Abstract
Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPβ regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPβ knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPβ-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPβ attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPβ knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPβ inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.
Similar content being viewed by others
Abbreviations
- C/EBPβ:
-
CCAAT/enhancer-binding protein beta
- ERS:
-
Endoplasmic reticulum stress
- ATG5:
-
Autophagy gene 5
- ATF4:
-
Activating transcription factor 4
- ATF6:
-
Activating transcription factor 6
- LC3-I & II:
-
Microtubule-associated protein 1 light chain 3-I & II
- CHOP:
-
CCAAT/enhancer-binding protein (C/EBP) homologous protein
- mTOR:
-
Mammalian target of rapamycin
- mTORC1:
-
mTOR complex 1
- mTORC2:
-
mTOR complex 2
- ox-LDL:
-
Oxidized low-density lipoproteins
References
Davies LC, Jenkins SJ, Allen JE, Taylor PR (2013) Tissue-resident macrophages. Nat Immunol 14:986–995. https://doi.org/10.1038/ni.2705
Lusis AJ (2000) Atherosclerosis. Nature 407:233–241. https://doi.org/10.1038/35025203
Mori M, Itabe H, Higashi Y, Fujimoto Y, Shiomi M, Yoshizumi M, Ouchi Y, Takano T (2001) Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum. J Lipid Res 42:1771–1781
Glass CK, Witztum JL (2001) Atherosclerosis. the road ahead. Cell 104:503–516
Hotamisligil GS (2010) Endoplasmic reticulum stress and atherosclerosis. Nat Med 16:396–399. https://doi.org/10.1038/nm0410-396
Oh J, Riek AE, Weng S, Petty M, Kim D, Colonna M, Cella M, Bernal-Mizrachi C (2012) Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation. J Biol Chem 287:11629–11641. https://doi.org/10.1074/jbc.M111.338673
Ross R (1999) Atherosclerosis–an inflammatory disease. N Engl J Med 340:115–126. https://doi.org/10.1056/nejm199901143400207
Bentzon JF, Otsuka F, Virmani R, Falk E (2014) Mechanisms of plaque formation and rupture. Circ Res 114:1852–1866. https://doi.org/10.1161/circresaha.114.302721
Tabas I (2005) Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency. Arterioscler Thromb Vasc Biol 25:2255–2264. https://doi.org/10.1161/01.ATV.0000184783.04864.9f
Seimon T, Tabas I (2009) Mechanisms and consequences of macrophage apoptosis in atherosclerosis. J Lipid Res 50(Suppl):S382–S387. https://doi.org/10.1194/jlr.R800032-JLR200
Arai S, Shelton JM, Chen M, Bradley MN, Castrillo A, Bookout AL, Mak PA, Edwards PA, Mangelsdorf DJ, Tontonoz P, Miyazaki T (2005) A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development. Cell Metab 1:201–213. https://doi.org/10.1016/j.cmet.2005.02.002
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42. https://doi.org/10.1016/j.cell.2007.12.018
Hewitt G, Korolchuk VI (2017) Repair, reuse, recycle: the expanding role of autophagy in genome maintenance. Trends Cell Biol 27:340–351. https://doi.org/10.1016/j.tcb.2016.11.011
Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132. https://doi.org/10.1146/annurev-cellbio-092910-154005
Gotoh T, Endo M, Oike Y (2011) Endoplasmic reticulum stress-related inflammation and cardiovascular diseases. Int J Inflamm 2011:8
Sheng R, Liu X-Q, Zhang L-S, Gao B, Han R, Wu Y-Q, Zhang X-Y, Qin Z-H (2012) Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning. Autophagy 8:310–325
Saitoh T, Fujita N, Jang MH, Uematsu S, Yang B-G, Satoh T, Omori H, Noda T, Yamamoto N, Komatsu M (2008) Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production. Nature 456:264
Levine B, Mizushima N, Virgin HW (2011) Autophagy in immunity and inflammation. Nature 469:323–335. https://doi.org/10.1038/nature09782
Anding AL, Baehrecke EH (2015) Autophagy in cell life and cell death. Curr Top Dev Biol 114:67–91. https://doi.org/10.1016/bs.ctdb.2015.07.012
Ouimet M, Franklin V, Mak E, Liao X, Tabas I, Marcel YL (2011) Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab 13:655–667. https://doi.org/10.1016/j.cmet.2011.03.023
Le Guezennec X, Brichkina A, Huang YF, Kostromina E, Han W, Bulavin DV (2012) Wip1-dependent regulation of autophagy, obesity, and atherosclerosis. Cell Metab 16:68–80. https://doi.org/10.1016/j.cmet.2012.06.003
Yao T, Ying X, Zhao Y, Yuan A, He Q, Tong H, Ding S, Liu J, Peng X, Gao E, Pu J, He B (2015) Vitamin D receptor activation protects against myocardial reperfusion injury through inhibition of apoptosis and modulation of autophagy. Antioxid Redox Signal 22:633–650. https://doi.org/10.1089/ars.2014.5887
Nussenzweig SC, Verma S, Finkel T (2015) The role of autophagy in vascular biology. Circ Res 116:480–488. https://doi.org/10.1161/circresaha.116.303805
Gatica D, Chiong M, Lavandero S, Klionsky DJ (2015) Molecular mechanisms of autophagy in the cardiovascular system. Circ Res 116:456–467. https://doi.org/10.1161/circresaha.114.303788
Tang QQ, Otto TC, Lane MD (2003) CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis. Proc Natl Acad Sci USA 100:850–855. https://doi.org/10.1073/pnas.0337434100
Giltiay NV, Karakashian AA, Alimov AP, Ligthle S, Nikolova-Karakashian MN (2005) Ceramide- and ERK-dependent pathway for the activation of CCAAT/enhancer binding protein by interleukin-1beta in hepatocytes. J Lipid Res 46:2497–2505. https://doi.org/10.1194/jlr.M500337-JLR200
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T (1990) A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:1897–1906
Natsuka S, Akira S, Nishio Y, Hashimoto S, Sugita T, Isshiki H, Kishimoto T (1992) Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood 79:460–466
Akira S, Kishimoto T (1997) NF-IL6 and NF-kappa B in cytokine gene regulation. Adv Immunol 65:1–46
Uematsu S, Kaisho T, Tanaka T, Matsumoto M, Yamakami M, Omori H, Yamamoto M, Yoshimori T, Akira S (2007) The C/EBPβ isoform 34-kDa LAP is responsible for NF-IL-6-mediated gene induction in activated macrophages, but is not essential for intracellular bacteria killing. J Immunol 179:5378–5386
Rahman SM, Janssen RC, Choudhury M, Baquero KC, Aikens RM, de la Houssaye BA, Friedman JE (2012) CCAAT/enhancer-binding protein beta (C/EBPbeta) expression regulates dietary-induced inflammation in macrophages and adipose tissue in mice. J Biol Chem 287:34349–34360. https://doi.org/10.1074/jbc.M112.410613
Rahman SM, Baquero KC, Choudhury M, Janssen RC, Becky A, Sun M, Miyazaki-Anzai S, Wang S, Moustaid-Moussa N, Miyazaki M (2016) C/EBPβ in bone marrow is essential for diet induced inflammation, cholesterol balance, and atherosclerosis. Atherosclerosis 250:172–179
(2019) Real-Time PCR, Applications Guide, Bio-Rad
Rahman SM, Choudhury M, Baquero K, Janssen RC, de la Houssaye BA, Miyazaki-anzai S, Miyazaki M, Majka S, Friedman JE (2010) Macrophage-specific deletion of ccaat/enhancer protein beta (c/ebpβ) in Apoe −/− Mice attenuates inflammation, atherosclerosis, and foam cell formation. Circulation 122:A18763
Tsukano H, Gotoh T, Endo M, Miyata K, Tazume H, Kadomatsu T, Yano M, Iwawaki T, Kohno K, Araki K (2010) The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques. Arterioscler Thromb Vasc Biol 30:1925–1932
Thorp E, Li G, Seimon TA, Kuriakose G, Ron D, Tabas I (2009) Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe −/− and Ldlr −/− mice lacking CHOP. Cell Metabol 9:474–481
Ouimet M (2013) Autophagy in obesity and atherosclerosis: interrelationships between cholesterol homeostasis, lipoprotein metabolism and autophagy in macrophages and other systems. Biochim et Biophys Acta (BBA) 1831:1124–1133
Zhang B-C, Zhang C-W, Wang C, Pan D-F, Xu T-D, Li D-Y (2016) Luteolin attenuates foam cell formation and apoptosis in Ox-LDL-stimulated macrophages by enhancing autophagy. Cell Physiol Biochem 39:2065–2076
Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P (2008) Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie 90:313–323. https://doi.org/10.1016/j.biochi.2007.08.014
Kim J, Kundu M, Viollet B, Guan K-L (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13:132
Kim YC, Guan KL (2015) mTOR: a pharmacologic target for autophagy regulation. J Clin Invest 125:25–32. https://doi.org/10.1172/jci73939
Hutchins PM, Heinecke JW (2015) Cholesterol efflux capacity, macrophage reverse cholesterol transport, and cardioprotective HDL. Curr Opin Lipidol 26:388
Chistiakov DA, Melnichenko AA, Myasoedova VA, Grechko AV, Orekhov AN (2017) Mechanisms of foam cell formation in atherosclerosis. J Mol Med 95:1153–1165
Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T (2005) Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169:425–434. https://doi.org/10.1083/jcb.200412022
Komatsu M, Waguri S, Chiba T, Murata S, Iwata J-i, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441:880–884
Sahani MH, Itakura E, Mizushima N (2014) Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids. Autophagy 10:431–441
Klionsky DJ, Cregg JM, Dunn WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M (2003) A unified nomenclature for yeast autophagy-related genes. Dev Cell 5:539–545
Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins. Autophagy 7:279–296
Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477
Zhang X, Evans TD, Jeong SJ, Razani B (2018) Classical and alternative roles for autophagy in lipid metabolism. Curr Opin Lipidol 29:203–211. https://doi.org/10.1097/mol.0000000000000509
Ouimet M, Ediriweera H, Afonso MS, Ramkhelawon B, Singaravelu R, Liao X, Bandler RC, Rahman K, Fisher EA, Rayner KJ, Pezacki JP, Tabas I, Moore KJ (2017) microRNA-33 regulates macrophage autophagy in atherosclerosis. Arterioscler Thromb Vasc Biol 37:1058–1067. https://doi.org/10.1161/atvbaha.116.308916
Qiao L, Zhang X, Liu M, Liu X, Dong M, Cheng J, Zhang X, Zhai C, Song Y, Lu H (2017) Ginsenoside Rb1 enhances atherosclerotic plaque stability by improving autophagy and lipid metabolism in macrophage foam cells. Front Pharmacol 8:727
Scull CM, Tabas I (2011) Mechanisms of ER stress-induced apoptosis in atherosclerosis. Arterioscler Thromb Vasc Biol 31:2792–2797
Kavurma MM, Rayner KJ, Karunakaran D (2017) The walking dead: macrophage inflammation and death in atherosclerosis. Curr Opin Lipidol 28:91–98. https://doi.org/10.1097/mol.0000000000000394
Li Y, Schwabe RF, DeVries-Seimon T, Yao PM, Gerbod-Giannone M-C, Tall AR, Davis RJ, Flavell R, Brenner DA, Tabas I (2005) Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-α and interleukin-6 model of NF-κB-and map kinase-dependent inflammation in advanced atherosclerosis. J Biol Chem 280:21763–21772
Hoseini Z, Sepahvand F, Rashidi B, Sahebkar A, Masoudifar A, Mirzaei H (2018) NLRP3 inflammasome: its regulation and involvement in atherosclerosis. J Cell Physiol 233:2116–2132. https://doi.org/10.1002/jcp.25930
Tabas I (2010) The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ Res 107:839–850. https://doi.org/10.1161/circresaha.110.224766
Tse G, Yan BP, Chan YW, Tian XY, Huang Y (2016) Reactive oxygen species, endoplasmic reticulum stress and mitochondrial dysfunction: the link with cardiac arrhythmogenesis. Front Physiol 7:313
Sun Y, Zhang D, Liu X, Li X, Liu F, Yu Y, Jia S, Zhou Y, Zhao Y (2018) Endoplasmic reticulum stress affects lipid metabolism in atherosclerosis via CHOP activation and over-expression of miR-33. Cell Physiol Biochem 48:1995–2010
Razani B, Feng C, Coleman T, Emanuel R, Wen H, Hwang S, Ting JP, Virgin HW, Kastan MB, Semenkovich CF (2012) Autophagy links inflammasomes to atherosclerotic progression. Cell Metab 15:534–544
Leng S, Iwanowycz S, Saaoud F, Wang J, Wang Y, Sergin I, Razani B, Fan D (2016) Ursolic acid enhances macrophage autophagy and attenuates atherogenesis. J Lipid Res 57:1006–1016
Shimobayashi M, Hall MN (2014) Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 15:155–162
Laplante M, Sabatini DM (2013) Regulation of mTORC1 and its impact on gene expression at a glance. The Company of Biologists Ltd, Cambridge
Buss SJ, Muenz S, Riffel JH, Malekar P, Hagenmueller M, Weiss CS, Bea F, Bekeredjian R, Schinke-Braun M, Izumo S (2009) Beneficial effects of Mammalian target of rapamycin inhibition on left ventricular remodeling after myocardial infarction. J Am Coll Cardiol 54:2435–2446
Sciarretta S, Zhai P, Shao D, Maejima Y, Robbins J, Volpe M, Condorelli G, Sadoshima J (2012) Rheb is a critical regulator of autophagy during myocardial ischemia: pathophysiological implications in obesity and metabolic syndrome. Circulation 125(9):1134–1146
Settembre C, Di Malta C, Polito VA, Arencibia MG, Vetrini F, Erdin S, Erdin SU, Huynh T, Medina D, Colella P (2011) TFEB links autophagy to lysosomal biogenesis. Science 332:1429–1433
Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Erdin S, Huynh T, Ferron M, Karsenty G, Vellard MC (2012) A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J 31:1095–1108
Emanuel R, Sergin I, Bhattacharya S, Turner J, Epelman S, Settembre C, Diwan A, Ballabio A, Razani B (2014) Induction of lysosomal biogenesis in atherosclerotic macrophages can rescue lipid-induced lysosomal dysfunction and downstream sequelae. Arterioscler Thromb Vasc Biol 114:303342
Acknowledgements
This research was funded by the American Heart Association (AHA) Beginning Grant In Aid and the Startup Fund (Texas Tech University), USA to SMR.
Author information
Authors and Affiliations
Contributions
SMR designed and supervised the research. MKZ, MR, and CP performed the experiments. MKZ, CP, and SMR analyzed the data. MKZ and SMR wrote the manuscript. MC, NMM, MR and SMR revised the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
All the authors declared that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zahid, M.K., Rogowski, M., Ponce, C. et al. CCAAT/enhancer-binding protein beta (C/EBPβ) knockdown reduces inflammation, ER stress, and apoptosis, and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells. Mol Cell Biochem 463, 211–223 (2020). https://doi.org/10.1007/s11010-019-03642-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-019-03642-4