From the Department of Health Chemistry, School of
Pharmaceutical Sciences, 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, August 29, 2000, and in revised form, October 6, 2000
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ABSTRACT |
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We analyzed the ability of a diverse set of
mammalian secreted phospholipase A2
(sPLA2) to release arachidonate for lipid mediator
generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA2-IIA, -IID, and -V
promote stimulus-dependent arachidonic acid release
and prostaglandin E2 production in a manner dependent on
the heparan sulfate proteoglycan glypican. In contrast,
sPLA2-IB, -IIC, and -IIE, which bind weakly or not at all
to heparanoids, fail to elicit arachidonate release, and addition of a
heparin binding site to sPLA2-IIC allows it to release
arachidonate. Heparin nonbinding sPLA2-X liberates
arachidonic acid most likely from the phosphatidylcholine-rich outer
plasma membrane in a glypican-independent manner. In rat mastocytoma
RBL-2H3 cells that lack glypican, sPLA2-V and -X, which are
unique among sPLA2s in being able to hydrolyze
phosphatidylcholine-rich membranes, act most likely on the
extracellular face of the plasma membrane to markedly augment
IgE-dependent immediate production of leukotriene
C4 and platelet-activating factor. sPLA2-IB,
-IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells.
These results are also supported by studies with sPLA2
mutants and immunocytostaining and reveal that
sPLA2-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent)
mechanisms in HEK293 and RBL-2H3 cells.
Phospholipase A2
(PLA2),1 which
catalyzes the hydrolysis of membrane glycerophospholipids to produce
free fatty acids and lysophospholipids, are a family of intracellular
and extracellular enzymes (1-5). Secreted PLA2
(sPLA2) comprises calcium-dependent interfacial enzymes with low molecular mass (typically 14-18 kDa) and multiple disulfides. To date, nine genes coding for structurally related and
enzymatically active sPLA2s have been identified in mammals (groups IB, IIA, IIC, IID, IIE, IIF, III, V, and X) (6-10).
Understanding the physiological functions of this diverse set of
sPLA2s is now a complex and challenging area of research in
the eicosanoid field, and the possibility that some of these enzymes
are involved in processes unrelated to eicosanoid generation should be
considered (11-21).
Group IB sPLA2 (sPLA2-IB), known as pancreatic
PLA2, is abundant in pancreatic juice, in which it
catalyzes the breakdown of dietary phospholipids, and is also expressed
in trace amounts in several tissues including lung and kidney (1, 22).
Group IIA sPLA2 (sPLA2-IIA), known as
inflammatory PLA2, is expressed in a variety of tissues and
hematopoietic cells, and its expression is markedly induced following
challenge with proinflammatory stimuli (1, 23-26). This
sPLA2 is thought to play a role in inflammation (1), host
defense against bacteria (13-15), tumor suppression (16), exocytosis
(17, 18), blood coagulation (19), and atherosclerosis (20, 21). Group
IIC sPLA2 (sPLA2-IIC) is expressed in rodent
testes, but only a pseudogene for this enzyme has been found in the
human genome (5, 27). Group V sPLA2 (sPLA2-V) is expressed mainly in rat and human heart (5, 28) and may in part
compensate for sPLA2-IIA particularly in the mouse, in which sPLA2-V is inducibly expressed in many tissues by
pro-inflammatory agents, whereas sPLA2-IIA expression is
largely restricted to mouse intestine (29-31). Group X
sPLA2 (sPLA2-X) possesses structural features
characteristic of both sPLA2-IB and sPLA2-IIA
and is highly expressed in organs associated with the immune response in humans (32).
More recently, several novel mammalian sPLA2s, groups IID,
IIE, IIF, and III, have been identified and cloned based on searching nucleic acid data bases for homologs to known mammalian and venom sPLA2s (6-10). Group IID (sPLA2-IID) and IIE
(sPLA2-IIE) sPLA2s are structurally most
related to sPLA2-IIA, and the genes for these three
isozymes as well as those for group IIC, IIF, and V sPLA2s
map to the same chromosome locus (4-9). Compared with other group II
sPLA2s, group IIF sPLA2 has a relatively long, proline-rich C-terminal extension containing a single cysteine residue
and is acidic (8). Group III sPLA2 is a homolog of the
group III enzyme originally detected in bee venom and possesses long
and unique N- and C-terminal extensions (10). Cellular functions of
these novel sPLA2s remain to be elucidated. Because the
sPLA2 family is diverse and the tissue distribution of each enzyme is unique, these enzymes are likely to have distinct
physiological functions.
In an effort to clarify the role of sPLA2s in the
regulation of arachidonic acid (AA) release from membrane
phospholipids, we have found that sustained expression of
sPLA2-IIA or sPLA2-V by forcible gene transfer
or by de novo induction following cytokine stimulation leads
to efficient stimulus-dependent but not spontaneous AA
release (24, 33-40). This liberated AA is functionally linked to
cyclooxygenase (COX)-mediated prostaglandin (PG) production in several
adherent cells (24, 33-40). In such cells, endogenously produced
sPLA2-IIA is captured by the heparan sulfate chains of the
glycosylphosphatidylinositol-anchored proteoglycan glypican and is
transferred to punctate and perinuclear compartments that colocalize
with caveolin (39). Such compartmentalization may allow
sPLA2-IIA to become in contact with its suitable substrates and to be more efficiently coupled to downstream AA-metabolizing enzymes.
On the other hand, mammalian cells are generally highly resistant to
exogenous sPLA2-IIA, with very high concentrations (greater than or equal to 10 µg/ml) usually being required to elicit AA release (41-43). This action has been reported to occur independently of the association of sPLA2-IIA with heparan sulfate
proteoglycan (43). Several hematopoietic cells are reportedly more
sensitive to exogenous sPLA2-V than sPLA2-IIA
(31, 44), and exogenous sPLA2-X is highly active in
releasing fatty acids, even from a variety of adherent cells that are
refractory to sPLA2-IB and -IIA (45, 46). These diverse
features of sPLA2 action may in part reflect their
different interfacial binding capacities to charge-neutral
phosphatidylcholine (PC) versus anionic phospholipid vesicles (43-47). Binding of sPLA2s to PC may be important
for the action on the external leaflet of mammalian cells because this
membrane surface is rich in charge-neutral PC and sphingomyelin. Indeed, sPLA2-V and -X are able to efficiently hydrolyze
PC-rich vesicles in vitro (40, 44-46), whereas PC-rich
vesicles are a very poor substrate for sPLA2-IIA because of
poor binding of this latter enzyme to the interface (43, 47).
sPLA2s display very distinct heparanoid and membrane
binding properties, and it is likely that these properties dictate
their behavior in various mammalian cells. To better understand the regulatory functions of sPLA2s in lipid mediator
biosynthesis, we have extended our gain-of-function studies by
transfecting human embryonic kidney 293 (HEK293) cells and rat
mastocytoma RBL-2H3 cells with a variety of sPLA2s. Studies
using sPLA2 mutants with altered heparanoid and interfacial
binding properties provide additional data that help us to formulate
models for the mechanisms of action of sPLA2s in these
mammalian cells. Moreover, using RBL-2H3 cells, we have demonstrated,
for the first time, the functional coupling between specific
sPLA2s and the leukotriene (LT) and platelet-activating
factor (PAF) biosynthetic pathways.
Materials--
HEK293 cells (Human Science Research Resources
Bank) and RBL-2H3 cells (Riken Cell Bank) were cultured in RPMI 1640 (Nissui Pharmaceutical Co.) containing 10% fetal calf serum (Bioserum) as described previously (18, 37-40). The cDNAs for mouse
sPLA2-IIA and its mutant IIA-KE4 (35), rat
sPLA2-V and its mutant V-G30S (37), rat
sPLA2-IIC, human sPLA2-X and its mutant X-G30S
(40), rat glypican-1 (39), human COX-1 and -2 (38), all of which were
subcloned into pcDNA3.1 (Invitrogen), were described previously. The cDNAs for mouse sPLA2-IID (7), human
sPLA2-IIA, and its mutants IIA-V3W and R7E/K10E/K16E (43,
47), and human sPLA2-V and its mutant V-W31A (44) were
subcloned into pCI-neo (Promega). Mouse sPLA2-IIE cDNA
(8) was subcloned into pcDNA3.1(+)/hygro (Invitrogen). Rat
sPLA2-IB cDNA was obtained by polymerase chain reaction
using rat stomach cRNA as a template with a set of 23-base pair
oligonucleotide primers corresponding to 5'- and 3'-nucleotide sequences of the open reading frame and subcloned into pCR3.1 (Invitrogen). C-terminally FLAG-tagged rat sPLA2-V, which
was subcloned into pCR3.1, was described previously (29). Mouse cytosolic PLA2 (cPLA2) cDNA was subcloned
into pBK-CMV (Stratagene) (37, 52). Site-directed mutagenesis was
carried out directly on the mammalian expression plasmids using the
QuickChange kit (Stratagene), and all plasmids were submitted to DNA
sequencing of the full sPLA2 insert to confirm their sequences.
Rabbit anti-human sPLA2-IIA antibody and the enzyme
immunoassay kits for PGE2 and LTC4 were
purchased from Cayman Chemicals. The rabbit anti-human COX-1, rabbit
anti-human cPLA2, and goat anti-human COX-2 antibodies were
purchased from Santa Cruz. Human IL-1 Preparation of Recombinant sPLA2s--
Recombinant
human sPLA2-IIA, mouse sPLA2-IID, and human
sPLA2-X were produced in Escherichia coli as
described (7, 43, 45). Methods for the recombinant expression in
E. coli, refolding, and purification of mouse
sPLA2-IIE will be reported
elsewhere.2 All recombinant
sPLA2s were found to be > 95% pure when analyzed by
SDS-polyacrylamide gel electrophoresis and to have the predicted mass
(within 0.5 atomic mass unit) when analyzed by electrospray mass
spectrometry (7, 43, 45). Because the resolution of the instrument in
this mass range is 0.5-1 atomic mass unit, mass spectrometry analysis
establishes that all sPLA2 have intact disulfides and thus
are likely to be properly folded.
Preparation of Antibodies against sPLA2s-IID, -IIE,
and -X--
Anti-sPLA2 antisera were prepared in rabbits
by Cocalico Biologicals Inc. (Reamstown, PA) using an initial injection
of 300 µg of sPLA2 in complete Freund's adjuvant
followed by a booster injection with 150 µg of sPLA2.
Antisera were screened by immunoblotting, and a second boost injection
was carried out as needed. Antisera were tested for sPLA2
cross-reactivity using the set of recombinant proteins (human and mouse
sPLA2-IB and -IIA, mouse sPLA2-IIC, -IID, -IIE,
and -IIF and human sPLA2-X). No cross-reactivity was observed by immunoblotting using 50 ng of each sPLA2 and
ECL detection (Amersham Pharmacia Biotech).
Establishment of Transfectants--
Establishment of various
HEK293 cell transformants was described previously (37-40). Briefly, 1 µg of plasmid was mixed with 2 µl of LipofectAMINE Plus 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) containing 0.5 ml of
Opti-MEM. After incubation for 6 h, the medium was replaced with 1 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 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 (Invitrogen) or 50 µg/ml hygromycin (Invitrogen). After culture for 3-4 weeks, wells containing a single colony were chosen, and the expression of each
protein was assessed by RNA blotting or immunoblotting. The established
clones were expanded and used for the experiments as described below.
To establish sPLA2-IID/COX double transformants, HEK293
transformants expressing each COX were subjected to a second
transfection with mouse sPLA2-IID cDNA that 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 and cloned by culturing in the presence of 50 µg/ml
zeocin (Invitrogen) to establish stable transformants. A similar
strategy was employed to produce sPLA2s/glypican-1 double
transformants, where cells expressing each sPLA2 were
transfected with glypican-1 cDNA in pcDNA3.1/Zeo (+) and
selected with zeocin.
RBL-2H3 cells were seeded into 150-mm diameter dishes and cultured for
2~3 days to subconfluency. The cells (107 cells) were
harvested, washed twice with Opti-MEM, and suspended in 400 µl of
Opti-MEM. The cells were mixed with each cDNA (2~5 µg) and
subjected to electroporation (BTX electroporator ECM600, at 200 V pulse
amplitude; capacitance, 900 microfarads). After culturing for 2 days,
the cells were resuspended in 10 ml of culture medium containing 800 µg/ml geneticin and seeded into 96-well plates. After culture for 2 weeks, single colonies were expanded into 12-well plates. After
reaching confluence, the expression of each PLA2 was
assessed by RNA blotting or immunoblotting. As a control, cells
transfected with the empty pcDNA3.1 vector were used.
Measurement of sPLA2 Activity--
Rates of
hydrolysis of phospholipid vesicles by sPLA2s in
vitro were obtained with the fatty acid binding protein assay as described (45). Reactions contained 30 µM
1-palmitoyl-2-oleoyl-phosphatidylglycerol vesicles (Avanti Polar Lipids
Inc.) as 100 nm unilamellar vesicles (prepared by extrusion) in 1.3 ml
of Hanks' balanced salt solution with 1 mM
CaCl2, 1 mM MgCl2, 9.7 µg of rat
liver fatty acid binding protein, and 1 µM
11-dansyl-undecanoic acid (Molecular Probes Inc.) with stirring at
37 °C.
Alternatively, sPLA2 activity was assayed by measuring the
amounts of free radiolabeled fatty acids released from the substrate 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine
(Amersham Pharmacia Biotech). 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 2 µM substrate. After incubation for 10-30 min at
37 °C, [14C]AA was extracted, and radioactivity was
quantified as described previously (35, 48).
Heparin Binding--
Affinity of recombinant mouse
sPLA2-IID and mouse sPLA2-IIE to
heparin-Sepharose was assessed as described previously (35, 37,
40).
RNA Blotting--
Approximately equal amounts (~10
µg) of total RNA obtained from transfected cells were applied to
separate lanes 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 Biomedicals). All
hybridizations were carried out as described previously (35).
SDS-Polyacrylamide Gel Electrophoresis
/Immunoblotting--
Lysates from 105 cells or culture
supernatants were subjected to SDS-polyacrylamide gel electrophoresis
using 15% (w/v) gels for sPLA2s and 10% gels for COXs
under nonreducing and reducing conditions, respectively. The separated
proteins were electroblotted onto nitrocellulose membranes (Schleicher
& Schuell) using a semi-dry blotter (MilliBlot-SDE system; Millipore).
The membranes were probed with the respective antibodies (1:2,000
dilutions for sPLA2s and 1:5000 dilutions for COXs) for
2 h, followed by incubation with horseradish peroxidase-conjugated
anti-rabbit IgG (1:5,000 dilution) (Amersham Pharmacia Biotech) for
2 h, and visualized using the ECL Western blot system (PerkinElmer
Life Sciences), as described previously (35).
Activation of HEK293 Cells--
HEK293 cells (5 × 104/ml) were seeded into each well of 24- or 48-well
plates. To assess AA release (37-40), 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 culturing was continued for another day. After three washes with
fresh medium, 250 µl (24-well plate) or 100 µl (48-well plate) of
RPMI 1640 with or without 10 µM A23187 (Calbiochem) with
1% FCS or 1 ng/ml IL-1 Activation of RBL-2H3 Cells--
The cells (5 × 104 cells/ml) were seeded into 24-well plates and cultured
for 2 days in 1 ml of culture medium. Then the cells were sensitized
with IgE anti-trinitrophenyl for 30 min, washed twice, and activated
for 10 min at 37 °C with 10 ng/ml trinitrophenyl-conjugated bovine
serum albumin as an antigen (Ag) (18). After harvesting the
supernatants, the remaining cells were collected and disrupted by two
freeze-thawing cycles. Release of LTC4 was assessed by enzyme immunoassay according to the manufacturer's instruction.
Detection of PAF--
RBL-2H3 cells (5 × 106
cells) were preincubated for 10 min with 25 µCi/ml
[3H]sodium acetate (PerkinElmer Life Sciences) and then
activated for various periods with IgE/Ag in the continued presence of
[3H]sodium acetate. After stopping the reaction by adding
0.1% sodium dodecyl sulfate, the lipids contained in the cells and/or
supernatants were extracted by the method of Bligh and Dyer (49) and
developed on thin layer chromatography plates, as described previously
(50). The spot corresponding to PAF was identified by comparison with an authentic PAF standard (Cayman Chemicals), and the silica was scraped from the plate and submitted to scintillation counting.
