Expression of COX-1 had a minimal effect on delayed
PGE2 generation, even when cPLA2 was
coexpressed (Fig. 2D). The expression of COX-2 alone
stimulated AA release, accompanied by a significant increase in
PGE2 generation. When cPLA2 and COX-2 were
coexpressed, they acted in synergy, resulting in marked augmentation of
AA release and PGE2 generation (Fig. 2D).
Kinetic experiments showed that IL-1/FCS-induced PGE2
generation, the typical delayed response, proceeded gradually and
continuously during culture of cPLA2/COX-2 double
transfectants for 8 h (Fig. 2E). IL-1 dependence of the delayed PGE2 generation, in which IL-1, in concert with
FCS, played a prerequisite role in the induction of endogenous COX-2
expression (32), by cells stably expressing COX-2 appeared less
obvious, although IL-1 exerted some augmentative effect on
FCS-dependent PGE2 generation (Fig.
2F) likely through enhancing cPLA2-mediated AA
release (32). A23187-induced immediate PGE2 generation by cPLA2/COX-1 and cPLA2/COX-2 transfectants was
suppressed selectively by valeryl salicyate and NS-398, respectively,
and IL-1/FCS-induced delayed PGE2 generation by
cPLA2/COX-2 transfectants was suppressed almost completely
by NS-398 (Fig. 2G).
PGE2 Generation by Transfectants Expressing
sPLA2 and COX--
Next, we established transformants
coexpressing sPLA2-IIA and either COX-1 or COX-2, and their
expression levels in each transfectant were verified by immunoblotting
(Fig. 4A, top).
sPLA2-IIA expression was also checked by determining the
enzymatic activities in the supernatants (Fig. 4A, bottom),
which correlated with the intensities of the sPLA2-IIA
protein bands visualized by immunoblotting (Fig. 4A, top).
Expression of sPLA2-IIA alone increased A23187-induced AA
release that was not accompanied by PGE2 generation,
whereas coexpression of sPLA2-IIA and COX-1 enhanced
PGE2 generation markedly (Fig. 4B). Coexpression
of sPLA2-IIA and COX-2 also resulted in significant
increase in PGE2 generation, although, unlike
cPLA2/COX-2 double transfectants (Fig. 2B), they
did not synergize at the level of AA release (Fig. 4B).
A23187-induced PGE2 generation by
sPLA2-IIA/COX-1 or -2 double transformants reached a
maximum within 10 min and plateaued thereafter (Fig. 4C).
Furthermore, the transfectants stably expressing both
sPLA2-IIA and BKR (Fig. 3D), in which
sPLA2-IIA expression level was comparable to that in cells
expressing sPLA2-IIA alone (Fig. 4A), released
more AA than the control cells following 30 min of exposure to BK (Fig. 3E), and the AA released by sPLA2-IIA in
response to BK stimulation was converted to PGE2 by COX-1
and COX-2 when each COX cDNA was transfected into the
sPLA2-IIA/BKR double transfectants (Fig. 3F).
Thus, sPLA2-IIA appeared to couple functionally with both COX-1 and COX-2 in the immediate response.
Expression of sPLA2-IIA alone increased IL-1/FCS-induced
delayed AA release, accompanied by a modest increase in
PGE2 generation, which depended on endogenous COX-2 (Fig.
4D), as reported previously (32). Although delayed
PGE2 generation by cells coexpressing sPLA2-IIA
and COX-1 was comparable to that by cells expressing sPLA2-IIA alone, leading further support to the hypothesis
that COX-1 is dissociated from the delayed response, it was augmented significantly when sPLA2-IIA and COX-2 were coexpressed
(Fig. 4D). Kinetic experiments showed that delayed
PGE2 generation by sPLA2-IIA/COX-2 double
transformants proceeded throughout the culture period (Fig.
4E). Valeryl salicylate and NS-398 reduced sPLA2-IIA/COX-1- and sPLA2-IIA/COX-2-mediated
A23187-induced immediate PGE2 generation, respectively
(Fig. 4F, top), and sPLA2-IIA/COX-2-mediated delayed PGE2 generation was suppressed almost completely by
NS-398 (Fig. 4F, bottom).
Our previous study (32), as well as those of others (26, 27),
demonstrated that sPLA2-V compensates for
sPLA2-IIA during stimulus-initiated AA release. The
expression profiles of sPLA2-V, COX-1, and COX-2 in 293 cells transfected with their cDNAs alone or in combination are
shown in Fig. 5A. The
expression levels of sPLA2-V were verified by RNA blotting
and enzyme assay (Fig. 5A, bottom). Expression of
sPLA2-V alone increased A23187-induced immediate and
IL-1/FCS-induced delayed AA release in a manner similar to
sPLA2-IIA (data not shown) (32). Coexpression of sPLA2-V and COX-1 markedly increased immediate (Fig.
