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Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

Gielen V, Sykes A, Zhu J, Chan B, Macintyre J, Regamey N, Kieninger E, Gupta A, Shoemark A, Bossley C, Davies J, Saglani S, Walker P, Nicholson SE, Dalpke AH, Kon OM, Bush A, Johnston SL, Edwards MR - J. Allergy Clin. Immunol. (2015)

Bottom Line: SOCS1 levels were also correlated with asthma-related clinical outcomes.Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors.Nuclear SOCS1 levels were also increased in BECs from asthmatic patients.

View Article: PubMed Central - PubMed

Affiliation: Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom; MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom; Centre for Respiratory Infection, Imperial College London, London, United Kingdom.

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Mutation of the SOCS1 NLS resulted in reduced suppression of rhinovirus-induced interferon promoter and IFN-β–induced minimal ISRE responsive promoter activation compared with SOCS1wt. A, All SOCS1 mutants expressed equally as assessed by anti-GFP detection when compared with anti–α-tubulin using Western blotting (WB). UT, Untransfected. B, The SOCS1 NLS mutant Δ6RA showed predominately cytoplasmic localization by using confocal microscopy. Left panel, GFP; right panel, 4′-6-Diamidino-2-phenylindole dihydrochloride. Evans blue and GFP overlay: bar = 10-μm scale. A diagram of SOCS1wt and the NLS mutant Δ6RA shows the 6 mutated amino acid residues in the NLS region. C, Δ6RA showed less suppression compared with SOCS1wt of RV1B-induced IFN-λ1. D, RV1B-induced IFN-β promoter activation. E, IFN-β (5 ng/mL) induced ISRE minimal promoter activation at 24 hours (n = 4 independent experiments). *P < .05, **P < .01, and ***P < .01, as indicated and versus RV1B-infected or IFN-β–treated GFP-transfected cells. +++P < .001 versus SOCS1wt-transfected, RV1B-infected, or IFN-β–treated cells by using 1-way ANOVA with the Bonferroni multiple comparison test. ns, Not significant (upper ns vs SOCS1wt, RV1B-infected, or IFN-β–treated cells; lower ns vs GFP, RV1B-infected, or IFN-β–treated cells).
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dfig6: Mutation of the SOCS1 NLS resulted in reduced suppression of rhinovirus-induced interferon promoter and IFN-β–induced minimal ISRE responsive promoter activation compared with SOCS1wt. A, All SOCS1 mutants expressed equally as assessed by anti-GFP detection when compared with anti–α-tubulin using Western blotting (WB). UT, Untransfected. B, The SOCS1 NLS mutant Δ6RA showed predominately cytoplasmic localization by using confocal microscopy. Left panel, GFP; right panel, 4′-6-Diamidino-2-phenylindole dihydrochloride. Evans blue and GFP overlay: bar = 10-μm scale. A diagram of SOCS1wt and the NLS mutant Δ6RA shows the 6 mutated amino acid residues in the NLS region. C, Δ6RA showed less suppression compared with SOCS1wt of RV1B-induced IFN-λ1. D, RV1B-induced IFN-β promoter activation. E, IFN-β (5 ng/mL) induced ISRE minimal promoter activation at 24 hours (n = 4 independent experiments). *P < .05, **P < .01, and ***P < .01, as indicated and versus RV1B-infected or IFN-β–treated GFP-transfected cells. +++P < .001 versus SOCS1wt-transfected, RV1B-infected, or IFN-β–treated cells by using 1-way ANOVA with the Bonferroni multiple comparison test. ns, Not significant (upper ns vs SOCS1wt, RV1B-infected, or IFN-β–treated cells; lower ns vs GFP, RV1B-infected, or IFN-β–treated cells).

