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*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    Liyong Zhang
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Wenpei Xiang
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

    Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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  • Guoliang Wang
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Zhengzheng Yan
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Zhaowei Zhu
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Zhong Guo
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Author Footnotes
    2 Present address: Dept. of Biochemistry and Biotechnology, Sardar Bhagwan Singh P. G. Institute, Dehradun, Uttarakhand, India.
    Rajib Sengupta
    Footnotes
    2 Present address: Dept. of Biochemistry and Biotechnology, Sardar Bhagwan Singh P. G. Institute, Dehradun, Uttarakhand, India.
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Alex F. Chen
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Patricia A. Loughran
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

    Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
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  • Ben Lu
    Affiliations
    Xiangya Third Hospital and Central South University School of Medicine, Changsha, China
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  • Qingde Wang
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Timothy R. Billiar
    Correspondence
    To whom correspondence should be addressed: Dept. of Surgery, University of Pittsburgh School of Medicine, 200 Lothrop St., Pittsburgh, PA 15213. Tel.:412-647-1749; Fax:412-647-5959;
    Affiliations
    Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants R37-GM-044100 and R01-GM-050441. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental Fig. S1.
    1 Both authors contributed equally to this work.
    2 Present address: Dept. of Biochemistry and Biotechnology, Sardar Bhagwan Singh P. G. Institute, Dehradun, Uttarakhand, India.
      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.

      Introduction

      The innate immune system responds rapidly to molecular patterns from invading micro-organisms or damaged tissue through pattern recognition receptors expressed by both immune cells and non-immune cell types (
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      Pathogen recognition and inflammatory signaling in innate immune defenses.
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      Innate immunity to virus infection.
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      Signaling in innate immunity and inflammation.
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      Role of toll-like receptors in changes in gene expression and NF-κ B activation in mouse hepatocytes stimulated with lipopolysaccharide.
      ). Currently, there are three classes of pattern recognition receptors involved in the recognition of pathogen-associated molecular pattern and damage-associated molecular pattern molecules, including toll-like receptors (TLRs),
      The abbreviations used are: TLR, toll-like receptor; poly(I:C), polyinosinic-polycytidylic acid; dsRNA, double-stranded RNA; iNOS, inducible NO synthase; PKR, double-stranded RNA-dependent protein kinase; TIR domain-containing adapter-inducing IFN B; SNAP, S-nitroso-N-acetylpenicillamine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; IRF, IFN-regulatory factor; FAK, focal adhesion kinase.
      RIG-I-like receptors, and NOD-like receptors (
      • Takeuchi O.
      • Akira S.
      Innate immunity to virus infection.
      ). Among these receptors, TLRs and RIG-I-like receptors are important for the production of type I interferons (IFN-I) (
      • Takeuchi O.
      • Akira S.
      Innate immunity to virus infection.
      ,
      • Yang S.
      • Deng P.
      • Zhu Z.
      • Zhu J.
      • Wang G.
      • Zhang L.
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      • Billiar T.R.
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      Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.
      ). IFN-I includes the IFN-α family and IFN-β, which exert a vast spectrum of biological functions. One of the well characterized functions is inhibition of the replication of viruses (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ), but IFN-I can also contribute to the pathogenesis of sterile liver injury (
      • Shen X.D.
      • Ke B.
      • Ji H.
      • Gao F.
      • Freitas M.C.
      • Chang W.W.
      • Lee C.
      • Zhai Y.
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      Disruption of type-I IFN pathway ameliorates preservation damage in mouse orthotopic liver transplantation via HO-1 dependent mechanism.
      ).
      Toll-like receptors recognize pathogen-associated molecular patterns and damage-associated molecular patterns and elicit proinflammatory signals, which lead to the activation of immune responses (
      • Moore T.C.
      • Petro T.M.
      IRF3 and ERK MAP-kinases control nitric oxide production from macrophages in response to poly-I:C.
      ). For example, TLR3 recognizes double-stranded RNA (dsRNA) and its analog poly(I:C) and induces the expression of antiviral genes and production of NO via the inducible NO synthase (iNOS, NO2) (
      • Moore T.C.
      • Petro T.M.
      IRF3 and ERK MAP-kinases control nitric oxide production from macrophages in response to poly-I:C.
      ,
      • Maa M.C.
      • Chang M.Y.
      • Li J.
      • Li Y.Y.
      • Hsieh M.Y.
      • Yang C.J.
      • Chen Y.J.
      • Li Y.
      • Chen H.C.
      • Cheng W.E.
      • Hsieh C.Y.
      • Cheng C.W.
      • Leu T.H.
      The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages.
      ,
      • Mehta D.R.
      • Ashkar A.A.
      • Mossman K.L.
      The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts.
      ). Phosphorylation of five tyrosine residues on TLR3 at Tyr733, Tyr756, Tyr759, Tyr764, and Tyr858 has been shown to participate in dsRNA-induced gene expression (
      • Sarkar S.N.
      • Smith H.L.
      • Rowe T.M.
      • Sen G.C.
      Double-stranded RNA signaling by Toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain.
      ,
      • Sarkar S.N.
      • Elco C.P.
      • Peters K.L.
      • Chattopadhyay S.
      • Sen G.C.
      Two tyrosine residues of Toll-like receptor 3 trigger different steps of NF-κ B activation.
      ). Recently, the importance of the tyrosine kinases Bruton's tyrosine kinase (
      • Lee K.G.
      • Xu S.
      • Kang Z.H.
      • Huo J.
      • Huang M.
      • Liu D.
      • Takeuchi O.
      • Akira S.
      • Lam K.P.
      Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response.
      ) and Src (
      • Yamashita M.
      • Chattopadhyay S.
      • Fensterl V.
      • Saikia P.
      • Wetzel J.L.
      • Sen G.C.
      Epidermal growth factor receptor is essential for Toll-like receptor 3 signaling.
      ) has been implicated in dsRNA-mediated TLR3 Tyr759 phosphorylation in different types of cells. In macrophages, dsRNA induces TLR3 Tyr759 phosphorylation to promote IFN-β secretion that is dependent on the up-regulation of iNOS, which activates Src (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ). The double-stranded RNA-dependent protein kinase (PKR) is also activated by dsRNA, leading to NFκB activation (
      • Jiang Z.
      • Zamanian-Daryoush M.
      • Nie H.
      • Silva A.M.
      • Williams B.R.
      • Li X.
      Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFκ B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR.
      ). Upon dsRNA binding to the TLR3 receptor, PKR is recruited by a TAK1-containing complex in transfected 293 cell lines to engage TLR3 signaling (
      • Jiang Z.
      • Zamanian-Daryoush M.
      • Nie H.
      • Silva A.M.
      • Williams B.R.
      • Li X.
      Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFκ B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR.
      ).
      Hepatocytes express TLR3 and rapidly up-regulate iNOS under a number of conditions, including TLR stimulation (
      • Liu S.
      • Gallo D.J.
      • Green A.M.
      • Williams D.L.
      • Gong X.
      • Shapiro R.A.
      • Gambotto A.A.
      • Humphris E.L.
      • Vodovotz Y.
      • Billiar T.R.
      Role of toll-like receptors in changes in gene expression and NF-κ B activation in mouse hepatocytes stimulated with lipopolysaccharide.
      ,
      • Broering R.
      • Lutterbeck M.
      • Trippler M.
      • Kleinehr K.
      • Poggenpohl L.
      • Paul A.
      • Gerken G.
      • Schlaak J.F.
      Long-term stimulation of Toll-like receptor 3 in primary human hepatocytes leads to sensitization for antiviral responses induced by poly I:C treatment.
      ,
      • Bogdan C.
      Nitric oxide and the immune response.
      ,
      • Chanthaphavong R.S.
      • Loughran P.A.
      • Lee T.Y.
      • Scott M.J.
      • Billiar T.R.
      A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes.
      ,
      • Deng M.
      • Loughran P.A.
      • Zhang L.
      • Scott M.J.
      • Billiar T.R.
      Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling.
      ,
      • Curran R.D.
      • Billiar T.R.
      • Stuehr D.J.
      • Hofmann K.
      • Simmons R.L.
      Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells.
      ). These cells are the target of several viruses (
      • El-Serag H.B.
      Epidemiology of viral hepatitis and hepatocellular carcinoma.
      ,
      • Wang K.
      Molecular mechanisms of hepatic apoptosis.
      ), and we recently provided evidence for a robust response to exogenous and endogenous dsRNA in hepatocytes for IFN-I production (
      • Yang S.
      • Deng P.
      • Zhu Z.
      • Zhu J.
      • Wang G.
      • Zhang L.
      • Chen A.F.
      • Wang T.
      • Sarkar S.N.
      • Billiar T.R.
      • Wang Q.
      Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.
      ,
      • Wang H.
      • Wang G.
      • Zhang L.
      • Zhang J.
      • Zhang J.
      • Wang Q.
      • Billiar T.R.
      ADAR1 suppresses the activation of cytosolic RNA-sensing signaling pathways to protect the liver from ischemia/reperfusion injury.
      ). Here we sought to define the relationship between the iNOS/cGMP/PKG, Src/TLR3/Trif, and PKR pathways in the hepatocyte response to dsRNA. We show that IFN-β production in response to poly(I:C) is entirely iNOS-dependent and that NO exerts a feedforward up-regulation on iNOS expression in a cGMP/PKG-dependent manner. iNOS/NO promotes TLR3 Tyr759 phosphorylation through an Src-dependent mechanism associated with a direct interaction of Src with TLR3 localized to the endosomal compartment. Both iNOS and Src are markedly up-regulated in a PKR-dependent mechanism that itself is dramatically induced in response to poly(I:C). TLR3 signaling through Trif synergistically interacts with PKR to up-regulate iNOS expression upon dsRNA stimulation. These findings demonstrate a robust production of IFN-I in hepatocytes exposed to dsRNA through synergistic signaling pathways dependent on iNOS/NO.

      Experimental Procedures

       Reagents and Antibodies

      Low molecular weight poly(I:C) (poly(I:C)LMW, 0.2–1 kb), high molecular weight poly(I:C) (poly(I:C)HMW, 1.5–8 kb), and ultrapure LPS (Escherichia coli 0111:B4) were purchased from InvivoGen (San Diego, CA). Williams' medium E, penicillin, streptomycin, l-glutamine, and HEPES were purchased from Invitrogen. Insulin (Humulin) was acquired from Eli Lilly and Co. (Indianapolis, IN), and fetal calf serum was purchased from Hyclone Laboratories (Logan, UT). Protein A/G plus agarose beads (sc-2003), Src kinase inhibitor (sc-204303), Src siRNA, PKR siRNA, PKGIβ siRNA, and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The selective imidazolo-oxindole PKR inhibitor C16 (19785) was from Sigma-Aldrich (St. Louis, MO). The transfection reagent GeneJammer was from Agilent Technologies (Santa Clara, CA).
      The following specific primary antibodies were used: iNOS (sc-651, NBP1–62139, and 610333), IRF-1 (sc-13041), IRF-3 (sc-9082), IRF-7 (sc-9083), PKR (sc-6282), TLR4 (sc-16240), FAK (sc-558), FAK-Tyr(P)861 (sc-16663), and proliferating cell nuclear antigen (sc-56) from Santa Cruz Biotechnology; TRIF (NB120-13810), TLR3-Tyr(P)759 (NBP2-24904) from Novus Biologicals (Littleton, CO); TLR3 (ab62566), Src (ab32102), IFN-β (ab85083), Rab5 (ab18211), and Rab7 (ab137029) from Abcam (Cambridge, MA); Src (05-184) from Upstate Biotechnology (Lake Placid, NY); and Src (LS-C96491) from LifeSpan BioSciences (Seattle, WA). Goat anti-rabbit, goat anti-mouse, and donkey anti-goat HRP-conjugated secondary antibodies were purchased from Promega (Madison, WI).

       Animals

      C57BL/6 WT 8- to 12-week-old mice were purchased from Charles River Laboratories (Wilmington, MA). TLR4 knockout (TLR4−/−, TLR4-KO), TRIF knockout (TRIF−/−, TRIF-KO), iNOS knockout (iNOS−/−, iNOS-KO), and PKR knockout (PKR−/−, PKR-KO) mice were bred in our facility. C57BL/6NJ TLR3 knockout (TLR3−/−, TLR3-KO) and wild-type mice of the same age and sex were from The Jackson Laboratory (Bar Harbor, ME). All experimental procedures involving animals were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

       Hepatocyte Culture and Poly(I:C) Treatment

      Hepatocytes were isolated from mice as described previously (
      • Yang S.
      • Deng P.
      • Zhu Z.
      • Zhu J.
      • Wang G.
      • Zhang L.
      • Chen A.F.
      • Wang T.
      • Sarkar S.N.
      • Billiar T.R.
      • Wang Q.
      Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.
      ,
      • Chanthaphavong R.S.
      • Loughran P.A.
      • Lee T.Y.
      • Scott M.J.
      • Billiar T.R.
      A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes.
      ,
      • Wang H.
      • Wang G.
      • Zhang L.
      • Zhang J.
      • Zhang J.
      • Wang Q.
      • Billiar T.R.
      ADAR1 suppresses the activation of cytosolic RNA-sensing signaling pathways to protect the liver from ischemia/reperfusion injury.
      ). The purity exceeded 99% as measured by flow cytometric assay, and viability was typically measured over 85% using trypan blue exclusion. Hepatocytes (1.5 × 105 cells/ml) were plated on gelatin-coated culture plates or seeded onto coverslips precoated with collagen I (BD Biosciences) in Williams' medium E with 10% calf serum, 15 mm HEPES,10−6 m insulin, 2 mm l-glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin. Hepatocytes were allowed to attach to plates overnight. Prior to treatments, cell culture medium was changed to medium containing 5% calf serum. After washing with PBS, hepatocytes were treated with 20 μg/ml poly(I:C) for stimulation for various durations. Culture medium and cell pellets were collected for further analysis.

       RNA Interference

      The Src siRNA and PKR siRNA were transiently transfected into hepatocytes using GeneJammer transfection reagent according to the instructions of the manufacturer's instructions. Twenty-four hours later, hepatocytes were stimulated with 20 μg/ml poly(I:C) for various durations. Culture medium and cell lysates collected at different time points were subject to Western blotting analysis.
      For hepatocytes transfected with PKGIβ siRNA, cells were treated with cytokines after washing with PBS and replenishment with complete William's E medium. Cells were harvested for detection of iNOS and NF-κB activity.

       Inhibitor Treatment

      Hepatocytes were pretreated with 50 nm Src kinase inhibitor or 250 nm imidazolo-oxindole PKR inhibitor C16 for 2 h after PBS washing. Media containing inhibitor and 20 μg/ml poly(I:C) were replenished after removing the media. Culture medium and cell lysates collected at various durations were subject to further analysis.

       Virus Infection

      Adenoviral vectors carrying bacterial β-galactosidase (Ad-LacZ) and iNOS (Ad-iNOS) were prepared as described previously (
      • Shears L.L.
      • Kawaharada N.
      • Tzeng E.
      • Billiar T.R.
      • Watkins S.C.
      • Kovesdi I.
      • Lizonova A.
      • Pham S.M.
      Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis.
      ). After overnight culture, hepatocytes were washed twice with PBS prior to infection at a multiplicity of infection of 3 in a volume of 1.5 ml of Opti-MEM (Life Technologies) using 6-well plates. Following a 2-h infection, the normal culture medium was changed for an incubation of 24 h. Cells were treated as designated in the figure legends. Samples were prepared at the indicated time points.

       Preparation of Total Lysates and Nuclear Extracts

      Hepatocytes were washed twice in PBS and harvested with lysis buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) on ice for 10 min. The supernatants were collected as whole cell extracts after centrifugation at 12,000 g for 30 min. The lysates from culture media were concentrated by adding less lysis buffer after centrifugation to remove the medium. The nuclear extracts were prepared as described previously (
      • Méndez J.
      • Stillman B.
      Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.
      ,
      • Zhang L.
      • Park C.H.
      • Wu J.
      • Kim H.
      • Liu W.
      • Fujita T.
      • Balasubramani M.
      • Schreiber E.M.
      • Wang X.F.
      • Wan Y.
      Proteolysis of Rad17 by Cdh1/APC regulates checkpoint termination and recovery from genotoxic stress.
      ). The protein concentrations were determined using the BCA protein assay kit (Pierce).

       Measurement of Nitrite Production

      The levels of nitrite (NO2) in culture supernatants and cell lysates were assessed to determine the amount of NO production according to the instructions of the Griess reagent system (G2930, Promega).

       IFN-β ELISA

      IFN-β levels in culture supernatants and cell lysates were detected using the IFN-β ELISA kit (R&D Systems) according to the instructions of the manufacturer.

       Western Blotting and Co-immunoprecipitation Analysis

      Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes, followed by incubation with optimized dilutions of primary antibodies at 4 °C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Proteins were detected with the ECL kit (Pierce). β-Actin, β-tubulin (Sigma), or proliferating cell nuclear antigen was used as a loading control.
      For co-immunoprecipitation, equal amounts of whole cell lysates were incubated with equal amounts of primary antibody rotating at 4 °C overnight, and immune complexes were then precipitated with protein A/G-agarose beads after rotating for 2 h at 4 °C. Electrophoresis loading buffer was added to the beads after washing with wash buffer (25 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1× protein inhibitor mixture) five times. After denaturing at 95 °C for 5 min, the supernatants were subjected to Western blotting.

       Immunofluorescent Staining

      Hepatocytes grown on coverslips were briefly washed in PBS and fixed in 2% paraformaldehyde at room temperature for 15 min and stained as described previously (
      • Eum H.A.
      • Vallabhaneni R.
      • Wang Y.
      • Loughran P.A.
      • Stolz D.B.
      • Billiar T.R.
      Characterization of DISC formation and TNFR1 translocation to mitochondria in TNF-α-treated hepatocytes.
      ). Hepatocytes were incubated with 2% BSA in PBS for 1 h, followed by five washes with PBS + 0.5% BSA (PBB). The samples were then incubated with antibodies diluted in PBB against iNOS, Src, and Tyr(P)759-TLR3 and organelle-specific markers, including Rab5 (an early endosome marker) and Rab7 (a late endosome marker) as described above. Samples were washed five times with PBB, followed by incubation in the appropriate Alexa Fluor 488 (1:500, Invitrogen) and Cy3 (1:1000, Jackson ImmunoResearch Laboratories) secondary antibodies diluted in PBB. Samples were washed three times with PBB, followed by a single wash with PBS before 30 s of incubation with Hoechst nuclear stain. The nuclear stain was removed, and samples were washed with PBS before covering the coverslip with Gelvatol (23 g of poly(vinyl alcohol 2000), 50 ml of glycerol, and 0.1% sodium azide to 100 ml of PBS). Positively stained cells in six random fields were imaged on a FluoView 1000 confocal scanning microscope (Olympus, Center Valley, PA). Imaging conditions were maintained at identical settings within each antibody labeling experiment, with original gating performed using the negative control.

       RT-PCR

      Total RNAs were extracted from hepatocytes using the RNeasy extraction kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. The quality of the RNA was assessed by 1% denaturing agarose gel electrophoresis and spectrophotometry. One microgram of total RNAs of each sample was reverse-transcribed to the first strand of cDNA with oligo(dT)12–18 primers by the OmniscriptTM reverse transcriptase kit (Qiagen). Real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was performed using forward and reverse primer pairs prevalidated and specific for iNOS, β-actin, and PKG (Qiagen). All samples were run in triplicates. The expression of the housekeeping gene β-actin was used as an internal control.

       EMSA

      For cell fractionation procedures, protease inhibitors (0.2 mg/ml Pefabloc, 0.01 mg/ml aprotinin, 0.01 mg/ml pepstatin, and 0.01 mg/ml leupeptin) were supplemented to all buffers. Briefly, cells were rinsed twice with ice-cold PBS and harvested in buffer A (10 mm HEPES, 1.5 mm MgCl2, 10 mm KCl, and 0.5% Nonidet P-40) and then incubated on ice for 10 min. Nuclei were pelleted at 5000 × g for 5 min at 4 °C. The pellets were resuspended in buffer A and washed twice. The pellets after the final wash were collected as nuclear extract and lysed in C+D buffer (20 mm HEPES, 1.5 mm MgCl2, 0.45 m KCl, 0.02 mm EDTA, and 25% Glycerol) for 1 h on ice. The supernatants were collected at 15,000 × g for 15 min and classified as the nuclear fraction. Equal amounts of nuclear extracts were incubated with 32P end-labeled, 45-mer, double-stranded NF-κB oligonucleotide for 30 min at room temperature. The DNA-protein complexes were separated from free oligonucleotide on 6.5% native polyacrylamide gels. Radioactivity in the gel was exposed to x-ray film (Eastman Kodak Co., Rochester, NY).

       Luciferase Activity Assay

      The mouse iNOS promoter-reporter plasmid (mouseiNOS (1.7) Luc) and the mouse iNOS promoter NF-кB specific mutant site-reporter plasmid (mouseiNOS-NF-кB Luc), which harbors 1.7 kb of upstream 5′-flanking DNA linked to the luciferase reporter gene, were constructed. Cells were transfected in Opti-MEM (Life Technologies) containing 1 μg of DNA and 10 μl of liposomes for 6 h. After washing with PBS, the cells were replenished with complete William's E medium. To control the transfection efficiency between groups, 0.2 μg of Renilla vector-pGL4.75 (hRluc/CMV) (Promega) was added to each well. Cells were treated with SNAP (100 μm), 8-pCPTcGMP (800 μm), and SNAP + 8-pCPTcGMP for 8 h. After treatment, cells were harvested and lysed in passive lysis buffer (Promega). Luciferase activity was detected with 20 μl of lysate in a Synergy Mx multimode reader (Biotek, Winooski, VT) using the Dual-Luciferase® reporter assay kit (Promega). Luciferase activity was normalized to Renilla activity.

       Statistical Analysis

      Each experiment was performed at least three times. The results were analyzed using analysis of variance in SigmaStat (Systat Software, San Jose, CA). Unless indicated, the results from a representative triplicate experiment are presented. Data are shown as mean ± S.D. p < 0.05 was considered statistically significant.

      Results

       Poly(I:C) Stimulates TLR3-Tyr759 Phosphorylation and IFN-β Production and Up-regulates Expression of Src and PKR in Hepatocytes

      It has been shown previously in macrophages that the iNOS/Src axis is involved in dsRNA-induced TLR3-Tyr759 phosphorylation and IFN-I up-regulation (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ,
      • Sarkar S.N.
      • Peters K.L.
      • Elco C.P.
      • Sakamoto S.
      • Pal S.
      • Sen G.C.
      Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.
      ). Hepatocytes also up-regulate IFNs when infected by a number of RNA and DNA viruses and also avidly express iNOS (
      • Heydtmann M.
      Macrophages in hepatitis B and hepatitis C virus infections.
      ,
      • Mihm S.
      • Fayyazi A.
      • Ramadori G.
      Hepatic expression of inducible nitric oxide synthase transcripts in chronic hepatitis C virus infection: relation to hepatic viral load and liver injury.
      ,
      • Guidotti L.G.
      • McClary H.
      • Loudis J.M.
      • Chisari F.V.
      Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice.
      ). Here we show that hepatocytes exposed to high (Fig. 1A) or low (supplemental Fig. S1) molecular weight preparations of the dsRNA mimic poly(I:C) exhibited a time-dependent increase in phosphorylation of TLR3-Tyr759 (Fig. 1A). TLR3-Tyr(P)759 first appeared at 2 h and continued to increase to the 24-h time point. In parallel, iNOS expression and NO production (measured as nitrite accumulation in the medium) increased (Fig. 1, A and B). The enhanced expression of iNOS and TLR3-Tyr(P)759 was also associated with increased PKR and Src expression as well as the appearance of the known Src phosphorylation target FAK-Tyr(P)861 (Fig. 1A). Furthermore, the levels of transcription factors known to be downstream of TLR3 signaling and involved in IFN-I production, including IRF3 and IRF7, increased in both the whole cell lysates and nuclear extracts in poly(I:C)-treated cells, whereas IRF1 levels remained unchanged (Fig. 1C). Both intracellular and medium levels of IFN-β increased in association with the up-regulation of the iNOS/Src/PKR/TLR3-Tyr(P)759 parameters (Fig. 1, C and D). The up-regulation of TLR3-Tyr(P)759, iNOS, Src, and IFN-β was observed in primary human hepatocytes (Fig. 1E) and a human hepatoma cell line (HepG2) following poly(I:C) treatment (Fig. 1F), indicating the conservation of the dsRNA response pathway in human liver cells.
      Figure thumbnail gr1
      FIGURE 1Poly(I:C)HMW induces a time-dependent increase of iNOS, iNOS activity, TLR3-Tyr(P)759, PKR, Src, Src activity, IRF3 and 7 expression, and nuclear accumulation as well as IFN-β production in cells and supernatant levels. A, mouse hepatocytes (WTMHC) were stimulated with poly(I:C)HMW for various durations (0, 0.17, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, iNOS, PKR, Src, FAK-Tyr(P)861, and FAK. β-Tubulin was used as a loading control. B, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. The nitrite concentrations in culture media and whole cell lysates were determined by Griess assay. Data are presented as individual data points and mean. *, p < 0.05. C, mouse hepatocytes were treated with poly(I:C)HMW for various durations (0, 0.17, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates, nuclear extracts, and culture media were separated by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, IRF1, IRF3, IRF7, and IFN- β. β-Tubulin, proliferating cell nuclear antigen, or β-actin was used as a loading control, respectively. D, mouse hepatocytes were exposed to poly(I:C)HMW for 24 h. IFN-β levels in culture media and whole cell lysates were examined by ELISA. Values came from at least three independent experiments and are presented as individual data points and mean. *, p < 0.05. E, human hepatocytes (HHC) were treated with poly(I:C)HMW for various durations (0, 1, 2, 6, 12, and 24 h). Whole cell lysates and culture media were resolved by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, iNOS, Src, and IFN-β. β-Tubulin or β-actin was used as a loading control. F, HepG2 cells were stimulated with poly(I:C)HMW for various durations (0, 1, 2, 6, 12, and 24 h). Whole cell lysates and culture media were fractionated by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, iNOS, Src, and IFN-β. β-Tubulin or β-actin was used as a loading control.