5B) but not delayed (Fig. 5C) PGE2
generation. Coexpression of sPLA2-V and COX-2 consistently
enhanced immediate (Fig. 5B) and delayed (Fig. 5C) PGE2 generation to approximately 1.5- and
2.7-fold, respectively, that by cells expressing COX-2 alone.
In previous studies, we showed that sPLA2-IIA and
sPLA2-V, but not sPLA2-IIC, each has a cluster
of cationic residues in the C-terminal domain that is essential for its
association with cell surface proteoglycan and for its AA-releasing
functions (32, 41). In order to gain further insight into the similar
actions of sPLA2-IIA and sPLA2-V, we
transfected a sPLA2-IIA mutant, G30S, which is
catalytically inactive but possesses normal cell binding capacity (32),
into 293 cells expressing sPLA2-V (Fig. 5D). We
reasoned that if sPLA2-IIA and sPLA2-V share
the common binding site on the plasma membrane,
sPLA2-IIA(G30S) would displace sPLA2-V from the
binding site, thereby solubilizing the cell surface-bound sPLA2-V and eventually leading to a reduction in
sPLA2-V-mediated AA release. As shown in Fig.
5D, about 65% of sPLA2-V, assessed by enzymatic
activity, was present as the cell surface-bound form. Coexpression of
sPLA2-IIA(G30S) altered the distribution of
sPLA2-V between the supernatant and cell surface, where the
cell surface-bound portion of sPLA2-V decreased to half
that in the nonmutant replicate cells. Accordingly, this reduced
sPLA2-V-mediated AA release in response to A23187 (data not
shown) or IL-1/FCS (Fig. 5D) by approximately 50%.
sPLA2-IIA(G30S) also reduced sPLA2-IIA-mediated
AA release in a plasmid concentration-dependent manner
(Fig. 5E), and this was accompanied by a decrease in the
amount of cell-associated native sPLA2-IIA (data not
shown). The dominant-negative effect of this particular
sPLA2-IIA mutant was confirmed by the observations that
another mutant, sPLA2-IIA(H48E), which also lacks catalytic activity but has intact cell binding ability (32), blocked
sPLA2-IIA-mediated AA release in a manner similar to
sPLA2-IIA(G30S), whereas sPLA2-IIA(KE4), which
has normal PLA2 activity and impaired cell binding ability, failed to do so (Fig. 5E). Thus, this experiment confirmed
that sPLA2-IIA and sPLA2-V exhibit their
AA-releasing effects through binding to a common component
(proteoglycan or alternative) on the cell surface.
PGE2 Generation by Transfectants Expressing
iPLA2 and COX--
Accumulating evidence suggests that
iPLA2 plays a crucial role in phospholipid remodeling (30,
31). In support of this, we showed recently that iPLA2
overexpression in 293 cells led to increased spontaneous fatty acid
release, which showed virtually no link with PGE2
generation via endogenous COX-2 (32). To ascertain whether
iPLA2 could participate in PG biosynthesis or not, we herein established 293 transfectants stably expressing both
iPLA2 and either COX-1 or COX-2, the expression of which
was assessed by RNA blotting and immunoblotting, respectively (Fig.
6A). Unexpectedly, we found
that treatment of iPLA2-expressing cells with A23187 induced marked AA release (Fig. 6B, top). Furthermore,
coexpression studies demonstrated that the AA released by
iPLA2 was efficiently metabolized to PGE2 by
COX-1 in marked preference to COX-2 (Fig. 6B, bottom). These
results suggest that although iPLA2 is a
Ca2+-independent enzyme when assayed in vitro,
it is subject to regulation by Ca2+ in vivo,
probably indirectly, thereby acting as a sort of signaling PLA2. In marked contrast, iPLA2-mediated AA
release failed to link with COX-1 for delayed PGE2
biosynthesis (Fig. 6C). Moreover, delayed PGE2
generation by cells coexpressing iPLA2 and COX-2 was almost
equal to that by cells expressing COX-2 alone (Fig. 6C),
providing further support for our previous observation (32) that
iPLA2 plays a minimal role in the delayed phase of
COX-2-dependent PGE2 biosynthesis.
Transcellular PGE2 Generation by
sPLA2
We also performed similar coculture experiments using cells expressing
each PLA2 and COX-1. After stimulation with A23187 for 30 min, PGE2 generation by COX-1-expressing cells was
potentiated more efficiently by sPLA2s than by
cPLA2. For instance, in a representative assay, the amount
of PGE2 generated by cocultured COX-1- and
cPLA2-expressing cells reached no more than 2 ng/well,
whereas that produced by COX-1- and sPLA2-V-expressing
cells in coculture reached 7 ng/well.
Although PLA2/COX coupling during the two phases of
the PG biosynthetic pathway in various cell types has been studied by several investigators, including us, the rapidly increasing number of
PLA2 isozymes, the limitations of the technologies used to distinguish them, and the occurrence of cell-specific events has led to
rather confusing results (4-11). Therefore, we recently carried out
reconstitution analysis of five distinct PLA2s
(cPLA2, sPLA2s-IIA, -V, -IIC, and
iPLA2) in 293 and CHO cells in an attempt to define their
roles in stimulus-induced and constitutive AA release (32). In the
present study, we introduced the two COX isoforms, COX-1 and COX-2,
into this system, in order to obtain information about general aspects
of the functional coupling and segregation of particular
PLA2 isozymes and COXs. Our results provided evidence that
the signaling PLA2s, namely cPLA2,
sPLA2-IIA and sPLA2-V, can each link
functionally with both COX-1 and COX-2 and predominantly with COX-2 in
the immediate and delayed PG-biosynthetic responses, respectively,
whereas iPLA2, a phospholipid remodeling PLA2
that dissociates from COX-2-dependent delayed PG
generation, can promote Ca2+-dependent
immediate AA release, which is preferentially linked with COX-1 (Fig.
8). Moreover, coculture experiments
revealed a distinct role of sPLA2 as a regulator of
transcellular PG biosynthesis, a role not played by the intracellular
PLA2 enzymes (Fig. 7D).
INTRODUCTION
Top
Abstract
Introduction
References
after the recent discovery of two
related isozymes cPLA2
and
(17)), a family of
secretory PLA2s (sPLA2s), and
Ca2+-independent PLA2 (iPLA2) have
been paid considerable attention. The increased cytoplasmic
Ca2+ concentration and activation of mitogen-activated
protein kinases, which are prerequisite for cPLA2
activation, occur rapidly and are often transient, implying that
cPLA2 plays a crucial role in immediate AA release
(18-20). Evidence is accumulating that cPLA2 is also
involved in the delayed response (9, 21, 22), although the mechanism
whereby cPLA2 is activated under such a Ca2+-free condition is poorly understood. Some studies
showed that sPLA2 couples selectively with COX-1 during the
immediate phase (6), whereas others demonstrated that
sPLA2, particularly type IIA (sPLA2-IIA),
functions during the delayed phase, as its expression is often induced
markedly in response to proinflammatory stimuli and correlates with
ongoing PG biosynthesis (10, 23-25). Several types of cell express
type V sPLA2 (sPLA2-V), which appears to mediate certain phases of PG biosynthesis (26, 27). Supportive (28, 29)
and contradictory (30, 31) evidence for the involvement of
iPLA2 in stimulus-dependent AA release has been
reported. The conflicting observations reported so far may be due to
the limitations of studies using chemical inhibitors, antibodies, and
even antisense oligonucleotides, which often cannot gain access to
certain cellular compartments and may cause cross-inhibition or
undesirable side effects, thereby leading to misinterpretation.
EXPERIMENTAL PROCEDURES
--
The cDNAs for mouse cPLA2,
mouse sPLA2-IIA, and its mutants (G30S, H48E, and KE4), rat
sPLA2-V, rat sPLA2-IIC, and hamster iPLA2 were described previously (32). Human COX-1 and COX-2 cDNAs were provided by Dr. S. Nagata (Osaka University) and
subcloned into pcDNA3.1 (Invitrogen). Rat bradykinin receptor (BKR)
B2 cDNA (33), obtained from a rat genomic library
(CLONTECH) by performing the reverse
transcription-polymerase chain reaction using a RNA polymerase chain
reaction kit (AMV) version-2 (Takara Shuzo), was subcloned into pCR3.1
(Invitrogen). Human embryonic kidney 293 cells were obtained from Riken
Cell Bank, and CHO-K1 cells stably expressing human COX-1 and COX-2
were provided by Dr. M. Sugimoto (Chugai Pharmaceutical Co. Ltd.). The
rabbit anti-human cPLA2 antibody and sPLA2
inhibitor LY311727 (34) were provided by Dr. R. M. Kramer (Lilly
Research). The rabbit anti-rat sPLA2-IIA antibody was
prepared as described previously (35). The goat anti-human COX-2
antibody was purchased from Santa Cruz. The rabbit anti-human COX-1
antibody and COX-1 inhibitor valeryl salicylate (36) were provided by
Dr. W. L. Smith (Michigan State University). The COX-2 inhibitor
NS-398 (37) was provided by Dr. J. Trzaskos (DuPont Merck
Pharmaceutical Co.). The cPLA2 inhibitor methyl arachidonylfluorophosphate (MAFP) (38), the iPLA2 inhibitor bromoenol lactone (39), AA, and the PGE2 enzyme immunoassay kit were purchased from Cayman Chemical. A23187 was purchased from
Calbiochem. BK was purchased from Sigma. Human and mouse interleukin
(IL)-1
s were purchased from Genzyme. LipofectAMINE PLUS reagent,
Opti-MEM medium, and Trizol reagent were obtained from Life
Technologies. RPMI 1640 medium was purchased from Nissui Pharmaceutical.
