Skip to main content

Main menu

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons

User menu

  • Subscribe
  • Log in

Search

  • Advanced search
The Journal of Immunology
  • Other Publications
    • American Association of Immunologists
    • ImmunoHorizons
  • Subscribe
  • Log in
The Journal of Immunology

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Next in The JI
    • Archive
    • Brief Reviews
    • Pillars of Immunology
    • Translating Immunology
    • Most Read
    • Top Downloads
    • Annual Meeting Abstracts
  • COVID-19/SARS/MERS Articles
  • Info
    • About the Journal
    • For Authors
    • Journal Policies
    • Influence Statement
    • For Advertisers
  • Editors
  • Submit
    • Submit a Manuscript
    • Instructions for Authors
    • Journal Policies
  • Subscribe
    • Journal Subscriptions
    • Email Alerts
    • RSS Feeds
    • ImmunoCasts
  • More
    • Most Read
    • Most Cited
    • ImmunoCasts
    • AAI Disclaimer
    • Feedback
    • Help
    • Accessibility Statement
  • Follow The Journal of Immunology on Twitter
  • Follow The Journal of Immunology on RSS

Calcineurin Negatively Regulates TLR-Mediated Activation Pathways

Young Jun Kang, Brenda Kusler, Motoyuki Otsuka, Michael Hughes, Nobutaka Suzuki, Shinobu Suzuki, Wen-Chen Yeh, Shizuo Akira, Jiahuai Han and Patricia P. Jones
J Immunol October 1, 2007, 179 (7) 4598-4607; DOI: https://doi.org/10.4049/jimmunol.179.7.4598
Young Jun Kang
*Department of Biological Sciences, Stanford University, Stanford, CA 94305;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brenda Kusler
*Department of Biological Sciences, Stanford University, Stanford, CA 94305;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Motoyuki Otsuka
†Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Hughes
*Department of Biological Sciences, Stanford University, Stanford, CA 94305;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nobutaka Suzuki
‡Advanced Medical Discovery Institute, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shinobu Suzuki
‡Advanced Medical Discovery Institute, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wen-Chen Yeh
‡Advanced Medical Discovery Institute, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shizuo Akira
§Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jiahuai Han
†Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Patricia P. Jones
*Department of Biological Sciences, Stanford University, Stanford, CA 94305;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • FIGURE 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 1.

    Calcineurin inhibitors activate TLR signaling. A, RAW 264.7 cells were stimulated by LPS (1 μg/ml), FK506 (FK, 10 μg/ml, obtained from Fujisawa), or CsA (50 μg/ml) for 4 h. Nuclear extracts were prepared, and NF-κB DNA-binding activity was determined by EMSA. Supershift assays were performed by incubating LPS-stimulated nuclear extract with anti-p50 or anti-p65 Abs. B, RAW 264.7 cells were stimulated by LPS, FK506, or CsA for the indicated time period, and cell lysates were prepared and subjected to SDS-PAGE, followed by Western blot with indicated Abs. C, IKK in vitro kinase assay. RAW 264.7 cells were stimulated by LPS or FK506 for the indicated time period, and cell lysates were prepared and subjected to kinase assay using GST-IκΒ-α (1–54) as a substrate. Kinase activity (KA) was visualized by autoradiography, and enzyme immunoprecipitation was confirmed by immunoblotting with the corresponding anti-IKK-α or IKK-β Abs (IB). D, Peritoneal macrophages were stimulated with 0, 1, or 10 μg/ml FK506 and 0, 0.01, or 0.1 μg/ml LPS for 24 h. Culture supernatants were collected to measure TNF-α levels by ELISA. E, RAW 264.7 cells were stimulated with medium (None), LPS (0.1 μg/ml), poly(I:C) (20 μg/ml), or FK506 (10 μg/ml), and harvested after 3 h of incubation, and total RNA was prepared and subjected to RT-PCR using IFN-β (ifn-b) and IP-10 (ip-10) primers. GAPDH (gapdh) primers were used as the internal control. F, RAW 264.7 cells were stimulated with medium (None), FK506 (10 μg/ml), poly(I:C) (20 μg/ml), or LPS (0.1 μg/ml), and incubated for 6 h, and cell lysates were subjected to native PAGE and immunoblotting using anti-IRF3 Ab. G, RAW 264.7 cells were stimulated by LPS, or the indicated amount of FK506, and incubated for 30 min. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated Abs. Results shown are representative of two to four separate experiments.

  • FIGURE 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 2.

    Knockdown of calcineurin enhances the activation of TLR-mediated signaling pathway. RAW 264.7 cells were transfected with control or calcineurin-targeting siRNA, and incubated for 3 days, and cells were cultured 4 h before treatment. A, Cell lysates were prepared and analyzed by immunoblot using anti-calcineurin or GAPDH Abs. B, Cells were treated with medium (None), LPS (0.1 μg/ml), peptidoglycan (PGN, 10 μg/ml), poly(I:C) (20 μg/ml), or CpG DNA (2.5 μg/ml) for 4 h, and nuclear extracts were prepared for EMSA. To provide a comparison with effects of FK506 on levels of activated NF-κB, nontransfected cells were stimulated with FK506 for 4 h. The x-ray films of the EMSAs from nontransfected FK506-treated or siRNA-transfected cells were exposed for 4 h, whereas the EMSA film from siRNA-transfected nonstimulated cells was exposed for 16 h. C′, Cold competition of FK506-treated cell nuclear extract using unlabeled NF-κB probe. C, Cells transfected with control or calcineurin siRNA were treated with medium (None), poly(I:C), LPS, or CpG DNA for 24 h. Culture supernatants were assayed for TNF-α by ELISA. D, Cells transfected with control or calcineurin siRNA were treated with medium, LPS, or poly(I:C) for 6 h, and RNA samples were prepared for RT-PCR using IFN-β or GAPDH primers. E, The same as C, except cells were treated with medium or FK506 (10 μg/ml). Data are shown as means ± SD of triplicates. ∗, p < 0.05, and ∗∗, p < 0.01 vs control. Results shown are representative of two to three independent experiments.

  • FIGURE 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 3.

    Calcineurin inhibits the IL-1R/TLR-mediated activation of NF-κB. A, RAW 264.7 cells were transfected with pNF-κB-luc (1.5 μg), pSV-β-gal (1.5 μg), and 4.5 μg of control or constitutively active calcineurin (ΔCnA) expression vectors. After 48 h, cells were stimulated by medium (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (20 μg/ml), LPS (0.1 μg/ml), CpG DNA (2.5 μg/ml), FK506 (10 μg/ml), IL-1β (20 ng/ml), or TNF-α (10 ng/ml). Cell lysates were prepared and subjected to luciferase and β-galactosidase assays. After normalization with β-galactosidase activity, fold activation was calculated as the fold increase in luciferase activity compared with the unstimulated cells. B, 293T cells were transfected with IRAK-1-FLAG, MyD88-AU-1, and GFP-ΔCnA vectors. Cell lysates were analyzed by immunoblotting using anti-IRAK-1 Ab to examine the phosphorylation of transfected IRAK-1, and also probed with anti-AU-1 or GFP Abs. pEGFP-C1 vector was cotransfected as the control. C, RAW 264.7 cells were cotransfected with pNF-κB-luc (1.5 μg), pSV-β-gal (1.5 μg), and control, dn TRAF6, dn IRAK-1, dn MyD88, N-IRAK-1, or KA-IRAK-4 expression vectors (4.5 μg each). After 48 h, cells were stimulated by LPS (1 μg/ml) or FK506 (10 μg/ml) and incubated for 6 h. Cell lysates were prepared for luciferase and β-galactosidase assays. D, Wild-type or MyD88−/− peritoneal macrophages were treated with medium (None), CpG DNA (2.5 μg/ml), poly(I:C) (25 μg/ml), or FK506 (indicated amount in μg/ml) for 4 h. Nuclear extracts were prepared, and DNA-binding activity was analyzed by EMSA. ∗, Indicates nonspecific binding. Results shown are representative of three to four independent experiments.

  • FIGURE 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 4.

    Targets of negative regulation by calcineurin. Peritoneal macrophages from wild-type, MyD88−/−, TRIF−/−, TLR2−/−, TLR3−/−, TLR4−/−, or TLR9 mutant TLR9CpG1/CpG1 mice were A, incubated with 10 μg/ml FK506 for the indicated time period and harvested for Western blot analysis using indicated Abs, or B, incubated with medium (None), Pam3CSK4 (Pam3, 200 ng/ml), poly(I:C) (25 μg/ml), LPS (0.1 μg/ml), CpG DNA (2.5 μg/ml), or FK506 (10 μg/ml) for 24 h, as indicated in each figure. Culture supernatants were assayed for TNF-α by ELISA. Data are shown as means ± SD of triplicates. ∗, p < 0.01; ∗∗, p < 0.005; and ∗∗∗, p < 0.001 vs control. Results shown are representative of two to three independent experiments.

