From the Instituto de Biología y Genética Molecular,
Universidad de Valladolid-Consejo Superior de Investigaciones
Científicas, 47005 Valladolid, Spain
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INTRODUCTION |
Phospholipases A2 (phosphatide
sn-2-acylhydrolases, EC 3.1.1.4) from mammalian tissues play
a role in physiological functions such as defense mechanisms and the
production of bioactive lipids (1-3). In the last years, purification
and molecular cloning of phospholipases A2
(PLA2)1 has
allowed the characterization of several enzymes displaying significant
differences in both structural and functional properties. On the one
hand, the 14-kDa type IIA PLA2 (sPLA2) behaves
as an acute phase protein whose production is induced in a variety of immunoinflammatory conditions, e.g. rheumatoid arthritis and
endotoxemia (4-8), although its causal role in these conditions has
not been ascertained, and there is no clear evidence about its
involvement in the release of arachidonic acid elicited by agonists.
Recent studies have shown the ability of sPLA2 to promote
mitogenesis by acting on a cell surface receptor (9, 10) and the
appearance of chronic epidermal hyperplasia and hyperkeratosis similar
to those observed in human dermopathies in mice hyperexpressing the human type IIA PLA2 gene (11). A similar histological
picture accompanied by inflammatory changes is produced by injection of sPLA2 in the skin of experimental animals (12, 13). In
addition, sPLA2 may initiate cell activation because of its
ability to generate the lipid mediator lysophosphatidic acid (14).
On the other hand, cytosolic phospholipase A2
(cPLA2) plays a central role in the release of arachidonic
acid (AA) triggered by growth factors and neurotransmitters (15-17),
and contains the consensus primary sequence (Pro-Leu-Ser-Pro) for
phosphorylation by mitogen-activated protein (MAP) kinases, which play
an important role in its regulation (18-20). Since sPLA2
is an ectoenzyme that first encounters the outer leaflet of the lipid
bilayers, two means of interaction leading to cell signaling should be
considered. (i) sPLA2 might interact with a binding
structure on the outer leaflet of the cell membrane, or (ii)
sPLA2 might generate both unesterified fatty acid and
lysophospholipid, e.g. lysophosphatidate (LPA) and
lysophosphatydylcholine, which could act on signaling either as
cofactors for protein kinase C or, in the case of LPA, by acting on
specific receptors. This poses as a likely possibility that
sPLA2 might ultimately lead to the activation of
cPLA2 by eliciting a signaling cascade mimicking the usual
transducing mechanism conveyed by the physiological activators of this
enzyme. In this connection, it should mentioned that cross-talk between cPLA2 and sPLA2 has been suggested in signal
transduction events in polymorphonuclear leukocytes and macrophages
(21, 22), and a recent study in neural cells has shown a complex
interplay between neurotransmitter-activated cPLA2 and
sPLA2 (23).
cPLA2 is expressed in human astrocytes of the gray matter
(24), and, in a recent study, we have observed coupling of this enzyme
to the activation of both muscarinic and thrombin receptors in the
1321N1 astrocytoma cell line (25, 26). This cell line displays thrombin
and muscarinic M3 receptors, and its pattern of responses
elicited by ligand binding includes activation of phospholipases
A2, C, and D (25-32) and induction of AP-1 transcriptional activity (30, 31). 1321N1 astrocytoma cells express high amounts of
cPLA2, and they do not contain sPLA2. Thus,
this cell line is a good model to study the biochemical responses
elicited by exogenously added sPLA2.
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EXPERIMENTAL PROCEDURES |
Materials--
Plasma from patients with septicemia was obtained
from venous blood anticoagulated with heparin.
[9,10-3H]Myristic acid (53 Ci/mmol),
[1-14C]oleate (53, 9 mCi/mmol), and
[3H]arachidonic acid (100 Ci/mmol) were from Amersham
International, Bucks, United Kingdom. Essentially fatty acid-free BSA
was from Miles Laboratories. Reagent for the measurement of proteins
according to the method of Bradford (33) was purchased from Bio-Rad.
Heparin-agarose type I,
p-aminophenyl-
-D-mannopyranoside-BSA
(mannose-BSA), and porcine pancreatic PLA2 were from Sigma.
A C127 mouse fibroblast line stably transfected with the coding
sequence of type IIA PLA2 from human placenta (34) was used
as a source of human recombinant type IIA PLA2. Rabbit
polyclonal anti-cPLA2 antibody was obtained as described
(35). Mouse monoclonal anti-MAP kinase antibody reacting with both p42
and p44 MAP/ERK was from Zymed Laboratories Inc., San
Francisco, CA. Rabbit polyclonal anti-p38 MAP kinase antibody was from
Santa Cruz Biotechnology Inc., Santa Cruz, CA. Monoclonal
anti-phosphotyrosine antibody clone 4G10 was from Upstate Biotechnology, Lake Placid, NY. The MAP kinase kinase (MEK) inhibitor PD-98059 was a gift from Dr. Alan R. Saltiel (Parke Davis
Pharmaceutical Research, Ann Arbor, MI) (36). The p38 MAP kinase
inhibitor SB 203580 was a gift from Dr. John C. Lee (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) (37). Glutathione
S-transferase (GST) fusion protein with amino acids 1-223
of the N-terminal portion of c-Jun protein (a kind gift of Dr. Carmen
Caelles, Instituto de Investigaciones Biomédicas, Madrid, Spain)
was expressed in bacteria using a pGEX-2T plasmid (Pharmacia Biotech
Inc.) and purified with glutathione-agarose beads from Sigma.
Purification of sPLA2--
sPLA2 was
purified from both plasma of patients with septicemia and culture
medium according to the protocol described in Ref. 38. Briefly,
heparin-agarose was used to bind sPLA2 from plasma.
Fractions showing PLA2 activity in the
[1-14C]oleate-labeled Escherichia coli assay
were concentrated and loaded into a HiLoad Superdex 75 column
(Pharmacia LKB, Uppsala, Sweden). Fractions containing PLA2
after this step were made in 0.1% trifluoroacetic acid, and applied
into a C1/C8 reverse-phase FPLC column (ProRPC
HR 5/2, Pharmacia LKB). Fractions showing PLA2 activity
were pooled and evaporated to dryness in a Speed-Vac concentrator.
Human recombinant type IIA phospholipase A2 was purified
from cultures at superconfluence of line C127 mouse fibroblasts stably
transfected with the coding sequence of type IIA PLA2 from human placenta (34).
Assay of sPLA2 Activity--
The assay was carried
out in a total volume of 0.1 ml, according to the procedure of Elsbach
et al. (39). Samples were incubated with
5,000 dpm of
[1-14C]oleate-labeled autoclaved E. coli of a
K12 strain, containing 10-20 nmol of phospholipid, as assessed by the
measurement of phospholipid-associated phosphate. The assay medium
contained 0.1 M Tris/HCl, 1 mg/ml fatty acid-free BSA, and
0.5 mM CaCl2, pH 7.4. The reaction proceeded
for 30 min and was stopped by addition of 0.04 ml of ice-cold 2 N HCl and 0.02 ml of 10% BSA, followed by centrifugation
for 5 min at 13,000 rpm in an Eppendorf microcentrifuge. The
radioactivity released into the supernatant was assayed by liquid
scintillation counting.
Cell Culture and Metabolic Labeling of 1321N1 Cells--
1321N1
astrocytoma cells were cultured in Dulbecco's modified Eagle's medium
containing 5% fetal calf serum at 37 °C in an atmosphere containing
5% CO2. Labeling with [3H]AA was performed
in cells in monolayer that had been deprived of fetal calf serum for
16 h to render them quiescent. Labeling with [3H]AA
was carried out for 2 h in the presence of 0.3 µCi of
[3H]AA/ml. Labeling with 0.3 µCi/ml
[1-14C]oleate and 1 µCi/ml [3H]myristic
acid was carried out under similar conditions but increasing the
labeling period to overnight incubation. After labeling, cells were
washed at 37 °C four or five times with medium, and finally allowed
to equilibrate at 37 °C before addition of agonists or vehicle
solution. The release of labeled [3H]AA and
[1-14C]oleic acid acid was assessed in 0.2-ml aliquots of
culture medium. Production of LPA was assessed from the incorporation
of [3H]myristic acid into phosphatidic acid and was
separated from the label incorporated in other phospholipid classes by
two-dimensional chromatography using a system of solvents consisting of
chloroform/methanol/28% ammonium hydroxide (6:4:1; v/v/v) in the first
dimension and chloroform/acetone/methanol/acetic acid/water (6:8:2:2:1;
v/v/v) in the second dimension (40). Experiments were carried out with
triplicate samples.
Measurement of DNA Synthesis Reinitiation--
Quiescent 1321N1
cells were treated in serum-free Dulbecco's modified Eagle's medium
for 24 h with different agonists in the presence of 0.5 µCi/ml
[3H]thymidine. At the end of this period, the incubation
was terminated with three washes with ice-cold 0.1 M
MgCl2, and the radioactivity incorporated into the
trichloroacetic acid-precipitable fraction measured.
