Prostacyclin-dependent Apoptosis Mediated by PPARδ
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m107180200
ISSN1083-351X
AutoresToshihisa Hatae, M. Wada, Chieko Yokoyama, Manabu Shimonishi, Tadashi Tanabe,
Tópico(s)Antiplatelet Therapy and Cardiovascular Diseases
ResumoProstacyclin (PGI2) plays important roles in hemostasis both as a vasodilator and an endogenous inhibitor of platelet aggregation. PGI2 functions in these roles through a specific IP receptor, a G protein-coupled receptor linked to Gs and increases in cAMP. Here, we report that intracellular prostacyclin formed by expressing prostacyclin synthase in human embryonic kidney 293 cells promotes apoptosis by activating endogenous peroxisome proliferator-activated receptor δ (PPARδ). In contrast, treatment of cells with extracellular prostacyclin or dibutyryl cAMP actually reduced apoptosis. On the contrary, treatment of the cells with RpcAMP (adenosine 3′,5′-cyclic monophosphothioate, Rp-isomer), an antagonist of cAMP, enhanced prostacyclin-mediated apoptosis. The expression of an L431A/G434A mutant of PPARδ completely blocked prostacyclin-mediated PPARδ activation and apoptosis. These observations indicate that prostacyclin can act through endogenous PPARδ as a second signaling pathway that controls cell fate. Prostacyclin (PGI2) plays important roles in hemostasis both as a vasodilator and an endogenous inhibitor of platelet aggregation. PGI2 functions in these roles through a specific IP receptor, a G protein-coupled receptor linked to Gs and increases in cAMP. Here, we report that intracellular prostacyclin formed by expressing prostacyclin synthase in human embryonic kidney 293 cells promotes apoptosis by activating endogenous peroxisome proliferator-activated receptor δ (PPARδ). In contrast, treatment of cells with extracellular prostacyclin or dibutyryl cAMP actually reduced apoptosis. On the contrary, treatment of the cells with RpcAMP (adenosine 3′,5′-cyclic monophosphothioate, Rp-isomer), an antagonist of cAMP, enhanced prostacyclin-mediated apoptosis. The expression of an L431A/G434A mutant of PPARδ completely blocked prostacyclin-mediated PPARδ activation and apoptosis. These observations indicate that prostacyclin can act through endogenous PPARδ as a second signaling pathway that controls cell fate. prostaglandins prostacyclin PGI2 synthase cyclooxygenase human embryonic kidney 293 cells peroxisome proliferator-activated receptor PPAR-response element bovine aortic endothelial cells dibutyryl cyclic AMP phosphate-buffered saline Tris-buffered saline Dulbecco's modified Eagle's medium fetal bovine serum enzyme-linked immunosorbent assay 5-bromo-4-chloro-3-indolyl β-galactoside polymerase chain reaction arachidonic acid carbaprostacyclin adenosine 3′,5′-cyclic monophosphothioate, Rp-isomer 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine hemagglutinating virus Japan prostaglandin endoperoxide H2 Prostaglandins (PGs)1are a diverse family of oxygenated fatty acids derived from arachidonic acid (AA). AA is converted to an intermediate, prostaglandin endoperoxide H2 (PGH2) by two isoforms of cyclooxygenase, COX-1 and COX-2. The COX product PGH2 is converted to one of several biologically important prostanoids, including PGE2, PGD2, PGF2α, thromboxane A2 and prostacyclin (PGI2) by specific synthases (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2464) Google Scholar, 2Tanabe T. Ullrich V. J. Lipid Mediat. Cell Signal. 1995; 12: 243-255Crossref PubMed Scopus (72) Google Scholar). PGs have wide-ranging effects in regulating aspects of homeostasis and pathogenesis. For example, PGE2modulates inflammation, pain, and fertility whereas PGI2 is important in hemostasis and also exerts bronchiectasis and proliferation inhibitory effect (3Sugimoto Y. Narumiya S. Ichikawa A. Prog. Lipid Res. 2000; 39: 289-314Crossref PubMed Scopus (168) Google Scholar, 4Samuelsson B. Goldyne M. Granstrom E. Hamberg M. Hammarstrom S. Malmsten C. Annu. Rev. 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A. 1998; 95: 8806-8811Crossref PubMed Scopus (760) Google Scholar), macrophages (25Chinetti G. Griglio S. Antonucci M. Torra I.P. Delerive P. Majd Z. Fruchart J.C. Chapman J. Najib J. Staels B. J. Biol. Chem. 1998; 273: 25573-25580Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar), and vascular endothelial cells (26Bishop-Bailey D. Hla T. J. Biol. Chem. 1999; 274: 17042-17048Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar); however, apoptosis is suppressed by activation of PPARγ in cerebellar granule cells (27Heneka M.T. Feinstein D.L. Galea E. Gleichmann M. Wullner U. Klockgether T. J. Neuroimmunol. 1999; 100: 156-168Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Although PGI2 and its analogs can activate PPARδ (28Gupta R. Tan J. Krause W.F. Geraci M.W. Willson T.M. Dey S.K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13275-13280Crossref PubMed Scopus (358) Google Scholar), it has not been known whether intracellular PGI2 can actually function via PPARs. In our previous study (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), we found that overexpression of PGI2 synthase (PGIS) in the human embryonic kidney epithelial 293 (HEK-293) cell line induces cell death with apoptotic characteristics. Here, we report that intracellular PGI2 produced by expressing PGIS in HEK-293 cells promotes apoptosis by activating the endogenous PPARδ. Iloprost, carbaprostacyclin (cPGI), arachidonic acid, and anti-COX-1 polyclonal antiserum were purchased from Cayman Chemical Co. (Ann Arbor, MI). RpcAMP (adenosine 3′,5′-cyclic monophosphothioate, Rp-isomer), dbcAMP (dibutyryl cyclic AMP), and H-7 (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine) were from Sigma Chemical Co. (St. Louis, MO). Texas Red-linked donkey anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Hoechst 33258 (bisbenzimide H33258 fluorochrome trihydrochloride) was obtained from Nacalai Tesque Co. (Kyoto, Japan). Anti-PPAPδ polyclonal antibody and anti-COX-1 goat polyclonal IgG were products from Santa Cruz Biotechnology (Santa Cruz, CA). Hemagglutinating virus Japan (HVJ) and liposome were provided by Dr. Y. Kaneda (Osaka University Medical School). All culture media were purchased from Life Technologies, Inc. (Rockville, MD). Synthetic peptides, P1: PGEPPLDLGSIPWLGYALDC corresponding to amino acid residues 27–45 in human PGIS, or P4: LMQPEHDVPVRYRIRP corresponding to amino acids 485–500 coupled to keyhole limpet hemocyanin were prepared by the Peptide Institute Inc. (Osaka, Japan). Japanese white rabbits were immunized with 1 mg of the conjugated peptide in Freund's complete adjuvant. Both P1 and P4 antisera were useful for immunoblotting. In this study, P1 antiserum was used for immunoblotting, and P4 antiserum was used for immunofluorescence staining. Monolayers of HEK-293 cells were seeded (3.0 × 105 cells/60-mm dish) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mm l-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin ("medium A"). After incubation for 24 h, the cells were transfected with 3 μg of the high expression vector for wild-type PGIS (pCMV/PGISWT) (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), the catalytically inactive mutant PGISC441A (pCMV/PGISC441A) (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), or the control vector (pCMV-7) (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar) plus 0.3 μg of pVA (a plasmid encoding adenovirus-associated RNA1) (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar) using 9.9 μl of LipofectAMINE (Life Technologies, Inc.) with serum-free DMEM, and cells were cultured for 5 h. Subsequently, DMEM containing 20% FBS ("medium B") was added, and cells were cultured with or without 100 μmaspirin or 100 μmU46619. After 36 h, the cells were rinsed with PBS, fixed in 3.7% formaldehyde for 10 min, and washed with PBS three times. The cells were then incubated with anti-PGIS antibody P4 for 2 h, and with anti-rabbit IgG-Texas Red for 1 h at 37 °C, washed three times with PBS containing 2% FBS, stained with 0.3 mm Hoechst 33258 and observed under fluorescent microscopy. Total cellular extracts from the transfected cells were prepared by lysing cells in 1% SDS, 5 mm EDTA, 5 mm EGTA, 1 mm dithiothreitol, 200 mm phenylmethanesulfonyl fluoride, and 100 mmleupeptin. Protein from 1.0 × 104 cells was separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred onto Immobilon-P polyvinylidene difluoride transfer membranes (Millipore, Bedford, MA). Filters were blocked overnight with Tris-buffered saline (TBS) containing 5% skim milk (Bio-Rad, Hercules, CA) and 3% bovine serum albumin (Seikagaku Kogyo Co., Tokyo, Japan) at 4 °C. Immunostaining steps were performed in TBS containing 0.05% Tween 20 and 3% bovine serum albumin at room temperature. Filters were incubated with primary and secondary antibodies for 1 h each. Filters were washed in TBS containing 0.05% Tween 20 four times for 10 min between each step and were developed with ECL reagent (Amersham Pharmacia Biotech). HEK-293 (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), CV-1, and bovine aortic endothelial cells (BAEC) (30Hara S. Miyata A. Yokoyama C. Inoue H. Brugger R. Lottspeic F. Ullrich V. Tanabe T. J. Biol. Chem. 1994; 269: 19897-19903Abstract Full Text PDF PubMed Google Scholar) (1.2 × 104 cells/cm2) were cultured in medium A. Caco-2 cells (1.0 × 105 cells/30-mm dish coated with collagen) were cultured in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. For expression of PGISwt, PGISC441A, COX-1, and COX-2, plasmid vectors pCMV/PGISWT (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), pCMV/PGISC441A (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar), pcDNACOX-1 (31Kinoshita T. Takahashi Y. Sakashita T. Inoue H. Tanabe T. Yoshimoto T. Biochim. Biophys. Acta. 1999; 1438: 120-130Crossref PubMed Scopus (114) Google Scholar), and pcDNACOX-2 (31Kinoshita T. Takahashi Y. Sakashita T. Inoue H. Tanabe T. Yoshimoto T. Biochim. Biophys. Acta. 1999; 1438: 120-130Crossref PubMed Scopus (114) Google Scholar) were used, respectively. HEK-293, Caco-2 and CV-1 cells were transfected with DNA using LipofectAMINE. BAEC were transfected using TransIT LT-1 (PanVera Co., Madison, WI). Transfections were performed in a ratio of 1 μg of DNA to 3.0 μl of reagent, and cells were incubated in serum-free medium at 37 °C for 5 h. Subsequently, medium B or RPMI 1640 containing 20% FBS was added, and cells were grown for 12–72 h before analysis of apoptosis. Cell viability was determined by trypan blue exclusion. 10 μl of a 0.5% solution of the dye was added to 100 μl of cell suspension (1.0 × 104cells/100 μl). The suspension was then applied to a hemacytometer, and both viable and nonviable cells were counted. A minimum of 300 cells was counted for each data point. A cell death detection enzyme-linked immunosorbent assay (ELISA) (Roche Molecular Biochemicals, Indianapolis, IN) was performed to determine the apoptotic index by detecting nucleosome breakdown, the histone-associated DNA fragments (mono- and oligonucleosomes) generated by apoptotic cells. HEK-293 cells (8.0 × 104 cells/well in 12-well plates) were plated in medium A and grown for 12 h. Cells were washed with PBS, transfected with PGISwt expression vector or mock vector, and cultured for 72 h. The cells were collected along with the floating cells and used to prepare the cytosol fractions. A volume of 10 μl of these cytosolic fractions were incubated in anti-histone antibody-coated wells (96-well plates), and the histones of the DNA fragments were detected by using 2,2′-azino-di-(3-ethylbenzathiazoline sulfonate). The reaction products in each of the 96 wells were read using a Bio-Rad microplate reader (model 3550-UV). To assess DNA ladder formation, low molecular weight DNA was extracted from 1.8 × 106 cells (both floating and adherent) using ApoLadder EX kit (Takara Shuzo Co., Tokyo). The extracted DNA fragments were applied to a 1.5% agarose gel, separated electrophoretically, and visualized with ethidium bromide. Apoptotic cells were distinguished by their characteristic morphological changes such as membrane blebbing and cell body shrinkage. HEK-293, CV-1, or Caco-2 cells (2.0 × 105 cells/well in 6-well plates) were plated with the respective serum-supplemented media and grown for 12 h. Cells were washed with PBS, cotransfected with 1 μg of β-galactosidase expression vector, and 2.5 μg of PGISwt or PGISC441A expression vector and cultured for 5 h in serum-free medium. Subsequently, medium B was added and cells were grown for 60 h. The cells were stained with X-gal (5-bromo-4-chloro-3-indolyl β-galactoside) solution (PBS containing 5 mm X-gal, 1 mmMgCl2, 10 mm KCl, 5 mmK3Fe(CN)6, 5 mmK4Fe(CN)6-3H2O) at 37 °C for 3 h and scored for apoptotic cells. For measurement of effects of reagents on apoptosis, cells (2.0 × 105 cells/well in 6-well plates) cotransfected with 1 μg of β-galactosidase and 2.5 μg of PGISwt or PGISC441A were cultured in serum-free medium containing several concentrations of iloprost, dbcAMP, H-7, or RpcAMP for 24 h at 37 °C, washed gently with PBS, and stained with X-gal. For analysis of effects of PPARδ on apoptosis, HEK-293 cells (8.0 × 104 cells/well in 12-well plates) were cotransfected with 0.1 μg of β-galactosidase expression vector and 0.7 μg of expression vector for PPARδ or mutant PPARδ and PGISwt, PGISC441A, or mock vector using LipofectAMINE. The total amount of DNA was kept at 2 μg by the addition of control DNA (pCMV-7). After 24 h of transfection, cells were stained with X-gal and apoptosis was measured. The cells underwent apoptosis were indicated by a percentage of the total number of the blue cells stained by X-gal. A minimum of 300 cells was counted for each data point. Apoptosis was monitored with the ApoAlert caspase (CPP-32) assay kit using the (acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acid α-(4-trifluoromethyl-coumacyl-7-amido)) substrate (CLONTECH, Palo Alto, CA). HEK-293 cells (3.0 × 105 cells/60-mm dish) were transfected with 22 μg of antisense or sense oligonucleotide for PPARδ using HVJ liposome (5Todaka T. Yokoyama C. Yanamoto H. Hashimoto N. Nagata I. Tsukahara T. Hara S. Hatae T. Morishita R. Aoki M. Ogihara T. Kaneda Y. Tanabe T. Stroke. 1999; 30: 419-426Crossref PubMed Scopus (67) Google Scholar), incubated for 48 h at 37 °C, and cotransfected with 3 μg of expression vector for PGISwt, PGISC441A, or mock vector in the presence of additional 3 μg of antisense or sense oligonucleotide for PPARδ. After 36 h, CPP-32 activity was determined according to the manufacturer's instructions using (acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspart-1-al)-pretreated lysate as a control. Monolayers of HEK-293 cells in a 225-cm2 flask (3.0 × 106 cells) were washed three times with PBS, pH 7.4, and suspended in 1 ml of PBS, pH 7.4, containing 10 mm EDTA, 0.1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μg/ml antipain, 10 μmpepstatin (buffer A), and sonicated three times at 150 watts for 5 s on ice. The homogenate was centrifuged at 10,000 × gfor 10 min, and the supernatant was centrifuged at 100,000 ×g at 4 °C for 1 h. Pellets were suspended in 0.5 ml of ice-cold buffer A containing 1% Tween 20, sonicated three times at 150 watts for 15 s on ice, and centrifuged at 100,000 ×g at 4 °C for 1 h. The solubilized protein (800 μg) was incubated with protein G-Sepharose (150 μl) preincubated with control IgG for 2 h at 4 °C as a preclearing step. After pelleting the protein G-Sepharose, COX-1 polyclonal IgG or control IgG coupled to protein G-Sepharose was added to a half of resulting supernatant and incubated for 3 h at 4 °C. Protein G-Sepharose was then washed five times with 1 ml of the same buffer, mixed with 50 μl of sample buffer under nonreducing condition, heated for 5 min at 100 °C, subjected to 10% SDS-polyacrylamide gel electrophoresis, and transferred onto Immobilon-P as described above. Membrane was then subjected to immunoblotting analysis using COX-1 polyclonal antiserum. PGI2 production was determined in HEK-293 cells (1.0 × 106/100-mm dish) cotransfected with expression vector for PGISwt, PGISC441A, or mock vector with or without COX-1 or COX-2 vector. After 24 h of transfection, medium was removed and serum-free medium containing 0 or 10 μm arachidonic acid was added. After 48 h, incubation media were collected and stored at −20 °C before assay. Amounts of 6-keto-PGF1α were measured using an ELISA kit (Cayman Chemical Co.). For cAMP assay, HEK-293 or BAEC (2.0 × 105 cells/well in 6-well plates) were washed once in serum-free DMEM, followed by incubation with several concentrations of iloprost or cPGI for 24 h at 37 °C. Reactions were terminated by aspiration of the medium and addition of 2 ml of 5% trichloroacetic acid. The cells were cooled to 4 °C, cAMP was extracted with extraction buffer (65% ethanol containing 5 mm isobutylmethylxanthine, Sigma), and lysates were cleared by spinning at 10,000 × g for 5 min and dried under vacuum. Samples were reconstituted in assay buffer, and cAMP was quantified using an ELISA kit (Amersham Pharmacia Biotech). Distribution of PGI2 analog, iloprost, in HEK-293 cells was measured using [3H]iloprost. HEK-293 cells (1.0 × 106 cells/100-mm dish) were cultured in medium A containing 10 nm [3H]iloprost (0.05 μCi/ml) for 0–180 min. Cells were washed with ice-cold PBS, harvested, and homogenized using a Dounce homogenizer in three volumes of homogenization buffer (10 mm Hepes, pH 7.5, 250 mm sucrose, 0.1 mm EDTA, 1.5 mmdithiothreitol, 10 μg/ml trypsin inhibitor, 10 μg/ml leupeptin, 2 μg/ml aprotinin, and 1.0 mg/ml phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 1450 × g for 10 min. The supernatant was used as the cytoplasm-containing fraction. Nuclei and plasma membranes were isolated from the resulting pellets. The pellet was resuspended in ice-cold homogenization buffer. The solution containing the resuspended pellet was adjusted to a final concentration of 1.6 m sucrose by addition of homogenization buffer with a high density sucrose solution. A two-layer step gradient was set up by layering 250 mm sucrose homogenization buffer over the 1.6 m sucrose suspension and centrifuged at 70,900 ×g for 60 min. The band at the gradient interface (250 mm and 1.6 m sucrose) was collected and used as the plasma membrane fraction. The pellet produced after the gradient centrifugation containing the nuclear fraction was collected and used for the nuclei fraction. The [3H]iloprost-derived radioactivity of each fraction or whole cells was determined by liquid scintillation counting. Oligonucleotides dS, 5′-AAGAGGAGGAGAAAGAGGA-3′ corresponding to the human PPARδ cDNA (32Schmidt A. Vogel R.L. Witherup K.M. Rutledge S.J. Pitzenberger S.M. Adam M. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar) sense sequence (nucleotide residues 38–56); dAS, 5′-TCCTCTTTCTCCTCCTCTT-3′, the antisense sequence; aS, 5′-CTCGGTGACTTATCCTGTG-3′ corresponding to the human PPARα cDNA sense sequence (nucleotide residues 237–255); and the antisense sequence aAS, 5′-CACAGGATAAGTCACCGAG-3′, were synthesized. These oligonucleotides were transfected into cells using the HVJ liposome method (3Sugimoto Y. Narumiya S. Ichikawa A. Prog. Lipid Res. 2000; 39: 289-314Crossref PubMed Scopus (168) Google Scholar). Each oligonucleotide (22 μg) was mixed with a nuclear protein, high mobility group-1. HVJ liposomes were prepared by mixing dried lipids (phosphatidylserine/phosphatidylcholine/cholesterol, 1:4.8:2, w/w/w) with UV light-inactivated HVJ virus. After an incubation and sucrose gradient centrifugation, the top layer was collected and used for transfection. After 48 h of transfection the cells were used for apoptosis assay and PPAR-responsive element (PPRE) reporter assay. The PPREx3-luciferase reporter vector, which contains three copies of PPRE for hydroxymethylglutaryl-CoA reductase (33Juge-Aubry C. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), was constructed by synthesis of the sense oligonucleotide, 5′-CGCGTAAAAACTGGGCCAAAGGTCTAAAAACTGGGCCAAAGGTCTAAAAACTGGGCCAAAGGTCTC-3′ and the antisense oligonucleotide 5′-TCGAGAGACCTTTGGCCCAGTTTTTAGACCTTTGGCCCAGTTTTTAGACCTTTGGCCCAGTTTTTA-3′. Both oligonucleotides were annealed and subcloned into theMluI-XhoI site of the pGL3-promoter vector (Promega, Madison, WI). BAEC (8.0 × 104 cells/well in 12-well plates) were cotransfected with the 0.2 μg of PPREx3-luciferase reporter vector and 0.1 μg of β-galactosidase expression vector using TransIT LT-1. After 12 h of transfection, cells were washed with PBS, incubated in serum-free DMEM containing several concentrations of iloprost or cPGI for 24 h at 37 °C, and subjected to luciferase assay. HEK-293 cells (8.0 × 104 cells/well in 12-well plates) treated with antisense or sense oligonucleotide were cotransfected with 0.2 μg of PPREx3-luciferase reporter vector, 0.1 μg of β-galactosidase expression vector, and 0.7 μg of expression vector for PGISwt, PGISC441A, or mock vector using LipofectAMINE. Nontreated HEK-293 cells (8.0 × 104 cells/well in 12-well plates) were cotransfected with 0.2 μg of PPREx3-luciferase reporter vector and 0.1 μg of β-galactosidase expression vector with 0.7 μg of expression vector for PPARδ or mutant PPARδ and PGISwt, PGISC441A, or mock vector using LipofectAMINE. The total amount of DNA was kept at 2 μg by the addition of control DNA (pCMV-7). After another 24 h, the cells were harvested and the luciferase and β-galactosidase activities were quantified. The luciferase activity of the extract was normalized with β-galactosidase activity. The cDNA for PPARδ was isolated by PCR amplification. The poly(A)+ RNA was extracted from 2.0 × 106 cells of mouse vascular smooth muscle-derived SVS30 cells using a FastTrack 2.0 kit (Promega). The poly(A)+ RNA was reverse-transcribed by extension with Superscript reverse transcriptase (Life Technologies, Inc.) and a specific antisense primer complementary to the sequence located in the 3′-region of the cDNA for PPARδ: 5′-TTAGTACATGTCCTTGTAGATTTC-3′; the reverse-transcribed cDNA was used for the following PCR amplification. The PPARδ cDNA was amplified using the primers Pdw5 (5′-GAAAGCTTGTCGACCCACCATGGAACAGCCACAG-3′) and Pdw3 (5′-GTCTAGAGGATCCTTAGTACATGTCCTTGTAGAT-3′). Pdw5 or Pdw3 has HindIII or BamHI restriction site (underlined, respectively). PCR was performed for 35 cycles with a temperature profile of 10 s at 94 °C, 30 s at 55 °C, and 4 min at 72 °C using KOD polymerase (Toyobo, Osaka, Japan). The amplification products were purified in 1% agarose gel using a Qiagen gel extraction kit. The isolated DNA was inserted into theHindIII-BamHI site of pBluescript II vector (Stratagene, La Jolla, CA) and sequenced using an Applied Biosystems model 310 genetic analyzer. For construction of an expression vector for PPARδ, PCR-derived cDNA for PPARδ was excised from the pBluescript vector with HindIII and BamHI and religated into the pcDNAIII vector (Invitrogen, Groningen, The Netherlands). The PPARδL431A/G434A double mutant was generated by site-directed mutagenesis of wild-type receptor. The oligonucleotide used in mutagenesis was Pdm3 (5′-GTACATGTCCTTGTAGATCGCCTGGAGCGCGGGGTGCAGC-3′), and the construct was verified by sequencing. Pdm3 has two mutated sites (underlined). The products were purified, subcloned into theHindIII-BamHI site of pBluescript II, and inserted into the HindIII-BamHI site of pcDNAIII vector. An expression vector encoding human PGIS (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar) or control vector was transfected into HEK-293 cells, and cell viability was determined. As shown in Fig. 1 A, we observed a significant decrease in the viability of the cells transfected with the PGIS expression vector, whereas no change in viability was seen in the cells transfected with a mock vector. Moreover, transfection of the PGIS expression vector into HEK-293 cells resulted in significant morphological changes, including membrane blebbing and cell body shrinkage (Fig. 1 B, left panel), features typical of apoptosis (34Yang X. Khosravi-Far R. Chang H.Y. Baltimore D. Cell. 1997; 89: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar). When the histone-associated DNA fragments (mono- and oligonucleosomes) in cytosol fractions of the cells were measured by ELISA, the amounts of DNA fragments in the cells transfected with the PGIS expression vector increased gradually after 36 h of transfection (Fig.1 C). However, cell morphology and contents of DNA fragments were not changed in cells transfected with the mock vector (Fig. 1,B and C). These data indicate that transfection with the PGIS expression vector causes a decline in cell viability and an increase in apoptotic cell death in HEK-293 cells. There is no detectable endogenous PGIS in HEK-293 cells (29Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (35) Google Scholar). A significant amount of endogenous COX-1, which synthesizes PGH2, a substrate for PGIS, was detected in the HEK-293 cells by immunoprecipitation (Fig. 1 D), although it has been reported that expression of both COX-1 and -2 in the cells is undetectable by immunoblotting (35Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (
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