Increased expression of Zinc finger protein 267 in non-alcoholic fatty liver disease

Introduction

The Kruppel-like transcription factor (KLF) family is involved in the regulation of diverse aspects of cellular development, differentiation, growth, and activation [1]. KLFs contain multiple zinc fingers, which represent one of the most common DNA binding domains. A zinc finger contains two cysteine and two histidine residues that coordinate a single zinc ion and fold the domain into a finger-like projection [2]. Seventeen mammalian KLFs have been identified and some of them have been recognized to be involved in numerous disorders such as cancer, obesity, cardiovascular disease, and inflammatory conditions [1].

KLF proteins are also involved in the development and progression of different chronic liver disease. For example, Kruppel-like factor 15 activates hepatitis B virus gene expression and replication [3]. KLF8 is up-regulated in hepatocellular cancer (HCC), promotes tumour invasion and indicates a poor prognosis for patients with liver cancer [4]. In contrast, KLF6 (also named human zinc finger 9 (ZF9) or COPEB) is a tumor suppressor gene for HCC [5,6]. Furthermore, KLF6 is up-regulated in association with increased steatosis, inflammation, and fibrosis in non-alcoholic fatty liver disease (NAFLD) [7,8].

One further member of the Kruppel-like zinc finger family is zinc finger protein 267 (ZNF267; also named human zinc finger 2 or HZF2) [9]. It contains a conserved Kruppel associated box (KRAB) domain in the amino terminal part, which is separated through a linker region from a clustered zinc finger domain [9]. The first zinc finger is followed by three degenerated fingers, and then continues with 13 zinc fingers at the carboxy terminus [9]. The function of ZNF267 is widely unknown; its mRNA is up-regulated upon nitric oxide treatment in venous endothelial cells [10], and previously, we have demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis compared to normal human liver tissue [11]. Furthermore, we found that ZNF267 expression is increased in human HCC-cells and tissue compared to primary human hepatocytes (PHH) and corresponding non-tumorous hepatic liver tissue, respectively [12].

Nonalcoholic fatty liver disease (NAFLD) is today recognized as the most common liver disease and one of the most frequent causes of liver cirrhosis [13,14]. Furthermore, NAFLD is a main risk factor for the development and progression of HCC [15]. NAFLD is characterized by lipid accumulation in the liver that may progress from simple hepatic steatosis towards inflammation (non-alcoholic steatohepatitis [NASH]) with progressive fibrosis [16,17,18].

NAFLD is strongly associated with the metabolic syndrome and its defining pathophysiological components obesity, diabetes and dyslipidemia. Thus, overeating delivers an excess of triglycerides to the liver. Further, peripheral lipolysis in obese and insulin resistant states leads to an increased hepatic influx of fatty acids (FA). In addition, hepatic FA synthesis is increased secondary to elevated glucose and insulin levels. Together, both uptake of exogenously derived FA and de novo hepatic synthesis of FA lead to an increase of hepatic lipid content, e.g. hepatic steatosis [19,20,21].

In the present study we aimed to assess the effect of hepatic steatosis on ZNF267 expression in an in vitro model of hepatocellular lipid accumulation [22] and in hepatic specimens of NAFLD patients.

Materials and methods

Results

Discussion

The aim of this study was to investigate the expression and function of ZNF267 in nonalcoholic fatty liver disease (NAFLD). Assessment of human liver specimens revealed that ZNF267 mRNA levels were significantly higher in NAFLD compared to normal hepatic tissue. Interestingly, ZNF267 mRNA levels did not differ significantly between simple steatosis and NASH. These findings indicate that hepatocellular lipid accumulation and not inflammation is the inducer of ZNF267 gene transcription in NAFLD. In accordance, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent upregulation of ZNF267 expression. In line with previous studies [31,32,33] we observed considerable ROS formation in response to the hepatocellular lipid accumulation, and moreover, we demonstrated that also chemically induced ROS formation led to a marked upregulation of ZNF267 mRNA expression in hepatoma cells. Most recently, we have identified the transcription factor Ets-1 as key regulator of basal as well as induced ZNF267 expression in liver cancer cells [12], and the release of free radicals as ROS has been shown to induce the transcription of Ets-1 in tumor cells [35]. In summary, these findings suggest that ROS-induced Ets-1 activation is at least one loop by which hepatic steatosis induces ZNF267 expression. With regards to this, we recently demonstrated that ZNF267 promotes proliferation and migration of HCC cells [12], and further, that Ets-1 is strongly linked to HCC progression [36,37,38]. Moreover, we have recently shown that ZNF267 promotes tumorigenicity of HCC cells in vitro [12]. Together these findings indicate that enhanced ZNF267 expression may be at least part of the mechanisms by which Ets-1 exhibits its pro-cancerogenic effect in NAFLD. Furthermore, it has to be once more emphasized that ZNF267 expression is already increased in steatotic livers in the absence of inflammation. This further underscores that simple steatosis cannot be considered as benign but per se is a risk factor for HCC development and progression [15]. Furthermore, it has to be considered that ZNF267 is already up-regulated in human cirrhotic liver tissue and might promote liver fibrosis through alteration of matrix degradation [11]. Since cirrhosis is the main risk factor for HCC development, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.