Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase
2011; Elsevier BV; Volume: 52; Issue: 4 Linguagem: Inglês
10.1194/jlr.m009639
ISSN1539-7262
AutoresAnnette Cronin, Martina Decker, Michael Arand,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoHepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A3 and B3 with a Vmax of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N'-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH−/− mice; and iv) liver homogenates of sEH−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling. Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A3 and B3 with a Vmax of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N'-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH−/− mice; and iv) liver homogenates of sEH−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling. Epoxide hydrolases (EC 3.3.2.7-11) catalyze the hydrolysis of oxiranes to the corresponding vicinal diols. To date, a number of mammalian epoxide hydrolases have been characterized that play diverse roles in the organism (1Decker M. Arand M. Cronin A. Mammalian epoxide hydrolases in xenobiotic metabolism and signalling.Arch. Toxicol. 2009; 83: 297-318Crossref PubMed Scopus (157) Google Scholar). The soluble epoxide hydrolase (sEH, EC 3.1.3.76; EC 3.3.2.10) is a bifunctional homodimeric enzyme composed of an epoxide hydrolase (EH) and a lipid phosphatase in each of its subunits (2Cronin A. Homburg S. Durk H. Richter I. Adamska M. Frere F. Arand M. Insights into the catalytic mechanism of human sEH phosphatase by site-directed mutagenesis and LC-MS/MS analysis.J. Mol. Biol. 2008; 383: 627-640Crossref PubMed Scopus (21) Google Scholar, 3Cronin A. Mowbray S. Durk H. Homburg S. Fleming I. Fisslthaler B. Oesch F. Arand M. 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The authors further discriminated hepoxilin hydrolase from other EHs due to its size (53 kDa) and substrates preference for hepoxilin A3 compared with leukotriene or styrene oxide (30Pace-Asciak C.R. Lee W.S. Purification of hepoxilin epoxide hydrolase from rat liver.J. Biol. Chem. 1989; 264: 9310-9313Abstract Full Text PDF PubMed Google Scholar). To date, the enzyme is only incompletely characterized and no structural or sequence information is available. Most enzymatic-derived endogenous lipid epoxides are of cis-configuration, but also some trans-substitutes lipid epoxides are formed within the organism, such as the inflammatory mediator leukotriene A4. The trans-epoxy hydroxy eicosanoids hepoxilin A3 and B3 (HxA3 and HxB3) are formed from arachidonic acid through the 12-lipoxygenase (LOX) pathway (Fig. 1) in various organs like liver, brain, lung, pancreas, and skin (9Newman J.W. Morisseau C. Hammock B.D. Epoxide hydrolases: their roles and interactions with lipid metabolism.Prog. 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Here, we report that 12S-LOX-derived HxA3 and HxB3 are efficiently converted to the corresponding trioxilins by sEHs. Our results suggest a biological relevance of sEH, rather than hepoxilin hydrolase, in hepoxilin metabolism, which opens a new branch in the numerous physiological functions of sEH. Human full length sEH containing an N-terminal Strep-Tag and rat sEH containing an N-terminal His-Tag were cloned, recombinantly expressed in Escherichia coli, and purified as described previously (3Cronin A. Mowbray S. Durk H. Homburg S. Fleming I. Fisslthaler B. Oesch F. Arand M. The N-terminal domain of mammalian soluble epoxide hydrolase is a phosphatase.Proc. Natl. Acad. Sci. USA. 2003; 100: 1552-1557Crossref PubMed Scopus (111) Google Scholar). Epoxide hydrolase activity was measured using 8(R,S)-Hydroxy-11S,12S-epoxyeicosa-5Z,9E,14Z-trienoic acid (HxA3) and 10(R,S)-Hydroxy-11S,12S-epoxyeicosa-5Z,8Z,14Z-trienoic acid (HxB3) as substrates by determining the formation of the corresponding diols 8(R,S)-Hydroxy-11,12-dihydroxyeicosa-5Z,9E,14Z-trienoic acid (TrxA3) and 10(R,S)-Hydroxy-11,12-dihydroxyeicosa-5Z,8Z,14Z-trienoic acid (TrxB3). Typically, 5–50 ng purified sEH or 10–100 µg organ extracts were incubated with various concentrations of HxA3 and HxB3 ranging from 0.1 µM to 30 µM with or without inhibitor in 50 mM Tris HCl, 50 mM NaCl, 2% glycerol, pH 7.4 in a final volume of 50 µl for 10 min at 37°C. The reaction was stopped by addition of 2 vols of methanol and samples were pelleted for 5 min at 13,000 rpm prior to LC-MS/MS analysis. Substrate turnover was determined using internal HxA3 and HxB3 standards. EH activity against EETs was performed as described previously (50Marowsky A. Burgener J. Falck J.R. Fritschy J.M. Arand M. Distribution of soluble and microsomal epoxide hydrolase in the mouse brain and its contribution to cerebral epoxyeicosatrienoic acid metabolism.Neuroscience. 2009; 163: 646-661Crossref PubMed Scopus (100) Google Scholar). Kinetic constants were calculated by kinetic modeling based on the equation V = E × CS/(CS+Km) (with V = % turnover of Vmax, E = total amount of enzyme, CS = substrate concentration, and Km = Michaelis Menten constant). Variations were calculated from four to five independent experiments using freshly prepared enzyme preparations. Lipid substrates were purchased from Biomol except for TrxA3 and TrxB3, which were synthesized biochemically using purified sEH. One microgram of HxA3 or HxB3 was turned over to the corresponding diol using 200 ng sEH in 50 mM Tris HCl, 50 mM NaCl, 2% glycerol, pH 7.4 in a final volume of 500 µl for 30 min at 37°C. The reaction products were extracted three times with 2 vols of ethyl acetate, dried under a stream of nitrogen, and reconstituted in methanol. Separation of analytes was performed on an Agilent eclipse XDB-C18 reverse phase column (4.6 × 150 mm, 5 μm pore size) with a corresponding opti-gard precolumn using an Agilent 1100 liquid chromatography system. The mobile phase consisted of (A) 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid at a flow rate of 400 μl/min using an injection volume of 20 μl. Starting conditions of 70% buffer B were maintained for 2 min followed by a linear gradient from 70 to 100% B within 7 min. An isocratic flow of 100% B was held for 1.5 min and finally the column was re-equilibrated for 2 min with 70% B. The HPLC system was coupled to a 4000 QTRAP hybrid quadrupole linear ion trap mass spectrometer (Applied Biosystems) equipped with a TurboV source and electrospray (ESI) interface. Analytes were recorded using multiple reaction monitoring in negative mode (−MRM) using the following source specific parameters: IS −4500V, TEM 450°C, curtain gas (CUR = 30), nebulizer gas (GS1 = 50), heater gas (GS2 = 70) and collision gas (CAD = 10). The compound specific parameters for the different substrates (as specified in supplemental material) were determined by direct infusion of standard solutions (100–300 ng/ml) in methanol at a flow rate of 10µl/min using the quantitative optimization function of the Analyst software 1.4.2. Samples were quantified by determining the peak area under the curve (AUC) with the quantification function of the Analyst software 1.4.2 using the transitions as specified in the supplementary online material. The background noise was assessed by analyzing blank samples and standard curves were generated using blank samples spiked with a series of control lipids ranging from 1 to 1,000 ng/ml. For HxA3, HxB3, TrxA3, and TrxB3, the limit of detection ranged from 4 to 20 pg and the limit of quantification from 15 to 65 pg, corresponding to a signal-to-noise ratio of 3 and 10, respectively. C57BL/6 mice were obtained from the Institute of Laboratory Animal Sciences, University of Zurich and C57BL/6 sEH−/− mice (51Sinal C.J. Miyata M. Tohkin M. Nagata K. Bend J.R. Gonzalez F.J. Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation.J. Biol. Chem. 2000; 275: 40504-40510Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) were kindly provided by Dr. F. J. Gonzales (National Institutes of Health, Bethesda, MD). Six- to ten-week-old male mice were euthanized by cervical dislocation and livers were excised and homogenized in ice-cold phosphate buffered saline, pH 7.4. All subsequent steps were performed at 4°C. Cytosolic and microsomal fractions were prepared by ultracentrifugation of the 9,000 × g supernatant of the liver homogenates at 100,000 × g for 45 min. Lipids were extracted from liver homogenates by addition of ethanol to a final concentration of 25% followed by solid phase extraction on C18 Bond Elut SPE columns (Varian, Palo Alto, CA). Extracts were applied to the SPE columns preconditioned with 2 ml acetonitrile and 2 ml ddH2O. Columns were washed with 1 ml ddH2O and evaporated to dryness. Lipids were eluted with 3 × 600 μl ethylacetate, dried under a stream of nitrogen, dissolved in acetonitrile, and pelleted for 2 min at 13,000 rpm prior to LC-MS/MS analysis as described above. In some cases, liver homogenates were treated with 30 μM arachidonic acid at 37°C for 30 min and lipids were isolated by solid phase extraction as described above. Protein samples in Laemmli buffer were subjected to SDS-PAGE and semi-dry blotting following standard procedures. Blots were incubated with