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Generation and characterization of epoxide hydrolase 3 ( EPHX3 )-deficient mice

View Article: PubMed Central - PubMed

ABSTRACT

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo.

No MeSH data available.


Disruption of Ephx3 does not alter mRNA levels of CYP epoxygenases.Quantitative RT-PCR of Cyp2c and Cyp2j isoforms in (A) lung, (B) skin, (C) stomach, (D) heart, (E) esophagus, (F) tongue, and (G) liver of Ephx3-/- and WT mice. (*p<0.05; n = 3–5, p = NS).
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pone.0175348.g003: Disruption of Ephx3 does not alter mRNA levels of CYP epoxygenases.Quantitative RT-PCR of Cyp2c and Cyp2j isoforms in (A) lung, (B) skin, (C) stomach, (D) heart, (E) esophagus, (F) tongue, and (G) liver of Ephx3-/- and WT mice. (*p<0.05; n = 3–5, p = NS).

Mentions: Mice with global disruption of the Ephx3 gene were generated using standard Cre-LoxP methodology which targeted the proximal promoter and first four exons of the gene (Fig 1A–1E). The first 4 exons contain the previously identified hydrolase domain, and encode 63% of the protein (270 of 425 AA). Proper targeting was confirmed by Southern blot analysis (Fig 1F). While there are no immunospecific antibodies to EPHX3, qPCR results showed that Ephx3 mRNA levels were low/undetectable in various tissues from the Ephx3-/- mice (Fig 2A). To determine if there were compensatory changes in other known epoxide hydrolases in Ephx3-/- mice, qPCR analysis was also performed using specific primer/probe sets for Ephx1, Ephx2, and Ephx4. Levels of the other epoxide hydrolases were similar in Ephx3-/- mice relative to WT controls (Fig 2B–2D). Cardiac Ephx1 expression was significantly lower in Ephx3-/- hearts relative to WT; however, Ephx1 expression in the heart is relatively low in WT mice and the 20% decrease in Ephx3-/- mice is unlikely to be physiologically relevant. We also examined expression of Cyp2j and Cyp2c genes in Ephx3-/- and WT tissues by qPCR. Expression levels of these CYP epoxygenases were largely unaffected by Ephx3 genetic disruption in the tissues analyzed (Fig 3A–3G).


Generation and characterization of epoxide hydrolase 3 ( EPHX3 )-deficient mice
Disruption of Ephx3 does not alter mRNA levels of CYP epoxygenases.Quantitative RT-PCR of Cyp2c and Cyp2j isoforms in (A) lung, (B) skin, (C) stomach, (D) heart, (E) esophagus, (F) tongue, and (G) liver of Ephx3-/- and WT mice. (*p<0.05; n = 3–5, p = NS).
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pone.0175348.g003: Disruption of Ephx3 does not alter mRNA levels of CYP epoxygenases.Quantitative RT-PCR of Cyp2c and Cyp2j isoforms in (A) lung, (B) skin, (C) stomach, (D) heart, (E) esophagus, (F) tongue, and (G) liver of Ephx3-/- and WT mice. (*p<0.05; n = 3–5, p = NS).
Mentions: Mice with global disruption of the Ephx3 gene were generated using standard Cre-LoxP methodology which targeted the proximal promoter and first four exons of the gene (Fig 1A–1E). The first 4 exons contain the previously identified hydrolase domain, and encode 63% of the protein (270 of 425 AA). Proper targeting was confirmed by Southern blot analysis (Fig 1F). While there are no immunospecific antibodies to EPHX3, qPCR results showed that Ephx3 mRNA levels were low/undetectable in various tissues from the Ephx3-/- mice (Fig 2A). To determine if there were compensatory changes in other known epoxide hydrolases in Ephx3-/- mice, qPCR analysis was also performed using specific primer/probe sets for Ephx1, Ephx2, and Ephx4. Levels of the other epoxide hydrolases were similar in Ephx3-/- mice relative to WT controls (Fig 2B–2D). Cardiac Ephx1 expression was significantly lower in Ephx3-/- hearts relative to WT; however, Ephx1 expression in the heart is relatively low in WT mice and the 20% decrease in Ephx3-/- mice is unlikely to be physiologically relevant. We also examined expression of Cyp2j and Cyp2c genes in Ephx3-/- and WT tissues by qPCR. Expression levels of these CYP epoxygenases were largely unaffected by Ephx3 genetic disruption in the tissues analyzed (Fig 3A–3G).

View Article: PubMed Central - PubMed

ABSTRACT

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified &gt;30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo.

No MeSH data available.