Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages
2014; Elsevier BV; Volume: 55; Issue: 10 Linguagem: Inglês
10.1194/jlr.m050864
ISSN1539-7262
AutoresMotohiro Sekiya, Daisuke Yamamuro, Taichi Ohshiro, Akira Honda, Manabu Takahashi, Masayoshi Kumagai, K Sakai, Shuichi Nagashima, Hiroshi Tomoda, Masaki Igarashi, Hiroaki Okazaki, Hiroaki Yagyu, Jun-ichi Osuga, Shun Ishibashi,
Tópico(s)Atherosclerosis and Cardiovascular Diseases
ResumoAn excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis. An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis. Atherosclerotic cardiovascular diseases are the leading cause of mortality in industrialized countries, despite advances in the management of coronary risk factors. Heart attacks arise from the thrombotic occlusion of coronary arteries following the rupture of plaques. Characteristic of these rupture-prone plaques is their lipid-rich nature due to the presence of cholesteryl ester (CE)-laden macrophage foam cells (1WeberC.ZerneckeA.LibbyP.. 2008. The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nat. Rev. Immunol., 8: 802–815.Google Scholar). 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Herein, we demonstrate that Nceh1-deficient macrophages are highly susceptible to apoptosis induced by 25-HC, and the underlying mechanism may involve the activation of ER stress signaling due to accumulation of 25-HC ester in the ER. 27-Hydroxycholesterol (27-HC) was purchased from Research Plus (Bayonne, NJ); all other oxysterols and MG-132 were purchased from Sigma (St. Louis, MO). Ca2+-free DMEM medium was purchased from Gibco (Carlsbad, CA). K-604 (24Ikenoya M. Yoshinaka Y. Kobayashi H. Kawamine K. Shibuya K. Sato F. Sawanobori K. Watanabe T. Miyazaki A. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels.Atherosclerosis. 2007; 191: 290-297Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and CS-505 (25Takahashi K. Kasai M. Ohta M. Shoji Y. Kunishiro K. Kanda M. Kurahashi K. Shirahase H. 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Mice used in this study were crossed onto the C57BL/6J background for more than five generations. All experimental procedures and handling of animals were conducted according to our institutional guidelines. Thioglycollate-elicited peritoneal macrophages (TGEMs) were obtained from 8-week-old mice as described (9Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. et al.Ablation of neutral cholesterol ester hydrolase 1 accelerates atherosclerosis.Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and incubated in DMEM containing 10% FCS or 10% LPDS. Cells were fixed in modified Karnovsky's phosphate-buffered (0.1 M) glutaraldehyde (2.5%)-paraformaldehyde (4%) mixture at room temperature for 12 h, postfixed for 2 h in 2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated in ethanol and propylene oxide, and embedded in Epon. The sections were cut and then counterstained with uranyl acetate and lead citrate for transmission electron microscopy (TEM). DNA (0.5 μg), which was extracted from the cells, was end-labeled with [α-32P]dCTP by Klenow and subjected to electrophoresis in a 1.5% agarose gel and then transferred to nylon membranes, as described previously (30Sekiya M. Osuga J. Okazaki H. Yahagi N. Harada K. Shen W.J. Tamura Y. Tomita S. Iizuka Y. Ohashi K. et al.Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice.J. Biol. Chem. 2004; 279: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Mouse thymocyte apoptotic DNA was used as a control (31Harada K. Ishibashi S. Miyashita T. Osuga J. Yagyu H. Ohashi K. Yazaki Y. Yamada N. Bcl-2 protein inhibits oxysterol-induced apoptosis through suppressing CPP32-mediated pathway.FEBS Lett. 1997; 411: 63-66Crossref PubMed Scopus (49) Google Scholar). 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Major A.S. Swift L.L. Gleaves L.A. Accad M. Linton M.F. Farese Jr, R.V. Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages.J. Clin. Invest. 2001; 107: 163-171Crossref PubMed Scopus (216) Google Scholar). Lipid was extracted from the cytosolic (100 μg of protein) and microsomal fraction (50 μg of protein), and was separated by TLC with toluene-ethyl acetate (67:33) as the solvent. Visualization was done with 10% sulfuric acid. Concentrations of oxysterols in subcellular fractions were measured using LC-MS/MS as described (32Honda A. Yamashita K. Hara T. Ikegami T. Miyazaki T. Shirai M. Xu G. Numazawa M. Matsuzaki Y. Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS.J. Lipid Res. 2009; 50: 350-357Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). After the addition of deuterated internal standards and butylated hydroxytoluene, each fraction was either hydrolyzed with 1 N ethanolic KOH and derivatized into picolinyl esters, or directly converted into picolinyl esters. Northern blot analyses were performed as described (9Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. et al.Ablation of neutral cholesterol ester hydrolase 1 accelerates atherosclerosis.Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Total RNA was reverse transcribed and amplified using a sense primer (5′-AAACAGAGTAGCAGCGCAGACTGC-3′) and an antisense primer (5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′). This fragment was further digested by PstΙ as described previously (33Hosogai N. Fukuhara A. Oshima K. Miyata Y. Tanaka S. Segawa K. Furukawa S. Tochino Y. Komuro R. Matsuda M. 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Other primer sequences were as follows: immunoglobulin heavy chain-binding protein (Bip) (sense 5′-TCATCGGACGCACTTGGAA-3′, antisense 5′-CAACCACCTTGAATGGCAAGA-3′), CCAAT/enhancer-binding protein-homologous protein (Chop) (sense 5′-GTCCCTAGCTTGGCTGACAGA-3′, antisense 5′-TGGAGAGCGAGGGCTTTG-3′), Nceh1 (sense 5′-AGCCTGCAGTTTGAGCTTA-3′, antisense 5′-AGAGTCGGTATTTCTGGAGACG-3′), Acat1 (sense 5′-GGAAGTTGGGTGCCACTTCG-3′, antisense 5′-GGTGCTCTCAGATCTTTGG-3′), Rplp0 (sense 5′-GAAGACAGGGCGACCTGGAA-3′, antisense 5′-TTGTGGCTCCCACAATGAAGC-3′), and Atcb (sense 5′-CGATGCCCTGAGGCTCTTT-3′, antisense 5′-TGGATGCCACAGGATTCCA-3′). TGEMs were homogenized in buffer A [50 mM Tris-HCl, 250 mM sucrose, 1 mM EDTA, 2 μg/ml leupeptin (pH 7.0)]. Ten micrograms of proteins of whole lysates were separated by SDS-PAGE on the NuPAGE 10% Bis-Tris gel and transferred to a nitrocellulose membrane. For detection of the proteins, the membranes were incubated with each anti-murine Akt (Abcam) or anti-murine GAPDH at a dilution of 1:1,000 in Hikari A solution (Nacalai Tesque). Specifically bound immunoglobulins were detected in a second reaction with a horseradish peroxidase-labeled IgG conjugate and visualized by ECL detection (GE Healthcare) with Image Quant LAS 4000 Mini (GE Healthcare). Statistical differences between groups were analyzed by one-way ANOVA and the post hoc Tukey-Kramer test or two-tailed Student's t-test, unless otherwise stated. While attempting to examine the intracellular structures of the Nceh1−/− TGEMs using TEM, we noticed a small number of apoptotic cells featuring condensed nuclei in Nceh1−/− TGEMs (Fig. 1A). Interestingly, while cellular cholesterol loading with acLDL did not alter the frequency of apoptotic nuclei (Fig. 1B), treatment of Nceh1-deficient TGEMs with 25-HC (Fig. 1C, D; Fig. 2A), 7-KC (Fig. 2A), and 27-HC (data not shown) augmented apoptosis. The Nceh1-dependent augmentation of apoptosis was not observed for other compounds including lipopolysaccharide, tunicamycin, staurosporin, 5α6α-epoxycholesterol, 5β6β-epoxycholesterol, 7β-hydroxycholesterol, and 24-hydroxycholesterol (data not shown). The effects were far more pronounced for 25-HC than for 7-HC and 27-HC, and detectable even at a concentration of 0.1 μg/ml (0.26 μM) (Fig. 1D), which is close to a physiological concentration of oxysterols (0.01–0.1 μM in plasma) (34Schroepfer Jr, G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar).Fig. 2Inhibition of ACAT1 suppresses the augmentation of 25-HC-induced apoptosis in Nceh1-deficient TGEMs. Four wells of TGEMs were incubated in DMEM containing 10% LPDS with vehicle or 25-HC (10 μg/ml) in the presence or absence of the ACAT inhibitors: K-604, PPPA, and CS-505 (10 μM) for 24 h. A: The apoptotic cells were detected by TUNEL. B: Expression of Chop was measured by RT-PCR. Data are expressed as the mean ± SEM. **P < 0.01; NS, a nonsignificant difference.