Immunoblots of cPLA2, p42 MAP Kinase, and
Immunoprecipitated p38 MAP Kinase--
Cell lysates from preconfluent
1321N1 cells were loaded into a 10% SDS-PAGE gel, and transferred to
polyvinyldifluoride membrane (Immobilon P, Millipore Corp., Bedford,
MA) using a liquid transfer module from CBS Laboratories. The membranes
were blocked with dry milk for 2 h, washed with Tris-buffered
saline, and used for immunoblot using a rabbit polyclonal
anti-cPLA2. When the purpose of the experiments was the
detection of p42 MAP kinase, a semidry transfer system was used and the
membrane was incubated with mouse monoclonal antibody. This was
followed by incubation with sheep anti-mouse IgG-horseradish
peroxidase-conjugated antibody, and detection with the Amersham ECL
system. For detection of tyrosine phosphorylation of p38 MAP kinase,
the endogenous kinase was immunoprecipitated from cell lysates using
anti-p38 MAP antibody. The immune complex was recovered using Gammabind
G-Sepharose. After washing three times with Nonidet-P-40-buffer and
twice with LiCl buffer, the beads were resuspended in Laemmli sample
buffer and subjected to SDS-PAGE. The extent of tyrosine
phosphorylation of the p38 MAP kinase immunoprecipitated was determined
by immunoblot with anti-phosphotyrosine mouse monoclonal antibody.
Assay of JNK Activity--
To obtain the substrate for the
kinase assay as a GST-c-Jun fusion protein, the procedure of Smith and
Corcoran (41) was followed. For this purpose, transformed XL1-blue
cells containing a pGEX-2T plasmid encoding residues 1-223 of the
N-terminal portion of c-Jun protein were grown in LB/ampicillin medium.
The expression of the fusion protein was induced by addition of 1 mM isopropyl-1-thio-
-D-galactoside. Cells
were lysed using a probe sonicator and the fusion protein purified with
glutathione-agarose beads. The cytosolic extracts for the kinase assay
were obtained from the lysis of 5 × 106 1321N1 cells
in 200 µl of a medium containing 25 mM Hepes, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 100 mM orthovanadate, 20 mM
-glycerophosphate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, pH 7.7. After centrifugation at 12,000 rpm at
4 °C, the supernatant was diluted in 600 µl of the above mentioned
medium without NaCl, and mixed with 10 µg of GST-c-Jun protein and
glutathione-agarose beads. The mixture was incubated under continuous
shaking for 3-5 h at 4 °C, and then washed to remove the fraction
not associated to the glutathione-agarose beads. The kinase reaction
was carried out with 20 mM ATP and 5 µCi of
[
-32P]ATP in a volume of 30 µl. The reaction was
diluted in buffer and centrifugated to discard supernantant and then
boiled in Laemmli SDS sample buffer and DTT. Phosphorylated GST-c-Jun
was resolved by 10% SDS-PAGE and detected by autoradiography.
Quantitation of the phosphorylation was carried out by densitometric
scanning.
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RESULTS |
sPLA2 Produces [3H]AA Release and
Mitogenesis in 1321N1 Astrocytoma Cells, but Does Not Release
[1-14C]Oleic Acid--
Incubation of 1321N1 cells with
sPLA2 at concentrations of 10 ng to 0.4 µg induced the
release of [3H]AA into the medium (Table
I, Fig.
1A). This release was similar to that produced by agonists acting on membrane receptors on this cell
line, namely carbachol (25), thrombin (26), and LPA (Fig. 1B). Astrocytes labeled with [1-14C]oleic acid
were treated with sPLA2 under the same conditions used for
the assay of [3H]AA release. As shown in Table I, no
significant release of [1-14C]oleic acid was observed.
Since sPLA2 produces mitogenesis in astrocytes (9), we
addressed whether this response was also elicited in quiescent 1321N1
cells, using 10% fetal calf serum as a positive control and thrombin
as a prototypic mitogenic agonist of this cell line (42). As shown in
Table II, sPLA2 behaved as a
mitogenic agonist somewhat more potent than thrombin.
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Table I
Effect of sPLA2 on [3H]AA and [1-14C]oleate
release by 1321N1 cells
1321N1 cells were labeled and incubated for 45 min at 37 °C with
different concentrations of sPLA2. At the end of this period, the radioactivity contained in 0.2 ml of cell medium was assayed. The
radioactivity incorporated in cell phospholipids under the conditions
used for labeling was 120,000 dpm for [3H]AA and 16,000
for [1-14C]oleate. Results represent mean ± S.E. of
three independent experiments in triplicate.
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Fig. 1.
Release of [3H]AA in response
to sPLA2 and LPA. 1321N1 cells were incubated in the
presence of 0.1 µg/ml sPLA2 (A) or 0.2 µM LPA (B) for the times indicated, and then
the release of [3H]AA assayed in the culture medium.
Closed circles indicate cells incubated in the presence of
agonist. Open circles indicate cells incubated in the
presence of vehicle. Data represent mean ± S.E. of six
independent experiments in duplicate. *, p < 0.05 as
compared with control cells incubated for the same time. The
closed triangle in A indicates cells preincubated
for 30 min with 25 µM PD-98959 prior to the addition of
sPLA2. The closed square in A
indicates cells pretreated with 25 µM SB 203580 under
identical conditions. The release of [3H]AA in the
presence of these drugs has been compared with that produced by
sPLA2 in the absence of these compounds.
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Table II
Incorporation of [3H]thymidine into acid-precipitable
material
Results are expressed as percent of the response elicited by 10% FCS
(83,000 dpm). Data represent mean ± S.E. of three experiments with duplicate samples.
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sPLA2 Induces the Phosphorylation of Both
cPLA2 and MAP Kinases--
Since cPLA2 is the
most specific enzyme that releases arachidonate from phospholipids, and
sPLA2 does not display selectivity for any unsaturated
fatty acid on the sn-2 position of phospholipids (43, 44),
sPLA2 responses might a priori reflect either a direct consequence of its catalytic activity or recruitment of the
arachidonate-selective enzyme cPLA2. Considering that
1321N1 cells contain cPLA2 as the main PLA2
activity detected in cell-free homogenates and the implication of this
activity in the mobilization of [3H]AA produced by
receptor stimulation (25, 26), we hypothesized that activation
cPLA2 could explain the release of [3H]AA
triggered by sPLA2. The increase in catalytic activity of the 85-kDa PLA2 has been linked to phosphorylation of the
enzyme, which results in reduced mobility upon electrophoresis. As
shown in Fig. 2A, 0.1 µg/ml
sPLA2 purified from the plasma of patients with septicemia
induced phosphorylation of cPLA2. The activation of this
protein shows a time course that clearly precedes [3H]AA
release. Maximal amount of P-cPLA2 was achieved within
10-15 min and was maintained up to 30 min after cellular stimulation. Interestingly, phosphorylation of the p42 MAP kinase preceded cPLA2 phosphorylation, since it was near maximal values at
5 min (Fig. 2A). In vitro kinase assay of c-Jun
kinase activity in lysates from cells stimulated with sPLA2
showed an increase of the activity that peaked about 10 min after
addition of sPLA2 (Fig.
3A). Blotting with
anti-phosphotyrosine antibody of the immunoprecipitate obtained with
anti-p38 MAP antibody in lysates from 1321N1 cells, showed an increase
of tyrosine phosphorylation of p38 MAP kinase of about 4-fold, 2 min
after addition of sPLA2 (Fig. 3B), thus
suggesting that sPLA2 activates all the subgroups of the
MAP kinase family following different time courses.

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Fig. 2.
Activation of cPLA2 and p42 MAP
kinase by sPLA2 (A) and LPA
(B). Cells were stimulated with 0.1 µg/ml
sPLA2 purified from patients with septicemia or 0.2 µM LPA, and at the times indicated washed and then lysed
in Laemmli s buffer. About 50 µg of protein from the same samples
were processed separately by SDS-PAGE (10% gel) with prolonged
electrophoresis to separate the phosphorylated
(P-cPLA2, P-p42-MAP) from the nonphosphorylated forms (cPLA2, p42-MAP) of cPLA2 and
p42 MAP. The proteins were electrophoretically transferred to
polivinyldifluoride membranes, and immunoblotting was carried out with
anti-cPLA2 antibody or with anti-MAP/ERK antibody.
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Fig. 3.
Effect of sPLA2 on c-Jun kinase
activity 2nd on p38 MAP kinase phosphorylation. 1321N1 cells were
incubated with sPLA2 for the times indicated, and at the
end of these periods, cell lysates were collected and used for in
vitro assay of c-Jun kinase activity using GST-c-Jun protein and
glutathione-agarose beads. Heat shock (HS) was used as
a positive control for c-Jun kinase activation. This was carried out by
incubating the cells for 2 min in medium preheated at 45 °C,
followed by incubation at 37 °C. Quantitative analysis of the
phosphorylation of GST-c-Jun was obtained by densitometric scanning and
is expressed as fold-increase of basal activity (A). The
experiment shown in B was carried out by immunoprecipitation
of p38 MAP kinase from cell lysates, SDS-PAGE separation of the
immunoprecipitate, and blotting with anti-phosphotyrosine antibody. The
histogram shows the densitometric scanning of the blots.
P-Y-p38-MAP indicates p38 MAP kinase phosphorylated in tyrosine.
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We also investigated the effect of human recombinant sPLA2
isolated from permanent transfected C127 fibroblasts. Stimulation of
astrocytes with concentrations of human recombinant sPLA2
above 0.1 µg/ml also resulted in a shift of the electrophoretic
mobility of cPLA2 (Fig. 4).
Similarly, the addition of type I PLA2 (pancreatic PLA2, 0.8-8 µg/ml) to 1321N1 astrocytoma cells also
increased the amount of P-cPLA2 detected upon
electrophoresis (Fig. 4). To confirm that the observed increase of the
cPLA2 phosphorylation was due to sPLA2 rather
than linked to a possible lipopolysaccharide contamination in the
sPLA2 preparation from septic patients, we treated our
cells with 10 µg/ml lipopolysaccharide. SDS-PAGE revealed that
lipopolysaccharide is not able to induce cPLA2
phosphorylation (data not shown), thus ruling out the view that the
observed activation of cPLA2 could be due to contamination
by lipopolysaccharide. Having established that addition of either of
the two secreted forms of PLA2 induced phosphorylation of
cPLA2, we hypothesized two possible mechanisms either a
direct action of sPLA2 on its receptor or an indirect
effect through lipid mediators generated as a consequence of its
catalytic activity.

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Fig. 4.
Activation of cPLA2 by pancreatic
PLA2, recombinant human sPLA2, and LPA: dose
dependence. 1321N1 cells were incubated with different
concentrations of agonists for 15 min. At the end of these periods, the
cell lysate was processed. Immunoblots are representative of at least
three blots with identical pattern.
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sPLA2 Elicits Its Effect Independently of
Lysophosphatidate Formation--
Since LPA is a mitogenic agonist
(reviewed in Ref. 45) and a product of sPLA2 (14), we put
forward the hypothesis that sPLA2 could elicit its effect
via the formation of this lipid mediator that acts via the interaction
with a G-protein-coupled receptor. To check this notion, we first
looked at the effect of LPA. As shown in Fig. 1B, a
concentration of LPA as low as 0.2 µM induced
[3H]AA release. To determine the time course of
LPA-induced phosphorylation of cPLA2, astrocytes were
exposed to 0.2 µM LPA for 0-60 min. As shown in Fig.
2B, the response is already evident by 5 min and is fully
developed by 10 min. cPLA2 band-shift induced by LPA was
preceded by p42 MAP kinase phosphorylation, which was already
significant at 1 min and maximal at 5 min (Fig. 2B). Fig. 4
shows the dose-dependent effect of LPA. Since both
sPLA2 and LPA produced a similar pattern of activation,
this finding could be considered as an initial hint that LPA could be
involved in the mediation of sPLA2 effect.
It has been shown that the LPA-induced MAP kinase activation is
sensitive to pertussis toxin inhibition (46, 47), thus indicating a
critical role for a pertussis toxin-sensitive Gi-protein. On this basis, if sPLA2 were acting through LPA generation,
cPLA2 and MAP kinase activation in response to
sPLA2 should show identical sensitivity to PTX. Then, in
astrocytes preincubated with or without 100 nM PTX, we
looked at the effect of sPLA2 on the phosphorylation of p42
MAP kinase and cPLA2. Whereas overnight incubation of
1321N1 cells with PTX inhibited the LPA-induced shift in
electrophoretic mobility of both cPLA2 and p42 MAP kinase,
this treatment did not affect the ability of sPLA2 to
phosphorylate cPLA2 or p42 MAP kinase (Fig.
5). This suggests not only that LPA is
not involved in the cellular response to sPLA2, but also
that sPLA2 acts through a pathway independent of
Gi-proteins.

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Fig. 5.
Effect of PTX on the activation of
cPLA2 and p42 MAP kinase elicited by LPA and
sPLA2. 1321N1 cells were incubated overnight with 100 ng/ml PTX, and then stimulated with vehicle or the indicated
concentrations of agonists for 15 min. At the end of this period, the
cell lysate was obtained and used to separate, by electrophoresis, the
phosphorylated and nonphosphorylated forms of cPLA2 and p42
MAP. Immunoblot is representative of two blots with identical
trend.
