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Am J Physiol Heart Circ Physiol 290: H55-H63, 2006. First published August 19, 2005; doi:10.1152/ajpheart.00427.2005
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14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-{alpha}

Xiang Fang,1 Shanming Hu,1 Bingkun Xu,1 Gary D. Snyder,1 Shawn Harmon,1 Jianrong Yao,1 Yi Liu,4 Bhavani Sangras,5 J. R. Falck,5 Neal L. Weintraub,2,3 and Arthur A. Spector1,2

Departments of 1Biochemistry and 2Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa; 3Veterans Administration Medical Center, Iowa City, Iowa; 4Department of Cell Biology and Neuroscience, University of California, Riverside, California; and 5Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas

Submitted 28 April 2005 ; accepted in final form 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPAR{alpha} activator in a COS-7 cell expression system. Incubation with 10 µM 14,15-DHET produced a 12-fold increase in PPAR{alpha}-mediated luciferase activity, an increase similar to that produced by the PPAR{alpha} agonist Wy-14643 (20 µM). Although 10 µM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPAR{alpha}. However, PPAR{alpha} was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPAR{alpha} with a Kd of 1.4 µM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPAR{alpha}-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPAR{alpha}.

cytochrome P-450; soluble epoxide hydrolase; 14,15-epoxyeicosatrieonic acid analogs

ARACHIDONIC ACID is converted to an epoxyeicosatrienoic acid (EET) through a monooxygenation catalyzed by NADPH-dependent cytochrome P-450 epoxygenases (4, 42, 45, 52). The four EET regioisomers that are formed in this reaction (5,6-; 8,9-; 11,12-; and 14,15-EET) function primarily as cardiovascular and renal biomediators (36, 42, 53). They produce vasodilation by activating membrane maxi-Ca2+-activated K+ (BKCa) channels and, therefore, function as endothelium-derived hyperpolarizing factors (3, 23, 45). In addition, there is accumulating evidence that EETs also produce anti-inflammatory effects (37), modulate smooth muscle proliferation (24), and inhibit smooth muscle migration (47).

EETs are converted to their corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (SEH) in many tissues, including vascular endothelial and smooth muscle cells (16, 20, 45), and this is generally considered to be an inactivation reaction (42, 52). DHETs have diminished activity in many systems compared with the corresponding EET, including relaxation of the preglomerular vasculature (30), inhibition of prostaglandin formation (18), calcium mobilization (22), stimulation of ADP ribosylation (44), and relaxation of the bovine coronary artery (13). However, small amounts of DHETs are incorporated into endothelial and smooth muscle lipids (17, 19, 48), and DHETs inhibit the hydrosmotic action of arginine vasopressin in the kidney (29), produce relaxation in porcine coronary rings and canine coronary arterioles (2, 15, 17, 38, 49), and activate the BKCa channel of rat coronary artery myocytes (33). These findings suggest that rather than being inactivation and excretion products, DHETs may have biological activity in some systems.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. PPARs play an important role in lipid metabolism, cell proliferation, and inflammatory signaling (40). PPAR{alpha}, a member of this class that is expressed in tissues exhibiting high rates of {beta}-oxidation, activates genes that are involved in the regulation of fatty acid {beta}-oxidation. Many hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids activate PPAR{alpha} (1, 25, 31, 50). Recently, the {omega}-hydroxylated derivative of 14,15-EET was shown to activate PPAR{alpha} (9). Therefore, we investigated the possibility that other EET derivatives might also function as endogenous PPAR{alpha} activators.

In the present study, we find that 14,15-DHET binds to the ligand binding domain (LBD) of PPAR{alpha} and activates PPAR{alpha}-mediated luciferase expression in a transfected COS-7 cell system. The response produced by 14,15-DHET is considerably greater than that of 14,15-EET or other EET and DHET regioisomers and is comparable to the effect of an equivalent concentration of Wy-14643, a widely used experimental PPAR{alpha} agonist. These results suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as a potent endogenous activator of PPAR{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The EET regioisomers and corresponding diols were purchased from Cayman Chemicals (Ann Arbor, MI), and 2-arachidonyl glycerol ester and pirinixic acid (Wy-14643) were obtained from BioMol (Plymouth Meeting, PA). 14,15-[3H]EET and 14,15-[3H]DHET (7.5 µCi/nmol) were synthesized from [5,6,8,9,11,12,14,15-3H]arachidonic acid as described previously (16, 48). N,N'-dicyclohexylurea (DCU), a selective SEH inhibitor, and a specific antibody against human SEH were kindly provided by Dr. Bruce D. Hammock (University of California, Davis, CA).

Preparation of HTDE and 14,15-GEET. 1 The synthesis of 14-hexyloxytetradec-5(Z)-enoic acid (HTDE) was initiated by incubating 1,8-octanediol with 1-bromohexane to form 8-hexyloxyoctan-1-ol. The product was converted to 1-bromo-8-hexyloxyoctane by addition of carbon tetrabromide and triphenylphosphine. After addition of n-butyl lithium to 6-(tert-butyldiphenylsilyloxy)hex-1-yne (51) in dry tetrahydrofuran and hexamethylphosphoramide, 1-(tert-butyldiphenylsilyloxy)-14-hexyloxytetradec-5-yne was purified by SiO2 column chromatography. This product was converted to 14-hexyloxytetradec-5-yn-1-ol with tetra-n-butylammonium fluoride and then to 14-hexyloxytetradec-5(Z)-en-1-ol by addition of NaBH4 and freshly distilled ethylenediamine. Jones reagent (12) was added, and after being filtrated, washed, and concentrated in vacuo, 14-HTDE was purified by SiO2 column chromatography.

The synthesis of 2-(14,15-epoxyeicosatrienoyl)glycerol (14,15-GEET) was initiated by adding 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to a solution of 14,15-EET (8) and 1,3-bis(triisopropylsilyl)-2-glycerol (28). The product, 1,3-bis(triisopropylsilyl)-2-(14,15-GEET), was incubated with a mixture of tetra-n-butylammonium fluoride and acetic acid. The 14,15-GEET that was formed was purified by flash chromatography over a short column of silica gel eluted with 30–50% ethyl acetate and hexane.

Cell culture. COS-7 cells were purchased from American Type Culture Collection (ATCC) and suspended in DMEM supplemented with MEM nonessential amino acids, MEM vitamin solution, 15 mmol/l HEPES, 2 mmol/l L-glutamine, 50 µmol/l gentamicin, and 10% FBS. 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% CO2-95% air. Stocks were subcultured weekly by trypsinization, and the cells were transferred into 60-mm tissue culture dishes or 6-well tissue culture plates for experiments. Cultures were used between passages 12 and 25 (14). In separate experiments, HepG2 cells, purchased from ATCC, were cultured in modified DMEM supplemented with 1 mM sodium pyruvate (14).

Transient transfection of COS-7 and HepG2 cells. PPAR{alpha} was overexpressed in COS-7 and HepG2 cells. The plasmids containing cDNA for mouse PPAR{alpha} and the PPAR-responsive luciferase reporter construct were kindly provided by Dr. Ronald M. Evans (Salk Institute, San Diego, CA) (25, 26). These expression vectors contained the cytomegalovirus immediated-early promoter/enhancer (pCMX) upstream of the wild-type mouse PPAR{alpha} (pCMX-mPPAR{alpha}) genes. The plasmids were further replicated and purified by using QIAprep Miniprep (Qiagen), and they were analyzed by restriction digest and agarose gel electrophresis.

The cells (60–70% confluent) in 60-mm dishes were transiently transfected by using SuperFect with 0.02 µg of PPAR{alpha}, 0.02 µg of the PPAR-responsive-luciferase (thymine kinase-peroxisome proliferator-activated response elements x3-luciferase) reporter construct, and 0.2 µg of a {beta}-galactosidase ({beta}-Gal)-expression plasmid. The {beta}-Gal plasmid was used as an internal control to normalize for transfection efficiency. After being incubated for 24 h, the medium containing the plasmids was removed and the cultures were incubated with various concentrations of EETs, DHETs, or related compounds. After the cells were collected and lysed, luciferase and {beta}-Gal activities were measured and the luciferase activity was normalized to the {beta}-Gal activity (6, 14).

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. Samples were denatured with a SDS loading buffer at 95°C for 5 min, and the proteins were separated in a 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% (wt/vol) nonfat milk in 0.02 M Tris and 0.15 M NaCl buffer, pH 7.45, containing 0.1% Tween 20 (TTBS) for 1 h. After an overnight incubation in TTBS buffer containing specific antibodies against human, murine, and rat PPAR{alpha} (1:1,000, Cayman Chemical) or human SEH, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Boehringer Mannheim) for 1 h at room temperature. The anti-PPAR{alpha} or anti-SEH antibodies were detected by using an enhanced chemiluminescence detection system (Pierce Chemical) and exposure to X-ray film. After this procedure, the membrane was stripped and reprobed with antibody against {beta}-actin (Sigma), and the density of the {beta}-actin band was used to normalize for protein loading (18).

