From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
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ABSTRACT |
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Fas-mediated apoptosis of human leukemic U937
cells was accompanied by increased arachidonic acid (AA) and oleic acid
release from membrane glycerophospholipids, indicating phospholipase
A2 (PLA2) activation. During apoptosis,
type IV cytosolic PLA2 (cPLA2), a
PLA2 isozyme with an apparent molecular mass of 110 kDa
critical for stimulus-coupled AA release, was converted to a 78-kDa
fragment with concomitant loss of catalytic activity. Cleavage of
cPLA2 correlated with increased caspase-3-like protease
activity in apoptotic cells and was abrogated by a caspase-3 inhibitor.
A mutant cPLA2 protein in which Asp522 was
replaced by Asn, which aligns with the consensus sequence of the
caspase-3 cleavage site (DXXDX), was
resistant to apo-ptosis-associated proteolysis. Moreover, a
COOH-terminal deletion mutant of cPLA2 truncated at
Asp522 comigrated with the 78-kDa fragment and exhibited no
enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage
eventually leads to destruction of a catalytic triad essential for
cPLA2 activity, thereby terminating its AA-releasing
function. In contrast, the activity of type VI
Ca2+-independent PLA2 (iPLA2), a
PLA2 isozyme implicated in phospholipid remodeling,
remained intact during apoptosis. Inhibitors of iPLA2, but
neither cPLA2 nor secretory PLA2 inhibitors,
suppressed AA release markedly and, importantly, delayed cell death
induced by Fas. Therefore, we conclude that iPLA2-mediated
fatty acid release is facilitated in Fas-stimulated cells and plays a
modifying although not essential role in the apoptotic cell death
process.
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INTRODUCTION |
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Release of free fatty acids from glycerophospholipids, a major component of cell membranes, is crucial for various cellular responses, such as signal transduction and membrane remodeling and is tightly regulated by a diverse family of phospholipase A2 (PLA2)1 enzymes. Type IV cytosolic PLA2 (cPLA2), which exhibits strict substrate specificity for arachidonic acid (AA)-containing phospholipids in the presence of submicromolar concentrations of Ca2+, is a key enzyme that triggers stimulus-initiated AA metabolism, leading to production of bioactive lipid mediators in activated cells (1, 2). Various Ca2+-mobilizing agonists and proinflammatory cytokines induce cPLA2 activation for immediate and delayed eicosanoid biosynthesis, respectively, through mechanisms involving Ca2+-dependent translocation to the perinuclear and endoplasmic reticular membranes and phosphorylation by kinases belonging to the mitogen-activated protein kinase (MAPK) family (3-7). Evidence is also accumulating that certain phases of stimulus-initiated AA release are regulated by types IIA or V secretory PLA2 (sPLA2) isozymes, which require millimolar concentrations of Ca2+ for catalytic activity (8-12). In contrast, type VI Ca2+-independent PLA2 (iPLA2) has been proposed to participate in fatty acid release associated with phospholipid remodeling and to play a minimal role in signal transduction (13-15).
The programmed cell death pathway called apoptosis plays a fundamental
role in tissue homeostasis, development, and host defense. The
discovery of increasing numbers of death factors (e.g. Fas ligand and tumor necrosis factor (TNF) ), their receptors, adapter molecules, and caspases have provided new insight into the molecular mechanisms leading to apoptosis that are highly conserved in eukaryotic cells (16, 17). Following ligation of these receptors by their cognate
ligands, several cytoplasmic signal-transducing adapters are recruited
to the receptors to form multimeric complexes, thereby leading to
either apoptosis or cellular activation, depending on the adapters
involved (18, 19). In the apoptotic pathway, these adapter molecules in
turn recruit particular caspases (20-22), which trigger the protease
cascade where multiple caspases are sequentially activated as a
consequence of their proteolytic processing at a specific Asp residue,
allowing them to become self-activated and activate one another.
