Proline Oxidase, a Proapoptotic Gene, Is Induced by Troglitazone

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), 2 also known as proline dehydrogenase, is a mitochondrial inner membrane enzyme that catalyzes the first step of proline degradation (1). POX converts proline to pyrroline 5-carboxy-late (P5C) and transfers electrons into mitochondrial electron transport with an intervening flavoprotein (1). 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 (1,2). Polyak et al. (3) 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 (4,5).
Peroxisome proliferator-activated receptor ␥ (PPAR␥) belongs to the nuclear hormone receptor superfamily and functions as a ligand-dependent transcription factor (6). It is thought that PPAR␥ plays important physiological roles in regulating lipid metabolism and homeostasis and also is involved in control of many cellular processes (7,8). PPAR␥ 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 (8). Recent reports have demonstrated that PPAR␥ and its ligands are also important in control of tumor cell growth (9). PPAR␥ is widely expressed in many malignant tissues, and PPAR␥ ligands induce terminal differentiation, cell growth inhibition, and apoptosis in a variety of cancer cells, including colon, gastric, breast, prostate, and lung (9 -14).
Putative endogenous ligands of PPAR␥ include the 15-deoxy-⌬12,14prostaglandin J 2 as well as several polyunsaturated fatty acids (6). High affinity synthetic ligands that selectively activate PPAR␥ 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 (6). 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 (15,16). PPAR␥ and its ligands have also been reported to induce intracellular oxidative stress, resulting in generation of reactive oxygen species (ROS) (17)(18)(19). Since the glitazones cause mitochondrial depolarization, the mitochondria are reported to be the most likely source of ROS, which have been implicated in mediating PPAR␥ ligand-induced growth arrest and apoptosis. However, the mechanism of generation of ROS by PPAR␥ ligands is not clearly defined.
The significance of ROS in intracellular signaling has now been relatively well documented (20,21). 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 (4). 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.
PPAR␥ 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 PPAR␥ 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 (8,22). Moreover, the signals that control bioenergetics have been linked to the regulation of cell survival and apoptosis (23). 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 PPAR␥ 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 PPAR␥ ligands is mediated, at least in part, by activating POX gene expression, resulting in increased ROS generation.
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% CO 2 . To test the effect of PPAR␥ ligands/antagonists, cells were refed with Dulbecco's modified Eagle's medium with or without ligands or GW9662 in Me 2 SO for various time periods. The final Me 2 SO concentration was 0.1%.
Plasmid Constructs-A 1.26-kb genomic fragment containing a portion of the POX promoter sequence from Ϫ1250 to ϩ10 relative to the translation initiation codon was amplified from genomic DNA. The primers used were as follows: forward, 5Ј-AAA CTC CGT GGG CCT TGG CAG CCC CT-3Ј; reverse, 5Ј-TCA GAG CCA TGG CGG GAC GGC GGT A-3Ј. The PCR amplification was performed in a final reaction volume of 50 l 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 promoterluciferase reporter construct (POX-Luc). The sequence was confirmed to be identical to the POX genomic sequences in the GenBank TM data base. The POX promoter sequence (Ϫ1250 to ϩ10) 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 (Ϫ1013 to Ϫ682) 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 ϳ900 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 PPAR␥ and p53 was carried out by isolation of total RNA followed by RT-PCR using RT-PCR beads (Amersham Biosciences). The sequences for the PPAR␥ forward and reverse primers were 5Ј-GAT CGG TAC CAT GAC CAT GGT TGA CAC AGA-3Ј and 5Ј-AGT CGT CGA CTA GTA CAA GTC CTT GTA GA-3Ј, and p53 forward and reverse primers were 5Ј-GAC ACT TTG CGT TCG GGC T-3Ј and 5Ј-CGG GAC AAA GCA AAT GGA AGT-3Ј. The amplified PPAR␥ 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 PPAR␥/p53 on POX promoter activity, equivalent amounts of PPAR␥/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 M troglitazone for 36 h. Nuclear extracts were prepared using NE-PER TM 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 PPAR␥ 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 doublestranded DNA, with end labeling of both complementary oligonucleotides separately, followed by annealing at room temperature. The consensus oligonucleotide sequences (5Ј to 3Ј) used were as follows: ATC ACA AGG TCA GGA GAT CAA GAC C (PPAR␥, forward) and GGT CTT GAT CTC CTG ACC TTG TGA T (PPAR␥, 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 l 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 PPAR␥ (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 (Ϫ1040 to Ϫ872) containing POX-PPRE: forward, 5Ј-CGT GGT GGC TCA CGC CTG TA-3Ј; reverse,  5Ј-ACG CCA TTC TCC CAC CTC AG-3Ј. 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 g of total RNA, 0.5 g of random primers, and 0.2 M specific primers in a 50-l 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, 5Ј-GCC ATT AAG CTC ACA GCA CTG GG-3Ј; reverse, 5Ј-CTG ATG GCC GGC TGG AAG TAG-3Ј) and 2) PPAR␥selective target gene keratin 20 (forward, 5Ј-AGT CAT GGC CCA GAA GAA CCT TCA-3Ј; reverse, 5Ј-TGG TCT CCT CTA GAG TGT GCT CCA AA-3Ј) 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 l 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 Trisbuffered 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 Trisbuffered 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 phosphatebuffered 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 OAB⅐P5C complex was quantified. A 200-l reaction mixture containing 0.1 M KPO 4 , pH 7.2, 1.2 mg/ml OAB, 0.12 mg/ml cytochrome c, 5 mM proline, and cell extract containing 50 g of protein was incubated for 30 min at 37°C. The reaction was terminated by the addition of 20 l of OAB (10 g/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/g 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 PPAR␥ ligands/proline, as specified, for an additional 36 h before analysis for ROS. For inhibition stud-ies, 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 serumfree, phenol red-free medium containing 50 M 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 Ϯ S.D.

