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CARDIOVASCULAR

Activation of Peroxisome Proliferator-Activated Receptor by Substituted Urea-Derived Soluble Epoxide Hydrolase Inhibitors

Departments of Biochemistry (X.F., S.H., G.D.S., J.Y., A.A.S.), Internal Medicine (N.L.W., A.A.S.), and Radiation Oncology (N.L.W.), Carver College of Medicine, University of Iowa, Iowa City, Iowa; Department of Entomology and Cancer Research Center (T.W., B.D.H.), University of California, Davis, California; Veterans Administration Medical Center (N.L.W.), Iowa City, Iowa; and Department of Cell Biology and Neuroscience (Y.L., J.Y.-J.S.), University of California, Riverside, California

Received March 2, 2005; accepted March 25, 2005.

	Abstract

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Soluble epoxide hydrolase (sEH) plays a major role in regulating vascular epoxyeicosatrienoic acid metabolism and function, and substituted urea derivatives that inhibit sEH activity reduce blood pressure in hypertensive rats. We found that substituted urea derivatives containing a dodecanoic acid group, besides effectively inhibiting sEH, increased peroxisome proliferator-activated receptor (PPAR) activity. In PPAR transfected COS-7 cells, treatment with 10 µM N-cyclohexyl-N'-dodecanoic acid urea (CUDA) or N-adamantanyl-N'-dodecanoic acid urea (AUDA) produced 6- and 3-fold increases, respectively, in PPAR activation. Neither CUDA nor AUDA activated PPAR or PPAR directly, indicating selectivity for PPAR. CUDA did not alter PPAR protein expression, and it competitively inhibited the binding of Wy-14643 (pirinixic acid) to the ligand binding domain of PPAR, suggesting that it functions as a PPAR ligand. CUDA and AUDA were metabolized to chain-shortened -oxidation products, a process that reduced their potency as sEH inhibitors and their ability to bind and activate PPAR. N,N'-Dicylclohexylurea and N-cyclohexyl-N'-dodecylurea, sEH inhibitors that do not contain a carboxylic acid group, did not activate PPAR. In HepG2 cells, CUDA increased the expression of the PPAR-responsive gene carnitine palmitoyltransferase 1A. We conclude that CUDA and AUDA, by virtue of their carboxylic acid substitution, activate PPAR in addition to potently inhibiting sEH. Further development of these compounds could lead to a class of agents with hypotensive and lipid-lowering properties that may be valuable for the prevention and treatment of cardiovascular disease.

Disruption of the sEH gene in male mice decreases blood pressure (Sinal et al., 2000), suggesting that sEH could be a novel target for the treatment of hypertension. This finding has generated considerable interest in developing effective sEH inhibitors, and derivatives of urea that are potent, selective, and stable sEH inhibitors recently have been synthesized (Morisseau et al., 1999). N,N'-Dicylclohexylurea (DCU) is a representative compound of this class. Treatment with DCU reduced blood pressure in spontaneously hypertensive rats (Yu et al., 2000), and related compounds inhibited the proliferation of cultured vascular smooth muscle cells (Davis et al., 2002). N-Cyclohexyl-N'-dodecylurea (CDU), in which a hydrocarbon chain replaces the N'-cyclohexyl group of DCU, increases the potency of sEH inhibition 8- to 16-fold. However, the potential pharmacological usefulness of CDU is limited by its low aqueous solubility. Therefore, the compound was structurally modified to produce more H₂O-soluble derivatives. Two of these derivatives that contain a carboxylic acid substitution, N-cyclohexyl-N'-dodecanoic acid urea (CUDA) and N-adamantidyl-N'-dodecanoic acid urea (AUDA), increase H₂O solubility without an appreciable reduction in the potency of sEH inhibition (Morisseau et al., 2002).

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone receptor superfamily that function to transduce a variety of nutritional and inflammatory signals. PPAR, a member of this class, activates the expression of genes that regulate lipid metabolism. Many hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids activate PPAR (Yu et al., 1995; Forman et al., 1997; Kliewer et al., 1997; Bishop-Bailey and Wray, 2003), and 14,15-EET and its hydroxylated derivatives recently were shown to also activate PPAR (Cowart et al., 2002). Because sEH inhibition substantially increases the intracellular accumulation and retention of 14,15-EET (Fang et al., 2001), we investigated whether this might produce an EET-mediated activation of PPAR.

All of the urea-derived selective sEH inhibitors that we tested had similar inhibitory effects on the conversion of 14,15-EET to DHET. However, we unexpectedly found that only the sEH inhibitors containing an N'-carboxylic acid substitution activated PPAR, indicating that the activation occurred through an EET-independent mechanism. This report describes the activation of PPAR by CUDA and AUDA in a transfected COS-7 cell model system and provides evidence that CUDA functions as a PPAR ligand. The metabolism of CUDA and AUDA also was investigated in the COS-7 cells to determine whether this might affect the ability of these sEH inhibitors to function as PPAR ligands.

	Materials and Methods

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Cell Culture. COS-7 cells were purchased from American Type Culture Collection (Manassas, VA) and suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with minimum essential medium nonessential amino acids, minimum essential medium vitamin solution, 15 mM HEPES, 2 mM L-glutamine, 50 µM gentamicin, and 10% fetal bovine serum. The suspended cells were counted with a hemocytometer and plated into 25-cm² flasks at the density of 4 x 10⁴ cells/ml, and the cultures were maintained until confluent at 37°C in a humidified atmosphere containing 5% CO₂. Stocks were subcultured weekly by trypsinization, and the cells were transferred into six-well plates for all experiments. Cultures were used between passage numbers 12 and 25.

Transient Transfection of COS-7 Cells. PPAR was overexpressed in COS-7 cells, which inherently have very low or absent expression of this gene (Wurch et al., 2002). For comparison, additional COS-7 cultures were transfected with the PPAR or PPAR genes. The plasmids containing cDNA for mouse PPAR, PPAR, PPAR, and the PPAR-responsive luciferase reporter construct were kindly provided by Dr. Ronald M. Evans (Salk Institute, San Diego, CA) (Forman et al., 1995, 1997; Wang et al., 2003). Briefly, these expression vectors contained the cytomegalovirus IE promoter/enhancer (pCMX) upstream of wild-type mouse PPAR (pCMX-mPPAR), mouse PPAR (pCMX-mPPAR), or mouse PPAR1 (pCMX-mPPAR) genes. The plasmids were further replicated and purified using QIAprep Miniprep (QIAGEN, Valencia, CA), and they were analyzed by restriction digest and agarose gel electrophoresis.

COS-7 cells (60-70% confluent) in 60-mm dishes were transiently transfected using SuperFect with 0.02 µg of PPAR, , or ; 0.02 µg of the PPAR-responsive-luciferase (tk-PPREx3-luc) reporter construct, and 0.2 µg of a -galactosidase (-Gal)-expression plasmid. The -Gal plasmid was used as an internal control to normalize for transfection efficiency. Following incubation for 24 h, the medium containing the plasmids was removed, and the cultures were incubated with various concentrations of sEH inhibitors for 18 h. These inhibitors were dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the medium was 0.1% (v/v). After the cells were collected and lysed, luciferase and -Gal activities were measured (Zhu et al., 2001; Chen et al., 2005), and the luciferase activity was normalized to the -Gal activity.

Western Blot Analysis. Cells were placed in an ice bath and lysed with three 20-s bursts of sonic irradiation (Tekar Sonic Disruptor). The protein content of the cell lysate was measured by the Bradford method (Bradford, 1976), using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). Samples were denatured with an SDS loading buffer at 95°C for 5 min, and the proteins were separated in an SDS-10% polyacrylamide gel with a 5% stacking gel in SDS-Tris-glycine running buffer. The proteins were transferred electrophoretically to a nitrocellulose membrane, which was then blocked with 5% (w/v) nonfat milk in 0.02 M Tris/0.15 M NaCl buffer, pH 7.45, with 0.1% Tween 20 for 1 h. After an overnight incubation in 0.02 M Tris/0.15 M NaCl buffer, pH 7.45, with 0.1% Tween 20 buffer containing specific rabbit anti-serum raised against a peptide corresponding to amino acids 22 to 36 of human/murine/rat PPAR (1:1000; Cayman Chemical, Ann Arbor, MI), the blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Roche Diagnostics, Indianapolis, IN) for 1 h at room temperature, and the anti-PPAR antibodies were detected using an ECL detection system (Pierce Chemical, Rockford, IL) and exposure to X-ray film. Following this, the membrane was stripped and reprobed with antibody against -actin (Sigma-Aldrich, St. Louis, MO) as described above, and the density of the -actin band was used to normalize for protein loading.

Carnitine Palmitoyltransferase 1A (CPT1A) mRNA Analysis by Real-Time RT-PCR. Total RNA from cultures was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA), and the RNA content was measured spectrophotometrically (Fang et al., 2000b). Two micrograms of total RNA from each sample was reversed transcribed. The resulting cDNAs were diluted (1/10-1/50), and equal amounts were aliquoted for real-time PCR analysis using a Stratagene Mx 3000P instrument (Stratagene (La Jolla, CA). Primers and FAM-labeled probes for CPT1A, GAPDH (housekeeping gene), and Universal Taqman master mix were purchased from Applied Biosystems (Foster City, CA). CPT1A mRNA was assayed by the comparative quantitation method, and the calculated differences in mRNA expression were determined according to User Bulletin 2 (10/2001; Applied Biosystems). Gene expression data are expressed as -fold differences from the control cells and have been normalized to the expression of GAPDH.

Binding Competition Assays. [³H]Wy-14643 (7.5 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO), together with CUDA or CUDA metabolites, was incubated with recombinant PPAR ligand binding domain (GST-mPPAR-LBD) in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM dithiothreitol, and 200 ng/ml ovalbumin (Forman et al., 1997). After being incubated at 25°C for 30 min and chilling on ice for 15 min, the free and bound ligands were separated by Sephadex G-25 (Sigma-Aldrich) columns in a buffer containing 15% glycerol, 25 mM Tris-HCl, pH 7.8, 0.05% Triton X-100, 0.5 mM EDTA, and 75 mM KCl. The quantity of bound ligands was determined by liquid scintillation counting.

EET Metabolism. COS-7 cells were incubated with sEH inhibitors in modified DMEM for 30 min, after which 1 µM [³H]14,15-EET was added, and the incubation continued in the presence of the inhibitor. After 1 h, the medium was collected, and the cells were washed twice with cold phosphate-buffered saline solution and harvested by scraping into methanol. Radioactivity in an aliquot of the medium and cell lipid extract was measured by liquid scintillation counting. The remainder of the medium was extracted twice with 4 ml of ice-cold ethyl acetate, and after the extracts were combined, the solvent was evaporated under N₂ and the lipid residue dissolved in acetonitrile. The lipids were separated by reverse-phase high-performance liquid chromatography (HPLC), and the column effluent was mixed with scintillator solution and passed through an in-line flow detector (IN/US System, Inc., Tampa, FL) to determine the distribution of radioactivity (Fang et al., 2001, 2004).

Uptake and Metabolism of CUDA and AUDA. The COS-7 cells in 75-cm² tissue culture flasks were incubated with either 10 µM CUDA or AUDA for various times. After incubation, the cells were harvested and extracted with a 2:1 (v/v) mixture of chloroform/methanol. The extracts were hydrolyzed for 1 h at 50°C in 0.5 ml of methanol containing 50 µl of 0.2 N NaOH and 10% H₂O, and the reactants were further extracted with ethyl acetate and analyzed by high-performance liquid chromatography combined with tandem mass spectrometry (LC/MS-MS). The LC/MS-MS analysis was carried out using a Micromass Quattro Ultima triple quadrupole tandem mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ionization interface. The HPLC system consisted of a Waters model 2790 separations module (Waters, Milford, MA) equipped with a Waters model 2487 dual-wavelength absorbance detector. The mass spectrometer was coupled to the outlet of the HPLC column (XTerra MS C₁₈ column, 30-x 2.1-mm i.d., 3.5 µm). Ten microliters of samples was injected onto the column, and the separation was done with solvents A and B containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. The mobile phases were mixed with a linear gradient from 40% B to 100% B over 0 to 5 min and then held for 8 min with 100% B at a flow rate of 0.3 ml/min. The electrospray ionization was performed in the positive mode. Identification of sEH inhibitor metabolites was carried out with full scan and/or daughter ion scan in the positive and negative mode. Data were acquired in the multichannel analysis mode and continuum mode, and quantitative analysis was performed in the multiple reaction monitoring mode. The data were processed with MassLynx software (version 3.5) (Watanabe and Hammock, 2001).

sEH Activity Measurement. The effect of CUDA and GW7647, an ureidothioisobutyric acid derivative that has improved lipid-lowering activity compared with fenofibrate (Brown et al., 2001), on recombinant murine sEH activity was determined as described previously (Widstrom et al., 2003). Briefly, a working stock of 13 nM sEH in assay buffer containing 0.75 µM bovine serum albumin was prepared. [³H]14,15-EET (0.6 µM) was added to 155 µl of assay buffer containing 5 nM bovine serum albumin in glass vials. Reactions were initiated by adding sEH to a final concentration of 0.4 nM and incubated for 5 min at 30°C. Reaction mixtures that included CUDA, GW7647, or DMSO (control) were incubated for 2 min prior to enzyme addition. The reactions were terminated by transfer into 5 ml of chloroform/methanol (2:1), followed by addition of 1 ml of 0.9% saline. Tubes were vortexed and centrifuged for 10 min at 4°C. After the bottom layer was removed and saved, the top layer was re-extracted with 1 ml of chloroform/methanol/0.9% saline (86:14:1). The bottom phases were combined, dried under nitrogen, resuspended in chloroform/methanol (2:1), and applied to Silica Gel G thin-layer chromatography (TLC) plates and developed to 4 cm in chloroform/methanol/acetic acid (60:30:1). The plate was dried and then further developed in hexane/ethyl acetate/acetic acid (70:30:1). Analysis of the TLC plates with a radioisotope scanner (BioScan, Washington DC) demonstrated the presence of two radioactive peaks that were identified as EET and DHET by comigration with standards.

Statistical Analysis. All data are expressed as mean ± S.D. Differences between mean values of two groups were analyzed by Student's t tests. Differences between mean values of multiple groups were analyzed by one-way analysis of variance with a Newman-Keul analysis. Probability values of 0.05 or less were considered to be statistically significant.

	Results

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PPAR Expression and Activation by Different Urea-Derived Selective sEH Inhibitors in COS-7 Cells. We first established a reliable assay system for PPAR activity. The PPAR protein was not detected in COS-7 cells under control conditions, whereas it was readily detected when the COS-7 cells were transfected with the plasmid containing the mouse PPAR gene. After 18 h of incubation, Wy-14643 (20 µM), a fibric acid derivative that is widely used as a PPAR activator (Devchand et al., 1996), did not increase the PPAR activity in control conditions but caused a 10-fold increase in PPAR activity in cells that overexpressed PPAR. We next determined the ability of different ureaderived selective sEH inhibitors to activate PPAR in the transfected COS-7 cells (Fig. 1, top left). Incubation of the cells with 10 µM CUDA for 18 h increased PPAR activity 5-fold as measured by the luciferase assay. By contrast, no increase in luciferase activity was produced by either 10 µM DCU or 10 µM CDU, which contains the dodecyl hydrocarbon chain but no terminal carboxyl group.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Activation of PPAR by sEH inhibitors. The COS-7 cultures were transfected with -Gal, PPAR, and the luciferase reporter genes in a 24-h incubation. The cultures were then incubated for 18 h with 1 ml of control medium or media containing 10 µM DCU, CDU, or CUDA, and the luciferase and -Gal activities in cell lysates were measured. The luciferase activity was normalized to the -Gal activity. The results are expressed as -fold activation relative to the vehicle control (top left). In the time-dependent study (top right), the cultures were incubated with 1 ml of control medium, 10 µM CUDA, or 20 µM Wy-14643. In the CUDA concentration study (bottom left) and AUDA concentration study (bottom right), the cultures were incubated with these compounds for 18 h. The data are presented as mean ± S.D. values obtained from at least three separate experiments. *, P < 0.05; **, P < 0.01 versus control.

Concentration-dependence studies indicated that CUDA produced an increase in luciferase activity in the transfected cells at a threshold concentration of 3 µM (Fig. 1, bottom left). A 6-fold increase in luciferase activity occurred when the cells were incubated for 18 h with 10 µM CUDA, the highest concentration tested. A 3-fold increase in luciferase activity also occurred when the transfected cells were incubated for 18 h with 10 µM AUDA (Fig. 1, bottom right), which, like CUDA, contains a dodecyl chain with a terminal carboxyl group.

PPAR and PPAR Activity. To determine whether the stimulatory effect of CUDA was selective for PPAR, additional studies were done with COS-7 cells transfected with plasmids containing the mouse PPAR or PPAR genes. No increase in luciferase activity was produced by incubation with 10 µM CUDA in the cells that expressed PPAR, whereas 10 µM ciglitazone, a selective PPAR agonist, increased luciferase activity 5-fold. Additional studies indicated that 10 µM CUDA also did not increase luciferase activity in COS-7 cells that expressed PPAR. Likewise, incubation with 10 µM AUDA did not increase luciferase activity driven by PPAR or PPAR in the transduced COS-7 cultures.

Effect of sEH Inhibitors on 14,15-EET Metabolism. To determine whether the difference in the ability of CUDA and AUDA compared with CDU to activate PPAR-dependent gene expression might be due to differences in their effects on EET metabolism, we investigated the effects of these inhibitors on [³H]14,15-EET metabolism by the COS-7 cells. When the cultures were incubated for 1 h with 1 µM [³H]14,15-EET, 48% of total radioactivity was incorporated into the cell lipids. As shown in Fig. 2, HPLC analysis of the culture medium demonstrated that [³H]14,15-DHET was the major metabolite formed by cultures incubated in a control medium. By contrast, the formation of [³H]14,15-DHET was reduced substantially when the cultures were incubated in media containing 10 µM CDU, CUDA, or AUDA. As shown in Table 1, the reductions produced by these inhibitors were between 90 and 95%, and the small differences were not statistically significant. These sEH inhibitors did not affect the uptake of [³H]14,15-EET by the cells, and transfection with the PPAR gene did not affect [³H]14,15-EET metabolism. The HPLC analysis of the medium demonstrated that in addition to 14,15-DHET, radiolabeled metabolites with retention times of 33, 27, and 11 min accumulated when 10 µM CDU, CUDA, or AUDA were added (Fig. 2). Only very small amounts of these radiolabeled metabolites were detected in the medium of control cultures. The metabolites with retention times of 33 and 27 min have been identified previously as the 14,15-EET -oxidation products 10,11-epoxyhexadecadienoic acid (10,11-epoxy-16:2) and 8,9-epoxytetradecaenoic acid (8,9-epoxy-14:1), respectively (Fang et al., 2000a). Consistent with these findings, previous studies with porcine coronary endothelial cultures demonstrated that inhibition of sEH by DCU also increased the conversion of [³H]14,15-EET to 10,11-epoxy-16:2 and 8,9-epoxy-14:1 (Fang et al., 2001). We have not been able to positively identify the metabolite with a retention time of 11 min.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of sEH inhibitors on 14,15-EET metabolism. COS-7 cells were incubated initially in 1 ml of DMEM medium containing 0.1 µM albumin at 37°C, with or without 10 µM sEH inhibitors. After 30 min, 1 µM[3H]14,15-EET was added, and the incubations continued for 1 h. The media were collected and extracted, the lipid extracts were separated by reverse-phase HPLC, and the radiolabeled compounds were detected with an in-line flow scintillation counter. Radiochromatograms from single cultures are shown, but similar results were obtained from two additional cultures in each case.

