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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:793–799, 2000
Vol. 294, No. 3
900006/843028
Printed in U.S.A.
Perspectives in Pharmacology
Phospholipase A2s in Cell Injury and Death
BRIAN S. CUMMINGS, JANE MCHOWAT, and RICK G. SCHNELLMANN
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (B.S.C., R.G.S.); and
Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri (J.M.)
Accepted for publication March 15, 2000
This paper is available online at http://www.jpet.org
Phospholipase A2s (PLA2s) represent a superfamily of esterases that hydrolyze the sn-2 ester bond in phospholipids
releasing free fatty acids and lysophospholipids. The ubiquitous nature of PLA2s highlights the important role they play
in many biological processes, including the generation of
proinflammatory lipid mediators such as prostaglandins and
leukotrienes, and the regulation of lipid metabolism (Glaser,
1995). Since 1997, PLA2s have been classified according to
their nucleotide sequence (Balsinde et al., 1999). At this time,
at least 10 groups have been described (I–X) with each group
having at least one member and a majority containing at
least two members. Individual members of each group are
designated by capital letters. There is significant confusion in
the field of PLA2 because many of the identified PLA2s are
not associated with specific cellular activities and functions,
and cellular activities and functions of PLA2s are not associated with identified PLA2s. A previous classification system
is based on whether the PLA2 is secreted from the cell
(sPLA2), Ca2⫹-dependent and cytosolic (cPLA2) or Ca2⫹-independent (iPLA2). This older classification system still remains and retains some value at this time (Table 1). sPLA2
isoforms require millimolar amounts of Ca2⫹ for activity,
have low molecular masses (14 –18 kDa), and demonstrate no
selectivity for arachidonylated phospholipids (Types I–III, V,
IX, and X). cPLA2 isoforms are found in the cytosol, have a
Received for publication January 3, 2000.
oncosis depends upon the PLA2 isoform, the cell type, and the
stimulus of injury. The purpose of this review is to discuss the
functions of iPLA2, cPLA2 and sPLA2 isoforms in oncosis and
apoptosis, including oxidant-induced and receptor-mediated
cell death. In addition, the measurement and modulation of
PLA2 is discussed.
higher molecular mass (⬃85 kDa), require micromolar
amounts of Ca2⫹ for translocation to membrane phospholipids, and are selective for arachidonylated phospholipids
(Types IVA and B). The iPLA2s are located in both the cytosol
(Balsinde and Dennis, 1996a) and membrane fractions
(McHowat and Creer, 1998) (Types IVC, VI, VIIB, and VIII).
They do not require Ca2⫹ for activity and have molecular
masses ranging from 29 to 85 kDa. Within certain PLA2
groups, such as iPLA2s, there exist multiple splice variants of
the same gene resulting in the expression of two “catalytically distinct” iPLA2 isoforms (Larsson et al., 1998; Ma et al.,
1999).
The involvement of PLA2s in inflammation is the result of
their ability to mobilize arachidonic acid from phospholipids.
Arachidonic acid serves as a substrate for prostaglandin H
synthase 1 and 2 (COX-1 and -2, respectively), resulting in
the production of prostaglandins. Prostaglandins activate
cellular receptors resulting in the subsequent initiation of
signal cascades involving G-proteins and cyclic AMP (Cirino,
1998). Thus, PLA2s have an important role in cellular injury
via their ability to mediate inflammatory responses.
Measurement and Chemical Inhibition of PLA2 Activity
Chemical inhibitors of PLA2s play an important role in
elucidating the actions of specific PLA2s. The study of PLA2
inhibitors is a critical area of investigation due to the potential pharmacological benefit of these compounds in the treat-
ABBREVIATIONS: PLA2, phospholipase A2; AACOCF3, arachidonyl trifluoromethyl ketone; BEL, bromoenol lactone; TNF␣, tumor necrosis factor
␣; PKC, protein kinase C; PKA, protein kinase A; MAPK, mitogen-activated protein kinase.
793
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ABSTRACT
Phospholipase A2s (PLA2s) represent a family of esterases that
hydrolyze the sn-2 ester bond in phospholipids, releasing free
fatty acids and lysophospholipids. PLA2s are important in the
signaling of several cellular processes and are known to play a
significant role in inflammation. Studies also show that PLA2s
are modulators of drug-, chemical-, and ischemia/reperfusioninduced cellular injury. The role of PLA2s in apoptosis and
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Cummings et al.
Vol. 294
that arachidonic acid is incorporated into are not usually
determined (e.g., plasmenylcholine, phosphatidylcholine,
and alkylacyl glycerophosphorylcholine). This should be determined under control conditions to determine whether arachidonic acid incorporation has reached equilibrium and to
determine whether the labeled arachidonic acid is preferentially incorporated into one specific phospholipid pool. Finally, care should be taken to ensure that arachidonic acid
release occurs before increases in markers of cell death.
Direct measurement of PLA2 activity using synthetic phospholipid substrates that are also the endogenous phospholipids for PLA2-catalyzed hydrolysis can alleviate some of the
above problems. Although some of these substrates are available commercially, many are not. However, they can be synthesized, and PLA2 activity measurements obtained using
these substrates can be determined in subcellular fractions.
Using plasmenylcholine, phosphatidylcholine, and alkylacyl
glycerophosphorylcholine substrates, it is possible to determine whether PLA2 activity is influenced by the covalent
linkage of the sn-1 fatty acid. The selectivity of PLA2 for
arachidonylated substrates can be determined using substrates with oleic acid or arachidonic acid at the sn-2 position.
Proper use of these substrates may indicate if a PLA2 isoform
has a preference for a specific phospholipid (i.e., those with
covalent linkages at the sn-1 or sn-2 position). When studying the effect of PLA2 inhibition on cellular injury and death,
careful selection of multiple inhibitors is key with special
attention paid to experiments verifying that PLA2 activity is
being inhibited. Activity should be measured using a method
that relies on the use of endogenous substrates of PLA2 (i.e.,
plasmenylcholine, phosphatidylcholine, alkylacyl glycerophosphorylcholine).
Molecular Modulation of PLA2 Activity
A number of studies have used advances in molecular
biology to overcome some of the problems involved with
chemical inhibitors of PLA2. For example, cell lines that
overexpress certain types of PLA2 isoforms, antisense oligonucleotides that decrease specific PLA2 isoforms, and transgenic mice that are deficient or “overexpress” PLA2 isoforms
have been developed. Overexpression of PLA2 isoforms allows one to study the effect of increased activity of a specific
PLA2 isoform. Sapirstein et al. (1996) overexpressed human
cPLA2 and sPLA2 in LLC-PK1 cells and used these cells to
study the role of these PLA2 isoforms in oxidant-induced cell
injury (see the section on oncosis).
Antisense oligonucleotide technology has been used to decrease specific PLA2 isoforms. Locati et al., (1996) used an
antisense oligonucleotide directed against codons 4 through 9
of human cPLA2 to produce a 57% decrease in cPLA2 protein
levels in cultures of human monocytes. When these cells were
exposed to monocyte chemotactic protein, arachidonate release was 19% of cells treated with the same oligonucleotide
TABLE 1
Classifications of PLA2 isoforms
Type
a
cPLA2
sPLA2
iPLA2
a
b
Groups
Molecular Mass
Location
Ca2⫹ Requirements
IVA–B
IA–B, IIA–C, III, V, IX, X
IVC, VI, VIIB, VIII
85–100 kDa
14–18 kDab
29–85 kDa
Cytosol
Secreted
Cytosol and membrane
Micromolar
Millimolar
None
Ca2⫹ is not needed for the catalytic activity of cPLA2 but rather to facilitate translocation to membrane phospholipids.
A sPLA2 isolated from human plasma has a molecular size of 45 kDa (Balsinde et al., 1999).
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ment of inflammation and cell injury, and as a tool to investigate the role of PLA2s in physiological functions and in
cellular injury and death. As the number and roles of PLA2s
have increased in recent years the need for isoform-selective
inhibitors has become critical. Many of the early inhibitors of
PLA2 (e.g., dibucaine, mepacrine) were neither isoform-specific nor potent. More recently, several new PLA2 inhibitors
have been developed. Table 2 lists various PLA2 inhibitors
including the isoforms they inhibit and the type of inhibition.
A review by Glaser (1995) discusses the kinetics of PLA2
inhibition and lists several criteria for potential PLA2 inhibitors.
