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LXRα pathway inhibits cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage
Biochemical and Biophysical Research Communications 514 (2019) 1093e1100
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p62/mTOR/LXRa pathway inhibits cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage Xiaofei Liang, Chao Wang, Yan Sun, Wei Song, Jing Lin, Jiashan Li, Xiuru Guan* First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China
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Article history: Received 29 March 2019 Accepted 18 April 2019 Available online 14 May 2019
Objective: Atherosclerosis is a disease characterized by abnormal lipid metabolism, and the formation of foam cells is considered an early event of atherosclerosis. Intracellular cholesterol efflux mediated by ABCA1 and ABCG1 helps to reduce lipid accumulation in foam cells. Related studies have shown that autophagy and mTOR are involved in cholesterol efflux, but the role of p62, an autophagy substrate protein, has not been evaluated. Methods: THP-1 derived macrophages were incubated with ox-LDL to establish a foam cell model and treated with different autophagy inducers. The effects of p62 on cholesterol efflux were investigated using overexpression vectors, gene silencing and western blotting. Results: This study showed a blockage of autophagy and decreased expression of ABCA1 and ABCG1 under the stress of excess ox-LDL in a concentration-dependent manner in THP-1 cells. Furthermore, the activation of autophagy led to increased expression of ABCA1 and ABCG1, as well as their upstream transcription factor LXRa, thereby promoting cholesterol efflux from foam cells. We also demonstrated that accumulated p62 played an important role during autophagy blockage, which was achieved by activating mTOR and then inhibited the expression of LXRa and its downstream target proteins ABCA1 and ABCG1. Conclusion: In conclusion, our experiments demonstrated that a p62/mTOR/LXRa signaling pathway was involved in cholesterol efflux mediated by ABCA1 and ABCG1 when autophagy blockage occurred. Our study offers a rationale for the development of autophagy and p62 as a new target for the treatment of atherosclerosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: ABCA1 ABCG1 Atherosclerosis Autophagy Cholesterol efflux LXRa mTOR p62/SQSTM1
1. Introduction Atherosclerosis (AS) is a chronic inflammatory condition that causes cardiovascular and cerebrovascular diseases and is considered to be the leading killer of humans worldwide [1,2]. Although the pathological mechanisms of AS are complex, abnormal lipid metabolism has been recognized as the foremost risk factor [3]. Macrophages phagocytose excess lipid droplets in the blood and gradually develop into foam cells d an early event of AS. Simultaneously, macrophages phagocytose cholesterol lipid droplets and also initiate cholesterol efflux. Intracellular cholesterol efflux helps
* Corresponding author. E-mail addresses: [email protected] (X. Liang), [email protected] (C. Wang), [email protected] (Y. Sun), [email protected] (W. Song), [email protected] (J. Lin), [email protected] (J. Li), [email protected] (X. Guan). https://doi.org/10.1016/j.bbrc.2019.04.134 0006-291X/© 2019 Elsevier Inc. All rights reserved.
to reduce lipid accumulation in foam cells and delay the development of AS [4]. The ATP binding cassette transporter (ABC) is a major component of the regulation of cholesterol efflux and is known as the gatekeeper of reverse cholesterol transport (RCT). ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) are important players of this protein family [5]. The cholesterol efflux mediated by ABCA1 and ABCG1 is mainly regulated by their upstream transcription factor, liver X receptor (LXR), which belongs to the nuclear hormone receptor superfamily [6]. The LXRa subtype is mainly expressed in adipose tissue such as liver, fat and macrophages, while LXRb is widely expressed in various tissues of the body. Studies have shown that autophagy and mammalian target of rapamycin (mTOR) participate in the regulation of LXRa and its downstream target proteins ABCA1 and ABCG1. Autophagy is a highly conserved biodegradation process that is responsible for the clearance of long-lived proteins and damaged organelles, and is also involved in lipid metabolism [7]. Ouimet
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et al. demonstrated that autophagy increases cholesterol efflux in macrophage-derived foam cells via lysosomal acid lipase, and cholesterol efflux mediated by autophagy is predominantly ABCA1dependent [8]. Other studies [9e12] also confirmed that autophagy can increase the expression of ABCA1 and ABCG1, and that mTOR is involved in the process [13]. mTOR is a receptor that binds ATP, free amino acids and hormones in cells, and plays important roles in nutrition balance and lipid metabolism in the body [14]. Some studies [15e17] have shown that mTOR also upregulates the expression of LXRa, ABCA1 and ABCG1. Although autophagy and mTOR are known to be involved in the cholesterol efflux mediated by ABCA1 and ABCG1, the role of p62, an autophagy substrate protein, has not been evaluated. In this study, we found that excess oxidized low-density lipoprotein (oxLDL) inhibited the expression of ABCA1 and ABCG1 and also disrupted autophagic flow in a concentration-dependent manner. Furthermore, we found that activation of autophagy upregulated the expression of ABCA1 and ABCG1 as well as LXRa, thereby promoting cholesterol efflux from foam cells. We also determined that accumulated p62 was a key regulator of this process, which was achieved by activating mTOR. In conclusion, we have demonstrated for the first time that a p62/mTOR/LXRa signaling pathway plays an important role in cholesterol efflux from foam cells during autophagy blockage. Our results suggest that p62 and autophagy may provide a new therapy target for AS. 2. Materials and methods
2.2.3. Gene overexpression The p62 overexpression reagent was prepared by our research group in advance. Induced THP-1 cells in 24-well plates were transfected with a control plasmid (pCMV-Myc) or a p62 overexpression plasmid (pMyc-p62) at 50 nM for 48 h. Western blotting was used to confirm gene overexpression, after which additional experiments were performed. 2.2.4. Western blot analysis The total protein was extracted using radioimmunoprecipitation assay lysis buffer containing the protease inhibitor PMSF (Beyotime, China), and the protein concentration was measured using the BCA kit (Solarbio, China). The denatured proteins were separated using SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and then blocked with 5% bovine serum albumin (BSA) for 1 h. Target bands were incubated with the primary antibody overnight at 4 C and then incubated with AP or HRP-conjugated secondary antibody for 1 h. The immune complexes were detected in an exposure apparatus using an enhanced chemiluminescence agent, and the target strip protein content was quantified using Tanon Gis software. Strips were re-incubated for proteins of similar molecular weight after treatment with membrane regeneration fluid. The dilution ratios for the various antibodies were as follows: ABCA1 (1:1000), ABCG1 (1:5000), mTOR (1:3000), Beclin1 (1:3000), p62/SQSTM (1:1000), LXRa (1:2000), LC3B (1:3000), b-actin (1:6000), goat anti-mouse secondary antibody (1:10000), and goat anti-rabbit secondary antibody (1:50000).
2.1. Materials Hiperfect Transfection Reagent was purchased from Qiagen (USA). Ox-LDL was from Guangzhou Yiyuan (China). RAPA, 3-MA and CQ were all from Sigma (USA). Antibodies for mTOR, ABCA1, ABCG1, LXRa, p62/SQSTM1, Beclin1, microtubule-associated protein 1 light chain 3 (LC3)B, and b-actin, horseradish peroxidase (HRP)labeled goat anti-mouse IgG þ IgM and alkaline phosphatase (AP)labeled goat anti-rabbit IgG secondary antibody were purchased from Abcam (USA). The intracellular total cholesterol and free cholesterol assay kits were purchased from Applygen (China). The cholesterol efflux detection kit was purchased from Biovision (USA).
2.2.5. Determination of intracellular cholesterol concentration The residual serum of the culture medium of treated cells was removed by washing twice with PBS; 0.1 ml lysate was added per 1 106 cells in proportion, and allowed to stand for 10 min. The appropriate amount of lysate was heated at 70 C for 10 min and then subjected to centrifugation at 2000g for 5 min. The supernatant was taken for enzymatic determination, and the remaining unheated lysate was assayed for protein content by the BCA method. The cholesterol content of each sample was measured according to the manufacturer's instructions, which was then corrected to the cholesterol content by/mg protein concentration or cell number.
2.2.1. Cell treatment THP-1 monocytes were purchased from the China Center for Type Culture Collection and cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) (both from Hyclone USA) at 37 C and 5% CO2. The cells were transferred to 6-well or 24-well plates at a cell density of about 5 105 to 1 106. Each well was incubated with PMA (Sigma, USA) at a final concentration of 100 ng/ml for 48 h to induce the differentiation of THP-1 cells into macrophages before being treated with the following: ox-LDL (0, 50, 100, 150, or 200 mg/mL) for 48 h; or ox-LDL (50 mg/mL) with RAPA (50 nM), 3-MA (2 mM) or CQ (30 mM) for 48 h.
2.2.6. Cholesterol efflux detection Cells were treated with the fluorescence labeling reagent according to the manufacturer's kit instructions in 96-well plates. After 16 h, the labeling reagent was discarded and the cells were gently washed with serum-free RPMI medium and then treated with cholesterol receptor for 4e6 h. At the end of the incubation, the supernatant was aspirated, 100 ml cell lysis buffer was added to each well and the plates were incubated on a shaker for 30 min to dissolve the cells. The fluorescence intensity of the supernatant and lysate were measured at an Ex/Em of 482/515 nm. The fluorescence intensity of the supernatant was divided by the total fluorescence intensity of the supernatant and the lysate, and the value was multiplied by 100 to obtain a % cholesterol efflux rate.
2.2.2. Small interfering RNA (siRNA) transfection After induction of THP-1 cells in 24-well plates, siRNA (Guangzhou Ruibo, China) was used to inhibit gene expression of human p62 and mTOR at a working concentration of 50 nM according to the manufacturer's instructions. An unrelated 21nucleotide-length siRNA was used as a negative control. After 72 h of transfection we used western blotting to detect whether the gene silencing was successful, after which additional experiments were performed.
2.2.7. Oil red O staining Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 5e10 min, then washed a further three times with PBS after removing the paraformaldehyde solution. The oil red O solution was added (2 ml to each well of a 6-well plate, or 0.5 ml to each well of a 24-well plate) and incubated at 37 C for 15 min. The cells were washed three times with PBS, differentiated with 60% isopropanol for 30 s, and washed another three times with PBS for microscopic observation and photography. The PBS in the plate
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was discarded and the dye was extracted with 100% isopropyl alcohol for 10 min. The OD value of the mixture was measured at 520 nm for quantitative analysis. 2.2.8. Statistical analysis Data were processed using GraphPad Prism 6 software and are expressed as mean ± standard deviation. The t-test was performed for two-group comparisons, and comparisons of three groups or more were analyzed by one-way analysis of variance. Differences were considered statistically significant at *p < 0.05.
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3. Results 3.1. Excess ox-LDL inhibited the expression of ABCA1 and ABCG1, and induced blockage of autophagy To simulate the formation of foam cells in vitro, THP-1 macrophages were treated with a range of concentrations of ox-LDL for 48 h. We found that ox-LDL induced the transformation of THP1 cells into foam cells starting at a concentration of 50 mg/mL, and the degree of cell foaming increased with increasing concentrations
Fig. 1. Excess ox-LDL inhibits the expression of ABCA1 and ABCG1 and induces autophagy blockage. (a) Oil red 0 staining of THP-1 cells treated with different concentrations of ox-LDL: 0, 50, 100, 150, and 200 mg/mL (40 magnification). (b) Immunoblotting experiments in each of the ox-LDL treatment groups. (c) Intracellular lipid titration detection from oil red O staining. (deg) Expression of ABCA1, ABCG1, p62 and LC3 under each concentration of ox-LDL. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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of ox-LDL (Fig. 1a, c). The expression of ABCA1 and ABCG1, which can regulate cholesterol efflux, gradually decreased with increasing concentrations of ox-LDL as shown by western blotting (Fig. 1b, d, e). We also found that autophagic flow was inhibited under the stress of excess ox-LDL as shown by increased levels of LC3II/I and p62, which indicated that the binding of autophagosomes and lysosomes were blocked (Fig. 1b, f, g). In summary, we found that excess ox-LDL inhibited the expression of ABCA1 with ABCG1 and induced autophagy blockage of THP-1 cells in a concentrationdependent manner.
3.2. Autophagy affected cholesterol efflux and participated in foam cell formation Although autophagy is associated with the expression of ABCA1 and ABCG1, the effect of autophagy on cholesterol efflux from foam cells specifically has not been determined. To demonstrate the relationship between autophagy and the cholesterol efflux mediated by ABCA1 and ABCG1, we used RAPA as an autophagy activator and 3-MA and CQ as autophagy inhibitors to examine THP-1 cells in a state of autophagy blockage. Compared with the control group,
Fig. 2. Autophagy affects cholesterol efflux and participates in foam cells formation. (a, b) Immunoblotting experiments in each of the following treatment groups: ox-LDL (50mg/mL), ox-LDL þ 3-MA, ox-LDL þ CQ, and ox-LDL þ RAPA. (c) Intracellular cholesterol efflux rate, (d, e) intracellular cholesterol concentrations, and (f, g) oil red 0 staining and intracellular lipid titration detection in each of the treatment groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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the expression of ABCA1, ABCG1 and LXRa were all increased in the RAPA-treated group (Fig. 2a and b), and cholesterol efflux in the foam cells was increased (Fig. 2c). In addition, the levels of total cholesterol and free cholesterol in foam cells were decreased (Fig. 2d and e), and foam cell formation was significantly reduced (Fig. 2f and g) with RAPA treatment. Conversely, treatment with either 3-MA or CQ yielded the opposite results. These results suggested that the upregulation of autophagy can promote intracellular cholesterol efflux and reduce foam cell formation, while further inhibition of autophagy during autophagy blockage may have opposite effects.
3.3. p62 overexpression accelerates foam cell formation during autophagy blockage 3-MA is an inhibitor of the initial stage of autophagy, while CQ inhibits the fusion of autophagosomes and lysosomes. Both inhibitors are known to cause a large accumulation of p62 in THP1 cells. To demonstrate the role of p62 in autophagy blockage, we transfected a lentiviral p62 vector into THP-1 cells to obtain a p62 overexpression cell model. Compared with the control group, the expression of ABCA1, ABCG1 and LXRa in the p62-overexpression group were all decreased (Fig. 3a and b) in parallel with a decrease in intracellular cholesterol efflux (Fig. 3d). The levels of total cholesterol and free cholesterol in foam cells were increased (Fig. 3e and f), and the formation of foam cells was significantly increased (Fig. 3c, g) in the p62-overexpression group. Given that intracellular cholesterol efflux was promoted in the group treated with RAPA, we hypothesized that mTOR may be involved because
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RAPA is a potent inhibitor of mTOR. As expected, p62 overexpression upregulated the activity of mTOR, thereby inhibiting autophagy activity and further aggravating autophagy blockage (Fig. 3a and b). In summary, our data indicated that p62 overexpression can inhibit intracellular cholesterol efflux and accelerate foam cell formation, which is likely to be achieved through an mTOR pathway. 3.4. A p62/mTOR/LXRa signaling pathway is involved in the formation of foam cells during autophagy blockage To verify the presence of a p62/mTOR/LXRa pathway in the cholesterol efflux mediated by ABCA1 and ABCG1, we observed the expression of LXRa, ABCA1 and ABCG1 under silencing of p62 and mTOR. We found that the expression of mTOR was decreased in the p62 knockdown group, while the expression of LXRa, ABCA1 and ABCG1 were all increased (Fig. 4a and b). Moreover, the expression of LXRa, ABCA1 and ABCG1 were all increased in the mTOR knockdown group as well (Fig. 4a and b). We also showed that p62 and mTOR knockdown promoted intracellular cholesterol efflux (Fig. 4d), and led to significant reductions in intracellular total cholesterol and free cholesterol (Fig. 4e and f). Furthermore, oil red O staining revealed a decreased formation of foam cells after p62 and mTOR were silenced (Fig. 4c, g). In addition, the silencing of mTOR increased autophagy activity to a certain extent, and reduced p62 accumulation, which in turn repressed the activity of mTOR (Fig. 4a and b). Taken together, these results indicated that a p62/ mTOR/LXRa pathway is involved in the cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage (Fig. 5).
Fig. 3. Overexpression of p62 accelerates foam cell formation during autophagy blockage. (a, b) THP-1 cells transfected with a control (pCMV-Myc) or p62 overexpression plasmid (pMyc-p62) and treated with 50mg/mL ox-LDL were subjected to (a, b) immunoblotting and (c) oil red 0 staining (40 magnification). (d) Intracellular cholesterol efflux rate, (e, f) intracellular cholesterol concentrations, and (g) intracellular lipid titration detection from oil red O staining for the two transfection groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. A p62/mTOR/LXRa signaling pathway is involved in the formation of foam cells during autophagy blockage. THP-1 cells were transfected with siRNAs. (a, b) Immunoblotting and (c) oil red 0 staining (40 magnification) in the control (si-con), p62 silencing (si-p62), and mTOR silencing (si-mTOR) groups. (d) Intracellular cholesterol efflux rate, (e, f) intracellular cholesterol concentrations, and (g) intracellular lipid titration detection from oil red O staining in the same three groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion The formation of foam cells is a process of continuous accumulation of lipids in cells. Our experiment simulated the formation of foam cells in vitro by gradually increasing the concentration of ox-LDL. Cholesterol efflux mediated by the ABC family protein plays an important role in inhibiting foam cell formation. Relevant study [18] indicated that ox-LDL can induce ABCA1 expression in a parabolic quantity relationship, and ox-LDL inhibits the expression of ABCA1 at a concentration of higher than 50 mg/ml, which is also proved by our experiment. In addition, we also found that excess ox-LDL can induce autophagy blockage in a concentrationdependent manner. Therefore, the above experimental results suggest that cholesterol efflux in foam cells may be associated with autophagy. While autophagy and cholesterol efflux are independent biological mechanisms, Ouimet et al. [8,19] demonstrated that autophagy can regulate cholesterol efflux and miR-33 promotes lipid droplet breakdown are related to autophagy. SIRT6 overexpression promoted cholesterol efflux [10] and several new therapies of AS [9,12] were all autophagy-dependent. We also showed that the cholesterol efflux mediated by ABCA1 and ABCG1 are regulated by autophagy through different autophagy inducers. However, Han et al. [11] found that photodynamic therapy (PDT) of autophagydependence increased the expression of ABCA1, there was no significant change in ABCG1, which suggests that cholesterol efflux may be mediated by ABCA1 alone. Leng et al. [20] found that ursolic
acid can promote cholesterol efflux to apoA1 by activating autophagy, but it did not alter the mRNA or protein levels of ABCA1 and ABCG1. Unlike our study, they used the RAW264.7 cell line, which may explain the differences in our results. In a study similar to ours, Dong et al. [13] demonstrated that inhibition of Akt can promote cholesterol efflux mediated by ABCA1 through its downstream targets, both mTOR and autophagy. Cholesterol efflux mediated by ABCA1 and ABCG1 are regulated by multiple signaling pathways, and research on the mTOR pathway has long been a hot spot in this field. Ellsso et al. [21] found that the level of high-density lipoprotein (HDL)-cholesterol in AS-prone apolipoprotein E-deficient (APOE/) mice treated with RAPA was increased by 30% compared with the control group. On this basis, Varghese et al. [15] first demonstrated that the ASprotective effect of RAPA is achieved by inhibiting cholesterol uptake and upregulating ABCA1 expression to increase cholesterol efflux. Subsequent studies also proved that this effect is the result of mTOR inhibition [16,17,22]. Nagao et al. [23] believe that RAPA can induce ABCA1 expression by inhibiting the mTOR pathway at a concentration of 10e100 nM; the concentration we used (50 nM) also reinforced this conclusion. In addition, we also confirmed the role of mTOR in cholesterol efflux by silencing its expression. Although related studies have demonstrated the role of autophagy and mTOR on cholesterol efflux, whether or not p62 can regulate cholesterol efflux has not been reported. The discovery of the p62-mTOR pathway provided a theoretical basis for this hypothesis. Duran et al. [24] found that p62 knockdown increased
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autophagy activity with decreased mTORC1 activity, an unexpected finding suggesting that p62 is degraded by autophagy but also regulates autophagy in return. While the physiological significance of this process is unclear, Moscat et al. [25] proposed that its role may be to ensure the irreversibility of cell starvation death. In a study of tumors, it was found that silencing the p62 gene can reduce mTOR activity, while overexpressing p62 can increase mTOR activity [26]. With an intention to draw lessons from this mechanism, we envisaged that p62 could enhance mTOR activity during autophagy blockage, and thereby inhibit LXRa and its downstream transcription proteins so as to inhibit cholesterol efflux. Our experiments have proven this hypothesis to be a reasonable one, we found for the first time that p62/mTOR/LXR pathway is involved in cholesterol efflux and the formation of foam cells. As a multifunctional protein, p62 is involved in the development of various diseases, and its role in AS is particularly complex [27]. Sergin et al. [28] found that inclusion bodies enriched for p62 can protect against atherosclerosis. However, they only studied the role of p62 in AS at physiological level, and did not explore the situation of autophagy blockage. Grootaert et al. [29] demonstrated that a deficiency of autophagy can accelerate the senescence of vascular smooth muscle cells (VSMCs) and promote neointima formation and AS in mice. Transfection of p62 in Atg7-overexpressed VSMCs induced similar results, suggesting that accumulated p62 is the main contributor. Guan et al. [30] obtained similar findings in which they treated macrophages with ox-LDL to induce autophagy blockage. The accumulation of p62 upregulated the expression of matrix metalloproteinase-9 (MMP-9) via the nuclear factor (NF)-kB signaling pathway. In another study [31], they also demonstrated that ox-LDL can induce p62 accumulation and promote IL-18 secretion and cell death, thereby accelerating the progression of AS. Consistent with the above conclusions, our study also demonstrated the role of p62 in AS in terms of cholesterol efflux. The accumulation of cholesterol in foam cells is the result of a combination of uptake, efflux and endogenous synthesis. However, we did not consider the effect of p62 on cholesterol uptake and synthesis, and our study was limited to experiments conducted in vitro. Our future research will include related experiments in animals, and will seek to verify the effect of p62 on the expression of inflammatory factors caused by abnormal lipid metabolism. The effects of autophagy and p62 on cholesterol uptake and synthesis in macrophages should also be investigated. In conclusion, we have shown that a p62/mTOR/LXRa pathway played a significant role in cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage, which may provide a new target for the prevention and treatment of AS. Disclosures No conflict of interest exists in the submission of this manuscript. Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Funding The research was supported by the National Natural Science
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Foundation of China (81672084). Acknowledgements We thank Michelle Kahmeyer-Gabbe, PhD, from Liwen Bianji, Edanz Editing China. (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.134. References [1] C. Weber, H. Noels, Atherosclerosis: current pathogenesis and therapeutic options, Nat. Med. 17 (2011) 1410e1422. [2] R. Lozano, M. Naghavi, K. Foreman, et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010, Lancet 380 (2012) 2095e2128. [3] P.N. Hopkins, Molecular biology of atherosclerosis, Physiol. Rev. 93 (2013) 1317e1542. [4] K.J. Moore, I. Tabas, Macrophages in the Pathogenesis of Atherosclerosis, CELL, 2011. [5] A.D. Attie, ABCA1: at the nexus of cholesterol, HDL and atherosclerosis, Trends Biochem. Sci. 32 (2007) 172e179. [6] Y. Wang, J. Viscarra, S.J. Kim, et al., Transcriptional regulation of hepatic lipogenesis, Nat. Rev. Mol. Cell Biol. 16 (2015) 678e689. [7] A.M. Choi, S.W. Ryter, B. Levine, Autophagy in human health and disease, New. Engl. J. Med. 368 (2013) 651e662. [8] M. Ouimet, V. Franklin, E. Mak, et al., Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase, Cell Metab. 13 (2011) 655e667. [9] X. Li, X. Zhang, L. Zheng, et al., Hypericin-mediated sonodynamic therapy induces autophagy and decreases lipids in THP-1 macrophage by promoting ROS-dependent nuclear translocation of TFEB, Cell Death Dis. 7 (2016) e2527. [10] J. He, G. Zhang, Q. Pang, et al., SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition, FEBS J. 284 (2017) 1324e1337. [11] X.B. Han, H.X. Li, Y.Q. Jiang, et al., Upconversion nanoparticle-mediated photodynamic therapy induces autophagy and cholesterol efflux of macrophage-derived foam cells via ROS generation, Cell Death Dis. 8 (2017), e2864. [12] Y. Tang, H. Wu, B. Shao, et al., Celosins inhibit atherosclerosis in ApoE -/- mice and promote autophagy flow, J. Ethnopharmacol. 215 (2018) 74e82. [13] F. Dong, Z. Mo, W. Eid, et al., Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1, PLoS One 9 (2014), e113789. [14] T. Schmelzle, M.N. Hall, TOR, a central controller of cell growth, Cell 103 (2000) 253e262. [15] Z. Varghese, R. Fernando, J.F. Moorhead, et al., Effects of sirolimus on mesangial cell cholesterol homeostasis: a novel mechanism for its action against lipid-mediated injury in renal allografts, Am. J. Physiol.-Renal. 289 (2005) F43eF48. [16] M. Kemmerer, I. Wittig, F. Richter, et al., AMPK activates LXRa and ABCA1 expression in human macrophages, Int. J. Biochem. Cell Biol. 78 (2016) 1e9. [17] B. Castella, J. Kopecka, P. Sciancalepore, et al., The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vgamma9Vdelta2 T cell activation by dendritic cells, Nat. Commun. 8 (2017) 15663. [18] S. Zhao, B. Yu, X. Xie, et al., Dual effects of oxidized low-density lipoprotein on LXR-ABCA1-apoA-I pathway in 3T3-L1 cells, Int. J. Cardiol. 128 (2008) 42e47. [19] M. Ouimet, H. Ediriweera, M.S. Afonso, et al., microRNA-33 regulates macrophage autophagy in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 37 (2017) 1058e1067. [20] S. Leng, S. Iwanowycz, F. Saaoud, et al., Ursolic acid enhances macrophage autophagy and attenuates atherogenesis, J. Lipid Res. 57 (2016) 1006e1016. [21] M.M. Elloso, N. Azrolan, S.N. Sehgal, et al., Protective effect of the immunosuppressant sirolimus against aortic atherosclerosis in apo E-deficient mice, Am. J. Transplant. 3 (2003) 562e569. [22] H. Zheng, Y. Fu, Y. Huang, et al., mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway, Mol. Med. Rep. 16 (2017) 3315e3323. [23] K. Nagao, M. Maeda, N.B. Manucat, et al., Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding, Biochim. Biophys. Acta 1831 (2013) 398e406. [24] A. Duran, R. Amanchy, J. Linares, et al., p62 is a key regulator of nutrient sensing in the mTORC1 pathway, Mol. Cell 44 (2011) 134e146. [25] J. Moscat, M.T. Diaz-Meco, Feedback on fat: p62-mTORC1-Autophagy connections, Cell 147 (2011) 724e727.
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[26] T. Valencia, J.Y. Kim, S. Abu-Baker, et al., Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis, Cancer Cell 26 (2014) 121e135. [27] X. Liang, X. Guan, p62/SQSTM1: a potential molecular target for treatment of atherosclerosis, Front. Lab. Med. 1 (2017) 104e106. [28] I. Sergin, S. Bhattacharya, R. Emanuel, et al., Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis, Sci. Signal. 9 (2016) ra2. [29] M.O. Grootaert, C.M.P. Da, N. Bitsch, et al., Defective autophagy in vascular
smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis, Autophagy 11 (2015) 2014e2032. [30] F. Zhou, D. Liu, H. Ning, et al., The roles of p62/SQSTM1 on regulation of matrix metalloproteinase-9 gene expression in response to oxLDL in atherosclerosis, Biochem. Bioph. Res. Co. 472 (2016) 451e458. [31] H. Ning, D. Liu, X. Yu, et al., Oxidized low-density lipoprotein-induced p62/ SQSTM1 accumulation in THP-1-derived macrophages promotes IL-18 secretion and cell death, Exp. Ther. Med. 14 (2017) 5417e5423.
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p62/mTOR/LXRa pathway inhibits cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage Xiaofei Liang, Chao Wang, Yan Sun, Wei Song, Jing Lin, Jiashan Li, Xiuru Guan* First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China
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a b s t r a c t
Article history: Received 29 March 2019 Accepted 18 April 2019 Available online 14 May 2019
Objective: Atherosclerosis is a disease characterized by abnormal lipid metabolism, and the formation of foam cells is considered an early event of atherosclerosis. Intracellular cholesterol efflux mediated by ABCA1 and ABCG1 helps to reduce lipid accumulation in foam cells. Related studies have shown that autophagy and mTOR are involved in cholesterol efflux, but the role of p62, an autophagy substrate protein, has not been evaluated. Methods: THP-1 derived macrophages were incubated with ox-LDL to establish a foam cell model and treated with different autophagy inducers. The effects of p62 on cholesterol efflux were investigated using overexpression vectors, gene silencing and western blotting. Results: This study showed a blockage of autophagy and decreased expression of ABCA1 and ABCG1 under the stress of excess ox-LDL in a concentration-dependent manner in THP-1 cells. Furthermore, the activation of autophagy led to increased expression of ABCA1 and ABCG1, as well as their upstream transcription factor LXRa, thereby promoting cholesterol efflux from foam cells. We also demonstrated that accumulated p62 played an important role during autophagy blockage, which was achieved by activating mTOR and then inhibited the expression of LXRa and its downstream target proteins ABCA1 and ABCG1. Conclusion: In conclusion, our experiments demonstrated that a p62/mTOR/LXRa signaling pathway was involved in cholesterol efflux mediated by ABCA1 and ABCG1 when autophagy blockage occurred. Our study offers a rationale for the development of autophagy and p62 as a new target for the treatment of atherosclerosis. © 2019 Elsevier Inc. All rights reserved.
Keywords: ABCA1 ABCG1 Atherosclerosis Autophagy Cholesterol efflux LXRa mTOR p62/SQSTM1
1. Introduction Atherosclerosis (AS) is a chronic inflammatory condition that causes cardiovascular and cerebrovascular diseases and is considered to be the leading killer of humans worldwide [1,2]. Although the pathological mechanisms of AS are complex, abnormal lipid metabolism has been recognized as the foremost risk factor [3]. Macrophages phagocytose excess lipid droplets in the blood and gradually develop into foam cells d an early event of AS. Simultaneously, macrophages phagocytose cholesterol lipid droplets and also initiate cholesterol efflux. Intracellular cholesterol efflux helps
* Corresponding author. E-mail addresses: [email protected] (X. Liang), [email protected] (C. Wang), [email protected] (Y. Sun), [email protected] (W. Song), [email protected] (J. Lin), [email protected] (J. Li), [email protected] (X. Guan). https://doi.org/10.1016/j.bbrc.2019.04.134 0006-291X/© 2019 Elsevier Inc. All rights reserved.
to reduce lipid accumulation in foam cells and delay the development of AS [4]. The ATP binding cassette transporter (ABC) is a major component of the regulation of cholesterol efflux and is known as the gatekeeper of reverse cholesterol transport (RCT). ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) are important players of this protein family [5]. The cholesterol efflux mediated by ABCA1 and ABCG1 is mainly regulated by their upstream transcription factor, liver X receptor (LXR), which belongs to the nuclear hormone receptor superfamily [6]. The LXRa subtype is mainly expressed in adipose tissue such as liver, fat and macrophages, while LXRb is widely expressed in various tissues of the body. Studies have shown that autophagy and mammalian target of rapamycin (mTOR) participate in the regulation of LXRa and its downstream target proteins ABCA1 and ABCG1. Autophagy is a highly conserved biodegradation process that is responsible for the clearance of long-lived proteins and damaged organelles, and is also involved in lipid metabolism [7]. Ouimet
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et al. demonstrated that autophagy increases cholesterol efflux in macrophage-derived foam cells via lysosomal acid lipase, and cholesterol efflux mediated by autophagy is predominantly ABCA1dependent [8]. Other studies [9e12] also confirmed that autophagy can increase the expression of ABCA1 and ABCG1, and that mTOR is involved in the process [13]. mTOR is a receptor that binds ATP, free amino acids and hormones in cells, and plays important roles in nutrition balance and lipid metabolism in the body [14]. Some studies [15e17] have shown that mTOR also upregulates the expression of LXRa, ABCA1 and ABCG1. Although autophagy and mTOR are known to be involved in the cholesterol efflux mediated by ABCA1 and ABCG1, the role of p62, an autophagy substrate protein, has not been evaluated. In this study, we found that excess oxidized low-density lipoprotein (oxLDL) inhibited the expression of ABCA1 and ABCG1 and also disrupted autophagic flow in a concentration-dependent manner. Furthermore, we found that activation of autophagy upregulated the expression of ABCA1 and ABCG1 as well as LXRa, thereby promoting cholesterol efflux from foam cells. We also determined that accumulated p62 was a key regulator of this process, which was achieved by activating mTOR. In conclusion, we have demonstrated for the first time that a p62/mTOR/LXRa signaling pathway plays an important role in cholesterol efflux from foam cells during autophagy blockage. Our results suggest that p62 and autophagy may provide a new therapy target for AS. 2. Materials and methods
2.2.3. Gene overexpression The p62 overexpression reagent was prepared by our research group in advance. Induced THP-1 cells in 24-well plates were transfected with a control plasmid (pCMV-Myc) or a p62 overexpression plasmid (pMyc-p62) at 50 nM for 48 h. Western blotting was used to confirm gene overexpression, after which additional experiments were performed. 2.2.4. Western blot analysis The total protein was extracted using radioimmunoprecipitation assay lysis buffer containing the protease inhibitor PMSF (Beyotime, China), and the protein concentration was measured using the BCA kit (Solarbio, China). The denatured proteins were separated using SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and then blocked with 5% bovine serum albumin (BSA) for 1 h. Target bands were incubated with the primary antibody overnight at 4 C and then incubated with AP or HRP-conjugated secondary antibody for 1 h. The immune complexes were detected in an exposure apparatus using an enhanced chemiluminescence agent, and the target strip protein content was quantified using Tanon Gis software. Strips were re-incubated for proteins of similar molecular weight after treatment with membrane regeneration fluid. The dilution ratios for the various antibodies were as follows: ABCA1 (1:1000), ABCG1 (1:5000), mTOR (1:3000), Beclin1 (1:3000), p62/SQSTM (1:1000), LXRa (1:2000), LC3B (1:3000), b-actin (1:6000), goat anti-mouse secondary antibody (1:10000), and goat anti-rabbit secondary antibody (1:50000).
2.1. Materials Hiperfect Transfection Reagent was purchased from Qiagen (USA). Ox-LDL was from Guangzhou Yiyuan (China). RAPA, 3-MA and CQ were all from Sigma (USA). Antibodies for mTOR, ABCA1, ABCG1, LXRa, p62/SQSTM1, Beclin1, microtubule-associated protein 1 light chain 3 (LC3)B, and b-actin, horseradish peroxidase (HRP)labeled goat anti-mouse IgG þ IgM and alkaline phosphatase (AP)labeled goat anti-rabbit IgG secondary antibody were purchased from Abcam (USA). The intracellular total cholesterol and free cholesterol assay kits were purchased from Applygen (China). The cholesterol efflux detection kit was purchased from Biovision (USA).
2.2.5. Determination of intracellular cholesterol concentration The residual serum of the culture medium of treated cells was removed by washing twice with PBS; 0.1 ml lysate was added per 1 106 cells in proportion, and allowed to stand for 10 min. The appropriate amount of lysate was heated at 70 C for 10 min and then subjected to centrifugation at 2000g for 5 min. The supernatant was taken for enzymatic determination, and the remaining unheated lysate was assayed for protein content by the BCA method. The cholesterol content of each sample was measured according to the manufacturer's instructions, which was then corrected to the cholesterol content by/mg protein concentration or cell number.
2.2.1. Cell treatment THP-1 monocytes were purchased from the China Center for Type Culture Collection and cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) (both from Hyclone USA) at 37 C and 5% CO2. The cells were transferred to 6-well or 24-well plates at a cell density of about 5 105 to 1 106. Each well was incubated with PMA (Sigma, USA) at a final concentration of 100 ng/ml for 48 h to induce the differentiation of THP-1 cells into macrophages before being treated with the following: ox-LDL (0, 50, 100, 150, or 200 mg/mL) for 48 h; or ox-LDL (50 mg/mL) with RAPA (50 nM), 3-MA (2 mM) or CQ (30 mM) for 48 h.
2.2.6. Cholesterol efflux detection Cells were treated with the fluorescence labeling reagent according to the manufacturer's kit instructions in 96-well plates. After 16 h, the labeling reagent was discarded and the cells were gently washed with serum-free RPMI medium and then treated with cholesterol receptor for 4e6 h. At the end of the incubation, the supernatant was aspirated, 100 ml cell lysis buffer was added to each well and the plates were incubated on a shaker for 30 min to dissolve the cells. The fluorescence intensity of the supernatant and lysate were measured at an Ex/Em of 482/515 nm. The fluorescence intensity of the supernatant was divided by the total fluorescence intensity of the supernatant and the lysate, and the value was multiplied by 100 to obtain a % cholesterol efflux rate.
2.2.2. Small interfering RNA (siRNA) transfection After induction of THP-1 cells in 24-well plates, siRNA (Guangzhou Ruibo, China) was used to inhibit gene expression of human p62 and mTOR at a working concentration of 50 nM according to the manufacturer's instructions. An unrelated 21nucleotide-length siRNA was used as a negative control. After 72 h of transfection we used western blotting to detect whether the gene silencing was successful, after which additional experiments were performed.
2.2.7. Oil red O staining Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 5e10 min, then washed a further three times with PBS after removing the paraformaldehyde solution. The oil red O solution was added (2 ml to each well of a 6-well plate, or 0.5 ml to each well of a 24-well plate) and incubated at 37 C for 15 min. The cells were washed three times with PBS, differentiated with 60% isopropanol for 30 s, and washed another three times with PBS for microscopic observation and photography. The PBS in the plate
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was discarded and the dye was extracted with 100% isopropyl alcohol for 10 min. The OD value of the mixture was measured at 520 nm for quantitative analysis. 2.2.8. Statistical analysis Data were processed using GraphPad Prism 6 software and are expressed as mean ± standard deviation. The t-test was performed for two-group comparisons, and comparisons of three groups or more were analyzed by one-way analysis of variance. Differences were considered statistically significant at *p < 0.05.
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3. Results 3.1. Excess ox-LDL inhibited the expression of ABCA1 and ABCG1, and induced blockage of autophagy To simulate the formation of foam cells in vitro, THP-1 macrophages were treated with a range of concentrations of ox-LDL for 48 h. We found that ox-LDL induced the transformation of THP1 cells into foam cells starting at a concentration of 50 mg/mL, and the degree of cell foaming increased with increasing concentrations
Fig. 1. Excess ox-LDL inhibits the expression of ABCA1 and ABCG1 and induces autophagy blockage. (a) Oil red 0 staining of THP-1 cells treated with different concentrations of ox-LDL: 0, 50, 100, 150, and 200 mg/mL (40 magnification). (b) Immunoblotting experiments in each of the ox-LDL treatment groups. (c) Intracellular lipid titration detection from oil red O staining. (deg) Expression of ABCA1, ABCG1, p62 and LC3 under each concentration of ox-LDL. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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of ox-LDL (Fig. 1a, c). The expression of ABCA1 and ABCG1, which can regulate cholesterol efflux, gradually decreased with increasing concentrations of ox-LDL as shown by western blotting (Fig. 1b, d, e). We also found that autophagic flow was inhibited under the stress of excess ox-LDL as shown by increased levels of LC3II/I and p62, which indicated that the binding of autophagosomes and lysosomes were blocked (Fig. 1b, f, g). In summary, we found that excess ox-LDL inhibited the expression of ABCA1 with ABCG1 and induced autophagy blockage of THP-1 cells in a concentrationdependent manner.
3.2. Autophagy affected cholesterol efflux and participated in foam cell formation Although autophagy is associated with the expression of ABCA1 and ABCG1, the effect of autophagy on cholesterol efflux from foam cells specifically has not been determined. To demonstrate the relationship between autophagy and the cholesterol efflux mediated by ABCA1 and ABCG1, we used RAPA as an autophagy activator and 3-MA and CQ as autophagy inhibitors to examine THP-1 cells in a state of autophagy blockage. Compared with the control group,
Fig. 2. Autophagy affects cholesterol efflux and participates in foam cells formation. (a, b) Immunoblotting experiments in each of the following treatment groups: ox-LDL (50mg/mL), ox-LDL þ 3-MA, ox-LDL þ CQ, and ox-LDL þ RAPA. (c) Intracellular cholesterol efflux rate, (d, e) intracellular cholesterol concentrations, and (f, g) oil red 0 staining and intracellular lipid titration detection in each of the treatment groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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the expression of ABCA1, ABCG1 and LXRa were all increased in the RAPA-treated group (Fig. 2a and b), and cholesterol efflux in the foam cells was increased (Fig. 2c). In addition, the levels of total cholesterol and free cholesterol in foam cells were decreased (Fig. 2d and e), and foam cell formation was significantly reduced (Fig. 2f and g) with RAPA treatment. Conversely, treatment with either 3-MA or CQ yielded the opposite results. These results suggested that the upregulation of autophagy can promote intracellular cholesterol efflux and reduce foam cell formation, while further inhibition of autophagy during autophagy blockage may have opposite effects.
3.3. p62 overexpression accelerates foam cell formation during autophagy blockage 3-MA is an inhibitor of the initial stage of autophagy, while CQ inhibits the fusion of autophagosomes and lysosomes. Both inhibitors are known to cause a large accumulation of p62 in THP1 cells. To demonstrate the role of p62 in autophagy blockage, we transfected a lentiviral p62 vector into THP-1 cells to obtain a p62 overexpression cell model. Compared with the control group, the expression of ABCA1, ABCG1 and LXRa in the p62-overexpression group were all decreased (Fig. 3a and b) in parallel with a decrease in intracellular cholesterol efflux (Fig. 3d). The levels of total cholesterol and free cholesterol in foam cells were increased (Fig. 3e and f), and the formation of foam cells was significantly increased (Fig. 3c, g) in the p62-overexpression group. Given that intracellular cholesterol efflux was promoted in the group treated with RAPA, we hypothesized that mTOR may be involved because
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RAPA is a potent inhibitor of mTOR. As expected, p62 overexpression upregulated the activity of mTOR, thereby inhibiting autophagy activity and further aggravating autophagy blockage (Fig. 3a and b). In summary, our data indicated that p62 overexpression can inhibit intracellular cholesterol efflux and accelerate foam cell formation, which is likely to be achieved through an mTOR pathway. 3.4. A p62/mTOR/LXRa signaling pathway is involved in the formation of foam cells during autophagy blockage To verify the presence of a p62/mTOR/LXRa pathway in the cholesterol efflux mediated by ABCA1 and ABCG1, we observed the expression of LXRa, ABCA1 and ABCG1 under silencing of p62 and mTOR. We found that the expression of mTOR was decreased in the p62 knockdown group, while the expression of LXRa, ABCA1 and ABCG1 were all increased (Fig. 4a and b). Moreover, the expression of LXRa, ABCA1 and ABCG1 were all increased in the mTOR knockdown group as well (Fig. 4a and b). We also showed that p62 and mTOR knockdown promoted intracellular cholesterol efflux (Fig. 4d), and led to significant reductions in intracellular total cholesterol and free cholesterol (Fig. 4e and f). Furthermore, oil red O staining revealed a decreased formation of foam cells after p62 and mTOR were silenced (Fig. 4c, g). In addition, the silencing of mTOR increased autophagy activity to a certain extent, and reduced p62 accumulation, which in turn repressed the activity of mTOR (Fig. 4a and b). Taken together, these results indicated that a p62/ mTOR/LXRa pathway is involved in the cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage (Fig. 5).
Fig. 3. Overexpression of p62 accelerates foam cell formation during autophagy blockage. (a, b) THP-1 cells transfected with a control (pCMV-Myc) or p62 overexpression plasmid (pMyc-p62) and treated with 50mg/mL ox-LDL were subjected to (a, b) immunoblotting and (c) oil red 0 staining (40 magnification). (d) Intracellular cholesterol efflux rate, (e, f) intracellular cholesterol concentrations, and (g) intracellular lipid titration detection from oil red O staining for the two transfection groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. A p62/mTOR/LXRa signaling pathway is involved in the formation of foam cells during autophagy blockage. THP-1 cells were transfected with siRNAs. (a, b) Immunoblotting and (c) oil red 0 staining (40 magnification) in the control (si-con), p62 silencing (si-p62), and mTOR silencing (si-mTOR) groups. (d) Intracellular cholesterol efflux rate, (e, f) intracellular cholesterol concentrations, and (g) intracellular lipid titration detection from oil red O staining in the same three groups. All experiments were repeated three times. Data are expressed as mean ± label difference. *p < 0.05 was considered statistically significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4. Discussion The formation of foam cells is a process of continuous accumulation of lipids in cells. Our experiment simulated the formation of foam cells in vitro by gradually increasing the concentration of ox-LDL. Cholesterol efflux mediated by the ABC family protein plays an important role in inhibiting foam cell formation. Relevant study [18] indicated that ox-LDL can induce ABCA1 expression in a parabolic quantity relationship, and ox-LDL inhibits the expression of ABCA1 at a concentration of higher than 50 mg/ml, which is also proved by our experiment. In addition, we also found that excess ox-LDL can induce autophagy blockage in a concentrationdependent manner. Therefore, the above experimental results suggest that cholesterol efflux in foam cells may be associated with autophagy. While autophagy and cholesterol efflux are independent biological mechanisms, Ouimet et al. [8,19] demonstrated that autophagy can regulate cholesterol efflux and miR-33 promotes lipid droplet breakdown are related to autophagy. SIRT6 overexpression promoted cholesterol efflux [10] and several new therapies of AS [9,12] were all autophagy-dependent. We also showed that the cholesterol efflux mediated by ABCA1 and ABCG1 are regulated by autophagy through different autophagy inducers. However, Han et al. [11] found that photodynamic therapy (PDT) of autophagydependence increased the expression of ABCA1, there was no significant change in ABCG1, which suggests that cholesterol efflux may be mediated by ABCA1 alone. Leng et al. [20] found that ursolic
acid can promote cholesterol efflux to apoA1 by activating autophagy, but it did not alter the mRNA or protein levels of ABCA1 and ABCG1. Unlike our study, they used the RAW264.7 cell line, which may explain the differences in our results. In a study similar to ours, Dong et al. [13] demonstrated that inhibition of Akt can promote cholesterol efflux mediated by ABCA1 through its downstream targets, both mTOR and autophagy. Cholesterol efflux mediated by ABCA1 and ABCG1 are regulated by multiple signaling pathways, and research on the mTOR pathway has long been a hot spot in this field. Ellsso et al. [21] found that the level of high-density lipoprotein (HDL)-cholesterol in AS-prone apolipoprotein E-deficient (APOE/) mice treated with RAPA was increased by 30% compared with the control group. On this basis, Varghese et al. [15] first demonstrated that the ASprotective effect of RAPA is achieved by inhibiting cholesterol uptake and upregulating ABCA1 expression to increase cholesterol efflux. Subsequent studies also proved that this effect is the result of mTOR inhibition [16,17,22]. Nagao et al. [23] believe that RAPA can induce ABCA1 expression by inhibiting the mTOR pathway at a concentration of 10e100 nM; the concentration we used (50 nM) also reinforced this conclusion. In addition, we also confirmed the role of mTOR in cholesterol efflux by silencing its expression. Although related studies have demonstrated the role of autophagy and mTOR on cholesterol efflux, whether or not p62 can regulate cholesterol efflux has not been reported. The discovery of the p62-mTOR pathway provided a theoretical basis for this hypothesis. Duran et al. [24] found that p62 knockdown increased
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autophagy activity with decreased mTORC1 activity, an unexpected finding suggesting that p62 is degraded by autophagy but also regulates autophagy in return. While the physiological significance of this process is unclear, Moscat et al. [25] proposed that its role may be to ensure the irreversibility of cell starvation death. In a study of tumors, it was found that silencing the p62 gene can reduce mTOR activity, while overexpressing p62 can increase mTOR activity [26]. With an intention to draw lessons from this mechanism, we envisaged that p62 could enhance mTOR activity during autophagy blockage, and thereby inhibit LXRa and its downstream transcription proteins so as to inhibit cholesterol efflux. Our experiments have proven this hypothesis to be a reasonable one, we found for the first time that p62/mTOR/LXR pathway is involved in cholesterol efflux and the formation of foam cells. As a multifunctional protein, p62 is involved in the development of various diseases, and its role in AS is particularly complex [27]. Sergin et al. [28] found that inclusion bodies enriched for p62 can protect against atherosclerosis. However, they only studied the role of p62 in AS at physiological level, and did not explore the situation of autophagy blockage. Grootaert et al. [29] demonstrated that a deficiency of autophagy can accelerate the senescence of vascular smooth muscle cells (VSMCs) and promote neointima formation and AS in mice. Transfection of p62 in Atg7-overexpressed VSMCs induced similar results, suggesting that accumulated p62 is the main contributor. Guan et al. [30] obtained similar findings in which they treated macrophages with ox-LDL to induce autophagy blockage. The accumulation of p62 upregulated the expression of matrix metalloproteinase-9 (MMP-9) via the nuclear factor (NF)-kB signaling pathway. In another study [31], they also demonstrated that ox-LDL can induce p62 accumulation and promote IL-18 secretion and cell death, thereby accelerating the progression of AS. Consistent with the above conclusions, our study also demonstrated the role of p62 in AS in terms of cholesterol efflux. The accumulation of cholesterol in foam cells is the result of a combination of uptake, efflux and endogenous synthesis. However, we did not consider the effect of p62 on cholesterol uptake and synthesis, and our study was limited to experiments conducted in vitro. Our future research will include related experiments in animals, and will seek to verify the effect of p62 on the expression of inflammatory factors caused by abnormal lipid metabolism. The effects of autophagy and p62 on cholesterol uptake and synthesis in macrophages should also be investigated. In conclusion, we have shown that a p62/mTOR/LXRa pathway played a significant role in cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage, which may provide a new target for the prevention and treatment of AS. Disclosures No conflict of interest exists in the submission of this manuscript. Conflict of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled. Funding The research was supported by the National Natural Science
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Foundation of China (81672084). Acknowledgements We thank Michelle Kahmeyer-Gabbe, PhD, from Liwen Bianji, Edanz Editing China. (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.134. References [1] C. Weber, H. Noels, Atherosclerosis: current pathogenesis and therapeutic options, Nat. Med. 17 (2011) 1410e1422. [2] R. Lozano, M. Naghavi, K. Foreman, et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010, Lancet 380 (2012) 2095e2128. [3] P.N. Hopkins, Molecular biology of atherosclerosis, Physiol. Rev. 93 (2013) 1317e1542. [4] K.J. Moore, I. Tabas, Macrophages in the Pathogenesis of Atherosclerosis, CELL, 2011. [5] A.D. Attie, ABCA1: at the nexus of cholesterol, HDL and atherosclerosis, Trends Biochem. Sci. 32 (2007) 172e179. [6] Y. Wang, J. Viscarra, S.J. Kim, et al., Transcriptional regulation of hepatic lipogenesis, Nat. Rev. Mol. Cell Biol. 16 (2015) 678e689. [7] A.M. Choi, S.W. Ryter, B. Levine, Autophagy in human health and disease, New. Engl. J. Med. 368 (2013) 651e662. [8] M. Ouimet, V. Franklin, E. Mak, et al., Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase, Cell Metab. 13 (2011) 655e667. [9] X. Li, X. Zhang, L. Zheng, et al., Hypericin-mediated sonodynamic therapy induces autophagy and decreases lipids in THP-1 macrophage by promoting ROS-dependent nuclear translocation of TFEB, Cell Death Dis. 7 (2016) e2527. [10] J. He, G. Zhang, Q. Pang, et al., SIRT6 reduces macrophage foam cell formation by inducing autophagy and cholesterol efflux under ox-LDL condition, FEBS J. 284 (2017) 1324e1337. [11] X.B. Han, H.X. Li, Y.Q. Jiang, et al., Upconversion nanoparticle-mediated photodynamic therapy induces autophagy and cholesterol efflux of macrophage-derived foam cells via ROS generation, Cell Death Dis. 8 (2017), e2864. [12] Y. Tang, H. Wu, B. Shao, et al., Celosins inhibit atherosclerosis in ApoE -/- mice and promote autophagy flow, J. Ethnopharmacol. 215 (2018) 74e82. [13] F. Dong, Z. Mo, W. Eid, et al., Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1, PLoS One 9 (2014), e113789. [14] T. Schmelzle, M.N. Hall, TOR, a central controller of cell growth, Cell 103 (2000) 253e262. [15] Z. Varghese, R. Fernando, J.F. Moorhead, et al., Effects of sirolimus on mesangial cell cholesterol homeostasis: a novel mechanism for its action against lipid-mediated injury in renal allografts, Am. J. Physiol.-Renal. 289 (2005) F43eF48. [16] M. Kemmerer, I. Wittig, F. Richter, et al., AMPK activates LXRa and ABCA1 expression in human macrophages, Int. J. Biochem. Cell Biol. 78 (2016) 1e9. [17] B. Castella, J. Kopecka, P. Sciancalepore, et al., The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vgamma9Vdelta2 T cell activation by dendritic cells, Nat. Commun. 8 (2017) 15663. [18] S. Zhao, B. Yu, X. Xie, et al., Dual effects of oxidized low-density lipoprotein on LXR-ABCA1-apoA-I pathway in 3T3-L1 cells, Int. J. Cardiol. 128 (2008) 42e47. [19] M. Ouimet, H. Ediriweera, M.S. Afonso, et al., microRNA-33 regulates macrophage autophagy in atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 37 (2017) 1058e1067. [20] S. Leng, S. Iwanowycz, F. Saaoud, et al., Ursolic acid enhances macrophage autophagy and attenuates atherogenesis, J. Lipid Res. 57 (2016) 1006e1016. [21] M.M. Elloso, N. Azrolan, S.N. Sehgal, et al., Protective effect of the immunosuppressant sirolimus against aortic atherosclerosis in apo E-deficient mice, Am. J. Transplant. 3 (2003) 562e569. [22] H. Zheng, Y. Fu, Y. Huang, et al., mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway, Mol. Med. Rep. 16 (2017) 3315e3323. [23] K. Nagao, M. Maeda, N.B. Manucat, et al., Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding, Biochim. Biophys. Acta 1831 (2013) 398e406. [24] A. Duran, R. Amanchy, J. Linares, et al., p62 is a key regulator of nutrient sensing in the mTORC1 pathway, Mol. Cell 44 (2011) 134e146. [25] J. Moscat, M.T. Diaz-Meco, Feedback on fat: p62-mTORC1-Autophagy connections, Cell 147 (2011) 724e727.
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[26] T. Valencia, J.Y. Kim, S. Abu-Baker, et al., Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis, Cancer Cell 26 (2014) 121e135. [27] X. Liang, X. Guan, p62/SQSTM1: a potential molecular target for treatment of atherosclerosis, Front. Lab. Med. 1 (2017) 104e106. [28] I. Sergin, S. Bhattacharya, R. Emanuel, et al., Inclusion bodies enriched for p62 and polyubiquitinated proteins in macrophages protect against atherosclerosis, Sci. Signal. 9 (2016) ra2. [29] M.O. Grootaert, C.M.P. Da, N. Bitsch, et al., Defective autophagy in vascular
smooth muscle cells accelerates senescence and promotes neointima formation and atherogenesis, Autophagy 11 (2015) 2014e2032. [30] F. Zhou, D. Liu, H. Ning, et al., The roles of p62/SQSTM1 on regulation of matrix metalloproteinase-9 gene expression in response to oxLDL in atherosclerosis, Biochem. Bioph. Res. Co. 472 (2016) 451e458. [31] H. Ning, D. Liu, X. Yu, et al., Oxidized low-density lipoprotein-induced p62/ SQSTM1 accumulation in THP-1-derived macrophages promotes IL-18 secretion and cell death, Exp. Ther. Med. 14 (2017) 5417e5423.