Mentions: SOCS1 can prevent nuclear factor κB (NF-κB) signaling by entering the nucleus through a C-terminal proximal nuclear localization sequence (NLS) and targeting NF-κB p65 for proteasomal degradation through the C-terminal SOCS box.32 Therefore we hypothesized that SOCS1 might suppress rhinovirus-induced interferon induction by translocating into the nucleus and initiating proteasomal degradation of transcription factors required for interferon induction. To investigate the role of nuclear translocation of SOCS1 and of the SOCS box, we used vectors expressing green fluorescent protein (GFP)–tagged full-length wild-type human SOCS1 (SOCS1wt) and 2 mutants. The mutants included SOCS1 truncations with both the NLS and the SOCS box deleted (Q108X) or with a deleted SOCS box alone, leaving the NLS intact (R172X; Fig 5, A).32 We found that the SOCS1 mutant that lacked the NLS (Q108X) was indeed unable to translocate to the nucleus; however, both SOCS1wt and R172X, which had a deleted SOCS box but intact NLS, were able to translocate to the nucleus (Fig 5, A). We then tested the ability of these constructs to suppress rhinovirus induction of interferons in BEAS-2B cells and found that the construct lacking the NLS (Q108X) had lost its ability to suppress rhinovirus-induced IFN-β and IFN-λ promoter activation, whereas fully intact SOCS1 (SOCS1wt containing both the NLS and the SOCS box) and R172X (containing the NLS but lacking the SOCS box) were still suppressive (Fig 5, B). Furthermore, SOCS1wt, but neither Q108X nor R172X, suppressed interferon-induced ISRE promoter activation. This definitively proves that SOCS1-mediated suppression of rhinovirus-induced interferon is NLS dependent but SOCS box independent and therefore distinct from interferon-induced ISRE activation, which is dependent on both the NLS and the SOCS box (Fig 5, B). Furthermore, the requirement for nuclear localization for both rhinovirus- and interferon-induced responses was supported with a full-length SOCS1 construct containing mutated NLS residues (Δ6RA), which was impaired in its ability to enter the nucleus and exhibited a less suppressive effect on interferon induction when compared with SOCS1wt (see Fig E6, B-D, in this article's Online Repository at www.jacionline.org). SOCS1wt, R172X, and Q108X proteins were expressed at similar levels, as determined by using Western blotting (see Fig E6, A).


Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

Gielen V, Sykes A, Zhu J, Chan B, Macintyre J, Regamey N, Kieninger E, Gupta A, Shoemark A, Bossley C, Davies J, Saglani S, Walker P, Nicholson SE, Dalpke AH, Kon OM, Bush A, Johnston SL, Edwards MR - J. Allergy Clin. Immunol. (2015)

