Original articles
Hui Qian, Qingyun Bai, Xiao Yang, Jephte Y. Akakpo, Lili Ji, Li Yang, Thomas Rülicke, Kurt Zatloukal, Hartmut Jaeschke, Hong-Min Ni, Wen-Xing Ding. Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice[J]. Acta Pharmaceutica Sinica B, 2021, 11(12): 3791-3805

Dual roles of p62/SQSTM1 in the injury and recovery phases of acetaminophen-induced liver injury in mice
Hui Qiana, Qingyun Baia,b,c, Xiao Yanga,b, Jephte Y. Akakpoa, Lili Jib, Li Yangb, Thomas Rülicked, Kurt Zatloukale, Hartmut Jaeschkea, Hong-Min Nia, Wen-Xing Dinga
a. Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, USA;
b. The MOE Key Laboratory for Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines and the SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
c. School of Chemistry and Bioengineering, Yichun University, Yichun 336000, China;
d. Department of Biomedical Sciences, University of Veterinary Medicine Vienna Veterinärplatz, Vienna 1210, Austria;
e. The Institute of Pathology, Medical University of Graz, Graz A-8036, Austria
Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.
Key words:    Autophagy    Coagulation    DILI    Liver regeneration    Macrophage    Hepatotoxicity    Platelet   
Received: 2021-10-30     Revised: 2021-11-05
DOI: 10.1016/j.apsb.2021.11.010
Funds: This study was supported by fundings U01 AA024733, R37 AA020518, R21 AA027250, R01 DK102142, and R01 AG072895 (USA).
Corresponding author: Wen-Xing Ding,E-mail:wxding@kumc.edu     Email:wxding@kumc.edu
Author description:
PDF(KB) Free
Hui Qian
Qingyun Bai
Xiao Yang
Jephte Y. Akakpo
Lili Ji
Li Yang
Thomas Rülicke
Kurt Zatloukal
Hartmut Jaeschke
Hong-Min Ni
Wen-Xing Ding

[1] Mowry JB, Spyker DA, Brooks DE, Zimmerman A, Schauben JL. 2015 annual report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 33rd annual report. Clin Toxicol (Phila) 2016;54:924-1109
[2] Lee WM. Acetaminophen (APAP) hepatotoxicity-isn’t it time for APAP to go away?. J Hepatol 2017;67:1324-1331
[3] Larson AM. Acetaminophen hepatotoxicity. Clin Liver Dis 2007;11:525-548
[4] Ostapowicz G, Fontana RJ, Schioedt FV, Larson A, Davern TJ, Han SH, et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 2002;137:947-954
[5] Lee WM. Acetaminophen-related acute liver failure in the United States. Hepatol Res 2008;38:S3-S8
[6] Budnitz DS, Lovegrove MC, Crosby AE. Emergency department visits for overdoses of acetaminophen-containing products. Am J Prev Med 2011;40:585-592
[7] Heard KJ. Acetylcysteine for acetaminophen poisoning. N Engl J Med 2008;359:285-292
[8] McGill MR, Sharpe MR, Williams CD, Taha M, Curry SC, Jaeschke H. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation. J Clin Invest 2012;122:1574-1583
[9] Hinson JA, Roberts DW, James LP. Mechanisms of acetaminophen-induced liver necrosis. Handb Exp Pharmacol 2010;(196):369-405
[10] Yan M, Huo Y, Yin S, Hu H. Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions. Redox Biol 2018;17:274-283
[11] Ramachandran A, Jaeschke H. Acetaminophen toxicity: novel insights into mechanisms and future perspectives. Gene Expr 2018;18:19-30
[12] Huebener P, Pradere JP, Hernandez C, Gwak GY, Caviglia JM, Mu X, et al. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis. J Clin Invest 2015;125:539-550
[13] He Y, Feng D, Li M, Gao Y, Ramirez T, Cao H, et al. Hepatic mitochondrial DNA/Toll-like receptor 9/microRNA-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice. Hepatology 2017;66:220-234
[14] Ganey PE, Luyendyk JP, Newport SW, Eagle TM, Maddox JF, Mackman N, et al. Role of the coagulation system in acetaminophen-induced hepatotoxicity in mice. Hepatology 2007;46:1177-1186
[15] Holt MP, Cheng L, Ju C. Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. J Leukoc Biol 2008;84:1410-1421
[16] Groeneveld D, Cline-Fedewa H, Baker KS, Williams KJ, Roth RA, Mittermeier K, et al. Von Willebrand factor delays liver repair after acetaminophen-induced acute liver injury in mice. J Hepatol 2020;72:146-155
[17] Ni HM, McGill MR, Chao X, Du K, Williams JA, Xie Y, et al. Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice. J Hepatol 2016;65:354-362
[18] Williams JA, Ni HM, Haynes A, Manley S, Li Y, Jaeschke H, et al. Chronic deletion and acute knockdown of Parkin have differential responses to acetaminophen-induced mitophagy and liver injury in mice. J Biol Chem 2015;290:10934-10946
[19] Wang H, Ni HM, Chao X, Ma X, Rodriguez YA, Chavan H, et al. Double deletion of PINK1 and Parkin impairs hepatic mitophagy and exacerbates acetaminophen-induced liver injury in mice. Redox Biol 2019;22:101148
[20] Katsuragi Y, Ichimura Y, Komatsu M. Regulation of the Keap1-Nrf2 pathway by p62/SQSTM1. Curr Opin Toxicol 2016;1:54-61
[21] Manley S, Williams JA, Ding WX. Role of p62/SQSTM1 in liver physiology and pathogenesis. Exp Biol Med (Maywood) 2013;238:525-538
[22] Katsuragi Y, Ichimura Y, Komatsu M. p62/SQSTM 1 functions as a signaling hub and an autophagy adaptor. FEBS J 2015;282:4672-4678
[23] Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007;282:24131-24145
[24] Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Nat Cell Biol 2010;12:213-223
[25] Lau A, Wang XJ, Zhao F, Villeneuve NF, Wu T, Jiang T, et al. A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p62. Mol Cell Biol 2010;30:3275-3285
[26] Jain A, Lamark T, Sjottem E, Larsen KB, Awuh JA, Overvatn A, et al. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J Biol Chem 2010;285:22576-22591
[27] Reisman SA, Csanaky IL, Aleksunes LM, Klaassen CD. Altered disposition of acetaminophen in Nrf2-null and Keap1-knockdown mice. Toxicol Sci 2009;109:31-40
[28] Gum SI, Cho MK. Recent updates on acetaminophen hepatotoxicity: the role of nrf2 in hepatoprotection. Toxicol Res 2013;29:165-172
[29] Ni HM, Boggess N, McGill MR, Lebofsky M, Borude P, Apte U, et al. Liver-specific loss of Atg5 causes persistent activation of Nrf2 and protects against acetaminophen-induced liver injury. Toxicol Sci 2012;127:438-450
[30] Sanchez-Martin P, Saito T, Komatsu M. p62/SQSTM1: 'Jack of all trades' in health and cancer. FEBS J 2019;286:8-23
[31] Moscat J, Karin M, Diaz-Meco MT. p62 in cancer: signaling adaptor beyond autophagy. Cell 2016;167:606-609
[32] Lahiri P, Schmidt V, Smole C, Kufferath I, Denk H, Strnad P, et al. p62/Sequestosome-1 is indispensable for maturation and stabilization of Mallory-Denk bodies. PLoS One 2016;11:e0161083
[33] Yang H, Ni HM, Guo F, Ding Y, Shi YH, Lahiri P, et al. Sequestosome 1/p62 protein is associated with autophagic removal of excess hepatic endoplasmic reticulum in mice. J Biol Chem 2016;291:18663-18674
[34] Chao X, Wang S, Zhao K, Li Y, Williams JA, Li T, et al. Impaired TFEB-mediated lysosome biogenesis and autophagy promote chronic ethanol-induced liver injury and steatosis in mice. Gastroenterology 2018;155:865-879.e12
[35] Jaeschke H. Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: the protective effect of allopurinol. J Pharmacol Exp Ther 1990;255:935-941
[36] Akakpo JY, Ramachandran A, Kandel SE, Ni H, Kumer SC, Rumack BH, et al. 4-Methylpyrazole protects against acetaminophen hepatotoxicity in mice and in primary human hepatocytes. Hum Exp Toxicol 2018;37:1310-1322
[37] R-Core-Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing 2020; Vienna
[38] R Studio-Team. Studio: integrated development for R. RStudio 2020; PBC
[39] Ichimura Y, Waguri S, Sou YS, Kageyama S, Hasegawa J, Ishimura R, et al. Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol Cell 2013;51:618-631
[40] Hanawa N, Shinohara M, Saberi B, Gaarde WA, Han D, Kaplowitz N. Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury. J Biol Chem 2008;283:13565-13577
[41] Ding WX, Guo F, Ni HM, Bockus A, Manley S, Stolz DB, et al. Parkin and mitofusins reciprocally regulate mitophagy and mitochondrial spheroid formation. J Biol Chem 2012;287:42379-42388
[42] Ding WX, Yin XM. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 2012;393:547-564
[43] Ding WX, Ni HM, Li M, Liao Y, Chen X, Stolz DB, et al. Nix is critical to two distinct phases of mitophagy, reactive oxygen species-mediated autophagy induction and Parkin-ubiquitin-p62-mediated mitochondrial priming. J Biol Chem 2010;285:27879-27890
[44] Zhong Z, Umemura A, Sanchez-Lopez E, Liang S, Shalapour S, Wong J, et al. NF-kappaB restricts inflammasome activation via elimination of damaged mitochondria. Cell 2016;164:896-910
[45] Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, et al. Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol 2002;15:1504-1513
[46] Yang R, Zou X, Koskinen ML, Tenhunen J. Ethyl pyruvate reduces liver injury at early phase but impairs regeneration at late phase in acetaminophen overdose. Critical Care 2012;16:1-9
[47] Miyakawa K, Joshi N, Sullivan BP, Albee R, Brandenberger C, Jaeschke H, et al. Platelets and protease-activated receptor-4 contribute to acetaminophen-induced liver injury in mice. Blood 2015;126:1835-1843
[48] Taniguchi K, Yamachika S, He F, Karin M. p62/SQSTM1-Dr. Jekyll and Mr. Hyde that prevents oxidative stress but promotes liver cancer. FEBS Lett 2016;590:2375-2397
[49] Xie Y, McGill MR, Du K, Dorko K, Kumer SC, Schmitt TM, et al. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes. Toxicol Appl Pharmacol 2015;289:213-222
[50] McGill MR, Williams CD, Xie Y, Ramachandran A, Jaeschke H. Acetaminophen-induced liver injury in rats and mice: comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity. Toxicol Appl Pharmacol 2012;264:387-394
[51] Chen Y, Liu K, Zhang J, Hai Y, Wang P, Wang H, et al. c-Jun NH2-terminal protein kinase phosphorylates the Nrf2-ECH homology 6 domain of nuclear factor erythroid 2-related factor 2 and downregulates cytoprotective genes in acetaminophen-induced liver injury in mice. Hepatology 2020;71:1787-1801
[52] He J, Chen J, Wei X, Leng H, Mu H, Cai P, et al. Mammalian target of rapamycin Complex 1 signaling is required for the dedifferentiation from biliary cell to bipotential progenitor cell in zebrafish liver regeneration. Hepatology 2019;70:2092-2106
[53] Ni HM, Bockus A, Boggess N, Jaeschke H, Ding WX. Activation of autophagy protects against acetaminophen-induced hepatotoxicity. Hepatology 2012;55:222-231
[54] Sun H, Ni HM, McCracken JM, Akakpo JY, Fulte S, McKeen T, et al. Liver-specific deletion of mechanistic target of rapamycin does not protect against acetaminophen-induced liver injury in mice. Liver Res 2021;5:79-87
[55] Ni HM, Chao X, Yang H, Deng F, Wang S, Bai Q, et al. Dual roles of mammalian target of rapamycin in regulating liver injury and tumorigenesis in autophagy-defective mouse liver. Hepatology 2019;70:2142-2155
[56] Umemura A, Park EJ, Taniguchi K, Lee JH, Shalapour S, Valasek MA, et al. Liver damage, inflammation, and enhanced tumorigenesis after persistent mTORC1 inhibition. Cell Metab 2014;20:133-144
[57] Jaeschke H, Ramachandran A. Pleiotropic roles of platelets and neutrophils in cell death and recovery during acetaminophen hepatotoxicity. Hepatology 2020;72:1873-1876
[58] Jaeschke H, Ramachandran A. Mechanisms and pathophysiological significance of sterile inflammation during acetaminophen hepatotoxicity. Food Chem Toxicol 2020;138:111240
[59] Fisher JE, McKenzie TJ, Lillegard JB, Yu Y, Juskewitch JE, Nedredal GI, et al. Role of Kupffer cells and toll-like receptor 4 in acetaminophen-induced acute liver failure. J Surg Res 2013;180:147-155
[60] Krenkel O, Mossanen JC, Tacke F. Immune mechanisms in acetaminophen-induced acute liver failure. Hepatobiliary Surg Nutr 2014;3:331-343
[61] Laschke MW, Dold S, Menger MD, Jeppsson B, Thorlacius H. Platelet-dependent accumulation of leukocytes in sinusoids mediates hepatocellular damage in bile duct ligation-induced cholestasis. Br J Pharmacol 2008;153:148-156
[62] Chauhan A, Adams DH, Watson SP, Lalor PF. Platelets: no longer bystanders in liver disease. Hepatology 2016;64:1774-1784
[63] Chauhan A, Sheriff L, Hussain MT, Webb GJ, Patten DA, Shepherd EL, et al. The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure. Nat Commun 2020;11:1939
[64] Torisu T, Torisu K, Lee IH, Liu J, Malide D, Combs CA, et al. Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor. Nature medicine 2013;19:1281
[65] Rastegarlari G, Pegon JN, Casari C, Odouard S, Navarrete AM, Saint-Lu N, et al. Macrophage LRP1 contributes to the clearance of von Willebrand factor. Blood 2012;119:2126-2134
Similar articles:
1.Layla Shojaie, Myra Ali, Andrea Iorga, Lily Dara.Mechanisms of immune checkpoint inhibitor-mediated liver injury[J]. Acta Pharmaceutica Sinica B, 2021,11(12): 3727-3739
2.Hartmut Jaeschke, Olamide B. Adelusi, Jephte Y. Akakpo, Nga T. Nguyen, Giselle Sanchez-Guerrero, David S. Umbaugh, Wen-Xing Ding, Anup Ramachandran.Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls[J]. Acta Pharmaceutica Sinica B, 2021,11(12): 3740-3755
Similar articles: