The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF-β signaling

J Hepatol. 2013 Nov;59(5):942-8. doi: 10.1016/j.jhep.2013.06.025. Epub 2013 Jul 2.

Abstract

Background & aims: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis.

Methods: The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-β signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression.

Results: Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-β signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-β signaling.

Conclusions: Thus reciprocal regulation between HCV and SNARK promotes TGF-β signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs.

Keywords: CsA; Fibrosis; HCC; HCV; HTAs; JFH1; Japanese fulminant hepatitis 1; Kinase; Metformin; NUAK2; SMAD; SNARK; SVR; TGF-beta; TGF-β; cyclosporin A; hepatocellular carcinoma; host-targeting antivirals; sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-related protein kinase; sustained virological response; transforming growth factor beta.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biopsy
  • Cell Line
  • Gene Expression Regulation / drug effects
  • Hepacivirus / physiology*
  • Hepatitis C / etiology*
  • Hepatitis C / physiopathology
  • Humans
  • Liver / pathology
  • Liver / virology
  • Metformin / pharmacology
  • Protein Kinase Inhibitors / pharmacology
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / physiology*
  • RNA, Small Interfering / pharmacology
  • Signal Transduction / physiology*
  • Transforming Growth Factor beta / physiology*
  • Virus Replication / physiology*

Substances

  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Transforming Growth Factor beta
  • Metformin
  • NUAK2 protein, human
  • Protein-Serine-Threonine Kinases