Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

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 V(max) 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.


Introduction
Epoxide hydrolases (EC 3.3.2.7-11) catalyse the hydrolysis of oxiranes to the corresponding vicinal diols. To date a number of mammalian epoxide hydrolases are characterized that play diverse roles in the organism (1).
The metabolism of these lipid epoxides by sEH to the corresponding diols is generally considered a deactivation process. For this reason the sEH is a promising target for the treatment of hypertension, inflammatory diseases, pain, diabetes and stroke (16,(21)(22)(23)(24)(25). A number of sEH inhibitors (sEHI) have been developed (26,27) for therapeutic applications.
Yet, the sEH also serves some function in xenobiotic metabolism by accepting certain transsubstituted epoxides, which are very poor substrates for microsomal epoxide hydrolase (mEH) (28,29).
Other epoxide hydrolases with rather narrow substrate selectivity have been identified in mammals. Of those, Hepoxilin A 3 epoxide hydrolase (Hepoxilin hydrolase, EC 3.3.2.7) was at SMAC Consortium -University of Zürich, on March 10, 2014 www.jlr.org Downloaded from partly purified from rat liver cytosol and identified as the main hydrolase of the endogenous lipid hepoxilin A 3 . The authors further discriminated hepoxilin hydrolase from other EHs due to its size (53 kDa) and substrates preference for hepoxilin A 3 , compared to leukotriene or styrene oxide (30). 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 A 3 and B 3 (HxA 3 and HxB 3 ) are formed from arachidonic acid through the 12-lipoxygenase pathway (figure 1) in various organs like liver, brain, lung, pancreas and skin (9,31). They can be transformed to the corresponding trihydroxy metabolites (trioxilins, Trx) or glutathione conjugates (32).
Early studies showed an hepoxilin mediated increased insulin release from pancreatic islets (33). In the brain hepoxilins modulate synaptic neurotransmission and neuronal excitabilitymostly through stimulation of intracellular calcium release or increased calcium influx into the cell (34)(35)(36)(37). Hepoxilin are considered pro-inflammatory because increased hepoxilins and trioxilin levels have been found in psoriatic lesions (38) and hepoxilin A 3 was shown to regulate neutrophil migration in response to inflammation (39, 40). Several reports suggest an involvement of these lipid mediators in epidermal differentiation and skin barrier function (41). Interestingly, mutations in the hepoxilin generating 12R-LOX pathway in the skin are associated with a congenital form of ichthyosis (42-46). A hepoxilin receptor has not been identified, but several reports (34,(47)(48)(49)) support its existence.
Here, we report that 12S-LOX derived hepoxilin A 3 and B 3 (HxA 3 and HxB 3 ), are efficiently converted to the corresponding trihydroxy metabolites (trioxilins) by soluble epoxide hydrolases (sEH). 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.
Lipid substrates were purchased from Biomol except for TxA 3 and TxB 3 , which were synthesized biochemically using purified soluble epoxide hydrolase. One µg HxA 3 or HxB 3 were turned over to the corresponding diol using 200 ng soluble epoxide hydrolase 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.  5. In addition, ACU inhibited hepoxilin metabolism by purified rat sEH and liver cytosolic preparations with an IC 50 value of approximately 1 nM (data not shown), which is in line with previously reported data (52). In microsomal preparations of sEH WT mice hepoxilin turnover amounted to 30% compared to 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 microsomal epoxide hydrolase which is highly abundant in the liver does not show any activity against hepoxilins (data not shown).
sEH is responsible for hepoxilin metabolism. To investigate the quantitative contribution of sEH to hepoxilin turnover we incubated protein extracts isolated from livers of sEH WT and sEH -/mice with HxA 3 and HxB 3 . Hepoxilin turnover to trioxilins was greatly abolished in sEH -/mice compared to the WT mice (figure 6). Specifically, in both cytosolic and microsomal liver preparations of sEH -/mice, the activity towards HxA 3 and HxB 3 was greatly reduced compared to the WT mice. Again, the activity towards hepoxilins in liver microsomal preparations of WT animals is explained by the presence of some sEH, while no sEH protein was detectable in the sEH -/mice by immunoblotting (data not shown).

Discussion
Here we report for the first time that trans-hydroxy-epoxy lipids, in particular the endogenous 12S-LOX derived lipid mediators HxA 3 and HxB 3 are excellent substrates for mammalian sEH and converted to the corresponding trioxilins. 12R-LOX derived hepoxilins which are specifically generated in skin are most likely preferred sEH substrates, although they have not been tested to date. sEH metabolises hepoxilins with a catalytic efficiency which is within the range of turnover of its previously identified physiological substrates epoxyeicosatrienoic acids (EETs) which are among the best endogenous substrates for sEH.
The activity of mammalian sEH against EETs lies in the range of 1-20 µmol/mg/min. We do not see a negative cooperativity with both hepoxilins, like it has been suggested for the EET turnover by sEH (50). Human sEH turns over hepoxilins less efficiently (by a factor of three) than rat sEH. This has been seen for other substrates and might be explained by a compensatory effect due to the lower expression level of sEH in rat liver compared to human liver. HxA 3 is a better substrate for mammalian sEH than HxB 3 . The hydroxy group positioned directly next to the epoxide might pose a sterical hindrance leading to less efficient turnover by sEH.
The sEH turns over hepoxilins orders of magnitudes more efficiently than the previously reported hepoxilin hydrolase that displayed a specific activity of 0.2 nmol/mg/min. Hepoxilin hydrolase was partly purified from rat liver and suggested to be distinct from other mammalian EHs (mEH, sEH, Leukotriene A4 Hydrolase) by its molecular weight as well as substrate and inhibitor spectrum. However, hepoxilin hydrolase is still only incompletely characterized and the amino acid sequence is not reported to date. The purification scheme used for the isolation of hepoxilin hydrolase (30) is quiet similar to the procedure used for the isolation of rat liver sEH (53). We assume that the hepoxilin hydrolase activity in the enzyme preparation published previously is due to an invisible contamination by sEH. The assignment at SMAC Consortium -University of Zürich, on March 10, 2014 www.jlr.org Downloaded from of the enzymatic activity to an incorrect polypeptide might be due to the obviously low abundance of sEH in livers of untreated rats, which would also explain the striking activity difference between the two enzymes.
These results suggested a physiological role of sEH in hepoxilin metabolism. Analysis of mouse liver cytosol by gel permeation chromatography followed by activity measurements against HxA3, 14,15-EET (an excellent sEH substrate) and 5,6-EET (a rather poor sEH substrate) and western blot revealed that the hepoxilin hydrolase activity is linked to sEH presence, showing a perfect match. The double peak in the activity profile can be explained by the presence of monomeric and dimeric sEH in liver cytosol. These results suggested that mammalian sEH -rather than hepoxilin hydrolase -is the key enzyme responsible for hepoxilin metabolism in mouse liver.
To strengthen our hypothesis we analysed liver extracts from sEH WT and sEH -/mice.
Hepoxilin turnover was greatly abolished compared to the WT animals. The activity against hepoxilins found in the liver microsomal preparation of WT animals can be explained by the presence of sEH due to its partial peroxisomal localisation in liver (54,55), which we confirmed by western blot analysis.
Only sEHIs but not mEHIs quantitatively inhibited hepoxilin turnover in cytosolic as well as microsomal liver preparations of WT animals. mEH shows a substrate preference for bulky, cis-substituted epoxides compared to the sEH, which accepts both cisand transsubstituted epoxides. Indeed, we have shown that purified microsomal epoxide hydrolase does not turnover hepoxilins. In addition, ACU inhibited hepoxilin metabolism by purified rat sEH as well as liver cytosolic preparations with an IC 50 value of approximately 1 nM. These acid (AA) presumably leads to a strong production of hepoxilin precursors such as 12HPETE.
In this case, both HxA 3 and HxB 3 significantly accumulate in the livers of sEH -/animals and only a slow turnover to trioxilins is detected ( figure 7b). An AA pre-treatment better reflects the actual enzyme capacity of the organ analysed, while in the basal state compensatory mechanisms of lipid metabolism might be of importance.
Quit unexpected were the large amounts of particularly HxB 3 in the livers of sEH -/mice while HxA 3 did not accumulated to that extent. HxA 3 has been shown to be a substrate for glutathione-S-transferases, and the glutathione conjugated metabolite maintains biologic activity (32,37). Due to the high expression level of GSTs in the liver, one would expect a lack of hepoxilin accumulation, which is only seen for the HxA 3 regioisomer (figure 7).
Therefore glutathione conjugation of HxB 3 does not seem to be an important pathway in the liver. Note that the glutathione derivative of HxB 3 has not been detected in vivo to date. In contrast, HxA 3 seem to be preferentially glutathionylated in livers of sEH -/animals, which might also be the case in other organs, when the epoxide hydrolysis pathway is blocked.
Taken together our results strongly suggest that mammalian sEH is the key enzyme responsible for hepoxilin metabolism and indeed identical to previously reported hepoxilin hydrolase. Other mammalian epoxide hydrolase contribute -if at all -only partly to this metabolic pathway, depending on the tissue analysed.
Our inhibitory analyses using sEHI clearly show a complete block of hepoxilin hydrolysis in the liver. Therefore possible undesirable effects of sEH inhibitors, which are in development for a number of applications, should be considered. Lipid signalling pathways other then the mostly targeted EET pathways might be affected, with -to our knowledgeunknown consequences. This is even more important as EETs and hepoxilins seem to have somewhat opposing effects. While the action of EETs are generally considered antiinflammatory (15)(16)(17), hepoxilins instead are suspected to have pro-inflammatory effects. In psoriatic lesions elevated levels of hepoxilins and trioxilins have been detected (38). In conclusion, hepoxilins are excellent substrates for mammalian sEH in vitro and in vivo.
Our findings suggest that sEH is identical to liver hepoxilin hydrolase and plays an important role in the physiological regulation of hepoxilins, with important implications in particular for inflammatory diseases.