RESULTS
Effect of PPAR␥ 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 (Ϫ1250 to ϩ10) 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, PPAR␥ was the most potent and resulted in a 6-fold activation of the POX promoter activity (Fig. 1A). Since PPAR␥ strongly activated the POX promoter, we characterized the interaction of PPAR␥ with POX in detail. PPAR␥ is a ligand-activated transcription factor; therefore, we next investigated if the PPAR␥ ligand troglitazone could further enhance the activation of POX promoter. HEK293 cells transfected with POX-Luc construct and PPAR␥ 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 M further increased the POX promoter activity over PPAR␥ alone.
High levels of PPAR␥ have been reported in several colon cancer cells, and the role of PPAR␥ and its ligands in cell growth arrest and apoptosis has been well documented (9,10). Therefore, we transfected various colon cancer cell lines with a POX-Luc construct, and analyzed whether activation of endogenous PPAR␥ 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 M). 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 PPAR␥ ligands in HCT116 cells (Fig. 1C). All of the PPAR␥ ligands tested increased the POX promoter activity, indicating that the activation may be mediated through PPAR␥.
Troglitazone Increases the Binding of PPAR␥ to the POX Promoter-Analysis of the POX promoter region revealed the presence of a putative PPAR/retinoid X receptor binding site (PPRE) between Ϫ982 and Ϫ969 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 PPAR␥ (24), we used these cells to study the functionality and binding of PPAR␥ 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 PPAR␥, as shown in Fig. 2. The intensity of the bands was increased by treatment with 25 M troglitazone for 36 h.
To confirm the binding of PPAR␥ 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 PPAR␥ 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 PPAR␥ and its ligands can effect the actual expression of endogenous POX. HCT116 cells expressing PPAR␥ were treated with troglitazone (25 M) 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 M (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 M 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 reaction mixture and incubated for 30 min at 37°C. In the ligand-stimulated cells, a time-and dose-dependent increase (2-3-fold) in POX catalytic activity was obtained in parallel to the increase in mRNA and protein expression (Fig. 4, A and B).
The Dependence of Activation of POX on PPAR␥-To test whether activation of POX was PPAR␥-dependent, we analyzed the effects of GW9662 on ligand-induced promoter activity. GW9662 is an inhibitor of PPAR␥ with high affinity and can selectively and fully inhibit PPAR␥dependent effects (25). GW9662 (10 M) only partially decreased the troglitazone-induced POX promoter activity, as evaluated by the luciferase assay (Fig. 5A, white bars), whereas GW9662 alone had no effect on POX promoter activity, ruling out the possibility of GW9662-medi- A, HCT116 cells were exposed to medium containing troglitazone (25 M) or Me 2 SO in controls and collected at various time points. Total RNA was isolated by the Trizol method, and RT-PCR was performed using POX-specific primers as described under "Materials and Methods." Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control primers were used in an identical RT-PCR with 20 cycles. The graph shows the densitometric analysis of the relative expression of POX mRNA normalized to glyceraldehyde-3phosphate dehydrogenase expression with time. Concentration-dependent effects of troglitazone on POX protein expression (B) and the time course of troglitazone-induced POX protein expression (C) are shown. HCT116 cells were exposed to medium containing different concentrations of the ligand for 36 h or treated with 25 M troglitazone and collected at various time periods. After treatment, all of the cells were harvested, and cell lysates were prepared. The protein concentration was determined, and equal amounts of cell lysates were electrophoresed on acrylamide denaturing gels and transferred by electroblotting onto nitrocellulose membrane. Western blots were performed using anti-POX antibody. Actin was used as a loading control (1:2000; Sigma). Blots were developed using the enhanced chemiluminscence kit (Amersham Biosciences). ated PPAR␥-independent POX activation. This result implies that the effects of troglitazone are not limited to mechanisms dependent on the interaction of PPAR␥ with the promoter and may involve some other mechanism(s) at least in regard to POX expression. To verify this view, we then checked the endogenous expression of POX by RT-PCR in troglitazone-treated cells in the presence and absence of GW9662. As a control, we also monitored the expression of keratin 20, a PPAR␥-selective target gene important in colon epithelial cell maturation (26). Increased expression of POX mRNA was observed with troglitazone, and the addition of only GW9662 had no effect (Fig. 5B). The troglitazone-induced POX expression was only slightly inhibited by GW9662, whereas expression of keratin 20 was suppressed, indicating that the concentration of GW9662 used was sufficient to inhibit ligand activation of PPAR␥ target genes (Fig. 5B). This result also supports the involvement of other non-PPAR␥ effects.
We further addressed this point by generating a deletion construct of POX-Luc. The stimulation of PPAR␥ by troglitazone in cells transfected with the truncated POX-Luc construct containing a deleted PPRE was reduced as compared with the untruncated construct (Fig. 5A, gray  bars). However, the luciferase response was not completely abrogated; furthermore, the presence of GW9662 had no effect on the activation obtained by the truncated POX-Luc excluding the possible activation by secondary PPAR␥ binding sites. These results further suggested the involvement of other PPAR␥-independent mechanisms.
Activation of POX by Troglitazone Also Involves the Induction of p53 Expression-Increased levels of p53 in response to troglitazone have been reported (27,28), and POX has been shown to be a downstream mediator in p53-induced apoptosis. Therefore, we considered whether the PPAR␥-independent activation of POX may be mediated by p53. We measured the levels of p53 after exposure to troglitazone in HCT116 cells, that have a wild type and functional p53. Troglitazone treatment in HCT116 cells resulted in a time-and concentration-dependent increase in p53 protein levels parallel to the increase obtained in POX expression (Fig. 6A). This suggests that the induction of POX in HCT116 cells is mediated through both PPAR␥ and p53.
To demonstrate the involvement of p53 in the troglitazone-induced POX expression, we used the HCT15 colon cancer cells, which showed no significant effect of troglitazone on the activation of POX promoter (Fig. 6B). The HCT15 cells have mutant p53 as well as mutant PPAR␥, as a result of which these cells have been shown to be resistant to PPAR␥ ligand-induced growth inhibition (29). To show the dependence of POX induction on both p53 and PPAR␥, we hyperexpressed both wild type p53 and PPAR␥ in the HCT15 cells and measured the POX promoter activity. The expression of either p53 or PPAR␥ stimulated the POX promoter, and an additional increase was obtained in the presence of troglitazone (Fig. 6B, a). The transfection of p53 or PPAR␥ also increased endogenous POX expression in HCT15 cells (Fig. 6B, b), indicating that both PPAR␥-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. In the HT29 cells that have a wild type PPAR␥ and a mutant p53, increased activation was observed with troglitazone treatment due to the presence of wild type PPAR␥. This activation was suppressed by the inhibitor GW9662, indicating the activation to be PPAR␥-dependent. Furthermore, an increase in POX promoter activity was also observed with forced expression of wild type p53 (Fig. 6C), and an additive effect was obtained in the presence of both troglitazone and p53. This result again attests to the involvement of both PPAR␥ and p53 in the troglitazone-induced POX induction.
Proline-dependent Generation of Intracellular ROS-The p53-induced expression of POX has been reported to be accompanied by proline-mediated ROS generation (4). To ascertain whether PPAR␥ ligand treatment also leads to the accumulation of intracellular ROS, HCT116 cells were loaded with the peroxide-sensitive fluorescent probe DCF-DA in the absence or presence of different PPAR␥ ligands, and fluorescence of 2Ј,7Ј-dichlorofluorescein was quantified by spectrofluorometry. As shown in Fig. 7A, treatment of HCT116 cells with PPAR␥ ligands increased intracellular ROS. An additional proline-dependent increase in ROS was obtained in the presence of ligands, whereas proline alone had no effect on ROS generation, indicating that induction of POX is necessary for this increase. Furthermore, when cells were pretreated with N-acetyl cysteine, a potent antioxidant, it reversed the effects of proline on ROS generation in the troglitazone-treated cells (Fig. 7B). The direct role of POX in the troglitazone-induced ROS generation was further validated by studies using POX antisense. The expression of POX antisense in HCT116 cells abolished the troglitazone-induced POX catalytic activity and also suppressed the increase in ROS obtained in the presence of added proline, corroborating the contribution of POX to the troglitazone-induced ROS generation (Fig. 7C).