Binding assays. The recombinant mouse gluthathione S-transferase (GST)-PPAR{alpha}-LBD protein was kindly provided by Dr. John Shyy (University of California, Riverside, CA). 14,15-[3H]DHET (7.5 µCi/nmol) was incubated with GST-mPPAR{alpha}-LBD in a buffer containing (in mM) 10 Tris·HCl (pH 8.0), 50 KCl, and 10 dithiothreitol (25). After being incubated at 25°C for 30 min and chilled on ice for 15 min, the free and bound ligands were separated by using Sephadex G-25 (Sigma) columns and a buffer solution containing 15% glycerol, 25 mM Tris·HCl (pH 7.8), 0.05% Triton X-100, 0.5 mM EDTA, and 75 mM KCl. The amount of bound radiolabeled ligand was determined by liquid scintillation counting.

14,15-EET and 14,15-DHET metabolism. COS-7 cells were incubated with 2 µM 14,15-[3H]EET or 14,15-[3H]DHET for various times. After being incubated, the medium was collected, and the cells were washed twice with cold phosphate-buffered saline and harvested by scraping into methanol. The radioactivity contained in an aliquot of the medium and in the cell lipid extract was assayed in a liquid scintillation counter. The remainder of the medium was extracted twice with 4 ml of ice-cold ethyl acetate; after the extracts were combined, the solvent was evaporated under N2, and the lipid residue was dissolved in acetonitrile. The lipids were separated by reverse-phase HPLC, and the column effluent was combined with scintillator solution and passed through an in-line flow detector (IN/US System, Tampa, FL) to determine the distribution of radioactivity (16, 20). The cell lipids were extracted from the COS-7 cells with chloroform-methanol (2:1). After the phases were separated and the solvent removed under N2, the lipids were dissolved in 200 µl of chloroform-methanol (2:1 vol/vol), and an aliquot of this mixture was dried under N2 and assayed for radioactivity in a liquid scintillation spectrometer (16, 21). To determine the distribution of the radioactivity in the cell lipids, aliquots of the extract were separated by TLC on Whatman LK5D silica gel plates obtained from Alltech Associates (Deerfield, IL) with a solvent system of chloroform, methanol, 40% methylamine (65:35:5 vol/vol/vol). The radioactivity contained in the separated lipids was assayed using a Bioscan gas flow proportional scanner with automatic peak search and integration (16, 17). Authentic phospholipid standards purchased from Avanti Polar Lipids (Naperville, IL) were added to each chromatogram and visualized by staining (19).

CPT1A mRNA analysis by real-time RT-PCR. Total RNA from the HepG2 cultures was isolated with TRIzol reagent (Life Technologies, Grand Island, NY), and the amount of RNA was measured spectrophotometrically (14). Two micrograms of total RNA from each sample were reversed transcribed. The resulting cDNAs were diluted, and aliquots containing equal amounts were taken for real-time PCR analysis using a Stratogene Mx 3000P instrument. Primers and carboxyfluorscein-labeled probes for carnitine palmitoyltransferase 1A (CPT1A), GAPDH, and Universal Taqman master mix were obtained from Applied Biosystems (Foster City, CA). The comparative quantitation method was used to assay for CPT1A mRNA (14). The gene expression data are expressed as fold differences from the control cells.

Statistical analysis. Data are means ± SD, except where otherwise indicated. Differences between mean values of two groups were analyzed by Student's t-tests. A one-way ANOVA with a Newman-Keuls test was used to analyze differences between mean values of multiple groups. Probability values of ≤0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation of PPAR{alpha} by EETs and DHETs in COS-7 cells. Using our previously established transfected COS-7 cell assay system for PPAR{alpha} activity (14), we compared the ability of EETs and their corresponding DHET metabolites to activate PPAR{alpha}. Incubation of the cells for 18 h with 10 µM of any of the EET regioisomers or with 10 µM of 5,6-; 8,9-; or 11,12-DHET, produced a two- to fourfold increase in PPAR{alpha} activity. In contrast, incubation with 14,15-DHET produced a 12-fold increase in PPAR{alpha} activity, an increase similar to that produced by 20 µM Wy-14643, a widely used PPAR{alpha} agonist. The degree of activation of PPAR{alpha} by 14,15-DHET was substantially greater than the threefold increase produced by 14,15-EET. No other DHET regioisomer produced a significantly larger increase in PPAR{alpha} activity compared with its corresponding EET (Fig. 1).



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  		Fig. 1. Activation of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) by epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs). COS-7 cultures were transfected with {beta}-galactosidase ({beta}-Gal), PPAR{alpha}, and luciferase reporter genes in 24-h incubation. Cultures were then incubated for 18 h with 1 ml control medium or media containing 10 µM EETs or corresponding DHETs. Luciferase and {beta}-Gal activities in cell lysates were measured. Results are expressed as fold activation relative to vehicle control. Data values are means ± SD obtained from at least 3 separate experiments. **P < 0.01 vs. control; #P < 0.01 vs. 14,15-EET.

 
A time-dependence study with 10 µM 14,15-DHET indicated that a fourfold increase in PPAR{alpha}-mediated luciferase activity occurred after 3 h of incubation with the COS-7 cells, the earliest time point tested (Fig. 2 A). Luciferase activity gradually increased as the incubation continued, and a 12-fold increase was observed at 18 h. A similar time-dependent response was observed with 20 µM Wy-14643. A fourfold increase in PPAR{alpha} activity was produced by 10 µM 14,15-EET after 6 h of incubation, and no further increase was observed when the incubation was extended to 18 h. No increase in luciferase activity occurred when the transfected COS-7 cells were incubated in a control medium that did not contain a PPAR{alpha} agonist (data not shown).



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  		Fig. 2. Time- and concentration-dependent effects of 14,15-EET and 14,15-DHET on PPAR{alpha} activity. COS-7 cultures were transfected with {beta}-Gal, PPAR{alpha}, and luciferase reporter genes for 24 h. Cultures were then incubated with 10 µM 14,15-EET or 14,15-DHET for various times (A) or with different concentrations of these compounds for 18 h (B). Luciferase and {beta}-Gal activities in cell lysates were measured. Each point represents average of results obtained from 2 separate cultures, and both values were within 10% agreement.

 
Concentration-dependence studies indicated that 14,15-EET and 14,15-DHET produced an increase in luciferase activity at a threshold concentration of 3 and 1 µM, respectively. A 12-fold increase in PPAR{alpha}-mediated luciferase activity also occurred in this experiment when the cells were incubated for 18 h with 10 µM 14,15-DHET, the highest concentration tested. By comparison, the maximum increase in PPAR{alpha} activity observed when the transfected COS-7 cells were incubated for 18 h with 10 µM 14,15-EET in this experiment was only threefold (Fig. 2B).

Effect of 14,15-EET analogs on PPAR{alpha} activity. To determine whether formation of 14,15-DHET is necessary for 14,15-EET-mediated activation of PPAR{alpha}, we investigated the effects of different 14,15-EET analogs on PPAR{alpha} activity in the transfected COS-7 cells. 14-HTDE (10 µM), which lacks an epoxy group and cannot be converted to a diol, failed to activate PPAR{alpha} (Fig. 3A). 2-Arachidonyl glycerol ester, which was not a substrate for conversion to 14,15-DHET, also failed to activate PPAR{alpha} in the COS-7 cells (data not shown). However, 10 µM 14,15-GEET produced a fourfold increase in PPAR{alpha} activation after an 18 h incubation (Fig. 3B). To investigate the mechanism of this finding, the COS-7 cells were incubated with 14,15-GEET that contained a 14C-labeled EET moiety. HPLC analysis of incubation medium without cells indicated that the radioactivity was present entirely as 14,15-GEET (Fig. 3C). After incubation for 18 h, HPLC analysis demonstrated that the radioactivity in the medium was present entirely as 14,15-DHET (Fig. 3D). These results indicate that the cells hydrolyzed the radiolabeled 14,15-GEET and converted the 14,15-EET to 14,15-DHET.



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  		Fig. 3. Effect of 14-hexyloxytetradec-5(Z)-enoic acid (14-HTDE) and 2-(14,15-epoxyeicosatrienoyl)glycerol (14,15-GEET) on PPAR{alpha} activity and metabolism of 14,15-GEET. COS-7 cultures were transfected with plasmids as described in Fig. 1. Cultures were then incubated for 18 h with 1 ml of control medium or media containing 10 µM 14-HTDE or 14,15-EET (A) or 10 µM 14,15-GEET or 10 µM Wy-14643 (B), and luciferase and {beta}-Gal activities in cell lysates were measured. Results are expressed as fold activation relative to vehicle control. Data values are means ± SD obtained from at least 3 separate experiments. **P < 0.01 vs. control. In a separate experiment, transfected COS-7 cells were incubated with 14,15-[1-14C]GEET for 18 h. After incubation, media were collected, extracted, and assayed for metabolites by reverse-phase HPLC. C: representative HPLC chromatogram obtained from incubation without cells. D: chromatogram obtained from incubation with cells. DPM, disintegrations/min.

