| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Institut für Immunologie, Christian-Albrechts-Universität Kiel, Kiel, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(8), focal adhesion kinase (9), the
inhibitor of caspase-activated DNase (ICAD) (10, 11), and gelsolin
(12), to name a few. The cleavage of other substrates abrogates their
function for cell survival or appears to be without a known consequence
for apoptosis (13). Knowledge about apoptosis induced by receptors belonging to the nerve growth factor/TNF receptor superfamily (for review, see 14 , which includes among others the p55 TNFR, CD95/Fas, death domain-containing receptor 3 (DR3 (15)), DR4 (16), and DR5 (17), has made remarkable progress during the last few years. It is now clear that members of the caspase family are the effectors of the apoptotic signaling pathway triggered by TNF and Fas ligand (for review, see 18 . Caspase-8 (FLICE, Mach), recruited to the p55 TNFR and CD95/Fas after ligand binding, appears to be the start point of a cascade of caspases finally leading to cell death.
Cytosolic phospholipase A2 (cPLA2) is an 85-kDa protein that preferentially liberates arachidonic acid (AA) from the sn-2 position of phospholipid generating lysophospholipid and free AA (for review, see Refs. 19 and 20). AA can be converted to potent inflammatory lipid mediators, the eicosanoids. This conversion occurs enzymatically through the lipoxygenase or cyclooxygenase pathways for the production of leukotrienes, lipoxins, thromboxanes, or prostaglandins (20). The important role of AA in the regulation of an inflammatory response requires that its levels be tightly controlled. cPLA2 plays a major role in maintaining AA levels, and its enzymatic activity is subject to complex mechanisms of regulation (21). At least two receptor-mediated events have been identified leading to full activation of cPLA2: the binding of calcium promotes translocation of cPLA2 to membranes (22), while phosphorylation at Ser505 of cPLA2 directly increases its activity (23). Activation of cPLA2 occurs in many cell types in response to various stimuli (19). In particular, cPLA2 can be activated by the p55 TNFR and CD95/Fas (24, 25), which also mediate apoptosis in certain cell types. Thus, in the same cell the induction of programmed cell death may coincide with the generation of inflammatory mediators.
In this report, we show that cPLA2 is cleaved during apoptosis and that this cleavage is most likely conducted by caspase-3 or a protease with similar substrate specificity. The processing of cPLA2 abolishes its catalytic activity, thereby inhibiting a proinflammatory signal potentially activated in parallel with the apoptotic signal and ensuring physiologic death without inflammation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Highly purified human TNF (3 x 107 U/mg) was provided by G. Adolf (Bender, Vienna, Austria). The mAb against human cPLA2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal anti-Fas Ab was purchased from Coulter/Immunotech (Hialeah, FL), and the mAb against PARP was originally obtained from Dr. G. Poirier (McGill University, Quebec, Canada). The caspase inhibitors z-VAD.fmk, Ac-DEVD. CHO, and Ac-YVAD.cmk were purchased from Bachem (Torrance, CA). The calcium ionophore A23187 (Sigma, St. Louis, MO) was dissolved in ethanol at 1 mM. Cycloheximide (CHX), etoposide, and daunomycin were purchased from Sigma. C2-ceramide and the cPLA2 inhibitors methylarachidonylfluorophosphonate (MAFP) and arachidonoyl trifluoromethyl ketone (AACOCF3) were obtained from Biomol (Hamburg, Germany).
