Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori
- 5 January 2010
- journal article
- Published by Springer Science and Business Media LLC in Zeitschrift für Krebsforschung und Klinische Onkologie
- Vol. 136 (6), 911-921
- https://doi.org/10.1007/s00432-009-0733-y
Abstract
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. 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. 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. 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.Keywords
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