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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Differential Roles for Cytosolic and Microsomal Ca²⁺-Independent Phospholipase A₂ in Cell Growth and Maintenance of Phospholipids

Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia

Received April 3, 2006; accepted June 7, 2006.

	Abstract

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Physiological roles of microsomal (iPLA₂) and cytosolic (iPLA₂)Ca²⁺-independent phospholipase A₂ were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA₂ inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA₂ and iPLA₂, respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 µM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA₂ siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA₂ siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA₂ and iPLA₂ siRNA on cell number and protein was also determined, and iPLA₂ siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA₂ siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA₂, but not iPLA₂, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA₂ inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA₂ inhibition results in greater decreases in cell growth and proliferation than iPLA₂, identifies specific phospholipids whose expressions are differentially regulated by iPLA₂ and iPLA₂, and suggests novel roles for iPLA₂ in cell growth.

PLA₂ take part in the "Lands pathway," in which a fatty acid is cleaved from glycerophospholipids by PLA₂ and reincorporated into new phospholipids by acyltransferases (Balsinde, 2002). It is hypothesized that this pathway is the major route by which cells release and reincorporate arachidonic acid, a 20-carbon-long fatty acid containing four double bonds (20:4), into various phospholipids. In fact, in a nonstimulated cell, the majority of arachidonic acid released is not converted to thromboxanes, prostacyclins, or even prostaglandins, but rather is reincorporated into the membrane, partially via the actions of PLA₂ (Balsinde, 2002).

Group VI PLA₂ represents some of the newest PLA₂ to be discovered. They are distinct from the two other classes of PLA₂, cytosolic Ca²⁺-dependent PLA₂ and secreted PLA₂ (Cummings et al., 2000; Balsinde, 2002). The first group VI PLA₂ identified was cytosolic Ca²⁺-independent phospholipase A₂ (iPLA₂) (or group VIA PLA₂), which was subsequently shown to exist in long and short forms via alternative splicing (Hazen et al., 1991; Ma et al., 1999), both of which were inhibited by bromoenol lactone (or BEL) (Balsinde et al., 1995; Balsinde and Dennis, 1996). Study of the human genome project identified microsomal Ca²⁺-independent phospholipase A₂ (iPLA₂), a microsomal-bound PLA₂ (group VIB PLA₂), which is inhibited by BEL (Mancuso et al., 2000), but is a distinctive gene product from iPLA₂. iPLA₂ is expressed in several tissues from several species, including rat, rabbit, and human brain, heart, lung, and kidney (Portilla et al., 1998; Balsinde et al., 1999; Ma et al., 1999; Yang et al., 1999; McHowat et al., 2001). These enzymes are also expressed in P388D1 macrophages, human neutrophils, rat stromal cells, rabbit kidney cells, human embryonic kidney (HEK) 293, and Caki-1 cells (Balsinde, 2002; Cummings et al., 2002; Kinsey et al., 2005; Zhang et al., 2005). Within cells, iPLA₂ is primarily expressed in the cytosol (Ma et al., 1999), whereas iPLA₂ has only been identified bound to membranes, including those of the peroxisomes (Yang et al., 2003), mitochondria (Mancuso et al., 2004), and the endoplasmic reticulum (Mancuso et al., 2000; Cummings et al., 2002).

Altering Ca²⁺-independent phospholipase A₂ (iPLA₂) expression and activity alters arachidonic acid incorporation and phospholipid remodeling (Balsinde et al., 1995, 1997a, b; Cummings et al., 2004a; Perez et al., 2004; Bao et al., 2006). However, many studies addressing the role of iPLA₂ in cell physiology used R/S-BEL or were performed before the discovery of iPLA₂. Thus, although these studies show roles for iPLA₂, and even iPLA₂, in phospholipid remodeling and arachidonic acid release, the contribution of iPLA₂ to these same functions is not fully known. Furthermore, few studies have studied the differential roles of iPLA₂ and iPLA₂ in phospholipid remodeling in a single cell model.

Recent studies show that R- and S-BEL selectively inhibit iPLA₂ and iPLA₂, respectively (Jenkins et al., 2002; Kinsey et al., 2005). Furthermore, these studies have been validated using siRNA (Su et al., 2004). Such studies show that iPLA₂ mediates arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells (Jenkins et al., 2002). However, the differential roles of iPLA₂ and iPLA₂ in phospholipid remodeling and in other processes of cell physiology are not known. Particularly, few data exist identifying the exact phospholipids altered during inhibition of iPLA₂ or iPLA₂. In the present work, we use both R- and S-BEL and siRNA against iPLA₂ and iPLA₂ to identify the effect of selective inhibition of iPLA₂ isoforms on phospholipid profiles and cell growth.

	Materials and Methods

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Materials. HEK293 and Caki-1 (kidney carcinoma), cell growth medium, and fetal bovine serum were purchased from American Type Culture Collection (Manassas, VA). Penicillin and streptomycin were obtained from Invitrogen (Carlsbad, CA). Annexin V-fluorescein isothiocyanate was obtained from R&D Systems (San Diego, CA). Cell stripper was obtained from Mediatech, Inc. (Herndon, VA). Propidium iodide (PI) and BEL were obtained from Cayman Chemical Co. (Ann Arbor, MI). All of the other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Production of iPLA₂ and iPLA₂ siRNA Plasmids. To generate plasmids containing siRNA against iPLA₂ and iPLA₂, chemically synthesized siRNA oligonucleotides were designed by screening the cDNA sequence of human iPLA₂ and iPLA₂ for unique 53- to 55-nucleotide sequences in the National Center for Biotechnology Information database using the BLAST search algorithm. Candidate sequences were chosen based on homology to each iPLA₂ isoform, probability of forming hairpin structures, and the internal stability profile. Primers were then designed from these sequences and cloned into the pSEC siRNA expression vector from Ambion (Austin, TX) using polymerase chain reaction (PCR). The oligonucleotides used were iPLA₂:5'-TAACGCCAGGGTTTTCCCAGTCACG-3' (sense) and 5'-TCATCCTTCAACTTGGTGAAGAAACTCTCC-3' (antisense), and iPLA₂:5'-TTCACCAAGTTGAAGGATGAAACTCTTCAG-3' (sense) and 5'-GGATAACCGTATTACCGCCTTTGAGTG-3' (antisense). PCR conditions were 94°C for 1.25 min, followed by 25 cycles at 50°C for 30 s, 72°C for 1 min, and 15 s at 94°C, and a final extension step at 68°C for 3 min. The presence of siRNA sequences in all of the plasmids was verified by DNA sequencing and restriction fragment digest gel assays.

Transfection of siRNA Plasmids into Cells. At 30% confluence, cells were transfected with siRNA plasmids by dilution of 1 µg of the respective plasmids in 3 µl of transfection agent (SiPORT XP-1, Ambion) and 100 µl of Opti-MEM I medium. Samples were mixed by gentle pipetting and incubated at room temperature for 10 min. The SiPORT XP-1/siRNA complex was added drop-wise onto the cells, which were gently rocked to evenly distribute the complexes. Cells were then allowed to grow for 48 h in normal cell culture conditions.

Analysis of iPLA₂ and iPLA₂ Expression Using Real-Time PCR. Total RNA was isolated from control, mock-transfected, vector only-transfected, and cells transfected with plasmids containing siRNA against iPLA₂ and iPLA₂ using the TRIzol reagent as directed by the manufacturer (Invitrogen, Carlsbad, CA). Following isolation, RNA was digested for 15 min at 37°C with DNase I in 40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl₂, and 10 mM CaCl₂. RNA was re-extracted and cDNA prepared using reverse transcriptase for 30 min at 50°C using 10 µM hexanucleotides and each deoxynucleoside-5'-triphosphate at 400 µM (Amersham Biosciences, Piscataway, NJ). cDNA was diluted in the range of 1.0 to 1.0 x 10^-6 µg/µl in sterile H₂O, and 25 µl of SuperMix SYBR (Bio-Rad, Hercules, CA) was added along with 1 µl of forward and reverse primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or the specific iPLA₂ isoform in question (see below) and 5 µl of each cDNA reaction along with sterile H₂O. Negative controls included the absence of cDNA. Each sample was run in duplicate (20 µl per reaction) using Bio-Rad ICycler sequencer in 96-well PCR plates under the conditions of 95°C for 3 min, followed by 40 cycles at 95°C for 10 s and 55°C for 1 min. For all of the cells, a separate GAPDH reaction was performed for normalization. A standard curve was generated for each set of primers and each set of cells. PCR efficiency was determined by analysis of serial dilutions of cDNA for each primer set. Quantification of RNA was based on comparison of the number of cycles required to reach reference and target threshold values (C_t) as normalized against GAPDH. The primers used for real-time PCR are as follows: GAPDH sense = 5'-AAGGTCGGAGTCAACGGAT-3', GAPDH antisense = 5'-TGGAAGATGGTGATGGGATT-3'; iPLA₂ sense = 5'-TCCTGAAGCGGGAGTTTG-3', iPLA₂ antisense = 5'-GACAGTTTCTGGAGCATCGTA-3'; and iPLA₂ sense = 5'-GGGCATTAGTTCAGGCATT-3', iPLA₂ antisense = 5'-CCCTTGTTCCTCCACCATC-3'.

Immunoblot Analysis. Microsomal and cytosolic fractions (40 µg) were mixed with SDS-sample buffer, heated to 70°C for 10 min, separated under reducing conditions on a 12% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. Nonspecific binding was blocked by incubating the membrane with 3% (w/v) bovine serum albumin in Tris-buffered saline (10 mM Tris-HCl, pH 7.5, and 150 mM NaCl) overnight at room temperature. Membranes were then incubated with a polyclonal rabbit anti-iPLA₂ antibody (Cayman Chemical Co.) or a peptide antibody against iPLA₂ (CZSKYIERNEHKMKKVAK) for 2 h (Zhang et al., 2005). Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary antibody for iPLA₂, whereas an anti-chicken secondary antibody was used for iPLA₂. Bands were detected by enhanced chemiluminescence (Amersham Biosciences).

Isolation of R- and S-BEL. Enantiomers were isolated from racemic BEL (R/S-BEL) (Cayman Chemical) using a chirex 3,5-dinitrobenzoyl-(R)-phenylglycine chiral high-performance liquid chromatography column (Phenomonex, Torrance, CA) following previously published methods (Jenkins et al., 2002; Kinsey et al., 2005). The column was equilibrated with hexane/dichloroethane/ethanol (150:15:1), and the optical enantiomers eluted isocratically at 2 ml/min. Elution was monitored by UV absorbance at 280 nm. Peaks were collected corresponding to retention time, dried under N₂, and stored at -20°C. The concentration of each enantiomer was determined spectrophotometrically based on its UV absorbance ( = 6130 cm/M in acetonitrile).

Measurement of iPLA₂ Activity. PLA₂ activity was determined under linear conditions in microsomes and cytosol as described previously (McHowat et al., 1998). Activity was measured using synthetic (16:0, [³H]18:1) phosphatidylcholine (PtdCho) substrates (100 µM) in the absence of Ca²⁺ (presence of 4 mM EGTA). These radiolabeled substrates were synthesized as described previously (Creer and McHowat, 1998).

Analyses of Cell Growth and Proliferation. Cell growth and proliferation were determined using MTT staining, and all of the experiments were carried out in the linear working range of MTT. Cells were seeded in six-well plates at a concentration of 3.0 x 10⁵ cells/well and allowed to grow for 24 h before treatment for 48 h. At the desired time points, 100 µl of 5 mg/ml MTT was added to each well, the cells were incubated for 2 h, after which the media were removed, 2 ml of dimethyl sulfoxide was added to dissolve the resulting purple formazan, and absorbance was read at 544 nm with FLUOstar OPTIMA plate reader (BMG Labtech, Inc., Durham, NC). In addition, cell growth was verified by cell count using trypan blue and a hemacytometer.

Measurement of Cell Death Using Flow Cytometry. Annexin V (apoptotic cell marker) and PI (necrotic cell marker) staining were determined using flow cytometry as described previously with modifications (Cummings and Schnellmann, 2002). In brief, media were removed, and cells were washed twice with phosphate-buffered saline (PBS) and incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂, pH 7.4) containing annexin V-fluorescein isothiocyanate (25 µg/ml) and PI (25 µg/ml) for 10 min. Cells were washed three times in binding buffer, released from the monolayers using a rubber policeman, and staining-quantified using a Becton Dickinson FACSCalibur flow cytometer. For each measurement, 10,000 events were counted.

Assessment of Cell Morphology. Cell morphology was determined using phase-contrast microscopy as described previously (Cummings and Schnellmann, 2002; Cummings et al., 2004b) with modifications. In brief, following treatment, cells were washed twice with PBS, fixed for 20 min using 10% buffered formalin/4% formaldehyde, washed with PBS, covered with mounting media, and coverslips were applied. Visualization of cell was performed using a Nikon TE300 Eclipse microscope (Nikon, Melville, NY).

Characterization and Quantitation of Cellular Phospholipids Using Electrospray Ionization-Mass Spectrometry. Cellular phospholipids were extracted according to the method of Bligh and Dyer (1959) at 4°C. Lipid extracts were dried under argon, and lipid phosphorus was quantified using malachite green (Zhou and Arthur, 1992). Mass spectrometry analysis was performed essentially as described previously (Taguchi et al., 2000). Lipid extracts from cells were analyzed using an LCT Premier time-of-flight mass spectrometer (Waters, Milford, MA) equipped with an electrospray ion source. Five microliters of sample (500 pmol/ml) dissolved in chloroform/methanol (2:1, v/v) was introduced, by means of a flow injector, into the electrospray ionization chamber at a flow rate of 0.2 ml/min. The elution solvent was acetonitrile/methanol/water (2:3:1, v/v/v) containing 0.1% (w/v) ammonium formate, pH 6.4. The mass spectrometer was operated in the positive ion scan mode. The nitrogen drying gas flow rate was 10 l/min, and its temperature was 80°C. The capillary voltage was set at 2.5 kV, and the cone voltage was set at 30 V. Identification of individual molecular species was based on theoretical monoisotopic mass values as given in previous publications (Taguchi et al., 2000). In accordance with other studies (Taguchi et al., 2000), phospholipid comparisons are relative to the expression of the most abundant phospholipid in each sample, which corresponded to an m/z ratio of 760 [34:1 (16:0-18:1) PtdCho].

Protein Determination. Protein concentrations were determined using the bicinchoninic acid assay method as described by Sigma.

Statistical Analysis. Cells isolated from a distinct passage of HEK293 or Caki-1 cells represented one experiment (n = 1). Data are represented as the average ± S.E.M. of at least three separate experiments (n = 3). The appropriate analysis of variance was performed for each data set using SAS software (SAS Institute, Cary, NC). Individual means were compared using Fisher's protected least significant difference test with P < 0.05 considered indicative of a statistically significant difference between mean values.

	Results

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Isolation and Characterization of Chiral Inhibitors of iPLA₂ and iPLA₂. R- and S-BEL were prepared from commercially available R/S-BEL (50/50 mixture) using methods first described by Jenkins et al. (2002). We subsequently verified these findings in HEK293 cells by showing that R-BEL selectively, and in a concentration-dependent manner, inhibits microsomal iPLA₂, but not cytosolic iPLA₂, activity with an IC₅₀ of approximately 2.5 µM (Kinsey et al., 2005). This same study also showed that S-BEL selectively inhibits cytosolic iPLA₂, but not microsomal iPLA₂, with a similar IC₅₀ (Kinsey et al., 2005). To verify the specificity of 2.5 µM R- and S-BEL against iPLA₂ and iPLA₂, respectively, we exposed HEK293 cells to R/S-BEL, R-BEL, or S-BEL for 30 min before their isolation and fractionation into microsomes and cytosol and analyses of iPLA₂ activity. As previously reported, treatment of HEK293 cells with R/S-BEL, R-BEL, or S-BEL (2.5 µM) for 30 min resulted in significant decreases in PtdCho cleavage compared with control cells (Fig. 1). R/S-BEL decreased PtdCho cleavage in both microsomal and cytosolic fractions (Fig. 1, A, and B), whereas S-BEL predominantly decreased cleavage in the cytosolic fraction (Fig. 1A). In contrast, R-BEL predominantly decreased PtdCho cleavage in the microsomal fraction and did not lower cleavage in the cytosolic fraction (Fig. 1B).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Effect of R/S-BEL, R-BEL, and S-BEL on PtdCho cleavage in HEK293 cells. HEK293 cells were exposed to 2.5 µM R/S-BEL, R-BEL, or S-BEL for 30 min before isolation and fractionation into cytosolic (A) and microsomal (B) fractions for analysis of iPLA2 activity as determined by PtdCho cleavage. Data are represented as the mean ± S.E.M. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Effect of R/S-BEL, R-BEL, and S-BEL on MTT staining in HEK293 and Caki-1 cells. HEK293 (A) and Caki-1 (B) cells at 30% confluence were exposed to R/S-BEL, R-BEL, or S-BEL and allowed to grow for 48 h before analysis of cell growth using MTT staining. Data are represented as the mean ± S.E.M. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

These data suggest that inhibition of iPLA₂ activity decreases cellular proliferation and that inhibition of iPLA₂ has a greater effect on cell growth than inhibition of iPLA₂. To confirm this hypothesis, studies were performed using siRNA against iPLA₂ and iPLA₂. For these studies, cells were transfected with plasmids containing siRNA against iPLA₂ or iPLA₂ at approximately 30% confluence, allowed to grow for 24 h, subcultured, seeded onto six-well plates at a concentration of 3.0 x 10⁵ cells/well, and allowed to grow for 48 h before analyses of cell proliferation using MTT staining, or iPLA₂ and iPLA₂ expression using real-time PCR or immunoblot analysis. Assessment of iPLA₂ or iPLA₂ expression using real-time PCR showed that transfection of cells with plasmids containing siRNA against iPLA₂ resulted in a 1.5-fold decrease in iPLA₂ expression compared with control cells (Fig. 3A), as normalized against the expression of GAPDH. Similar results were seen when cells were transfected with plasmids containing siRNA against iPLA₂ (Fig. 3B). Decreasing the expression of iPLA₂ using siRNA did not alter the expression of iPLA₂, nor did decreasing the expression of iPLA₂ alter the expression of iPLA₂ (data not shown). The ability of siRNA to decrease the expression of iPLA₂ and iPLA₂ was verified using immunoblot analysis. Transfection of siRNA against iPLA₂ into HEK293 cells decreased the expression of this protein compared with cells transfected with vector alone (Fig. 3C). Similar findings were seen when immunoblot analysis of microsomal fractions from cells transfected with siRNA against iPLA₂ was performed (Fig. 3D).

View larger version (74K): [in this window] [in a new window]   Fig. 3. Effect of siRNA on iPLA2 and iPLA2 expression in HEK293 cells. HEK293 cells at 30% confluence were transfected with DNA plasmids containing siRNA against either iPLA2 (A and C) or iPLA2 (B and D), allowed to grow to confluence, subcultured, and allowed to grow for 48 h before isolation of RNA and analysis of expression using real time-PCR (A and B) or immunoblot analysis of iPLA2 (C) and iPLA2 (D) expression. For C and D, 1 refers to control fractions, and 2 refers to fractions isolated from siRNA-treated cells. Data are representative of at least three separate experiments.

Transfection of HEK293 and Caki-1 cells with siRNA against iPLA₂ significantly altered cell growth compared with control cells, as determined by phase contrast microscopy (Fig. 4, A and B). Transfection of cells with siRNA against iPLA₂ also decreased cell growth compared with control cells, as assessed by phase contrast microscopy (Fig. 4, A and C). To verify the effect of siRNA against iPLA₂ and iPLA₂ on cell growth, MTT staining in HEK293 and Caki-1 cells was determined (Fig. 5, A and B). Transfection of cells with siRNA against iPLA₂ decreased MTT staining 15 to 25% in both HEK293 and Caki-1 cells compared with control cells. In contrast, transfection of cells with siRNA against iPLA₂ did not decrease MTT staining in either HEK293 or Caki-1 cells compared with control cells (Fig. 5, A and B). In addition, transfection of cells with siRNA against iPLA₂ significantly decreased cell number (Fig. 5C) and cellular protein (Fig. 5D) in HEK293 cells compared with control cells. Transfection of HEK293 cells with siRNA against iPLA₂ also slightly decreased cell number compared with controls, but did not decrease cellular protein (Fig. 5, C and D). Collectively, these data support the hypothesis that iPLA₂ inhibition decreases cell proliferation and growth and suggest that inhibition of iPLA₂ has a greater effect on cell growth than inhibition of iPLA₂.

View larger version (133K): [in this window] [in a new window]   Fig. 4. Effect of siRNA against iPLA2 and iPLA2 on cell growth. HEK293 cells at 30% confluence were mock-transfected (A), transfected with DNA plasmids encoding for siRNA against either iPLA2 (B) or iPLA2 (C), grown to confluence, subcultured, and allowed to grow for 48 h before analysis of cell morphology and growth assessed by phase contrast microscopy. Data are representative of at least three separate experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of siRNA against iPLA2 and iPLA2 on MTT, cell number, and cellular protein. Cells at 30% confluence either were mock-transfected or transfected with DNA plasmids encoding for siRNA against either iPLA2 or iPLA2, grown to confluence, subcultured, and allowed to grow for 48 h before analysis of MTT staining in HEK293 (A) or Caki-1 (B) cells, or analysis of cell number (C) or cellular protein (D) in HEK293 cells. Data are represented as the mean ± S.E.M. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

Effect of iPLA₂ and iPLA₂ Inhibition on Cellular Phospholipids. Arachidonic acid, in low amounts, is hypothesized to enhance cell growth (Teslenko et al., 1997), and iPLA₂ is suggested to mediate the release of arachidonic acid and the level of arachidonic acid-containing phospholipids (Balsinde et al., 1997a, b; Teslenko et al., 1997; Perez et al., 2004). To identify the effect of inhibition of individual iPLA₂ isoforms on cellular phospholipids, HEK293 cells were exposed to 2.5 µM R-or S-BEL for 24 h, and the phospholipid profile was analyzed using electrospray ionization-mass spectrometry (ESI-MS). Exposure of cells to R-BEL significantly decreased 14:0-16:0 PtdCho and increased 16:0-20:1 PtdCho compared with solvent-treated control cells (Fig. 6A). In contrast, exposure of cells to S-BEL had no effect on either of these phospholipids but decreased the amount of 16:0/20:4, 16:1-20:4, 18:0-20:4, and 18:1-20:4 PtdCho compared with control cells (Fig. 6A). Exposure of cells to R-BEL did not decrease these same phospholipids compared with control.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Effect of iPLA2 and iPLA2 inhibition on cellular phospholipid profiles. HEK293 cells at 30% confluence were exposed to either solvent control or 2.5 µM R- or S-BEL for 24 h before isolation of phospholipids (B) or transfected with siRNA against iPLA2 or iPLA2, grown to confluence, subcultured, and allowed to grow for 48 h. Data are represented as the mean ± S.E.M. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

	Discussion

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Although several studies show roles for iPLA₂ activity in the maintenance of phospholipids, the functional consequence of this activity on cell physiology is not fully understood. Previous studies suggest roles for iPLA₂ in arachidonic acid-mediated macrophage cell spreading (Teslenko et al., 1997), but the specific iPLA₂ isoform and phospholipids involved were not identified. Recent reports show differential roles for iPLA₂ and iPLA₂ in arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells (Jenkins et al., 2002), but phospholipid remodeling was not addressed. Finally, although several studies suggest that iPLA₂ mediates cell death via its ability to release arachidonic acid (Atsumi et al., 2000; Cummings et al., 2002, 2004c; Perez et al., 2004), few have defined the differential roles of individual iPLA₂ isoforms in nonpathological conditions.

Data reported above support the hypothesis that inhibition of iPLA₂ activity decreases cell proliferation and growth in two different epithelial cell models. A recent study suggested that iPLA₂ mediates -cell selection and proliferation (Bao et al., 2006), but the role of iPLA₂ was not addressed. We previously reported that both HEK293 and Caki-1 cells express iPLA₂ and iPLA₂ and that these isoforms are inhibited by BEL. We, along with others, also showed that that R-BEL inhibits iPLA₂ activity, whereas S-BEL inhibits iPLA₂ activity (Jenkins et al., 2002; Kinsey et al., 2005). Using these inhibitors, plus siRNA, we show herein the novel finding that iPLA₂ inhibition results in greater decreases in HEK293 and Caki-1 cell growth and proliferation than inhibition of iPLA₂. These data support the hypothesis that iPLA₂ isoforms have differential roles in cell physiology.

Inhibition of iPLA₂ inhibited cell growth approximately 40%. This number is similar to, if not greater than, that reported by Bao et al. (2006) in cells using siRNA against iPLA₂. It is doubtful that the level of inhibition is caused by low transfection efficiency of siRNA plasmids because pharmacological inhibition of iPLA₂ activity with R/S-BEL or S-BEL resulted in a similar level of inhibition. Furthermore, immunoblot analysis showed that both iPLA₂ and iPLA₂ expression were significantly decreased.

Inhibition of cell growth by R- and S-BEL was not caused by increases in cell death because neither of these compounds increased annexin V or PI staining at any time point. Furthermore, cells treated with either compound reached confluence approximately 24 h later than control cells (data not shown). Thus, inhibition of iPLA₂, at the very least, delays cell growth. Inhibition of cell growth was also not an artifact of the transfection protocol or plasmids used because cells exposed to vector control or mock-transfected cells displayed no alteration in growth compared with nontreated controls.

Inhibition of cell growth was verified using phase-contrast microscopy, MTT staining, cell number, and cell protein. In all of these cases, inhibition of iPLA₂ resulted in greater decreases in cell growth than inhibition of iPLA₂. The decrease in cellular protein and number, in addition to MTT staining, supports the conclusion that decreased cell growth is not a result of a simple decrease in cell size. Furthermore, it is unlikely that decreases in cell growth are caused by inhibition of phosphatidic acid phosphohydrolase-1 by BEL because this enzyme is only inhibited 50% by 25 µM BEL (Balsinde and Dennis, 1996), which is 10-fold higher than the concentrations used in this study. Furthermore, siRNA against iPLA₂ and iPLA₂ gave the same exact results of R- and S-BEL.

Arachidonic acid release is hypothesized to mediate cell spreading, adhesion, or proliferation in macrophages and cancer cells (Teslenko et al., 1997; Avis et al., 2005). Furthermore, PLA₂ activity is hypothesized to mediate the release of arachidonic acid during these events (Guthridge et al., 1994; Longo et al., 1999), and some studies have suggested roles for iPLA₂ (Shen et al., 1998). However, few studies have identified the actual arachidonic acid-containing phospholipids involved. Data reported above show that inhibition of iPLA₂ significantly decreased the basal levels of several arachidonic acid-containing phospholipids in HEK293 cells. In contrast, inhibition of iPLA₂ had little effect on any arachidonic acid-containing phospholipid. Furthermore, inhibition of iPLA₂, using R-BEL, selectively decreased 14:0-16:0 PtdCho and increased 16:0-20:1 PtdCho. In contrast, inhibition of iPLA₂ had no effect on these same phospholipids. Thus, these data support the novel hypothesis that iPLA₂ isoforms differentially regulate the phospholipid profile of HEK293 cells.

The ability of iPLA₂ inhibition to selectively decrease cell growth may be linked to its ability to selectively decrease the above arachidonic acid-containing phospholipids. This hypothesis is supported by recent studies in cells, which showed that decreased iPLA₂ expression correlated with decreased levels of 18:0-20:4 PtdCho and cell proliferation. Together, these lines of evidence suggest that 18:0-20:4 PtdCho plays a key role in proliferation of multiple cell types. Decreases in the amount of 18:0-20:4 PtdCho could decrease the overall amount of arachidonic acid release, which could decrease cell growth. Alternatively, the cytosolic location of iPLA₂ may result in it being the predominant isoform responsible for arachidonic acid release during cell growth. Furthermore, decreased arachidonic acid-containing phospholipids, paired with decreases in the enzyme responsible for arachidonic acid release, may result in the overall inhibition of cell growth. In either scenario, once released arachidonic acid could facilitate cell growth through a number of well established mechanisms, including activation of epidermal growth factor receptors (Choudhury et al., 2000) or activation of kinase signaling cascades (Nony et al., 2005). The decrease in phospholipids induced by iPLA₂ inhibition is probably caused by a decreased role of this enzyme in the Land's cycle as shown others (Balsinde et al., 1997a). In this pathway, iPLA₂ acts as the major enzyme that produces lysophosphatidylcholine, which acts as an acceptor for arachidonic acid derived from various sources (Balsinde et al., 1995, 1997a; Ramanadham et al., 1999). In support of this hypothesis, all of the arachidonic acid-containing phospholipids decreased by S-BEL or siRNA against iPLA₂ were PtdCho in origin.

In summary, data reported above suggest that iPLA₂ and iPLA₂ have differential roles in the cell growth and proliferation. Furthermore, these data suggest that these differential roles are a result of selective inhibition of the expression of select arachidonic acid-containing phospholipids. Finally, specific phospholipids were identified whose expression is mediated by either iPLA₂ or iPLA₂. The identification of roles for iPLA₂ in cell growth and proliferation will have significant impacts on several fields, including cancer cell physiology, wound healing, and cell repair.

	Acknowledgements

 
We thank Dr. Jane Mchowat for her assistance in performing the PLA₂ activity assays and Drs. Mchowat and Rick Schnellmann for their gift of the antipeptide iPLA₂ antibody.

	Footnotes

 
doi:10.1124/jpet.106.105650.

This work was supported by a Georgia Cancer Coalition Distinguished Scholar Grant, a University of Georgia Junior Faculty Grant (B.S.C.), and a University of Georgia Fellowship and an American Foundation of Pharmaceutical Education Fellowship (B.P.).

ABBREVIATIONS: PLA₂, phospholipase A₂; iPLA₂ , cytosolic Ca²⁺-independent phospholipase A₂; BEL, bromoenol lactone [(E)-6-(bromoethylene)-3-(1-naphthaleny)-2H-tetrahydropyran-2-one]; iPLA₂ , microsomal Ca²⁺-independent phospholipase A₂; HEK, human embryonic kidney; iPLA₂,' Ca²⁺-independent phospholipase A₂; PI, propidium iodide; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PtdCho, phosphatidylcholine; MTT, 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; ESI-MS, electrospray ionization-mass spectrometry.

Address correspondence to: Dr. Brian S. Cummings, Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602. E-mail: bsc{at}rx.uga.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Atsumi G, Murakami M, Kojima K, Hadano A, Tajima M, and Kudo I (2000) Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2 inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. J Biol Chem 275: 18248-18258.[Abstract/Free Full Text]

Avis I, Martinez A, Tauler J, Zudaire E, Mayburd A, Abu-Ghazaleh R, Ondrey F, and Mulshine JL (2005) Inhibitors of the arachidonic acid pathway and peroxisome proliferator-activated receptor ligands have superadditive effects on lung cancer growth inhibition. Cancer Res 65: 4181-4190.[Abstract/Free Full Text]

Balsinde J (2002) Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipid incorporation and remodelling. Biochem J 364: 695-702.[CrossRef][Medline]

Balsinde J, Balboa MA, and Dennis EA (1997a) Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages. J Biol Chem 272: 29317-29321.[Abstract/Free Full Text]

Balsinde J, Balboa MA, and Dennis EA (1997b) Inflammatory activation of arachidonic acid signaling in murine P388D1 macrophages via sphingomyelin synthesis. J Biol Chem 272: 20373-20377.[Abstract/Free Full Text]

Balsinde J, Balboa MA, Insel PA, and Dennis EA (1999) Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol 39: 175-189.[CrossRef][Medline]

Balsinde J, Bianco ID, Ackermann EJ, Conde-Frieboes K, and Dennis EA (1995) Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages. Proc Natl Acad Sci USA 92: 8527-8531.[Abstract/Free Full Text]

Balsinde J and Dennis EA (1996) Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages. J Biol Chem 271: 31937-31941.[Abstract/Free Full Text]

Bao S, Bohrer A, Ramanadham S, Jin W, Zhang S, and Turk J (2006) Effects of stable suppression of group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells. J Biol Chem 281: 187-198.[Abstract/Free Full Text]

Bligh EG and Dyer WJ (1959) A rapid method of total lipid extraction and purification. J Biochem Physiol 37: 911-917.

Choudhury QG, McKay DT, Flower RJ, and Croxtall JD (2000) Investigation into the involvement of phospholipases A(2) and MAP kinases in modulation of AA release and cell growth in A549 cells. Br J Pharmacol 131: 255-265.[CrossRef][Medline]

Creer MH and McHowat J (1998) Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation. Am J Physiol 275: C1498-C1507.[Medline]

Cummings BS, Gelasco AK, Kinsey GR, McHowat J, and Schnellmann RG (2004a) Inactivation of endoplasmic reticulum bound Ca²⁺-independent phospholipase A2 in renal cells during oxidative stress. J Am Soc Nephrol 15: 1441-1451.[Abstract/Free Full Text]

Cummings BS, Kinsey GR, Bolchoz LJ, and Schnellmann RG (2004b) Identification of caspase-independent apoptosis in epithelial and cancer cells. J Pharmacol Exp Ther 310: 126-134.[Abstract/Free Full Text]

Cummings BS, McHowat J, and Schnellmann RG (2000) Phospholipase A(2)s in cell injury and death. J Pharmacol Exp Ther 294: 793-799.[Abstract/Free Full Text]

Cummings BS, McHowat J, and Schnellmann RG (2002) Role of an endoplasmic reticulum Ca(2+)-independent phospholipase A(2) in oxidant-induced renal cell death. Am J Physiol 283: F492-F498.

Cummings BS, McHowat J, and Schnellmann RG (2004c) Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced renal cell apoptosis. J Pharmacol Exp Ther 308: 921-928.[Abstract/Free Full Text]

Cummings BS and Schnellmann RG (2002) Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther 302: 8-17.[Abstract/Free Full Text]

Guthridge CJ, Stampfer MR, Clark MA, and Steiner MR (1994) Phospholipases A2 in ras-transformed and immortalized human mammary epithelial cells. Cancer Lett 86: 11-21.[CrossRef][Medline]

Hazen SL, Zupan LA, Weiss RH, Getman DP, and Gross RW (1991) Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J Biol Chem 266: 7227-7232.[Abstract/Free Full Text]

Jenkins CM, Han X, Mancuso DJ, and Gross RW (2002) Identification of calcium-independent phospholipase A2 (iPLA2) beta, and not iPLA2gamma, as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s. J Biol Chem 277: 32807-32814.[Abstract/Free Full Text]

Kinsey GR, Cummings BS, Beckett CS, Saavedra G, Zhang W, McHowat J, and Schnellmann RG (2005) Identification and distribution of endoplasmic reticulum iPLA2. Biochem Biophys Res Commun 327: 287-293.[CrossRef][Medline]

Longo WE, Grossmann EM, Erickson B, Panesar N, Mazuski JE, and Kaminski DL (1999) The effect of phospholipase A2 inhibitors on proliferation and apoptosis of murine intestinal cells. J Surg Res 84: 51-56.[CrossRef][Medline]

Ma Z, Wang X, Nowatzke W, Ramanadham S, and Turk J (1999) Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1. J Biol Chem 274: 9607-9616.[Abstract/Free Full Text]

Mancuso DJ, Jenkins CM, and Gross RW (2000) The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2). J Biol Chem 275: 9937-9945.[Abstract/Free Full Text]

Mancuso DJ, Jenkins CM, Sims HF, Cohen JM, Yang J, and Gross RW (2004) Complex transcriptional and translational regulation of iPLAgamma resulting in multiple gene products containing dual competing sites for mitochondrial or peroxisomal localization. Eur J Biochem 271: 4709-4724.[Medline]

McHowat J, Liu S, and Creer MH (1998) Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes. Am J Physiol 274: C1727-C1737.[Medline]

McHowat J, Tappia PS, Liu S, McCrory R, and Panagia V (2001) Redistribution and abnormal activity of phospholipase A(2) isoenzymes in postinfarct congestive heart failure. Am J Physiol 280: C573-C580.

Nony PA, Kennett SB, Glasgow WC, Olden K, and Roberts JD (2005) 15S-Lipoxygenase-2 mediates arachidonic acid-stimulated adhesion of human breast carcinoma cells through the activation of TAK1, MKK6, and p38 MAPK. J Biol Chem 280: 31413-31419.[Abstract/Free Full Text]

Perez R, Melero R, Balboa MA, and Balsinde J (2004) Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide. J Biol Chem 279: 40385-40391.[Abstract/Free Full Text]

Portilla D, Crew MD, Grant D, Serrero G, Bates LM, Dai G, Sasner M, Cheng J, and Buonanno A (1998) cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities. J Am Soc Nephrol 9: 1178-1186.[Abstract]

Ramanadham S, Hsu FF, Bohrer A, Ma Z, and Turk J (1999) Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells. J Biol Chem 274: 13915-13927.[Abstract/Free Full Text]

Shen Z, Belinson J, Morton RE, and Xu Y (1998) Phorbol 12-myristate 13-acetate stimulates lysophosphatidic acid secretion from ovarian and cervical cancer cells but not from breast or leukemia cells. Gynecol Oncol 71: 364-368.[CrossRef][Medline]

Su X, Mancuso DJ, Bickel PE, Jenkins CM, and Gross RW (2004) Small interfering RNA knockdown of calcium-independent phospholipases A2 beta or gamma inhibits the hormone-induced differentiation of 3T3-L1 preadipocytes. J Biol Chem 279: 21740-21748.[Abstract/Free Full Text]

Taguchi R, Hayakawa J, Takeuchi Y, and Ishida M (2000) Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry. J Mass Spectrometry 35: 953-966.[CrossRef][Medline]

Teslenko V, Rogers M, and Lefkowith JB (1997) Macrophage arachidonate release via both the cytosolic Ca(2+)-dependent and -independent phospholipases is necessary for cell spreading. Biochim Biophys Acta 1344: 189-199.[Medline]

Yang HC, Mosior M, Ni B, and Dennis EA (1999) Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A2 from rat brain. J Neurochem 73: 1278-1287.[CrossRef][Medline]

Yang J, Han X, and Gross RW (2003) Identification of hepatic peroxisomal phospholipase A(2) and characterization of arachidonic acid-containing choline glycerophospholipids in hepatic peroxisomes. FEBS Lett 546: 247-250.[CrossRef][Medline]

Zhang L, Peterson BL, and Cummings BS (2005) The effect of inhibition of Ca²⁺independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells. Biochem Pharmacol 70: 1697-1706.[CrossRef][Medline]

Zhou X and Arthur G (1992) Improved procedures for the determination of lipid phosphorus by malachite green. J Lipid Res 33: 1233-1236.[Abstract]

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