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Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori

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Abstract

Purpose

Tumor necrosis factor-α inducing protein (Tipα) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipα specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-α and chemokine genes mediated through NF-κB activation, resulting in tumor development. To elucidate mechanism of action of Tipα, we studied a binding protein of Tipα in gastric epithelial cells.

Methods

Tipα binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipα into the cells was determined by Western blotting and expression of TNF-α gene was quantified by RT-PCR.

Results

Nucleolin was co-precipitated with Tipα-FLAG, but not with del-Tipα-FLAG (an inactive mutant). After treatment with Tipα-FLAG, incorporated Tipα was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipα to recombinant His-tagged nucleolin fragment (284–710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipα-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipα and expression of TNF-α gene.

Conclusions

All the results indicate that nucleolin acts as a receptor for Tipα and shuttles Tipα from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipα.

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Abbreviations

H. pylori :

Helicobacter pylori

Tipα:

TNF-α inducing protein

CagA:

Cytotoxin associated antigen

LC–MS:

Liquid chromatography–mass spectrometry

RBD:

RNA binding domain

NF-κB:

Nuclear factor-kappa B

NEMO:

NF-κB essential modulator

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Acknowledgments

We thank Ms. Kaori Suzuki and Ikuko Shiotani, Research Institute for Clinical Oncology, Saitama Cancer Center for their technical assistance. We also thank Dr. Masae Tatematsu for supplying of MGT-40 cells. This work was supported by Japan Society for the Promotion of Science, and Smoking Research Fund.

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Authors and Affiliations

  1. Saitama Cancer Center, Research Institute for Clinical Oncology, Kitaadachi-gun, Saitama, 362-0806, Japan

    Tatsuro Watanabe, Atsushi Takahashi & Masami Suganuma

  2. Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama, 338-8570, Japan

    Tatsuro Watanabe & Atsushi Takahashi

  3. Institute for Health Sciences, Tokushima Bunri University, Tokushima, 770-8514, Japan

    Hideaki Tsuge & Takahito Imagawa

  4. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, 770-8514, Japan

    Daisuke Kise & Hirota Fujiki

  5. Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392, Japan

    Kazuya Hirano & Masatoshi Beppu

  6. Hospital, Saitama Cancer Center, Saitama, 362-0806, Japan

    Kensei Yamaguchi

Authors
  1. Tatsuro Watanabe
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  2. Hideaki Tsuge
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  3. Takahito Imagawa
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  9. Hirota Fujiki
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  10. Masami Suganuma
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Correspondence to Masami Suganuma.

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Watanabe, T., Tsuge, H., Imagawa, T. et al. Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori . J Cancer Res Clin Oncol 136, 911–921 (2010). https://doi.org/10.1007/s00432-009-0733-y

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  • DOI: https://doi.org/10.1007/s00432-009-0733-y

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