p53/p66Shc-mediated signaling contributes to the progression of non-alcoholic steatohepatitis in humans and mice Kengo Tomita 1,2,⇑,  , Toshiaki Teratani 2,  , Takahiro Suzuki 2 , Tetsuya Oshikawa 2 Hirokazu Yokoyama 3 , Katsuyoshi Shimamura 2 , Kiyoshi Nishiyama 4 , Norikazu Mataki 1 , Rie Irie 5 Tohru Minamino 6 , Yoshikiyo Okada 1 , Chie Kurihara 1 , Hirotoshi Ebinuma 2 , Hidetsugu Saito 7 Ippei Shimizu 6 , Yohko Yoshida 6 , Ryota Hokari 1 , Kazuo Sugiyama 2 , Kazuo Hatsuse 4 Junji Yamamoto 4 , Takanori Kanai 2 , Soichiro Miura 1 , Toshifumi Hibi 2 1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa-shi, Saitama 359-8513, Japan; 2 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; 3 Health Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; 4 Department of Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa-shi, Saitama 359-8513, Japan; 5 Department of Pathology, Kawasaki Municipal Hospital, 12-1 Shinkawadori, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-0013, Japan; 6 Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan; 7 Graduate School of Pharmaceutical Sciences, Keio University Faculty of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan Background & Aims: The tumor suppressor p53 is a primary sen- sor of stressful stimuli, controlling a number of biologic pro- cesses. The aim of our study was to examine the roles of p53 in non-alcoholic steatohepatitis (NASH). Methods: Male wild type and p53-deficient mice were fed a methionine- and choline-deficient diet for 8 weeks to induce nutritional steatohepatitis. mRNA expression profiles in normal liver samples and liver samples from patients with non-alcoholic liver disease (NAFLD) were also evaluated. Results: Hepatic p53 and p66Shc signaling was enhanced in the mouse NASH model. p53 deficiency suppressed the enhanced p66Shc signaling, decreased hepatic lipid peroxidation and the number of apoptotic hepatocytes, and ameliorated progression of nutritional steatohepatitis. In primary cultured hepatocytes, transforming growth factor (TGF)-b treatment increased p53 and p66Shc signaling, leading to exaggerated reactive oxygen species (ROS) accumulation and apoptosis. Deficient p53 signal- ing inhibited TGF-b-induced p66Shc signaling, ROS accumulation, and hepatocyte apoptosis. Furthermore, expression levels of p53, p21, and p66Shc were significantly elevated in human NAFLD liver samples, compared with results obtained with normal liver samples. Among NAFLD patients, those with NASH had signifi- cantly higher hepatic expression levels of p53, p21, and p66Shc compared with the group with simple steatosis. A significant cor- relation between expression levels of p53 and p66Shc was observed. Conclusions: p53 in hepatocytes regulates steatohepatitis pro- gression by controlling p66Shc signaling, ROS levels, and apopto- sis, all of which may be regulated by TGF-b. Moreover, p53/ p66Shc signaling in the liver appears to be a promising target for the treatment of NASH. Ó 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction Non-alcoholic fatty liver disease (NAFLD) afflicts as much as 20% of the US adult population [1]. Non-alcoholic steatohepatitis (NASH)—part of the spectrum of NAFLD—is the most prevalent liver disease in the US, affecting approximately 3–4% of the population [1]. NAFLD and NASH are often co-morbid with disorders charac- terized by insulin resistance, such as diabetes and obesity. Thus, these liver diseases can be considered hepatic manifestations of metabolic syndrome. Given the growing number of patients with metabolic syndrome, the incidences of NAFLD and NASH are expected to increase further, particularly in North America, Eur- ope, Asia, and countries in the Western Pacific. NASH is a progressive disease. In a study that followed NASH patients for ten years, the disease progressed to cirrhosis in 20% of the patients and led to fatal liver disease in 8% of the cases [2]. A population-based cohort study demonstrated that Journal of Hepatology 2012 vol. 57 j 837–843 Keywords: P53; Non-alcoholic steatohepatitis; P66Shc; Reactive oxygen species; Transforming growth factor-b. Received 9 December 2011; received in revised form 14 May 2012; accepted 21 May 2012; available online 26 May 2012 * Corresponding author. Address: Division of Gastroenterology and Hepatology, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa-shi, Saitama 359-8513, Japan. Tel.: +81 4 2995 1211x2369; fax: +81 4 2996 5201. E-mail address: kengo@ndmc.ac.jp (K. Tomita).   These authors contributed equally to this work. Abbreviations: NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; ROS, reactive oxygen species; MCD, methionine-deficient and choline-deficient; ALT, alanine aminotransferase; HE, haematoxylin-eosin; HNE, hydroxynonenal; TUNEL, terminal deoxy-nucleotidyl transferase-mediated nick end-labeling; PCR, polymerase chain reaction; TGF-b, transforming growth factor- b; PFT, pifithrin; Col11, 1 (I) collagen; Col12, 2 (I) collagen; SMA, smooth muscle actin; MDA, malondialdehyde. Research Article