J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Journal of Lipid Research, Vol. 42, 716-724, May 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes

Yan J. Jianga, Grant M. Hatcha, David Mymina, Thomas Dembinskia, Edwin A. Kroegera, and Patrick C. Choya
a The Lipid Research Group, Department of Biochemistry and Medical Genetics, Faculty of Medicine, 770 Bannatyne Avenue, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3

Correspondence to: Patrick C. Choy, To whom correspondence should be addressed.


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Cytosolic phospholipase A2 (cPLA2) is responsible for the release of arachidonic acid, a precursor for eicosanoid biosynthesis, from cellular phospholipids. The objective of this study is to examine the regulation of cPLA2 by peroxisome proliferator-activated receptor (PPAR) activators in preadipocyte SW872 (SW) cells. PPAR belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor. In this study, the presence of both PPAR{alpha} and PPAR{gamma} was confirmed in SW cells by positive identification of their mRNA in the cellular homogenate. Clofibrate, a PPAR{alpha} activator, caused an enhancement of ionophore A-23187-induced arachidonate release in SW cells. This increase resulted from an enhancement of cPLA2 activity, which was caused by an increase in enzyme protein. Clofibrate at lower concentrations (10;–200 µM) produced increases in the mRNA levels of cPLA2 in a dose-response manner. At higher concentrations (>400 µM), clofibrate treatment resulted in the attenuation of the cPLA2 mRNA level and protein expression.

We postulate that clofibrate, acting through the PPAR{alpha}, caused an induction in the transcription of cPLA2 gene, which led to an increase in the cPLA2 protein. The observed increase in arachidonate release in SW cells appeared to be a direct result of the enhanced cPLA2 activity.—Jiang, Y. J., G. M. Hatch, D. Mymin, T. Dembinski, E. A. Kroeger, and P. C. Choy. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes. J. Lipid Res. 2001. 42: 716–724.

Supplementary key words: cPLA2, PPAR{alpha}, clofibrate, preadipocytes, arachidonic acid, SW cells, COX-2, peroxisome proliferator-activated receptor


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Phospholipase A2 (PLA2) (EC 3.1.1.4) belongs to a group of enzymes that catalyzes the hydrolysis of the phospholipids at the sn-2 position. This group of enzymes plays a central role in a variety of cellular processes, and the products of the reaction are involved in host defense and signal transduction (1) (2). PLA2 are classified into several major types according to their primary structure, subcellular localization, calcium requirement, and substrate specificity (reviewed in (3) (4) (5) (6)). In mammalian cells, the 14-kDa secretory PLA2 (type II) (7) and the 85;–110-kDa cytosolic PLA2 (type IV) (8) (9) (10) are the prominent types. The cytosolic PLA2 (cPLA2) is different from other PLA2 types in that it is an intracellular enzyme with high molecular weight and has a high degree of specificity for the arachidonoyl group. The arachidonic acid released by the action of PLA2 may be converted into eicosanoids via the cyclooxygenase (COX) pathway, leading to the synthesis of prostaglandins and thromboxanes (11) (12). In view of its importance in lipid homeostasis, the activity of cPLA2 is regulated via several tiers of control. The post-translational modification is responsible for the rapid activation of cPLA2 in response to a variety of physiological stimuli, including the calcium-dependent translocation to the membrane fraction via the CaLB domain (13) (14). Alternatively, the enzyme activity is enhanced by phosphorylation, a process mediated by mitogen-activated protein (MAP) kinase, as well as by the indirect activation by protein kinase C and G-protein-coupled receptors (15). For prolonged responses, the expression of cPLA2 is regulated at the transcriptional level by mediators such as cytokines and growth factors, including interferon-gamma (16), macrophage colony stimulating factor (17), tumor necrosis factor-alpha (18) (19), epidermal growth factor (20), and glucocorticoid (21). The regulation of cPLA2 activity by peroxisome proliferator-activated receptors (PPAR), however, has not been fully explored.

PPAR belong to a subgroup of the nuclear receptor superfamily of transcription factors. The functional DNA binding of these factors would require the ligand-induced heterodimerization with retinoid X receptor (22). Three different types of PPAR, {alpha}, {gamma}, and {delta}, have been identified. They are encoded from separate genes, have distinct tissue distribution, and carry out different functions. The PPAR are activated by a variety of fatty acids and arachidonic acid metabolites, as well as by hypolipidemic agents such as clofibrate (23). Although the direct effect of PPAR in phospholipid catabolism has not been demonstrated, the activation of PPAR{alpha} has been shown to affect lipid metabolism in liver, intestine, and kidney (22) (24). For example, the activation of PPAR{alpha} causes an enhancement in lipid oxidation, an alteration in lipoprotein metabolism, and an inhibition of vascular inflammation (22) (24) (25). In addition, the activation of PPAR{gamma} has been implicated as a mediator for the differentiation of preadipocytes into adipocytes and provides a mechanism for thiazolidinedione to exert its effect during in vivo insulin sensitization (26). Recently, PPAR{gamma} has been shown to be associated with the inhibition of human colorectal tumorigenesis mediated by nonsteroid anti-inflammatory drugs (27). Because preadipocytes contain both lipogenesis and lipolysis gene sets, they serve as a good model to study the modulation of cPLA2 expression on PPAR ligand stimulation.

We reported previously that lysophosphatidylcholine caused an elevation of cellular Ca2+ and the activation of protein kinase C, which resulted in an increased release of arachidonate in endothelial cells (28) and in H9c2 cells (29). In the current study, the regulation of cPLA2 for the release of arachidonic acid by PPAR{alpha} was investigated. Clofibrate, an activator of PPAR{alpha}, was found to up-regulate the expression of cPLA2 and COX-2 in preadipocytes.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
Dulbecco's modified Eagle's medium (DMEM), Ham's F12 nutrition mixture (F12), phosphate-buffered saline, and clofibrate were obtained from Sigma Chemical Company (St. Louis, MO). Heat-inactivated newborn calf serum, TRIZOL Reagent, Moloney murine leukemia virus reverse transciptase, Taq DNA polymerase, gentamycin, and trypsin were purchased from Life Technologies, Inc. Ribonuclease inhibitor and deoxynucleotides were obtained from Promega Inc. (Madison, WI). Both the cPLA2 polyclonal antibody and the cPLA2 protein standard were generous gifts from the Genetics Institute (Boston, MA). Polyclonal (rabbit) anti-PPAR{alpha} antibody was purchased from Affinity BioReagents, Inc. (Golden, CO). 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) was purchased from Roche Inc. (Quebec, Canada). 5-Deoxy-{Delta}12,14-prostaglandin J2 (PGJ2,) was obtained from Cayman Chemical Co. (Ann Arbor, MI). Calcium ionophore A-23187 was obtained from Biomol Inc. (Plymouth Meeting, PA). [5,6,8,9,11,12,14,15-3H]- arachidonic acid (209.6 Ci/mmol), 1-stearoyl-2-[1-14C]arachidonyl-sn-glycerol-3-phosphocholine (55 mCi/mmol) were obtained from Amersham Corp. All lipid standards were purchased from Serdary Research Laboratory (Ontario, Canada). Thin-layer chromatography (TLC) plates (silica gel G) were obtained from Fisher Scientific.

Cell culture and radiolabeling
Preadipocyte SW 872 (SW) cells, derived from human liposarcoma, were acquired from American Type Culture Collection. These cells were cultured at 37°C with 5% CO2 and 95% air in DMEM/F12 nutrition mixture at a ratio of 3:1 and supplemented with 5% heat-inactivated fetal bovine serum (FBS), 50-µg/ml gentamycin, and 20 mM HEPES. Cells were allowed to reach 30% confluence in a monolayer and were then incubated in a medium containing either clofibrate, 5-deoxy-{Delta}12,14-PGJ2, or vehicle alone (<0.05% dimethyl sulfoxide, <0.05% ethanol) for the prescribed amount of time. The medium was changed every 48 h until the end of the experiment.

Subsequent to ligand treatment, cells were maintained in the DMEM/F12 medium (3:1) for the prescribed period of time. After three washes with HEPES-buffered saline (HBS), cells were incubated for 16 h in a DMEM/F12 medium containing 5% FBS, various concentrations of ligand, and 0.88;–1.0 µCi/ml [5,6,8,9,11,12,14,15-3H]arachidonic acid (209.6 Ci/mmol) (30). HBS contained 140 mM NaCl, 4 mM KCl, 5.5 mM glucose, 10 mM HEPES, 1.5 mM CaCl2, 1.0 mM MgCl2, and 0.025% (w/v) bovine serum albumin (BSA), with a final pH of 7.4.

Determination of arachidonate release
The arachidonate released from the cells was determined as described previously (28). At the end of the labeling with [5,6,8,9,11,12,14,15-3H]arachidonic acid, cells were rinsed three times with HBS containing 0.025% BSA to remove any nonspecific binding of arachidonic acid to the cell surface. Cells were then challenged with 10 µM calcium ionophore A-23187 for 10 min. The labeled arachidonate released into the medium was determined by scintillation counting. The viability of cells under this treatment was examined by Trypan blue exclusion. Our result indicates that agonist stimulation had no deteriorating effect on the viability of these cells. Additional cell viability assays were performed with the WST-1 assay.

Electrophoresis and Western blot analyses
Samples containing 50 µg of protein were subjected to sodium dodecylsulfate/7.5% polyacrylamide gel electrophoresis (SDS-PAGE) with cPLA2 protein standard (15;–20 ng) as a reference. Protein fractions in the gel were transferred to the hybond nitrocellulose membrane. The membrane was incubated with the polyclonal anti-cPLA2 antibodies (1:1,500;–2,000) dissolved in tris(hydroxymethyl)aminomethane (Tris)-buffered saline containing 0.1% Tween-20 and 2% skim milk overnight at 4°C. Subsequently, the membrane was washed and incubated with the peroxidase-labeled anti-rabbit secondary antibodies (1:3,000) for 30 min at room temperature. Protein bands in the membrane were visualized by enhanced chemiluminescence. The relative intensities of the bands were analyzed by scanning the film and subsequently determined by Scion Image software.

For the detection of PPAR{alpha} in SW cells, 30;–80 µg protein samples were used. Following the transfer, the membrane con- taining protein samples was incubated with polyclonal anti-PPAR{alpha} antibodies (1:500;–1,000). The membrane was then washed and incubated with the peroxidase-labeled anti-rabbit secondary antibodies (1:2,000) for 30 min at room temperature.

Determination of phospholipase A2 activity
After clofibrate (10;–400 µM) treatment, SW cells were lysed by sonication in a buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA), 10 µM leupetin, 10 µM aprotinin, 20 mM NaF, 10 mM Na2HPO4, and 1 mM dithiothreitol. The cell lysate was centrifuged at 100,000 g for 1 h. The supernatant was designated as the cytosolic fraction, and the pellet was designated as the membrane fraction. PLA2 activity in the subcellular fractions was determined by the hydrolysis of 1-stearoyl-2-[1-14C]arachidonyl-sn-glycerol-3-phosphocholine to yield the labeled arachidonate. The assay mixture contained 50 mM Tris-HCl (pH 8.0), 3.0 mM CaCl2, 0.9 nmol of 1-stearoyl-2-[1-14C]arachidonyl-sn-glycerol-3-phosphocholine (100,000 dpm/assay), and 10-µg protein in a final volume of 100 µl. The mixture was incubated for 30 min at 37°C, and the reaction was terminated by the addition of 1.5 ml of chloroform/methanol (2:1, v/v). Oleic acid was added as a fatty acid carrier. The fatty acid was extracted and separated by TLC in a solvent system containing hexane/diethyl ether/acetic acid (70:30:1, v/v/v). The fatty acid fraction was visualized by exposure to iodine vapor, and the radioactivity of the arachidonic acid released was determined by liquid scintillation counting.

Isolation of total RNA
Total RNA was isolated using the TRIZOL Reagent according to the manufacturer's instruction. Briefly, cells were lysed with 1 ml of TRIZOL Reagent in each 100-mm dish, extracted with phenol-chloroform, and precipitated with ethanol. The RNA pellet was suspended in autoclaved, double-distilled water (31) and quantified by absorbance at 260 nm, using the 260:280 nm ratio as an index of purity. The integrity of the RNA was confirmed by denaturing agarose gel electrophoresis on the isolated RNA sample.

Polymerase chain reaction primers and reverse transcriptase polymerase chain reactions
The cDNA for cPLA2, COX-1, COX-2, acyl-CoA oxidase (ACO), PPAR{alpha}, or PPAR{gamma} was amplified with a pair of specific primers synthesized by Gibco Life Technology, Inc. The sequence of primers and the length of predicted polymerase chain reaction (PCR) products are listed in Table 1.


 
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  		Table 1. Polymerase chain reaction primers

The first-strand cDNA from 1-µg total RNA was synthesized by employing 150 U of moloney murine leukemia virus reverse transcriptase (RT), 25 pmol of random hexamer primer, 20 U of ribonuclease inhibitor, 1 mM dithiothreitol, and 10 pmol each of the four deoxynucleotides, in a total volume of 15 µl (32). The reaction mixture was incubated at 37°C for 1 h and terminated by boiling the sample at 95°C for 5 min. An aliquot of 1.0;–1.2 µl of the resultant cDNA preparation was used directly for each amplification reaction.

PCR was performed in 20-µl reaction mixtures containing 8 pmol of each primer, 8 pmol of each dNTP, and 0.4-U Taq DNA polymerase. The mixture was overlaid with 30-µl mineral oil to prevent evaporation and was incubated in a Perkin-Elmer DNA Thermal Cycler under the following conditions. For the PCR of glyceraldehyde 3-phosphate dehydrohenase (GAPDH), PPAR{alpha} or PPAR{gamma}: 25;–30 cycles, denaturation, 30 s at 94°C; annealing, 30 s at 55;–60°C; extension, 30 s at 72°C. For the PCR of cPLA2, COX-1 or COX-2: 25;–32 cycles, denaturation, 60 s at 94°C; annealing, 60 s at 55;–60°C; extension, 2 min at 72°C. The amplified RT-PCR product was analyzed by 1.2;–1.8% agarose gels electrophoresis in 1x TAE buffer (40 mM Tris acetate, 2 mM sodium EDTA) and visualized by staining with 0.5-µg/ml ethidium bromide. The GAPDH band was used as an internal control.

An increase in the level of a specific mRNA is caused by either an increase in its rate of synthesis, a decrease in its rate of degradation, or a combination of these two processes. Hence, mRNA stability assays were conducted using actinomycin D as an inhibitor for RNA synthesis. SW cells were treated with clofibrate (50;–200 µM) for 48 h, and actinomycin D (5 µg/ml) was added after clofibrate treatment. The mRNA levels for cPLA2 and GAPDH were determined by RT-PCR at 4-h intervals following the actinomycin D treatment. No apparent changes in mRNA degradation were observed within a 24-h period, indicating that clofibrate did not cause any change in the degradation of cPLA2 mRNA.

SYBR Green I real-time PCR analysis
Real-time quantitative PCR was performed using a fluorescence temperature cycler (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany). The primers for amplification of cPLA2 cDNA were the same as previously described. Amplification was done according to the manufacturer's general guidelines after optimization of the various reaction parameters. The reaction mixture (20 µl) was placed in a glass capillary and contained 2 µl of LightCycler-DNA Master SYBR Green I mix (Taq DNA polymerase, 10x PCR buffer, deoxynucleotide triphosphate mix, and SYBR Green I dye), 3 mM MgCl2, and 0.4 pmol of each primer. PCR-grade H2O instead of cDNA was used as a negative control.

The amplification program included the initial denaturation step at 95°C for 30 s. It was followed by 55 cycles of denaturation at 95°C for 0 s, annealing at 55°C for 10 s, and extension at 72°C for 20 s. The temperature transition rate was 20°C/s. Fluorescence was determined at the end of each extension step. After amplification, a melting curve was acquired by heating the product at 20°C/s to 95°C, cooling it at 20°C/s to 60°C, keeping it at 60°C for 30 s, and then slowly heating it at 0.2°C/s to 97°C. Fluorescence was determined throughout the slow heating phase. Melting curves were used to determine the specific PCR products, which were further confirmed using conventional gel electrophoresis. Absolute copy numbers of the target transcripts per ng of transcribed total RNA were determined with cloned plasmid DNA for cPLA2.

Sequence analysis of cDNA
The identity of each PCR product generated from RT-PCR was verified and confirmed by direct DNA sequencing. The gel fraction containing the target DNA, including COX-1, COX-2, cPLA2, PPAR{alpha}, or PPAR{gamma}, was excised and submitted for sequencing analysis. The sequence analysis was performed using the Perkin-Elmer Applied Biosystems ABI 310 Genetic Analyzer (Institute of Cell Biology, University of Manitoba, Winnipeg, Canada). The sequences obtained were used for homologous search by the Basic Local Alignment Search Tool (BLAST; NIH, Bethesda, MD). The purified cPLA2 fragment was subcloned into One Shot TOPO10 competent cells (Invitrogen Corp., Carlsbad, CA), and the cPLA2 plasmid was used to generate a standard curve for real-time PCR analysis.

Statistical analysis
The data were analyzed with a two-tailed independent Student's t-test. In all cases, the level of significance was defined as P < 0.05. Results are presented as the mean ± standard deviation.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

The effect of clofibrate on A-23187-induced arachidonic acid release in SW cells
SW cells were cultured in DMEM/F12 (3:1) medium containing 5% FBS and 50;–200 µM clofibrate for either 8 or 32 h. [3H]arachidonate (0.88 µCi/ml) was added to the incubating mixture, and the cells were incubated for another 16 h. Under such experimental conditions, SW cells were exposed to clofibrate for a total of 24 or 48 h. Initial experiments revealed that incubation with arachidonate for more than 16 h would cause the breakdown of the labeled arachidonic acid and/or result in chain elongation of arachidonate into adrenate (33) and that the formation of adrenate would inhibit the agonist-induced release of arachidonate from phospholipids (33). Under the current experimental conditions, about 33% of the labeled arachidonic acid was taken up by the cells, and the majority of the label was associated with phosphatidylcholine. No morphological change in the shape or size of cells was apparent during the course of treatment. In a control study, the preincubation of clofibrate for 0, 8, or 32 h did not significantly alter the total arachidonic acid uptake. Subsequent to labeling, the cells were challenged with calcium ionophore A-23187 (10 µM) (34) for 10 min, and the arachidonate released into the medium was determined. An increase in arachidonate release (40%) was observed at 24 h of incubation with 200 µM clofibrate, whereas a higher increase (50%) was obtained at 48 h of incubation. In a dose-response study, a 34% increase in arachidonate release was observed when cells were treated with 50 µM of clofibrate for 48 h. Maximum increase in arachidonate release (50%) was obtained when the cells were treated with 200 µM of clofibrate under the same conditions ( Table 2). Our results indicate that clofibrate treatment caused the enhancement of arachidonic acid release in human preadipocytes.


 
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  		Table 2. The effect of clofibrate on A-23187-induced arachidonic acid release in SW 872 preadipocytes

The effect of clofibrate on cPLA2 activity
Because clofibrate caused an increase in arachidonic acid release, it would be of interest to examine whether the increase was associated with a change in cPLA2 activity. Hence, the activity of the enzyme was determined after 24 and 48 h of clofibrate (200 µM) treatment. The cPLA2 activity was found to increase in the cell homogenate after cells were treated with clofibrate for 24 h (110%) and reached a maximum (240%) at 48 h of incubation. The activity of the phospholipase A2 was not significantly changed when assayed in the presence of 1 mM of dithiothreitol, suggesting that there was not a significant amount of secretory phospholipase A2 in the homogenate. Despite the increase in cPLA2 activity induced by clofibrate, the ratio of enzyme activity remained constant between the cytosolic and membrane fractions, suggesting that no significant translocation of enzyme occurred during clofibrate treatment. In a dose-response study, cells were incubated with 50;–400 µM of clofibrate for 48 h ( Fig 1). Maximum activation of the cPLA2 activity was obtained at 200 µM clofibrate, but the enzyme activity was attenuated from its maximal value when cells were incubated with 400 µM clofibrate.



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  		Figure 1. The effect of clofibrate on cPLA2 activity in human preadipocytes. SW cells were incubated in Dulbecco's modified Eagle's medium/Ham's F12 nutrition mixture (DMEM/F12) (3:1) containing 5% fetal bovine serum with increasing concentrations of clofibrate for 48 h. Cells were lysed, and cPLA2 activity was assayed in the total homogenate. Results are means ± standard deviation of three separate sets of experiments. * P < 0.05. CON: vehicle control.

The effect of clofibrate on the protein level of cPLA2
The enhancement of the cPLA2 activity might be caused by an increase in the enzyme protein level. Hence, the level of cPLA2 protein in SW cells after clofibrate treatment was determined by Western blot analysis. The change in enzyme protein corresponded to the change in enzyme activity at 24 and 48 h of clofibrate treatment. Incubation of cells with increasing concentrations of clofibrate (up to 200 µM) for 48 h resulted in progressive increases in cPLA2 protein levels ( Fig 2). Similar to the enzyme activity study, the enzyme protein level was attenuated from its maximal value when cells were incubated with 500 µM clofibrate.



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  		Figure 2. The effect of clofibrate on the protein level of cPLA2. SW cells were incubated in DMEM/F12 (3:1) with increasing concentrations of clofibrate as described in Fig 1. Western blot analyses of cPLA2 protein were performed using a rabbit polyclonal cPLA2 antibodies. A: A representative blot is depicted. Std: cPLA2 standard. B: Densitometry plot of A.

Up-regulation of cPLA2 and COX-2 mRNA by clofibrate
The role of clofibrate in the expression of cPLA2 mRNA was examined. Cells were treated with 200 µM clofibrate for 0;–72 h, the mRNA for cPLA2 and GAPDH were amplified by RT-PCR, and the PCR products were analyzed by agarose gel electrophoresis. The relative intensity of each band in the agarose gel was quantified by scanning and then normalized by the intensity of GAPDH band. As depicted in Fig 3, the maximum cPLA2 mRNA level was observed at 48 h of incubation. In a dose-response study, cells were incubated with 0;–1000 µM clofibrate for 48 h, and the cPLA2 mRNA level was determined. The cPLA2 mRNA level was elevated by a broad range of clofibrate concentrations (100;–1000 µM), with the maximal effect obtained at 200 µM ( Fig 4). The cPLA2 mRNA level was attenuated from its maximal value when the cells were incubated with 500 or 1,000 µM clofibrate.