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Attempts to demonstrate formation of LPA by sPLA2 were
carried out by labeling the phospholipid pools with
[3H]myristic acid and analysis of the cellular culture
medium. The lipid fraction was analyzed by a two-dimensional TLC
system, which allows LPA to be separated from other polar lipids with a
high degree of resolution. However, upon sPLA2 treatment,
[3H]LPA accumulation was not detected, even though a high
concentration of lipid-free BSA was added to the medium to trap LPA
because of its strong binding to albumin (48).
Inactivation of sPLA2 Catalytic Activity Does Not Block
the Ability to Induce Phosphorylation of cPLA2--
As we
failed to find accumulation of LPA or any other fatty acid but
[3H]AA in the cell culture medium, we addressed the
possibility of regarding sPLA2 as the direct responsible
for cPLA2 phosphorylation. To determine whether blockade of
sPLA2 catalytic activity may affect its ability to induce
cPLA2 activation, the actions of known sPLA2
inhibitors were examined. We first looked at the effect of
p-bromophenacyl bromide (BPB), a compound that inactivates the enzyme by alkylating a histidine residue located in the active site
(49). Pretreatment of sPLA2 for 30 min with different doses of the inhibitor resulted in a dose-dependent lose of its
catalytic activity on the E. coli membrane system, reaching
a complete blockage at 100 µM. However, even in the
presence of this doses of BPB, the cPLA2 band-shift induced
by sPLA2 was not affected (Fig.
6). It should be noted that 1 mM BPB (but not the other doses) alters agonist-induced
cPLA2 band-shift, thus suggesting a toxic effect of this
compound, which we could confirm by the appearance of the cell culture;
this agrees with the report by Lister et al. (50), who have
suggested that the inhibitory effects of high concentrations of BPB is
nonspecific, as it is due to the hydrophobicity of the compound.
Incubation of sPLA2 for 30 min with the thiol reagent DTT,
dramatically reduced the catalytic activity of this enzyme (50% with
0.1 mM, 90% with 1 mM, and 100% with 10 mM); however, this treatment did not affect the ability of
sPLA2 to phosphorylate cPLA2 upon addition to
1321N1 cells (Fig. 6). Taken together, the above results show that both
cPLA2 phosphorylation and AA mobilization induced by
sPLA2 are events independent of the catalytic activity of
the enzyme.

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Fig. 6.
Effect of BPB, DTT, and heparin on the
activation of cPLA2 by sPLA2. BPB was
added to the cells in the range of concentrations shown, 10 min before
the addition of sPLA2. In the case of experiments with DTT,
the enzyme was incubated with the indicated concentrations of DTT for
30 min at room temperature, and then added to the cells to reach a
final 100-fold dilution of DTT, and the indicated concentration of
sPLA2. The left lane shows the effect of the
maximal concentration of DTT on 1321N1 cells. Heparin was added to
1321N1 cells 10 min before sPLA2.
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Compounds Blocking Binding of sPLA2 to Cell Membrane
Surface Inhibit the Ability to Phosphorylate cPLA2--
In
contrast to the aforementioned data, previous treatment of 1321N1 cells
with mannose-BSA prior to sPLA2 addition blocked cPLA2 band-shift (Fig. 7)
with an EC50 similar to that described for the inhibition
of binding of sPLA2 to its receptor (51). The same effect
was observed when the same samples were used to study the effect on p42
MAP kinase band-shift (Fig. 7). Since it has been described that
sPLA2 may trigger mast cell activation through binding of
its heparin-binding domain to the cell surface (52), the effect of
exogenous heparin on sPLA2-induced cPLA2 phosphorylation was also tested. As shown in the lower panel
in Fig. 6, concentrations of heparin similar to those active on mast cells inhibited the cPLA2 band-shift, without affecting
significantly sPLA2 catalytic activity on
[1-14C]oleate-labeled autoclaved E. coli (data
not shown). All these findings suggesting that both cPLA2
and p42 MAP kinase activation can be explained by interaction of
sPLA2 with a binding structure on 1321N1 cell surface.

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Fig. 7.
Effect of mannose-BSA on the activation of
cPLA2 and p42 MAP kinase elicited by
sPLA2. 1321N1 cells were incubated for 10 min with the
indicated concentrations of mannose-BSA prior to the addition of
sPLA2. This immunoblot is representative of three blots
with identical pattern.
