Interferon β (IFN-β) Production during the Double-stranded RNA (dsRNA) Response in Hepatocytes Involves Coordinated and Feedforward Signaling through Toll-like Receptor 3 (TLR3), RNA-dependent Protein Kinase (PKR), Inducible Nitric Oxide Synthase (iNOS), and Src Protein

J Biol Chem. 2016 Jul 15;291(29):15093-107. doi: 10.1074/jbc.M116.717942. Epub 2016 May 17.


The sensing of double-stranded RNA (dsRNA) in the liver is important for antiviral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here we sought to establish the pathways involved in the production of type I interferons (IFN-I) in response to extracellular poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and, unlike most immune cells that readily die after activation with dsRNA, are not viewed as cells with robust antimicrobial capacity. We found that poly(I:C) leads to rapid up-regulation of inducible nitric oxide synthase (iNOS), double-stranded RNA-dependent protein kinase (PKR), and Src. The production of IFN-β was dependent on iNOS, PKR, and Src and partially dependent on TLR3/Trif. iNOS and Src up-regulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-β production in an iNOS- and Src-dependent manner, and Src was found to directly interact with TLR3 in the endosomal compartment of poly(I:C)-treated cells. Furthermore, we identified a robust NO/cGMP/PKG-dependent feedforward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-β production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR.

Keywords: TIR domain-containing adapter-inducing interferon B (TRIF); Toll-like receptor (TLR); hepatocyte; immunology; inflammation; nitric oxide; nitric oxide synthase.

Publication types

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

MeSH terms

  • Adaptor Proteins, Vesicular Transport / deficiency
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Animals
  • Cells, Cultured
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Hepatocytes / drug effects
  • Hepatocytes / immunology*
  • Hepatocytes / metabolism*
  • Interferon-beta / biosynthesis*
  • Lipopolysaccharides / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Models, Biological
  • Nitric Oxide Synthase Type II / deficiency
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism*
  • Phosphorylation / drug effects
  • Poly I-C / pharmacology
  • RNA Interference
  • RNA, Double-Stranded / immunology*
  • RNA, Double-Stranded / pharmacology*
  • Signal Transduction / drug effects
  • Toll-Like Receptor 3 / deficiency
  • Toll-Like Receptor 3 / genetics
  • Toll-Like Receptor 3 / metabolism*
  • Tyrosine / chemistry
  • Up-Regulation / drug effects
  • eIF-2 Kinase / deficiency
  • eIF-2 Kinase / genetics
  • eIF-2 Kinase / metabolism*
  • src-Family Kinases / antagonists & inhibitors
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism*


  • Adaptor Proteins, Vesicular Transport
  • Lipopolysaccharides
  • RNA, Double-Stranded
  • TICAM-1 protein, mouse
  • TLR3 protein, mouse
  • Toll-Like Receptor 3
  • Tyrosine
  • Interferon-beta
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • src-Family Kinases
  • eIF-2 Kinase
  • protein kinase R, mouse
  • Poly I-C