DISCUSSION
Previously, we reported that the hyperexpression of POX in DLD-1 colon cancer cells generated ROS, which was sufficient to initiate the  ). B, effect of GW9662 on troglitazone-induced POX expression. HCT116 cells were exposed to medium containing troglitazone (25 M) or GW9662 (10 M) or both and collected after 36 h. Total RNA was isolated by the Trizol method, and RT-PCR was performed using specific primers for POX and keratin-20, a PPAR␥-selective target as described under "Materials and Methods." Glyceraldehyde-3-phosphate dehydrogenase control primers were used in an identical RT-PCR reaction with 20 cycles. apoptotic pathway in the absence of functional p53 (4,30). Despite the demonstrated role of POX in growth inhibition and apoptosis (4, 30 -32), the molecular mechanisms regulating its expression are not defined. To gain an understanding of these mechanisms, we analyzed the human POX promoter region for potential transcription factor binding sites. Among the different transcription factors that were tested, PPAR␥ significantly stimulated the POX promoter activity. Also, in various colon cancer cell lines, activation of PPAR␥ by its ligand troglitazone was found to induce POX promoter activity. Although PPAR␥ is classically considered to be a regulator of metabolism in adipose tissues, it is increasingly considered an important regulator of growth and programmed cell death. PPAR␥ is expressed in various cancer cell types including breast, prostate, colon, and others (9). The colon cells are of particular interest, because PPAR␥ is expressed at high levels, comparable with those in adipose tissue (33). In a number of colon cancer cell lines, PPAR␥ activated by its agonists induces growth arrest and apoptosis, expression of markers of differentiation, and inhibition of anchorage-independent growth (34 -38). Loss-of-function mutations of PPAR␥ in tumor tissue have been detected in some patients with adenocarcinoma of the colon (39), suggesting that PPAR␥ could function as a tumor suppressor. PPAR␥ thus plays a key modulatory role in signaling, especially in the colon and cultured colon cancer cells, although the mechanisms by which PPAR␥ agonists cause apoptosis remain to be clearly defined.
Since transfected PPAR␥ significantly stimulated POX promoter activity, the effect of endogenous PPAR␥ on POX expression became a physiologically important question. We therefore used the HCT116 colon cancer cells that are known to express a wild type and functional PPAR␥. In these cells, the PPAR␥ ligand troglitazone not only stimulated POX promoter activity but also enhanced binding of PPAR␥ to the POX promoter. Furthermore, troglitazone up-regulated expression of endogenous POX as evidenced by increased mRNA and protein levels and increased POX catalytic activity, demonstrating that POX may be playing a functional role in troglitazone-induced effects. In addition, other PPAR␥ ligands also induced POX promoter activity, indicating that the activation may be mediated through PPAR␥. However, our results also showed that the inductive effect of troglitazone on POX expression was not totally PPAR␥-dependent, since the POX promoter activity was only partially suppressed in the presence of PPAR␥ antagonist, GW9662, and also in the presence of a truncated POX promoter containing a deleted PPRE. This suggested that there may be other factors in addition to PPAR␥ activation contributing to the induction of POX expression in response to troglitazone. PPAR␥ ligands have been demonstrated to activate various pathways independent of PPAR␥ (40,41), and therefore, the troglitazone-mediated POX expression also appears to involve PPAR␥-independent mechanisms. Troglitazone-induced apoptosis has been demonstrated to be mediated via the p53 pathway both dependently and independently of PPAR␥ (11,27,28,42), and the p53-mediated apoptosis has been shown to be accompanied by the induction of POX (4,31,32). We observed increased levels of p53 when exposed to troglitazone, suggesting that the induction of p53 may also indirectly result in the induction of POX expression, which explains why there was only a partial suppression of POX activation although PPAR␥ was inhibited. The involvement of p53 was confirmed by increased POX activation in the presence of troglitazone by forced expression of wild type p53 in cell lines with mutant p53. Thus, in response to troglitazone the expression of POX is up-regulated through activation of PPAR␥ and also induction of p53.