Many PLA2 inhibitors originally thought to be selective for
a specific PLA2 are now known to inhibit other isoforms. For
example, methyl arachidonyl fluorophosphonate, an inhibitor of cPLA2 with an IC50 of ⬃0.5 ␮M for purified cPLA2, also
inhibits iPLA2 purified from murine macrophage-like
P388D1 cells, exhibiting half-maximal inhibition at 0.5 ␮M
(Lio et al., 1996). In fact, most cPLA2 inhibitors also inhibit
iPLA2 (Balsinde et al., 1999). One protocol that has been used
to overcome this problem is to use a combination of inhibitors
to differentiate between PLA2 isoforms. For example, if a
process is blocked by methyl arachidonyl fluorophosphonate
and arachidonyl trifluoromethyl ketone (AACOCF3) but not
bromoenol lactone (BEL, a specific inhibitor of iPLA2) then it
is likely that cPLA2 but not iPLA2 isoforms are involved in
the process (Balsinde et al., 1999). The reason most inhibitors of cPLA2 can inhibit iPLA2 is both isoforms have a serine
in their active site and these inhibitors contain a serinereactive group. In contrast, BEL does not react with this
serine group but interacts with other amino acids in the
active site of iPLA2. Unfortunately, many studies have used
these inhibitors to examine the role of PLA2 isoforms without
verifying that selective inhibition of PLA2 activity occurs or
have just used one inhibitor. Thus, the use of chemical inhibitors of PLA2 requires careful characterization to ensure that
selective inhibition occurs in a given model.
Numerous studies have used arachidonic acid release as a
marker for PLA2 activity. Typically this method involves
prelabeling cells with [3H]arachidonic acid followed by the
measurement of [3H] release from cells upon exposure to an
agonist or toxicant. There are several problems with this
method. First, arachidonic acid release is an indirect measure of PLA2 activity. Second, multiple pathways may cause
free arachidonate production, resulting in an overestimation
of PLA2 activity. Arachidonic acid release may be due to
activation of intracellular phospholipid metabolism independent of PLA2, such as arachidonoyl-CoA synthetase, CoAdependent acyltransferase, and CoA-independent transacylase, and contribute to [3H] release (Lio et al., 1996).
Apoptotic bodies that are released as a result of apoptosis
also contain arachidonic acid and can contribute to [3H] release. Another potential problem is the phospholipid pools
2000
Phospholipase A2s in Cell Injury and Death
fluids and organs when compared with mice lacking group II
PLA2.
The use of over-expression, antisense oligonucleotides, and
knockout models should increase our knowledge of the roles
and mechanisms of specific PLA2 in cell injury and death.
Future efforts will undoubtedly focus on applying the technologies perfected with cPLA2 and sPLA2 to other isoforms to
elucidate the overlapping roles of these enzymes in mediating both oncosis and apoptosis.
The Role of PLA2 Isoforms in Oncosis
Although the role of PLA2 in oncosis (cell death characterized by cell and organelle swelling, ATP depletion, increased
plasma membrane permeability, release of macromolecules,
and inflammation) has been studied over the past 20 years,
much remains unknown. It was originally postulated that
during oncosis, PLA2 activity increased, accelerated membrane phospholipid hydrolysis, and, in turn, increased
plasma membrane permeability and cell lysis (Sevanian,
1988). Typically, experiments demonstrated that the PLA2
inhibitors, mepacrine or dibucaine, decreased cell lysis following an injurious insult. Unfortunately, in many cases the
investigators did not document increases in PLA2 activity or
verify that PLA2 inhibitors were indeed inhibiting PLA2.
Furthermore, because the number of PLA2 isoforms known
and their characteristics were limited, the experiments were
crude in nature. Still some useful information can be gained
from these studies. For example, dibucaine and mepacrine
decreased the toxicity of tert-butyl hydroperoxide (a model
oxidant), and the reduced toxicity correlated with their inhibition of arachidonic acid release (Schnellmann et al., 1994).
The PLA2 inhibitors did not decrease the ability of antimycin
A (a mitochondrial inhibitor) nor carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative
phosphorylation) to cause toxicity. Thus, even though specific
PLA2 inhibitors were not used in this study it was determined that the role of PLA2 in renal cell oncosis depends on
the stimulus.
The development of more selective inhibitors has increased
our knowledge of the role of PLA2s in oncosis. For example,
TABLE 2
Inhibitors of commonly used PLA2 isoforms
Inhibitor
Isoform Specificity
Manoalide
p-Bromophenacyl bromide
3-(3-Acetamide-1-benzyl-2ethylindolyl-5-oxy) propane sulfonic
acid (LY311727)
Arachidonyl trifluoromethyl ketoneb
General
General
sPLA2
Low millimolar range
Low millimolar range
20–40 nMa
Irreversible
Irreversible
Competitive
Glaser, 1995; Balsinde et al., 1999
Glaser 1995; Balsinde et al., 1999
Balsinde et al., 1999
cPLA2
Low micromolar range
Reversible
iPLA2
cPLA2
1–10 ␮Mc
Low micromolar range
Low micromolar range
Riendeau et al., 1994; Ackermann et al.,
1995; Kohjimoto et al., 1999
Irreversible
Riendeau et al., 1994; Ackermann et al.,
1995
Irreversible
Hazen et al., 1991; Balsinde et al., 1996a
Tithof et al., 2000
Balsinde and Dennis, 1996b
Methyl arachidonyl fluorophosphonate
BEL
a
iPLA2
iPLA2
Reported IC50
0.5 ␮M
60 nM
1–20 ␮Me
8 ␮Mf
Type of Inhibition
Sources
d
Determined for IIA and V sPLA2 (Chen and Dennis, 1998).
Can also inhibit cyclooxygenase (Riendeau et al., 1994).
Concentrations used to inhibit oxalate-stimulated increases in cPLA2-mediated [3H]arachidonic acid release in Madin-Darby canine kidney cells.
d
Determined with iPLA2 purified from murine macrophage-like P388D1 cells (Balsinde et al., 1996b).
e
Concentrations used to inhibit [3H]arachidonic acid release in rat neutrophils stimulated with dieldrin and linadane.
f
Reported IC50 for iPLA2 in P388D1 macrophage-like cells.
b
c
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with four mismatched bases or an unrelated antisense oligonucleotide. Woo et al. (2000) also showed that the same
antisense oligonucleotide to cPLA2 inhibited Rac-mediated
c-Jun N-terminal kinase activation in Rat-2 fibroblast cells if
it is cotransfected with the Rac plasmid. Interestingly, the
effect of the antisense oligonucleotide was similar to cells
treated with AACOCF3. Thus, antisense oligonucleotide
technology provides an additional mechanism by which levels of a specific PLA2 isoform can be decreased to explore its
role in cell injury and death.
“Knockout” mice that lack specific PLA2 isoforms are another model that can be used to study specific PLA2 functions. A homozygous null (cPLA2⫺/⫺) mouse has been produced that develops normally and has weight gain and life
span equal to that of wild type mice (cPLA2⫹/⫹) (Bonventre,
1999). The cPLA2⫺/⫺ mice did display abnormal reproduction, resulting in small litters and a high death rate. Removal
of offspring after 18 days of pregnancy resulted in normal
mice, indicating that cPLA2 plays a critical role in parturition. This model has been used to study the role of cPLA2 in
ischemic injury to the kidney, brain, and other organs (Bonventre, 1999). In general, studies demonstrate that deletion
of cPLA2 results in decreased postischemic injury. Deletion of
cPLA2 also protects against 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP)-induced injury in the brain as
cPLA2⫺/⫺ mice were able to resist the dopamine-depleting
effects of MPTP compared to wild type mice (Klivenyi et al.,
1998). The decrease in injury seen in these studies is hypothesized to result from decreased production of lipid mediators
of injury such as eicosanoids, lysophospholipids, and oxidative species that are derived from metabolism of arachidonic
acid by cyclooxygenases and lipooxygenases.
In contrast to knockout mice, investigators have over expressed specific PLA2 isoforms in mice to study its role in
mediating injury. Laine et al. (2000) over-expressed group II
PLA2 (sPLA2) in sPLA2 deficient mice and reported that mice
expressing sPLA2 were more resistant to Staphylococcus aureus and Escherichia coli infection than sPLA2-deficient
mice. Specifically, mice over-expressing group II PLA2 had
lower rates of mortality and less bacterial growth in body
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Cummings et al.
and during toxic injury would greatly aid in elucidating the
site of action of cPLA2.
