Hepatitis B Virus Middle Protein Enhances IL-6 Production via p38 MAPK/NF-κB Pathways in an ER Stress-Dependent Manner

Abstract

During hepatitis B virus (HBV) infection, three viral envelope proteins of HBV are overexpressed in the endoplasmic reticulum (ER). The large S protein (LHBs) and truncated middle S protein (MHBst) have been documented to play roles in regulating host gene expression and contribute to hepatic disease development. As a predominant protein at the ultrastructural level in biopsy samples taken from viremic patients, the role of the middle S protein (MHBs) remains to be understood despite its high immunogenicity. When we transfected hepatocytes with an enhanced green fluorescent protein (EGFP)-tagged MHBs expressing plasmid, the results showed that expression of MHBs cause an upregulation of IL-6 at the message RNA and protein levels through activating the p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB) pathways. The use of specific inhibitors of the signaling pathways can diminish this upregulation. The use of BAPTA-AM attenuated the stimulation caused by MHBs. We further found that MHBs accumulated in the endoplasmic reticulum and increased the amount of glucose regulated protein 78 (GRP78/BiP). Our results provide a possibility that MHBs could be involved in liver disease progression.

Fig 1. MHBs accumulated in Huh7 cells.

(a) The intracellular accumulation of MHBs was determined with anti-preS2 antibody. (b) Proteins were collected from cells at 24 hours after transfection and were analyzed by western blot using an anti-preS2 antibody.

Fig 2. MHBs stimulates IL-6 production in hepatocytes and hepatoma cells.

Huh7, SMMC-7721 and L-02 cells were transfected with MHB as well as with GFP plasmid to detect IL-6 mRNA levels (a) and IL-6 protein levels (b-d).(e) Huh7 and L-02 cells were transfected with SHB as well as with GFP plasmid to detect IL-6 mRNA levels.

Fig 3. MHBs stimulates the phosphorylation of p38 but not ERK1/2.

Huh7, L02 and SMMC-7721 cells were transfected with MHB or GFP. (a) The phosphorylation of p38 and ERK1/2 were determined using specific antibodies and western blot. (b and c) The data from three independent experiments were analyzed with a paired t-test.

Fig 4. MHBs activates NF-κB.

Huh7, L-02 and SMMC-7721 cells were transfected with MHB or GFP. (a) The degradation of Iκ-B was detected by western blot. (b) The data from three independent experiments were analyzed using paired t-tests. (c) The nucleus-location of the p65 subunit was determined by immunostainig.

Fig 5. Activation of p38 and NF-κB pathway is necessary for MHBs-induced IL-6 production.

Huh7, L-02 and SMMC-7721 cells were transfected with MHB or GFP, cells were treated with SB203580 and QNZ individually or both, the IL-6 protein levels were analyzed.

Fig 6. ER stress is necessary for MHBs-induced p38/NF-κB activation and an increase in IL-6.

Huh7, L-02 and SMMC-7721 cells were transfected with MHB or GFP, the cells were treated with BAPTA-AM. (a) The phosphorylation of p38 MAPK and degradation of Iκ-B were detected by western blot. (b) The IL-6 protein level in the supernatants was detected by ELISA.

Fig 7. MHBs stimulates ER stress.

(a) Huh7, L02 and SMMC-7721 cells were transfected with MHB or GFP. The expression of BiP was determined with a specific antibody and western blot. (b) SMMC-7721 cells were cotransfected with ELP-1-CFP and MHB, the colocalization of the ELP-1 and MHB was observed.