Secretory phospholipase A2 induces apoptosis via a mechanism involving ceramide generation

Biochimica et Biophysica Acta 1581 (2002) 75 – 88 www.bba-direct.com

Secretory phospholipase A2 induces apoptosis via a mechanism involving ceramide generation Sheng Zhao a,b, Xiao-Yan Du b, Min-Qiang Chai a,b, Jun-Song Chen a,b, Yuan-Chong Zhou b, Jian-Guo Song a,b,* a

Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Box 25, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China b Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Box 25, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China Received 3 October 2001; received in revised form 8 January 2002; accepted 17 January 2002

Abstract Secretory phospholipase A2 (sPLA2) plays important roles in cellular signaling and various biological events. In this study, we examined the biological effects and the potential signaling mechanism of purified sPLA2 in MV1Lu cells. Three types of snake venom sPLA2 were purified and their enzymatic activities were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by determining their effects on the induction of arachidonic acid (AA) release. The purified sPLA2 induced apoptosis in Mv1Lu cells in a doseand time-dependent manner, and was associated with a rapid increase in the intracellular ceramide level. Similar apoptotic effects were observed in Mv1Lu cells treated with exogenous ceramide analog, C2- and C8-ceramide. Moreover, treatment of cells with sphingomyelinase (SMase), which reduced the intracellular SM level, enhanced the apoptotic response to sPLA2s. sPLA2s also displayed an inhibitory effect on bradykinin-induced phospholipase D (PLD) activity, which can be imitated by exogenous ceramide. Our data indicate that sPLA2 induces cell apoptosis via a mechanism involving increased ceramide generation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Apoptosis; Ceramide; Phospholipase D; Secretory phospholipase A2; Sphingomyelinase; Sphingomyelin

1. Introduction Phospholipase A2 (PLA2) is a diverse class of enzymes that catalyzes the hydrolysis of sn-2 fatty acyl bond of phospholipids with the concomitant generation of lysophospholipids and free fatty acids [1,2]. PLA2 plays important roles in various biological events, including phospholipid metabolism, immune defense, and signal transduction [3]. Hydrolysis of phospholipids by PLA2 generates arachidonic acid (AA) and lysophospholipids that are important members of the lipid signaling molecules. AA is a precursor for the synthesis of prostaglandin, leukotriene, and thromboxane. It Abbreviations: AA, arachidonic acid; sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; LPA, lysophosphatidic acid; PA, phosphatidic acid; PBut, phosphatidylbutanol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PLD, phospholipase D; PMA, phorbol-12myristate-13-acetate; PS, phosphatidylserine; SM, sphingomyelin; SMase, sphingomyelinase; TGF-h, transforming growth factor beta * Corresponding author. Box 25, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Tel./fax: +86-21-643-17660. E-mail address: [email protected] (J.-G. Song).

has been reported that introduction of arachidonic acid into heart mitochondria causes the uncoupling of succinate-supported respiration and cytochrome c release [4]. In human liver, arachidonic acid mediates co-oxidation of all-transretinoic acid in the microsomal fractions [5]. Exposure of spinal cord neurons to free AA induces activation of caspase and initiates apoptotic cell death [6]. Lysophosphatidic acid is an important lipid mediator that is implicated in a variety of cellular functions including stimulation of cell proliferation and differentiation, Ca2 + mobilization, tumor cell invasion, chemotaxis, and other functions [7]. Snake venom is known as one of the richest sources of secretory phospholipase A2 (sPLA2) and contains several types of sPLA2 that are structurally homologous to the type II sPLA2 found in mammalian species and contribute to some physiological effects of the venom. The function of the sPLA2 may result from its direct effect by hydrolysis of membrane phospholipids and from its indirect effect by liberation of bioactive lipid signaling molecules or their precursors. AA has been reported to mediate ceramide formation through a sphingomyelinase (SMase)-dependent pathway [8]. Ceramide has been shown to inhibit PLD activity both

1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 2 ) 0 0 1 2 2 - 1

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in vitro and in intact cells [9,10]. Functional cross-talk between either cytosolic PLA2 or sPLA2 and PLD in HEK293 cells has also been reported recently [11]. Phosphatidylcholine (PC)-specific PLD catalyzes the hydrolysis of PC to generate choline and phosphatidic acid (PA); the latter can be further hydrolyzed to diacylglycerol (DAG) by the action of a PA phosphatase. PA itself is an intracellular bioactive molecule, which is involved in the cell growth, differentiation, and other cellular functions [12 – 14]. A number of lines of evidence have indicated that ceramide plays an important role in the induction of apoptosis by various stimuli including cancer chemotherapy reagents, ultraviolet exposure, ionizing radiation, cytokines (e.g., TNF-a and interferon-g), and heat shock, etc. [15 – 17]. Hydrolysis of sphingomyelin (SM) by SMase has been reported as the most common pathway that leads to the increase in the generation of ceramide, while other pathways may also influence the intracellular level of ceramide [18,19]. Recent studies show that oxalate-induced increase in ceramide can be attenuated by pretreatment with AACOCF3, a specific cytosolic PLA2 (cPLA2) inhibitor [20], suggesting a role for cytosolic PLA2. It has been reported that PLA2 activity is modulated by SM, cholesterol, and annexin VI [21,22], which can change the membrane susceptibility to PLA2 or competitively inhibit PLA2. In this report, we purified three types of sPLA2, previously described as acidic PLA2, basic PLA2, and neutral PLA2. The hydrolytic activities of these enzymes on phospholipids were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by measuring their effects on the induction of cellular AA release. The effects of sPLA2 on the induction of apoptotic response in Mv1Lu and A-431 cells were examined and compared. The potential signaling mechanism for sPLA2induced apoptosis in Mv1Lu cells was further investigated.

Biotech Inc. Mink lung epithelial cell line (Mv1Lu cell) was purchased from American Type Culture Collection (ATCC, Manassas, VA). Cell culture reagents and fetal bovine serum (FCS) were purchased from Life Technologies (Grand Island, NY). EN 3HANCEk spray, [ 3H]-myristic acid (12.5 Ci/mmol), [3H]-AA (100 Ci/mmol), and [methyl-3H]-thymidine (83.60 Ci/mmol) were bought from NEN Life Science Products (Boston, MA). [3H]-serine (1.04 TBq/ mmol), [N-methyl-14C]-SM (47.0 mCi/mmol), and BCS scintillation cocktail were bought from Amersham Pharmacia Biotech (Buckinghamshire, UK). Phosphatidylbutanol (PBut) standard was bought from Avanti Polar Lipids (Alabaster, AL). 1-Butanol was bought from Aldrich Chemical Company, Inc. (Milwaukee, WI). PMA, bradykinin, were purchased from Calbiochem (La Jolla, CA). Silica Gel ˚ thin layer chromatography (TLC) plates were pur60 A chased from Whatman (Clifton, NJ). The neutral SMase (from Bacillus cereus) and all other reagents were bought form Sigma (St. Louis, MO) unless otherwise indicated. 2.2. Purification and isoelectric focusing electrophoresis of sPLA2s Three different types of sPLA2 from the venom of Agkistrodon halys pallas were purified as previously described [23]. They were designated as acidic PLA2 (APLA2), neutral PLA2 (NPLA2), and basic PLA2 (BPLA2) according to their isoelectric points, which are 4.5, 6.9, and 9.3, respectively. The isoelectric points of sPLA2s were determined by isoelectric focus in 7.5% gel with pharmalyte (pH 3 –10) using the Model 111 Mini IEF Cell of Bio-Rad and then stained as per manufacturers instructions. The pH value was referenced by the IEF standard. The concentrations of the purified sPLA2s were determined using the protein assay kit with BSA as standard. 2.3. Cell culture and labeling

2. Materials and methods 2.1. Materials Model 111 Mini Isoelectric Focusing (IEF) Cell, IEF standard (Cat. No. 161-0310) and protein assay kit (based on Bradford method) were purchased from Bio-Rad. Pharmalyte (pH 3– 10) was bought from Amersham Pharmacia

The Mv1Lu cells were cultured in MEM and A-431 cells cultured in DMEM supplemented with 10% FCS, 100 AM non-essential amino acid (for Mv1Lu), 100 units/ml of penicillin, and 100 Ag/ml of streptomycin. Cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 jC. At 80% confluence, cells were incubated in serumstarved medium containing 0.5% bovine serum albumin

Fig. 1. Enzymatic activities of purified sPLA2s. (A) Isoelectric points of three types of sPLA2. The purified sPLA2s were dissolved in H2O and loaded onto the isoelectrofocusing polyacrylamide gel. The electrophoresis was first conducted for 15 min each at 100 and 200 V, respectively to prevent overeating and subsequent dehydration of the gel. Finally, the voltage was increased to 450 V for an additional 60 min. (B) [3H]-PE, -PA, -PS, -PC (0.1 ACi for each reaction) prepared from [3H]-myristate labeled Mv1Lu cells, and [14C]-sphingomyelin were used as the substrates for sPLA2. Final concentration of each sPLA2 was 1 Ag/ml. The products [3H]-LPE, LPA, LPS, LPC, and [14C]-phosphocholine generated were determined by the methods as described in Materials and methods. For determining whether any SMase contamination exists in the purified sPLA2s, sphingomyelin was used as substrate. The acidic, neutral plus Mg2 + , and neutral without Mg2 + reaction buffers were used to determine acidic, neutral Mg2 + -dependent and -independent SMase activity. One representative result from three independent experiments was shown. (C) and (D) Mv1Lu cells were labeled with [3H]-arachidonic acid overnight and the cells were washed three times by PBS following 30 min incubation in 1.5 ml serum-starved medium before addition of sPLA2 (1 Ag/ml). The extracellular and intracellular arachidonic acids (AA) released were counted by liquid scintillation counter. Data are the means F SD of at least three independent experiments performed in duplicate. The average cpm for untreated controls for (C) and (D) were 7806 and 493, respectively.

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(BSA) (for Mv1Lu) or 1% new born calf serum (NCS) (for A-431), and labeled with radioactive compound for 16 h as indicated. For labeling glycerol phospholipids, 2 ACi/ml [3H]-AA or 3 ACi/ml [3H]-myristic acid was used. For labeling ceramide, 4 ACi/ml [3H]-serine was used. When necessary, the original labeling materials in ethanol were concentrated through Speed-Vac concentrator to make the final concentration of ethanol in the medium less than 0.1%. 2.4. Extraction and analysis of lipids Cells were incubated with reagents for the indicated time, the treatment was terminated and the total lipids in the organic phase were extracted and then loaded onto TLC plates for analysis as previously described [24]. Ceramide

was separated on TLC plates by the solvent system of hexane/diethyl ether/methanol/acetic acid (90:20:4:3, v/v). PLD activity was determined by measuring the formation of PBut, a unique product of transphosphatidylation reaction catalyzed by PLD [25]. Cells were incubated in the presence of 0.35% 1-butanol added 5 min before the treatment with other reagents. PBut was separated on TLC plate by the upper phase of a solvent system of ethyl acetate/trimethylpentane/acetic acid/water (90:50:20:100, v/v). Lipids were visualized by iodine staining and referenced with the lipid standards. The TLC plate was sprayed with EN3HANCEk, wrapped with Saran Wrap and exposed by autoradiography with a Kodak film at 80 jC for 5 – 8 days. Ceramide and phosphatidylbutanol were scrapped from TLC plates into scintillation vials. Lipid molecules were eluted out of the gel

Fig. 2. sPLA2 induces cell death in Mv1Lu Cells. Mv1Lu cells were incubated in serum-starved medium overnight and then treated with (1 Ag/ml) sPLA2. (A) Cells were treated with indicated concentration of sPLA2 for 36 h. (B) Cells were treated with sPLA2 for the times as shown. The viable cells were determined by at least three independent crystal violet assays in quadruplicate. (C) AO/EB stained the cells after 24 h sPLA2 treatment. Arrows point some of the cells that showed chromatin condensation. (D) DNA content analysis was conducted after 24-h treatment with sPLA2s by flow cytometry. The data of (A) and (B) are the means F range of two independent experiments in triplicate.

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by adding 500 Al of methanol and then mixed with 2.5 ml of BCS scintillation cocktail. The radioactivity was determined by liquid-scintillation spectrometry.

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CaCl2, 50 mM Tris – HCl, pH 7.0) containing [3H]-myristatelabeled lipid substrate (0.1 ACi for each reaction). After 30min incubation at 37 jC, the reaction was stopped and lipids were extracted and analyzed.

2.5. In vitro analysis of sPLA2 activity 2.6. SMase activity assay The [3H]-myristate lipid substrates for determination of PLA2s activities, including phosphatidylethanolamine (PE), PA, phosphatidylserine (PS), and PC, were prepared by extracting [3H]-myristate-labeled cellular lipids from Mv1Lu cells. The lipids were separated by TLC and re-extracted with CH3Cl3/MeOH and redissolved in ethanol. To determine the inhibitory effect of SM on sPLA2 activity, SM and purified [3H]-labeled PC were added together to the reaction buffer with 0.1% Triton-X-100 for higher solubility. The activity of each PLA2 was determined in a 100-Al reaction buffer (2 mM

SMase activity was quantified as previously described [26,27] with minor modifications. Purified sPLA2s were assayed in buffers for the three different SMase as follows: (1) neutral Mg2 + -dependent: 0.1% Triton X-100, 5 mM MgCl2, and 20 mM Hepes (pH 7.4); (2) neutral Mg2 + independent: 0.1% Triton X-100, and 20 mM Hepes (pH 7.4); (3) acidic: 0.1% Triton X-100, and 0.1 M sodium acetate (pH 5.0). Each reaction system contained 200 AM [14C]-SM (0.1 ACi/ml).

Fig. 3. sPLA2 stimulates ceramide generation. Mv1Lu cells cultured in 60-mm plates were labeled with [3H]-serine overnight, and then treated with indicated reagents for indicated times. Lipids were extracted and analyzed as described in Materials and methods. (A) Three purified sPLA2 (1 Ag/ml) rapidly induced the cellular ceramide generation. (B) Cells were treated with AA or LPA for 2 h, and the ceramide generation was determined. (C) Ceramide generation within a period of 2 h was determined after cells were treated with 50 AM AA or 500 nM LPA. (D) Ceramide generation within period of 16 h was determined after cells were treated with 50 AM AA or 500 nM LPA. Data for (A, B, and C) represent the means F SD of five independent experiments in duplicate. Data for (D) represent the means F range of two independent experiments in duplicate. The average cpm of [3H]-serine-labeled ceramide for untreated controls (for A, B, C, and D) were 337, 196, 196, and 268, respectively.

80 S. Zhao et al. / Biochimica et Biophysica Acta 1581 (2002) 75–88 Fig. 4. Effects of exogenous ceramide on the apoptosis of Mv1Lu cells. Mv1Lu cells were incubated in serum-starved medium overnight and then treated with indicated reagents. (A) Cells were treated with various concentrations of C2- or C8-ceramide for 48 h. (B) Cells were treated with C2- or C8-ceramide (10 AM) for the indicated times. Data represent two independent experiments in quadruplicate. (C) AO/EB stained the cells after 24 h C2- or C8-ceramide (25 AM) treatment, arrows point some of the cells that appeared chromatin condensation. (D) DNA content analysis was taken as a measure of the apoptotic rate after indicated treatment of cells with C2- or C8-ceramide (25 AM) for 24 h. (E) The effect of AA or LPA on the viability of cells. Mv1Lu cells were treated with 25 AM AA or 500 nM LPA for the indicated time. The viable cells remaining were determined. Data of (A), (B), and (E) are the means F SD of three independent crystal violet assays in quadruplicate.

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rate of the cell population. After indicated treatment, cells were trypsinized and fixed with 70% ethanol for over an hour. Cells were then pelleted and washed with PBS plus 20 mM EDTA. RNA was removed by adding RNase (1 mg/ml) at 37 jC for at least 2 h. Finally, the cells were stained with propidium iodide (final concentration: 30 Ag/ml) and analyzed the DNA content by FACS (Becton Dickinson FACSCalibur, the excitation wavelength is 488 nm). 2.11. Statistical analysis Results are presented as means F standard deviations (SD) for the number of experiments indicated. For statistical analysis, Student’s t-test was performed. Differences were considered significant at a level of P < 0.05. Fig. 4 (continued).

3. Results 2.7. Determination of arachidonic acid release Cells were labeled with [3H]-AA overnight, then washed for three times with phosphate-buffered saline (PBS) and further incubated in serum-starved medium for 30 min before treatment. For measuring the extracellular AA released from cells in response to sPLA2 treatment, the medium was collected, mixed with BCS scintillation cocktail and counted by liquid scintillation counter. To measure the intracellular AA released, the reaction was stopped and the lipids were extracted and separated on TLC.

3.1. Characterization of purified secretory PLA2 sPLA2 acts on several kinds of glycerol phospholipid both in vivo and in vitro. To study the role of purified

2.8. Cell viability assay The cell viability was detected by crystal violet in 96well plate [28]. Briefly, the quiescent medium was removed and the attached viable cells were fixed with 4% formalin for 30 min, then washed three times with water and stained with 0.5% crystal violet for 30 min. The plates were washed again with water for three times, then incubated with 100 Al of 10% acetic acid for 3 h. Optical density (OD) was measured at 570 nm. 2.9. Acridine orange/ethidium bromide (AO/EB) staining for chromatin condensation AO/EB staining for morphologic examination was conducted as described [29] with minor modifications. Cells were cultured in 35-mm dishes and treated as indicated. The cells were stained with AO/EB solution (the final concentration of AO and EB was 2 Ag/ml) and photographs were taken under a fluorescent microscope. 2.10. Flow cytometry analysis Cell apoptotic rate was quantitatively determined by flow cytometry analysis [29,30]. The percentage of cells with a sub-G1 DNA content was taken as a measure of the apoptotic

Fig. 5. The effects of sPLA2 and ceramide on PLD activity. Mv1Lu cells cultured in 60-mm plates were labeled with [3H]-myristate overnight, and then treated with PLA2 (1 Ag/ml) or with ceramide (25 AM) for indicated time. Cells were then stimulated with bradykinin (1 AM) (A) or PMA (100 ng/ ml) (B) for 30 min. PLD activity was measured as described in Materials and methods. Data are the means F SD of at least three independent experiments in duplicate. The average cpm of PBut labeled with [3H]-myristate for the untreated controls was 618 cpm. (**) Significant at P < 0.001.

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Fig. 6. The effects of SMase on sPLA2-induced cell death. Quiescent Mv1Lu and A-431 cells were pre-treated with SMase (5 mU/ml) for 10 min and then coincubated with sPLA2 (1 Ag/ml). The concentration of C2-ceramide or TGF-beta1 was 25 AM or 10 ng/ml. The viability of Mv1Lu (A) or A-431 (B) cells was determined after incubation for 36 h. Data are the means F SD of three independent experiments in quadruplicate. (C) AO/EB stained the A-431 cells after 24 h treatment, the arrows point out the cells which appeared after chromatin condensation. (D) DNA content analysis was conducted after 24 h indicated treatment. (E) The cellular background SMase activity of Mv1Lu and A-431 cells were determined as described in the Materials and methods. Data are the mean F range or SD of at least two independent experiments in duplicate.

PLA2s, we first examined their purity by isoelectric focusing. The purified secretory PLA2s were distinguished by their different isoelectric points, which were 4.5, 6.9 and 9.3 (Fig. 1A). They were therefore designated as acidic sPLA2 (APLA 2 ), neutral sPLA 2 (NPLA 2 ), and basic sPLA 2 (BPLA2), respectively. We then examined their enzymatic

activities by using the [3H]-myristate-labeled PE, PA, PS, and PC prepared from Mv1Lu cells as the substrates. sPLA2 catalyzes the hydrolysis of PE, PA, PS, and PC catalyzed to generate lysophosphatidylethanolamine (LPE), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and lysophosphatidylcholine (LPC), respectively. The levels of

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the hydrolytic products, which reflect the enzymatic activity of each sPLA2, were determined. As shown in Fig. 1B, APLA2, BPLA2, and NPLA2 showed similar hydrolytic activity on PS and PC. The activities of BPLA2 and NPLA2 on PE and PA were about double that of the APLA2. Moreover, none of the three sPLA2s show any hydrolytic activity on SM in various reaction buffers, which precluded the possibility that the purified enzymes were contaminated with any SMase. To further determine the enzymatic activities of these sPLA2s, their effects on inducing AA release in intact cells were measured. Fig. 1C showed that all three sPLA2s could induce a rapid increase in extracellular AA, which peaked at 10 min after the treatment. All sPLA2 also stimulated a rapid increase in intracellular AA level (Fig. 1D). Unlike the increase in extracellular AA, the increase in intracellular AA occurred more rapidly, peaking at 1 min after the treatment. APLA2 seemed to be more potent than BPLA2 and NPLA2 in stimulating the release of intracellular arachidonic acid. The differences in their ability to hydrolyze the phospholipid substrates and in their effects on the AA release among the three sPLA2s may contribute to their different effects or potency on signaling events in Mv1Lu cells. 3.2. sPLA2 induces apoptosis in Mv1Lu cells To investigate the biological effects of the purified sPLA2, cells at 90% confluence were treated with different concentrations of sPLA2 for the times indicated. Results shown in Fig. 2A demonstrated concentration-dependent apoptosis of Mv1Lu cells in response to sPLA2 treatment. At the concentration of 5 Ag/ml, sPLA2s treatment resulted in about 60% decrease in the number of viable cells. Longer exposure of cells to sPLA2s caused an increase in the death of cells, indicating a time-dependent manner of cytotoxicity induced by sPLA2s (Fig. 2B). Fig. 2C shows the sPLA2sinduced cell apoptosis as examined by AO/EB staining, in which condensed chromatins of cells were observed. Each sPLA2-induced apoptosis was also examined and quantified by measuring the DNA content using flow cytometry (Fig. 2D). It appeared from this result that BPLA2 and NPLA2 are more potent than the APLA2, which is consistent with the report that BPLA2 of snake venom is more poisonous than the APLA2 [31]. 3.3. sPLA2s increase intracellular ceramide formation To investigate the potential mechanism of sPLA2sinduced apoptosis, we examined ceramide levels in Mv1Lu cells treated with different types of sPLA2. As shown in Fig. 3A, sPLA2s induced a rapid increase of ceramide levels in Mv1Lu cells, which could be observed at 1 h and continued for over 10 h. Among the three types of purified sPLA2, the BPLA2 and NPLA2 showed relatively higher ability in inducing ceramide generation than APLA2. The results demonstrated that ceramide is a possible mediator in

sPLA2-induced cell apoptosis in Mv1Lu cells. Treatment of cells with exogenous AA or LPA does not affect the intracellular ceramide level (Fig. 3B, C, D), indicating that increased intracellular ceramide was not resulted from these direct products of sPLA2s. 3.4. Exogenous ceramide analogs mimic the effects of sPLA2 As an intracellular lipid signaling molecule, ceramide is well known for its ability to induce cell death. To further investigate whether ceramide plays a role in sPLA2sinduced apoptosis in Mv1Lu cells, we examined the effect on Mv1Lu cells of cell permeable exogenous ceramide analogs, C2- and C8-ceramides. As shown in Fig. 4A and B, both C2- and C8-ceramide treatment of cells caused a decrease in the viability of Mv1Lu cells, in a dose- and time-dependent manner with a pattern similar to that induced by sPLA2. AO/EB staining and DNA content analysis as shown in Fig. 4C and D further demonstrate that both C2- and C8-ceramide were able to induce apoptotic cell death in Mv1Lu cells. Consistent with the results shown in Fig. 3B and C, neither exogenous arachidonic acid nor LPA induced significant apoptotic response in Mv1Lu cells (Fig. 4E), indicating that the apoptotic effect of sPLA2 is unlikely to have resulted from the direct hydrolytic products, AA and LPA. 3.5. sPLA2 and ceramide inhibit PLD activity The interaction between the glycerol phospholipid signaling pathways and the sphingolipid signaling pathways is implicated in many physiological and pathological events. Since sPLA2 induces ceramide generation in Mv1Lu cells, which may also result in the inhibition of PLD activity, we therefore investigated the effects of the three purified sPLA2s on bradykinin- and PMA-induced PLD activities within the early treatment time. As shown in Fig. 5A, C2ceramide, BPLA2, and NPLA2 inhibited the bradykinininduced PLD activity. NPLA2, as well as ceramide, also has an inhibitory effect on PMA-induced PLD activity. APLA2, which was less potent in inducing the intracellular ceramide formation, showed no obvious effect on the PLD activity induced by either bradykinin or PMA (Fig. 5A and B). The results indicate that ceramide may be involved in the inhibition of agonist-induced PLD activity by the sPLA2, further suggesting that ceramide generation is an early event in sPLA2-induced signal transduction. 3.6. SMase enhances the apoptotic effects of sPLA2 SMase can increase ceramide level by catalyzing the hydrolysis of SM. We therefore investigated the effects of sPLA2 in the presence of the neutral SMase. Treatment of cells with neutral SMase alone slightly reduced the viability of Mv1Lu cells but not in A-431 cells, a carcinoma epithelia

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cell line, which is insensitive to sPLA2 treatment. However, when BPLA2 and NPLA2 were used in combination with SMase, the apoptotic effect of sPLA2 was greatly enhanced (Fig. 6A). Addition of SMase to cells treated with acidic sPLA2 only exhibited an additive effect, indicating a subtle difference in different types of the sPLA2. Transforming growth factor-h1 (TGF-h1), which inhibits the proliferation of epithelial cells including Mv1Lu cells, also showed an additive effect on the apoptotic effect of sPLA2, whereas, wortmanin, an inhibitor of phosphatidylinositol-3 kinase that functions as a survival factor, has no effect on the sPLA2-induced death of Mv1Lu cells (data not shown). However, when A-431 cells were treated with SMase together with BPLA2 or NPLA2, the cell viability was decreased greatly (Fig. 6B). AO/EB staining and the FACS analysis showed the same enhancement of SMase on the sPLA2-induced apoptosis (Fig. 6C and D). In contrast to SMase, ceramide itself also induced the apoptosis in A-431 cells. Furthermore, the background activity of Mg2 + dependent neutral SMase in Mv1Lu cells was much higher than that of A-431 (Fig. 6E), which may render the Mv1Lu cells more sensitive to sPLA2. 3.7. SM inhibits the sPLA2 activity

Fig. 7. Sphingomyelin inhibits the sPLA2 activity and the AA release. (A) The activity of purified sPLA2 (1 Ag/ml) was assayed in the reaction buffer containing indicated amounts of sphingomyelin and 25,000 cpm [3H]-PC purified from labeled Mv1Lu cells. The reaction was carried out for 30 min at 37 jC. (B) A-431 and Mv1Lu cells were incubated with 5 mU/ml neutral SMase for 4 h, and the cellular sphingomyelin levels were then determined. The data are the means F range of two independent experiments in duplicate. The average cpm of the background [3H]-Lyso-PC for the untreated controls is 585. (C) Mv1Lu cells were labeled with [3H]-AA overnight and then pretreated with 5 mU/ml SMase for 2 h. The cells were washed three times following 30 min incubation in 1.5 ml serum-starved medium. sPLA2 (1 Ag/ml) was then added and the cells were further incubated for 10 min. The extracellular AA released was counted by liquid scintillation counter. The data are the means F range of two independent experiments in triplicate. The average cpm of untreated controls was 1027.

To understand how SMase enhances the sPLA2-induced apoptosis, we investigated the effect of SM on the enzymatic activity of sPLA2. By using [3H]-PC extracted from Mv1Lu cells as substrate and by in vitro assay, we found that SM, the substrate of SMase, strongly and dose-dependently inhibited the enzyme activities of BPLA2 and NPLA2 (Fig. 7A). The inhibitory effect of SM on APLA2 was much less than that on BPLA2 and NPLA2. This may explain why SMase failed to enhance the effect of APLA2 on inducing cell apoptosis. The inhibitory effect of SM on sPLA2s activity suggests that intracellular level of SM may act as a regulatory factor on the activity of sPLA2. We therefore further determined the relative levels of SM in Mv1Lu and A-431 cells. Cells were labeled with [3H]-myristic acid, total lipids were extracted and SM was separated and measured. In both cells, the addition of exogenous SMase caused a pronounced decrease in cellular SM levels (Fig. 7B). The inhibitory effect of SM on sPLA2 activity was also conformed by AA release assay as it was shown in Fig. 7C, where pretreatment of cells with SMase significantly enhanced the BPLA2- and NPLA2-induced extracellular AA release. This effect was much less pronounced in cells treated with APLA2.

4. Discussion The purified sPLA2 exhibit some differences in their enzymatic activities on different substrates. The preferences of these sPLA2s for different substrates are probably due to the structural differences of these enzymes, as discussed

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previously [32]. We also determined the enzymatic activity of these sPLA2s on intact cells by measuring the intracellular and extracellular AA released by the action of sPLA2s, and found that all three types of the purified sPLA2 can stimulate rapid release of cellular arachidonic acids. Treatment of Mv1Lu cells with the purified sPLA2s caused dose- and time-dependent apoptosis as determined by measuring the cell viability, chromatin condensation, and flow cytometry analysis. Exogenous ceramide analogues, but not AA or LPA, showed a similar effect on Mv1Lu cells. We further found that sPLA2-induced death of Mv1Lu cells was associated with an increase in intracellular ceramide level. These results indicated that ceramide was associated with sPLA2s-induced cell apoptosis. In comparison to BPLA2 and NPLA2, APLA2 is less potent in inducing ceramide formation, but more potent in inducing the intracellular AA release. This may due to the differences in their hydrolytic activity and substrate specificity. These differences may contribute to the subtle differences in their apoptotic effects on Mv1Lu cells. It has been reported that treatment of L929 cells with tumor necrosis factor-a (TNFa) results in the release of AA through activating cytosolic PLA2, followed by a prolonged accumulation of ceramide [8,27]. In this study, neither AA nor LPA showed any effect on the generation of ceramide in Mv1Lu cells. Furthermore, addition of exogenous AA or LPA to the cell culture didn’t induce apoptosis. Therefore, it is unlikely that sPLA2sinduced ceramide formation and apoptotic response in Mv1Lu cells resulted from AA and LPA, the direct hydrolytic products of sPLA2-catalyzed reaction. This difference may result from the possibility that the effects of cPLA2 and sPLA2 or different subtypes of them are mediated via different mechanisms and the effects of these PLA2 may vary with cell types. Ceramide has been previously shown by in vitro study to inhibit sPLA2 activity, and the inhibitory activity correlated with the length of its fatty acid chain [33]. It has been also reported that ceramide inhibits PLD activity in vitro and in intact cells [10,11]. These findings suggest a ‘‘cross-talk’’ between glycerol phospholipid and sphingolipid signaling pathways. The inhibition of agonist-induced PLD activity by ceramide and sPLA2 therefore indicate a relevancy between the cellular effect of sPLA2 and the generation of intracellular ceramide. However, it is not clear whether the differences in inhibiting bradykinin- and PMA-induced PLD activity by snake venom sPLA2 can be explained by the existence of different PLD subtypes that are differentially regulated in intact cells. Regulation of intracellular level of ceramide has been implicated in signaling events in various stress-induced cellular responses and other biological processes [15,34 – 37]. Many factors that regulate sphingolipid metabolism can cause the alternation of intracellular levels of ceramide and other sphingolipid metabolites, while hydrolysis of SM by SMase has been known as a major pathway that leads to the increased generation of ceramide. We previously demon-

strated that treatment of A-431 cells with exogenous permeable C2-ceramide analogue for a short period of time (less than 6 h) does not induce cell apoptosis [38]. In this report, we found that with longer periods of treatment (till 24 h) ceramide did induce apoptosis in this cell line. However, treatment of cells with exogenous SMase, even for a longer period of time, failed to induce cell apoptosis. This could be explained by the possibility that the ceramide generated via exogenous SMase-catalyzed hydrolysis of SM is different from the ceramide formed through a special process in vivo and therefore has different biological significance. Exogenous SMase-catalyzed hydrolysis of the SM on membrane produces various ceramides with different fatty acid compositions, while the ceramide that act as intracellular second messenger in vivo is more likely to be a special one. It has been suggested that the site(s) of ceramide generation can influence its role in signaling [39]. Short-acyl chain ceramides are more likely to undergo interbilayer movement and passing through the bilayer membrane than long-acyl chain ceramide, suggesting the former can be more efficient in acting as signaling molecules. Previous reports have shown that short-acyl chain ceramides, like C2-ceramide, do exist in nature [40,41]. Thus, ceramide-induced apoptotic response is likely a tissue- and cell type-dependent process, and is dependent on the subtypes of the ceramide and their relative abundance. The biological effects of ceramide might be confined to the site(s) of its generation within the membrane lipid bilayer. The differences in the sensitivity to sPLA2 in different cells demonstrate that the physiological effect of the sPLA2 is also an event depending on the cellular context and their environment. It has been reported that SMase could enhance the catalytic activity of sPLA2 by damaging the membrane asymmetry [42] and changing the surface pressure of the membrane [43]. A recent report shows that the activity of cPLA2 can be inhibited by SM but reactivated by ceramide as well as cholesterol [21,22]. In this study, SM showed a strong inhibitory effect on sPLA2 activity, indicating that SM may also play an important role in signal transduction by modulating the activity of sPLA2. Since treatment of cells with SMase resulted in large decrease in the cellular SM levels in both cell lines, the SMase-enhanced cell death is likely due to the release of sPLA2 activity inhibited by membrane SM. Therefore, the existence of SMase may not be sufficient to trigger cell death. It needs to act together with sPLA2 at the special site in the cellular environment. In summary, our data show that three purified sPLA2s catalyze the hydrolysis of phospholipids with differential preference and potency, which can be strongly inhibited by SM. sPLA2 induces dose- and time-dependent apoptotic response in Mv1Lu cells, which is associated with increased ceramide formation. sPLA2-induced cell apoptosis is greatly enhanced by SMase. This is due to the pronounced increase in the degradation of cellular SM, which subsequently reduced the inhibition of sPLA2. AA and LPA, the direct products of sPLA2, have no effect on intracellular ceramide

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generation and cell viability. These findings provide an insight in understanding the signaling mechanism of sPLA2-mediated biological events.

Acknowledgements We are grateful to Dr. Chafen Lu, Dr. Jie Liu, and Dr. Wei Qu for critic reading and comments on this manuscript. This work was supported by research grants 396250007 and 39870396 from the Chinese National Natural Sciences Foundation, research grants KJ951-B1-608 from the Chinese Academy of Sciences.

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