       Involvement of TLR3/iNOS/NO/Src in Poly(I:C)-induced IFN-β Production in Hepatocytes

      Endosomal sensing of dsRNA occurs through TLR3 in immune cells (
      • Johnsen I.B.
      • Nguyen T.T.
      • Ringdal M.
      • Tryggestad A.M.
      • Bakke O.
      • Lien E.
      • Espevik T.
      • Anthonsen M.W.
      Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.
      ). To determine whether hepatocytes also utilize TLR3 to react to poly(I:C) applied to the outside of the cell, hepatocytes isolated from wild-type or TLR3−/− mice were exposed to poly(I:C) for varying periods of time. Hepatocytes from TLR3−/− mice, which completely lacked TLR3 protein (Fig. 2A), showed a partial suppression of iNOS expression, NO production, and IFN-β production compared with wild-type hepatocytes following poly(I:C) treatment (Fig. 2, B–D). However, the up-regulation of PKR and Src expression was only modestly reduced in TLR3−/− hepatocytes exposed to poly(I:C). These data show that TLR3 accounts for some but not all of the responses to extracellular dsRNA exposure in hepatocytes.
      Figure thumbnail gr2
      FIGURE 2TLR3 is indispensable for poly(I:C)HMW-stimulated IFN-β secretion in hepatocytes. A, TLR3 KO mouse hepatocytes were lysed for immunoblotting with TLR3. β-Tubulin was used as a loading control. B, TLR3 KO mouse hepatocytes were stimulated with poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates and culture media were separated by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, PKR, Src, and IFN-β. C, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. The nitrite concentration in culture media and whole cell lysates was determined by Griess assay. Data are presented as individual data points and mean. *, p < 0.05. D, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. IFN-β levels in culture media and whole cell lysates were examined by ELISA. Data are presented as individual data points and mean. *, p < 0.05.
      We next confirmed that Src expression was required for TLR3 phosphorylation in response to poly(I:C). Both Src knockdown (Fig. 3A) and Src inhibition (Fig. 3B) blocked the formation of TLR3-Tyr(P)759 in response to poly(I:C) without altering total TLR3 levels. Src inhibition also blocked iNOS up-regulation and the up-regulation of IFN-β expression in hepatocytes. To seek evidence that Src had a direct interaction with TLR3-Tyr(P)759, immunoprecipitation experiments were performed (Fig. 3C). Immunoprecipitation of Src effectively pulled down TLR3, but only in the absence of Src inhibition or suppression. Immunoprecipitation of TLR3 led to the pulldown of Src, and this association increased over time in poly(I:C)-treated cells (Fig. 3D). Thus, Src is required for enhanced iNOS expression and TLR3-Tyr759 phosphorylation as well as IFN-β up-regulation in hepatocytes exposed to dsRNA.
      Figure thumbnail gr3
      FIGURE 3Src is important in poly(I:C)HMW-stimulated IFN-β secretion in hepatocytes. A, mouse hepatocytes (WTMHC) were transfected with Src siRNA for 24 h and exposed to poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against Src, iNOS, TLR3-Tyr(P)759, TLR3, and IFN-β. β-Tubulin was used as a loading control. B, mouse hepatocytes were pretreated with Src inhibitor for 2 h and exposed to poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against Src, iNOS, TLR3-Tyr(P)759, TLR3, and IFN-β. β-Tubulin was used as a loading control. C, Src immunoprecipitation (IP) was performed from hepatocytes stimulated with poly(I:C)HMW for 0, 1, or 12 h and analyzed by immunoblotting (IB) with antibodies against TLR3-Tyr(P)759 and Src. D, TLR3 IP was performed from hepatocytes stimulated with poly(I:C)HMW for 0, 1, 6, 12, or 24 h and analyzed by immunoblotting with antibodies against TLR3-Tyr(P)759 and TLR3.
      To establish the importance of iNOS to this signaling cascade, iNOS−/− hepatocytes or wild-type hepatocytes treated with an NO synthase inhibitor (l-N5-(1-iminoethyl)ornithine) were exposed to poly(I:C). Deletion of iNOS completely prevented TLR3-Tyr759 phosphorylation and the up-regulation of IFN-β (Fig. 4, A and B) but only partially suppressed PKR and Src up-regulation following poly(I:C) treatment. Overexpression of iNOS using an adenoviral vector (AdiNOS, Fig. 4, C–E) increased iNOS expression and NO production but enhanced TLR3-Tyr759 phosphorylation and IFN-β formation only when the cells were also exposed to poly(I:C). The effect of AdiNOS was blocked by inhibiting iNOS activity with the NO synthase inhibitor. An NO donor (SNAP) also overcame the impact of iNOS deletion on poly(I:C)-induced IFN-β production (Fig. 4E). Somewhat unexpected was the observation that iNOS expression was enhanced by NO donor exposure and blocked by NOS inhibitors (Fig. 4, C, D, and F). Hepatocytes up-regulate cGMP levels in response to iNOS or NO (
      • Billiar T.R.
      • Curran R.D.
      • Harbrecht B.G.
      • Stadler J.
      • Williams D.L.
      • Ochoa J.B.
      • Di Silvio M.
      • Simmons R.L.
      • Murray S.A.
      Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes.
      ). We confirmed that hepatocytes express the β1 subunit of soluble guanylyl cyclase (Fig. 4I). The iNOS/NO-dependent increase in iNOS expression appeared to be at least partially cGMP-dependent because the soluble guanylate cyclase inhibitor ODQ also suppressed poly(I:C)-induced iNOS expression (Fig. 4G). This potential feedforward system for iNOS up-regulation is further addressed below.
      Figure thumbnail gr4
      FIGURE 4Phosphorylation of TLR3 Tyr759 depends on iNOS, whereas NO amplifies iNOS expression and induces a feedforward mechanism through cGMP. A, iNOS KO mouse hepatocytes (MHC) were stimulated with poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates and culture media were resolved by SDS-PAGE and immunoblotted with antibodies against iNOS, TLR3-Tyr(P)759, TLR3, PKR, Src, and IFN-β. β-Tubulin or β-actin was used as a loading control. B, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. IFN-β levels in whole cell lysates were examined by ELISA. Data are presented as individual data points and mean. *, p < 0.05. C, iNOS KO mouse hepatocytes were infected with Ad-LacZ or Ad-iNOS. After 2 h of infection, normal culture medium with SNAP was added back for 24-h recovery. Poly(I:C)HWM and SNAP were added for 12-h treatment after two PBS washes. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against iNOS, TLR3-Tyr(P)759, and TLR3. β-Tubulin was used as a loading control. D, iNOS KO mouse hepatocytes were infected with Ad-LacZ or Ad-iNOS. After 2 h of infection, normal culture medium with l-N5-(1-iminoethyl)ornithine (L-NIO) was added back for 24-h recovery. Poly(I:C) with l-N5-(1-iminoethyl)ornithine was added for 12-h treatment after two PBS washes. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with specific antibodies that recognize iNOS, TLR3-Tyr(P)759, and TLR3. β-Tubulin was used as a loading control. E, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. The nitrite in whole cell lysates was determined by Griess assay. Data are presented as individual data points and mean. *, p < 0.05. F, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. The IFN-β levels in whole cell lysates were determined by ELISA. Data are presented as individual data points and mean. *, p < 0.05. G, wild-type mouse hepatocytes (WTMHC) were treated with the iNOS inhibitor l-N5-(1-iminoethyl)ornithine for 1, 6, or 12 h. *, p < 0.05. β-Tubulin was used as a loading control. H, wild-type mouse hepatocytes were treated with the cGMP inhibitor ODQ for 1, 6, or 12 h. Whole cell lysates were resolved by SDS-PAGE and immunoblotted by antibodies against iNOS, TLR3-Tyr(P)759, and TLR3. β-Tubulin was used as a loading control. I, lysates from whole liver and isolated mouse hepatocytes were immunoblotted with an antibody against the β1 subunit of soluble guanylyl cyclase.