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The augmentation of the 25-HC-induced apoptosis, which was clearly observed in Nceh1−/− TGEMs, was not detectable in nonelicited macrophages; treatment with 25-HC significantly increased the number of apoptotic cells as well as the expression of Chop, even in nonelicited WT macrophages. Although the nonelicited Nceh1−/− cells showed slightly higher expression of Chop than the nonelicited WT cells (supplementary Fig. IA), there was no significant difference in the number of apoptotic cells between the two types of cells after treatment with 25-HC (supplementary Fig. IB). These results indicate that nonelicited macrophages are more susceptible to 25-HC-induced ER stress and apoptosis than elicited TGEMs. The susceptibility may be conferred by both the lower expression of Nceh1 and higher expression of Acat1 (supplementary Fig. IC, D). Membrane-bound enzyme ACAT, which is responsible for the intracellular esterification of cholesterol, is known to be strongly activated by oxysterols (35Chang T.Y. Chang C.C. Ohgami N. Yamauchi Y. Cholesterol sensing, trafficking, and esterification.Annu. Rev. Cell Dev. Biol. 2006; 22: 129-157Crossref PubMed Scopus (441) Google Scholar). Inasmuch as we have reported that Nceh1 catalyzes the intracellular hydrolysis of CE (2Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we speculated that Nceh1 also catalyzes the intracellular hydrolysis of esterified oxysterols and that cycles of esterification-hydrolysis could play a pivotal role in the underlying molecular processes. Indeed, Nceh1 hydrolyzed 25-HC oleate in vitro (supplementary Fig. II). Km of Nceh1 for 25-HC oleate was comparable to that for cholesteryl oleate: 6.4 ± 0.9 μM versus 6.9 ± 2.3 μM. As expected, nonselective ACAT inhibitor, CS-505, significantly inhibited the Nceh1-dependent augmentation of enhanced 25-HC-induced apoptosis (Fig. 2A). There are two ACAT isozymes: ACAT1 and ACAT2. To determine which isozyme mediated the 25-HC-induced apoptosis, we further compared the effects of ACAT1-specific inhibitor, K-604, and ACAT2-specific inhibitor, PPPA. As expected, the augmentation of the 25-HC-induced apoptosis was specifically inhibited by K-604, but not by PPPA, corroborating the fact that ACAT1 is the major isozyme of TGEMs (36Sakashita N. Miyazaki A. Chang C.C. Chang T.Y. Kiyota E. Satoh M. Komohara Y. Morganelli P.M. Horiuchi S. Takeya M. Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) is induced in monocyte-derived macrophages: in vivo and in vitro studies.Lab. Invest. 2003; 83: 1569-1581Crossref PubMed Scopus (44) Google Scholar). A similar phenomenon was also observed for 7-KC. Because inhibition of ACAT successfully reduced the augmentation of the 25-HC-induced apoptosis in Nceh1−/− TGEMs, we initially focused on the intracellular esterified oxysterol content to explore the underlying mechanism. Intracellular free 25-HC was already detectable after 1 h of incubation, and thereafter the free 25-HC content appeared to remain relatively constant (Fig. 3A). A small amount of 25-HC ester was detected after 1 h of incubation mainly in the microsomal fraction of Nceh1−/− TGEMs, and thereafter the intracellular 25-HC ester content increased gradually in Nceh1−/− TGEMs. 25-HC ester was not detectable in cells coincubated with CS-505. The isolated microsomal fraction contains the ER, where Nceh1 resides, and changes in ER lipid composition are known to initiate ER stress (13Seimon T. Tabas I. Mechanisms and consequences of macrophage apoptosis in atherosclerosis.J. Lipid Res. 2009; 50: S382-S387Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 37Ron D. Walter P. Signal integration in the endoplasmic reticulum unfolded protein response.Nat. Rev. Mol. Cell Biol. 2007; 8: 519-529Crossref PubMed Scopus (4865) Google Scholar). The prolonged activation of ER stress signaling is also known to lead to apoptotic cell death (38Kim I. Xu W. Reed J.C. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities.Nat. Rev. Drug Discov. 2008; 7: 1013-1030Crossref PubMed Scopus (1481) Google Scholar). Therefore, we examined whether ER stress mediates the 25-HC-induced apoptosis in Nceh1−/− TGEMs. Indeed, the accumulation of 25-HC ester preceded increased expression of ER stress markers such as Chop, Bip, and the spliced form of X-box-binding protein 1 (Xbp-1) (Fig. 3B). Oxysterols inhibit the processing of sterol regulatory element binding protein (Srebp)-2 by binding to an anchor protein called insulin induced genes (Insigs), and block cholesterol biosynthesis (39Radhakrishnan A. Ikeda Y. Kwon H.J. Brown M.S. Goldstein J.L. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.Proc. Natl. Acad. Sci. USA. 2007; 104: 6
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