Mutation of the SOCS1 NLS resulted in reduced suppression of rhinovirus-induced interferon promoter and IFN-β–induced minimal ISRE responsive promoter activation compared with SOCS1wt. A, All SOCS1 mutants expressed equally as assessed by anti-GFP detection when compared with anti–α-tubulin using Western blotting (WB). UT, Untransfected. B, The SOCS1 NLS mutant Δ6RA showed predominately cytoplasmic localization by using confocal microscopy. Left panel, GFP; right panel, 4′-6-Diamidino-2-phenylindole dihydrochloride. Evans blue and GFP overlay: bar = 10-μm scale. A diagram of SOCS1wt and the NLS mutant Δ6RA shows the 6 mutated amino acid residues in the NLS region. C, Δ6RA showed less suppression compared with SOCS1wt of RV1B-induced IFN-λ1. D, RV1B-induced IFN-β promoter activation. E, IFN-β (5 ng/mL) induced ISRE minimal promoter activation at 24 hours (n = 4 independent experiments). *P < .05, **P < .01, and ***P < .01, as indicated and versus RV1B-infected or IFN-β–treated GFP-transfected cells. +++P < .001 versus SOCS1wt-transfected, RV1B-infected, or IFN-β–treated cells by using 1-way ANOVA with the Bonferroni multiple comparison test. ns, Not significant (upper ns vs SOCS1wt, RV1B-infected, or IFN-β–treated cells; lower ns vs GFP, RV1B-infected, or IFN-β–treated cells).
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dfig6: Mutation of the SOCS1 NLS resulted in reduced suppression of rhinovirus-induced interferon promoter and IFN-β–induced minimal ISRE responsive promoter activation compared with SOCS1wt. A, All SOCS1 mutants expressed equally as assessed by anti-GFP detection when compared with anti–α-tubulin using Western blotting (WB). UT, Untransfected. B, The SOCS1 NLS mutant Δ6RA showed predominately cytoplasmic localization by using confocal microscopy. Left panel, GFP; right panel, 4′-6-Diamidino-2-phenylindole dihydrochloride. Evans blue and GFP overlay: bar = 10-μm scale. A diagram of SOCS1wt and the NLS mutant Δ6RA shows the 6 mutated amino acid residues in the NLS region. C, Δ6RA showed less suppression compared with SOCS1wt of RV1B-induced IFN-λ1. D, RV1B-induced IFN-β promoter activation. E, IFN-β (5 ng/mL) induced ISRE minimal promoter activation at 24 hours (n = 4 independent experiments). *P < .05, **P < .01, and ***P < .01, as indicated and versus RV1B-infected or IFN-β–treated GFP-transfected cells. +++P < .001 versus SOCS1wt-transfected, RV1B-infected, or IFN-β–treated cells by using 1-way ANOVA with the Bonferroni multiple comparison test. ns, Not significant (upper ns vs SOCS1wt, RV1B-infected, or IFN-β–treated cells; lower ns vs GFP, RV1B-infected, or IFN-β–treated cells).
Mentions: SOCS1 can prevent nuclear factor κB (NF-κB) signaling by entering the nucleus through a C-terminal proximal nuclear localization sequence (NLS) and targeting NF-κB p65 for proteasomal degradation through the C-terminal SOCS box.32 Therefore we hypothesized that SOCS1 might suppress rhinovirus-induced interferon induction by translocating into the nucleus and initiating proteasomal degradation of transcription factors required for interferon induction. To investigate the role of nuclear translocation of SOCS1 and of the SOCS box, we used vectors expressing green fluorescent protein (GFP)–tagged full-length wild-type human SOCS1 (SOCS1wt) and 2 mutants. The mutants included SOCS1 truncations with both the NLS and the SOCS box deleted (Q108X) or with a deleted SOCS box alone, leaving the NLS intact (R172X; Fig 5, A).32 We found that the SOCS1 mutant that lacked the NLS (Q108X) was indeed unable to translocate to the nucleus; however, both SOCS1wt and R172X, which had a deleted SOCS box but intact NLS, were able to translocate to the nucleus (Fig 5, A). We then tested the ability of these constructs to suppress rhinovirus induction of interferons in BEAS-2B cells and found that the construct lacking the NLS (Q108X) had lost its ability to suppress rhinovirus-induced IFN-β and IFN-λ promoter activation, whereas fully intact SOCS1 (SOCS1wt containing both the NLS and the SOCS box) and R172X (containing the NLS but lacking the SOCS box) were still suppressive (Fig 5, B). Furthermore, SOCS1wt, but neither Q108X nor R172X, suppressed interferon-induced ISRE promoter activation. This definitively proves that SOCS1-mediated suppression of rhinovirus-induced interferon is NLS dependent but SOCS box independent and therefore distinct from interferon-induced ISRE activation, which is dependent on both the NLS and the SOCS box (Fig 5, B). Furthermore, the requirement for nuclear localization for both rhinovirus- and interferon-induced responses was supported with a full-length SOCS1 construct containing mutated NLS residues (Δ6RA), which was impaired in its ability to enter the nucleus and exhibited a less suppressive effect on interferon induction when compared with SOCS1wt (see Fig E6, B-D, in this article's Online Repository at www.jacionline.org). SOCS1wt, R172X, and Q108X proteins were expressed at similar levels, as determined by using Western blotting (see Fig E6, A).

Bottom Line: SOCS1 levels were also correlated with asthma-related clinical outcomes.Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors.Nuclear SOCS1 levels were also increased in BECs from asthmatic patients.

View Article: PubMed Central - PubMed

Affiliation: Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom; MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom; Centre for Respiratory Infection, Imperial College London, London, United Kingdom.

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Related in: MedlinePlus