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Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

Graumann F, Churin Y, Tschuschner A, Reifenberg K, Glebe D, Roderfeld M, Roeb E - PLoS ONE (2015)

Bottom Line: The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks.In addition lower HBs-expression went on with decreased ER-stress.Thus, targeted modulation of HBs expression may offer new therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Justus Liebig University, Giessen, Germany.

ABSTRACT

Objective: The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice.

Methods: Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR.

Results: From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome.

Conclusions: Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

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Spontaneous loss of HBs expression reduced ER-stress and apoptosis.A) Representative Western blots of p-eIF2α, p-PERK, and CHOP demonstrated the correlation between HBs expression and activation of ER-stress. Loading controls: ß-Actin, eIF2α, and PERK. (B) Co-immunostaining of HBs (red) and CHOP (black) in transgenic mouse liver. The micrographs demonstrate that HBs expressing hepatocytes undergo apoptosis only. Scale bars 160/80 μm. Arrowheads indicate CHOP stained apoptotic hepatocytes. (C) Co-immunostaining of HBs (red) and GRP78 (black) in transgenic mouse liver. IHC demonstrated strong expression of GRP78 in selected hepatocytes in centrilobular areas. Scale bars 50 μm. Arrowheads indicate GRP78 stained hepatocytes.
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pone.0146099.g002: Spontaneous loss of HBs expression reduced ER-stress and apoptosis.A) Representative Western blots of p-eIF2α, p-PERK, and CHOP demonstrated the correlation between HBs expression and activation of ER-stress. Loading controls: ß-Actin, eIF2α, and PERK. (B) Co-immunostaining of HBs (red) and CHOP (black) in transgenic mouse liver. The micrographs demonstrate that HBs expressing hepatocytes undergo apoptosis only. Scale bars 160/80 μm. Arrowheads indicate CHOP stained apoptotic hepatocytes. (C) Co-immunostaining of HBs (red) and GRP78 (black) in transgenic mouse liver. IHC demonstrated strong expression of GRP78 in selected hepatocytes in centrilobular areas. Scale bars 50 μm. Arrowheads indicate GRP78 stained hepatocytes.

Mentions: Transgenic mice of both gender were sacrificed at week 5, 12, 19, 26, and 33 (n = 8–10 per group). All mice included were identified to be transgenic for HBs by genotyping. The spontaneous reduction in expression of HBs was observed in immunohistochemically stained liver samples of some elder mice (Fig 1A). HBs free areas appeared irregular in shape. The effect appeared in 20% of mice aged 12 weeks. The relative number of mice with reduced HBs-expression was 45% at the age of 26 weeks and 56% in 33 weeks old mice. Younger mice at the age of 5 weeks did not show any loss of HBs expression (Fig 1B). In order to define mouse groups with reduced transgene expression, 19–33 weeks old mice were split into three groups: high (n = 9, all hepatocytes were stained positive for HBs), medium (n = 5, 11–99% of all hepatocytes were stained positive for HBs), and low (n = 4, ≤10% of hepatocytes were stained positive for HBs) expression of HBs protein (Fig 1A). Significant differences in HBs expression were validated by ELISA and Western blotting (Figs 1C and 2A). Quantitative ELISA data revealed significant differences in HBs protein expression between all three groups. This clearly defines each group and furthermore underlines the distinguished character of the ´high´, ´median´, and ´low´ group. No gender specific effects were observed in this model.


Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes.

Graumann F, Churin Y, Tschuschner A, Reifenberg K, Glebe D, Roderfeld M, Roeb E - PLoS ONE (2015)

Spontaneous loss of HBs expression reduced ER-stress and apoptosis.A) Representative Western blots of p-eIF2α, p-PERK, and CHOP demonstrated the correlation between HBs expression and activation of ER-stress. Loading controls: ß-Actin, eIF2α, and PERK. (B) Co-immunostaining of HBs (red) and CHOP (black) in transgenic mouse liver. The micrographs demonstrate that HBs expressing hepatocytes undergo apoptosis only. Scale bars 160/80 μm. Arrowheads indicate CHOP stained apoptotic hepatocytes. (C) Co-immunostaining of HBs (red) and GRP78 (black) in transgenic mouse liver. IHC demonstrated strong expression of GRP78 in selected hepatocytes in centrilobular areas. Scale bars 50 μm. Arrowheads indicate GRP78 stained hepatocytes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696744&req=5

pone.0146099.g002: Spontaneous loss of HBs expression reduced ER-stress and apoptosis.A) Representative Western blots of p-eIF2α, p-PERK, and CHOP demonstrated the correlation between HBs expression and activation of ER-stress. Loading controls: ß-Actin, eIF2α, and PERK. (B) Co-immunostaining of HBs (red) and CHOP (black) in transgenic mouse liver. The micrographs demonstrate that HBs expressing hepatocytes undergo apoptosis only. Scale bars 160/80 μm. Arrowheads indicate CHOP stained apoptotic hepatocytes. (C) Co-immunostaining of HBs (red) and GRP78 (black) in transgenic mouse liver. IHC demonstrated strong expression of GRP78 in selected hepatocytes in centrilobular areas. Scale bars 50 μm. Arrowheads indicate GRP78 stained hepatocytes.
Mentions: Transgenic mice of both gender were sacrificed at week 5, 12, 19, 26, and 33 (n = 8–10 per group). All mice included were identified to be transgenic for HBs by genotyping. The spontaneous reduction in expression of HBs was observed in immunohistochemically stained liver samples of some elder mice (Fig 1A). HBs free areas appeared irregular in shape. The effect appeared in 20% of mice aged 12 weeks. The relative number of mice with reduced HBs-expression was 45% at the age of 26 weeks and 56% in 33 weeks old mice. Younger mice at the age of 5 weeks did not show any loss of HBs expression (Fig 1B). In order to define mouse groups with reduced transgene expression, 19–33 weeks old mice were split into three groups: high (n = 9, all hepatocytes were stained positive for HBs), medium (n = 5, 11–99% of all hepatocytes were stained positive for HBs), and low (n = 4, ≤10% of hepatocytes were stained positive for HBs) expression of HBs protein (Fig 1A). Significant differences in HBs expression were validated by ELISA and Western blotting (Figs 1C and 2A). Quantitative ELISA data revealed significant differences in HBs protein expression between all three groups. This clearly defines each group and furthermore underlines the distinguished character of the ´high´, ´median´, and ´low´ group. No gender specific effects were observed in this model.

Bottom Line: The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks.In addition lower HBs-expression went on with decreased ER-stress.Thus, targeted modulation of HBs expression may offer new therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Justus Liebig University, Giessen, Germany.

ABSTRACT

Objective: The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice.

Methods: Liver and serum of HBs transgenic mice aged 5-33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR.

Results: From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome.

Conclusions: Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation.

Show MeSH
Related in: MedlinePlus