Int J Clin Exp Pathol 2011;4(7):661-666 www.ijcep.com /IJCEP1108019 Original Article Increased expression of Zinc finger protein 267 in non-alcoholic fatty liver disease Bernd Schnabl1,2, Barbara Czech1, Daniela Valletta1, Thomas S Weiss3, Georgi Kirovski1, Claus Hellerbrand1 1Department of Internal Medicine I, University Hospital Regensburg, Germany, 2Department of Medicine, University of California San Diego, La Jolla, CA, USA, 3Center for Liver Cell Research, Department of Pediatrics and Juvenile Medicine, University Hospital Regensburg, Germany Received August 25, 2011; accepted September 10, 2011; Epub September 22, 2011; published October 31, 2011 Abstract: Hepatocellular lipid accumulation is a hallmark of non-alcoholic fatty liver disease (NAFLD), which encom- passes a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up- regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in stea- totic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Further- more, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign. Keywords: Non-alcoholic fatty liver disease, Kruppel-like factor, ZNF267 Introduction activates hepatitis B virus gene expression and replication [3]. KLF8 is up-regulated in hepato- The Kruppel-like transcription factor (KLF) family cellular cancer (HCC), promotes tumour inva- is involved in the regulation of diverse aspects sion and indicates a poor prognosis for patients of cellular development, differentiation, growth, with liver cancer [4]. In contrast, KLF6 (also and activation [1]. KLFs contain multiple zinc named human zinc finger 9 (ZF9) or COPEB) is a fingers, which represent one of the most com- tumor suppressor gene for HCC [5,6]. Further- mon DNA binding domains. A zinc finger con- more, KLF6 is up-regulated in association with tains two cysteine and two histidine residues increased steatosis, inflammation, and fibrosis that coordinate a single zinc ion and fold the in non-alcoholic fatty liver disease (NAFLD) domain into a finger-like projection [2]. Seven- [7,8]. teen mammalian KLFs have been identified and some of them have been recognized to be in- One further member of the Kruppel-like zinc volved in numerous disorders such as cancer, finger family is zinc finger protein 267 (ZNF267; obesity, cardiovascular disease, and inflamma- also named human zinc finger 2 or HZF2) [9]. It tory conditions [1]. contains a conserved Kruppel associated box (KRAB) domain in the amino terminal part, KLF proteins are also involved in the develop- which is separated through a linker region from ment and progression of different chronic liver a clustered zinc finger domain [9]. The first zinc disease. For example, Kruppel-like factor 15 finger is followed by three degenerated fingers, ZNF267 in NAFLD and then continues with 13 zinc fingers at the obtained by complexing the appropriate amount carboxy terminus [9]. The function of ZNF267 is of palmitate to BSA. PHH were grown in DMEM widely unknown; its mRNA is up-regulated upon supplemented with penicillin (400 U/ml), strep- nitric oxide treatment in venous endothelial tomycin (50 mg/ml), L-glutamine (300 mg/ml), cells [10], and previously, we have demon- 0.2% FCS and palmitate for 24h in different strated that ZNF267 mRNA is up-regulated in concentrations as indicated. FFA-free-BSA- liver cirrhosis compared to normal human liver treated cells served as controls. tissue [11]. Furthermore, we found that ZNF267 expression is increased in human HCC-cells and To induce ROS formation cells were incubated tissue compared to primary human hepatocytes with 10µM arsenic trioxide (As2O3; Sigma Al- (PHH) and corresponding non-tumorous hepatic drich) as described [12]. liver tissue, respectively [12]. Human tissue Nonalcoholic fatty liver disease (NAFLD) is today recognized as the most common liver disease Human liver specimens from patients were ob- and one of the most frequent causes of liver tained during resection of metastasis of non- cirrhosis [13,14]. Furthermore, NAFLD is a main hepatic tumors. Tissue samples were immedi- risk factor for the development and progression ately snap frozen after surgical resection and of HCC [15]. NAFLD is characterized by lipid stored at -80°C until subsequent analysis. For accumulation in the liver that may progress this study, the following exclusion criteria were from simple hepatic steatosis towards inflam- applied: 1. chronic alcohol abuse (more than 30 mation (non-alcoholic steatohepatitis [NASH]) g/day for men and 20g/day for women), 2. with progressive fibrosis [16,17,18]. medications known to cause hepatic steatosis (at present or within the last 2 years), 3. signifi- NAFLD is strongly associated with the metabolic cant weight loss (more than 3 kg within the last syndrome and its defining pathophysiological 3 months), 4. hepatobiliary diseases, 5. ascites components obesity, diabetes and dyslipidemia. on ultrasound, 6. inflammatory bowel disease, Thus, overeating delivers an excess of triglyc- 7. infection with the human immunodeficiency erides to the liver. Further, peripheral lipolysis in virus or Hepatitis B and C virus, and 8. chemo- obese and insulin resistant states leads to an therapy prior to partial hepatectomy. increased hepatic influx of fatty acids (FA). In addition, hepatic FA synthesis is increased sec- Hepatic steatosis and inflammation was scored ondary to elevated glucose and insulin levels. applying a modified histological score according Together, both uptake of exogenously derived to the recommendations of the Nonalcoholic FA and de novo hepatic synthesis of FA lead to Steatohepatitis Clinical Research Network [26]. an increase of hepatic lipid content, e.g. hepatic Briefly, four histological features were evaluated steatosis [19,20,21]. semi-quantitatively: steatosis (0-3), lobular in- flammation (0-2), hepatocellular ballooning (0- In the present study we aimed to assess the 2), and fibrosis (0-4). Simple steatosis was diag- effect of hepatic steatosis on ZNF267 expres- nosed in the presence of steatosis but the ab- sion in an in vitro model of hepatocellular lipid sence of inflammation, fibrosis and hepatocellu- accumulation [22] and in hepatic specimens of lar ballooning. Steatohepatitis was diagnosed NAFLD patients. when the sum of the scores was 4 or more. Materials and methods Human liver tissue was obtained and experi- mental procedures were performed according to Cells and cell culture the guidelines of the charitable state controlled foundation HTCR, with the informed patient's Isolation and culture of primary human hepato- consent, and the study was approved by the cytes (PHH) were performed as described [23]. local ethics committee of the University Regens- The HCC cell line HepG2 (ATCC HB-8065) was burg. cultured as described previously [24,24]. Hepa- tocellular lipid accumulation was induced as Expression analysis described [25]. Briefly, a 10 mM palmitate/1% bovine serum albumin (BSA) stock solution was Isolation of total cellular RNA from cultured cells 662 Int J Clin Exp Pathol 2011;4(7):661-666 ZNF267 in NAFLD and tissue and reverse transcription were per- formed as described previously [27]. Quantita- tive real-time PCR was performed with primers specific for ZNF267 (forward: 5’- ATG GGA GCT GTG ATC TTG AGA; reverse: 5’-GCA ATG ATG AAT GAG TAA AGA CC), employing LightCycler tech- nology (Roche, Mannheim, Germany) [28]. Detection of ROS Cells were plated in 96-well-plates and cultured in DMEM without phenol red (PAN Biotech GmbH, Aidenbach, Germany). Three hours after treatment with As2O3 or palmitate cells were washed with PBS and were incubated for 30min at 37°C with the redox-sensitive dye 2',7'- dichlorodihydrofluorescein diacetate (H2DCFDA, Figure 1. ZNF267 expression in NAFLD: Expression Invitrogen, Darmstadt, Germany; 100μM). Sub- of ZNF267 mRNA in normal, non steatotic human sequently, ROS formation was detected using a liver tissue compared to steatotic liver tissue without inflammation and NASH specimens analyzed by multiwell fuorescence plate reader (Spectra quantitative real-time PCR (*:p<0.05 compared to Fuor Plus, Tecan, Männedorf, Switzerland) with normal liver tissue). excitation and emission filters of 485 and 535nm, respectively. Statistical analysis lyzed ZNF267 mRNA expression in an in vitro model of hepatocellular lipid accumulation Statistical analyses were performed using which we