One of the demonstrated mediators of apoptosis is ROS, which are generated as by-products of a number of metabolic reactions. Mitochondria are believed to be a major site of ROS production (43). Our findings show that PPAR␥ ligands induce a proline-dependent production of intracellular ROS. Troglitazone was able to enhance ROS concomitant with an increase in POX activity in HCT116 cells, and ROS scavengers such as N-acetyl cysteine block the stimulatory effect of troglitazone. Furthermore, we found that in the presence of POX antisense, the additional effect of proline on ROS generation was suppressed, demonstrating the direct role of POX in troglitazone-induced ROS increase. Since POX antisense did not completely block ROS generation, decreasing only the proline-dependent ROS generation, it is likely that ROS-generating pathways other than POX are targeted by troglitazone. In this regard, we showed that troglitazone induces the expression of p53. The apoptotic effects of p53 involves the induction of treated with N-acetylcysteine in the presence of troglitazone/proline. C, effect of POX antisense on troglitazone-induced ROS generation. The cells were transfected with POX antisense or control vector. Troglitazone (25 M) and/or proline (5 mM) were added after 10 h, as indicated. After 36 h, the cell lysates were harvested, and the POX enzymatic activity was determined. **, p Ͻ 0.005; *, p Ͻ 0.01 is for comparison of troglitazone or troglitazone ϩ proline-treated cells versus cells transfected with POX antisense. For ROS measurement in A-C, all of the cells were treated for 36 h as indicated, followed by incubation for 30 min with the dye DCF-DA (50 M), and 2Ј,7Ј-dichlorofluorescein fluorescence was determined. Data shown are the averages from at least three separate determinations. various redox-related genes, leading to the formation of ROS (3). In addition, there may be other yet unknown ROS generating pathways induced by PPAR␥. Nevertheless, the results reported here clearly show that POX is one of the redox enzymes targeted for ROS generation by troglitazone. In line with the observed results, Perez-Ortiz et al. (17) have demonstrated ROS generation to be the major mechanism mediating glitazone-induced glial cell death. Ciglitazone and rosiglitazone were found to cause mitochondrial depolarization and increase the steady-state ROS levels in glioma C6 cells and primary astrocytes. Recently, many studies have shown that cyclopentanone prostaglandins, such as 15-deoxy-⌬12,14-prostaglandin J 2 , an endogenous PPAR␥ ligand, induce intracellular stress through generation of ROS, and this may be the mechanism underlying its antiproliferative and antitumor effects (18,19,44). In this context, POX may be one of the mediators of PPAR␥ ligand-induced apoptosis through the production of ROS. Earlier work in our laboratory has demonstrated that cytotoxic agents induce POX expression, resulting in increased generation of ROS (4). The overexpression of POX is accompanied by the hallmarks of apoptosis, including cytochrome c release and caspase activation (4, 30 -32). The localization of POX to inner membrane of mitochondria, an organelle central to apoptosis, allows direct channeling of electrons from substrate to ROS-generating mechanisms during the oxidation of proline to P5C. Under normal conditions, electrons are transferred to substrates such as cytochrome c. However, under substrate-limiting conditions, electrons can leak from this shuttling system and generate superoxide radicals (45). POX generates ROS, specifically superoxide (30), which can mediate apoptosis by influencing the mitochondrial membrane potential (4,30,32). Therefore, ROS may be the intermediate mediator for the action of troglitazone through up-regulating expression of POX.
To summarize, we have demonstrated that PPAR␥ ligands, such as troglitazone, up-regulate the expression of POX with concomitant increase in POX catalytic activity, resulting in generation of intracellular ROS and leading to apoptosis. Our findings provide evidence that the troglitazone-stimulated POX expression is mediated through both PPAR␥-dependent and independent mechanisms via induction of p53. Whether the involvement of both PPAR␥ and p53 in the induction of POX expression is true for all PPAR␥ ligands remains to be determined.
Amino acid degradation, in general, is correlated with vegetative states in which protein synthesis has been halted; the degradation of proline may be unique in that it serves special functions. First, proline can donate electrons directly for generation of ROS (4); the cycling of P5C and proline can transfer reducing potential derived either from glycolysis or the pentose phosphate shunt into ROS or ATP (46); and the metabolism of proline and its metabolites can regulate redox balance (47). Furthermore, the activation of the pentose phosphate shunt will contribute to the production of ribose (48) to replenish the NAD pool consumed during ADP-ribosylation in the apoptotic cascade. Thus, proline degradation may bioenergetically support a number of necessary functions that couple cellular oxidative metabolism to apoptosis; therefore, enzymes of the proline degradation pathway are important targets for apoptosis-initiating proteins, such as p53 and PPAR␥.