 
Effect of DCU on EET-mediated PPAR{alpha} activation. The SEH protein was detected in cultured COS-7 cells by Western blot analysis (Fig. 4A). Because EETs are converted to DHETs by SEH, we further investigated the role of 14,15-DHET in 14,15-EET-mediated PPAR{alpha} activation using the selective SEH inhibitor DCU. Because of the relatively short half-life of DCU inhibition of SEH activity in cultured cells (16), the transfected COS-7 cells were incubated with PPAR{alpha} ligands in the absence or presence of DCU for only 6 h. The basal PPAR{alpha} activity was not affected by 10 µM DCU, but DCU completely inhibited 14,15-EET-induced PPAR{alpha} activation (Fig. 4B). In contrast, DCU did not affect 14,15-DHET-induced PPAR{alpha} activity (data not shown). When the cells were incubated with 2 µM 14,15-[3H]EET, 14,15-DHET was the major metabolite in the incubation medium, and the amount of 14,15-DHET that accumulated was time dependent (Fig. 4C). DCU (10 µM) effectively inhibited the formation of 14,15-DHET for up to 6 h when the cells were incubated with 2 µM 14,15-[3H]EET.



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  		Fig. 4. Presence of soluble epoxide hydrolase (SEH) and effect of N,N'-dicyclohexylurea (DCU) on 14,15-EET-induced PPAR{alpha} activation. A: presence of SEH in a COS-7 cell protein extract was detected by Western blot analysis. B: results were obtained when COS-7 cultures were transfected with plasmids as described in Fig. 1. Cultures were then incubated for 6 h in 1 ml medium or media containing 10 µM 14,15-EET or 20 µM Wy-14643 in absence or presence of 10 µM DCU. Luciferase and {beta}-Gal activities in cell lysates were measured. Results are expressed as fold activation relative to vehicle control, and data values are means ± SD obtained from at least 3 separate experiments. **P < 0.01 vs. control. In a separate experiment, cells were incubated with 2 µM 14,15-[3H]EET in presence or absence of 10 µM DCU for various times. Media were collected, extracted, and assayed for metabolites by reverse phase-HPLC. Data were converted to picomole values using the specific activity of radiolabeled substrates added to cultures. C: values are means ± SE and are results obtained from 3 separate cultures.

 
Incorporation and distribution of 14,15-EET and 14,15-DHET in COS-7 cells. To investigate the reason that 14,15-DHET was more potent than 14,15-EET in activating PPAR{alpha}-mediated gene expression, we compared the metabolism of 14,15-EET and 14,15-DHET in the COS-7 cells. When the cells were incubated with 2 µM 14,15-[3H]EET, radioactivity was rapidly incorporated into the cells and reached a maximum after only 1 h of incubation, the earliest time tested. At this time, 45% of the 14,15-[3H]EET initially present in the medium was taken up by the cells. The amount of radiolabeled lipid recovered in the cells declined slightly during the remainder of the 6-h incubation (Fig. 5A). A similar time-dependent uptake pattern also was observed when the cells were incubated with 2 µM 14,15-[3H]DHET, but the cells took up a total of only 6% of the 14,15-[3H]DHET initially present in the medium.



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  		Fig. 5. Time-dependent uptake and distribution of 14,15-EET and 14,15-DHET by COS-7 cells. COS-7 cells were incubated in medium containing 2 µM 14,15-[3H]EET or 14,15-[3H]DHET for various times. After being incubated, cells were washed and extracted, and lipid-soluble radioactivity was assayed (A). Data were converted to picomole values using specific activity of radiolabeled substrates added to the cultures. Each point represents average of results obtained from 2 separate cultures, and both values were within 10% agreement. Incorporated radiolabeled materials were separated by thin-layer chromatography, and distribution was determined with gas-flow proportional scanner. Representative chromatograms were obtained after 3-h incubation with 14,15-[3H]EET (B) and 14,15-[3H]DHET (C). The main radiolabeled lipid fractions are phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), free 14,15-DHET, and neutral lipids.

 
The cell lipids were extracted and analyzed by TLC to determine the distribution of the incorporated EET and DHET. When the cells were incubated with 2 µM 14,15-[3H]EET for 3 h, 90% of the incorporated radioactivity was recovered in phospholipids, including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (Fig. 5B). No free 14,15-[3H]EET or 14,15-[3H]DHET was detected in unsaponified cell lipids. In contrast, when the cells were incubated with 2 µM 14,15-[3H]DHET for 3 h, ~50% of the radioactivity was incorporated into neutral lipid and only 24% into phospholipids. Furthermore, 20% of incorporated DHET radioactivity remained as free 14,15-[3H]DHET (Fig. 5C).

To determine how much of the incorporated 14,15-[3H]EET or 14,15-[3H]DHET remained in the form of EET and DHET, respectively, or was converted to metabolites that were retained in the cells, the extracted cell lipids were hydrolyzed by saponification and analyzed by reverse-phase HPLC. When the cells were incubated with 2 µM 14,15-[3H]EET for 3 h, 78% of the incorporated radioactivity remained as 14,15-EET and 4% was present as 14,15-DHET. Two additional unidentified metabolites with retention times of 39 and 57.5 min also were detected (Fig. 6A). However, HPLC analysis of the medium demonstrated that 65% of radioactivity was present as 14,15-DHET. Therefore, ~95% of the 14,15-DHET produced from 14,15-[3H]EET by the COS-7 cells was released into extracellular fluid. When the cells were incubated with 2 µM 14,15-[3H]DHET for 3 h, 84% of radioactivity present in the cells remained as 14,15-DHET and 8% was contained in a metabolite with a retention time of 18 min (Fig. 6B). Throughout the 6-h incubation, the cells incubated with 14,15-[3H]DHET contained substantially more radiolabeled 14,15-DHET than those incubated with 14,15-[3H]EET (Fig. 6C).



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  		Fig. 6. Identification of metabolites incorporated into cells after incubation with 14,15-[3H]EET or 14,15-[3H]DHET. COS-7 cells were incubated in medium containing 2 µM 14,15-[3H]EET or 14,15-[3H]DHET for various times. Lipids contained in isolated chloroform phase were hydrolyzed by saponification, and radioactivity contained in resulting lipid extract was analyzed by reverse-phase HPLC. A and B: representative chromatograms obtained after 3-h incubation with 14,15-[3H]EET or 14,15-[3H]DHET, respectively. C: relative amount of 14,15-DHET retained in the cells (pmol) was calculated by using specific activity of radiolabeled substrates added to cultures. Each point represents average of results obtained from 2 separate cultures, and both values were within 10% agreement.

 
Effect of 14,15-DHET on PPAR{alpha} protein expression and binding of 14,15-DHET to PPAR{alpha} protein. The presence of intracellular 14,15-DHET suggested that it might have a direct effect on PPAR{alpha}. Therefore, we determined whether incubation with 14,15-DHET would alter the expression of the PPAR{alpha} protein. An incubation for 18 h with 10 µM 14,15-DHET did not induce PPAR{alpha} protein in the control cells (data not shown) or in transfected COS-7 cells. Also, neither 10 µM 14,15-EET nor 20 µM Wy-14643 increased the expression of PPAR{alpha} protein in the transfected COS-7 cells (Fig. 7).



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  		Fig. 7. Effect of 14,15-DHET on expression of PPAR{alpha} protein and binding of 14,15-DHET to PPAR{alpha} protein. COS-7 cultures (60% confluent) were incubated for 24 h at 37°C with or without 0.02 µg of cytomegalovirus immediated-early promoter/enhancer (pCMX)-mPPAR{alpha} expression vector. Cultures were then incubated for additional 18 h in 1 ml medium containing vehicle alone, 10 µM 14,15-EET or 14,15-DHET, or 20 µM Wy-14643. The PPAR{alpha} and {beta}-actin proteins in cell lysates were detected by Western blot analysis using specific antibody against mouse PPAR{alpha} or {beta}-actin. For binding assay, 2 µg gluthathione S-transferase (GST)-PPAR{alpha}-ligand binding domain (LBD) fusion protein was incubated with various concentrations of 14,15-[3H]DHET in total volume of 50 µl buffer in presence of 2% ethanol. Free and bound ligands were separated on Sephadex G-25 column, and amount of bound 14,15-[3H]DHET was determined by liquid scintillation counting. Binding assays were repeated in quadruple sets, and data are means ± SD. Scatchard analysis was performed by replotting data (inset). C, control; Wy, Wy-14643; CPM, counts/min.

 
To determine whether 14,15-DHET can bind to the LBD of PPAR{alpha}, the mPPAR{alpha}-LBD-GST fusion protein was incubated with various concentrations of 14,15-[3H]DHET. Binding occurred, and a Scatchard analysis of the data indicated that 14,15-DHET binds to the LBD of mPPAR{alpha}-LBD with a Kd of 1.4 µM (Fig. 7).

Effect of 14,15-DHET on expression of PPAR{alpha} response gene CPT1A in HepG2 cells. We determined whether activation of PPAR{alpha} by 14,15-DHET will upregulate CPT1A, a PPAR{alpha} responsive gene in HepG2 cells. 14,15-DHET (10 µM) caused a threefold increase in PPAR{alpha} activity, as measured by luciferase activity in transfected HepG2 cells, and Wy-14643 (10 µM) increased the PPAR{alpha} activity fourfold (Fig. 8A). A real-time PCR assay indicated that like Wy-14643, 14,15-DHET increased the level of CPT1A mRNA in the transfected HepG2 cells (Fig. 8B).



