The phosphatase activity of soluble epoxide hydrolase regulates ATP‐binding cassette transporter‐A1‐dependent cholesterol efflux

Abstract The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E‐deficient mice, primarily in macrophage foam cells. Oxidized low‐density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH −/−) in macrophages markedly exacerbated oxLDL‐induced lipid accumulation and decreased the expression of ATP‐binding cassette transporters‐A1 (ABCA1) and apolipoprotein AI‐dependent cholesterol efflux following oxLDL treatment. The down‐regulation of ABCA1 in sEH −/− macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH‐ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL‐induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL‐induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

causes an increase in the accumulation of EETs and leads to the attenuation of angiotensin II-induced hypertension and cardiac hypertrophy and lipopolysaccharide-induced inflammation in vitro and in vivo. [5][6][7][8] Oral administration with inhibitors targeting EH activity of sEH or genetic disruption of sEH significantly retards the progression of atherosclerosis in hyperlipidemic mouse models. 9,10 Multiple lines of evidence demonstrate that the PT domain of sEH also has a crucial role in the regulation of cholesterol metabolism and cell growth in hepatocytes. 11,12 Moreover, the sEH Glu287Arg mutant, which has reduced PT activity, is known to be a risk factor for the development of coronary artery disease (CAD). 13,14 Additionally, this sEH polymorphism is closely associated with the increase in plasma cholesterol and triglyceride in familial hypercholesterolaemia patients. 15 However, the role of the PT activity of sEH in the cholesterol metabolism of macrophage foam cells remains to be investigated.
Atherosclerosis is a chronic inflammatory process, caused by the deregulation of lipid metabolism of macrophages within arterial walls, ultimately leading to the clinical complications of CADs in humans. 16 Although the detailed mechanisms of this disease are not yet fully defined, it has been believed that regulation of cholesterol metabolism and the inflammatory response by lipidladen macrophages are critical steps in the initiation and progression of atherosclerosis. 17,18 Several lines of evidence suggest that inhibition of foam cell formation retards the progression of atherosclerosis in experimental animal models. [19][20][21] The formation of foam cells is primarily caused by uncontrolled uptake of oxidized low-density lipoprotein (oxLDL) or impaired cholesterol efflux in macrophages, which result in excessive oxLDL-derived lipid accumulation inside macrophages. 19,22 Scavenger receptors (SRs) class A SR (SR-A) and CD36 are responsible for internalization of oxLDL. 23,24 By contrast, the efflux of accumulated cholesterol in macrophages is mediated through reverse cholesterol transporters (RCTs) including SR-BI and the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1). 25,26 Therefore, the lipid content of foam cells is dynamically regulated by these SRs and cholesterol efflux transporters. However, less is known about the interplay between sEH and foam cells. To this end, further investigation delineating the expression and the mechanisms of sEH on the formation of foam cells is warranted.
Given the importance of sEH in the development of CADs, in this study we investigated the role and the mechanism of sEH in regulating the cholesterol metabolism of macrophage foam cells.
We first investigated the expression and distribution of sEH in atherosclerotic lesions of apolipoprotein E null (Apoe −/− ) mice.
Our second aim was to characterize the role of sEH in oxLDL-deregulated cholesterol metabolism and the underlying molecular mechanism in bone marrow-derived macrophage (BMDM) foam cells by use of loss-of-function and gain-of-function strategies.
Here, we report that the PT activity, but not the EH activity, of sEH plays a crucial role in regulating ABCA1-dependent cholesterol efflux during the transformation of macrophage foam cells.
Our findings provide a novel explanation for the anti-atherogenic properties of sEH and suggest a molecular target for the treatment of atherosclerosis.

| Animals
All animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University. C57BL/6 mice were purchased from the National Laboratory Animal Center, National Science Council, and Apoe −/− mice and EPXH2 −/− sEH-deficient (sEH KO) mice were obtained from the Jackson Laboratory.
Mice were housed in barrier facilities on a 12-hour light/12-hour dark cycle and fed with regular chow (4.5% fat by weight, 0.02% cholesterol; Newco Distributors).

| Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue blocks were cut into 8 µM sections. Sections were deparaffinized, rehydrated and covered with 3% H 2 O 2 for 10 minutes. After blocking with BSA, slides were incubated with primary antibodies for 1 hour at 37°C and with corresponding secondary antibodies for an additional 1 hour. Antigenic sites were visualized by the addition of DAB. Slides were counterstained with haematoxylin.

