Hepatitis B Surface Antigen Activates Unfolded Protein Response in Forming Ground Glass Hepatocytes of Chronic Hepatitis B

Abstract

Ground glass hepatocytes (GGHs), a histological hallmark of chronic hepatitis B virus (HBV) infection, contain excessive hepatitis surface antigen (HBsAg) in the endoplasmic reticulum (ER), which is linked to unfolded protein response (UPR). The mechanism by which HBV activates UPR has not been fully defined. To investigate this, HepG2-NTCP cells and primary human hepatocytes (PHHs) were either infected with HBV or transduced with adenoviral vectors expressing replication-competent HBV genome or individual HBV genes. UPR markers were evaluated by qPCR, Western blotting, and immunofluorescence. Apoptosis and cell viability were measured by Caspase-3/7 and ATPlite assay respectively. We found that UPR markers were induced by the overexpression of HBsAg in HepG2-NTCP cells and PHHs. Elevation of UPR-induced genes showed a dose-dependent correlation with HBsAg levels. In HBV-infected livers, GGHs also demonstrated excessive accumulation of HBsAg associated with increased BIP/GRP78 staining, a marker of UPR. Prolonged activation of UPR by HBsAg overexpression induced signs of apoptosis. Overexpression of HBsAg can induce ER stress through protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in vitro, and may be linked to the appearance of GGHs. The activation of UPR by HBsAg may sensitize hepatocytes to cell death and result in possible subsequent cellular changes leading to a premalignant phenotype.

Keywords: apoptosis; chronic hepatitis B; endoplasmic reticulum stress; ground glass hepatocyte; hepatocellular carcinoma; liver disease.

Figure 1

Activation of PERK pathway genes by HBsAg overexpression. (A) HepG2-NTCP cells and (B) primary human hepatocyte cells were transduced with recombinant AdVs in triplicate (MOI of 1). Protein transportation inhibitor (PTI) was used as a positive control. mRNAs of PERK genes were determined by RT-qPCR three days after AdVs transduction. Relative mRNA expression was normalized to housekeeping gene TBP and mock control. The Student’s unpaired two-tailed t test was applied for data analysis. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Expression of HBsAg and BIP/GRP78 were determined by Western blotting in AdVs-transduced HepG2-NTCP cells. (D) HepG2-NTCP cells were infected with infectious HBV (MOI of 300). UPR markers were analyzed at different days post infection (DPI). Relative level to reference TATA-binding protein (TBP) was calculated. All results were confirmed by three independent experiments.

Figure 2

Dose-dependent induction of PERK pathway genes by HBsAg expression. Ad-GFP, Ad-HBsAg and Ad-HBeAg were transduced into HepG2-NTCP cells with increasing MOIs of 0.2, 1.0 and 5.0 in triplicate. Protein transportation inhibitor (PTI) was used as a positive control. (A) Three days after transduction, mRNAs of BIP/GRP78, ATF4, CHOP and GADD34 were determined by RT-qPCR. The Student’s unpaired two-tailed t test was applied for data analysis. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Expression of HBsAg and ER stress marker BIP/GRP78 were measured by Western blotting (left panel) and signals quantified (right panel). All results were confirmed by three independent experiments.

Figure 3

Co-localization of ER stress marker BIP/GRP78 and HBsAg. HepG2-NTCP cells were paraffin-embedded after transduction with Ad-GFP, Ad-HBsAg or treatment with PTI for six hours. (A) Immunofluorescent staining with anti-HBsAg and anti-BIP/GRP78 antibodies were performed three days after transduction. Nuclei were stained with Hoechst 33342. Scale bar represents 20 μm. (B) In Ad-HBsAg-transduced cells, the HBsAg and BIP/GRP78 intensities in each cell were measured, and their correlation was evaluated. (C) Ad-HBsAg transduced cells were grouped by HBsAg staining and analyzed for BIP/GRP78 signals. The ratio of BIP/GRP78 positive vs. negative cells was determined and shown. All results were confirmed by three independent experiments. *** p < 0.001.

Figure 4

Induction of apoptosis by HBsAg overexpression. HepG2-NTCP cells and PHHs were transduced by AdVs with an MOI of 1 or treated with Thapsigargin (Tg, 1 µM for 24 h) as a positive control. (A) Five days after transduction, apoptosis was evaluated by the Caspase-3/7 activity assay. (B) Cell viability was measured by ATPlite. The Student’s unpaired two-tailed t test was applied for data analysis. *p < 0.05, ***p < 0.001. (C) In AdVs-transduced HepG2-NTCP cells, immunofluorescent staining for HBsAg (red) or cleaved-caspase-3 (green) was performed. Nuclei were stained with Hoechst 33342. Scale bar: 20 μm. (D) Ad-HBsAg transduced cells were grouped by HBsAg staining and measured for cleaved-caspase-3 signals. The ratio of cleaved-caspase-3 positive vs. negative cells was determined and shown. ** represents p < 0.01.

Figure 5

H&E and immunofluorescent staining of HBV-infected livers. (A) Paraffin-embedded liver specimens were evaluated by H&E and immunofluorescent staining. Staining of representative fields of normal liver and three HBV patients are shown. Areas with GGHs are magnified and marked (white arrowheads). HBsAg (red), BIP/GRP78 (green) and nuclei (blue) are shown. Scale bar represents 50 μm. (B) Quantifications were performed based on the staining of HBV patients shown above. The ratio of HBsAg-positive to negative cells (left) or BIP/GRP78 -positive vs. negative cells (middle) is shown. Similarly, groups based on HBsAg staining and the ratio of BIP/GRP78 -positive vs. negative cells were determined (right). *** p < 0.001. (C) In immunofluorescent staining on liver samples from chronic hepatitis B patients, the HBsAg and BIP/GRP78 intensities in each cell were measured, and their correlation was evaluated.