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  		Fig. 8. Activation of PPAR{alpha} by 14,15-DHET and effect on CPT1A in HepG2 cells. HepG2 cultures were transfected with {beta}-Gal, PPAR{alpha}, and luciferase reporter gene in 24-h incubation. Cultures were then incubated for 18 h with 1 ml control medium or media containing 10 µM 14,15-DHET or Wy-14643, and luciferase and {beta}-Gal activities in cell lysates were measured. Luciferase activity was normalized to {beta}-Gal activity. Results are expressed as fold activation relative to vehicle control (A). In a separate experiment under the same conditions, RNA was extracted from HepG2 cells and CPT1A mRNA was assayed by real-time RT-PCR. Data are means ± SE and are expressed as fold differences from control cells and have been normalized to expression of GAPDH (B). **P < 0.01 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that 1) among the EET and DHET regioisomers, 14,15-DHET is the most potent PPAR{alpha} activator; 2) the 14,15-EET analog 14-HTDE, which cannot be converted to a DHET, failed to activate PPAR{alpha}, whereas 14,15-GEET, which is hydrolyzed and then converted to 14,15-DHET, increased PPAR{alpha} activity; 3) SEH protein was detected in COS-7 cells, and the selective SEH inhibitor DCU blocked the ability of 14,15-EET to increase PPAR{alpha} activity; 4) 14,15-DHET was incorporated by the cells, and a small amount of the uptake remained as free 14,15-DHET; 5) 14,15-DHET binds to the LBD of mPPAR{alpha}-LBD; and 6) 14,15-DHET increased the expression of the PPAR{alpha}-responsive gene CPT1A in transfected HepG2 cells. These findings suggest that 14,15-DHET, which is produced from 14,15-EET by the action of SEH, may contribute to the regulation of PPAR{alpha} activity.

Many polyunsaturated fatty acids and their eicosanoids products are ligands for PPAR{alpha} (25, 31, 35, 50), and EETs have been shown previously to be weak ligands for PPAR{alpha} (9). Consistent with this report, we also found that EETs are weak activators of PPAR{alpha} in the COS-7 cell expression system. Each of the four EET regioisomers produced small increases in PPAR{alpha} activity, and the increases produced by 11,12-; 8,9-; and 5,6-DHET were similar to those produced by the corresponding EET regioisomers. However, we found that 14,15-DHET is a potent activator of PPAR{alpha} and that it increased PPAR{alpha}-mediated luciferase expression to the same extent as the widely used PPAR{alpha} agonist Wy-14643. The positional importance of the diol group in activating PPAR{alpha} is indicated by the fact that the other DHET regioisomers were much less effective than 14,15-DHET. Positional specificity is also important for HETE potency, because the lipoxygenase product 8(S)-HETE is a potent activator of PPAR{alpha} in a transfected human osteosarcoma cell line, whereas 5-, 12-, and 15-HETE are not active (34, 50).

Although nuclear receptors can be activated through indirect mechanisms (32), most fatty acids and eicosanoids activate PPAR{alpha} by directly binding to the receptor (1, 35, 50). The present results suggest that 14,15-DHET may also act through a ligand-dependent mechanism. As was observed previously with endothelial cells (48), the COS-7 cells took up a small amount of 14,15-DHET, and the fact that 20% of the uptake remained unesterified suggests that intracellular 14,15-DHET might be available to target the PPAR{alpha} protein. Furthermore, a binding assay demonstrated that 14,15-DHET binds to the LBD of mPPAR{alpha}-LBD with a Kd of 1.4 µM. However, the physiological relevance of this is uncertain because the Kd value is probably too high for an eicosanoid mediator. An alternative possibility is that PPAR{alpha} activation was initiated through an interaction of the high concentration of extracellular 14,15-DHET with a cell-surface receptor. In this regard, a 1 µM concentration of (20-OH)-14,15-EET was necessary to activate PPAR{alpha} in transfected RK13 cells, even though a parinaric acid displacement assay indicated that (20-OH)-14,15-EET binds to PPAR{alpha} with high affinity (Ki = 3 ± 1 nM) (9).

