title: 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.
abstract: Double-stranded RNA (dsRNA) induces phosphorylation of Toll-like receptor 3 (TLR3) at tyrosine 759 and subsequently triggers signaling pathways to promote interferon-β (IFN-β) production. In this study, we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages. In addition to the immediate TLR3 Tyr-759 phosphorylation, we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-β transcription in the later phase of dsRNA stimulation. Interestingly, Src phosphorylated TLR3 Tyr-759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-β production. Reintroduction of Src restored all of these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Tyr-759 phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-β generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-β production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-β expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-β transcription. Taken together, these results suggested an essential role of the iNOS/Src/TLR3 axis in IFN-β production in macrophages.
authors: From the Institute of Basic Medical Sciences.
authors: Ming-Yu
authors: MY
authors: Hsieh
authors: Miao Ying
authors: MY
authors: Chang
authors: Yen-Jen
authors: YJ
authors: Chen
authors: Yung Kuo
authors: YK
authors: Li
authors: Tsung-Hsien
authors: TH
authors: Chuang
authors: Guann-Yi
authors: GY
authors: Yu
authors: Chun Hei Antonio
authors: CH
authors: Cheung
authors: Hui-Chen
authors: HC
authors: Chen
authors: Ming-Chei
authors: MC
authors: Maa
authors: Tzeng-Horng
authors: TH
authors: Leu
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral; D005786:Gene Expression Regulation; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D050839:Interferon Regulatory Factor-7 / Q000378:metabolism; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics; D017737:Macrophages, Peritoneal / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D015398:Signal Transduction; D051196:Toll-Like Receptor 3 / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; C494235:Irf7 protein, mouse; D012330:RNA, Double-Stranded; D051196:Toll-Like Receptor 3; D014443:Tyrosine; D016899:Interferon-beta; D052247:Nitric Oxide Synthase Type II; D016392:Proto-Oncogene Proteins pp60(c-src)
keywords: Double-stranded RNA; Interferon; Lipopolysaccharide (LPS); Macrophages; Src; Toll-like Receptors (TLR); iNOS