Nucleolin as cell surface receptor for tumor necrosis factor-alpha inducing protein: a carcinogenic factor of Helicobacter pylori.
OBJECTIVE
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.
Watanabe
Tatsuro
T
Saitama Cancer Center, Research Institute for Clinical Oncology, Kitaadachi-gun, Saitama 362-0806, Japan.
Tsuge
Hideaki
H
Imagawa
Takahito
T
Kise
Daisuke
D
Hirano
Kazuya
K
Beppu
Masatoshi
M
Takahashi
Atsushi
A
Yamaguchi
Kensei
K
Fujiki
Hirota
H
Suganuma
Masami
M
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002273:Carcinogens / Q000378:metabolism*; D002853:Chromatography, Liquid; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000378:metabolism* / Q000473:pathology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D000097796:Nucleolin
D001426:Bacterial Proteins; D002273:Carcinogens; D016328:NF-kappa B; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D014409:Tumor Necrosis Factor-alpha; D000097796:Nucleolin; C505332:Tipalpha protein, Helicobacter pylori