| Cell culture
Bone marrow-derived macrophages were prepared as previously described. 27 Briefly, mice were killed by CO 2 inhalation and mononuclear cells from their femurs were harvested by Percoll (1.073 g/cm 3 ) density-gradient centrifugation. The cells were then seeded in MEMα supplemented with 50 ng/mL macrophage colony-stimulating factor, 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) for differentiation for 5 days at 37°C. The differentiated macrophages were then subjected to further experiments. HEK293 cells and Huh7 hepatoma cells were cultured in DMEM supplemented with 10% FBS and penicillin (100 U/mL)/streptomycin (100 µg/mL) at 37°C.

| Low-density lipoprotein modification
The oxLDL was prepared as described previously. 28 LDL was exposed to 5 µM CuSO 4 for 24 hours at 37°C, and Cu 2+ was then removed by extensive dialysis. The extent of modification was determined by measuring thiobarbituric acid-reactive substances (TBARs).
OxLDL containing approximately 30-60 nmol of TBARs, defined as malondialdehyde equivalents per mg of LDL protein, was used for experiments.

| PT activity assay
Bone marrow-derived macrophages with or without indicated treatments were collected in phosphate-buffered saline (PBS), sonicated, and the supernatant was collected by centrifugation at 10 000 g for 10 minutes. This cell lysate was added to 4-nitrophenyl phosphate to 2 mM and incubated at 37°C for 1 hour. The yellow colour product was detected by OD 405 nm to determine PT activity.

| Oil red O staining
Cells were fixed with 4% paraformaldehyde and then stained by 0.5% oil red O. Haematoxylin was used as a counterstain. The intracellular lipid content was evaluated by alcohol extraction after oil red O staining. The absorbance at 540 nm was measured using a microplate reader (BioTek Instruments).

| Cholesterol and triglyceride measurement
Cellular cholesterol and triglyceride were extracted by hexane/isopropanol (3/2, v/v). After removing cellular debris, the supernatant was dried under nitrogen. The levels of cholesterol and triglyceride were measured using cholesterol and triglyceride assay kits (Randox).
Eluted protein samples were separated by 8% or 10% SDS-PAGE.
After transfer to membranes, the samples were incubated with primary antibodies, washed and then incubated with secondary antibodies conjugated with horseradish peroxidase. Bands were revealed using an enzyme-linked chemiluminescence detection kit (PerkinElmer), and signals were quantified using Imagequant 5.2 software (Healthcare Bio-Sciences).

| Cholesterol efflux assay
Macrophages were equilibrated with NBD cholesterol (1 µg/mL) for 12 hours. These cells were washed with PBS and incubated with oxLDL (50 µg/mL) in RPMI 1640 medium for another 12 hours in the presence of apoAI (10 µg/mL) or HDL (50 µg/mL). The fluorescence-labelled cholesterol released from the cells into the medium was analysed using a multilabel counter (PerkinElmer) with 485 nm excitation and 535 nm emission.

| Statistical analysis
Data are presented as mean ± SEM from 5 mice or 5 independent cell experiments. Data from animal studies were evaluated by parametric tests. One-way ANOVA followed by the LSD test was used for multiple comparisons. Data from cell studies were evaluated by non-parametric tests. The Mann-Whitney U test was used to compare 2 independent groups. The Kruskal-Wallis followed by Bonferroni post hoc tests was used to account for multiple comparisons. SPSS v 20.0 (SPSS Inc) was used for analysis. Differences were considered statistically significant at P < 0.05. of Apoe −/− mice relative to those of control WT mice ( Figure 1A).

| Expression of sEH is increased in atherosclerotic lesions of Apoe −/− mice
In addition, sEH was predominantly expressed in macrophage foam cells as revealed by immunohistochemistry ( Figure 1B).

| Genetic deletion of sEH amplifies oxLDLinduced lipid accumulation in macrophages
We found that oxLDL, the most important atherogenic factor, up-regulated the expression of sEH protein (Figure 2A). Thus, we have been suggested that sEH is involved in the regulation of lipid metabolism of macrophage foam cells. As shown in Figure 2B,C, oxLDL-induced lipid accumulation was significantly higher in EPXH2 −/− sEH-deficient macrophages than in WT cells, as evidenced by an increase in intracellular levels of cholesterol and triglycerides.

| Deficiency of sEH impairs cholesterol efflux by down-regulating ABCA1 expression following oxLDL challenge
The

| Deficiency of sEH promotes the turnover of ABCA1 protein
We further delineated the molecular mechanisms underlying the effect of sEH deficiency on the down-regulation of ABCA1 by measuring the gene expression and protein stability of ABCA1 in the presence of oxLDL. Our results showed that loss of function of sEH did not affect oxLDL-induced ABCA1 gene expression ( Figure 4A); however, the degradation rate of ABCA1 protein was promoted by oxLDL treatment (Figure 4B). These results suggest that sEH may play a key role in the stability of ABCA1 protein in response to oxLDL challenge.

| PT activity of sEH regulates ABCA1 stability and cholesterol metabolism in oxLDL-treated macrophages
We next investigated whether the PT activity or the EH activity of sEH participates in regulating expression of ABCA1 upon oxLDL treatment. Bone marrow-derived macrophages were pre-treated with AUDA (an inhibitor of EH activity of sEH) or ebselen (an inhibitor of sEH PT activity). As shown in Figure 5A-D, treatment with ebselen attenuated the oxLDL-induced increase in the protein expression of ABCA1, apoAI-dependent cholesterol efflux and cholesterol accumulation in macrophages. However, AUDA treatment failed to produce such effects, suggesting that the PT activity of sEH is crucial in regulating oxLDL-induced up-regulation of ABCA1.

| sEH interacts with ABCA1 and decreases ABCA1 phosphorylation
Previous studies have reported the phosphorylation status of ABCA1 protein is crucial for its stability. [30][31][32] We thus determined whether sEH regulates the phosphorylation and turnover of ABCA1 protein in macrophages. We demonstrated that oxLDL time-dependently increased the activity of phosphatase ( Figure 6A).
Immunoprecipitation assays revealed that oxLDL increased the interaction of sEH and ABCA1 in a time-dependent manner, peaking at 3 hours and gradually decreasing to basal levels 18 hours after treatment ( Figure 6B). In parallel, oxLDL increased ABCA1 phosphorylation at threonine residues, with maximal effect at 3 hours and decreased thereafter ( Figure 6B).
To address whether hydrolase activity or PT activity of sEH is involved in the changes of ABCA1 phosphorylation caused by oxLDL challenge, we amplified the EH activity or PT activity of sEH by overexpressing the PT or EH domains of sEH using an adenovirus system. As shown in Figure 6C,

| D ISCUSS I ON
In this study, we identified a novel role of PT activity of sEH in cholesterol homeostasis during the transformation of macrophage foam cells. We first determined that sEH was significantly increased in atherosclerotic aortas and in particular, in intralesional macrophage foam cells. In addition, we found that oxLDL, the most critical ath- F I G U R E 4 Genetic disruption of sEH promotes the degradation of ABCA1 protein in oxLDL-treated macrophages. A, Total RNA was isolated at 6 h after oxLDL incubation and ABCA1 and GAPDH mRNA was determined by semi-quantitative RT-PCR analysis. B, Macrophages were incubated in the presence of 2 µg/mL cycloheximide (CHX) to inhibit protein translation with oxLDL (50 µg/mL) for the time periods indicated. Data shown are mean ± SD from 5 independent experiments. *P < 0.05 vs oxLDL-treated WT macrophages of ABCA1 is one of the factors critical for its protein stability. [30][31][32] Indeed, our co-IP assays revealed that treatment with oxLDL promoted the interaction between sEH and ABCA1, and regulated the  In conclusion, we provide new evidence for PT activity of sEH in the regulation of cholesterol metabolism in macrophage foam cells.
The PT activity of sEH reduces oxLDL-induced ABCA1 phosphorylation and stabilizes ABCA1, which resulted in increased cholesterol efflux and decreased lipid accumulation in macrophage foam cells.
Activation of the PT of sEH may be a pharmacological target for atherosclerosis and related cardiovascular diseases.

ACK N OWLED G EM ENTS
The authors thank Dr Li-Chieh Ching and Dr Chien-Yu Chen for their helpful technical assistance. This study was supported by grants from F I G U R E 7 Schematic illustration of proposed mechanism underlying the sEH PT activity-mediated regulation of ABCA1 expression and cholesterol metabolism in macrophages. As shown, challenge with oxLDL increases sEH PT activity and promotes the formation of sEH-ABCA1 complex, which in turn decreases ABCA1 phosphorylation at threonine (Thr) residues, leading to the inhibition of ABCA1 degradation and consequently increases cholesterol efflux and decreases lipid accumulation in macrophages