From the Department of Health Chemistry, School of
Pharmaceutical Sciences, and the ¶ First Department of Internal
Medicine, School of Medicine, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142-8555, Japan, the ** Departments of
Chemistry and Biochemistry, University of Washington, Seattle,
Washington 98195-1700, and the
Institut de Pharmacologie
Moleculaire et Cellulaire, CNRS-UPR 411, 660 route des Lucioles, Sophia
Antipolis, 06560 Valbonne, France
Received for publication, November 6, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Here we report cellular arachidonate (AA) release
and prostaglandin (PG) production by novel classes of secretory
phospholipase A2s (sPLA2s), groups III
and XII. Human group III sPLA2 promoted spontaneous AA
release, which was augmented by interleukin-1, in HEK293 transfectants.
The central sPLA2 domain alone was sufficient for its
in vitro enzymatic activity and for cellular AA release at
the plasma membrane, whereas either the unique N- or C-terminal domain
was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA2 was constitutively expressed in two human
cell lines, in which other sPLA2s exhibited different
stimulus inducibility. Human group XII sPLA2 had a weak
enzymatic activity in vitro and minimally affects cellular
AA release and PG production. Cells transfected with group XII
sPLA2 exhibited abnormal morphology, suggesting a unique
functional aspect of this enzyme. Based on the present results as well
as our current analyses on the group I/II/V/X sPLA2s,
general properties of cellular actions of a full set of mammalian
sPLA2s in regulating AA metabolism are discussed.
Secretory phospholipase A2
(sPLA2)1
comprises a family of Ca2+-dependent lipolytic
enzymes with a conserved Ca2+-binding loop and His-Asp dyad
at the catalytic site (1, 2). To date, 10 sPLA2 enzymes
(groups IB, IIA, IIC, IID, IIE, IIF, V, X, III, and XII) have been
identified in mammals (1, 2). In general, sPLA2s exhibit
tissue- and species-specific expression, which suggests that their
cellular behaviors and functions differ.
The group I/II/V/X sPLA2s represent a class of enzymes with
a molecular mass of 14-18 kDa and 6-8 conserved disulfides (1, 2).
sPLA2-IB and -X, but not the other enzymes, have an
N-terminal prepropeptide, and the proteolytic cleavage of this
prepropeptide is a regulatory step for generation of an active enzyme
(3, 4). sPLA2-IB is abundantly present in pancreatic juice,
and its main function has been thought to be the digestion of dietary phospholipids, although recent data with sPLA2-IB knockout
mice have demonstrated no appreciable defects in this process (5). sPLA2-IIA, a prototypic inflammatory PLA2, and
other group II subfamily sPLA2s (IID, IIE, IIF, and V) are
inducible in various tissues with inflammation or damage (6-13). On
the bases of current biochemical and cell biological studies,
sPLA2s in the I/II/V/X branch may participate in various
biological events, including arachidonate (AA) release from cellular
membranes (see below) (9-18), host defense against bacteria (19, 20),
atherosclerosis (21, 22), blood coagulation (23), and cancer (24).
Beyond the essential role of cytosolic PLA2 Besides the I/II/V/X branch, two distinct classes of sPLA2,
namely the group III and group XII branches, have been recently identified in mammals (36-38). Structurally, these two novel
sPLA2s show homology with the I/II/V/X sPLA2s
only within the Ca2+ loop and catalytic site His-Asp dyad.
Human sPLA2-III is a 56-kDa protein containing a long
N-terminal domain, a central sPLA2 domain that is
homologous to bee venom group III sPLA2, and a long
C-terminal domain (36). sPLA2-XII, which harbors an unusual
Ca2+ loop, is distantly related to other classes of
sPLA2s (37, 38). This enzyme is expressed in
antigen-activated helper T cells in the mouse (38). However, cellular
functions of these two novel sPLA2s have not yet been
described. To expand our current understanding of the sPLA2
actions on cells, we studied the cellular AA-releasing and
prostaglandin (PG)-biosynthetic properties of human
sPLA2-III and -XII by expressing these enzymes in HEK293 cells, as we have previously done with the I/II/V/X sPLA2s
(9-12, 14-18).
Materials--
Human embryonic kidney 293 (HEK293) cells (Human
Science Research Resources Bank), human lung epithelial BEAS-2B cells
(American Type Cell Collection, Manassas, VA), and human colon
adenocarcinoma HCA-7 cells (a generous gift from Dr. M. Tsujii (Osaka
University) and Dr. R. DuBois (Vanderbilt University Medical Center and
VA Medical Center)) were cultured in RPMI 1640 medium (Nissui
Pharmaceutical Co.) containing 10% (v/v) fetal calf serum (FCS;
Bioserum). The cDNAs for human sPLA2-III (36), human
sPLA2-XII (37), human COX-1 and COX-2 (15), and rat
glypican-1 (16) were described previously. HEK293 cells stably
expressing human sPLA2-V, human sPLA2-IIF, and
human COX-2 were described previously (11, 14, 15). The enzyme
immunoassay kits for PGE2 and the COX-2 inhibitor NS-398
were purchased from Cayman Chemicals. The goat anti-human COX-1 and
anti-human COX-2 antibodies were purchased from Santa Cruz Biosciences,
Inc. (Santa Cruz, CA). A23187 was purchased from Calbiochem. Human
interleukin (IL)-1 Establishment of Transfectants--
Establishment of HEK293
transformants was performed as described previously (12-16). Briefly,
1 µg of plasmid (sPLA2 cDNAs subcloned into the
pRc-CMV or pCR3.1 vector) was mixed with 2 µl of LipofectAMINE 2000 in 100 µl of Opti-MEM medium for 30 min and then added to cells that
had attained 40-60% confluence in 12-well plates (Iwaki Glass)
containing 0.5 ml of Opti-MEM. After incubation for 6 h, the
medium was replaced with 1 ml of fresh culture medium. After overnight
culture, the medium was replaced with 1 ml of fresh medium, and culture
was continued at 37 °C in an incubator flushed with 5%
CO2 in humidified air. The cells were cloned by limiting
dilution in 96-well plates in culture medium supplemented with 1 mg/ml
geneticin. After culture for 3-4 weeks, wells containing a single
colony were chosen, and the expression of each protein was assessed
by RNA blotting. The established clones were expanded and used for the
experiments as described below.
In order to establish double transformants expressing sPLA2
and glypican, cells expressing each sPLA2 were subjected to
a second transfection with glypican cDNA subcloned into
pCDNA3.1/Zeo(+) using LipofectAMINE 2000. Three days after the
transfection, the cells were used for the experiments or seeded into
96-well plates and cloned by culturing in the presence of 50 µg/ml
zeocin to establish stable transformants.
To assess functional coupling between sPLA2 and either of
the two COX isozymes, cells stably expressing sPLA2 were
transfected with COX-1 or COX-2 subcloned into pCDNA3.1 using
LipofectAMINE 2000. Three days after the transfection, the cells were
activated with A23187 to measure PGE2 generation and were
subjected to immunoblotting to examine COX-1 or COX-2 expression (see below).
Measurement of sPLA2 Activity--
sPLA2
activity was assayed by measuring the amounts of radiolabeled fatty
acids released from the substrate
1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine
(2-AA-PE), 1-palmitoyl-2-[14C]linoleoyl-PE (2-LA-PE),
2-AA-PC, or 2-LA-PC (Amersham Biosciences). Each substrate in ethanol
was dried up under N2 stream and was dispersed in water by
sonication. Each reaction mixture (total volume 250 µl) consisted of
appropriate amounts of the required sample, 100 mM Tris-HCl
(pH 7.4), 4 mM CaCl2, and 10 µM
substrate. After incubation for 10-30 min at 37 °C,
[14C]AA or [14C]LA was extracted, and
radioactivity was quantified, as described previously (14-18).