Sevanian (1988) proposed that cellular insults result in
prolonged activation of PLA2 isoforms (Fig. 1). Consequently,
many of the products formed by hydrolysis of phospholipids
(free fatty acids, lysophospholipids) may decrease membrane
integrity by acting as detergents and altering membrane
fluidity. In addition, the release of membrane phospholipids
as a result of oxidant-induced lipid peroxidation and/or PLA2
metabolism may decrease membrane integrity independent
of free fatty acids or lysophospholipids. The free fatty acid
and lysophospholipids may serve as precursors for biologically active metabolites and promote inflammation and may
further increase the activity of PLA2s by themselves (see
below) (McHowat et al., 1993). Alternatively, or perhaps in
tandem with released membrane phospholipids, increased
cytosolic-free Ca2⫹ can increase cPLA2 activity. If the accumulation of free fatty acids and lysophospholipids or the loss
of membrane phospholipids contributes directly to cellular
injury and death, then inhibition of PLA2s would be protective.
In contrast to the above scenario it has been proposed that
PLA2 isoforms may serve to decrease free radical-induced
membrane phospholipid damage by hydrolyzing oxidized
phospholipids from the membrane. The hydrolysis of oxidized
phospholipids by PLA2s facilitates the removal of these damaged lipids from the cells and decreases toxicity (Fig. 1)
(Salgo et al., 1993). Furthermore, subsequent reacylation (by
enzymes such as CoA-dependent acyltransferase, and CoAindependent transacylase) of the phospholipids results in the
return of normal functions. This cycle is analogous to DNA
repair of damaged bases, but for membrane phospholipids. If
PLA2s that hydrolyze damaged phospholipids from membranes are inhibited then cellular injury would increase. In
support of this hypothesis, oxidized phospholipids are sub-
Fig. 1. Potential roles of PLA2s in cell
injury. 1, an injury stimulus may increase cytosolic free Ca2⫹ ([Ca2⫹]i), activate PLA2 or disrupt membrane
phospholipids directly; 2, increases in
[Ca2⫹]i facilitate the translocation of
cPLA2 to the cellular membrane
where it, as well as membrane bound
and other PLA2 isoforms, metabolizes
phospholipids to arachidonic acid and
a lysophospholipid. All of these processes can lead to decreased membrane phospholipids. 3, the injury
stimulus may also cause membrane
lipid peroxidation leading to the formation of oxidized arachidonylated
and other phospholipids; 4, PLA2 may
function to remove peroxidized phospholipids and minimize membrane
dysfunction; 5, the loss of membrane
phospholipids, increases in lysophosphatidyl lipids acting as detergents,
and lipid peroxidation may all decrease membrane integrity and/or
function.
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Kohjimoto et al. (1999) showed that preincubation of MadinDarby canine kidney cells with AACOCF3 (5–10 ␮M) significantly reduced the toxicity of oxalate. Arachidonic acid release occurred before cell death and was inhibited by the
cPLA2 inhibitor AACOCF3, but not inhibitors of sPLA2 (oleyloxyethyl phosphorylcholine; 20 ␮M) and iPLA2 (BEL; 10
␮M). Thus, this study used multiple inhibitors of PLA2 and
the measurement of PLA2 activity (albeit, an indirect measure) to suggest that cPLA2 is involved in renal cell oncosis.
Sapirstein et al. (1996) showed that cPLA2 was involved in
oxidant-induced oncosis by over-expressing cPLA2 or sPLA2
in LLC-PK1 cells and demonstrating that cells over-expressing cPLA2 were more susceptible to H2O2 toxicity, whereas
over-expression of sPLA2 did not increase H2O2 toxicity.
Briefly, cPLA2 and sPLA2 were over-expressed in LLC-PK1
cells, and activity was determined by measurement of both
1-steroyl-2-[1-14C]arachidonyl phosphatidylcholine as a substrate in vitro and release of [3H]arachidonic acid from cells.
It was shown that over-expression of cPLA2, but not sPLA2,
increased H2O2 toxicity. The increase in H2O2 toxicity was
not due to decreases in the activity of the antioxidant defense
enzymes, superoxide dismutase, catalase, or glutathione peroxidase. Interestingly, chelation of cytosolic-free Ca2⫹ protected cells from H2O2 toxicity, suggesting a key role for Ca2⫹
in the mediation of cPLA2-mediated oncosis in renal cells.
Data from many studies implicate cPLA2 as an important
mediator of oxidant damage in cells; however, the exact
mechanism of cPLA2-mediated cellular injury has yet to be
determined. Sapirstein et al. (1996) hypothesized that oxidant-induced damage may direct cPLA2 activity to a specific
subcellular location where it produces injury. An oxidantinduced rise in cytosolic free Ca2⫹ and, in turn, Ca2⫹ binding
to the Ca2⫹-lipid-binding domain of cPLA2 would initiate
translocation to intracellular membranes. Experiments to
determine alterations in cPLA2 cellular localization before
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2000
Role of PLA2 Isoforms in Apoptosis
In contrast to oncosis, apoptosis is characterized by cell
shrinkage, chromatin condensation, plasma membrane bud-
797
ding, caspase activation, and is ATP-dependent. Similar to
the role of PLA2s in oncosis, the role of PLA2s in apoptosis
appears to be dependent on the stimulus of apoptosis and the
cell type being targeted. For example, Atsumi et al. (1998)
suggested that cPLA2 does not have a role in Fas-induced
apoptosis as caspase-3 cleaved and inactivated cPLA2 in human leukemic U937 cells exposed to Fas (Fig. 2). Enari et al.
(1996) supported this hypothesis by demonstrating that
cPLA2 was not needed for Fas-induced apoptosis in mouse
L929 cells expressing human Fas. Finally, cPLA2 has been
shown to be a substrate for human caspase-1 and -8, as both
caspases degraded and inactivated cPLA2 in vitro (AdamKlages et al., 1998; Luschen et al., 1998). Thus, cPLA2 does
not appear to play a significant role in Fas-mediated apoptosis. Despite these studies several questions remain. For example, is cleavage and inactivation of cPLA2 by caspases a
required event for Fas-induced apoptosis or is cPLA2 inactivated to decrease the formation of proinflammatory prostanoids during apoptosis?
Although cPLA2 is not needed for Fas-induced apoptosis,
several studies report that iPLA2 mediates several signal
transduction processes associated with apoptosis such as
Fas-induced arachidonic acid release and membrane remodeling (Fig. 2) (Balsinde and Dennis, 1996; Atsumi et al.,
1998). One study showed that Fas-induced arachidonic acid
release in U937 cells undergoing apoptosis is mediated by
iPLA2 and inhibition of iPLA2 decreased Fas-induced cell
death (Atsumi et al., 1998). Furthermore, iPLA2 levels did
not decrease while cPLA2 inactivation occurred via caspasemediated cleavage. Other functions for iPLA2 in apoptosis
may include the generation of lipid signaling molecules that
regulate ion channel activity (Ma et al., 1997). Although
many of these processes have been proposed as key events in
apoptosis, very little work has been done correlating these
events to the genesis of apoptosis and the activity of iPLA2.
In contrast to the hypothesis that cPLA2 does not play a
role in apoptosis, several studies have reported that cPLA2 is
Fig. 2. Differences in the roles of
PLA2s in TNF␣ and Fas-induced
apoptosis. It has been proposed
that activation of caspases (e.g.,
caspase-3) results in the activation of cPLA2 in TNF␣-induced
apoptosis. cPLA2 may be needed
for the activation of downstream
caspases, arachidonic acid production, and the progression of
TNF␣-induced apoptosis. It is
not known if cPLA2-mediated
activation of caspases is a direct
result of arachidonic acid production or lysophospholipids and
other metabolites. The role of
iPLA2 during TNF␣-induced apoptosis is not known. Fas-induced apoptosis does not require
cPLA2 activity and cPLA2 is degraded by caspase-3. Caspase-1
and -8 can also degrade and inactivate cPLA2 in vitro. Inhibition of iPLA2 during Fas-induced
apoptosis
decreased
arachidonic acid release and reduced cell death, but the mechanism of such events and the role
of caspases in these processes
are not known.
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strates for cPLA2 and can decrease the Ca2⫹ requirement for
purified cPLA2, thereby enhancing its activity (Rashba-Step
et al., 1997). Key determinants of whether PLA2s are increasing or decreasing toxicity is the location of the membrane
phospholipids being released, the type of phospholipid (phosphatidylcholine, phosphatidyethanolamine, phosphatidylserine, etc.), the PLA2 isoforms responsible, and the stimulus of
injury. For oxidative injury, incurred by H2O2 or other toxicants, cPLA2 activity appears to increase toxicity and most
studies report a central role for Ca2⫹ in mediating oncosis.
Very little work has examined the role of iPLA2 in oncosis.
