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Proline Oxidase, a Proapoptotic Gene, Is Induced by Troglitazone

2005; Elsevier BV; Volume: 281; Issue: 4 Linguagem: Inglês

10.1074/jbc.m507867200

1083-351X

Jui Pandhare, Sandra K. Cooper, James M. Phang,

Lipid metabolism and biosynthesis

Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor γ (PPARγ) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARγ ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARγ, troglitazone enhanced the binding of PPARγ to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARγ activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARγ-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARγ-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARγ, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARγ, increased activation was observed by ligand stimulation, indicating that both PPARγ-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARγ-dependent and -independent mechanisms. Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor γ (PPARγ) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARγ ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARγ, troglitazone enhanced the binding of PPARγ to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARγ activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARγ-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARγ-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARγ, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARγ, increased activation was observed by ligand stimulation, indicating that both PPARγ-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARγ-dependent and -independent mechanisms. Proline oxidase (POX), 2The abbreviations used are: POX, proline oxidase; PPARγ, peroxisome proliferator-activated receptor γ; PPRE, peroxisome proliferator-responsive element; POX-Luc, POX promoter-luciferase reporter construct; ROS, reactive oxygen species; DCF-DA, 2′,7′-dichlorofluorescein diacetate; RT, reverse transcription; OAB, O-aminobenzaldehyde; P5C, pyrroline 5-carboxylate. also known as proline dehydrogenase, is a mitochondrial inner membrane enzyme that catalyzes the first step of proline degradation (1Phang J.M. Curr. Top. Cell Regul. 1985; 25: 91-132Crossref PubMed Scopus (232) Google Scholar). POX converts proline to pyrroline 5-carboxylate (P5C) and transfers electrons into mitochondrial electron transport with an intervening flavoprotein (1Phang J.M. Curr. Top. Cell Regul. 1985; 25: 91-132Crossref PubMed Scopus (232) Google Scholar). These interconversions form a metabolic shuttle of redox equivalents between cytosol and mitochondria, couple the oxidation of NADPH to mitochondrial electron transport, and serve as a mechanism for energy production (1Phang J.M. Curr. Top. Cell Regul. 1985; 25: 91-132Crossref PubMed Scopus (232) Google Scholar, 2Hagedorn C.H. Phang J.M. Arch. Biochem. Biophys. 1983; 225: 95-101Crossref PubMed Scopus (79) Google Scholar). Polyak et al. (3Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar) showed that POX is a p53-induced gene; we and others have shown that hyperexpression of POX in cancer cells is sufficient for initiating the apoptotic cascade (4Donald S.P. Sun X.Y. Hu C.A. Yu J. Mei J.M. Valle D. Phang J.M. Cancer Res. 2001; 61: 1810-1815PubMed Google Scholar, 5Maxwell S.A. Davis G.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13009-13014Crossref PubMed Scopus (156) Google Scholar). Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear hormone receptor superfamily and functions as a ligand-dependent transcription factor (6Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1706) Google Scholar). It is thought that PPARγ plays important physiological roles in regulating lipid metabolism and homeostasis and also is involved in control of many cellular processes (7Kersten K. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1678) Google Scholar, 8Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2746) Google Scholar). PPARγ forms a heterodimer with the retinoid X receptor and activates target genes by binding to specific peroxisome proliferator-responsive elements (PPREs) located in the promoter regions of these genes. These PPREs usually consist of a direct repeat of the hexanucleotide AGGTCA sequence separated by one or two nucleotides (8Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2746) Google Scholar). Recent reports have demonstrated that PPARγ and its ligands are also important in control of tumor cell growth (9Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (513) Google Scholar). PPARγ is widely expressed in many malignant tissues, and PPARγ ligands induce terminal differentiation, cell growth inhibition, and apoptosis in a variety of cancer cells, including colon, gastric, breast, prostate, and lung (9Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (513) Google Scholar, 10Yang W.L. Frucht H. Carcinogenesis. 2001; 22: 1379-1383Crossref PubMed Scopus (191) Google Scholar, 11Nagamine M. Okumura T. Tanno S. Sawamukai M. Motomura W. Takahashi N. Kohgo Y. Cancer Sci. 2003; 94: 338-343Crossref PubMed Scopus (63) Google Scholar, 12Elstner E. Muller C. Koshizuka K. Williamson E.A. Park D. Asou H. Shintaku P. Said J.W. Heber D. Koeffler H.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8806-8811Crossref PubMed Scopus (761) Google Scholar, 13Kubota T. Koshizuka K. Williamson E.A. Asou H. Said J.W. Holden S. Miyoshi I. Koeffler H.P. Cancer Res. 1998; 58: 3344-3352PubMed Google Scholar, 14Keshamouni V.G. Reddy R.C. Arenberg D.A. Joel B. Thannickal V.J. Kalemkerian G.P. Standiford T.J. Oncogene. 2004; 23: 100-108Crossref PubMed Scopus (184) Google Scholar). Putative endogenous ligands of PPARγ include the 15-deoxy-Δ12,14-prostaglandin J2 as well as several polyunsaturated fatty acids (6Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1706) Google Scholar). High affinity synthetic ligands that selectively activate PPARγ include the glitazones or thiazolidinediones (i.e. ciglitazone, troglitazone, pioglitazone, and rosiglitazone), a class of insulin-sensitizing drugs, some of which are presently used for the treatment of type 2 diabetes mellitus due to their effectiveness in controlling hyperglycemia (6Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1706) Google Scholar). Apart from their antidiabetic activity, glitazones have potent anti-inflammatory effects and are of special interest, since they induce growth arrest and apoptosis in a broad spectrum of tumor cells (15Grommes C. Landreth G.E. Heneka M.T. Lancet. 2004; 5: 419-429Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 16Theocharis S. Margeli A. Vielh P. Kouraklis G. Cancer Treatment Rev. 2004; 30: 545-554Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). PPARγ and its ligands have also been reported to induce intracellular oxidative stress, resulting in generation of reactive oxygen species (ROS) (17Perez-Ortiz J.M. Tranque P. Vaquero C.F. Domingo B. Molina F. Calvo S. Jordan J. Cena V. Llopis J. J. Biol. Chem. 2004; 279: 8976-8985Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Kondo M. Oya-Ito T. Kumagai T. Osawa T. Uchida K. J. Biol. Chem. 2001; 276: 12076-12083Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 19Li L. Tao J. Davaille C. Feral C. Mallat A. Rieusset J. Vidal H. Lotersztajn S. J. Biol. Chem. 2001; 276: 38152-38158Abstract Full Text Full Text PDF PubMed Google Scholar). Since the glitazones cause mitochondrial depolarization, the mitochondria are reported to be the most likely source of ROS, which have been implicated in mediating PPARγ ligand-induced growth arrest and apoptosis. However, the mechanism of generation of ROS by PPARγ ligands is not clearly defined. The significance of ROS in intracellular signaling has now been relatively well documented (20Finkel T. J. Leukocyte Biol. 1999; 65: 337-340Crossref PubMed Scopus (238) Google Scholar, 21Fleury C. Mignotte B. Vayssière J.L. Biochimie (Paris). 2002; 84: 131-141Crossref PubMed Scopus (880) Google Scholar). Accumulating evidence has shown that diverse stimuli can increase intracellular oxygen radicals that evoke many cellular events, such as proliferation, gene activation, cell cycle arrest, and apoptosis. The generation, transmission, and targeting of ROS signals may be essential events in the induction of apoptosis. In our earlier work, we have shown that the induction of p53 was accompanied by the induction of POX and by proline-mediated ROS generation (4Donald S.P. Sun X.Y. Hu C.A. Yu J. Mei J.M. Valle D. Phang J.M. Cancer Res. 2001; 61: 1810-1815PubMed Google Scholar). POX was demonstrated to generate ROS, which can initiate apoptosis by directly acting on the mitochondrial permeability core complex and affecting the mitochondrial permeability transition. PPARγ and its ligands have been shown to be important in several biological processes ranging from differentiation, regulation of metabolism, and maintenance of insulin sensitivity to control of cellular proliferation and inflammation. Therefore, pathways activated by PPARγ and