       Src Co-localizes with TLR3-Tyr(P)759 in Endosomes following Poly(I:C) Stimulation in Hepatocytes

      Our findings show that Src interacts directly with TLR3 following poly(I:C) stimulation (Fig. 5, A and B). TLR3 localizes to endosomes in unstimulated immune cells, such as dendritic cells (
      • Johnsen I.B.
      • Nguyen T.T.
      • Ringdal M.
      • Tryggestad A.M.
      • Bakke O.
      • Lien E.
      • Espevik T.
      • Anthonsen M.W.
      Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.
      ), and translocates to dsRNA-containing endosomes following the uptake of dsRNA into cells (
      • Johnsen I.B.
      • Nguyen T.T.
      • Ringdal M.
      • Tryggestad A.M.
      • Bakke O.
      • Lien E.
      • Espevik T.
      • Anthonsen M.W.
      Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.
      ). Confocal imaging following immunofluorescence staining was carried out to determine where Src and TLR3-Tyr(P)759 interact. As shown in Fig. 5, A and B, Src co-localized with TLR3-Tyr(P)759 in both early (Rab5-expressing) and late (Rab7-expressing) endosomes at 12 and 24 h following poly(I:C) exposure. Although it is not clear whether phosphorylation takes place in the endosome, the appearance of Src with its phosphorylation target suggests that this could be the case.
      Figure thumbnail gr5
      FIGURE 5Phosphorylated Tyr759-TLR3 associates with Src on endosomes. Wild-type mouse hepatocytes were stimulated with poly(I:C)HMW for 0, 12, and 24 h and fixed for immunofluorescent staining. A, antibodies against Src, iNOS, and TLR3-Tyr(P)759 proteins. Red, Src; green, Rab5; blue, TLR3-Tyr(P)759; white, DAPI. B, red, Src; green, Rab7; blue, TLR3-Tyr(P)759; white, DAPI.

       dsRNA Stimulation Leads to IFN-β Expression through Both Trif and PKR in Hepatocytes

      TLR3 is known to signal through the adaptor protein Trif (
      • Zorde-Khvalevsky E.
      • Abramovitch R.
      • Barash H.
      • Spivak-Pohis I.
      • Rivkin L.
      • Rachmilewitz J.
      • Galun E.
      • Giladi H.
      Toll-like receptor 3 signaling attenuates liver regeneration.
      ,
      • McAllister C.S.
      • Lakhdari O.
      • Pineton de Chambrun G.
      • Gareau M.G.
      • Broquet A.
      • Lee G.H.
      • Shenouda S.
      • Eckmann L.
      • Kagnoff M.F.
      TLR3, TRIF, and caspase 8 determine double-stranded RNA-induced epithelial cell death and survival in vivo.
      ,
      • Yamamoto M.
      • Sato S.
      • Hemmi H.
      • Hoshino K.
      • Kaisho T.
      • Sanjo H.
      • Takeuchi O.
      • Sugiyama M.
      • Okabe M.
      • Takeda K.
      • Akira S.
      Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.
      ,
      • Vercammen E.
      • Staal J.
      • Beyaert R.
      Sensing of viral infection and activation of innate immunity by toll-like receptor 3.
      ). Downstream signaling can also include the up-regulation of the serine and threonine kinase PKR via IFN (
      • Borden E.C.
      • Sen G.C.
      • Uze G.
      • Silverman R.H.
      • Ransohoff R.M.
      • Foster G.R.
      • Stark G.R.
      Interferons at age 50: past, current and future impact on biomedicine.
      ,
      • Solis M.
      • Goubau D.
      • Romieu-Mourez R.
      • Genin P.
      • Civas A.
      • Hiscott J.
      Distinct functions of IRF-3 and IRF-7 in IFN-α gene regulation and control of anti-tumor activity in primary macrophages.
      ,
      • García M.A.
      • Gil J.
      • Ventoso I.
      • Guerra S.
      • Domingo E.
      • Rivas C.
      • Esteban M.
      Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action.
      ,
      • He Y.
      • Franchi L.
      • Núñez G.
      The protein kinase PKR is critical for LPS-induced iNOS production but dispensable for inflammasome activation in macrophages.
      ). However, PKR can also respond directly to dsRNA for NFκB activation (
      • García M.A.
      • Gil J.
      • Ventoso I.
      • Guerra S.
      • Domingo E.
      • Rivas C.
      • Esteban M.
      Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action.
      ). We next assessed the role of Trif in hepatocytes exposed to poly(I:C). As shown in Fig. 6, A–C, hepatocytes from Trif−/− mice had lower iNOS expression and less TLR3-Tyr759 phosphorylation compared with wild-type hepatocytes following poly(I:C) treatment. Src up-regulation was only slightly lower in Trif−/− hepatocytes relative to wild-type cells exposed to poly(I:C). Nitrite and IFN-β accumulation in cells was significantly suppressed but not completely inhibited in Trif−/− hepatocytes. Interestingly, PKR deletion or suppression by siRNA almost completely blocked poly(I:C)-induced iNOS expression, Src up-regulation, and IFN-β expression in hepatocytes (Fig. 6, D and E). These data, combined with data from Fig. 2, suggest that PKR acts synergistically with TLR3 to up-regulate iNOS expression and in parallel with TLR3 to regulate Src expression in response to poly(I:C).
      Figure thumbnail gr6
      FIGURE 6iNOS expression, phosphorylation of TLR3 Tyr759, Src expression, and IFN-β production are PKR- dependent but partially TRIF-dependent in response to poly(I:C). A, TRIF KO mouse hepatocytes were lysed for immunoblotting with TRIF. B, TRIF KO mouse hepatocytes (MHC) were stimulated with poly(I:C)HMW for various durations (0, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against iNOS, TLR3-Tyr(P)759, TLR3, Src, and IFN-β. C, mouse hepatocytes were stimulated with poly(I:C)HMW for 24 h. The nitrite concentration and IFN-β levels in whole cell lysates were determined. Data are presented as individual data points and mean. *, p < 0.05. D, PKR KO mouse hepatocytes were stimulated with poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PKR, iNOS, TLR3-Tyr(P)759, TLR3, Src, and IFN-β. E, wild-type mouse hepatocytes were transfected with PKR siRNA for 24 h and stimulated with poly(I:C)HMW for various durations (0, 0.5, 1, 2, 6, 12, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PKR and iNOS. β-Tubulin was used as a loading control.

