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

Ming Yu Hsieh, Miao Ying Chang, Yen Jen Chen, Yung Kuo Li, Tsung Hsien Chuang, Guann Yi Yu, Chun-Hei Cheung, Hui Chen Chen, Ming Chei Maa, Tzeng-Horng Leu

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)9208-9220
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number13
DOIs
Publication statusPublished - 2014 Mar 30

Fingerprint

Toll-Like Receptor 3
Signal transduction
Phosphorylation
Macrophages
Nitric Oxide Synthase Type II
Interferons
Signal Transduction
Double-Stranded RNA
Transcription
Interferon Regulatory Factor-7
Interferon Regulatory Factor-3
3-tyrosine
Phenylalanine
Tyrosine
Down-Regulation
Ligands

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hsieh, Ming Yu ; Chang, Miao Ying ; Chen, Yen Jen ; Li, Yung Kuo ; Chuang, Tsung Hsien ; Yu, Guann Yi ; Cheung, Chun-Hei ; Chen, Hui Chen ; Maa, Ming Chei ; Leu, Tzeng-Horng. / 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. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 13. pp. 9208-9220.
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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.",
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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. / Hsieh, Ming Yu; Chang, Miao Ying; Chen, Yen Jen; Li, Yung Kuo; Chuang, Tsung Hsien; Yu, Guann Yi; Cheung, Chun-Hei; Chen, Hui Chen; Maa, Ming Chei; Leu, Tzeng-Horng.

In: Journal of Biological Chemistry, Vol. 289, No. 13, 30.03.2014, p. 9208-9220.

Research output: Contribution to journalArticle

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AU - Hsieh, Ming Yu

AU - Chang, Miao Ying

AU - Chen, Yen Jen

AU - Li, Yung Kuo

AU - Chuang, Tsung Hsien

AU - Yu, Guann Yi

AU - Cheung, Chun-Hei

AU - Chen, Hui Chen

AU - Maa, Ming Chei

AU - Leu, Tzeng-Horng

PY - 2014/3/30

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N2 - 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.

AB - 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.

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