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  		Figure 3. Induction of cPLA2 and COX-2 mRNA by clofibrate over time. Preconfluent SW cells were incubated up to 72 h in DMEM/F12 medium containing 200 µM clofibrate. The total RNA was isolated, and reverse transcriptase polymerase chain reaction (RT-PCR) on the cPLA2 and COX-2 mRNA was performed. Results are means ± standard deviation of three separate experiments.



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  		Figure 4. The effect of clofibrate on cPLA2, COX-2, and acyl-CoA oxidase (ACO) mRNA levels. SW cells were incubated in DMEM/F12 with increasing concentrations of clofibrate (10;–1,000 µM) for 48 h. Total RNA was isolated, and RT-PCR on cPLA2, COX-2, and ACO mRNA was performed. Results are means ± standard deviation of three separate experiments. A typical gel analysis is depicted. M: molecular marker. NC: negative control.

To analyze the increase of cPLA2 mRNA level by clofibrate treatment in a more quantitative manner, the SYBR Green I real-time PCR was performed. The cPLA2 plasmid was serially diluted, and real-time data were plotted with template copy numbers versus threshold cycle values. The linear relationship was obtained when the amplification was plotted against the initial template concentration, and all samples tested were located within the linear range. The results obtained from the real-time PCR study for cPLA2 expression were similar to those obtained from the PCR study described in the preceding section (data not shown).

To confirm that the action of clofibrate was mediated via PPAR{alpha}, the mRNA level of a known PPAR{alpha} target gene, ACO, was also studied. As predicted, clofibrate (10;–500 µM) caused the increase in the gene expression of ACO in SW cells ( Fig 4D). The effect of clofibrate on ACO, however, was less prominent than that obtained from cPLA2 or COX-2. The effect of other PPAR{alpha} ligands, such as WY14643 on the cPLA2 mRNA level, was also studied. As depicted in Fig 5, the cPLA2 mRNA level was elevated when cells were incubated with 10 µM of WY14643 for 48 h.



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  		Figure 5. The effect of WY14643 on the cPLA2 and COX-2 mRNA levels. Preconfluent SW cells were incubated for 48 h in DMEM/F12 medium containing 1;–10 µM WY14643. RT-PCR was performed as described in Materials and Methods. Results are means ± standard deviation of three independent experiments. * P < 0.05.

COX-1 and -2 are integral membrane proteins. COX-1 is responsible mainly for the biosynthesis of prostaglandins for homeostatic or "housekeeping" regulation (35), whereas COX-2 produces prostaglandins that act through nucleoplasmic targets in association with cell differentiation and replication through the nuclear receptor. Fibrates have been shown to regulate the gene expression of COX-2 via its interaction with PPAR (36). A functional coupling and differential regulation of cPLA2 and COX-2 in inflammation has recently been demonstrated (36). In addition, cPLA2 is functionally coupled with COX-2 to involve in both the immediate and delayed PG-biosynthetic responses (37) (38). Hence, the effect of clofibrate on the COX gene expression in SW cells was also examined. As predicted, clofibrate exhibited an effect on COX-2 similar to that observed in cPLA2. Specifically, clofibrate elicited a ~2.0- to 3.8-fold increase in the COX-2 mRNA level when compared with the control ( Fig 4). The effect of clofibrate on COX-2 was found to be concentration dependent, and the maximum effect was obtained at 200 µM. The enhancement of the COX-2 mRNA was also found to be attenuated from the maximal level when 500 µM of clofibrate was used. WY14643, another PPAR{alpha} ligand, showed a similar effect on COX-2 gene expression when treating cells at the 1;–10 µM range ( Fig 5). Taken together, these results demonstrate that clofibrate has the ability to up-regulate mRNA levels of both COX-2 and cPLA2, suggesting that there is a coordinated regulation of both enzymes upon stimulation in SW cells. No change in COX-1 mRNA level was observed in SW cells treated with clofibrate (data not shown), which is consistent with the postulation that COX-1 functions as a housekeeping enzyme.

In another set of experiments, cells were treated with PGJ2, which is a known PPAR{gamma} ligand. Cells were incubated with PGJ2 (1;–10 µM) for 48 h, and the cPLA2 activity, enzyme protein, and cPLA2 mRNA level were determined. The treatment of cells with 1;–3 µM PGJ2 did not elicit any significant effect on cPLA2 activity, changes in enzyme protein, or mRNA level (data not shown). When cells were treated with 5 µM of PGJ2 under the same conditions, only 50% of the cells survived after the incubated period. Incubation with 10 µM of PGJ2 caused the complete death of all cells within 24 h.

The expression of PPAR{alpha} and PPAR{gamma} in cultured SW cells
The action of fibrates on the regulation of transcription of lipid metabolic enzymes is mediated via the PPAR. Specifically, the interaction between clofibrate and PPAR{alpha} has been shown to regulate the transcription of a variety of lipid metabolic enzymes including ACO, lipoprotein lipase, and HMG-CoA synthetase. To show that PPAR{alpha} is a participant in the control of transcription of cPLA2 in SW cells, the total RNA was isolated from the cells, and the mRNA for PPAR{alpha}, PPAR{gamma}, and GAPDH was amplified by RT-PCR. As depicted in Fig 6A, mRNA for both PPAR{alpha} and PPAR{gamma} were identified in SW cells. Interestingly, both the wild-type fragment of PPAR{alpha} (433 bp) and its naturally occurring variant fragment (330 bp) were detected. The existence of PPAR{alpha} was confirmed by Western blot analysis using specific polyclonal anti-PPAR{alpha} antibodies ( Fig 6B).



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  		Figure 6. Expression of mRNA and protein for PPAR in cultured human preadipocytes. A: The total RNA was isolated from cultured SW cells, and the mRNAs for PPAR{alpha} and PPAR{gamma} were analyzed by RT-PCR. A typical gel analysis is depicted. The expression of PPAR{alpha} mRNA with a wild-type fragment (433 bp), a variant fragment (330 bp), and that of PPAR{gamma} mRNA with a 289-bp fragment are shown. M: molecular marker. NC: negative control. B: Western blot analysis of PPAR{alpha} expression. SW cells were grown in DEME/F12 containing 5% FBS. The expression of PPAR{alpha} in the whole cell lysate was examined by using polyclonal anti-PPAR{alpha} antibodies (1:500 dilution).

Sequencing of PCR products
To verify the identity of the PCR products in this study, the double-stranded DNA obtained from RT-PCR was subjected to sequence analysis. Each DNA fragment obtained, including cPLA2, COX-1, COX-2, PPAR{alpha}, or PPAR{gamma}, was separated, extracted, purified, and sequenced. The results from the sequence analysis were compared with published sequences for cPLA2, COX-1, COX-2, PPAR{gamma}, and PPAR{alpha} variant. The amplified cDNA fragments from SW cells were found to have a high degree of identity (>95%) with the published sequences ( Table 3 and related references). Unfortunately, the sequence of the DNA sample for the PPAR{alpha} wild type (433 bp) could not be obtained. This failure might be caused by a small amount of the variant form presented as contamination in the wild-type fragment after agarose gel separation.


 
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  		Table 3. Sequence comparison and homology


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

Arachidonic acid is an important precursor for ecoisanoid synthesis. Eicosanoids, which include prostaglandins, leukotrienes, and thromboxanes, have been implicated in biological processes as diverse as signal transduction (39) (40), the regulation of cell growth, and the maintenance of vascular integrity ( (41), and references therein). Under normal physiological conditions, the vast majority of the arachidonic acid is stored in esterified form in phospholipids and can be released by the action of PLA2. Ionophores are widely used to stimulate PLA2 for the release of arachidonate in experimental studies (42) because the use of other adrenergic agents may produce many effects innate to the physiological properties of these agents. Among the different types of PLA2 found in the mammalian cell, cPLA2 is the intracellular enzyme that directs the release of arachidonic acid under stimulation (43). The activation of cPLA2 can be regulated by many mechanisms at both the transcriptional and post-translational levels. The enzyme is regulated at the transcriptional level by cytokines and growth factors (17) (20), which is regarded as a prolonged process for the activation of the enzyme during tissue development and differentiation (16) (19). Alternatively, the post-translational regulation would provide the cell with a rapid process for the activation of cPLA2. Several mechanisms for the post-translational regulation of the enzyme have been identified, including the Ca2+-dependent translocation of the enzyme-to-membrane and phosphorylation-dependent activation of the enzyme mediated by MAP kinase and protein kinase C (44). An additional mechanism involved in cPLA2 activation is mediated via the G-proteins, which regulate the MAP kinase cascade (14). We have reported previously that lyso-PC is a potent activator of the cPLA2 activity in both human endothelial cells and rat cardiac myoblastic H9c2 cells (28) (29). In these studies, we have shown that the lyso-PC exerted its effect through mechanisms of post-transcriptional regulation. These observations led us to explore the regulation of this enzyme at the transcriptional level.

The nuclear hormone receptor PPAR{alpha} is a member of the thyroid/vitamin D/retinoid superfamily of transcription factors that require heterodimerization with the retinoid X receptor for optimal DNA binding (45). Whereas the activation of cPLA2 by PPAR{alpha} activators has not been previously reported, the ligand/activator of other hormone receptors in this superfamily, such as the analog or precursor of vitamin D, have been shown to activate cPLA2 or COX-2 (46) (47). Clofibrate is a classical peroxisome proliferator and PPAR{alpha} activator (22) (23), and it has been shown to stimulate the transcription of the lipoprotein lipase gene via peroxisome proliferator response element in the cultured murine 3T3 preadipocytes (24). As the first generation of lipid-lowering drugs, clofibrate has been approved for therapeutic use in the USA for the last three decades. Treatment with clofibrate results in a substantial decrease in plasma triglyceride-rich lipoproteins and an increase in the high density lipoprotein (HDL) (48) (49). These alterations are correlated with decreased levels of apolipoprotein-B (apoB), apoC-III, and apoE in triglyceride-rich lipoproteins and increased levels of apoA-I and apoA-II in HDL. The effect of fibrate on human apoA-I has been shown to be mediated by the transcription factor PPAR{alpha}, which interacts with a positive PPAR-response element at its promoter region (50).

The human liposarcoma cell line SW872 has been shown to express and secrete high levels of cholesteryl ester transfer protein and therefore has been effectively used for studies on lipid metabolism (51) (52). We have shown that this cell line has the ability to express high levels of PPAR{alpha} mRNA and protein and therefore is subjected to modulation by PPAR{alpha} ligands. In view of the key importance of cPLA2 in the release of arachidonate, it is of interest to elucidate the modulation of cPLA2 expression at the transcriptional level upon PPAR ligand stimulation in these cells.

Long-term stimulation of SW cells by insulin (1 µM) caused the transformation of these cells into adipocytic phenotypes (53). Prolonged stimulation of 3T3-L1 preadipocytes with fibrates (5;–7 days) caused the conversion into adipocytes via the activation of PPAR (54) (55). In this study, treatment of SW cells with clofibrate or PGJ2 was limited to 48 h to avoid any cell transformation. Transformation into adipocytes would be detected by the presence of fat globules, which would be verified by red oil O staining (53) (54). No adipocyte formation was observed throughout the course of clofibrate treatment (data not shown). It is interesting that PGJ2 at low concentrations did not produce any effect on the gene expression of cPLA2 or COX-2. The potency of PGJ2 to SW cells, however, was clearly demonstrated because treatment with 10 µM PGJ2 caused 100% cell death within 24 h.

Although the direct involvement of the PPAR pathway in the activation of cPLA2 has not been demonstrated in this study, there is ample evidence that the action of clofibrates is mediated via the PPAR{alpha} (22) (23). Clofibrates have been shown to regulate the gene expression of COX-2 via its interaction with PPAR (37), and a functional coupling and differential regulation of cPLA2 and COX-2 in inflammation has recently been demonstrated (38). Although PPAR{alpha} ligands have the ability to induce the ACO gene in rodents, their ability to induce the ACO gene in human cells has not been established (56). The relatively weak induction of ACO mRNA in SW cells by clofibrate treatment is in agreement with this notion. Nevertheless, the presence of PPAR in SW cells has been firmly established because these cells have the ability to express both the mRNA encoding PPAR{alpha} and the receptor protein itself. Interestingly, both PPAR{alpha} and PPAR{gamma} are expressed in the SW cells. This is not surprising, because this cell line is derived from the human liposarcoma and contains specific gene sets, which are responsible for lipogenesis and lipolysis upon stimulation. The presence of the wild-type fragment and the naturally occurring variant of PPAR{alpha} mRNA indicate that the SW cell has the ability to synthesize the full length PPAR{alpha} receptor as well as the truncated PPAR{alpha} receptor (57). The function of the PPAR{alpha} variant in preadipocytes, however, remains undefined.


  ACKNOWLEDGMENTS

This study was supported by a grant from the Heart and Stroke Foundation of Manitoba. Yan J. Jiang is a Scientist of the Centre for Research and Treatment of Atherosclerosis.

Manuscript received August 16, 2000; and in revised form January 8, 2001

Abbreviations: COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PPAR, peroxisome proliferator-activated receptor; SW cells, human preadipocyte SW872 cells


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