Effect of CUDA on PPAR Protein Expression and Competitive Binding of CUDA to the Ligand Binding Domain of PPAR. To determine whether CUDA altered expression of the PPAR protein, the control cells or cells transfected with the PPAR gene were incubated with 10 µM CUDA or 20 µM Wy-14643 for 18 h. CUDA did not induce PPAR protein in the control cells or cause an increase in the amount of PPAR protein expressed by the transfected cells. A similar result was obtained with Wy-14643 (Fig. 3, top). To determine whether CUDA can bind to the ligand binding domain of PPAR, the mPPAR-LBD-GST proteins were incubated with 300 nM [³H]Wy-14643 and 5 or 25 µM CUDA or 5 µM nonlabeled Wy-14643. The binding of [³H]Wy-14643 to the ligand binding domain of mPPAR-LBD was competitively displaced by Wy-14643 or CUDA (Fig. 3, bottom). A 56% reduction in [³H]Wy-14643 binding was produced by 5 µM nonlabeled Wy-14643. CUDA (5 µM) decreased [³H]Wy-14643 binding by 28%, and 25 µM CUDA decreased the binding by 46%.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Effect of CUDA on expression of PPAR protein and binding of CUDA to PPAR protein. COS-7 cultures (60% confluent) were incubated for 24 h at 37°C with or without 0.02 µg of the pCMX-mPPAR expression vector. The cultures were then incubated for an additional 18 h with 1 ml of control medium containing vehicle alone, 10 µM CUDA, or 20 µM Wy-14643. The PPAR and -actin proteins in the cell lysates were detected by Western blotting (top). For binding assays, 2 µg of GST-PPAR-LBD fusion protein was incubated with 300 nM [3H]Wy-14643 in a total volume of 50 µl in the presence of ethanol, unlabeled Wy-14643, or various concentrations of CUDA. The free and bound ligands were separated on a Sephadex G-25 column, and the amount of bound [3H]Wy-14643 was then determined by liquid scintillation counting (bottom). The binding assays were repeated in quadruple sets and presented as mean ± S.D. **, P < 0.01 versus controls.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Identification of CUDA and AUDA metabolites. COS-7 cells in 75-cm2 flasks were incubated with 5 ml of medium containing either 10 µM CUDA or AUDA. After incubation, the media were collected, extracted with ethyl acetate, and assayed for metabolites by LC/MS-MS. To obtain sufficient quantities of products for structural identification, samples from two separate cultures subjected to identical incubation conditions were combined. The tandem mass chromatograms and the structures of compounds contained in the medium after incubation for 6 h are shown. The n value indicates the number of carbon atoms contained in the fatty acid chain of the CUDA and AUDA metabolites.

Figure 5 shows the distribution of these compounds in the cells and medium at the end of the 6-h incubation. The media contained 90 to 170 times more of these compounds than the cells. -Oxidation products accounted for almost all of the material present in the media, whereas approximately half of the material contained in the COS-7 cells was either unmodified CUDA or AUDA. The most abundant CUDA metabolite recovered from the medium contained a 10-carbon fatty acid chain, whereas the most abundant AUDA metabolite contained an 8-carbon fatty acid chain. The main AUDA -oxidation product present in the cells also contained an eight-carbon fatty acid chain, whereas metabolites with 10- and 6-carbon fatty acid chains were the main CUDA derivatives recovered in the cells. These findings indicate that both CUDA and AUDA are substrates for partial -oxidation in COS-7 cells and that although most of the -oxidation products are released and accumulate in the medium, small amounts of these chain-shortened products are retained in the cells.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Distribution of CUDA and AUDA and their products in COS-7 cells and medium. The incubation conditions and methods of analysis were the same as described in Fig. 4.

Effect of CUDA Metabolites on PPAR Activity and Binding to the Ligand Binding Domain of PPAR. Additional studies were done to determine whether CUDA metabolism affects its ability to activate PPAR. The COS-7 cells were incubated with 10 µM CUDA, and after 18 h, the medium was collected and transferred to other transfected COS-7 cells that has not been exposed to CUDA. The incubation was then continued for an additional 18 h. Activation of PPAR by the postincubation medium was decreased by 50% compared with medium containing CUDA [6.34 ± 1.2 (10 µM CUDA) versus 3.16 ± 0.15 (medium from cells that had been incubated with 10 µM CUDA), P < 0.01]. These results suggested that CUDA metabolites have less capacity to activate PPAR. This observation was confirmed by incubating synthetic CUDA metabolites with the transfected cells. N-Cyclohexyl-N'-octanoic acid urea, a metabolic product of CUDA formed following two cycles of -oxidation, caused only a 3-fold increase in PPAR activity, and N-cyclohexyl-N'-hexanoic acid urea, a metabolic product of CUDA formed following three cycles of -oxidation, did not increase the PPAR activity compared with the control (Fig. 6, top). Competitive binding analyses indicated that the chain-shortened metabolite N-cyclohexyl-N'-octanoic acid urea displaced less [³H]Wy-14643 from the ligand binding domain of mPPAR-LBD compared with CUDA, a 20% decrease compared with a 50% decrease produced by CUDA. Moreover, N-cyclohexyl-N'-hexanoic acid urea did not reduce the binding of [³H]Wy-14643 to PPAR (Fig. 6, bottom).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Activation and the binding of PPAR by CUDA metabolites. The COS-7 cultures were transfected with -Gal, PPAR, and the luciferase reporter genes in a 24-h incubation. The cultures then were incubated for 18 h with 1 ml of control medium or media containing either 10 µM CUDA or the chain-shortened CUDA metabolites. The luciferase and -Gal activities in the cell lysates were measured, and the results are expressed as -fold activation relative to the vehicle control (mean ± S.D., n = 4). **, P < 0.01 versus control. For binding assays, 2 µg of GST-PPAR-LBD fusion protein was incubated with 300 nM [3H]Wy-14643 in a total volume of 50 µl in the presence of ethanol, 25 µM CUDA, or CUDA metabolites. The free and bound ligands were separated on a Sephadex G-25 column, and the amount of bound [3H]Wy-14643 was determined by liquid scintillation counting. The binding assays were repeated in quadruplicate sets and presented as mean ± S.D. *, P < 0.05; **, P < 0.01 versus controls. CUOA, cyclohexyl-octanoic acid urea; CUHA, cyclohexyl-hexanoic acid urea.

Effect of CUDA on Expression of the PPAR Response Gene CPT1A in HepG2 Cells. We determined whether activation of PPAR by CUDA will up-regulate a PPAR-responsive gene. Because PPAR-responsive genes have been identified in human HepG2 cells (Hsu et al., 2001), this cell line was used for these studies. CUDA (10 µM) caused a 2-fold increase of PPAR activity as measured by luciferase activity in transfected HepG2 cells, and Wy-14643 (10 µM) increased the PPAR activity 3-fold (Fig. 7, top). A real-time-PCR assay indicated that CUDA, as well as Wy-14643, significantly increased the level of CPT1A mRNA, a PPAR-responsive gene, in transfected HepG2 cells (Fig. 7, bottom).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Activation of PPAR by CUDA and effect on CPT1A in HepG2 cells. HepG2 cultures were transfected with -Gal, PPAR, and the luciferase reporter genes in a 24-h incubation. The cultures were then incubated for 18 h with 1 ml of control medium or media containing 10 µM CUDA or Wy-14643, and the luciferase and -Gal activities in cell lysates were measured. The luciferase activity was normalized to the -Gal activity. The results are expressed as -fold activation relative to the vehicle control (top). In a separate experiment under the same conditions, the RNA was extracted from the HepG2 cells, and CPT1A mRNA was assayed by real-time RT-PCR. The data are expressed as -fold differences from the control cells and have been normalized to the expression of GAPDH (bottom).

Effect of PPAR Agonists on sEH Activity.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Effect of PPAR agonists on cellular sEH activity. COS-7 cells were incubated initially in 1 ml of DMEM medium containing 0.1 µM albumin at 37°C, with or without various concentration of PPAR agonists. After 30 min, 1 µM [3H]14,15-EET was added, and the incubations were continued for 1 h. The media were then collected and extracted, the lipid extracts were separated by reverse-phase HPLC, and the radiolabeled compounds were detected with an in-line flow scintillation counter. The values are results obtained from three separate cultures, expressed as mean ± S.D. The specific activity of the added [3H]14,15-EET was used to calculate the pmol amounts (left). **, P < 0.01 versus control. Representative HPLC radiochromatograms from single cultures are shown on the right, but similar results were obtained from two additional cultures in each case.

View larger version (17K): [in this window] [in a new window]   Fig. 9. Inhibition of recombinant mouse sEH by CUDA and GW7647. [3H]14,15-EET (0.6 µM) was incubated with vehicle, CUDA, or GW7647 for 2 min prior to addition of recombinant mouse sEH. After a 5-min incubation, the reaction was terminated, and the lipids were extracted and analyzed by TLC. Representative radioactivity scans of TLC plates are shown. A, [3H]14,15-DHET production in the absence of inhibitors. B, [3H]14,15-DHET production in the presence of 1 µM CUDA. C, [3H]14,15-DHET production in the presence of 1 µM GW7647.