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 3% bovine serum albumin
(for blocking) and 1% saponin (for permeabilization) in PBS for 1 h, with antibodies against each sPLA2 (1:500 dilution) or
anti-FLAG antibody (1:200 dilution) for 1 h, and then with
fluorescein isothiocyanate goat anti-rabbit or -mouse IgG (1:100
dilution) for 1 h. After six washes with PBS, the cells were
mounted on glass coverslips using Perma Fluor (Japan Tanner), and the
sPLA2 signal was visualized using a laser scanning confocal
microscope (IX70; Olympus), as described previously (18, 39).
Statistical Analysis--
Data were analyzed by Student's
t test. Results are expressed as the means ± S.E.,
with p = 0.05 as the limit of significance.
AA Releasing Function of sPLA2s in HEK293 Cells
Heparanoid Binding--
We have recently reported the distinct
roles of mouse and human sPLA2-IIA, rat
sPLA2-IIC, rat sPLA2-V, and human
sPLA2-X in regulating AA metabolism by transfection
analyses using HEK293 and Chinese hamster ovary cells as model systems
(35, 37-40). To gain more insight into general aspects of the
regulatory functions of mammalian sPLA2s, two recently
identified sPLA2 enzymes, mouse sPLA2-IID and
sPLA2-IIE, were transfected into HEK293 cells. The expression levels of these sPLA2s in stable transfectants,
as assessed by RNA blotting, are shown in Fig.
1A. When the culture supernatant of cells transfected with mouse sPLA2-IID was
applied to a heparin-Sepharose column, the enzyme was recovered from
heparin-binding fractions and eluted with a buffer containing 0.7 M NaCl. In contrast, the affinity of mouse
sPLA2-IIE for heparin-Sepharose was very weak, a major
portion being eluted in the flow-through fraction and only a minor
portion (<10%) being eluted from the column with a buffer containing
0.2 M NaCl. The heparin binding affinities of these and
other mammalian sPLA2s are compared in Table
I. These results are roughly consistent
with the calculated pI values for the sPLA2s (IIA > IID ~ V > IIC > IIE > IB > X) (Table I) and thus the degree of positive charge on the sPLA2, which
is required for binding to anionic heparanoids. The exception is rat
sPLA2-IIC, which has a higher pI value than rat
sPLA2-V and yet binds weaker to heparin, showing that the
arrangement of basic residues on the surface of the sPLA2
is also important for heparin binding.
More than half of secreted mouse sPLA2-IID was detected in
the cell surface-associated fraction that was solubilized with 1 M NaCl as assessed by enzymatic assay and immunoblotting
(Fig. 1B), indicative of its binding to cell surface heparan
sulfate proteoglycan, as reported previously for sPLA2-IIA
and -V (Table I and Refs. 35 and 37-40). In contrast, most of the
secreted mouse sPLA2-IIE was detected in the supernatant
fraction (Fig. 1B). Data for these and other
sPLA2s are compared in Table I. Treatment of
sPLA2-IID-expressing cells with heparin or heparinase solubilized the enzyme into the supernatant (see below), as also observed with mouse sPLA2-IIA (39).
AA Release--
When mouse sPLA2-IID-expressing clones
were prelabeled with [3H]AA and then stimulated for 30 min with A23187, there was a marked increase in [3H]AA
release compared with control cells (Fig. 1C). Similarly, when the sPLA2-IID-expressing cells were cultured for
4 h with IL-1 in the presence of FCS,
stimulus-dependent but not spontaneous [3H]AA
release increased markedly (Fig. 1D). In contrast, none of the mouse sPLA2-IIE-expressing clones exhibited increased
[3H]AA release even after stimulation with A23187 (Fig.
1C) or IL-1/FCS (Fig. 1D). The AA releasing
properties of these and other sPLA2s are compared in Table
I. As reported previously (37), catalytic activity is essential for the
AA-releasing functions of sPLA2s in HEK293 cells.
PGE2 Biosynthesis--
sPLA2-IIA and -V
can efficiently couple to stimulus-induced PG biosynthesis via two
regulatory steps; enhanced supply of the substrate AA and induction of
endogenous COX-2, both of which are required for optimal delayed PG
biosynthesis (Refs. 35-40 and Table I). As shown in Fig.
2A, mouse
sPLA2-IID-transfected, but not control cells, produced a
significant amount of PGE2 after stimulation for 4 h
with IL-1/FCS. RNA blot analysis showed that sPLA2-IID
augmented endogenous COX-2 expression in IL-1-stimulated cells (Fig.
2B). Mouse sPLA2-IIE did not elicit
PGE2 generation appreciably, most probably because it
failed to supply AA and to induce COX-2 (data not shown). The
PGE2-generating capacity of these and other
sPLA2s in HEK293 cells are compared in Table I.
To investigate functional coupling between mouse sPLA2-IID
and COX isozymes further, we carried out cotransfection experiments. The expression levels of sPLA2-IID, COX-1, and COX-2 are
shown in Fig. 2C. Cells cotransfected with
sPLA2-IID and COX-1 produced more PGE2 than
those expressing sPLA2-IID or COX-1 alone in response to
A23187 (Fig. 2D). Similarly, A23187-induced PGE2
production by COX-2-expressing cells was markedly elevated when
sPLA2-IID was coexpressed (Fig. 2D). When the
cells were stimulated with IL-1, PGE2 generation by cells
expressing both sPLA2-IID and COX-2 was significantly
higher than those expressing sPLA2-IID alone, which linked
to endogenously induced COX-2 (Fig. 2B), or those expressing
COX-2 alone (Fig. 2E). COX-1 was not utilized in the delayed
response even when combined with sPLA2-IID (Fig.
2E). In contrast, no appreciable augmentation of
PGE2 generation was observed when mouse
sPLA2-IIE was cotransfected with each of the two COX
isozymes (data not shown), consistent with the fact that sPLA2-IIE expression did not lead to AA release (Fig. 1,
C and D). The functional coupling between these
and other sPLA2s with COX isozymes are compared in Table I.
Collectively, AA release and COX coupling of sPLA2-IID in
the immediate and delayed PGE2-biosynthetic responses are
similar to that of the heparin-binding sPLA2-IIA and -V
(Refs. 38-40 and Table I).
Gain-of-Function Mutation of sPLA2-IIC--
Comparison
of the C-terminal domains between sPLA2-IIC and
sPLA2-V from various species reveals that the former lacks
several basic residues that are conserved in sPLA2-V (Fig.
3A). These basic amino acid
clusters are important for rat sPLA2-V to bind heparan
sulfate proteoglycan on the cell surface and accordingly sPLA2-V-mediated AA release from the transfectants (37). We introduced basic amino acids into the corresponding positions in rat
sPLA2-IIC by replacing Leu95 and/or
Glu102 with Arg and Lys, respectively (IIC-L95R and
IIC-L95R/E102K), and transfected these mutants into HEK293 cells.
Whereas most of the native sPLA2-IIC and IIC-L95R were
secreted into the extracellular fluid, 40% of IIC-L95R/E102K was
detected in the cell surface-bound fraction (Fig. 3B).
In vitro enzyme activities of native and mutant enzymes did
not differ significantly (data not shown). When these transfectants
were stimulated with A23187, immediate [3H]AA release by
cells expressing IIC-L95R/E102K, but not native enzyme and IIC-L95R,
increased markedly (Fig. 3C). Furthermore, culturing
IIC-L95R/E102K transfectants, but not native enzyme or IIC-L95R
transfectants, with IL-1 in combination with FCS resulted in a marked
increase in delayed [3H]AA release (Fig. 3C).
When IIC-L95R/E102K transfectants were cotransfected with either COX-1
or COX-2, A23187-induced immediate PGE2 generation occurred
via both COX isozymes, and IL-1-induced delayed PGE2
generation occurred via COX-2 (Fig. 3D). There was an
increase in PGE2 synthesis in cells expressing
IIC-L95R/E102K alone after IL-1 stimulation that was similar to that
seen in cells expressing both IIC-L95R/E102K and COX-1 (Fig.
3D), suggesting that IIC-L95R/E102K expression induces
endogenous COX-2 expression. Thus, IIC-L95R/E102K, which acquired the
ability to associate with the cell surface, behaves like
sPLA2-IIA, -IID, and -V in regulating PGE2
biosynthesis in HEK293 cells.
Interaction of sPLA2s with Glypican--
Functional
similarities among the three heparin-binding group II subfamily of
sPLA2s (IIA, IID and V) in HEK293 cells suggest that they
utilize a common regulatory machinery for AA metabolism. Because the
function of sPLA2-IIA depends on its interaction with the
glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican in these cells (39), we examined whether sPLA2-IID and -V also utilize the glypican-dependent pathway. For
this purpose, we transfected glypican-1 cDNA into HEK293 cells
expressing mouse sPLA2-IID (Fig.
4) or rat sPLA2-V (Fig.
5A). Because the augmentative effect of glypican on sPLA2-IIA function is particularly
evident when sPLA2-IIA expression is suboptimal (39), we
chose clones expressing low levels of sPLA2-IID (Fig. 4)
and -V (Fig. 5A) in this experiment.
The expression of sPLA2-IID and glypican-1 in HEK293 cells
transfected with their cDNAs, alone or in combination, is shown in
Fig. 4A. The amount of sPLA2-IID bound on the
cell surface increased about 2-fold following glypican-1 coexpression
(data not shown). IL-1-stimulated delayed AA release by cells
coexpressing sPLA2-IID and glypican was significantly
higher than that by cells expressing sPLA2-IID alone (Fig.
4B). A similar increase in A23187-induced immediate AA
release was also observed following introduction of glypican into
sPLA2-IID-expressing cells (data not shown). PGE2 produced by sPLA2-IID/glypican
cotransfectants 4 h after IL-1 stimulation reached ~15-fold
higher levels than that produced by cells expressing
sPLA2-IID alone (Fig. 4C). IL-1-induced COX-2 expression, which was elevated modestly in the
sPLA2-IID-transfected cells relative to control cells, was
further increased in cells coexpressing sPLA2-IID and
glypican (Fig. 4D), whereas no significant augmentation of COX-2 induction occurred when
sPLA2-IID/glypican cotransfectants were cultured in the
absence of IL-1 (data not shown). These results imply that enhanced AA
release (Fig. 4B) and COX-2 induction (Fig. 4D)
converge on synergistic augmentation of PGE2 generation
following IL-1 stimulation (Fig. 4C). In further support of
the functional interaction between sPLA2-IID and glypican, we found that treating the cells with heparin or heparinase markedly reduced IL-1-stimulated PGE2 generation by both
sPLA2-IID single and sPLA2-IID/glypican double
transfectants (Fig. 4E), accompanied by solubilization of
sPLA2-IID into the extracellular culture medium (Fig.
4F).
As shown in Fig. 5A, IL-1-stimulated AA release by cells
expressing suboptimal level of rat sPLA2-V was also
enhanced markedly by glypican coexpression. This result shows that
sPLA2-V, a heparin-binding sPLA2, also utilizes
the glypican-dependent pathway in HEK293 cells. In
contrast, FCS-dependent AA release by human
sPLA2-X, a heparin-nonbinding isozyme, was not influenced
appreciably by glypican coexpression (Fig. 5B). Thus, the
augmentation by glypican is confined to the heparin-binding group II
subfamily of sPLA2s and is not a reflection of a
nonspecific action on cells.
Immunocytostaining--
We have previously shown that
sPLA2-IIA overexpressed in HEK293 cells and
cytokine-induced endogenous sPLA2-IIA in rat fibroblastic 3Y1 and hepatic BRL-3A cells resides in punctate and perinuclear compartments that colocalize with caveolin (39). Immunocytostaining of
mouse sPLA2-IID-expressing 293 cells with its specific
antibody revealed positive signals in punctate compartments throughout the cell and in the perinuclear area (Fig.
6, top panels). These signals
were largely abrogated when cells were incubated with heparin (Fig. 6,
middle panels). Because heparin is cell impermeable, this
result shows that the intracellular punctate domain enzyme is in
exchange with secreted enzyme and is not the result of intracellular aggregation of overexpressed sPLA2-IID. These results are
indistinguishable from the subcellular localization of
sPLA2-IIA (39). In contrast, punctate signals were
virtually undetectable when human sPLA2-X-transfected cells
were immunostained with its specific antibody (Fig. 6, bottom panel). These results are consistent with the idea that
sPLA2-IID, as is the case for sPLA2-IIA (39),
is localized in caveolae-derived compartments through binding to
glycosylphosphatidylinositol-anchored glypican, whereas
heparin-nonbinding sPLA2-X is mainly, if not exclusively,
released into the extracellular medium. Positive signals near the
perinuclear area in sPLA2-X-expressing cells appear to
correspond to Golgi, reflecting the sPLA2-X secretion process.
Studies Using Interfacial Binding Site Mutants of
sPLA2s--
To explore whether the
glypican-dependent group II subfamily of sPLA2s
(IIA, IID, and V) acts on the PC-rich outer plasma membrane or on
another compartmentalized membrane that is assumed to be rich in
anionic lipids, we transfected HEK293 cells with human
sPLA2-IIA and V mutants that have altered interfacial
binding to zwitterionic PC vesicles in vitro (43, 44, 47).
When assayed in vitro using PC as a substrate, IIA-V3W, in
which Val3 is replaced by Trp, is about 300-fold more
active than wild type enzyme (IIA-WT), most likely because the mutant
binds more tightly than wild type to PC vesicles (43, 47). Likewise,
V-W31A, in which Trp31 is replaced by Ala, is about
200-fold less active than V-WT (44). Enzymatic activities of IIA-WT and
IIA-V3W toward anionic phospholipids are virtually identical because
both bind tightly to anionic vesicles, and the same is true for V-WT
and V-W31A (43, 44, 47).
The expression levels of the native and mutant sPLA2s were
compared by RNA blotting (Fig.
7A), immunoblotting (Fig.
7B), and enzyme activity toward anionic vesicles (Fig.
7C). These analyses showed that IIA-WT and IIA-V3W were
expressed at a similar level (Fig. 7, A-C), as were V-WT
and V-W31A (Fig. 7, A and C). These transfectants
were prelabeled with [3H]AA and stimulated with A23187
for 30 min (Fig. 7D) or with IL-1/FCS for 4 h (Fig.
7E). Release of [3H]AA by cells expressing the
mutant enzymes did not differ significantly from that released by the
cells expressing the respective wild type enzymes. Enzyme assay
revealed that more than 90% of IIA-WT and IIA-V3W, and nearly 60% of
V-WT and V-W31A were recovered from the cell surface-associated
fraction, indicating that these mutations did not significantly alter
binding to glypican. Thus, these studies support the idea that the
glypican-dependent sPLA2s do not act on the
PC-rich extracellular face of the plasma membrane but in a compartment
enriched in anionic phospholipids.
We also established transfectants that express another human
sPLA2-IIA mutant, IIA-R7E/K10E/K16E, which displays weak
heparin and heparan sulfate affinity but unaltered enzymatic activity on anionic vesicles (43). More than 90% of the enzyme activity was
released into the supernatant (Fig. 7C), indicative of its impaired heparan sulfate binding. IL-1/FCS-stimulated
[3H]AA release increased minimally in IIA-R7E/K10E/K16E
transfectants compared with IIA-WT expressing cells (Fig.
7E), despite the fact that the expression level of this
mutant was almost equal to that of IIA-WT (Fig. 7, A-C).
This result reinforces the importance of heparanoid-binding for
sPLA2-IIA function and shows that in addition to the
C-terminal lysine cluster (35), several basic residues in the
N-terminal helix of sPLA2-IIA can also contribute significantly to functional interaction of sPLA2-IIA with
heparan sulfate (43).
AA Releasing Function of sPLA2s in Mast Cells
Granule Accumulation and Secretion--
Our previous studies
established that heparin-binding (IIA, IID, and V) and nonbinding (IIC
and IIE) sPLA2s are expressed endogenously in mouse bone
marrow-derived mast cells (18). To explore the AA releasing function of
these sPLA2s in mast cells and to examine possible
correlations of in vitro properties of sPLA2s
with functional properties in mast cells, we overexpressed different
mammalian sPLA2s in rat mastocytoma RBL-2H3 cells (the expression of each sPLA2 in the established transfectants
is shown in Fig. 8, insets).
These sPLA2s were rapidly released (within 5 min) after
cross-linking of the high affinity IgE receptor by multivalent Ag. The
release of sPLA2-IB, -IIA, -IIC, -IID, -V, and -X into the
supernatants after IgE/Ag activation reached 50, 5, 45, 15, 23, and
55% (relative to total sPLA2 content in cells), respectively (Table II), whereas
spontaneous release of all of these enzymes was minimal, as assessed by
enzymatic assay. Lower percentage release of sPLA2-IIA,
-IID, and -V versus a higher percentage release of -IB,
-IIC, and -X appears to reflect the association of the former enzymes
with heparanoids or other anionic components on the surface of cells;
an idea supported by the observation that IgE/Ag-induced release of the
heparin weak binding mouse sPLA2-IIA mutant, KE4 (35),
reached a level comparable with that of sPLA2-X (18).
Consistent with the idea that caveolae are poorly developed in cells of
hematopoietic origin (54-56), no caveolae-like structures were
observed by immunocytochemical studies (see below), and the expression
of caveolin-2 and glypican-1 was undetectable in RBL-2H3 cells by
immunoblotting (data not shown). Therefore, sPLA2-IIA,
-IID, and -V may bind to heparanoid proteoglycans other than glypican
or to nonheparanoid anionic components on the extracellular surface of
activated RBL-2H3 cells.
LTC4 Biosynthesis--
Fig. 8 illustrates the
functional coupling between sPLA2s and endogenous
5-lipoxygenase for IgE/Ag-induced LTC4 biosynthesis in
RBL-2H3 transfectants. LTC4 generation by the transfectants expressing rat sPLA2-IB (Fig. 8A), mouse (Fig.
8B), rat, and human sPLA2-IIA (data not shown),
rat sPLA2-IIC (Fig. 8C), mouse
sPLA2-IID (Fig. 8D), and mouse
sPLA2-IIE (Fig. 8E) increased only minimally compared with that produced by mock transfected cells. In contrast, cells expressing rat sPLA2-V (Fig. 8F) and human
sPLA2-X (Fig. 8G) produced significant amounts
of LTC4, reaching levels comparable with that produced by
cells transfected with cPLA2 (Fig. 8H), which
has been shown by gene disruption to be crucial for LTC4 generation in mast cells (57). The expression of endogenous 5-lipoxygenase, 5-lipoxygenase-activating protein and LTC4
synthase, as assessed by RNA blotting and immunoblotting, did not
differ significantly among the transfectants used (data not shown),
indicating that the LTC4-biosynthetic effect of
sPLA2-V and -X expression was not due to an alteration in
the expression of downstream enzymes in the 5-lipoxygenase pathway.
Catalytic site mutants V-G30S (Fig. 9A) and X-G30S (Fig.
9B) with very low enzymatic activity, in which
Gly30 in the Ca2+ binding loop of rat
sPLA2-V (37) and human sPLA2-X (40), respectively, is replaced by Ser, failed to augment LTC4
generation, indicating that a functional catalytic site is
essential.
Studies Using Interfacial Mutants of
sPLA2s--
Because sPLA2-V and -X show high
interfacial binding to PC vesicles in vitro (44, 45),
whereas the other sPLA2s do not,2 we reasoned
that their potent LTC4-biosynthetic activity might be a
reflection of their action on the PC-rich outer plasma membrane after
exocytosis. To explore this hypothesis, we examined the effect of the
human sPLA2-V mutant V-W31A with impaired PC vesicle binding (44) on LTC4 generation. As shown in Fig.
9C, cells transfected with V-W31A, the expression of which
was even higher than that of native sPLA2-V, produced
minimal LTC4. Conversely, transfection of the human
sPLA2-IIA mutant IIA-V3W, which has increased affinity for
PC (43, 47), led to a 5-fold increase in IgE/Ag-induced
LTC4 biosynthesis relative to that produced in wild type
sPLA2-IIA-expressing cells (0.10 and 0.51 ng/106 cells in IIA-WT and IIA-V3W-transfected cells,
respectively). Thus, unlike PGE2 generation in HEK293 cells
shown above, LTC4 generation by sPLA2s in mast
cells correlates with their ability to bind PC vesicles and does not
correlate with their heparin-binding affinity.
PAF Biosynthesis--
IgE/Ag-induced activation of mast cells
leads to production of another lipid mediator PAF via the
sn-2 ester hydrolysis of 1-O-alkyl-PC by
PLA2 and subsequent acetylation by PAF acetyltransferase. When RBL-2H3 cells transfected with various sPLA2s were
stimulated with IgE/Ag, the production of PAF in cells overexpressing
the PC-hydrolyzing isozymes rat sPLA2-V and human
sPLA2-X increased, reaching a level comparable with that in
cells overexpressing cPLA2 (Fig.
10). cPLA2 has been shown
to be involved in PAF biosynthesis from studies using
cPLA2-null mice (51). In contrast, expression of mouse
sPLA2-IIA, -IID (Fig. 10), and -IIE, rat
sPLA2-IB and -IIC, and the human sPLA2-V mutant
V-W31A (data not shown) did not lead to augmentation of PAF generation.
These results collectively suggest that the hydrolysis of PC by
sPLA2-V or -X leads to release of AA and lyso-PAF, which
are supplied to 5-lipoxygenase as a substrate for LTC4
biosynthesis and to PAF acetyltransferase for PAF biosynthesis,
respectively. The ability of sPLA2s to promote immediate
generation of PGD2, LTC4, and PAF and to
augment degranulation in RBL-2H3 cells is summarized in Table II.
Immunocytostaining--
We have recently shown by confocal and
electron microscopic analyses that sPLA2-IIA is stored in
secretory granules of unstimulated RBL-2H3 transfectants and moves in
close proximity to the plasma membrane after IgE/Ag activation, the
area corresponding to opening perigranular membranes where fusion
between the plasma and granule membranes is occurring (18). This
particular localization is dependent on the binding of
sPLA2-IIA to an unidentified anionic cell component,
possibly a heparan sulfate proteoglycan other than glypican. This
compartmentalization of sPLA2-IIA may lead to spatially
segregated lysophospholipid production, which may enhance membrane
fusion leading to degranulation (18).
To further elucidate the sites of action of sPLA2-V and -X
in mast cells, RBL-2H3 cells were transiently transfected with C-terminally FLAG-tagged rat sPLA2-V and native human
sPLA2-X, and transfectants were examined by confocal laser
immunofluorescent microscopy using anti-FLAG antibody and
anti-sPLA2-X antiserum, respectively. Like
sPLA2-IIA (18), sPLA2-V also resides in
granular components in the cytoplasm of cells before IgE/Ag activation (Fig. 11), confirming its localization
in secretory granules. After cell activation, sPLA2-V gave
a signal somewhat different from that of sPLA2-IIA (18).
Only the outline of the sPLA2-V expressing cells was
intensely stained by the anti-FLAG antibody (Fig. 11). This result
implies that sPLA2-V is exocytosed and then bound over the
entire plasma membrane surface. Possibly, the weaker affinity of
sPLA2-V for heparan sulfate compared with that of sPLA2-IIA may allow sPLA2-V to disperse from
the perigranular membrane, to which sPLA2-IIA binds (18),
onto the plasma membrane surface, where it may associate with PC and
with some other unknown proteoglycan species or anionic components.
Immunofluorescence studies with sPLA2-X-transfected cells
revealed that this enzyme is also stored in granular components prior
to IgE/Ag activation (Fig. 11). After cell activation, weak staining of
the plasma membrane was observed. This staining pattern is in line with
the observation that a large portion of sPLA2-X is secreted
extracellularly (see above) and with the idea that its association with
the cell surface depends on its interfacial binding to the PC-rich
membrane. Collectively, these immunocytochemical studies further
support the idea that in activated mast cells, exocytosed
sPLA2-V and -X interact with the PC-rich outer leaflet of
the plasma membrane to liberate AA and lysoPAF, which are supplied to
downstream enzymes for lipid mediator biosynthesis.
