The enhanced expression of death receptor 5 (DR5) mediated by HBV X protein through NF-kappaB pathway is associated with cell apoptosis induced by (TNF-α related apoptosis inducing ligand) TRAIL in hepatoma cells - PubMed (original) (raw)

Fanyun Kong et al. Virol J. 2015.

Abstract

Background: HBV X protein (HBX) is associated with cell apoptosis mediated by TNF-α related apoptosis inducing ligand (TRAIL), while the role of HBX on the expressions of TRAIL receptors death receptor 4 (DR4) and DR5 are unclear. In this study, we detected the cell apoptosis induced by TRAIL as well as gene and protein expressions of DR4 and DR5 in Huh-7 cells steadily transfected with HBX (Huh-7-HBX cells). In addition, we investigated the activation of different pathways associated with the expressions of TRAIL receptors in Huh-7-HBX cells.

Methods: The apoptosis of Huh-7-HBX cells induced by TRAIL was evaluated by flow cytometry analysis. The levels of DR4 and DR5 expression in cells were determined by real-time PCR and western blotting analysis. The activities of JNK pathway and NF-kappaB (NF-κB) pathway were demonstrated by western blotting assay.

Results: Compared to control cells, the percentage of cell apoptosis was increased in Huh-7-HBX cells. The increased expressions of DR4 and DR5 on gene and protein levels were observed in Huh-7-HBX cells. Further researches suggested that activation of JNK pathway was increased but not involved in the expression of TRAIL receptors in HBX positive cells. The activation of NF-κB pathway increased and was responsible for DR5 expression and cell apoptosis in HBX positive cells.

Conclusions: These results demonstrate that increased apoptosis induced by TRAIL is associated with increased expression of DR5 that mediated by HBX through NF-κB pathway. This finding provides a critical insight into the mechanism of hepatocyte apoptosis mediated by HBX in HBV infection.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

The apoptosis of Huh-7-HBX cells induced by TRAIL. a, b The expression of HBX in HepG2 cells was determined by RT-PCR and western blotting analysis. c After the cells treated with 100 ng/mL TRAIL for 24 h, the detection of apoptosis of Huh-7-HBX cells and control cells by flow cytometry

Fig. 2

Fig. 2

The expressions of DR4 and DR5 mediated by HBX. a The expressions of DR4 and DR5 mRNA detected by real-time PCR in Huh-7-HBV cells and control cells. b The expressions of DR4 and DR5 proteins determined by western blotting analysis in Huh-7-HBV cells and control cells. To detect the role of HBV on DR4 and DR5 expressions, Huh-7 cells were transfected with pUC18-HBV1.2 or control plasmid. Forty-eight hours after transfection, the expressions of HBX and HBsAg genes (c), the expressions of DR4 and DR5 were detected on mRNA levels (d) and protein levels (e) were measured. The Huh-7 cells transfected with pUC18-HBV1.2 was named as Huh-7-HBV cells, and the cells transfected with control plasmid was as Huh-7-HBV-CON cells. In order to test the role of HBX on the expressions of DR4 and DR5 mediated by HBV, Huh-7 cells were cotransfected pUC18-HBV1.2 with pSilencer3.1-shHBX or control plasmid. Forty-eight hours after transfection, the expressions of HBX, HBsAg as well as DR4 and DR5 were detected on mRNA levels with detected by RT-PCR (f) and real-time PCR (g), and protein levels measured by western blotting analysis (h). The Huh-7 cells cotransfected with pUC18-HBV1.2 with pSilencer3.1-shHBX were named as Huh-7-HBV-siHBX cells. The Huh-7 cells cotransfected with pUC18-HBV1.2 with control plasmid of pSilencer3.1-shHBX were as Huh-7-HBV-siCON cells

Fig. 3

Fig. 3

The roles of JNK and NF-κB pathways on expressions of DR4 and DR5 proteins in Huh-7-HBX cells. a The activation of JNK pathways induced by HBX. b The activation of NF-κB pathways induced by HBX. c The role of JNK pathways on expression of DR4 and DR5 proteins induced by HBX. Huh-7-HBX and control cells were cultured in 0.01 % dimethyl sulfoxide (DMSO) in the absence or presence of 20 μM SP600125 for 24 h. Then the expressions of DR4 and DR5 proteins were detected by western blotting analysis. d The role of NF-κB pathways on expressions of DR4 and DR5 proteins induced by HBX. Huh-7-HBX and control cells were cultured in 0.01 % dimethyl sulfoxide (DMSO) in the absence or presence of 10 μM BAY11-7082 for 24 h, and then the expressions of DR4 and DR5 proteins were detected by western blotting analysis

Fig. 4

Fig. 4

The role of NF-κB pathways on apoptosis of Huh-7-HBX cells induced by TRAIL. a Huh-7-HBX cells was transfected with shRNA plasmids for DR4 and DR5, and 48 h post-transfection, the expression of DR4 and DR5 proteins was measured by western blotting analysis. Huh-7-HBX cells transfected with control plasmid of shRNA were named as siCON cells, Huh-7-HBX cells transfected with shRNA plasmids for DR4 and DR5 (pGPU6/GFP/Neo-DR4 and pGPU6/GFP/Neo-DR5) were as siRNA cells. b After Huh-7-HBX cells transfected with shRNA plasmids for DR4 and DR5, and control plasmids for 48 h, the apoptosis was detected in Huh-7-Mock cells, Huh-7-HBX cells, and Huh-7-HBX cells transfected with different plasmids. c After the cells treated with 100 ng/mL TRAIL and 10 μM BAY11-7082 for 24 h, the apoptosis of cells was detected by flow cytometry analysis

References

    1. Iavarone M, Colombo M. HBV infection and hepatocellular carcinoma. Clin Liver Dis. 2013;17:375–397. doi: 10.1016/j.cld.2013.05.002. - DOI - PubMed
    1. Yang ST, Yen CJ, Lai CH, Lin YJ, Chang KC, Lee JC, Liu YW, Chang-Liao PY, Hsu LS, Chang WC, et al. SUMOylated CPAP is required for IKK-mediated NF-kappaB activation and enhances HBx-induced NF-kappaB signaling in HCC. J Hepatol. 2013;58:1157–1164. doi: 10.1016/j.jhep.2013.01.025. - DOI - PubMed
    1. You X, Liu F, Zhang T, Li Y, Ye L, Zhang X. Hepatitis B virus X protein upregulates oncogene Rab18 to result in the dysregulation of lipogenesis and proliferation of hepatoma cells. Carcinogenesis. 2013;34:1644–1652. doi: 10.1093/carcin/bgt089. - DOI - PubMed
    1. Zhang F, Wang Q, Ye L, Feng Y, Zhang X. Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear factor-kappaB/p65 in hepatoma cells. J Med Virol. 2010;82:920–928. doi: 10.1002/jmv.21753. - DOI - PubMed
    1. Klein NP, Schneider RJ. Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras. Mol Cell Biol. 1997;17:6427–6436. doi: 10.1128/MCB.17.11.6427. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources