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• W2982697290 endingPage "2033" @default.
• W2982697290 startingPage "2020" @default.
• W2982697290 abstract "CXC chemokine ligand 12 (CXCL12) is a member of the CXC chemokine family and mainly acts on cell chemotaxis. CXCL12 also elicits a proatherogenic role, but the molecular mechanisms have not been fully defined yet. We aimed to reveal if and how CXCL12 promoted atherosclerosis via regulating lipid metabolism. In vitro, our data showed that CXCL12 could reduce ABCA1 expression, and it mediated cholesterol efflux from THP-1-derived macrophages to apoA-I. Data from the luciferase reporter gene and chromatin immunoprecipitation assays revealed that transcription factor 21 (TCF21) stimulated the transcription of ABCA1 via binding to its promoter region, which was repressed by CXCL12. We found that CXCL12 increased the levels of phosphorylated glycogen synthase kinase 3β (GSK3β) and the phosphorylation of β-catenin at the Thr120 position. Inactivation of GSK3β or β-catenin increased the expression of TCF21 and ABCA1. Further, knockdown or inhibition of CXC chemokine receptor 4 (CXCR4) blocked the effects of CXCL12 on TCF21 and ABCA1 expression and the phosphorylation of GSK3β and β-catenin. In vivo, the overexpression of CXCL12 in Apoe−/− mice via lentivirus enlarged the atherosclerotic lesion area and increased macrophage infiltration in atherosclerotic plaques. We further found that the overexpression of CXCL12 reduced the efficiency of reverse cholesterol transport and plasma HDL-C levels, decreased ABCA1 expression in the aorta and mouse peritoneal macrophages (MPMs), and suppressed cholesterol efflux from MPMs to apoA-I in Apoe−/− mice. Collectively, these findings suggest that CXCL12 interacts with CXCR4 and then activates the GSK-3β/β-cateninT120/TCF21 signaling pathway to inhibit ABCA1-dependent cholesterol efflux from macrophages and aggravate atherosclerosis. Targeting CXCL12 may be a novel and promising strategy for the prevention and treatment of atherosclerotic cardiovascular diseases. CXC chemokine ligand 12 (CXCL12) is a member of the CXC chemokine family and mainly acts on cell chemotaxis. CXCL12 also elicits a proatherogenic role, but the molecular mechanisms have not been fully defined yet. We aimed to reveal if and how CXCL12 promoted atherosclerosis via regulating lipid metabolism. In vitro, our data showed that CXCL12 could reduce ABCA1 expression, and it mediated cholesterol efflux from THP-1-derived macrophages to apoA-I. Data from the luciferase reporter gene and chromatin immunoprecipitation assays revealed that transcription factor 21 (TCF21) stimulated the transcription of ABCA1 via binding to its promoter region, which was repressed by CXCL12. We found that CXCL12 increased the levels of phosphorylated glycogen synthase kinase 3β (GSK3β) and the phosphorylation of β-catenin at the Thr120 position. Inactivation of GSK3β or β-catenin increased the expression of TCF21 and ABCA1. Further, knockdown or inhibition of CXC chemokine receptor 4 (CXCR4) blocked the effects of CXCL12 on TCF21 and ABCA1 expression and the phosphorylation of GSK3β and β-catenin. In vivo, the overexpression of CXCL12 in Apoe−/− mice via lentivirus enlarged the atherosclerotic lesion area and increased macrophage infiltration in atherosclerotic plaques. We further found that the overexpression of CXCL12 reduced the efficiency of reverse cholesterol transport and plasma HDL-C levels, decreased ABCA1 expression in the aorta and mouse peritoneal macrophages (MPMs), and suppressed cholesterol efflux from MPMs to apoA-I in Apoe−/− mice. Collectively, these findings suggest that CXCL12 interacts with CXCR4 and then activates the GSK-3β/β-cateninT120/TCF21 signaling pathway to inhibit ABCA1-dependent cholesterol efflux from macrophages and aggravate atherosclerosis. Targeting CXCL12 may be a novel and promising strategy for the prevention and treatment of atherosclerotic cardiovascular diseases. Atherosclerosis is a chronic vascular disease that has been identified as one of the pathogenesis of cardiovascular disease. It is well known that atherosclerosis is driven by the dysregulation of cholesterol metabolism, leading to the formation of foam cells, a hallmark of atherosclerosis (1.Yu X.H. Zhang D.W. Zheng X.L. Tang C.K. C1q tumor necrosis factor-related protein 9 in atherosclerosis: mechanistic insights and therapeutic potential.Atherosclerosis. 2018; 276: 109-116Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Reverse cholesterol transport (RCT) is an important approach in preventing lipid accumulation during atherogenesis (2.Marques L.R. Diniz T.A. Antunes B.M. Rossi F.E. Caperuto E.C. Lira F.S. Gonçalves D.C. Reverse cholesterol transport: molecular mechanisms and the non-medical approach to enhance HDL cholesterol.Front. Physiol. 2018; 9: 526-537Crossref PubMed Scopus (70) Google Scholar, 3.Yu X.H. Zhang D.W. Zheng X.L. Tang C.K. Cholesterol transport system: an integrated cholesterol transport model involved in atherosclerosis.Prog. Lipid Res. 2019; 73: 65-91Crossref PubMed Scopus (120) Google Scholar). ABCA1 locates in the plasma membrane, serving as a cholesterol transporter to mediate excessive cholesterol efflux from macrophages to apoA-I. This is believed to be the first and most important step of RCT. Indeed, several lines of evidence have demonstrated that ABCA1 expression and cholesterol efflux are inversely associated with the development and progression of atherosclerosis (4.Gao J.H. Zeng M.Y. Yu X.H. Zeng G.F. He L.H. Zheng X.L. Zhang D.W. Ouyang X.P. Tang C.K. Visceral adipose tissue-derived serine protease inhibitor accelerates cholesterol efflux by up-regulating ABCA1 expression via the NF-κB/miR-33a pathway in THP-1 macrophage-derived foam cells.Biochem. Biophys. Res. Commun. 2018; 500: 318-324Crossref PubMed Scopus (16) Google Scholar, 5.Zhang M. Li L. Xie W. Wu J.F. Yao F. Tan Y.L. Xia X.D. Liu X.Y. Liu D. Lan G. Apolipoprotein A-1 binding protein promotes macrophage cholesterol efflux by facilitating apolipoprotein A-1 binding to ABCA1 and preventing ABCA1 degradation.Atherosclerosis. 2016; 248: 149-159Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 6.Zhang M. Zhao G.J. Yin K. Xia X.D. Gong D. Zhao Z.W. Chen L.Y. Zheng X.L. Tang X.E. Tang C.K. Apolipoprotein A-1 binding protein inhibits inflammatory signaling pathways by binding to apolipoprotein A-1 in THP-1 macrophages.Circ. J. 2018; 82: 1396-1404Crossref PubMed Scopus (29) Google Scholar, 7.Zhao Z.W. Zhang M. Chen L.Y. Gong D. Xia X.D. Yu X.H. Wang S.Q. Ou X. Dai X.Y. Zheng X.L. Heat shock protein 70 accelerates atherosclerosis by downregulating the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.Biochim Biophys Acta Mol Cell Biol Lipids. 2018; 1863: 806-822Crossref PubMed Scopus (32) Google Scholar). Thus, understanding how ABCA1 expression is regulated has always been an important research topic. Chemokines are a group of molecules that mainly function on cell chemotaxis and are extensively expressed in vascular cells, such as macrophages, endothelial cells (ECs), and smooth muscle cells (SMCs). CXC chemokine ligand 12 (CXCL12) belongs to the CXC subclass of the chemokine family. Several clinical reports have shown that higher serum levels of CXCL12 are closely related to an increase in atherosclerotic risk (8.Mehta N.N. Li M. William D. Khera A.V. DerOhannessian S. Qu L. Ferguson J.F. McLaughlin C. Shaikh L.H. Shah R. et al.The novel atherosclerosis locus at 10q11 regulates plasma CXCL12 levels.Eur. Heart J. 2011; 32: 963-971Crossref PubMed Scopus (59) Google Scholar, 9.Camnitz W. Burdick M.D. Strieter R.M. Mehrad B. Keeley E.C. Dose-dependent effect of statin therapy on circulating CXCL12 levels in patients with hyperlipidemia.Clin. Transl. Med. 2012; 1: 23-28Crossref PubMed Google Scholar, 10.Farouk S.S. Rader D.J. Reilly M.P. Mehta N.N. CXCL12: a new player in coronary disease identified through human genetics.Trends Cardiovasc. Med. 2010; 20: 204-209Crossref PubMed Scopus (39) Google Scholar, 11.Tavakolian Ferdousie V. Mohammadi M. Hassanshahi G. Khorramdelazad H. Falahati-Pour S.K. Mirzaei M. Tavakoli M.A. Kamiab Z. Ahmadi Z. Vazirinejad R. Serum CXCL10 and CXCL12 chemokine levels are associated with the severity of coronary artery disease and coronary artery occlusion.Int. J. Cardiol. 2017; 233: 23-28Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The overexpression of CXCL12 can aggravate atherosclerosis progression in Apoe−/− mice (12.Schober A. Knarren S. Lietz M. Lin E.A. Weber C. Crucial role of stromal cell-derived factor-1alpha in neointima formation after vascular injury in apolipoprotein E-deficient mice.Circulation. 2003; 108: 2491-2497Crossref PubMed Scopus (186) Google Scholar, 13.Gao J-H. Yu X-H. Tang C-K. CXC chemokine ligand 12 (CXCL12) in atherosclerosis: an underlying therapeutic target.Clin. Chim. Acta. 2019; 495: 538-544Crossref PubMed Scopus (24) Google Scholar). CXC chemokine receptor 4 (CXCR4), a specific receptor for CXCL12, is strongly correlated with plaque stability, lesion area, and risk for cardiovascular disease (14.Weiberg D. Thackeray J.T. Daum G. Sohns J.M. Kropf S. Wester H.J. Ross T.L. Bengel F.M. Derlin T. Clinical molecular imaging of chemokine receptor CXCR4 expression in atherosclerotic plaque using (68)Ga-pentixafor PET: correlation with cardiovascular risk factors and calcified plaque burden.J. Nucl. Med. 2018; 59: 266-272Crossref PubMed Scopus (71) Google Scholar, 15.Zernecke A. Schober A. Bot I. von Hundelshausen P. Liehn E.A. Mopps B. Mericskay M. Gierschik P. Biessen E.A. Weber C. SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells.Circ. Res. 2005; 96: 784-791Crossref PubMed Scopus (323) Google Scholar, 16.Liu Z. Han Y. Li L. Lu H. Meng G. Li X. Shirhan M. Peh M.T. Xie L. Zhou S. et al.The hydrogen sulfide donor, GYY4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E(−/−) mice.Br. J. Pharmacol. 2013; 169: 1795-1809Crossref PubMed Scopus (138) Google Scholar). Merckelbach et al. (17.Merckelbach S. Epc V.D.V. Kallmayer M. Rischpler C. Burgkart R. Döring Y. J. G. Schwaiger M. Eckstein H.H. Weber C. Expression and cellular localization of CXCR4 and CXCL12 in human carotid atherosclerotic plaques.Thromb. Haemost. 2018; 118: 195-206Crossref PubMed Scopus (37) Google Scholar) reported that CXCL12 and CXCR4 were abundantly expressed in macrophages within human carotid artery atherosclerosis. It has been shown that CXCL12 stimulates the formation of macrophage-derived foam cells via CXCR4 (18.Chatterjee M. von Ungern-Sternberg S.N. Seizer P. Schlegel F. Buttcher M. Sindhu N.A. Muller S. Mack A. Gawaz M. Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4-CXCR7.Cell Death Dis. 2015; 6: e1989Crossref PubMed Scopus (110) Google Scholar). However, the role of CXCL12 in cholesterol efflux is unclear. Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine kinase that is primarily involved in cellular glucose metabolism (19.Eldar-Finkelman H. Glycogen synthase kinase 3: an emerging therapeutic target.Trends Mol. Med. 2002; 8: 126-132Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). The activation and knockdown of GSK3β accelerate and inhibit atherosclerosis, respectively (20.McAlpine C.S. Bowes A.J. Khan M.I. Shi Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3beta activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 82-91Crossref PubMed Scopus (46) Google Scholar, 21.Choi S.E. Jang H.J. Kang Y. Jung J.G. Han S.J. Kim H.J. Kim D.J. Lee K.W. Atherosclerosis induced by a high-fat diet is alleviated by lithium chloride via reduction of VCAM expression in Apoe-deficient mice.Vascul. Pharmacol. 2010; 53: 264-272Crossref PubMed Scopus (37) Google Scholar). Although GSK3β has been implicated in dyslipidemia during atherogenesis, the role of GSK3β in ABCA1-mediated cholesterol efflux has yet to be determined. β-catenin belongs to the catenin family and commonly acts as a nuclear transcriptional activator to activate the transcription of target genes when Wnt/β-catenin is activated (22.Valenta T. Hausmann G. Basler K. The many faces and functions of beta-catenin.EMBO J. 2012; 31: 2714-2736Crossref PubMed Scopus (1028) Google Scholar). GSK3β-mediated change in protein phosphorylation regulates the translocation of β-catenin from the cytoplasm to the nucleus and the subsequent GSK3β/β-catenin pathway. This has been implicated in the development of cardiovascular disease (23.Lin H. Li Y. Zhu H. Wang Q. Chen Z. Chen L. Zhu Y. Zheng C. Wang Y. Liao W. et al.Lansoprazole alleviates pressure overload-induced cardiac hypertrophy and heart failure in mice by blocking the activation of beta-catenin.Cardiovasc. Res. January 24, 2019; (Epub ahead of print.)doi:10.1093/cvr/cvz016PubMed Google Scholar, 24.Xu B. Wang T. Xiao J. Dong W. Wen H.Z. Wang X. Qin Y. Cai N. Zhou Z. Xu J. et al.FCPR03, a novel phosphodiesterase 4 inhibitor, alleviates cerebral ischemia/reperfusion injury through activation of the AKT/GSK3beta/ beta-catenin signaling pathway.Biochem. Pharmacol. 2019; 163: 234-249Crossref PubMed Scopus (31) Google Scholar, 26.Sharma M. Chuang W.W. Zijie S. Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation.J. Biol. Chem. 2002; 277: 30935-30941Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Transcription factor 21 (TCF21) is a member of the basic helix-loop-helix transcription factor (TCF) family and affects the development of coronary vasculature (27.Hidai H. Bardales R. Goodwin R. Quertermous T. Quertermous E.E. Cloning of capsulin, a basic helix-loop-helix factor expressed in progenitor cells of the pericardium and the coronary arteries.Mech. Dev. 1998; 73: 33-43Crossref PubMed Scopus (92) Google Scholar). Lu et al. (28.Lu X. Laiyuan W. Shufeng C. Lin H. Xueli Y. Yongyong S. Jing C. Liang Z. Charles G.C. Jianfeng H. Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease.Nat. Genet. 2012; 44: 890-894Crossref PubMed Scopus (250) Google Scholar) found that TCF21 was associated with the risk of coronary artery disease (CAD) in a Chinese Han population. The inhibition of TCF21 expression dramatically increases CAD risk (29.Miller C.L. Ulrike H. Roxanne D. Leeper N.J. Kundu R.K. Bhagat P. Assimes T.L. Kaiser F.J. Ljubica P. Ulf H. Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation.PLoS Genet. 2014; 10: e1004263Crossref PubMed Scopus (88) Google Scholar). However, the underlying mechanisms remain to be addressed. In this study, we found that the binding of CXCL12 to CXCR4 activated the GSK3β/β-cateninT120/TCF21pathway. This downregulated ABCA1 expression and reduced cholesterol efflux. Further, our data confirmed the proatherogenic role of CXCL12 in Apoe−/− mice. Apoe−/− male mice from a C57BL/6 background (8 weeks old) were purchased from Cavens Animal Research Laboratories, Changzhou, China. Apoe−/− mice were injected with 1 × 1011 viral particles of