Activated caspases then cleave their respective substrates (23-30),
which are key regulatory and structural proteins such as protein
kinases and proteins involved in DNA repair and cytoskeleton integrity,
thereby contributing to the demise of the cell.
The changes in glycerophospholipid metabolism, unlike those in protein
and DNA levels, during apoptosis are rather obscure. Although the
involvement of particular PLA2 enzymes in stimulus-coupled eicosanoid biosynthesis has been studied extensively as discussed above, relatively little is known about their roles in fatty acid release associated with apoptotic signaling. Recently, we and others
showed that the plasma membrane phospholipids of apoptotic cells are
the preferred substrates for type IIA sPLA2 (31, 32), which
is induced by proinflammatory stimuli and has been implicated in the
pathogenesis of inflammation (10, 33). It has been speculated that the
AA thus liberated is taken up by surrounding live cells to be
metabolized to eicosanoids, representing a particular transcellular
pathway for AA metabolism that contributes to propagation of
inflammation. cPLA2 has also been implicated in AA release during certain cell death processes, such as TNF-induced apoptosis (34-36) and hydroperoxide-induced cytotoxicity (37). In these studies,
the use of cPLA2 inhibitors, overexpression, and antisense technologies revealed significant correlations between the
cPLA2 expression level, amounts of AA released, and cell
death. However, the role of cPLA2 is uncertain in
Fas-mediated apoptosis (38).
In order to explore the role of particular PLA2 isozymes in fatty acid release and glycerophospholipid metabolism alterations associated with the apoptotic pathway, we chose the Fas system, which strongly promotes apoptosis but barely elicits cell activation signals. We now provide evidence that Fas-mediated apoptosis of human leukemic U937 cells is accompanied by gradual fatty acid release, which appears to be mediated by iPLA2 but not by cPLA2 or sPLA2. During apoptosis, cPLA2 is inactivated by caspase-3-dependent proteolytic cleavage at Asp522. Inhibition of iPLA2-evoked fatty acid release results in slight but significant retardation of cell death, suggesting that disturbed phospholipid turnover renders cells more susceptible to Fas-mediated apoptotic signaling.
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EXPERIMENTAL PROCEDURES |
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Materials--
Mouse cPLA2 cDNA and a rabbit
antiserum against human cPLA2 were provided by Drs. M. Tsujimoto (RIKEN Institute) and J. D. Clark (Genetics Institute),
respectively. The agonistic anti-Fas antibody (CH-11) (39) was
purchased from MBL. The cPLA2 inhibitor arachidonoyl
trifluoromethyl ketone (AACOCF3) (40) was purchased from
Calbiochem. Methyl arachidonylfluorophosphonate (MAFP), which inhibits
both cPLA2 and iPLA2 (14), and the
iPLA2 inhibitor bromoenol lactone (13-15) were purchased
from Cayman Chemical. The type IIA sPLA2 inhibitor LY311727
(41) was donated by Dr. R. M. Kramer (Lilly Research). Two caspase
inhibitors (Ac-YVAD-CHO and Ac-DEVD-CHO) and chymostatin were obtained
from the Peptide Institute, and caspase substrates were obtained from
Takara Biomedicals. LipofectAMINE PLUS reagent, Opti-MEM medium, and
TRIzol reagent were obtained from Life Technologies. Etoposide,
p-bromophenacyl bromide, 4',6'-diamidino-2-phenylindole
(DAPI), leupeptin, antipain, pepstatin, cytochalasin B,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and
N-benzoyl-arginine ethyl ester were purchased from Sigma. All the other reagents used were of analytical grade and purchased from
Wako, unless stated otherwise. The human monocytic leukemia cell lines
U937 and HL-60 and human embryonic kidney 293 cells (RIKEN Cell Bank)
were maintained in RPMI 1640 medium (Nissui Pharmaceutical)
supplemented with 10% (v/v) fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at
37 °C in humidified air with 5% CO2.
Fatty Acid Release from U937 Cells Undergoing Apoptosis--
In
order to investigate AA and oleic acid release, U937 cells were
preincubated with 0.1 and 0.5 µCi/ml [3H]AA and
[3H]oleic acid (NEN Life Science Products), respectively,
for 24 h. Then the cells were washed three times, resuspended in
culture medium to produce 1 × 107 cells/ml, and
incubated for various periods with or without the anti-Fas antibody. In
the experiments to determine the effect of etoposide treatment, the
cells were incubated with etoposide for 1 h, washed three times,
and then incubated for a further 10 h in culture medium without
etoposide. In some experiments, various PLA2 inhibitors
were added to the cells during incubation. The process of cell death
was monitored by observing the morphological changes, assessing the
cell viability by trypan blue dye exclusion, and quantifying DNA
fragmentation fluorometrically using DAPI as described previously (42).
The free 3H-labeled fatty acids released were extracted by
the method of Dole and Meinertz (43), and the associated radioactivity
was counted using a liquid -scintillation counter (Aloka). The
amount of each fatty acid released, expressed as a percentage, was
calculated using the formula [S/(S + P)] × 100, where S and P are the
radioactivities of equal portions of supernatant and cell pellet,
respectively.
Immunoblotting-- Cells were washed with phosphate-buffered saline and then lysed in phosphate-buffered saline containing 100 µM p-4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 µM iodoacetamide, 5 mM EDTA, 1 µM pepstatin, 1 mg/ml soybean trypsin inhibitor, and 100 µM leupeptin by sonication for 1 min with a Branson Sonifer (power 30, 50% pulse cycle). The samples (10 µg protein equivalents/lane) were subjected to 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electroblotted onto nitrocellulose membranes (Schleicher & Schuell), which were probed with the antibody against human cPLA2 and visualized with the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (8). The protein contents were quantified using a BCA protein assay kit (Pierce).
Assessment of PLA2 Activity-- In order to assess cPLA2 activity, cells were washed once with phosphate-buffered saline, suspended in buffer comprising 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl (Tris-buffered saline), and lysed by sonication as described above. The resulting lysates were incubated in 250 µl of buffer comprising 100 mM Tris-HCl (pH 9.0), 4 mM CaCl2, and 2 µM 1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine (NEN Life Science Products) as the substrate at 37 °C for 30 min.
In order to assess iPLA2 activity, the lysates, prepared in 10 mM HEPES (pH 7.5) containing 1 mM EDTA, 1 mM dithiothreitol, and 0.34 M sucrose, were incubated in 250 µl of buffer comprising 100 mM HEPES (pH 7.5), 5 mM EDTA, 0.4 mM Triton X-100, 0.1 mM ATP, and 10 µM 1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine at 40 °C for 30 min (13, 15). The [14C]AA released was extracted by the method of Dole and Meinertz (43), and the associated radioactivity was counted.Thin Layer Chromatography--
The total lipids were extracted
from [3H]AA-labeled cells and their supernatants by the
method of Bligh and Dyer (44) and developed by two-dimensional thin
layer chromatography on silica 60 gel plates (Merck), according to a
method described previously with a slight modification (31). The first
solvent system consisted of chloroform/methanol/acetic acid/water
(65/25/4/2, v/v/v), and the second comprised chloroform/methanol/formic
acid (65/25/8.8, v/v/v). The zones on the silica gel corresponding to
AA and phospholipids were identified by comparison with the mobilities
of authentic standards and visualized with iodine vapor. Each zone was
scraped into a vial, and its radioactivity was counted using a liquid -scintillation counter. To separate free fatty acids and other neutral lipids, including triacylglyceride, the neutral lipid fraction
was developed further on fresh plates with a solvent system of
hexane/ether/acetic acid (80/30/1, v/v/v).
Preparation of Mouse Recombinant
cPLA2--
Sf9 cells (Invitrogen) were transfected
with mouse cPLA2 cDNA that had been subcloned into the
pVL1392 baculovirus transfection vector (Pharmingen), as described
previously (8). The cells were harvested, suspended in a buffer
comprising 250 mM sucrose, 10 mM Tris-HCl (pH
7.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-benzoyl-arginine ethyl
ester, 10 µg/ml leupeptin, and 10 µg/ml antipain, and disrupted by
sonication for 1 min, as described above. The lysates were centrifuged
at 100,000 × g for 1 h at 4 °C, and the
resulting supernatants were applied to a DEAE-Sephacel column (Amersham
Pharmacia Biotech) that had been pre-equilibrated with Tris-buffered
saline. After washing the column with Tris-buffered saline, the bound
proteins were eluted with a linear NaCl gradient from 0.15 to 0.5 M in 10 mM Tris-HCl (pH 7.4). The eluted
fractions containing cPLA2 activity were pooled, applied to
a phenyl-Superose column (Amersham Pharmacia Biotech) that had been
pre-equilibrated with 10 mM Tris-HCl (pH 7.4) containing 1 M NaCl, and the bound cPLA2 was eluted stepwise with 10 mM Tris-HCl (pH 7.4).
Assessment of Caspase Activity-- Cells were washed with phosphate-buffered saline, resuspended (108 cells/ml) in an extraction buffer comprising 50 mM PIPES-NaOH (pH 7.0), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 20 µM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 50 µg/ml antipain, and 10 mg/ml chymostatin, and disrupted by freeze-thawing twice. Aliquots (10 µg of protein equivalents) were incubated with 1 µM Mca-YVADAPK(Dnp)-OH or Mca-DEVDAPK(Dnp)-OH, substrates for caspase-1 and caspase-3, respectively (45), in a buffer containing 100 mM HEPES (pH 7.5), 10% (w/v) sucrose, 0.1% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM dithiothreitol, and 0.1 mg/ml ovalbumin, at 30 °C for 30 min. Then the fluorescence of each cleaved substrate was determined using a spectrofluorometer (Hitachi) at excitation and emission wavelengths of 325 and 392 nm, respectively.
Construction of Mutant cPLA2-- Substitution of Asp522 of mouse cPLA2 with Asn (cPLA2-D522N) was performed by altering the codon GTC to GTT using the QuikChangeTM site-directed mutagenesis kit (Stratagene) with two synthetic oligonucleotides for the sense and antisense strands: 5'-C CTT CGA TGA CGA GCT CAA CGC AGC GGT AGC AG-3' and 5'-C TGC TAC CGC TGC GTT GAG CTC GTC ATC GAA GG-3', respectively (altered codons are underlined). Briefly, mouse cPLA2 cDNA subcloned into pBK-CMV (Stratagene) at the EcoRI site was denatured and annealed with the two oligonucleotide primers containing the desired mutation. After incubation with Pfu DNA polymerase followed by treatment with DpnI, the resulting mutated plasmid was transfected into Escherichia coli XL1-Blue supercompetent cells (Stratagene). A cPLA2 deletion mutant, cPLA2(1-522), was constructed by polymerase chain reaction amplification of the cPLA2 coding sequence with ex Taq polymerase (Takara) using the oligonucleotide pair 5'-ATG TCA TTT ATA GAT CCT TAC-3' and 5'-TCA GTC GAG CTC GTC ATC GAA-3'. The polymerase chain reaction product was ligated into pCRTM3.1 (Invitrogen) and was transfected into Top10F' supercompetent cells (Invitrogen). Colonies were picked up, and the plasmids were isolated and sequenced using a Taq cycle sequencing kit (Takara) and an auto-fluorometric DNA sequencer (DSQ-1000L, Shimadzu) to confirm the mutation.
In Vitro Transcription and Translation of cPLA2-- [35S]Methionine-labeled cPLA2 and its mutants were synthesized using a PROTEINscriptTM kit (Ambion). Briefly, plasmids containing mouse cPLA2, cPLA2-D522N, or cPLA2(1-522) cDNA were transcribed using RNA polymerase and then incubated with [35S]methionine (NEN Life Science Products) and rabbit reticulocyte lysate. The products were subjected to SDS-PAGE and visualized autoradiographically.
Cleavage of cPLA2 Protein by Apoptotic Cell Lysates in Vitro-- Aliquots of [35S]cPLA2 or baculovirus-derived recombinant cPLA2 proteins prepared as described above were incubated with the cytosolic fraction obtained from U937 cells that had been treated for 12 h with or without the anti-Fas antibody in the presence or absence of a caspase inhibitor (Ac-YVAD-CHO or Ac-DEVD-CHO) (45). The reaction products were analyzed by SDS-PAGE followed by autoradiography and immunoblotting, respectively.
Transfection of 293 Cells with cDNAs for Native and Mutant cPLA2-- The cDNAs for mouse cPLA2 and its mutants cPLA2-D522N and cPLA2(1-522) that had been subcloned into pBK-CMV and PCR 3.1TM, respectively, were each transfected into 293 cells using LipofectAMINE PLUS reagent according to the manufacturer's instructions as described previously (46). The cells were harvested 48 h after transfection, lysed, and subjected to immunoblotting and PLA2 assay.
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RESULTS |
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AA Release from Anti-Fas Antibody-treated U937 Cells-- Human monocytic leukemia U937 cells were prelabeled with [3H]AA for 24 h, washed, and then exposed to various concentrations of the agonistic anti-Fas antibody for various periods to assess the changes in the free [3H]AA levels (Fig. 1A). AA release by cells cultured without the anti-Fas antibody increased minimally over 24 h, whereas that by replicate cells treated with the anti-Fas antibody increased significantly after culture for 3-24 h in a concentration-dependent manner. AA release by U937 cells treated with 10, 50, and 100 ng/ml anti-Fas antibody for 24 h was about 3.5, 5.0, and 6.5 times higher, respectively, than that by the cells before anti-Fas antibody addition (Fig. 1A). AA release was almost parallel to the reductions in cell viability and DNA fragmentation, assessed by trypan blue dye exclusion and fluorometric quantification with DAPI (33), respectively (Fig. 1, B and C), as well as DNA rudder formation, assessed by agarose gel electrophoresis (data not shown).
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Proteolysis of cPLA2 in Cells Undergoing Apoptosis-- Initially, we thought that the increased Fas-mediated AA release from apoptotic U937 cells was due to cPLA2 activation. Unexpectedly, however, the cPLA2 catalytic activity toward exogenous substrate gradually declined during the Fas-mediated apoptotic process: the cPLA2 activities of cells after treatment with 10 and 100 ng/ml anti-Fas antibody for 12 h were about one-half and one-ninth, respectively, of that of untreated cells (Fig. 3A). The cPLA2 inhibitor AACOCF3, at 1 µM, abolished the cPLA2 activity of U937 cells, whereas [3H]AA release by apoptotic cells incubated with and without AACOCF3 at 10 µM, the concentration at which it inhibits cPLA2 but not other PLA2s (Ref. 13 and data not shown), did not differ significantly (Fig. 3B). Moreover, net amounts of [3H]oleic acid comparable with those of [3H]AA were released by anti-Fas antibody-treated cells prelabeled with these fatty acids (Fig. 3C), arguing against the AA selective property of cPLA2.
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Cleavage of Recombinant cPLA2 by Apoptotic Cell Lysates in a Cell-free System-- In order to verify that apoptotic but not live U937 cells possessed protease activity that cleaved cPLA2 to produce a 78-kDa fragment, recombinant cPLA2, either prepared by in vitro transcription/translation or expressed by baculovirus-infected Sf9 insect cells, was incubated with lysates of U937 cells treated with or without the anti-Fas antibody for 12 h (Fig. 5). Detailed time course studies revealed that a reduction in the amount of 110-kDa [35S]cPLA2 translated in vitro was detected as early as 30 min after adding apoptotic U937 cell lysate, which correlated with the time when the 78-kDa fragment appeared (Fig. 5A). Cleavage of [35S] cPLA2 also depended on the amount of apoptotic cell lysate (Fig. 5B). Thus, all the 110-kDa [35S]cPLA2 was converted to the 78-kDa cleavage product by incubation with adequate apoptotic cell lysate within 4 h. In contrast, [35S]cPLA2 remained intact when incubated without the lysate (Fig. 5A) and with the lysate of nonapoptotic cells (Fig. 5B). Similar results were obtained in the experiment using baculovirus-derived recombinant cPLA2 (Fig. 5C). After mixing with the lysate prepared from cells treated with the anti-Fas antibody for 12 h, in which endogenous intact cPLA2 had been already degraded to a 78-kDa fragment (Fig. 4A), immunoblotting showed that recombinant cPLA2 disappeared gradually (Fig. 5C).
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Involvement of Caspase-3-like Protease(s) in the Site-specific Cleavage of cPLA2-- Because the cleavage of cPLA2 and onset of apoptosis occurred in parallel, we reasoned that caspases might cleave cPLA2 proteolytically. In order to explore this, we monitored the changes in the caspase activities in U937 cells after anti-Fas antibody treatment. As shown in Fig. 6A, the caspase-3-like activity increased during culture with the anti-Fas antibody for 3-12 h, showing good correlations with the apoptotic process (Fig. 1, B and C), AA release (Fig. 1A), and cPLA2 cleavage (Fig. 4A). The caspase-1-like activity increased transiently during Fas stimulation for 1-6 h and was much weaker than the caspase-3-like activity throughout (data not shown). When [35S]cPLA2 was incubated with apoptotic cell lysate in the presence of the caspase-1 and caspase-3 inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO, respectively, Ac-DEVD-CHO, at a concentration as low as 0.1 µM, inhibited cleavage of [35S]cPLA2 markedly, whereas Ac-YVAD-CHO was at least 100-fold weaker (Fig. 6B).
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Possible Participation of iPLA2 in Fas-mediated AA Release and Apoptosis-- The results shown above support the hypothesis that cPLA2 is not involved in fatty acid release from cells undergoing Fas-mediated apoptosis. In an attempt to establish which PLA2 isozymes are involved in Fas-mediated AA release, the effects of various PLA2 inhibitors were tested. Because both AA and oleic acid were released from Fas-stimulated cells in parallel (Fig. 3C), we focused on sPLA2 and iPLA2, which exhibit no fatty acid preference in in vitro PLA2 assays (10, 14). No appreciable inhibition of [3H]AA release from anti-Fas antibody-treated U937 cells was observed with LY311727, a sPLA2-selective inhibitor (Fig. 8A), or p-bromophenacyl bromide, which inactivates all known sPLA2s by binding covalently to the catalytic center histidine (33) (data not shown). Consistent with these findings, sPLA2 expression was not detected in U937 cells (data not shown). In contrast, Fas-mediated [3H]AA release was significantly suppressed by MAFP, which inhibits both cPLA2 and iPLA2 (14), as well as by bromoenol lactone, a fairly selective iPLA2 inhibitor (13-15) (Fig. 8A). MAFP blocked [3H]AA release from phosphatidylethanolamine in apoptotic cells (Fig. 8B), indicating that this agent indeed blocked the PLA2-mediated reaction. The iPLA2 activities in lysates of U937 cells treated for 12 h with increasing concentrations of anti-Fas antibody were almost equal to that of control cells (Fig. 8C) and were suppressed by 85% by 1 µM MAFP. Verification that MAFP inhibits iPLA2-dependent [3H]AA release at the in vivo level is provided by the observation that increased spontaneous [3H]AA release by iPLA2-overexpressing 293 cells was attenuated by MAFP.2 These results imply that iPLA2 or a related MAFP-sensitive PLA2 is responsible for Fas-mediated AA release.