	Discussion

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We have observed that CUDA and AUDA, two potent urea-derived sEH inhibitors that contain a carboxylic acid substitution, are activators of PPAR. The effects are specific for PPAR because neither compound activated PPAR or PPAR, and the activation of PPAR by CUDA and AUDA seems to be independent of sEH inhibition. The dual function of these compounds, inhibition of sEH and activation of PPAR, suggests a unique pharmacological profile that may be of considerable value in treating cardiovascular disease.

CDU, which differs from CUDA only by the absence of a carboxyl group at the end of the N'-dodecyl chain, did not activate PPAR. This suggests that the carboxylic acid group present in CUDA and AUDA plays a key structural role in the activation process, a finding consistent with the previous observation that long-chain fatty acids activate PPAR but the corresponding fatty alcohols do not (Forman et al., 1997). The fact that CUDA is more efficacious than AUDA in activating PPAR in the transduced COS-7 cells suggests that the N-cyclohexyl group facilitates the interaction more effectively than the corresponding N-adamantanyl group. In this regard, CUDA is structurally similar to GW7647, a potent PPAR agonist that also is a urea derivative containing an N-cyclohexyl group (Brown et al., 2001). However, in addition to structural features, differences in the biopharmaceutical properties of these compounds also may account for their relative potency in activating PPAR.

The possibility that CUDA and AUDA do not directly bind to PPAR but instead facilitate the binding of an endogenous ligand was initially considered. For example, the 19- and 20-hydroxylated derivatives of EETs are PPAR ligands (Cowart et al., 2002), suggesting that sEH inhibition might activate PPAR by increasing the intracellular accumulation of EET metabolites. However, CDU inhibited the conversion of 14,15-EET to DHET to about the same extent as CUDA and AUDA but did not increase PPAR activity, indicating that the activation of PPAR by CUDA and AUDA is independent of their inhibitory effect on the conversion of EET to DHET. Likewise, the increased accumulation of 14,15-EET partial -oxidation metabolites that occurred when the inhibitors were added seems to be unrelated to PPAR activation because CDU also increased the accumulation of these products.

Our data suggest that CUDA and AUDA most likely activated PPAR through a ligand-dependent mechanism. As was observed with Wy-14643, CUDA did not increase the amount of PPAR protein in the COS-7 cells and only increased PPAR-dependent luciferase activity in those cultures that expressed PPAR. Furthermore, small amounts of unmodified CUDA and AUDA were recovered in the cells in incubations lasting 6 h, indicating that these compounds probably are available intracellularly for an extended period. Many fatty acids and eicosanoids activate PPAR through a binding mechanism (Yu et al., 1995; Willson and Wahli, 1997; Murakami et al., 1999; Bishop-Bailey and Wray, 2003), and the fact that CUDA and AUDA are fatty acid derivatives suggests that this structural property enables these compounds to also bind to PPAR. Consistent with this notion, CUDA competitively inhibited the binding of Wy-14643 to the ligand binding domain of mPPAR-LBD, indicating that direct binding to PPAR can occur.

Activation of PPAR mediates induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, and the CPT1A gene is a PPAR-responsive gene in human HepG2 cells (Hsu et al., 2001). Overexpression of mPPAR in HepG2 cells does not increase the induction of CPT1A, but it causes a significant increase when the cells are treated with Wy-14643 (Hsu et al., 2001). We found that, similar to Wy-14643, CUDA increased the PPAR activity as measured by the luciferase activity, and it caused a 2-fold induction of CPT1A mRNA. These results suggest that CUDA can function as a PPAR agonist in human cells.

Analysis by tandem mass spectrometry revealed that most of the CUDA and AUDA added to the cultures was converted to -oxidation products during the first 6 h of incubations with the COS-7 cells. The carboxylic acid chains of these -oxidation products, which accumulated primarily in the medium, contained 4 to 10 carbons. This -oxidation process most likely is the catabolic mechanism that inactivates CUDA and AUDA. Indeed, the chain-shortened CUDA metabolites have decreased abilities to activate PPAR and bind weakly to the ligand binding domain of mPPAR-LBD compared with CUDA (Fig. 6). This is supported by previous studies with sEH inhibitors in which the terminal carboxylic acid is esterified. The inhibition of sEH produced by these esters, which are as potent as the corresponding carboxylic acid compounds, decreases as the chain becomes shorter, and the four-carbon derivative is essentially inactive (Morisseau et al., 2002).

The concentrations of CUDA and AUDA that produced appreciable activation of PPAR in the COS-7 cells, although in the same range as the effective concentration of Wy-14643, are substantially higher than those needed to inhibit sEH. For example, the IC₅₀ values for CUDA and AUDA inhibition of mouse and human recombinant sEH are between 10 and 100 nM (Morisseau et al., 2002). However, CUDA and AUDA were designed for selective sEH inhibition, without regard to PPAR activation. Many structurally related compounds retain a high degree of sEH inhibitory activity (Morisseau et al., 1999, 2002), making it quite likely that either CUDA or AUDA could be structurally modified to increase its effectiveness for PPAR activation without a substantial loss of sEH inhibitory activity. For example, because -oxidation is an inactivation process for CUDA, adding methyl groups to the -carbon is likely to block this process (Spector et al., 1965), thereby enhancing the intracellular activity of CUDA as a PPAR activator. Similar methyl-branched structures are present in two PPAR activators, ciprofibric acid and clofibric acid (Forman et al., 1997).

