The X Protein of Hepatitis B Virus Inhibits Apoptosis in Hepatoma Cells through Enhancing the Methionine Adenosyltransferase 2A Gene Expression and Reducing S-Adenosylmethionine Production

Lu Ma,Yingle Liu,Li Liu,Jianguo Wu,Jiwei Chen,Jun Zhang,Quanyan Liu,Zhisu Liu,Yueming He,Dongfeng Wang

doi:10.1074/jbc.m110.167783

Abstract

The X protein (HBx) of hepatitis B virus (HBV) is involved in the development of hepatocellular carcinoma (HCC), and methionine adenosyltransferase 2A (MAT2A) promotes the growth of liver cancer cells through altering S-adenosylmethionine homeostasis. Thus, we speculated that a link between HBx and MAT2A may contribute to HCC development. In this study, the effects of HBx on MAT2A expression and cell apoptosis were investigated, and the molecular mechanism by which HBx and MAT2A regulate tumorigenesis was evaluated. Results from immunohistochemistry analyses of 37 pairs of HBV-associated liver cancer tissues/corresponding peritumor tissues showed that HBx and MAT2A are highly expressed in most liver tumor tissues. Our in vitro results revealed that HBx activates MAT2A expression in a dose-dependent manner in hepatoma cells, and such regulation requires the cis-regulatory elements NF-κB and CREB on the MAT2A gene promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) further demonstrated that HBx facilitates the binding of NF-κB and CREB to MAT2A gene promoter. In addition, overexpression of HBx or MAT2A inhibits cell apoptosis, whereas knockdown of MAT2A expression stimulates apoptosis in hepatoma cells. Furthermore, we demonstrated that HBx reduces MAT1A expression and AdoMet production but enhances MAT2β expression. Thus, we proposed that HBx activates MAT2A expression through NF-κB and CREB signaling pathways to reduce AdoMet production, inhibit hepatoma cell apoptosis, and perhaps enhance HCC development. These findings should provide new insights into our understanding how the molecular mechanisms underline the effects of HBV infection on the production of MAT2A and the development of HCC.

Highlights

Reduce AdoMet production, inhibit hepatoma cell apoptosis, and perhaps enhance hepatocellular carcinoma (HCC) development
Our results demonstrated that HBx activated methionine adenosyltransferase 2A (MAT2A) expression through NF-␬B and CRE-binding site (CREB) signaling pathways, resulting in the decrease of AdoMet production and the inhibition of hepatoma cell apoptosis
Results from Electrophoretic mobility shift assay (EMSA) using the CREB probe showed that one faint band of protein-DNA complex was detected in the absence of HBx (Fig. 5A, lane 1), but the shift bands were enhanced in the presence of HBx, and the level of shift band increased as the concentration of HBx increased (Fig. 5A, lanes 2 and 3)

Summary

EXPERIMENTAL PROCEDURES

Patients and Tissue Specimens—Thirty seven cases of HBVassociated HCC were collected from Zhongnan Hospital, Wuhan University, between January 2008 and January 2009. The cells were transiently co-transfected with the pRL-TK plasmid (Promega, Madison, WI) containing the Renilla luciferase gene, which is used for internal normalization, and various constructs containing different lengths of the MAT2A 5Ј-flanking region. Western Blotting—Nuclear and cytoplasmic protein extracts were prepared from transfected cells and used for Western blot analysis using rabbit polyclonal antibodies. Protein extracts were obtained from transfected cells by sonication and centrifugation at 13,000 ϫ g for 15 min. AdoMet and AdoHcy contents were determined by reverse phase-HPLC, as described previously [19]. 2 ␮g of purified polyclonal antibody directed against NF-␬B subunit p65 (anti-p65), CREB (antiCREB), or IgG was incubated with nuclear extracts on ice for 30 min before being added to the binding buffer. All statistical analyses were performed using professional statistical software (SPSS 15.0 for Windows, SPSS Inc., Chicago)

RESULTS

HBV II

HBx Staining intensity

HBx AdoMet

DISCUSSION