Stimulation of cardiac cardiolipin biosynthesis by PPARα activation
2004; Elsevier BV; Volume: 45; Issue: 2 Linguagem: Inglês
10.1194/jlr.m300314-jlr200
ISSN1539-7262
AutoresYan Jiang, Biao Lü, Fred Y. Xu, Jennifer Gartshore, William A. Taylor, Andrew J. Halayko, Frank J. Gonzalez, Jun Takasaki, Patrick C. Choy, Grant M. Hatch,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe role of peroxisome proliferator-activated receptor α (PPARα)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARα-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARα activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo.These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARα activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARα activation. The role of peroxisome proliferator-activated receptor α (PPARα)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARα-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARα activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo. These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARα activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARα activation. Phospholipids are important structural and functional components of the biological membrane (1White D.A. The phospholipid composition of mammalian tissues.in: Ansell G.B. Hawthorne J.N. Dawson R.M.C. Form and Function of Phospholipids. Elsevier Science Publishers, Amsterdam1973: 441-483Google Scholar). Structurally, as major components of the biological membrane, they define the compartmentalization of organelles and the protective barrier, the cell membrane, that surrounds cells. An important class of phospholipids are the polyglycerophospholipids. The major polyglycerophospholipid found in most mammalian tissues is bis-(1,2-diacyl-sn-glycero-3-phospho)-1′,3′-sn-glycerol or cardiolipin (CL) (2Hostetler K.Y. Polyglycerophospholipids.in: Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier Science Publishers, Amsterdam1982: 215-261Google Scholar). CL was first isolated from beef heart in 1942 by Pangborn (3Pangborn M.C. Isolation and purification of a serologically active phospholipid from beef heart.J. Biol. Chem. 1942; 143: 247-256Abstract Full Text PDF Google Scholar). In rat liver, CL was initially shown to be associated exclusively with the inner mitochondrial membrane (4Stoffel W. Schiefer H-G. Biosynthesis and composition of phosphatides in outer and inner mitochondrial membranes.Hoppe-Seyler's Z. Physiol. Chem. 1968; 349: 1017-1026Crossref PubMed Scopus (103) Google Scholar). However, using various fractionation techniques, CL was identified also in the mitochondrial outer membrane of rat liver mitochondria (5Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane.Biochim. Biophys. Acta. 1990; 1021: 217-226Crossref PubMed Scopus (311) Google Scholar). In rat heart, CL is a major phospholipid component that constitutes ∼15% of the entire phospholipid phosphorus mass of the organ (6Poorthuis B.J.H.M. Yazaki P.J. Hostetler K.Y. An improved two-dimensional thin layer chromatography system for the separation of phosphatidylglycerol and its derivatives.J. Lipid Res. 1976; 17: 433-437Abstract Full Text PDF PubMed Google Scholar, 7Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Crossref PubMed Scopus (66) Google Scholar). CL is involved in modulation of the activity of a number of mitochondrial membrane enzymes involved in the generation of ATP (8Hoch F.L. Cardiolipins and biomembrane function.Biochim. Biophys. Acta. 1992; 1113: 71-133Crossref PubMed Scopus (546) Google Scholar). It is well documented that alteration in the content and the molecular species composition of CL alters mitochondrial enzyme activity and oxygen consumption in mammalian mitochondria (9Yamaoka-Koseki S. Urade R. Kito M. Cardiolipins from rats fed different dietary lipids affect bovine heart cytochrome c oxidase activity.J. Nutr. 1991; 121: 956-958Crossref PubMed Scopus (42) Google Scholar, 10Ohtsuka T. Nishijima M. Suzuki K. Akamatsu Y. Mitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant defective in cardiolipin.J. Biol. Chem. 1993; 268: 22914-22919Abstract Full Text PDF PubMed Google Scholar). Recently, CL has been implicated as an important mitochondrial factor that may be involved in the regulation of apoptosis (11Esposti M.D. Lipids, cardiolipin and apoptosis: a greasy license to kill.Cell Death Differ. 2002; 9: 234-236Crossref PubMed Scopus (61) Google Scholar, 12McMillann Dowhan W. Cardiolipin and apoptosis.Biochim. Biophys. Acta. 2002; 1585: 97-107Crossref PubMed Scopus (239) Google Scholar). Thus, maintenance of the appropriate content of CL in cardiac mitochondria is essential for proper cardiac function. The de novo biosynthesis of CL in the rat heart occurs via the CDP-1,2-diacyl-sn-glycerol (CDP-DG) pathway (2Hostetler K.Y. Polyglycerophospholipids.in: Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier Science Publishers, Amsterdam1982: 215-261Google Scholar). Phosphatidic acid (PA) is converted to CDP-DG by CDP-DG synthase (CDS; EC 2.2.7.41) (13Kiyasu J.Y. Pieringer R.A. Paulus H. Kennedy E.P. The biosynthesis of phosphatidylglycerol.J. Biol. Chem. 1963; 238: 2293-2298Abstract Full Text PDF PubMed Google Scholar). In the rat heart and in embryonic rat heart-derived H9c2 myoblast cells, CDS may be a rate-limiting enzyme for CL biosynthesis (7Hatch G.M. Cardiolipin biosynthesis in the isolated heart.Biochem. J. 1994; 297: 201-208Crossref PubMed Scopus (66) Google Scholar, 14Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The CDP-DG formed condenses with glycerol-3-phosphate to form phosphatidylglycerol (PG) catalyzed by PG phosphate (PGP) synthase (EC 2.7.8.5) and PGP phosphatase (EC 3.1.3.27) (13Kiyasu J.Y. Pieringer R.A. Paulus H. Kennedy E.P. The biosynthesis of phosphatidylglycerol.J. Biol. Chem. 1963; 238: 2293-2298Abstract Full Text PDF PubMed Google Scholar). In the final step, PG is converted to CL by condensation with CDP-DG catalyzed by CL synthase (15Hostetler K.Y. Van den Bosch H. Van Deneen L.L. Biosynthesis of cardiolipin in rat liver mitochondria.Biochim. Biophys. Acta. 1971; 239: 113-119Crossref PubMed Scopus (95) Google Scholar). CL may be hydrolyzed by mitochondrial phospholipase A2 (PLA2) (2Hostetler K.Y. Polyglycerophospholipids.in: Hawthorne J.N. Ansell G.B. Phospholipids. Elsevier Science Publishers, Amsterdam1982: 215-261Google Scholar). Previously, we demonstrated that mitochondrial PLA2 in rat embryonic H9c2 myoblastic cells could be activated by short-chain ceramides or tumor necrosis factor-α (TNFα) (16Xu F. Kelly S.L. Hatch G.M. N-Acetylsphingosine stimulates phosphatidylglycerolphosphate synthase activity in H9c2 cardiac cells.Biochem. J. 1999; 337: 483-490Crossref PubMed Scopus (25) Google Scholar). This activation of mitochondrial PLA2 led to an increase in CL biosynthesis that was mediated by stimulation of mitochondrial PGP synthase activity. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily (17Braissant O. Foufelle F. Scotto C. Dauca M. Wahil W. Differential expression of peroxisome proliferator-activated receptors (PPAR's): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (0) Google Scholar, 18Bishop-Bailey D. Peroxisome proliferator-activated receptors in the cardiovascular system.Br. J. Pharmacol. 2000; 129: 823-834Crossref PubMed Scopus (324) Google Scholar, 19Staels B. Auwerx J. Regulation of apoA-1 gene expression by fibrates.Atherosclerosis. 1998; 137: 19-23Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Three forms have been identified: PPARα, PPARβ, and PPARγ. It is well documented that the rat heart contains PPARα. Ethyl-p-chlorophenoxyisobutyrate (clofibrate) is a synthetic lipid-lowering agent used in clinical practice. The effect of clofibrate on lipid metabolism in rodent heart is mediated by PPARα after heterodimering with the retinoid X receptor and interacting with PPAR response elements in the promotor regions of target genes. A recent study indicated that the mRNA of uncoupling protein-2 was elevated in neonatal cardiomyocytes and embryonic rat heart-derived H9c2 myoblast cells, probably through PPARα activation (20Van der Lee K.A. Willemsen P.H. Van der Vusse G.J. Van Bilsen M. Effects of fatty acids on uncoupling protein-2 expression in the rat heart.FASEB J. 2000; 14: 495-502Crossref PubMed Scopus (52) Google Scholar). However, the presence of PPARα receptors in H9c2 cells was not examined in that study. Because PPARα activation stimulates PLA2 activity (21Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. Potentiation of TNF-alpha-stimulated group IIA phospholipase A2 expression by peroxisome proliferator-activated receptor alpha activators in rat mesangial cells.J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar, 22Jiang Y.J. Hatch G.M. Mymin D. Dembinski T. Kroeger E.A. Choy P.C. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes.J. Lipid Res. 2001; 42: 716-724Abstract Full Text Full Text PDF PubMed Google Scholar), the role of this receptor in CL biosynthesis was examined in embryonic rat heart H9c2 myoblastic cells and mouse heart. The results show that activation of PPARα mediates increased CL biosynthesis and metabolism via an increase in mitochondrial PLA2 and PGP synthase activities. However, activation of CDS-2, a rate-limiting enzyme of CL biosynthesis in rat heart embryonic H9c2 cells, is not necessary for the PPARα agonist-mediated stimulation of de novo CL biosynthesis. [14C]Glycerol-3-phosphate, [5-3H]CTP, [1,3-3H]glycerol, and [1-14C]oleate were obtained from either Dupont (Mississauga, Ontario, Canada) or Amersham (Oakville, Ontario, Canada). [14C]phosphatidylglycerol was synthesized from [14C]glycerol-3-phosphate (14Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). DMEM and FBS were products of Canadian Life Technologies (GIBCO; Burlington, Ontario, Canada). Lipid standards were obtained from Serdary Research Laboratories (Englewood Cliffs, NJ). Thin-layer plates (silica gel G; 0.25 mm thickness) were obtained from Fisher Scientific (Winnipeg, Canada). Ecolite scintillant was obtained from ICN Biochemicals (Montreal, Canada). Rat heart H9c2 myoblastic cells were obtained from the American Type Culture Collection. Rabbit anti-rat PPARα polyclonal antibody and anti-rat actin antibody were purchased from Affinity BioReagents, Inc. (Golden, CO). Monoclonal anti-cPLA2 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). For confocal immunocytochemistry, secondary FITC-conjugated goat anti-rabbit antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rabbit anti-rat PLA2-IIA polyclonal antibody (protein A purified) was a generous gift from Dr. J. Takasaki (Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd.). The RPN 2108 ECL Western blotting analysis system was used for PPARα and PLA2 expression studies and was obtained from Amersham Pharmacia Biotech UK, Ltd. (Buckinghamshire, UK). Kodak X-OMAT film was obtained from Eastman Kodak Co. (Rochester, NY). All other biochemicals were certified American Chemical Society grade or better and obtained from either Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific. Male C57BL/6N × Sv/129 mice (25–35 g) were obtained from Central Animal Care Services (University of Manitoba, Winnipeg, Manitoba, Canada). PPARα-null (−/−) mice were a generous gift from Dr. Frank Gonzalez (National Institutes of Health). Treatment of mice conformed to the guidelines of the Canadian Council on Animal Care. The mice were maintained on rat chow and water ad libitum. All animals were kept in identical housing units on a cycle of 12 h of light and 12 h of darkness. In treatment groups, mice were fed 0.5% clofibrate in a chow diet for 14 days (23Pan D.A. Mater M.K. Thelen A.P. Peters M. Gonzalez F.J. Jump D.J. Evidence against the peroxisome proliferator-activated receptor alpha (PPARalpha) as the mediator for polyunsaturated fatty acid suppression of hepatic L-pyruvate kinase gene transcription.J. Lipid Res. 2000; 41: 742-751Abstract Full Text Full Text PDF PubMed Google Scholar). Rat heart H9c2 myoblastic cells were grown and incubated in DMEM containing 10% FBS until 90% confluence was achieved. Cells were then incubated for 48 h with DMEM containing 10% FBS in the absence or presence of 200 μM clofibrate or 5 μM WY14,643. The medium was changed once after 24 h. In radiolabeling experiments, cells were then incubated with DMEM containing 10% FBS for up to 3 h with 0.1 μM [1,3-3H]glycerol (10 mCi/ml medium). The medium was removed and the cells were washed twice with ice-cold saline and then harvested from the dish with 2 ml of methanol-water (1:1, v/v) for lipid extraction. CL was separated from other phospholipids by two-dimensional TLC, and radioactivity incorporated into CL and other phospholipids was determined as described (14Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In some experiments, H9c2 cells were preincubated with 0.1 μM [1-14C]oleate (1.0 mCi/ml medium) for 24 h and then incubated in the absence or presence of 200 μM clofibrate for up to 8 h, and the radioactivity incorporated into CL was determined. H9c2 cells were incubated for 48 h with DMEM containing 10% FBS in the absence or presence of 200 μM clofibrate. The medium was changed once after 24 h. Subsequently, the cells were washed twice with ice-cold saline and harvested with 2 ml of homogenization buffer (10 mM Tris-HCL, pH 7.4, and 0.25 M sucrose). The cells were homogenized with 15 strokes of a Dounce A homogenizer. The homogenate (designated the cell lysate) was centrifuged at 1,000 g for 5 min, and the resulting supernatant was centrifuged at 10,000 g for 15 min. The pellet was resuspended in 0.5 ml of homogenization buffer and designated the mitochondrial fraction. CDS, PGP synthase, and CL synthase activities were determined as described (14Hatch G.M. McClarty G. Regulation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells by cytidine-5′-triphosphate.J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In some experiments, 200 μM clofibrate was added directly to the enzyme incubation mixture of mitochondrial fractions prepared from untreated H9c2 cells, and CDS, PGP synthase, and CL synthase activities were determined. PLA2 activity was determined as described (24Hatch G.M. Cao S.G. Angel A. Decrease in cardiac phosphatidylglycerol in streptozotocin-induced diabetic rats: evidence for distinct pools of phosphatidylglycerol in the heart.Biochem. J. 1995; 306: 759-764Crossref PubMed Scopus (46) Google Scholar). PA phosphohydrolase activity also was determined as described (25Xu F.Y. Taylor W.A. Hurd J.A. Hatch G.M. Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells.J. Lipid Res. 2003; 44: 415-423Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). H9c2 cells were incubated for 48 h with DMEM containing 10% FBS in the absence or presence of 50 μM clofibrate. The medium was changed once after 24 h. The cells were harvested and homogenized, and mitochondrial fractions were prepared as described above. A 50 μg aliquot of the homogenate or mitochondrial fraction was subjected to SDS-7.5% PAGE with molecular weight standards using a Bio-Rad (Hercules, CA) Mini-Protean® II Dual Slab Cell electrophoresis unit. Proteins were transferred from the gel onto polyvinylidene difluoride (PVDF) membranes by incubation for 30 min at 15 V using a Bio-Rad Trans-Blot SD Semi-Dry Transfer Cell. Expression of PPARα was examined by incubating the PVDF membrane with the polyclonal anti-rat PPARα antibody (1:1,000 dilution) dissolved in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 2% skim milk overnight at 4°C. Subsequently, the membrane was washed and incubated with peroxidase-labeled anti-rabbit secondary antibody (1:5,000) for 5–30 min at room temperature. Protein bands in the membrane were visualized by enhanced chemiluminescence. For detection of 14 kDa PLA2, after transfer to PVDF membranes, the membranes containing protein samples were incubated with monoclonal anti-PLA2 antibody (1:1,000). The membrane was then washed and incubated with the peroxidase-labeled anti-rabbit secondary antibodies (1:5,000) for 5–30 min at room temperature. The relative intensities of the bands were analyzed by scanning the film and subsequently determined using Scion Image software (22Jiang Y.J. Hatch G.M. Mymin D. Dembinski T. Kroeger E.A. Choy P.C. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes.J. Lipid Res. 2001; 42: 716-724Abstract Full Text Full Text PDF PubMed Google Scholar). Sterilized glass coverslips were transferred to individual wells of 12-well culture plates. H9c2 cells were seeded on the plates (40,000 cells/well) for 24 h. Cells were then incubated for 48 h with DMEM containing 10% FBS in the absence or presence of 50 μM clofibrate (40,000 cells/well). The medium was changed once after 24 h. The medium was then removed, and the cells were fixed with 3% paraformaldehyde for 15 min and then permeabilized using 3% paraformaldehyde and 0.3% Triton X-100 for 5 min at room temperature. The cells were next rinsed twice with buffer (10 mM MES, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, and 5 mM glucose, pH 6.1), stored in sealed culture chambers with 1 ml of TBS buffer (20 mM Tris-HCl, 150 mM NaCl, 2.0 mM EGTA, and 2.0 mM MgCl2, pH 7.2), and stored. Cells could be stored for up to 2 weeks before analysis. The cells were then incubated with TBS containing 1% BSA and 2% sheep serum for 1 h. Coverslips were removed and inverted onto 20 μl of the primary anti-cPLA2 antibody and incubated overnight at 4°C in a humidified chamber. The coverslip was then placed cell side up in 12-well culture dishes that were then immediately filled with 3 ml of TBS buffer containing 0.1% Tween 20. After 10 min, the buffer was removed and washed twice with TBS buffer containing 0.1% Tween 20. Coverslips were removed, inverted onto 20 μl of the secondary antibody (diluted 1:100), and incubated in the dark for 2–3 h at room temperature in a humidified chamber. The coverslips were then washed three times as described above with TBS buffer containing 0.1% Tween 20. DNA of the cells was stained with propidium iodide (1 μg/ml) in TBS buffer containing RNase (40 μg/ml). After staining, coverslips were washed three times with double- distilled water and then twice with TBS buffer containing 0.1% Tween 20. Coverslips were then washed three times with double-distilled water and mounted cell side down onto glass slides prespotted with ProLong AntiFade mounting medium (Molecular Probes, Inc., Eugene, OR). Confocal image stacks, consisting of three to five sections obtained at 0.5 mm vertical intervals, were obtained using an Olympus (Tokyo, Japan) LX70 inverted microscope equipped with FluoView Laser Scanning hardware, including argon and krypton lasers to generate 488 and 568 nm lines for the detection of FITC and propidium iodide fluorescence, respectively. The cDNA for CDS-2 was amplified with a pair of specific primers synthesized by Invitrogen™ Life Technologies (Carlsbad, CA). The rat CDS-2 primers used for the RT-PCR experiments were previously described (26Volta M. Bulfone A. Gattuso C. Rossi E. Mariani M. Consalez G.G. Zuffardi O. Ballabio A. Banfi S. Franco B. Identification and characterization of CDS2, a mammalian homolog of the Drosophila CDP-synthase gene.Genomics. 1999; 55: 68-77Crossref PubMed Scopus (36) Google Scholar): CDS-2 (U), 5′-GAATCAGAGTCGGAAGCA; and CDS-2 (L), 5′-ACCAGGGCAAGTCGTAG. H9c2 cells were incubated for up to 72 h with 200 μM clofibrate, and total RNA was isolated using the Trizol reagent according to the manufacturer's instructions. The RNA pellet was suspended in autoclaved, double-distilled water and quantitated 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 of the isolated RNA sample. The first-strand cDNA from 1 μg of total RNA was synthesized using 150 U of Moloney murine leukemia virus RT, 25 pmol of random hexamer primer, 20 U of RNase inhibitor, 1 mM dithiothreitol, and 10 pmol each of the four deoxynucleotides, in a total volume of 15 μl. 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 the resulting cDNA preparation was used directly for each amplification reaction. PCR was performed in 20 μl reaction mixtures containing 8 pmol of primer, 8 pmol of each deoxynucleotide trisphosphate, and 0.4 U of Taq DNA polymerase. The mixture was overlaid with 30 μl of mineral oil to prevent evaporation and was incubated in a Perkin-Elmer (Foster City, CA) DNA Thermal Cycler under the following conditions. For the PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) CDS-2: 25–30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55–60°C, and extension for 2 min at 72°C. The amplified RT-PCR product was analyzed by 1.2–1.8% agarose gel electrophoresis in 1× TAE buffer (40 mM Tris acetate and 2 mM sodium EDTA) and visualized by staining with 0.5 μg/μl ethidium bromide. The GAPDH band was used as an internal control. An increase in the level of a specific mRNA is caused by 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 of RNA synthesis. H9c2 cells were incubated with 200 μM clofibrate for 48 h, and 5 μg/ml actinomycin D was added after clofibrate treatment. The mRNA levels for CDS-2 and GAPDH were determined by RT-PCR at 4 h intervals after the actinomycin 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 CDS-2 mRNA. Based on the primer pair used for rat CDS-2, a homologous primer pair for mouse, 5′-GAATCAGAGTCCGAAGCA and 5′-CTACGACCTGCCCTGGT (GenBank identifier gi31542373), was designed for RT-PCR of CDS-2 in mouse heart (GenBank identifier gi31542373). Primers for β-actin were 5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTG-3′ The conditions for amplification were 25 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 30 s at 72°C. The amplified RT-PCR product was analyzed as described above. Protein was determined by the method of Lowry et al. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Student's t-test was used to determine statistical significance. The level of significance was defined as P < 0.05. Acute activation of mitochondrial PLA2 by TNFα or short-chain ceramide resulted in the stimulation of CL biosynthesis in rat heart H9c2 cardiac myoblast cells (16Xu F. Kelly S.L. Hatch G.M. N-Acetylsphingosine stimulates phosphatidylglycerolphosphate synthase activity in H9c2 cardiac cells.Biochem. J. 1999; 337: 483-490Crossref PubMed Scopus (25) Google Scholar). Previous studies indicated that PPARα activation resulted in an increase in PLA2 activity in human premonocytic cells and in rat mesangial cells (21Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. Potentiation of TNF-alpha-stimulated group IIA phospholipase A2 expression by peroxisome proliferator-activated receptor alpha activators in rat mesangial cells.J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar, 22Jiang Y.J. Hatch G.M. Mymin D. Dembinski T. Kroeger E.A. Choy P.C. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes.J. Lipid Res. 2001; 42: 716-724Abstract Full Text Full Text PDF PubMed Google Scholar). Because the heart contains PPARα, we examined whether activation of PLA2 through a TNFα- and ceramide-independent mechanism stimulated CL biosynthesis. Initially, we examined the nuclear expression of PPARα in H9c2 cells with polyclonal anti-rat PPARα antibody. H9c2 cells were grown to 90% confluence and harvested, and nuclear fractions were prepared. PPARα receptor was expressed in the nuclear fraction of H9c2 cells (Fig. 1). PPARα activation in rat Morris cells by ciprofibrate resulted in increased nuclear association of PPARα (28Rodriguez C. Noe V. Caberero A. Ciudad C.J. Laguna J.C. Differences in the formation of PPARalpha-RXR/acoPPRE complexes between responsive and non-responsive species upon fibrate activation.Mol. Pharmacol. 2000; 58: 185-193Crossref PubMed Scopus (23) Google Scholar). We examined whether the addition to H9c2 cells of clofibrate, a known pharmacological activator of PPARα, resulted in increased nuclear association of PPARα. H9c2 cells were incubated for up to 4 h with 200 μM clofibrate and then harvested, and nuclear fractions were prepared. We previously demonstrated that this concentration of clofibrate was shown to result in PPARα activation in SW872 preadipocytes (22Jiang Y.J. Hatch G.M. Mymin D. Dembinski T. Kroeger E.A. Choy P.C. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes.J. Lipid Res. 2001; 42: 716-724Abstract Full Text Full Text PDF PubMed Google Scholar). Incubation of H9c2 cells with clofibrate resulted in a rapid increase in nuclear PPARα compared with untreated control cells (Fig. 1). It is known that clofibrate may be toxic to tissue culture cells in high concentrations. We observed that 48 h of incubation of H9c2 cells with 200 μM clofibrate did not affect the ability of these cells to exclude Trypan blue (98% exclusion). Thus, H9c2 cells contain PPARα, and clofibrate addition to H9c2 cells resulted in the activation of PPARα. To determine whether the activation of PPARα in H9c2 cells increased PLA2 activity, cells were incubated in the absence or presence of 200 μM clofibrate for 48 h and lysed, and PLA2 activity was determined. As seen in Table 1, treatment of H9c2 cells with clofibrate resulted in a 32% increase (P < 0.05) in membrane PLA2 activity compared with control cells. We examined the mechanism for the increase in PLA2 activity. We previously demonstrated that the expression of cytosolic PLA2 activity and protein expression were upregulated by PPARα in human premonocytic cells (22Jiang Y.J. Hatch G.M. Mymin D. Dembinski T. Kroeger E.A. Choy P.C. Modulation of cytosolic phospholipase A2 by PPAR activators in human preadipocytes.J. Lipid Res. 2001; 42: 716-724Abstract Full Text Full Text PDF PubMed Google Scholar). H9c2 cells were incubated for 48 h with 200 μM clofibrate, and the presence of cytosolic PLA2 was examined by immunofluorescence confocal microscopy. Clofibrate incubation of H9c2 cells resulted in an increase in cytosolic PLA2 that was chiefly marked by the appearance of numerous dense foci of cytosolic staining near the periphery of each cell (Fig. 2). Thus, PPARα activation by clofibrate resulted in an increase in the expression of cytosolic PLA2. However, because cytosolic PLA2 does not
Referência(s)