lentiviral vector (LV)-CXCL12 or LV via the tail vein and then fed the Western-type diet for 12 weeks. Afterward, mice were euthanized using pentobarbital sodium. Blood samples and tissues were collected. Prior to euthanasia, mice were intraperitoneally injected with 4% thioglycollate broth, followed by an injection of 5 ml PBS for the collection of peritoneal macrophages. All procedures were conducted in accordance with the Institutional Animal Ethics Committee and the University of South China Animal Care Guidelines for the Use of Experimental Animals. Aortas were dissected from mice with all adventitia removed. The quantification of lesion size and composition is in agreement with Arteriosclerosis, Thrombosis, and Vascular Biology guidelines and previous studies (30.Daugherty A. Tall A.R. Mjap D. Falk E. Fisher E.A. Garcíacardeña G. Lusis A.J. Rd O.A. Rosenfeld M.E. Virmani R. Recommendation on design, execution, and reporting of animal atherosclerosis studies: a scientific statement from the American Heart Association.Arterioscler. Thromb. Vasc. Biol. 2017; 37: e131-e157Crossref PubMed Scopus (204) Google Scholar, 31.Zhang M. Zhao G.J. Yao F. Xia X.D. Gong D. Zhao Z.W. Chen L.Y. Zheng X.L. Tang X.E. Tang C.K. AIBP reduces atherosclerosis by promoting reverse cholesterol transport and ameliorating inflammation in Apoe −/− mice.Atherosclerosis. 2018; 273: 122-130Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Aortas were then unfolded along the longitudinal axis, stained with Oil Red O, and photographed with a CASIO EX-ZR3700 digital camera en face to measure the percentage of total atherosclerotic lesions on the aortic surface. Mouse hearts were sectioned perpendicular to the axis of the aorta once the aortic root was identified by the appearance of aortic valve leaflets. Eight serial sections (10 μm intervals) of aortic sinus were obtained per mouse. We randomly selected three sections from each mouse per group for Oil Red O, H&E, and Masson staining. The lesion areas were quantified with Oil Red O staining, and the collagen contents were quantified with Masson staining. All analyses were quantified using Image J software. Plasma lipid levels were analyzed as previously described (7.Zhao Z.W. Zhang M. Chen L.Y. Gong D. Xia X.D. Yu X.H. Wang S.Q. Ou X. Dai X.Y. Zheng X.L. Heat shock protein 70 accelerates atherosclerosis by downregulating the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.Biochim Biophys Acta Mol Cell Biol Lipids. 2018; 1863: 806-822Crossref PubMed Scopus (32) Google Scholar). Blood samples were collected from the retro-orbital plexus of Apoe−/− mice injected with or without LV-CXCL12 or saline and fed the Western-type diet for 12 weeks. Plasma levels of total cholesterol (TC), LDL-C, HDL-C, and triglyceride (TG) levels were analyzed by enzymatic methods using their specific kits (BioSino Bio-Technology and Science Inc.). The sections of aortic roots were washed with 1× PBS three times and blocked with 5% goat serum for 30 min at room temperature. THP-1-derived macrophages were incubated with or without CXCL12, washed with PBS, and then fixed with methanol. Afterward, the sections and cells were incubated with anti-ABCA1 antibody (mouse monoclonal antibody; 1:200; Abcam), anti-CD68 antibody (mouse monoclonal antibody; 1:200; Abcam), or anti-CXCR4 antibody (mouse monoclonal antibody; 1:200; Abcam) overnight at 4°C, followed by Cy3-labeled goat anti-mouse IgG (1:200; Beyotime) for 1 h at room temperature in a dark place. Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Fluorescence microscopy of ABCA1 and CD68 in the sections and ABCA1 and CXCR4 in cells were performed using an EVOS FL AUTO 2 (Thermo Fisher Scientific). The mean fluorescence intensity on the stained sections of aortic roots and cells were quantified using Image J software. Mice aorta root sections were incubated with UV block (Thermo Fisher Scientific) containing 10% goat serum (Abcam) for 30 min and then with mouse monoclonal anti-CD68 antibody (1:1000; Abcam) overnight in a humid chamber at 4°C. Afterward, sections were incubated with a biotin-conjugated secondary antibody for 1 h at 37°C, followed by streptavidin-HRP for 30 min. Sections were then counterstained with hematoxylin for 15 s, differentiated with a hydrochloric acid/alcohol mixture, and then stained with 3,3′-diaminobenzidine for 30 s. After washing with water for 15 min, sections were imaged using an EVOS FL AUTO 2 (Thermo Fisher Scientific). The efficiency of RCT was determined as previously described (7.Zhao Z.W. Zhang M. Chen L.Y. Gong D. Xia X.D. Yu X.H. Wang S.Q. Ou X. Dai X.Y. Zheng X.L. Heat shock protein 70 accelerates atherosclerosis by downregulating the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.Biochim Biophys Acta Mol Cell Biol Lipids. 2018; 1863: 806-822Crossref PubMed Scopus (32) Google Scholar). J774.1 macrophages were loaded with 50 μg/ml ac-LDL and 5 μCi/ml [3H]cholesterol for 24 h in DMEM. The labeled J774 cells were injected into the abdominal cavity of individual mice. Plasma was collected at 6, 24, and 48 h after injection. The feces were continuously collected from 0 to 48 h until the endpoint of the experiment. The feces were weighed and dissolved in 50% ethanol. Aliquots (20 μl) were used for scintillation counting after shaking overnight. The liver was collected from euthanized mice, washed in ice-cold PBS, blotted up with filter papers, weighed, and stored at −20°C. Frozen liver tissue (80 mg) was added into n-hexane and isopropanol at a proportion of 3:2 with shaking for 10 min. Afterward, the samples were vacuum-dried for the extraction of liver lipids, and radioactivity was counted with a liquid scintillation counter. The results were calculated as the percentage of injected = cpm (plasma, liver, or feces)/total cpm (5 μCi/ml [3H]cholesterol). Human THP-1 monocytes were purchased from the Chinese Academy of Sciences cell bank and cultured in RPMI-1640 supplemented with 0.1% nonessential amino acids, penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% FBS. HEK293T cells were purchased from the Chinese Academy of Sciences cell bank and cultured in DMEM containing 10% FBS. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. The differentiation of THP-1 monocytes into macrophages was induced by 160 nM phorbol-12-myristate acetate for 24 h. THP-1-derived macrophages and HEK293T cells were cultured in 12-well plates for different treatments. β-catenin agonist SKL2001 (MedChemExpress), GSK3β inhibitor TWS119 (MedChemExpress), CXCR4 inhibitor LY2510924 (MedChem­Express), or recombinant human CXCL12 protein (Abcam) was incubated with cells. CXCR4 siRNA (Abcam) and TCF21 (Santa Cruz Biotechnology) were introduced to cells by transfection and LV, respectively. Approximately 6 to 12 h later, the medium was changed to DMEM containing antibiotics and 10% FBS. Tissues and cell-derived total proteins were extracted by the regular method. Nuclear and cytoplasmic proteins were extracted with a nuclear and cytoplasmic protein extraction kit (Sangon Biotech). β-actin was used as the loading control for total proteins derived from tissues and cells and cytoplasmic proteins. H3 was used as the loading control for nuclear fractions (32.Li B. He J. Lv H. Liu Y. Lv X. Zhang C. Zhu Y. Ai D. c-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow.J. Clin. Invest. 