title: 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.
abstract: 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.
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Liyong
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, the Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Wenpei
authors.initials: W
authors.lastname: Xiang
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Wang
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Zhengzheng
authors.initials: Z
authors.lastname: Yan
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Zhaowei
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Guo
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Rajib
authors.initials: R
authors.lastname: Sengupta
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Alex F
authors.initials: AF
authors.lastname: Chen
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, the Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, and.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Loughran
authors.affiliation: the Xiangya Third Hospital and Central South University School of Medicine, Changsha, China.
authors.forename: Ben
authors.initials: B
authors.lastname: Lu
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
authors.forename: Qingde
authors.initials: Q
authors.lastname: Wang
authors.affiliation: From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, billiartr@upmc.edu.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D016899:Interferon-beta / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008954:Models, Biological; D052247:Nitric Oxide Synthase Type II / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011070:Poly I-C / Q000494:pharmacology; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000276:immunology* / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051196:Toll-Like Receptor 3 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D015854:Up-Regulation / Q000187:drug effects; D019892:eIF-2 Kinase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D016899:Interferon-beta; D008070:Lipopolysaccharides; D052247:Nitric Oxide Synthase Type II; D011070:Poly I-C; D012330:RNA, Double-Stranded; D051196:Toll-Like Receptor 3; D014443:Tyrosine; D019892:eIF-2 Kinase; D019061:src-Family Kinases; C476286:Ticam1 protein, mouse; C519703:TLR3 protein, mouse; C496317:Nos2 protein, mouse; C000605978:protein kinase R, mouse
keywords: TIR domain-containing adapter-inducing interferon B (TRIF); Toll-like receptor (TLR); hepatocyte; immunology; inflammation; nitric oxide; nitric oxide synthase