Effects of human soluble epoxide hydrolase polymorphisms on isoprenoid phosphate hydrolysis

Soluble epoxide hydrolase (sEH) is highly expressed in human liver and contains a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Endogenous C-terminal hydrolase substrates include arachidonic acid epoxides, however, data are limited regarding possible endogenous substrates for the N-terminal phosphatase. Possible sEH N-terminal substrates include isoprenoid phosphate precursors of cholesterol biosynthesis and protein isoprenylation. Here, we report the kinetic analysis for a range of sEH isoprenoid substrates. We also provide an analysis of the effects of human sEH polymorphisms on isoprenoid hydrolysis. Interestingly, the Arg287Gln polymorphism recently suggested to be involved in hypercholesterolemia was found to possess a higher isoprenoid phosphatase activity than the wild type sEH. Consistent with the finding of isoprenoid phosphates as substrates for sEH, we identified isoprenoid-derived N-terminal inhibitors with IC50 values ranging from 0.84 (+/-0.9) to 55.1 (+/-30.7) microM. Finally, we evaluated the effects of the different isoprenoid compounds on the C-terminal hydrolase activity.     

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.     

Lipid sulfates and sulfonates are allosteric competitive inhibitors of the N-terminal phosphatase activity of the mammalian soluble epoxide hydrolase

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with K(I) in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH.     

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.     

可溶性表氧化物水解酶与脂代谢

可溶性表氧化物水解酶(soluble epoxide hydrolase,sEH)在哺乳动物体内广泛存在。研究发现,sEH可以降解表氧二十碳三烯酸(epoxyeicosatrienoic acids,EETs)以及其他脂肪酸表氧化物。EETs具有广泛的心血管系统保护及抗炎作用,故sEH因其与心血管疾病的关系而受到关注。新近研究显示,sEH与脂质代谢密切相关,并与其C端水解酶区域和N端磷酸酶区域的不同活性有关。进一步深入探讨sEH的作用机制,将为研究脂质调节紊乱相关的代谢疾病提供一个新的治疗靶点。本文对sEH两端酶活性,及其与脂质代谢调节的研究进展予以综述。     

Distribution of soluble epoxide hydrolase, cytochrome P450 2C8, 2C9 and 2J2 in human malignant neoplasms

Summary Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Arachidonic acid epoxides, previously suggested to be involved in apoptosis, oncogenesis and cell proliferation, are generated by cytochrome P450 epoxygenases and are good substrates of the sEH C-terminal domain. In addition, the N-terminal phosphatase domain hydrolyzes isoprenoid mono- and pyrophosphates, which are involved in cell signaling and apoptosis. Here we provide a comprehensive analysis of the distribution of sEH, CYP2C8, 2C9 and 2J2 in human neoplastic tissues using tissue micro-arrays. The human neoplastic tissue micro-arrays provide a well-controlled side by side analysis of a wide array of neoplastic tissues and their surrounding normal tissue controls. Many of the neoplastic tissues showed altered expression of these enzymes as compared to normal tissues. Altered expression was not limited to the neoplastic tissues but also found in the surrounding non-neoplastic tissues. For example, sEH expression in renal and hepatic malignant neoplasms and surrounding non-neoplastic tissues was found to be significantly decreased, whereas expression was found to be increased in seminoma as compared to normal tissues. Our study warrants further investigation of the role of altered expression of these enzymes in neoplastic tissues.     

Soluble epoxide hydrolase homologs in Strongylocentrotus purpuratus suggest a gene duplication event and subsequent divergence

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Genetic disruption of soluble epoxide hydrolase is protective against streptozotocin-induced diabetic nephropathy

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important roles in regulating cardiovascular functions. The anti-inflammatory, antiapoptotic, proangiogenic, and antihypertensive properties of EETs suggest a beneficial role for EETs in diabetic nephropathy. Endogenous EET levels are maintained by a balance between synthesis by CYP epoxygenases and hydrolysis by epoxide hydrolases into physiologically less active dihydroxyeicosatrienoic acids. Genetic disruption of soluble epoxide hydrolase (sEH/EPHX2) results in increased EET levels through decreased hydrolysis. This study investigated the effects of sEH gene disruption on diabetic nephropathy in streptozotocin-induced diabetic mice. Streptozotocin-induced diabetic manifestations were attenuated in sEH-deficient mice relative to wild-type controls, with significantly decreased levels of Hb A(1c), creatinine, and blood urea nitrogen and urinary microalbumin excretion. The sEH-deficient diabetic mice also had decreased renal tubular apoptosis that coincided with increased levels of antiapoptotic Bcl-2 and Bcl-xl, and decreased levels of the proapoptotic Bax. These effects were associated with activation of the PI3K-Akt-NOS3 and AMPK signaling cascades. sEH gene inhibition and exogenous EETs significantly protected HK-2 cells from TNFα-induced apoptosis in vitro. These findings highlight the beneficial role of the CYP epoxygenase-EETs-sEH system in the pathogenesis of diabetic nephropathy and suggest that the sEH inhibitors available may be potential therapeutic agents for this condition.     

Role of soluble epoxide hydrolase phosphatase activity in the metabolism of lysophosphatidic acids

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of epoxides from arachidonic acid and other unsaturated fatty acids, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, inflammation and pain. While recent findings suggested complementary biological roles for Nterm-phos, its mode of action is not well understood. Herein, we demonstrate that lysophosphatidic acids are excellent substrates for Nterm-phos. We also showed that sEH phosphatase activity represents a significant (20-60%) part of LPA cellular hydrolysis, especially in the cytosol. This possible role of sEH on LPA hydrolysis could explain some of the biology previously associated with the Nterm-phos. These findings also underline possible cellular mechanisms by which both activities of sEH (EH and phosphatase) may have complementary or opposite roles.     

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice

Aim

20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI).

Methods

Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry.

Results

Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice.

Conclusion

These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.     

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.     

Soluble Epoxide Hydrolase as an Anti-inflammatory Target of the Thrombolytic Stroke Drug SMTP-7

Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy.     

Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis

The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Dürk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction.     

Inhibition of soluble epoxide hydrolase attenuates high-fat-diet-induced hepatic steatosis by reduced systemic inflammatory status in mice

Non-alcoholic fatty liver disease is associated with obesity and considered an inflammatory disease. Soluble epoxide hydrolase (sEH) is a major enzyme hydrolyzing epoxyeicosatrienoic acids and attenuates their cardiovascular protective and anti-inflammatory effects. We examined whether sEH inhibition can protect against high-fat (HF)-diet-induced fatty liver in mice and the underlying mechanism. Compared with wild-type littermates, sEH-null mice showed lower diet-induced lipid accumulation in liver, as seen by Oil-red O staining and triglycerides levels. We studied the effect of sEH inhibition on diet-induced fatty liver by feeding C57BL/6 mice an HF diet for 8 weeks (short-term) or 16 weeks (long-term) and administering t-AUCB, a selective sEH inhibitor. sEH inhibition had no effect on the HF-diet-increased body and adipose tissue weight or impaired glucose tolerance but alleviated the diet-induced hepatic steatosis. Adenovirus-mediated overexpression of sEH in liver increased the level of triglycerides in liver and the hepatic inflammatory response. Surprisingly, the induced expression of sEH in liver occurred only with the long-term but not short-term HF diet, which suggests a secondary effect of HF diet on regulating sEH expression. Furthermore, sEH inhibition attenuated the HF-diet-induced increase in plasma levels of proinflammatory cytokines and their mRNA upregulation in adipose tissue, which was accompanied by increased macrophage infiltration. Therefore, sEH inhibition could alleviate HF-diet-induced hepatic steatosis, which might involve its anti-inflammatory effect in adipose tissue and direct inhibition in liver. sEH may be a therapeutic target for HF-diet-induced hepatic steatosis in inhibiting systemic inflammation.     

Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants

Endogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal lie of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent protein soluble epoxide hydrolase-SKI was strictly cytosolic, indicating that -SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase-SKI into peroxisomes in plant cells revealed that the context of -SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C-terminal -SKI is a non-functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

Mechanism of Protection by Soluble Epoxide Hydrolase Inhibition in Type 2 Diabetic Stroke

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice.