Yoneda J, Saiki I, Igarashi Y, Kobayashi H, Fujii H, Ishizaki Y, Kimizuka F, Kato I, Azuma I
Institute of Immunological Science, Hokkaido University, Sapporo, Japan.
Exp Cell Res. 1995 Mar;217(1):169-79. doi: 10.1006/excr.1995.1076.
Cellular responses to fibronectin (FN) are likely to have a complex molecular basis involving the interactions between multiple functional domains of FN and specific cell surface molecules. We have utilized several types of recombinant FN fragments and their chimeric fragments to examine the regulatory mechanisms of the spreading and chemotactic migration of HT1080 human fibrosarcoma cells on FN. The CH-271 fusion fragment, in which the cell-binding domain (C-274) of FN is adjacent to the heparin-binding domain (H-271), promoted cell spreading more efficiently than C-274, H-271, or their mixture (C-274 + H-271) over a 60-min incubation. The CH-271 variants containing various modules in the heparin-binding domain (CHV peptide) showed different promotion of cell migration, spreading, and vinculin accumulation at focal adhesion, respectively. The preincubation of the cells with heparitinase I resulted in significant inhibition of chemotactic migration to FN and its fragments containing the III13 and/or III14 modules of the heparin-binding domain. Additionally, migration to CH-271 was inhibited by the presence of the RGDS peptide in a concentration-dependent fashion. Thus, the spread and migration responses of HT1080 cells onto FN fusion peptides require the adjacent coexistence of cell- and heparin-binding domains and are mediated by the interactions between cell surface heparan sulfate and the heparin-binding domain, in concert with the interaction between cell surface integrin and the cell-binding domain. In conclusion, our present study demonstrated that fusion peptides of fibronectin can efficiently induce two signals from the cell-binding and heparin-binding domains required for the completion of cell spreading, the formation of focal contact, and motility at the early stage of the culture, suggesting that the III13 or III14 modules within the heparin-binding domain are responsible for the initiation of different cellular responses.
细胞对纤连蛋白（FN）的反应可能具有复杂的分子基础，涉及FN多个功能域与特定细胞表面分子之间的相互作用。我们利用了几种类型的重组FN片段及其嵌合片段，来研究HT1080人纤维肉瘤细胞在FN上的铺展和趋化迁移的调控机制。CH - 271融合片段中，FN的细胞结合域（C - 274）与肝素结合域（H - 271）相邻，在60分钟的孵育过程中，它比C - 274、H - 271或它们的混合物（C - 274 + H - 271）更有效地促进细胞铺展。在肝素结合域中含有各种模块的CH - 271变体（CHV肽）分别对细胞迁移、铺展以及粘着斑处纽蛋白的积累表现出不同程度的促进作用。用肝素酶I对细胞进行预孵育，可显著抑制细胞对FN及其含有肝素结合域III13和/或III14模块的片段的趋化迁移。此外，RGDS肽的存在以浓度依赖的方式抑制细胞向CH - 271的迁移。因此，HT1080细胞在FN融合肽上的铺展和迁移反应需要细胞结合域和肝素结合域相邻共存，并由细胞表面硫酸乙酰肝素与肝素结合域之间的相互作用介导，同时还与细胞表面整合素和细胞结合域之间的相互作用协同发挥作用。总之，我们目前的研究表明，纤连蛋白融合肽可以在培养早期有效地诱导细胞铺展、粘着斑形成和运动所需的来自细胞结合域和肝素结合域的两种信号，这表明肝素结合域内的III13或III14模块负责引发不同的细胞反应。