       LPS Also Induces IFN-β Production through an Src-dependent Manner in Hepatocytes

      LPS is a potent inducer of iNOS expression in mouse hepatocytes (
      • Chanthaphavong R.S.
      • Loughran P.A.
      • Lee T.Y.
      • Scott M.J.
      • Billiar T.R.
      A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes.
      ,
      • Deng M.
      • Loughran P.A.
      • Zhang L.
      • Scott M.J.
      • Billiar T.R.
      Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling.
      ) and is known to signal through Trif via TLR4 (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ,
      • Maa M.C.
      • Chang M.Y.
      • Li J.
      • Li Y.Y.
      • Hsieh M.Y.
      • Yang C.J.
      • Chen Y.J.
      • Li Y.
      • Chen H.C.
      • Cheng W.E.
      • Hsieh C.Y.
      • Cheng C.W.
      • Leu T.H.
      The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages.
      ). To determine whether the signaling events observed with poly(I:C) also occurred following LPS stimulation, we exposed wild-type hepatocytes to LPS (100 ng/ml) with or without poly(I:C) for 12 h (Fig. 7, A–D). LPS induced a time-dependent increase in TLR3-Tyr759 phosphorylation and an associated increase in iNOS, PKR, Src, and IFN-β expression. Levels of these parameters were less than that seen with poly(I:C), and the combination of LPS with poly(I:C) showed no additional increase over poly(I:C) alone. LPS also increased the phosphorylation of the Src target FAK and increased nuclear IRF3 and IRF7 levels. The increase in LPS-induced IFNβ production was prevented by Src suppression or inhibition (Fig. 7D). Taken together, these data indicate that LPS also induces Src-dependent induction of IFN-β production by hepatocytes through a pathway that may include signaling through iNOS and PKR.
      Figure thumbnail gr7
      FIGURE 7TLR4 is indispensable for NO-mediated induction of TLR3 tyrosine 759 phosphorylation in LPS- or poly(I:C)-treated hepatocytes. A, wild-type mouse hepatocytes (WTMHC) were stimulated with 100 ng/ml LPS, 20 μg/ml poly(I:C)HMW, or a combination of LPS and poly(I:C)HMW for various durations (0, 1, and 12 h). Whole cell lysates, and culture media were resolved by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, iNOS, Src, PKR, and IFN-β. β-Tubulin or β-actin was used as a loading control. B, IFN-β levels in whole cell lysates from A were determined by ELISA. Data are presented as individual data points and mean. *, p < 0.05. C, wild-type mouse hepatocytes were stimulated with 100 ng/ml LPS or 20 μg/ml poly(I:C)HMW following transfection with Src siRNA for 24 h or treatment with Src inhibitor for 2 h. Whole cell lysates, nuclear extracts, and culture media were resolved by SDS-PAGE and immunoblotted with antibodies against TLR3-Tyr(P)759, TLR3, Src, IRF3, IRF7, and IFN-β. β-Tubulin, proliferating cell nuclear antigen (PCNA), or β-actin were used as loading controls, respectively. D, IFN-β levels in whole cell lysates from B were determined by ELISA. Data are presented as individual data points and mean. *, p < 0.05.

       Nitric Oxide Induces Feedforward Up-regulation of iNOS through a cGMP/PKG/NFκB-dependent Pathway

      Our observations that both NOS and soluble guanylate cyclase inhibitors suppressed poly(I:C)-induced iNOS expression (Fig. 4, D, F, and G) suggest that the production of NO by iNOS may promote the expression of iNOS through a feedforward mechanism to stimulate more NO production. To pursue this possibility, we assessed iNOS expression in cultured hepatocytes following exposure to the NO donor SNAP, the cell-permeable cGMP analog 8-pCPTcGMP, or the potent iNOS inducer IL-1β (
      • Geller D.A.
      • de Vera M.E.
      • Russell D.A.
      • Shapiro R.A.
      • Nussler A.K.
      • Simmons R.L.
      • Billiar T.R.
      A central role for IL-1 β in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase: IL-1 β induces hepatic nitric oxide synthesis.
      ). As shown in Fig. 8A, SNAP, 8-pCPTcGMP, or IL-1β alone induced iNOS expression. SNAP- and IL-1β-induced iNOS expression was suppressed by the soluble guanylate cyclase inhibitor ODQ whereas 8-pCPTcGMP-induced expression was not. Both SNAP- and 8-pCPTcGMP-induced time- and concentration-dependent increases in iNOS expression (Fig. 8, B–D). The PKG inhibitor KT5823 suppressed both the SNAP- and 8-pCPTcGMP induction of iNOS (Fig. 8E), indicating the stimulation of iNOS involved the cGMP target PKG.
      Figure thumbnail gr8
      FIGURE 8The expression of iNOS is regulated by nitric oxide via the cGMP/PKGIβ pathway. A, wild-type mouse hepatocytes were treated with ODQ, SNAP, IL-1β, and/or cGMP analog 8-pCPTcGMP for 8 h. The expression of iNOS was detected by Western blotting. Results are shown as mean ± S.D. of at least three independent experiments. *, p < 0.01 versus control; #, p < 0.01 versus SNAP; &, p < 0.01 versus IL-1β; @ p > 0.05 versus SNAP+8-pCPTcGMP. B, mouse hepatocytes were stimulated with 100 μm SNAP for various durations (0, 1, 2, 4, 6, 8, 16, and 24 h). Whole cell lysates were resolved by SDS-PAGE and immunoblotted with iNOS. β-Actin was used as a loading control. C, wild-type mouse hepatocytes were treated with different concentrations of SNAP for 4 h. The expression of iNOS was assessed by Western blotting. D, wild-type mouse hepatocytes were treated with different concentrations of 8-pCPTcGMP for 8 h. Whole cell lysates were resolved by SDS-PAGE and immunoblotted with iNOS. β-Actin was used as a loading control. E, mouse hepatocytes were treated with 8-pCPTcGMP, SNAP, and KT5823 as indicated. Whole cell lysates were separated by SDS-PAGE and immunoblotted with iNOS. β-Actin was used as a loading control. Results are shown as mean ± S.D. of at least three independent experiments. *, p < 0.01 versus control; $, p < 0.01 versus SNAP; &, p < 0.01 versus SNAP+8-pCPTcGMP. F, PKGIβ, PKGIα, and PKGII protein levels were determined by Western blotting in mouse hepatocytes after treatment with SNAP. *, p < 0.01 versus SNAP 0 h; #, p > 0.05 versus SNAP 0 h. Results are shown as mean ± S.D. of at least three independent experiments. G, iNOS protein levels were detected by Western blotting in mouse hepatocytes treated with PKGIβ siRNA. Results are shown as mean ± S.D. of at least three independent experiments. *, p < 0.01 versus control; #, p < 0.01 versus control siRNA.
      At baseline, hepatocytes expressed the PKG subunits PKGIβ, PKGIα, and PKGII (Fig. 8F). Remarkably, NO exposure further up-regulated PKGIβ within 4 h, and the suppression of PKGIβ expression using siRNA prevented the up-regulation of iNOS induced by SNAP or 8-pCPTcGMP (Fig. 8, F and G). The transcriptional up-regulation of iNOS is known to involve NFκB (
      • Peng H.B.
      • Libby P.
      • Liao J.K.
      Induction and stabilization of I κ B α by nitric oxide mediates inhibition of NF-κ B.
      ). To determine whether SNAP or cGMP could up-regulate NFκB activation in hepatocytes, EMSA was performed. Both SNAP (100 μm) and 8-pCPTcGMP increased NFκB activation in hepatocytes (Fig. 9, A and C). This could be suppressed by ODQ or KT5823 (Fig. 9, B and D) or by PKGIβ-siRNA treatment (Fig. 9D). To implicate regulation at the transcriptional level, hepatocytes were transiently transfected with a 1.7-kb mouse iNOS promoter-luciferase construct or a construct expressing the iNOS promoter with mutated NFκB binding sites (mutant-pro). As shown in Fig. 9E, both SNAP and 8-pCPTcGMP induced increased luciferase expression in cells transfected with the wild type promoter construct but not the mutant construct. Together, our findings indicate that NO/cGMP/PKG/NFκB signaling is involved in a feedforward mechanism to promote iNOS expression in hepatocytes.
      Figure thumbnail gr9
      FIGURE 9Regulation of NO on iNOS expression is orchestrated by the cGMP/PKGIβ/NF-κB pathway. A, wild-type mouse hepatocytes were treated with 100 μm SNAP for various durations (0, 1, 2, 4, 6, and 8 h). NFκB activity was determined by EMSA. B, wild-type mouse hepatocytes were treated with SNAP and KT5823 for various durations (0, 1, 2, 4, 6, and 8 h). NFκB activity was determined by EMSA. C, mouse hepatocytes were treated with different concentrations of 8-pCPTcGMP for 2 h. NFκB activity was determined by EMSA. D, NFκB activity was detected by EMSA in mouse hepatocytes after treatment with control siRNA and PKGIβ siRNA. E, mouse hepatocytes were transfected with plasmids containing the miNOS luciferase reporter and murine iNOS-NFκB mutant luciferase reporter, respectively. Luciferase activities were detected by a luminometer after stimulation with SNAP alone or in combination with 8-pCPTcGMP. Results are shown as individual data points and mean. *, p < 0.01 versus WT-Pro.

