Hepatitis B virus is a major causative agent of acute and chronic hepatitis in humans and is closely associated with the incidence of human HCC (20
). The X protein of HBV is essential for transactivation of the viral and some cellular genes (21
). Epidemiological and molecular evidence indicates that HBx
is involved in the development of primary HCC (23
is reported to induce liver cancer growth in transgenic mice and to activate several host genes important for cell proliferation and transformation, such as c-fos
, and β-interferon. HBx
is a trans-activating protein that alters gene expression by interacting with transcription factors or stimulating signaling pathways that promote cell growth and survival. HBx
also binds to and inactivates tumor suppressors and senescence-related factors (25
). However, the mechanisms behind the role of HBx
in the regulation of HCC development remain largely unknown.
In this study, we identified MAT2A as a new target of HBx. HBx activates the MAT2A promoter and induces its expression in hepatic cell line (L02) and hepatoma cell lines (HepG2 and BEL-7404) in a dose-dependent manner, indicating HBx is capable of regulating MAT2A expression. To gain insight into the mechanism behind the function of HBx in the regulation of MAT2A expression, a series of mutations of MAT2A promoter were constructed and assayed. Our results indicated that the sequence between nt −548 and −108 of the promoter of the MAT2A gene along with NF-κB2 and CREB recognition elements are required for the function of HBx in the activation of MAT2A expression. HBx facilitates the binding of NF-κB and CREB to the MAT2A promoter. In addition, we further confirmed that NF-κB and CREB signal transduction pathways are involved in the activation of MAT2A expression regulated by HBx in hepatoma cells. These results suggested that one of the mechanisms in which HBx regulates HCC development is through activating MAT2A expression.
In mammals, two genes (MAT1A
) encode two homologous catalytic subunits of the MAT enzyme. Normal liver expresses MAT1A
, whereas MAT2A
is induced in human HCC and facilitates cancer cell growth (27
). Studies have shown that a switch in gene expression from MAT1A
in human liver cancer is pathogenetically important, as stimulation of MAT2A
expression in cancer cells enhances cell growth through DNA hypomethylation, whereas inhibition of MAT2A
expression in hepatoma cells has a reverse phenotype (29
). Thus, MAT2A
functions as a positive regulator in hepatoma growth, suggesting that manipulating MAT2A
expression may have therapeutic potential for the treatment of hepatoma.
MAT2A is transcriptionally induced in human HCC and in rodents during rapid liver growth and dedifferentiation. Many studies have shown that increased MAT2A expression provides liver cancer cells a growth advantage by inhibiting cellular apoptosis. HBx protein also can inhibit cell apoptosis during the development of HBV-associated HCC. The anti-apoptotic effect shared by HBx and MAT2A prompted us to examine the effects of HBx on MAT2A activation and cell apoptosis in hepatoma cells. Our results showed that both HBx and MAT2A inhibit cell apoptosis in HepG2 cells. In contrast, knockdown of MAT2A expression by shRNA induces cell apoptosis in hepatoma cell, even in the presence of HBx. These results suggested that MAT2A is a downstream target of HBx and may partially explain the anti-apoptotic effect of HBx on the development of HBV-associated HCC.
However, the roles of HBx
and its regulatory effect on apoptosis are still controversial, with some studies showing pro-apoptotic effects (31
). In fact, HBx
is known to localize in both the mitochondria and the nuclei of cells and subsequently modulates mitochondrial membrane potential and transcription of certain genes, suggesting that the status and localization of HBx
for the regulation of NF
activation and apoptosis may be changed according to the processing phase of its functional activity within a cell.
MAT enzyme activity is required for the production of AdoMet, because AdoMet is synthesized from methionine and ATP in a reaction catalyzed by the enzyme (34
). In normal liver cells, two mechanisms to maintain the high cellular level of AdoMet are as follows: 1) up-regulation of MAT1A
expression by AdoMet with the increase in MAT I/III activity, and 2) the high capacity of MAT I/III to convert dietary methionine and ATP into AdoMet. Because AdoMet down-regulates MAT2A
expression and inhibits MAT II activity, the contribution of MAT II to the production of AdoMet is minimal in liver cells (15
). AdoMet has rapidly moved from being a methyl donor to a key metabolite that regulates hepatocyte growth, death, and differentiation (35
). In hepatocytes, AdoMet levels are related to the differentiation status of the cells. AdoMet levels are at high in quiescent and at low in proliferating hepatocytes during liver regeneration. The high level of hepatic AdoMet is transient. If the high level of hepatic AdoMet was persisting, it would favor a proliferative phenotype and ultimately the development of HCC (39
Liver injury caused by hepatotoxins or partial hepatectomy initiates a cellular response that involves a vast number of growth factors and cytokines (such as HGF, TNF
-α, and IL-6
) and generation of oxidative stress (NO
), which leads to the inactivation of MAT I/III and a reduction of hepatic AdoMet level. This reduction in AdoMet level induces MAT2A
expression and MAT II activity, which results in a new and lower steady state level of AdoMet. The reduced level of hepatic AdoMet releases the inhibitory effect of this molecule on the proliferative activity of hepatocyte growth factor, which facilitates liver regeneration. If the conditions leading to oxidative stress persist (e.g.
chronic HBV infection), the hepatic levels of AdoMet are continuously low, which predisposes the liver to develop steatohepatitis, cirrhosis, and ultimately HCC (39
). In the cirrhotic liver, MAT1A
expression is progressively silenced by a mechanism that involves the methylation of the gene promoter and its association with hypoacetylated histones (15
In cancerous liver cells, MAT activity is very high when methionine is at low physiological concentrations (50–80 μm
). However, in cultured normal rat and human hepatocytes, MAT activity is also very high when methionine concentrations are high (5 mm
). This led us to speculate that MAT2
β, a regulatory subunit of MAT2A
, is activated in response to HBx stimulation. MATII can reduce the Km
value for methionine and the Ki
value for AdoMet, making the subunit more susceptible to feedback inhibition (42
). The steady state AdoMet level should be lower when the level of MAT2A is higher due to this feedback regulation. Our results showed that a sharp decrease of MAT1A
protein levels and increases of MAT2A and MAT2β protein levels were detected in the presence of HBx
in hepatocytes. In accordance with changes of the expression of MAT
genes, the intracellular AdoMet levels were remarkably decreased in response to HBx stimulation.
AdoMet not only controls liver growth but also regulates apoptosis, with an anti-apoptotic role in normal hepatocytes and a pro-apoptotic role in liver cancer cells (43
inhibits apoptosis of liver cancer cells, contributing to a reduction of steady state AdoMet levels. The reduction in hepatic AdoMet levels can feed into a vicious cycle that favors a switch in MAT expression and liver dedifferentiation (29
). In an effort to investigate the effect of MAT2A
on the changes in AdoMet levels and cell apoptosis regulated by HBx, we demonstrated that the changes in hepatoma cell apoptosis were concomitant with changes in AdoMet production. Knockdown of MAT2A
expression by shRNA stimulated AdoMet production, and thus abolished the inhibitory effect of HBx on apoptosis in HepG2 cells. These results suggested that MAT2A
and AdoMet act oppositely to the inhibition of apoptosis in hepatoma cells regulated by HBx.
In summary, we demonstrated that the X protein of HBV directly regulates the expression of MAT2A gene in hepatoma cells by enhancing the binding of transcription factors NF-κB and CREB to the promoter of the MAT2A gene. We proposed that during HBV infection, the viral protein HBx stimulates the expression of MAT2A gene, resulting in an increase of MAT II enzyme activity, a decrease of AdoMet production, and the inhibition of apoptotic cell death in cancer cells (). Our results suggested that HBx-induced MAT2A expression may play an important role in HBV-mediated HCC progression, which would provide new insights into our understanding the molecular mechanisms involved in the development of HCC caused by HBV infection.
FIGURE 8. Proposed model for the role of HBx in MAT2A expression, AdoMet production, cell apoptosis, and perhaps HCC development. Top, in normal liver cells, two mechanisms to maintain the high cellular AdoMet level are as follows: ① up-regulation of MAT1A (more ...)