Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages

Abstract

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

FIGURE 1.

Structure of NCEH. Sequence alignment of murine NCEH with human, rat, and mouse HSL. The deduced amino acid sequence of murine NCEH protein (NCEH) is aligned and compared with murine, rat, and human HSL (murHSL, ratHSL, and humHSL). The first Met residue is numbered as 1. HSL has four functional domains as follows: 1) the N-terminal 300 residues domain, containing the ALBP-binding site (boldface); 2) catalytic core domain (line), containing the HG dipeptide oxyanion motif (shaded box) and GXSXG active serine motif (shaded box); 3) regulatory module domain, containing five serine residues, Ser-557, Ser-559, Ser-591, Ser-650, and Ser-65, which can be phosphorylated by cAMP-dependent protein kinase (solid circle) or other kinases such as AMP-activated protein kinase or extracellular signal-regulated kinase (ERK) (open circle); 4) lipid-binding domain (dashed line), containing a putative lipid-binding site and two of the catalytic triad, i.e. Asp-694 and His-724 (shaded box). The predicted secondary structure was shown as follows: α-helix is indicated by a line; β-sheet is indicated by an arrow; secondary structure of HSL and NCEH is shown in gray and black, respectively. The mature NCEH protein is highly conserved during evolution and has homology to HSL in these motifs and secondary structures, except for the regulatory domain and the N-terminal 300 residue domain. NCEH has an N-terminal transmembrane region (outlined letters). Identical amino acids are shown by asterisks. Amino acids considered as strongly conserved are as follows: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW, and are indicated by colons. Amino acids considered as weakly conserved are as follows: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY, and are indicated by dots.

FIGURE 2.

Tissue and subcellular distribution of NCEH expression. Total RNA (10 μg) from adipose tissues and 3T3-L1 adipocytes at various stages of differentiation and murine peritoneal macrophages (A), various murine tissues (B), and human monocytes/monocyte-derived macrophages (C) were subjected to Northern blot analysis. Specific mRNAs were detected with radiolabeled murine cDNAs for NCEH, HSL, and TGH-1. Ethidium bromide staining of the gels is shown. Abbreviations used are as follows: WAT, white adipose tissue; BAT, brown adipose tissue; MPM, murine peritoneal macrophages; ed, epididymal; sc, subcutaneous. Subcellular distribution of nCEH activity and NCEH and HSL proteins. MPM were sonicated and centrifuged at 100,000 × g and the supernatant (S-100) or microsomal (Ms) fractions along with whole cell lysate (whole) were subjected to the measurements of nCEH activity (D) and Western blot analysis (E) using anti-HSL and anti-NCEH antisera. F, quantification of NCEH or HSL proteins in macrophages. Ten micrograms of proteins of whole cell lysates from RAW264.7 and MPM were separated by SDS-PAGE on the same gel as the indicated amounts of GST fusion proteins (supplemental Fig. S2). Immunoblotting was performed, and the densities of bands from HSL or NCEH of RAW264.7 or MPM were quantified using NIH Image. Blots for ACAT1 and F4/80 are shown as controls. The moles of HSL or NCEH in 10 μg of protein from RAW264.7 or MPM were calculated from equations relating moles and densities of GST fusion proteins.

FIGURE 3.

Enzymological characteristics of NCEH. HEK293 cells were infected with Ad-LacZ, Ad-HSL, or Ad-NCEH and used for the experiments. A, expression of NCEH or HSL was confirmed by Western blot analysis. Effects of various concentrations of NaCl on the nCEH activity of whole cell lysate from cells infected with Ad-NCEH (B) or of MPM (C). D, effects of various pH on the nCEH activity of whole cell lysate from cells infected with Ad-NCEH using 50 mm acetate buffer (pH 5), 50 mm phosphate buffer (5 < pH < 7.6), or 50 mm Tris-HCl buffer (pH 7.6). Shown are the effects of various concentrations of cholesterol oleate (E) or triolein (F) on the nCEH or triglyceride lipase activities, respectively, of whole cell lysate from cells infected with Ad-NCEH or Ad-HSL. Values for Ad-LacZ were subtracted from those for Ad-NCEH or Ad-HSL and plotted against the concentrations of substrates. K_m values were calculated by fitting lines in Lineweaver-Burk plots.

FIGURE 4.

Effects of RNA interference of NCEH to inhibit protein expression and nCEH activity in murine peritoneal macrophages. Recombinant adenoviruses coding for shRNA against LacZ (Ad-shLacZ) or NCEH (Ad-shNCEH) were used to infect murine peritoneal macrophages at 300 and 600 m.o.i. Two days after infection, whole cell lysates were subjected to Western blotting (A) and the measurement of nCEH activity (B). This is a representative result of two independent experiments.

FIGURE 5.

Effects of overexpression of NCEH on cholesterol ester accumulation or cholesterol ester formation in THP-1 macrophages. Recombinant adenoviruses coding for LacZ (Ad-LacZ), HSL (Ad-HSL), or NCEH (Ad-NCEH) were used to infect THP-1 macrophages, and experiments were performed 3 days after infection. The cells were sonicated and used for Western blot analysis (A) and measurement of nCEH activity (B). Twenty four h after THP-1 macrophages were infected with recombinant adenovirus carrying NCEH, HSL, or LacZ as a control, the cells were incubated with 100 μg/ml acLDL. On day 3 after infection, intracellular content of CE (C) and CE formation from [¹⁴C]oleate (D) were measured. Data are presented as means ± S.E. of four measurements (C and D) (^*, p < 0.001, Ad-HSL versus Ad-LacZ, or Ad-NCEH versus Ad-LacZ).

FIGURE 6.

Expression of NCEH in murine peritoneal macrophages and in foamy macrophages in atherosclerotic plaques. Expression of NCEH protein in MPM (A and B) and the aorta of apoE^-/- mice (C and D) was localized by immunohistochemistry using affinity-purified anti-NCEH rabbit IgG (visualized in brown) (B and D) or by combined immunohistochemical staining for NCEH (brown) and F4/80 (in blue, for macrophages) (E). Peroxidase activity was visualized using 3,3′-diaminobenzidine (brown) or Vector blue (blue), and sections were counterstained with 3% methyl green. As a control experiment, staining of MPM (A) and the aorta (C) with preimmune rabbit IgG as a primary antibody did not produce any horseradish peroxidase cross-reactivity. Objective magnifications are ×400 (A and B) and ×100 (C–E).