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DISCUSSION |
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In the present study, we attempted to establish the roles of particular PLA2 enzymes in AA release and changes in glycerophospholipid metabolism in cells stimulated with Fas, a receptor that transduces death signal. We found that treatment of U937 cells with an agonistic anti-Fas antibody enhanced delayed AA release, which occurred in parallel with the hallmarks of apoptosis. Unexpectedly, however, we found that cPLA2 lost its catalytic activity during apoptosis. Furthermore, the cPLA2 inhibitor AACOCF3 failed to suppress AA release, and oleic acid was also released in comparable amounts to AA, suggesting that Fas-induced AA release is largely cPLA2-independent. Detailed analysis revealed that intact cPLA2 disappeared as a result of proteolytic cleavage in apoptotic cells. Pharmacological studies suggest that iPLA2 is the most likely PLA2 isozyme responsible for liberation of fatty acids from apoptotic cell membranes.
Activation of cPLA2 by post-translational modifications,
such as Ca2+-dependent translocation and
MAPK-dependent phosphorylation, is essential for the
initiation of stimulus-initiated AA release (1-7). The results of this
study throw light on a novel post-translational modification of
cPLA2, proteolytic cleavage by caspase-3, which leads to
inactivation of cPLA2. We found that Fas-induced apoptosis of human leukemic cells led to cPLA2 cleavage, producing a
catalytically inactive fragment with an apparent molecular mass of 78 kDa. Several lines of evidence indicate that this cPLA2
cleavage is mediated by a caspase-3-like protease(s): (i)
cPLA2 cleavage in vivo and increased
caspase-3-like activity occurred in parallel; (ii) apoptosis induced by
the anti-cancer agent etoposide, which activates caspase-3 (49), was
also accompanied by cPLA2 cleavage; (iii) cPLA2
cleavage in vitro was suppressed by the caspase-3 inhibitor
Ac-DEVD-CHO; (iv) a mutant cPLA2 protein in which
Asp522 was replaced by Asn, which is located within the
putative consensus sequence of the caspase-3 cleavage site
(DELD522A), was not cleaved by the apoptotic cell
lysate; and (v) another cPLA2 mutant truncated at
Asp522, cPLA2(1-522), had the same apparent
molecular mass, 78 kDa, as the catalytically inactive fragment. Thus,
cPLA2 can be categorized as a physiological target of the
death effector proteases. Further support for this mechanism of
cPLA2 inactivation is provided by the finding that this
cleavage process destroyed a catalytic triad (Arg200,
Ser228, and Arg549) that is essential for
cPLA2 function (50).
Several workers have suggested that cPLA2 is involved in
apoptosis or cytotoxicity. Sapirstein et al. (37) reported
that cPLA2-overexpressing LLC-PK1 renal epithelial cells
were more susceptible to oxidant damage than their parental cells.
Voelkel-Johnson et al. (34, 51) showed, in studies employing
cPLA2 overexpression and cPLA2 antisense
introduction, that cPLA2 was both necessary for and
rate-limiting in TNF-induced apoptosis of various melanoma cells.
Hayakawa et al. (35) showed that the TNF
-resistant
subline of L929 cells, which showed reduced cPLA2
expression compared with the parental line, regained a TNF
-sensitive
phenotype after overexpression of transfected cPLA2.