2019; 129: 1167-1179Crossref PubMed Scopus (63) Google Scholar). Total proteins were quantified and subjected to SDS-PAGE, followed by immunoblotting. Primary antibodies included anti-GSK3β (1:1000; Santa Cruz Biotechnology), anti-phospho-GSK3β (1:1000; Santa Cruz Biotechnology), anti-β-catenin (1:1000; Santa Cruz Biotechnology), anti-phospho-β-cateninY654 (1:1000; Abcam), anti-phospho-β-cateninT120 (1:2000; Santa Cruz Biotechnology), anti-CXCR4 (1:200; Santa Cruz Biotechnology), anti-ABCA1 (1:200; Santa Cruz Biotechnology), anti-TCF21 (1:10000; Santa Cruz Biotechnology), anti-histone H3 (1:500; Santa Cruz Biotechnology), and anti-β-actin (1:2000; Santa Cruz Biotechnology). Secondary antibodies were HRP-labeled goat anti-mouse IgG (1:1000; Santa Cruz Biotechnology). Antibody binding was visualized with a Tanon 5500 and BeyoECL Plus (Beyotime). THP-1-derived macrophages were incubated with CXCL12 and then homogenized and lysed in RIPA buffer. Protein A plus-agarose (Thermo Fisher Scientific) was washed in PBS three times. Lysates were incubated with antibodies overnight at 4°C, followed by an incubation with prewashed protein A plus-agarose overnight at 4°C. Beads were then washed three times in PBS. Immunoprecipitated proteins were eluted from the bead with 60 μl 2× SDS sample buffer (Beyotime) containing 2-mercaptoethanol at 60°C for 5 min and then subjected to immunoblotting. Cholesterol efflux was performed as described previously (4.Gao J.H. Zeng M.Y. Yu X.H. Zeng G.F. He L.H. Zheng X.L. Zhang D.W. Ouyang X.P. Tang C.K. Visceral adipose tissue-derived serine protease inhibitor accelerates cholesterol efflux by up-regulating ABCA1 expression via the NF-κB/miR-33a pathway in THP-1 macrophage-derived foam cells.Biochem. Biophys. Res. Commun. 2018; 500: 318-324Crossref PubMed Scopus (16) Google Scholar). THP-1-derived macrophages were incubated with or without CXCL12 for 12 h and then cultured in 0.1% BSA and RPMI 1640 medium for 24 h. Peritoneal macrophages were isolated from Apoe−/− mice and cultured in RPMI 1640 medium containing 0.1% BSA for 24 h. Mouse peritoneal macrophages (MPMs) and THP-1-derived macrophages were incubated with 50 μg/ml ox-LDL and then labeled with 0.5 μCi/ml [3H]cholesterol for 24 h in serum-free medium. Afterward, the cells were washed in PBS and then cultured in RPMI 1640 medium containing 0.1% BSA and 25 μg/ml apoA-I or 50 μg/ml HDL (Sigma-Aldrich) for 24 h. Radioactivity in culture medium and cells was counted in a liquid scintillation counter separately. Cholesterol efflux was calculated as the ratio of [3H]cholesterol in medium to total [3H]cholesterol in cells and medium. The lipid content in THP-1-derived macrophages was examined as previously described (4.Gao J.H. Zeng M.Y. Yu X.H. Zeng G.F. He L.H. Zheng X.L. Zhang D.W. Ouyang X.P. Tang C.K. Visceral adipose tissue-derived serine protease inhibitor accelerates cholesterol efflux by up-regulating ABCA1 expression via the NF-κB/miR-33a pathway in THP-1 macrophage-derived foam cells.Biochem. Biophys. Res. Commun. 2018; 500: 318-324Crossref PubMed Scopus (16) Google Scholar). THP-1-derived macrophages were washed three times with PBS. The cells were homogenized in 1 ml 0.9% NaCl on ice using the ultrasonic processor (Cole-Parmer). Protein concentrations were measured using the BCA protein assay kit (Abcam). Isopropanol (1 mg cholesterol/ml) was added to extract cholesterol and then stored at 20°C. The stock solution was diluted as cholesterol standard liquid and then supplied with 10 μl reaction mixture containing 500 mM MgCl2, 500 mM Tris-HCl (pH 7.4), 10 mM dithiothreitol, and 5% NaCl to 0.1 ml cholesterol standard liquid. Afterward, 0.4 U cholesterol oxidase in 10 μl 0.5% NaCl was added into each sample to examine free cholesterol or 0.4 U cholesterol oxidase together with 0.4 U cholesterol esterase for the measurement of TC. Samples were incubated at 37°C for 30 min. The reaction was then terminated by adding 100 ml methanol-ethanol (1:1). After cooling for 30 min, samples were centrifuged at 1,500 rpm for 10 min at 15°C. Ten microliters of supernatant was applied to a 2790 chromatographer (Waters Corporation) for chromatographic analysis. Absorbance at 216 nm was monitored, with cholesterol content indicated by the peak area. THP-1-derived macrophages, MPMs, and tissues were lysed for RNA extraction using TRIzol. The sequences of the RT-PCR primers were as follows: human ABCA1, 5′-GTCCTCTTTCCCGATTATCTGG-3′ and 5′-CACTCACTCTCGCTCGCAAT-3′; human TCF21, 5′-GGTTAGTTAGGAGGGGAAGTA-3′ and 5′-ACACCCAA­AACAAAATAATCTTA-3′; and mouse ABCA1, 5′-GGGTGGTGTTCTTCCTCATTAC-3′ and 5′-GAATGACGAGGATGAGGATGTG-3′. mRNA levels were analyzed with an ABI PRISM 7900 sequence detection system (Applied Biosystems). The TCF21 expression vector was acquired from Genechem. The luciferase vectors with the promoter region of 3,000 bp of the human ABCA1 were prepared by Genechem. HEK293T cells were treated with CXCL12 or transfected with 0.5 μg TCF21 overexpression vector using Lipofectamine 2000 (Invitrogen). The cells were then cotransfected with 0.5 μg of the ABCA1 promoter reporter construct and renilla luciferase control reporter vector. After 12 h, cells were washed in PBS and incubated in medium containing 0.1% BSA and RPMI 1640. The luciferase activity was detected using the Dual-Glo Luciferase Assay System (Promega). The results were standardized in the corresponding luciferase activity and plotted as a percentage of the control. THP-1-derived macrophages transfected with CXCL12 were cross-linked in 1% formaldehyde for 15 min at 37°C. The reaction was stopped by adding glycine solution. SDS lysis buffer (Beyotime) and PMSF (Beyotime) were then added to the cells before sonication with an ultrasonic processor (Sonics) for 14 bursts of 4.5 s with 9 s intervals under 60 W on the ice. Cell lysates were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant containing sheared chromatin was kept on ice. DNA fragment sizes were measured using agarose gel electrophoresis. Afterward, the samples were subjected to immunoprecipitation using a chromatin immunoprecipitation assay kit (Abcam) and antibody against TCF21 (Abcam) or antibodies against IgG (Abcam). The DNA was eluted and collected for analysis of quantitative RT-PCR using human ABCA1 primers (forward: 5′-CTCGGTGCAGCCGAATCTAT-3′; reverse: 5′-CACTCACTCTCGCTCGCAAT-3′). All data are presented as means ± SDs. The distribution of all data was analyzed by an F-test and showed no significant difference (P > 0.05). Statistical significance was evaluated by either one-way ANOVA or Student's t-test. Student's t-test was used for assessing the differences between two groups, while one-way ANOVA was used for analyzing the difference between multiple groups. Scheffe's test was used for specific comparisons. P < 0.05 was considered statistically significant. To investigate the role of CXCL12 in lipid metabolism, we treated THP-1-derived macrophages with CXCL12. As shown in Fig. 1A, CXCL12 reduced the mRNA and protein levels of ABCA1. To confirm this finding, we detected ABCA1 in THP-1-derived macrophages using immunofluorescence micros" @default.
• W2982697290 created "2019-11-08" @default.
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• W2982697290 date "2019-12-01" @default.
• W2982697290 modified "2023-10-18" @default.
• W2982697290 title "CXCL12 promotes atherosclerosis by downregulating ABCA1 expression via the CXCR4/GSK3β/β-cateninT120/TCF21 pathway" @default.
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