Many studies have demonstrated that sPLA2s in snake or
bee venom are responsible for cellular injury. There is a large
amount of evidence to suggest a role for inflammation, but
studies have shown that the toxicity of sPLA2s may be independent of its ability to produce arachidonic acid. Furthermore, investigators have shown that sPLA2 requires specific
membrane phospholipids to mediate cellular injury. For example, recombinant human and venom-derived sPLA2 are
indirectly cytolytic to human erythrocytes, erythroleukemia,
and U937 cells in a manner dependent on the presence of
liposomal phospholipids (phosphatidylcholine and phosphatidylethanolamine) (Vadas, 1997). Interestingly, human
sPLA2 was cytolytic only in the presence of phosphatidylethanolamine. Thus, phospholipid metabolites of PLA2 other
than arachidonic acid can be mediators of cellular injury (i.e.,
lysophospholipids). However, sPLA2 is believed to be responsible for the bulk of arachidonic acid released into the extracellular milieu as a result of its extracellular location/action
(Balsinde and Dennis, 1996b). This is also believed to occur
because of the increase in oxidized phospholipids being translocated to the extracellular surface of the membrane, secondary to cellular injury, which make them more accessible to
sPLA2 (Balsinde and Dennis, 1996b).
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Activation of PLA2
A key determinant of the role of PLA2s in oncosis and
apoptosis is the mechanism of PLA2 regulation/activation
during these processes. It is likely that events that cause
oncosis elicit a set of signals that activate PLA2s differentially than the signals elicited when apoptosis is induced.
Increased PLA2 activity can be caused by agents that produce
alterations in membrane phospholipids that result in the
exposure of preferential lipid substrates (Sevanian, 1988;
Salgo et al., 1993; Sapirstein et al., 1996). For example,
oxidative stress may lead to the rearrangement of membrane
phospholipids such that the sn-2 fatty acids are more accessible to PLA2 (Balsinde and Dennis, 1996b). Furthermore,
excessive toxicant and/or oxidant injury may result in the
release of intact membrane phospholipids themselves exposing the sn-2 ester bond. As the result of either of the above
processes, PLA2 activity increases and any agent that inhibits the access of PLA2 to either exposed or released mem-
brane phospholipids would decrease PLA2 activity. In support of this hypothesis, agents that bind to phospholipids
such as lipocortins and annexins can inhibit the ability of
PLA2 to hydrolyze phospholipids (Buckland and Wilton,
1998).
Alterations in cellular membrane phospholipid integrity
may be one process that results in modifications in PLA2
activity but several studies have reported that increased
PLA2 activity occurs independently of significant phospholipid alterations. One hypothesis is that cellular injury may
cause a rise in intracellular Ca2⫹, activation of protein kinase C (PKC) and PKC-mediated activation of PLA2. In support of this hypothesis, Chen et al. (1999) demonstrated a
link between increases in Ca2⫹, PKC-⑀ activation, and the
activation of cPLA2, and Akiba et al. (1999) reported that
zymosan stimulated iPLA2-mediated arachidonic acid release via a PKC-dependent mechanism. In vitro, both PKC
and protein kinase A (PKA) can phosphorylate cPLA2 but the
phosphorylation did not lead to a corresponding increase in
activation (Leslie, 1997). In vivo it is not known whether PKC
and PKA regulate cPLA2 by direct phosphorylation.
The signaling cascade involved in activation of cPLA2 by
mitogen-activated protein kinase (MAPK) has been studied
also. Nemenoff et al. (1993) demonstrated that p42 MAPK
phosphorylated cPLA2 and increased its activity in vitro, and
later studies demonstrated this event in cell lines (Leslie,
1997). The phosphorylation of cPLA2, at serine 505, occurs
before the increases in intracellular Ca2⫹ that facilitate the
binding of the lipid-binding domain of cPLA2 to phospholipids, promoting its translocation to cellular membranes and
arachidonic acid release. Recently, a negative feedback loop
for cPLA2 activation by MAPK has been proposed (Xing et al.,
1999). In this model, purinergic receptor activation results in
MAPK activation followed by activation of cPLA2 and an
increase in arachidonic acid. The increase in cellular arachidonate levels is followed by an increase in prostaglandin E2,
which in turns activates adenyl cyclase and PKA. Activation
of these enzymes decreases MAPK and cPLA2 activity. This
feedback loop only was seen with an agonist of purinergic
receptors and was not seen in adrenergic receptor activation
of MAPK, suggesting that cPLA2 regulation can occur by
multiple processes that appear to depend on the stimuli.
cPLA2 can be activated by pathways independent of MAPK
also as studies have shown that okadaic acid can increase
cPLA2 activity in a Ca2⫹-independent manner and induce
phosphorylation of cPLA2 at serine 727 rather than serine
505 (de Carvalho et al., 1996). Serine 727 is not within a
consensus site of MAPK but appears to be a site for a basotrophic kinase (Leslie, 1997).