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Blockade of MAP/ERK Kinase Activation Inhibits cPLA2
Phosphorylation and [3H]AA Release--
As shown
previously, prolonged SDS-PAGE and immunoblotting of 1321N1 cell
lysates, with a monoclonal antibody that recognizes an epitope found in
both the 42- and 44-kDa isoforms of MAP/ERK kinases, only showed
positive staining of a 42-kDa protein in resting cells, suggesting that
this is the main isoform of the ERK subfamily of MAP kinases expressed
in 1321N1 cells. Preincubation of 1321N1 cells with the compound
PD-98059 (36), which inhibits MAP/ERK kinase activation by interfering
with the upstream kinase MEK, inhibited both cPLA2 and p42
MAP kinase activation over the same range of concentrations (Fig.
8), as well as the release of
[3H]AA (Fig. 1A), thus suggesting the
involvement of the MAP/ERK subgroup of MAP kinases in the
phosphorylation of cPLA2 elicited by sPLA2.
Pretreatment of the cells with the p38 inhibitor SB 203580 at the
concentration of 25 µM also caused inhibition of [3H]AA release (Fig. 1A), thus suggesting that
this subfamily of MAP kinases could be involved in the pathway for
cPLA2 activation elicited by sPLA2.

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Fig. 8.
Effect of the MEK inhibitor PD-98059 on the
activation of cPLA2 and p42 MAP kinase elicited by
sPLA2. 1321N1 cells were incubated for 10 min with
different concentrations of PD-98059 prior to the addition of
sPLA2. About 10 min after addition of sPLA2,
the cell lysate was collected for immunoblotting. This immunoblot is
representative of two blots with identical trend.
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DISCUSSION |
We have selected 1321N1 astrocytoma cells to study the effect of
sPLA2 because these cells do not express this enzyme, but do contain high amounts of cPLA2, which is the form of
enzyme most usually involved in the release of [3H]AA
coupled to receptor stimulation. Analysis of the physiological effects
of sPLA2 indicates several possible mechanisms through which they might be exerted. One of them takes into account the lysophospholipids formed as a consequence of the catalytic properties of the enzyme. In this connection, analysis of the involvement of LPA
is of central importance, since this is a multifunctional signaling
phospholipid that elicits cell responses by binding to a receptor,
which couples to both PTX-sensitive and PTX-insensitive G-proteins
(Gi and Gq, respectively) to trigger various
effector pathways. At least four G-protein-mediated signaling pathways have been identified in the action of LPA (revised in Ref. 45): (i)
stimulation of phospholipases C, D, and A2 (this report); (ii) inhibition of adenylyl cyclase; (iii) activation of Ras and the
downstream Raf/MAP kinase pathway; and (iv) protein-tyrosine phosphorylation. This is relevant to the present study since LPA is
detected in human serum at concentrations in the range 2-70 µM (45-47), and the effect of sPLA2 on
platelets incubated with lipid microvesicles has been related to the
production of LPA (14). Some of our findings agree with this mechanism
of signaling in view of the ability of exogenous LPA to trigger
biochemical signals in 1321N1 cells resembling a portion of the effect
of sPLA2; however, a careful appraisal of the results shows
the existence of several differences, e.g. the involvement
of a PTX-sensitive G-protein in LPA signaling, which is not involved in
sPLA2 signaling. Moreover, direct attempts to assay LPA
formation upon sPLA2 did not show the production of this
mediator. Generation of unesterified fatty acids by sPLA2
could be another mechanism through which this enzyme conveys cell
responses. This point is a matter of considerable debate, since there
is a number of mammalian cells where there has not been possible to
trigger AA release by sPLA2 (38, 53, 54), unless a membrane
rearrangement of phospholipids is produced (55). Separation by TLC of
cell-associated lipids and assay of supernatants of cells in culture
stimulated with sPLA2 showed no evidence of unesterified
[1-14C]oleate, but did show [3H]AA. Since
unlike cPLA2, sPLA2 does not have a
preferential effect on AA-containing membrane phospholipids as compared
with those containing other fatty acids, our results should be
explained on the basis of the activation by sPLA2 of a
selective mechanism for AA release that would implicate a signaling
cascade leading to cPLA2 activation. Selective release of
AA by sPLA2 has already been reported in mice bone marrow
mast cells (56). In this study, concentrations of
1 µg/ml
PLA2 from different sources, including human recombinant
type IIA sPLA2 and Naja naja type I
PLA2, elicited the release of AA in a similar way to that
observed in response to immunological challenge by specific antigen.
Since other unsaturated fatty acids were not detected in the
supernatant, this finding also points to the recruitment by
sPLA2 of a selective mechanism of AA release.