its ligands are very important. Recently, there has been an increased awareness about the possible link between obesity, diabetes, and cancer, and also alterations in metabolic pathways may lead to cancer susceptibility (8Desvergne B. Wahli W. Endocr. Rev. 1999; 20: 649-688Crossref PubMed Scopus (2746) Google Scholar, 22Evans R.M. Barish G.D. Wang Y.X. Nat. Med. 2004; 10: 355-361Crossref PubMed Scopus (1293) Google Scholar). Moreover, the signals that control bioenergetics have been linked to the regulation of cell survival and apoptosis (23Hammerman P.S. Fox C.J. Thompson C.B. Trends Biochem. Sci. 2004; 29: 586-592Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). POX is one of the mitochondrial metabolic redox enzymes; the increased cycling of proline through POX may alter the redox balance critical for regulation of cell growth and apoptosis. In the present paper, we have studied the regulation of POX gene expression. We have demonstrated that PPARγ ligand troglitazone can up-regulate the expression of POX, which is accompanied by increased ROS. From our results, we hypothesize that the induction of apoptosis by PPARγ ligands is mediated, at least in part, by activating POX gene expression, resulting in increased ROS generation. Reagents—Troglitazone, rosiglitazone, ciglitizone, and pioglitazone were purchased from Cayman Chemical Co. (Ann Arbor, MI); GW9662 was obtained from Sigma. 2′,7′-Dichlorofluorescein diacetate was purchased from Molecular Probes, Inc. (Eugene, OR). Cell Culture—The human colon cancer cell lines HCT116, HCT15, HT29, KM12, HCC2998, and SW620 were provided by the NCI cell line repository. Two colon cancer cell lines RKO and LoVo and the HEK293 cell line were obtained from the American Type Culture Collection. All of the cells were cultured in Dulbecco's modified Eagle's medium (Quality Biologicals, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), penicillin and streptomycin, 2 mm glutamine at 37 °C, and 5% CO2. To test the effect of PPARγ ligands/antagonists, cells were refed with Dulbecco's modified Eagle's medium with or without ligands or GW9662 in Me2SO for various time periods. The final Me2SO concentration was 0.1%. Plasmid Constructs—A 1.26-kb genomic fragment containing a portion of the POX promoter sequence from –1250 to +10 relative to the translation initiation codon was amplified from genomic DNA. The primers used were as follows: forward, 5′-AAA CTC CGT GGG CCT TGG CAG CCC CT-3′; reverse, 5′-TCA GAG CCA TGG CGG GAC GGC GGT A-3′. The PCR amplification was performed in a final reaction volume of 50 μl under the following conditions: denaturation at 95 °C for 5 min, 30 cycles of 30 s at 94 °C, and 3 min at 68 °C. The PCR product was cloned into the NheI and HindIII restriction sites of the pGL3 vector (Promega, Madison, WI) to generate the POX promoter-luciferase reporter construct (POX-Luc). The sequence was confirmed to be identical to the POX genomic sequences in the GenBank™ data base. The POX promoter sequence (–1250 to +10) was analyzed for potential transcription factor binding sites by the Transcription Element Search System (TESS) of the Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania (available on the World Wide Web at www.cbil.upenn.edu/tess/) by using a 5-bp minimum element size limit. Another construct of the POX-Luc plasmid was also generated in which the POX promoter region (–1013 to –682) containing the PPRE was deleted by restriction digestion with PstI and BstXI. After digestion, the resulting overhangs were filled with Klenow and religated to generate a truncated POX-Luc construct containing a length of ∼900 bp of the POX promoter. A proline oxidase antisense vector was constructed by amplifying a part of the proline oxidase cDNA from bp 694–1782 and cloning it in the antisense orientation in the mammalian expression vector pCI (Promega). The POX antisense construct was validated to block the expression of POX mRNA by RT-PCR. The cloning of PPARγ and p53 was carried out by isolation of total RNA followed by RT-PCR using RT-PCR beads (Amersham Biosciences). The sequences for the PPARγ forward and reverse primers were 5′-GAT CGG TAC CAT GAC CAT GGT TGA CAC AGA-3′ and 5′-AGT CGT CGA CTA GTA CAA GTC CTT GTA GA-3′, and p53 forward and reverse primers were 5′-GAC ACT TTG CGT TCG GGC T-3′ and 5′-CGG GAC AAA GCA AAT GGA AGT-3′. The amplified PPARγ and p53 cDNAs were cloned into the mammalian expression vector pCI (Promega). Luciferase Assay—POX transcriptional activity was measured using the Dual-Luciferase Reporter Assay (Promega, Madison, WI) according to the manufacturer's protocol. The cells were co-transfected with the POX-Luc construct and pRL-null, a Renilla construct for normalizing transfection efficiency. To determine the effect of PPARγ/p53 on POX promoter activity, equivalent amounts of PPARγ/p53 cDNA or vector plasmid were transfected using Lipofectamine 2000 (Invitrogen). Transfected cells were lysed, and luciferase activity was measured with equal amounts of cell extract using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) and normalized with the Renilla activity. Electrophoretic Mobility Shift Assay—HCT116 cells were treated with 25 μm troglitazone for 36 h. Nuclear extracts were prepared using NE-PER™ nuclear and cytoplasmic extraction reagents (Pierce) following the manufacturer's instructions. The protein concentration of the extracts was determined using the BCA protein assay (Pierce), and samples were stored at –70 °C. The binding of PPARγ to the PPRE in the POX promoter region was studied, employing the Lightshift® chemiluminescent electrophoretic mobility shift assay kit (Pierce) as per the manufacturer's protocol. This method employs a nonisotopic method to detect DNA-protein interaction and uses biotin end-labeled DNA. Electrophoretic mobility shift assays conducted using LightShift assays require biotin-labeled double-stranded DNA, with end labeling of both complementary oligonucleotides separately, followed by annealing at room temperature. The consensus oligonucleotide sequences (5′ to 3′) used were as follows: ATC ACA AGG TCA GGA GAT CAA GAC C (PPARγ, forward) and GGT CTT GAT CTC CTG ACC TTG TGA T (PPARγ, reverse). The biotin end-labeled DNA was detected using streptavidin horseradish peroxidase conjugate and a chemiluminescent substrate. The membrane was exposed to x-ray film (XAR-5; Amersham Biosciences) and developed with an Eastman Kodak Co. film processor. Chromatin Immunoprecipitation Assay—HCT116 cells treated with troglitazone for 36 h were incubated with 1% formaldehyde to fix protein-DNA complexes. Cells were resuspended in 200 μl of SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1, complete proteinase inhibitor mixture) and sonicated on ice to shear DNA to an average length between 200 and 1000 bp. Sonicated samples were centrifuged to spin down cell debris, and the soluble chromatin was immunoprecipitated using a reagent kit (Upstate Biotechnology, Inc., Lake Placid, NY) as recommended by the manufacturer. A portion of the sonicated chromatin was used as DNA input control and a no antibody control; the remaining DNA was then precipitated using specific antibody against PPARγ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The purified DNA from the immunoprecipitated complexes of antibody-protein-DNA was detected by PCR (30 cycles) using the specific primer pair spanning the POX promoter region (–1040 to –872) containing POX-PPRE: forward, 5′-CGT GGT GGC TCA CGC CTG TA-3′; reverse, 5′-ACG CCA TTC TCC CAC CTC AG-3′. RT-PCR—Total RNA was isolated from harvested cells using Trizol (Invitrogen) and quantified using a Beckman DU-65 spectrophotometer. A two-step RT-PCR (0.5 μg of total RNA, 0.5 μg of random primers, and 0.2 μm specific primers in a 50-μl volume) was performed using RT-PCR beads (Amersham Biosciences). The reaction mixture was incubated at 42 °C for 30 min. Specific oligomers unique to human 1) POX (forward, 5′-GCC ATT AAG CTC ACA GCA CTG GG-3′; reverse, 5′-CTG ATG GCC GGC TGG AAG TAG-3′) and 2) PPARγ-selective target gene keratin 20 (forward, 5′-AGT CAT GGC CCA GAA GAA CCT TCA-3′; reverse, 5′-TGG TCT CCT CTA GAG TGT GCT CCA AA-3′) were designed to amplify a product of 478 and 212 bp, respectively. The glyceraldehyde-3-phosphate dehydrogenase control primers (Clontech) were used in a reaction with identical conditions, except that the reaction was performed for 20 cycles. All reaction products (15 μl with glycerol loading buffer) were run on a 2% agarose gel and stained with ethidium bromide, and the products were recorded and quantified using the Electrophoresis Documentation and Analysis System (Kodak Digital Science, Rochester, NY). Western Blotting—Cell lysates were prepared and quantified according to established methods. Equal amounts of cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad). Membranes were blocked using Tris-buffered saline with 3% nonfat milk (pH 8.0; Sigma). Blots were then probed with the primary anti-POX or anti-p53 antibody (Santa Cruz Biotechnology) in blocking buffer and subsequently by a secondary antibody conjugated to horseradish peroxidase (1:2000). All blots were washed in Tris-buffered saline with Tween 20 (pH 8.0; Sigma) and developed using the ECL procedure (Amersham Biosciences). Blots were routinely stripped by the Encore blot stripping kit (Novus Molecular, Inc., San Diego, CA) and reprobed with anti-actin monoclonal antibody (Sigma) to serve as loading controls. Anti-rabbit or anti-mouse antibody (Santa Cruz Biotechnology) was used as secondary antibody. POX Enzyme Assay—HCT116 cells were grown in the appropriate medium, after which cells were rinsed and scraped in cold phosphate-buffered saline, pelleted, and resuspended in cold sucrose buffer (0.250 m sucrose, 3.5 mm Tris, and 1 mm EDTA (pH 7.4)). Suspensions were then sonicated for 20 s at a setting of 25% (Branson Sonifier 450; Branson Ultrasonics Corp., Danbury, CT). Total protein was determined using the BCA protein assay (Pierce). P5C formed was detected using a specific spectrophotometric method. Briefly, P5C formed from the substrate proline was reacted with O-aminobenzaldehyde (OAB) and the resultant OAB·P5C complex was quantified. A 200-μl reaction mixture containing 0.1 m KPO4, pH 7.2, 1.2 mg/ml OAB, 0.12 mg/ml cytochrome c, 5 mm proline, and cell extract containing 50 μg of protein was incubated for 30 min at 37 °C. The reaction was terminated by the addition of 20 μl of OAB (10 μg/ml in 6 n HCl). The samples were centrifuged, and the absorbance of the supernatants was measured at 440 nm. All of the reactions were performed in triplicate, and proper protein controls were included for each measurement. A standard calibration curve was generated using P5C, and the P5C formed (nmol/min/μg of protein) was determined. Generation and Measurement of Intracellular ROS—HCT116 cells were cultured in 6-well plates in the growth medium for 24 h before treatment. Cells were then treated with PPARγ ligands/proline, as specified, for an additional 36 h before analysis for ROS. For inhibition studies, cells were treated with 1 mm N-acetyl cysteine 24 h before and concurrent with troglitazone/proline treatment. Cells were transfected with POX antisense or vector control and treated with troglitazone/proline, as specified before measurement of ROS. 2,7-Dichlorohydrofluorescein diacetate (DCF-DA; Sigma) was used as an indicator of the amount of intracellular ROS. On the day of the experiment, treatment medium was removed, and the monolayer was exposed to serum-free, phenol red-free medium containing 50 μm DCF-DA. Cells were exposed to the dye for 30 min in the dark to allow for equilibration. After two washes with phosphate-buffered saline, cells were solubilized with 0.5% SDS and 5 mm Tris HCl (pH 7.5). The fluorescent intensity of the lysate was determined using a spectrofluorometer (Jovin Yvon Specs2) with excitation and emission wavelengths of 485 and 530 nm, respectively. Samples were assayed in triplicate. Data are shown as arbitrary units of fluorescence ± S.D. Effect of PPARγ Ligands on POX Promoter Activity—For investigating the transcriptional regulatory mechanisms involved in the basal expression of POX gene, we cloned the human POX promoter region (–1250 to +10) and produced a POX promoter/luciferase reporter construct. Analysis of the promoter nucleotide sequence revealed the presence of a number of potential transcription factor binding sites, including CCAAT/enhancer-binding protein, AP-1, Sp1, RAR, PPAR, and retinoid X receptor, which became the basis for our initial studies. By cotransfecting the POX-Luc construct with expression constructs of the several transcription factors in HEK293 cells, we determined which transcription factors stimulated POX promoter activity (data not shown). Among the different transcription factors tested, PPARγ was the most potent and resulted in a 6-fold activation of the POX promoter activity (Fig. 1A). Since PPARγ strongly activated the POX promoter, we characterized the interaction of PPARγ with POX in detail. PPARγ is a ligand-activated transcription factor; therefore, we next investigated if the PPARγ ligand troglitazone could further enhance the activation of POX promoter. HEK293 cells transfected with POX-Luc construct and PPARγ were treated with troglitazone for 24–36 h, and the POX promoter activity was monitored. As seen in Fig. 1A, troglitazone at a concentration of 25 μm further increased the POX promoter activity over PPARγ alone. High levels of PPARγ have been reported in several colon cancer cells, and the role of PPARγ and its ligands in cell growth arrest and apoptosis has been well documented (9Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (513) Google Scholar, 10Yang W.L. Frucht H. Carcinogenesis. 2001; 22: 1379-1383Crossref PubMed Scopus (191) Google Scholar). Therefore, we transfected various colon cancer cell lines with a POX-Luc construct, and analyzed whether activation of endogenous PPARγ by treatment with its ligand stimulated POX promoter activity. As shown in Fig. 1B, all of the cell lines tested except for the HCT15 and SW620 cell lines activated the POX promoter activity in the presence of troglitazone (25 μm). Maximum activation of POX promoter activity was obtained in the HCT116 cells. To verify that the increase in POX activation was not only a troglitazone-specific effect, we measured the activation of POX in the presence of other PPARγ ligands in HCT116 cells (Fig. 1C). All of the PPARγ ligands tested increased the POX promoter activity, indicating that the activation may be mediated through PPARγ. Troglitazone Increases the Binding of PPARγ to the POX Promoter—Analysis of the POX promoter region revealed the presence of a putative PPAR/retinoid X receptor binding site (PPRE) between –982 and –969 bp relative to the transcription start site. Since the POX promoter activity was stimulated by troglitazone in the HCT116 cells and previous studies have shown that HCT116 cells express wild type and functional PPARγ (24Tsujie M. Nakamori S. Okami J. Hayashi N. Hiraoka N. Nagano H. Dono K. Umeshita K. Sakon M. Monden M. Exp. Cell Res. 2003; 289: 143-151Crossref PubMed Scopus (33) Google Scholar), we used these cells to study the functionality and binding of PPARγ to the PPRE in the POX promoter region by an electrophoretic mobility shift assay using an oligonucleotide probe of 25 bp containing the PPAR binding site. Incubation of nuclear extracts from HCT116 cells with the POX-PPRE probe increased the DNA binding activity of PPARγ, as shown in Fig. 2. The intensity of the bands was increased by treatment with 25 μm troglitazone for 36 h. To confirm the binding of PPARγ to the POX-PPRE in vivo, we also performed a chromatin immunoprecipitation assay. In HCT116 cells treated with troglitazone, we observed a significant amplification of the POX promoter region containing the PPAR binding site (POX-PPRE) by a chromatin immunoprecipitation assay, demonstrating directly the interaction of PPARγ with POX-PPRE. Up-regulation of POX mRNA and Protein Expression by Troglitazone in HCT116 Cells—After the initial studies using the POX-Luc construct, we further investigated whether PPARγ and its ligands can effect the actual expression of endogenous POX. HCT116 cells expressing PPARγ were treated with troglitazone (25 μm) for various time periods, after which RNA was harvested, and the level of POX mRNA expression was determined by RT-PCR. A significant increase in the concentration of POX mRNA was observed in troglitazone-treated as compared with vehicle-treated cells (Fig. 3A). This effect occurred in a time-dependent manner. The expression of POX mRNA was observed within 12–24 h and peaked at 36 h of treatment, resulting in a 3–4-fold increase in POX transcript levels. To investigate whether changes in POX mRNA levels were associated with induction of the corresponding POX protein expression, HCT116 cells were treated with troglitazone at various concentrations and examined by Western analysis. Our results revealed that POX protein expression dose-dependently increased (3–4-fold) with troglitazone after 36 h of treatment, with maximal effects seen at 20–30 μm (Fig. 3B). Thus, mRNA induction correlates with increased POX protein expression in ligand-treated cells. To assess the time course of POX expression in response to troglitazone, HCT116 cells were treated with 25 μm troglitazone for various time periods, after which the level of POX protein expression was determined. Western blot analysis revealed that the POX protein expression was induced by 24 h and increased with time, with a maximum POX expression observed after 36 h (Fig. 3C). Increase in POX Enzymatic Activity Concomitant to POX Expression—A spectrophotometric assay that detects an o-aminobenzaldehyde-P5C complex formed by conversion of proline to P5C was used to determine whether troglitazone-induced POX expression results in increased POX catalytic activity. After troglitazone treatment at various concentrations and for various durations, HCT116 cells were harvested, and lysates were added to the reactio