If PLA2 activation in a given model depends on PKC, PKA,
cAMP, or MAPK activation then inhibition of these compounds may inhibit PLA2 isoforms during cellular injury.
Understanding of the signaling pathways involved in the
activation/deactivation of PLA2 during cellular injury will
point to key events that can be used to prevent the cellular
injury. Furthermore, to date, there is limited information
regarding the regulation of iPLA2 or sPLA2 by these pathways.
Future Directions
The role of PLA2 in cell injury and the potential benefits of
pharmacological inhibition have been studied for over 20
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
needed for apoptosis and that inhibition of cPLA2 decreased
apoptosis. cPLA2 is required and appears to be the ratelimiting step in tumor necrosis factor ␣ (TNF␣)-induced apoptosis in several cell types (Enari et al., 1996; Ilic et al.,
1998). In this case, cPLA2 is activated by caspase-3 and is
needed to activate downstream caspases (Wissing et al.,
1997), but the mechanism of action of PLA2 on caspases
downstream of caspase-3 is not known (Fig. 2). These studies,
as well as those listed above, demonstrate that Fas and
TNF␣ cause apoptosis by different pathways and that cPLA2
has distinctly different roles in each pathway (Fig. 2). In a
system where apoptosis was caused by removal of extracellular matrix survival factors (fibronectin) or focal adhesion
kinases, inhibition of cPLA2 with AACOCF3 significantly
improved the survival of cell lines undergoing apoptosis (Ilic
et al., 1998). Interestingly, the activation of caspase and
protein kinase C␭ in this model were thought to occur downstream of cPLA2 activation. Although cPLA2 inhibition has
been shown to decrease apoptosis, several studies demonstrate that inhibition of PLA2 increases apoptosis. For example, Miao et al. (1997) reported that apoptosis in human
umbilical vein endothelial cells induced by deprivation of
fibroblast growth factor and serum was increased by preincubation of the cultures with PLA2 inhibitors (manoalide,
3-(4-octadecyl)benzoylacrylic acid, and oleyloxyethylphosphorylcholine).
Whether cPLA or iPLA2 has no role in apoptosis, is required for apoptosis, or inhibit apoptosis depends on the
stimuli of injury and the model system studied. To increase
our knowledge of the role of these PLA2s in apoptosis, a
precise examination of their activity using endogenous substrates and correlation to the activation of the apoptotic
cascade (caspase-9 activation, cytochrome c release, etc.) is
needed. Balsinde and Dennis (1996b) have hypothesized that
iPLA2 and cPLA2 have distinct roles, with iPLA2 responsible
for membrane remodeling (maintenance of membrane integrity and phospholipid content) and cPLA2 responsible for
arachidonic acid production. Similarly, PLA2 localization
also may be a key determinant in the role of PLA2 isoforms in
cellular injury. For example, membrane-bound PLA2 isoforms may serve to regulate membrane fluidity/integrity during apoptosis, although cPLA2 isoforms may respond to
fluxes in Ca2⫹ and the release of membrane phospholipids.
Vol. 294
2000
years. Within the last 5 years additional PLA2 isoforms have
been identified and characterized, including the discovery of
catalytically different splice variants of iPLA2, but the role of
these new isoforms in cell injury needs to be explored. The
use of over-expression and knockout mice models and antisense technology has increased our knowledge of these enzymes but these advances need to be expanded to encompass
more PLA2 isoforms and cell injury studies. This information
along with transgenic mice models can be used to design
therapeutic treatments for organ (e.g., brain, kidney) injury,
develop more potent anti-inflammatory inhibitors, and study
the mechanisms of apoptosis and oncosis. Careful analysis of
PLA2 isoforms in general and in specific models must be
considered at every step.
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