Expression of Recombinant sPLA2s by the Baculovirus
System--
Baculovirus expression of recombinant sPLA2
proteins was performed using the BAC-to-BAC baculovirus expression
system (Invitrogen). Briefly, sPLA2 cDNAs were
subcloned into the baculovirus expression vector pFASTBAC1 (Invitrogen)
at appropriate restriction enzyme sites. Recombinant sPLA2
proteins were first expressed in Sf9 insect cells and then
amplified in High Five insect cells (Invitrogen) according to the
manufacturer's instructions. Culture supernatants and cell lysates
(4-5 days after infection) were used for subsequent experiments.
Sf9 cells and High Five cells were maintained in Grace's insect
medium (Invitrogen) supplemented with 10% FCS and Express Five SFM
serum-free medium (Invitrogen), respectively.
Heparin Binding--
Recombinant sPLA2s (culture
supernatants from baculovirus-infected High Five cells) were incubated
with various amounts of heparin-Sepharose beads in 10 mM
Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS) for 2 h
at 4 °C, and PLA2 activities remaining in the
supernatants were assayed.
RNA Blotting--
Approximately equal amounts (~5 µg) of
total RNA obtained from the cells were applied to separate lanes of
1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred
to Immobilon-N membranes (Millipore Corp.). The resulting blots were
then probed with the respective cDNA probes that had been labeled
with [32P]dCTP (Amersham Biosciences) by random priming
(Takara Biomedicals). All hybridizations were carried out as described
previously (14-18).
SDS-PAGE/Immunoblotting--
Lysates from
105 cells were subjected to SDS-PAGE using 7.5-12.5% gels
under reducing conditions. The separated proteins were electroblotted
onto nitrocellulose membranes (Schleicher and Schuell) using a semidry
blotter (MilliBlot-SDE system; Millipore). After blocking with 3%
(w/v) skim milk in TBS containing 0.05% Tween 20 (TBS-Tween), the
membranes were probed with the respective antibodies (1:20,000 dilution
for COX-1, 1:5,000 dilution for COX-2, and 1:20,000 for FLAG epitope in
TBS-Tween) for 2 h, followed by incubation with horseradish
peroxidase-conjugated anti-goat (for COXs) or anti-mouse (for FLAG) IgG
(1:5,000 dilution in TBS-Tween) for 2 h and were visualized using
the ECL Western blot system (PerkinElmer Life Sciences) (14-18).
Reverse Transcription (RT)-PCR--
Synthesis of cDNA was
performed using 0.5 µg of total RNA from human cell lines and avian
myeloblastosis virus reverse transcriptase, according to the
manufacturer's instructions supplied with the RNA PCR kit (Takara
Biomedical). Subsequent amplification of the cDNA fragments was
performed using 1 µl of the reverse-transcribed mixture as a template
with specific primers for each sPLA2. For amplification of
sPLA2-IB, -IIA, -IID, -IIE, -IIF, -V, -X, and -XII, a set
of 23-bp oligonucleotide primers corresponding to 5'- and 3'-nucleotide
sequences of their open reading frames were used as primers (40-45).
For amplification of sPLA2-III, primers directed for the
sPLA2 domain and N-terminal domain were used (see below).
The PCR condition was 94 °C for 30 s and then 35 cycles of
amplification at 94 °C for 5 s and 68 °C for 4 min, using
the Advantage cDNA polymerase mix (Clontech).
The PCR products were analyzed by 1% agarose gel electrophoresis with
ethidium bromide. The gels were further subjected to Southern blot
hybridization using sPLA2 cDNAs as probe.
Construction of sPLA2-III
Mutants--
sPLA2-III mutants were produced by PCR with
the Advantage cDNA polymerase mix using
sPLA2-III/pRc-CMV as a template. The condition of PCR was
25 cycles at 94, 55, and 72 °C for 30 s each. The primers used
were as follows: III-5' primer (5'-ATGGGGGTTCAGGCAGGGCTG-3'), III-3'
primer (TCACTGGCTCCAGGACTTCTG-3'), III-S-S primer
(5'-GATGGACCATGCCTGGCACAC-3'), III-S-AS primer
(TCAAGTTGGGGAGGTGGCCCG-3'), III-HQ-S primer
(5'-TGCCGGGAACAAGACCGCTGC-3'), and III-HQ-AS primer
(5'-GCAGCGGTCTTGTTCCCGGCA-3'). In order to obtain sPLA2-III
wild type (WT) and the truncated mutants III-S, III-N+S, and III-S+C
(see "Results"), the primer sets III-5' and III-3', III-S-S and
III-S-AS, III-5' and III-S-AS, and III-S-S and III-3', respectively,
were used. In order for the mutants III-S and III-S+C to be secreted
when expressed in cells, the signal sequence for human group IIA
sPLA2 (40) was linked to the 5'-end of the III-S-S primer
and PCR-amplified. To attach the FLAG epitope at the C terminus, the
FLAG antisense oligonucleotide 5'-TTACTTGTGATCGTCGTCCTTGTAGTC-3' were
directly linked to the 5'-ends of the antisense primers. In order to
construct the catalytically inactive mutant III-N+S-HQ (see
"Results"), the first PCR was conducted with III-5' and III-HQ-AS
primers or with III-HQ-S and III-S-AS primers using
sPLA2-III-WT cDNA as a template. The resulting two
primary PCR fragments were mixed, denatured at 94 °C for 5 min,
annealed at 37 °C for 30 min and then 55 °C for 2 min, and extended at 72 °C for 4 min during each cycle. The secondary PCR product with specific mutation was obtained after 25 additional PCR
cycles with III-5' and III-S-AS primers. A similar strategy was used to
prepare III-S-HQ. Each PCR product was ligated into the pCR3.1 and was
transfected into Top10F' supercompetent cells (Invitrogen). The
plasmids were isolated and sequenced using a Taq cycle
sequencing kit (Takara Biomedicals) and an autofluorometric DNA
sequencer 310 Genetic Analyzer (Applied Biosystems) to confirm the sequences.
Activation of HEK293 Cells--
HEK293 cells (5 × 104/ml) were seeded into each well of 48-well plates. To
assess fatty acid release (14-18), [3H]AA or
[3H]oleic acid (OA) (both from Amersham Biosciences) (0.1 µCi/ml) was added to the cells in each well on day 3, when they had
nearly reached confluence, and culturing was continued for another day. After three washes with fresh medium, 100 µl of RPMI 1640 with or
without 10 µM A23187 with 1% FCS or 1 ng/ml IL-1
To assess transcellular PGE2 biosynthesis (11, 15), two
cell populations (2.5 × 104 cells/ml for each) were
added to the same wells of 48-well plates (100 µl/well) and cultured
for 4 days. Then the cells were stimulated with IL-1 Exogenous sPLA2 Assay--
Subconfluent cells grown
in 48-well plates were incubated with recombinant sPLA2s
(culture supernatants from baculovirus-infected High Five cells) for
1 h, and PGE2 released into the supernatants was quantified.
Confocal Laser Microscopy--
Cells grown on collagen-coated
cover glasses (Iwaki Glass) were fixed with 3% paraformaldehyde for 30 min in phosphate-buffered saline (PBS). After three washes with PBS,
the fixed cells were sequentially treated with 1% (w/v) bovine serum
albumin (for blocking) and 0.2% (v/v) Triton X-100 (for
permeabilization) in PBS for 1 h, with anti-FLAG antibody (1:500
dilution) for 1 h in PBS containing 1% albumin and then with
fluorescein isothiocyanate-goat anti-mouse IgG (1:500 dilution) for
1 h in PBS containing 1% albumin. After six washes with PBS, the
cells were mounted on glass slides using Perma Fluor (Japan Tanner),
and the sPLA2 signal was visualized using a laser-scanning
confocal microscope (IX70; Olympus), as described previously (16,
18).
Statistical Analysis--
Data were analyzed by Student's
t test. Results are expressed as the mean ± S.E., with
p = 0.05 as the limit of significance.