polyclonal sEH rabbit antiserum for 2 h (dilution of 1:1000) in Tris-buffered saline containing 0.5% Tween-20. An alkaline phosphatase conjugated anti-rabbit antibody (Sigma Aldrich) was applied at a dilution of 1:30000 followed by colorimetric detection using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium chloride (NBT). Human and rat sEH were cloned, recombinantly expressed in E. coli, and purified to homogeneity using affinity chromatography as described previously (3Cronin A. Mowbray S. Durk H. Homburg S. Fleming I. Fisslthaler B. Oesch F. Arand M. The N-terminal domain of mammalian soluble epoxide hydrolase is a phosphatase.Proc. Natl. Acad. Sci. USA. 2003; 100: 1552-1557Crossref PubMed Scopus (111) Google Scholar). To determine the effect of purified recombinant rat or human sEH on hepoxilin metabolism, we used LC-MS/MS analysis followed by kinetic evaluation. Human or rat sEH was incubated with various concentrations of HxA3 and HxB3 with or without inhibitor and samples were analyzed by LC-MS/MS. HxA3 was efficiently hydrolyzed by purified rat soluble epoxide hydrolase with a Vmax of 1.7 µmol/mg/min, a Km of 5 µM, and a catalytic efficiency of 4.5 × 105 as shown in Fig. 2. Both HxA3 and HxB3 are substrates for purified rat sEH and also human sEH (Fig. 2) and a summary of the kinetic parameters is presented in Table 1.TABLE 1Summary of kinetic parameters for hepoxilin turnover by sEHsEH SubstrateVmaxKmkcatkcat/Kmnmol/mg/minµMs−1Ms−1rsEH HxA31739 ± 5394.6 ± 2.31.88 ± 0.584.5 x 10 ± 1.6 × 10rsEH HxB3550 ± 26114.7 ± 5.30.60 ± 0.285.1 x 10 ± 1.4 × 10hsEH HxA3385 ± 957.3 ± 3.30.42 ± 0.105.8 x 10 ± 9.5 × 10hsEH HxB395 ± 4310.8 ± 3.40.10 ± 0.051.2 x 10 ± 8.4 × 10Kinetic constants were calculated by simulation of the Michaelis Menten kinetic as described in the Methods section. Variations were calculated from four to five independent experiments. Open table in a new tab Kinetic constants were calculated by simulation of the Michaelis Menten kinetic as described in the Methods section. Variations were calculated from four to five independent experiments. To evaluate the physiological contribution of sEH to hepoxilin metabolism, we separated mouse liver cytosolic fractions using gel permeation chromatography and tested each elution fraction for the metabolism of HxA3, 14,15-EET, and 5,6-EET. Each fraction was further assayed for the presence of sEH by Western blot analysis (Fig. 3). The hepoxilin hydrolase and sEH activities show 100% coelution from the column. Moreover, the fraction with the highest hepoxilin hydrolase activity also contains the highest amount of sEH (Fig. 3, lower panel). To characterize the physiological contribution of sEH to hepoxilin metabolism, we used a selection of epoxide hydrolase inhibitors. The hepoxilin turnover by purified sEH was effectively inhibited by sEHIs as shown in Fig. 4. Hepoxilin metabolism in liver protein extracts from wild-type (WT) animals was inhibited by ACU and AUDA but not the mEH inhibitor valpromide, as shown in Fig. 5. In addition, ACU inhibited hepoxilin metabolism by purified rat sEH and liver cytosolic preparations with an IC50 value of ∼1 nM (data not shown), which is in line with previously reported data (52Hwang S.H. Tsai H.J. Liu J.Y. Morisseau C. Hammock B.D. Orally bioavailable potent soluble epoxide hydrolase inhibitors.J. Med. Chem. 2007; 50: 3825-3840Crossref PubMed Scopus (207) Google Scholar). In microsomal preparations of sEH, WT mice hepoxilin turnover amounted to 30% compared with the cytosolic fraction. Western blot analysis of microsomal and cytosolic liver preparations confirmed the presence of sEH protein in both liver fractions, although to significantly lower amount in microsomes (data not shown). Furthermore, purified mEH, which is highly abundant in the liver, does not show any activity against hepoxilins (data not shown).Fig. 5Inhibition of hepoxilin metabolism. Protein extracts from liver (cytosolic and microsomal preparations) of WT mice were incubated with 3 µM HxA3 and HxB3 in the presence of inhibitors (2 µM ACU, AUDA, 2 mM valpromide), and samples were analyzed by LC-MS/MS. The representations show the fraction (%) of substrate turnover compared with the cytosolic preparation of WT animals, which is adjusted to 100% turnover. Error bars indicate SD. Unpaired, one-sided Student's t-tests were performed on each set of inhibited versus noninhibited samples. Two stars indicate a significant statistical difference with a p-value < 0.01.View Large Image Figure ViewerDownload Hi-res image
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