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

Abstract

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.

Fig. 1

Identification of nucleolin as Tipα binding protein. a Schematic representation of Tipα-FLAG, del-Tipα-FLAG and C5A/C7A-Tipα-FLAG proteins (top). Induction of TNF-α gene expression with Tipα-FLAG and with del-Tipα-FLAG in MGT-40 cells (bottom). Total RNAs were isolated from MGT-40 cells 1 h after treatment with Tipα-FLAG and with del-Tipα-FLAG, and the levels of TNF-α and GAPDH mRNAs were determined by semi-quantitative RT-PCR, as described in Materials and methods. b Representative results of FLAG pull-down assay. After incubation of MGT-40 cell lysates with Tipα-FLAG (Tip) and with del-Tipα-FLAG (del), Tipα-FLAG and del-Tipα-FLAG were immunoprecipitated with anti-FLAG antibody. The polypeptides that co-immunoprecipitated with Tipα-FLAG and with del-Tipα-FLAG were resolved in 4–12% NuPAGE and then stained with Quick CBB (left panel) and immunoblotted with anti-nucleolin antibody (IB: right panel). c Amino acid sequence of mouse nucleolin. Amino acids with red characters are assigned to the sequences determined by LC–MS analysis. Underlined sequences are recognition sites of anti-NUC295

Fig. 2

Direct interaction of nucleolin with Tipα. a Tipα was immunoprecipitated with endogenous human nucleolin in MKN-1 and THP-1 cells. MKN-1 and THP-1 cells were treated with 100 μg/ml Tipα-FLAG (Tip) and with del-Tipα-FLAG (del) at 37°C for 1 h. Tipα-FLAG and del-Tipα-FLAG significantly incorporated into the cells (left panels). Each cell lysate was immunoprecipitated with anti-nucleolin antibody (NUC) and with rabbit IgG (as a control, IgG). Immunoprecipitates were resolved in 12% SDS-PAGE and immunobotted (IB) with anti-FLAG antibody and anti-nucleolin antibody (right panels). b Direct interaction of recombinant human nucleolin fragment with Tipα in vitro. His-tag removed Tipα-FLAG (Tip) and His-tag removed C5A/C7A-FLAG (C5A), which were prepared as described in Experimental procedures, were incubated with a 6-His-tag fused recombinant human nucleolin fragment containing 284–710 amino acid residues (NUC284) and then subjected to pull-down assay using Ni²⁺ chelating resins. The precipitates were resolved in 12% SDS-PAGE and analyzed by Western blotting with anti-Tipα antibody and with anti-nucleolin antibody

Fig. 3

Localization of nucleolin on cell surface of MGT-40 and THP-1 cells. a Subcellular localization of nucleolin analyzed by cell fractionation. MGT-40 and THP-1 cells were fractionated into membrane (M), cytosolic (C) and nuclear (N) fractions, and each fraction was immunoblotted with anti-nucleolin antibody. Each fraction was confirmed by Western blotting with antibodies for fractionation-marker proteins: EGFR for membrane of MGT-40 cells, TNF-R2 for membrane of THP-1 cells, HSP90 for cytosol and lamin B for nuclei. b Detection of nucleolin on cell surface shown by flow cytometry. MGT-40 cells were incubated with 1 μg/ml anti-NUC295 (Anti-NUC295) and with pre-immune serum (Pre-serum) as a control in the presence of 10 μg/ml Alexa Fluor 488-conjugated goat rabbit IgG on ice for 30 min. Preincubation of Anti-NUC295 with recombinant nucleolin fragment (Anti-NUC+GST-NUC284) significantly reduced fluorescence. c Strong induction of TNF-α gene expression with Tipα in THP-1 cells (filled circle) and MGT-40 cells (open circle). One hour after treatment with Tipα at various concentrations, expression of TNF-α and GAPDH genes was determined by semi-quantitative RT-PCR. Relative expression of TNF-α gene is shown as fold change compared with control after normalization of GAPDH mRNA levels. The results are the averages of three independent experiments. Bars indicate standard deviation. Statistical levels between non-treated and Tipα-treated cells were shown to be significant *P < 0.01

Fig. 4

Significant enhancement of Tipα-induced TNF-α gene expression and Tipα incorporation in cells induced by anti-NUC295. a MGT-40 cells were previously incubated with rabbit IgG, with anti-NUC H-250 and anti-NUC295 antibodies at 4°C for 1 h, and further treated with 50 μg/ml Tipα at 37°C for 1 h. Relative TNF-α gene expression is shown as fold change compared with that of cells treated with 50 μg/ml Tipα after normalization of GAPDH gene expression levels. The results are the averages of three independent experiments. Bars indicate standard deviation. Statistical significance of effects of anti-NUC295 in TNF-α induction by Tipα compared with non-treated were shown as *P < 0.01 and **P < 0.05, and the difference between 2 and 5 μg/ml of anti-NUC295 was significant at the level of **P < 0.05. b Incorporation of Tipα was determined by Western blotting with anti-Tipα antibody. Nucleolin levels were also determined by anti-nucleolin antibody

Fig. 5

Inhibition of Tipα-induced TNF-α gene expression and Tipα incorporation in cells induced by down-regulation of cell surface nucleolin. a MGT-40 cells were treated with or without 5 μg/ml tunicamycin in DMSO at 37°C for 5 h. The cell surface nucleolin on MGT-40 cells was visualized using flow cytometry with anti-NUC295, as described in Materials and methods. b Inhibition of TNF-α gene expression induced by Tipα after pretreatment with tunicamycin. After pretreatment of MGT-40 cells with or without 5 μg/ml tunicamycin in DMSO at 37°C for 5 h, the cells were treated with various concentrations of Tipα at 37°C for 1 h. The levels of TNF-α and GAPDH gene expression in MGT-40 cells were determined by semi-quantitative RT-PCR. The results are the averages of three independent experiments. Bars indicate standard deviation. Statistical levels were significant *P < 0.01 and **P < 0.05. c Inhibition of Tipα-incorporation into MGT-40 cells by pretreatment with tunicamycin. After treatment with tunicamycin, both MGT-40 cells treated with various concentrations of Tipα and cell lysates were resolved in 12% SDS-PAGE solution and analyzed by Western blotting with anti-Tipα antibody and anti-nucleolin antibody (IB)