Plasmids
The cDNA encoding human cPLA2 was subcloned into the
eukaryotic expression vector pRK5 (26). The truncated form of
cPLA2 (PLA2
523) was obtained by cloning the
N-terminal SalI-PvuII fragment of the
cPLA2 cDNA into the expression vector pEF.Bos (27). The
expression plasmids for caspase-1 and caspase-8 were generously
provided by Dr. D. Goeddel (Tularik, South San Francisco, CA). cDNAs
encoding caspase-3, -4, and -7 were obtained by PCR using a cDNA
library from U937 cells and subcloned into pRK5. Oligonucleotides used
for PCR were: for caspase-3, 5'-ATAAAGGTATCCATGGAGAACACTG-3'
and 5'-CCACCAACCAACCATTTCTTTAGTG-3'; for caspase-4,
5'-AGAGGCTGTTCCCTATGGCAGAAGG-3' and
5'-CTTGTGGCTTCCATTTTCAATTGCC-3'; and for caspase-7,
5'-TGGGAACGATGGCAGATGATCAGGG-3' and 5'-TGGCTATTGACTGAAGTAGAGTTCC-3'.
Cell culture and transfection
HEK 293 cells and HeLa cells were originally obtained from the American Type Culture Collection (Manassas, VA). Both cell types were grown in DMEM without HEPES (Biochrom, Berlin, Germany) supplemented with 10% FCS, 2 mM glutamine, and 50 µg/ml each of streptomycin and penicillin. Transfection was performed using the calcium phosphate precipitation method (28).
Western blot analysis
About 16 h after transfection, cells were detached using EDTA, lysed in TNE buffer (20 mM Tris (pH 8.0), 140 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, and protease inhibitor mix Complete (Boehringer, Mannheim, Germany). After precipitating cell debris for 5 min at 14,000 rpm, protein concentrations were determined in the cytosolic supernatants using a Coomassie reagent (Pierce, Rockford, IL). From each lysate, 20 µg of total protein was separated on a 10% SDS-PAGE and transferred to Porablot nitrocellulose filters (0.45 µM; Macherey-Nagel, Düren, Germany). Filters were blocked overnight in PBST (PBS containing 0.1% Tween 20) supplemented with 5% milk powder. After incubation at room temperature with either the mAb against human cPLA2 or the mAb against PARP, filters were washed three times with PBST. Filters were incubated with a 1:5000 dilution of a peroxidase-conjugated rabbit anti-mouse antiserum (Dianova, Hamburg, Germany), washed five times with PBST and developed with the ECL detection reagent (Amersham, Arlington Heights, IL).
Expression in Escherichia coli
The cDNA encoding caspase-3 was subcloned by PCR (oligonucleotides: 5'-ACGGATCCATGGAGAACACTGAAAACTC-3' and 5'-ACGTCGACTTCGTGATAAAAATAGA-3') in frame with the N-terminal T7 tag into the bacterial expression vector pET21a (Novagen, Madison, WI). The plasmid was transformed into the provided E. coli strain BL21(DE3)pLysS (Novagen). Expression of the T7/caspase-3 fusion protein was induced with 0.8 mM isopropylthiogalactoside (IPTG) according to the instructions provided by the manufacturer.
In vitro cleavage assays
Human PLA2-WT cDNA or the PLA2-D/A mutant cloned into the expression vector pRK5 were used for in vitro transcription/translation employing the SP6-coupled TNT Reticulocyte Lysate System (Promega, Madison, WI) and [35S]methionine (Amersham).
To obtain cytosolic extracts from HEK 293 or HeLa cells containing active caspases, cells were detached using EDTA 18–24 h after transient transfection and lysed in ACE buffer (10 mM HEPES (pH 7.4), 50 mM NaCl, 5 mM EGTA, 1 mM DTT, 1 mM Pefabloc SC (Boehringer)) by four cycles of freezing/thawing followed by repeated passaging through a 23-gauge needle. Cell debris was removed by centrifugation at 4°C for 30 min at 14,000 rpm. Protein concentrations in the cytosolic supernatants were determined using the Coomassie reagent. Total protein (7.5 µg) from the ACE extracts were incubated with 1.5 µl of in vitro-translated cPLA2 in a final volume of 15 µl ACE assay buffer (20 mM Tris (pH 7.5), 0.1 mM EDTA, 10 mM DTT, 1 mg/ml Pefabloc SC) for 2 h at 30°C. The reaction was stopped by adding 5 µl of 4x SDS sample buffer. Radioactive cPLA2 proteins were analyzed on a 12.5% SDS-PAGE. The gels were fixed in 10% acetic acid, dried, and exposed on Kodak BioMAX films.