Subsequently, cPLA2 was shown to participate in
TNF
-induced apoptosis of this cell line through the
ceramide-dependent pathway (52). However, our present
results suggest that AA release by cPLA2 is not necessary
for the apoptotic pathway and that the involvement of cPLA2
in apoptosis observed in previous studies (34-37, 51, 52) may reflect
secondary or cell type- or stimulus-specific events. While this study
was underway, Wissing et al. (36) reported that
cPLA2 was cleaved by caspase-3, presumably at the same site
we identified, during TNF
-induced apoptosis of MCF-7S1 breast
carcinoma cells and stated that this proteolytic process led to
cPLA2 activation based on the observations that AA release
from TNF
-treated cells was inhibited by AACOCF3. In
their study, however, they did not measure cPLA2 activity
directly and, even though cPLA2 cleavage occurred to some
extent, the major portion of cPLA2 remained uncleaved. In
view of our findings that both caspase-3-cleaved cPLA2 and
recombinant cPLA2(1-522) were catalytically inactive, it
is more likely that the TNF
-induced AA release Wissing et
al. observed was the result of the activation of intact, not
cleaved, cPLA2, which may undergo some other
post-translational modifications, such as phosphorylation by MAPK
family protein kinases downstream of the TNF-specific signal
transduction pathway.
Nonetheless, our results reinforce the hypothesis that TNF- and
Fas-mediated signaling pathways are segregated; cPLA2 is involved in AA release by the former but not the latter. This proposal
is consistent with previous reports that the cPLA2
expression level correlated with TNF
-dependent but not
Fas-induced apoptosis of L929 cells (35, 38). In this respect, the JNK
and p38 subfamilies of MAPKs, which are capable of phosphorylating
cPLA2 (53, 54), are known to be strongly activated by type
1 TNF receptors but only minimally activated by Fas (27).
Alternatively, a unique death domain-containing adapter molecule, MADD,
which links type 1 TNF receptors but not Fas to the ERK subfamily MAPKs
and cPLA2 activation (55), may facilitate the linkage of
cPLA2 to the TNF-specific apoptotic pathway through an as
yet unidentified mechanism. These TNF-specific signalings may be
coupled with cPLA2 activation, which modulates apoptosis
under certain conditions.
Dissociation of cPLA2 from Fas-mediated AA release prompted us to examine the effects of various inhibitors for other PLA2 isoforms. Of the PLA2 inhibitors examined so far, only iPLA2 inhibitors inhibited Fas-mediated AA release, suggesting that iPLA2 (or related enzymes), an 85-kDa cytosolic protein that exists as a multimeric complex of ~300 kDa, that exhibits no fatty acid selectivity, and that contains eight ankyrin motifs (56), is involved at least in part in apoptosis-associated fatty acid release from membrane phospholipids. Ongoing studies by Dennis and his colleagues (14, 15) have so far provided support for the notion that iPLA2 is involved in phospholipid remodeling, further support for which was provided by our recent overexpression studies (10). Because iPLA2 activity in vitro remained unchanged during Fas-mediated apoptosis, we surmise that apoptotic signals may render membrane phospholipids more susceptible to iPLA2 or that some regulatory proteins in the iPLA2 complex may be modified in vivo, leading to increased fatty acid release. Interestingly, treating the cells with an iPLA2 inhibitor retarded cell death significantly. Chilton and his colleagues (57) demonstrated that uneven AA redistribution between glycerophospholipid subclasses, which is caused by inhibition of CoA-independent transacylase, correlated with apoptosis. Thus, we speculate that perturbed phospholipid remodeling may affect the structures, dynamics, integrity, or asymmetry of bilayer membranes, thereby influencing apoptotic changes of the cell.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. D. Clark and M. Tsujimoto for providing antiserum and cDNA for cPLA2, respectively. We thank Dr. R. M. Kramer for providing the sPLA2 inhibitor LY311727.
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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 and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-3-3784-8196;
Fax: 81-3-3784-8245; E-mail: kudo{at}pharm.showa-u.ac.jp.
1 The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; TNF, tumor necrosis factor; AA, arachidonic acid; AACOCF3, arachidonoyl trifluoromethyl ketone; MAFP, methyl arachidonylfluorophosphonate; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; DAPI, 4',6'-diamidino-2-phenylindole; PIPES, 1,4-piperazinediethanesulfonic acid.
2 M. Murakami, T. Kambe, S. Shimbara, and I. Kudo, unpublished observation.
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