In this study, we have analyzed the AA-releasing function and
attendant lipid mediator-producing capacity of a collection of
mammalian sPLA2s in two transfected cell lines where
sPLA2s display different profiles of secretion and
localization. In HEK293 cells, sPLA2s enter the
constitutive secretory process. This pathway appears to be reminiscent
of fibroblasts (24), hepatocytes (34), mesangial cells (23, 25), smooth
muscle cells (58, 59), and endothelial cells (33), in which expression
of sPLA2-IIA for example, is up-regulated by
proinflammatory stimuli and is maintained over a long culture period.
In this system, heparin-binding sPLA2s (IIA, IID, and V)
bind to glycosylphosphatidylinositol-anchored heparan sulfate
proteoglycan glypican and possibly other cell surface components and
accumulate in caveolin-rich and perinuclear compartments (Ref. 39 and
Fig. 6), where they augment stimulus-induced AA release and
PGE2 generation. This heparan sulfate
proteoglycan-dependent action occurs independently of
their interfacial affinity for PC vesicles (Fig. 7). In contrast, in
mastocytoma RBL-2H3 cells, sPLA2-IIA (18), -V, and -X (Fig.
11) are stored in secretory granules and are released immediately after
cell activation through the degranulation pathway of secretion. The
fact that sPLA2-IB, -IIC, and -IID are also rapidly
released from IgE/Ag-stimulated RBL-2H3 cells suggests that these
enzymes also reside in secretory granules. This route often takes place
in hematopoietic cells such as mast cells (18, 60), platelets (61), and
neutrophils (62). In RBL-2H3 cells, only sPLA2-V and -X are
capable of augmenting LTC4 and PAF generation for reasons
discussed below. In some cells, both degranulation and constitutive
secretion may occur. The schematic models for the two pathways are
illustrated in Fig. 12.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from Genzyme.
LipofectAMINE Plus reagent, Opti-MEM medium and TRIzol reagent were
obtained from Life Technologies, Inc. RPMI 1640 medium was purchased
from Nissui Pharmaceuticals. Heparin and Flavobacterium
heparinum heparinase III were purchased from Sigma. Fluorescein
isothiocyanate-conjugated goat anti-rabbit and -mouse IgG antibodies
were purchased from Zymed Laboratories Inc. Mouse
monoclonal anti-FLAG antibody was from Sigma. Mouse IgE
anti-trinitrophenyl and trinitrophenyl-conjugated bovine serum albumin
were provided by Dr. H. Katz (Harvard Medical School).
and/or 10% FCS was added to each well, and
the amount of free [3H]AA released into the supernatant
during culturing for 0.5 and 4 h, respectively, was measured. The
percentage release of AA was calculated using the formula [S/(S + P)] × 100, where S and P are the radioactivity measured in equal portions
of the supernatant and cell pellet, respectively. The supernatants from
replicate cells were subjected to the PGE2 enzyme immunoassay.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Expression of sPLA2-IID and -IIE
and their ability to release AA in HEK293 cells. A, RNA
blotting of mouse sPLA2-IID (mIID) and mouse
sPLA2-IIE (mIIE) in HEK293 transfectants and
parental cells (Control). B, the distribution of
sPLA2s in the supernatants (S) and cell
surface-associated fractions expressed as a percentage of the total
secreted sPLA2 (cell associated plus culture medium;
C) of transfected HEK293 cells, as determined by enzymatic
activity assays. Cells expressing mIID and mIIE, were cultured for 4 days, and then the supernatants containing the secreted enzymes were
collected. The cells were then washed for 15 min with 1 M
NaCl to solubilize cell surface proteoglycan-associated enzymes (37).
Inset, immunoblotting of mIID and mIIE in the supernatants
(S) and cell surface-associated fractions (C) of
the respective transfectants. C and D, HEK293
cells transfected with each sPLA2 were prelabeled with
[3H]AA and then stimulated for 30 min with 10 µM A23187 (C) or for 4 h with or without
1 ng/ml IL-1 and 10% FCS (D) to assess [3H]AA
release. A representative result of three to six independent
experiments is shown.
Properties and eicosanoid generating functions of mammalian
sPLA2s in HEK293 cells
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Fig. 2.
PGE2 biosynthesis by mouse
sPLA2-IID in HEK293 transfectants. A, cells
expressing mouse sPLA2-IID (mIID) and control
cells were cultured for the indicated periods with 1 ng/ml IL-1 in the
presence of 10% FCS in 24-well plates. PGE2 released into
the supernatants was quantified. B, after 4 h of
incubation with (+) or without ( ) IL-1/FCS, COX-2 mRNA expression
was assessed by RNA blotting. C, expression of
sPLA2-IID, COX-1 and COX-2 in the transfectants as assessed
by RNA blotting. D and E, PGE2
generation by cells expressing sPLA2-IID and COX-1 or COX-2
alone or in combination. Cells were stimulated for 30 min with A23187
(D) or for 4 h with IL-1/FCS (E) in 48-well
plates, and PGE2 released into the supernatants was
quantified. A representative result of three to six independent
experiments is shown.
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Fig. 3.
Site-directed mutagenesis of
sPLA2-IIC. A, alignment of the C-terminal
regions of sPLA2-V and -IIC. The conserved cationic amino
acids are surrounded by boxes. Residues Lsu95
and Glu102 in rat sPLA2-IIC were mutated to Arg
and Lys, respectively. The amino acid numbers are based on the
alignment with the sPLA2-IB sequence. B, the
percentage distribution of sPLA2-IIC and its mutants in the
supernatants (S) and cell surface-associated fractions
(C), as assessed by enzymatic activity assay. WT,
wild type. C, cells prelabeled with [3H]AA
were stimulated for 30 min with A23187 or for 4 h with or without
IL-1/FCS, and AA release was quantified. D, cells expressing
native or mutant sPLA2-IIC were transiently transfected
with either COX-1 or COX-2. Three days after transfection, cells were
stimulated for 30 min with A23187 or for 4 h with IL-1/FCS, and
PGE2 generation was quantified. Expression of COX-1 and
COX-2 was verified by immunoblotting (not shown). A representative
result of three independent experiments is shown.
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Fig. 4.
Functional interaction of
sPLA2-IID with glypican in HEK293 cells. A,
expression of mouse sPLA2-IID and glypican in HEK293 cells
as assessed by RNA blotting. sPLA2-IID single transfectants
and two independent sPLA2-IID/glypican transfectants
(clones 5 and 11) are shown. B-D, cells were stimulated for
4 h with IL-1/FCS. AA (B) and PGE2
(C) released into the supernatants were measured. The
remaining cells were harvested and subjected to RNA blotting to examine
the expression of COX-2 mRNA. E and F, cells
preincubated for 2 days with 1 mg/ml heparin or 0.5 unit/ml heparinase
were stimulated for an additional 4 h with IL-1/FCS, and
PGE2 activity (E) and PLA2 activity
(F) in the supernatants were quantified. A representative
result of three to five independent experiments is shown.
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Fig. 5.
Effects of glypican overexpression on the
AA-releasing function of sPLA2-V and -X in HEK293
cells. Glypican was coexpressed with rat sPLA2-V
(A) and human sPLA2-X (B), and AA
release from the transfectants in response to IL-1/FCS (A)
or FCS alone (B) was measured. Expression levels of each
sPLA2 and glypican in the transfectants, as assessed by RNA
blotting, are shown in the top panel. A representative
result of three independent experiments is shown. Previous studies
showed that IL-1 addition does not enhance AA release in HEK293 cells
expressing sPLA2-X (40).
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Fig. 6.
Immunocytostaining of sPLA2-IID
and -X in HEK293 transfectants. Cells expressing mouse
sPLA2-IID (top and middle panels) and human
sPLA2-X (bottom panel) were fixed,
permeabilized, and subjected to immunostaining using specific
antibodies as detailed under "Experimental Procedures." In
the middle panel, sPLA2-IID-expressing cells are
cultured for 4 h in the presence of 1 mg/ml heparin.
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Fig. 7.
Effects of mutation of the interfacial
binding surfaces of sPLA2-IIA and -V on AA release in
HEK293 cells. A, expression of wild type
(WT) and mutant human sPLA2-IIA and -V in HEK293
cells, as assessed by RNA blotting. B and C,
distribution of sPLA2-IIA-WT and three mutants in the
supernatants (S) and cell surface-associated fractions
(C), as assessed by immunoblotting using
anti-sPLA2-IIA antibody (B) and enzymatic
activity toward phosphatidylglycerol vesicles measured by the fatty
acid binding protein assay (C). D and
E, cells expressing wild type or mutant
sPLA2-IIA and -V were stimulated for 30 min with A23187
(D) or for 4 h with IL-1/FCS (E), and AA
release was quantified. Values are the means ± s.e. of three to five
experiments
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Fig. 8.
Effects of various sPLA2s on
IgE/Ag-mediated LTC4 generation in RBL-2H3
transfectants. RBL-2H3 cells were stably transfected with rat
sPLA2-IB (rIB; A), mouse
sPLA2-IIA (mIIA; B), rat
sPLA2-IIC (rIIC; C), mouse
sPLA2-IID (mIID; D), mouse
sPLA2-IIE (mIIE; E), rat
sPLA2-V (rV; F), human
sPLA2-X (hX; G), and mouse
cPLA2 (H). The cells were sensitized with IgE
and stimulated for 10 min with Ag as described under "Experimental
Procedures." LTC4 released into the supernatants was
quantified. The expression levels of PLA2s, assessed by RNA
blotting (for sPLA2s) and immunoblotting (for
cPLA2), are shown in the insets. Values are the
means ± S.E. of three to seven independent experiments.
Properties and functions of mammalian sPLA2s in RBL-2H3 cells
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Fig. 9.
Effects of sPLA2-V and -X mutants
on LTC4 generation in RBL-2H3 cells. Cells were
transfected with wild type or mutant sPLA2s, and human
sPLA2-X (B), and IgE/Ag-dependent
LTC4 generation was quantified. Values are the means ± S.E. of three to six independent experiments.
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Fig. 10.
Effects of various PLA2s on PAF
generation in RBL-2H3 cells. PAF generation by parental cells and
cells expressing mouse sPLA2-IIA, mouse
sPLA2-IID, rat sPLA2-V, human
sPLA2-X, and mouse cPLA2 (same transfectants as
shown in Fig. 8) was quantified as described under "Experimental
Procedures." Values are the means ± S.E. of three independent
experiments.
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Fig. 11.
Immunocytostaining of sPLA2-V
and -X in RBL-2H3 transfectants. Cells expressing FLAG-tagged rat
sPLA2-V and human sPLA2-X before and 10 min
after IgE/Ag activation were fixed, permeabilized, and then subjected
to immunostaining using anti-FLAG and anti-sPLA2-X
antibodies, as detailed under "Experimental Procedures."
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ABSTRACT
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DISCUSSION
REFERENCES
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Fig. 12.
Two sPLA2-dependent
eicosanoid-biosynthetic pathways. A, the
glypican-shuttling mechanism. In fibroblasts and several other adherent
cells, intracellularly produced heparin-binding sPLA2s
(IIA, IID, and V) may be directly channeled to heparan sulfate chains
of glypican inside secretory vesicles and prior to release to the
extracellular space. Glypican-bound sPLA2s are then
delivered into caveolae signalsomes, which shuttle between the plasma
membrane and intracellular membrane compartments through potocytosis
(54). Glypican-bound sPLA2s release AA selectively from
these compartmentalized membrane microdomains (37, 39) that may be
enriched in anionic phospholipids. sPLA2-X, which does not
enter the glypican-shuttling route, is released into the extracellular
medium and acts on the PC-rich outer surface of the plasma membrane to
release AA and other fatty acids (40). AA released by these
sPLA2s is supplied to the delayed
PGE2-biosynthetic route that involves the two inducible
perinuclear enzymes, COX-2 and membrane-bound PGE2 synthase
(mPGES) (64, 65). Preferential coupling of
sPLA2s with COX-2 to COX-1 may result from the fact that
COX-2 is favored over COX-1 when the AA supply is limited (38) and that
mPGES is preferentially coupled with COX-2 (65). sPLA2-IB,
IIC, and IIE, which show low interfacial binding to PC-rich membranes
and do not bind glypican, do not release AA in this setting.
B, the glypican-independent, plasma membrane mechanism. In
mast cells and probably other hematopoietic cells in which caveolae are
poorly developed and glypican is poorly expressed, if at all,
sPLA2s are stored in secretory granules and undergo rapid
exocytosis after cell activation. sPLA2s with high heparin
affinity (IIA and IID) are associated with membranous sites where
fusion between plasma membrane and granule membrane occurs and
contribute to local production of lysophospholipids, which further
facilitates membrane fusion leading to enhanced degranulation (18).
sPLA2-V, which shows intermediate heparin affinity, is
distributed uniformly on the plasma membrane surface. These
distributions of sPLA2-IIA, -IID, and -V may be mediated by
different sets of heparan sulfate proteoglycans or other anionic
components. sPLA2-X, -IB, -IIC, and -IIE are released into
the extracellular medium. Only the two PC-hydrolyzing enzymes,
sPLA2-V and -X, are capable of releasing AA from the outer
plasma membrane. The AA released is supplied sequentially to
constitutive COX-1 and hematopoietic PGD2 synthase
(H-PGDS) for immediate production of PGD2 and to
5-lipoxygenase and LTC4 synthase (LTCS) for
immediate production of LTC4 (70, 71). This action of
sPLA2-V and -X is similar to that seen when these enzymes
are added exogenously (44-46). sPLA2-V and -X also have
the ability to augment PAF biosynthesis through generation of
lysoPAF.
Heparanoid-dependent Action-- The following consistent picture is emerging for the action of heparin-binding sPLA2s (IIA, IID, and V) in promoting eicosanoid generation in cells that utilize the glypican shuttling mechanism. Binding of heparin-binding sPLA2s to the heparan sulfate chains of glypican allows accumulation of the enzyme on the cell surface and also promotes enzyme internalization into punctate domains that are rich in caveolin.
On the other hand, sPLA2-IB, -IIC, and -IIE, which have low affinity for heparin, are found mainly in the culture medium rather than bound to the cell surface, and they failed to elicit AA release under the conditions employed here. Clusters of basic amino acids near the C and N termini of sPLA2-IIA and -V form the binding sites for negatively charged heparin or heparan sulfate (35, 37, 43, 63). sPLA2-IIA and -V mutants in which these basic amino acids are mutated show reduced heparin affinity and lose their ability to elicit AA release (35, 37, 43). sPLA2-IID contains this basic amino acid cluster in the C-terminal domain (7), whereas fewer basic residues are found in the corresponding portions of sPLA2-IIC (27) and -IIE (8). Our ability to functionally manipulate the behavior of sPLA2s by protein engineering, i.e. loss-of-function by removal of the glypican binding (sPLA2-IIA and -V) and gain-of-function by addition of a heparin binding site (sPLA2-IIC), provides very strong circumstantial evidence for the functional requirement of glypican shuttling in IL-1/FCS-dependent AA liberation and PGE2 production in the HEK293 cell model.
We cannot rule out the possibility that the heparan sulfate mechanism only occurs as a result of sPLA2 overexpression in transfected cells. However, the physiological significance of heparan sulfate-dependent action of sPLA2-IIA is supported by the observations that with other cells such as human umbilical vein endothelial cells (33), rat hepatocytic BRL-3A cells (34), and rat fibroblastic 3Y1 cells (24), solubilization of membrane surface-associated endogenous sPLA2-IIA by exogenous heparin or heparinase greatly reduced cytokine-stimulated prolonged PG biosynthesis. Furthermore, in BRL-3A and 3Y1 cells, cytokine-induced endogenous sPLA2-IIA is colocalized with caveolin (39).
As a result of glypican shuttling in these cells, endogenously expressed sPLA2-IIA is delivered into a caveolae-like compartment and internalized through potocytosis to reach the perinuclear area (39), where the downstream PG-biosynthetic enzymes (COX and PGE2 synthase) are located (64, 65). This sPLA2-IIA sorting appears to be crucial for its proper function. Exogenously added sPLA2-IIA, which is poorly active on mammalian cells (1, 41-43), does not access this shuttling process for reasons that are not yet clear. It has been reported that in certain cells, exogenously added sPLA2-IIA deposits poorly on cell surfaces and binds tightly to extracellular matrix proteins including decorin (66). Perhaps intracellularly produced sPLA2-IIA is directly channeled to glypican inside secretory vesicles prior to release to the extracellular compartment (Fig. 12). Further work is needed to understand why exogenously added sPLA2-IIA is poorly active on mammalian cells, but a key factor is its poor ability to bind directly to the PC-rich outer layer of the plasma membrane (45).