      Discussion

      Hepatocytes respond to both endogenous and exogenous sources of dsRNA with a robust IFN-I response (
      • Li K.
      • Chen Z.
      • Kato N.
      • Gale Jr., M.
      • Lemon S.M.
      Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-β production in hepatocytes.
      ,
      • Kanda T.
      • Steele R.
      • Ray R.
      • Ray R.B.
      Hepatitis C virus infection induces the β interferon signaling pathway in immortalized human hepatocytes.
      ). We have shown previously that this response is restrained in hepatocytes by a rapid up-regulation of adenosine deaminase acting on RNA 1 (ADAR1) (
      • Wang H.
      • Wang G.
      • Zhang L.
      • Zhang J.
      • Zhang J.
      • Wang Q.
      • Billiar T.R.
      ADAR1 suppresses the activation of cytosolic RNA-sensing signaling pathways to protect the liver from ischemia/reperfusion injury.
      ). Here we show that iNOS/NO are required for IFN-β production in response to poly(I:C) in both murine and human hepatocytes. iNOS is rapidly up-regulated in response to poly(I:C) through a pathway that involves the synergistic actions of TLR3/Trif, Src, and PKR. iNOS/NO promoted Src-dependent phosphorylation of TLR3 at Tyr759, and Src was required for both IFN-β and iNOS expression. Src up-regulation was, in turn, entirely dependent on PKR and partially dependent on TLR3/Trif and iNOS/NO. We also identified a robust feedforward mechanism where NO promotes iNOS expression through a cGMP/PKG-dependent pathway that further enhances NO and IFN-β production. These data provide evidence that iNOS/NO are an integral component of innate immune signaling pathways leading to IFN-I production in hepatocytes.
      Both microbial and self-dsRNA can be sensed by receptors of the innate immune system (
      • Mogensen T.H.
      Pathogen recognition and inflammatory signaling in innate immune defenses.
      ,
      • Jensen S.
      • Thomsen A.R.
      Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion.
      ). These include TLR3 in the endosomal compartment (
      • Johnsen I.B.
      • Nguyen T.T.
      • Ringdal M.
      • Tryggestad A.M.
      • Bakke O.
      • Lien E.
      • Espevik T.
      • Anthonsen M.W.
      Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.
      ,
      • Borden E.C.
      • Sen G.C.
      • Uze G.
      • Silverman R.H.
      • Ransohoff R.M.
      • Foster G.R.
      • Stark G.R.
      Interferons at age 50: past, current and future impact on biomedicine.
      ) and PKR as well as Rig-I like receptors, including DExDH box helicases (Rig-I and MDA 5) (
      • Jiang Z.
      • Zamanian-Daryoush M.
      • Nie H.
      • Silva A.M.
      • Williams B.R.
      • Li X.
      Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFκ B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR.
      ,
      • He Y.
      • Franchi L.
      • Núñez G.
      The protein kinase PKR is critical for LPS-induced iNOS production but dispensable for inflammasome activation in macrophages.
      ), in the cytosol. Signaling triggered by the interaction of dsRNA with TLR3 or PKR activates the transcriptional expression of a number of genes through NFκB and the IFN regulatory factor family of transcription factors (IRFs). Among the genes induced by dsRNA are type I IFNs, including IFN-α cytokines, and IFN-β, which sensitizes cells for the detection of invading pathogens, inhibits protein synthesis, and suppresses viral replication (
      • Borden E.C.
      • Sen G.C.
      • Uze G.
      • Silverman R.H.
      • Ransohoff R.M.
      • Foster G.R.
      • Stark G.R.
      Interferons at age 50: past, current and future impact on biomedicine.
      ). TLR3 has been shown to respond to dsRNA from necrotic cells and perhaps transcription, leading to immune activation in the setting of sterile inflammation (
      • Borden E.C.
      • Sen G.C.
      • Uze G.
      • Silverman R.H.
      • Ransohoff R.M.
      • Foster G.R.
      • Stark G.R.
      Interferons at age 50: past, current and future impact on biomedicine.
      ). Activation of TLR3 leads to signaling through TRIF/TRAF6 and TBK1/IκB-kinase ϵ for the up-regulation of IRF3 and 7 as well as of NFκB (
      • Kawai T.
      • Akira S.
      Signaling to NF-κB by Toll-like receptors.
      ). PKR has been shown to participate in TLR3 signaling through association with TBK1 (
      • Mogensen T.H.
      Pathogen recognition and inflammatory signaling in innate immune defenses.
      ) and can also activate NFκB following binding to dsRNA (
      • Zamanian-Daryoush M.
      • Mogensen T.H.
      • DiDonato J.A.
      • Williams B.R.
      NF-κB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-κB-inducing kinase and IκB kinase.
      ).
      In hepatocytes, TLR3 plays a central role during hepatitis B virus and hepatitis C virus infection (
      • Ma Z.
      • Zhang E.
      • Yang D.
      • Lu M.
      Contribution of Toll-like receptors to the control of hepatitis B virus infection by initiating antiviral innate responses and promoting specific adaptive immune responses.
      ). Studies in primary human hepatocytes establish that TLR3 triggers antiviral responses (
      • Wang N.
      • Liang Y.
      • Devaraj S.
      • Wang J.
      • Lemon S.M.
      • Li K.
      Toll-like receptor 3 mediates establishment of an antiviral state against hepatitis C virus in hepatoma cells.
      ). Results from experiments using hepatoma cell lines show that PKR activation restrains HCV1a replication through the regulation of NFκB expression (
      • Zhang L.
      • Alter H.J.
      • Wang H.
      • Jia S.
      • Wang E.
      • Marincola F.M.
      • Shih J.W.
      • Wang R.Y.
      The modulation of hepatitis C virus 1a replication by PKR is dependent on NF-κB mediated interferon β response in Huh7.5.1 cells.
      ). TLR3−/− mice have been used to implicate dsRNA responses in the immunopathology associated with concanavalin A-induced hepatitis (
      • Xiao X.
      • Zhao P.
      • Rodriguez-Pinto D.
      • Qi D.
      • Henegariu O.
      • Alexopoulou L.
      • Flavell R.A.
      • Wong F.S.
      • Wen L.
      Inflammatory regulation by TLR3 in acute hepatitis.
      ) and acetaminophen-induced liver damage (
      • Cavassani K.A.
      • Moreira A.P.
      • Habiel D.
      • Ito T.
      • Coelho A.L.
      • Allen R.M.
      • Hu B.
      • Raphelson J.
      • Carson 4th, W.F.
      • Schaller M.A.
      • Lukacs N.W.
      • Omary M.B.
      • Hogaboam C.M.
      • Kunkel S.L.
      Toll like receptor 3 plays a critical role in the progression and severity of acetaminophen-induced hepatotoxicity.
      ). The demonstration that IRF3 (
      • Loi P.
      • Yuan Q.
      • Torres D.
      • Delbauve S.
      • Laute M.A.
      • Lalmand M.C.
      • Pétein M.
      • Goriely S.
      • Goldman M.
      • Flamand V.
      Interferon regulatory factor 3 deficiency leads to interleukin-17-mediated liver ischemia-reperfusion injury.
      ) and IFN-β (
      • Zhai Y.
      • Qiao B.
      • Gao F.
      • Shen X.
      • Vardanian A.
      • Busuttil R.W.
      • Kupiec-Weglinski J.W.
      Type I, but not type II, interferon is critical in liver injury induced after ischemia and reperfusion.
      ) are involved in ischemia/reperfusion injury in the liver provides further support for the importance of pathways leading to IFN-I production as part of the inflammatory response in the liver. We have shown that iNOS is also involved in ischemia/reperfusion injury in the liver (
      • Lee V.G.
      • Johnson M.L.
      • Baust J.
      • Laubach V.E.
      • Watkins S.C.
      • Billiar T.R.
      The roles of iNOS in liver ischemia-reperfusion injury.
      ), raising the possibility that iNOS and IFN-β could be linked in this relevant model of sterile inflammation. Taken together, these studies provide evidence that hepatocytes respond to dsRNA and that dsRNAs of both microbial and self-sources are important to the immune responses in the liver.
      In this study, we extend these observations to show that the response to exogenous dsRNA for the production of IFN-β in murine and human hepatocytes requires iNOS/NO, TLR3/TRIF, PKR, and Src. The role of Src-TLR3 interactions as well as iNOS/NO/cGMP in promoting TLR3 phosphorylation has been shown in macrophages (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ,
      • Maa M.C.
      • Chang M.Y.
      • Li J.
      • Li Y.Y.
      • Hsieh M.Y.
      • Yang C.J.
      • Chen Y.J.
      • Li Y.
      • Chen H.C.
      • Cheng W.E.
      • Hsieh C.Y.