Bacterial extracts were prepared 5 h after IPTG induction by resuspending the cell pellet from 50 ml of culture in 3 ml of caspase buffer (20 mM PIPES (pH 7.2), 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 10 mM DTT, 1 mg/ml Pefabloc SC, 1 µg/ml leupeptin) and lysing the bacteria by four cycles of freezing/thawing followed by sonication for 30 s. Debris was removed by centrifugation at 4500 rpm for 20 min at 4°C. Protein concentration in the supernatant was determined using the Coomassie reagent. In vitro cleavage of 35S-labeled cPLA2 by bacterially expressed caspase-3 was performed essentially as described for cleavage by HEK 293 cell lysates with the following modifications: 1 µg of bacterial proteins was used with an incubation time of 0.5 h.
Site-directed mutagenesis
The cDNA encoding human cPLA2 was subcloned into the
mutagenesis vector pALTER-1 (Promega). Using the
oligonucleotide-directed in vitro mutagenesis kit (Promega) a single
amino acid (aspartic acid at position 522) was replaced by alanine (GAT
GCT, oligonucleotide 5'-GATGAACTGGCTGCAGCTGTA-3') to generate
PLA2-D/A following the protocols provided by the supplier.
The introduced point mutation was verified by DNA sequencing.
Arachidonic acid release
To test different mutant forms of human cPLA2 for enzymatic activity AA release assays were performed essentially as described (23). HEK 293 cells were transiently transfected in triplicates with expression constructs for various cPLA2 proteins and labeled 6 h after transfection with 1 µl/ml medium [5,6,8,9,11,12,14,15-3H]AA (150–230 Ci/mmol, 1 mCi/ml; Amersham). After overnight incubation, cells were washed two times with medium and stimulated as indicated in the figure legends. Released radioactivity from the supernatants was quantified by liquid scintillation counting.
Determination of cPLA2 activity using phosphatidylcholine vesicles
The phosphatidylcholine vesicle assay was performed as described (23). Briefly, HEK 293 cells overexpressing cPLA2 or its mutant forms were detached, washed once with PBS, and lysed by sonication in buffer containing 10 mM Tris (pH 7.4), and 150 mM NaCl. Protein content was measured, and equal amounts were incubated in reaction buffer (100 mM Tris, (pH 8.8), 4 mM CaCl2, 2 µM 1-stearoyl-2-[14C]arachidonylphosphatidylcholine; Amersham) for 30 min at 37°C. The [14C]AA released was extracted by the method of Dole and Meinertz (29). The associated radioactivity was quantified by scintillation counting.
Cell cycle analysis
HeLa cells were transiently transfected with expression constructs for various cPLA2 proteins and incubated for 24 h to allow for expression of cPLA2 proteins. Cells were detached using EDTA, washed twice with cold PBS/5 mM EDTA, and resuspended in 1 ml PBS/5 mM EDTA. Cells were fixed by adding 1 ml of ethanol and incubated for 30 min at room temperature. Cells were harvested and resuspended in 0.5 ml of PBS/5 mM EDTA. RNA was removed by digestion with 20 µl of RNase A (1 mg/ml) for 30 min at room temperature. After 1 h of incubation with 0.5 ml of staining solution (500 µg/ml propidium iodide in PBS/5 mM EDTA), cell cycle analysis was performed by flow cytometry using a FACSCalibur Analyzer (Becton Dickinson, Heidelberg, Germany).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Analysis of the amino acid sequence of human cPLA2
revealed the presence of a putative cleavage site identified for
cysteine proteases. This sequence motif (DELD at amino acids 519–522;
Fig. 1
A) is very similar to
the characteristic cleavage site for caspase-3 within its substrate
PARP (DEVD; 13 . We used HeLa cells stimulated with diverse cell
death-inducing agents to investigate whether cleavage of
cPLA2 could be detected during apoptosis. Western blot
analysis with a mAb directed against the N-terminal 200 amino acids of
human cPLA2 revealed the appearance of a cleavage product
of about 70 kDa molecular mass (Fig. 1B). Given the fact
that the cPLA2 protein migrates on SDS-PAGE higher than
predicted from its calculated molecular mass (30), the size of this
protein fragment is in agreement with a cleavage at the putative
caspase-3 motif, DELD. The amount of the 70-kDa cleavage product and,
in parallel, the disappearance of the intact cPLA2
correlated well with the number of apoptotic cells determined by cell
cycle analysis using flow cytometry (Fig. 1C). Cleavage of
the typical caspase substrate PARP was also detected in comparable
proportions (Fig. 1B).
|
D). This
indicated that a caspase with the specificity of caspase-3 might be
responsible for cPLA2 cleavage. Caspase-mediated cleavage of cPLA2 occurs in intact cells and in vitro
To further investigate the involvement of distinct caspases in
the observed apoptotic cleavage of cPLA2, we used
HEK 293 cells overexpressing different types of caspases.
Since cPLA2 expression in HEK 293 cells is
extremely low (data not shown), we cotransfected an expression
construct for human cPLA2. Proteolytic processing of
cPLA2 yielding the 70-kDa cleavage product could be
detected in HEK 293 cells overexpressing cPLA2 after
stimulation with TNF (Fig. 2A). HEK 293 cells are
resistant to TNF-induced apoptosis, but appear to activate endogenous
caspases after TNF treatment (data not shown). To identify specific
caspases that can use cPLA2 as substrate, we used HEK 293
cells overexpressing caspase-1, -3, and -8, belonging to different
subfamilies (6), in combination with cPLA2. As shown in
Fig. 2B, overexpression of caspase-3 and -8 resulted in the
generation of the 70-kDa cleavage product detected in apoptotic HeLa
cells or after TNF treatment. Overexpression of caspase-1, however, led
to a different cleavage product of 
58 kDa molecular mass (Fig. 2B).
|
A). Extracts from control
cells did not lead to cPLA2 processing (Fig. 3A). The generated cleavage products (70 and 32 kDa)
correspond to both protein fragments expected after cleavage at the
DELD motif. The 32-kDa fragment was not detected in Western blots with
the mAb against cPLA2, indicating that it corresponds to
the C-terminal portion of cPLA2. In vitro cleavage of
cPLA2 by cell extracts containing caspase-3 was also seen
using HeLa cells (data not shown). In vitro cleavage of
cPLA2 after incubation with extracts from cells
overexpressing caspase-3 was inhibited by the caspase-3-specific
inhibitor Ac-DEVD.CHO, but not by Ac-YVAD.cmk (data not shown).
|
A). The size of the major
cleavage products was identical to that seen with extracts from HEK 293
or HeLa cells. Additional protein fragments seen in lower quantities
might be due to processing of alternatively started translation
products or to nonspecific cleavage at related sites. No cleavage could
be detected with E. coli lysates containing the expression
vector (Fig. 4A). The activity of caspase-3 in the E.
coli lysates was confirmed by an enzymatic assay using the
caspase-3-specific fluorescence peptide substrate, Ac-DEVD.AMC.
Detectable nonspecific cleavage was also seen using the
caspase-1-specific substrate, Ac-YVAD.AMC (Fig. 4B).
In vitro cleavage of cPLA2 could be inhibited by the
caspase-3-specific inhibitor, Ac-DEVD.CHO, but not by the
caspase-1-specific inhibitor, Ac-YVAD.cmk (data not shown).
|
Mutational analysis of cPLA2
To prove that the major caspase cleavage site used in apoptosis is
indeed the proposed DELD sequence, we constructed a mutant form of
human cPLA2 replacing aspartic acid (residue 522) with
alanine (PLA2-D/A). Furthermore, we generated a truncated
form of human cPLA2 (PLA2523) that
terminates after residue 523 to compare the migration pattern of this
truncated cPLA2 with the observed 70-kDa cleavage product.
Both of the mutant cPLA2 proteins were expressed in HEK 293
cells. After treatment with TNF to induce cleavage, the cells were
lysed, and the different cPLA2 forms were detected in
immunoblots. The cPLA2 protein and consequently the
cleavage products migrate in SDS-PAGE in a slightly different manner
from that predicted by their calculated molecular mass (29). The
truncated form, PLA2523, showed a migration pattern
indistinguishable from the 70-kDa cleavage product of wild-type
cPLA2 generated in cells treated with TNF (Fig. 5). In contrast to wild-type
cPLA2 (PLA2-WT), the mutant form,
PLA2-D/A, was not cleaved in cells treated with TNF (Fig. 5). Identical results were obtained using HeLa cells overexpressing all
three of the cPLA2 proteins (data not shown). In
vitro-translated PLA2-D/A was also not cleaved by lysates
from cells overexpressing various caspases (Fig. 3B) or by
E. coli extracts containing active caspase-3 (data not
shown). These data clearly show that cPLA2 processing in
apoptotic cells, presumably by caspase-3, occurs at residue 522, as
predicted from the amino acid sequence.
|
To determine the effects of cPLA2 cleavage on its
enzymatic activity, we first measured AA release in HEK 293 cells
overexpressing PLA2-WT, PLA2-D/A, or
PLA2523 after stimulation with TNF, the calcium
ionophore A23187, or a combination of both. In this system, increased
AA release after stimulation was observed in cells overexpressing
PLA2-WT or PLA2-D/A, but not in
vector-transfected cells (Fig. 6A), while treatment with a
specific inhibitor of cPLA2 (AACOCF3)
completely inhibited the stimulated AA release (data not shown). This
result indicates that the observed release of AA is due to the activity
of overexpressed cPLA2. Overexpression of the cleavage
resistant PLA2-D/A led to an increased basal activity.
After stimulating the cells with TNF and, more prominently, with a
combination of TNF and A23187, the rise in AA release was greater
compared with wild-type cPLA2 (Fig. 6A). This
indicates that resistance to the caspase-mediated cPLA2
cleavage leads to an increased enzymatic activity. In contrast, cells
overexpressing the truncated cPLA2 protein showed no
increased AA release after stimulation. In addition, overexpression of
PLA2523 exhibited a dominant negative effect on the
basal level of AA release (Fig. 6A). Equal expression of
cPLA2 proteins was demonstrated by immunoblot analysis
(Fig. 6A).
|
B, PLA2-D/A exhibited
a catalytic activity slightly higher than PLA2-WT, while
PLA2523 demonstrated no enzymatic activity at all. The
activity of PLA2-WT and PLA2-D/A could be
completely blocked by the cPLA2 inhibitor
AACOCF3 at a concentration of 10 µM. These data confirm
that cleavage of cPLA2 leading to the 70-kDa fragment
inactivates its function.
To further corroborate that inhibition of cPLA2 cleavage
indeed leads to enhanced enzymatic activity as proposed by the higher
activity of PLA2-D/A, we inhibited caspase activity
using z-VAD.fmk, an inhibitor that blocks a broad range of caspases. In
Western blots, we proved that z-VAD.fmk blocked cPLA2
cleavage. Fig. 7A shows that
the appearance of the 70-kDa product was inhibited in a dose-dependent
manner by z-VAD.fmk. In parallel, we performed an AA release assay
that revealed a marked increase in the stimulated AA release in cells
treated with an amount of z-VAD.fmk that completely blocked
cPLA2 processing (Fig. 7B).
|
cPLA2 has been implicated in the induction of
apoptosis by TNF (31, 32, 33, 34). Moreover, Wissing et al. (35) reported that
cPLA2 is activated by caspase-dependent cleavage
and speculated that the presumably activated
cPLA2 cleavage product might be involved in the initiation
of apoptosis. To examine whether cleavage of cPLA2 by
caspases influences the induction or execution of the apoptotic
program, we overexpressed wild-type cPLA2 or the truncated
cleavage product in HeLa cells. After stimulation with TNF/CHX, the
amount of apoptotic cells was determined by cell cycle analysis using
flow cytometry. Overexpression of neither PLA2-WT nor
PLA2523 led to induction of apoptosis by itself. In
addition, the amount of apoptotic cells after treatment with TNF/CHX
was not changed significantly (Fig. 8A). Thus, in HeLa cells,
cleavage of cPLA2 does not appear to alter the course of
apoptosis.
|
B). This confirms that
cPLA2 function is unlikely to be a prerequisite for
apoptosis in HeLa cells. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The generation of cPLA2 mutants (PLA2-D/A and
PLA2523) allowed us to examine the consequence of the
observed cPLA2 processing on its enzymatic function. We
used HEK 293 cells overexpressing the cPLA2 mutants to test
for their catalytic function, because these cells express very little
endogenous cPLA2 and show almost no cPLA2
activity or increased AA release in response to stimulation (Fig. 6).
We could show that overexpression of PLA2-D/A leads to
slightly enhanced enzymatic activity after stimulation, while the
70-kDa cleavage product demonstrated no enzymatic activity at all. The
increased basal activity of PLA2-D/A might result from an
increase of apoptotic cells after transfection leading to cleavage of
PLA2-WT, but not PLA2-D/A. However, we cannot
rule out the possibility that the PLA2-D/A mutant is
intrinsically more active than PLA2-WT. Taken together, our
data clearly indicate that cPLA2 belongs to the group of
caspase substrates that become inactivated by cleavage.
This finding is in agreement with previously published studies on
structure and function of human cPLA2 identifying residues
200, 228, 331, and 549, located N-terminally as well as C-terminally of
the cleavage site at residue 522 (Fig. 1A), to be essential
for the catalytic function of cPLA2 (20, 36). Therefore, it
is highly unlikely that either one of the cPLA2 fragments
resulting from the caspase-mediated cleavage at residue 522 (the
N-terminal 70-kDa or the C-terminal 32-kDa fragment) can be
catalytically active. Earlier data demonstrating that the cytosol from
apoptotic cells had less cPLA2 activity than cytosol from
control cells (37) also provided evidence for an inactivation mechanism
of cPLA2 in apoptosis. There is, however, a contradiction
to a previous report of a caspase-dependent activation mechanism of
cPLA2 (35). These authors have generated all functional
data on cPLA2 activation by caspase cleavage in two cell
lines that are highly sensitive to the cytotoxic activity of TNF (35).
Thus, their AA release data are derived from apoptotic cells. In our
hands, cells undergoing apoptosis appear to release AA nonspecifically
even before cell death becomes apparent (data not shown). HEK 293 cells
used in this study are resistant to apoptosis induced by TNF.
Overexpression of the 70-kDa cleavage product of cPLA2 not
only showed no detectable enzymatic activity, but exhibited a dominant
negative effect on the slight activation of endogenous
cPLA2 (Fig. 6A). A possible explanation might be
that the truncated cPLA2 could compete for binding of
cofactors necessary for the activation process. Such cofactors are
calcium ions stimulated for by the ionophore A23187 or protein kinases
phosphorylating cPLA2 activated by TNF. Since both the
calcium-binding domain (CaLB domain) and the phosphorylation site (Fig. 1) are still contained in the functionally inert PLA2523
protein, the highly overexpressed truncated cPLA2 would
efficiently compete for binding of calcium ions or kinase activity.
Thus, a quantitative cleavage of cPLA2 after stimulation of
caspase activity during apoptosis leading to high levels of truncated
cPLA2 might down-regulate the activity of the remaining
intact cPLA2. Ligands such as TNF or FasL can induce
apoptosis but also activate cPLA2 (24, 25). If both of
these actions took place in the same cell, an inflammatory signal would
coincide with the apoptotic program that does not lead to an
inflammatory response. The cleavage of cPLA2
followed by the competitive inhibition of the remaining
cPLA2 molecules by the cleavage product, as proposed by
this study, might be an additional mechanism to block an inflammatory
response during physiologic cell death.
Several studies have implied that activation of cPLA2 is a necessary step in the signaling pathway leading to TNF-induced apoptosis. Hayakawa et al. (31) showed that a TNF-resistant clone of L929 cells showed reduced cPLA2 expression and became TNF sensitive upon cPLA2 overexpression. Other groups have demonstrated that apoptosis of various tumor cell lines was dependent on the activity of cPLA2 (32, 33). Palombella and Vilcek (34) reported that PLA2 activity is essential for cytotoxicity, but is also essential for growth stimulation of TNF. Our data, however, clearly show that cPLA2 is cleaved and thereby inactivated in HeLa cells during apoptosis induced by different stimuli, including TNF. In addition, neither overexpression nor pharmacologic inhibition of cPLA2 demonstrated any influence on TNF/CHX-induced cell death in HeLa cells. Thus, a causative role for cPLA2 during TNF-mediated apoptosis in HeLa cells appears to be rather unlikely. In summary, the involvement of cPLA2 in TNF-induced apoptosis observed in previous studies (31, 32, 33, 34) may thus reflect secondary or cell type-specific events.
Recently published studies of cPLA2 knockout mice did not reveal any phenotypic signs that would suggest irregularities in cell death involving processes during development, thus questioning the general involvement of cPLA2 in apoptosis (38, 39). However, cPLA2 knockout mice showed decreased brain infarct sizes after ischemic injury (39), which could reflect decreased neuronal apoptosis after ischemia, again pointing to a potential role of cPLA2 in apoptosis in select cell types.
In summary, our data indicate that cPLA2 is cleaved during the apoptotic process in HeLa cells by caspase-3 and/or a related caspase, leading to a functionally inactive, dominant negative inhibitor of its own enzymatic function. This cleavage does not appear to be involved in apoptosis but might ensure that a potentially proinflammatory enzyme is inactivated in physiologic cell death, which does not lead to an inflammatory response.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Sabine Adam-Klages, Institut für Immunologie, Christian-Albrechts-Universität Kiel, Brunswiker Strasse 4, 24105 Kiel, Germany. E-mail address:
3 Abbreviations used in this paper: ICE, IL-1ß-converting enzyme; AA, arachidonic acid; AACOCF3, arachidonoyltrifluoromethyl ketone; CHX, cycloheximide; cPLA2, cytosolic phospholipase A2; MAFP, methylarachidonylfluorophosphonate; PARP, poly(ADP-ribose) polymerase; WT, wild-type; IPTG, isopropylthiogalactoside; CHAPS, 3-[(3-cholamidolpropyl)dimethylammonio]-1-propanesulfonate; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
Received for publication March 19, 1998. Accepted for publication July 20, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in induction of apoptosis. J. Biol. Chem. 272:20317.
in malignant gynecological cell lines is associated with the expression of protein(s) that prevent the activation of phospholipase A2 by tumor necrosis factor . Cancer Res. 15:866.
This article has been cited by other articles:
|
|
 |
|
  S. Tashiro, T. Sumi, N. Uozumi, T. Shimizu, and T. Nakamura B-Myb-Dependent Regulation of c-Myc Expression by Cytosolic Phospholipase A2 J. Biol. Chem., April 23, 2004; 279(17): 17715 - 17722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Foghsgaard, U. Lademann, D. Wissing, B. Poulsen, and M. Jaattela Cathepsin B Mediates Tumor Necrosis Factor-induced Arachidonic Acid Release in Tumor Cells J. Biol. Chem., October 11, 2002; 277(42): 39499 - 39506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Grewal, E. E. Morrison, S. Ponnambalam, and J. H. Walker Nuclear localisation of cytosolic phospholipase A2-{alpha} in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation J. Cell Sci., January 12, 2002; 115(23): 4533 - 4543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Fonteh, C. R. Marion, B. J. Barham, M. B. Edens, G.-i. Atsumi, J. M. Samet, K. P. High, and F. H. Chilton Enhancement of Mast Cell Survival: A Novel Function of Some Secretory Phospholipase A2 Isotypes J. Immunol., October 15, 2001; 167(8): 4161 - 4171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. R. Panini, L. Yang, A. E. Rusinol, M. S. Sinensky, J. V. Bonventre, and C. C. Leslie Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol J. Lipid Res., October 1, 2001; 42(10): 1678 - 1686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N.-E. El Mahdani, M. Ameyar, Z. Cai, O. Colard, J. Masliah, and S. Chouaib Resistance to TNF-Induced Cytotoxicity Correlates with an Abnormal Cleavage of Cytosolic Phospholipase A2 J. Immunol., December 15, 2000; 165(12): 6756 - 6761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Walev, J. Klein, M. Husmann, A. Valeva, S. Strauch, H. Wirtz, O. Weichel, and S. Bhakdi Potassium Regulates IL-1{beta} Processing Via Calcium-Independent Phospholipase A2 J. Immunol., May 15, 2000; 164(10): 5120 - 5124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-i. Atsumi, M. Murakami, K. Kojima, A. Hadano, M. Tajima, and I. Kudo Distinct Roles of Two Intracellular Phospholipase A2s in Fatty Acid Release in the Cell Death Pathway. PROTEOLYTIC FRAGMENT OF TYPE IVA CYTOSOLIC PHOSPHOLIPASE A2alpha INHIBITS STIMULUS-INDUCED ARACHIDONATE RELEASE, WHEREAS THAT OF TYPE VI Ca2+-INDEPENDENT PHOSPHOLIPASE A2 AUGMENTS SPONTANEOUS FATTY ACID RELEASE J. Biol. Chem., June 9, 2000; 275(24): 18248 - 18258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Fonteh, T. LaPorte, D. Swan, and M. A. McAlexander A Decrease in Remodeling Accounts for the Accumulation of Arachidonic Acid in Murine Mast Cells Undergoing Apoptosis J. Biol. Chem., January 5, 2001; 276(2): 1439 - 1449. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
523, or PLA2-D/A. After a 6-h
incubation, cells were labeled with [3H]AA for an
additional 16 h. cPLA2 activity was stimulated for
6 h with 50 ng/ml TNF, 1 µM A23187, or both, as indicated.
Released [3H]AA was measured in a scintillation counter.
Shown are values of one representative experiment
(n = 4); the bars indicate the respective SDs from
triplicate determinations. Statistical analysis was performed using
Student’s t test; *, p < 0.05 vs
untreated cells; **, p < 0.001 vs untreated
cells. Equivalent expression of all cPLA2 proteins is shown
in an immunoblot using anti-cPLA2 Ab that has been
performed in parallel (inset). B, Lysates
from HEK 293 cells overexpressing the indicated cPLA2
proteins were used for in vitro measurement of cPLA2
activity. Where indicated, incubation of lysates with the substrate was
conducted in the presence of 10 µM AACOCF3. Each lysate
was used for triplicate determination. cPLA2 activity is
shown as percentage of a control triplicate containing reaction buffer
instead of cellular lysate. The bars indicate the respective SDs from
triplicate determinations.