GW7464, which is a very potent PPAR agonist (EC50 = 6 nM for PPAR activation), contains a cyclohexyl urea group. Therefore, we tested whether it also can inhibit sEH activity. Although no sEH inhibition was observed with 100 nM GW7464, a concentration that activates PPAR, GW7464 inhibited sEH activity at high concentrations in cultured COS-7 cells and when incubated with recombinant sEH. However, the inhibitory effect of GW7464 on sEH was weaker than the inhibition produced by CUDA. These observations further suggest that structural modification of either CUDA or GW7464 is likely to produce novel compounds that combine potent PPAR activation with selective sEH inhibition.

Activation of PPAR decreases cholesterol esterification in macrophages and increases the removal of cholesterol from human macrophage foam cells (Chinetti et al., 2001, 2003; Ricote et al., 2004). In addition to these effects on lipids, PPAR activation inhibits vascular smooth muscle activation and has anti-inflammatory effects (Staels et al., 1998; Delerive et al., 1999, 2000; Fruchart et al., 1999). It also produces many favorable effects on vascular function, such as diminishing oxidative stress, antagonizing the actions of angiotensin II, and reducing blood pressure in rodent models of hypertension (Schiffrin et al., 2003). Therefore, in addition to preserving EETs, activation of PPAR by CUDA or AUDA could have favorable effects on vascular function, the regulation of lipid metabolism, and inflammatory processes that facilitate atherosclerosis (Plutzky, 2003).

Another potential benefit of PPAR activation by CUDA or AUDA, as opposed to activators that do not inhibit sEH activity, is suggested by the fact that PPAR increases sEH expression in rodent species. For example, sEH mRNA levels in mouse liver, heart, and kidneys are increased by clofibrate, a hypolipidemic drug that activates PPAR (Hammock and Ota, 1983; Johansson et al., 1995). Because sEH activity is associated with blood pressure elevations in some experimental systems (Sinal et al., 2000; Yu et al., 2000; Imig et al., 2002; Spector et al., 2004), increased sEH expression is a potentially undesirable consequence of PPAR activation. However, any increase in sEH expression produced by CUDA or AUDA should be compensated by their potent inhibitory effect on sEH activity.

Hypertension is a major risk factor for atherosclerotic cardiovascular disease, and selective sEH inhibitors are being tested in animal models of hypertension (Yu et al., 2000). The use of sEH inhibitors like CUDA or AUDA may have the added benefit of PPAR activation. Therefore, further development of this novel class of compounds that has combined effects on sEH and PPAR could represent a useful new pharmacological approach for the prevention and treatment of cardiovascular disease.

	Acknowledgements

 
We thank Shawn D. Harmon and Bingkun Xu for technical assistance, Joseph Dillon for valuable discussion, and Ronald M. Evans at the Salk Institute, Howard Hughes Medical Institute, for providing the PPAR plasmids.

	Footnotes

 
The work at the University of Iowa was supported in part by American Heart Association Scientist Development Grant 0230096N (to X.F.), National Institutes of Health Grants HL072845 (to A.A.S.), HL070860 (to N.L.W.), and HL062984 (to N.L.W. and A.A.S.), and a Veterans Administration Merit Award (to N.L.W.). The work at the University of California (Davis) was supported by National Institutes of Health Grants HL59699 and ES02710, the Superfund Basic Research Program P42 ES04699, and National Institute of Environmental Health Sciences Centers P30 ES05707 and P01 ES11269 (to B.D.H.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.085605.

ABBREVIATIONS: EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; sEH, soluble epoxide hydrolase; DCU, N,N'-dicylclohexylurea; CDU, N-cyclohexyl-N'-dodecylurea; CUDA, N-cyclohexyl-N'-dodecanoic acid urea; AUDA, N-adamantidyl-N'-dodecanoic acid urea; PPAR, peroxisome proliferator-activated receptor; DMEM, Dulbecco's modified Eagle's medium; -Gal, -galactosidase; DMSO, dimethyl sulfoxide; CPT1A, carnitine palmitoyltransferase 1A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Wy-14643, pirinixic acid; HPLC, high-performance liquid chromatography; LC/MS-MS, liquid chromatography combined with tandem mass spectrometry; GW7647, 2-(4-(2-(1-cyclohexanebutyl-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid; TLC, thin-layer chromatography.

Address correspondence to: Dr. Xiang Fang, Department of Biochemistry, 4-403 BSB, University of Iowa Carver College of Medicine, Iowa City, IA 52242. E-mail: xiang-fang{at}uiowa.edu

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