The physiological relevance of these findings is uncertain because the range of 14,15-DHET concentrations required to activate PPAR{alpha} in the intact COS-7 cells (3–10 µM) is higher than those likely to occur under ordinary conditions. However, platelets stimulated with thrombin or platelet-activating factor release unesterified EETs that can reach a concentration in the range of 1 µM (53), and this presumably would transiently generate a high extracellular concentration of DHET. In addition, the concentration of circulating DHETs is increased in pregnancy-induced hypertension (5), and an increased release of EETs and DHETs occurs when vascular endothelium is damaged by trauma (43). The aorta of cholesterol-fed rabbit also releases increased amounts of DHETs (39). Therefore, it is possible that DHET might transiently reach levels that are sufficient to activate PPAR{alpha} in some pathological conditions.

Because 14,15-EET is rapidly taken up by cells and is a weak ligand for PPAR{alpha} (9), the possibility that it also activates PPAR{alpha} through a binding mechanism cannot be excluded. However, the present findings suggest that the 14,15-EET-mediated PPAR{alpha} activation occurs as a result of its conversion to 14,15-DHET. Although most of the newly formed DHET was released into the medium, a small amount was retained in the cells. Thus the DHET produced from 14,15-EET could affect PPAR{alpha} through either a cell surface or intracellular mechanism. DCU, a selective inhibitor of SEH that substantially reduced the conversion of 14,15-EET to DHET, blocked the 14,15-EET-induced activation of PPAR{alpha}. This is consistent with a role for DHET in the activation process. Moreover, the 14,15-EET analog 14-HTDE, which is not a substrate for SEH and cannot be converted to 14,15-DHET, failed to activate PPAR{alpha}. In contrast, 14,15-GEET, the glycerol ester of 14,15-EET that was hydrolyzed and converted to DHET, activated PPAR{alpha}. Although these results do not exclude the possibility of a direct effect of 14,15-EET, they strongly suggest that 14,15-DHET formation plays a major role in 14,15-EET-mediated activation of PPAR{alpha}. It should be noted that even though 14,15-DHET is the major metabolite that accumulated in the extracellular fluid when the cells were incubated with 14,15-EET or 14,15-GEET, the absolute intracellular and extracellular concentrations of 14,15-DHET were lower than that when the cells were incubated directly with 14,15-DHET. This difference might explain why 14,15-DHET is more potent than 14,15-EET or 14,15-GEET in activation of PPAR{alpha}.

The CPT1A gene is a PPAR{alpha}-responsive gene in human HepG2 cells (14). We found that, similar to Wy-14643, 14,15-DHET activated PPAR{alpha} as measured by luciferase activity in transfected HepG2 cells, and it produced a twofold induction of CPT1A mRNA. These results indicate that 14,15-DHET increases the transcription of an endogenous downstream PPAR{alpha} target gene in a human cell.

The conversion of an EET to a DHET is generally considered to be an inactivation process (45). However, the present studies suggest that 14,15-DHET might be an endogenous metabolite that activates PPAR{alpha}. Consistent with this possibility, other biological activities have been observed with DHETs (15, 17, 29, 38, 49). The present findings suggest that 14,15-DHET may have additional vascular actions as a result of its effect on PPAR{alpha}. Activation of PPAR{alpha} decreases cholesterol esterification in macrophages and increases cholesterol removal from human macrophage foam cells (7, 41). It also inhibits vascular smooth muscle proliferation and has anti-inflammatory effects (10, 11, 27, 46). Therefore, 14,15-EET formation and subsequent conversion to 14,15-DHET by SEH could have beneficial effects on lipid metabolism, neointimal formation, and vascular inflammatory processes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The studies at the University of Iowa were supported in part by American Heart Association Scientist Development Grant 0230096N (to X. Fang); National Institutes of Health Grants HL-072845 (to A. A. Spector), HL-070860 and HL-076684 (to N. L. Weintraub), HL-062984 (to N. L. Weintraub and A. A. Spector), and DK-038226 and GM-31278 (to J. R. Falck); and the Robert A. Welch Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Joseph Dillon for helpful discussions; Dr. Ronald M. Evans (Salk Institute, Howard Hughes Medical Institute) for providing PPAR plasmids; Dr. Bruce D. Hammock (University of California, Davis) for providing the SEH inhibitor DCU and SEH antibody; and Dr. John Shyy (University of California, Riverside) for providing the GST-mPPAR{alpha}-LBD protein.


   FOOTNOTES
 

Address for reprint requests and other correspondence: X. Fang, Dept. of Biochemistry, Univ. of Iowa Carver College of Medicine, Iowa City, IA 52242 (e-mail: xiang-fang{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Detailed methods for the synthesis of 14-HTDE and 14,15-GEET may be found at http://ajpheart.physiology.org/cgi/data/00427.2005/DC1/1. Back


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