      • Cheng C.W.
      • Leu T.H.
      The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages.
      ). Like macrophages, we show that Src-TLR3 interactions in hepatocytes take place in the endosomal compartment and lead to an iNOS/NO-dependent phosphorylation of TLR3 at Tyr759. We show that PKR is expressed at baseline in hepatocytes and that its expression is strongly induced by dsRNA and LPS. Increases in Src and iNOS/NO as well as TLR3 Tyr759 phosphorylation and IFN-β production in response to poly(I:C) were entirely dependent on PKR expression but only partially dependent on TLR3. This suggests that PKR plays roles in promoting TLR3 signaling but also roles independent of TLR3 in the dsRNA response in hepatocytes. A similar Src-dependent up-regulation of TLR3 Tyr759 phosphorylation, IRF3 and 7 levels, and IFN-β production was seen after LPS treatment. Cooperativity between TLR3 and TLR4 has been shown in macrophages (
      • Hsieh M.Y.
      • Chang M.Y.
      • Chen Y.J.
      • Li Y.K.
      • Chuang T.H.
      • Yu G.Y.
      • Cheung C.H.
      • Chen H.C.
      • Maa M.C.
      • Leu T.H.
      The inducible nitric-oxide synthase (iNOS)/Src axis mediates Toll-like receptor 3 tyrosine 759 phosphorylation and enhances its signal transduction, leading to interferon-β synthesis in macrophages.
      ,
      • Jiang W.
      • Sun R.
      • Wei H.
      • Tian Z.
      Toll-like receptor 3 ligand attenuates LPS-induced liver injury by down-regulation of toll-like receptor 4 expression on macrophages.
      ) and may also take place in hepatocytes. The overlap in the responses to poly(I:C) and LPS is most likely explained by the involvement of Trif in TLR3 and TLR4 signaling shared in common downstream of these receptors.
      Hepatocytes are capable of rapid and sustained up-regulation of iNOS across a range of species, including humans and rodents (
      • Curran R.D.
      • Billiar T.R.
      • Stuehr D.J.
      • Hofmann K.
      • Simmons R.L.
      Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells.
      ,
      • Nussler A.K.
      • Di Silvio M.
      • Billiar T.R.
      • Hoffman R.A.
      • Geller D.A.
      • Selby R.
      • Madariaga J.
      • Simmons R.L.
      Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.
      ,
      • Geller D.A.
      • Lowenstein C.J.
      • Shapiro R.A.
      • Nussler A.K.
      • Di Silvio M.
      • Wang S.C.
      • Nakayama D.K.
      • Simmons R.L.
      • Snyder S.H.
      • Billiar T.R.
      Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes.
      ). iNOS/NO leads to cGMP-dependent and -independent actions in hepatocytes, such as regulation of cell death pathways (
      • Kim Y.M.
      • de Vera M.E.
      • Watkins S.C.
      • Billiar T.R.
      Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-α-induced apoptosis by inducing heat shock protein 70 expression.
      ,
      • Kim Y.M.
      • Talanian R.V.
      • Billiar T.R.
      Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms.
      ) and activation of TNFα-converting enzyme (TACE/ADAM17) (
      • Chanthaphavong R.S.
      • Loughran P.A.
      • Lee T.Y.
      • Scott M.J.
      • Billiar T.R.
      A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes.
      ,
      • Deng M.
      • Loughran P.A.
      • Zhang L.
      • Scott M.J.
      • Billiar T.R.
      Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling.
      ). In this study, we show that iNOS/NO is also involved in the production of IFN-β by hepatocytes in response to TLR ligands. We also found that inhibition of iNOS activity or the soluble guanylate cyclase suppressed iNOS expression in response to dsRNA (Fig. 4). This led us to further characterize the roles of NO and cGMP in promoting iNOS expression in hepatocytes as a feedforward mechanism to amplify NO production. NO and cGMP lead to a PKG-dependent up-regulation of iNOS in hepatocytes. Hepatocytes were found to express all three PKG subunits at baseline; however, only the expression of PKGIβ was induced by NO exposure. NO/cGMP-induced increases in iNOS expression were dependent on PKGIβ expression. Transcription of the iNOS gene is NFκB-dependent (
      • delaTorre A.
      • Schroeder R.A.
      • Punzalan C.
      • Kuo P.C.
      Endotoxin-mediated S-nitrosylation of p50 alters NF-κ B-dependent gene transcription in ANA-1 murine macrophages.
      ). It is likely that one of the mechanisms for enhancement of iNOS expression by NO/cGMP/PKG is through amplification of iNOS transcription. NO and cGMP induced a sustained increased in NFκB activation and also increased iNOS promoter activation. How the cGMP/PKG pathway promotes NFκB activation is unknown. It is reasonable to speculate that a PKG phosphorylation target regulates NFκB signaling. A number of posttranscriptional pathways are also known to be involved in the regulation of iNOS, and a role for these in iNOS/NO induction of iNOS cannot be excluded.
      In summary, this series of experiments provides evidence that iNOS/NO can be rapidly up-regulated by dsRNA through TLR3/PKR/Src-dependent mechanisms and is amplified further through an iNOS/NO/cGMP/PKG feedforward mechanism. Importantly, we establish that iNOS expression is required for IFN-β production in hepatocytes following stimulation with dsRNA. A proposed model for dsRNA sensing leading to IFN-β production by hepatocytes is shown in Fig. 10, which takes into account these new observations as well as recently published findings on the role of ADAR1 in suppressing dsRNA sensing through RIG-I (
      • Yang S.
      • Deng P.
      • Zhu Z.
      • Zhu J.
      • Wang G.
      • Zhang L.
      • Chen A.F.
      • Wang T.
      • Sarkar S.N.
      • Billiar T.R.
      • Wang Q.
      Adenosine deaminase acting on RNA 1 limits RIG-I RNA detection and suppresses IFN production responding to viral and endogenous RNAs.
      ,
      • Wang H.
      • Wang G.
      • Zhang L.
      • Zhang J.
      • Zhang J.
      • Wang Q.
      • Billiar T.R.
      ADAR1 suppresses the activation of cytosolic RNA-sensing signaling pathways to protect the liver from ischemia/reperfusion injury.
      ). Although gaps in our knowledge of the interactions between these pathways persist, our findings provide insight into the intricate signaling and regulatory pathways controlling the sensing of dsRNA in hepatocytes and thus have important implications for understanding the mechanisms of antiviral immune responses and sterile inflammation in the liver.
      Figure thumbnail gr10
      FIGURE 10Schematic of the proposed model for production of IFN-β in response to extracellular dsRNA. In this figure, we incorporate our new findings into a diagram that depicts known pathways for the sensing of extracellular dsRNA. Uptake of dsRNA engages signaling through specific dsDNA receptors, including TLR3, PKR, and RIG-I (
      • Mogensen T.H.
      Pathogen recognition and inflammatory signaling in innate immune defenses.
      ). In this study, we show that iNOS, PKR, Src, and PKGIβ are all up-regulated at the protein level early after dsRNA addition. We have shown previously that ADAR1 is also rapidly up-regulated and suppresses dsDNA sensing through the RIG-I pathway (
      • Wang H.
      • Wang G.
      • Zhang L.
      • Zhang J.
      • Zhang J.
      • Wang Q.
      • Billiar T.R.
      ADAR1 suppresses the activation of cytosolic RNA-sensing signaling pathways to protect the liver from ischemia/reperfusion injury.
      ). We now show that TLR3/Trif and PKR act together to up-regulate iNOS and Src to promote TLR3-Tyr(P)759 and IFN-β production. PKR plays a dominant role in the up-regulation of both iNOS and Src, and we propose both TLR3-dependent and -independent roles for PKR based on our findings. iNOS expression is promoted by a feedforward mechanism that depends on cGMP/PKG.

      Author Contributions

      L. Z. contributed to the experimental design, performed the experiments, and wrote the manuscript. W. X. performed the experiments and contributed to the writing of the manuscript. G. W. and Z. Y. helped collect samples. Z. Z. and A. F. C. provided the PKR KO mice. Z. G. provided technical assistance. P. A. L. performed all immunofluorescence experiments. R. S., B. L., and Q. W. provided critical discussion of the manuscript. T. R. B. designed most of the research project, provided oversight of the experiments, and edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Dr. David Geller for the human hepatocytes.

      Supplementary Material

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