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. 2001 Jan;75(1):215-25.
doi: 10.1128/JVI.75.1.215-225.2001.

Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein

F Su  1 , C N Theodosis, R J Schneider
Affiliations

Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein

F Su et al. J Virol. 2001 Jan.

Abstract

Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-alpha) and etopocide. Paradoxically, HBx activates members of the NF-kappaB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-alpha. We therefore examined the roles of NF-kappaB, c-Myc, and TNF-alpha in apoptotic killing of cells by HBx. RelA/NF-kappaB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-kappaB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-kappaB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-kappaB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-alpha. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis.

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Figures

FIG. 1
FIG. 1
Induction of NF-κB DNA-binding activity by HBx and TNF-α in NIH 3T3 cells. Serum-starved (A) WT 3T3 cells and (B) RelA KO 3T3 cells were transduced by Ad-HBx or Ad-HBxo vector for 8 h or treated with mouse TNF-α (10 ng/ml) for 30 min. Nuclear extracts were prepared, and 10 μg was used to measure NF-κB DNA-binding activity by EMSA with a double-stranded 32P-labeled oligonucleotide probe containing one NF-κB binding site. For competition studies, a 100-fold molar excess of unlabeled competitor (c. comp.) probe was added. Protein-DNA complexes were resolved by electrophoresis in 4.5% polyacrylamide gels and visualized by autoradiography, and radioactivity was quantitated by digital densitometry. Control cells were neither transduced nor treated. Note that 10-fold less TNF-α sample was loaded in panel A.
FIG. 2
FIG. 2
Composition of NF-κB DNA-binding complexes induced by HBx protein and TNF-α in WT and RelA KO 3T3 cells. Serum-starved (A) WT and (B) RelA KO cells were transduced with the Ad-HBx vector for 8 h or treated with mouse TNF-α (10 ng/ml) for 30 min. Nuclear extracts were prepared, and 10 μg of protein extract was used to detect NF-κB DNA-binding activity by EMSA as described in the legend to Fig. 1. Specific polyclonal antibodies (Ab) were added to binding reactions corresponding to p50 (NF-κB1), p52 (NF-κB2), RelA, and c-Rel proteins by addition 15 min prior to that of 32P-labeled oligonucleotide probe. Control refers to extracts from untreated quiescent cells. n. serum, normal serum.
FIG. 3
FIG. 3
Cell killing by HBx and TNFα in WT and RelA KO 3T3 cells. Serum-starved (A) WT and (B) RelA KO 3T3 cells were transduced by Ad-HBx or Ad-HBxo vector for 8 h, followed by 16 h of mock treatment or treatment with murine TNF-α (10 ng/ml) or TNF-α (10 ng/ml) and cycloheximide (cycl.) (10 μg/ml). Cell death was quantified spectrophotometrically by trypan blue dye exclusion. Results represent the means of three independent experiments, with derived standard errors shown. Control (Ctrl) refers to cells not transduced by Ad vectors.
FIG. 4
FIG. 4
Combined HBx expression and TNF-α treatment induce death of 3T3 cells by apoptosis. WT and RelA KO 3T3 cells were grown on coverslips, transduced by Ad-HBx, and either mock treated or treated with TNF-α as described in the legend to Fig. 3. Cells were processed for staining with Hoechst DNA dye 33258 and then photographed under UV light using a Zeiss Axiophot photomicroscope at ×600 magnification. Apoptotic cells contain condensed nuclei that stain brightly, compared to diffuse and pale staining of normal cells.
FIG. 5
FIG. 5
Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.
FIG. 6
FIG. 6
Effect of c-Myc overexpression on HBx-induced cell killing. (A) WT and RelA KO 3T3 cells were transfected with a plasmid that expresses the human c-myc gene under the control of the SV40 promoter, which is only slightly induced by HBx (68). Equal amounts of whole-cell extracts were resolved by SDS-polyacrylamide gel electrophoresis, and c-Myc protein was identified by immunoblotting with a specific antibody. Cell treatment with TNF-α was carried out as described below. (B) WT 3T3 cells were transfected by 1 μg or 10 μg (10×) of the Myc expression plasmid, followed 8 h later by transduction with Ad-HBxo (vec) or Ad-HBx (HBx). At 10 h following transduction, cells were treated with TNF-α (10 ng/ml) for 16 h or mock treated. Cell killing was quantified by trypan blue dye exclusion assay. (C) Cells grown on coverslips were transfected as above, followed by transduction with an Ad-HBx-Flag vector (vec) (27), which expresses HBx protein containing the Flag foreign epitope. HBx-Flag behaves identically to HBx protein (27). Cells were processed for staining with Hoechst 33258 DNA dye, and indirect immunofluorescence was performed using FITC-conjugated M2 anti-Flag antibodies to visualize HBx. Cells were photographed as described in the legend to Fig. 4.
FIG. 7
FIG. 7
Apoptotic killing of 3T3 cells expressing c-Myc and HBx by TNF-α is blocked by IGF-II. WT 3T3 cells were grown on coverslips, transfected by a human c-myc expression plasmid, and transduced by Ad-HBx, as described in the legend to Fig. 6. Cells were treated with either murine TNF-α (10 ng/ml) for 16 h, or TNF-α (10 ng/ml) and IGF-II simultaneously. (A) Cells were photographed under UV light as described in the legend to Fig. 4. (B) Quantification of cell killing was performed by the dye exclusion assay.
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