Human sPLA2-III
Enzymatic Properties--
Human sPLA2-III-WT and its
truncated mutants, III-S, III-N+S, and III-S+C, (structures illustrated
in Fig. 1A) were each
transfected into HEK293 cells. Stable transfectants expressing WT and
truncated enzymes, with or without C-terminal FLAG epitope, were
screened by Northern blotting using a specific sPLA2-III
cDNA probe (Fig. 1B) or immunoblotting using an
anti-C-terminal FLAG tag antibody (Fig. 1C), and clones in
which their expression levels were almost comparable with one another
were used in subsequent studies. As shown in Fig. 1C,
III-WT, III-N+S, III-S+C, and III-S were expressed as major
immunoreactive proteins with predicted molecular masses of 56, 32, 42, and 17 kDa, respectively. Flanking the C terminus with the FLAG epitope
did not significantly affect in vitro and cellular
functions, as described below.
Culture supernatants of these transfectants were assayed for
PLA2 activity using PE and PC bearing AA or LA at their
sn-2-position as substrates. Under our PLA2
assay condition, the WT and truncated enzymes exhibited comparable
PLA2 activity with similar substrate specificity (Fig.
1D). Of the four substrates tested, 2-LA-PE was the best
substrate, being hydrolyzed 2-3 and 6-8 times more efficiently than
2-LA-PC and 2-AA-PE, respectively. 2-AA-PC was hydrolyzed ~2-fold
faster than 2-AA-PE, whereas 2-LA-PE was hydrolyzed ~2-fold faster
than 2-LA-PC.
Cellular Functions--
To assess the fatty acid-releasing
function of sPLA2-III in cells,
sPLA2-III-WT-transfected and control HEK293 cells were preincubated overnight with [3H]AA or
[3H]OA, washed, and then cultured for 4 h with or
without 10% FCS and/or IL-1. As shown in Fig.
2A, in the presence of 10%
FCS, the WT enzyme significantly increased the release of both
[3H]AA and [3H]OA almost in parallel. The
further addition of IL-1 resulted in an increase in
[3H]AA, but not [3H]OA, release (Fig.
2A). Comparing these properties of sPLA2-III with other sPLA2s reported so far,
FCS-dependent, fatty acid nonselective release is similar
to that by the plasma membrane-acting enzymes, such as
sPLA2-X and -V, and IL-1 augmentation of AA release is reminiscent of that by the HSPG-shuttled enzymes, such as
sPLA2-IIA, -IID, -IIE, and -V (9, 10, 14-18).
In agreement with the in vitro enzymatic activity (Fig. 1),
the release of [3H]AA (Fig. 2B) and
[3H]OA (data not shown) by cells transfected with the
three truncated enzymes (III-S, III-N+S, and III-S+C) was similar to
that by cells transfected with the WT enzyme. As shown in Fig.
2C, [3H]AA release proceeded gradually over
8 h of culture. Thus, the sPLA2 domain alone is
essential and sufficient for cellular fatty acid release. As a notable
difference, [3H]AA release by cells expressing III-WT,
III-N+S, or III-S+C was significantly augmented by IL-1, whereas this
augmentation was not observed appreciably in cells expressing III-S
(i.e. the sPLA2 domain alone) (Fig.
2B). When III-N+S-HQ and III-S-HQ, in which the putative
catalytic center His in III-N+S and III-S was, respectively, replaced by Gln, were transfected into HEK293 cells, no in
vitro PLA2 activity was detected (data not shown), and
an increase in cellular [3H]AA release was not observed
(Fig. 2D), despite their reasonable expression levels (data
not shown). This result indicates that the catalytic activity is an
absolute requirement for the enzymatic action of sPLA2-III
on both phospholipid vesicles and cellular membranes.
The AAs released by III-WT, -N+S, and -S+C following IL-1 stimulation
were each efficiently converted to PGE2 (Fig.
3A). This PGE2
production was ablated by the COX-2 inhibitor NS-398 (data not shown),
revealing functional coupling between sPLA2-III and COX-2
in the IL-1-stimulated delayed response. Remarkably, PGE2 production by cells transfected with III-S was less than that by cells
transfected with III-WT, -N+S, and -S+C (Fig. 3A), despite the fact that all four proteins produced similar amounts of AA (Fig.
2B). Kinetic experiments demonstrated that PGE2
production by III-WT increased linearly over 1-8 h, thus lagging
behind AA release (Fig. 2C), whereas PGE2
production by III-S was increased only modestly over the whole culture
period (Fig. 3B). As shown in the top
panel of Fig. 3A, III-WT, III-N+S, and III-S+C
augmented IL-1-induced expression of COX-2 markedly relative to
replicate control cells, whereas COX-2 induction by III-S was less than that by III-WT, -N+S, and -S+C (although it was still higher than control cells). Time course experiments showed that COX-2 mRNA induction in III-WT-expressing cells peaked at 1 h and declined to
a plateau level after 4-8 h, whereas that in control and
III-S-expressing cells reached a peak by 1 h and disappeared
thereafter (Fig. 3B, inset). These results
suggest that sPLA2-III has the ability to enhance COX-2
expression, as in the case of several HSPG-binding group II subfamily
sPLA2s (16-18), and that the poor
PGE2-biosynthetic action of the sPLA2 domain
alone is, at least in part, due to its poor ability to induce
COX-2.
To elucidate functional coupling between sPLA2-III and COX
enzymes more directly, we performed cotransfection experiments, in
which HEK293 cells expressing sPLA2-III were subsequently
transfected with either COX-1 or COX-2, and PGE2 production
following A23187 stimulation was examined. Expression of COX-1 and
COX-2 was verified by immunoblotting (data not shown). As shown in Fig.
3C, PGE2 production by either COX-1 or COX-2 was
markedly augmented by coexpression of sPLA2-III-WT. When
the PGE2-biosynthetic activities of III-WT and -S were
compared in this cotransfection analysis, PGE2 production
by III-WT via the overexpressed COX-1 (Fig. 3D) or COX-2
(data not shown) was significantly higher than that produced by III-S,
although A23187-induced immediate [3H]AA releases by
III-WT and -S were comparable over 30 min of incubation period (Fig.
3E). These results suggest that the sPLA2 domain
alone is coupled with downstream COX enzymes less efficiently than is
III-WT, even if COX enzymes are equivalently expressed in cells.
III-N+S and III-S+C were coupled with the overexpressed COX-1 with the
same potency as III-WT (data not shown), indicating that either the N-
or C-terminal domain can confer onto the sPLA2 domain the
ability to efficiently couple to COX enzymes.
We next performed the transcellular PGE2-biosynthetic assay
(11, 15), in which sPLA2-III-expressing and
COX-2-expressing HEK293 cells were cocultured. As was observed with
sPLA2-IIF used as a positive control (11), coculture of
sPLA2-III-WT-expressing cells with COX-2-expressing cells
resulted in a marked increase in the production of PGE2
(Fig. 3F), indicating that the WT enzyme is capable of
supplying AA to COX-2 in neighboring cells to propagate PGE2 production in a paracrine manner. Coculturing
sPLA2-III-S-expressing cells with COX-2-expressing cells
also increased PGE2 production significantly, yet the
amount of PGE2 produced by III-S was again reproducibly
lower than that produced by the WT enzyme (Fig. 3F).
Heparanoid Dependence--
When recombinant FLAG-tagged
sPLA2-III-S, -N+S, and -S+C, which were expressed by the
baculovirus/insect cell system (see below), were incubated with
incremental amounts of heparin-conjugated beads, III-N+S and III-S+C
were more efficiently absorbed than III-S to the beads (Fig.
4A, left) under the
condition where they were equally precipitated by anti-FLAG
antibody-conjugated beads (Fig. 4A, right),
indicating that both N- and C-terminal domains facilitate the binding
of sPLA2-III to heparanoids. To assess whether
sPLA2-III action on cells depends on cell surface HSPG, as
does the HSPG-shuttled group II subfamily sPLA2s (14-18),
the effect of exogenous heparin, which solubilizes the HSPG-bound pool
of sPLA2s and thereby suppresses their cellular functions (14-18), was examined. As shown in Fig. 4B, AA release by
sPLA2-III-WT in the absence of IL-1 (i.e.
FCS-dependent release) was insensitive to exogenous
heparin, whereas IL-1-augmented AA release was reversed by heparin to a
level comparable with FCS-dependent release (Fig. 4B, left). This heparin effect resembled its
effect on sPLA2-V-mediated AA release, where
IL-1-stimulated (reflection of the HSPG-shuttling route), but not
FCS-dependent (reflection of the plasma membrane route), AA
release was suppressed by heparin (Fig. 4B,
right). AA release by III-S, which was largely unaffected by
IL-1 as noted above (Fig. 2), was unaffected by exogenous heparin (Fig.
4B, middle). Moreover, IL-1-stimulated
PGE2 production by WT-expressing cells was reduced to a
level comparable with that by III-S-expressing cells following heparin
treatment (Fig. 4C). In contrast, the inhibitory effect of
heparin on PGE2 production by III-S-expressing cells was
only minimal (Fig. 4C).
Since the function of the HSPG-shuttled group II subfamily
sPLA2s is augmented by overexpression of glypican, a
glycosylphosphatidylinositol-anchored HSPG that acts as a functional
adaptor for these sPLA2s (16, 18), we next assessed the
effect of glypican coexpression on sPLA2-III-mediated AA
metabolism. Expression of glypican in III-WT- or III-S-expressing cells
was verified by Northern blotting (Fig. 4D). As shown in
Fig. 4E, coexpression of glypican markedly enhanced AA
release and PGE2 production by the WT enzyme. AA release
and PGE2 production by III-S were increased by glypican to
a much lesser extent (Fig. 4E). A small augmentation of
III-S function by glypican overexpression may be due to its weak
affinity for heparanoids (Fig. 4A, left) or to
some other unknown mechanisms. Overexpression of glypican alone did not
affect AA release, as previously reported (16).
The function of the HSPG-shuttled group II subfamily
sPLA2s, but not that of the plasma membrane-acting
sPLA2s, is markedly attenuated by
12/15-lipoxygenase-inhibitable antioxidants (such as NDGA), leading to
the suggestion that stimulus-induced membrane modification involves
lipid-oxidative events (11, 46). In agreement with this notion,
IL-1-augmented AA release (data not shown) and PGE2
production (Fig. 4F) by sPLA2-III-WT were
reduced partially, to the level of those by III-S, by treatment
of the transfectants with NDGA, whereas NDGA failed to affect
PGE2 production by III-S. This suggests that III-WT, as has
been proposed for the other HSPG-shuttled sPLA2s (11,
46-48), is accessible to particular membrane compartments that undergo
oxidative modification after cytokine signaling.
Effect of Exogenous sPLA2-III on AA
Metabolism--
The ability of the sPLA2 domain of
sPLA2-III to elicit spontaneous (FCS-dependent)
AA release suggests that it can act on the external plasma membrane, as
does sPLA2-X and -V (17, 25-27). To explore this
possibility further, we aimed to examine the effects of exogenous
sPLA2-III on AA metabolism in mammalian cells. To this end,
we expressed FLAG-tagged recombinant sPLA2-III in High Five
insect cells using the baculovirus system in order to obtain recombinant enzyme in abundance. As assessed by immunoblotting using
the anti-FLAG antibody, recombinant III-S, III-N+S, and III-S+C were
expressed abundantly in the insect High Five cells as the expected
sizes (Fig. 5A). The
expression levels of III-S and III-N+S were comparable, whereas that of
III-S+C was approximately one-tenth that of III-S and III-N+S. III-WT
was not expressed appreciably in High Five cells (possibly due to rapid
proteolytic degradation). Similar results were obtained when the
enzymes were expressed in Sf9 insect cells (data not shown). The
in vitro PLA2 activity of III-S was comparable
with that of III-N+S and was about 10 times higher than III-S+C (Fig.
5B), in agreement with their expression levels (Fig.
5A). Enzymatic properties (e.g. substrate
specificity, pH dependence, and Ca2+ requirement) of these
insect cell-derived truncated enzymes were similar to those of enzymes
expressed in HEK293 cells (data not shown).
We then investigated the effects of recombinant III-S and III-N+S,
which were secreted from the baculovirus-infected High Five cells at
comparable levels (Fig. 5, A and B), on
PGE2 production by HEK293 cells. Thus, culture supernatants
of control (i.e. no baculovirus infection) and of III-S- or
III-N+S-expressing High Five cells were diluted 10 times with RPMI 1640 plus 10% FCS and added to COX-2-transfected HEK293 cells. As shown in
Fig. 5C, both exogenous III-N+S and III-S markedly increased
PGE2 production by COX-2-transfected HEK293 cells after a
1-h incubation.
Subcellular Localization--
We next performed indirect
immunofluorescent confocal microscopy to assess subcellular
distribution of sPLA2-III-WT and truncated mutants in
HEK293 transfectants. As shown in Fig. 6,
signals for III-WT, III-N+S, and III-S+C were detected in the
cytoplasmic punctate regions that excluded the nucleus, whereas III-S
was distributed mainly on the plasma membrane but not in the cytosol. In addition, intense staining was seen at the spindle edges of cell
adhesion sites in cells transfected with III-WT or III-N+S and to a
lesser extent with III-S+C (Fig. 6).
Expression in Human Cell Lines--
We next looked for human cell
lines that expressed sPLA2-III endogenously and found that
BEAS-2B, a human lung epithelial cell line (Fig.
7, A and B), and
HCA-7, a human adenocarcinoma cell line (Fig. 7C), expressed
this enzyme in addition to several other sPLA2s. RT-PCR for
sPLA2-III was carried out using two different sets of
primers (III-5'/III-HQ-AS and III-5'/III-S-AS), each of which amplified
a single band with a predicted size (Fig. 7A). Subsequent
Southern hybridization using a sPLA2-III-specific cDNA probe confirmed that these bands indeed corresponded to the expected portions of the enzyme (data not shown). In BEAS-2B cells,
sPLA2-III was constitutively expressed and decreased after
stimulation with cytokines (TNF-
sPLA2-III was also constitutively expressed in HCA-7 cells,
in which its expression was unaffected by IL-1 (Fig. 7C). In
this cell line, sPLA2-IIA was absent in unstimulated cells
and was strongly induced by IL-1; sPLA2-IID, -IIF, -V, and
-X were constitutively expressed, among which only sPLA2-V
was up-regulated by IL-1; and other sPLA2s (IB, IIE, and
XII) were undetectable (Fig. 7C). When
sPLA2-III-S and sPLA2-N+S, which were produced
by the baculovirus system (Fig. 5), were exogenously added to HCA-7
cells, there was a substantial increase in PGE2 production
(Fig. 7D).
Human sPLA2-XII
Enzymatic Properties--
Human sPLA2-XII cDNA was
transfected into HEK293 cells, and the expression of the enzyme in
stable transfectants obtained after drug selection was assessed by
Northern blotting (Fig. 8A, inset) and Western blotting (see below). In our
PLA2 assay using the four substrates (PE and PC bearing
sn-2-AA or -LA), sPLA2-XII-expressing cells
displayed no detectable PLA2 activity in both culture
supernatants and cell lysates. The enzyme activity was still below the
detection limit even when recombinant sPLA2-XII was
overexpressed by the baculovirus/High Five cell system (data not
shown). It is unlikely that sPLA2-XII was inappropriately
expressed in our system, since it was detected by Northern and Western
blottings as readily as other sPLA2s transfected in HEK293
or insect cells. Thus, sPLA2-XII does not exhibit
detectable enzymatic activity toward PE and PC in our PLA2
assay, in line with a previous report that the activity of this enzyme
is extremely low as compared with most other sPLA2s (38).
Effects on AA Metabolism--
[3H]AA and
[3H]OA release by sPLA2-XII-expressing cells
in the presence of 10% FCS was increased minimally relative to that by
control cells, even in the presence of IL-1 (Fig. 8A). There was no appreciable increase in A23187-stimulated immediate [3H]AA release in sPLA2-XII-expressing cells
(data not shown). sPLA2-XII-expressing cells did not
display increased PGE2 production relative to control cells
even when cells were cotransfected with COX-1 (Fig. 8B) or
COX-2 (data not shown), although sPLA2-III and -IIF showed marked functional COX-1 coupling under the same experimental condition. sPLA2-XII also failed to increase PGE2
production when HEK293 cells expressing this enzyme were cocultured
with those expressing COX-2 in the transcellular assay (data not shown).
Microscopic Analyses--
Our antibody raised against human
sPLA2-XII specifically recognized a single 18-kDa protein
in HEK293 transfectants on Western blotting (Fig. 8C). Using
this antibody, we performed immunostaining of sPLA2-XII
expressed in HEK293 cells by confocal microscopy. We noted that a
population of sPLA2-XII-expressing cells exhibited unusual
morphology; some cells appeared thin and long (Fig. 8D, panel b), whereas other cells were giant and multinucleated
(Fig. 8D, panel c). In both situations,
immunoreactivity of sPLA2-XII was detected throughout the
intracellular regions excluding the nucleus. These unique morphological
features were not observed in cells transfected with other
sPLA2s (9-11, 14-18).
To complete our current understanding of the AA-releasing capacity
of the full set of mammalian sPLA2s in transfected cells (HEK293), we have herein examined the AA-releasing and
PGE2-biosynthetic functions of the two recently discovered
sPLA2s, group III and XII. These two sPLA2s
show homology with other sPLA2s only in the catalytic site
and Ca2+-binding loop (36, 38) and thus appear to have
diverged from the group I/II/V/X sPLA2s at early
evolutional stages. Group III enzymes were originally identified in bee
venom (49); subsequently in venom from scorpion (imperatoxin I and
phospholipin) (50, 51), lizard (52), and jellyfish (53); and more
recently in Drosophila (in which five distinct group III
sPLA2-related genes have been found) by scanning of public
databases (2). Bee venom group III sPLA2 has been shown to
induce AA release when added exogenously to several cell types and
elicits various biological effects in vivo (54-58).
Imperatoxin I, a scorpion venom group III sPLA2, inhibits
ryanodine binding to Ca2+ release channels probably
dependent upon its catalytic activity (50). However, it has remained
unknown whether its mammalian homolog exerts a similar biological
effect and, if so, how its function is regulated. Group XII
sPLA2s, first cloned from humans (37) and mice (38), have
also been found in the genomic databases of various low vertebrate
species, yet there has been no functional assessment of this group of enzymes.
sPLA2-III--
Human sPLA2-III is made up
of a central group III sPLA2 domain flanked by N- and
C-terminal regions (36). Although no data base entries with significant
homology to the N- and C-terminal domains can be found, both domains
are highly cationic and are predicted to fold separately from the
sPLA2 domain (36). Assessment of in vitro
enzymatic activities of sPLA2-III-WT and the truncated mutants (III-S, III-N+S, and III-S+C) expressed in HEK293 cells (Fig.
1) and High Five insect cells (Fig. 5) demonstrates that the central
sPLA2 domain alone is sufficient for catalytic function and
neither N- nor C-terminal domain profoundly modulates the catalytic
function of the sPLA2 domain. This is in line with the observation that recombinant bee venom sPLA2 expressed as
an N-terminal fusion protein exhibits the same catalytic activity as
the recombinant protein after removal of the N-terminal fusion peptide
(59) and implies that the presence of the N-terminal extension (and presumably the C-terminal region, which is also not part of the catalytic site (36, 60)) does not interfere with the catalytic activity
of sPLA2-III. This contrasts with sPLA2-IB and
-X, for which proteolytic removal of the prepropeptide is essential for full enzymatic activity (3, 4). This difference exists most likely
because, unlike the I/II/V/X sPLA2s, which contain a
hydrogen bond network linking the N terminus to catalytic residues (1, 2), the N terminus of bee venom group III enzyme (and probably human
sPLA2-III) does not form part of the active site structure (60).
The present cellular study suggests that sPLA2-III-WT, as
well as the mutants harboring either the N- or C-terminal domain, can
act on cells through the HSPG pathway after IL-1 stimulation. The
highly cationic nature of the N-terminal (pI 9.1) and C-terminal (pI
11.3) domains, in contrast to the central sPLA2 domain that is acidic (pI 5.4) (36), may allow their electrostatic interaction with
anionic heparin (or other anionic components). Although the affinity of
III-N+S and III-S+C for heparin is weaker than that of
sPLA2-IIA, sPLA2-IID, and sPLA2-V
(data not shown), it appears to be still sufficient to promote cellular
function. Indeed, the IL-1-augmented components of AA release and
PGE2 production by sPLA2-III-WT are
sensitive to heparin treatment, and, conversely, overexpression of
glypican, a glycosylphosphatidylinositol-anchored HSPG to which
HSPG-shuttled sPLA2s bind (16, 18), results in marked
increases in AA release and PGE2 production by III-WT (Fig.
4), providing strong support for a functional link between sPLA2-III-WT and cellular HSPG. Moreover, the
immunocytostaining study suggests that either the N- or C-terminal
domain is essential for intracellular localization of the enzyme (Fig.
6). This is reminiscent of the previous finding that the
heparin-binding group II subfamily sPLA2s (IIA, IID, IIE,
and V) can be internalized into cells in HSPG- and caveolae- or
raft-dependent manners, followed by cytoplasmic vesicle
formation and intracellular membrane hydrolysis (16, 18, 28).
Without cell stimulation, sPLA2-III-WT and all of the
truncated mutants elicit spontaneous (FCS-dependent),
nonselective fatty acid release (Fig. 2). This pattern is very similar
to that of sPLA2-X, which acts on the PC-rich outer leaflet
of the plasma membrane (4, 17, 25, 27), and suggests that
sPLA2-III, via its sPLA2 domain, can act on
cells through the external plasma membrane pathway. The facts that
sPLA2-III (and its truncated mutants) has significant
activity toward PC (Fig. 1D), that the cellular AA-releasing
function of III-S is insensitive to exogenous heparin treatment or is
poorly augmented by glypican overexpression (Fig. 4), that III-S is
distributed on the plasma membrane (Fig. 6), and that exogenous enzyme
is capable of increasing PGE2 production in two cell types
(Figs. 5C and 7D) support this notion.
Collectively, we conclude that sPLA2-III can utilize the
HSPG-shuttling pathway, where its unique N- and C-terminal domains play
a role in entering the HSPG-shuttling route (and thereby affecting the
targeting of the enzyme) and its core sPLA2 domain exerts
cellular membrane hydrolysis (Fig. 9).
Although the N- and C-terminal domains have no sequence homology, both
of them contribute to heparanoid binding based on the presence of basic residues. By comparison, the group II subfamily sPLA2s have
clusters of heparin-binding cationic residues on their core surfaces
(61, 62). Strikingly, the N-terminal (and probably C-terminal) domain of sPLA2-III also facilitates the distribution of this
enzyme into the spindle edges of cell adhesion sites (Fig. 6). So far, none of the other sPLA2s exhibit this unique localization
(10, 11, 16-18). This suggests that some anionic components or binding molecules that preferentially associate with the N-terminal domain may
exist in the spindle edges of HEK293 cells, although functional consequences of this localization remain to be elucidated. Note that
the N- and C-terminal domains found in the five Drosophila group III enzymes have no sequence homology with those of human enzyme
(2), suggesting that their roles are different.
Since group III enzyme purified from bee venom consists of only the
sPLA2 core (49), it may be anticipated that human
sPLA2-III also undergoes proteolytic processing and
maturation in mammalian cells. The presence of a basic doublet KE at
the end of the N-terminal domain and several basic residues including
basic doublets in the C-terminal domain (36) suggests that their
proteolytic removal by subtilisin-like protease in the Golgi (63).
Indeed, besides the major bands with expected sizes, several shorter
products are faintly detected in HEK293 cells transfected with
sPLA2-III (e.g. 30-kDa band in III-WT and
III-N+S) in our immunoblot analysis (Fig. 1C). In addition,
sPLA2-III appears to be susceptible to endogenous
protease(s) in High Five insect cells, in which III-S+C and III-N+S
(and probably III-WT, which was almost undetectable) are rapidly
degraded during storage (data not shown). At present, the maturation
process of sPLA2-III in mammalian cells remains obscure. It
would be important to determine which forms of the enzyme (III-WT,
III-N+S, III-S+C, III-S, or other processed forms) are truly present
and functioning in vivo.
Interestingly, coexpression experiments demonstrate that III-WT (as
well as III-N+S and III-S+C) is more efficiently coupled with COX than
III-S (Fig. 3, D-F). Although the precise reasons for this
result are unclear, it reminds us of the central dogma in the
eicosanoid field that subcellular location of the biosynthetic enzymes
is a critical determinant for their optimal functional coupling. It is
now obvious that the perinuclear co-localization of sequential
biosynthetic enzymes, including cPLA2
The sPLA2-III transcript is detected in the kidney, heart,
liver, and skeletal muscle by Northern blotting (36). In two cell lines
that endogenously express sPLA2-III, sPLA2-III
expression is constitutive and not cytokine-inducible, unlike the group
II subfamily sPLA2s, sPLA2-IIA and
sPLA2-V, that are induced by diverse sets of cytokines
(Fig. 7). This implies that the transcriptional regulations and
possibly functions of these sPLA2s are distinct in these
cells. Although exogenous sPLA2-III-S modestly increased PGE2 production by HCA-7 cells (Fig. 7D), it
remains to be elucidated whether endogenous sPLA2-III also
participates in PGE2 production or exhibits other
functions. Expression of multiple sPLA2s in a colon
adenocarcinoma cell line is intriguing to note, since the COX-2-derived
PGE2 has been implicated in the exacerbation of colorectal
cancer (70-72). Although targeted disruption of cPLA2
Bee venom group III enzyme has been shown to evoke several cellular and
in vivo responses via the N-type sPLA2 receptor
independently of its catalytic activity (2, 32). It is tempting to
speculate that human sPLA2-III may represent an endogenous
ligand for the N-type receptor. Although the molecular entity of the
N-type sPLA2 receptor is still obscure, it has been
recently shown that some neurotoxic sPLA2s, including bee
venom sPLA2, bind to calmodulin with high affinity (73).
Since calmodulin is a cytosolic protein, this finding supports the view
that neurotoxic sPLA2s have to be internalized to exert
their effect. Whether calmodulin indeed acts as a functional binding
protein for sPLA2-III and whether another high affinity
binding site(s) for sPLA2-III exists need to be addressed.
Further elucidation of the N-type receptor may provide insights into
the regulatory functions of sPLA2-III. Additionally, a
recent finding that a peptide derived from bee venom sPLA2
inhibits replication of human immunodeficiency virus by blocking the
virus entry into host cells (74) suggests a possible role of human counterpart in this process.
sPLA2-XII--
sPLA2-XII has an unusual
structure among the sPLA2 family members in that only 3 of
its 11 cysteines correspond to cysteines of other sPLA2s
(37, 38). Here we show that sPLA2-XII fails to increase
cellular fatty acid release and PGE2 production despite its
expression in HEK293 cells (Fig. 8, A and B). It
is thus likely that sPLA2-XII is incapable of mobilizing
cellular AA due to its weak catalytic activity, although we cannot rule
out the possibility that at higher expression levels it can influence
AA metabolism. The putative sPLA2-XII in zebrafish
represented in genomic databases contains a leucine in place of
histidine in the catalytic center, strongly suggesting that the
zebrafish sPLA2-XII has little or no catalytic activity
(37). This supports the idea that the catalytic activity of
sPLA2-XII may not be critical for its cellular function.
The appearance of multinucleated giant cells in a population of
sPLA2-XII-transfected cells (Fig. 8D) is
noteworthy, which suggests a potential role of this enzyme in membrane
fusion or cell division, although the molecular mechanisms are unclear. In these multinucleated cells, the main sPLA2-XII
immunoreactivity appears to be enriched in endoplasmic reticulum.
Similar intracellular localization of mouse sPLA2-XII has
been reported in baby hamster kidney cells transfected with this enzyme
(38). Immunohistochemistry using our anti-sPLA2-XII
antibody to determine cell types that endogenously express this enzyme
will help us to understand its physiological and pathological functions.
Using the strategy of overexpression of AA-metabolic enzymes
in HEK293 cells and several other cell lines, we have uncovered some of
the functional properties of the full set of mammalian sPLA2s. The ability of sPLA2s to release AA
from quiescent cells is highly dependent upon their interfacial binding
to PC enriched in the outer leaflet of the plasma membrane (X > V > IIF ~ III > IB Although this series of studies has provided useful information about
sPLA2 behaviors and functions, much work is still needed to
determine the complete set of functions of each sPLA2
in vivo. Each sPLA2 displays different tissue
distribution and stimulus inducibility, implying that the
sPLA2 members exhibit nonredundant functions in each
tissue. Some sPLA2s may act as ligands (like cytokines),
rather than enzymes, to transduce signals via the distinct classes of
sPLA2 receptors (32). Some sPLA2s may play roles in defense against bacterial infection (which appears to be true
for sPLA2-IIA) (19, 20), lipoprotein metabolism (21, 22,
75, 76), and other biological events (23, 24, 77). Determining the
precise localization of each sPLA2 in various physiological
and pathological tissues, developing inhibitors specific for each
sPLA2, and targeted disruption or transgenic expression of
each sPLA2 will yield more informative and conclusive answers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
(cPLA2
) in the initiation of stimulus-coupled AA
metabolism, the I/II/V/X class of sPLA2s also has the
ability to augment AA metabolism by multiple mechanisms (1).
sPLA2s that show high interfacial binding to zwitterionic
phosphatidylcholine (PC), such as sPLA2-X and -V, are
capable of releasing AA from the PC-rich outer leaflet of the plasma
membrane of quiescent cells (the external plasma membrane pathway) (17, 25-27). Cationic, heparin-binding, group II subfamily sPLA2s, such as sPLA2-IIA, -IID, -IIE, and -V,
show marked preference for anionic phospholipids over PC and utilize
the heparan sulfate proteoglycan (HSPG)-shuttling pathway (14-18, 28).
In this regulatory pathway, these enzymes are captured by HSPGs
(typically glypican, a glycosylphosphatidylinositol-anchored HSPG) in
caveolae or rafts on activated cells and then internalized
into vesicular membrane compartments that are enriched in the
perinuclear area, where downstream cyclooxygenases (COXs) are located
(16, 17, 28). This spatiotemporal co-localization of sPLA2s
and COXs in the perinuclear compartments may allow efficient supply of
AA between these enzymes. Recent evidence implies that the
clathrin-independent, caveolae/raft-mediated endocytosis, and
associated vesicular traffic is directed toward a rapid cycling pathway
via the Golgi and endoplasmic reticulum (29). Occurrence of the
HSPG-shuttling pathway appears to be cell type- and stimulus-specific,
and in certain cases HSPGs exhibit a negative regulatory effect on the
heparin-binding sPLA2s by facilitating their
internalization and subsequent lysosomal degradation (30, 31).
sPLA2-IIF, an anionic group II subfamily sPLA2
with poor affinity for HSPG, may interact stably with the plasma
membrane through its unique C-terminal extension and releases AA (11),
although the possibility that this enzyme also functions after
internalization cannot be ruled out. In addition, cellular actions of
several sPLA2s can be mediated by sPLA2
receptors independent of their enzymatic activity (32). Targeted
disruption of the M-type sPLA2 receptor gene results in
reduced inflammatory response in mice (33). The M-type
sPLA2 receptor can also act as a negative regulator for
sPLA2s by inhibiting their enzymatic functions in serum and
by promoting their internalization and subsequent degradation (34,
35).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
, interferon (IFN)-
, and tumor necrosis factor
(TNF-
) were purchased from Genzyme. LipofectAMINE 2000 reagent,
Opti-MEM medium, TRIzol reagent, geneticin, zeocin, and mammalian
expression vectors (pCR3.1, pRc-CMV, and pcDNA3.1 series of vectors
containing a neomycin- or zeocin-resistant gene) were obtained from
Invitrogen. Fluorescein isothiocyanate-conjugated anti-mouse and
anti-rabbit IgGs and horseradish peroxidase-conjugated anti-goat IgG
were purchased from Zymed Laboratories Inc.. Mouse monoclonal anti-FLAG antibody, anti-FLAG antibody-conjugated agarose, and heparin were from Sigma. The lipoxygenase-inhibitory antioxidant nordihydroguaiaretic acid (NDGA) was purchased from BIOMOL.
Heparin-Sepharose was purchased from Amersham Biosciences. Rabbit
antiserum for human sPLA2-XII was prepared as described
previously (39).
and/or 10% FCS was added to each well, and the amount of free
[3H]AA or [3H]OA released into the
supernatant was measured. The percentage release was calculated using
the formula (S/(S + P)) × 100, where S and P represent the radioactivity
measured in the supernatant and cell pellet, respectively. The
supernatants from replicate cells were subjected to the
PGE2 enzyme immunoassay.
in medium
containing 10% FCS for 4 h, and PGE2 released into
the supernatants was quantified.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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Fig. 1.
Expression of the WT and truncated forms of
sPLA2-III in HEK293 cells. A, structures of
the WT and truncated forms (S, N+S, and S+C) of sPLA2-III.
In the case of III-S and III-S+C, a signal peptide for human
sPLA2-IIA was fused at their N termini. The C terminus of
each protein was tagged with the FLAG epitope as required for the
experiments. B and C, expression levels of the WT
and truncated forms of sPLA2-III in their HEK293
transfectants were assessed by Northern blotting (5 µg of total RNA
per lane) (B) and in the case of FLAG-tagged enzymes by
SDS-PAGE/immunoblotting (105 cell equivalents/lane)
(C). D, in vitro enzymatic activity of
the WT and truncated forms of sPLA2-III expressed in HEK293
transfectants. Aliquots (5-20 µl) of the culture supernatants were
taken for PLA2 assay using 2-AA-PE, 2-AA-PC, 2-LA-PE, and
2-LA-PC as substrates. Values (percentage of substrate hydrolysis
equivalent to 107 cells) are means ± S.E. of five
independent experiments.
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Fig. 2.
Cellular fatty acid release by
sPLA2-III. A, control or
sPLA2-III-WT-expressing cells, which were prelabeled with
[3H]AA or [3H]OA, were incubated for 4 h with 1% ( ) or 10% (+) FCS with (+) or without (
) 1 ng/ml
IL-1
to assess the release of these fatty acids. B and
C, comparison of the [3H]AA-releasing property
between the WT and truncated (S, N+S, and S+C) sPLA2-III.
[3H]AA-prelabeled cells were cultured for 4 h in
varied combinations of FCS and IL-1
(B) or for the
indicated periods in the presence of 10% FCS plus 1 ng/ml IL-1
(IL-1/FCS) (C). D, [3H]AA release
by III-N+S, III-S, and their point mutants (HQ), in which the catalytic
center His was replaced with Gln, following 4-h incubation with
IL-1/FCS. Values are mean ± S.E. of 3-5 independent experiments
in A, B, and D, and a representative
result of three independent experiments is shown in C.
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Fig. 3.
Coupling between sPLA2-III and
COXs. A, PGE2 generation by parent HEK293
cells and cells transfected with the WT or truncated (S, N+S, and S+C)
sPLA2-III after 4 h of incubation with IL-1/FCS.
Endogenous COX-2 mRNA expression in the presence (+) or absence
( ) of IL-1
was assessed by Northern blotting (top).
B, time course of PGE2 production by parental
cells (open circles) and cells transfected with
III-WT (closed circles) or III-S
(closed squares) after incubation with IL-1/FCS.
COX-2 mRNA expression in these cells at each time point, assessed
by Northern blotting, is shown in the inset. C, parental and
III-WT-transfected cells were transfected with mock (
) and COX-1 or
COX-2 (+) plasmids (1 µg). Three days after transfection, the cells
were stimulated with 10 µM A23187 for 30 min to assess
PGE2 release. D, parental cells and cells
expressing III-WT or III-S were transfected with the indicated amounts
of the COX-1 plasmid. Three days after transfection, A23187-stimulated
PGE2 production was assessed. COX-1 protein expression, as
assessed by immunoblotting, is shown in the top panel.
E, [3H]AA release by parental cells
(open circles) and cells expressing III-WT
(closed circles) or III-S (closed
squares) after stimulation with A23187 over 30 min.
F, transcellular PGE2 production. Parental cells
and cells transfected with III-WT, III-S, or human
sPLA2-IIF were cocultured with COX-2-transfected (+) or
parental (
) HEK293 cells, and PGE2 production after 4-h
incubation with IL-1/FCS was assessed. Values are mean ± S.E. of
three independent experiments in A, E, and
F, and a representative result of 2-4 reproducible
experiments is shown in B-D.
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Fig. 4.
Heparanoid dependence of
sPLA2-III-mediated AA metabolism. A,
absorption of insect cell-derived recombinant FLAG-tagged
sPLA2-III (S, N+S, and S+C) by heparin- or anti-FLAG
antibody-conjugated beads. The culture supernatants of
baculovirus-infected High Five cells were diluted with TBS (5-fold for
S+C and 50-fold for S and N+S to adjust their enzyme activities to be
equivalent) and mixed with heparin- or anti-FLAG antibody-conjugated
beads. After 2-h incubation, remaining PLA2 activities in
the supernatants were measured. B and C, effect
of exogenous heparin on cellular AA release.
[3H]AA-prelabeled HEK293 cells expressing
sPLA2-III-WT or sPLA2-III-S and human
sPLA2-V were incubated for 5 h with 0.5 mg/ml heparin
and then cultured for an additional 4 h in medium containing 10%
FCS with (+) or without ( ) IL-1 in the continued presence of heparin
to assess [3H]AA release (B) and
PGE2 production (C). D and
E, effect of glypican coexpression. HEK293 cells expressing
suboptimal levels of sPLA2-III-WT or III-S were subjected
to second transfection with glypican. The expression of III-WT, III-S,
and glypican was as- sessed by Northern blotting (D). These cells were
prelabeled with [3H]AA and analyzed for
IL-1/FCS-dependent [3H]AA release
(top) and PGE2 production (bottom)
after 4-h treatment with IL-1/FCS. F, effect of NDGA. HEK293
cells expressing III-WT or III-S were cultured for 4 h with
IL-1/FCS in the presence (+) or absence (
) of 10 µM
NDGA, and PGE2 released into the supernatants was
quantified. Values are mean ± S.E. of 3-5 independent
experiments.
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Fig. 5.
Recombinant expression of truncated
sPLA2-III mutants by the baculovirus system and their
effects on PGE2 production by HEK293 cells.
A, expression of the FLAG-tagged, truncated forms of
sPLA2-III in High Five insect cells. High Five cells grown
in 12-well plates were infected with baculovirus bearing the truncated
sPLA2-III cDNAs for 5 days, harvested, and lysed in 1 ml of PBS, and 10-µl aliquots were subjected to
SDS-PAGE/immunoblotting using anti-FLAG antibody. B,
in vitro enzymatic activity of recombinant
sPLA2-III expressed in High Five cells. The indicated
amounts of the culture supernatants of the baculovirus-infected High
Five cells were taken for PLA2 assay. A representative
result of two reproducible experiments is shown. C, effects
of recombinant III-S and III-N+S on PGE2 production by
COX-2-expressing HEK293 cells. The cells were incubated for 1 h
with the culture supernatants of control or
sPLA2-expressing High Five cells that were diluted (1:10)
with RPMI 1640 containing 10% FCS, and PGE2 released into
the supernatants was measured. A representative result of three
independent preparations is shown. Values are mean ± S.E. of
three independent experiments.
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Fig. 6.
Subcellular distribution of
sPLA2-III. HEK293 cells transfected with the
FLAG-tagged sPLA2-III-WT and truncated forms (III-S,
III-N+S, and III-S+C) were subjected to immunofluorescent staining
using anti-FLAG antibody and fluorescein
isothiocyanate-conjugated anti-IgG. Detailed procedures are described
under "Experimental Procedures."
and IFN-
) (Fig. 7B).
In comparison, the expression of sPLA2-IIA and
sPLA2-V was tightly controlled by cytokines in that
sPLA2-IIA was induced by TNF-
and suppressed by IFN-
,
and sPLA2-V expression required both TNF-
and IFN-
(Fig. 7B). sPLA2-X was constitutively expressed
(Fig. 7B), and other sPLA2s (IB, IID, IIE, IIF,
and XII) were undetectable (data not shown).
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Fig. 7.
Expression of various endogenous
sPLA2s in human cell lines. A, detection of
endogenous sPLA2-III in BEAS-2B cells by RT-PCR. Two
different primer sets were used to amplify sPLA2-III
cDNA fragments in lanes 1 (III-5' and
III-HQ-AS) and 2 (III-5' and III-S-AS). Specific bands were
visualized by ethidium bromide in agarose gels. B and
C, expression of various sPLA2s in
BEAS-2B (B) and HCA-7 (C) cells with or
without stimulation with 10 ng/ml IL-1 , IFN-
, or TNF-
for
24 h. Specific bands were detected by RT-PCR followed by Southern
hybridization. A-C, representative results of two
reproducible experiments are shown. D, effects of
recombinant III-S and III-N+S on PGE2 production by HCA-7
cells (n = 3). The procedure is the same as in Fig.
5C.
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[in a new window]
Fig. 8.
Properties of HEK293 cells transfected with
sPLA2-XII. A, cellular fatty acid release.
Control and sPLA2-XII-transfected cells were prelabeled
with [3H]AA or [3H]OA, and the release of
these fatty acids after 4-h incubation with 1% ( ) or 10% (+) FCS
with (+) or without (
) 1 ng/ml IL-1
was assessed. The expression
of sPLA2-XII was assessed by Northern blotting
(inset). B, PGE2 production. Control
cells and cells stably transfected with sPLA2-XII,
sPLA2-III, or sPLA2-IIF (positive control) were
transfected with the indicated concentrations of the COX-1 plasmid.
Three days after transfection, the cells were stimulated for 30 min
with 10 µM A23187 to assess PGE2 release.
COX-1 expression was assessed by immunoblotting (top).
C, detection of sPLA2-XII protein in the
sPLA2-XII-transfected (+) and parental (
) HEK293 cells by
immunoblotting using anti-sPLA2-XII antibody. D,
immunofluorescent staining on sPLA2-XII-transfected or
control cells using anti-sPLA2-XII antibody. Two typical
versions of sPLA2-XII staining, in which the cells
exhibited abnormal morphologies, are shown (b and
c). Control cells did not show positive signals
(a).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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[in a new window]
Fig. 9.
Diagram of domain-function relationship of
human sPLA2-III. A central sPLA2 domain is
essential for the enzyme function and the plasma membrane action of the
enzyme. Either the N- or C-terminal domain mediates HSPG binding,
intracellular distribution, COX-2 induction, and efficient COX
coupling. These domains, the N-terminal domain in particular,
facilitate unique localization of this enzyme to the spindle edges of
cell adhesion sites.
, COXs, and terminal PG synthases in the COX pathway and cPLA2
,
5-lipoxygenase, 5-lipoxygenase-activating protein, and terminal
leukotriene synthases in the lipoxygenase pathway, is crucial for their
functional coupling in activated cells (64-68). Indeed, our
preliminary experiments have shown that the native
cPLA2
, which translocates to the perinuclear membrane,
is more efficiently coupled with COX than the cPLA2
mutant, which moves to the plasma
membrane.2 In human
neutrophils, the AA released by cPLA2
at the perinuclear membrane, but not that released by sPLA2-V at the plasma
membrane, can be metabolized to leukotriene by the perinuclear
5-lipoxygenase (69). Considering the scenario that the HSPG-shuttled
sPLA2s can be internalized and cause membrane hydrolysis in
the perinuclear region (16, 18, 28), it would be speculated that the AA released by the internalized sPLA2s can be more efficiently
supplied to adjacent COX in the perinuclear membrane than the AA
released from cell surface by the plasma membrane-acting
sPLA2s (including III-S), although the latter AA can be
accessible to the perinuclear COX (possibly by diffusion across the
cytosol or with the aid of fatty acid transfer proteins), as has been
observed with sPLA2-X (4, 17, 25, 27) and even III-S (Fig.
3, D and F). Although intracellular membrane
hydrolysis by the HSPG-shuttled sPLA2s will need further
study, our present data may shed light on the intracellular action of
these sPLA2s and its importance in efficient coupling with
downstream enzymes.
(70), COX-2 (71), or the PGE2 receptor EP2 (72) each
reduces the incidence of colorectal cancer in Apc mutant
mice, the phenotype of the cPLA2
knockout mice is milder
than that of the COX-2 or EP2 knockout mice, suggesting that some other
PLA2s can contribute to supplying AA to COX-2 in colorectal
cancer. Possible involvement of sPLA2s in colon cancer
development is now under investigation.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
IIA; IIC, IID, IIE, and XII
are almost inactive in this route). These sPLA2s induce
stimulus-independent, nonselective fatty acid release.
sPLA2s that poorly act on the PC-rich membrane can promote
AA release from activated cells with support of HSPG as an adapter
(IIA ~ V > IID > III > IIE; IB, IIC, X, and
XII are nonfunctional in this route). These sPLA2s prefer
anionic membranes, bind heparanoids, and promote
stimulus-dependent, AA-selective release and COX-2
induction. These sPLA2s (an exception is
sPLA2-IIF, whose function does not depend on HSPG (11)) may
be sorted into caveolae/rafts and internalized into particular membrane
compartments that are assumed to be rich in AA and anionic membrane
surfaces. Certain lipid-hydrolyzing products (fatty acids,
lysophospholipids, or their derivatives) spatiotemporally generated by
these sPLA2s in these compartments may be linked to COX-2
induction. The occurrence of these distinct regulatory pathways depends
on cell types and stimuli.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Tsujii and R. DuBois for providing HCA-7 cells.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan.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.: 81-3-3784-8197; Fax: 81-3-3784-8245; E-mail: mako@pharm.showa-u.ac.jp.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211325200
2 M. Murakami, W. Cho, and I. Kudo, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
sPLA2, secretory phospholipase A2;
PLA2, phospholipase
A2;
cPLA2, cytosolic PLA2
;
COX, cyclooxygenase;
AA, arachidonic acid;
OA, oleic acid;
LA, linoelic
acid;
PG, prostaglandin;
PGE2, prostaglandin
E2;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
HSPG, heparan sulfate proteoglycan;
FCS, fetal calf serum;
NDGA, nordihydroguaiaretic acid;
RT-PCR, reverse transcriptase-PCR;
IL-1, interleukin-1;
TNF-
, tumor necrosis factor
;
IFN-
, interferon-
;
PBS, phosphate-buffered saline;
TBS, Tris-buffered
saline;
WT, wild type;
2-LA-PE, 1-palmitoyl-2-[14C]linoleoyl-PE;
2-LA-PC, 1-palmitoyl-2-[14C]linoleoyl-PC;
2-AA-PC, 1-palmitoyl-2-[14C]arachidonoyl-PC;
2-AA-PE, 1-palmitoyl-2-[14C]archidonoyl-PE.
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