The characterization of the binding site in cell membrane involved in
the triggering of the response to sPLA2 herein described requires a detailed discussion in view of the different structures that
could be involved. Thus, there is some evidence associating many
effects of sPLA2 to the activation of a membrane surface receptor, which shows significant homology with the macrophage mannose-binding receptor (6, 7, 51), and is also activated by the
pancreatic type of PLA2, thus suggesting that endogenous PLA2(s) might be its physiological ligands. In fact,
stimulation of prostaglandin synthesis by pancreatic type
PLA2 acting through a receptor-binding reaction has been
shown in rat mesangial cells (57) and in mouse osteoblastic cells (58).
Moreover, inflammatory factors stimulate expression of type IIA
PLA2 in astrocytes in culture (59), and brain tissue is
rich in N-type PLA2 receptors (60). However, previous
reports do not support the involvement of PLA2 receptors in
our system, since unlike the rabbit receptor (10), the human 180-kDa
receptor expressed in COS cells binds neither type IIA PLA2
nor mannose-BSA (61). Interaction of sPLA2 with heparan
sulfate proteoglycans is another possibility, in view of a recent
report where sPLA2 expressed endogenously and anchored on
cell surfaces via its C-terminal heparin-binding domain was shown to be
involved in the biosynthesis of prostaglandins elicited by growth
factors and cytokines (62). Our attempts to unveil the binding
structure by using pharmacological agents such as heparin and
mannose-BSA have shown inhibition by either compound, thus suggesting
more than one binding structure or, alternatively, a scarce selectivity
for these compounds. Therefore, additional studies of binding and
receptor expression are required to characterize these structures more
precisely.
Irrespective of the nature of the membrane structure involved in
sPLA2 binding, the overall response induced by
sPLA2 in 1321N1 cells is in keeping with a mechanism
dependent on the occupancy of the physiological binding sites for
secreted PLA2. Little is known about the biochemical
signaling triggered by sPLA2 receptor binding. Murakami
et al. (52) have proposed the involvement of
protein-tyrosine phosphorylation reactions in
sPLA2-mediated mast cell activation in view of the blockade
of the response by inhibitors of this reactions such as genistein and
herbimycin A. In view of the important interactions of protein-tyrosine
phosphorylation signaling and activation of the MAP kinase cascade,
this report agrees with our finding of the activation of the MAP kinase
cascades by sPLA2, including p42 MAP kinase, p38, and c-Jun
kinase. Based on the different time courses of the activation of these
kinases, and the results obtained with the MEK inhibitor PD-98959 as
well as with SB 203580, which inhibits p38 MAP kinase activity, our data suggest the implication of both kinases in the signaling pathway
leading to [3H]AA release, although characterization of
the actual kinase(s) implicated in cPLA2 phosphorylation
requires additional studies. Studies on the regulation of
cPLA2 have stressed the requirement of
Ca2+-dependent translocation to the cell
membrane for elicitation of its catalytic effect (63). In keeping with
this mechanism, we have observed that both pancreatic type
PLA2 and type IIA sPLA2 elicit Ca2+
mobilization in fura-2-labeled 1321N1
cells2 showing a pattern
similar to that elicited by LPA, thus suggesting a mechanism of action
compatible with the occupancy of a binding site. However, since
mechanisms other than Ca2+ mobilization have been
implicated in the translocation of cPLA2 (25, 64), we
cannot establish a direct link between Ca2+ mobilization
and the activation of cPLA2 as yet.
As to the pathophysiological significance of our findings, it should be
pointed out that the effect of sPLA2 has been obtained with
concentrations of enzyme below those detected in human plasma in a
number of clinical conditions, including septic shock (65), salicylate
intoxication (66), and severe Plasmodium falciparum malaria
(67).
In summary, our data show that sPLA2 elicits biochemical
signaling in 1321N1 astrocytoma cells by a mechanism that is best explained by interaction with a membrane receptor similar to the macrophage mannose receptor or, alternatively, via engagement of
heparan sulfate proteoglycans. The set of responses observed includes
phosphorylation of cPLA2, most probably involving the p42
MAP kinase route, release of AA, and mitogenesis. These findings might
be of interest to explain some of the controversial findings regarding
release of AA by sPLA2.
We thank Dr. G. Bereziat for providing the
transfected fibroblast cell line, Dr. Victor Calvo for help in assaying
MAP kinase, Dr. J. Castañeda for selecting patients with multiple
organ failure and high levels of sPLA2 to obtain plasma,
Dr. Joan Heller Brown for providing the 1321N1 cell line, and Dr.
Carmen Caelles for providing the GST-c-Jun fusion protein.