PURPOSE: Tumor necrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS: Tipalpha 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 Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS: Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha 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 Tipalpha-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 Tipalpha and expression of TNF-alpha gene. CONCLUSIONS: All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.
PURPOSE:Tumornecrosis factor-alpha inducing protein (Tipalpha) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipalpha specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-alpha and chemokine genes mediated through NF-kappaB activation, resulting in tumor development. To elucidate mechanism of action of Tipalpha, we studied a binding protein of Tipalpha in gastric epithelial cells. METHODS: Tipalpha binding protein was found in cell lysates of mousegastric 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 Tipalpha into the cells was determined by Western blotting and expression of TNF-alpha gene was quantified by RT-PCR. RESULTS:Nucleolin was co-precipitated with Tipalpha-FLAG, but not with del-Tipalpha-FLAG (an inactive mutant). After treatment with Tipalpha-FLAG, incorporated Tipalpha was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipalpha 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 Tipalpha-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 Tipalpha and expression of TNF-alpha gene. CONCLUSIONS: All the results indicate that nucleolin acts as a receptor for Tipalpha and shuttles Tipalpha from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipalpha.