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

Phospholipase A₂ (PLA₂) are a family of esterases that cleave glycerophospholipids at the sn-2 position of the ester bond, resulting in the release of a free fatty acid and a lysophospholipid. The fatty acid released varies in size from 14 to 24 carbons and contains zero to six double bonds (Cummings et al., 2000). The lysophospholipid contains a polar head group (a choline, ethanolamine, serine, or inositol) and the sn-1 fatty acid. The released fatty acid can be metabolized to potent lipid signals that may induce cell death, such as thromboxanes, prostacyclins, and prostaglandins (Cummings et al., 2000). The lysophospholipid can form lysophosphatidic acid, a potent lipid signal in its own right, or it can be reacylated, combined with a re-esterfied fatty acid, and reinserted into the plasma membrane. This later step is crucial for the maintenance of cellular phospholipids and membrane integrity (Balsinde, 2002).

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 (iPLA₂β) (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 (iPLA₂γ), a microsomal-bound PLA₂ (group VIB PLA₂), which is inhibited by BEL (Mancuso et al., 2000), but is a distinctive gene product from iPLA₂β. iPLA₂β 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, iPLA₂β is primarily expressed in the cytosol (Ma et al., 1999), whereas iPLA₂γ 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 iPLA₂γ. Thus, although these studies show roles for iPLA₂, and even iPLA₂β, in phospholipid remodeling and arachidonic acid release, the contribution of iPLA₂γ to these same functions is not fully known. Furthermore, few studies have studied the differential roles of iPLA₂β and iPLA₂γ in phospholipid remodeling in a single cell model.

Recent studies show that R- and S-BEL selectively inhibit iPLA₂γ and iPLA₂β, 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 iPLA₂β mediates arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells (Jenkins et al., 2002). However, the differential roles of iPLA₂β and iPLA₂γ 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 iPLA₂β or iPLA₂γ. In the present work, we use both R- and S-BEL and siRNA against iPLA₂β and iPLA₂γ to identify the effect of selective inhibition of iPLA₂ isoforms on phospholipid profiles and cell growth.

Materials and Methods

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 iPLA₂β and iPLA₂γ siRNA Plasmids. To generate plasmids containing siRNA against iPLA₂β and iPLA₂γ, chemically synthesized siRNA oligonucleotides were designed by screening the cDNA sequence of human iPLA₂β and iPLA₂γ 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 iPLA₂β:5′-TAACGCCAGGGTTTTCCCAGTCACG-3′ (sense) and 5′-TCATCCTTCAACTTGGTGAAGAAACTCTCC-3′ (antisense), and iPLA₂γ: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 iPLA₂β and iPLA₂γ Expression Using Real-Time PCR. Total RNA was isolated from control, mock-transfected, vector only-transfected, and cells transfected with plasmids containing siRNA against iPLA₂β and iPLA₂γ 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 × 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′; iPLA₂β sense = 5′-TCCTGAAGCGGGAGTTTG-3′, iPLA₂β antisense = 5′-GACAGTTTCTGGAGCATCGTA-3′; and iPLA₂γ sense = 5′-GGGCATTAGTTCAGGCATT-3′, iPLA₂γ 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-iPLA₂β antibody (Cayman Chemical Co.) or a peptide antibody against iPLA₂γ (CZSKYIERNEHKMKKVAK) for 2 h (Zhang et al., 2005). Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary antibody for iPLA₂β, whereas an anti-chicken secondary antibody was used for iPLA₂γ. 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 (ϵ = 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 × 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

Isolation and Characterization of Chiral Inhibitors of iPLA₂β and iPLA₂γ. 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 iPLA₂γ, but not cytosolic iPLA₂β, activity with an IC₅₀ of approximately 2.5 μM (Kinsey et al., 2005). This same study also showed that S-BEL selectively inhibits cytosolic iPLA₂β, but not microsomal iPLA₂γ, with a similar IC₅₀ (Kinsey et al., 2005). To verify the specificity of 2.5 μM R- and S-BEL against iPLA₂γ and iPLA₂β, 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).

Effect of Inhibition of iPLA₂β and iPLA₂γ on Cell Growth. To test the effect of iPLA₂ inhibition on cell growth and proliferation, HEK293 and Caki-1 cells were seeded in six-well plates, allowed to grow for 24 h, and then exposed to R/S-BEL, R-BEL, or S-BEL (0-5 μM) for 48 h (Fig. 2). Exposure of cells to R/S-BEL resulted in a concentration-dependent decrease in cell growth as determined by decreases in the staining of cells for MTT. Similar to R/S-BEL, exposure of cells to R-BEL decreased MTT staining 15 to 20% compared with controls. In contrast, exposure of cells to S-BEL induced significantly greater decreases in MTT staining compared with either R/S-BEL or R-BEL at comparable concentrations. Decreases in cell growth were not caused by cell death because treatment of cells with R/S-BEL, R-BEL, or S-BEL did not increase either annexin V or PI staining as determined by flow cytometry (data not shown).

These data suggest that inhibition of iPLA₂ activity decreases cellular proliferation and that inhibition of iPLA₂β has a greater effect on cell growth than inhibition of iPLA₂γ. To confirm this hypothesis, studies were performed using siRNA against iPLA₂β and iPLA₂γ. For these studies, cells were transfected with plasmids containing siRNA against iPLA₂β or iPLA₂γ at approximately 30% confluence, allowed to grow for 24 h, subcultured, seeded onto six-well plates at a concentration of 3.0 × 10⁵ cells/well, and allowed to grow for 48 h before analyses of cell proliferation using MTT staining, or iPLA₂β and iPLA₂γ expression using real-time PCR or immunoblot analysis. Assessment of iPLA₂β or iPLA₂γ expression using real-time PCR showed that transfection of cells with plasmids containing siRNA against iPLA₂β resulted in a 1.5-fold decrease in iPLA₂β 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 iPLA₂γ (Fig. 3B). Decreasing the expression of iPLA₂β using siRNA did not alter the expression of iPLA₂γ, nor did decreasing the expression of iPLA₂γ alter the expression of iPLA₂β (data not shown). The ability of siRNA to decrease the expression of iPLA₂β and iPLA₂γ was verified using immunoblot analysis. Transfection of siRNA against iPLA₂β 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 iPLA₂γ was performed (Fig. 3D).

Transfection of HEK293 and Caki-1 cells with siRNA against iPLA₂β 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 iPLA₂γ 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 iPLA₂β and iPLA₂γ on cell growth, MTT staining in HEK293 and Caki-1 cells was determined (Fig. 5, A and B). Transfection of cells with siRNA against iPLA₂β 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 iPLA₂γ 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 iPLA₂β 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 iPLA₂γ 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 iPLA₂β has a greater effect on cell growth than inhibition of iPLA₂γ.

Effect of iPLA₂β and iPLA₂γ 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.

To verify the differential effect iPLA₂β and iPLA₂γ inhibition had on the arachidonic acid-containing phospholipids, lipids were extracted from control cells, and cells were transfected with siRNA against iPLA₂β or iPLA₂γ and subjected to ESI-MS analysis. Transfection of cells with siRNA against iPLA₂β caused slight decreases in 16:0-20:4 and 16:1-20:4 PtdCho compared with controls; however, the decreases were not significantly different from controls. In contrast, siRNA against iPLA₂β resulted in significant decreases in 18:0-20:4 and 18:1-20:4 PtdCho compared with control cells (Fig. 6B). Transfection of cells with siRNA against iPLA₂γ had no effect on these same phospholipids.

Discussion

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 iPLA₂β and iPLA₂γ 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 iPLA₂β mediates β-cell selection and proliferation (Bao et al., 2006), but the role of iPLA₂γ was not addressed. We previously reported that both HEK293 and Caki-1 cells express iPLA₂β and iPLA₂γ and that these isoforms are inhibited by BEL. We, along with others, also showed that that R-BEL inhibits iPLA₂γ activity, whereas S-BEL inhibits iPLA₂β activity (Jenkins et al., 2002; Kinsey et al., 2005). Using these inhibitors, plus siRNA, we show herein the novel finding that iPLA₂β inhibition results in greater decreases in HEK293 and Caki-1 cell growth and proliferation than inhibition of iPLA₂γ. 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 β cells using siRNA against iPLA₂β. 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 iPLA₂β and iPLA₂γ 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 iPLA₂β resulted in greater decreases in cell growth than inhibition of iPLA₂γ. 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 iPLA₂β and iPLA₂γ 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 iPLA₂β significantly decreased the basal levels of several arachidonic acid-containing phospholipids in HEK293 cells. In contrast, inhibition of iPLA₂γ had little effect on any arachidonic acid-containing phospholipid. Furthermore, inhibition of iPLA₂γ, using R-BEL, selectively decreased 14:0-16:0 PtdCho and increased 16:0-20:1 PtdCho. In contrast, inhibition of iPLA₂β 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 iPLA₂β 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 β 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 iPLA₂β 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 iPLA₂β 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 iPLA₂β were PtdCho in origin.

In summary, data reported above suggest that iPLA₂β and iPLA₂γ 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 iPLA₂β or iPLA₂γ. The identification of roles for iPLA₂β in cell growth and proliferation will have significant impacts on several fields, including cancer cell physiology, wound healing, and cell repair.