Cholesteryl ester hydrolase activity is abolished in HSL-/- macrophages but unchanged in macrophages lacking KIAA1363

Abstract

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363(-/-) and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL(-/-) mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363(-/-) but unchanged in HSL(-/-) mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL(-/-) macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Fig. 1.

Esterase activity of recombinant HSL and KIAA1363. (A) KIAA1363 and HSL protein expressions were determined by Western blotting using anti-KIAA1363 and anti-HSL antibodies. (B) COS-7 cell lysates transiently overexpressing HSL and KIAA1363 were assayed for PNPV activity. LacZ was used as negative control. Data are presented as mean values ± SEM of three independent experiments performed in triplicate. ^**P ≤ 0.01; ^***P ≤ 0.001. HSL, hormone-sensitive lipase; LacZ, β-galactosidase; PNPV, p-nitrophenolvalerate.

Fig. 2.

CE, TG, DG, and AcMAGE hydrolase activities of HSL and KIAA1363. COS-7 cells were transiently transfected with HSL, KIAA1363, or LacZ (negative control). A: CE hydrolase activities were determined in cell lysates of transfected cells by adding 35.5 µg mixed micelles of PC and PI (3:1, w:w) and 4 µM Na-taurocholate to the substrate. B: TG and (C) DG hydrolase activities were determined in cell lysates of transfected cells by adding 15 µg mixed micelles of PC and PI (3:1, w:w) and 4 µM Na-taurocholate to the respective substrates. D: CE hydrolase activities were determined in cell lysates of transfected cells by adding 35.5 µg mixed micelles of PC and PI (3:1, w:w) or 35.5 µg PC and 2 µM Na-taurocholate to the substrate. E: COS-7 cell lysates transiently overexpressing HSL and KIAA1363 were incubated with the fluorescent activity recognition probes NBD-sn1-TGP (lane 1), NBD-sn3-TGP (lane 2), and NBD-CP (lane 3) and separated by SDS-PAGE. The lack of HSL binding to NBD-sn3-TGP is consistent with previous results, demonstrating that most known lipases react poorly with that probe (32, 33). However, enantioselectivity of lipases directed to TG sn-1 or sn-3 acyl ester bonds is still under investigation as appropriately labeled substrates are needed for this purpose but are not yet commercially available. LacZ was used as negative control. Visualization was performed by laser scanning on a BioRad FY Pro laser scanner. F: AcMAGE hydrolase activities were determined in cell lysates of transfected cells. Data are presented as mean values ± SEM of four independent experiments performed in triplicate. ^***P ≤ 0.001. AcMAGE, 2-acetyl monoalkylglycerol ether; CE, cholesteryl ester; DG, diacylglycerol; HSL, hormone-sensitive lipase; LacZ, β-galactosidase; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NBD-sn1-TGP, enantiomeric TG analog; NBD-sn3-TGP, enantiomeric TG analog; PC, phosphatidylcholine; PI, phosphatidylinositol; TG, triacylglycerol.

Fig. 3.

CE and AcMAGE hydrolase activities in HSL^−/− and KIAA1363^−/− tissues. (A, B) Neutral CE and (C, D) AcMAGE hydrolase activities were determined in tissues isolated from HSL^−/−, KIAA1363^−/−, and control mice (WT). Data are presented as mean values ± SEM of three independent experiments (using three mice in each experiment). ^***P ≤ 0.001. AcMAGE, 2-acetyl monoalkylglycerol ether; BAT, brown adipose tissue; CE, cholesteryl ester; CM, cardiac muscle; HSL, hormone-sensitive lipase; SM, skeletal muscle; WAT, white adipose tissue; WT, wild-type.

Fig. 4.

TG, DG, and CE hydrolase activities in HSL^−/− and KIAA1363^−/− macrophages. Western blotting of macrophages from (A, F) HSL^−/−, KIAA1363^−/−, and control mice (WT), and of (E, J) cell lysate, cytosol, and membrane fractions from WT macrophages using (A, E) anti-HSL or (F,J ) anti-KIAA1363 antibodies. (B, G) TG, (C, H) DG, and (D, I) neutral CE hydrolase activities were determined in cell lysates, cytosolic, and membrane fractions of HSL^−/−, KIAA1363^−/−, and WT littermate macrophages by adding (B, C, G, H) 15 µg or (D, I) 35.5 µg mixed micelles of PC and PI (3:1, w:w) and 4 µM Na-taurocholate to the respective substrates. Data are presented as mean values of three independent experiments ± SEM (using three mice in each experiment). ^***P ≤ 0.001. CE, cholesteryl ester; DG, diacylglycerol; HSL, hormone-sensitive lipase; LacZ, β-galactosidase; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NBD-sn1-TGP, enantiomeric TG analog; NBD-sn3-TGP, enantiomeric TG analog; PC, phosphatidylcholine; PI, phosphatidylinositol; TG, triacylglycerol; WT, wild-type.

Fig. 5.

CE hydrolase activities in HSL^−/− and KIAA1363^−/− macrophages using alternative mixed micelles and AcMAGE hydrolase activities. (A, B) Neutral CE hydrolase activities were determined in cell lysates from (A) HSL^−/− and (B) KIAA1363^−/− macrophages after adding 35.5 µg mixed micelles of PC and PI (3:1, w:w) or 35.5 µg PC and 2 µM Na-taurocholate to the substrate. (C, D) AcMAGE hydrolase activities were determined in cell lysates, cytosolic, and membrane fractions of (C) HSL^−/− and (D) KIAA1363^−/− macrophages and compared with their WT littermates. Data are presented as mean values (n = 3) ± SEM. ^***P ≤ 0.001. AcMAGE, 2-acetyl monoalkylglycerol ether; CE, cholesteryl ester; HSL, hormone-sensitive lipase; PC, phosphatidylcholine; PI, phosphatidylinositol; WT, wild-type.

Fig. 6.

CE turnover in HSL^−/− and KIAA1363^−/− macrophages. MPM from HSL^−/− and KIAA1363^−/− mice and WT littermates were loaded with [³H]oleate for 24 h. After equilibration overnight, CE turnover was assayed in the presence of the ACAT inhibitor 58035 (5 µg/ml) at the indicated time points. Radioactivity in the cells before CE turnover was set at 100%. Data represent mean values (n = 8) ± SEM of two independent experiments. CE, cholesteryl ester; HSL, hormone-sensitive lipase; MPM, mouse peritoneal macrophage; WT, wild-type.

Fig. 7.

Cholesterol efflux from HSL^−/− and KIAA1363^−/− macrophages. Macrophages from HSL^−/− and KIAA1363^−/− mice and WT littermates were labeled with [³H]cholesterol and cholesterol efflux to (A, B, E) 100 µg HDL₃ and (C, D, F) 15 µg apoA1 was assessed in the (A-D) presence or (E, F) absence of 8-bromo cAMP. Cholesterol efflux was expressed as the percentage (%) of [³H]cholesterol transferred from cells to medium. Data are presented as mean values ± SEM of two independent experiments of 6–10 mice per genotype performed in triplicate. (G, I) ABCA1 and (H, J) ABCG1 mRNA levels in macrophages and foam cells of (G, H) HSL^−/− and (I, J) KIAA1363^−/− mice and WT littermates normalized to cyclophilin A. Expression levels in WT macrophages were arbitrarily set to 1. Western blotting of macrophages and foam cells incubated in the absence or presence of cAMP from HSL^−/−, KIAA1363^−/−, and WT mice using (K) anti-ABCA1 and (L) anti-ABCG1 antibodies. Anti-β-actin (42 kDa) was used as a control. ^*P < 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001. ABC, ATP-binding cassette; apo, apolipoprotein; HSL, hormone-sensitive lipase; MPM, mouse peritoneal macrophage; WT, wild-type.

Fig. 8.

Foam cell formation of HSL^−/− and KIAA1363^−/− macrophages. Representative fluorescent microscopy after Nile red staining of HSL^−/−, KIAA1363^−/−, and WT macrophages and foam cells. Foam cell formation was achieved by loading macrophages with 100 µg acLDL for 48 h and 72 h. Images were taken on a Zeiss LSM 510 Meta microscope. HSL, hormone-sensitive lipase; MPM, mouse peritoneal macrophage; WT, wild-type.