Date on Master's Thesis/Doctoral Dissertation

12-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

Barve, Shirish Shrikrishna

Author's Keywords

Interferon-alpha; Hepatitis C; S-adenosylmethionine; Epigenetics

Subject

Hepatitis C virus--Research; Interferon--Therapeutic use; Adenosylmethionine

Abstract

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease in the United States and is a huge burden on the US healthcare system. The FDA-approved traditional standard of care for HCV is pegylated interferon-alpha (lFNa) combined with ribavirin, which is effective in about 50% of patients. The molecular mechanisms involved in resistance to IFNa therapy remain unclear. Recent data strongly suggest that histone deacetylases (HDACs) and methylation play critical roles in the regulation of IFNa anti-HCV signaling and gene expression. The present work was carried out to elucidate the roles of HDACs and S-adenosylmethionine (SAM) metabolism in regulating IFNa anti-HCV signaling in human hepatoma cells. Inhibition of HDACs, by pharmacologic HDAC inhibitors or siRNA, significantly suppressed IFNa-mediated antiviral gene expression and partially reversed the anti-HCV action of IFNa in human hepatoma cells. The decrease in antiviral gene expression correlated with decreased retention time of activated STATs in the nucleus, an increase in STAT acetylation, inhibition of the STAT1 :HDAC1 complex, and decreased occupancy of STAT1 on antiviral gene promoters. We used siRNA to specifically identify HDACs 1 and 3 as being critical for IFNa-mediated anti-HCV activity. Finally, we showed that boosting HDAC gene expression by theophylline supplementation improved IFNa-mediated antiviral gene expression and anti-HCV activity, thus supporting the hypothesis that HDACs are critical for IFNa anti-HCV signaling. Impaired SAM metabolism, as a result of increased intracellular S-adenosylhomocysteine, markedly reduced IFNa-mediated antiviral gene expression and anti-HCV activity, which correlated with a decrease in STAT phosphorylation and an increase in association between STAT1 and its negative regulator PIAS 1. We also showed that impaired SAM metabolism downregulated expression of several HDACs, which may also impact IFNa antiviral signaling. Importantly, SAM supplementation restored the antiviral and anti-HCV properties of IFNa. Acrolein, an environmental pollutant, significantly inhibited antiviral gene expression, which correlated to impaired STAT phosphorylation, decreased induction of class I HDAC mRNAs, and reduced HDAC activity in human hepatoma cells. The results presented herein reveal a critical role for HDACs and SAM metabolism in IFNa-mediated anti-HCV activity and support the use of SAM and/or inducers of HDACs as adjunct therapy in managing HCV infection.

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