and/or 10% FCS was added to
each well and the amount of free [3H]AA released into
each supernatant during culture (up to 30 min after the addition of
A23187 and BK and up to 8 h after IL-1
) was measured. The
percentage release of AA was calculated using the formula
(S/(S + P)) × 100, where S
and P are the radioactivities measured in equal portions of
the supernatant and cell pellet, respectively. The supernatants from
replicate cells cultured without added radiolabeled fatty acids were
subjected to the PGE2 enzyme immunoassay. Conversion of
exogenous AA to PGE2 was determined by treating cells with
AA for 30 min and immunoassaying the PGE2 produced.
RESULTS
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Fig. 1.
Effects of COX overexpression.
A, expression of COX-1 and COX-2 proteins, assessed by
immunoblotting, in 293 cells transfected with each cDNA.
B, PGE2 generation from endogenous AA by control
293 cells and COX-1 and COX-2 transfectants following stimulation with
10 µM A23187 for 30 min (top) and 1 ng/ml
human IL-1 in the presence of 10% FCS for 4 h
(bottom). C, conversion of exogenous AA to
PGE2 by COX-1 (circles) and COX-2 (closed
squares) transfectants and control cells (open
squares). The cells were incubated with various concentrations of
AA in RPMI 1640 medium for 30 min. D, effects of the COX-1
inhibitor valeryl salicylate and the COX-2 inhibitor NS-398 on the
conversion of exogenous AA to PGE2. The cells were
preincubated for 5 h with each inhibitor, washed, and then
incubated for 30 min with 50 µM AA in the continued
presence of each inhibitor. E, COX-2 itself stimulated AA
release. Control and COX-2-expressing 293 cells prelabeled with
[3H]AA were treated for 2 h with 10 µM
MAFP, 50 µM bromoenol lactone (BEL), or 5 ng/ml NS-398, washed, and then cultured for a further 4 h in RPMI
1640 medium in the continued presence of each inhibitor to assess
[3H]AA release (top) and PGE2
generation (bottom). F, expression of COX-1 and
COX-2 proteins, assessed by immunoblotting, in parental CHO cells and
CHO cells transfected with each cDNA (top) and that of
endogenous cPLA2 protein in parental 293 and CHO cells
(bottom). G, PGE2 generation by
control CHO cells and COX-1 or COX-2 transfectants after treatment with
10 µM A23187 for 30 min (left), 1 ng/ml mouse
IL-1
in the presence or absence of 10% FCS for 4 h
(middle), or various concentrations of exogenous AA for 30 min (right). Means ± S.E. of three to five experiments
are shown in B, C, and E, and representative
results of three independent experiments are shown in A, D,
F, and G.
the amount produced by COX-1
transfectants. No appreciable conversion of AA to PGE2 by
the control cells was observed. Conversion of exogenous AA to
PGE2 by cells expressing COX-1 and COX-2 was inhibited by the COX-1-specific inhibitor valeryl salicylate and the COX-2-specific inhibitor NS-398, respectively, but not vice versa (Fig.
1D). Collectively, these data suggest that COX-1 is favored
over COX-2 when excess AA is supplied, whereas COX-2 is more active
than COX-1 when the supply of AA is limited.
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Fig. 2.
Coexpression of cPLA2 and COX-1
or COX-2. A, expression of cPLA2, COX-1 and
COX-2 proteins, assessed by immunoblotting, in 293 cells transfected
with each cDNA alone or in combination. B, AA release
(top) and PGE2 generation (bottom) by
cPLA2, COX-1, and COX-2 single transfectants and
cotransfectants after stimulation for 30 min with 10 µM
A23187. C, time course of A23187-induced PGE2
generation in cells expressing cPLA2/COX-1 (solid
squares), cPLA2/COX-2 (solid circles), and
parental cells (open circles). D, AA release
(top) and PGE2 generation (bottom) by
the cPLA2, COX-1, and COX-2 single transfectants and
cotransfectants after stimulation for 4 h with 1 ng/ml IL-1
plus 10% FCS. E, time course of IL-1/FCS-induced
PGE2 generation in cells expressing cPLA2/COX-1
(solid triangles), cPLA2/COX-2 (solid
circles), COX-2 (solid squares), and parental cells
(open circles). F, dependence of delayed
PGE2 generation on IL-1 and FCS. Control cells and
transfectants expressing COX-2 alone or cPLA2/COX-2 were
cultured for 4 h in the presence or absence of 1 ng/ml IL-1
plus 10% FCS. G, effects of COX inhibitors. Cells
expressing cPLA2/COX-1 and cPLA2/COX-2 were
pretreated for 2 h with 1 µg/ml valeryl salicylate
(VS) or 5 ng/ml NS-398 (NS), washed, and then
activated for 30 min with A23187 (top) or for 4 h with
IL-1/FCS (bottom) to assess PGE2 generation.
Means ± S.E. of six to seven independent experiments are shown in
B, D, and F, and representative results of three
to four independent experiments are shown in A, C, E, and
G.
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Fig. 3.
Immediate response induced by BK.
A, expression of cPLA2 and BKR assessed by RNA
blotting. B, AA release by BKR and cPLA2/BKR
transfectants after stimulation with 10 µM BK for 30 min.
C, PGE2 generation by cPLA2/COX/BKR
triple transfectants. BKR- and cPLA2/BKR-expressing 293 cells were transfected transiently with either COX-1 or COX-2 after
stimulation with BK for 30 min. Three days after COX transfection, the
cells were stimulated with 10 µM BK for 30 min to assess
immediate PGE2 generation. D, expression of
sPLA2-IIA and BKR assessed by RNA blotting. E,
AA release by BKR and sPLA2-IIA/BKR transfectants in
response to BK. F, PGE2 generation by
sPLA2-IIA/COX/BKR triple transfectants in response to BK.
Representative results for two (A, C, D, and F)
and four (B and E) independent experiments are
shown.
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Fig. 4.
Coexpression of sPLA2-IIA and
COX-1 or COX-2. A, expression of sPLA2-IIA,
COX-1 and COX-2 proteins, assessed by immunoblotting, in 293 cells
transfected with each cDNA alone or in combination (top)
and sPLA2 activity released into the supernatants
(bottom). B, AA release (top) and
PGE2 generation (bottom) by
sPLA2-IIA, COX-1, and COX-2 single transfectants and
cotransfectants after stimulation for 30 min with 10 µM
A23187. C, time course of A23187-induced PGE2
generation in transfectants expressing sPLA2-IIA/COX-1
(solid squares) or sPLA2-IIA/COX-2 (solid
circles) and control cells (open circles).
D, AA release (top) and PGE2
generation (bottom) by the sPLA2-IIA, COX-1 and
COX-2 single transfectants and cotransfectants after stimulation for
4 h with 1 ng/ml IL-1 plus 10% FCS. E, time course
of IL-1/FCS-induced PGE2 generation by control cells
(open circles) and transfectants expressing
sPLA2-IIA/COX-1 (solid triangles),
sPLA2-IIA/COX-2 (solid circles), and COX-2 alone
(solid squares). F, effects of COX inhibitors.
Cells expressing sPLA2-IIA/COX-1 and
sPLA2-IIA/COX-2 were pretreated for 2 h with 1 µg/ml
valeryl salicylate (VS) or 5 ng/ml NS-398 (NS),
washed, and then activated for 30 min with A23187 or for 4 h with
IL-1/FCS to assess PGE2 generation. Means ± S.E. of
six to seven independent experiments are shown in B and
D, and representative results of three to four independent
experiments are shown in A, C, E, and F.
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Fig. 5.
Coexpression of sPLA2-V and COX-1
or COX-2. A, expression of COX-1 and COX-2, assessed by
immunoblotting, in 293 cell transfectants (top) and that of
sPLA2-V, assessed by enzyme activity released into the
supernatants (bottom) and RNA blotting (Inset).
B, PGE2 generation by the sPLA2-V,
COX-1 and COX-2 single transfectants and cotransfectants after
stimulation for 30 min with 10 µM A23187. C,
PGE2 generation by the sPLA2-V and/or COX-1 or
-2 transfectants after stimulation for 4 h with 1 ng/ml IL-1
plus 10% FCS. D, dominant negative effect of the
catalytically inactive sPLA2-IIA mutant (G30S) on
sPLA2-V-mediated AA release. The sPLA2-IIA
mutant G30S was coexpressed in 293 cells stably expressing
sPLA2-V, and the distributions of sPLA2-V
activity in the supernatant (S) and cell surface-associated
fraction (C) (left) and IL-1/FCS-stimulated AA
release (right) were compared with those of replicate cells
not subjected to G30S cotransfection. E, dominant negative
effect of the catalytically inactive sPLA2-IIA mutants
(G30S and H48E) on sPLA2-IIA-mediated AA release. The
indicated amounts of plasmids containing cDNAs for the
sPLA2-IIA mutants G30S, H48E, and KE4 were each
cotransfected into 293 cells stably expressing sPLA2-IIA,
and IL-1/FCS-stimulated delayed AA release was examined. Means ± S.E. of four independent experiments are shown in B and
C, and representative results of three independent
experiments are shown in A, D, and E.
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Fig. 6.
Coexpression of iPLA2 and COX-1
or COX-2. A, expression of COX-1 and COX-2 in 293 cell
transfectants, assessed by immunoblotting (top), and that of
iPLA2, assessed by RNA blotting (bottom).
B, AA release (top) and PGE2
generation (bottom) by iPLA2, COX-1, and COX-2
single transfectants and cotransfectants after stimulation for 30 min
with 10 µM A23187. C, AA release
(top) and PGE2 generation (bottom) by
the iPLA2, COX-1, and COX-2 single transfectants and
cotransfectants after stimulation for 4 h with 1 ng/ml IL-1
plus 10% FCS. Means ± S.E. of five to eight independent
experiments are shown in B and C, and
representative results of three independent experiments are shown in
A.
--
The fact that sPLA2s represent
the only group of PLA2 enzymes that are able to gain access
to other cells in microenvironments prompted us to ask whether
sPLA2s promote PG biosynthesis by neighboring cells, namely
the transcellular PG-biosynthetic response. To explore this, 293 cells
expressing each PLA2 and those expressing COX-2 were
cocultured, and the PGE2 produced by the COX-2-expressing cells after stimulation with IL-1/FCS for 4 h was quantified. Whereas PGE2 generation increased only in an additive
manner when COX-2-expressing cells and cPLA2-expressing
(Fig. 7A) or
iPLA2-expressing (Fig. 7B) cells were
cocultured, it increased synergistically when COX-2-expressing cells
and sPLA2-V-expressing (Fig. 7A) or sPLA2-IIA-expressing (Fig. 7B) cells were
cocultured, even though the amounts of AA released by the individual
PLA2s were comparable in each set of experiments. The
failure of cPLA2 and iPLA2 to participate in
transcellular PGE2 generation in this setting would appear
to eliminate the possibility that AA released from
PLA2-expressing cells was incorporated into
COX-2-expressing cells to be converted to PGE2. Neither the
sPLA2-IIA mutant G30S nor KE4 increased PGE2 generation by COX-2-expressing cells, even though the expression levels
of the mutants and the native enzyme were similar (32), indicating that
both catalytic activity and cell surface binding ability are essential
for promotion of the transcellular PG-biosynthetic pathway by
sPLA2-IIA (Fig. 7C).
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Fig. 7.
Transcellular PG biosynthesis by
sPLA2s. COX-2-expressing 293 cells and 293 cells
expressing cPLA2, sPLA2-V (A),
sPLA2-IIA, and iPLA2 (B) were
cocultured for 3 days and then stimulated with IL-1/FCS for 4 h to
assess AA release and PGE2 generation. C, cells
expressing COX-2 and those expressing native or mutated
sPLA2-IIA in coculture were stimulated with IL-1/FCS.
Representative results of four independent experiments are shown.
D, schematic model of transcellular PG biosynthesis
following IL-1 stimulation. cPLA2 couples only with COX-2
expressed in the same cell, whereas COX-2 may activate
cPLA2 through an unknown mechanism. sPLA2-IIA
and -V not only couple with COX-2 expressed in the same cell in a
autocrine manner, but also act on another COX-2-expressing cell in a
paracrine manner. Similarly,
sPLA2/COX-1-dependent transcellular
PGE2 generation occurs when cells are stimulated with
A23187 (see text).
DISCUSSION
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Fig. 8.
Diagram of PLA2/COX
coupling. cPLA2, sPLA2-IIA, and
sPLA2-V release AA rather specifically, and the AA is, in
turn, metabolized to PGE2 via COX-1 and COX-2 during the
immediate responses induced by A23187 and BK and via COX-2, but not
COX-1, during the delayed response induced by IL-1 plus FCS.
iPLA2 releases both AA and oleic acid (OA) in
the presence of FCS and plays a role in phospholipid remodeling.
Furthermore, iPLA2-derived AA is utilized for the immediate
PG biosynthesis via COX-1. The function of sPLA2-IIC is
unknown.
Several studies have shown that utilization of the constitutive isozyme COX-1 is often, but not always, associated with immediate PG biosynthesis (4-6, 10, 11, 14), and that the induction of COX-2 expression is accompanied by a concomitant increase in PG generation throughout the culture period (4-6, 9-11, 15) and by the priming for enhancement of the immediate response (8, 11, 42), even though both isozymes exhibited some compensatory functions under certain conditions (43). Nevertheless, the closer association of COX-2 than COX-1 with inflammation, development, and tumorigenesis, which has been recently verified by studies on mice subjected to targeted disruption of either COX gene (14, 15, 44, 45), appears to agree with the proposal that COX-2 plays a prominent role in the prolonged generation of PGs, which would modify the nuclear events associated with cell differentiation and replication.
Analyses of COX transfectants have provided some clues about the mechanisms responsible for the functional differences between the two isoforms. Our results confirmed that utilization of COX-1 is restricted to the immediate response, whereas COX-2 is quite active in both the immediate and delayed responses, when AA is supplied endogenously. Comparison of the conversion of exogenous AA to PGE2 by COX-1 and COX-2 transformants revealed that low concentrations of AA are utilized predominantly by COX-2, whereas high concentrations of AA are utilized preferentially by COX-1. The latter finding is reminiscent of that of Reddy and Herschman (12), who were the first to demonstrate that COX-1 and COX-2 utilize exogenous and endogenous AA, respectively. While our study was underway, Shitashige et al. (13) reported a similar result that only COX-2 was functional at low AA concentrations and further showed that intracellular peroxides may contribute to COX-2 activation under conditions of low AA concentrations. Our results also appear to be compatible with the recent subtle kinetic studies on purified enzymes in that COX-2 has a lower threshold for hydroperoxide activation than COX-1, thereby enabling COX-2 to oxygenate AA in the presence of low peroxide concentrations (46, 47), and that negative allosteric regulation of COX-1 by low AA concentrations has the overall effect of increasing the rate of COX-2-mediated PG formation severalfold (48).
The different sensitivities of the two COX isozymes to high and low concentrations of AA could account, at least in part, for their segregated utilization during immediate and delayed PG biosynthesis from endogenous AA. It seems reasonable to speculate that during the immediate response, when a burst of AA is released in a short time, the local concentration of AA reaches a level high enough to activate both COX-1 and COX-2, whereas limited amounts of AA may be supplied gradually during the delayed phase, under which conditions only COX-2 remains active. This hypothesis is further supported by the finding that COX-1-dependent immediate PGE2 generation by 293 cells transfected with COX-1 alone was only modest, but it was enhanced considerably when COX-1 and the signaling PLA2s, which markedly increased the free AA levels, were coexpressed. We presume that the failure of several earlier studies to demonstrate the COX-1-dependent immediate response in several cell types, irrespective of the presence of COX-1 (12, 13, 42), was due to limitation of the supply of AA by PLA2 under the experimental conditions used. Mouse mast cells (4) and rat 3Y1 fibroblasts (10) utilize COX-1 predominantly in the immediate PG-biosynthetic response even when both COX isozymes are copresent, probably because cPLA2, which is abundantly expressed in these cells, may liberate in an intracellular local area rather high amounts of AA that reach a level enough to elicit preferred COX-1-dependent PG biosynthesis. Although the functional difference between COX-1 and COX-2 was suggested to be due to their different subcellular locations (49), recent immunohistochemical studies by Smith and co-workers (16) clearly demonstrated that any specific connection between COX-2 and the generation of products that might function in the nucleus would result from differences in the expression of activities of COX-1 and COX-2, not from gross differences in their subcellular distributions. Our preliminary immunocytostaining study also showed that the subcellular distributions of COX-1 and COX-2 in 293 transfectants are almost identical.2
We observed that transfection of cells with COX-2 alone induced spontaneous PGE2 generation, with a concomitant increase in AA release. This AA release was suppressed by the cPLA2 inhibitor MAFP, but not by the other PLA2 or COX inhibitors tested, and cPLA2 is the only PLA2 isozyme detectable in the parental 293 cells examined so far. Moreover, coexpression of COX-2 and cPLA2, but no other PLA2s, led to a marked increase in AA release during both the immediate and delayed phases. These observations led us to formulate the hypothesis that COX-2 has the ability to activate cPLA2 through an as yet unknown, COX activity-independent, positive feedback mechanism. This may provide new insight into the mechanism responsible for cPLA2 activation in the delayed response, which is not accompanied by cytoplasmic Ca2+ signaling. A direct physical interaction between cPLA2 and COX-2 is unlikely, because cPLA2 is located in the cytosol (18), whereas COXs face the luminal sides of the perinuclear and endoplasmic reticular membranes (50). There may be an accessory protein that affects the functional cPLA2/COX-2 interaction. However, the possibility that MAFP inhibits an unidentified PLA2 isozyme, which is activated by and functionally linked with COX-2, cannot be ruled out at present.
In our previous study, we classified cPLA2, sPLA2-IIA, and sPLA2-V as signaling PLA2s, the AA-releasing effects of which were dependent upon the cellular activation state, and each of them, if properly expressed, could mediate both immediate and delayed AA release (32). We found here that AA released by these three signaling PLA2s was metabolized to PGE2 through COX-1 and COX-2 in the immediate response and COX-2 in the delayed response. Segregated utilization of cPLA2 and sPLA2 during these two PG-biosynthetic phases, which was observed in some previous studies (5, 6, 10, 11), may, therefore, depend on the expression levels or subcellular localizations of these PLA2s, which probably differ in different cell types, at the moment PG generation takes place.
A remarkable difference between cPLA2 and sPLA2 is the ability of the latter, but not the former, to mediate transcellular PG biosynthesis. In contrast to cPLA2, which contributes to PG biosynthesis only when COX coexists in the same cell, sPLA2-IIA and sPLA2-V are secreted extracellularly, bind to the cell surfaces of neighboring COX-expressing cells, and then contribute to remote stimulus-dependent PG biosynthesis. This property reflects a distinct role of the secretory group of PLA2s as an amplifier of a signal from a single cell to its surrounding microenviromnent. This paracrine route of sPLA2 action may be compatible with the role of sPLA2 seen in mast cell-fibroblast interactions (51, 52) and may also apply to other cell systems. Given that abundant sPLA2-IIA (and probably sPLA2-V in the mouse, in which sPLA2-V is distributed in a wide variety of tissues, whereas sPLA2-IIA expression is restricted to the intestine)3 accumulates in inflammatory exudates and exacerbates the process of inflammation (53), COX-2-expressing cells would appear to be the major targets of sPLA2 responsible for the propagation of sustained PG biosynthesis at inflamed sites.
The concentrations of sPLA2-IIA produced by 293 transfectants, estimated by the enzyme activities and the intensities of the immunoblot bands, reached as high as 10 to ~50 ng/ml, comparable to those produced by cytokine-stimulated cells, such as fibroblasts (10), macrophages (11) and liver cells (54), and sufficient to promote PG generation in these cells. In contrast, a single addition of purified or recombinant sPLA2-IIA to target cells generally requires concentrations of the order of µg/ml to exert its actions (9, 24, 25, 52, 55-60), as we also observed with our 293 transfectant system.2 The striking difference between the amounts of sPLA2-IIA required by these different systems, i.e. endogenously produced (ng/ml) versus exogenously added (µg/ml), implies that the continued supply of sPLA2-IIA, which occurs in the former situation, may be an important factor for its adequate actions during cellular (particularly prolonged) responses. As sPLA2-IIA is believed to be produced continuously at inflamed sites, it is likely that the former case is most relevant to pathophysiologic conditions in vivo. Moreover, the higher sensitivity of the cells to endogenously produced than exogenously added sPLA2-IIA predicts the presence of particular machinery, through which newly produced sPLA2-IIA is readily transported into the compartments where AA-rich phospholipid pools are present. Therefore, caution should be exercised in interpreting the results of studies involving single addition of excess sPLA2-IIA, which may not always reflect in vivo circumstances.
We previously observed that iPLA2 overexpression did not induce endogenous COX-2-dependent delayed PGE2 generation (32). We have now confirmed this observation by demonstrating that iPLA2 failed to mediate the delayed response even when COX-2 was overexpressed. To our surprise, A23187 elicited marked iPLA2-induced AA release, and this AA was metabolized to PGE2 via COX-1 in marked preference to COX-2. Gross and co-workers (28, 29) showed that iPLA2-dependent AA release was activated by A23187 in smooth muscle cells and suggested that calmodulin might be involved in this regulatory step, and our results of AA release appear to be in line with their observations. However, the apparently preferred coupling between iPLA2 and COX-1 in the immediate response, despite the fact that the amount of AA released induced by iPLA2 was comparable to that released by other PLA2s, cannot be simply explained by any of the theories we have proposed. We suppose that iPLA2 releases AA in closer proximity to COX-1 than COX-2 and/or iPLA2-derived AA is virtually inaccessible to COX-2. Because iPLA2 is known to form a multimeric complex (61) and even exists as multiple splice variants that affect enzyme activity (62) in cells, it may bind to a certain cofactor that facilitates iPLA2/COX-1 interaction following Ca2+ signaling. Nonetheless, these results reveal an unexplored bifunctional mode of iPLA2 actions in the regulation of phospholipid remodeling and signaling pathways.
Finally, although the approaches used have yielded valuable
information, we have to be careful of the limitation of this study that
the cell model may still not fully mimic other cells that produce COX
products. Important components may be missing from the cell machinery
that could alter the interactions under study. For instance, specific
fatty acid-binding proteins might be expressed and play a role,
undefined receptors might be involved, specific protein kinase cascades
could vary, and subcellular disposition of AA stores might differ,
according to cell types.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. A. Tischfield, S. S. Jones, J. Trzaskos, W. L. Smith, and R. M. Kramer for providing cDNAs, antibodies, and inhibitors. We thank Drs. H. Naraba, Y. Kosugi, A. Ueno, and S. Oh-ishi for the cloning of rat BKR B2 cDNA.
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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, Sports and Culture of Japan and 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.
The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; PG, prostaglandin; COX, cyclooxygenase; IL-1, interleukin-1; FCS, fetal calf serum; MAFP, methyl arachidonylfluorophosphate; CHO, Chinese hamster ovary; BK, bradykinin; BKR, bradykinin receptor.
2 T. Kambe, M. Murakami, S. Yamamoto, H. Kuwata, R. Takamiya, Y. Wakabayashi, M. Suematsu, and I. Kudo, unpublished observations.
3 H. Sawada, M. Murakami, and I. Kudo, unpublished observations.
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REFERENCES |
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