Saitama Cancer Center, Research Institute for Clinical Oncology, Kitaadachi-gun, Saitama 362-0806, Japan.
J Cancer Res Clin Oncol. 2010 Jun;136(6):911-21. doi: 10.1007/s00432-009-0733-y. Epub 2010 Jan 5.
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.
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.
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.
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.
肿瘤坏死因子-α诱导蛋白（Tipalpha）是一种从幽门螺杆菌（H. pylori）中释放的独特致癌因子。Tipalpha 特异性结合细胞，并被纳入细胞质和细胞核，在那里它通过 NF-κB 激活强烈诱导 TNF-α和趋化因子基因的表达，导致肿瘤的发展。为了阐明 Tipalpha 的作用机制，我们研究了胃上皮细胞中 Tipalpha 的结合蛋白。
通过 FLAG 下拉测定法在小鼠胃癌细胞系 MGT-40 的细胞裂解物中发现了 Tipalpha 结合蛋白，并通过使用抗核仁蛋白抗体的流式细胞术将其鉴定为细胞表面核仁蛋白。通过 Western blot 测定 Tipalpha 的掺入，通过 RT-PCR 定量 TNF-α 基因的表达。
核仁蛋白与 Tipalpha-FLAG 共沉淀，但与 del-Tipalpha-FLAG（无活性突变体）不沉淀。在用 Tipalpha-FLAG 处理后，用抗核仁蛋白抗体共免疫沉淀了内源性核仁蛋白与掺入的 Tipalpha。还证实了 Tipalpha 与重组 His 标记的核仁蛋白片段（284-710）的直接结合。尽管核仁蛋白是核仁中丰富的非核糖体蛋白，但我们发现核仁蛋白存在于 MGT-40 细胞的细胞表面上。用抗核仁蛋白抗体预处理可通过核仁蛋白内化增强 Tipalpha 掺入细胞。此外，用衣霉素预处理，一种 N-糖基化抑制剂，可减少细胞表面核仁蛋白的量，并抑制 Tipalpha 的内化和 TNF-α 基因的表达。
所有结果表明核仁蛋白作为 Tipalpha 的受体起作用，并将 Tipalpha 从细胞表面转运到细胞质和细胞核。这些发现为 Tipalpha 诱导的胃癌发展提供了新的机制见解。
