Zhang Shu, Liu Tao, Liang Huifang, Zhang Hailong, Yan Dan, Wang Nidan, Jiang Xiaodan, Feng Wei, Wang Jing, Li Pinlan, Li Zhuoya
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan, Hubei 430030, China.
Mol Immunol. 2009 Apr;46(7):1551-60. doi: 10.1016/j.molimm.2009.01.001. Epub 2009 Feb 8.
Since transmembrane tumor necrosis factor-alpha (tmTNF-alpha) has been reported to have a palmitoylated site at Cys(-47), and therefore its functions may be linked to lipid raft membrane microdomains. The present study tested a hypothesis that lipid rafts may serve as a signaling platform to mediate the bioactivity of tmTNF-alpha. We found that destruction of lipid rafts with methyl-beta-cyclodextrin (MCD) in Raji cells almost completely blocked the cytotoxicity of tmTNF-alpha, as did an anti-TNF-alpha antibody. Although a proportion of tmTNF-alpha was colocated with lipid rafts, either the replacement of Cys at -47 by Ala, destructing its possible lipid rafts-attaching site or the displacement of its cytoplasmic domain by the C-terminal sequence (131-157) of caveolin-1, making all tmTNF-alpha target to lipid rafts, had no effect on tmTNF-alpha cytotoxicity. The data suggest that the cytotoxicity of tmTNF-alpha is not associated with its lipid rafts location. Unparallel to decreased cytotoxicity, moreover, MCD significantly increased tmTNF-alpha expression on the cell surface, and these increased tmTNF-alpha molecules were capable of binding to sTNFR1. To further explore the mechanism of lipid rafts-mediated cytotoxicity of tmTNF-alpha, we demonstrated that MCD led to a marked decrease in adhesion of Raji cells to T24 cells, which was due to dissociation of adhesion molecule ICAM-1 from lipid rafts. These results indicate that lipid rafts importantly participate in the cytotoxicity of tmTNF-alpha through ICAM-1 clustering and consequent enhancement of the cell-cell contact. The data suggest that lipid rafts are essential for the killing of tmTNF-alpha through the cell-cell contact mediated by ICAM-1. However, lipid rafts may limit exposure of tmTNF-alpha to the cell surface.
由于据报道跨膜肿瘤坏死因子-α（tmTNF-α）在Cys（-47）处有一个棕榈酰化位点，因此其功能可能与脂筏膜微区相关。本研究验证了一个假说，即脂筏可能作为一个信号平台来介导tmTNF-α的生物活性。我们发现，用甲基-β-环糊精（MCD）破坏Raji细胞中的脂筏几乎完全阻断了tmTNF-α的细胞毒性，抗TNF-α抗体也有同样的效果。尽管一部分tmTNF-α与脂筏共定位，但将-47位的Cys替换为Ala（破坏其可能的脂筏附着位点）或用小窝蛋白-1的C末端序列（131-157）取代其胞质结构域（使所有tmTNF-α靶向脂筏），对tmTNF-α的细胞毒性均无影响。数据表明，tmTNF-α的细胞毒性与其在脂筏中的定位无关。此外，与细胞毒性降低不同的是，MCD显著增加了细胞表面tmTNF-α的表达，并且这些增加的tmTNF-α分子能够与sTNFR1结合。为了进一步探究脂筏介导的tmTNF-α细胞毒性的机制，我们证明MCD导致Raji细胞与T24细胞的黏附显著降低，这是由于黏附分子ICAM-1从脂筏上解离所致。这些结果表明，脂筏通过ICAM-1聚集以及随之增强的细胞间接触，重要地参与了tmTNF-α的细胞毒性作用。数据表明，脂筏对于通过ICAM-1介导的细胞间接触杀伤tmTNF-α至关重要。然而，脂筏可能会限制tmTNF-α暴露于细胞表面。
