Structural and Functional Analysis of New Type Extracellular Matrix Receptor syndecan-2

小鼠Lewis肺癌来源的低转移性P29克隆的肿瘤发生依赖于富含纤维连接蛋白的基质，而高转移性LM66-H11克隆的生长依赖于基底膜。P29细胞在体外粘附纤维连接蛋白基质时表现为应激纤维形成，而LM66-H11细胞则表现为皮质作用结构。P29细胞的表型与整合素α5β1和syndrecan-2的表达密切相关，这些整合素α5β1和syndrecan-2具有对cooh末端肝素结合域具有特异性亲和力的硫酸肝素侧链。LM66-H11细胞表达整合素的水平与P29细胞相同，但syndecan-2的表达水平明显低于P29细胞。在体外用syndecan-2的反义寡核苷酸处理P29细胞，选择性抑制其生物合成，细胞表型与LM66-H11细胞无明显区别。P29细胞的表型是通过附着在含有RGD细胞结合域（C-274）和含有RGD细胞结合域（C-274）和c -末端肝素结合域（H271）的纤维连接蛋白重组融合多肽（CH-271）上复制的，而LM66-H11细胞的表型仅由C-274多肽诱导，这表明整合素α5β1和syndecan2对多肽的信号传导是必不可少的，可以被大量的H-271多肽取代。或者是针对辛迪甘-2或硫酸肝素的抗体。此外，当接种于含有C-274多肽和对syndecan-2外结构域具有强亲和力的碱性FGF的融合多肽时，即使是LM66-H11细胞也会形成应激纤维。这些发现表明这两种受体的聚集对于肌动蛋白细胞骨架组织的信号传导是必要的。

Mouse Lewis lung carcinoma-derived low metastatic P29 clone exhibits tumorigenesis dependent on the fibronectin-rich stromal matrix, whereas the growth of highly metastatic LM66-H11 clone depends on the basement membranes. On adhesion to the fibronectin substratum in vitro, P29 cells show stress fiber formation, whereas LM66-H11 cells form cortex action structure. The phenotype of P29 cells is closely correlated to the expression of integrin α5β1 and syndrecan-2 having heparan sulfate side chains with specific affinity to COOH-terminal heparin-binding domain. LM66-H11 cells express the integrin at the same level to that of P29 cells, but syndecan-2 at a significantly lower level. On treatment of P29 cells in vitro with antisense oligonucleotide for syndecan-2, causing selective inhibition of its biosynthesis, the cells turn out to be indistinguishable from LM66-H11 cells as regards the phenotype. The phenotype of P29 cells is reproduced by adhesion to fibronectin recombinant fusion polypeptide (CH-271) comprising RGD cell-binding (C-274) and C-terminal heparin-binding (H271) comprising RGD cell-binding (C-274) and C-terminal heparin-binding (H271) domains whereas the phenotype of LM66-H11 cells in induced only by C-274 polypeptide, indicating that signaling through integrin α5β1 and syndecan-2 is essential for the polypeptide can be replaced by a large amount of H-271 polypeptide, or antibodies specific to syndecan-2 or heparan sulfate. Furthermore, when inoculated on a fusion polypeptide comprising C-274 polypeptide and basic FGF with strong affinity to syndecan-2 ectodomain, even LM66-H11 cells form stress fibers. These findings indicate the necessity of clustering of the two receptors for signaling for the organization of the actin cytoskeletons.