  • FIGURE 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 5.

    Calcineurin is associated with MyD88, TRIF, TLR2, and TLR4, not with TLR3 and TLR9. A, 293T cells were transfected with TLR4-FLAG, MyD88-AU-1, and GFP-ΔCnA or control GFP vectors. Cell lysates were immunoprecipitated with anti-AU-1 Ab and subjected to immunoblotting with indicated Abs. *, IgH. B, 293T cells were transfected with FLAG-TRIF, FLAG-IRAK-4, and GFP-ΔCnA or control GFP vectors. Cell lysates were immunoprecipitated with anti-FLAG Ab and subjected to immunoblotting with indicated Abs. C, 293T cells were transfected with FLAG-tagged TLR 2, 3, 4, or 9, and GFP-ΔCnA expression vectors. Cell lysates were immunoprecipitated with anti-FLAG Ab and subjected to immunoblotting with indicated Abs. D, RAW 264.7 cells were untreated or treated with FK506 (10 μg/ml) for 30 min, and cell lysates were immunoprecipitated with isotype or indicated Abs, and the immunoprecipitate was resolved in SDS-PAGE, followed by immunoblotting using indicated Abs. Results shown are representative of two to four independent experiments.

  • FIGURE 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    FIGURE 6.

    Model for the negative regulation of TLR signaling by calcineurin. A, In nonactivated (resting) macrophages, calcineurin interacts and negatively regulates the adaptor proteins MyD88 and TRIF, and TLRs expressed on the plasma membrane such as TLR2 and TLR4, not TLRs that are only expressed in endosomes (TLR3 and TLR9 have been tested), the initial components of the pathways leading to NF-κB, MAPK, and IRF3 activation. B, Calcineurin inhibitors or ligand binding to the TLRs lead to the activation of NF-κB and other transcription factors (TFs) via MyD88-dependent and MyD88-independent pathways. The filled and open symbols denote the inactive and active forms of the molecules, respectively. Endosomes are indicated as double-lined circles. See Discussion.

PreviousNext
Back to top

In this issue

The Journal of Immunology: 179 (7)
The Journal of Immunology
Vol. 179, Issue 7
1 Oct 2007
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Advertising (PDF)
  • Back Matter (PDF)
  • Editorial Board (PDF)
  • Front Matter (PDF)
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word about The Journal of Immunology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Calcineurin Negatively Regulates TLR-Mediated Activation Pathways
(Your Name) has forwarded a page to you from The Journal of Immunology
(Your Name) thought you would like to see this page from the The Journal of Immunology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Calcineurin Negatively Regulates TLR-Mediated Activation Pathways
Young Jun Kang, Brenda Kusler, Motoyuki Otsuka, Michael Hughes, Nobutaka Suzuki, Shinobu Suzuki, Wen-Chen Yeh, Shizuo Akira, Jiahuai Han, Patricia P. Jones
The Journal of Immunology October 1, 2007, 179 (7) 4598-4607; DOI: 10.4049/jimmunol.179.7.4598

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Calcineurin Negatively Regulates TLR-Mediated Activation Pathways
Young Jun Kang, Brenda Kusler, Motoyuki Otsuka, Michael Hughes, Nobutaka Suzuki, Shinobu Suzuki, Wen-Chen Yeh, Shizuo Akira, Jiahuai Han, Patricia P. Jones
The Journal of Immunology October 1, 2007, 179 (7) 4598-4607; DOI: 10.4049/jimmunol.179.7.4598
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Disclosures
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
  • Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
  • Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Show more CELLULAR IMMUNOLOGY AND IMMUNE REGULATION

Similar Articles

Navigate

  • Home
  • Current Issue
  • Next in The JI
  • Archive
  • Brief Reviews
  • Pillars of Immunology
  • Translating Immunology

For Authors

  • Submit a Manuscript
  • Instructions for Authors
  • About the Journal
  • Journal Policies
  • Editors

General Information

  • Advertisers
  • Subscribers
  • Rights and Permissions
  • Accessibility Statement
  • Public Access
  • Privacy Policy
  • Disclaimer

Journal Services

  • Email Alerts
  • RSS Feeds
  • ImmunoCasts
  • Twitter

Copyright © 2021 by The American Association of Immunologists, Inc.

Print ISSN 0022-1767        Online ISSN 1550-6606