Distinct Roles of Two Intracellular Phospholipase A2s in Fatty Acid Release in the Cell Death Pathway
2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês
10.1074/jbc.m000271200
ISSN1083-351X
AutoresGen‐ichi Atsumi, Makoto Murakami, Kayoko Kojima, Atsuyoshi Hadano, Masae Tajima, Ichiro Kudo,
Tópico(s)Drug-Induced Hepatotoxicity and Protection
ResumoCytosolic phospholipase A2α (cPLA2α; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp522 during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA2(1–522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A2s (PLA2s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA2s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA2(1–522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca2+-independent PLA2(iPLA2; type VI), a phospholipid remodeling PLA2, contributes to the cell death-associated increase in fatty acid release. iPLA2 was cleaved at Asp183by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA2functions. iPLA2 had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA2α behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA2 accelerates phospholipid turnover that may lead to apoptotic membranous changes. Cytosolic phospholipase A2α (cPLA2α; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp522 during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA2(1–522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A2s (PLA2s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA2s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA2(1–522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca2+-independent PLA2(iPLA2; type VI), a phospholipid remodeling PLA2, contributes to the cell death-associated increase in fatty acid release. iPLA2 was cleaved at Asp183by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA2functions. iPLA2 had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA2α behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA2 accelerates phospholipid turnover that may lead to apoptotic membranous changes. phospholipase A2 cytosolic phospholipase A2 secretory PLA2 Ca2+-independent PLA2 tumor necrosis factor α interleukin-1β arachidonic acid oleic acid phosphate-buffered saline methylarachidonyl fluorophosphate bromoenol lactone cycloheximide human embryonic kidney fetal calf serum phosphatidylserine polymerase chain reaction polyacrylamide gel electrophoresis enhanced green fluorescent protein hemagglutinin Phospholipase A2(PLA2)1 comprises a growing family of distinct enzymes that exhibit different substrate specificity, cofactor requirements, subcellular localization, and cellular functions (1.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. 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Deletion of the N-terminal first ankyrin repeat by the action of caspase-3 renders iPLA2 more active than the uncleaved form. In contrast, the catalytically inactive N-terminal cPLA2α fragment produced by caspase-3 shows a higher affinity for membranes than does the intact enzyme and behaves like a dominant-negative inhibitor of stimulus-induced AA release by cPLA2α as well as by sPLA2-IIA. The latter observation provides further support for a functional linkage between cPLA2α and signaling sPLA2. A23187 was obtained from Calbiochem. The agonistic anti-Fas antibody CH-11 (37.Yonehara S. Ishii A. Yonehara M. J. Exp. Med. 1989; 169: 1747-1756Crossref PubMed Scopus (1428) Google Scholar) and caspase-3/CPP32 Colorimetric protease assay kit were purchased from Medical & Biological Laboratories. Human recombinant tumor necrosis factor α (TNFα) and human recombinant interleukin-1β (IL-1β) were purchased from Genzyme. Methylarachidonyl fluorophosphonate (MAFP), which inhibits both iPLA2 and cPLA2α (38.Lio Y.C. Reynolds L.J. Balsinde J. Dennis E.A. Biochim. Biophys. Acta. 1996; 1302: 55-60Crossref PubMed Scopus (15) Google Scholar), bromoenol lactone (BEL), an iPLA2 inhibitor (39.Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), and rabbit polyclonal anti-iPLA2 antibody were obtained from Cayman Chemical. Rabbit polyclonal anti-cPLA2α antibody was from Santa Cruz Biotechnology. The anti-FLAG antibody M2, cycloheximide (CHX), and etoposide were purchased from Sigma. cDNAs for mouse cPLA2α and hamster iPLA2 have been described previously (16.Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Human caspase-3 cDNA was a generous gift from Dr. K. Takahashi (Showa University, Tokyo, Japan). Phospholipids and neutral lipids used as standards for thin layer chromatography (TLC) were purchased from Avanti. MACS apoptotic cell isolation kit was purchased from Miltenyi Biotec. Protease inhibitors and all other regents, which were of analytical grade, were obtained from Wako. Human monocytic U937 cells, human cervix epithelioid carcinoma HeLa cells, and mouse fibroblastic L929 cells were obtained from RIKEN Cell Bank. Human embryonic kidney (HEK) 293 cells were from Health Science Research Resources Bank. U937 cells were maintained in RPMI 1640 medium (Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum (FCS) (Intergen), 2 mmglutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in humidified air containing 5% CO2. The cells were preincubated with 0.1 μCi/ml [3H]AA (NEN Life Science Products) for 24 h, washed three times, resuspended at 1 × 107 cells/ml in cultured medium, and then treated with 50 or 100 ng/ml anti-Fas antibody or 100 units/ml TNFα in the presence or absence of 10 μg/ml CHX for various periods. In some experiments, MAFP or BEL was added to the cells during incubation. The [3H]AA contents of neutral lipids were assessed by counting the radioactivity of a fraction extracted using the method of Dole and Meinertz (40.Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar). Cell viability was assessed by the trypan blue dye exclusion test. The total lipids in radiolabeled cells and their supernatants were extracted using the method of Bligh and Dyer (41.Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (43132) Google Scholar) and developed by two-dimensional TLC on Silica Gel 60 plates (Merck), as described previously (34.Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The first and second solvent systems consisted of chloroform/methanol/acetic acid/water (65/25/4/2, v/v) and chloroform/methanol/formic acid (65/25/8.8, v/v), respectively. The zones on the silica gel corresponding to neutral lipids (a mixture of free fatty acids and other neutral lipids) and phospholipids were identified by comparing their mobilities with those of authentic standards, which were visualized with iodine vapor. The zones were each scraped into a vial, and the radioactivity was counted using a liquid β-scintillation counter (Aloka). In order to separate further the free fatty acids from other neutral lipids, the total lipids were developed on Silica Gel 60 plates using a solvent system of hexane/ether/acetic acid (80/30/1, v/v). The radioactivity associated with each lipid was expressed as a percentage of that associated with the total lipids. The cells were washed once with a buffer comprising 10 mm HEPES (pH 7.5), 1 mm EDTA, and 340 mm sucrose, suspended (3.3 × 107 cells/ml) in the same buffer containing 1 mm dithiothreitol, and lysed by sonication for 1 min with a Branson Sonifier (power 30, 50% pulse cycle). The cell lysates were centrifuged at 100,000 × g for 1 h at 4 °C, and the resulting supernatants were incubated at 40 °C for 30 min 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 15 μm1-palmitoyl-2-[14C]arachidonyl phosphatidylethanolamine as a substrate (42.Ackermann E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). The [14C]AA released was extracted using the method of Dole and Meinertz (40.Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar), and the associated radioactivity was counted. Cells were magnetically labeled with annexin V conjugated with MACS colloidal super-paramagnetic MicroBeads, according to the instructions for the MACS apoptotic cell isolation kit, and a population of cells that expressed PS on the outer leaflets of their plasma membranes was collected by passing them through a separation column. Briefly, 5 × 106 cells prelabeled with 0.1 μCi/ml [3H]AA were incubated with annexin V MicroBeads in a binding buffer containing Ca2+ for 15 min at 10 °C and washed with the binding buffer. The magnetically labeled apoptotic cells were applied to the MS+ separation column, which was placed in the magnetic field of a MiniMACS magnet separator, and the non-apoptotic cells were passed through the column. The column was rinsed with the binding buffer and removed from the magnet separator. The apoptotic cells, which were magnetically retained on the column, were eluted with the binding buffer, and their radioactivity was counted. Caspase-3 activity was assayed using the CPP32/Caspase-3 colorimetric protease assay kit, which is based on spectrophotometric detection of the chromophorep-nitroanilide that is produced after cleavage of the labeled substrate DEVD-p-nitroanilide. Briefly, cells were lysed with the lysis buffer and incubated on ice for 10 min. The lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the protein concentrations of the supernatants were determined using the BCA protein assay kit (Pierce). Then, 50 μg of protein equivalents were incubated with DEVD-p-nitroanilide for 2 h at 37 °C, and the absorbance of each solution at 405 nm was measured spectrophotometrically using a microtiter plate reader (Bio-Rad). Cells were washed with phosphate-buffered saline (PBS) and then lysed in PBS containing 100 μm p-4(2-aminoethyl)-benzenesulfonyl fluoride, 5 μm iodoacetamide, 5 mm EDTA, 1 μm pepstatin, 1 μg/ml soybean trypsin inhibitor, and 100 μm leupeptin by sonication for 1 min with a Branson Sonifier (power 30, 50% pulse cycle). The samples (10 μg of 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 antibodies and visualized with the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (13.Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). A cPLA2α deletion mutant, cPLA2(1–522), was constructed by polymerase chain reaction (PCR) of the cPLA2α coding sequence with exTaq polymerase (Takara) using the oligonucleotide pair 5′-ATGTCATTTATAGATCCTTAC-3′ and 5′-TCAGTCGAGCTCGTCATCGAA-3′, as described previously (34.Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The PCR product was ligated into pCR™3.1 (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). Construction of iPLA2 mutants was carried out by PCR with KlenTaq polymerase (CLONTECH) using hamster iPLA2 cDNA (Dr. S. Jones, Genetics Institute) as a template. An iPLA2 deletion mutant, iPLA2(184-C), was generated using the oligonucleotide pair 5′-ATG TATCCGTATGATGTTCCTGATTATGCTAGCCTC AAC AAA GGA GAG ACG G-3′ (HA epitope underlined) and 5′-TCA CTTGTCATCGTCGTCCTTGTAGTC TGA TGA GGG CGA CAG CAG C-3′ (FLAG epitope underlined, which was attached as required for the experiments). HA-tagged iPLA2 was constructed using the primers 5′-ATG TATCCGTATGATGTTCCTGATTATGCTAGCCTC ATG CAG TTC TTC GGA C-3′ (HA epitope underlined) and 5′-TCA GGG CGA CAG CAG CAT TTG-3′. FLAG-tagged iPLA2 was constructed using the primers 5′-ATG CAG TTC TTC GGA CGC C-3′ and 5′-TCA CTTGTCATCGTCGTCCTTGTAGTC TGA TGA GGG CGA CAG CAG C-3′ (FLAG epitope underlined). PCR conditions were 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, for 25 cycles. The products of the expected size were subcloned into the pCR3.1 vector and sequenced as noted above. HEK293 cells stably expressing each PLA2 were established as described previously (16.Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 17.Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). To obtain cells expressing cPLA2(1–522) and epitope-tagged or truncated iPLA2, their cDNAs subcloned into pCR3.1 were transfected into 293 cells using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instruction. Briefly, 1 μg of plasmid was mixed with 5 μl of LipofectAMINE Plus in 200 μl of Opti-MEM medium, left for 15 min, and then added to cells that had attained 40–60% confluency in 6-well plates (Iwaki) in 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium (RPMI 1640 containing 10% FCS). After overnight culture, the medium was replaced again with 2 ml of fresh medium, and culture was continued at 37 °C in a CO2incubator flushed with 5% CO2 in humidified air. For transient expression analyses, the cells were harvested 3 days after transfection and used immediately. In order to establish stable transfectants, the cells were cloned by limiting dilution in 96-well plates in culture medium supplemented with 1 mg/ml G418 (Life Technologies, Inc.). After culture for 2–4 weeks, wells containing a single colony were chosen, and the expression was assessed by RNA blotting as well as immunoblotting using appropriate probes or antibodies. These established clones were expanded and used in the experiments described below. To establish double transformants, 293 cells stably expressing cPLA2(1–522) were subjected to a second transfection with cPLA2α, sPLA2-IIA, or iPLA2cDNA, which had been subcloned into pcDNA3.1/Zeo(+) (Invitrogen). Three days after transfection, the cells were seeded into 96-well plates in the presence of 50 μg/ml zeocin (Invitrogen) in order to establish stable transformants expressing both cPLA2(1–522) and either PLA2 isozyme. Approximately equal amounts (∼10 μg) of total RNA obtained from the transfected cells were applied to individual lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the relevant cDNA probes, which had been labeled with [32P]dCTP (Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All hybridizations were carried out as described previously (15.Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). HEK293 transfectants (5 × 104 cells in 1 ml of culture medium) were seeded into 24-well plates. In order to assess AA release, 0.1 μCi/ml [3H]AA was added to the cells on day 3, when they had nearly reached confluent, and culture was continued for another day. After two washes with fresh medium, 250 μl of RPMI 1640 with or without 10 μm A23187, 1 ng/ml IL-1β, and/or 10% FCS was added to each well, and the amount of free [3H]AA released into the supernatant during culture (30 min with A23187 and up to 8 h with IL-1β) was measured. The percentage release of AA was calculated using the formula (S/(S +P)) × 100, where S and P are the radioactivities measured in equal portions of the supernatant and cell pellet, respectively. All of these procedures are described previously in detail (16.Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 17.Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 18.Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 19.Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). HEK293 transfectants (5 × 104 cells in 1 ml of culture medium) were seeded into 24-well plates. 0.1 μCi/ml [3H]AA was added to the cells on day 2, when they are 50% confluent, and culture was continued for another day. After two washes with fresh medium without FCS, 250 μl of RPMI 1640 containing 1% FCS with or without CHX or etoposide was added to each well, and the amount of radioactivity released into the supernatant during culture was measured. MAFP or BEL was added to cells during treatment with CHX or etoposide as required for the experiments. To assess oleic acid (OA) release, 0.5 μCi/ml [3H]OA (NEN Life Science Products) was added to the cells instead of [3H]AA. [35S]Methionine-labeled cPLA2α and its truncated mutant cPLA2(1–522) were synthesized using a PROTEINscript™ kit (Ambion). Briefly, plasmids containing mouse cPLA2α 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. The procedure was described in our previous report (34.Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). cDNAs for native and truncated forms of cPLA2α and those for HA-tagged native and truncated forms of iPLA2 were subcloned into the pEGFP-C3 and -C1 vectors (CLONTECH), respectively, at the EcoRI site. These plasmids were transfected into 293 cells seeded onto collagen-coated coverglasses (Iwaki Glass) using LipofectAMINE 2000 (Life Technologies, Inc.). After culture for 3 days, the cells were fixed with 2% (w/v) paraformaldehyde in PBS for 30 min at room temperature. The coverslips were mounted on glass slides using Perma Fluor (Japan Tanner) and examined using a FLUOVIEW laser fluorescence microscope (Olympus). All values shown are means ± S.E. for three to six separate experiments. Differences between means were determined by Student's t test, and those atp < 0.05 were considered significant. We have previously reported that cPLA2α is cleaved at Asp522 by caspase-3 in U937 cells undergoing Fas-mediated apoptosis (34.Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). This proteolytic process destroys the catalytic dyad (Ser228 and Asp549) essential for cPLA2α activity, thereby leading to its enzymatic inactivation. Similar cPLA2α degradation was observed when U937 (see below), HeLa (35.Adam-Klages S. Schwandner R. Luschen S. Ussat S. Kreder D. Kronke M. J. Immunol. 1998; 161: 5687-5694PubMed Google Scholar), or L929 cells (data not shown) were killed with TNFα in combination with CHX. Thus, it is likely that the caspase-directed cleavage of cPLA2α is an event generally occurring in the apoptotic process. In order to investigate whether the cleaved cPLA2α plays some roles in cellular AA metabolism, death, survival, or proliferation, we prepared cDNA for the mutant cPLA2α truncated at Asp522 (cPLA2(1–522)) and transfected it into HEK293 cells. When a sonicate of HEK293 cells expressing native cPLA2α was centrifuged at 100,000 × g and then subjected to immunoblotting using anti-cPLA2α antibody, most of the enzyme, as expected, was found to be recovered mainly in the supernatant cytosolic fraction (Fig. 1 A). On the other hand, overexpressed c
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