The precise membrane compartment where glypican-shuttled sPLA2s liberate AA for eicosanoid production in HEK293 cells remains to be established. The fact that sPLA2-IIA binds extremely poorly to PC-rich vesicles and to the PC-rich outer face of the plasma membrane of mammalian cells but binds more than one million-fold tighter to anionic vesicles (43, 45) argues that glypican shuttling may bring this enzyme in contact with a membrane surface that is more enriched in acidic phospholipids than is the outer face of the plasma membrane. This is supported by the results obtained with sPLA2-IIA and -V mutants, which retain high affinity for anionic phosphatidylglycerol vesicles but display altered affinity for PC vesicles (Fig. 7). Expression of sPLA2-IIA in HEK293 cells leads to preferential AA release over oleic acid (37, 40) despite the fact that this enzyme shows virtually no sn-2 fatty acyl chain specificity in vitro (1, 67). This suggests that glypican shuttling brings the enzyme in contact with phospholipids that are enriched in AA. It is also noteworthy that AA release by the glypican-shuttled sPLA2s occurs only in agonist-stimulated and not in unstimulated HEK293 cells. Perturbed membrane structures evoked by various cellular events, such as cPLA2-directed membrane hydrolysis (37, 53, 68), lipid oxidation (68), and loss of lipid bilayer asymmetry (40), during cell activation may facilitate exposure of anionic phospholipids to these sPLA2s in the sorted compartment. Finally, endogenous COX-2 induction in HEK293 cells is limited to glypican-binding sPLA2s (Refs. 39 and 40 and Table I). The fact that significant IL-1-dependent PGE2 production occurred in cells transfected with IIC-L95R/E102K alone argues that this mutant is also able to induce COX-2 expression.
The inability of the heparin-nonbinding sPLA2-IIC, -IIE, and -IB to elicit AA release may be due to their poor affinity for heparan sulfate. Because these enzymes are mostly secreted into the culture medium, they could release AA from the outer surface of the plasma membrane. However, sPLA2-IIC, -IIE, and -IB bind poorly to PC-rich vesicles and show very low activity when added exogenously to mammalian cells (45).2 sPLA2-X efficiently produces AA in transfected HEK293 cells by a mechanism that is distinct from that used by sPLA2-IIA, -IID, and -V (Ref. 40 and Table I). Like sPLA2-IIC and -IIE, this enzyme does not bind to heparanoids and accumulates in the culture medium. However, sPLA2-X binds very efficiently to PC-rich membranes and is highly potent in releasing AA when added exogenously to a variety of mammalian cells (45). This and the fact that sPLA2-X releases both oleic acid and AA (40) argues that this enzyme acts in a different HEK293 cell membrane compartment than does the heparanoid-binding sPLA2s; it probably acts on the external face of the plasma membrane.
Lipid Interface-dependent Action-- In contrast to the seemingly redundant functions of the three heparin-binding sPLA2s (IIA, IID, and V) in AA release and PGE2 generation in the HEK293 cell system, these enzymes display distinct roles in the regulation of immediate LTC4 biosynthesis in rat mastocytoma RBL-2H3 cell transfectants. Among the sPLA2s examined, only sPLA2-V and -X, but not catalytic site mutants with poor enzymatic activity, exerted a potent enhancing effect on stimulus-dependent immediate production of LTC4 (Figs. 8 and 9 and Table II). The same pattern was found for PGD2 generation in these cells (18). These results are compatible with the previous observation that introduction of sPLA2-V antisense DNA into MMC-34 mast cells reduced immediate PGD2 generation (69). Failure of sPLA2-IB, -IIA, -IIC, -IID, and -IIE to augment LTC4 generation in activated mast cells implies that this event does not correlate with the heparanoid-binding tendency of sPLA2s. Among these seven sPLA2s, sPLA2-V and -X are unique in being able to bind efficiently to PC-rich vesicles (44, 45). Thus, within the limits inherent in the method of forcible gene expression by transfection, the results suggest that in RBL-2H3 cells, which lack glypican, sPLA2-V and -X could be acting on the PC-rich outer layer of the plasma membrane.
The plasma membrane target for sPLA2-V and -X action in RBL-2H3 cells is further supported by immunocytochemical studies, which show that these two enzymes are associated with the plasma membrane after IgE/Ag activation (Fig. 11). sPLA2-V provides a more intense signal than does sPLA2-X on the plasma membrane of activated RBL-2H3 cells, suggesting that the former may not only bind directly to the PC-rich membrane, as does the latter, but also it may bind to specific proteoglycan species or other anionic components, which may be distributed uniformly on the external surface of the plasma membrane. The molecular entity of this putative cell surface component that may act as an adapter for sPLA2-V on the plasma membrane of RBL-2H3 cells remains to be elucidated.
PC hydrolysis by sPLA2-V and -X in activated mast cells is also supported by the observation that only these two sPLA2s augment the production of PAF (Fig. 10), which is derived from 1-O-alkyl-PC. It is therefore likely that lyso-PAF produced by these PC-hydrolyzing sPLA2s is supplied to lyso-PAF acetyltransferase for PAF production. These studies provide the first evidence that specific sPLA2s can be coupled to 5-lipoxygenase and lyso-PAF acetyltransferase for the biosynthesis of LT and PAF, respectively. However, the possibility that this coupling occurred as a result of overexpression cannot be ruled out, and therefore this should be verified in a cell model with endogenous physiological levels of these enzymes in a future study.
Conclusions and Future Prospects--
The gain-of-function studies
reported here have revealed diverse aspects of the regulatory
mechanisms for sPLA2 function in two mammalian cell lines.
Both heparan sulfate-dependent sorting into intracellular
membrane compartments and interfacial binding to PC-rich membranes
critically affect the mechanism of cellular action of
sPLA2s. Both of these parameters are influenced not only by
the structural properties of each sPLA2 but also by the presence of distinct secretory pathways in different cell types that
are regulated by different proinflammatory stimuli. Remaining key
questions include the generality of the two sPLA2
regulatory mechanisms in other mammalian cell types, the role of
sPLA2 receptors in modulating the functions of a diverse
set of sPLA2s (11, 12), and the precise mechanism by which
differential cell trafficking of sPLA2s is coupled to
differential sn-2 fatty acyl chain specificity for
phospholipid hydrolysis, COX-2 induction, and interfacial binding of
those sPLA2s that cannot bind to PC-rich membranes. The
fact that sPLA2-IB, -IIC, and -IIE are not involved in AA release, at least in HEK293 and RBL-2H3 cells studied under the conditions reported here, suggest that these sPLA2s may
have novel functions which remain to be elucidated. Several studies
have established that sPLA2-IB from several animal species
acts as a potent ligand for the M-type sPLA2 receptor
(11, 12).
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Note Added in Proof |
---|
While this paper was under review, several important sPLA2 papers related to this work have been reported. Cho and co-workers (72) have confirmed that exogenous human sPLA2-V acts on human neutrophils through the heparanoid-independent, PC interface-dependent external plasma membrane mechanism. Moreover, they have shown that certain heparan sulfate proteoglycans on the neutrophil surface facilitates the internalization and subsequent proteolytic degradation of sPLA2-V (72), the event also found by us for exogenous SPLA2-IIA in mouse bone marrow-derived mast cells (73). Thus, in these cases, heparan sulfate proteoglycan works as a negative regulator of heparin-binding sPLA2s. Hanasaki and his co-workers (74) have demonstrated that human sPLA2-X releases AA from colon cancer cell lines through the external plasma membrane mechanism and proposed that this isozyme may be involved in COX-2-dependent exacerbation of colon tumorigenesis. Finally, the tenth member of mammalian sPLA2, group XII, has been very recently identified (75), the functions of which remain to be elucidated.
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FOOTNOTES |
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* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, Grant HL36235 from the National Institutes of Health, and by CNRS and the Association pour le Recherche sur le Cancer.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@pharm.showa-u.ac.jp.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007877200
2 M. H. Gelb, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; Ag, antigen; COX, cyclooxygenase; cPLA2, cytosolic PLA2; FCS, fetal calf serum; IL-1, interleukin-1; LT, leukotriene; PAF, platelet-activating factor; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PG, prostaglandin; sPLA2, secreted PLA2; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
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