Hepatitis B Virus X Protein Modulates Apoptosis in Primary Rat Hepatocytes by Regulating both NF-κB and the Mitochondrial Permeability Transition Pore

Abstract

The hepatitis B virus (HBV) X protein (HBx) is a multifunctional protein that regulates numerous cellular signal transduction pathways, including those that modulate apoptosis. However, different HBx-dependent effects on apoptosis have been reported; these differences are likely the consequence of the exact conditions and cell types used in a study. Many of the previously reported studies that analyzed HBx regulation of apoptosis were conducted in immortalized or transformed cells, and the alterations that have transformed or immortalized these cells likely impact apoptotic pathways. We examined the effects of HBx on apoptotic pathways in cultured primary rat hepatocytes, a biologically relevant system that mimics normal hepatocytes in the liver. We analyzed the effects of HBx on apoptosis both when HBx was expressed in the absence of other HBV proteins and in the context of HBV replication. HBx stimulation of NF-B inhibited the activation of apoptotic pathways in cultured primary rat hepatocytes. However, when HBx-induced activation of NF-B was blocked, HBx stimulated apoptosis; blocking the activity of the mitochondrial permeability transition pore inhibited HBx activation of apoptosis. These results suggest that HBx can be either proapoptotic or antiapoptotic in hepatocytes, depending on the status of NF-B, and confirm previous studies that link some HBx activities to modulation of the mitochondrial permeability transition pore. Overall, our studies define apoptotic pathways that are regulated by HBx in cultured primary hepatocytes and provide potential mechanisms for the development of HBV-associated liver cancer.

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca 2+ increase and cell death in Caenorhabditis elegans

“…Importantly, CED-9-dependent Ca 2+ elevation and necrosis induced by HBx can both be suppressed by CsA, an inhibitor of MPT. These results are consistent with the observations in human cells that HBx acts through MPT to control intracellular calcium, cell death, and HBV replication (13)(14)(15)17) and indicate that CED-9 is the cellular target of HBx in elevating intracellular Ca 2+ (Fig. 2B).…”

“…These include key regulators of apoptosis (CED-9, CED-4, and CED-3) and critical components in the necrosis pathway, especially those involved in regulating Ca 2+ signaling, including ER Ca 2+ -binding proteins and channels (CRT-1, CNX-1, UNC-68, and ITR-1), Ca 2+ -dependent proteases (CLP-1 and TRA-3), and MPT, many of which previously have not been implicated in HBV or HBx-induced pathogenesis. MPT, however, has been implicated in HBx-induced cell killing and HBV replication in humans (13)(14)(15)17). CED-9, a key apoptosis regulator, was discovered unexpectedly to be a host target of HBx in cell death and Ca 2+ signaling in C. elegans, which led to identification of human Bcl-2 proteins as conserved host targets of HBx-mediated Ca 2+ stimulation and HBV replication in human hepatocytes (companion article, ref.…”

“…Although the cellular target of HBx-mediated calcium stimulation is unknown, HBx has been proposed to target mitochondria to effect permeability transition (13)(14)(15)17), which plays an important role in regulating intracellular Ca 2+ homeostasis (38). In this study, we show that HBx interacts directly with CED-9, a mitochondrial protein, to increase cytosolic Ca 2+ , which then triggers activation of necrosis in C. elegans through Ca 2+ -dependent proteases (CLP-1 and TRA-3) (Figs.…”

“…Additionally, HBx has been shown to modulate cell death by altering cytosolic Ca 2+ (20). Although HBx has been suggested to target mitochondria and mitochondria permeability transition (MPT) in Ca 2+ regulation (14,15,17), the cellular target(s) with which HBx interacts to alter cytosolic Ca 2+ is unknown and could be at the crux of HBx biology and HBV therapy.…”

“…HBx can sensitize liver cells to cell death by various insults, including tumor necrosis factor-α (TNF-α) and growth factor deprivation (10,11). HBx induces mitochondria aggregation, loss of mitochondrial membrane potential, and cytochrome c release, indicating that HBx may act through mitochondria to induce cell death (12)(13)(14)(15). Apoptosis and necrosis are also early events of liver pathogenesis in HBx transgenic mice and may lead to development of cirrhosis and HCC (9,10,12).…”