Secretory phospholipase A2 induces apoptosis through TNF-α and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells

European Journal of Pharmacology 536 (2006) 47 – 53 www.elsevier.com/locate/ejphar

Secretory phospholipase A2 induces apoptosis through TNF-α and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells ChuHee Lee a , Dae-Weon Park a , Jingu Lee a , Tae-Il Lee b , Young-Jo Kim b , Yun-Sik Lee c , Suk-Hwan Baek a,⁎ a

Aging-Associated Vascular Disease Research Center, Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717, South Korea b Department of Internal Medicine, College of Medicine, Yeungnam University, Daegu 705-717, South Korea c Division of Endocrinology, University of Pennsylvania, Philadelphia, PA 19014, USA Received 12 September 2005; received in revised form 9 January 2006; accepted 21 February 2006 Available online 28 February 2006

Abstract Phospholipase A2 (PLA2) is an esterase that cleaves the sn-2 ester bond in glycerophospholipids, thereby releasing free fatty acids and lysophospholipids. In addition to the apoptotic activity of cytosolic PLA2 and Ca2+-independent PLA2, recent studies showed that secretory PLA2 (sPLA2) also play a role in apoptosis. However, the details of molecular mechanism have not been fully elucidated. Our data demonstrated that group IB PLA (IB PLA2)-exposed murine macrophage 264.7 cells showed characteristic features of apoptosis such as morphological changes, DNA laddering, staining positive for propidium iodide (PI) as well as Annexin V and activation of caspases and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in dose- and time-dependent manner. Moreover, IB PLA2 was found to elicit tumor necrosis factor (TNF)-α production and release of cytochrome c, suggesting that IB PLA2 exerts its apoptotic activity via the induction of TNF-α production and cytochrome c release, which results in triggering the activation of caspase cascade and PARP cleavage. © 2006 Elsevier B.V. All rights reserved. Keywords: Secretory phospholipase A2; Apoptosis; Caspase; Poly (ADP-ribose) polymerase; TNF-α; Cytochrome c

1. Introduction In multi-cellular organisms, programmed cell death is required to control normal development and avoid inappropriate cell survival. Excessive or inappropriate apoptotic cell death is causally involved in a various human diseases, including sepsis and cancer (Cauwels et al., 2003). Apoptosis of macrophages infected with bacteria is considered an important innate defense mechanism that prevents the spread of infection by sequestering pathogens within apoptotic bodies and protecting the surrounding tissue from their harmful effects (Gan et al., 2005; Lopez et al., 2003). Causes of apoptotic cell death include growth factor deprivation, environmental stress and death receptor triggering. In

⁎ Corresponding author. Tel.: +82 53 620 3981; fax: +82 53 623 8032. E-mail address: [email protected] (S.-H. Baek). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.02.043

death receptor-mediated apoptosis, both initiation and execution phases are centrally regulated and mediated by a family of aspartate-specific cysteine proteases called caspases (Cauwels et al., 2003; Pleskoff et al., 2005). On the other hand, apoptotic stimuli by death signal lead to mitochondrial disruption and the release of death-promoting factors such as cytochrome c. Once released, cytochrome c initiates a self-amplifying cascade of proteolysis among cytosolic caspases, which trigger the biochemical and morphological changes occurring during cell dismantling. Cleavage of specific substrates by caspases during apoptosis promotes the degradation of key structural proteins, including poly (ADP-ribose) polymerase (PARP), and leads to external display of phosphatidylserine, DNA fragmentation, and cellular condensation (Slee et al., 2001). Phospholipase A2 (PLA2) comprises a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn2 fatty acyl bond of phospholipids to yield free fatty acids and

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lysophospholipids. The various groups of PLA2 can be divided into three categories: secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2) and Ca2+-independent PLA2 (iPLA2). At present, mammalian sPLA2s are classified into 10 different groups depending on the primary structure characterized by the number and positions of cysteine residues (Kuwata et al., 2000). The biological activity of sPLA2 has been attributed to its enzymatic capacity to hydrolyze membrane phospholipids. However, some studies have provided evidences that sPLA2 exerts its function through specific receptor (Hanasaki and Arita, 2002; Kim et al., 2001). It has been reported that cPLA2 is involved in various cytotoxic reactions, such as UV-induced cell injury in skin cells, oxidant-induced cytotoxicity, and TNF-α-mediated cell death (Leslie, 1997). The iPLA2 is also associated with apoptosis such as FasL-induced arachidonic acid release and membrane remodeling (Akiba and Sato, 2004; Atsumi et al., 1998). Recent reports demonstrated sPLA2-induced apoptosis in neuronal cells via DNA damage (Daniel and DeCoster, 2004; DeCoster et al., 2002). One of the potential mechanisms of how sPLA2 exerts its apoptotic activities is binding of sPLA2s to a cell surface receptor and generation of apoptotic signaling. Hernandez et al. suggested that the engagement of M-type receptor by sPLA2 activates mitogen-activated protein kinase (MAPK) cascade, and upregulates the cell surface expression of Fas (APO-1/ CD95) ligand (FasL) (Fuentes et al., 2002). Group IIA PLA2 has been shown to bind to apoptotic cell surface heparan sulfate proteoglycan with high affinity, which may enhance their ability to generate inflammatory signals (Boilard et al., 2003). However, the precise apoptotic processes still remain to be elucidated. The aim of the current study, therefore, is to investigate the apoptosis mechanism of group IB PLA2 in macrophages. Our data showed apoptosis of macrophages in response to IB PLA2, accompanying with activation of caspase cascade and PARP cleavage. We also found upregulation of TNF-α and cytochrome c from IB PLA2-treated cells, implying that they might function as secondary mediators for IB PLA2-mediated apoptosis. These findings are important for us to understand diverse cellular activities of IB PLA2. 2. Materials and methods 2.1. Antibodies and reagents Group IB PLA2 form porcine pancreas, PI and Annexin Vphycoerythirin (PE) were purchased from the Sigma-Aldrich (St. Louis, MO). The commercial PLA2 was purified with C18 column chromatography and the endotoxin content was determined using Lymulus amoebocyte lysate (LAL) kit (BioWhittaker, Walkersville, MD); the ECL reagent from NEN Life Science (Boston, MA); RPMI 1640 from Life Technologies (Grand Island, NY); TNF-α ELISA kit from R&D (Minneapolis, MN); fetal calf serum from Hyclone (Logan, UT). Antibodies against procaspase-3, -7, -9, -10 and PARP were obtained from Cell Signaling Technology Inc. (Beverly,

MA), and antibody against cytochrome c was from Santa Cruz Biotechnology (Santa Cruz, CA). 2.2. Cell culture RAW 264.7 macrophage cell line was obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum, grown at 37 °C, in 5% CO2 fully humidified air and subcultured twice a week. They were then seeded in a plate, and stimulated with IB PLA2 in the presence or absence of inhibitors, for an indicated time, ranging from a few minutes to 24 h. 2.3. Cell viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay is another standard method used to assess cell viability. Cells were seeded in 96-well plate (Nunc, Denmark). After exposure to IB PLA2 for indicated time, 50 μl MTT (Sigma) solution (2 mg/ml in PBS) was added to each well and the plates were incubated for additional 4 h at 37 °C. MTT solution in medium was aspirated off. To achieve solubilization of the formazan crystal formed in viable cells, 200 μl dimethyl sulfoxide (DMSO) was added to each well. The absorbance was read at 540 nm on microwell plate reader with DMSO as the blank. 2.4. Apoptosis assay Detection of apoptotic oligonucleosomal DNA fragmentation was performed. Cells were exposed to IB PLA2 for 24 h and the cells were collected by centrifugation. Cell pellets were resuspended in lysis buffer (5 mM Tris, pH 7.4, 20 mM EDTA, and 0.5% Triton X-100) and treated with RNase A at 37 °C for 1 h. Lysates were extracted with an equal volume of phenol/ chloroform/isoamyl alcohol (25:24:1, v:v:v) and DNA precipitated with 2 volumes of ice-cold absolute ethanol. The DNA was collected in a centrifuge and resuspended in 20 μl of 10 mM Tris–HCl, pH 8.0 and 10 mM EDTA. After digesting RNA with RNase (20 μg/ml) at 37 °C for 30 min, DNA samples were subjected to electrophoresis in 2% agarose gel. Confirmation of apoptosis was quantified by measurement of externalized phosphatidylserine residues as detected using Annexin V-PE. After exposure to IB PLA2, cells were collected and washed with ice-cold PBS and then suspended in 500 μl of Annexin V binding buffer. A 100-μl aliquot was taken, 5 μl of Annexin V-PE was added, and the mixture was incubated for 15 min at room temperature in the dark. After the addition of 400 μl of binding buffer, the cells were acquired on a FACS Calibur Flow Cytometer and analyzed using CellQuest software. The results are shown as a histogram with Annexin V-positive cells calculated as apoptotic cells. For the quantification of apoptotic nuclei, cells were incubated with 50 μg/ml PI and Rnase A for 30 min at room temperature followed by washing and the samples were processed by flow cytometer.

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2.7. Western blot analysis RAW 264.7 cells were plated in 6-well plates and treated with IB PLA2. The cells were washed with cold-PBS, scraped off and pelleted at 700×g at 4 °C. Cell pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1 mM PMSF, and protease inhibitor cocktail), the preparation cleared by centrifugation, and the supernatant retained as a whole-cell lysate. Protein (30– 50 μg) was separated by 8% reducing SDS-PAGE and transferred to nitrocellulose membrane in 20% methanol, 25 mM Tris, and 192 mM glycine, which was then blocked with 5% non-fat dry milk in Tween–Tris buffered saline (TTBS) (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.05% Tween-20) and subsequently incubated with the indicated antibodies for 4 h. The membrane was then washed and incubated for 1 h with

Fig. 1. Effect of IB PLA2 on survival of RAW 264.7 macrophages. A. RAW 264.7 cells were treated with the indicated concentrations of group IB PLA2. MTT reducing activity was determined 24 h later. B. Macrophages were treated with 1 μM IB PLA2. MTT reducing activity was determined at the indicated time points. The values for MTT assay are represented as mean ± S.D.

2.5. TNF-α measurements Cells were treated with IB PLA2 for the indicated time and conditioned media from cells were collected. The level of TNFα in culture media was measured by enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions (R&D Systems). 2.6. Isolation of cytosol and mitochondrial fractions Cells were plated in Φ60 mm dishes at a density of 2 × 106 cells/dish. After treatment of IB PLA2, the cells were scraped off and collected by centrifugation. The cell pellets were washed once using ice-cold PBS followed by an additional centrifugation. Cell pellets were resuspended in 200 μl of cold lysis buffer (20 mM HEPES–KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA/EGTA, 0.1 mM Phenylmethylsulphonylfluoride (PMSF), and protease inhibitor cocktail). Homogenizer lysed cells and the homogenates were centrifuged at 700×g for 5 min at 4 °C. The supernatants were then centrifuged at 10,000×g for 15 min at 4 °C. The resulting mitochondrial pellets were resuspended in 50 μl of cold lysis buffer. The remaining supernatant was again subjected to centrifugation at 100,000×g for 1 h to obtain the cytosolic fractions which were used for detection of cytochrome c.

Fig. 2. FACS analysis of apoptosis using PI and Annexin V, and DNA fragmentation. A and B. Macrophages were treated with the indicated concentrations of IB PLA2 for 24 h and analyzed for apoptosis by PI staining (A) and Annexin V-PE binding (B) using flow cytometry. C. Cells were cultured in the presence or absence of 1 μM IB PLA2 for 24 h and DNA fragmentation was analyzed as described under Materials and methods. Data obtained from one representative experiment performed in quadruplicate are shown.

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cells which are in apoptotic phase (Fig. 2B). Moreover, a ladder of DNA fragment which is nucleosomal-sized was observed from genomic DNA preparation of IB PLA2-treated RAW 264.7 cells (Fig. 2C). IB PLA2 is a very well known protein which is too stable to be denatured by any treatments such as chemicals and heat. We found that enzymatic activity and apoptotic effect of IB PLA2 were remained even after heat treatment. Therefore, we fail to see loss of apoptotic activity by heat-treated IB PLA2 (data not shown). This result reminds us that there is no association between enzymatic activity and apoptotic effect. 3.2. Caspases-3/7 activation and PARP degradation in cells treated with IB PLA2 Because apoptosis is accompanied by caspase activation, we analyzed whether IB PLA2 treatment gives rise to the activation of caspase-3 and -7, two key executioners of apoptosis. Western

Fig. 3. Caspase-3 and -7 activation and PARP degradation by IB PLA2. A and B. Macrophages were treated with the indicated concentrations of IB PLA2 for 24 h (A, C) or treated with 1 μM IB PLA2 for various times (B). The cells were harvested and whole cell lysates were subjected to Western blot analysis with antibodies against cleaved caspase-3, procaspase-3, -7 or PARP. Western blot data shown are representative of four independent experiments.

secondary antibodies conjugated to HRP, rewashed and developed using an ECL system. 3. Results 3.1. Group IB PLA2 induced apoptosis in RAW 264.7 macrophages To investigate the effect of IB PLA2 on cell survival, we have used murine macrophages, RAW 264.7 cell. Following treatment of cells with IB PLA2, cell viability was determined by MTT assay. Addition of IB PLA2 reduced the total number of viable cells in a dose- and time-dependent manner (Fig. 1A and B). Of note, endotoxin content was determined using LAL kit and we found that its level was less than 1 EU. This result excludes a possible contamination of endotoxin which might affect IB PLA2-induced apoptosis. We also observed IB PLA2 effect on apoptosis in the presence of polymyxin B which is known to bind endotoxin and neutralize it. IB PLA2-induced apoptosis was not influenced by polymyxin B, confirming that there is no possibility of endotoxin contamination of IB PLA2 and effect on its apoptotic activity. Next, we examined if IB PLA2-treated cells show apoptotic features. IB PLA2-treated cells exhibited morphologic changes which are characteristic of apoptosis such as cell shrunken, rounded and detached from the dish (data not shown). Flow cytometry analysis using PI and Annexin V-PE revealed that IB PLA2 treatment induced dose-dependent increment of nonviable PI-positive cells (Fig. 2A) as well as Annexin V-positive

Fig. 4. Effect of IB PLA2 on TNF-α production, cytochrome c release and caspase-9/-10 activation. Macrophages were treated with 1 μM IB PLA2 for various times. Culture media were collected and TNF-α production was determined by ELISA (A). Total RNA was isolated from each sample and subjected to RT-PCR with specific primers for TNF-α and β-actin (B). C and E. Cells were treated with the indicated concentrations of IB PLA2 for 24 h. Whole cell lysates were prepared and electrophoresed on 10% SDS-PAGE, and membranes were used for Western blot analysis using a procaspase-9 or -10 antibody. D. Macrophages were treated with 1 μM IB PLA2 for the indicated times. Cytosolic fraction was prepared and used to detect cytochrome C. Western blot data shown are representative of three independent experiments. Values for the TNF-α production are represented as mean ± S.D.

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Fig. 5. Effects of TNF-α neutralizing antibody and caspase inhibitor on the IB PLA2-induced apoptosis. Macrophages were pre-treated with indicated concentrations of TNF-α neutralizing antibody (A) or caspase-3/7 inhibitor Ac-DNLD-CHO for 1 h and then stimulated with 1 μM IB PLA2 for 24 h. After incubation, cells were analyzed for apoptosis by PI staining. The values for the PI-positive cells are represented as mean ± S.D.

blot analysis showed that procaspases which are enzymatically inactive were disappeared. We found that as low as 0.25 μM of IB PLA2 could decrease the level of caspase-3 and -7 and reduction of caspases by IB PLA2 was dose- and timedependent. Both caspase-3 and -7 were gradually decreased within 24 h post treatment (Fig. 3A, B). Fig. 3C showed the increment of cleaved form of caspase-3, which would be expected that the level of the procaspase decreases with the concomitant appearance of a cleaved, active form of caspase. Since one of the major substrates for activated caspase-3 and -7 is known to be PARP, PARP degradation is a good marker for caspase-dependent apoptosis. Along with the activation of caspase-3 and -7, significant cleavage of the 116-kDa, fulllength PARP to the 85-kDa, cleaved PARP was observed in cells treated with IB PLA2, indicating a sequential event within IB PLA2-treated cells which is procaspase cleavage, activation of caspase, and degradation of PARP. 3.3. IB PLA2-induced apoptosis occurred via both TNF-αcaspase-10 and cytochrome c-caspase-9 pathways Apoptotic effect of IB PLA2 on macrophages could be exerted by a secondary mediator such as cytokines which can be induced by IB PLA2 treatment (Hernandez et al., 2002). Based on previous report, we monitored the amount of TNF-α in response to IB PLA2. IB PLA2 induced secretion of TNF-α into culture supernatants as detected using a specific ELISA. The effect of IB PLA2 was time-dependent (Fig. 4A). TNF-α

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mRNA level was also increased by IB PLA2 (Fig. 4B), suggesting that TNF-α is upregulated at transcriptional level. Caspase-10, one of the initiator caspases, is closely coupled to proapoptotic activity of TNF-α, although it is not the sole effector to deliver TNF-α-mediated apoptotic signal (Nunez et al., 1998). We observed activation of caspase-10 using Western blot to see the involvement of caspase-10-dependent proteolysis in IB PLA2-induced apoptosis. As we expected, significant degradation of procaspase-10 was observed in the IB PLA2treated macrophages (Fig. 4C), suggesting TNF-α and caspase10-dependent apoptosis in these cells. In the apoptotic signal transduction pathway, mitochondria play an essential role by releasing apoptogenic factors such as cytochrome c (Li et al., 1997; Marsden et al., 2002). Cytochrome c released into the cytosol is thought to trigger caspase activation downstream of mitochondria through binding to Apaf-1 and autoactivation of caspase-9. Activated caspase-9 also can activate additional caspases responsible for PARP cleavage. As shown in Fig. 4D, IB PLA2 increased the release of cytochrome c from mitochondria into cytoplasm at 6 h with further increment being observed at 18 h. In the same fashion, activated caspase-9 was generated after IB PLA2 stimulation (Fig. 4E). These data suggest that cytochrome c is one of the factors in IB PLA2-induced apoptosis and that its release may be related to the activation of caspase-9, which subsequently triggers the activation of caspase-3 and -7, and the cleavage of PARP. 3.4. IB PLA2-induced apoptosis was blocked by TNF-α neutralizing antibody and caspase-3 inhibitor To confirm TNF-α-mediated apoptotic effect of IB PLA2, we tested the effect of TNF-α neutralizing antibody on apoptosis in response to IB PLA2. As shown in Fig. 5A, the number of PIpositive apoptotic cells was significantly reduced by TNF-α neutralizing antibody. Moreover, the effect of TNF-α neutralizing antibody was concentration-dependent. We also tested the effect of caspase-3 inhibitor Ac-DNLDCHO on apoptosis in response to IB PLA2. IB PLA2-induced apoptosis was suppressed in the presence of caspase-3 inhibitor, demonstrating that caspase-3 activation is required for the IB PLA2-mediated apoptosis in RAW 264.7 cells. 4. Discussion It has been suggested that sPLA2 is one of the regulators in apoptosis. However, the mechanism by which sPLA2 acts upon apoptosis remains unclear. Here, we examined the effect of group IB PLA2 on the apoptosis in macrophages. IB PLA2 induced a dose- and time-dependent increase of apoptosis. The apoptotic activity of the IB PLA2 was associated with the release of TNF-α and cytochrome c release. In addition, both caspase-9 and -10-dependent cascades were involved in IB PLA2-induced apoptosis. PLA2 is the enzyme involved in the first step of arachidonic acid metabolism. Various forms of PLA2 have been characterized, purified, and cloned from diverse sources. Some reports

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indicate that sPLA2 plays a role in apoptosis. Yagami et al. observed apoptosis of cortical neuronal cells after treatment of group IIA PLA2, which is associated with prostaglandin D2 (PGD2) (Yagami et al., 2002). In addition, DeCoster et al. reported that sPLA2 potentiates glutamate-induced cell death in primary neuronal cells (DeCoster, 2003). On the other hand, another report suggests that sPLA2 suppresses apoptosis of certain cells including mast cells (Fonteh et al., 2001). Although sPLA2 displays apoptotic or anti-apoptotic activities in a variety of cell types, evidence for a link between sPLA2 and apoptosis has been shown in a few studies. It appears that the discrepancies among these reports were due largely to the different experimental conditions. In our experiment, group IB PLA2 exerted significant toxic effects on macrophage viability. Our data from several experiments such as PI staining, Annexin V staining, DNA fragmentation, and caspase activation demonstrated that IB PLA2 induced apoptosis in RAW 264.7 cell. To our knowledge, this is the first study showing a direct link between IB PLA2 and the mechanism of apoptosis in macrophages. Our finding is in conflict with the report by Arita et al. who demonstrated that IB PLA2 has an effect on the proliferation of the fibroblast (Arita et al., 1991). The basis for this disparity is not clear but it could be due to differences in cell types, the concentrations, or other unknown variables. In fact, the IB PLA2-mediated decrease of apoptosis at the low concentrations was reproducibly observed in different experiments of RAW 264.7 cells (Fig. 2A). Except during severe inflammation such as sepsis, rheumatoid arthritis, and multiple organ failure syndromes, in vivo concentration of sPLA2 was shown under the nanomolar range (Jamal et al., 1998; Partrick et al., 2001; Yokota et al., 1999). This raised the question why the concentration of IB PLA2 required in vitro was higher than that expressed in vivo. This discrepancy could be explained by the role of cell surface molecules, the presence of cofactors, and the sensitivity variation between endogenous sPLA2 and exogenous sPLA2 (Murakami et al., 1996). There are two major pathways for the induction of apoptosis: the receptor-mediated pathway and the mitochondria-dependent pathway. In both, a hallmark and downstream target of these apoptotic pathways is the activation of caspase-3 and -7 (Kominsky et al., 2002). Therefore, we examined caspase-3 and -7 activation in cells treated with IB PLA2 and found them to be dramatically decreased. We subsequently tested caspase-9 and -10 activation; the former is a downstream indicator of the mitochondrial apoptotic pathway, whereas the latter is a downstream indicator of activation of the receptor-mediated apoptotic pathway. We found that IB PLA2 treatment induced both caspase-9 and -10 activation, suggesting that IB PLA2-induced apoptosis in RAW 264.7 cells is mediated through both pathways, mitochondrial-mediated caspase-9 activation and receptor-mediated caspase-10 activation. Many studies suggested that sPLA2 stimulates the TNF-α production in monocytic cells (Hernandez et al., 2002; Triggiani et al., 2002) and increased FasL expression as a kind of intermediator of apoptosis (Peter and Krammer, 2003; Scaffidi et al., 1998). Therefore, we monitored changes in the expression of FasL and the production of TNF-α after IB PLA2 treatment.

There was no upregulation of FasL expression over 24 h while IB PLA2 induced TNF-α production into the culture media as detected by using a highly specific ELISA. These results suggest that TNF-α-mediated pathway is involved in IB PLA2induced apoptosis. Increased expression and release of sPLA2 have been found in many diseases including inflammation (Jamal et al., 1998; Partrick et al., 2001; Yokota et al., 1999). These conditions are usually associated with high blood levels of TNF-α (Triggiani et al., 2002). TNF-α is considered a mediator of the acute phase of diseases and their blood concentration is used as an indicator of the extent of disease (Akira and Kishimoto, 1992). Furthermore, macrophage is thought to be one of the major producers of circulating TNF-α (Hart et al., 1996). Thus, our results indicate that this is an effective stimulus for macrophage activation and it may contribute to the production of TNF-α, resulting in apoptosis. Besides death receptor-mediated activation of caspases, mitochondria-dependent caspase cascade is another important pathway of apoptosis. The natural procaspase-9 activation is related to the release of cytochrome c from mitochondria, which form a complex with Apaf-1. The Apaf-1 and cytochrome c complex recruits procaspase-9 and induces its autoactivation by aggregation (Li et al., 1997; Marsden et al., 2002). We observed that IB PLA2 induces translocation of cytochrome c from mitochondria to cytoplasm, indicating that during IB PLA2mediated apoptosis, mitochondrial integrity is changed. By making the mitochondria membrane permeable, IB PLA2 would facilitate the release of apoptotic proteins such as cytochrome c, which ultimately affects caspase-9 and later cleaves caspase-3/7. The role of sPLA2 in cell function has been explained through two mechanisms: (i) its catalytic activity; (ii) interaction with membrane receptor analogues. To date, most biological activities of sPLA2 have been attributed to its enzymatic capacity to hydrolyze membrane phospholipids and a requirement for relatively high concentrations of Ca2+ for catalysis. To determine whether catalytic activity is required to induce apoptosis, the effects of Ca2+ chelation on IB PLA2-induced apoptosis were examined. In the presence of EGTA, IB PLA2 did not affect its ability to induce apoptosis (data not shown), suggesting that its enzymatic activity is not required for the induction of apoptosis. The presence of PLA2 receptors in a variety of cells including smooth muscle, fibroblasts, and astrocytes has been documented (Hanasaki and Arita, 2002). Two distinct PLA2 receptors have been identified: the M-type and N-type. Next, we asked whether M-type PLA2 receptor mediates apoptosis, because this receptor is known to be highly expressed in RAW 264.7 cells and binds to IB PLA2 with high affinity (Cupillard et al., 1999). We established the stable cells overexpressing Mtype receptor and tested susceptibility of these cells to IB PLA2induced apoptosis. Overexpression of M-type receptor had no effect on apoptosis by sPLA2 (data not shown), suggesting that M-type PLA2 receptor might not be involved in IB PLA2mediated apoptosis in RAW 264.7 cells. In summary, our results demonstrated that IB PLA2 promotes apoptosis in RAW 264.7 cells via the TNF-α-caspase-10 and cytochrome c-caspase-9 pathways. Although biological relevance

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